Group Title: effects of antioxidant, chloroquine and noise exposure on auditory brainstem response threshold and distortion product otoacoustic emission amplitude in guinea pigs
Title: The effects of antioxidant, chloroquine and noise exposure on auditory brainstem response threshold and distortion product otoacoustic emission amplitude in guinea pigs
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Title: The effects of antioxidant, chloroquine and noise exposure on auditory brainstem response threshold and distortion product otoacoustic emission amplitude in guinea pigs
Physical Description: Book
Language: English
Creator: Babeu, Lorraine Reid
Publisher: University of Florida
Place of Publication: Gainesville Fla
Gainesville, Fla
Publication Date: 2001
Copyright Date: 2001
 Subjects
Subject: Communication Sciences and Disorders thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Communication Sciences and Disorders -- UF   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
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non-fiction   ( marcgt )
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Summary: ABSTRACT: The purpose of this study was to evaluate if an antioxidant (U74389G) has a protective effect against the combined ototoxicity of exposure to noise and chloroquine. Fifty-six pigmented guinea pigs were divided into eight groups. Groups were treated with the following: 1 ml subcutaneous injection of chloroquine diphosphate in saline (35mg/kg), a .3 ml oral solution of antioxidant U74389G in citrate NaCl, or exposed to 93 dB SPL of broadband noise for 48 hours, other groups served as controls. The treatments were administered in the following manner: once daily chloroquine subcutaneous injection for five consecutive days, antioxidant given orally two times per day for three days and noise exposure began on the second day of the antioxidant treatment and continued for 48 hours. Baseline auditory brainstem response (ABR) to tone bursts at 4, 8, 12 & 16 kHz and clicks and distortion product otoacoustic emissions (DPOAE) at 4 and 8 kHz were recorded at three periods. The first was baseline, which occurred prior to exposure to any of the factors. The second measurement (postexposure) was taken at the end of the 48 hours of noise exposure and the third measurement (recovery) was taken 48 hours after the end of the noise exposure. The ABR measurements indicated that chloroquine appeared to create some hearing loss that was greater in the higher frequencies. The antioxidant appeared to have a protective effect against the combined ototoxic effects of chloroquine and noise exposure. In the antioxidant and noise exposure condition the antioxidant did not decrease the amount of the threshold shift but it appeared to aid in the recovery from noise exposure. The DPOAE measurement also indicated that chloroquine caused hearing loss and that the antioxidant was protective against chloroquine and noise exposure.
Summary: KEYWORDS: chloroquine, antioxidant, noise exposure, ABR, DPOAE
Thesis: Thesis (Ph. D.)--University of Florida, 2001.
Bibliography: Includes bibliographical references (p. 202-212).
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System Details: Mode of access: World Wide Web.
Statement of Responsibility: by Lorraine Reid Babeu.
General Note: Title from first page of PDF file.
General Note: Document formatted into pages; contains xvi, 213 p.; also contains graphics.
General Note: Vita.
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Bibliographic ID: UF00100742
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: oclc - 49231815
alephbibnum - 002766288
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THE EFFECTS OF ANTIOXIDANT, CHLOROQUINE AND NOISE EXPOSURE ON
AUDITORY BRAINSTEM RESPONSE THRESHOLD AND DISTORTION
PRODUCT OTOACOUSTIC EMISSION AMPLITUDE IN GUINEA PIGS













By

LORRAINE REID BABEU


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


2001




























Copyright 2001

by

Lorraine Reid Babeu






















Dedicated

to

Yves Gerard Babeu

whom my soul knew before he was born

and

Andrew Gerard Babeu

my inspiration















ACKNOWLEDGMENTS

"Oh, that You would bless me indeed. .. is the first line of the Jabez Prayer

(Wilkinson, 2000). In this prayer Jabez is in wonder that he could and would be blessed.

I believe that I know how Jabez must have felt because I am in awe of the blessings that I

have received. The blessings have largely come in the form of people that have nagged,

tolerated, supported and taught me. To those people words can never truly express my

gratitude but from my heart I say thank you.

The United States Army is acknowledged for making obtaining a doctorate degree

a possibility. Many military and civilian personnel whom I have met through the years

have helped in my personal and professional development. These are just a few of the

people that have supported me throughout my career. Colonel Dorene Hurt taught

important management and soldier skills, also gave me my mantra for life, "nothing beats

a failure but a try." Colonel Linda Pierson was there during the beginning of my military

career. When I told her that I was thinking of applying for the doctorate program, she

encouraged me to move forward. Linda's support has been continual. One day during

my data collection phase when things were not going too well, she seemed to sense my

panic and said the word (variability) I needed to hear. I was able to calm down and move

forward. Colonel Nancy Vause is so dynamic; she energizes people to accomplish good

things. Lieutenant Colonel Michael Moul was my CFY supervisor and mentor; always

encouraged me to do my best and be a career soldier. And last but not the least, is









Colonel Rich Dennis, whose support during one of the toughest years of my life, was

deeply appreciated.

The successful completion of the doctorate degree is dependent upon the caliber

of one's advisor and committee chair. I learnt from other students that the best person for

that job would be Dr. Kenneth Gerhardt. Ever since I have been at the University of

Florida, he has been an excellent instructor, committee chair and mentor. He has always

been patient and willing to answer questions that I probably should have known the

answer to already. His calm spirit is infectious and helps to make tough times bearable. I

admire his style of management and have learned so much from him.

Overall, my committee members are experts in their respective fields so I had no

doubt that with their guidance the dissertation would be of top quality. Dr. Scott Griffiths

has always been extremely patient and giving and when problems arose he was ready and

available to assist in resolving them. Dr. Patrick Antonelli was very giving and ensured

that equipment or other needs were always met. He asked really good questions (some

kept me awake at night) that always increased my knowledge, which led to making the

dissertation better. Dr. Patricia Kricos has the reputation in the department of being the

instructor who makes speech pathology majors switch to audiology. After taking her

audiological rehabilitation course, I could see why she has that reputation Her

knowledge and love of what she does is inspiring. And for someone who has been in the

field for a while, she reaffirms why one wanted to become an audiologist in the first

place. Dr. Jill Varnes' knowledge of worksite health promotion has given me concrete

things to do to help my colleagues provide health education for hearing conservation.









In addition to the faculty, there have been various support staff members who

have been extremely helpful. I would like to thank Angela Prevatt, whose talents as a

research assistant were excellent. She is bright and gutsy and I believe she has a great

future ahead of her. She taught what I needed to know to work in the laboratory and then

gave me space and tough love" when I needed it. I would also like to acknowledge

Idella King and Virginia Dawson for their encouragement and support. A special thank

you to fellow student Samantha Lewis for being a great study partner and friend.

Last but certainly not the least, I would like to give very special thanks to my

parents, Elaine and Gladstone Reid. They taught me to believe in myself and encouraged

me to be a willing worker and achiever. As a parent I am finding out how difficult it can

be to instill those ideals in a child. To my brother Bryan, thank you for the late night

timely pep talks to keep my spirits up. My seven-year-old son Andrew was also very

supportive. Whenever I was discouraged or really tired, Andrew who is my inspiration,

would always tell me "You can do this Mom, just believe in yourself."

When I met my husband Yves, I thought he was such a nice person that even if

we did not stay together, I wanted him for a friend. Well I got lucky and was able to

marry my best friend. He has always been there for me cheerfully and especially for this

project. The help he provided ranged from setting up equipment to data entry. I cherish

and appreciate his loving support at all times.
















TABLE OF CONTENTS

page


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

LIST OF TABLES ............................... ...................... .......... x

LIST OF FIGURES .................................................. .......................xi

A B ST R A C T ............. ............................................................. ......... xv

CHAPTERS

1 IN TRODUCTION .................................................. .. ................. 1

2 LITER A TU R E R EV IEW ................................................................ ....................... 3

Problem of N oise ........................................ ....... .... .... ........ .......... 3
Individual Susceptibility to Hearing Loss From Noise Exposure............................ 4
Damage to the Auditory System From Noise Exposure................... ........ .......... 6
Tem porary Threshold Shift.................... ................................... ......................... 12
Perm anent Threshold Shift..................... .................................... ......................... 15
Synergistic Effects of Noise and Drugs ..................................................... 15
O to to x ic D ru g s .................................................................. ................................ 17
R active O xygenated Species................................................................................. 19
M a la ria ..................................................................... 2 4
Q u in in e ......................................................................................... 2 9
C hloroquine ....................................................................................................... 30
Antioxidant U74389G .......................................... 37
Hearing Measurement ........................... ........ ................... 37
Auditory Evoked Response........................................ ...37
Electrophysiological Measures in the Guinea Pig ........................................ 39
Otoacoustic Emissions .. ... ........ ................... .............. 41

3 M E T H O D ..............................................................................4 8

Experimental Design......................................................... 48
Animal Preparation ................................. ........................... ... ...... 51
Pilot Study................................... .............. .............. 51
Sam ple Extraction Procedure ................................................................................ 52









Serology Testing ..................... ...... ... ............... .. .......... .. 52
Results HPLC ................. ......... ...................... 52
Auditory Procedures and Equipment................. .......... ................... 54
A uditory Brainstem R response .............. ......................................................... 54
Distortion Product Otoacoustic Em mission .............................................. .............. 55
N oise G generation. ........................ ............ ........................ .. .............. .. ................ 56
Preparation of Antioxidant U-73489G..... .................. .............. 56
S ta tistic s ....................... ............................................................................... 5 6

4 R E S U L T S ..................................................................5 9

A uditory B rainstem R response ............................................... ........................... 59
M orphology and L agency ............................................................. ......... .............. 59
Average ABR Thresholds by Groups ....................................................... 62
Statistical M methods .................. .............................. ... .... .............. .. 67
G raphic Plots of the D ata .............. ... ........... .............. ............................. .............. 69
Response variable--baseline to post-treatment .............................................. 72
Response variable--baseline to recovery............................... 72
Response variable--post-treatment to recovery........................... ..... ........... 74
ABR Results All Stimulus Conditions-Tabular................................................ 75
Distortion Product Otoacoustic Emissions ......................................... ..... ......... 78
M orphology ......... ......................... ...... .... .. ........... 78
Average DPOAE Amplitudes by Groups .......................................................... 79
Statistical M methods ....... .......................... ........ ..... .............. .......... 80
Graphic Plots of the Data ............. ..... .... .. ................................. ......... 84
Response variable--baseline to post-treatment .............................................. 85
Response variable--baseline to recovery............................... 86
Response variable--post-treatment to recovery......................................... 87
DPOAE Results All Stimulus Conditions--Tabular.............................. 88

5 DISCU SSION AND SUM M ARY ............................................................ ............... 93

N o ise ....................................93.............................
C hloroquine ......... ................................... ........................... 96
A n tio xid an t ............................................................. ........ ...... 9 8
Synergism .............. ............................................................................... 99
Noise and Chloroquine ............... ......................... ..... .............. 99
Noise/ Chloroquine and Antioxidant .............. ............ ........... ........ 100
Summary ................ ... ............................ 102

APPENDICES

A SU B JEC T D A TA ..... .. ......................................................... ........... ........ 103

B N OISE EXPO SURE D A TA .............................................. ............................. 122

C STATISTICAL SUBJECT DATA......................................... ......................... 123


viii









R E F E R E N C E S ...................................... ........................................................... .. 2 0 2

BIOGRAPHICAL SKETCH ............................................................. ..................213
















LIST OF TABLES


Table Page

1: Electrophysiology Measurements of Threshold in the Guinea Pig .................................40

2:Treatment groups of 7 animals per group for a total of 56 subjects .............................49

3: Tim line of Treatm ent. .......................... ........................ .. ............. .. ......50

4: Auditory Brainstem Response (ABR) Baseline to Post-Treatment for all Stimulus
C on edition s .........................................................................76

5: ABR Baseline to Recovery for all Stimulus Conditions................ ...............77

6: Post-Treatment to Recovery for all Stimulus Conditions ..............................................78

7: Baseline to Post-Treatment Distortion Product Otoacoustic Emissions (DPOAE) 8 kHz 90

8: Baseline to Recovery- DPOAE 8 kHz ............. ... .......................................91

9: Post-Treatment to Recovery DPOAE 8 kHz .......................................... ............... 92

10: Noise Levels of the Cage Prior to and at the End of a Noise Exposure Period.............122















LIST OF FIGURES


Figure Pag(

1: Partial recovery of Distortion Product Otoacoustic Emissions (DPOAE) after exp. to
industrial noise for one anim al........................................ ......................... 47

2:Auditory Brainstem Response (ABR) waveforms to click stimuli from one animal.
Four observable peaks were present that decreased in amplitude and increased
in latency to decreasing intensity in dB increments. In this series threshold
w as 20 dB ............................................. ........ ................. 60

3: Average baseline latency values of the control group. Panels A and B represents
Waves I & II. Panels C and D represents Waves III & IV for frequencies 4, 8,
12, 16 kHz and click. The x-axis represents dB dial on the Tucker Davis
S y stem ......................................................... ................ 6 1

4: Average thresholds for non-noise exposed groups for treatment periods of baseline,
post-treatment and recovery. Chloroquine creates changes in threshold at the
post-treatm ent and recovery tim e periods................................. ............... 64

5: Average thresholds for noise exposed groups for baseline, post-treatment and recovery.
The N-Ch-A group had the least change from baseline at post-treatment and
recovery tim e periods............ .............................................. .. .... ............... 66

6: Change in threshold (in decibels) from baseline to post-treatment for ABR click. For
the non-noise exposed groups only the chloroquine group had a significant
change from baseline to post-treatment and an antioxidant effect.....................71

7: Baseline to recovery threshold (in dB) results for the ABR click. The chloroquine
group continued to show the significant increase seen in previous time period.
The slant of the lines for the N-Ch and N-Ch-A group indicate that the N-Ch-
A had more recovery that can be attributed to the antioxidant. .........................73

8: Post treatment to recovery thresholds (in dB) for the ABR click. For the non-noise
groups no significant change between the two time periods. For the noise-
exposed groups, the presence of antioxidant appears helpful in the recovery
p ro c e ss ................................. ....................................................... ............... 7 4

9: DPOAE at 8 kHz 60 dB for one control animal. .................................... .................79









10: Average DPOAE amplitudes for the non-noise exposed groups over the time periods
of baseline, post-treatment and recovery. Chloroquine group showed a
noteworthy change in amplitude and had the least amount of recovery ............81

11: Average DPOAE amplitudes for the noise exposed groups over the time periods of
baseline, post-treatment and recovery. The N-Ch-A group had least amplitude
change for the post-treatment and recovery time periods..................................83

12: Amplitude change from baseline to post treatment at 8 kHz at 60 dB. The
chloroquine group has a significant decrease in amplitude, while the Ch-A
d o se n ot. ........................................................ ................ 8 6

13: Baseline to recovery DPOAE for all groups. Noise exposed group the presence of
antioxidant (N-Ch & N-Ch-A) assisting in the recovery of amplitude ..............87

14: Post treatment to recovery DPOAE for all groups. Looking across the groups,
presence of antioxidant for the Ch-A and N-A aids in recovery of amplitudes...88

15: DPOAE data during the baseline time period at 4 kHz for groups one through four......104

16: DPOAE data during the baseline time period at 8 kHz for groups one through four ......105

17: DPOAE data during the post-treatment time period at 4 kHz for groups one through
fou r ......... ...... ............ ...................................... ........................... 10 6

18: DPOAE data during the post-treatment time period at 8 kHz for groups one through
fou r ......... ...... ............ ...................................... ........................... 10 7

20: DPOAE data during the recovery time period at 8 kHz for groups one through four.....109

21: ABR data during the baseline time period for the click, 4, 8, 12 & 16 kHz for groups
one through four........... ...... ............................... ........ .. .. ........ .. .. 110

22: ABR data during the post-treatment time period for the click, 4, 8, 12, & 16 kHz for
groups one through four ............ ............ ...... ............... 11

23: ABR data during the recovery time period for the click, 4, 8, 12 & 16 kHz for groups
one through four........... ...... ............................... ........ .. .. ........ .. .. 112

24: DPOAE data during the baseline time period at 4 kHz for groups five through eight. ...113

25: DPOAE data during the baseline time period at 8 kHz for groups five through eight. ...114

26: DPOAE data during the post-treatment time period at 4 kHz for groups five through
e ig h t ...................................... .................................................. 1 1 5

27: DPOAE data during the post-treatment period at 8 kHz for groups five through eight. .116

28: DPOAE during the recovery time period at 4 kHz for groups five through eight...........117









29: DPOAE during the recovery time period at 8 kHz for groups five through eight...........118

30: ABR data during the baseline time period for the click, 4, 8, 12, & 16 kHz for groups
five through eight. ........ .. ........ ............ ............ .. ........ .. .. .119

31: ABR data during the post-treatment time period for the click, 4, 8, 12, & 16 kHz for
groups five through eight. ............................................ ............................ 120

32: ABR data during the recovery time period for the click, 4, 8, 12, & 16 kHz for groups
five through eight. ........................................... ........................ 121

33: Confidence Interval Plot for DPOAE at 4 kHz at 70 dB baseline to post-treatment.......125

34: Confidence Interval Plot for DPOAE at 4 kHz at 60 dB baseline to post-treatment.......127

35: Confidence Interval Plot for DPOAE at 4 kHz at 50 dB baseline to post-treatment.......129

36: Confidence Interval Plot for DPOAE at 4 kHz at 40 dB baseline to post-treatment.......131

