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Effects of Hyperbilirubinemia on Cisplatin Nephrotoxicity


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1 EFFECTS OF HYPERBILIRUBINEMIA ON CISPLATIN NEPHROTOXICITY By KARRI ANN BARABAS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 Karri Ann Barabas

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3 ACKNOWLEDGMENTS I would like to thank my family for their support and love thro ughout my education. I would like to also thank Dr. Ch ristopher Adin, Dr. Rowan Milner Dr. Jim Farese, and Dr. Chris Baylis for their mentorship and guidance during the pursuit of this degree. Additional thanks go to Dr. Dan Lewis for providing the position that allowed me to achieve this degree. Lastly, I would like to thank Linda Archer and Marc Sa lute for their technica l help throughout this project.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES................................................................................................................ .........7 ABSTRACT....................................................................................................................... ..............8 CHAPTER 1 CISPLATIN: A REVIEW.....................................................................................................11 Introduction................................................................................................................... ..........11 Chemistry...................................................................................................................... ..........11 Pharmacokinetics............................................................................................................... .....12 Pharmacodynamics............................................................................................................... ..13 Mechanisms of Resistance......................................................................................................14 Toxicities..................................................................................................................... ...........15 Nephrotoxicity.................................................................................................................15 Hypomagnesemia and Hypocalcemia.............................................................................19 Gastrointestinal and Myelosuppression...........................................................................19 Ototoxicity.................................................................................................................... ...20 Neurotoxicity.................................................................................................................. .20 Syndrome of Inappropriate Secretion of Antidiuretic Hormone (SIADH).....................20 Protective Measures for Nephrotoxicity.................................................................................21 Saline Diuresis................................................................................................................ .21 Diuretics...................................................................................................................... ....21 Hypertonic Saline............................................................................................................22 Pharmaceuticals...............................................................................................................22 Enzymatic and Molecular Alterations.............................................................................23 Cisplatin Use in Animals....................................................................................................... .24 Dogs........................................................................................................................... ......24 Cats........................................................................................................................... .......29 Horses......................................................................................................................... .....29 Other Platinum Drugs........................................................................................................... ..30 Summary........................................................................................................................ .........30 2 HYPERBILIRUBINEMIA PROTECTS AGAINST CISPLATIN NEPHROTOXICITY IN THE GUNN RAT..............................................................................................................36 Introduction................................................................................................................... ..........36 Materials and Methods.......................................................................................................... .37 Animals........................................................................................................................ ....37 Cisplatin-Induced Acute Renal Failure...........................................................................38

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5 Assays......................................................................................................................... .....39 Histologic Grading..........................................................................................................39 Cell Lines..................................................................................................................... ....40 Cell Viability Assay........................................................................................................40 Statistical Analysis..........................................................................................................41 Results........................................................................................................................ .............42 Renal Functional Parameters...........................................................................................42 Light Microscopy............................................................................................................43 Cell Culture Studies.........................................................................................................44 Discussion..................................................................................................................... ..........44 3 CONCLUSION..................................................................................................................... ..54 LIST OF REFERENCES............................................................................................................. ..57 BIOGRAPHICAL SKETCH.........................................................................................................72

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6 LIST OF TABLES Table page 1-1 Canine diuresis protocols when administering cisplatin....................................................32 2-1 Serum bilirubin concentrations were significantly higher for the Gunn j/j when compared to the other groups.............................................................................................49

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7 LIST OF FIGURES Figure page 1-1 Two-dimensional structures for cisplati n, carboplatin, oxaliplatin, and lobaplatin. While the core structure (Pt) is the same for each drug, the leaving groups are different for each compound..............................................................................................33 1-2 Aquation reaction and adduct formation at the N-7 position of guanine on 2 sites of DNA. These adducts result in DNA damage resulting in cell kill....................................34 1-3 Four pathways exist for cisplatin resistance......................................................................35 2-1 Serum BUN concentrations on D0, 3, and 5. No significant difference between any groups on D0. The most significant diffe rence in BUN occurred when comparing the Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a significantly higher BUN .................................................................................................................... ...49 2-2 Serum creatinine concentrations on D0, 3, and 5. No significant difference between any groups on D0. The most significant difference in creatinine occurred when comparing the Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a significantly creatinine.......................................................................................................50 2-3 Histologic grading showed significant preservation of the OSOMPT in homozygous Gunn rats when compared to heterozygous Gunn rats and Wistar rats given cisplatin...................................................................................................................... ........52 2-4 Viable cell count for one of the canine osteosarcoma cell lines utilized (POS)................53

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8 Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF HYPERBILIRUBINEMIA ON CISPLATIN NEPHROTOXICITY By Karri Ann Barabas May 2007 Chair: Rowan Milner Major: Veterinary Medical Sciences Cisplatin is a powerful chemotherapeutic agen t used in a variety of malignancies in many species. Systemic dose-related toxicities associat ed with cisplatin have precluded its use in many species. Of these toxicities, nephrotoxicity is the most frequent a nd clinically significant toxicity. No specific compound has yet ameliora ted cisplatin nephrotoxic ity. Recent research has shown that the heme oxygenase-1 (HO-1) enzy me provides beneficial effects in mitigating cisplatin nephrotoxicity in a rodent model. HO-1 is normally induced in response to cellular stress and converts the heme molecule into equimolar quantities of biliverdin (BV), carbon monoxide (CO), and iron. Biliverdin is then converted to bilirubin (BR) by the enzyme biliverdin reductase. Many previous studies s uggest that HO and its products are important endogenous mechanisms for cytoprotection. These products were once cons idered to be toxic metabolites but have been shown to have dose-de pendent vasodilatory, anti-oxidant, and antiinflammatory properties that are desirable for tis sue protection during a to xic insult. Our first objective was to review the lite rature regarding cisplatin and, in particular, nephrotoxicity associated with cisplatin administration and cu rrent methods to prevent nephrotoxicity. Our second objective was to determine if hyperbi lirubinemia would ameliorate cisplatin

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9 nephrotoxicity in a rat model. Our third object ive was to determine if bilirubin would prevent cisplatins ability to kill neoplastic cells in vitro The in vivo cisplatin model involved 3 groups of rats (n=6 rats/group): homozygous Gunn rats (j/j), heterozygous Gunn rats (j/+) and conge nic Wistar (+/+) rats. Homozygous Gunn rats lack the UDPGT enzyme needed to conjugate bilirubin resulting in an unconjugated hyperbilirubinemia while hetero zygous Gunn rats lack normal levels of UDGPT, but can conjugate bilirubin to a certain de gree. On Day 0, all rats were an esthetized and administered 4 mg/kg cisplatin IP. Blood was sampled on Da y 0, 3 and 5 for comparison of serum BR, creatinine (Cr), and BUN. On Day 5, kidney tissue samples were obtained prior to euthanasia. Cell culture studies were then performed us ing 4 canine osteosarcoma cell lines (POS, HMPOS, COS, D17) incubated with the average c oncentrations of BR for j/j rats at Day 0 and 3. We added BR to all cell lines (alone and with ci splatin) and cell viabilit y was assessed using the CellTiter BlueTM assay. Serum BR levels were 72 16 M/L in homozygous Gunn rats, 7 3 M/L in heterozygous Gunn rats, and 0 0 M/L in Wistar rats at Day 0. The BR provided a dosedependent nephroprotective effect, with significantly lower BUN (24 5 mg/dL) and Cr (0.35 0.05 mg/dL) in homozygous Gunn rats when compar ed to Wistar rats (BUN79 17 mg/dL, Cr1.4 0.4 mg/dL) at Day 5 (P <0.05). An interm ediate level of nephroprotection was noted in the heterozygous Gunn rats, although BUN ( 38 10 mg/dL) and Cr (0.4 0.06 mg/dL ) remained significantly lower than Wistar ra ts at Day 5 (P <0.05). Histological grading demonstrated preservation of the S3 segment in homozygous Gunn rats when compared to heterozygous Gunn rats and Wistar rats (P <0.05). The BR had no significant effect on the

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10 antineoplastic effect of cisplatin at either c oncentration in the 4 osteosarcoma cell lines (P <0.001). Hyperbilirubinemia in the homozygous Gunn ra t provided nearly complete preservation of renal function and pathology in this model of cisplatin nephrotoxicity. Addition of exogenous BR did not interfere with the antineoplastic activ ity of this chemotherapy agent in cell culture.

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11 CHAPTER 1 CISPLATIN: A REVIEW Introduction Cisplatin is one of the most potent chemothe rapy agents used in human and veterinary medicine. Its use in veterinary medicine bega n more than 2 decades ago and was prompted by the success of cisplatin in trea ting human malignancies. Unfort unately, cisplatin administration was associated with numerous adverse side effect s including: nephrotox icity, severe nausea and vomiting, myelosuppression, ototoxic ity, and neurotoxicity. Of th ese, the most clinically significant and common toxicity is nephrotoxicity. Despite the ne phrotoxicity, many veterinary oncologists are of the opinion that cisplatin is more potent than its other platinum counterparts with regard to its anti-neoplas tic activity. This has resulted in its continued use throughout veterinary hospitals around the world for malignanc ies such as osteosarcoma (OSA), transitional cell carcinoma (TCC), intralesional th erapy, and radiati on sensitization. Since cisplatins development, research has centered on mitigating nephrotoxicity to allow cisplatin to be delivered at therapeutic doses without adversely affecting the kidneys. Recent studies have discovered new prot ocols, compounds, enzymes, and molecular alterations that reduce the nephrotoxicity of cisplatin. This review will focus on cisplatins chemistry, pharmacokinetics, pharmacodynamics, mechanisms of resistance, toxicity, prevention of nephrotoxicity, and use in animals. Also included is a section on the other platinum drugs th at have been used in veterinary medicine to date. Chemistry Cisplatin was inadvertently discovered while studying the growth characteristics of Escherichia coli (1) Initially, research ers observed that platin um compounds exhibited

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12 antibacterial properties and s ubsequently it was discovered that they also possessed antineoplastic properties. (1, 2) The molecular stru cture of cisplatin compri ses a central platinum atom surrounded by two chlorine atoms and two ammonia groups in a cis configuration. (3) Other platinum compounds have th e same core platinum compound, and cis configuration, however, their leaving groups are different (Figure 1-1). (4) Th e bond angles for the platinum core are fixed resulting in DNA bending to ac commodate the structure of the drug. (4) Pharmacokinetics Plasma platinum has been shown to be highly protein bound. (5) Most cisplatin present in the cell is found in the cytosol and is not prot ein bound. (6) Cisplatins clearance in the dog is biphasic in nature with a rapid pha se half-life of 22 minutes and a slow phase half-life of 5 days. (7) Significant amounts of platinum are still de tectable in plasma 12 days after intravenous injection. (7) A study performed in humans de monstrated that plasma platinum levels corresponded with nephrotoxicity. (8 ) Plasma platinum levels may serve as a marker for risk of nephrotoxicity in certain patients. Urinary levels of platinum in the dog elevat e rapidly after administ ration with 60% of the dose recovered in the urine within the first 4 hou rs and 76% of the administered dose recovered by 48 hours after treatment. (7, 9) Only small amounts of platinum were detected in bile suggesting minimal fecal excretion. (7) Free platinum clearance has been shown to exceed the creatinine clearance by 156% suggesting that in a ddition to excretion by filtration, cisplatin or a metabolite is secreted by the kidney. (5) It is thought that secretion i nvolves active accumulation of secreted substances in the renal cells and passi ve transport into the tubu le of the lumen. (10) The pars recta of the proximal tubule is the mo st active site of secre tion and also the most damaged site of the kidney during cisplatin neph rotoxicity. (5) Renal accumulation of platinum is dependent on the presence of normal oxygen ut ilization and the organic base transport

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13 system. (11) Thus, concurrent administration of drugs known to be transported by this system can significantly reduce cisplatin uptake in the kidney. (11) Cisplatin is initially distribut ed to all tissues, however, in the first hour, it tends to accumulate in the kidney, liver, muscle, and skin. (7) The localization in the kidney and liver is protracted, with high renal tissue concentrations present as long as 12 days after treatment in the dog. (7) The highest tissue platinum concentrati ons occur in those tissues where the drug exerts its most potent antineoplastic activity, such as th e ovary and uterus. (7) It is thought that the presence of tumor may alter the toxicity and pha rmacokinetics of drugs. (12) One study in tumor bearing rats showed that the di stribution half-time was longer fo r the tumor bearing rats than their controls, while the terminal elimination ha lf-time was the same for both groups. (12) Based on this study, it is unlikely that tumor presence would alter toxicity or other distributiondependent drug parameters. (12) Pharmacodynamics Cisplatin is activated by an a quation reaction involving the exchange of the two chloride leaving groups with water or hydr oxyl ligands. (13) When a high concentration of chloride is present, as in isotonic saline or extracellular fluid, the aquation reaction does not occur and the drug remains neutral. (13) The neutral form is believed to be biologi cally inactive. (14) Intracellular fluid has approximate ly one-thirteenth the chloride concentration of extracellular fluid and it is under these conditi ons that the aquation reaction pr oceeds, leading to eventual DNA damage. (13) The primary e ffect produced by cisplatin in ca ncerous cells is inhibition of DNA synthesis. (15, 16) The ability to inhibit DNA synthesis occu rs at much lower doses than that necessary to inhibit RNA and protein synthe sis. (15) DNA damage induced by cisplatin is similar to that caused by alkylating agents. (1 7) With aquation of the platinum compound the two chloride groups are replaced wi th water and will bind to two s ites in DNA. (4) Generally, if

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14 the two sites are on the same DNA strand, the lesi on is referred to as a DNA adduct and if the sites are on different strands, the lesion is referred to as a DNA cross-link. (4) Cisplatin has been noted to bind to all DNA bases but has a prefer ence for the N-7 positions of adenine and guanine due to the high nucleophilicity of the N-7 sites of these purine bases (Figure 1-2). (4, 13, 18) Cisplatin forms bifunctional adducts >90% of the time with crosslinks being < 2% of the lesions formed. (4) These adducts and crosslinks inhi bit DNA template replication in mammalian cells. (19) DNA crosslinks and ad ducts increase with time afte r the drug is removed and are repaired slowly. (20) In vitro studies have also indicated that interaction between the cisplatin molecule and DNA may contribute to the genera tion of superoxide radicals, causing further toxicity to cancer cells. (21, 22) Mechanisms of Resistance Four generic pathways for cisplatin resistance have been uncovered. They include: altered cellular accumulation, cyto solic inactivation of cisplatin, DNA repair, and altered apoptosis (Figure 1-3). (4) In vitro studies have described active effl ux particularly as mediated by CuH transporters, ATP7A and ATP7B, and other less we ll-defined systems. (4) Covalent binding of proteins or peptides with incr eased levels of sulfhydryl groups to cisplatin may confer cellular resistance. (4) These compounds include glut athione (GSH) and metallothionein. (23-26) MRP2 (multidrug resistance-associated protein 2) may also play a role in cisplatin resistance by removing the cisplatin-GSH complex from the cells (27) Platinum-DNA repair occurs by the nucleotide-excision repair (NER) and NER is increased in cispla tin-resistant cells. (4, 28, 29) Mismatch repair (MMR) mediates apoptosis in response to cisplatin. (30-32) Defects in MMR result in altered cell sensitivity to cisplatin, most likely resulting in greater resistance. (4) Reports indicate that where altera tions in MMR exist, concurrent enhancement of the activity of NER exists. (4)

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15 Toxicities Nephrotoxicity Cisplatin is associated with several systemic toxicities, but is most frequently associated with nephrotoxicity. Cisplatin induced nephrotoxicity occurs in a number of species including mice, rats, dogs, and humans. An estimated 28 to 36% of human patient s receiving an initial dose of 50-100 mg/m2 of cisplatin develop acute renal failur e. (33, 34) Cisplatin nephrotoxicity is dose and duration of treatment dependent and is enhanced by the use of other nephrotoxins such as aminoglycosides. (35, 36) Most patien ts who develop some de gree of renal dysfunction never fully recover. (37) Howe ver, one study evaluating the long-te rm renal effects of cisplatin in human patients showed that renal dysfunction may not be progressive provided further insult is avoided. In this study, an initial increase in cr eatinine and a decrease in GFR and renal plasma flow were noted directly after treatment, but these levels rema ined stable for up to 12 to 24 months after discontinuing ci splatin treatment. (38) The morphologic alterations in the kidney attributed to cispla tin administration occur in the pars recta of the proximal tubule situated in the outer stripe of the medulla. (39) Histological changes are consistent with both apoptosis and necrosis. (40) One of the earliest histopathological changes noted is the swelling of mitochondria. (1 4) Most of the pathological changes start 3 days after cisplatin admini stration, including clum ped nuclear chromatin, increased number of cytoplasmic vesicles, focal loss of microvillus brush border, and completely necrotic cells sloughed in the tubular lumen. (39) The most severe damage is seen 5 days after cisplatin administration and cons ists of widespread tubular n ecrosis in the pars recta, desquamation of necrotic epithe lia cells resulting in a denuded basement membrane, necrotic cells and debris in the tubular lumen, and the changes seen in 3 days after cisplatin

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16 administration for non-necrotic cells. (39) By 7 days after cisplatin administration, extensive regeneration in the pars rect a is noted with necrotic cells still present. (39) Research into the mechanism of cisplatin nephr otoxicity is an importa nt step in developing methods for renal protection. One theory invo lves DNA crosslinks and the position in the cell cycle. It was thought that cisp latin-DNA crosslinks could be the cause of cytotoxicity in the renal cell, but the proximal tubule cells selectively killed by cispla tin are relatively quiescent and therefore should not be as sensi tive to the toxicity of DNA damaging agents. (41) However, there is a fall in DNA turnover that precedes necrosis in the proximal tubule and the later increase in DNA turnover in those cells coincides with the timelin e of regeneration. (42) Both the outer cortex and outer stripe of the outer medulla (pars recta) have decreased DNA synthesis 1 day after cisplatin administrati on yet only the cells in the pars recta undergo necrosis. (42) Three possibilities have been cons idered regarding this theory in cluding: i nhibition of DNA synthesis is irrelevant to cytotoxicity in the kidney; cells in the pars recta cannot repair the damage as cells elsewhere can; or the levels of DNA adducts in th e pars recta are lethal whereas those produced in other segments are not. (42, 43 ) Interestingly, recent studies have related cisplatin administration to altera tions in the cell cycle. Cells in the kidney enter the cell cycle after cisplatin administration a nd genes for the p21 and 14-3-3 cell cycle inhibitors are simultaneously upregulated. (44-46) Mice with a deleted p21 gene were more sensitive to cisplatin injury. (46) Price et al. showed that the addition of p21 adenovirus and the pharmacological inhibitor of cyclin dependent ki nases, roscovitine both protected kidney cells from cisplatin induced nephrotoxicity in vitro. (47) p21 inhibits caspase activation which is discussed later as a mechanism of apoptos is in the renal tubule. (47)

