Immunosuppression by Aflatoxin B 1 in C57BL/5 Mice and its Relationship with Neuroendocrine ...

118 Pages·2016·18.27 MB·English

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All Graduate Theses and Dissertations Graduate Studies
5-1990
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Yasuhiko Hatori
Utah State University
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Hatori, Yasuhiko, "Immunosuppression by Aflatoxin B1 in C57BL/6 Mice and its Relationship with
Neuroendocrine Mechanisms" (1990). All Graduate Theses and Dissertations. 4647.
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IMMUNOSUPPRESSION BY AFLATOXIN B, IN C57BU6 MICE AND ITS
RELATIONSHIP WITH NEUROENDOCRINE MECHANISMS
by
Yasuhiko Hatori
A thesis submitted in partial fulfillment
of the requirements for the degree
of
MASTER OF SCIENCE
in
Toxicology
Approved:
UTAH STATE UNIVERSITY
Logan, Utah
1990
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my major professor, Dr.
Raghubir P. Sharma, for his professional guidance, encouragement, patience, and
support. His wisdom and kindness will be deeply missed.
I also want to thank Dr. Akiyo Shigematsu, the president and director of the
Institute of the Whole Body Metabolism in Japan for his support and advice. He
is the one who provided me the opportunity to undertake the graduate work and the
necessary help. All our colleagues at the institute are always appreciated for their
friendship.
I also want to thank Drs. Reed P. Warren and LeGrande C. Ellis for serving
on my supervisory committee and for their valuable advice. I would especially like
to mention the valuable cooperation of Dr. Warren in allowing me to use the
instruments in his laboratory.
I am also thankful to Roger Burger and Michael Huie for their timely technical
advice and help in performing various experiments and using computers.
I also extend my thanks to my laboratory mates and friends: Yang Won
Kim, Gagandeep Suri, Michael Chen, Akiko Hatori, Michael Taylor, Brian Hughes,
Gin-Chang Hsieh, Sunil Jayasekara, Brian Howard, Evert Brown, Charles Healey,
Hind Abdul-kareem, and Andrzej Jureck for helping in various immunotoxicology
methods, telling me to correct my English, assisting in my experiments, and giving
their friendship.
I owe my parents, Tadao Hatori and Kazuko Hatori, and parents-in-law,
Sadao Suzuki and Kichiyo Suzuki, for their sincere help, understanding, and their
great affection.
Special thanks to my wife, Akiko, who studied in the toxicology program at
iii
the same time. Her encouragement and help was great in our home and in the
laboratory.
I owe a lot to my lovely son, Akihiro; he helped me to relax when I was
tired, and he was patient when I was busy.
I am thankful to all the professors and faculty at U.S.U. for their help in
developing my knowledge. I will always miss the beautiful surroundings of Logan.
I am sure to miss everybody I met and everything I experienced during my stay in
Utah.
Yasuhiko Hatori
iv
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
LIST OF ACRONYMS USED IN TEXT . . . . . . . . . . . . . . . . . . . . . . . . . vi
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
ABSTRACT xii
Chapter
I. INTRODUCTION
II. LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Aflatoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Aflatoxin B, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
lmmunotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
The effects of Aflatoxin B, exposure on the immune system . . . . . . 15
Neurochemical approaches to toxicology . . . . . . . . . . . . . . . . . . . 16
Neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
The effects of Aflatoxin B, exposure on neurotransmitters . . . . . . . 18
Relationships of the immune, endocrine, and neurotransmitter
systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Hypothalamus-pituitary-adrenal gland (HPA) axis . . . . . . . . . . . . . 21
Ill. MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Aflatoxin B, exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Evaluation of immune responses after AFB1 treatment . . . . . . . . . 28
Assay of neurotransmitters and their metabolites . . . . . . . . . . . . . 34
Endocrine hormone measurements . . . . . . . . . . . . . . . . . . . . . . 35
Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
IV. RESULTS 39
SET I. Effects of AFB1 on C57BU6 mice .......... . .. . 39
SET II. Effects of adrenalectomy on the response of C57BU6
mice to AFB, ............... . ... ........ . 57
v
TABLE OF CONTENTS (Continued)
Page
