Induction of heat shock proteins in liver during hepatotoxicant exposure

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Induction of heat shock proteins in liver during hepatotoxicant exposure
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Salminen, William F
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Research   ( mesh )
Chaperonins -- metabolism   ( mesh )
Chaperonins -- physiology   ( mesh )
Heat-Shock Proteins -- metabolism   ( mesh )
Heat-Shock Proteins -- physiology   ( mesh )
Gene Expression Regulation   ( mesh )
Acetaminophen -- toxicity   ( mesh )
Acetaminophen -- pharmacology   ( mesh )
Bromobenzenes -- toxicity   ( mesh )
Bromobenzenes -- pharmacology   ( mesh )
Cocaine -- toxicity   ( mesh )
Cocaine -- pharmacology   ( mesh )
Carbon Tetrachloride -- toxicty   ( mesh )
Carbon Tetrachloride -- pharmacology   ( mesh )
Acetylcysteine -- pharmacology   ( mesh )
Liver   ( mesh )
Mice   ( mesh )
Toxicity Tests   ( mesh )
Department of Pharmacology and Therapeutics thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pharmacology and Therapeutics -- UF   ( mesh )
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non-fiction   ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1997.
Bibliography:
Bibliography: leaves 118-129.
Statement of Responsibility:
by William F. Salminen, Jr.
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Typescript.
General Note:
Vita.

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INDUCTION OF HEAT SHOCK PROTEINS IN LIVER
DURING HEPATOTOXICANT EXPOSURE












By

WILLIAM F. SALMINEN, JR.














A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA

1997



























I dedicate this dissertation to my wife and three boys who supported me throughout my

doctoral work. My wife worked three hours away, two days a week so that our boys could

stay at home during their first years of life and I could work on my dissertation at the same

time. Without her support this research would not have been possible. My three boys

(Tyler, Alec, and Jake) gave me tremendous inspiration for my accomplishments. Besides,

it's great when your son says, "My daddy is a scientist just like Bill Nye the science guy."














ACKNOWLEDGMENTS


I would like to thank the many people who contributed to my work. Their help and

advice not only streamlined the presented work, but also gave me insight into the

significance of performing good and sound research.

Stephen M. Roberts, Ph.D. provided tremendous support and advice during my

time in his laboratory. He provided the necessary insight into problems when I was stuck,

but let me try to solve most problems on my own. Steve let me design most experiments

which not only improved my experimental design skills, but made me think critically about

the research I was performing. Steve is the type of doctoral advisor I think every candidate

should be blessed with.

Richard Voellmy, Ph.D. is thanked for many insights into the possible implications

of the results obtained from my research and possible paths to direct further research. Many

avenues of research presented in this dissertation came from discussions between Steve

Roberts, Richard, and myself.

Susan Frost, Ph.D., Margaret James, Ph.D., Thomas Rowe, Ph.D., and Kathleen

Shiverick, Ph.D. provided outstanding guidance in ensuring that the presented research

was top quality and are thanked for reviewing this dissertation.

John Munson is thanked for all the help he provided during my time in Steve

Roberts laboratory. Without John, my work would have taken much longer than it did.

Judy Adams is thanked for guiding me through the University of Florida

bureaucracy and ensuring that all paper work was filed and deadlines were met at the

appropriate times throughout my doctoral training.















TABLE OF CONTENTS


ACKNOWLEDGMENTS.............................................. ...................... iii

LIST OF TABLES ................................................................ vi

LIST OF FIGURES ..............................................................................vii

APPENDIX..................................................................................... ix
Abbreviations and Definitions............................................................ ix

ABSTRACT ...................... .............................................. x

CHAPTERS

1. BACKGROUND AND OBJECTIVES ...................................................... 1
Background and Objectives...................................... ..................... 1

2. INDUCTION OF HSP70 IN HEPG2 CELLS IN RESPONSE TO
HEPATOTOXICANTS.................................................................... 8
Introduction...................................... ......................................... 8
Materials and Methods ........................................................................10
Results ......................................................................................15
Discussion..................................................................................21

3. HEAT SHOCK PROTEIN INDUCTION IN MURINE LIVER AFTER ACUTE
TREATMENT WITH COCAINE ...........................................................25
Introduction........................................ .......................................25
Materials and Methods ........................................................................27
Results ......................................................................................31
Discussion .................................................................................41

4. DIFFERENTIAL HEAT SHOCK PROTEIN INDUCTION BY
ACETAMINOPHEN AND A NON-HEPATOTOXIC REGIOISOMER, 3'-
HYDROXYACETANILIDE, IN MOUSE LIVER .......................................45
Introduction................................................................................45
Materials and Methods ........................................................................47
Results ......................................................................................52
Discussion..................................................................................66

5. PROTECTION AGAINST HEPATOTOXICITY BY A SINGLE DOSE OF
AMPHETAMINE: THE POTENTIAL ROLE OF HEAT SHOCK PROTEIN
INDUCTION...............................................................................70
Introduction................................................................................70
Materials and Methods ........................................................................71
Results ................................... ................................................75
Discussion..................................................................................91


iv









6. EFFECT OF N-ACETYL-CYSTEINE ON HEAT SHOCK PROTEIN
INDUCTION BY ACETAMINOPHEN IN MOUSE LIVER: THE ROLE OF
PROTEIN ARYLATION...................................................................95
Introduction................................................................................ 95
Materials and Methods ........................................................................97
Results .......................................................................................99
Discussion..................................................................................... 108

7. CONCLUSIONS .............................................................................. 113
Conclusions ................................................................................... 113

LIST OF REFERENCES ........................................ .................... 118

BIOGRAPHICAL SKETCH ........................................ ................... 130









































V














LIST OF TABLES


pableag


Table 2-1. Irrevserible Binding of Bromobenzene, Carbon Tetrachloride, or
Cocaine to Protein in HepG2 Cells........................................................21

Table 3-1. Effect of SKF-525A pretreatment on serum ALT activities after cocaine
administration. .................................................................... .......37

Table 5-1. Irrevserible Binding of Hepatotoxicants to Protein.............................90

Table 5-2. Liver Glutathione Levels After Hepatotoxicant Exposure......................91














LIST OF FIGURES
Figure g

Figure 1-1. Mechanism of heat shock protein induction by denatured proteins ........... 4

Figure 2-1. Immunochemical detection of hsp 70i in HepG2 cells at various times
after a sub-lethal heat shock (430C for one hour). ..................................... 16

Figure 2-2. Scanning densitometry analysis of the level of hsp 70i in HepG2 cells
24 hours after hepatotoxicant treatment. ................................................. 17

Figure 2-3. Northern blot showing the level of hsp 70i mRNA in HepG2 cells 24
hours after hepatotoxicant treatment...................................... ............ 18

Figure 2-4. The effect of a 24 hour prior sub-lethal heat shock (sub-LHS= 430C for
one hour) on hepatotoxicant-induced cell lethality...................................... 19

Figure 2-5. The effect of a 24 hour prior sub-lethal heat shock (sub-LHS= 430C for
one hour) on hepatotoxicant-induced cell lethality....................................... 20

Figure 3-1. Heat shock protein (hsp) levels in murine liver 0, 3, 6, or 24 hours
after treatment with 50 mg/kg cocaine...................................... ............. .. 32

Figure 3-2. Imunohistochemical detection of hsp25 in marine liver 24 hours after
treatment of naive mice with saline. ...................................................... 33

Figure 3-3. Imunohistochemical detection of hsp25, hsp70i, and cocaine-adducted
cellular macromolecules in murine liver 24 hours after treatment of naive mice
with 50 mg/kg cocaine. .................................................. .................. 34

Figure 3-4. Immunohistochemical detection of hsp25 or hsp70i induction and
cocaine adduction of cellular macromolecules in murine liver 24 hours after
treatment of B-naphthoflavone-pretreated mice with 50 mg/kg cocaine .............. 38

Figure 4-1. Heat shock protein (hsp) induction in mouse liver 0, 3, 6, or 24 hours
after treatment with 200 mg/kg APAP............................. ............. 53

Figure 4-2. Immunohistochemical detection of hsp25 and hsp70i accumulation and
APAP adduction of cellular macromolecules in mouse liver 24 hours after
treatment of naive mice with 200 mg/kg APAP..........................................55

Figure 4-3. Immunohistochemical detection of hsp25 and hsp70i levels and AMAP
adduction of cellular macromolecules in mouse liver three hours after treatment
of naive mice with 1,000 mg/kg AMAP. ............................................... 58

Figure 4-4. The effect of APAP, AMAP, and butathionine sulfoximine (BSO) +
AMAP on the liver levels of non-protein sulfhydryls (NPSH)........................ 62









Figure 4-5. Immunohistochemical detection of hsp25 and hsp70i accumulation in
mouse liver 24 hours after treatment of naive mice with butathionine
sulfoximine (BSO) + AMAP........................................ .............. .. 63

Figure 5-1. Amphetamine-induced hyperthermia......................................76

Figure 5-2. Heat shock protein (hsp) induction in murine liver 0, 6, 24, 48, 72, or
96 hours after treatment with amphetamine (15 mg/kg, i.p.)..........................77

Figure 5-3. Immunohistochemical detection of hsp25 and hsp70i accumulation in
murine liver after amphetamine (15 mg/kg, i.p.) treatment.............................78

Figure 5-4. The effect of amphetamine pretreatment on acetaminophen,
bromobenzene, carbon tetrachloride, or cocaine hepatotoxicity....................... 81

Figure 5-5. Liver histopathology of saline or amphetamine pretreated mice
administered a single dose of bromobenzene or acetaminophen ..................... 86

Figure 6-1. The effect of N-acetyl-cysteine (NAC) or diallyl sulfide (DAS) on
acetaminophen (APAP) hepatotoxicity.................................................... 100

Figure 6-2. Effect of N-acetyl-cysteine (NAC) on acetaminophen (APAP) arylation
of protein 6 and 24 hours after treatment with 250 mg/kg APAP..................... 102

Figure 6-3. Effect of N-acetyl-cysteine (NAC) on acetaminophen (APAP) arylation
of protein 24 hours after treatment with 250 mg/kg APAP............................104

Figure 6-4. Effect of N-acetyl-cysteine (NAC) on acetaminophen (APAP) induction
of hsp25 and hsp70i in mouse liver 24 hours after treatment with 250 mg/kg
A PA P .................................... ..................................................... 105

Figure 6-5. Effect of diallyl sulfide (DAS) on acetaminophen (APAP) arylation of
protein and induction of hsp25 and hsp70i in mouse liver 24 hours after
treatment with 250 mg/kg APAP............................................................ 106

Figure 6-6. The effect of N-acetyl-cysteine (NAC= 300 mg/kg) on the liver levels
of glutathione (GSH) with and without acetaminophen (APAP= 250 mg/kg)
treatm ent. .................................................................................... 107














APPENDIX


ABBREVIATIONS AND DEFINITIONS

ALT= Serum alanine aminotransferase activity

AMAP= 3'-Acetamidophenol

APAP= Acetaminophen

IBNF= Beta-naphthoflavone

BSO= L-Buthionine-[S,R]-sulfoximine

DEN= Diethylnitrosamine

DMN= Dimethylnitrosamine

HPLC= High performance liquid chromatography

HSE= Heat shock element

HSF= Heat shock transcription factor

HSP= Heat shock protein

LDH= Lactate dehydrogenase

PB= Phenobarbital

Proteotoxicity= Any stress or insult that alters the native state of a protein

sub-LHS= Sub-lethal heat shock














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INDUCTION OF HEAT SHOCK PROTEINS IN LIVER
DURING HEPATOTOXICANT EXPOSURE

By

William F. Salminen, Jr.

May, 1997



Chairman: Stephen M. Roberts, Ph.D.
Major Department: Pharmacology and Therapeutics

Heat shock proteins (hsps) are ubiquitous proteins expressed in both prokaryotic

and eukaryotic organisms. They are believed to play a role in maintaining protein

homeostasis by trafficking and refolding proteins throughout the cell. In addition, elevated

levels of hsps have been correlated with protection against a variety of stressors. Since

many hepatotoxicants disrupt protein homeostasis, it was hypothesized that hsp induction

might occur in the liver after hepatotoxicant exposure as a mechanism to help the liver cope

with the toxicant-induced proteotoxicity. A series of studies was conducted to determine if

hepatotoxicants induce hsps in mouse liver and if elevated levels of hsps provide protection

against the toxicant-induced damage. A variety of toxicants were able to induce hsp25 and

hsp70i, without affecting the level of hsp60, hsc70, or hsp90 in vivo. Similar results were

obtained in the HepG2 cell line in which some toxicants were potent inducers of hsp70i

while others were not. Elevated levels of hsps produced by amphetamine-induced

hyperthermia in vivo, or mild heat treatment in the HepG2 cell line, were correlated with

protection from some, but not all of the toxicants. The intralobular pattern of hsp25 and









hsp70i induction in mouse liver was determined immunohistochemically for several

toxicants to see what cells induced hsps during toxicant exposure. For acetaminophen

(APAP) and bromobenzene, hsp25 induction was predominantly on the periphery of the

lesions, whereas hsp25 induction after cocaine or carbon tetrachloride exposure was

uniform throughout the lesions with no induction observed on the periphery. Experiments

were conducted in vivo to determine if protein adduction without concurrent toxicity was

sufficient to trigger hsp induction. N-Acetyl-cysteine (NAC) prevented APAP

hepatotoxicity, but did not significantly affect APAP arylation of protein. The NAC

treatment did not inhibit APAP-induced hsp accumulation indicating that protein binding

without concurrent toxicity may be sufficient to trigger hsp induction. These results show

that some toxicants are potent inducers of hsps and that elevated levels of hsps may provide

protection against some toxicants indicating that hsp induction during toxicant exposure

might be a protective mechanism to deal with the toxicant-induced injury.














CHAPTER 1
BACKGROUND AND OBJECTIVES


Background and Objectives

The experiments outlined in this dissertation were devised to test the hypothesis that

hsp induction occurs in the liver after hepatotoxicant exposure as a result of the toxicant-

induced proteotoxicity. In addition, some of the following experiments address the

hypothesis that hsp induction during toxicant exposure is a protective mechanism to help

the liver repair or eliminate toxicant-damaged proteins. Most of the results presented in this

dissertation were obtained from experiments conducted in the whole animal. This is a

unique aspect of this study since very few past investigations have looked at the role hsps

play during stress in the whole animal. Through the use of a variety of experimental

approaches in vitro and in vivo the previously mentioned hypotheses were tested and the

data gathered are presented and interpreted in this dissertation.

The heat shock response was discovered over 30 years ago when a study by

Ritossa (1962) noted a new heat-inducible puffing pattern along the salivary gland

chromosomes of Drosophila melanogaster. The puffs corresponded to increased heat

shock protein (hsp) gene transcription. Since this first discovery, many types of hsps have

been discovered and grouped into families based on their apparent molecular weight and

amino acid sequence relationships: small hsps, hsp60, hsp70, hsp90, and high molecular

weight hsp families. As their name implies, cells induce hsps when they are exposed to

mild hyperthermia; however, exposure to other types of stress can also cause hsp induction

(Parsell and Lindquist, 1994; Voellmy, 1994; Welch, 1992). In addition, some hsps are

synthesized at very low levels and only induced upon exposure to a stress or stressorr." In

the liver of naive mice, for example, hsp90, hsc70, and hsp60 are expressed constitutively









at appreciable levels, very low levels of hsp 70i are observed, and hsp25 is virtually

undetectable (Wilkinson and Pollard, 1993; Klemenz et al., 1993; Bardella et al., 1987).

In animals subjected to thermal stress, levels of hsp70i and hsp25 are increased

dramatically (Lu and Das, 1993; Blake et al., 1990).