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17 Platinum drugs are similar to other heavy metals such as mercury. Another possible mechanism for nephrotoxicity incorporates the known mechanism of ne phrotoxicity in these other heavy metals. (48) This toxicity occurs due to the binding of cisplatin to sulfhydryl (SH) groups in the kidney which are necessary for en zyme function and depletion of intracellular glutathione. (48, 49) The decrease in SH groups occurred before the rise in BUN and creatinine and was not seen with acute renal failure cause d by glycerol, another potent nephrotoxin. (49) Proximal tubular cell death was initially believe d to occur mostly by necrosis. However, another mechanism of proximal tubule cell death is apoptosis. A previous study showed that the type of cell death was concentra tion dependent with high concentra tions of cisplatin leading to necrosis and low concentrations causing apoptos is. (50) Reactive oxygen species (ROS) and mitochondria are thought to play a role in the apoptotic cascade. (51) Mitochondrial dysfunction occurs early in cisplatin-induced renal tubular toxicity and is potentially mediated by ROS. (52, 53) It was shown in a previous study that ove rexpression of manganese superoxide dismutase, an antioxidant enzyme found in mitochondria, prot ected renal epithelial cells in vitro from cisplatin toxicity. (54) In vitro studies performed on renal proximal tubule cells examined the role of caspases and p53 in apoptos is related to cisplatin. Lau et al. showed that caspase 3 was activated in vitro in response to cisplatin bu t the initiators of the activat ion were not found. (55) The tumor suppressor gene p53 is activated in response to DNA damage, a lterations in the cell cycle, and hypoxia. (56) Another study found th at 50% of cisplatin induced renal proximal tubular cell apoptosis was mediated by p53 and that p53 activates caspase 3 independent of other caspases or mitochondrial dysfunction. (57) The other 50% is me diated by additional mechanisms independent of p53 and caspases 3, 8, and 9. (57) Interestingly, a different in vitro

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18 study performed by Xiao et al. showed that when a caspase in hibitor that has no effect on p53 was applied to renal tubular cells, cisplati n-induced apoptosis did not occur. (58) Recent studies in rats and mice have shown th at the nephrotoxicity of cisplatin can be blocked by inhibiting either of two enzymes e xpressed in proximal tubules, gamma-glutamyl transpeptidase (GGT) or cysteine-S-conjugate beta-l yase. (59-61) This suggested that metabolic activation of cisplatin to a nephrot oxin occurred in the kidney. (41) For this reaction to occur, cisplatin must form a conjugate with glutathione which has been shown to occur spontaneously in solution. (62, 63) Selective inhibition of each enzyme resulted in a decrease in toxicity, in vitro (41) Interestingly, conjugati on of cisplatin with glutathione reduces cisplatin crosslinks with DNA resulting in decreased toxicity to dividing cells, suggesti ng decreased antitumor activity. (64) Also, GGT expression in tumors ha s been shown to decrease the antitumor activity of cisplatin. (65) These c onflicting reports show that th ere are many areas regarding mechanisms of cisplatin-induced apoptosis and necros is that still need to be investigated. The physiologic alterations seen with cisplatin are relatively consiste nt. Renal failure is gradual and usually occurs 3 to 5 days after administration. (66) Polyuria may be due to a reduction in normal cortical-papillary solute gradie nt in association with a failure to recycle urea. (67) Whole kidney GFR, single nephron GFR, and renal pl asma flow are all decreased after cisplatin administration. (38, 38, 42, 68) Initially, it was t hought that the renin-angiotensin system may play a role in ci splatin-induced acute renal failure although experimental studies failed to confirm this hypothesis. (42, 69) In rats, decreases in GFR are related to afferent vasoconstriction and possibly an al tered ultrafiltration coefficient, both of which occur before evidence of tubular obstruction. (70) It should be restated that histopathologically, the glomerulus is minimally affected by cisplatin.

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19 Hypomagnesemia and Hypocalcemia Other physiologic changes related to cisplatin administration include hypomagnesemia and hypocalcemia. Hypomagnesemia has been reported to occur in more than ha lf of human patients receiving cisplatin chemotherapy. (7 1) The persistent excretion of magnesium in the presence of declining magnesium levels suggests that the hypomagnesemia is due to a renal defect in magnesium reabsorption. (71) This is not necessa rily associated with overt renal insufficiency and may be a more common manifestat ion than renal failure. (71) The mechanism for this is still slightly unclear but studies in rats suggest that ab normal magnesium excretion may be due to a defect in magnesium transport in juxtamedulla ry nephrons or collecti ng ducts. (72) When clinical manifestations of hypomagnesemia such as neuromuscular, CNS, and cardiac function abnormalities occur usually seen with serum levels less than 1 mEq/L, parenteral replacement of magnesium sulfate should be administered. (34) Hypomagnesemia is usually complicated by hyp ocalcemia that is probably secondary to diminished PTH release and/or end-organ re sistance to parathyroid hormone induced by hypomagnesemia. (73, 74) Hypocalcemia resolv es when magnesium is replaced and is unresponsive to calcium re placement alone. (34) Gastrointestinal and Myelosuppression Gastrointestinal toxicity and myelosuppression appear to be as sociated with the death of the rapidly dividing cells in the lining of the ga strointestinal tract and in the bone marrow. Cisplatin also activates the chemoreceptor trigge r zone to induce vomiting. (3) The use of antiemetics such as metoclopramide, butorphanol, do lasetron, ondansetron, a nd chlorpromazine can be given prior to, during, and after cisplatin infusi on to decrease nausea and vomiting. (75, 76)

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20 Ototoxicity Ototoxicity has been reported more commonly in human patie nts than the dog. Ototoxicity has been observed in 7-90% of human patients receiving doses of up to 120 mg/m2 per course. (77, 78) Hearing loss is in the high-frequency range and is dose-related, cumulative, and frequently irreversible. (79, 80) Concurrent cranial irradiation enha nces the ototoxicity. (81) In one study with high-dose cisplatin, in spite of significantly lower hearing levels, no human patient suffered a disabling hear ing loss requiring a hearing aid and the use of hypertonic saline and vigorous hydration was not found to minimize ot otoxiciy. (82) While humans manifest this toxicity in hearing a high-pitched tinnitus (ringi ng in ears) and hearing sounds differently, small animals express this as an inappropriate respons e or an unusually strong response to an auditory stimulus, such as hyperactivity or excessive bark ing. (83) The mechanism for this toxicity is unclear but may involve spontaneously recruitin g adjacent neurons, aberrant cochlear fluid currents, or undermodulation of membrane movement. (83) Neurotoxicity Neurotoxicity is described as a peripheral neuropathy and usually develops in human patients that receive a cu mulative dose of 400 mg/m2 or higher. (84) In studies using high dose cisplatin with protective measures, mild to mode rate paresthesias have occurred in some human patients. (81, 85) Syndrome of Inappropriate Secretion of Antidiuretic Hormone (SIADH) SIADH has been reported in the human literatu re with the administra tion of cisplatin and other cytotoxic drugs. (86) SIADH is characterized by hyponatremia with concurrent hypoosmolality of the serum, continued renal excr etion of sodium, no clinical evidence of volume depletion, urine osmolality greater than th at appropriate for concurrent osmolality of serum, and normal function of the kidneys, supr arenal glands, and thyroid glands. (86)

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21 Protective Measures for Nephrotoxicity Saline Diuresis The most common protocol for administering cisplatin consis ts of preand post-hydration with concurrent saline diuresis. The mainte nance of adequate hydration is important for decreasing nephrotoxicity, but the mech anism of protection is unknown. Diuretics Other common methods for decreasing the nephrot oxicity of cisplatin include mannitol or furosemide administration. The exact mechanism behind diuretics ameliora ting cisplatin toxicity is unknown, but postulated mechanisms include: accelerating the passage of cisplatin through the renal tubules by increased urinary excretion, reversing the osmotic gradient in tubules by mannitol, and blocking of sodium and water reabso rption by furosemide. (87) It has also been suggested that these diuretics may also atte nuate cisplatin nephrotoxicity reducing the concentration of platinum in the urine. (9, 88) However, in the study by Pera et al. it was noted that, while diuretic administra tion significantly improved renal f unction, some degree of tubular necrosis was still present. (88) Another st udy showed neither mannitol nor furosemide was superior to the other in reduci ng nephrotoxicity in the human pa tients involved. (87) However, there have been conflicting studies comparing hydration with or without mannitol. In one study, mannitol ameliorated nephrotoxicity better than hydration alone. (89) The conflicting study used the same dose of cisplatin but stated there was no difference between gr oups receiving mannitol and hydration or hydration alone. (90) In spite of these conflicting repo rts, administration of mannitol or furosemide along with continuous salin e diuresis has become standard practice when using cisplatin chemotherapy in human cancer patients.

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22 Hypertonic Saline Protection from nephrotoxicity wa s also seen when cisplatin was dissolved in a hypertonic NaCl solution (4.5%) relative to distilled wate r with no effect on the antitumor action of cisplatin. (91) It is postulated that the presence of the high concen tration of NaCl in the vehicle was great enough to force the aquatio n reaction far to the left thus favoring the presence of the parent cis molecule decreasing binding to plasma pr oteins and tissue binding sites. (91) Pharmaceuticals Additional drugs have been administered in conjunction with cisplatin to reduce nephrotoxicity. Amifostine (WR-2721) is a SH-c ontaining compound that when injected before cisplatin in rats, decreased nephrotoxicity by a factor of 1.7 without in hibiting its antitumor effect. (92-94) Diethyldithiocar bamate (DDTC) is a chelating agent that potentially removes platinum bound to renal tubules. (95) However, several side effects (hypertension, agitation, flushing, and diaphoresis) that required patients to receive sedation during administration and the failure of the drug to ameliorate gastrointestin al side effects and otot oxicity, have limited the clinical application of this dr ug. (81) Probenecid is thought to partially inhibit platinum renal secretion and subsequently decreas es the platinum concentration in the renal tubules, decreasing nephrotoxicity without affecting cisplatins antitumor activity. (85) This drug is nontoxic, inexpensive, and readily available, but failed to protect against the other side effects of cisplatin such as myelosuppression, gastrointestinal toxicit y, and ototoxicity. (85) Sodium thiosulfate is an antioxidant in the thiol family. (96) It is used most commonl y in conjunction with intracavitary cisplatin to reduce toxi city and allows the dose of cispla tin to be delivered to be as high as 270 mg/m2. (97) In a recent study performed on a rat model, sodium thiosulfate was found to provide protection from cisplatin otot oxicity when delivered at 4 to 8 hours after cisplatin, but was not consisten tly protective against nephrotoxic ity. (98) Procainamide, an

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23 antiarrhythmic agent, has also been shown to protect against cisplatin nephrotoxicity without altering its antitumor effects. (99) Procainamide and cisplatin form a complex that increases the amount of platinum bound to DNA and may prevent metabolism of cisplatin to a nephrotoxin by GGT through the formation of a cisplatin-glut athione complex. (41, 100) Methimazole, an antithyroid drug, was given intraperitoneally 30 mi nutes prior and 4 hours after cisplatin infusion without saline prehydration to normal dogs and was found to significantly decrease nephrotoxicity. (101) Methimazole is thought to exert an antioxi dative effect to protect the kidney, but it is unknown whether this compound a ffects cisplatin tumori cidal activity. (101) Liposome-encapsulation of cisplatin has also b een proven to allow an increase in dose of cisplatin that can be safely administered withou t increasing the nephrotoxic ity in dogs and cats. (102-104) Enzymatic and Molecular Alterations Reduction of nephrotoxicity has also been asso ciated with some enzymes or agents that control or prevent the formation of free radicals. One study demons trated a decrease in cisplatin nephrotoxicity in rats treated w ith a superoxide dismutase mime tic, orgotein. (105) Another rat model study used N-acetylcysteine, an antioxida nt, delivered at 400 mg/kg IV 15 minutes prior to cisplatin injection and found that treated rats had normal BUN, creatinine, and histologically normal kidneys 3 days after injection. (98) Many studies investigating the mechanism of cisplatin nephrotoxicity have been using agents such as caspase inhibito rs and antioxidants to view their role in apoptosis of renal tubular cells. (47, 54, 58) Also, upregulation of the genes p21 and manganese superoxide dismutase is consider ed as a potential futu re therapy, as studies done in vitro have demonstrated that upregulation of these two genes resulted in protection against cisplatin nephro toxicity. (47, 54) Unfortunately, many of these studies have been in vitro

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24 on kidney tubule cells and these genes effect on cisplatins in vivo effects in higher mammals and antitumor effect is still unknown. Another recent development has been w ith the endogenous enzyme heme oxygenase-1 (HO-1). HO-1 is an inducible enzyme that de grades heme and produces carbon monoxide (CO), iron, and biliverdin. Biliverd in is reduced to bilirubin in vivo and is a powerful antioxidant. (106, 107) Carbon monoxide possesses vasodilatory, an ti-inflammatory, and antiapoptotic properties. (108-111) HO-1 upregulation occu rs after cisplatin administra tion and upregulation protects against cisplatin nephrot oxicity. (112) Tayem et al. recently showed that a water-soluble carbon monoxide releasing molecule protected rena l tubular cells from cisplatin injury in vitro and in vivo in rats. (113) Again, as with other re cent studies mentioned previously, it is unknown whether HO-1 interferes with cisplatins antitumo r effect. Recently, in our laboratory, we have reported that hyperbilirubinemia ameliorates nephrot oxicity in rats receivi ng cisplatin. (114) Cisplatin Use in Animals Dogs Cisplatin has been used as a systemic or lo cal chemotherapy agent in dogs via intravenous, intraarterial, intramedullary, intralesional and intracavitary routes. Two short-term diuresis protocols have been utilized in the dog (Table 1-1). The incide nce of nephrotoxicity was similar between the studies and survival times for the dog s developing nephrotoxicosis were similar to those that did not develop nephrotoxicosis in bo th studies. (115, 116) In human patients, the cisplatin dose can be divided over 5 days with the patient receiving conc urrent NaCl diuresis during cisplatin administration, adequate prean d posthydration, and possibl e use of the diuretics mannitol and furosemide. (117) Administering ci splatin over many days under constant diuresis is not utilized in veterinary medicine because it is not cost effective in animals.

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25 Cisplatin is most commonly used as a single ag ent or as part of combination chemotherapy with doxorubicin to treat osteosarcoma. Median su rvival times in dogs treated with cisplatin as the sole chemotherapeutic agent range from 262 to 413 days. (118-122) Various combination protocols have been inves tigated. Doxorubicin (30 mg/m2 IV on day 1) and cisplatin (60 mg/m2 IV on day 21) repeated for 2 treatment cycles resulted in median survival times of 300 days. (123) A more recent st udy investigated the use of doxorubi cin and cisplatin administered within 24 hours of each other. (124) Cisplatin (50 mg/m2) was administered IV followed by doxorubicin (15 mg/m2) 24 hours later with the intent of co mpleting 4 treatment cycles. (124) Median survival times were equivalent to studies where cisplatin and doxorubicin were administered 3 weeks apart at 300 days, but sign ificant toxicity was en countered with this protocol. (124) Renal toxicity was present in 11% of patients, which is higher than the reported incidence in the diuresis studi es utilizing cisplatin as a so le treatment agent. (124) Gastrointestinal toxicity and my elosuppression were comparable to larger studies utilizing only cisplatin. (124-126) The use of STEALTH liposome-encapsulated cisplatin versus carboplatin has found that while the use of the STEALTH cispla tin allowed safe administration of five times the maximally tolerated dose of free cisplatin, this did not translate into pr olonged disease-free or overall survival. (127) Other methods of administration of cisplatin fo r the treatment of osteosarcoma that have been investigated include intralesional (with implants or injection), intraarterial, and intramedullary administration. Amputation is usually recommended for dogs that have appendicular osteosarcoma to achieve local tumor control and palliation, but concurrent orthopedic or neurological disease may make amput ation less feasible in ce rtain dogs. When this occurs, limb-spare procedures and palliative radiation have been utilized as acceptable

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26 alternatives. Cisplatin-containi ng implants have been utilized in limb-spare procedures for additional local control. (128) Dogs receiving cisplatin implants were 53.5% less likely to develop local recurrence than the control groups, although this effect did not reach statistical significance. (128) Percent tumor necrosis was found to be sta tistically significant when predicting local tumor control. (129) Significantly greater tumor n ecrosis is observed after intraarterial cisplatin administration when compared to systemic cispla tin administration using an intravenous route. (129) The use of radiation therapy in addition to intraarterial cisplatin further increased percent of tumor necrosis. (129) One study investigated the use of intraarterial cisplatin and radiation in dogs for local tumor control. (130) Radiation wa s administered in 10 equal fractions, 3 days a week while cisplatin (70 mg/m2) was given intraarterial in the affected leg on the first and last treatment days over 2 hours with appropriate diur esis. (130) Eighty-nine percent of dogs had an improvement in limb function with no toxicity no ted as a result of therap y. (130) Intraarterial cisplatin did not improve survival times by preven ting metastasis as intrav enous cisplatin does. (130) Intrame dually cisplatin (60 mg/m2 over 20 minutes) administered with a Jamshidi biopsy needle inserted into the tumor provided effective local control in 50% (2/4) of dogs unable to undergo amputation or a limb-spare procedure. (131) Cisplatin has also been utilized for transi tional cell carcinoma (TCC) and squamous cell carcinomas (SCC). At a dose of 50 mg/m2 given IV every 28 days, cisplatin was found to have a palliative effect for dogs with SCC and TCC. ( 118) No complete responses were observed in this study, but partial remission and stable disease were observed in the majority of dogs without significant toxicity noted. (118) In another study in which cisplatin was given at 60 mg/m2 IV every 3 weeks to dogs with a variety of malignant tumors that included TCC and SCC, an overall