V. DISCUSSION 74
General effects of AFB, exposure . . . . . . . . . . . . . . . . . . . . . . . 74
The effects of AFB, on immune system . . . . . . . . . . . . . . . . . . . 74
The effects of AFB, on brain amine . . . . . . . . . . . . . . . . . • . . . . 76
The effects of AFB, on endocrine hormones . . . . . . . . . . . . . . . . 77
Strain susceptibility . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . 79
The effects of adrenalectomy . . . . . . . . . . . . . . . . . . . . . . . . . . 80
General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
VI. CONCLUSIONS . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . 82
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . 83
APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
vi
LIST OF ACRONYMS AND ABBREVIATIONS USED IN TEXT
A.C .: adrenalectomized animal control group
ACTH: adrenocorticotropic hormone
A.D.V.S.: Animal, Dairy, and Veterinary Sciences
AFB,: Aflatoxin B,
AFB2: Aflatoxin B2
AFG,: Aflatoxin G,
AFG2: Aflatoxin G2
AFM,: Aflatoxin M,
A. H.: adrenalectomized animal group treated with high dose AFB,
ANOVA: analysis of variance
CMI: cell mediated immunity
ConA: concanavalin A
CRF: corticotropin releasing factor
DA-P-hydroxylase: dopamine-P-hydroxylase
DTH: delayed-type hypersensitivity
DA: 3,4-dihydroxyphenethylamine (dopamine)
DNA: deoxyribonucleic acid
EDTA: ethylenediaminetetraacetic acid
ELISA: enzyme linked immunosorbent assay
molar absorptivity
EPI: epinephrine
FAGS: fluorescence activated cell sorting
FBS: fetal bovine serum
5-HIAA: 5-hydroxyindole-3-acetic acid
vii
LIST OF ACRONYMS AND ABBREVIATIONS USED IN TEXT (Continued)
5-HT: 5-hydroxytryptamine
GST: glutathione S-transferase
HPA axis: hypothalamus-pituitary-adrenal gland axis
HPLC: high-performance liquid chromatography
HVA: 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid)
lgM: immunoglobulin M
IFNa: interferon
IL-2: lnterleukin-2
LARC: Laboratory Animal Research Center
LD median lethal dose
50
LPS: lipopolysaccharide
LSD: Fisher's least significant differences
ME: mercaptoethnol
MLC: mixed lymphocyte culture
N.C.: non-operated animal control group
NE: [-]-norepinephrine
N.H.: non-operated animal group treated with high dose AFB,
NK cell: natural killer cell
PFC: plaque forming cell
PHA: phytohemagglutinin
PNMT: phenylethanolamine N-methyl transferase
PWM: pokeweed mitogen
RES: reticuloendothelial system
RIA: radioimmunoassay
viii
LIST OF ACRONYMS AND ABBREVIATIONS USED IN TEXT (Continued)
RNA: ribonucleic acid
SRBC: sheep red blood cells
3H-TdR: [methyl-3H]-Thymidine
VMA: dl-4-hydroxy-3-methoxy-mandelic acid
SE: standard error
S.l.: stimulation index
S.C.: sham-operated animal control group
S.H.: sham-operated animal group treated with high dose AFB,
TLC: thin-layer chromatography
UV: ultra violet
ix
LIST OF TABLES
Table Page
11-1 PHYSICOCHEMICAL CHARACTERISTICS OF AFB, . . . . . . . . . . . . 7
11-2 TOXICITY OF AFB, IN VERTEBRATES . . • . • . • . . . . . . . . . . . . . 9
11-3 IMMUNE FUNCTION ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
IV-1 EFFECTS OF AFB, FOUR-WEEK EXPOSURE ON BODY WEIGHT
GAIN OF C57BU6 MICE .. .. ........ ......... ... .... . . 40
IV-2 EFFECTS OF AFB, FOUR-WEEK EXPOSURE ON ORGAN WEIGHTS
AND PERIPHERAL BLOOD COUNTING OF C57BU6 MICE . ..... 41
IV-3 EFFECTS OF AFB, FOUR-WEEK EXPOSURE ON PHENOTYPIC
DISTRIBUTION OF SPLENOCYTES OF C57BU6 MICE ANALYZED
BY FLOW CYTOMETRY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
IV-4 EFFECTS OF AFB, FOUR-WEEK EXPOSURE ON MIXED
LYMPHOCYTE RESPONSE OF C57BU6 MICE MEASURED BY MLC
ASSAY . .. . . .. . . .. ... ... .. . . ......... . .... . .. . ... 45
IV-5 EFFECTS OF AFB, FOUR-WEEK EXPOSURE ON NATURAL KILLER
CELL ACTIVITY OF C57BU6 MICE MEASURED BY 51Cr RELEASE
ASSAY ......... ...... .. . . .. .. ............ ... . . .. 46
IV-6 EFFECTS OF ADRENALECTOMY ON BODY WEIGHT GAIN OF
C57BU6 MICE FOLLOWING FOUR-WEEK AFB, EXPOSURE . .. . . 58
IV-7 EFFECTS OF ADRENALECTOMY ON ORGAN WEIGHTS AND
PERIPHERAL BLOOD COUNTING OF C57BU6 MICE FOLLOWING
FOUR-WEEK AFB, EXPOSURE . . . . . . . . . . . . . . . . . . . . . . . . . 59
IV-8 EFFECTS OF ADRENALECTOMY ON PHENOTYPIC DISTRIBUTION
OF SPLENOCYTES OF C57BU6 MICE FOLLOWING FOUR-WEEK
AFB, EXPOSURE ANALYZED BY FLOW CYTOMETRY . . . . . . . . . 60
IV-9 EFFECTS OF ADRENALECTOMY ON MIXED LYMPHOCYTE
RESPONSE OF C57BU6 MICE FOLLOWING FOUR-WEEK AFB,
EXPOSURE MEASURED BY MLC ASSAY .... ... .. . . ... ... . 63
IV-10 EFFECTS OF ADRENALECTOMY ON NATURAL KILLER CELL
ACTIVITY OF C57BU6 MICE FOLLOWING FOUR-WEEK AFB,
EXPOSURE MEASURED BY 51Cr RELEASE ASSAY . .. ........ 64