Hsps are grouped under the broad category of stress proteins which includes

glucose regulated proteins (grps), ubiquitin, metallothioneins, and heme oxygenases. All

of these proteins share the common feature of being induced during stress. Grps are

readily induced by glucose deprivation and metallothioneins are readily induced by

exposure to heavy metals. However, induction of a stress protein is not necessarily

restricted to one type of stress, and induction of several types of stress proteins may occur

simultaneously. Hsps and grps can be induced by heavy metals such as cadmium, and

grps are slightly induced in some cells during exposure to hyperthermia (Goering et al.,

1993; Joslin et al., 1991). Often, only a subset of hsps are induced during exposure to a

given stress and the magnitude of induction often varies among those hsps that are induced

(Sanchez et al., 1992; Mirkes et al., 1994). For example, Wiegant et al. (1994) noted

dramatic induction of hsp70i during treatment of rat hepatoma cells with cadmium with no

change in the level of hsp60. In addition, some hsps, most notably hsp25 and hsp70i,

appear to be induced to a greater extent during exposure to many types of stresses

compared to other hsps (Bernelli-Zazzera et al., 1992; Goering et al., 1993; Van Laack et

al., 1993; Lin et al., 1994; Roberts et al., 1996).

Several observations suggest that accumulation of non-native protein may trigger

hsp induction. Overexpression of mammalian proteins unable to fold properly caused

activation of hsp genes in bacteria (Goff and Goldberg, 1985), and the injection of

chemically-denatured proteins into vertebrate cells increased stress protein synthesis while

injection of the corresponding native protein did not (Ananthan et al., 1986). Indeed, many

of the agents that induce hsps also bind, denature, and/or alter the redox status of proteins

(Lee and Dewey, 1988; Lee and Hahn, 1988; Lumpkin et al., 1988; Ciocca et al., 1993;






3

Lin et al., 1994). A widely accepted scenario for induction of hsp70 by denatured

proteins, as described by Morimoto et al. (1994), is diagrammed in figure 1-1. In a normal

eukaryotic cell, hsp70 is constitutively bound to heat shock transcription factor (hsf).

Upon protein denaturation, hsp70 releases hsf and binds denatured proteins for which it

has a higher affinity (Rothman, 1989; Burel et al., 1992; Beckman et al., 1992). The

released hsf forms a homotrimer and translocates to the nucleus where it binds the heat

shock element (hse), which is a sequence of DNA within the promoter of hsp genes (Amin

et al., 1988; Sarge et al., 1993). Binding of hsf to the hse increases the rate of hsp gene

transcription (Femandes et al., 1994). Once additional hsp70 is produced and/or denatured

proteins are refolded by hsps, the level of free hsp70 increases to such a level that it can

once again bind hsf, returning the rate of hsp gene transcription to constitutive levels (Baler

et al, 1992).









Normal Cell:


protein


Stressed Cell:
non-native


Figure 1-1. Mechanism of heat shock protein induction by denatured proteins.


Two other factors play a role in producing elevated levels of hsps during stress. In

addition to the increased rate of hsp gene transcription, the hsp mRNA is preferentially

translated and its half life is increased. During hyperthermia, the synthesis of normal

proteins is down regulated while the translation of hsp mRNA is increased and the rate of

hsp mRNA degradation is decreased (Theodorakis and Morimoto, 1987; Petersen and

Lindquist, 1988; De Maio et al, 1993). It is through increased hsp gene transcription,








preferential translation of hsp mRNA, and stabilization of the hsp mRNA during stress that

hsp accumulation occurs.

As mentioned above, hsp70 binds denatured proteins and releases the

constitutively bound hsf. The binding of hsp70 to denatured proteins is believed to aid

protein solubility by preventing the incorrect aggregation of exposed hydrophobic areas of

non-native proteins (Beckman et al., 1992; Ciocca et al, 1993; Beck and De Maio, 1994).

Hsp70 hydrolyzes ATP once the protein refolds to its native form using the energy derived

from ATP hydrolysis to release the protein, not to actively refold the protein (Beckman et

al., 1990). It is interesting to note that hsp70 also aids the translocation of proteins into the

endoplasmic reticulum and mitochondria by binding hydrophobic areas of the proteins,

keeping the proteins in a translocationally competent form (Chirico, 1992). Recent

evidence also suggests that hsps may help target proteins for degradation if they are

damaged beyond repair (Parag et al., 1987; Craig et al., 1994).

Many studies have correlated elevated levels of hsps with cytoprotection from a

variety of stresses, the most notable being extreme hyperthermia (Hahn and Li, 1990; Feige

and Mollenhauer, 1992). Recent studies have provided direct evidence that it is hsps, not a

by-product of the inducing treatment, that provides cytoprotection. Li et al. (1991)

transfected recombinant human hsp70 into rat fibroblasts and found that it protected the

cells from thermal stress, and injection of antibodies against hsp70 into fibroblasts

decreased their ability to survive short exposures of hyperthermia (Riabowol et al., 1988).

The mechanism of hsp cytoprotection is believed to be due to the ability of hsps to bind

denatured proteins and maintain their solubility until they can assume their native form.

This process prevents protein precipitation and conserves valuable resources since protein

can be recovered instead of degraded (Brown et al., 1993; Parsell and Lindquist, 1994).

Most of the studies investigating the protective effect of elevated levels of hsps have been in

vitro ; however, elevated levels of hsps have been shown to provide protection from a

handful of stressors n vivo suggesting that the cytoprotective function of hsps is not unique









to cultured cells and is applicable to the whole animal (Villar et al., 1993; Hotchkiss et al.,

1993; Currie et al., 1988; Saad et al., 1995).

Given the mechanism of action of hsps, it is reasonable to hypothesize that any

stress that damages or denatures proteins should elicit a heat shock response. Indeed,

many of the stimuli that induce hsps are known to alter the native form of proteins. Heavy

metals such as cadmium and arsenic are strong inducers of hsps in many cell types and in

vivo (Lee and Dewey, 1988; Goering et al., 1993; Bauman et al., 1993; Abe et al., 1994;

Ovelgonne et al., 1994). These metals bind free sulfhydryl groups of proteins, altering

their native state (Jacobson and Turner, 1980). Many agents that organisms are exposed to

alter the native form of proteins either directly or indirectly. Toxicants such as

bromobenzene and carbon tetrachloride covalently bind protein which presumably

denatures the target protein (Sipes and Gandolfi, 1982; Hanzlik et al, 1989). Toxicants

may also alter protein conformation by altering the redox status of a cell. Diamide rapidly

oxides reduced glutathione in a cell which leads to increased formation of protein-mixed

disulfides (Grimm et al., 1985; Collison et al., 1986). It is conceivable that any xenobiotic

that alters protein homeostasis should induce hsps.

The liver presents a unique model to investigate the role of the heat shock response

during toxicant insult since the liver is the site of metabolism of the majority of xenobiotics.

In mammals, the majority of cytochrome P450 mixed-function oxygenase activity is

present in the liver along with the conjugating enzymes necessary to form toxicant

metabolites that can be eliminated from the body (Plaa, 1993). Often, toxicants are

metabolized to reactive intermediates that can overwhelm endogenous defense mechanisms

ultimately resulting in cell damage (Lindamood, 1991). A typical example is

acetaminophen (APAP) hepatotoxicity. Normally, a small fraction of the acetaminophen

exposed to the liver is metabolized to the reactive N-acetyl-p-benzoquinoneimine (NAPQI)

by cytochrome P450 mixed-function oxygenases. This metabolite is highly reactive and

can bind cellular macromolecules; however, the metabolite is conjugated with glutathione









before it can attack nucleophilic cell molecules (Plaa, 1993). Only when glutathione levels

are depleted does NAPQI bind cellular constituents and cause cell damage (Rashed et al.,

1990).

Given the diversity of hepatotoxicants, it follows that cell damage occurs through a

vast array of mechanisms in the liver. A few examples are lipid peroxidation, covalent

binding to cell macromolecules, free radical production, inhibition of protein synthesis, and

lipid accumulation (Marzella and Trump, 1991). The availability of a wide array of

hepatotoxicants that cause damage through different mechanisms was employed in studies

to help refine what role(s) hsps play during toxicant exposure in the liver. A variety of

approaches and models were used to determine if hsps were able to recognize and alleviate

toxicant induced damage in the liver.

The following experiments were devised to help determine what role hsps play in

the liver during toxicant exposure. First, Western blot analysis of hsp levels in HepG2

cells and in mouse liver after toxicant exposure was used to determine whether or not hsps

were induced by toxicants. Second, the pattern of hsp accumulation in mouse liver after

toxicant exposure was determined immunohistochemically to detect which cells in the liver

(i.e., normal or damaged) induced hsps after toxicant exposure. Third, the correlation

between elevated levels of hsps and protection against hepatotoxicty was measured in

HepG2 cells and in the whole animal to try and determine if elevated levels of hsps could

provide protection against toxicant-induced damage. Finally, the ability of toxicant

adducted protein to trigger hsp induction was measured in the whole animal to try and

determine if toxicant adducted protein alone could trigger hsp induction independent of

toxicity. The following results provide strong evidence that toxicants are able to induce

hsps in liver, toxicant adduction of protein plays a major role in triggering hsp induction,

and elevated levels of hsps may provide protection against some toxicants.














CHAPTER 2
INDUCTION OF HSP70 IN HEPG2 CELLS IN RESPONSE TO HEPATOTOXICANTS

Introduction


Prokaryotic and eukaryotic cells respond to a variety of stresses by enhancing the

transcription of a specific set of genes that encode heat shock proteins (hsp). In eukaryotic

cells, increased levels of hsps occur as a result of the activation of a heat shock

transcription factor (HSF) that is normally in an inactive form (reviewed by Voellmy,

1994), apparently as a heterooligomer with hsp70 (Baler, 1992; Wu et al., 1994). Stresses

including heat shock causes protein unfolding and nonnative proteins have a higher affiity

for hsp70 than native proteins (Flynn et al., 1989). Titration of hsp70 through such

binding may cause the release of hsp70 from HSF, allowing HSF to assemble DNA

binding homotrimers (Baler et al., 1992a; Westwood et al., 1992). The HSF homotrimers

bind to promoters of hsp genes, and after a further stress-induced activation step become

competent to enhance the transcription of the genes (Zuo et al., 1995). After removal of the

stress, HSF returns to an inactive state, and transcription of hsp genes decreases at a rate

that depends on the severity of the stress (Zuo et al., 1995).

The binding of hsp70 to damaged proteins is believed to assist in preventing their

aggregation and promoting correct refolding ("molecular chaperoning"), as well as

facilitating their degradation (Parsell and Lindquist, 1994). As such, hsp70 and other

stress proteins represent an important mechanism by which cells prevent accumulation of

aberrant proteins. Further, experimental manipulations that result in elevated levels of hsps

(e.g., thermal treatment or use of an inducible expression vector) have been observed to

result in cytoprotection from a variety of subsequent stresses such as a lethal heat shock (Li

et al., 1990; Kampinga et al., 1995; Parsell and Lindquist, 1994). These observations








suggest that induction of hsp70 and other hsps may represent an important cellular defense

mechanism against proteotoxicity from a variety of stressors.

Among the stressors that have been demonstrated to result in hsp induction is a

rather extensive list of chemical toxicants (Goering et al., 1993; Nover, 1991; Levinson et

al., 1980). The number and variety of toxicants shown, in one experimental system or

another, to result in increased expression of hsps have led some to suggest that hsp

induction may be a universal response to cytotoxicity (e.g., Sanders et al., 1993; Anderson

et al., 1987; Blom et al., 1992). However, a recent study of stress protein induction in rat

hepatoma cells in response to four chemical agents (viz., arsenite, cadmium, dinitrophenol,

and ethanol) found marked differences among these agents in the ability to induce specific

stress proteins. In fact, cytotoxic concentrations of two of these toxicants (dinitrophenol

and ethanol) failed to induce any of the battery of hsps examined. The results of this study

suggest that hsp induction in response to chemical toxicants may be more complex than

originally envisioned, with some, but not all, toxicants producing proteotoxicity triggering

an hsp response. Further, though not explicitly addressed in this study, it may be possible

that the cytoprotection against chemical toxicants afforded by hsp induction observed in

some studies (e.g., Ovelgonne et al., 1995; Kampinga et al., 1995; Steels et al., 1992) may

not be applicable to all toxicants.

In the present study, we have examined the inducible 70-kDa hsp (hsp 70i)

response to a variety of hepatotoxic agents in HepG2 cells. The HepG2 cell line, derived

from a human liver hepatoma, was chosen as the model system since it is reported to retain

many of the properties of primary cells, including the ability to metabolize a wide variety of

toxicants (Doostdar et al., 1993; Neuman et al., 1993). This aspect was regarded as

particularly important since most hepatotoxicants require bioactivation to produce their

characteristic effects on the liver. Also, HepG2 cells have been shown to display the

classical heat shock response, i.e. mild hyperthermia leads to induction of hsps, and the

resulting elevated levels of hsps provide protection from subsequent severe hyperthermic









treatment (De Maio et al., 1993). Using this model system, the effect of each

hepatotoxicant on hsp 70i expression was evaluated. Additionally, the ability of elevated

levels of hsps to afford cytoprotection from each hepatotoxicant was examined and

compared with its ability to induce hsps.


Materials and Methods

Cell culture and treatments. HepG2 cells, obtained from the American Type Culture

Collection (ATCC No. HB 8065, Rockville, Maryland), were cultured in Earle's salt-based

MEM supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan,
Utah), 1 mM sodium pyruvate, and 50 mg/L gentamicin in a humidified, 5% C02

atmosphere maintained at 370 C. Cells were grown to confluency in 60 x 15 mm tissue

culture plates or T25 tissue culture flasks (Coming Glass Works, Coming, NY) before all

treatments and assays. The T25 flasks were used for carbon tetrachloride or bromobenzene

exposure so that the volatile compounds did not escape the flasks. In some experiments,

cells were made hyperthermic by floating plates of cells in a circulating water bath

maintained at the desired temperature within 0.10 C. Sub-lethal heat shock was induced by

increasing the culture medium temperature to 430 C for one hour. Lethal heat shock was

produced by increasing the culture medium temperature to 46.50 C for two hours. After

heat treatment the culture medium was replaced and the plates and/or flasks returned to the

incubator. Cadmium acetate, cocaine hydrochloride, cyclophosphamide monohydrate, or

N-nitrosodiethylamine (diethylnitrosamine) (Sigma Chemical Co., St. Louis, MO) were

dissolved in culture medium to the desired concentration. The culture medium was then

sterile-filtered before being placed on the cells. Preliminary experiments found that adding

bromobenzene or carbon tetrachloride to the culture medium resulted in at least a fivefold

decrease in medium concentration over the first 30 minutes as these volatile agents

evaporated into the headspace of the flask. Increasing the amount of carbon tetrachloride or

bromobenzene added to the flask to achieve the desired final concentration resulted in








unacceptably high, transient concentrations of toxicant. To avoid this problem,

bromobenzene or carbon tetrachloride were introduced to the culture medium by placing a

measured amount into a 9 mm diameter x 3 mm height polypropylene container that was

placed on the sloping edge of a T25 flask containing 3 mls of culture medium and sealing

the flask. Pilot studies found that both toxicants quickly volatilized from the vessel, and an

equilibrium between the headspace and culture medium was achieved within 30 minutes. A

series of experiments were conducted to determine the appropriate amounts to add to the

vessel to achieve the desired concentrations in the culture medium. With the flask sealed,

the concentration of carbon tetrachloride or bromobenzene in the culture medium decreased

no more than 25% over 24 hours at 370 C.

Gas chromatoraphy. To measure carbon tetrachloride and bromobenzene

concentrations in the culture medium, one milliliter of culture medium was added to 2 mis

of pentane. One hundred microliters of a 20 pg/ml trichloroethylene solution was added to

each sample to serve as an internal standard. The mixture was vortexed for one minute and

centrifuged at 3,000 x g at 50 C for ten minutes to separate the layers. A 3 tl aliquot of the

top (pentane) layer was injected onto an Econo-cap SE-54 column, 15 m x 0.54 mm ID

(Alltech, Deerfield, IL). The following conditions were used: helium carrier gas flow rate

of 5 ml/min.; nitrogen makeup gas flow rate of 50 ml/min.; injector port temperature was

110C, and detector temperature was 2000C. The oven temperature was 50C for carbon

tetrachloride analysis and 120C for bromobenzene analysis. The eluted compounds were

detected by an electron capture detector. Carbon tetrachloride and bromobenzene were

quantitated using standard curves prepared from culture medium spiked with known

amounts of reference compound.