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27 response rate (complete or partial remissi on) of 19% was observed. (126) Those dogs demonstrating progressive disease did so by 42 days after the start of trea tment, suggesting that dogs should be evaluated at this time to determine response to treatment. (126) The use of cisplatin concurrently with radiati on therapy is a source of interest as cisplatin reportedly inhances radiation-i nduced cell kill. (132-136) A study performed on dogs with nasosinus carcinomas compared coba lt radiation alone with radiati on in addition to cisplatin at 7.5mg/m2 IV bolus before every other radiation treatment. (137) The mean and median tumor control and survival times were not significantly different be tween treatment groups; however, there was a trend toward longer tumor control an d survival times in the cisplatin treatment group. (137) Another study regardin g nasal tumors utilized an OPLA-Pt implant concurrently with radiation and found that th e implant was clinically tolera ble and yielded comparable or perhaps improved survival times when compared to other published protocols. (138) The use of radiation with cisplain has also been researched with regard to oral melanomas. One study gave cisplatin 10-30 mg/m2 IV with diuresis or carboplain 90 mg/m2 IV prior to 6 weekly 6-Gy radiation fractions. (139) The use of low dose ch emotherapy resulted in a median survival of 363 days which was longer than the previously re ported survival times for surgery or radiation therapy alone. (139) It should be noted that the dogs in this study ha d small initial tumor size and no lymph node or lung metastasis at the start possibly positively affecting survival times. (139) Intralesional cisplatin has also become a po ssibility for local cisplatin administration. Implants containing cisplatin, viscous gel, and a vasoactive modifier have been used as primary treatment for melanomas. (140) Implants were injected until tumor saturation was visualized and these treatments occurred weekly until complete tumor resolution was observed. (140) Seventy

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28 percent of dogs had a >50% decrease in volum e and 55% of these dogs had a complete response. (140) Tumors that re sponded received a mean of 2 .6 treatments. (140) The most common side effect was local necrosis seen in 85% of patients and was associated with tumor response. (140) Systemic toxico sis was minimal with no dog exhi biting renal toxicosis. (140) Patient survival was comparable to other forms of local treatment such as radiation and surgery. (140) Although there are no current published reports in dogs, intralesional cisplatin is also being utilized for other tumors using sesame oil instead of the viscous gel described above. Intracavitary cisplatin is the last well-know n use of cisplatin in dogs. In one study, 50 mg/m2 of cisplatin was administered every 28 days for a median of 2.5 treatments to dogs with mesothelioma and carcinomatosis. (141) When using intracavitary chemotherapy, the tumor is exposed by the capillary blood supply to a concen tration equivalent to that achieved by IV administration, and the surface cell layers are exposed to a concentration th at is 1-3 logs higher. (142, 143) Although intracavitary cisp latin administration is a local method of chemotherapy delivery, significant systemic absorp tion does occur and the dose limiting toxicity for intracavitary cisplatin is re nal toxicity.(141) Animals in th is study underwent diuresis and 66.7% of dogs received additional treatment with sodium thiosulf ate preventing toxicity during the study. (141) This method of cisplatin delivery was associated with palliation and control of malignant pleural and/or abdominal effusion in 5 of 6 dogs and this palliation lasted 129 to greater than 807 days. (141) A lthough results for the previous study were promising, a more recent study found that the use of intracavitary carbopl atin or mitoxantrone was just as effective for dogs with similar diseases. (144) The ease of administration for carboplatin and mitoxantrone is superior to cisp latin since diuresis is not requir ed and minimal side effects were

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29 noted with the former two chemotherapy agents suggesting that the use of cisplatin in intracavitary infusions will become obsolete in veterinary medicine. (144) Cats Cisplatin is unable to be administered to cats due to its acute drug toxicity in this species. Cats receiving 60 mg/m2 of cisplatin became dyspneic and died 48-96 hours after administration. (145) Postmortem findings incl uded severe hydrothorax, pulmonary edema, and mediastinal edema. (145) A group of cats undergoing equivalent saline diur esis as the cisplatin group did not show these signs, causing the beli ef that cisplatin had induced the changes mentioned. (145) Lowering the dose of cisplatin to 40 mg/m2 resulted in similar but less severe pulmonary changes, while decreasing the dose to 20 mg/m2 showed no pulmonary changes. (145) The tumoricidal activity of cisp latin alone at such doses is not known. Using repetitive low dosing (10 mg/m2 3 times a week for 10 txt), cispla tin use resulted in reversible pulmonary edema and renal insufficiency. (146) The use of liposome encapsulated cisplatin has been researched in cats. Studies with this form of cisplatin showed no renal or pulmonary toxicity but all cats had transient pyrexia and/ or lethargy, vomiting, inappetence, and an acute infusion reaction prevented by administering at ropine-diphenhydramine. (102, 103) When this formulation was looked at in cats with squamous cell carcinoma, the liposome encapsulated cisplatin was found to be an ineffective treatment since none of the cats had complete or partial remissions. (147) Horses Intratumoral cisplatin in oily emulsion ha s proven efficacious in treatment of cutaneous tumors in horses such as squamous cell carc inoma and sarcoids. (148, 149) Intralesional cisplatin can be used alone for treatment of sm all tumors or in combination with surgery for larger tumors. (150) Treatments are given at 2 w eek intervals at a dose of 1 mg cisplatin for each

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30 cm3 of tissue in the target field. (151) When surgery is performed, cisplatin should be administered perioperatively or earl y in the postoperative period. (151) Other Platinum Drugs The most commonly used alternative platinum dr ug to cisplatin is carboplatin. Carboplatin has been shown to be less nephrotoxic than ci splatin and can be admi nistered without saline diuresis. Similarly, nausea and vomiting are common side effects w ith carboplatin as they are with cisplatin. Carboplatin alone or in combin ation with doxorubicin has been utilized to treat dogs with osteosarcoma with su rvival times at 321 days and 320 days respectively which are similar to survival times achieved to cisplatin alone or in combination with doxorubicin. (152, 153) However, in regards to treating TCC, car boplatin was not as effective as cisplatin in producing a clinical response. (154) Other tumors in whic h carboplatins use has been researched include: malignant melanoma, nasa l tumors, and anal sac adenocarcinoma. As mentioned previously, carboplatin has additional uses as an intr acavitary chemotherapeutic and as a radiosensitizer. (139, 144) Lobaplatin, another platinum analog, was found to result in a one-year survival fraction of 31.8% when administered every 3 weeks to dogs with appendicular osteosarcoma. (155) Clinical signs related to toxi cosis were uncommon and usually were vomiting and depression. (155) Unlike cisplati n, lobaplatin did not require pr etreatment infusions. Summary Cisplatin has long been utilized in both human and veterinary medicine Cisplatin is still used widely in many protocols in human patient s affected with head and neck, lung, and germ cell tumors, as well as OSA. The use of cisplati n in veterinary medicine is not as widespread which is most likely due to cisp latins severe adverse side eff ects. In addition, carboplatin, a platinum analog, has shown fewer severe side effects and has recently become a more cost

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31 effective alternative to cisplatin. It is still likely that cisplatin will continue to be utilized in veterinary medicine for treatment of OSA, intr alesional chemotherapy for a variety of tumors, and as a sensitizer prior to ra diation therapy.

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32 Table 1-1: Canine diuresis pr otocols when administering cispla tin. These protocols utilized 70 mg/m2 given with 0.9% NaCl IV over 20 minutes. Duration of diuresis Fluid rate (ml/kg/hr) Length of diuresis before cisplatin Length of diuresis after cisplatin Incidence of nephrotoxicity Reference 4 hours 25 3 hours 1 hour 7.8% (116) 6 hours 18.3 4 hours 2 hours 6.6% (115)

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33 Figure 1-1: Two-dimensional st ructures for cisplatin, carbopla tin, oxaliplatin, and lobaplatin. While the core structure (Pt) is the same for each drug, the leaving groups are different for each compound. Pt N H2 N H2 O O C O O CH2 CH2 CH2 Pt O O C C O NH2 NH2 Pt O O C C O O NH2 NH2 Pt Cl NH2 NH2 Cl Cisplatin Carboplatin Oxaliplatin Lobaplatin

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34 Figure 1-2: Aquation reaction a nd adduct formation at the N-7 position of guanine on 2 sites of DNA. These adducts result in DNA damage resulting in cell kill.

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35 Figure 1-3: Four pathways exis t for cisplatin resistance. They include: (1)decreased cellular accumulation, (2) inactivation of the drug, (3) DNA repair, and (4) prevention of apoptosis. Cisplatin Pt Cl Cl N H2 N H2 Cisplatin Active Cisplatin Inactivated by GSH and Metallothionein Pt-SG DNA adduct Excision repair Cancer CellReduced uptake Resistance to Apoptosis 1 2 3 4 MRP

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36 CHAPTER 2 HYPERBILIRUBINEMIA PROTECTS AGAINST CISPLATIN NEPHROTOXICITY IN THE GUNN RAT Introduction Cisplatin is one of the most commonly used antineoplastic agents in human patients. Cisplatin is currently a front line drug used in chemotherapy protocols to treat a wide variety of tumors including ovarian, cervical, testicular, head and neck tumors, transitional cell carcinomas, osteosarcomas, small cell lung, and esophageal cancer s. Cisplatin is also utilized as a rescue agent in the treatment of ot her solid tumors. (4, 156-161) Systemic dose-related toxicitie s associated with cisplatin are well-documented and have precluded its use in many patients. Of these, ne phrotoxicity is the most frequently observed and most clinically significant toxicity. The mech anism for cisplatin nephrotoxicity has not been completely elucidated; however, many theories have been developed. (42, 43, 48-50) One theory is that reactive oxygen species (ROS) and mitoc hondria play a role in the apoptotic cascade involved in cisplatin nephrotoxic ity. (51) The morphologic alterati ons in the kidney ascribed to cisplatin occur in the pars recta of the proximal t ubule situated in the oute r stripe of the medulla and the maximum damage is seen by day 5 after administration. (39) In vitro studies have shown that necrosis and apoptosis can occur, with the form of ce ll death being dependent on the concentration of cisplatin the cells are exposed to. (50) Recently, the endogenous enzyme heme-oxygenase 1 (HO-1) has been investigated for its role in protecting organ systems from various insults. HO-1 is induced in res ponse to cellular stress, and converts the pro-oxida nt heme molecule into eqimola r quantities of biliverdin (BV), carbon monoxide (CO), and iron. (162) BV is converted to unconjugate d bilirubin (BR) via bilirubin reductase. (163) Unc onjugated BR is then converted to conjugated BR by the hepatic microsomal enzyme, uridine diphosphate gluc uronyltransferase (UDPGT). (164) These

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37 molecules of heme degradation were once consider ed to be toxic metabolites, but have recently been shown to have dose-dependent vasodilatory anti-oxidant, and anti-inflammatory properties that may be useful in protection of various organ systems from toxic insult. (165) Specifically, the HO-1 enzyme and its products ha ve been studied in association with toxic acute renal failure. Hyperbilirub inemia has been shown to result in protection against acute renal failure caused by the nephrotoxin glycerol. ( 166) With regards to cisplatin mediated nephrotoxicity, depletion of the HO-1 enzyme resulte d in more significant renal failure and renal injury in one study (112) and the administration of CO along with cisplatin ameliorated signs of renal failure in another study. ( 113) However, upregulation of HO-1 or its products may affect cisplatins antineoplastic activity. The objectives of this study we re to investigate the protectiv e effect of hyperbilirubinemia in vivo in the rat model against cisplatin-indu ced nephrotoxicity. Hyperbilirubinemia in vivo was achieved using both the homozygous and heteroz ygous Gunn rat. The homozygous Gunn rat is unable to induce UDPGT as a resu lt of an autosomal recessive de ficiency in this enzyme. (167, 168) The lack of induction of UDPGT results fr om an alteration in the coding region of the mRNA which results in an instability of the mR NA and a synthesis of a truncated, functionally inactive UDPGT. (168) In additi on, an intermediate level of b ilirubin can be obtained using heterozygous Gunn rats that have varying degree s of functional UDPGT. We also investigated the effect of bilirubin on the an tineoplastic activity of cisplatin using four established canine osteosarcoma cell lines (POS, HMPOS, COS31, D17). Materials and Methods Animals This study was approved by the University of Florida Institutional An imal Care and Use Committee and was performed in accordance with th e Institute for Lab Animal Research Guide

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38 for the Care and Use of Laboratory Animals. Male Wistar, homozygous Gunn (j/j) and heterozygous Gunn (j/+) rats weighing 200-400 g we re purchased from Harlan Sprague Dawley, Inc (Indianapolis, IN) and mainta ined in a temperature controlle d room with alternating 12 hour light/12 hour dark cycles in an an imal facility at the University of Florida. Animals were fed standard rat chow and allowe d free access to water. Cisplatin-Induced Acute Renal Failure Three groups of male rats (n =6 rats/group) were used: (1) Wistar (2) homozygous Gunn (3) heterozygous Gunn. Rats were weighed and observed for changes in attitude during the course of the experiment. On Days 0, 3, and 5, rats were anestheti zed using 5% inhalant isoflurane in 100% oxygen and maintained with 23% isoflurane by mask. Animals were placed on a warm water heating pad to maintain normal body temperature. Prior to blood sampling on Days 0 and 3, the rats tail was soaked in 40-42 C water for 3-5 minutes to facilitate vasodilation. One milliliter of blood was sample d from the tail veins or the lateral saphenous veins. After blood sampling was complete on Da y 0, all rats were given an intraperitoneal injection of cisplatin (American Pharmaceutical Partners Inc, Schaumburg, IL) at 4 mg/kg. Once the IP injection was complete, all rats were recovered. Upon recovery, an injection of 0.01 mg/kg buprenorphine hydrochlorid e (Reckitt Benckiser Healthcar e (UK) Ltd, Hull, England) was given SQ. Blood was sampled on Days 0, 3, a nd 5 for evaluation of BUN, serum creatinine, and serum bilirubin concentrations. On Day 5, once the rats were anesthetized a midline incision was performed and blood was sampled from the ca udal vena cava. Both kidneys were isolated and harvested and the rats were euthanized by an overdose of sodium pe ntobarbital (Euthasol, Diamond Animal Health, Inc., Des Moines, IA). Three groups of male rats (n=4 rats/group) were used as sh am control rats. The groups consisted of: (1) Wistar (2) homozygous G unn (3) heterozygous Gunn. These groups were

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39 treated as described above for the cisplatin induced acute rena l failure groups; however, instead of receiving 4 mg/kg cisplatin, the rats received the equivalent amount of 0.9% sodium chloride IP. The sham control rats for each group were intended to prove that equivalent times of anesthesia would have no effect on the ki dney functional parameters and histopathology. Assays BUN and serum bilirubin (BR) concentrations were determined using an automated chemistry analyzer (Hitachi 911 Chemistry Anal yzer). Serum creatinin e (Cr) concentrations were determined using a dry chemistry analyz er (Johnson & Johnson Vitros DT6011) due to the potential effect of icterus on the standard Jaffe methodology for measurement of Cr. (169) Histologic Grading Both kidneys were placed in 10% buffered form alin for at least 24 hours before processing. Transverse sections of the left kidney for all rats were processed using hematoxylin and eosin (H&E) staining and periodic-acid-Schiff (PAS ) staining. Histological examination was performed by a renal pathologist who was blinde d with respect to the treatment groups. Renal tissue was divided into 4 regions for analysis: cortical proximal tubules (CPT), S3 segment of the outer stripe of the outer medullary proxima l tubule (OSOMPT), medullary thick limb in the inner stripe (ISOM mTAL), and co llecting ducts (CD). Renal injury was graded in 7 different categories: normal, cellular swelling/vacuoliz ation, brush border loss, nuclear condensation, karyolysis/apoptosis/necrosis (most severe form of injury), regeneration, and capillaritis. Each category was assigned a numerical score: 0= none, 1 =<10% 2= 10-25%, 3= 25-50%, 4= 5075%, 5= 75-100% based on the percentage of ce lls in each region displaying the described injury.

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40 Cell Lines Osteosarcoma is a highly-aggressive tumo r in dogs and affected dogs are commonly treated with cisplatin. Four canine osteosarcoma cell lines which have been well characterized in our laboratory were utilized in this study. The POS (parent osteosarcoma) cell line was originally developed from a primary osteosarcoma affecting the left proximal femur of a 1 and a half year-old male mongrel dog (Dr. Tsuyoshi Ka dosawa, University of Sapporo, Japan). (170) The HMPOS (highly metastatic parent osteos arcoma) cell line is a pulmonary metastatic derivative of POS cell line (Dr. Tsuyoshi Kadosaw a, University of Sapporo, Japan). (171) D17 is another established canine osteosarcoma ce ll line (American Type Tissue Culture Collection, Manassas, Virginia). The COS31 cell line wa s established from a dog with spontaneously occurring osteosarcoma (Dr. Ah med Shoieb, University of Tennessee, College of Veterinary Medicine, Knoxville, TN). Cells were cultured at 37 C under 5% CO2 and 95% room air with their respective media. POS and HMPOS medi a consisted of RMPI 1640 media supplemented with 10 % heat inactivated feta l calf serum, vitamins, sodi um pyruvate, non-essential amino acids, L-glutamine, and antibiotics (penicillin (0.0 625 g/L) and streptomycin (0.1 g/L)). D17 and COS31 media consisted of Dulbeccos Modifed Eagl es medium with 10% heat inactivated fetal calf serum, L-glutamine, and antibiotics (penici llin (0.0625 g/L) and streptomycin (0.1 g/L)). The cells were grown to confluence, washed w ith physiological buffered saline, and detached from the flasks with trypsin. Cells were stained with Trypan blue and counted with a hemacytometer. Cell Viability Assay An assessment of cell viability was performed with the CellTiter BlueTM Cell Viability Assay (Promega Corporation, Madison, WI). A ssays were performed in 96-well flat-bottomed black microtiter plates. All cell lines were seeded at 10,000 cells/well with 50 L of media and

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41 placed in the incubator at 37 C under 5% CO2 and 95% room air for 24 hours. The IC50 for each cell line with cisplatin was de termined prior to treating the cells with bilirubin and cisplatin. All cells were treated with 50 L of bilirubin at co ncentrations of 71 M and 128 M alone and combined with each cell types IC50 concentration of cisplatin. Bilirubin was dissolved in the respective media to achieve appr opriate concentrations The micromolar concentrations of bilirubin used in this study were taken from the average serum bilirubin levels on Day 0 and 3 that provided functional nephroprotection of th e homozygous Gunn rats receiving cisplatin. After incubation for 72 hours under the conditions described previously, 20 L of CellTiter BlueTM reagent, resazurin, was added to each well. Viable cells retain the ability to reduce resazurin into resorufin, which is pink and highl y fluorescent. (172) Pl ates were placed on a low-speed shaker for 10 seconds and then incubated for 4 hours. The amount of fluorescence was recorded with a fluorescen ce plate reader at 530/590 nm. Statistical Analysis Statistical calculations were performed using a computer software pr ogram (SigmaStat for Windows, version 3.00, and SigmaPlot for Window s, version 8.02, SPSS Inc, Chicago, Ill.) Data was tested for normality and equal variance using the Kolmogorov-Smirnov test. The comparisons between groups used ANOVA for parametric data and ANOVA on Ranks for nonparametric data. Differences between groups were identified using (post-hoc) pair wise multiple comparison procedures (Holm-Sidak method or D unn's Method). Parametric data is reported as mean SD and nonparametric data as median with an inter-quartile range ([IQR], 25% to 75%). P value < 0.05 was considered significant.