37: Confidence Interval Plot for DPOAE at 8 kHz at 70 dB baseline to post-treatment.......133

38: Confidence Interval Plot for DPOAE at 8 kHz at 60 dB baseline to post-treatment.......135

39: Confidence Interval Plot for DPOAE at 8 kHz at 50 dB baseline to post-treatment.......137

40: Confidence Interval Plot for DPOAE at 8 kHz at 40 dB baseline to post-treatment.......139

41: Confidence Interval Plot for ABR at 4 kHz baseline to post-treatment ........................141

42: Confidence Interval Plot for ABR at 8 kHz baseline to post-treatment ........................143

43: Confidence Interval Plot for ABR at 12 kHz baseline to post-treatment ......................145

44: Confidence Interval Plot for ABR at 16 kHz baseline to post-treatment ......................147

45: Confidence Interval Plot for ABR click baseline to post-treatment............................149

46: Confidence Interval Plot for DPOAE at 4 kHz 70 dB baseline to recovery.................. 151

47: Confidence Interval Plot for DPOAE at 4 kHz 60 dB baseline to recovery...................153

48: Confidence Interval Plot for DPOAE at 4 kHz 50 dB baseline to recovery.................. 155

49: Confidence Interval Plot for DPOAE at 4 kHz 40 dB baseline to recovery...................157

50: Confidence Interval Plot for DPOAE at 8 kHz 70 dB baseline to recovery................159

51: Confidence Interval Plot for DPOAE at 4 kHz 60 dB baseline to recovery ...................161









52: Confidence Interval Plot for DPOAE at 8 kHz 50 dB baseline to recovery...................163

53: Confidence Interval Plot for DPOAE at 8 kHz 40 dB baseline to recovery.................. 165

54: Confidence Interval Plot for ABR at 4 kHz dB baseline to recovery..........................167

55: Confidence Interval Plot for ABR at 8 kHz baseline to recovery...............................169

56: Confidence Interval Plot for ABR at 12 kHz baseline to recovery..............................171

57: Confidence Interval Plot for ABR at 16 kHz baseline to recovery..............................173

58: Confidence Interval Plot for ABR click baseline to recovery ......................................175

59: Confidence Interval Plot for DPOAE at 4 kHz 70 dB post-treatment to recovery..........177

60: Confidence Interval Plot for DPOAE at 4 kHz 60 dB post-treatment to recovery.......... 179

61: Confidence Interval Plot for DPOAE at 4 kHz 50 dB post-treatment to recovery..........181

62: Confidence Interval Plot for DPOAE at 4 kHz 40 dB post-treatment to recovery.......... 183

63: Confidence Interval Plot for DPOAE at 8 kHz 70 dB post-treatment to recovery.......... 185

64: Confidence Interval Plot for DPOAE at 8 kHz 60 dB post-treatment to recovery.......... 187

65: Confidence Interval Plot for DPOAE at 8 kHz 50 dB post-treatment to recovery.......... 189

66: Confidence Interval Plot for DPOAE at 8 kHz 40 dB post-treatment to recovery.......... 191

67: Confidence Interval Plot for ABR at 4 kHz post-treatment to recovery........................193

68: Confidence Interval Plot for ABR at 8 kHz post-treatment to recovery........................195

69: Confidence Interval Plot for ABR at 12 kHz post-treatment to recovery......................197

70: Confidence Interval Plot for ABR at 16 kHz post-treatment to recovery......................199

71: Confidence Interval Plot for ABR click post-treatment to recovery .............................201















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

THE EFFECTS OF ANTIOXIDANT, CHLOROQUINE AND NOISE EXPOSURE ON
AUDITORY BRAINSTEM RESPONSE THRESHOLD AND DISTORTION
PRODUCT OTOACOUSTIC EMISSIONS AMPLITUDE IN GUINEA PIGS

By

LORRAINE REID BABEU

August 2001

Chairman: Kenneth J. Gerhardt, Ph. D.
Major Department: Communication Sciences and Disorders

The purpose of this study was to evaluate if an antioxidant (U74389G) has a

protective effect against the combined ototoxicity of exposure to noise and chloroquine.

Fifty-six pigmented guinea pigs were divided into eight groups. Groups were treated with

the following: 1 ml subcutaneous injection of chloroquine diphosphate in saline (35

mg/kg), a .3 ml oral solution of antioxidant U74389G in citrate NaC1, or exposed to 93

dB SPL of broadband noise for 48 hours, other groups served as controls. The treatments

were administered in the following manner: once daily chloroquine subcutaneous

injection for five consecutive days, antioxidant given orally two times per day for three

days and noise exposure began on the second day of the antioxidant treatment and

continued for 48 hours. Baseline auditory brainstem response (ABR) to tone bursts at 4,

8, 12 & 16 kHz and clicks and distortion product otoacoustic emissions (DPOAE) at 4

and 8 kHz were recorded at three periods. The first was baseline, which occurred prior to

exposure to any of the factors. The second measurement (post-treatment) was taken at









the end of the 48 hours of noise exposure and the third measurement (recovery) was

taken 48 hours after the end of the noise exposure. The ABR measurements indicated that

chloroquine appeared to create some hearing loss that was greater in the higher

frequencies. The antioxidant appeared to have a protective effect against the combined

ototoxic effects of chloroquine and noise exposure. In the antioxidant and noise exposure

condition the antioxidant did not decrease the amount of the threshold shift but it

appeared to aid in the recovery from noise exposure. The DPOAE measurement also

indicated that chloroquine caused hearing loss and that the antioxidant was protective

against chloroquine and noise exposure.














CHAPTER 1
INTRODUCTION

For years the medical community has been informing the public that there is no

medical cure for noise-induced hearing loss (NIHL). However, recent research has found

success in reducing the ototoxicity of noise through the use of therapeutic antioxidants.

The studies base their treatment hypothesis on the grounds that antioxidants reduce

cellular oxidization, thus reducing the damaging effects of noise exposure (Quirk,

Schivapuja, Schwimmer & Seidman, 1994; Lui, 1992).

Noise is not the only agent of toxicity to the auditory system. Other agents

include certain classes of drugs and industrial vibration. In industry and military settings,

people can be exposed to recognized and as yet unknown ototoxic agents. For example,

vibration alone does not cause hearing loss, but in combination with noise it increases the

severity of hearing loss (Boettcher, Henderson, Gratton, Danielson & Byrne, 1987).

Since there has been some success in the treatment and prevention of NIHL with

antioxidants, the next step would be to determine if the protective effect could be

extended to synergistic ototoxicity.

In the case of the military, exposure to noise and some ototoxic drugs are quite

common. Chloroquine, which is used in the prevention and treatment of malaria, has

ototoxic side effects (Toone, Hayden, & Ellman, 1965; Matz & Naunton, 1968).

Military personnel deployed to parts of the world where malaria is endemic, are at risk of

exposure to both noise and chloroquine. For example, from 1997 to 2000 there were 184

reported cases of malaria in US service members, thus proving that malaria continues to









be a threat to the combat readiness of the armed forces (Medical Surveillance Monthly

Report, 1999, & Medical Surveillance Monthly Report, 2001). The risk for exposure to

malaria is not only an issue for the military; there have been cases of outbreaks among

civilians in New York and Texas (Isaacson, 1989).

Although noise is a major source of hearing loss in military and industry, the

extent that other ototoxic agents contribute to the severity of the hearing loss cannot be

overlooked. Any treatments or preventive measures for NIHL should consider the

synergistic components. The purpose of this study is to evaluate if an antioxidant,

U74389G, will have a protective effect against the ototoxicity of noise and chloroquine.














CHAPTER 2
LITERATURE REVIEW


Problem of Noise

According to the National Institutes of Health (1991), there are over 28 million

adult Americans with hearing loss. Of the 28 million, 10 million of those cases were

caused by noise exposure. A noise-induced hearing loss (NIHL) can occur at any age.

The most common cause of a NIHL is exposure to the high-intensity noise levels in the

workplace (some examples of noise hazardous occupations include automotive mechanic,

factory work, construction work, farming and music). Noise hazards can be found also at

home and during some recreational activities. Hazards at home include carpentry and

amplified noise. Recreational hazards can include hunting and motorcar racing.

Noise can be classified as either continuous or impulsive noise. An example of

continuous noise is like that of the sound emitted from an engine. An example of

impulsive noise is the sound produced by weapons discharge. Continuous noise can

cause damage that leads to a hearing loss at intensity levels of 85 dBA or greater

(Boettcher et al, 1987). The severity of the hearing loss varies, based on the length of

exposure and the species. In the case of impulse noise, levels in excess of 140 dBP

(sound pressure level peak, single exposure) have been found to produce hearing loss

(Boettcher et al, 1987; Hamernik, Ahroon, Hsuch, Lei & Davis, 1993; Pekkarinen, 1995).

The acoustic energy of a 141-dBP signal can cause immediate damage to the

Organ of Corti because of the sudden, rapid change in air pressure. The damage to the









ear is usually mechanical in nature (blast over pressure creates instantaneous damage to

tissue of the inner ear including both sensory and support cells) and is referred to as an

acoustic trauma. Some examples of things capable of producing noise levels greater than

140 dBP are: a toy cap gun fired close to the ear (155 dBP), or a firecracker detonating

near the head (170 dBP). Hearing loss produced by impulsive levels between 85 and 140

dBA can cause permanent damage but factors such as duration of exposure and

individual susceptibility determine the extent of the damage (Hamernik et al., 1993).

Although NIHL is preventable, it continues to be one of the leading causes of

preventable occupational disease in dustry and in the military (National Institute of

Health, 1991; Pekkarinen, 1995). One of the reasons for this high ranking among

occupational diseases is how the NIHL loss develops. NIHL is painless, progressive and

permanent. In most cases, by the time the individual is aware of hearing loss, significant

damage has already occurred (Clark, 1992).

By-products of a NIHL include a wide range of hearing deficits such as tinnitus,

recruitment, poor frequency selectivity, poor temporal processing and poor speech

perception. These deficits are a problem for rehabilitation because they persist even when

rehabilitative efforts such as hearing aids are used. Hearing aids will amplify sound but

will not help with any of the aforementioned problems (Katz, 1985).

Individual Susceptibility to Hearing Loss From Noise Exposure

There have been numerous studies that have tried to determine individual

susceptibility to NIHL. This is part of the continuing effort to tailor educational programs

to help reduce the risk of NIHL. There have been several investigators (Carlin &

McCroskey, 1980;Loeb & Flectcher, 1963; Ward, 1995;) who have tried to determine if

intrinsic non-auditory factors such as age, melanin, gender, or smoking make an









individual more susceptible to NIHL. Generally, attempts to predict susceptibility based

on the above criteria have yielded equivocal results and only accounted for minor

variability (Henderson, Subramaniam & Boettcher, 1993).

Price (1976) compared the susceptibility of kittens and cats to NIHL and found

that there was little difference in susceptibility up to a certain point beyond which the

kittens were more susceptible to hearing loss from noise. This suggests that there is a

crucial time period in the development of the auditory system when the young are more

susceptible to noise. Lalande, Hetu and Lambert (1986) explored the possibility that the

human fetus was susceptible to NIHL. They examined 131 children whose mothers while

pregnant had worked in noise conditions that ranged from 65 to 95 dBA. The results

indicated that mothers who had exposures of 85 to 95 dBA had children who were more

at risk for hearing loss at 4 kHz. So there is the possibility that susceptibility to NIHL

may be age related for the young of a species and less so for the adults of the same

species. As for susceptibility for the old, there has been no conclusive evidence that aging

increases susceptibility to noise induced hearing loss (Henderson et al., 1993).

There have been reports that melanin may serve as a protector against noise

damage. Reports that have used melanin in eye color as the determining factor have

produced conflicting results. Tota and Bocci (cited in Ward, 1995) reported that brown-

eyed young Italian males developed much less temporary threshold shift (TTS) than did

blue-eyed males. But Karlovich (1975) repeated the study using American youths and his

results conflicted with Tota and Bocci's results. However, when using skin melanin as

the determining factor, Henderson, Shadoan, Subramaniam, Saunders and Ohlin (1995)

found that black soldiers did not seem to be as affected by noise exposure when









compared to white soldiers. It is possible that the melanin in skin is a better susceptibility

predictor than melanin of the iris.

For several years the common belief was that women were less susceptible to

NIHL than men. However Mueller and Richardz (as cited Ward, 1995) found that age

and noise exposure matched men and women had no difference in hearing sensitivity,

thus no difference in susceptibility. There have been attempts to determine if smoking,

because of the problems it causes to the circulatory system, could increase susceptibility

to NIHL. Thus far, studies have indicated that smoking is not a predictor of susceptibility

(Pyykko, Pekkarinen & Starck, 1987; Starck, Toppila, & Pyykko, 1999). Preexisting

hereditary hearing loss has variable effects on the extent of NIHL. The size of temporary

threshold shift has also proven equivocal in predicting susceptibility to a NIHL (Borg,

Canlon & Engstrom, 1995; Ward, 1995).

Studies have been unable to prove a susceptibility or vulnerability link between

noise exposure and silicosis, leprosy, vitamin deficiencies, menstrual cycle or mental

attitude toward the noise (Axelsson, Borg & Hornstrand, 1983; Ivanstan, 1960; Parrot,

1984). This confirms what is already known in the field that there is not one factor that

increases susceptibility to hearing loss from noise exposure (Ward, 1995).

Damage to the Auditory System From Noise Exposure

The contributions of the external and middle ear systems to the impact of NIHL

should not be overlooked. The resonant frequency of the external auditory meatus

(EAM) is about 3,200 Hz (Henderson & Hamemik, 1995). Depending on the frequency

of the sound and direction of the source of the noise, the EAM can amplify sound by as

much as 20 dB as it travels to the tympanic membrane. The acoustic characteristics of

the EAM are primarily responsible for the appearance of the 4 kHz notches that is seen in









patients with NIHL (Henderson & Hamemik, 1995). Studies ofbasilar membrane

vibrations show that the point of maximum displacement occurs half to one octave above

the frequency of stimulation. Since most industrial noises are relatively broadband, the

EAM creates a band pass noise centered at 3 kHz; thus the place of maximal

displacement on the basilar membrane is in the 4 kHz regions. So the notch that is seen

on the audiogram is not the result of weakness in the 4kHz region of the cochlea but due

to the acoustic characteristics of the EAM (Henderson & Hamemik, 1995). Pierson,

Gerhardt, Rodriguez, and Yanke (1994) studied the relationship between the frequency of

peak outer ear resonance and the frequency of maximum hearing loss in patients

(comprised of 24 men) with a history of noise exposure. Analysis of the frequency of

maximum hearing loss was completed with sweep frequency Bekesy audiometry with a

resolution of 100 Hz. The outer ear resonant frequency was determined with a resolution

of 175 Hz. They found that the mean frequency of maximum hearing loss was 4,481 Hz

and the mean outer ear resonant frequency was 2814 Hz. The authors concluded that

although there were differences in the type and duration of noise exposure reported by the

subjects, the positive correlation between outer ear resonant frequency and frequency of

maximum hearing loss, emphasized the function that the properties of the external ear

plays in the development of the 4 kHz audiometric notch.

The middle ear serves as an impedance matcher and ensures the transmission of

sound (including the gain at the resonant frequency) to the inner ear. The acoustic reflex,

when activated, serves to reduce the intensity of sound transmitted. However, this is only

helpful for frequencies 2 kHz and below. Transmission of higher frequencies is in fact

enhanced the acoustic reflex and can create damage (Henderson & Hamernik, 1995).









In order to determine whether the acoustic properties of the outer and middle ears

influenced susceptibility to noise exposure, Dancer (1995) measured TTS in 40 cats, 84

chinchillas and 128 guinea pigs. Five to 10 animals were exposed to a 2, 4 or 8 kHz pure

tone for 20 minutes. The level of the pure tone ranged from 84 to 132 dB SPL in front of

the tympanic membrane. Compound action potential audiograms (1-32 kHz) were

obtained prior to exposure and 20 minutes post exposure. In each species, the maximum

TTS was toward the high frequencies when the stimulus level increased. The increase

toward the high frequencies was approximately from a 14 to two-octave jump when

compared to the exposure frequency. There was an increase in auditory susceptibility

from cats to guinea pigs and from guinea pigs to chinchillas. It was concluded that the

difference in susceptibility could only be attributed to conditions of transmission from the

peripheral auditory system.

Noise tends to affect the Organ of Corti, a delicate structure in the cochlea. The

cochlea houses the sensory part of the auditory system. Noise exposure will usually have

a greater impact on outer hair cells (OHC) than on inner hair cells (IHC). One of the

reasons is that the OHCs encounter a direct shearing force at their stereocilia (the top of

the OHC stereocilia are embedded in the "B" fibers of the tectorial membrane) as

opposed to the IHCs, which are stimulated by viscous drag (Henderson & Hamernik,

1995). Another reason is the long axes of the OHCs are unprotected from mechanical

stress, while the IHCs are supported on all surfaces with supporting cells. Lastly OHCs

are closer to the place of maximal displacement on the basilar membrane of the traveling

wave than the IHC (Henderson & Hamernik, 1995). Damage to the sensory epithelium

eventually leads to atrophy of the auditory nerve and higher auditory centers perhaps due









to a lack of stimulation (Strominger, Bohne, & Harding, 1995). Noise has the ability to

affect sensory cells, supporting cells, nerve fibers and the cochlea vascular supply

system. Noise can damage the auditory system in two ways, metabolic changes and

direct mechanical alterations.