Polvacrylamide eel electrophoresis. Cells were homogenized in sample buffer (0.05

M Tris[hydroxymethyl]aminomethane (Tris-HC1, pH 6.8), 2% sodium dodecyl sulfate, 10

mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride). Each sample was

boiled for five minutes, passed through a 22 ga. needle three times to sheer DNA, and








stored at -800C until use. Twenty micrograms of protein from each sample was aliquoted to

separate tubes, and bromphenol blue was added to a final concentration of 0.0025%. Each

aliquot was boiled for five minutes, loaded onto separate lanes of a 10% SDS-PAGE gel,

and resolved by electrophoresis (Laemmli, 1970).

Protein blotting and immunostaining. Proteins separated by SDS-PAGE were

immediately blotted to supported nitrocellulose (Bio-Rad Laboratories, Hercules, CA)

using a semi-dry blotting apparatus (Millipore, Bedford, MA) and one-half strength

Towbin buffer (10 mM Tris-base, 96 mM glycine, and 10% methanol). Blotting was done

at 320 mA for 1.5 hours. Upon completion, the membrane was blocked in TBS (20 mM

Tris-HC1, pH 7.5, 500 mM sodium chloride) containing 3% gelatin. The membrane was

washed two times for five minutes each in TTBS (TBS containing 0.05%

polyoxyethylenesorbitan monolaurate) and probed with an antibody specific for hsp 70i

(Stressgen, Victoria, BC, Canada) at a 1:1000 dilution in TTBS containing 1% gelatin.

Incubation was for 18 hours at 240C with continuous shaking. The membrane was washed

two times for five minutes each with TTBS and then a goat anti-mouse alkaline

phosphatase-conjugated antibody (Bio-Rad Laboratories, Hercules, CA) at a 1:3000

dilution in TTBS containing 1% gelatin was incubated with the membrane for one hour at

240C with continuous shaking. Next the membrane was washed two times for five minutes

each in TTBS and once for five minutes in TBS. The colorimetric substrate BCIP/NBT

(100 mM Tris-base, pH 9.5, 165 ig/ml 5-bromo-4-chloro-3-indolyl phosphate, 330 jg/ml

nitro blue tetrazolium, 100 mM sodium chloride, 5 mM magnesium chloride) was added to

the membrane to localize antibody binding.

Lactate dehydrogenase (LDH) leakage assay. Two hundred microliters of culture

medium from each plate of cells was removed and the amount of LDH activity present

determined spectrophotometrically using the LD-L assay (Sigma Chemical Co., St. Louis,

MO). All samples were analyzed at 340 nm using a kinetic microplate reader (Molecular

Devices, Menlo Park, CA). The cells were then lysed by adding Triton X-100 to the culture








medium to a final concentration of 0.7%. Two hundred microliters of the culture medium

containing the lysed cells was removed and the amount of LDH activity determined. The

percent of LDH released into the culture medium during incubation was determined by

dividing the culture medium-only LDH activity by the total LDH activity (i.e. after cell

lysis). The percent of LDH leakage was used as an indicator of cell viability.

HSP 70i mRNA analysis. Total RNA was isolated from treated cells using the acid

guanidinium-phenol-chloroform method as described previously (Chomczynski and

Sacchi, 1987). Twenty micrograms of RNA from each sample was denatured in 6.5%

formaldehyde, IX MOPS running buffer (0.02 M 3-[N-morpholino]propanesulfonic acid,

8 mM sodium acetate, 1 mM [ethylenedinitrilo]tetraacetic acid), and 50% formamide by

heating at 650C for 15 minutes and then cooling on ice. Loading buffer was added to each

sample to a final concentration of 5% glycerol, 0.1 mM EDTA (pH 8.0), and 0.025%

bromphenol blue and loaded onto separate lanes of a 1.2% agarose/ 1X MOPS running

buffer/ 6.6% formaldehyde gel. After resolving the RNA by electrophoresis, the RNA was

transferred to a positively charged nylon membrane by capillary transfer using 20X SSC (3

M sodium chloride, 0.3 M sodium citrate, pH 7.0). RNA was fixed to the membrane using

120 mJ/cm2 of 254 nm UV light. Prehybridization was performed using 5X SSC, 50%

formamide, 0.02% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 2% blocking

reagent (Genius System, Boehringer Mannheim, USA) at 420C for two hours.

Hybridization was carried out in freshly prepared prehybridization solution containing 20

ng/ml of a digoxigenin-labeled hsp 70i DNA (ATCC No. 57494, American Type Culture

Collection, Rockville, Maryland). Hybridization was at 420C for 18 hours. The membrane

was washed in 2X SSC, 0.1% sodium dodecyl sulfate, pH 7.0 at 240C and then in 0.5X

SSC, 0.1% sodium dodecyl sulfate, pH 7.0 at 650C. Detection of bound probe was

accomplished with an alkaline phosphatase-conjugated anti-digoxigenin antibody and the

chemiluminescent substrate Lumiphos 530 (Boehringer Mannheim, USA) according to the

manufacturer's directions. Luminescence was detected using standard X-ray film. To








ensure equal transfer of the RNA to the membrane, the membrane was stripped by boiling

in 0.1% SDS and reprobed using a digoxigenin-labeled 8-actin cDNA (ATCC No. 65128,

American Type Culture Collection, Rockville, Maryland).

Covalent binding. [14C]-Carbon tetrachloride (New England Nuclear, Boston,

MA), ring-labeled [14C]-bromobenzene (ICN Radiochemicals, Irvine, CA) or tropine ring-

labeled [3H]-cocaine hydrochloride (National Institute on Drug Abuse, Rockville, MD)

were used to measure covalent binding of toxicant to proteins. Radiolabeled toxicant was

added to unlabeled toxicant such that each culture plate received 1 .Ci at the desired total

toxicant concentration (5 mM for cocaine, 0.8 mM for carbon tetrachloride, and 1.5 mM in

the case of bromobenzene). As a control, incidental binding was measured for each

radiolabeled toxicant using cells pretreated with trichloroacetic acid (6% w/v) to inhibit

metabolism. After exposure, cells were scraped from the plate, transferred to a 15 ml

conical tube, and centrifuged at 2000 x g for five minutes. The cell pellet was rinsed with

phosphate buffered saline (10 mM potassium phosphate, 2.7 mM potassium chloride, 120

mM sodium chloride, pH 7.4) and the protein precipitated with 1 ml of 6% trichloroacetic

acid (w/v). The solution was transferred to a 1.5 ml centrifuge tube, and protein was

pelleted by centrifugation at 14000 x g for five minutes. The pellet was washed

extensively with 1 ml aliquots of methanol/ether (3:1) until [14C]- or [3H]-radioactivity in

one-half volume of the supernatant was indistinguishable from background. The pellets

were air dried and resuspended in 1 N sodium hydroxide. [14C]- or [3H]-Radioactivity in

each sample was detected by liquid scintillation spectrometry. The protein concentration of

each sample was measured as described below and used to normalize the detected

radioactivity to the protein content.

Protein determination. Protein concentration was measured by the method of

Bradford (Bradford, 1976) using the Micro Protein Determination assay (Sigma Chemical

Co., St. Louis, MO). Bovine serum albumin was used as standard.









Statistical Analysis. Data were analyzed by a one-way ANOVA followed by a

Student Neuman-Keuls post-hoc test. The level of significant difference was defined as the

0.05 level of probability.


Results

In initial experiments, the time course of hsp 70i induction in HepG2 cells was

determined using the classical hsp inducer, sub-lethal heat shock (sub-LHS; 430C for 1

hour) (see Figure 2-1). Induction of hsp 70i, as determined by Western blotting, could be

seen as early as one hour post sub-LHS with maximal accumulation occurring at 24 hours.

Although somewhat diminished, Hsp 70i was still substantially elevated above control

levels at 72 hours post sub-LHS. Based on this information, the time to peak

accumulation, 24 hours, was chosen as the best interval to evaluate the presence of hsp 70i

induction after toxicant exposure.

The extent of induced accumulation of hsp 70i after hepatotoxicant exposure is

shown in Figure 2-2. The hsp 70i signal on Western blots after hepatotoxicant exposure

was quantitated using scanning densitometry and compared with the signal produced by

untreated cells. In this experiment, hepatotoxicant concentrations were selected, as

determined by LDH leakage, to produce approximately 40-70% cell mortality within 24

hrs. Under these conditions, diethylnitrosamine (50 mM), cadmium acetate (50 gM),

bromobenzene (1.5 mM), and cyclophosphamide (20 mM) significantly induced the level

of hsp 70i, while carbon tetrachloride (0.8 mM) and cocaine (5 mM) produced no change

in the level of hsp 70i compared to untreated cells. Lower concentrations of each

hepatotoxicant (concentrations that caused 8- 15% cell mortality) were also tested to ensure

that hsp 70i induction was not being inhibited by excessive cytotoxicity in some cases.

Cadmium acetate was the only hepatotoxicant with greater hsp 70i expression at the lower

concentration. The other hepatotoxicants displayed no detectable hsp 70i induction at the

reduced concentration (data not shown).
















Time post sub-LHS (hrs.)
I I
C 0 1 2 3 5 24 48 72

HSP 701- -











Figure 2-1. Immunochemical detection of hsp 70i in HepG2 cells at various times after a
sub-lethal heat shock (430C for one hour). Protein was resolved on a 10% SDS-PAGE
gel, and hsp 70i was detected by Western blotting using a monoclonal antibody specific for
hsp 70i. Equal amounts of protein (i.e., 50 gig) from each sample were loaded onto
separate lanes. Control cells were maintained at 370C.


A further set of experiments was conducted to determine whether the apparent
absence of an hsp 70i induction response to carbon tetrachloride and cocaine was due to an
inhibition of transcription. The levels of hsp 70i mRNA in HepG2 cells after treatment
with the hepatotoxicants for 24 hours were measured by Northern blotting (Figure 2-3).
Consistent with measurements of protein expression, diethylnitrosamine, cadmium acetate,
bromobenzene, and cyclophosphamide all induced the level of hsp 70i mRNA, while
carbon tetrachloride and cocaine produced no change. These observations suggest that the
absence of an hsp 70i response to carbon tetrachloride and cocaine is not due to inhibition
at the transcriptional level.


















0 500-


b 400-

U
C)
i- 300-
0

t 200-


100



w U U Q .S
U0U
0 d u




Treatment
Figure 2-2. Scanning densitometry analysis of the level of hsp 70i in HepG2 cells 24 hours
after hepatotoxicant treatment. Cells were exposed for 24 hours to various hepatotoxicants
at the indicated concentrations. Protein was resolved on a 10% SDS-PAGE gel, and hsp
70i detected by Western blotting using a monoclonal antibody specific for hsp 70i. A goat
anti-mouse IgG antibody conjugated with alkaline phosphatase was used to detect the
primary antibody binding. DEN= diethylnitrosamine; Cd= cadmium acetate; BB=
bromobenzene; CC14= carbon tetrachloride; CP= cyclophosphamide. Values represent
mean SEM (n=3 plates). denotes significantly different from control by Student
Neuman-Keuls post-hoc test using p<0.05.




















HSP 70-





B-Actin-




Figure 2-3. Northern blot showing the level of hsp 70i mRNA in HepG2 cells 24 hours
after hepatotoxicant treatment. Cells were exposed for 24 hours to various hepatotoxicants
at the indicated concentrations. Total RNA was isolated, resolved and blotted as described
in Methods. Hsp 70i mRNA was detected by hybridizing the immobilized RNA with
digoxigenin-labeled hsp 70i DNA. Hybridizing probe was located using an anti-
digoxigenin antibody and a chemiluminescent substrate. The blot was stripped and
reprobed with a digoxigenin-labeled B-actin probe. Sub-LHS= sub-lethal heat shock (430C
for one hour); DEN= diethylnitrosamine; Cd= cadmium acetate; BB= bromobenzene;
CCI4= carbon tetrachloride; CP= cyclophosphamide.


Previous studies have shown that exposure to a sub-LHS may provide protection
from other types of subsequent stresses. It has been inferred, but not shown, that the

induction of hsps is responsible for this effect. It is possible that the induction of hsp 70i

during exposure to some of the hepatotoxicants tested is a cytoprotective response of the

cell; however, it may also be a by-product of cellular damage and serve no cytoprotective

function. To examine these possibilities, the effect of a sub-LHS on hepatotoxicant

potency was measured. Cells were subjected to sub-LHS, followed 24 hours later by








exposure to hepatotoxicant. The effect of sub-LHS on hepatotoxicant-induced cell
lethality, as measured by LDH leakage, is shown in Figures 2-4 and 2-5. As a positive
control, sub-LHS pretreatment diminished the cell mortality from lethal heat shock (46.50C
for two hours) exposure, as expected. Sub-LHS also significantly diminished the
cytolethality of diethylnitrosamine, cadmium acetate, bromobenzene, and
cyclophosphamide (Figures 2-4 and 2-5). Interestingly, sub-LHS pretreatment caused no
significant change in the hepatotoxicant-induced cell lethality from carbon tetrachloride or
cocaine (Figure 2-4).
100 A Cntrol

[O sub-LHS
r /lm __EIIIZ


Treatment
Figure 2-4. The effect of a 24 hour prior sub-lethal heat shock (sub-LHS= 430C for one
hour) on hepatotoxicant-induced cell lethality. Cells were heated in a water bath maintained
at 430C 0.10C. Twenty four hours after the sub-LHS, hepatotoxicants were incubated
with the cells for an additional 24 hours and the percent of LDH activity released into the
culture medium was determined. BB= bromobenzene; CC14= carbon tetrachloride; CP=
cyclophosphamide. Values represent mean SEM (n=5 plates). denotes significantly
different from control by Student Neuman-Keuls post-hoc test using p<0.05.












0 75 sub-LHS
con
-75-



50-



25-





CIO.
00





Treatment
Figure 2-5. The effect of a 24 hour prior sub-lethal heat shock (sub-LHS= 430C for one
hour) on hepatotoxicant-induced cell lethality. Cells were heated in a water bath maintained
at 430C 0.10C. Twenty four hours after the sub-LHS, hepatotoxicants were incubated
with the cells for an additional 24 hours and the percent of LDH activity released into the
culture medium was determined. LHS= lethal heat shock (46.50C for two hours); DEN=
diethylnitrosamine; Cd= cadmium acetate. Values represent mean SEM (n=5 plates). *
denotes significantly different from control by Student Neuman-Keuls post-hoc test using
p<0.05.


One mechanism by which the hepatotoxicants in this study might stimulate hsp 70i
induction is through adduction of proteins by reactive metabolites. Among the
hepatotoxicants tested, carbon tetrachloride, cocaine, diethylnitrosamine,
cyclophosphamide, and bromobenzene each produce reactive metabolites in vivo (Evans,
1983; Sipes and Gandolfi, 1982; Hanzlik et al., 1989; Kanekal et al., 1992; Osterman-
Golkar and Bergmark, 1988; Plaa, 1993). In order to interpret the apparent absence of hsp








70i induction by cocaine and carbon tetrachloride in HepG2 cells in the context of this

potential mechanism, it was important to establish whether or not reactive metabolites were,

or were not, being formed under the incubation conditions employed. To test this, the

formation of reactive metabolites was evaluated through measurement of covalent (i.e.,

irreversible) binding to proteins following incubation with radiolabeled [14]C-carbon

tetrachloride, [14]C-bromobenzene, or [3]H-cocaine. As shown in Table 2-1, among these

three hepatotoxicants, only bromobenzene produced detectable binding to protein.


Table 2-1. Irreversible Binding of Bromobenzene, Carbon Tetrachloride, or Cocaine to
Protein in HepG2 Cells.

pmoles bound
Treatment Duration (hours) per pg protein

1.5 mM Bromobenzene 4 13.36 6.66
24 20.78 5.67
0.8 mM Carbon tetrachloride 4 ND
24 ND

5 mM Cocaine 4 ND
24 ND


Note. HepG2 cells were treated with [14C]-bromobenzene, [14C]-carbon tetrachloride, or
[3H]-cocaine (1 gCi/plate) at the indicated concentrations. As a control, incidental binding
was measured using the same exposure regimen in cells pretreated with trichloroacetic acid
(6% w/v). Cells were harvested at the indicated times and washed extensively as described
in Methods. ND= no detectable irreversible binding observed. Values represent mean +
SEM (n=3 plates) after subtracting incidental binding.