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42 Results Renal Functional Parameters Bilirubin levels were significantly higher fo r all days in the homozygous Gunn rats when compared to the heterozygous Gunn rat and the Wist ar rat (Table 2-1). Statistically, bilirubin levels were not significantly hi gher in the heterozygous Gunn rat compared to the Wistar rat at any day. However, the Wistar rats had a mean of 0 mg/dL and the heterozygous rats had a mean of 0.4 to 0.1 mg/dL, indicating that a mild degree of hyperbilirubinemia existed in the heterozygous Gunn rats. The homozygous and heterozygous Gunn rats we re protected from the nephrotoxic effects of cisplatin based on functional kidney parameters. There was no significant difference in BUN or Cr on Day 0 between any of the groups of rats (Figures 2-1 and 2-2). Importantly, in the face of a nephrotoxic dose of cisplatin, the hyperbil irubinemia present in the homozygous Gunn rat resulted in a significant nephropr otective effect when compared with the hetero zygous Gunn rat and the Wistar rat (means BUN homozygous: Day 523.83 5.49 mg/dL, p<0.05; means Cr homozygous: Day 50.35 0.05 mg/dL). Th e protection was clini cally significant as the homozygous rats BUN and Cr values re mained within the normal range. While, the intermediate level of hyperbi lirubinemia provided by the hete rozygous Gunn rat still provided nephroprotective effects when compar ed to the Wistar rat, the prot ection was not as effective as for the homozygous Gunn rat. When comparing he terozygous Gunn rat a nd Wistar rat BUN and Cr levels with each other, the differences in BUN were not significant until Day 5 but were present on Day 3 and 5 with regard to Cr (means BUN heterozygous: Day 538.17 10.34 mg/dL; means Cr heterozygous: Da y 50.40 0.06 mg/dL; means BUN Wistar: Day 578.67 16.92 mg/dL; means Cr Wistar: Day 51.4 0.43 mg/dL)

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43 When comparing the homozygous Gunn rat to the heterozygous Gunn rat in regards to functional kidney parameters, it was found that there was no significant difference in BUN on Day 3 and Cr on Day 3 and 5 (Figure 2-1 and 2-2). However, there was a st atistically significant difference in BUN between the groups on Day 5, with the homozygous Gunn rats showing a lower BUN than the heterozygous rats. When all groups of sham rats (homozygous Gunn, heterozygous Gunn, Wistar rats) were compared to the control group of rats receiving cisplatin, it was found that there was no significant difference in BUN or Cr on Day 0. There were signifi cant differences for the sham groups when compared to the control Wistar group in BUN and Cr for Day 3 and 5. Between the sham groups, there were no statisti cally significant differences in BUN and Cr at Day 0, 3, or 5. Importantly, there was also no significant difference between the homozygous Gunn rat receiving cisplatin and any of the sham groups in BUN and Cr at Day 0, 3, or 5. With regard to bilirubin levels, there was a signi ficant difference between the cont rol Wistar rats and the sham homozygous Gunn rats, but no sign ificant difference between the control Wistar rats and the sham heterozygous Gunn rats. This is equivale nt to what was reported for the groups receiving cisplatin. Light Microscopy For all treatment groups, the cortical proximal tubules and collecting ducts were graded as 75-100% of cells being normal. The area of most in terest with regard to cisplatin nephrotoxicity is the S3 segment of the outer stripe of the outer medullary proximal tubule (OSOMPT). Homozygous Gunn rats had signifi cantly decreased karyolysis/a poptosis/necrosis than the heterozygous Gunn rats and the Wist ar rats in the OSOMPT (Figur e 2-3A and 2-3B). There was no significant difference in ka ryolysis/apoptosis/necrosis between heterozygous Gunn rats and Wistar rats in the OSOMPT. The homozygous G unn rats had a significa ntly greater proportion

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44 of normal cells and cells without ce llular swelling in the medullary thick limb in the inner stripe (ISOM mTAL) than the Wistar rats. The sham groups had identical normal histological scores for all segments of the kidney and were thus grouped together for statistical analysis. Even though the homozygous Gunn rats receiving cisplatin had significantly decreased hi stologic damage to the OSOMPT than the other groups receiving cisplatin, ther e was significantly greater degr ee of injury in the homozygous Gunn rats receiving cisplatin with regard to karyolysis/apoptosis/ necrosis in the OSOMPT when compared to the sham groups. There was also a significant difference in cellular swelling, brush border loss, and regeneration in the OSOMPT between the homozygous Gunn rats receiving cisplatin and the sham groups. A signifi cant difference was also noted between the heterozygous Gunn rats receivi ng cisplatin and the sham groups in cellular swelling, brush border loss, and karyolysis/apopto sis/necrosis in the OSOMPT. Cell Culture Studies In vitro bilirubin had mild cytotoxic effect on COS31 cells at 72 and 128 M and D17 cells at 128 M versus control cells No significant cytotoxic effect s of bilirubin were seen with HMPOS and POS cells at either concentrati on or D17 cells at 72 M. Bilirubin had no significant effect on the antineoplasti c effect of cisplatin at either concentration in any of the four canine osteosarcoma cell lines (Figure 2-4). Discussion While the toxic properties of bilirubin, particularly kernicterus and neonatal hyperbilirubinemia are well documen ted, the therapeutic pr operties of bilirubin are just recently being discovered. (173-176) This study demonstrated that hype rbilirubinemia found in the Gunn rat had a nephroprotective effect when the neph rotoxic, anti-neoplastic agent cisplatin was

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45 administered, as evidenced by the maintenance of normal renal function values and markedly less histologic ev idence of tubular necrosis. Protective effects of bilirubin against to cellular injury have been studied previously but never in the cisplatin mode l of nephrotoxicity. Leung et al. showed that ligation of the common bile duct effectively protect ed against glycerol indu ced acute renal failure in the rat model. (166) Results of our study and the one performed by Leung suggest that sustained hyperbilirubinemia protects the kidney from known pot ent nephrotoxins. (166) Our la b has also reported protection from ischemia-reperfusion injury with exogenous bilirubin when deliver ed into the isolated, perfused rat kidney. (177) The same effects, however, were not noted when the equivalent in vivo rat study was performed. (165) This suggests that the protective effects of bilirubin may only be applicable to nephrotoxins or perhaps th e dose of exogenous bilirubi n required to protect the kidney from various insults has yet to be ascertained. Bilirubin has also shown a protective effect in other organ systems such as the liver (178, 179), intestine (180), and neural ti ssue (181). Clinical trials have shown that the incidence of coronary and ischemic heart disease is lower in humans with hyperbilirubinemia. (182, 183) Bilirubin is a product of HO-1. The benefici al effects of HO-1 induction have been well established in multiple organ systems. (106, 166, 178, 184, 185) In fact, Shirashi et al demonstrated that mice deficient in HO-1 (-/-) developed more severe renal failure and renal injury than wild type mice (+ /+) when cisplatin was admini stered. (112) These findings prompted a look at the individual agents produced by HO-1 to determine if bilirubin or CO alone could mimic this effect. A study by Tayem et al. showed that treatment with a water-soluble carbon monoxide-releasing molecule protected the kidney function and improved histology of rats treated with cisplatin. (113) The results of our study indicate that hyperbilirubinemia in the

PAGE 46

46 homozygous Gunn rat was completely nephroprot ective with regards to functional renal parameters and partially prot ective histologically. Partial protection of renal functional parameters were also provided to the heterozy gous Gunn rat, whose bilirubin levels were not statistically higher than the Wist ar rat at any day, although histol ogically there was no significant difference in the amount of most severe kidney da mage to the S3 segment. An improvement in functional kidney parameters is still important and a trend was present at day 0 for higher bilirubin levels in the heterozygous Gunn rat than the Wistar rat. This suggests that the protective effects of bilir ubin may be exerted on the day the nephrot oxin is administered (Day 0). The exact concentration of bilirubin in serum needed to prevent severe nephrotoxicity due to cisplatin remains in question. Higher av erage serum bilirubin values present in the homozygous Gunn rat resulted in significantly le ss kidney injury than th e heterozygous Gunn rat suggesting that the value of bilirubin required fo r any histological protection may lie somewhere in between those two groups. For full histologic al protection, serum bilirubin values may need to be higher than those seen in the homozygous Gunn rat. Un fortunately, a higher dose may result in bilirubin toxicity. Most likely, individual variation in serum bilirubin levels exist even between the homozygous and heterozygous Gunn rats due to a variation in the levels of the enzyme UDPGT. Homozygous Gunn rats have a defi ciency of this enzyme needed to conjugate bilirubin but UDPGT may still be present in varying small amounts in these rats. The sham group of rats served as a negative control group in this experiment. The purpose of the sham group was to verify that the anes thesia episodes required for venipuncture had no functional or histological effect on the kidneys that would skew our re sults. All of the rats in this group maintained normal BUN and Cr and had hist ologically normal kidneys substantiating that anesthesia did not contri bute to renal pathology.

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47 Another aim of our study was to determine if a dding cisplatin with bilirubin would have an additive, inhibitory, or no effect on neoplastic cells in culture. Fo r this experiment, we utilized four different canine osteosarcoma cell lines for completeness. Bilirubin did not adversely effect cisplatins in vitro antineoplatic activity in any of the four cell lines. This is a very important aspect to view for clinical application of this study. Agents such as bilirubin that proved nephroprotection cannot interfere with a chemotherapeu tic agents efficacy to be useful clinically. Bilirubins antineoplastic effect needs to be tested in other neoplastic ce ll populations and in an in vivo setting before any definitive conclusions can be drawn. Bilirubin has been shown to have some cytotoxic effects on human colon adenocar cinoma cells and human carcinoma cell lines. (186, 187) While two of the cell lines showed some cytotoxi city caused by bilirubin alone (COS31 at 72 M and 128 M and D17 at 128 M), these effects were minimal and had no additional impact on the cells after cisplatin wa s added. Bilirubins cytotoxicity against neoplastic cells may only pertain to certain type s of neoplasia as evidenced by our cell culture results. Future studies would be helpful to determine if the same effects could be duplicated using rats with a sustained conjugated hyperbilirubinemia It is unknown if pr otection is afforded by unconjugated or conjugated bilirub in. Future studies are also warranted to determine if administration of exogenous bilirubin can mitigate cisplatin induced nephro toxicity. Delivering a bilirubin dose high enough to exceed the seru m concentrations seen in the homozygous Gunn rat without toxicity with resulti ng complete histological protection of the S3 segment of the outer medulla may be possible. Another area to rese arch may include concurrent administration of exogenous bilirubin and the water-s oluble carbon monoxide releasi ng molecule to determine if the combination could prevent any histologi c nephrotoxicity caused by cisplatin.

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48 In conclusion, hyperbilirubinemia present in the homozygous Gunn rat resulted in complete nephroprotection with functional re nal parameters and pa rtial nephroprotection histologically. Only partial functional nephroprot ection was seen in heterozygous Gunn rats that did not demonstrate significant hyperbilirubinemia when compared to the control Wistar rats. The anesthetic episodes each rat underwent did not impact functional or histologic kidney parameters. In the four canine osteosarcoma cell lines utilized in our la b, bilirubin did not have an adverse effect on the antineo plastic activity of cisplatin. Our findings suggest that HO-1, more specifically HO-1s products especially bilirubin, may protect the kidney from toxic nephropathy caused by cisplatin.

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49 Table 2-1. Serum bilirubin concentrations were significantly higher for the Gunn j/j when compared to the other groups as noted by th e *. No significant difference was noted between Gunn j/+ and Wistar rats but a tr end toward higher bilirubin values was noted on D0. Means standard deviations serum bilirubin concentrations (mg/dL ) in rats receiving cisplatin Wistar Gunn j/+ Gunn j/j Day 0 0 0 0.41 0.2 4.2 0.9* Day 3 0.07 0 0.1 0 7.5 1.2* Day 5 0 0 0.12 0 5.5 1.1* Figure 2-1. Serum BUN concentrations on D0, 3, and 5. No significant difference between any groups on D0. The most significant differe nce in BUN occurred when comparing the Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a significantly higher BUN

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50 Figure 2-2. Serum creatinine concentrations on D 0, 3, and 5. No significant difference between any groups on D0. The most significant difference in creatinine occurred when comparing the Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a significantly creatinine.

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51 A Figure 2-3..Histologic grading s howed significant preservation of the OSOMPT in homozygous Gunn rats when compared to heterozygous G unn rats and Wistar rats given cisplatin. Although functionally heterozygous Gunn rats were signific antly different than the Wistar rat, histologically, there was no signi ficant difference. A) Outer stripe of the outer medulla (center) with necrotic a nd sloughed tubular epit helial cells coupled with regeneration in the Wistar rats (PAS stain, 50X). B) This is compared to the remarkably well-preserved tubules at the j unction between the inner and outer stripes of the outer medulla with occasional apopt otic and sloughed cells seen at a higher magnification in the homozygous Gunn rats (PAS stain, 50X).

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52 B Figure 2-3. continued

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53 Figure 2-4 Viable cell count for one of the canine osteosarcoma ce ll lines utilized (POS). With this cell line, no significant difference was noted between cisplatin alone and either concentration of bilirubin alone. As w ith all cell lines test ed, bilirubin had no significant difference on cisplatins anti-neoplastic activity in culture.

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54 CHAPTER 3 CONCLUSION The current lifetime risk for a human de veloping cancer in the United States is approximately 40%. Additionally, our pets are living longer lives making them more prone to developing cancer also. One of the most powerful treatments for cancer in any species is chemotherapy but these agents have countless adverse side effects. Specifically, cisplatin is a chemotherapeutic used in humans and animals to treat a variety of malignancies. The most clinically significant side effect associat ed with cisplatin is nephrotoxicity. Chapter 1 discussed the phamacokinetics, m echanism of anti-tumor action, toxicities (especially nephrotoxicity), current protective m easures against nephrotoxicity, and the use of cisplatin in animals. Many theories have been developed regarding the mechanism of cisplatin nephrotoxicity but none have proven to be the sole, underlying cause for this toxicity. Perhaps this is why finding a compound to ameliorate cispla tin nephrotoxicity has been so difficult. Numerous studies have shown that certain compo unds and enzyme alterations can protect against cisplatin nephrotoxicity but not without disadv antages of their own, including worsened side effects of the compound, expense, or difficulty in administering the compound or enzyme. While recent studies about HO regarding protection of various organ systems have been performed, no studies utilizing th e end-product BR to protect agai nst cisplatin nephrotoxicity exist. Chapter 2 of this thesis describes an expe riment designed to inve stigate if sustained unconjugated hyperbilirubinemia in the Gunn rat woul d protect against cisplatin nephrotoxicity. An experiment was also performed to determine if BR had any effect on the ability of cisplatin to kill canine osteosarcoma cell in culture. Homozygous Gunn rats lack the enzyme UDPGT necessary to conjugate bilirubi n. Heterozygous Gunn rats lack normal levels of UDPGT but can

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55 still conjugate bilirubin to a certain extent. Th e homozygous Gunn rats were utilized to achieve a state of unconjugated hyperbilirubinemia. The c ongenic Wistar rats were the positive control with normal levels of UDPGT. Cisplain was administered to all ra ts and functional renal parameters were monitored for 5 days. On da y 5, kidneys were harvested for histological evaluation. To ensure that multiple anesth etic episodes had no negative impact on kidney function and histology, sham rats from each group also underwent the same procedures except they did not receive cisplatin. Cell culture studies were then performed in 4 canine osteosarcoma cell lines using the average concentrations of BR fo r homozygous Gunn rats at day 0 and 3. BR was added to all cell lines alone and with cisplati n and cell viability was asse ssed using the CellTiter BlueTM assay. Results of this study demonstrate comple te functional and partial histologic nephroprotection with the homozygous Gunn rats wh en compared to the heterozygous Gunn rats and Wistar rats. While the heterozygous Gunn ra ts lacked histologic nephroprotection from cisplatin, they had improved functional parameters when compared to the Wistar rats. The sham rats showed no significant differe nce in their functional or histologic parameters when compared to one another. This suggests that multiple anesthetic episodes had no negative impact on renal function or histology. With regard to bilirubins effect on cisp latin in cell culture, there was no significant difference in the cell viability of any of the four canine osteosarcoma cell lines when bilirubin was added with cisplatin. Cisplatin nephrotoxicity has provided a difficu lt obstacle to administering the drug safely to chemotherapy patients of all species. HO a nd its products have proven to be beneficial in protecting a variety of organ systems from assorted insults. The results obtained from this thesis

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56 have provided directions for future applicatio ns regarding the products of HO-1 in cisplatin nephrotoxicity. Future studies using a rodent model with conjugated hyperbilirubinemia and administering exogenous BR are warranted. Pote ntially, a canine and human model could be developed in the future. Additionally, the admi nistration of BR and CO together may provide complete functional and histological nephroprotection from cisplatin.

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72 BIOGRAPHICAL SKETCH Karri Barabas began her collegiate education at the University of Florida in 1996 and received her Bachelor of Science in animal science with highest honors in May 2000. She was accepted to the University of Florida College of Veterinary Medicine and graduated Magna Cum Laude with a Doctor of Veteri nary Medicine in May 2003. Karri completed a one-year rotating small animal medicine and surgical internship at Texas A&M University College of Veterinary Medicine immediately after graduation. She th en completed an oncology internship at Regional Veterinary Referral Center in Springfield, Virginia after finishing at Texa s A&M and returned to the University of Florida to pursue a combined Master of Science and small animal medical oncology residency at the College of Veterinary Medicine. Karri will complete her oncology residency in July 2009.


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Permanent Link: http://ufdc.ufl.edu/UFE0020667/00001

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Title: Effects of Hyperbilirubinemia on Cisplatin Nephrotoxicity
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Copyright Date: 2008

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Material Information

Title: Effects of Hyperbilirubinemia on Cisplatin Nephrotoxicity
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
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EFFECTS OF HYPERBILIRUBINEMIA ON CISPLATIN NEPHROTOXICITY


By

KARRI ANN BARABAS
















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007



































2007 Karri Ann Barabas









ACKNOWLEDGMENTS

I would like to thank my family for their support and love throughout my education. I

would like to also thank Dr. Christopher Adin, Dr. Rowan Milner, Dr. Jim Farese, and Dr. Chris

Baylis for their mentorship and guidance during the pursuit of this degree. Additional thanks go

to Dr. Dan Lewis for providing the position that allowed me to achieve this degree. Lastly, I

would like to thank Linda Archer and Marc Salute for their technical help throughout this

project.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ...............................................................................................................3

L IST O F T A B L E S ..................................................................................................... . 6

LIST O F FIG U RE S ................................................................. 7

ABSTRAC T .........................................................................................