Metabolic damage can involve the exhaustion of key enzymes or metabolites in

the cells during prolonged noise exposure. At low noise levels, damage can occur to all

the components of the sensory cell body (such as the mitochondria, the subsurface

cistemase and the smooth endoplasmic reticulum) and the nucleus. The changes include a

distortion or swelling of the cells, increase in osmiophilic (lysosomes) bodies, and

disruptions of membrane-attached structures such as the Henson bodies. In the OHC,

disarray, fusion or swelling of stereocilia can occur. As the length of time after the

exposure is increased, the damage to OHCs starts to show necrotic changes such as

expansion and eventual rupture of the smooth endoplasmic reticulum and destruction of

the nucleus. Changes to membranous structures can lead to ballooning of cell walls and

possible leaking of cell contents. This impairs the cell's capability to regulate its ionic

composition, which results in swelling and improper cell function (Bohne, 1976;

Henderson & Hamemik, 1995).

Robertson and Johnstone (1980) combined scanning electron microscopy (SEM)

and measurement of N1 audiograms for their investigation of the early effects of intense

tones on the guinea pig cochlea. Seventeen pigmented guinea pigs were exposed to pure

tones ranging from 96 to 129 dB SPL for one hour. N1 audiograms were used to measure

auditory threshold pre-exposure and within four minutes post-exposure. The first changes

to the cochlea observed using SEM was the collapse and/or fusion of the stereocilia of the









innermost row of outer hair cells (OHC1) at intensities of 106 dB SPL and higher. As the

intensity increased above 106 dB SPL the damage to the OHC1 became more acute and

extended over a greater length of the organ of Corti. At 117 dB the damage spread more

basally of the 10 kHz point than apically, but frequencies below 10 kHz eventually began

to show signs of damage.

At first, IHC damage was a mild separation and derangement of the upright and

regular bundle of the tallest stereocilia. As intensity increased the stereocilia flopped

outwards away from the modiolus. The most severe damage to the IHC was the loss of

the tall stereocilia and in some instances actual extrusion of the top of the IHC from the

reticular lamina. The N1 audiograms at the lowest intensity of 96 dB SPL showed slight

threshold shifts at 10 kHz (20 dB) but increased to a maximum loss of 55 dB at 15 and 18

kHz.

As noise levels increase, there is a point where mechanism for damage is no

longer metabolic but instead is mechanical. At high noise levels, the damage can include

fractures of the tight cell junctions of the Organ of Corti and separation of entire

segments of cells from their basilar membrane attachments. The cilia of the OHCs have

delicate filamentous connections to the tectorial membrane, which serves as a

disarticulation detector. These are the first to be damaged as a result of excessive

displacement due to over stimulation. Noise damage to the cilia includes fusing of the

cilia, splayed, disarrangement and floppiness (Borg, 1995; Henderson & Hamernik,

1995). The vascular system of the cochlea, namely the stria vascularis, has not been

implicated as a major player in NIHL. For moderate exposure levels, the general

impression is that stria vascularis is unaffected. However, as the intensity increases, noise









can cause lesions in stria vascularis. The levels needed to create lesions also create

mechanical damage to the cochlea, so as a whole, the stria vascularis does not appear to

be affected by noise levels that create metabolic damage (Henderson & Hamernik, 1995).

In order to explore the method of cell death after noise exposure, Hu, Guo, Wang,

Henderson and Jiang (2000) examined morphological changes in guinea pig cochlear hair

cell nuclei to help them decipher if hair cell death was apoptic (cell kills itself) or necrotic

(death result of injury). They felt that previous studies that have associated necrosis with

HC death from noise exposure should be reviewed. They indicated that with the increase

in knowledge about apoptosis it was worth looking for any possible indication the noise

exposed HC death could be related to apoptosis.

They stained the hair cell (HC) nuclei with Hoechst 33342, a fluorescent dye used

to label nuclear DNA. The animals were exposed to narrow band noise centered at 4 kHz

for four hours at levels of 110, 115 and 120 dB SPL. The cochleae were examined at

intervals of three hours; three and 14 days post exposure. They found that there were two

major types of nuclear changes. One was the nuclear condensation, which appeared as

karyorrhexis that has been associated with apoptosis, and the second was nuclear

swelling. Nuclear swelling was found in animals that had exposures of 110 and 115 dB,

while karyorrhexis and nuclear swelling was found in animals with exposure of 120 dB.

These findings have led the investigators to conclude that nuclear swelling and nuclear

condensation (karyorrhexis) originates from two distinct biological processes. They also

indicated that the karyorrhexis observed was similar to morphological changes described

from cell apoptosis. They concluded that at high noise levels, apoptosis plays a

significant role in HC death.









The neural effects of noise include an increase in the number of synaptic vesicles

in the efferent endings under the OHCs. Swelling has been observed at the dendritic

endings under the IHCs, which is indicative of high metabolic activity. As noise levels

increase and the loss of OHCs occur, severe swelling and eventual loss of dendritic

endings can take place. Noise that is intense enough to cause IHC loss will lead to

degeneration of VIII nerve fibers (Henderson & Hamemik, 1995). In order to observe

damage to the afferent neural system after noise exposure, Omata and Schatzle (1980)

exposed rabbits to 100 dB SPL for two hours with pure tones and studied changes in the

afferent and efferent nerve terminals. They found that about 50% of the animals showed

abnormalities and changes in the postsynaptic cisterna and in most animals the synaptic

vesicles in the efferent terminals disappeared.

According to Spoendlin (1976) there is no simple relationship between intensity

levels and the extent of structural damage. For example, he found that a five-minute

exposure of 120 dB of continuous noise produced slight structural changes that were

more metabolic in nature. One week of 110 dB of continuous noise produced barely

detectable changes. He found that at exposure levels of 90-130 dB, metabolic damage

was greatest with minimal mechanical damage. He also found that damage associated

with exposures less than 130 dB mainly affected OHCs. He found that at intensities

above 130 dB there was always major structural damage (mechanical). His findings may

suggest that the damage occurring immediately after exposure was probably mechanical

but later manifestations of the insult may be metabolic in nature.

Temporary Threshold Shift

Initial exposure to high-intensity noise creates what is called a temporary

threshold shift (TTS). Common complaints associated with TTS include tinnitus,









decreased sensitivity and aural stuffiness/fullness. The structural changes associated with

TTS can include small intracellular changes in the sensory (hair) cells and enlargement of

the auditory nerve. Other changes include vascular changes, metabolic exhaustion and

chemical changes within the cochlea (National Institute of Health, 1991).

Gao, Ding, Zhen, and Ruan (1991) observed the changes in the stereocilia with

the scanning electron microscope (SEM) at different phases after exposure to high-level

impulse noise, and investigated the correlation between these changes and the temporary

threshold recovery pattern. They exposed 38 albino guinea pigs to 165 dBSPL of impulse

noise. All the animals suffered a 20-60 dB permanent decrease in hearing sensitivity.

The cochlea observed at .5-hour post exposure showed disorders in the arrangement of

stereocilia such as bent, loose, askew and disarrayed hair bundles. At four hours post

exposure, hair bundles became more severed and partial fusion occurred at the tips of the

stereocilia in certain parts of the cochlea. At eight hours post exposure, complete fusion

throughout the length of the stereocilia had occurred. The area, in which all three rows of

the outer hair cells stereocilia fused, extended from the basal to the second turn of the

cochlea. Many outer hair cells were also missing. Eight hours after exposure, most

exposed animals showed a slow recovery of temporary threshold shift; however, no

improvement in structural changes were found in the stereocilia with the SEM.

The magnitude of TTS often decreases during the first minute after exposure,

increases to its maximum amount during the next 2-3 minutes and then gradually

decreases. In order to avoid the early fluctuation, TTS is usually measured about 2

minutes after the exposure. The frequencies affected depend on the spectrum of the

stimulus. If the stimulus is a moderate level, pure tone or narrow-band noise, the TTS









will usually be restricted to a narrow range of frequencies near the exposure frequency.

At higher exposure levels, the TTS spreads predominantly toward the high frequencies

and the maximum hearing loss typically shifts to a point that is one-half octave above the

center frequency of the stimulus. Broadband noise with approximately equal energy at

all frequencies usually will affect the 3-5 kHz range (Quaranta, Portalatini & Henderson,

1998).

During the course of the noise exposure, the magnitude of the TTS grows over the

first eight hours of exposure. It reaches its maximum point of growth and then stops even

if the exposure continues for 18-24 hours. The point where there is no further growth in

TTS is called asymptotic threshold shift. If the TTS is approximately 25-40 dB and the

exposure duration is less than 8 hours, then threshold shift decreases approximately

linearly in log time and usually recovers to its pre-exposure level in 16 to 24 hours.

When the exposure lasts more than a few days and the threshold shift is larger than 40 dB

then the recovery is generally slow, especially during the first 12 hours and may last a

few weeks (Quaranta et al., 1998).

When monitoring for TTS, consideration should be given to the delayed or

rebound TTS observed in the following study. Dancer, Grateau, Cabanis, Vaillant &

Lafont (1991) looked at the issue of delayed temporary threshold shift induced by

impulse noise in soldiers. Three groups of soldiers wearing no hearing protection were

exposed to rifle discharge in a free field over a two-day period. Audiometric evaluations

were conducted at 5 minutes; 1 hour and 4 hours post exposure. In the majority of the

cases, the TTS pattern was consistent with conventional recovery patterns. In 29% of the

TTS cases a delayed TTS was observed either 1 or 4 hours post exposure. The authors









concluded that under the customary procedure of measuring TTS at 2 minutes post

exposure, instances of delayed TTS may be missed and thus resulting in an under

estimate of the acoustic hazard.

Permanent Threshold Shift

According to Quaranta et al (1998), temporary threshold shift that remains for

more than four weeks past the exposure is considered to be permanent. Permanent

threshold shift (PTS) tends to begin at the 3-6 kHz frequency regions. The rate of growth

of hearing loss decreases over 20 years of exposure. The rate of growth is greatest in the

first 10 years. For impact noise, Henderson and Hamernik (1995) found that the PTS is

related to the level for intensities above 125 dB SPL. Below that level, the hearing loss is

proportional to the total acoustic energy. An explanation for this finding is that high-

level impulse noise can mechanically damage the cochlea, while below the critical level

the damage is due to metabolic factors.

Rosler (as cited in Quaranta et al, 1998) found that the effects of chronic noise

exposure were more evident in subjects younger than 30 years of age. Beyond the age of

50, permanent threshold shift created by noise does not grow significantly; however, the

effects of aging continue to create hearing loss.

Synergistic Effects of Noise and Drugs

There are several agents that have the capability to produce temporary and

permanent damage to the auditory system. When these agents are combined they cause

more damage than would be predicted that the agents would produce separately. The

agents include noise, equipment vibration and drugs. At sufficient levels all of these

agents (with the exception of vibration) are capable of producing damage to the inner ear,









but when combined with noise cause more damage than the agents would be predicted to

produce separately (Boettcher et al., 1987).

Industrial noise is usually a combination of two types of noise, impulse and

continuous. Hamernik, Henderson, Crossley and Salvi (1974) exposed groups of

chinchillas to impulse and continuous noise separately and in combination. They found

that the combination exposure caused more hearing loss than the separate exposure.

Researchers have also found the relative distribution of energy and temporal relationship

in the impulse and background noise is critical to the interaction process (Boettcher et al

1987).

An individual exposed to hand or whole body vibrations are at risk for an increase

in severity of NIHL. The most common injury from whole-body vibration is back pain.

Hand-arm vibration syndrome can lead to nerve damage in the fingers and can include

symptoms such as finger blanching (white finger disease or Raynaud's phenomenon) and

numbness (Siedel, 1993). Vibration alone is not enough to create hearing loss; however,

noise and vibration has been noted to increase threshold shifts of workers exposed to

both. It has been reported that forestry workers with occupational induced white-finger

disease have greater degrees of hearing loss than their non-white finger co-workers (Iki,

Kurumantani, Hirata, & Moriyama, 1985; Miyakita, Miura, & Futatsuka, 1987). Animal

studies confirm that the combination of vibration and noise created more hearing loss

than each exposure separately (Hamerink, Ahroon, Davis & Axelsson, 1989). There

could be countless more agents that have a synergistic effect with noise but at this

juncture there have been no studies to make that clear determination (Boettcher et al,

1987).









Ototoxic Drugs

The five classifications of ototoxic drugs include aminoglycoside antibiotics, anti-

neoplastic agents, loop-inhibiting diuretics, salicylates and quinine compounds (quinine

will be discussed in more detail in the quinine and chloroquine sections).

Aminoglycosides are antibacterial drugs such as streptomycin, kanamycin, gentamicin

and neomycin (to name a few) used to treat life-threatening infections caused by bacterial

agents such as Escherichia. coli, Proteus, Enterobacter aerogenes and Klebsiella

pneumoniae. Hearing loss caused by aminoglycosides results from damage to the outer

and inner hair cells, and the stria vascularis. It is believed that the mechanism for

ototoxicity is the disruption of the lipid portion of the hair cell membrane that normally

occurs after the administration of the drug (Boettcher et al., 1987). Physiological and

histological data show that NIHL and hair cell losses are increased by simultaneous

administration of gentamicin, kanamycin and neomycin (Ryan & Bone; 1982; Jauhianien,

Kohoren & Jauhianien, 1972). Ryan and Bone (1982) found that noise exposure

followed by the administration of kanamycin caused more histological damage than the

noise or kanamycin alone. The same increase in ototoxicity was observed for the

combination of noise and neomycin (Brown, Brummett, Meikle, & Vernon, 1978).

Anti-neoplastic drugs such as cisplatin are used in the treatment of cancers of the

head, neck and urogenital systems. The mechanism of ototoxicity starts in the stria

vascularis and then progresses to the hair cells. Audiometric damage does not usually

become apparent until the cumulative dosage of 200 mg is exceeded (Boettcher et al.,

1987). Gratton, Salvi, Kamen and Saunders (1990) studied the effects on noise and

cisplatin on chinchillas. One group was exposed to cisplatin alone; another exposed to

noise alone and finally one group was exposed to combined effects of noise and cisplatin.









In the cisplatin only group, the hearing loss was minimal but in the combination group

the hearing loss was significantly greater, thus leading to the conclusionthat cisplatin

increased the severity of the hearing loss.

Loop-inhibiting diuretics are used to treat conditions such as pulmonary edema,

renal edema and hepatic cirrhosis. It affects the auditory system by causing edema of the

marginal cells and pooling of erythrocytes in the vessels of the stria vascularis. The

effects on hearing are probably due to the interference of the sodium-potassium pump in

the marginal cells of the stria vascularis. It does not cause damage to the hair cells

directly. The hearing loss created by loop-inhibiting diuretics is temporary in nature as

opposed to the previously mentioned drugs.

Salicylates are analgesics and anti-inflammatory agents. The most common form

of salicylates is aspirin and heat rubs. The administration of more than 2.5 grams of

aspirin per day is sufficient to cause tinnitus and temporary hearing loss. The mechanism

for ototoxicity is unclear but some have proposed that the drug alters the function of the

mitochondria of cells of the stria vascularis (Covell, 1936). Other theories include the

inhibition of prostaglandin synthesis (which is part of the analgesic effect of salicylates)

that may lead to decreased sodium and potassium ATPase activity, thus changing the

ionic balances of the cochlear fluid (Miller, 1985). The additive potential of salicylates is

inconclusive; the general feeling is that salicylates do not have a synergistic effect on

noise induced hearing loss (Woodford, Henderson & Hamernik, 1978; Bancroft,

Boettcher, Salvi & Wu, 1991).









Reactive Oxygenated Species

During normal cellular metabolism, radicals are produced when molecular oxygen

is converted to water. Radicals are molecules that have an open bond or a half bond and

are highly reactive (Jaeschke, 1995). In order to convert oxygen into water, four

electrons must be added to the oxygen molecule. About 95% of the time, this activity is

done with the addition of all four electrons at the same time. About 5% of the time, the

oxygen is converted into water by adding one electron at a time.

Normally, one pair of electrons occupies every orbital encircling the nucleus of an

atom. If an orbital in the outer shell of a molecule loses an electron, the molecule

becomes a free radical. The unpaired electron makes the molecule unstable and volatile.

This free radical starts to react in a nonspecific manner with any near-by molecule. They

achieve this by pulling necessary electrons from whatever sources available such as

cellular molecules found in lipids, proteins, nucleic acids and carbohydrates (Jaeschke,

1995). The free radical's goal is to become paired at any cost. The free radical reacts

with other molecules creating more free radicals, which can create an almost endless

chain reaction. The chain reaction is stopped by either the free radical bonding to another

free radical or by reacting with antioxidants or an antioxidant enzyme or both. The role of

antioxidants will be discussed later on in this section.

As indicated previously, in 5% of the cases, oxygen is converted into water

through the addition of one electron at a time. In these cases where the oxygen molecule

is added one at a time, unstable intermediates called reactive oxygen free radicals or

reactive oxygen species (ROS) are formed. The most common reactive oxygen species

formed are the oxygen radical superoxide (2-), hydroxyl radical (OH) and the non-

radical hydrogen peroxide (H202). These radicals have useful functions especially as part









of the immune system but become a problem when they are produced in excess amounts

(Barber & Harris, 1994; Halliwell, 1992; Jaeschke, 1995). Under normal circumstances

free radicals are useful in the fight against invading microorganisms. When there is an

invading microorganism present in the body, large amounts of oxygen free radicals are

produced by various phagocyctic cells in response to the invading microorganism (Barber

& Harris, 1994; Halliwell, 1992; Jaeschke, 1995). However in the case of inflammatory

disease such as rheumatoid arthritis, phagocyticaly produced free radicals are produced in

excess that tend to initiate a self-propagating cycle that only serves to aggravate the

inflammatory process.