Discussion

A common theme among agents that induce the heat shock response is the ability to

disrupt protein homeostasis (Ananthan et al., 1986; Hightower, 1991). Many studies have

focused on the induction of hsp 70i since it appears to be universally induced during stress.

Consistent with recent reports by several investigators, however, the present study








suggests there are exceptions to this rule, even though cell injury occurs (Wiegant et al.,

1994; Mirkes et al., 1994; Goodman and Sloviter, 1993).

A strong correlation between hepatotoxicant induction of hsp 70i and the ability of

sub-LHS pretreatment to provide protection from those hepatotoxicants was observed.

Diethylnitrosamine, cadmium acetate, bromobenzene, and cyclophosphamide induced hsp

70i and showed decreased cytotoxicity in cells pretreated with sub-LHS. In contrast, no

change in the level of hsp 70i was evident after either carbon tetrachloride or cocaine

treatment, nor was their cytotoxicity diminished in cells pretreated with sub-LHS. In this

study, the extent of hsp 70i induction was evaluated at only one time point, and the relative

ability of the various hepatotoxicants to increase hsp 70i levels cannot therefore be

determined with confidence. To the extent that the 24 hour data reflect overall induction,

however, the magnitude of induction of hsp 70i appears to be correlated with the ability of

a prior sub-LHS to provide protection from the hepatotoxicants. Diethylnitrosamine and

cadmium strongly induced hsp 70i and showed the greatest decrease in cytotoxicity in cells

given a prior sub-LHS. Cyclophosphamide and bromobenzene mildly induced hsp 70i at

24 hours and showed only minimal decreases in cytotoxicity. The apparent correlation

between the ability of a hepatotoxicant to increase hsp 70i levels and cytoprotection

afforded by elevated levels of hsps argues that hsp 70i may play an important role in

providing cytoprotection from hepatotoxicants. Information regarding similar correlations

for other toxicants is extremely limited. Li and coworkers, however, in a study of hsp

induction and cytotoxicity of a series of membrane-active agents (viz., solvents and local

anesthetics), also found a strong correlation between the ability to stimulate hsp synthesis

and cytoprotection afforded by prior hsp induction in Chinese hamster cells (Hahn et al.,

1985).

Since carbon tetrachloride and cocaine did not induce hsp 70i and showed no

decrease in cytotoxicity in cells pretreated with sub-LHS, it was necessary to investigate the

possibility that the concentrations of toxicants used may have precluded the expression of








hsp 70i through inhibition of transcription, translation or some other means. To address

these questions, Northern blots measuring hsp 70i mRNA were used to determine if

transcription of the hsp 70i gene was inhibited by the toxicants. In addition, the effect of

lower hepatotoxicant concentrations on the level of hsp 70i was measured. Neither of these

experimental approaches indicated that carbon tetrachloride or cocaine were preventing the

expression of hsp 70i at the concentrations used. The levels of hsp 70i mRNA after carbon

tetrachloride or cocaine treatment were, in fact, the same as in untreated cells. These

observations suggest that either carbon tetrachloride and cocaine are not producing cellular

changes triggering hsp induction (in contrast to the other toxicants), or that they are

inhibiting expression prior to, or at a level of, transcription of the hsp 70i gene.

Induction of hsps is believed to be triggered by denatured proteins (Ananthan et al.,
1986; Baler et al., 1992). Many hepatotoxicants are metabolized to reactive intermediates

that bind cellular proteins, and it is possible that the adducted proteins are recognized by

hsps as nonnative, thereby triggering induction. This type of mechanism has been

proposed by Chen et al. (1992) for hsp induction by nephrotoxic cysteine conjugates.

Among the toxicants tested in the present study, all are capable of producing reactive

metabolites which bind to protein in vivo, with the exception of cadmium, which can bind

to proteins directly through interaction with sulfhydryls (Jacobson and Turner, 1980).

Assuming that each of the toxicants also bound to protein in the HepG2 model system, the

absence of hsp 70i induction following exposure of HepG2 cells to cytotoxic

concentrations of carbon tetrachloride and cocaine appeared initially to argue against a

reactive metabolite binding mechanism. Subsequent experiments, however, served to

reinforce a correlation between covalent binding and hsp induction. Though HepG2 cells

have been reported to metabolize many toxicants through cytochrome P-450 dependent

mixed function oxidation (Belisario et al., 1991), reactive metabolite binding was found to

be absent in the HepG2 cells during the carbon tetrachloride and cocaine exposures.

Bromobenzene concentrations that induced hsp 70i expression in the HepG2 cells, on the









other hand, resulted in readily detectable covalent binding to proteins. Thus, HepG2 cells

may be incapable of bioactivating carbon tetrachloride and cocaine. It is worthwhile noting

that in mice, where administration of cocaine or carbon tetrachloride results in significant

covalent binding, hsp 70i induction has been observed (unpublished observations).

In conclusion, experiments conducted using the HepG2 human hepatoma cell line

and a battery of hepatotoxicant chemicals indicate that cytotoxicity is not always

accompanied by hsp 70i induction. Most of the toxicants tested produced at least moderate

induction, but some appeared to be completely ineffective. It is logical to suspect that

differences in induction reflect differences in interactions between the toxicants and cellular

proteins, such as through the formation of reactive metabolites, although this has not yet

been clearly demonstrated. Perhaps one of the most interesting observations was the

strong apparent correlation between the ability of the hepatotoxicants to induce hsp 70i and

the extent of cytoprotection against their effects afforded by sub-LHS pretreatment. This

suggests that, at least for some hepatotoxicants, hsp 70i induction may be an important

cellular defense mechanism.














CHAPTER 3
HEAT SHOCK PROTEIN INDUCTION IN MURINE LIVER AFTER ACUTE
TREATMENT WITH COCAINE


Introduction

Heat shock proteins (hsps) are ubiquitous in nature and can be grouped into a

number of distinct families based on size and amino acid sequence relationships. Most

widely recognized are the hsp90, hsp70, hsp60, and hsp25 families. Several families

include members that are constitutively expressed as well as members whose synthesis is

induced by heat stress and by a variety of other adverse stimuli (Parsell and Lindquist,

1994; Voellmy, 1994; Welch, 1992; Linquist and Craig, 1988). In the liver of naive

mice, for example, hsp90, hsc70, and hsp60 are expressed constitutively at appreciable

levels. Levels of hsp70i are relatively low, and hsp25 is virtually undetectable (Wilkinson

and Pollard, 1993; Klemenz et al., 1993; Bardella et al., 1987). In animals subjected to

heat stress, levels of hsp70i and hsp25 are increased dramatically (Lu and Das, 1993;

Blake et al, 1990).

A number of observations suggest that stress induction of hsp synthesis is

mediated by the presence of non-native proteins. For example, overexpression of

mammalian proteins unable to fold properly causes activation of hsp genes in bacteria

(Goff and Goldberg, 1985), and the injection of chemically-denatured proteins into

vertebrate cells increases stress protein synthesis, while injection of the corresponding

native protein does not (Ananthan et al., 1986). Proteins of the hsp70 family are known to

be capable of binding non-native proteins (Palleros et al., 1991; Hightower et al., 1994)

and are thought to chaperone their refolding or elimination. During this binding, the heat

shock transcription factor normally bound to hsp70 is released, whereupon it rapidly








oligomerizes to become transcriptionally active (Baler et al., 1992; Abravaya et al., 1992;

Baler et al., 1993).

Many hepatotoxicants are bioactivated to reactive intermediates that bind cellular

macromolecules including proteins (Plaa, 1993; Lindamood, 1991). It is conceivable that

adducted proteins resulting from reactive metabolite binding are physiologically equivalent

to proteins unfolded as a consequence of exposure to classical inducers of the heat stress

response (e.g., heat, heavy metals, etc.), thereby activating hsp gene expression. Limited

studies have suggested that hepatotoxicants forming reactive metabolites may increase the

expression of hsps (Salminen et al., 1996), but there has been little direct examination of

the relationship between hepatic reactive metabolite formation and hsp gene activation.

This report describes the results of a study of hsp induction in mice treated with

hepatotoxic doses of cocaine. Cocaine, like a number of other hepatotoxicants, is

metabolized to a reactive metabolite which binds to proteins (Evans, 1983). The role of

adducted proteins in the cytotoxicity of cocaine in the liver is unclear, but previous studies

have shown that reactive metabolite binding occurs only in the region of the lobule where

necrosis subsequently develops zone 2 in naive ICR mice (Roth et al., 1992). Unlike

other classical hepatotoxicants, the location of reactive metabolite binding and toxicity

within the hepatic lobule can be altered by pretreatment with hepatic enzyme inducing

agents. In mice pretreated with phenobarbital (PB), protein binding and necrosis occur in

zone 1, while in b-naphthoflavone (bNF)-pretreated mice they are localized primarily in

zone 3 (Roth et al., 1992). In this study, we show a strong correlation between the

intralobular location of cocaine reactive metabolite binding and the location of hsp

accumulation, suggesting that cocaine reactive metabolite binding to protein plays a role in

triggering hsp induction.










Materials and Methods

Animals and treatments. Adult ICR male mice (Harlan Sprague-Dawley,

Indianapolis, IN) weighing 25-30 g were used for these studies. Mice were housed on

corn cob bedding in temperature- and humidity-controlled animal quarters with a 12-h

light/dark cycle and allowed free access to food and water before and during the

experiments. The following pretreatment regimens were used to induce or inhibit

cytochrome P-450 activity in the liver: sodium phenobarbital, 80 mg/kg i.p., daily for four

days; b-naphthoflavone, 40 mg/kg i.p., once daily for three days; or SKF-525A, 50

mg/kg i.p., 30 minutes before treatment with cocaine. After pretreatment, mice were

administered a single i.p. dose of cocaine HCI (Sigma Chemical Co., St. Louis, MO) in

saline. All pretreatment and cocaine doses were given with an injection volume of 10

ml/kg body weight. Mice were killed by carbon dioxide asphyxiation. Before initiation of

the study, all procedures were assessed and approved by the Institutional Animal Care and

Use Committee.

Polvacrylamide gel electrophoresis. Four hundred milligrams of liver was

homogenized in 5 ml of sample buffer (0.05 M Tris[hydroxymethyl] aminomethane

(Tris), 2% sodium dodecyl sulfate, 10 mM dithiothreitol, 10% glycerol, 1 mM

phenylmethylsulfonyl fluoride, pH 6.8). Each sample was boiled for five minutes,

passed through a 22 ga. needle three times to shear DNA, and stored at -800C until use.

Two hundred micrograms of protein from each sample was aliquoted to separate tubes and

bromphenol blue was added to a final concentration of 0.0025%. Each aliquot was boiled

for five minutes, loaded onto separate lanes of a 12.5% SDS-PAGE gel, and resolved by

electrophoresis (Laemmli, 1970).

Protein blotting and immunostaining. Proteins separated by SDS-PAGE were

immediately blotted to Hybond-ECL Western membrane (Amersham, England) using a

semi-dry blotting apparatus (Millipore, Bedford, MA) and one-half strength Towbin








buffer (10% methanol, 96 mM glycine, and 10 mM Tris-base). Blotting was done at 320

mA for 1.5 hours. Upon completion, the membrane was blocked in TBS (20 mM Tris,

500 mM sodium chloride, pH 7.5) containing 3% gelatin. The membrane was washed

two times for five minutes each in TTBS (TBS containing 0.05% polyoxyethylenesorbitan

monolaurate) and probed with one of the following antibodies: anti-hsp25 (rabbit

polyclonal), anti-hsp60 (mouse monoclonal), anti-hsc70 (mouse monoclonal), anti-hsp70i

(mouse monoclonal), or anti-hsp90 (mouse monoclonal). Each of these antibodies were

obtained from Stressgen (Victoria, B.C., Canada) and used at a 1:1000 dilution in TTBS

containing 1% gelatin. Incubation was for 18 hours at 24C with continuous shaking. The

membrane was washed two times for five minutes each with TTBS and then a sheep anti-

mouse or donkey anti-rabbit (depending upon the primary antibody used) horseradish

peroxidase-conjugated antibody (Amersham, England) at a 1:3000 dilution in TTBS

containing 1% gelatin was incubated with the membrane for one hour at 240C with

continuous shaking. Next, the membrane was washed three times for five minutes each in

TTBS and once for five minutes in TBS. The chemiluminescent horseradish peroxidase

substrate Luminol (Amersham, England) was added to the membrane and the membrane

exposed to standard X-ray film to localize antibody binding.

Immunohistochemical detection ofhsp25. hsp70i. and cocaine adducts in murine

liver. Five millimeter thick sections from several lobes of each liver were placed in tissue

cassettes and fixed in neutral buffered formalin for three hours. The livers were rinsed and

stored in saline, processed routinely, and embedded in paraffin. Four sequential sections,

4-6 pm thick, were cut from the same block to facilitate, to the extent possible, cell-by-cell

comparisons of cocaine reactive metabolite binding, histopathology, and induced

accumulation of hsp25 and hsp70i. One section was stained with hematoxylin and eosin

and examined for histopathology by light microscopy, while the remaining sections were

immunohistochemically stained with an anti-hsp25 antibody, an anti-hsp70i monoclonal

antibody, or an anti-cocaine antibody as follows. Preliminary experiments were








performed to ensure that an adequate blocking reagent was used that would prevent

nonspecific binding ofimmunoglobulins to necrotic areas. TBS containing 25% (v/v)

bovine serum plus 3% (w/v) purified bovine serum albumin (BSA) (blocking solution)

was found to prevent the non-specific binding to necrotic areas of normal rabbit serum,

mouse IgG, or normal sheep serum used as negative controls for the anti-hsp25, anti-

hsp70i, or anti-cocaine antibodies, respectively. Fab fragment goat anti-mouse IgG

(H+L) (Jackson Immuno Research Laboratories, Inc., West Grove, Pennsylvania) was

added to the blocking solution (10 tg/ml final concentration) before blocking slides

subsequently probed for hsp70i induction. The latter addition blocked any endogenous

mouse IgG that was present in the sections and prevented false positive signals when

probing with the biotinylated anti-mouse IgG secondary antibody. Secondary antibody-

only treated slides exhibited no binding.

The following procedure was used for immunostaining the sections. Paraffin

embedded sections were deparaffinized by passing through three changes of xylene for

five minutes each. The sections were passed through 100% ethanol two times for one
minute each, 95% ethanol for one minute, and double distilled water (ddH20) two times

for two minutes each. Endogenous peroxidase activity was quenched by submerging the

slides in 3% hydrogen peroxide containing 0.1% sodium azide for ten minutes. The
slides were then washed in ddH20 three times for two minutes each and then equilibrated

in TBS for at least two minutes. All the following incubations were performed in a

humidified chamber. Blocking solution was placed on each section and incubated at 370C

for one hour. The slides were washed two times in TBS for two minutes each. Anti-

hsp25 antibody (rabbit polyclonal, Stressgen, B.C., Canada) was diluted 1:100 in

blocking solution and placed on the appropriate slides, while anti-hsp70i (mouse

monoclonal, Stressgen, B.C., Canada) was diluted 1:100 in blocking solution devoid of

the Fab fragment goat anti-mouse IgG antibody and placed on the appropriate slides.

Anti-cocaine antibody (sheep polyclonal, JEM Research Products, Inc., Arlington,








Virginia) was diluted 1:100 in blocking solution and placed on the appropriate slides. The

antibodies were incubated with the sections at 370C for one hour and then at 240C for 18

hours. The sections were washed three times in TBS for two minutes each. Biotinylated

goat anti-mouse, goat anti-rabbit, or rabbit anti-sheep (Southern Biotechnology

Associates, Inc., Birmingham, AL), depending upon the primary antibody used, was

diluted 1:500 in TBS containing 3% BSA, placed on the slides and incubated at 370C for

thirty minutes. The sections were washed three times in TBS for two minutes each.