CHAPTER

1 CISPLATIN : A REVIEW ...................................... ................ ...... ................ 11

In tro d u c tio n .............................................................. ................................................ 1 1
C h e m istry ................................................................ ................................................1 1
P harm acokinetics ............................................................................... 12
P h arm ac o d y n am ic s ............................................................................................................ 13
Mechanisms of Resistance................... ....... .. ...... ... ..................14
Toxicities .......................................15
N ephrotoxicity .....................................................................................................15
H ypom agnesem ia and H ypocalcem ia ........................................................................ 19
Gastrointestinal and Myelosuppression.......................... .......... 19
O totoxicity .......................................... .......... .............................. .20
N eurotoxicity ............................. .. .. ......... ................................... ............... ...... 20
Syndrome of Inappropriate Secretion of Antidiuretic Hormone (SIADH) .....................20
Protective M measures for Nephrotoxicity ................................... ..................... ... .... 21
S a lin e D iu re sis .................................................................................................................2 1
D iu retic s ................................................................2 1
H ypertonic Saline ................................................................22
P h arm aceu ticals ................... ............................................................................ 2 2
Enzym atic and M molecular A lterations............................................ .................... ......23
Cisplatin U se in Anim als............................................................................24
D ogs ................. ................. ....... ... .. 24
C a ts ................... ...................2...................9..........
H horses .................. ................... ......................... .............. 29
O their P platinum D rugs ............................................................................... 30


2 HYPERBILIRUBINEMIA PROTECTS AGAINST CISPLATIN NEPHROTOXICITY
IN TH E G U N N RA T ............................................................................................... ....... 36

In tro d u ctio n ................... ...................3...................6..........
M materials and M methods ............................................................................................... 37
Animals.............................. ... ....................37
Cisplatin-Induced Acute Renal Failure ..................................................................38


4









A ssay s............... ...........................................................3 9
Histologic Grading ................... ......................... ........... 39
C e ll L in e s .................................................................................................................... 4 0
Cell Viability A ssay .................................... ..... .......... ....... .... 40
Statistical Analysis .................... .......................... ........ 41
R e su lts ................ ............. .... ............................................................................................... 4 2
R enal Functional Param eters ............................................................. ..................42
Light Microscopy ....................... ........................... ......43
C ell C u ltu re Stu dies............. ...................................................................... ..... .. ..... .. 44
D iscu ssio n .............. ..... ............ ................. .......................................... 4 4

3 CON CLU SION .......... ........................................................ .. .. ...... ........ 54

L IST O F R E FE R E N C E S ......... ................. ..................................... ...................... ............... 57

B IO G R A PH IC A L SK E T C H ............................................................................... .....................72









LIST OF TABLES


Table page

1-1 Canine diuresis protocols when administering cisplatin............. ........... ...... .............32

2-1 Serum bilirubin concentrations were significantly higher for the Gunn j/j when
com pared to the other groups................................................... .............................. 49









LIST OF FIGURES


Figure page

1-1 Two-dimensional structures for cisplatin, carboplatin, oxaliplatin, and lobaplatin.
While the core structure (Pt) is the same for each drug, the leaving groups are
different for each com pound ...................................................................... ..................33

1-2 Aquation reaction and adduct formation at the N-7 position of guanine on 2 sites of
DNA. These adducts result in DNA damage resulting in cell kill.................................34

1-3 Four pathways exist for cisplatin resistance.. ........................................ ...............35

2-1 Serum BUN concentrations on DO, 3, and 5. No significant difference between any
groups on DO. The most significant difference in BUN occurred when comparing
the Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a significantly
h ig h er B U N ............................................................................ 4 9

2-2 Serum creatinine concentrations on DO, 3, and 5. No significant difference between
any groups on DO. The most significant difference in creatinine occurred when
comparing the Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a
significantly creatinine....... ..................................................................... ........ .. ... 50

2-3 Histologic grading showed significant preservation of the OSOMPT in homozygous
Gunn rats when compared to heterozygous Gunn rats and Wistar rats given
cisp latin ................... .......................................................... ................ 52

2-4 Viable cell count for one of the canine osteosarcoma cell lines utilized (POS). ...............53









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF HYPERBILIRUBINEMIA ON CISPLATIN NEPHROTOXICITY

By

Karri Ann Barabas

May 2007

Chair: Rowan Milner
Major: Veterinary Medical Sciences

Cisplatin is a powerful chemotherapeutic agent used in a variety of malignancies in many

species. Systemic dose-related toxicities associated with cisplatin have precluded its use in many

species. Of these toxicities, nephrotoxicity is the most frequent and clinically significant

toxicity. No specific compound has yet ameliorated cisplatin nephrotoxicity. Recent research

has shown that the heme oxygenase-1 (HO-1) enzyme provides beneficial effects in mitigating

cisplatin nephrotoxicity in a rodent model. HO-1 is normally induced in response to cellular

stress and converts the heme molecule into equimolar quantities of biliverdin (BV), carbon

monoxide (CO), and iron. Biliverdin is then converted to bilirubin (BR) by the enzyme

biliverdin reductase. Many previous studies suggest that HO and its products are important

endogenous mechanisms for cytoprotection. These products were once considered to be toxic

metabolites but have been shown to have dose-dependent vasodilatory, anti-oxidant, and anti-

inflammatory properties that are desirable for tissue protection during a toxic insult. Our first

objective was to review the literature regarding cisplatin and, in particular, nephrotoxicity

associated with cisplatin administration and current methods to prevent nephrotoxicity. Our

second objective was to determine if hyperbilirubinemia would ameliorate cisplatin









nephrotoxicity in a rat model. Our third objective was to determine if bilirubin would prevent

cisplatin's ability to kill neoplastic cells in vitro.

The in vivo cisplatin model involved 3 groups of rats (n=6 rats/group): homozygous Gunn

rats (j/j), heterozygous Gunn rats (j/+) and congenic Wistar (+/+) rats. Homozygous Gunn rats

lack the UDPGT enzyme needed to conjugate bilirubin resulting in an unconjugated

hyperbilirubinemia while heterozygous Gunn rats lack normal levels of UDGPT, but can

conjugate bilirubin to a certain degree. On Day 0, all rats were anesthetized and administered

4 mg/kg cisplatin IP. Blood was sampled on Day 0, 3 and 5 for comparison of serum BR,

creatinine (Cr), and BUN. On Day 5, kidney tissue samples were obtained prior to euthanasia.

Cell culture studies were then performed using 4 canine osteosarcoma cell lines (POS,

HMPOS, COS, D17) incubated with the average concentrations of BR for j/j rats at Day 0 and 3.

We added BR to all cell lines (alone and with cisplatin) and cell viability was assessed using the

CellTiter BlueTM assay.

Serum BR levels were 72 16 tM/L in homozygous Gunn rats, 7 3 JM/L in

heterozygous Gunn rats, and 0 + 0 [M/L in Wistar rats at Day 0. The BR provided a dose-

dependent nephroprotective effect, with significantly lower BUN (24 5 mg/dL) and Cr (0.35 +

0.05 mg/dL) in homozygous Gunn rats when compared to Wistar rats (BUN- 79 17 mg/dL,

Cr- 1.4 0.4 mg/dL) at Day 5 (P <0.05). An intermediate level of nephroprotection was noted in

the heterozygous Gunn rats, although BUN (38 10 mg/dL) and Cr (0.4 0.06 mg/dL)

remained significantly lower than Wistar rats at Day 5 (P <0.05). Histological grading

demonstrated preservation of the S3 segment in homozygous Gunn rats when compared to

heterozygous Gunn rats and Wistar rats (P <0.05). The BR had no significant effect on the









antineoplastic effect of cisplatin at either concentration in the 4 osteosarcoma cell lines (P

<0.001).

Hyperbilirubinemia in the homozygous Gunn rat provided nearly complete preservation of

renal function and pathology in this model of cisplatin nephrotoxicity. Addition of exogenous

BR did not interfere with the antineoplastic activity of this chemotherapy agent in cell culture.









CHAPTER 1
CISPLATIN: A REVIEW

Introduction

Cisplatin is one of the most potent chemotherapy agents used in human and veterinary

medicine. Its use in veterinary medicine began more than 2 decades ago and was prompted by

the success of cisplatin in treating human malignancies. Unfortunately, cisplatin administration

was associated with numerous adverse side effects including: nephrotoxicity, severe nausea and

vomiting, myelosuppression, ototoxicity, and neurotoxicity. Of these, the most clinically

significant and common toxicity is nephrotoxicity. Despite the nephrotoxicity, many veterinary

oncologists are of the opinion that cisplatin is more potent than its other platinum counterparts

with regard to its anti-neoplastic activity. This has resulted in its continued use throughout

veterinary hospitals around the world for malignancies such as osteosarcoma (OSA), transitional

cell carcinoma (TCC), intralesional therapy, and radiation sensitization.

Since cisplatin's development, research has centered on mitigating nephrotoxicity to allow

cisplatin to be delivered at therapeutic doses without adversely affecting the kidneys. Recent

studies have discovered new protocols, compounds, enzymes, and molecular alterations that

reduce the nephrotoxicity of cisplatin.

This review will focus on cisplatin's chemistry, pharmacokinetics, pharmacodynamics,

mechanisms of resistance, toxicity, prevention of nephrotoxicity, and use in animals. Also

included is a section on the other platinum drugs that have been used in veterinary medicine to

date.

Chemistry

Cisplatin was inadvertently discovered while studying the growth characteristics of

Escherichia coli. (1) Initially, researchers observed that platinum compounds exhibited









antibacterial properties and subsequently it was discovered that they also possessed anti-

neoplastic properties. (1, 2) The molecular structure of cisplatin comprises a central platinum

atom surrounded by two chlorine atoms and two ammonia groups in a cis configuration. (3)

Other platinum compounds have the same core platinum compound, and cis configuration,

however, their leaving groups are different (Figure 1-1). (4) The bond angles for the platinum

core are fixed resulting in DNA bending to accommodate the structure of the drug. (4)

Pharmacokinetics

Plasma platinum has been shown to be highly protein bound. (5) Most cisplatin present in

the cell is found in the cytosol and is not protein bound. (6) Cisplatin's clearance in the dog is

biphasic in nature with a rapid phase half-life of 22 minutes and a slow phase half-life of 5 days.

(7) Significant amounts of platinum are still detectable in plasma 12 days after intravenous

injection. (7) A study performed in humans demonstrated that plasma platinum levels

corresponded with nephrotoxicity. (8) Plasma platinum levels may serve as a marker for risk of

nephrotoxicity in certain patients.

Urinary levels of platinum in the dog elevate rapidly after administration with 60% of the

dose recovered in the urine within the first 4 hours and 76% of the administered dose recovered

by 48 hours after treatment. (7, 9) Only small amounts of platinum were detected in bile

suggesting minimal fecal excretion. (7) Free platinum clearance has been shown to exceed the

creatinine clearance by 156% suggesting that in addition to excretion by filtration, cisplatin or a

metabolite is secreted by the kidney. (5) It is thought that secretion involves active accumulation

of secreted substances in the renal cells and passive transport into the tubule of the lumen. (10)

The pars recta of the proximal tubule is the most active site of secretion and also the most

damaged site of the kidney during cisplatin nephrotoxicity. (5) Renal accumulation of platinum

is dependent on the presence of normal oxygen utilization and the organic base transport









system. (11) Thus, concurrent administration of drugs known to be transported by this system

can significantly reduce cisplatin uptake in the kidney. (11)

Cisplatin is initially distributed to all tissues, however, in the first hour, it tends to

accumulate in the kidney, liver, muscle, and skin. (7) The localization in the kidney and liver is

protracted, with high renal tissue concentrations present as long as 12 days after treatment in the

dog. (7) The highest tissue platinum concentrations occur in those tissues where the drug exerts

its most potent antineoplastic activity, such as the ovary and uterus. (7) It is thought that the

presence of tumor may alter the toxicity and pharmacokinetics of drugs. (12) One study in tumor

bearing rats showed that the distribution half-time was longer for the tumor bearing rats than

their controls, while the terminal elimination half-time was the same for both groups. (12) Based

on this study, it is unlikely that tumor presence would alter toxicity or other distribution-

dependent drug parameters. (12)

Pharmacodynamics

Cisplatin is activated by an equation reaction involving the exchange of the two chloride

leaving groups with water or hydroxyl ligands. (13) When a high concentration of chloride is

present, as in isotonic saline or extracellular fluid, the equation reaction does not occur and the

drug remains neutral. (13) The neutral form is believed to be biologically inactive. (14)

Intracellular fluid has approximately one-thirteenth the chloride concentration of extracellular

fluid and it is under these conditions that the equation reaction proceeds, leading to eventual

DNA damage. (13) The primary effect produced by cisplatin in cancerous cells is inhibition of

DNA synthesis. (15, 16) The ability to inhibit DNA synthesis occurs at much lower doses than

that necessary to inhibit RNA and protein synthesis. (15) DNA damage induced by cisplatin is

similar to that caused by alkylating agents. (17) With equation of the platinum compound the

two chloride groups are replaced with water and will bind to two sites in DNA. (4) Generally, if









the two sites are on the same DNA strand, the lesion is referred to as a DNA adduct and if the

sites are on different strands, the lesion is referred to as a DNA cross-link. (4) Cisplatin has been

noted to bind to all DNA bases but has a preference for the N-7 positions of adenine and guanine

due to the high nucleophilicity of the N-7 sites of these purine bases (Figure 1-2). (4, 13, 18)

Cisplatin forms bifunctional adducts >90% of the time with crosslinks being < 2% of the lesions

formed. (4) These adducts and crosslinks inhibit DNA template replication in mammalian

cells. (19) DNA crosslinks and adducts increase with time after the drug is removed and are

repaired slowly. (20) In vitro studies have also indicated that interaction between the cisplatin

molecule and DNA may contribute to the generation of superoxide radicals, causing further

toxicity to cancer cells. (21, 22)

Mechanisms of Resistance

Four generic pathways for cisplatin resistance have been uncovered. They include: altered

cellular accumulation, cytosolic inactivation of cisplatin, DNA repair, and altered apoptosis

(Figure 1-3). (4) In vitro studies have described active efflux particularly as mediated by CuH

transporters, ATP7A and ATP7B, and other less well-defined systems. (4) Covalent binding of

proteins or peptides with increased levels of sulfhydryl groups to cisplatin may confer cellular

resistance. (4) These compounds include glutathione (GSH) and metallothionein. (23-26)

MRP2 (multidrug resistance-associated protein 2) may also play a role in cisplatin resistance by

removing the cisplatin-GSH complex from the cells. (27) Platinum-DNA repair occurs by the

nucleotide-excision repair (NER) and NER is increased in cisplatin-resistant cells. (4, 28, 29)

Mismatch repair (MMR) mediates apoptosis in response to cisplatin. (30-32) Defects in MMR

result in altered cell sensitivity to cisplatin, most likely resulting in greater resistance. (4)

Reports indicate that where alterations in MMR exist, concurrent enhancement of the activity of

NER exists. (4)









Toxicities


Nephrotoxicity

Cisplatin is associated with several systemic toxicities, but is most frequently associated

with nephrotoxicity. Cisplatin induced nephrotoxicity occurs in a number of species including

mice, rats, dogs, and humans. An estimated 28 to 36% of human patients receiving an initial

dose of 50-100 mg/m2 of cisplatin develop acute renal failure. (33, 34) Cisplatin nephrotoxicity

is dose and duration of treatment dependent and is enhanced by the use of other nephrotoxins

such as aminoglycosides. (35, 36) Most patients who develop some degree of renal dysfunction

never fully recover. (37) However, one study evaluating the long-term renal effects of cisplatin

in human patients showed that renal dysfunction may not be progressive provided further insult

is avoided. In this study, an initial increase in creatinine and a decrease in GFR and renal plasma

flow were noted directly after treatment, but these levels remained stable for up to 12 to 24

months after discontinuing cisplatin treatment. (38)

The morphologic alterations in the kidney attributed to cisplatin administration occur in the

pars recta of the proximal tubule situated in the outer stripe of the medulla. (39) Histological

changes are consistent with both apoptosis and necrosis. (40) One of the earliest

histopathological changes noted is the swelling of mitochondria. (14) Most of the pathological

changes start 3 days after cisplatin administration, including clumped nuclear chromatin,

increased number of cytoplasmic vesicles, focal loss of microvillus brush border, and completely

necrotic cells sloughed in the tubular lumen. (39) The most severe damage is seen 5 days after

cisplatin administration and consists of widespread tubular necrosis in the pars recta,

desquamation of necrotic epithelia cells resulting in a denuded basement membrane, necrotic

cells and debris in the tubular lumen, and the changes seen in 3 days after cisplatin









administration for non-necrotic cells. (39) By 7 days after cisplatin administration, extensive

regeneration in the pars recta is noted with necrotic cells still present. (39)

Research into the mechanism of cisplatin nephrotoxicity is an important step in developing

methods for renal protection. One theory involves DNA crosslinks and the position in the cell

cycle. It was thought that cisplatin-DNA crosslinks could be the cause of cytotoxicity in the

renal cell, but the proximal tubule cells selectively killed by cisplatin are relatively quiescent and

therefore should not be as sensitive to the toxicity of DNA damaging agents. (41) However,

there is a fall in DNA turnover that precedes necrosis in the proximal tubule and the later

increase in DNA turnover in those cells coincides with the timeline of regeneration. (42) Both

the outer cortex and outer stripe of the outer medulla (pars recta) have decreased DNA synthesis

1 day after cisplatin administration yet only the cells in the pars recta undergo necrosis. (42)

Three possibilities have been considered regarding this theory including: inhibition of DNA

synthesis is irrelevant to cytotoxicity in the kidney; cells in the pars recta cannot repair the

damage as cells elsewhere can; or the levels of DNA adducts in the pars recta are lethal whereas

those produced in other segments are not. (42, 43) Interestingly, recent studies have related

cisplatin administration to alterations in the cell cycle. Cells in the kidney enter the cell cycle

after cisplatin administration and genes for the p21 and 14-3-30 cell cycle inhibitors are

simultaneously upregulated. (44-46) Mice with a deleted p21 gene were more sensitive to

cisplatin injury. (46) Price et al. showed that the addition of p21 adenovirus and the

pharmacological inhibitor of cyclin dependent kinases, roscovitine both protected kidney cells

from cisplatin induced nephrotoxicity in vitro. (47) p21 inhibits caspase activation which is

discussed later as a mechanism of apoptosis in the renal tubule. (47)