The first free radical formed (during the single addition of an electron at a time

process) is the superoxide anion. The next radical formed by the addition of another

electron is hydrogen peroxide, which is parried but has such strong oxidation ability that

it is grouped with the free radicals. The next radical formed is the hydroxyl radical which

is extremely destructive. Free radicals are capable of severely damaging cells by

oxidizing proteins and deoxyribonucleic acid (DNA).

Free radicals have been implicated as playing a major role in several biological

processes such as aging, bacterial infection, tissue ischemia, cancer, rheumatoid arthritis,

cardiovascular disease and tissue reoxygenation (Barber & Harris, 1994; Ikeda, Sunose &

Takasaka, 1993). ROS causes damage by reducing circulation and oxygen flow in tissue.

The amount of ROS in living tissue is determined by the symmetry between the

production of ROS and the tissue's ability to produce antioxidants.

Antioxidants exist in two forms, one as an enzyme, and the other as vitamins.

The antioxidant enzymes are superoxide dismutase catalase and gluthatione peroxidase.









Superoxide dismutase exists in two forms, one as copper/zinc superoxide dismutase and

is found in the cell cytoplasm. The second is in the form of manganese superoxide

dismutase, which is found in the mitochondria. Catalase exists in one form but is found

throughout the body. Glutathione peroxidase exists in the blood or in cell membranes.

The numerous forms and locations of these enzymes provide protection wherever damage

from free radicals occurs. Vitamins A, E, C and reduced gluthatione are called intrinsic

antioxidants and exist throughout the body. The release of antioxidant enzymes can be

influenced based on the amount of free radical activity (Jaeschke, 1995; Barber & Harris,

1994).

Antioxidants can act to stop or change the oxidative process in several of the

following ways. They can decrease oxygen concentrations, remove catalytic metal ions,

scavenge initiating radicals, remove key reactive oxygenated species such as O2-, H202,

break the chain of initiated sequence or by scavenging singlet oxygen (Gutteridge, 1994).

Antioxidants that reduce lipid peroxidation by decreasing oxygen concentrations,

removing catalytic metal ions, scavenging initiating radicals and quenching singlet

oxygen are called preventative antioxidants. The antioxidant used for this study would

fall in the category of a preventative antioxidant.

Antioxidants that remove key ROS are preventive as well but if they are enzymes

(catalase, SOD and glutathione peroxidase) they are not consumed by the reaction. Chain

breaking antioxidants (singlet oxygen quenchers and metal chelators) will be consumed

in the process of conducting their protective roles. In addition to the functions listed

previously, antioxidant can aid the cell by repairing oxidative damage, increasing the rate

of elimination of damaged molecules and providing non-repair recognition of extremely









damaged molecules in order to prevent cell mutations from occurring (Gutteridge, 1994;

Jaeschke, 1995; Barber & Harris, 1994).

Adenosine is produced during normal metabolic activity and it works by

activating adenosine receptors. Activation of the adenosine receptors leads to the release

of antioxidants. Between the intrinsic antioxidants and those created by adenosine, the

cell has the ability to fight the negative effects of ROS. The amount of adenosine

available increases whenever the tissue is experiencing ischemia or poor circulation

(Ramakumar, Nie, Rybak & Magginwar, 1995).

Animal studies have found that levels of ROS were increased considerably in the

cochlea after noise damage. The assumption is that this condition leads to hair cell

damage and changes in the cochlea microcirculation (Lui, 1992 Yamane, Nakai,

Takayama, Konishi, Iguchi, Nakagawa, Shibata, Kato, Sunami & Kawakatsu, 1995).

The theory behind recent reports of drug therapy for NIHL is that if there is a way to

increase the activation of adenosine receptors, thus increasing the release of antioxidants,

then that would lead to an increase in cellular protection against ROS. R-

phenylisopropyladenosine (R-PIA) is an adenosine agonist that increases the release of

antioxidants. In fact, R-PIA causes a two to three time increase in the release of

antioxidants superoxide dismutase, cattalos, glutathione peroxidase and glutathione

reductase. There is evidence that drugs such as R-PIA and other oxidation inhibitors

decrease threshold shift associated with noise exposure.

Quirk, et al (1994) studied whether or not a lipid peroxidation inhibitor

(U74389F) could decrease temporary threshold shift in male Wistar-Kyoto rats. The

U74389F drug is from a class of drugs called Lazaroids. These drugs work in the









following manner; they inhibit lipid peroxidation, they scavenge free radicals and they

help the cell maintain vitamin E levels even in poor circulation conditions. The rats were

exposed to continuous wideband noise at 90 dB SPL for 60 hours. The experimental

group of rats received the U74389F at 24 and 12 hours prior to noise exposure and every

12 hours until noise exposure was completed. The drug was injected into the rats so the

entire body of the rat was exposed to the U74389F. The threshold shift for the drug-

treated group was less than the non-treated noise exposed animals. It appeared that the

lipid perioxidation inhibitor had a protective effect against the noise.

Hu, Zheng, McFadden, Kopke and Henderson (1997) stated that in order to

achieve the best results from drug therapy it is best to have the concentrated effects of the

drug in the cochlea. In their study they used R-PIA on chinchillas. The R-PIA was

applied to the round window of the right ear and saline was applied to the left ear thus

allowing the animal to be used as its own control. The animals were then exposed to

octave-band noise centered at 4 kHz at 105 dB SPL for 4 hours. Recovery from noise-

induced damage over a 20-day period was more complete in the R-PIA treated ears than

in the control ears for frequencies between 4 and 16 kHz. The most important finding

was that the permanent threshold shift measured after the 20-day period was 10-15 dB

better in the R-PIA treated ears than in the saline treated ears. Hight, Henderson,

McFadden and Zheng (1999,) found that using another antioxidant, glutathione

monoethyl ester, applied to the round window, decreased hair cell loss and attenuated

hearing loss from impulse noise. Ohinata, Yamasoba, Schact and Miller (1999) found

that interperitoneal injection of the antioxidant gluthathione attenuated noise-induced

cochlea damage.









Clerici, Hensley, DiMartino and Butterfield (1996) found that ototoxic drugs

produced reactive oxygen species in cochlear explants. They studied the ability of

ototoxic agents such as gentamicin sulfate, kanamycin, cisplatin, trimethyltin,

furosemide, ethacrynic acid and quinine to produce ROS in cochlea tissue. They found

that all the drugs were capable of producing reactive oxygen species. The next question

is whether antioxidants can provide protection to the cochlea against ototoxic drugs.

Kopke, Liu and Gabaizadeh (1997) found that antioxidants have a protective effect

against cisplatin ototoxic effects on cochlea hair cells in vitro.


Malaria

Malaria (the disease treated with chloroquine) also appears responsive to

treatments that include antioxidants. Goldring and Ramoshebi (1999) found that

antioxidants and anti-inflammatory drugs reduced the adherence between monocytes and

malaria infected erythrocytes. Monocytes play an integral role in immune response

during infections with parasites such as orchestrating cytokine secretions and cell-to-cell

recognition. Their study was undertaken in order to observe the role monocyte adhesion

molecules might have in malaria infections. The adherence of malaria-parasitised red

cells to monocytes was determined after the monocytes were incubated for 24 hours in

the presence of glucocorticoids (dexamethasone and cortisol), antioxidants (probucol and

ambroxol) and danazol and staurosporine. At the 24-hour time point, all the drugs had

reduced the adherence of parasitised red cells in a dose-dependent manner.

In the same vein, Siddiqui, Puri, Dutta, Maheshwari and Pandey (1999) wanted to

determine if the use of chloroquine and poly ICLC (double-stranded RNA which is a

known producer of cytokines proven to restrict parasite multiplication during malaria









infection) had an effect on antioxidant defense system of mice infected with Plasmodium

yoelii nigeriensis (P.y. nigeriensis). The animals were divided into the following four

groups: control, chloroquine alone, poly ICLC alone, and chloroquine and poly ICLC.

The experimental animals were injected i.p. with P.y. nigeriensis infected erythrocytes.

The drugs were administered to the animals from day two to day five after the infection.

The chloroquine treated group had a decrease in blood parasitaemia and oxidative stress,

the poly ICLC group had no change in the level of blood parasitaemia or oxidative stress

but an increase in antioxidant indexes and the combination of chloroquine and poly ICLC

resulted in clearance of blood parasitaemia and the return of oxidative stress and

antioxidant indexes to normal levels. It was concluded that the poly ICLC acted as an

antioxidant and when combined with chloroquine worked the best of the three conditions.

It appeared that the performance of chloroquine in the presence of antioxidants was not

affected.

The outlook for the control of malaria is dismal. The disease, which is caused by

mosquito borne parasites, is present in 102 countries and is responsible for over 100

million clinical cases and 1 to 2 million deaths annually. Over the last few decades,

efforts to eradicate malaria have not been successful. For instance, in areas where malaria

transmission had been eliminated, the disease has made a surprising come back (Pan

American Health Organization, 1984; World Health Organization, 2000). Malaria is

primarily a disease of the tropics, but can be found in many temperate regions of the

world including parts of the Middle East and Asia. Malaria in humans is caused by four

species of a protozoan parasite of the genus Plasmodium. The four species are P.

falciparum, P. vivax, P. ovale and P. malariae. P. vivax is responsible for most of the









malaria infections globally. However, the most severe form of malaria is caused by P.

falciparum. P. vivax is the most common variety observed in temperate regions of the

world (Oaks, Mitchell, Pearson & Carpenter, 1991). The severity of malaria depends

more on the immunological status of the person infected than on the strain of the disease.

It is important to note that there is a difference between active malaria disease and

malaria infection. Many infected people who live in areas where the disease is endemic

have no symptoms. However these individuals (who are reservoirs) without symptoms

are the major contributors to the transmission of the disease. In the case of active disease,

the individual will have the symptoms of malaria (Ho & White, 1999).

The clinical manifestations of malaria vary from person to person. The general

description of an individual with malaria is periodic shaking chills through severe fevers,

to drenching sweats. In some cases, malaria-infected individuals may have symptoms

that mimic other diseases, which can lead to a misdiagnosis. As stated earlier, the most

severe form of the disease is P. falciparum, which is considered a medical emergency and

is characterized by fever, chills, headache, anemia and splendomegaly. These symptoms

generally respond to appropriate antimalarial drugs; however, if left untreated patients

can develop serious complications. The complications can be manifested as coma

(cerebral malaria), metabolic acidosis, hypoglycemia, and severe anemia and in adult's

renal failure and pulmonary edema (Ho & White, 1999). The overall mortality from

severe malaria is between 15 to 30%. The highest mortality rate results from cerebral

malaria, metabolic acidosis and pulmonary edema. Malaria during pregnancy can cause

miscarriages, fetal death, intrauterine growth retardation, low birth weight and premature

delivery.









The pathogenicity of P. falciparum occurs because of its ability to place high

parasite burden and the unique ability to adhere to capillary and post capillary venular

endothelium during the second half of the 48-hour life cycle. The ability to adhere to the

capillary venular endothelium is called cytoadherence (Ho & White, 1999. The

adherence leads to alterations in microculatory blood flow and metabolic dysfunction that

eventually leads to the symptoms of malaria. Oxidative stress is associated with acute

malaria; it is believed that the parasite in its developmental stages in the erythrocytes

produce reactive oxygenated species (Golenser, Peled-Kamar, Schwartz, Friedman,

Groner, & Pollack, 1998; Mishra, Kabilan, & Sharma, 1994).

There are approximately 2,500 known species of mosquitoes worldwide, but only

a group of 50-60 species in the genus Anopheles are capable of transmitting malaria.

Female anophelines require blood meals to reproduce. The life cycle of the malaria

parasite has three phases in the mosquito and two in the human host. The parasite is

transmitted to humans by the sporozoite that forms in the saliva of infected female

anophelines. After entering the human host, the sporozoites invade liver cells where,

during the next five to 15 days, daughter parasites called merozoites are released and

invade the red blood cells. Once inside the red blood cells, each merozoite matures into a

schizont containing 8 to 32 new merozoites. The red blood cell eventually ruptures and

releases the merozoites, which are then free to invade additional red blood cells. The

rupturing of the blood cells is associated with the fever and clinical onset of malaria

(Oaks et al, 1991; Pan American Health Organization, 1984, World Health Organization,

2000).









Merozoites separate into two sexual forms. A mosquito ingests the gametocytes,

which are in the blood cells, during its next blood meal from the human host. Once in the

mosquito, the sexual forms leave the blood cells and male and female gametes fuse to

form a zygote. Over the next 12 to 48 hours, the zygote becomes an ookinete. The

ookinete penetrates the wall of the mosquito's stomach and becomes an oocyst. During

the next three weeks the oocyst enlarges and forms more than 10,000 sporozoites. When

the oocyst ruptures, the sporozoites migrate to the mosquito salivary glands where it can

be injected into the human host. Malarial illness may recur months to years after

successful treatment. In patients with P. vivax and P. ovale this is called relapse. The

dormant liver-stage forms of the parasites that have resumed their developmental cycle

and release merozoites into the bloodstream are the cause of relapse (Oaks et al, 1991;

Pan American World Health Organization, 2000, World Health Organization, 1984).

Malaria control efforts through the years have relied on anti-malarial drugs,

environmental sanitation, and the use of pesticides (Oaks, et al., 1991). Anti-malarial

drugs have been used to prevent the onset of the disease, treat the disease and prevent

disease transmission within populations. Drugs such as doxycycline, proguanil,

pyrimethamine, and primaquine attack the liver state of malaria by preventing the release

of parasites into the bloodstream. Other drugs such as chloroquine, quinine, sulfaoxine-

pyrimethamine and mefloquine kill the parasite within the red blood cells. The problem

with drug use is that there has been noted resistance to chloroquine of the P. falciparum

and P. vivax parasite. This phenomenon of drug resistance is starting to affect more of the

anti-malarial drugs (World Health Organization, 2000).









Environmental control methods such as eliminating the larval development sites

by draining standing water or the use of pesticides are helpful. However, mosquitoes

have been showing resistance to most pesticides. Protective clothing, insect repellent,

mosquito coils and bed nets are all methods of reducing the contact between humans and

mosquitoes. The use of pyrethrin impregnated bed nets have been shown to reduce up to

50% of clinical malaria in some areas. However, patient non-compliance with

environmental control methods continue to be the biggest contribution to the failure of

eradication efforts, thus making medical methods an integral component of malaria

control (Oaks et al, 1991; Pan American Health Organization, 1984, World Health

Organization, 2000).

Quinine

Cinchona, which is derived from the bark of the Chinchona tree, has been used

since the 16th century in South America, for the treatment of fevers. These fevers were

probably associated with malaria. However, the malaria was not known at that time.

Quinine is derived from cinchona and is an anti-protozoal drug that has been used in the

treatment of malaria. Quinine's ototoxic effects can include vertigo, tinnitus, and

deafness. Additional side effects include headaches, blurred vision, vasodilation,

seizures, acute hemolysis (leading to black water fever and renal failure) and coma. In

most cases, the visual and auditory effects are temporary and are linked to prolonged

dose levels of 200 to 300 mg. However, at higher doses and prolonged use, quinine can

cause permanent hearing loss. Congenital deafness and hyperplasia of the cochlea

(excessive amounts of normal cells in the normal tissue arrangement of the organ) have

been observed in children whose mothers took quinine during their pregnancies (Miller,

1985). In the study where quinine-dihydrochloride was administered to Caucasians (12









healthy and 10 with malaria), nine of the healthy and all of the malaria subjects

developed temporary hearing loss. In cases when hearing loss was developed the plasma

concentrations of the drug was only 2 mg/1 (Classen, van Boxtel, Perenboom, Tange,

Wetsteijn & Kager, 1998).

Ruedi, Furrer, Luthy, Nager, and Tschirren (1952) found that prolonged doses of

50 to 100 mg/kg quinine for periods of 35 to 522 days causes degenerative changes in the

Organ of Corti, cochlear neurons and stria vascularis with more pronounced damage in

the basal portion. The damage ranged from complete loss of outer hair cells to destruction

of supporting structures. The stria vascularis was atrophic with a decrease in cellular

tissue and the formation of large vascular spaces. Although the auditory and visual side

effects were temporary, an alternative to quinine was needed because malaria had

become resistant to it, and because of quinine's life-threatening side effects (Classen,

Boxtel, Perenboom, Tange, Wetseijn & Kager, 1998).

Chloroquine

Chloroquine was developed in 1946 as part of the search for an alternative to

quinine. Chloroquine is structurally similar to quinine. It is also used in the treatment of

rheumatoid arthritis, chronic discoid lupus erythematosus and dermatological conditions

caused by or irritated by light (Sykes, 1984; Adelusi & Salako, 1982). Chloroquine is

well known as a fast acting inhibitor of nucleic acid and protein synthesis in susceptible

strains of the malaria parasite. It is thought that chloroquine acts on the parasite's

digestive pathway for haemoglobin (Krishna and White, 1996). It has also been

suggested that chloroquine forms molecular complexes with DNAs that inhibit DNA

dependent nucleic acid polymerase reactions. It is believed that the drug binds to the

plasmodial DNA and inhibits DNA synthesis in malaria schizonts (Kinglhardt, (1978).