Streptavidin-linked horseradish peroxidase (Southern Biotechnology Associates, Inc.,

Birmingham, AL) was diluted 1:200 in TBS containing 3% BSA, placed on the slides and

incubated at 370C for thirty minutes. The sections were washed three times in TBS for two

minutes each. The horseradish peroxidase colorimetric substrate 3,3'-diaminobenzidine
(DAB) (Sigma Chemical Co., St. Louis, MO) supplemented with 0.03% NiCl2 (w/v)

was incubated with each section for fifteen minutes at 240C to provide a permanent

location of antibody binding. The sections were then counterstained with hematoxylin and

dehydrated by passing through graded alcohols and xylene in the reverse order as for

deparaffinizing the sections. The sections were mounted using Permount (Fisher

Scientific, Orlando, FL) and a glass cover slip. As a negative control, no binding of the

anti-cocaine antibody, using the above procedure, was observed in livers from mice

administered necrogenic doses of carbon tetrachloride, bromobenzene, or acetaminophen.

Protein determination. Protein was measured with the Micro Protein Determination

assay (Sigma Chemical Co., St. Louis, MO) using BSA as standard.

Serum alanine aminotransferase (ALT) activity. Blood for measurement of serum

ALT activity was collected by cardiac puncture immediately after carbon dioxide

asphyxiation. Serum ALT activity was determined according to the method of Bergmeyer

et al. (1978) using a commercially available kit (Sigma Diagnostics, St. Louis, MO).








Statistical analysis. Data were analyzed by a one-way ANOVA followed by a

Student Neuman-Keuls post-hoc test. The level of significant difference was defined as

the 0.05 level of probability.


Results

Initial experiments used Western blot analysis to identify specific hsps induced in

response to an hepatotoxic dose of cocaine. Representative Western blots showing the

hepatic levels of hsp25, hsp60, hsc70, hsp70i, and hsp90 at 0 (control), 3, 6, and 24

hours after treatment of naive, male ICR mice with cocaine, 50 mg/kg, i.p., are shown in

Figure 3-1. The levels of hsp60, hsc70, and hsp90 in the liver were unaffected by

cocaine treatment at any of these time points. Levels of hsp25, however, were elevated at

6 and 24 hours, and levels of hsp70i were higher than those in controls at each of the

observation times. Concentrations of hsp25 and hsp70i were both greatest at 24 hours.

In a subsequent set of experiments, livers were removed for immunostaining and

histopathologic evaluation 0, 3, 6, or 24 hours after a cocaine dose (50 mg/kg, i.p.).

Based on the results of the Western blot experiments (described above), immunostaining

for hsps was restricted to hsp25 and hsp70i. Following cocaine treatment, swelling of

midzonal (zone 2) and centrilobular (zone 3) hepatocytes was observed at the earliest time

point 3 hours and midzonal necrosis was evident by 6 hours after the cocaine dose

(not shown). At 24 hours, the midzonal necrosis was extensive. Immunoreactive cocaine

bound to protein was present beginning with the 3 hour specimens and was restricted to

cells with altered morphology (i.e., cell swelling). Positive immunostaining for hsp25

and hsp70i was observed 3 hours after the cocaine dose. Hsp25 and hsp70i levels

increased over time with the greatest accumulation observed 24 hours post-administration

based on the intensity of immunostaining. As with the intralobular distribution of cocaine

reactive metabolite binding, hsp25 and hsp70i expression was limited to cells with altered

morphology. The extent of immunostaining appeared to be relatively uniform within the









lesion. An example of the relationship between morphology, reactive metabolite binding,

and hsp25 and hsp70i levels is shown in Figure 3-3, which is from a liver specimen taken

24 hours after the cocaine dose. Hsp25 and hsp70i accumulation and cocaine binding

(Figures 3-3b, c, & d) were superimposable upon the hepatocytes with altered

morphology in the midzonal regions (Figure 3-3a). Liver sections from vehicle-treated

control mice displayed normal histology and no detectable immunostaining for cocaine

reactive metabolite, hsp25, or hsp70i (Figure 3-2 and not shown).


OH 3H 6H 24H






-w -i


HSP9O


HSP701


HSC70

HSP60


HSP25






Figure 3-1. Heat shock protein (hsp) levels in murine liver 0, 3, 6, or 24 hours after
treatment with 50 mg/kg cocaine. Liver protein was resolved on a 12.5% SDS-PAGE gel,
and the relative levels of various hsps estimated by Western blotting using antibodies
specific for the indicated hsps. All samples were from the same experiment, with each
lane showing hsp levels in a single mouse liver. Equal amounts of protein (i.e., 200 .tg)
from each sample were loaded onto separate lanes.












































Figure 3-2. Imunohistochemical detection of hsp25 in murine liver 24 hours after
treatment of naive mice with saline. The photomicrograph is of an immunohisto-
chemically stained liver section using an anti-hsp25 antibody. The slide was
counterstained with hematoxylin. Arrow indicates central vein and arrow head indicates
portal triad. Liver histology is unremarkable and no positive immunostaining is evident
Field magnification 200X.









Figure 3-3. Imunohistochemical detection of hsp25, hsp70i, and cocaine-adducted cellular
macromolecules in murine liver 24 hours after treatment of naive mice with 50 mg/kg
cocaine. Sequential sections were stained as follows: a) hematoxylin and eosin stain, b)
immunohistochemical stain using an anti-hsp25 antibody, c) immunohistochemical stain
using an anti-hsp70i antibody, d) immunohistochemical stain using an anti-cocaine
antibody. All immunostained slides were counterstained with hematoxylin. Arrow
indicates central vein and arrow head indicates portal triad. Midzonal (zone 2) necrosis is
evident with some injury occurring closer to the central vein (zone 3). Field magnification
200X.





35












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"-r, 2
"N




|.











To test if hsp accumulation following cocaine was a function of reactive metabolite

binding and/or hepatotoxicity, rather than a direct effect of cocaine, the ability of the

cytochrome P-450 inhibitor SKF 525A to inhibit cocaine binding and cocaine-induced hsp

accumulation was measured through immunostaining. Consistent with previous

observations (Roth et al, 1992), SKF 525A pretreated animals had little or no hepatic

necrosis from cocaine, and the increase in serum alanine aminotransferase (ALT) activity

associated with cocaine treatment, indicative of hepatic damage, was abolished (Table 3-

1). Also lost in SKF 525A pretreated mice was any detectable immunostaining for

cocaine reactive metabolite, hsp25, or hsp70i (not shown).


Table 3-1. Effect of SKF-525A pretreatment on serum ALT activities after cocaine
administration.
Treatment Serum ALT Activity (IU/L)

Saline 54.4 13.3

SKF 525A alone 58.0 6.2

Cocaine alone 917.5 410.3 *

SKF 525A then Cocaine 21.8 9.8

Note. SKF-525A (50 mg/kg, i.p.) was administered 30 minutes prior to administration of
cocaine (50 mg/kg, i.p.). Serum ALT activities were measured 24 hours after
administration of cocaine. Data expressed as mean SEM (n = 5 animals per treatment
group). indicates significantly different from saline treated mice (p<0.05).

In an additional experiment, mice were pretreated with PB or 6NF to determine if

the association between cocaine reactive metabolite binding and hsp accumulation would

be retained if the site of the cocaine-induced lesion was shifted. As with naive mice,

preliminary experiments using Western blotting confirmed that among the hsps tested,

only hsp25 and hsp70i were strongly induced by cocaine in either PB- or BNF-pretreated











Figure 3-4. Immunohistochemical detection of hsp25 or hsp70i induction and cocaine
adduction of cellular macromolecules in marine liver 24 hours after treatment of B-
naphthoflavone-pretreated mice with 50 mg/kg cocaine. Sequential sections were stained
as follows: a) hematoxylin and eosin stain, b) immunohistochemical stain using an anti-
hsp25 antibody, c) immunohistochemical stain using an anti-hsp70i antibody, d)
immunohistochemical stain using an anti-cocaine antibody. All immunostained slides were
counterstained with hematoxylin. Arrow indicates central vein and arrow head indicates
portal triad. Extensive centrilobular (zone 3) necrosis is evident. Field magnification
200X.






39

















S(B"f~~'' '
o, j' '..:
r *i, .,J6
C/r-Now


''C .f.~',4













r-e "4 i
~i


, ,"i *. .
4^ '/i" '7









mice (not shown). Consistent with previous observations (Roth et al., 1992), INF

pretreatment caused an apparent shift in the intralobular site of necrosis, as well as reactive

metabolite binding, to the centrilobular region (zone 3) (Figure 3-4). This was

accompanied by an identical shift in the intralobular site of accumulation of hsp25 and

hsp70i. As was the case with mice without pretreatment, hsp accumulation occurred only

in cells with detectable reactive metabolite binding and altered morphology. Mice

pretreated with PB, in contrast, displayed hepatic necrosis and reactive metabolite binding

predominantly in the peripheral lobular region (zone 1) (not shown). Again, the

intralobular sites of accumulation of hsp25 and hsp70i corresponded precisely with the

localization of the reactive metabolite binding and lesion.


Discussion

Heat stress, the prototypical stimulus for increased hsp synthesis, characteristically

induces the synthesis of an array of hsps. A survey of the response of the most common

hsp families from 25- to 90-kDa to a series of hepatotoxicants in mice found that only

hsp25 and hsp70i were increased significantly (Voellmy et al., 1994). The results

observed here with cocaine are consistent with this earlier report. The variability

observed in the magnitude of hsp induction among mice at a given time point (see Figure

3-1) has been observed previously for other toxicants. Since the degree of toxicity varies

also among individual animals within a given treatment group, this is perhaps not

surprising.

Hsp70i induction has been observed in rats treated with halothane (VanDyke et

al., 1992). Effects of this hepatotoxicant on other hsps is unknown, since this study

focused specifically on hsp70i. Goering et al. (1993) incubated liver slices from

cadmium-treated rats with 35S-methionine and observed increased de novo synthesis of

proteins with relative molecular masses of 70-kDa, 90-kDa, and 110-kDa. Western blots

probed with specific antibodies revealed the 70-kDa protein to be hsp70i (termed hsp72 in








their report) and the 90-kDa protein to be grp94. We have found no increase in grp94

levels following cocaine treatment in mice (unpublished observations), an apparent

difference in response that could be related to differences in either hepatotoxicant action

between cocaine and cadmium, or to fundamental species differences in response to

hepatotoxicants. It is unclear whether there was also a difference in hsp25 response.

Goering and colleagues did not observe increased synthesis of proteins with a molecular

mass at or near 25-kDa following cadmium treatment as was seen with cocaine. Induction

of hsp25 synthesis could conceivably have been missed using 35S-methionine labeling in

their study, however, since rodent hsp25 lacks methionine (Kim et al., 1983).

Metabolism of many hepatotoxic compounds leads to the formation of reactive

metabolites, and binding of these metabolites with proteins has been postulated to play a

role in their cytotoxic effects. In general, very little is known about the effects of

adduction with these relatively small molecular weight metabolites on the conformation of

target proteins. It is plausible, however, that adduction of target proteins could lead to

their recognition as non-native, causing activation of hsp genes. Cocaine is among the

hepatotoxicants whose oxidative metabolism leads to a reactive metabolite (Evans, 1983),

and it has been shown previously that immunohistochemistry can be used to identify the

intraacinar sites of cocaine reactive metabolite binding (Roth et al., 1992). In order to

explore the relationship between reactive metabolite binding and increased hsp synthesis,

this technique was used to compare the localization of cocaine-adducted proteins relative to

accumulation of hsp25 and hsp70i.

The spatial correlation within the lobule between cocaine reactive metabolite

binding, hsp25 and hsp70i accumulation, and cytotoxicity was found to be remarkably

consistent. That is, immunostaining of sequential sections revealed that only cells with

detectable cocaine metabolite binding had altered morphology and increased concentrations

of hsp25 and hsp70i. This correlation existed whether the site of cocaine metabolite

binding was in zone 2 (naive mice), zone 1, (PB-induced mice), or zone 3 (bNF








pretreated mice). Cocaine reactive metabolite formation and hepatotoxicity result from

cytochrome P-450 mediated oxidation of cocaine (Boelsterli and Goldlin, 1991), and both

can be prevented by pretreatment of mice with the cytochrome P-450 inhibitor SKF 525A

(Roth et al., 1992). When reactive metabolite formation and binding was prevented by

pretreatment with SKF 525A, no increases in hsp25 or hsp70i concentrations were

observed. These observations are consistent with a mechanism in which cocaine-adducted

protein provides a stimulus for activation of hsp25 and hsp70i genes. Of course, because

reactive metabolite binding is closely correlated with cytotoxicity, the possibility cannot be

ruled out that hsp induction occurs in response to some facet of toxicity rather than as a

direct consequence of protein adduction. The difficulty in experimentally separating

cocaine reactive metabolite binding and toxicity makes this possibility difficult to address.

Another interesting observation arising from this study was the relative uniformity

of the immunostaining within the affected areas, both for cocaine reactive metabolite

binding and the hsps. To the extent that the intensity of staining is reflective of

intracellular concentrations, relatively equivalent levels of hsps were observed in cells

within the lesions, up to the margins of the affected areas. Cells immediately adjacent to

cells in the affected area had no apparent staining. This suggests that hsp induction in

response to cocaine may be an "all or none" phenomenon. The absence of a graded hsp

response may be a function of a threshold for cocaine reactive metabolite binding to

proteins. This would be consistent with the hypothesis that cocaine reactive metabolite

binding to protein triggers hsp induction, and would explain why cells lacking cocaine

reactive metabolite binding (i.e. cells surrounding and away from the necrotic lesions) did

not exhibit hsp induction. The existence of a threshold for reactive metabolite binding is

well established for other hepatotoxicants such as acetaminophen and bromobenzene

(Casini et al., 1985; Birge et al., 1990; Roberts et al., 1990), and is related to the capacity

of intracellular detoxification mechanisms such as glutathione conjugation. The presence

of a similar threshold for cocaine has not been explicitly demonstrated, but the apparent








absence of a graded response among cells for cocaine reactive metabolite binding in this

study implies that such a threshold exists.

Increased levels of hsp25 and hsp70i were detectable in cocaine-treated mice at the

earliest time point examined, 3 hours post-administration, and the hsp concentrations

continued to increase during the 24-hour period following the cocaine dose (Figure 3-1

and data not shown). The apparent continued synthesis of the hsps between 6 and 24

hours post-administration was somewhat surprising, given the severity of toxicity

experienced by the cells within the lesion. By the 6-hour time point, a number of the cells

within the affected zone were necrotic, and by 24 hours severe coagulation necrosis was

evident. Continued increases in hsp levels during this time frame suggest that hepatocytes

are capable of sustaining hsp synthesis until very near cell death, at least in response to

cocaine intoxication.

In conclusion, the results of this study indicate that cocaine hepatotoxicity results

in a strong, but selective induction of hsp accumulation in the mouse. Induction of hsp

accumulation, as well as cytotoxicity, is confined to cells with detectable cocaine

metabolite binding, suggesting that this binding may be responsible for both phenomena.

The role of this induced hsp accumulation in the hepatotoxicity of cocaine and other

compounds remains to be determined. On one hand, hsp induction may constitute a

cellular defense mechanism to enhance the correct refolding of damaged proteins and/or

their targeting for degradation (Salminen et al., 1996; Parsell and Lindquist, 1994;

Ovelgonne et al., 1995). On the other hand, the continued synthesis of these proteins may

contribute to toxicity by further perturbing metabolism of cells attempting to respond to a

toxicant insult (Goering et al., 1993). Delineation of the role of hsps in toxicant action

will be an important step in understanding the fundamental mechanisms of hepatotoxicity.














CHAPTER 4
DIFFERENTIAL HEAT SHOCK PROTEIN INDUCTION BY ACETAMINOPHEN
AND A NON-HEPATOTOXIC REGIOISOMER, 3'-HYDROXYACETANILIDE, IN
MOUSE LIVER


Introduction

Recent studies have shown that levels of certain heat shock proteins (hsps) are

increased in the liver in response to hepatotoxicants. The livers of rats treated with

halothane have elevated levels of hsp70i (also termed hsp72) (VanDyke et al., 1992), and

cadmium treatment in rats results in increased synthesis of hsp70i, grp94 and a 110-kDa

protein (Goering et al., 1993). Mice treated with hepatotoxic doses of cocaine have

elevated hepatic levels of hsp25 and hsp70i (Salminen et al., 1997). The significance of

increased de novo synthesis of these proteins in response to hepatotoxicants is unclear,

although hsp induction has been correlated with protection from some hepatotoxicants

(Salminen et al., 1996). These observations, when taken together with the fact that hsps

function as chaperones (for reviews see Jaattela and Wissing, 1992; Welch, 1992;

Lindquist and Craig, 1988), suggest that hsp induction may constitute an important cell

defense mechanism against proteotoxic chemicals.