Platinum drugs are similar to other heavy metals such as mercury. Another possible

mechanism for nephrotoxicity incorporates the known mechanism of nephrotoxicity in these

other heavy metals. (48) This toxicity occurs due to the binding of cisplatin to sulfhydryl (SH)

groups in the kidney which are necessary for enzyme function and depletion of intracellular

glutathione. (48, 49) The decrease in SH groups occurred before the rise in BUN and creatinine

and was not seen with acute renal failure caused by glycerol, another potent nephrotoxin. (49)

Proximal tubular cell death was initially believed to occur mostly by necrosis. However,

another mechanism of proximal tubule cell death is apoptosis. A previous study showed that the

type of cell death was concentration dependent with high concentrations of cisplatin leading to

necrosis and low concentrations causing apoptosis. (50) Reactive oxygen species (ROS) and

mitochondria are thought to play a role in the apoptotic cascade. (51) Mitochondrial dysfunction

occurs early in cisplatin-induced renal tubular toxicity and is potentially mediated by ROS. (52,

53) It was shown in a previous study that overexpression of manganese superoxide dismutase,

an antioxidant enzyme found in mitochondria, protected renal epithelial cells in vitro from

cisplatin toxicity. (54) In vitro studies performed on renal proximal tubule cells examined the

role of caspases and p53 in apoptosis related to cisplatin. Lau et al. showed that caspase 3 was

activated in vitro in response to cisplatin but the initiators of the activation were not found. (55)

The tumor suppressor gene p53 is activated in response to DNA damage, alterations in the cell

cycle, and hypoxia. (56) Another study found that 50% of cisplatin induced renal proximal

tubular cell apoptosis was mediated by p53 and that p53 activates caspase 3 independent of other

caspases or mitochondrial dysfunction. (57) The other 50% is mediated by additional

mechanisms independent of p53 and caspases 3, 8, and 9. (57) Interestingly, a different in vitro









study performed by Xiao et al. showed that when a caspase inhibitor that has no effect on p53

was applied to renal tubular cells, cisplatin-induced apoptosis did not occur. (58)

Recent studies in rats and mice have shown that the nephrotoxicity of cisplatin can be

blocked by inhibiting either of two enzymes expressed in proximal tubules, gamma-glutamyl

transpeptidase (GGT) or cysteine-S-conjugate beta-lyase. (59-61) This suggested that metabolic

activation of cisplatin to a nephrotoxin occurred in the kidney. (41) For this reaction to occur,

cisplatin must form a conjugate with glutathione which has been shown to occur spontaneously

in solution. (62, 63) Selective inhibition of each enzyme resulted in a decrease in toxicity, in

vitro. (41) Interestingly, conjugation of cisplatin with glutathione reduces cisplatin crosslinks

with DNA resulting in decreased toxicity to dividing cells, suggesting decreased antitumor

activity. (64) Also, GGT expression in tumors has been shown to decrease the antitumor activity

of cisplatin. (65) These conflicting reports show that there are many areas regarding

mechanisms of cisplatin-induced apoptosis and necrosis that still need to be investigated.

The physiologic alterations seen with cisplatin are relatively consistent. Renal failure is

gradual and usually occurs 3 to 5 days after administration. (66) Polyuria may be due to a

reduction in normal cortical-papillary solute gradient in association with a failure to recycle

urea. (67) Whole kidney GFR, single nephron GFR, and renal plasma flow are all decreased

after cisplatin administration. (38, 38, 42, 68) Initially, it was thought that the renin-angiotensin

system may play a role in cisplatin-induced acute renal failure, although experimental studies

failed to confirm this hypothesis. (42, 69) In rats, decreases in GFR are related to afferent

vasoconstriction and possibly an altered ultrafiltration coefficient, both of which occur before

evidence of tubular obstruction. (70) It should be restated that histopathologically, the

glomerulus is minimally affected by cisplatin.









Hypomagnesemia and Hypocalcemia

Other physiologic changes related to cisplatin administration include hypomagnesemia and

hypocalcemia. Hypomagnesemia has been reported to occur in more than half of human patients

receiving cisplatin chemotherapy. (71) The persistent excretion of magnesium in the presence of

declining magnesium levels suggests that the hypomagnesemia is due to a renal defect in

magnesium reabsorption. (71) This is not necessarily associated with overt renal insufficiency

and may be a more common manifestation than renal failure. (71) The mechanism for this is still

slightly unclear but studies in rats suggest that abnormal magnesium excretion may be due to a

defect in magnesium transport in juxtamedullary nephrons or collecting ducts. (72) When

clinical manifestations of hypomagnesemia such as neuromuscular, CNS, and cardiac function

abnormalities occur usually seen with serum levels less than 1 mEq/L, parenteral replacement of

magnesium sulfate should be administered. (34)

Hypomagnesemia is usually complicated by hypocalcemia that is probably secondary to

diminished PTH release and/or end-organ resistance to parathyroid hormone induced by

hypomagnesemia. (73, 74) Hypocalcemia resolves when magnesium is replaced and is

unresponsive to calcium replacement alone. (34)

Gastrointestinal and Myelosuppression

Gastrointestinal toxicity and myelosuppression appear to be associated with the death of

the rapidly dividing cells in the lining of the gastrointestinal tract and in the bone marrow.

Cisplatin also activates the chemoreceptor trigger zone to induce vomiting. (3) The use of anti-

emetics such as metoclopramide, butorphanol, dolasetron, ondansetron, and chlorpromazine can

be given prior to, during, and after cisplatin infusion to decrease nausea and vomiting. (75, 76)









Ototoxicity

Ototoxicity has been reported more commonly in human patients than the dog. Ototoxicity

has been observed in 7-90% of human patients receiving doses of up to 120 mg/m2 per

course. (77, 78) Hearing loss is in the high-frequency range and is dose-related, cumulative, and

frequently irreversible. (79, 80) Concurrent cranial irradiation enhances the ototoxicity. (81) In

one study with high-dose cisplatin, in spite of significantly lower hearing levels, no human

patient suffered a disabling hearing loss requiring a hearing aid and the use of hypertonic saline

and vigorous hydration was not found to minimize ototoxiciy. (82) While humans manifest this

toxicity in hearing a high-pitched tinnitus (ringing in ears) and hearing sounds differently, small

animals express this as an inappropriate response or an unusually strong response to an auditory

stimulus, such as hyperactivity or excessive barking. (83) The mechanism for this toxicity is

unclear but may involve spontaneously recruiting adjacent neurons, aberrant cochlear fluid

currents, or undermodulation of membrane movement. (83)

Neurotoxicity

Neurotoxicity is described as a peripheral neuropathy and usually develops in human

patients that receive a cumulative dose of 400 mg/m2 or higher. (84) In studies using high dose

cisplatin with protective measures, mild to moderate paresthesias have occurred in some human

patients. (81, 85)

Syndrome of Inappropriate Secretion of Antidiuretic Hormone (SIADH)

SIADH has been reported in the human literature with the administration of cisplatin and

other cytotoxic drugs. (86) SIADH is characterized by hyponatremia with concurrent

hypoosmolality of the serum, continued renal excretion of sodium, no clinical evidence of

volume depletion, urine osmolality greater than that appropriate for concurrent osmolality of

serum, and normal function of the kidneys, suprarenal glands, and thyroid glands. (86)









Protective Measures for Nephrotoxicity


Saline Diuresis

The most common protocol for administering cisplatin consists of pre- and post-hydration

with concurrent saline diuresis. The maintenance of adequate hydration is important for

decreasing nephrotoxicity, but the mechanism of protection is unknown.

Diuretics

Other common methods for decreasing the nephrotoxicity of cisplatin include mannitol or

furosemide administration. The exact mechanism behind diuretics ameliorating cisplatin toxicity

is unknown, but postulated mechanisms include: accelerating the passage of cisplatin through

the renal tubules by increased urinary excretion, reversing the osmotic gradient in tubules by

mannitol, and blocking of sodium and water reabsorption by furosemide. (87) It has also been

suggested that these diuretics may also attenuate cisplatin nephrotoxicity reducing the

concentration of platinum in the urine. (9, 88) However, in the study by Pera et al. it was noted

that, while diuretic administration significantly improved renal function, some degree of tubular

necrosis was still present. (88) Another study showed neither mannitol nor furosemide was

superior to the other in reducing nephrotoxicity in the human patients involved. (87) However,

there have been conflicting studies comparing hydration with or without mannitol. In one study,

mannitol ameliorated nephrotoxicity better than hydration alone. (89) The conflicting study used

the same dose of cisplatin but stated there was no difference between groups receiving mannitol

and hydration or hydration alone. (90) In spite of these conflicting reports, administration of

mannitol or furosemide along with continuous saline diuresis has become standard practice when

using cisplatin chemotherapy in human cancer patients.









Hypertonic Saline

Protection from nephrotoxicity was also seen when cisplatin was dissolved in a hypertonic

NaCl solution (4.5%) relative to distilled water with no effect on the antitumor action of

cisplatin. (91) It is postulated that the presence of the high concentration of NaCl in the vehicle

was great enough to force the equation reaction far to the left thus favoring the presence of the

parent cis molecule decreasing binding to plasma proteins and tissue binding sites. (91)

Pharmaceuticals

Additional drugs have been administered in conjunction with cisplatin to reduce

nephrotoxicity. Amifostine (WR-2721) is a SH-containing compound that when injected before

cisplatin in rats, decreased nephrotoxicity by a factor of 1.7 without inhibiting its antitumor

effect. (92-94) Diethyldithiocarbamate (DDTC) is a chelating agent that potentially removes

platinum bound to renal tubules. (95) However, several side effects (hypertension, agitation,

flushing, and diaphoresis) that required patients to receive sedation during administration and the

failure of the drug to ameliorate gastrointestinal side effects and ototoxicity, have limited the

clinical application of this drug. (81) Probenecid is thought to partially inhibit platinum renal

secretion and subsequently decreases the platinum concentration in the renal tubules, decreasing

nephrotoxicity without affecting cisplatin's antitumor activity. (85) This drug is nontoxic,

inexpensive, and readily available, but failed to protect against the other side effects of cisplatin

such as myelosuppression, gastrointestinal toxicity, and ototoxicity. (85) Sodium thiosulfate is

an antioxidant in the thiol family. (96) It is used most commonly in conjunction with

intracavitary cisplatin to reduce toxicity and allows the dose of cisplatin to be delivered to be as

high as 270 mg/m2. (97) In a recent study performed on a rat model, sodium thiosulfate was

found to provide protection from cisplatin ototoxicity when delivered at 4 to 8 hours after

cisplatin, but was not consistently protective against nephrotoxicity. (98) Procainamide, an









antiarrhythmic agent, has also been shown to protect against cisplatin nephrotoxicity without

altering its antitumor effects. (99) Procainamide and cisplatin form a complex that increases the

amount of platinum bound to DNA and may prevent metabolism of cisplatin to a nephrotoxin by

GGT through the formation of a cisplatin-glutathione complex. (41, 100) Methimazole, an

antithyroid drug, was given intraperitoneally 30 minutes prior and 4 hours after cisplatin infusion

without saline prehydration to normal dogs and was found to significantly decrease

nephrotoxicity. (101) Methimazole is thought to exert an antioxidative effect to protect the

kidney, but it is unknown whether this compound affects cisplatin tumoricidal activity. (101)

Liposome-encapsulation of cisplatin has also been proven to allow an increase in dose of

cisplatin that can be safely administered without increasing the nephrotoxicity in dogs and

cats. (102-104)

Enzymatic and Molecular Alterations

Reduction of nephrotoxicity has also been associated with some enzymes or agents that

control or prevent the formation of free radicals. One study demonstrated a decrease in cisplatin

nephrotoxicity in rats treated with a superoxide dismutase mimetic, orgotein. (105) Another rat

model study used N-acetylcysteine, an antioxidant, delivered at 400 mg/kg IV 15 minutes prior

to cisplatin injection and found that treated rats had normal BUN, creatinine, and histologically

normal kidneys 3 days after injection. (98) Many studies investigating the mechanism of

cisplatin nephrotoxicity have been using agents such as caspase inhibitors and antioxidants to

view their role in apoptosis of renal tubular cells. (47, 54, 58) Also, upregulation of the genes

p21 and manganese superoxide dismutase is considered as a potential future therapy, as studies

done in vitro have demonstrated that upregulation of these two genes resulted in protection

against cisplatin nephrotoxicity. (47, 54) Unfortunately, many of these studies have been in vitro









on kidney tubule cells and these genes' effect on cisplatin's in vivo effects in higher mammals

and antitumor effect is still unknown.

Another recent development has been with the endogenous enzyme heme oxygenase-1

(HO-1). HO-1 is an inducible enzyme that degrades heme and produces carbon monoxide (CO),

iron, and biliverdin. Biliverdin is reduced to bilirubin in vivo and is a powerful antioxidant. (106,

107) Carbon monoxide possesses vasodilatory, anti-inflammatory, and anti-apoptotic properties.

(108-111) HO-1 upregulation occurs after cisplatin administration and upregulation protects

against cisplatin nephrotoxicity. (112) Tayem et al. recently showed that a water-soluble carbon

monoxide releasing molecule protected renal tubular cells from cisplatin injury in vitro and in

vivo in rats. (113) Again, as with other recent studies mentioned previously, it is unknown

whether HO-1 interferes with cisplatin's antitumor effect. Recently, in our laboratory, we have

reported that hyperbilirubinemia ameliorates nephrotoxicity in rats receiving cisplatin. (114)

Cisplatin Use in Animals

Dogs

Cisplatin has been used as a systemic or local chemotherapy agent in dogs via intravenous,

intraarterial, intramedullary, intralesional and intracavitary routes. Two short-term diuresis

protocols have been utilized in the dog (Table 1-1). The incidence of nephrotoxicity was similar

between the studies and survival times for the dogs developing nephrotoxicosis were similar to

those that did not develop nephrotoxicosis in both studies. (115, 116) In human patients, the

cisplatin dose can be divided over 5 days with the patient receiving concurrent NaCl diuresis

during cisplatin administration, adequate pre- and posthydration, and possible use of the diuretics

mannitol and furosemide. (117) Administering cisplatin over many days under constant diuresis

is not utilized in veterinary medicine because it is not cost effective in animals.









Cisplatin is most commonly used as a single agent or as part of combination chemotherapy

with doxorubicin to treat osteosarcoma. Median survival times in dogs treated with cisplatin as

the sole chemotherapeutic agent range from 262 to 413 days. (118-122) Various combination

protocols have been investigated. Doxorubicin (30 mg/m2 IV on day 1) and cisplatin (60 mg/m2

IV on day 21) repeated for 2 treatment cycles resulted in median survival times of 300

days. (123) A more recent study investigated the use of doxorubicin and cisplatin administered

within 24 hours of each other. (124) Cisplatin (50 mg/m2) was administered IV followed by

doxorubicin (15 mg/m2) 24 hours later with the intent of completing 4 treatment cycles. (124)

Median survival times were equivalent to studies where cisplatin and doxorubicin were

administered 3 weeks apart at 300 days, but significant toxicity was encountered with this

protocol. (124) Renal toxicity was present in 11% of patients, which is higher than the reported

incidence in the diuresis studies utilizing cisplatin as a sole treatment agent. (124)

Gastrointestinal toxicity and myelosuppression were comparable to larger studies utilizing only

cisplatin. (124-126) The use of STEALTH liposome-encapsulated cisplatin versus carboplatin

has found that while the use of the STEALTH cisplatin allowed safe administration of five times

the maximally tolerated dose of free cisplatin, this did not translate into prolonged disease-free or

overall survival. (127)

Other methods of administration of cisplatin for the treatment of osteosarcoma that have

been investigated include intralesional (with implants or injection), intraarterial, and

intramedullary administration. Amputation is usually recommended for dogs that have

appendicular osteosarcoma to achieve local tumor control and palliation, but concurrent

orthopedic or neurological disease may make amputation less feasible in certain dogs. When this

occurs, limb-spare procedures and palliative radiation have been utilized as acceptable









alternatives. Cisplatin-containing implants have been utilized in limb-spare procedures for

additional local control. (128) Dogs receiving cisplatin implants were 53.5% less likely to

develop local recurrence than the control groups, although this effect did not reach statistical

significance. (128)

Percent tumor necrosis was found to be statistically significant when predicting local

tumor control. (129) Significantly greater tumor necrosis is observed after intraarterial cisplatin

administration when compared to systemic cisplatin administration using an intravenous route.

(129) The use of radiation therapy in addition to intraarterial cisplatin further increased percent

of tumor necrosis. (129) One study investigated the use of intraarterial cisplatin and radiation in

dogs for local tumor control. (130) Radiation was administered in 10 equal fractions, 3 days a

week while cisplatin (70 mg/m2) was given intraarterial in the affected leg on the first and last

treatment days over 2 hours with appropriate diuresis. (130) Eighty-nine percent of dogs had an

improvement in limb function with no toxicity noted as a result of therapy. (130) Intraarterial

cisplatin did not improve survival times by preventing metastasis as intravenous cisplatin

does. (130) Intramedually cisplatin (60 mg/m2 over 20 minutes) administered with a Jamshidi

biopsy needle inserted into the tumor provided effective local control in 50% (2/4) of dogs

unable to undergo amputation or a limb-spare procedure. (131)

Cisplatin has also been utilized for transitional cell carcinoma (TCC) and squamous cell

carcinomas (SCC). At a dose of 50 mg/m2 given IV every 28 days, cisplatin was found to have a

palliative effect for dogs with SCC and TCC. (118) No complete responses were observed in

this study, but partial remission and stable disease were observed in the majority of dogs without

significant toxicity noted. (118) In another study in which cisplatin was given at 60 mg/m2 IV

every 3 weeks to dogs with a variety of malignant tumors that included TCC and SCC, an overall









response rate (complete or partial remission) of 19% was observed. (126) Those dogs

demonstrating progressive disease did so by 42 days after the start of treatment, suggesting that

dogs should be evaluated at this time to determine response to treatment. (126)

The use of cisplatin concurrently with radiation therapy is a source of interest as cisplatin

reportedly inhances radiation-induced cell kill. (132-136) A study performed on dogs with naso-

sinus carcinomas compared cobalt radiation alone with radiation in addition to cisplatin at

7.5mg/m2 IV bolus before every other radiation treatment. (137) The mean and median tumor

control and survival times were not significantly different between treatment groups; however,

there was a trend toward longer tumor control and survival times in the cisplatin treatment

group. (137) Another study regarding nasal tumors utilized an OPLA-Pt implant concurrently

with radiation and found that the implant was clinically tolerable and yielded comparable or

perhaps improved survival times when compared to other published protocols. (138) The use of

radiation with cisplain has also been researched with regard to oral melanomas. One study gave

cisplatin 10-30 mg/m2 IV with diuresis or carboplain 90 mg/m2 IV prior to 6 weekly 6-Gy

radiation fractions. (139) The use of low dose chemotherapy resulted in a median survival of

363 days which was longer than the previously reported survival times for surgery or radiation

therapy alone. (139) It should be noted that the dogs in this study had small initial tumor size

and no lymph node or lung metastasis at the start possibly positively affecting survival

times. (139)

Intralesional cisplatin has also become a possibility for local cisplatin administration.