Chloroquine is absorbed through the gastrointestinal tract and reaches high

plasma concentrations quickly. It is well absorbed whether it is given orally

bioavailabilityy is about 80%), subcutaneously, intramuscularly and even rectally

(Krishna and White, 1996). Very little of the drug is excreted; consequently it stays in

tissue for a considerable amount of time (Sheffield & Turner, 1971). In fact in human

subjects given subcutaneous administration of chloroquine the chloroquine reached its

peak plasma concentrations levels on an average of 30 minutes after injection (Phillips,

Warrell, Edwards, Galagedera, Theakston, Abeysekera, & Dissanayaka, 1986).

Gustafsson, Walker, Alvan, Beermann, Estevez, Gleisner, Lindstrom and Sjoquist

(1983) gave 300 mg single doses of chloroquine in three forms, i.v. infusion, an oral

solution and tablets at intervals of at least 56 days to 11 healthy Caucasian males.

Chloroquine was detectable in plasma up to 23 days after administration and in some

cases as long as 52 days. The terminal half-life of chloroquine in the plasma ranged from

146 to 333 hours. It took from fifteen minutes for the i.v. method to obtain peak plasma

concentrations, while it took one and a half to six hours to reach peak plasma

concentrations for the oral solution. The same study was conducted on healthy Nigerian

subjects (Walker, Salako, Alvan, Ericsson, & Sjoquist (1987) with similar results. The

terminal half-life was 144 to 298 hours. Wetsteyn, DeVries, Oosterhuis and Van Boxtel

(1995) studied the pharmacokinetics of chloroquine after giving one of three multiple

dose regiments for three weeks. One group of the adult subjects was given once a week a

300mg/kg dose, another group was given two times a week a 200mg dose and the third

group was given a 50mg dose daily. After the first week all three-dose levels reached









peak and trough concentration levels sufficient (16[tg 11) to suppress malaria. They

found that the elimination of half-life ranged from 144 to 479 hours.

Auto radiographic observations of chloroquine in rats (adults and fetus) found that

chloroquine left the blood quickly and was localized in melanized tissue such as the

kidney, liver, lung, stomach, salivary glands, eyes, Harder's gland, skin and hair follicles.

It was also noted that the chloroquine crossed the placenta and accumulated in the fetal

eye (Lindquist, 1973). Dencker and Lindquist (1975) conducted autoradiography of the

inner ear after the injection of intravenous injection of chloroquine (10 micro curies)

tagged with carbon 14 in hooded Lister rats. There was a strong accumulation of

chloroquine in the stria vascularis 48 hours after the injection. The high concentration

remained up to 13 days. There was no buildup found in the endolymph or perilymph.

They hypothesized that the buildup and long retention of chloroquine in the stria

vascularis may lead to vascular injuries, which could eventually compromise the

composition of the endolymph possibly leading to metabolic malfunction. The stria

vascularis helps to maintain the ionic composition of the endolymphatic fluid and an

imbalance in endolymph could seriously alter the sensitivity of the inner ear (Gerhardt,

Ma, Rybak, & Rarey, 1998).

Chloroquine has also been implicated as a cause of permanent hearing loss

(Physician's Desk Reference, 1999, Dewar & Mann, 1954; Dwivedi & Mehra, 1978;

Hadi, Nuwayhid & Hasbinin, 1996). However, the literature is mixed on the ototoxic

effects of this drug. The lack of documentation of chloroquine's ototoxicity may be due

to the fact that chloroquine's toxicity is usually visual first, so the monitoring of patients









on chloroquine is usually an ophthalmological examination while hearing is rarely

monitored (Bernard, 1985).

Studies of cochlear outer hair cells in chloroquine-treated and non-treated animals

found no significant differences. The work by Sykes (1984) investigated whether

treatment with chloroquine causes permanent damage to the sensory hair cells of the

spiral organ in the guinea pig. He did not measure hearing but rather studied hair cell

damage. Albino and pigmented animals were given seven daily subcutaneous injections

of either 80mg/kg or 60mg/kg of chloroquine. The animals were killed 21 days after the

last injection. Neither treatment group showed significantly greater hair cell loss than the

control group. The author compared damage from chloroquine to aminoglycosides and

indicated that chloroquine caused minimal outer hair cell loss. He noted that doses

greater than 80mg/kg might cause more hair cell loss. However at these doses,

chloroquine did not create significant outer hair cell loss or obvious damage to the stria

vascularis.

In that same vein, Barrenas (1997) studied the relationship between hair cell loss

and pigmentation. Albino, red and black guinea pigs were divided into controls and

chloroquine-treated groups. Four days prior to the start of noise exposure, the treatment

animals were given a single injection into the jugular vein of 2 or 20-mg/kg chloroquine

diphosphate. The animals were then exposed to 105 dB SPL of 1 kHz-filtered noise for

72 hours. The author found that the mean total OHC loss for the control animals was

781(s.d.322), for the low dose chloroquine 895(s.d. 361) and for the high dose

650(s.d.376). It appeared that chloroquine did not have a synergistic effect with noise

exposure. The author, in an attempt to explain the lack of ototoxicity, indicated that only









a single dose was given, whereas treatment with chloroquine in humans can continue for

weeks. The lack of ototoxicity may be related to the fact that melanin-binding drugs

accumulate in the melanized tissues and once overloaded, the melanin molecule releases

the toxic drug into the system.

Barrenas and Holgers (2000) exposed red, black and albino guinea pigs to noise

(1 kHz, 105 dB SPL for 72 hours) after giving them a 2, 20 mg/kg or no intravenous dose

of chloroquine. They found that the high dose of chloroquine significantly affected the

amplitude of the DPOAE more than the low dose or control group. These results indicate

the possibility of synergism between noise and chloroquine because the higher dose

created more change in amplitude. In a majority of the early animal studies of

chloroquine, no attempt was made to determine auditory thresholds, thus in animal

models it is still undetermined whether or not chloroquine causes hearing loss. However,

in the human model there are several reports of chloroquine ototoxicity.

Matz and Naunton (1968) report that chloroquine phosphate can be ototoxic to the

fetal cochlea. A 7-year-old boy with profound bilateral sensorineural hearing loss

(SNHL) was one of three affected children whose mother took chloroquine phosphate

during the first trimester of pregnancy. She was taking chloroquine for the treatment of

systemic lupus erythematosus. The boy was the first child, the second child also deaf with

a Wilm's tumor (cancerous tumor of the kidney that generally occurs in children), and the

third pregnancy ended in miscarriage. Three subsequent pregnancies without

chloroquine resulted in normal children with normal hearing sensitivity. Sataloff &

Vassallo (1970) reported on a former Peace Corp worker who developed a precipitous

high-frequency hearing loss with ringing tinnitus after taking antimalarial drugs









containing chloroquine phosphate. Hadi, Nuwayhid, and Hasbini (1996) report on a 2-1/2

year old boy who was given a single intramuscular dose of chloroquine for the treatment

of malaria. The boy's parents noticed that he had an unsteady gait within a few hours of

the injection and by the second day he was deaf. Prior to this incident there had been no

concern about the child's auditory status.

Mukherjee (1979) reported on a case of a six-year-old girl diagnosed with malaria

and treated with seven daily injections of chloroquine phosphate. She complained of

deafness and dizziness on the fourth day of injection but that was thought to be weakness

from the disease and not the medication and so the injections continued until the seventh

day. Audiometric evaluation after the completion of the injections revealed that she had

a moderately severe sensorineural hearing loss in both ears.

Dwivedi and Mehra (1968) report on the case of the 52-year-old man who had

taken four chloroquine tablets (.25g) for treatment of malaria. Within one and a half

hours after taking the tablets he developed severe vomiting, vertigo, blurred vision and

tinnitus. Audiometric evaluation indicated that he had a severe sensorineural hearing loss

in both ears. The vertigo and blurred vision subsided but the hearing loss remained.

Seckin, Ozoran, Ikinciogullari, Borman and Bostan (2000) reported on a 34-year-old

woman who developed a sensorineural hearing loss after five months of treatment with

hydroxychloroquine. The hearing loss completely resolved two weeks after treatment

with hydroxychloroquine stopped. Johansen and Gran (1998) reported on two patients

being treated with hydroxychloroquine for lupus erythematosus, developed irreversible

sensorineural after several years of treatment.









Barrenas (1993) found greater TTS in patients who were treated with chloroquine

for rheumatoid arthritis and exposed to noise than when they were not receiving

treatment and were exposed to noise. The objective of the study was to determine if

melanin-binding drugs increased susceptibility to TTS. Twelve patients who were being

treated with chloroquine for rheumatoid arthritis were exposed to one-third-octave band

filtered noise presented at 105 dB SPL for 10 minutes. They were also exposed to the

same noise when they had completed their treatment. Thresholds were measured pre and

post exposure, using Bekesy audiometry. The investigator's results demonstrated that

subjects on chloroquine had greater TTS than when they were not on the medication. She

concluded that chloroquine increased susceptibility to TTS.

Chloroquine is also toxic to other auditory structures, including parts of the

central nervous system. At extremely high doses it can produce neurological, respiratory

and cardiovascular effects and with a mortality rate of 35% in cases of overdoses.

Retinopathy, pruritus (severe itching), and seizures are some of the major toxic effects of

chloroquine. Minor effects include nausea, vomiting, dizziness, headache, rashes and

abdominal pain (Luzzi & Peto, 1993, Phillips-Howard & Kuile, 1995). The rising

incidences of chloroquine resistant malaria have limited the use of chloroquine in some

parts of the world. However, chloroquine is now used in combination with proguanil,

which has no ototoxic effects (Rosenthal, 1998). As indicated previously, these reports

indicate that chloroquine under some conditions is ototoxic; however the exact nature of

the conditions for ototoxicity are not known. And as Bernard (1985) indicated, since the

side effects associated with chloroquine are visual and chloroquine monitoring is usually

visual examinations, the extent of chloroquine's ototoxicity has not been investigated.









Antioxidant U74389G

Twenty-one-aminosteriods form a new group of compounds made from

glucocorticoids that has been proven to be a strong inhibitor of membrane lipid

perioxidation because of their abilities to scavenge lipid peroxyl radicals and inhibit iron-

dependent lipid perioxidation. U74389G is part of that 21-aminosteriod group. Free

radical induced lipid perioxidation of cell membranes is considered a major component in

myocardial reperfusion damage. One study observed the effects of U74389G in the

protection of the rat myocardium after ischemia and reperfusion. Treatment with

U74389G caused a reduction of necrotic and myeloperoxidase activity, which increased

the survivability of the animals (Campo, Squadrito, Alatavilla, Squadrito, Avenoso,

Canale, loculano, Sperandeo & Caputi, 1996). It has also been proven effective in

preventing arachidonate-induced lipid peroxidation and permeability alteration in brain

micro vessel endothelia cells, which form part of the blood brain barrier system (Shi,

Cavitt, & Audus, 1995).


Hearing Measurement

Auditory Evoked Response

The auditory evoked response (AER), recorded from most mammals, has been an

excellent tool in the assessment of auditory activity. It provides a reliable, objective

measure of threshold when testing animals (Greene, Giddings, & Jacobson, 1992). The

trans-membrane ionic current flow of a cell and specifically the auditory neurons are the

source of voltage potentials that underlie the AER. The voltage are generated when the

auditory system is stimulated with sounds such as clicks, tone bursts or even speech. The

brain activity for the AER is a very small voltage (less than one micro volt) that requires









computer averaging and filtering for extraction from the on-going electrical activity of

the brain. There are several types of electrophysiological methods to measure the AER.

For example electrocochleography (ECochG) is the response that arises from the cochlea

and the eighth nerve, and it occurs within the first 1.5-2 milliseconds after acoustic

stimulation. It is usually recorded from electrodes placed on the round window of the

cochlea. The ECochG has three components; the first is the cochlea microphonic (CM),

which is generated at the hair cell level in the cochlea. The next two components are the

action potential (AP) and the summating potential (SP). Other terms for the AP are N1

and auditory brainstem response Wave I (Hall, 1992).

Another AER is the auditory brainstem response (ABR), which is the most widely

used AER in audiology clinics. The ABR is the AER that will be used for this project.

The response is generally recorded through the use of electrodes placed at various

locations on the scalp. Generally in humans five peaks (represented by the Roman

numerals I through V) of the waveform are analyzed. Wave I represents 8th nerve

activity. Wave II represents 8th nerve activity as the fibers enter the brainstem. Wave III

represents activity in the caudal portion of the auditory pons. Wave IV is thought to

represent activity in the superior olivary complex with contributions from the cochlear

nucleus. Wave V is thought to represent activity from the lateral leminiscus and inferior

colliculus (Hall, 1992). The time period in which Wave I occurs is 1.5 ms after stimulus

onset and the other waveforms follow at approximately one ms intervals (Hall & Mueller,

1997).

The click is the most common auditory stimulus used to elicit the ABR; however,

tone bursts can also be used. The click delivers a signal that stimulates a broad area of









the cochlea mostly in the 1-4 kHz region. Stimulation leads to multiple synchronous

neural activities. Increased neural activity leads to increased voltage activity, which leads

to a better overall recording of the response. The drawback to using the click to elicit the

ABR is that the click is comprised of a broad range of frequencies. The stimulation of a

wide area of the cochlea results in a loss of frequency specificity. In order to overcome

this problem, tone bursts are used to obtain frequency specific ABRs (Hall, 1992).

Auditory thresholds can be obtained by reducing the intensity level of the stimulus until

the waveforms are no longer distinguishable from on-going EEG.

Other AER measurements include the middle latency response (MLR), the late

latency response (LLR) and the P300. These responses are thought to represent the

auditory thalamus and primary auditory cortex, and the hippocampus. These methods are

generally not used for threshold determination.

Electrophysiological Measures in the Guinea Pig

Researchers have used several types of AERs to determine auditory thresholds in

the guinea pig. AER have been found to be a reliable and valid method of assessing

hearing sensitivity (Ingham, Thornton, Comis, & Withington, 1998; Dum, Schmidt, &

Von Wedel, 1981; Subramaniam, Henderson & Songr, 1994). Ingham et al (1998)

looked at the ABR response in the guinea pig with regard to aging. They observed four

positive-peak responses rather than the five peaks normally found in humans. The

amplitudes of the responses decreased with decreasing stimulus intensity and the

latencies increased with decreasing stimulus intensity, as with the human AER response

(Greene, Giddings, & Jacobson 1992). Table 1 illustrates the general auditory thresholds

in the guinea pig using different AER testing methods. Both normal hearing levels (dB

nHL) and sound pressure level (SPL) are used as reference in the studies cited.









Table 1: Electrophysiology Measurements of Threshold in the Guinea Pig


Greene, Giddings & Jacobson,
1992


Ingham, 1998 ABR Click 20 dB HL

Shi & Martin, 1997 ABR- Click 20-30 dB SPL

Cody & Robertson, 1983 N1- Tone Bursts 4K- 40 dB SPL
8K-25 dB SPL
12K- 20 dB SPL
16K- 15 dB SPL

Conlee, 1986 CM Tone Bursts 4K- 15-32 dB SPL

Cody, Robertson, Bredberg & N1- Tone Bursts 4K 45 dB SPL
Johnston, 1980 8K 25 dB SPL
10K 20 dB SPL
Yamasoba, 1998 ABR Tone Bursts 4K- 10-25 dB SPL
8K- 10-25 dB SPL
12K- 5-15 dB SPL
16K 5-15 dB SPL
Canlon & Fransson, 1994 ABR Tone Bursts 1K 27 dB SPL
2K 32 dB SPL
HL-The level of sound relative to dB HL and is based on the average hearing threshold
for young adults with normal hearing sensitivity. It is the dB reference on audiometers.
SPL- a dB value on a scale that has 0 dB equal to 20 [tPa and is measured by a sound
level meter (Katz, 1985).


Please note that SPL values are always higher than HL values for the same stimulus.

The effects of noise on the auditory threshold of the guinea pig have been well

documented. Walger, Schmidt and von Wedel (1985) have stated that the flaw in

previous studies had been that the experiments were conducted under acute conditions so

the noise exposures tended to be brief and at extremely high levels. Their experiment

looked at frequency specific changes in the threshold of the cochlear action potential

(CAP) in response to longer-term exposure to broadband noise at moderate intensities.


ABR


Click


9.3 dB HL









The guinea pigs were exposed to continuous noise at intensity levels of 80, 90 and 100

dB SPL for 120 hours. A total of five CAP measurements were made during quiet

intervals during the continuous exposure. Post exposure and recovery measurements

were also completed. They found that exposure to noise caused threshold shifts for all

frequencies tested with the expected variation based on frequency and noise exposure

levels (80, 90 or 100 dB). The change in threshold first became apparent 2 hours after

exposure in the frequency range of 4-12 kHz and then continued exponentially until

reaching an asymptote at 48 to 72 hours. At the lower frequencies of .5, 1 and 2 kHz

asymptote occurred at 24 to 48 hours and at the higher frequencies of 16, 20 24 kHz

asymptote occurred 72 to 96 hours during the exposure.