Activation of heat shock transcription factor (HSF), the factor responsible for the

activation of hsp genes during stress (Baler et al., 1993; Abravaya et al., 1992), appears to

result from the presence within the cell of non-native proteins (Ananthan et al., 1986; Zuo

et al., 1995). It is reasonable to postulate that adduction of proteins by reactive metabolites

of hepatotoxicants may render them sufficiently "non-native" to enable them to trigger the

activation of HSF, resulting in upregulation of hsp synthesis. Indirect evidence is provided

by recent studies comparing the intralobular localization of hsp accumulation and reactive

metabolite binding from cocaine (Salminen et al., 1997). In mice treated with an








hepatotoxic dose of cocaine, elevated hsp levels were observed only in cells with detectable

cocaine reactive metabolite binding, and shifting the location within the lobule of metabolite

binding through pretreatment with phenobarbital or B-naphthoflavone produced a

corresponding shift in cells expressing the hsps. However, because reactive metabolite

binding was also correlated with cytotoxicity, the possibility could not be ruled out that

increased hsp expression occurred in response to some manifestation of toxicity rather than

as a direct consequence of protein adduction.

In an effort to discriminate between protein adduction versus some unidentified

facet of cytotoxicity as a stimulus for hsp synthesis, additional studies were conducted

using acetaminophen (APAP) and its regioisomer 3'-hydroxyacetanilide (AMAP). APAP

is a commonly used analgesic/antipyretic agent that can cause liver necrosis in overdose

situations (Boyer and Rouff, 1971; Ambre and Alexander, 1977; Hinson, 1980).

Cytochrome P-450-mediated oxidation of APAP results in the formation of an electrophilic

intermediate, N-acetyl-p-quinone imine (NAPQI), that is detoxified by conjugation with

glutathione (GSH) (Plaa, 1993; Corcoran et al., 1980; Dahlin et al., 1984). Following

hepatotoxic doses of APAP, hepatocellular GSH becomes depleted, permitting

unconjugated NAPQI to bind cellular macromolecules (Manautou et al., 1996; Rashed et

al., 1990). Though many possible nucleophilic targets exist in the cell, certain proteins are

preferentially bound by NAPQI and their adduction has been proposed to play a role in

causing cell death (Bartolone et al., 1992; Bulera et al., 1995).

AMAP also undergoes oxidation to produce electrophilic intermediates which bind

to proteins. Based on GSH conjugates identified following AMAP administration to mice,

at least three AMAP metabolites (viz., 2-acetamido-p-benzoquinone, 4-acetamido-o-

benzoquinone, and N-acetyl-3-methoxy-p-benzoquinone imine) have been postulated to

account for this binding (Rashed and Nelson, 1989). While equimolar doses of APAP and

AMAP produce similar levels of covalently bound metabolites, AMAP is not hepatotoxic

(Roberts et al., 1990). Even when the AMAP dose was increased to its highest non-lethal









amount, and peak reactive metabolite binding was nearly twice that of an hepatotoxic dose

of APAP, no liver injury from AMAP was observed, and biochemical effects associated

with APAP were absent (i.e., perturbation of calcium homeostasis, inhibition of

mitochondrial function, and inhibition of glutathione peroxidase and thioltransferase

activity) (Tirmenstein and Nelson, 1989; Rashed et al., 1990; Tirmenstein and Nelson,

1990).

In the study reported here, APAP and AMAP were compared with respect to

toxicity, reactive metabolite binding, and effects on hepatic hsp levels. The difference

between these two structurally related compounds in terms of hepatotoxic effect was

verified. Total hepatic hsp responses were evaluated using SDS-PAGE and Western blots,

and potential differences in responses among cells within the hepatic lobule were examined

using immunohistochemistry. Also, the distribution of cells expressing hsps was

compared with the intralobular distribution of reactive metabolite binding.

Materials and Methods

Animals and treatments. Adult B6C3F1 male mice (Harlan Sprague-Dawley,

Indianapolis, IN) weighing 22-25 g were used. Mice were housed on corn cob bedding in

temperature- and humidity-controlled animal quarters with a 12-h light/dark cycle, and

allowed free access to water before and during the experiments. Mice were fasted for 16

hours before APAP (4'-hydroxyacetanilide) or AMAP (3'-hydroxyacetanilide) (Sigma

Chemical Co., St. Louis, MO) doses and then given food for the duration of the

experiments. Mice were administered a single i.p. dose of 200 mg/kg APAP or 1000

mg/kg AMAP in warm saline. The dose for each compound was determined in preliminary
experiments to be the maximum tolerated dose that allowed survival for the duration of the

experiments. Some mice were pretreated one hour prior to the AMAP dose with L-

butathionine-[S,R]-sulfoximine (BSO; 222 mg/kg, i.p.) dissolved in saline. APAP and

BSO were given with an injection volume of 10 ml/kg body weight. AMAP was given with








an injection volume of 20 ml/kg since its limited solubility precluded using a smaller

injection volume. Mice were killed by carbon dioxide asphyxiation.

Polvacrylamide gel electrophoresis. protein blotting, and immunostaining. Hsp

levels in total liver protein were detected by SDS-PAGE and Western blotting as described

previously (Salminen et al., 1996b) with the following modifications. Four hundred

milligrams of liver was homogenized in sample buffer (0.05 M

Tris[hydroxymethyl]aminomethane (Tris), 2% sodium dodecyl sulfate, 10 mM

dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, pH 6.8), boiled for five

minutes, passed through a 22 ga. needle three times to shear DNA, and stored at -800C

until use. Two hundred micrograms of protein from each sample was aliquoted to separate

tubes and bromphenol blue added to a final concentration of 0.0025%. Each aliquot was

boiled for five minutes, loaded onto separate lanes of a 12.5% SDS-PAGE gel, and

resolved by electrophoresis. Proteins separated by SDS-PAGE were immediately blotted

to Hybond-ECL Western membrane (Amersham, England). Upon completion, the

membrane was blocked in TBS (20 mM Tris, 500 mM sodium chloride, pH 7.5)

containing 3% gelatin and then probed with one of the following antibodies: anti-hsp25

(rabbit polyclonal), anti-hsp60 (mouse monoclonal), anti-hsc70 (mouse monoclonal), anti-

hsp70i (mouse monoclonal), or anti-hsp90 (mouse monoclonal). Each of these antibodies

were obtained from Stressgen (Victoria, B.C., Canada) and used at a 1:1000 dilution in

TTBS (TBS containing 0.05% polyoxyethylenesorbitan monolaurate) containing 1%

gelatin. Incubation was for 18 hours at 24C with continuous shaking. Primary antibody

binding was detected using a sheep anti-mouse or donkey anti-rabbit (depending upon the

primary antibody used) horseradish peroxidase-conjugated antibody (Amersham, England)

at a 1:3000 dilution in TTBS containing 1% gelatin. The chemiluminescent horseradish

peroxidase substrate Luminol (Amersham, England) was added to the membrane and the

membrane exposed to standard X-ray film to localize antibody binding.








Immunohistochemical detection of hsp25. hsp70i. and APAP adducts in mouse

livr. Five millimeter thick sections from several lobes of each liver were placed in tissue

cassettes and fixed in neutral buffered formalin for three hours. The livers were rinsed and

stored in saline, processed routinely, and embedded in paraffin. Four sequential sections 4-
6 nim thick were cut from the same block to facilitate comparison of localization of hsp

induction, APAP or AMAP reactive metabolite binding, and morphologic changes. One

section was stained with hematoxylin and eosin and examined for histopathology by light

microscopy, while the remaining sections were immunohistochemically stained with an

anti-hsp25 antibody, an anti-hsp70i monoclonal antibody, or an anti-APAP antibody as

follows. Paraffin embedded sections were deparaffinized by passing through three

changes of xylene for five minutes each. The sections were passed through 100% ethanol

two times for one minute each, 95% ethanol for one minute, and double distilled water
(ddH20) two times for two minutes each. Endogenous peroxidase activity was quenched

by submerging the slides in 3% hydrogen peroxide containing 0.1% sodium azide for ten
minutes. The slides were then washed in ddH20 three times for two minutes each and then

equilibrated in TBS for at least two minutes. All the following incubations were performed

in a humidified chamber. Blocking solution (TBS containing 25% v/v bovine serum plus

3% w/v purified bovine serum albumin) was placed on each section and incubated at 370C

for one hour. Fab fragment goat anti-mouse IgG (H+L) (Jackson Immuno Research

Laboratories, Inc., West Grove, Pennsylvania) was added to the blocking solution (10

Ig/ml final concentration) before blocking slides subsequently probed for hsp70i
induction. The latter addition blocked any endogenous mouse IgG that was present in the

sections and prevented false positive signals when probing with the biotinylated anti-mouse
IgG secondary antibody. The slides were washed two times in TBS for two minutes each.

Anti-hsp25 antibody (rabbit polyclonal, Stressgen, B.C., Canada) was diluted 1:100 in

blocking solution and placed on the appropriate slides, while anti-hsp70i (mouse

monoclonal, Stressgen, B.C., Canada) was diluted 1:100 in blocking solution devoid of








the Fab fragment goat anti-mouse IgG antibody and placed on the appropriate slides. Anti-

acetaminophen antibody was diluted 1:100 in blocking solution and placed on the

appropriate slides. The antibodies were incubated with the sections at 370C for one hour

and then at 240C for 18 hours. The sections were washed three times in TBS for two

minutes each. Biotinylated goat anti-mouse or goat anti-rabbit (Southern Biotechnology

Associates, Inc., Birmingham, AL), depending upon the primary antibody used, was

diluted 1:500 in TBS containing 3% BSA, placed on the slides, and incubated at 370C for

thirty minutes. The sections were washed three times in TBS for two minutes each.

Streptavidin-linked horseradish peroxidase (Southern Biotechnology Associates, Inc.,

Birmingham, AL) was diluted 1:200 in TBS containing 3% BSA, placed on the slides and

incubated at 370C for thirty minutes. The sections were washed three times in TBS for two

minutes each. The horseradish peroxidase colorimetric substrate 3,3'-diaminobenzidine
(DAB) (Sigma Chemical Co., St. Louis, MO) supplemented with 0.03% NiC12 (w/v) was

incubated with each section for fifteen minutes at 240C to provide a permanent location of

antibody binding. The sections were then counterstained with hematoxylin and dehydrated

by passing through graded alcohols and xylene in the reverse order as for deparaffinizing

the sections. The sections were mounted using Permount (Fisher Scientific, Orlando, FL)

and a glass cover slip. Using this procedure, no binding of normal rabbit serum or mouse

IgG, used as negative controls for the anti-hsp25 and anti-APAP, or anti-hsp70i

antibodies, respectively, was observed. In addition, secondary antibody-only treated slides

exhibited no binding.

The anti-APAP antibody used in this study has been characterized extensively in

two past reports (Matthews et al., 1996; Matthews et al., 1997). It was raised against a

protein conjugate of the arylacetamide, N-acetyl-p-aminobenzoic acid, and has a high

affinity and specificity for the acetamide and ring portions of the APAP molecule. Due to

this specificity, it can recognize the parent compound (APAP), NAPQI, AMAP, and

reactive metabolites formed from AMAP that retain the arylacetamide portions of the parent








molecule. We have shown that cytochrome P-450 inhibitors can prevent the

immunostaining in liver from mice treated with AMAP or APAP indicating that the detected

binding is due to covalently bound metabolites and not due to unbound parent compound

(unpublished observations). The anti-APAP antibody exhibited no binding in livers from

mice given an acute necrogenic dose of carbon tetrachloride, bromobenzene, or cocaine

indicating that antibody binding could not be attributed to unmasking of an endogenous

antigen during necrosis. Further, preincubation of the anti-APAP antibody with 1 mM

APAP at 370C for one hour before its addition to the slides prevented the binding of the

antibody to the livers from APAP or AMAP treated mice. This observation provided

additional confirmation that the antibody specifically recognized protein-bound APAP and

AMAP metabolites.

Liver non-protein sulfhydryl (NPSH) depletion. Liver NPSH levels were

measured as the total acid soluble thiols according to the method of Ellman (1959). Each

liver was homogenized in 5 ml of 6% trichloroacetic acid (w/v) and 1 mM

[ethylenedinitrilo]tetraacetic acid and then centrifuged at 1000 x g at 40C for fifteen minutes.

Eighty microliters of supernatant solution was added to 2 ml of phosphate buffer (0.1M,

pH 8.0). After addition of 40 pl of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB: 4 mg/ml

in 95% ethanol), the solution was vortex mixed and allowed to stand at 240C for five

minutes. The absorbance at 412 nm was then measured and the corresponding NPSH

concentrations determined using an extinction coefficient of 13,100 M_' cm'.

Protein determination, Protein was measured with the Micro Protein Determination

assay (Sigma Chemical Co., St. Louis, MO) using BSA as standard.

Statistical analysis. NPSH data were analyzed by a one-way ANOVA followed by a

Student Neuman-Keuls post-hoc test. The level of significant difference was defined as the

0.05 level of probability.











Results

Western blot analysis was used to measure the induction of hsp accumulation in

mouse liver by APAP or AMAP. Representative Western blots in Figure 4-1 show the

hepatic levels of hsp25, hsp60, hsc70, hsp70i, and hsp90 at 0 (control), 3, 6, and 24

hours after treatment of naive, male B6C3F1 mice with APAP, 200 mg/kg, i.p. The levels

of hsp60, hsc70, and hsp90 in the liver were unaffected by APAP treatment, while hsp25

levels were increased at 6 and 24 hours and hsp70i levels were increased at each time point.

Maximal accumulation was observed at 24 hours for both hsps. No increase in any of the

hsps was observed in response to AMAP, 1000 mg/kg, i.p. (not shown).

Using information gained from Western blots identifying the hsps increased by

APAP treatment, subsequent experiments utilized immunohistochemistry to determine the

distribution of hsp25 and hsp70i accumulation within the lobule. In addition, the

intralobular location of covalently bound APAP or AMAP was determined

immunohistochemically using an anti-APAP antibody. Visible increases in hsp25 and

hsp70i immunostaining were observed at the earliest observation time, 3 hours after the

dose (not shown). Consistent with the Western blot experiments, hsp25 and hsp70i levels

were greatly increased at 6 and 24 hours. Hsp25 and hsp70i accumulation at 6 hours was

evident throughout the centrilobular region, and the distribution of cells with increased

hsps, covalently bound APAP, and mild morphological changes (i.e. cell swelling) were

essentially superimposable (not shown). By 24 hours, the pattern ofhsp25 induction had

changed dramatically. At that time, hsp70i accumulation was still uniform throughout the

centrilobular region and restricted to necrotic hepatocytes (Figure 4-2). In contrast, hsp25

accumulation was minimal within the most affected centrilobular hepatocytes and strongest









in hepatocytes on the periphery and surrounding the lesions. Similar to observations at 6

hours, hsp25 and hsp70i accumulation was restricted to hepatocytes that had covalently-

bound APAP.


0 3 hrs. 6 hrs. 24 hrs.
I I I I I


-HSP90

-HSP70i


-HSC70

-HSP60


-HSP25








Figure 4-1. Heat shock protein (hsp) induction in mouse liver 0, 3, 6, or 24 hours after
treatment with 200 mg/kg APAP. Liver protein was resolved on a 12.5% SDS-PAGE gel,
and the level of various hsps determined by Western blotting using an antibody specific for
the indicated hsp. The Western blots are from a single experiment with each lane
containing a liver sample from a single mouse. Equal amounts of protein (i.e., 200 g.g)
from each sample was loaded onto separate lanes.