Implants containing cisplatin, viscous gel, and a vasoactive modifier have been used as primary

treatment for melanomas. (140) Implants were injected until tumor saturation was visualized and

these treatments occurred weekly until complete tumor resolution was observed. (140) Seventy









percent of dogs had a >50% decrease in volume and 55% of these dogs had a complete

response. (140) Tumors that responded received a mean of 2.6 treatments. (140) The most

common side effect was local necrosis seen in 85% of patients and was associated with tumor

response. (140) Systemic toxicosis was minimal with no dog exhibiting renal toxicosis. (140)

Patient survival was comparable to other forms of local treatment such as radiation and

surgery. (140) Although there are no current published reports in dogs, intralesional cisplatin is

also being utilized for other tumors using sesame oil instead of the viscous gel described above.

Intracavitary cisplatin is the last well-known use of cisplatin in dogs. In one study, 50

mg/m2 of cisplatin was administered every 28 days for a median of 2.5 treatments to dogs with

mesothelioma and carcinomatosis. (141) When using intracavitary chemotherapy, the tumor is

exposed by the capillary blood supply to a concentration equivalent to that achieved by IV

administration, and the surface cell layers are exposed to a concentration that is 1-3 logs

higher. (142, 143) Although intracavitary cisplatin administration is a local method of

chemotherapy delivery, significant systemic absorption does occur and the dose limiting toxicity

for intracavitary cisplatin is renal toxicity.(141) Animals in this study underwent diuresis and

66.7% of dogs received additional treatment with sodium thiosulfate preventing toxicity during

the study. (141) This method of cisplatin delivery was associated with palliation and control of

malignant pleural and/or abdominal effusion in 5 of 6 dogs and this palliation lasted 129 to

greater than 807 days. (141) Although results for the previous study were promising, a more

recent study found that the use of intracavitary carboplatin or mitoxantrone was just as effective

for dogs with similar diseases. (144) The ease of administration for carboplatin and

mitoxantrone is superior to cisplatin since diuresis is not required and minimal side effects were









noted with the former two chemotherapy agents, suggesting that the use of cisplatin in

intracavitary infusions will become obsolete in veterinary medicine. (144)

Cats

Cisplatin is unable to be administered to cats due to its acute drug toxicity in this species.

Cats receiving 60 mg/m2 of cisplatin became dyspneic and died 48-96 hours after

administration. (145) Postmortem findings included severe hydrothorax, pulmonary edema, and

mediastinal edema. (145) A group of cats undergoing equivalent saline diuresis as the cisplatin

group did not show these signs, causing the belief that cisplatin had induced the changes

mentioned. (145) Lowering the dose of cisplatin to 40 mg/m2 resulted in similar but less severe

pulmonary changes, while decreasing the dose to 20 mg/m2 showed no pulmonary

changes. (145) The tumoricidal activity of cisplatin alone at such doses is not known. Using

repetitive low dosing (10 mg/m2 3 times a week for 10 txt), cisplatin use resulted in reversible

pulmonary edema and renal insufficiency. (146) The use of liposome encapsulated cisplatin has

been researched in cats. Studies with this form of cisplatin showed no renal or pulmonary

toxicity but all cats had transient pyrexia and/or lethargy, vomiting, inappetence, and an acute

infusion reaction prevented by administering atropine-diphenhydramine. (102, 103) When this

formulation was looked at in cats with squamous cell carcinoma, the liposome encapsulated

cisplatin was found to be an ineffective treatment since none of the cats had complete or partial

remissions. (147)

Horses

Intratumoral cisplatin in oily emulsion has proven efficacious in treatment of cutaneous

tumors in horses such as squamous cell carcinoma and sarcoids. (148, 149) Intralesional

cisplatin can be used alone for treatment of small tumors or in combination with surgery for

larger tumors. (150) Treatments are given at 2 week intervals at a dose of 1 mg cisplatin for each









cm3 of tissue in the target field. (151) When surgery is performed, cisplatin should be

administered perioperatively or early in the postoperative period. (151)

Other Platinum Drugs

The most commonly used alternative platinum drug to cisplatin is carboplatin. Carboplatin

has been shown to be less nephrotoxic than cisplatin and can be administered without saline

diuresis. Similarly, nausea and vomiting are common side effects with carboplatin as they are

with cisplatin. Carboplatin alone or in combination with doxorubicin has been utilized to treat

dogs with osteosarcoma with survival times at 321 days and 320 days respectively which are

similar to survival times achieved to cisplatin alone or in combination with doxorubicin. (152,

153) However, in regards to treating TCC, carboplatin was not as effective as cisplatin in

producing a clinical response. (154) Other tumors in which carboplatin's use has been

researched include: malignant melanoma, nasal tumors, and anal sac adenocarcinoma. As

mentioned previously, carboplatin has additional uses as an intracavitary chemotherapeutic and

as a radiosensitizer. (139, 144)

Lobaplatin, another platinum analog, was found to result in a one-year survival fraction of

31.8% when administered every 3 weeks to dogs with appendicular osteosarcoma. (155)

Clinical signs related to toxicosis were uncommon and usually were vomiting and

depression. (155) Unlike cisplatin, lobaplatin did not require pretreatment infusions.

Summary

Cisplatin has long been utilized in both human and veterinary medicine. Cisplatin is still

used widely in many protocols in human patients affected with head and neck, lung, and germ

cell tumors, as well as OSA. The use of cisplatin in veterinary medicine is not as widespread

which is most likely due to cisplatin's severe adverse side effects. In addition, carboplatin, a

platinum analog, has shown fewer severe side effects and has recently become a more cost









effective alternative to cisplatin. It is still likely that cisplatin will continue to be utilized in

veterinary medicine for treatment of OSA, intralesional chemotherapy for a variety of tumors,

and as a sensitizer prior to radiation therapy.









Table 1-1: Canine diuresis protocols when administering cisplatin. These protocols utilized 70
mg/m2 given with 0.9% NaCl IV over 20 minutes.
Duration Fluid rate Length of Length of Incidence of Reference
of diuresis (ml/kg/hr) diuresis before diuresis after nephrotoxicity
cisplatin cisplatin
4 hours 25 3 hours 1 hour 7.8% (116)
6 hours 18.3 4 hours 2 hours 6.6% (115)











O

SNH~2 O-C
Pt"o
""NH / O-C

Oxalipla
Oxaliplatin


,- NH


SO-C
Ptoc
O-C

O


Lobaplatin


0
O
H2N, OC
Pt CH2
H2N O \ H2
OH2


Carboplatin


Cl Cli
Pt
NH2 NH2


Cisplatin


Figure 1-1: Two-dimensional structures for cisplatin, carboplatin, oxaliplatin, and lobaplatin.
While the core structure (Pt) is the same for each drug, the leaving groups are
different for each compound.











H2N p
/Pt
H2N

pKa= 74







SH,
CI H


OH


H2
H2
H,


OH

OH


H2N p CI

H2N CI
Cisplatin



pKa=74


DNA
3' 5'
DNA Adduct

HN Guanine C
Pt
H2N Guanine C


5' 3'


Figure 1-2: Aquation reaction and adduct formation at the N-7 position of guanine on 2 sites of
DNA. These adducts result in DNA damage resulting in cell kill.


N OH;,
+-
2N OH2
Pt


SpKa=5 6

N CI

N OH;


H2N,

HN











Pt
H2N Cl
Cisplatin


Reduced uptake


Cancer Cell


Figure 1-3: Four pathways exist for cisplatin resistance. They include: (1)decreased cellular
accumulation, (2) inactivation of the drug, (3) DNA repair, and (4) prevention of
apoptosis.









CHAPTER 2
HYPERBILIRUBINEMIA PROTECTS AGAINST CISPLATIN NEPHROTOXICITY IN THE
GUNN RAT

Introduction

Cisplatin is one of the most commonly used antineoplastic agents in human patients.

Cisplatin is currently a front line drug used in chemotherapy protocols to treat a wide variety of

tumors including ovarian, cervical, testicular, head and neck tumors, transitional cell carcinomas,

osteosarcomas, small cell lung, and esophageal cancers. Cisplatin is also utilized as a rescue

agent in the treatment of other solid tumors. (4, 156-161)

Systemic dose-related toxicities associated with cisplatin are well-documented and have

precluded its use in many patients. Of these, nephrotoxicity is the most frequently observed and

most clinically significant toxicity. The mechanism for cisplatin nephrotoxicity has not been

completely elucidated; however, many theories have been developed. (42, 43, 48-50) One theory

is that reactive oxygen species (ROS) and mitochondria play a role in the apoptotic cascade

involved in cisplatin nephrotoxicity. (51) The morphologic alterations in the kidney ascribed to

cisplatin occur in the pars recta of the proximal tubule situated in the outer stripe of the medulla

and the maximum damage is seen by day 5 after administration. (39) In vitro studies have shown

that necrosis and apoptosis can occur, with the form of cell death being dependent on the

concentration of cisplatin the cells are exposed to. (50)

Recently, the endogenous enzyme heme-oxygenase 1 (HO-1) has been investigated for its

role in protecting organ systems from various insults. HO-1 is induced in response to cellular

stress, and converts the pro-oxidant heme molecule into eqimolar quantities of biliverdin (BV),

carbon monoxide (CO), and iron. (162) BV is converted to unconjugated bilirubin (BR) via

bilirubin reductase. (163) Unconjugated BR is then converted to conjugated BR by the hepatic

microsomal enzyme, uridine diphosphate glucuronyltransferase (UDPGT). (164) These









molecules of heme degradation were once considered to be toxic metabolites, but have recently

been shown to have dose-dependent vasodilatory, anti-oxidant, and anti-inflammatory properties

that may be useful in protection of various organ systems from toxic insult. (165)

Specifically, the HO-1 enzyme and its products have been studied in association with toxic

acute renal failure. Hyperbilirubinemia has been shown to result in protection against acute renal

failure caused by the nephrotoxin glycerol. (166) With regards to cisplatin mediated

nephrotoxicity, depletion of the HO-1 enzyme resulted in more significant renal failure and renal

injury in one study (112) and the administration of CO along with cisplatin ameliorated signs of

renal failure in another study. (113) However, upregulation of HO-1 or its products may affect

cisplatin's antineoplastic activity.

The objectives of this study were to investigate the protective effect of hyperbilirubinemia

in vivo in the rat model against cisplatin-induced nephrotoxicity. Hyperbilirubinemia in vivo was

achieved using both the homozygous and heterozygous Gunn rat. The homozygous Gunn rat is

unable to induce UDPGT as a result of an autosomal recessive deficiency in this enzyme. (167,

168) The lack of induction of UDPGT results from an alteration in the coding region of the

mRNA which results in an instability of the mRNA and a synthesis of a truncated, functionally

inactive UDPGT. (168) In addition, an intermediate level ofbilirubin can be obtained using

heterozygous Gunn rats that have varying degrees of functional UDPGT. We also investigated

the effect of bilirubin on the antineoplastic activity of cisplatin using four established canine

osteosarcoma cell lines (POS, HMPOS, COS31, D17).

Materials and Methods

Animals

This study was approved by the University of Florida Institutional Animal Care and Use

Committee and was performed in accordance with the Institute for Lab Animal Research Guide









for the Care and Use of Laboratory Animals. Male Wistar, homozygous Gunn (j/j) and

heterozygous Gunn (j/+) rats weighing 200-400 g were purchased from Harlan Sprague Dawley,

Inc (Indianapolis, IN) and maintained in a temperature controlled room with alternating 12 hour

light/12 hour dark cycles in an animal facility at the University of Florida. Animals were fed

standard rat chow and allowed free access to water.

Cisplatin-Induced Acute Renal Failure

Three groups of male rats (n=6 rats/group) were used: (1) Wistar (2) homozygous Gunn

(3) heterozygous Gunn. Rats were weighed and observed for changes in attitude during the

course of the experiment. On Days 0, 3, and 5, rats were anesthetized using 5% inhalant

isoflurane in 100% oxygen and maintained with 2-3% isoflurane by mask. Animals were placed

on a warm water heating pad to maintain normal body temperature. Prior to blood sampling on

Days 0 and 3, the rat's tail was soaked in 40-42C water for 3-5 minutes to facilitate

vasodilation. One milliliter of blood was sampled from the tail veins or the lateral saphenous

veins. After blood sampling was complete on Day 0, all rats were given an intraperitoneal

injection of cisplatin (American Pharmaceutical Partners Inc, Schaumburg, IL) at 4 mg/kg. Once

the IP injection was complete, all rats were recovered. Upon recovery, an injection of 0.01

mg/kg buprenorphine hydrochloride (Reckitt Benckiser Healthcare (UK) Ltd, Hull, England)

was given SQ. Blood was sampled on Days 0, 3, and 5 for evaluation of BUN, serum creatinine,

and serum bilirubin concentrations. On Day 5, once the rats were anesthetized a midline incision

was performed and blood was sampled from the caudal vena cava. Both kidneys were isolated

and harvested and the rats were euthanized by an overdose of sodium pentobarbital (Euthasol,

Diamond Animal Health, Inc., Des Moines, IA).

Three groups of male rats (n=4 rats/group) were used as sham control rats. The groups

consisted of: (1) Wistar (2) homozygous Gunn (3) heterozygous Gunn. These groups were









treated as described above for the cisplatin induced acute renal failure groups; however, instead

of receiving 4 mg/kg cisplatin, the rats received the equivalent amount of 0.9% sodium chloride

IP. The sham control rats for each group were intended to prove that equivalent times of

anesthesia would have no effect on the kidney functional parameters and histopathology.

Assays

BUN and serum bilirubin (BR) concentrations were determined using an automated

chemistry analyzer (Hitachi 911 Chemistry Analyzer). Serum creatinine (Cr) concentrations

were determined using a dry chemistry analyzer (Johnson & Johnson Vitros DT6011) due to the

potential effect of icterus on the standard Jaffe methodology for measurement of Cr. (169)

Histologic Grading

Both kidneys were placed in 10% buffered formalin for at least 24 hours before processing.

Transverse sections of the left kidney for all rats were processed using hematoxylin and eosin

(H&E) staining and periodic-acid-Schiff (PAS) staining. Histological examination was

performed by a renal pathologist who was blinded with respect to the treatment groups. Renal

tissue was divided into 4 regions for analysis: cortical proximal tubules (CPT), S3 segment of

the outer stripe of the outer medullary proximal tubule (OSOMPT), medullary thick limb in the

inner stripe (ISOM mTAL), and collecting ducts (CD). Renal injury was graded in 7 different

categories: normal, cellular swelling/vacuolization, brush border loss, nuclear condensation,

karyolysis/apoptosis/necrosis (most severe form of injury), regeneration, and capillaritis. Each

category was assigned a numerical score: 0= none, 1 =<10%, 2= 10-25%, 3= 25-50%, 4= 50-

75%, 5= 75-100% based on the percentage of cells in each region displaying the described

injury.









Cell Lines

Osteosarcoma is a highly-aggressive tumor in dogs and affected dogs are commonly

treated with cisplatin. Four canine osteosarcoma cell lines which have been well characterized in

our laboratory were utilized in this study. The POS (parent osteosarcoma) cell line was

originally developed from a primary osteosarcoma affecting the left proximal femur of a 1 and a

halfyear-old male mongrel dog (Dr. Tsuyoshi Kadosawa, University of Sapporo, Japan). (170)

The HMPOS (highly metastatic parent osteosarcoma) cell line is a pulmonary metastatic

derivative of POS cell line (Dr. Tsuyoshi Kadosawa, University of Sapporo, Japan). (171) D17

is another established canine osteosarcoma cell line (American Type Tissue Culture Collection,

Manassas, Virginia). The COS31 cell line was established from a dog with spontaneously

occurring osteosarcoma (Dr. Ahmed Shoieb, University of Tennessee, College of Veterinary

Medicine, Knoxville, TN). Cells were cultured at 37C under 5% CO2 and 95% room air with

their respective media. POS and HMPOS media consisted of RMPI 1640 media supplemented

with 10 % heat inactivated fetal calf serum, vitamins, sodium pyruvate, non-essential amino

acids, L-glutamine, and antibiotics (penicillin (0.0625 g/L) and streptomycin (0.1 g/L)). D17 and

COS31 media consisted of Dulbecco's Modifed Eagle's medium with 10% heat inactivated fetal

calf serum, L-glutamine, and antibiotics (penicillin (0.0625 g/L) and streptomycin (0.1 g/L)).

The cells were grown to confluence, washed with physiological buffered saline, and detached

from the flasks with trypsin. Cells were stained with Trypan blue and counted with a

hemacytometer.

Cell Viability Assay

An assessment of cell viability was performed with the CellTiter BlueTM Cell Viability

Assay (Promega Corporation, Madison, WI). Assays were performed in 96-well flat-bottomed

black microtiter plates. All cell lines were seeded at 10,000 cells/well with 50 [tL of media and









placed in the incubator at 37C under 5% C02 and 95% room air for 24 hours. The IC50 for

each cell line with cisplatin was determined prior to treating the cells with bilirubin and cisplatin.

All cells were treated with 50 pL of bilirubin at concentrations of 71 [M and 128[M alone and

combined with each cell type's IC50 concentration of cisplatin. Bilirubin was dissolved in the

respective media to achieve appropriate concentrations. The micromolar concentrations of

bilirubin used in this study were taken from the average serum bilirubin levels on Day 0 and 3

that provided functional nephroprotection of the homozygous Gunn rats receiving cisplatin.

After incubation for 72 hours under the conditions described previously, 20tL of CellTiter

BlueTM reagent, resazurin, was added to each well. Viable cells retain the ability to reduce

resazurin into resorufin, which is pink and highly fluorescent. (172) Plates were placed on a

low-speed shaker for 10 seconds and then incubated for 4 hours. The amount of fluorescence

was recorded with a fluorescence plate reader at 530/590 nm.

Statistical Analysis

Statistical calculations were performed using a computer software program (SigmaStat for

Windows, version 3.00, and SigmaPlot for Windows, version 8.02, SPSS Inc, Chicago, Ill.)

Data was tested for normality and equal variance using the Kolmogorov-Smimov test. The

comparisons between groups used ANOVA for parametric data and ANOVA on Rank's for non-

parametric data. Differences between groups were identified using (post-hoc) pair wise multiple

comparison procedures (Holm-Sidak method or Dunn's Method). Parametric data is reported as

mean + SD and nonparametric data as median with an inter-quartile range ([IQR], 25% to 75%).