At 72-96 hours post exposure, CAP thresholds for all frequencies had completely

recovered for all exposure intensity levels. In the frequency range where the threshold

shift was the greatest (4, 8, 12 kHz), the recovery was faster during the first 24 hours,

then more gradual after. Overall, these results are consistent with previous studies where

the audiogram initially shows hearing loss in the most sensitive region (4-12 kHz), then

progresses to higher frequencies (16-24 kHz), and last to the lower frequencies (.5-2 kHz)

(Taylor, Pearson, Mair, & Burns, 1964; Passchier-Vermeer & Eijk, 1974). Given the

results of the preceding research it is expected that a 93 dB SPL noise exposure for 48

hours is sufficient to create the desired temporary threshold shift in the guinea pig animal

model.

Otoacoustic Emissions

Otoacoustic emissions (OAEs) are acoustical signals generated by the cochlea that

can be recorded in the ear canal. As an auditory signal travels from the base to the apex

of the cochlea, some of that energy travels backwards through the middle ear system to









the ear canal. Some believe that the discovery of OAEs was one of a few events that led

to a revolution in thinking about how the auditory system works (Robinette & Glattke,

1997; Hall 2000; Hall & Mueller, 1997). Although von Beskesy's traveling wave theory

laid the foundation of cochlea mechanics, it did not explain the boost in sensitivity and

sharpness in frequency tuning that came out of the cochlea. Kemp's discovery of OAEs

in the 1970s and subsequent work by Kim (1980) supported strong evidence of the

mechanical non-linearity and bi-directional interaction between hair cells and cochlear

mechanics (Kemp, 1986). The cochlea is considered non-linear because when the

magnitude of the input is increased, the magnitude of the response does not grow directly

in proportion to the magnitude of the input (Moore, 1998).

Brownell's (1983) demonstration of outer hair cell motility (length changes of the

hair cell in response to stimulation) suggested that the generator site of otoacoustic

emissions was the outer hair cells. By 1985 OAEs were accepted as a by-product of what

seemed to be a biological amplifier. Brownell (1983) believed that hearing loss is a loss

in active biomechanical responsiveness in the cochlea rather than a basic loss of hair cell

sensitivity.

OAEs are present only when the cochlea is in near normal condition and if the

middle ear system is operating normally. The sounds generated by the cochlea are small

but potentially audible sometimes, amounting to as much as 30 dB SPL. OAEs can

emerge spontaneously but are generally elicited with acoustic stimulation. Electrodes are

not required to record OAEs since they are not an electrical response. The response is

detected by a microphone and then converted to an electrical signal for processing

(Robinette & Glattke, 1997). It is possible to obtain poor OAE results but have a normal









audiogram because the test is very site specific and provides information about the status

of the OHC. Although the audiogram provides information about the middle ear,

cochlea, brainstem and higher auditory processing centers of the auditory system, OAEs

only assess the peripheral auditory system up through the outer hair cells (Robinette &

Glattke, 1997). There are instances where the patient will have a normal OAE but an

abnormal or absent ABR, which is usually the seen in auditory neuropathy (disorder of

auditory nerve function) (Hall, 2000; Robinette & Glattke, 1997). The general rule for

ABR and OAE comparisons is that with a normal middle ear, if OAEs are present and

ABR absent, then the lesion is beyond the outer hair cells. If there is no OAEs and

elevated ABR, then it is a sensorineural hearing loss (Robinette & Glattke, 1997).

OAEs fall into two general categories, spontaneous and evoked. Spontaneous

OAEs occur without acoustic stimulation and are comprised of energy at one or more

frequencies. Evoked OAEs require some type of auditory stimulation to be detected.

There are three types of evoked OAE responses but since the distortion product

otoacoustic emissions (DPOAE) will be used for this project the following discussion

will focus only on DPOAEs (Robinette & Glattke, 1997).

The DPOAE is elicited when two tones, which are close in frequency, are

concurrently presented. When delivered to the ear, a third tone is created which is called

the distortion product (DP). If the tones used to elicit the DP are too far apart in

frequency then there will not be a distortion product. The tones used to create the DP are

called primary tones designated by the symbols fi and f2. The f2 frequency is higher than

fi. The response skirt of fi overlaps into the response skirt of f2, which leads to the

generation of the DP (Robinette & Glattke, 1997; Hall, 2000). The DP response is best









recorded when the intensity is less than 70 dB and there is a 10 dB difference between the

two primary tones. It is always best to have fi primary tone higher than f2 primary tone

since fi is more important to the amplitude of the response. The amplitude of the

response is also influenced by the ratio of the f2 frequency to the fi frequency, which is

derived by f2/fl. The most widely used ratio is 1.20-1.22 (result of f2/fl) (Robinette &

Glattke, 1997; Hall, 2000; Hall & Mueller, 1997). It is generally believed that the DP

response is more reflective of the f2 region of the cochlea rather than at the region thought

to be associated with the distortion product. The formula used to determine the response

is 2fi-f2 (also known as the cubic method) (Robinette & Glattke, 1997; Hall, 2000; Hall

& Mueller, 1997).

As with all biological testing it is important to ensure that the response is

reflective of testing and not just the artifact. In the case of the DPOAE the artifact

activity is a combination of electrical and thermal noise in the equipment, ambient

environmental noise or physiological noise (also referred to as the noise floor). In order

to determine if there is a DP response the amplitude of the response should be at least a

minimum of 3 dB above the noise floor. A DP response with amplitude of 3 dB doesn't

mean that the response is normal but that there is simply a response. It has been

suggested that clinics might use 6 dB amplitude above noise floor as a response criteria

because it ensures that what is recorded is not some type of artifact (Robinette & Glattke,

1997; Hall, 2000).

The DP has remarkable frequency specificity. The clinician has the ability to look

at the response of the cochlea in regions used on the audiogram (meaning that the

frequencies used on the audiogram can also be used for the DP), which allows the DP to









be used as an adjunctive test to the audiogram. As with all evoked OAEs, the DP is a

measurement of the health of the OHCs. The DP response is present nearly 100% of the

time in healthy ears. If hearing sensitivity is between 0-15 dB HL (hearing level) then

there is an excellent chance that a DP will be present. At thresholds above 20-25 dB HL,

the chances of obtaining a DP response are greatly reduced and thresholds above 30 dB

HL the response will not be present (Robinette & Glattke, 1997; Hall, 2000; Hall &

Mueller, 1997).

Because the DP is a reflection of OHC function, it is particularly useful when

testing for things known to affect the OHC. The DP response will not be present if there

is damage to the OHC. So for example, in monitoring patients on ototoxic drugs the

absence of the DP could serve as the first indication of a problem. The same would hold

true for noise exposure, where the absence or depression of DPOAE, amplitudes could

serve as an early indicator of damage to the OHCs. It is important to point out that the

DP is frequency specific and will reflect the state of OHC based on frequency. For

example if damage is in the 4 kHz region, the DP in that region will be affected (Harris,

1990). Thus distortion products will be used in this project, as an initial indicator of the

condition of the OHC.

The use of DPOAE as an early detector of cochlea damage was borne out by Shi

and Martin (1997) who compared the effectiveness of the click ABR and DPOAE in

detecting cochlear damage caused by gentamicin treatment in the guinea pig. The guinea

pigs received daily injections of gentamicin for four weeks. Their hearing status was

evaluated periodically through the four-week period using ABRs and DPOAEs. Changes

in the DPOAE input/output functions were observed after two weeks of treatment while









changes to the ABR threshold were not observed until three weeks after treatment. This

information was valuable for this project because there were conditions where

observations of the effects of chloroquine alone on hearing sensitivity were made. The

initial hypothesis was that it was possible that the ABR would not show a change in

hearing sensitivity but the DPOAEs would show a change.

DPOAEs have also been used to determine the effects of noise on the cochlea

response. Skellett, Cullen, Fallon & Bobbin (1998) found that noise conditioned guinea

pigs exposed to 105 dB SPL for 72 hours had the biggest decrease in DPOAE amplitude

(12-18 dB SPL) in the 1414 to 4000 Hz range. Kemp (1996) had similar findings in

humans where the amplitude of the DP was most affected in the 3-5 kHz regions of the

cochlea after exposure to narrow-band noise (center frequency 2 kHz) at an intensity of

102 dB SPL for 10 minutes.

The DPOAEs' recovery following noise exposure is similar to other

electrophysiological measures where the response over time recovers to or close to pre-

exposure amplitude values. Emmerich, Richter, Reinhold, Linss and Linss (2000)

observed the effects of industrial noise exposures on the DPOAEs of guinea pigs.

Twelve guinea pigs were exposed to two hours of tape-recorded industrial noise (105 dB

SPL). DPOAEs (1.5 to 6 kHz) were recorded pre-exposure and at five minute intervals

for a total period of 2 hours. The recordings were repeated daily for 3 days post exposure

and then at 2-day intervals until the DPOAE amplitudes stabilized. They found that DP

measures taken within the first 2 hours post exposure were absent in a majority of the

animals. On one to two days post exposure, recovery to near pre-exposure levels was

seen at frequencies other than 2.5 kHz. Pre-exposure recovery of DPs at lower






47

frequencies took three to nine days. Frequencies above 5 kHz recovered the quickest

(See Figure 1).


A



DPOAE


D wl l
(dB SPLJ





lo +- 3.0
u10 -- -- -,'----
A5 U f4Hz
57.

e20r n d 10 d 7w
B ine po t exposure




Figure 1: Partial recovery of Distortion Product Otoacoustic Emissions (DPOAE) after
exposure to industrial noise for one animal.



Therefore, it is apparent that the DPOAE is a valid instrument that can be used to

determine cochlea response to noise and ototoxic drug exposure. The DPOAE will

provide an opportunity to observe the cochlear response at the pre-synaptic level (pre 8th

nerve) to antioxidant, chloroquine and noise.














CHAPTER 3
METHOD

The overall goal of this project was to determine if the antioxidant U74389G has a

protective effect against the ototoxic consequences of noise exposure and chloroquine.

The three experimental factors, noise (N), antioxidant (A) and chloroquine (Ch) were

given in isolation or in combination with each other. The change in auditory function was

evaluated using two methods, the auditory brainstem response and the distortion product

otoacoustic emission. Fifty-six pigmented guinea pigs (Kuiper's Ranch, Gary, IN)

weighing at least 500 grams (age approximately greater than 3.5 months) served as

subjects for this project. The Institutional Animal Care and Utilization Committee

approved this protocol (approval number 0602).


Experimental Design

Pigmented guinea pigs were randomly assigned to eight treatment groups with

seven animals per group. The paired experimental factors included noise exposure/no

noise exposure, chloroquine/injectable saline, and antioxidant/saline. The eight treatment

groups and observed effects can be found in Table 2.

By way of an example, animals exposed to noise were compared to animals

exposed to only ambient noise of 40 dBA. Likewise, animals given the antioxidant were

compared to animals given flavored saline.










Table 2:Treatment groups of 7 animals per group for a total of 56 subjects

Group Effect Observed
Group 1 Control Effects of just saline on hearing sensitivity
No Noise
Injectable Saline
Flavored Saline
Group 2 Noise/Saline (N) Effect of noise on hearing sensitivity
Noise
Injectable Saline
Flavored Saline
Group 3 Noise/Chloroquine/Antioxidant (N- Protective effect of antioxidant against
Ch-A) noise and chloroquine
Noise
Chloroquine
Antioxidant
Group 4 Noise/Chloroquine N-Ch) Effect of noise and chloroquine on hearing
Noise sensitivity
Chloroquine
Flavored Saline
Group 5 Chloroquine/antioxidant (Ch-A) Protective effect of antioxidant against
No Noise chloroquine
Chloroquine
Antioxidant
Group 6 Chloroquine (Ch) Effect of chloroquine on hearing
No Noise sensitivity
Chloroquine
Flavored Saline
Group 7 Noise/antioxidant (N-A) Protective effect of antioxidant against
Noise noise exposure
Injectable Saline
Antioxidant
Group 8 Antioxidant (A) Effect of antioxidant on hearing
No Noise
Injectable Saline
Antioxidant


For five consecutive days animals from treatment groups N-Ch-A and N-Ch

received daily injections of 35mg/kg (Sykes, 1984) of chloroquine diphosphate (Fisher

Scientific, Plymouth Meeting, PA) in saline subcutaneously. Dosage was determined

during pilot testing of 2, 20, 25, 30 and 35 mg/ml of chloroquine diphosphate in saline

(Barrenas, 1997; Grundman et al, 1972; Sykes, 1984; Filkins, 1969). The dosage of 35

mg/ml was found to create a change in auditory function (reduce ABR threshold). After









administration of chloroquine they received antioxidant treatment. Ten milligrams per

kilogram of the antioxidant U74389G in citrate NaCl (.3 ml volume) was administered by

mouth utilizing a feeding syringe at 24 hours and 12 hours prior to the start of the noise

exposure and every 12 hours during a 48-hour noise exposure.

The noise exposure was continuous broadband noise at 93 dB SPL (+/- 1 dB).

During the time of the exposure the animals were housed in a cage located in a 7' x 7'

room and given food and water ad libitum. Immediately after noise exposure, ABR and

DPOAE measures were completed to assess threshold shifts. Only data for the left ear

were reported. The ABR and DPOAE were repeated 24 hours later to observe recovery

of thresholds. Temporary threshold shifts that have not recovered 30 days after exposure

are considered permanent hearing loss. As such this study only observed TTS that has

occurred and recovered within 48 hours post exposure. Baseline ABR and DPOAE

measures were obtained a minimum of three days prior to start of the administration of

chloroquine. This assured that the animal has recovered from the anesthesia required for

baseline testing (See Table 3, Protocol Timeline).




Table 3: Timeline of Treatment.
Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Anesthesia
ABR/DPOAE
Recovery
Chloroquine
Antioxidant
Noise

Timeline of when different procedures were conducted throughout the study are
presented. For example on Day 1, baseline measurements were followed by three days of
recovery before the start of the chloroquine.









Animal Preparation

All animals were tested in an IAC double-walled sound booth with an ambient

noise level of less than 40 dB SPL (Bruel and Kjaer Type 1613 sound level meter). The

sound level meter was calibrated with a sound calibrator (Bruel and Kjaer type 4220).

Prior to testing, the guinea pigs were anesthetized with a mixture of Ketamine (Ketaset,

40 mg/kg) and xylazine (Xyla-jet, 5 mg/kg) and placed on a heating pad so that body

temperature was maintained at 370 F. The condition of the ear canals and tympanic

membrane were evaluated with an operating microscope. Animals with obvious

pathologies were eliminated from the study.

Pilot Study

A pilot study was conducted to determine the following; the feasibility of the

project, to determine dosage of chloroquine, and to assay chloroquine levels in the blood

stream. Ten animals were put through the protocol as stipulated in Table 3. Two animals

were given daily subcutaneous injections of chloroquine for five consecutive days at the

following dose levels: 2, 20, 25, 30 and 35 mg/ml (Barrenas, 1997; Grundman et al,

1972; Sykes, 1984; Filkins, 1969). Auditory brainstem response testing was used to

determine changes in auditory activity and was conducted prior to the start of treatment

and on the fifth day of treatment. Additionally, on the fifth day of treatment, two blood

samples (1 ml each time) were taken from the animals. The animals were sedated, ABR

testing was conducted, and then 1 ml of blood was drawn from the anterior vena cava

vein. Following the blood draw, the fifth injection of chloroquine was administered.

Thirty minutes later 1 ml of blood was drawn from the same site.









Sample Extraction Procedure

The blood samples collected were immediately placed into heparinized bottles.

The samples were centrifuged 3,000 g for 10 minutes. The purpose of the centrifuging

was to obtain the plasma from the blood. The samples were immediately frozen and

stored in an -800C freezer for storage. The 0.3 ml of plasma and 1.2 ml of diethyl ether

were placed into a ten ml extraction tube. The mixture of plasma and diethyl ether was

basified with 0.3 ml of 2 M NaOH and whirl mixed for one minute. The organic layer

was then transferred into a fresh tube to which 100 pl of 0.1 N HCI was added. This was

whirl mixed for one minute and centrifuged for ten minutes. A 25-1l aliquot of the

aqueous layer was injected into the Waters Model 440 chromatographic machine for

processing (Walker & Ademowo, 1996).

Serology Testing

High-pressure liquid chromatography (HPLC), which is a commonly used

method of separating chemical compounds, was used to test the chloroquine levels in the

blood samples obtained from the guinea pigs. Briefly, chemical separation was

accomplished when the sample was injected into the mobile phase of an assay through

the injector port of the chromatographic equipment. As the sample flowed through a

column, the components of the sample migrated according to the non-covalent

interactions of the compounds within the column. The chemical interactions of the

sample and the column determined the degree of migration and thus the separation of the

components in the sample (Walker & Ademowo, 1996).

Results HPLC

Recall that the HPLC was used to determine the levels of chloroquine in the

plasma of the guinea pigs. The animals were given daily subcutaneous injections of 35









mg/ml (total dosel75 mg) of chloroquine diphosphate in saline. The plasma sample

results indicated that the concentration of chloroquine prior to the administration of the

5th injection, which represents the trough data, was 32.10 ng/ml. This value was similar to

the trough values of 20 to 40 pg per liter for humans given a 0.5 g daily dose of

chloroquine (Goodman & Gilman, 1985). It takes 30 minutes for subcutaneous injections

of chloroquine to reach peak plasma levels (Goodman & Gilman, 1985). The peak

plasma value for the guinea pigs was 42.17 ng/ml.

Westin et al., (1995) gave healthy subjects three different dose regimens of

chloroquine. For three weeks subjects were given one of the following doses of

chloroquine. Group one received once a week 300 mg base for a total dose 900 mg.