In contrast to the hepatocellular swelling and necrosis produced by APAP, no

significant morphological changes were observed in mice treated with AMAP, 1,000

mg/kg. i.p., up to 24 hours after the dose (Figure 4-3). To confirm reactive metabolite

binding from AMAP, liver sections from AMAP-treated mice were immunostained. As has

been observed elsewhere (Salminen et al., 1997- submitted for publication), the pattern of

binding was panlobular rather than localized in the centrilobular region as is the case with

APAP. As reflected by the intensity of immunostaining, AMAP binding was greatest one

hour after the dose and somewhat reduced at 3 and 6 hours post-treatment. AMAP binding

was barely detectable 24 hours after the dose. It is possible that Western blotting (as

shown for APAP in Figure 4-1) could have been insufficiently sensitive to detect

overexpression of hsps in a small subset of cells. For this reason, liver sections from

AMAP-treated mice were also immunostained for hsp25 and hsp70i. No immunostaining

for hsp25 or hsp70i was detected anywhere in the lobule at any of the time points (Figure

4-3).

Previous reports that cellular thiol status may be important in hsp gene activation

prompted an additional experiment in which the effect of APAP and AMAP on hepatic non-

protein sulfhydryls (NPSH) was measured. APAP caused a rapid decline in NPSH to

21% of control values by one hour post-administration (Figure 4-4). NPSH levels began

rising by 3 hours and exceeded control levels at 24 hours. The depression in hepatic










Figure 4-2. Immunohistochemical detection of hsp25 and hsp70i accumulation and APAP
adduction of cellular macromolecules in mouse liver 24 hours after treatment of naive mice
with 200 mg/kg APAP. Four sequential sections were cut to facilitate comparison of hsp
induction with APAP adduction and morphological changes. The slides were treated as
follows: a) hematoxylin and eosin stain; b) immunohistochemical stain using an anti-hsp25
antibody; c) immunohistochemical stain using an anti-hsp70i antibody; d)
immunohistochemical stain using an anti-APAP antibody. All immunostained slides were
counterstained with hematoxylin. Arrow indicates portal triad; arrow head indicates central
vein.






56











d 4
.G ".























4 i r?: ,m '.









Figure 4-3. Immunohistochemical detection of hsp25 and hsp70i levels and AMAP
adduction of cellular macromolecules in mouse liver three hours after treatment of naive
mice with 1,000 mg/kg AMAP. Four sequential sections were cut to facilitate comparison
of hsp induction with AMAP adduction and morphological changes. The slides were
treated as follows: a) hematoxylin and eosin stain; b) immunohistochemical stain using an
anti-hsp25 antibody; c) immunohistochemical stain using an anti-APAP antibody. All
immunostained slides were counterstained with hematoxylin. Arrow indicates portal triad;
arrow head indicates central vein.






59






60








NPSH produced by AMAP was much less pronounced, with a nadir at 46% of control

observed 6 hours after the dose. The depression of hepatic NPSH was more protracted

than after APAP, however. These data suggested that the lower efficacy of AMAP in

depleting NPSH levels might be a possible explanation of its inability to trigger hsp

induction.

In order to produce an hepatic NPSH depression in AMAP-treated mice similar to

that observed in animals treated with APAP, mice were pretreated with the glutathione

synthesis inhibitor butathionine sulfoximine (BSO) one hour prior to the AMAP dose. The

dose of AMAP was lowered to 600 mg/kg for these experiments since greater than 50%

mortality was observed in mice administered BSO + 1,000 mg/kg AMAP. Despite using a

lower AMAP dose, the BSO + AMAP treatment resulted in only a slightly lower level of

binding of AMAP to liver protein compared to the 1,000 mg/kg AMAP dose with no

change in the pattern of binding (i.e. binding was still panlobular with the greatest binding

in the single layer of hepatocytes surrounding the central veins), as measured by

immunostaining (not shown). The BSO + AMAP treatment produced a similar decrease in

NPSH as APAP at one hour (26% for BSO + AMAP vs. 21% for APAP) with a nadir of

14% at three hours (Figure 4-4). BSO alone caused a maximal decline of NPSH of 52 and

46% of control levels 1 and 3 hours after the dose, respectively (not shown). Similar to

AMAP alone, BSO + AMAP failed to cause hepatotoxicity detectable by light microscopy

at any of the observation times (Figure 4-5). Western blot analysis of hsp25 and hsp70i

induction by BSO + AMAP was inconclusive, but suggested that a slight induction of

hsp25 might have occurred by 24 hours (not shown). Immunohistochemical detection of

hsp25 accumulation confirmed that BSO + AMAP did produce hsp25 accumulation by 24

hours in a small fraction of cells surrounding the central veins of the liver with no

concurrent induction of hsp70i (Figure 4-5). BSO treatment alone failed to cause induction

of either hsp as measured by Western blotting and immunohistochemistry (not shown).






















120-
0
100

80 *o-, **.-

60-
Z
40

20


0 3 6 9 12 15 18 21 24

Time (hours)

Figure 4-4. The effect of APAP, AMAP, and butathionine sulfoximine (BSO) + AMAP on
the liver levels of non-protein sulfhydryls (NPSH). Mice were administered 200 mg/kg
APAP or 1,000 mg/kg AMAP and the level of NPSH in the liver measured 0, 1, 3, 6, or
24 hours after the dose as described in Materials and Methods. Some mice were pretreated
with BSO (222 mg/kg) one hour prior to the AMAP dose. The AMAP dose was lowered
to 600 mg/kg for the combined BSO + AMAP treatment since the 1,000 mg/kg AMAP
dose produced an unacceptably high level of mortality. Results are expressed as percentage
of the mean NPSH concentration in concurrently euthanized, saline-treated controls. Data
are displayed as the mean SEM (n = 3 4 mice). denotes a statistically significant
decrease/increase in NPSH caused by the APAP or BSO + AMAP treatments compared to
control levels; ** denotes a statistically significant decrease in NPSH caused by all three
treatments compared to control levels (p < 0.05).










Figure 4-5. Immunohistochemical detection of hsp25 and hsp70i accumulation in mouse
liver 24 hours after treatment of naive mice with butathionine sulfoximine (BSO) + AMAP.
Mice were pretreated with BSO (222 mg/kg) one hour prior to the AMAP dose (600
mg/kg). Three sequential sections were cut to facilitate comparison of hsp induction with
morphological changes. The slides were treated as follows: a) hematoxylin and eosin stain;
b) immunohistochemical stain using an anti-hsp25 antibody; c) immunohistochemical stain
using an anti-hsp70i antibody. All immunostained slides were counterstained with
hematoxylin. Arrow indicates portal triad; arrow head indicates central vein.







64




































.






65










Discussion

The results of this study show that toxicity from APAP, like that from a number of

other hepatotoxicants, is accompanied by increased hepatic concentrations of hsps.

Consistent with previous observations with cocaine, bromobenzene, and carbon

tetrachloride (Salminen et al., 1997, Roberts et al., 1996), induction of hsps selectively

involved hsp25 and hsp70i no changes in hsp60, hsc70, and hsp90 were detected.

Western blot analysis appeared to suggest that accumulation of hsp70i may precede that of

hsp25 (see Figure 4-1). This distinction is probably an artifact resulting from the limited

sensitivity of measuring whole tissue hsps by this method. Using immunohistochemical

staining, both hsp25 and hsp70i concentrations were increased at the earliest time point

examined, i.e., 3 hours after APAP administration. Observations in this study suggest that

hsp25 may, in fact, be a more sensitive indicator of cytotoxicity. At 24 hours after the

APAP dose, cells at the margin of the lesions had detectable reactive metabolite binding and

appeared to be only minimally affected. Within these cells, hsp25 but not hsp70i was

elevated (see Figure 4-2). Additional evidence is provided by the observation that

combined BSO + AMAP treatment caused induction of only hsp25 (see Figure 4-5).

The strong correlation in intralobular distribution of APAP reactive metabolite

binding and increased levels of hsp25 and hsp70i is consistent with protein adduction as a

stimulus for hsp synthesis. On the other hand, the absence of increased levels of hsps in

response to AMAP, despite extensive reactive metabolite binding, argues that protein

adduction alone may not be an effective trigger for hsp upregulation. The latter observation

must be interpreted cautiously, as there are a number of potential explanations for the lack

of an hsp response. One is that it may not be protein adduction per se, but adduction of

certain critical proteins that leads to increased hsp synthesis. There is ample evidence that

the reactive metabolites of APAP and AMAP adduct different proteins (Tirmenstein and

Nelson, 1989; Myers et al. 1995), and it has been postulated that the greater reactivity of








AMAP metabolites leads to binding with more proximal, and less critical, cellular protein

targets than is the case with APAP (Rashed et al., 1990; Ramsay et al., 1989). Differences

in hsp induction between APAP and AMAP may reflect differences in the intracellular

site(s) of the adducted proteins, or perhaps the nature of the adducted proteins themselves.

Tirmenstein et al. (1991) showed that pretreatment with BSO caused an increase in binding

of AMAP to mitochondrial proteins. Even though we did not explicitly show increased

binding of AMAP to mitochondrial proteins by BSO pretreatment in our study, the

combined BSO + AMAP treatment's ability to trigger hsp25 induction might be

representative of the increased binding of AMAP to critical cellular targets that were not

accessible without BSO pretreatment.

Another possibility relates to the amount or concentration of adducted protein that

may be required to trigger increased hsp synthesis. Previous studies have shown the

overall extent of binding of AMAP and APAP metabolites to hepatic protein to be similar

(Roberts et al., 1990; Rashed et al., 1990). Immunohistochemical staining shows that

AMAP reactive metabolite binding occurs in hepatocytes throughout the liver, however,

while binding of APAP metabolites is confined to the centrilobular region (see Figures 4-2

and 4-3). The more diffuse binding of AMAP metabolites may result in a lower amount or

concentration of adducted protein per cell, perhaps less than the threshold required to

trigger hsp synthesis. Increasing adducted protein concentrations by increasing the AMAP

dose in order to test this hypothesis is unfortunately precluded by the respiratory

depression which occurs at higher dosages of AMAP (Rashed et al., 1990). The ability of

BSO + AMAP to trigger hsp25 induction around the central veins is compatible with the

notion that the concentration of adducted protein may play a critical role in triggering hsp

induction. While AMAP binding occurs panlobularly, the greatest binding occurs in the

single layer of hepatocytes surrounding the central veins (see Figure 4-3) which is where

hsp25 induction occurs after BSO + AMAP treatment.








Another possible explanation for the absence of hsp induction following AMAP

treatment is that protein adduction alone is an insufficient stimulus. Chen et al. (1992)

found that binding of an electrophilic metabolite of nephrotoxic cysteine conjugates to

protein in LLC-PKl cells was associated with induction of hsp70 mRNA and increased

hsp70 synthesis. Treatment of cells with the thiol-reducing agent dithiothreitol did not

affect protein adduction, but nonetheless inhibited induction of hsp70 mRNA. Based on

these observations, the authors have proposed that a combination of protein adduction and

alterations in cellular non-protein thiols may be needed to activate hsp transcription factor.

Huang et al. (1994) found that dithiothreitol prevented induction of hsps by hyperthermia,

further suggesting that oxidation of cellular thiols is an important trigger of hsp induction.

In the present study, both APAP and AMAP diminished hepatocellular NPSH

content, but the effect of AMAP was relatively modest compared with APAP. When

NPSH depletion from AMAP was enhanced by BSO pretreatment to produce declines in

NPSH levels comparable to those caused by APAP, accumulation of hsp25 was observed,

albeit only in cells surrounding the central vein in which AMAP binding was greatest.

These results are consistent with a requirement for both protein adduction and a substantial

loss of reduced thiols in triggering hsp25 induction. It is unclear why the hsp25 response

in BSO + AMAP treated mice was restricted to cells surrounding the central vein, but there

are several possible explanations. As discussed above, the level of protein adduction in

other cells may have been below some threshold requirement, the nature of the protein

adducts may be different in different regions of the liver, the extent of NPSH depletion in

other cells may have been lesser and insufficient to permit the hsp25 response, or some

combination of these factors. The absence of an hsp70i response in BSO + AMAP treated

animals suggests that there may be somewhat different requirements in terms of cellular

events for hsp70i versus hsp25 induction. This hypothesis is supported by the observed

differences in localization of hsp25 and hsp70i within the hepatic lobule after hepatotoxic

doses of APAP (see Figure 4-2).






69

In conclusion, hepatotoxic doses of APAP in mice lead to increased levels of hsp25

and hsp70i in the liver. As has been observed previously with cocaine, there is a close

temporal and spatial correlation within the lobule of reactive metabolite binding,

cytotoxicity, and accumulation of these hsps. The absence of similar increases in hsps in

response to AMAP, despite reactive metabolite binding similar in extent to that from APAP

(at least on a whole organ basis), implies that the simple presence of adducted protein may

not be a sufficient stimulus for increased hsp gene expression. It appears that, at least for

hsp25, a reduction in NPSH levels may have to occur simultaneously with protein

adduction in order to trigger hsp induction.














CHAPTER 5
PROTECTION AGAINST HEPATOTOXICITY BY A SINGLE DOSE OF
AMPHETAMINE: THE POTENTIAL ROLE OF HEAT SHOCK PROTEIN
INDUCTION.


Introduction

Amphetamine and methamphetamine are commonly abused drugs that cause

peripheral adrenergic and central nervous system stimulation. A common side effect of

amphetamine and similar compounds in humans and rodents is hyperthermia, during which

core body temperatures often exceed 400C in overdose situations (Bushnell and Gordon,

1987; Seale et al., 1985; Nowak, 1988; Callaway and Clark, 1994). Amphetamine-

induced hyperthermia is believed to be due to dopamine receptor activation and not

excessive motor movement (Callaway and Clark, 1994). Amphetamine treatment in mice

has been shown to increase liver and brain levels of a 70 kDa heat shock protein (hsp70i),

presumably as a result of increased body temperature (Nowak, 1988). The induction of

hsp70i by amphetamine is similar to that observed in cultured cells in which the media

temperature is raised 3-50C above normal culture temperature, which causes induction of a

variety of heat shock proteins (hsps; for reviews see Parsell and Lindquist, 1994; Voellmy,

1994; Welch, 1992).

Many studies have documented that increased levels of hsps in cultured cells

induced by mild hyperthermic treatment can result in tolerance to severe hyperthermia and

other stressors including chemical inducers of hsps such as arsenic and diamide (Kampinga

et al, 1995; Li and Werb, 1982; Hahn and Li, 1982). It has been proposed that the

apparent ability of hsps to provide protection from various adverse stimuli in these

experiments is due to their ability to bind non-native proteins (Palleros et al., 1991;

Hightower et al., 1994) and chaperone their refolding or elimination. Only a handful of








studies have addressed the question whether hsps provide protection from various insults

in whole animals (Villar et al., 1993; Hotchkiss et al., 1993; Currie et al., 1988; Saad et

al., 1995), in part because of the difficulty of inducing hsps in vivo. In such studies,

hyperthermia was typically produced by heating animals in incubators or water baths. The

animals often had to be anesthetized, however, and a large variability in the response of the

animals was noted (Myers et al., 1992; DeMaio et al., 1993; Blake et al., 1990; Abe et al.,

1993). Amphetamine treatment offers a novel alternative to increase hsps in a variety of

tissues, including the liver.

The first goal of this study was to confirm and extend the previous observation that

hyperthermic treatment by amphetamine administration increases hsp levels in mice,

specifically in liver tissue. The second objective was to determine whether increases in

hepatic hsps resulting from amphetamine treatment are associated with a diminished

susceptibility to hepatotoxicants. To test this, mice with elevated hsps from amphetamine

treatment were challenged with one of four different hepatotoxicants- acetaminophen,

bromobenzene, carbon tetrachloride, or cocaine. The effects of amphetamine pretreatment

on liver injury, hepatotoxicant bioactivation and hepatic glutathione status were assessed.


Materials and Methods

Animals and treatments. Adult ICR male mice (Harlan Sprague-Dawley,

Indianapolis, IN) weighing 25-30 g were housed on corn cob bedding in temperature- and

humidity-controlled animal quarters with a 12-h light/dark cycle. The animals were

allowed free access to food and water before and during the experiments, with the

exception of the fasting of mice for 16 hours before acetaminophen (APAP) treatment.