P value < 0.05 was considered significant.









Results


Renal Functional Parameters

Bilirubin levels were significantly higher for all days in the homozygous Gunn rats when

compared to the heterozygous Gunn rat and the Wistar rat (Table 2-1). Statistically, bilirubin

levels were not significantly higher in the heterozygous Gunn rat compared to the Wistar rat at

any day. However, the Wistar rats had a mean of 0 mg/dL and the heterozygous rats had a mean

of 0.4 to 0.1 mg/dL, indicating that a mild degree of hyperbilirubinemia existed in the

heterozygous Gunn rats.

The homozygous and heterozygous Gunn rats were protected from the nephrotoxic effects

of cisplatin based on functional kidney parameters. There was no significant difference in BUN

or Cr on Day 0 between any of the groups of rats (Figures 2-1 and 2-2). Importantly, in the face

of a nephrotoxic dose of cisplatin, the hyperbilirubinemia present in the homozygous Gunn rat

resulted in a significant nephroprotective effect when compared with the heterozygous Gunn rat

and the Wistar rat (means BUN homozygous: Day 5- 23.83 5.49 mg/dL, p<0.05;

means Cr homozygous: Day 5- 0.35 0.05 mg/dL). The protection was clinically significant as

the homozygous rats' BUN and Cr values remained within the normal range. While, the

intermediate level of hyperbilirubinemia provided by the heterozygous Gunn rat still provided

nephroprotective effects when compared to the Wistar rat, the protection was not as effective as

for the homozygous Gunn rat. When comparing heterozygous Gunn rat and Wistar rat BUN and

Cr levels with each other, the differences in BUN were not significant until Day 5 but were

present on Day 3 and 5 with regard to Cr (means BUN heterozygous: Day 5-

38.17 10.34 mg/dL; means Cr heterozygous: Day 5- 0.40 + 0.06 mg/dL; means BUN Wistar:

Day 5- 78.67 16.92 mg/dL; means Cr Wistar: Day 5- 1.4 + 0.43 mg/dL)









When comparing the homozygous Gunn rat to the heterozygous Gunn rat in regards to

functional kidney parameters, it was found that there was no significant difference in BUN on

Day 3 and Cr on Day 3 and 5 (Figure 2-1 and 2-2). However, there was a statistically significant

difference in BUN between the groups on Day 5, with the homozygous Gunn rats showing a

lower BUN than the heterozygous rats.

When all groups of sham rats homozygouss Gunn, heterozygous Gunn, Wistar rats) were

compared to the control group of rats receiving cisplatin, it was found that there was no

significant difference in BUN or Cr on Day 0. There were significant differences for the sham

groups when compared to the control Wistar group in BUN and Cr for Day 3 and 5. Between the

sham groups, there were no statistically significant differences in BUN and Cr at Day 0, 3, or 5.

Importantly, there was also no significant difference between the homozygous Gunn rat

receiving cisplatin and any of the sham groups in BUN and Cr at Day 0, 3, or 5. With regard to

bilirubin levels, there was a significant difference between the control Wistar rats and the sham

homozygous Gunn rats, but no significant difference between the control Wistar rats and the

sham heterozygous Gunn rats. This is equivalent to what was reported for the groups receiving

cisplatin.

Light Microscopy

For all treatment groups, the cortical proximal tubules and collecting ducts were graded as

75-100% of cells being normal. The area of most interest with regard to cisplatin nephrotoxicity

is the S3 segment of the outer stripe of the outer medullary proximal tubule (OSOMPT).

Homozygous Gunn rats had significantly decreased karyolysis/apoptosis/necrosis than the

heterozygous Gunn rats and the Wistar rats in the OSOMPT (Figure 2-3A and 2-3B). There was

no significant difference in karyolysis/apoptosis/necrosis between heterozygous Gunn rats and

Wistar rats in the OSOMPT. The homozygous Gunn rats had a significantly greater proportion









of normal cells and cells without cellular swelling in the medullary thick limb in the inner stripe

(ISOM mTAL) than the Wistar rats.

The sham groups had identical normal histological scores for all segments of the kidney

and were thus grouped together for statistical analysis. Even though the homozygous Gunn rats

receiving cisplatin had significantly decreased histologic damage to the OSOMPT than the other

groups receiving cisplatin, there was significantly greater degree of injury in the homozygous

Gunn rats receiving cisplatin with regard to karyolysis/apoptosis/necrosis in the OSOMPT when

compared to the sham groups. There was also a significant difference in cellular swelling, brush

border loss, and regeneration in the OSOMPT between the homozygous Gunn rats receiving

cisplatin and the sham groups. A significant difference was also noted between the

heterozygous Gunn rats receiving cisplatin and the sham groups in cellular swelling, brush

border loss, and karyolysis/apoptosis/necrosis in the OSOMPT.

Cell Culture Studies

In vitro, bilirubin had mild cytotoxic effect on COS31 cells at 72 and 128 iM and D17

cells at 128 [iM versus control cells. No significant cytotoxic effects of bilirubin were seen with

HMPOS and POS cells at either concentration or D17 cells at 72 riM. Bilirubin had no

significant effect on the antineoplastic effect of cisplatin at either concentration in any of the four

canine osteosarcoma cell lines (Figure 2-4).

Discussion

While the toxic properties of bilirubin, particularly kernicterus and neonatal

hyperbilirubinemia are well documented, the therapeutic properties of bilirubin are just recently

being discovered. (173-176) This study demonstrated that hyperbilirubinemia found in the Gunn

rat had a nephroprotective effect when the nephrotoxic, anti-neoplastic agent cisplatin was









administered, as evidenced by the maintenance of normal renal function values and markedly

less histologic evidence of tubular necrosis.

Protective effects of bilirubin against to cellular injury have been studied previously but

never in the cisplatin model of nephrotoxicity. Leung et al. showed that ligation of the common

bile duct effectively protected against glycerol induced acute renal failure in the rat model. (166)

Results of our study and the one performed by Leung suggest that sustained hyperbilirubinemia

protects the kidney from known potent nephrotoxins. (166) Our lab has also reported protection

from ischemia-reperfusion injury with exogenous bilirubin when delivered into the isolated,

perfused rat kidney. (177) The same effects, however, were not noted when the equivalent in

vivo rat study was performed. (165) This suggests that the protective effects ofbilirubin may

only be applicable to nephrotoxins or perhaps the dose of exogenous bilirubin required to protect

the kidney from various insults has yet to be ascertained.

Bilirubin has also shown a protective effect in other organ systems such as the liver (178,

179), intestine (180), and neural tissue (181). Clinical trials have shown that the incidence of

coronary and ischemic heart disease is lower in humans with hyperbilirubinemia. (182, 183)

Bilirubin is a product of HO-1. The beneficial effects of HO-1 induction have been well

established in multiple organ systems. (106, 166, 178, 184, 185) In fact, Shirashi et al

demonstrated that mice deficient in HO-1 (-/-) developed more severe renal failure and renal

injury than wild type mice (+/+) when cisplatin was administered. (112) These findings

prompted a look at the individual agents produced by HO-1 to determine if bilirubin or CO alone

could mimic this effect. A study by Tayem et al. showed that treatment with a water-soluble

carbon monoxide-releasing molecule protected the kidney function and improved histology of

rats treated with cisplatin. (113) The results of our study indicate that hyperbilirubinemia in the









homozygous Gunn rat was completely nephroprotective with regards to functional renal

parameters and partially protective histologically. Partial protection of renal functional

parameters were also provided to the heterozygous Gunn rat, whose bilirubin levels were not

statistically higher than the Wistar rat at any day, although histologically there was no significant

difference in the amount of most severe kidney damage to the S3 segment. An improvement in

functional kidney parameters is still important and a trend was present at day 0 for higher

bilirubin levels in the heterozygous Gunn rat than the Wistar rat. This suggests that the

protective effects of bilirubin may be exerted on the day the nephrotoxin is administered (Day 0).

The exact concentration of bilirubin in serum needed to prevent severe nephrotoxicity due

to cisplatin remains in question. Higher average serum bilirubin values present in the

homozygous Gunn rat resulted in significantly less kidney injury than the heterozygous Gunn rat

suggesting that the value of bilirubin required for any histological protection may lie somewhere

in between those two groups. For full histological protection, serum bilirubin values may need

to be higher than those seen in the homozygous Gunn rat. Unfortunately, a higher dose may

result in bilirubin toxicity. Most likely, individual variation in serum bilirubin levels exist even

between the homozygous and heterozygous Gunn rats due to a variation in the levels of the

enzyme UDPGT. Homozygous Gunn rats have a deficiency of this enzyme needed to conjugate

bilirubin but UDPGT may still be present in varying small amounts in these rats.

The sham group of rats served as a negative control group in this experiment. The purpose

of the sham group was to verify that the anesthesia episodes required for venipuncture had no

functional or histological effect on the kidneys that would skew our results. All of the rats in this

group maintained normal BUN and Cr and had histologically normal kidneys substantiating that

anesthesia did not contribute to renal pathology.









Another aim of our study was to determine if adding cisplatin with bilirubin would have an

additive, inhibitory, or no effect on neoplastic cells in culture. For this experiment, we utilized

four different canine osteosarcoma cell lines for completeness. Bilirubin did not adversely effect

cisplatin's in vitro antineoplatic activity in any of the four cell lines. This is a very important

aspect to view for clinical application of this study. Agents such as bilirubin that proved

nephroprotection cannot interfere with a chemotherapeutic agents efficacy to be useful clinically.

Bilirubin's antineoplastic effect needs to be tested in other neoplastic cell populations and in an

in vivo setting before any definitive conclusions can be drawn. Bilirubin has been shown to have

some cytotoxic effects on human colon adenocarcinoma cells and human carcinoma cell

lines. (186, 187) While two of the cell lines showed some cytotoxicity caused by bilirubin alone

(COS31 at 72 gtM and 128 giM and D17 at 128 giM), these effects were minimal and had no

additional impact on the cells after cisplatin was added. Bilirubin's cytotoxicity against

neoplastic cells may only pertain to certain types of neoplasia as evidenced by our cell culture

results.

Future studies would be helpful to determine if the same effects could be duplicated using

rats with a sustained conjugated hyperbilirubinemia. It is unknown if protection is afforded by

unconjugated or conjugated bilirubin. Future studies are also warranted to determine if

administration of exogenous bilirubin can mitigate cisplatin induced nephrotoxicity. Delivering

a bilirubin dose high enough to exceed the serum concentrations seen in the homozygous Gunn

rat without toxicity with resulting complete histological protection of the S3 segment of the outer

medulla may be possible. Another area to research may include concurrent administration of

exogenous bilirubin and the water-soluble carbon monoxide releasing molecule to determine if

the combination could prevent any histologic nephrotoxicity caused by cisplatin.









In conclusion, hyperbilirubinemia present in the homozygous Gunn rat resulted in

complete nephroprotection with functional renal parameters and partial nephroprotection

histologically. Only partial functional nephroprotection was seen in heterozygous Gunn rats that

did not demonstrate significant hyperbilirubinemia when compared to the control Wistar rats.

The anesthetic episodes each rat underwent did not impact functional or histologic kidney

parameters. In the four canine osteosarcoma cell lines utilized in our lab, bilirubin did not have

an adverse effect on the antineoplastic activity of cisplatin. Our findings suggest that HO-1,

more specifically HO-i's products especially bilirubin, may protect the kidney from toxic

nephropathy caused by cisplatin.









Table 2-1. Serum bilirubin concentrations were significantly higher for the Gunn j/j when
compared to the other groups as noted by the *. No significant difference was noted
between Gunn j/+ and Wistar rats but a trend toward higher bilirubin values was
noted on DO.
Means standard deviations serum bilirubin concentrations (mg/dL) in rats receiving
cisplatin


Day 0
Day 3
Day 5


Wistar
00
0.07 0
00


Gunn j/+
0.41 + 0.2
0.1 0
0.120


Gunn j/j
4.2 + 0.9*
7.5 1.2*
5.5 + 1.1*


Wistar
I Gunn j/+
80 Gunn jlj


Day 0 Day 3 Day 5
Days


Figure 2-1. Serum BUN concentrations on DO, 3, and 5. No significant difference between any
groups on DO. The most significant difference in BUN occurred when comparing the
Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a significantly
higher BUN.












I(o P
1.6
1.6 Wistar

1.4 / Gunn j/+
/ Gunn j/j
S1.2 -
0i
E 1.0 P<0.05





0.8

0.2

0.0 I --
Day 0 Day 3 Day 5
Days


Figure 2-2. Serum creatinine concentrations on DO, 3, and 5. No significant difference between
any groups on DO. The most significant difference in creatinine occurred when
comparing the Wistar rat to both the Gunn j/j and Gunn j/+. The Wistar rats had a
significantly creatinine.

































UWIM Mia "G& ....... WNALA.L _7.r%0VW,..T'V A

Figure 2-3..Histologic grading showed significant preservation of the OSOMPT in homozygous
Gunn rats when compared to heterozygous Gunn rats and Wistar rats given cisplatin.
Although functionally heterozygous Gunn rats were significantly different than the
Wistar rat, histologically, there was no significant difference. A) Outer stripe of the
outer medulla (center) with necrotic and sloughed tubular epithelial cells coupled
with regeneration in the Wistar rats (PAS stain, 50X). B) This is compared to the
remarkably well-preserved tubules at the junction between the inner and outer stripes
of the outer medulla with occasional apoptotic and sloughed cells seen at a higher
magnification in the homozygous Gunn rats (PAS stain, 50X).



































B

Figure 2-3. continued












6000 -
Grp 1 Control
Grp 2 Bilirubin 71 umol/ml
5000 Grp 3 Bilirubin 128 umol/ml
Grp 4 Cisplatin 0.5 umollml
Grp 5 Cisplatin + Bili 71 umol/ml
c 4000 Grp 6 Cisplatin + Bill 128 umollml
0
O (P<0.05)
3000
0

5 2000


1000 -


0
1 2 3 4 5 6

Treatment Groups
POS ostesarcoma cell line

Figure 2-4 Viable cell count for one of the canine osteosarcoma cell lines utilized (POS). With
this cell line, no significant difference was noted between cisplatin alone and either
concentration ofbilirubin alone. As with all cell lines tested, bilirubin had no
significant difference on cisplatin's anti-neoplastic activity in culture.









CHAPTER 3
CONCLUSION

The current lifetime risk for a human developing cancer in the United States is

approximately 40%. Additionally, our pets are living longer lives making them more prone to

developing cancer also. One of the most powerful treatments for cancer in any species is

chemotherapy but these agents have countless adverse side effects. Specifically, cisplatin is a

chemotherapeutic used in humans and animals to treat a variety of malignancies. The most

clinically significant side effect associated with cisplatin is nephrotoxicity.

Chapter 1 discussed the phamacokinetics, mechanism of anti-tumor action, toxicities

(especially nephrotoxicity), current protective measures against nephrotoxicity, and the use of

cisplatin in animals. Many theories have been developed regarding the mechanism of cisplatin

nephrotoxicity but none have proven to be the sole, underlying cause for this toxicity. Perhaps

this is why finding a compound to ameliorate cisplatin nephrotoxicity has been so difficult.

Numerous studies have shown that certain compounds and enzyme alterations can protect against

cisplatin nephrotoxicity but not without disadvantages of their own, including worsened side

effects of the compound, expense, or difficulty in administering the compound or enzyme.

While recent studies about HO regarding protection of various organ systems have been

performed, no studies utilizing the end-product BR to protect against cisplatin nephrotoxicity

exist.

Chapter 2 of this thesis describes an experiment designed to investigate if sustained

unconjugated hyperbilirubinemia in the Gunn rat would protect against cisplatin nephrotoxicity.

An experiment was also performed to determine if BR had any effect on the ability of cisplatin to

kill canine osteosarcoma cell in culture. Homozygous Gunn rats lack the enzyme UDPGT

necessary to conjugate bilirubin. Heterozygous Gunn rats lack normal levels of UDPGT but can









still conjugate bilirubin to a certain extent. The homozygous Gunn rats were utilized to achieve

a state of unconjugated hyperbilirubinemia. The congenic Wistar rats were the positive control

with normal levels of UDPGT. Cisplain was administered to all rats and functional renal

parameters were monitored for 5 days. On day 5, kidneys were harvested for histological

evaluation. To ensure that multiple anesthetic episodes had no negative impact on kidney

function and histology, sham rats from each group also underwent the same procedures except

they did not receive cisplatin.

Cell culture studies were then performed in 4 canine osteosarcoma cell lines using the

average concentrations of BR for homozygous Gunn rats at day 0 and 3. BR was added to all cell

lines alone and with cisplatin and cell viability was assessed using the CellTiter BlueTM assay.

Results of this study demonstrate complete functional and partial histologic

nephroprotection with the homozygous Gunn rats when compared to the heterozygous Gunn rats

and Wistar rats. While the heterozygous Gunn rats lacked histologic nephroprotection from

cisplatin, they had improved functional parameters when compared to the Wistar rats. The sham

rats showed no significant difference in their functional or histologic parameters when compared

to one another. This suggests that multiple anesthetic episodes had no negative impact on renal

function or histology.

With regard to bilirubin's effect on cisplatin in cell culture, there was no significant

difference in the cell viability of any of the four canine osteosarcoma cell lines when bilirubin

was added with cisplatin.

Cisplatin nephrotoxicity has provided a difficult obstacle to administering the drug safely

to chemotherapy patients of all species. HO and its products have proven to be beneficial in

protecting a variety of organ systems from assorted insults. The results obtained from this thesis









have provided directions for future applications regarding the products of HO-1 in cisplatin

nephrotoxicity. Future studies using a rodent model with conjugated hyperbilirubinemia and

administering exogenous BR are warranted. Potentially, a canine and human model could be

developed in the future. Additionally, the administration of BR and CO together may provide

complete functional and histological nephroprotection from cisplatin.









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BIOGRAPHICAL SKETCH

Karri Barabas began her collegiate education at the University of Florida in 1996 and

received her Bachelor of Science in animal science with highest honors in May 2000. She was

accepted to the University of Florida College of Veterinary Medicine and graduated Magna Cum

Laude with a Doctor of Veterinary Medicine in May 2003. Karri completed a one-year rotating

small animal medicine and surgical internship at Texas A&M University College of Veterinary

Medicine immediately after graduation. She then completed an oncology internship at Regional

Veterinary Referral Center in Springfield, Virginia after finishing at Texas A&M and returned to

the University of Florida to pursue a combined Master of Science and small animal medical

oncology residency at the College of Veterinary Medicine. Karri will complete her oncology

residency in July 2009.