Group two received two times a week 200 mg base for a total dose of 1200 mg. Group

three received daily dose of 50 mg base for a total dose of 1050 mg. On day four the

trough values for chloroquine concentration was 25 ng/ml, 15 ng/ml and 10 ng/ml for

groups one, two and three respectively. The 300 mg dose once per week is similar to

what is used for suppressive therapy with chloroquine. The trough value was similar to

what was observed in this study. Overall the HPLC provided two findings. The first was

that the use of subcutaneous injections of chloroquine in saline produced measurable

chloroquine levels in the plasma. The second was that the chloroquine levels found in the

plasma were consistent with levels found in humans (the dose used in this study was not

excessive).









Auditory Procedures and Equipment

Auditory Brainstem Response

ABR and DPOAE measurements were performed with a Tucker

Davis Technologies (TDT) System II operated by a personal computer that is equipped

with the TDT Auditory evoked potentials and otoacoustic emissions software. ABR

thresholds were determined using clicks and tone bursts at 4, 8, 12 and 16 kHz. The tone

bursts will be centered at a given frequency with a 2-cycle rise and fall times and a 1-

cycle plateau. The presentation rate was 21 per second for 1024 samples.

The electrical signals were picked up by active alligator clip electrodes placed

behind the auricle and the ground on the lower back. Alternating polarity (. Ims) clicks

were calibrated in the following manner. The peak-to-peak voltage for a 90 dB relative

level click was determined using an oscilloscope (Leader Model 8020). A 4 kHz tone

burst (500 ms duration) was adjusted until the peak-to-peak voltage was the same as the

click. The sound pressure level of the tone burst at this setting was measured with a

sound level meter (Bruel and Kjaer Type 1613). The peak equivalent dB pe SPL of the

click stimulus at a relative level of 90 dB was determined to be 75 dB pe SPL. The tone

bursts were calibrated by routing the output of the ER-2 earphones to the input of a 1/8"

Bruel and Kjaer microphone (Type UA0036) fitted in a 1/8" I.D. tube. The sound

pressure level of frequencies 4, 8, 12 and 16 kHz was determined separately. The

distance between the tip of the ER-2 earphone tubing and the diaphragm of the

microphone was 5 mm, which is the approximate distance between the ER-2 tubing and

the tympanic membrane of the guinea pigs.

Sample signals were amplified, digitally averaged, filtered and analyzed on a

computer. The detection criteria was the lowest level at which a visually reproducible









response was obvious. In order to reduce investigator bias, two independent judges

evaluated the ABR tracings for threshold determination. Threshold shift was calculated

based on the difference between pre-exposure and post exposure thresholds. The initial

stimulus intensity was 70 dB SPL and dropped in 10 dB increments for high levels, and 5

dB increments at lower levels until reproducible waveforms were no longer present

(Ekborn, Laurell, Andersson, Eksborg & Ehrsson, 2000; Greene et al, 1992).

Distortion Product Otoacoustic Emission

Distortion product otoacoustic emissions (DPOAE) were elicited using two tones

(as mentioned previously the tones used to create the DP are called primary tones

designated by the symbols fi and f2) at frequencies fi and f2 such that f2/fl=1.22. The

intensity of f was set to be 10 dB higher than f2. The pairs of primary tones selected to

obtain the emission represent the function of the basilar membrane at frequencies 4 and 8

kHz. The DPOAE was considered present if the amplitude of the response is 6 dB or

greater above the averaged noise floor. The intensity of fi was decreased from 70 dB SPL

to 40 dB SPL in order to determine the change in DPOAE amplitude in response to the

decrease in intensity (Khvoles, Freeman & Sohmer, 1999). The Tucker-Davis System II

audio processing unit was used to generate the primary tones. The tones were sent to two

Etymotic Research ER-2 earphones and coupled to an Etymotic Research ER-10B

microphone. The ER-10B microphone detected the DPOAE response and the voltage

was routed back to the computer and expressed in millivolts. One hundred rms averages

were collected, fast Fourier transformed and analyzed by independent judges.

The calibration of the primary tones was accomplished by coupling the acoustic

probe assembly to a 1/8" condenser microphone (Briel and Kjaer type UA 0036,









Melbourne, Australia) and then checking the output of the two speakers. The placement

of the earphone tubing was positioned 5 mm from the diaphragm as described above.

Noise Generation

Broadband noise, generated by the Tucker-Davis Systems II wave generator, and

routed to SA1 speaker amplifier and TWEET speakers, was used for the noise exposure.

Four speakers were suspended in the cage so that noise radiated down toward the position

of the guinea pigs. The approximate distance from the speakers to the guinea pigs was

ten inches. A Bruel and Kjaer (Type 1613) sound level meter and 1/2" microphone

(Bruel and Kjaer, Type 4165) was used to assure a uniform level of 93 dB SPL

throughout the entire area of the cage. The microphone (Bruel and Kjaer type 4165) was

positioned at the level of the guinea pigs' ears, and measurements were made at various

locations within the cage. Noise levels were measured at the beginning and ending of

each exposure.

Preparation of Antioxidant U-73489G

The antioxidant U-73489G was purchased from Upjohn Company, Kalamazoo,

Michigan. The antioxidant was dissolved according to the directions provided by Upjohn

Company, which helped to ensure the potency of the product. The citrate acid

formulation of U-73489G used for this project was prepared in the following manner.

Two mg/ml of U-73489G, .02 M (Molar) citric acid monohydrate, .0032 M sodium

citrate dihydrate and .077M NaCl was combined and mechanically stirred for eight hours.

The solution was refrigerated when not in use.

Statistics

This study had three experimental factors (noise, chloroquine and antioxidant)

that were measured over several time periods (Baseline, Post-Exposure and Recovery).









Because the experimental factors were given in isolation or combined together, a 3-Way

Analysis of Variance (ANOVA), was used to analyze the results. A covariate was added

to control for variability in animal sensitivity at baseline. Use of the 3-Way ANOVA

provided the greatest statistical power that allowed for the observation of variability

created by each of the experimental factors. Being able to observe the variability caused

by the experimental factors allows conclusions to be drawn about the specific influence

of antioxidant among the various treatment groups.

Sample size determination was accomplished using a power analysis based on the

parameters of the ABR. The power analysis was comprised of the following parameters:

two-tailed hypothesis test, a 5 dB change in threshold was used as the critical value,

power of .80, and alpha of 0.05. Based on the results of the power analysis it was

determined that a sample size of seven animals per group would lead to statistically

significant results.

The independent variables were the experimental factors each animal received

(see table 1). The dependent variables were the thresholds of the ABR and the

amplitudes of the DPOAE. The ANOVA was used to determine whether or not the

means of different experimental factors were significant (alpha = .05). This design

allowed for the following hypotheses to be tested:

A 48-hour, 93 dB SPL broadband noise exposure will produce significant

changes in both ABR thresholds and DPOAE amplitudes.

A 35-mg/kg dose of chloroquine for five days will produce significant

changes in both ABR thresholds and DPOAE amplitudes.









* Antioxidant treatment will alter the effect on the ABR thresholds and

DPOAE amplitudes produced by chloroquine.

* Antioxidant treatment will alter the effect on the ABR thresholds and

DPOAE amplitudes produced by noise exposure.

* The combined exposure to noise and chloroquine will produce more

change in ABR thresholds and DPOAE amplitudes than either exposure

alone.

* Antioxidant treatment will produce changes in the ABR thresholds and

DPOAE amplitudes to the combined exposure of noise and chloroquine.

* Antioxidant treatment will produce changes in ABR thresholds and

DPOAE amplitudes in the absence of noise exposure and/or chloroquine.

* Noise exposure will result in changes to post exposure and recovery for

ABR thresholds and DPOAE amplitudes compared to pre-exposure

thresholds/amplitudes.

* Antioxidants will result in changes to post exposure and recovery for ABR

thresholds and DPOAE amplitudes compared to pre-exposure

thresholds/amplitudes.

* Chloroquine will result in changes to post exposure and recovery for ABR

thresholds and DPOAE amplitudes compared to pre-exposure

thresholds/amplitudes.














CHAPTER 4
RESULTS

This project was designed to determine if the antioxidant U74389G has a

protective effect against noise exposure and chloroquine. Pigmented guinea pigs were

randomly assigned to eight treatment groups, with seven animals in each group. The

administered experimental factors were chloroquine, antioxidant and noise exposure

separately and in combinations (see Table 2 for treatment groups). Changes in auditory

function were measured using the threshold of the auditory brainstem response and the

amplitudes of the distortion product otoacoustic emissions.


Auditory Brainstem Response

Morphology and Latency

The ABR responses obtained during this study were consistent in morphology and

latency as found by Greene et al., (1992) and (Ingham et al., (1998). Figures two and

three are representative of the data obtained for the ABR. ABR morphology presented in

Figure 2 was consistent with the results obtained by other experimenters within this

species (Greene et al, 1992; & Ingham et al, 1998). In the guinea pig, there were four

prominent peaks labeled Waves I through IV. The response behaved as expected with a

decrease in amplitude and an increase in latency as the intensity of the stimuli decreased.

In this instance the response was last observable (threshold) at 20 dB (the peak equivalent

sound pressure level of the click stimulus at a relative level of 90 dB was 75 dB pe SPL).







60



S85dB
75dB
65dB

2SdB



4520dB


15d5



Figure 2:Auditory Brainstem Response (ABR) waveforms to click stimuli from one
animal. Four observable peaks were present that decreased in amplitude and increased in
latency to decreasing intensity in dB increments. In this series threshold was 20 dB



Figure 3 represents the decrease in latency as a function of intensity. The longer

latencies are generally associated with the lower frequencies. This finding was expected

and represented the additional travel time along the basilar membrane. These data were

used to make judgments about the reliability of the system and the sensitivity of the

animals. Wave II is present at the lowest stimulus levels followed by Wave I and then by

Wave III and Wave IV. This effect was likely the result of the electrode montage

(inverting and non-inverting electrodes behind the auricles and the ground on the lower

back). This horizontal montage would enhance the contributions of the dipole generators

oriented within a frontal place, while minimizing contributions from generators that have

more rostral-caudal orientation (Hall, 1992).









61





Control Group- Baseline
ABR Latency/Intensity





2 90


2 70
S-Click Wave I
---4k Wave I
2 508k Wave I
12k Wave I
o 12k Wave I
2 30 16k Wave I


2 10


1 90


170

10 15 20 25 30 35 45 55 65 75 85

dB

A




ABR- Control Group Baseline
Latency/Intensity




3 80


3 60

3 40 --ClickWave II
---4k Wave II
3 20 8k Wave II

12k Wave II
3 00
3 00 16k Wave II

2 80


2 60


240
10 15 20 25 30 35 45 55 65 75 85

dB

B



Figure 3: Average baseline latency values of the control group. Panels A and B represents

Waves I & II. Panels C and D represents Waves III & IV for frequencies 4, 8, 12, 16 kHz

and click. The x-axis represents dB dial on the Tucker Davis System.









62




ABR-Control Group Baseline
Latency/Intensity



3.90

3.80
-3.0 Click Wave III
3.70 -- --4k Wave III

S 3.60 8k Wave III
3. 12k Wave III
3 .50 ------------- ~ a i
3.0 --16k Wave III
3.40

3.30

3.20

10 15 20 25 30 35 45 55 65 75 85

dB


C



ABR-Control Group Baseline
Latency Intensity


4 90
4 80
4 70
--Click Wave IV
4 60
4 60 -----4k Wave IV

8k Wave IV
440 8k Wave IV
E 440
S12k Wave IV
4 30
S16k Wave IV
4 20
4 10
4 00
10 15 20 25 30 35 45 55 65 75 85

dB

D



Figure 3---continued


Average ABR Thresholds by Gro ups


The eight treatment groups were divided into two broad categories: non-noise


exposed which consisted of control, chloroquine (Ch-), antioxidant (A-), and chloroquine


and antioxidant (Ch-A) and, the noise exposed group consisted of the noise (N-), noise


and chloroquine (N-Ch-), noise and antioxidant (N-A-) and noise, chloroquine and


antioxidant (N-Ch-A). The two broad categories were then compared over time. The









time periods represented are baseline, (prior to receiving any treatment) post-treatment

(immediately after treatment) and recovery (24 hours post treatment).

Figure 4 depicts the averaged thresholds for the time periods of baseline, post-

treatment and recovery for the non-noise exposed groups. The baseline results (Figure 4)

indicate there were no major differences between the groups prior to treatment. The

thresholds were lowest at 8 and 12 kHz, which were the most sensitive frequencies for

the animals (Walger et al, 1985; Heffner, Heffner & Masterton, 1971; Cody et al, 1980).

When comparing the baseline to the post-treatment condition, the Ch- thresholds were

elevated and separated (approximately 10 dB) from the control, -A, and Ch-A thresholds.

The Ch-A group had some change from baseline but not of the magnitude of the Ch-

group. The elevation in threshold was maintained to the recovery time period.

Figure 5 depicts the averaged thresholds for the time periods of baseline, post-

treatment and recovery for the noise exposed groups. When comparing the noise-

exposed (Figure 5) groups across the time periods, again the baseline results were similar

across groups to the non-noise exposure baseline results. In the post-treatment time

period the N-Ch-A group had the least amount of change in threshold from baseline of all

the groups. The N-A group had the most change in threshold from baseline of all the

groups. During the recovery time period, the N-Ch-A group still had the least amount of

threshold change from baseline. Also note that at post-treatment the N-A group had the

most change in threshold from baseline; however, in the recovery time period the N-A

had a decrease in threshold that was now at the same level as the N-Ch group.



































Non-Noise Exposed: Post Treatment
90
80
70 -- Control
60 -Ch-
-A
50 -A
50 Ch-A
440
30 -
20
10 ---
0

4K 8K 12K 16K Click

Frequency



Figure 4: Average thresholds for non-noise exposed groups for treatment periods of
baseline, post-treatment and recovery. Chloroquine creates changes in threshold at the
post-treatment and recovery time periods.


Non-Noise Exposed:Baseline

90
80
70 -- Control
60- + Ch-
50 -A
m 40- Ch-A
30 --~
20
10 -
0

4K 8K 12K 16K Click

Frequency

































Figure 4---continued







66



Noise Exposed:Baseline

90
80
70 ---N-
60- --- N-Ch
N-A
0 50-
c N-Ch-A
a 40 --------------------_________ _____
m 40
30 -- -
20 ------
10 l
0

4K 8K 12K 16K Click

Frequency






Noise Exposed:Post Treatment
90


70 -___- N

60 ---N-Ch
_N-A
0 -50
m N-Ch-A
M 40

30

20

10 -
10

0 I-I I I
4K 8K 12K 16K Click
Frequency


Figure 5: Average thresholds for noise exposed groups for baseline, post-treatment and
recovery. The N-Ch-A group had the least change from baseline at post-treatment and
recovery time periods.

































Figure 5---continued

Statistical Methods

In the previous section the mean data were presented according to the three time

periods during which measurements were obtained. To assess the extent to which a

treatment influences the individual threshold for an animal, the baseline threshold for

each stimulus condition was subtracted from the post-treatment threshold. In most noise

exposure literature the resulting difference is referred to as temporary threshold shift

(Henderson et al, 1993). In addition to this manipulation, baseline threshold was

subtracted from the recovery threshold, and post-treatment threshold was subtracted from

the recovery threshold. These three numeric manipulations, baseline to post-treatment,

baseline to recovery and post-treatment to recovery, form the partially correlated sets of

response variables.









For the entire stimulus conditions tested (ABR click, 4, 8, 12, & 16 kHz and

DPOAE 8 kHz 70, 60, & 50 dB), each of their respective response variables was

analyzed separately using 3-way analysis of variance (ANOVA). In analyzing Baseline to

Post-Treatment and Baseline to Recovery response variables, baseline threshold levels

were included as a covariate in the 3-way ANOVA in order to control for variation in

baseline sensitivity among individual animals across the eight treatment groups (Box,

Hunter, & Hunter, 1978; Fleiss, 1986).

Least-square means (adjusted means), standard errors, and 95% confidence

intervals for the true mean were estimated for each treatment group as part of the

ANOVA. Each mean change in threshold over the time periods were also tested to

determine if they differed significantly from zero by taking the ratio of the least-square

mean to its standard error and comparing it to a t-distribution parameterized by the

appropriate degrees of freedom determined from the ANOVA. This ensured that data

met the key assumptions of an ANOVA, such as normally distributed observations.

In the case of the response variables that involved change from baseline, least-

square response means were estimated with the baseline threshold covariate set to the

mean baseline threshold level for all animals included in the ANOVA. F-tests based on

Type 3 sums of squares were used to evaluate the significance of main effects and

interactions among the effects of the 3 experimental factors (Box et al, 1978). ANOVA

tables are located in appendix c.

After fitting each ANOVA model, residuals were assessed for normality and

outliers by examining histograms and plots of residuals versus predicted values. Box-Cox

analysis was also used to determine if the data being analyzed in a given ANOVA should









be transformed to improve model fit (Box & Cox, 1964). In all instances, these

assessments indicated no significant lack of fit when models were fit using untransformed

observations. Several moderate outliers were detected. To determine if these outliers

were influential, ANOVA models were refit excluding the outliers and the results of these

ANOVAs were then compared to results that were obtained with the outliers included.