ACS grade bromobenzene or carbon tetrachloride (Fisher Scientific, Orlando, FL) was

administered i.p. in corn oil with an injection volume of 5 ml/kg body weight. APAP (4'-

hydroxyacetanilide), D-amphetamine sulfate, and cocaine hydrochloride (Sigma Chemical








Co., St. Louis, MO) were administered i.p. in saline with an injection volume of 10 ml/kg

body weight.

For most experiments, mice were pretreated with amphetamine (15 mg/kg) or saline
and returned to the animal quarters, which were maintained at 24-250C. Seventy-two hours

after pretreatment, mice were administered one of the four hepatotoxicants (APAP,

bromobenzene, carbon tetrachloride, or cocaine) and liver injury was assessed through

measurement of serum ALT activity and by histopathology 24,48, and 72 hours after the

dose. In an additional experiment, mice were administered APAP or bromobenzene 144

hours after the amphetamine treatment and liver injury assessed as mentioned above.

Animals were killed by carbon dioxide asphyxiation.

Rectal temperatures were measured with a digital thermometer using a YSI 400
Series rectal thermistor probe (Fisher Scientific, Orlando, FL).

Polvacrylamide gel electrophoresis. protein blotting and immunostaining. Sodium
dodecyl sulfate- polyacrylamide gel electrophoresis and Western blotting of liver protein

was as described previously (Salminen et al., 1997). Briefly, 200 gig of total liver protein

from each sample was resolved by electrophoresis on a 12.5% SDS-PAGE gel. The

proteins separated by SDS-PAGE were immediately blotted to Hybond-ECL Western

membrane (Amersham, England) and then blocked in TBS (20 mM Tris, 500 mM sodium

chloride, pH 7.5) containing 3% gelatin. After blocking, the membrane was washed in

TTBS (TBS containing 0.05% polyoxyethylenesorbitan monolaurate) and then probed with

antibodies to the various hsps (StressGen, Victoria, B.C., Canada) diluted 1:1000 in TTBS

containing 1% gelatin. Incubation was for 18 hours at 240C with continuous shaking. The

membrane was washed with TTBS and incubated with continuous shaking for one hour at

240C with a horseradish peroxidase-conjugated secondary antibody (Amersham, England)

at a 1:3000 dilution in TTBS containing 1% gelatin. Next, the membrane was washed in

TTBS, the chemiluminescent horseradish peroxidase substrate Luminol (Amersham,








England) was added, and the membrane was exposed to standard X-ray film to localize

antibody binding.

Immunohistochemical detection of hsp25 and hsp70i. Immunohistochemical

detection of hsp25 and hsp70i accumulation in the liver was as described previously

(Salminen et al., 1997). Briefly, sections 4-6 pm thick were cut from formalin-fixed,

paraffin-embedded liver specimens. Paraffin embedded sections were deparaffinized and

endogenous peroxidase activity was quenched by submerging the slides in 3% hydrogen

peroxide containing 0.1% sodium azide. Slides were incubated with blocking solution

(TBS containing 25% v/v bovine serum plus 3% w/v purified bovine serum albumin

(BSA)) for one hour at 370C and then washed with TBS. In the case of slides

subsequently probed for hsp70i, Fab fragment goat anti-mouse IgG (H+L) (Jackson

Immuno Research Laboratories, Inc., West Grove, Pennsylvania) was added to the

blocking solution (10 pg/ml final concentration). After the blocking step, slides were

incubated with anti-hsp25 antibody (rabbit polyclonal, Stressgen, B.C., Canada) diluted

1:100 in blocking solution or anti-hsp70i (mouse monoclonal, Stressgen, B.C., Canada)

diluted 1:100 in blocking solution [devoid of the Fab fragment goat anti-mouse IgG

antibody] for one hour at 370C and then for 18 hours at 240C. The slides were washed in

TBS and then incubated for 30 minutes at 370C with a biotinylated secondary antibody

diluted 1:500 in TBS containing 3% BSA. The sections were washed again and then

incubated for 30 minutes at 370C with streptavidin-linked horseradish peroxidase diluted

1:200 in TBS containing 3% BSA. Finally, the sections were washed and incubated for 15

minutes with the horseradish peroxidase colorimetric substrate 3,3'-diaminobenzidine
(DAB) (Sigma Chemical Co., St. Louis, MO) supplemented with 0.03% NiCl2 (w/v).

The sections were then counterstained with hematoxylin and dehydrated with graded

alcohols and then xylene.

Radiolabeled toxicant binding to protein. Ring-labeled [3H]-APAP (Dupont NEN,

Boston, MA), [14C]-carbon tetrachloride (Dupont NEN, Boston, MA), ring-labeled








[14C]-bromobenzene (ICN Radiochemicals, Irvine, CA) or tropine ring-labeled [3H]-

cocaine hydrochloride (National Institute on Drug Abuse, Rockville, MD) were used to

measure covalent binding of toxicant to proteins. Radiolabeled toxicant was added to

unlabeled toxicant such that each animal received 3 gCi at the desired total toxicant dose (50

mg/kg cocaine, 0.04 ml/kg carbon tetrachloride, 350 mg/kg APAP, and 0.45 ml/kg

bromobenzene, respectively). At varying times after the dose, the mice were killed and the

livers removed, rinsed in saline, weighed, and transferred to 15 ml conical tubes on ice

containing 5 ml of 6% (w/v) trichloroacetic acid and 1 mM [ethylenedinitrilo]tetraacetic

acid. Liver was homogenized on ice and a 0.5 ml aliquot of the homogenate was

transferred to a 1.5 ml centrifuge tube for measurement of covalent binding of radiolabeled

toxicant to protein. The remaining 4.5 ml of homogenate were used to assess glutathione

(GSH) levels as described below. The solution in the 1.5 ml centrifuge tube was

sonicated with an Ultratip Labsonic sonicator (Lab-Line Instruments, Inc., Melrose Park,

IL) for 10 seconds at 30-40 watts and the protein pelleted by centrifugation at 8000 x g for

5 minutes. The pellet was washed extensively with 1 ml aliquots of methanol/ether (3:1)

until [14C]- or [3H]-radioactivity in the supernatant was indistinguishable from

background. The pellet was air dried and resuspended in 1 N sodium hydroxide.

Radioactivity in each sample was determined by liquid scintillation spectrometry. The

protein concentration of each sample was measured as described below and used to

normalize detected radioactivity to protein content.

GSH depletion. Hepatic GSH was measured as total acid soluble thiols according

to the method of Ellman (1959). A 4.5 ml aliquot of liver homogenate was centrifuged at

2000 x g at 40C for fifteen minutes. Eighty microliters of the supernatant solution were

added to 2 ml of phosphate buffer (0.1M, pH 8.0), followed by addition of 40 .l of 5,5'-
dithiobis-(2-nitrobenzoic acid) (DTNB: 4 mg/ml in 95% ethanol). The resulting solution

was then vortex mixed and allowed to stand for five minutes at room temperature. GSH








was calculated from the absorbance of the solution at 412 nm using an extinction coefficient

of 13,100 (Sedlak and Lindsay, 1968).

Serum alanine aminotransferase activity. Blood for the determination of alanine

aminotransferase (ALT) activity was collected by cardiac puncture immediately following

carbon dioxide asphyxiation 24, 48, or 72 hours after the hepatotoxicant dose. Serum ALT

activities were determined by the method of Bergmeyer, et al. (1978) using a commercially

available kit (ALT 20; Sigma Diagnostics, St. Louis, MO).

Protein determination. Protein was measured with the Micro Protein Determination

assay (Sigma Chemical Co., St. Louis, MO), based on the method of Bradford (1976),

using BSA as standard.

Statistical analysis. Serum ALT activity values and hepatic GSH concentrations

were analyzed using a one-way ANOVA followed by a Student Neuman-Keuls post-hoc

test. Differences among groups were considered significant at p < 0.05. ALT data were

log transformed prior to statistical analysis.


Results

Initial experiments were conducted to characterize the time course of hyperthermia

resulting from a test dose of amphetamine (15 mg/kg, i.p.). Core body temperature in

amphetamine-treated mice rose quickly and peaked near 400C 30 minutes after the

amphetamine dose (Figure 5-1). After two hours, the mean core body temperature dropped

to a value that was slightly, but not significantly, lower than the pretreatment ("0 time")

core body temperature.












^. 40


39-






37-


o 36-
U

35

0 1 2 3 4 5 6

Time After Amphetamine Injection (hours)
Figure 5-1. Amphetamine-induced hyperthermia. Rectal temperatures were measured at the
indicated times after a single dose of amphetamine (15 mg/kg, i.p.). Data are expressed as
mean SEM (n = 5 mice). denotes a statistically significant elevation of body
temperature above normal body temperature (p < 0.05).


Amphetamine had been shown to induce hsp70i in murine liver; however, hsp
accumulation was only measured two hours after amphetamine treatment (Nowak, 1988).

In order to better characterize the time course of hsp accumulation and disappearance in

response to amphetamine-induced hyperthermia, mice were euthanized at various intervals
from 6 to 144 hours after an amphetamine dose (15 mg/kg, i.p.) and hepatic hsp levels

were estimated by SDS-PAGE and Western blot analysis. Amphetamine treatment

increased hsp25 and hsp70i levels at 6, 24, 48, and 72 hours, with maximal accumulation

of both hsps at 24-48 hours (Figure 5-2). Both hsps returned to pretreatment
of both hsps at 24-48 hours (Figure 5-2). Both hsps returned to pretreatment








(undetectable) levels by 96 hours post-administration in some experiments (Figure 5-2),
and by 144 hours in all experiments. The levels of other hsps (hsp60, hsc70, and hsp90)
were not altered by amphetamine treatment at any time point When the animals were
housed at 19-20C instead of the usual 24-250C, the same 15 mg/kg dose of amphetamine
failed to cause hyperthermia. This effect of ambient temperature on amphetamine-induced
hyperthermia had been noted previously (Nowak, 1988; Yehuda and Wurtman, 1972). No
increase in hsp25 or hsp70i levels following amphetatamine treatment was observed by
Western blotting or immunohistochemistry when hyperthermia failed to occur (not shown).





e

S 6 hrs. 24hrs. 48 hrs. 72 hrs. 96 hrs.
I HI I I


-HSC70
-HSP60


-HSP25


Figure 5-2. Heat shock protein (hsp) induction in murine liver 0, 6, 24, 48, 72, or 96
hours after treatment with amphetamine (15 mg/kg, i.p.). Liver protein was resolved on a
12.5% SDS-PAGE gel, and the levels of various hsps were determined by Western
blotting using antibodies specific for the indicated hsps. Equal amounts of protein (i.e.,
200 ig) from each sample was loaded onto separate lanes.


Mill








Figure 5-3. Immunohistochemical detection of hsp25 and hsp70i accumulation in murine
liver after amphetamine (15 mg/kg, i.p.) treatment. Hsp25 and hsp70i accumulation was
measured 72 hours after amphetamine treatment The immunostained slides were
counterstained with hematoxylin. Arrow indicates central vein; arrow head indicates portal
triad. A) hsp25 accumulation; B) hsp70i accumulation.






79










A subsequent series of experiments was conducted to determine whether increased

hepatic hsp25 and hsp70i levels from amphetamine treatment are associated with increased

resistance or tolerance to hepatotoxicants. To avoid having results confounded by acute

effects of amphetamine, hepatotoxicant administration was delayed until 72 hours after the

amphetamine dose. This corresponded to the latest time point after amphetamine treatment

at which elevated hsp25 and hsp70i levels were observable by the Western blot

experiments described above. Immunohistochemical staining for hsps at this time point

showed that hsp25 accumulation was restricted to hepatocytes within the centrilobular

regions of the liver (i.e., zone 3), while hsp70i was elevated uniformly throughout the

lobule (Figure 5-3).

Amphetamine pretreatment markedly decreased hepatic necrosis from

bromobenzene and APAP as measured by serum ALT activities (Figure 5-4).

Histopathological examination of liver sections taken at 24, 48, and 72 hours corroborated

the ALT activity data. Representative sections from bromobenzene- and APAP-treated

mice, with and without amphetamine pretreatment, are shown in Figure 5-5. Elevated

serum ALT activities from carbon tetrachloride or cocaine were not affected by

amphetamine pretreatment (Figure 5-4), and histopathological examination of liver sections

confirmed the absence of any protection against hepatotoxicity from these compounds (not

shown). As a follow-up experiment, the interval between amphetamine and hepatotoxicant

dose was extended to 144 hours, at which time hsp25 and hsp70i levels had returned to

normal (see above). Under these conditions, amphetamine pretreatment afforded no

protection against the hepatotoxicity of either APAP or bromobenzene (not shown).








Figure 5-4. The effect of amphetamine pretreatment on acetaminophen, bromobenzene,
carbon tetrachloride, or cocaine hepatotoxicity. A single dose of saline or amphetamine (15
mg/kg, i.p.) was administered 72 hours prior to hepatotoxicant challenge. Serum alanine
aminotransferase activity was assessed at the indicated times after a single dose of A)
APAP (350 mg/kg, i.p.), B) bromobenzene (0.45 ml/kg, i.p.), C) carbon tetrachloride
(0.04 ml/kg, i.p.), or D) cocaine (50 mg/kg, i.p.) as an indicator of liver injury. Data are
expressed as the means SEM (n = 8- 15 mice per group). denotes a statistically
significant difference between the two means at a given time point (p < 0.05). NOTE: y-
axis values shown are log normal. ALT= alanine aminotransferase; AMPH= amphetamine;
APAP= acetaminophen.












































Time After APAP Injection (hours)


-a- APAP

----- Amph + APAP


3

. 1000-




E


2 100-















10000






S 1000






E 100
2CO o


0 12 24 36 48 60 72

Time After Bromobenzene Injection (hours)


-0- Bromobenzene

*...-..... Amph +
Bromobenzene
















- --- Carbon Tetrachloride

*-..--.--. Amph +
Carbon Tetrachloride


1000





100
S100
E


12 24 36 48 60 72

Time After Carbon Tetrachloride Injection (hours)
















10000.






1000.




1

E 100oo-
2


12 24 36 48 60 72

Time After Cocaine Injection (hours)


SCocaine

-.-...-- Amph + Cocaine








Figure 5-5. Liver histopathology of saline or amphetamine pretreated mice administered a
single dose of bromobenzene or acetaminophen. A single acute dose of saline or
amphetamine (15 mg/kg, i.p.) was administered 72 hours prior to the bromobenzene (0.45
ml/kg, i.p.) or acetaminophen (350 mg/kg, i.p.) dose. A) saline + bromobenzene, B)
amphetamine + bromobenzene, C) saline + acetaminophen, and D) amphetamine +
acetaminophen represent typical sections from mice 24 hours after the bromobenzene or
acetaminophen dose. Arrow indicates central vein; arrow head indicates portal triad.
Sections are stained with hematoxylin-eosin.






87



























Fl. ".P










In an attempt to explore alternative explanations for the protective effects of a 72-

hour amphetamine pretreatment, the extent of reactive metabolite formation and binding

was evaluated in experiments in which mice were administered radiolabeled doses of the

toxicants and the extent of covalent binding to protein was measured. Previous studies

have shown relatively rapid covalent binding to proteins after APAP, carbon tetrachloride,

and cocaine administration to mice (Roberts et al., 1995; Evans, 1983; Rashed et al.,

1990). Consequently, covalent binding was assessed 1 and 4 hours after administration of

these toxicants. Reactive metabolite binding of bromobenzene was known to occur over a

more protracted period of time (Casini et al., 1985). Therefore, bromobenzene binding to

protein was assessed 6 and 24 hours after the radiolabeled dose. As shown in Table 5-1,

the 72-hour amphetamine pretreatment had no appreciable effect on covalent binding of

radiolabel from any of the hepatoxicants. The only statistically significant difference was

an increase in covalent binding of bromobenzene in amphetamine pretreated mice 6 hours

after the dose.

As had been observed previously, covalent binding of APAP and bromobenzene

was accompanied by a marked depression in hepatic GSH content, while comparatively

little change in GSH concentration resulted from treatment with carbon tetrachloride or

cocaine (Table 5-2). Consistent with the absence of an effect on covalent binding,

amphetamine pretreatment also had no discernable effect on hepatic GSH status of the liver,

before or during hepatotoxicant exposure.




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