These comparisons indicated that none of the outliers significantly influenced the

ANOVA results. All of the estimation and testing carried out within the framework of

each 3-Way ANOVA were performed using the PROC MIXED procedure in the SAS

statistical analysis software package, Version 8.1 (Littell, Milliken, Stroup & Wolfinger,

1996).

Four specific contrasts among treatment-group means were performed within the

framework of the ANOVA to assess the effect of antioxidant on mean change in

threshold over time. Each of the contrasts involved the comparison of a treatment group

that received antioxidant to a group that did not receive antioxidant treatment. In each

comparison, both of the groups being compared shared one of the four possible

combinations of chloroquine and noise experimental factors. Thus, the statistical

significance of the antioxidant was evaluated under all possible conditions involving the

presence or absence of chloroquine and noise exposure (Box & Cox, 1964).

Graphic Plots of the Data

The analyzed data are presented in two forms graphic and tabular. The graphic

figures allow for an in depth review of the results for one type of stimulus condition

(ABR click) across the groups. The tabular presentation depicts the results for all

treatment conditions (click, 4, 8, 12, & 16 kHz) and groups.









The following information provides an orientation to Figure 6, which depicts the

statistical results for the ABR threshold change from Baseline to Post-Treatment. Recall

that the first response variable represents the change in hearing function from baseline to

post-treatment. The second response variable represents the change in hearing function

from baseline to recovery (48 hours post noise exposure) and the third response variable

represents change in hearing function from post-treatment to recovery. On the x-axis, the

groups are divided into two broad categories, non-noise exposed (Noise-N) and noise

exposed (Noise-Y). The sub-category of chloroquine (Chloro-N) further divides the

graph and finally the presence or absence of antioxidant is indicated. The data points on

the figure represent the mean change from baseline to post-treatment for each group. The

vertical bars that bracket each mean represent the 95% confidence interval. The y-axis

represents the response variable in dB (change in ABR threshold from baseline to post-

treatment). The dotted line at zero on the y-axis indicates no change from baseline. So if

the interval intersects the dotted line then the potential change from baseline was not

significant.

In addition to determining if the treatments caused a significant change from

baseline, an analysis was done to determine if there was an antioxidant effect between

two treatment conditions. When looking at two groups, the antioxidant effect was

defined as the change in the response variable that could only be attributed to the absence

or presence of the antioxidant. Two asterisks next to the word antiox (**) indicated

significance defined as p < 0.05. One asterisk next to the word antiox (*) indicated trends

associated with p values of p=< 0.1 but > 0.05. To further highlight the statistically

significant presence of an antioxidant effect the lines between the means are solid. The







71









Change from BASELINE to POST-TREATMENT
controlling for BASELINE
ABR Click
(Least-squares means with 95% confidence intervals)


N Y N Y
Antiox Antiox *
Chloro-N Chloro-Y
Noise-N


N Y N Y
Antlox Antlox *
Chloro-N Chloro-Y
Noise-Y


Figure 6: Change in threshold (in decibels) from baseline to post-treatment for ABR
click. For the non-noise exposed groups only the chloroquine group had a significant
change from baseline to post-treatment and an antioxidant effect.




dashed (--) lines between the means indicate that no significant difference in


antioxidant effect was present.


The p values less than .1 are mentioned in this report because in complex designs,


which include multiple factors and interactions, statistical significance of 0.05 to 0.1,


should not be completely ignored. The reason for this is that interactions can be complex


and higher order interaction tends to have the least statistical power. So higher p values


may still show trends that may provide valuable information (Marks, 2000).


0
LID
0
0
I

0 0
E N


0
EL0


.............. --... ... ... ... .









Response variable--baseline to post-treatment

The first two data points on figure 6 represent the control group and antioxidant

group. Both confidence intervals intersect zero, which indicates that there was no

significant change in threshold from baseline to post-treatment. Thus, the antioxidant

alone made no significant change to auditory function.

The next two data points are for the Ch- and Ch-A groups. The chloroquine group

was significantly (p=. 017) different from baseline but not so for the Ch-A group. These

results indicate that the change from baseline to post-treatment for the chloroquine group

was significant. However, when antioxidant is added to the chloroquine, antioxidant

tends (p=. 076) to reduce the negative effects of chloroquine

The change from baseline to post-treatment was significant for all the noise-

exposed groups, (none of the intervals intersect zero) which means that the noise

exposure created a significant increase in threshold as expected. When looking at the N-

group and the N-A, the -A group experienced greater change than the N- group, which

was not expected. Comparing the N-Ch and the N-Ch-A groups, the TTS for the N-Ch-A

group was less. The antioxidant effect was present (p= .054) which indicate that the

antioxidant was providing some type of a protective effect.

Response variable--baseline to recovery

In Figure 7, the confidence interval lines of the control and antioxidant groups

intersect zero therefore there was no significant change from baseline for both groups.

When looking at the Ch- group and the Ch-A groups, the chloroquine group maintained

the significant increase in threshold (basically the same increase that was seen in Figure

6). The Ch-A had no significant change to threshold and thresholds were better than the

chloroquine group.
















Change from BASELINE to RECOVERY
controlling for BASELINE
ABR Click
(Least-squares means with 95% confidence intervals)

Co
Co















Antiox Antiof* Antiox Ant\of*



Figure 7: Baseline to recovery threshold (in dB) results for the ABR click. The
0)

-Q


0





N Y N Y NY NY
Antlox Antlox* Antlox Antlox*
Chloro-N Chloro-Y Chloro-N Chloro-Y
Noise-N Noise-Y

Figure 7: Baseline to recovery threshold (in dB) results for the ABR click. The
chloroquine group continued to show the significant increase seen in previous time
period. The slant of the lines for the N-Ch and N-Ch-A group indicate that the N-Ch-A
had more recovery that can be attributed to the antioxidant.




The antioxidant effect was significant (p= .007) for the Ch- versus Ch-A group


comparison, which indicated that the antioxidant was providing some protection against


the negative effects of chloroquine.


As in the baseline to post-treatment time period, all four of the noise-exposed


groups, maintained a significant increase in threshold from baseline to recovery.


Comparing the N- and N-A groups, the N-A group had more recovery than the N- group,


although the antioxidant effect was not significant. Comparing the N-Ch to the N-Ch-A


groups, the N-Ch-A group had more recovery from baseline and the antioxidant effect










was significant (p=. 007). This indicates that antioxidant was assisting during the baseline

to recovery time period.

Response variable--post-treatment to recovery

For all the non-noise exposed groups (Figure 8), the change in threshold from post

treatment to recovery was not significant and no antioxidant effects were present. The

noise exposed group continued to have a significant threshold change. Comparing the N-

and N-A groups, the antioxidant effect was significant which indicated that the

antioxidant assisted in the recovery from the noise exposure. Although not significant

when comparing the N-Ch to N-Ch-A groups, the antioxidant was apparently still aiding

in the recovery process.






Change from POST-TREATMENT to RECOVERY

ABR Click
(Least-squares means with 95% confidence intervals)
o


E o ... .. ..... ....... ... ............. ............ ............
4--










NY NY NY NY
Antiox Antiox Antiot Antiox
Chloro-N Chloro-Y Chloro-N Chloro-Y
Noise-N Noise-Y


Figure 8: Post-treatment to recovery thresholds (in dB) for the ABR click. For the non-
noise groups no significant change between the two time periods. For the noise-exposed
groups, the presence of antioxidant appears helpful in the recovery process.









ABR Results All Stimulus Conditions-Tabular

It is important to note that in reviewing the three previous figures in most cases

the presence of antioxidant was associated with smaller shifts from baseline. Generally,

the trend of antioxidant providing a protective effect was evident in the rest of the ABR

stimulus conditions as evidenced by the following tables.

This is an orientation to the tables that will be used to for the presentations of all

of the ABR data. A double plus sign (++) indicated that the change of the response

variable for a given interval i.e. baseline to post-treatment was in a positive direction and

indicated significance defined as p < 0.05. A single plus (+) indicated trends associated

with p values of p< 0.1 but > 0.05. In the case of the ABR a positive direction change

would mean an increase (worse) in threshold.

The minus (-) or (- -) indicates that the change between two intervals was negative

and level of significance is the same as above, and for the ABR, would indicate a

decrease (better) in threshold. One or two asterisks indicate that the significance as define

above of the antioxidant effect was present for that treatment group.

Again the treatment groups are divided into two broad categories of non-noise

exposed and noise exposed. In the non-noise exposed condition (Table 4) the response

variables for the intervals of baseline to post-treatment, had no significant change except

for the chloroquine group that had a trend towards significance (p< 0.1) for 8-16 kHz. No

antioxidant effect was present for the previously mentioned stimulus conditions. The

noise-exposed groups all had the expected significant change in threshold and no

significance for the antioxidant effect









Table 4: Auditory Brainstem Response (ABR) Baseline to Post-Treatment for all
Stimulus Conditions
Experimental Factors ABR
Noise Chloroquine Antioxidant 4 kHz 8 kHz 12 kHz 16 kHz Click
N N N
N N Y
N Y N + + + ++
*
N Y Y
Y N N ++ ++ ++ ++ ++
Y N Y ++ ++ ++ ++ ++

Y N Y ++ ++ ++ ++ ++

Y Y N ++ ++ ++ ++ ++
*
Y Y Y ++ ++ ++ ++ ++

Y= received treatment. N= did not receive treatment; (-) = p> 0.05 but < 0.1, (- -)= p<
0.05 (*) presence of antioxidant effect.

The response variables for non-noise exposed groups for the intervals of baseline

to recovery (Table 5) continue to show no significant change from baseline except for the

Ch- group. The chloroquine group was significant for threshold increase across all the

stimulus conditions and significant antioxidant effects were present for 12 kHz (p=.007),

16 kHz (p=.000) and the click(p=.007). An antioxidant trend was noted at 4 kHz (p=

.050). This suggests that the antioxidant provided a protective effect against the

chloroquine. The noise exposed groups continued to show a significant change in

threshold from baseline, again as expected. An antioxidant effect was only present at 4

kHz. For the non-noise exposed groups for the interval of post-treatment to recovery

(Table 6), no significant change was noted for the groups except at 4 kHz for the control

group, which had a significant decrease in threshold.













Table 5: ABR Baseline to Recovery for all Stimulus Conditions
Experimental Factors ABR
Noise Chloroquine Antioxidant 4 kHz 8 kHz 12 16 Click
kHz kHz
N N N
N N Y
N Y N ++ ++ ++ ++ ++
** ** **

N Y Y
Y N N ++ ++ ++ ++ ++
Y N Y ++ ++ ++ ++ ++
Y Y N ++ ++ ++ ++ ++
**

Y Y Y ++ ++ ++ ++ ++



Y= received treatment. N= did not receive treatment; (-) = p> 0.05 but < 0.1, (- -)= p<
0.05 (*) presence of antioxidant effect.


The noise exposed groups all had significant decreases in threshold indicating that

recovery of threshold was continuing from post-treatment to recovery. A comparison of

the N-Ch and N-A groups indicated that there was an antioxidant trend that appeared to

assist with recovery at 12 kHz (p=. 064) and 16 kHz (p=. 070). (p=.094). It appeared that

although the antioxidant in the N-A treatment condition was not able to reduce the

amount of threshold shift it was able to aid in recovery.












Table 6: Post-Treatment to Recovery for all Stimulus Conditions
Experimental Factors ABR
Noise Chloroquine Antioxidant 4 kHz 8 kHz 12 kHz 16 kHz Click
N N N --
N N Y
N Y N
N Y Y
Y N N -- -- -- -- --
*
Y N Y -- -- -- -- --
Y Y N -- -- -- -- --

Y Y Y -- -- -- -- --


Y= received treatment. N= did not receive treatment; (-)
0.05 (*) presence of antioxidant effect.


p> 0.05 but < 0.1, (- -)= p<


Distortion Product Otoacoustic Emissions

Morphology

Distortion product otoacoustic emi ssions were observed in response to intensity

levels of 50, 60, and 70 dB SPL for 8 kHz. The response was considered to be present if

the amplitude of the response was 3 dB or greater than the noise floor. The noise floor

was calculated as the average of the two positive peaks on either side of the DPOAE

response. An increase in amplitude indicated that the response was stronger (more

robust) and a decrease indicated the response was weaker. In this example (Figure 9) the

amplitude of the noise floor was -22.5 dB SPL and the amplitude of the response was 17

dB SPL. The difference of 31 dB between the two indicated that the response was well

above the 3 dB criteria. The stimulus tones were indicated as fi and f2.










-1 OdBv

fi
-30dBv f2


-SOdBv
DPOAE


-70dBv


-90dBv



5396 8Hz 6222.2Hz 7047.6Hz 7873 OHz 8698 4Hz 9523.8Hz



Figure 9: DPOAE at 8 kHz 60 dB for one control animal.




Average DPOAE Amplitudes by Groups

As with the ABR, the eight treatment groups were divided into two broad

categories: non-noise exposed which consisted of control, chloroquine (Ch-), antioxidant


(-A), and chloroquine and antioxidant (Ch-A) and, the noise exposed group consisted of

the noise (N-), noise and chloroquine (N-Ch), noise and antioxidant (N-A) and noise,

chloroquine and antioxidant (N-Ch-A). The two broad categories were then compared

over time. The time periods represented are baseline, (prior to receiving any treatment)

post-treatment (immediately after treatment) and recovery (24 hours post treatment).

The results of the DPOAE at 4 kHz were weaker and variable which made it

difficult to analyze. A determination was made to drop the results of 4 kHz from the

study. The data presented in Figure 10 are the mean baseline amplitudes for the non-

noise exposed groups at 8 kHz (60 dB). The amplitude values were consistent with

ranges of up to 30 dB as cited in the literature (Robinette & Glattke, 1997; Hall, 2000).









For the baseline time period, the amplitudes were closely aligned across groups. At the

post-treatment time period, the chloroquine group showed a clear separation (decrease in

amplitude) from the other groups. At the recovery time period, the chloroquine group

continued to have a decrease in amplitude. The noise exposed group at baseline (Figure

11) had amplitudes that were closely aligned and similar to baseline for the non-noise

exposed groups. During the post treatment time period the N-Ch-A group had the least

amount of change in amplitude. The N-, N-A, N-Ch groups were closely associated. The

N- group showed the greatest decrease in amplitude from baseline. At the recovery time

period, the N-Ch-A, N-A, and N-Ch groups had more recovery of amplitude than the N-

group. The N-Ch-A group had the most recovery. As with the ABR the antioxidant

appeared to provide a protective effect against the ototoxicity of chloroquine and assisted

with the recovery from noise exposure.

Statistical Methods

The same statistical methods used to analyze the ABR were used to analyze the

DPOAE. For each of the stimulus conditions, 8 kHz at 50, 60 & 70 dB, three "change in

emission amplitudes over time" response variables were defined. The first represents the

change in amplitudes from baseline to post-treatment. The second represents the change

in amplitudes from baseline to recovery. The third represents the change in amplitudes

from post-treatment to recovery. For each of the stimulus conditions tested, each of their

respective response variables were analyzed separately using 3-way analysis of variance

(ANOVA). In analyzing the first two response variables that involved change from

baseline, baseline amplitude levels were included as a covariate in the 3-way ANOVA in

order to control for variation in baseline sensitivity among individual animals across all











treatment groups. As with the ABR, contrasts between groups were made to determine


the influence of the antioxidant on the results.


Figure 10: Average DPOAE amplitudes for the non-noise exposed groups over the time
periods of baseline, post-treatment and recovery. Chloroquine group showed a
noteworthy change in amplitude and had the least amount of recovery.


Baseline: Non-Noise DP 8k


25

20 -
-1 Control
15 Ch-


5 Ch-A
0 -A


ca
-5 70 60 50

-10

-15


dB SPL


Post-Treatment: Non-Noise DP 8k


-*- Control
--- Ch-
-A
Ch-A


dB SPL







82



Recovery:Non-Noise DP 8K


25

20
15 -- Control
15 -- Ch-
10 -A

5 Ch-A
0
-5 70 60 s0

-10

-15


dB SPL


Figure 10---continued







































































Figure 11: Average DPOAE amplitudes for the noise exposed groups over the time
periods of baseline, post-treatment and recovery. The N-Ch-A group had least amplitude
change for the post-treatment and recovery time periods.


Baseline: Noise DP8k

25
20 ------ N -
15 ---N-Ch
10 N-A
5 N-Ch-A
0
S -5 70 60 50

-10
-15
dB SPL


Post Treatment: Noise DP8k
25

20
15 -- N-
---N-Ch
10
N-A
5
N-Ch-A
C,
- 5 -- - -_ -- 6 0 0 _

-10
-15


dB SPL





























Figure 11---continued

Graphic Plots of the Data

The results are presented in two forms, graphic and tabular. The graphic figures

allow for an in depth review of the results for one type of stimulus condition (8 kHz at 60

dB) across the groups. The tabular presentation of the results, depict the results for all

treatment conditions and groups.

Recall that the response variable represents the change in hearing function for the

following intervals: baseline to post treatment, baseline to recovery (48 hours post noise

exposure), and post-treatment to recovery. For the x-axis on Figure 12, the groups are

divided into two broad categories, non-noise exposed (Noise-N) and noise exposed

(Noise-Y). The sub-category of chloroquine (Chloro-N) further divides the graph and

finally the presence or absence of antioxidant in indicated. The data points on the figure

represent the mean change from baseline to post treatment for each group. The vertical

bars that bracket each mean represent the 95% confidence interval. The y-axis represents




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