EPA-600/1-77-042
September 1977 Environmental Health Effects Research Series
METABOLIC INTERACTIONS OF HORMONAL
STEROIDS AND CHLORINATED
HYDROCARBONS-Effects of Neonatal
Treatment with o,p'-DDT on the
Development of the Steroidogenic
Endocrine System of the Male Rat
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-77-042
September 1977
METABOLIC INTERACTIONS OF HORMONAL STEROIDS
AND CHLORINATED HYDROCARBONS
Effects of Neonatal Treatment with o.p'-DDT on the Development
of the Steroidogenic Endocrine System of the Male Rat
by
Kenneth Lyle Campbell
The University of Michigan Medical School
Ann Arbor, Michigan 48104
Principal Investigator
Merle Mason, Ph.D.
Associate Professor of Biological Chemistry
The University of Michigan Medical School
Ann Arbor, Michigan 48104
Grant Number R-800637
Project Officer
Laurence Rosenstein, Ph.D.
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
The study described herein was initiated to meet program requirements
concerning the improvement of in vivo measurements involved in determining
the potential toxicity of xenobiotics on reproduction. The information
gained from this study has permitted the evaluation of the effects on the
developing endocrine system of a rat following neonatal exposure to a
model compound, o,p'-DDT. It is anticipated that the model developed in
this study can be applied to other compounds and species and thus serve
as a useful adjunct to toxicology research.
ljohn H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
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ACKNOWLEDGEMENTS
This project was part of the doctoral dissertation research
of Kenneth Lyle Campbell, under the mentorship of Dr. Merle Mason.
Support of Mr. Campbell during the tenure of his program was derived
from fellowships from The University of Michigan Institute for
Environmental Quality and the Albert Euclid Hinsdale Memorial
Fellowship Fund and from a traineeship from the National Institutes
of Health (Grant No. 5-T01-GM-00187-15) and is gratefully acknowledged.
Experimental assistance and help in the interpretation of results
was obtained from the Reproductive Endocrinology Program, The
University of Michigan. Dr. Fred Karsch of that program was
especially helpful in interpreting the radioimmunoassays and in
designing some experiments.
Maija Mitzens provided invaluable support and assistance in
writing the MetFLX program and in many other aspects of research and
writing.
This document is submitted as a final report under U.S.
Environmental Protection Grant No. R-800637 with Dr. Merle Mason.
iv
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TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF APPENDICES xii
LIST OF ABBREVIATIONS xiii
CHAPTER 1. INTRODUCTION 1
I. Steroidogenic Endocrine Functiont 2
II. Steroidogenic Endocrine Developments
Imprinting 12
III. Steroidogenic Endocrine Development: The
Normal Time-Course 18
IV. Metabolism and Physiological Effects of DDT: 2k
CHAPTER 2. MATERIALS AND METHODS 4l
I. Animals: *H
II. Chemicals:
III. Treatments:
IV. Measurements: 50
A. Organ Weights: 50
B. Organ Histologies: 50
C. Serum Corticosterone Measurements: 51
D. Radioimmunoassays: 58
1. The Assay 58
2. Effect of Sampling Time and Ether 6*4-
E. Estimation of o,p'-DDT Derived Residues
in Rat Pups 69
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TABLE OP CONTENTS (Cont.)
P. Testicular Incubationsi 81
1. Protocol 81
2. Estimation of Steroids by Gas
Chromatography and Scintillation
Counting 89
CHAPTER 3. EXPERIMENTS ON THE EFFECTS OP DIRECT
INJECTION OP o,p'-DDT.INTO NEONATAL
MALE RATS 101
I. Organ Weights in Intact Male Rats and in
Neonatally Castrated Adult Male Rats Neo-
natally Injected with o,p'-DDT.i 101
II. Developmental Time Course and Dose-Response
to Neonatally Administered o.p'-DDT: 109
A. Body and Organ Weights» 111
B. Organ Histologies! 135
C. Serum Corticosterone: 137
D. Serum LH: 150
III. Serum LH Response to Adult Castration: 156
CHAPTER 1*. EXPERIMENTS ON THE EFFECTS OF ADMIN-
ISTERING o.p'-DDT TO NEONATAL MALE RATS
VIA THEIR MOTHER'S MILK 169
I. Administration of o.p'-DDT to Neonatal Rats
Via the Dam: 169
II. Organ Weights in Adult Rats Neonatally
Treated with o,p'-DDT Via Their Dams: 183
III. Periodicity of LH Release in Adult Rats
Neonatally Exposed to DDT: 188
IV. Serum LH Response to Adult Castration: 193
V. Serum LH Response, to Exogenous LHRH: 199
VI
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TABLE OF CONTENTS (Cont.)
CHAPTER 5. DISCUSSION OF ANIMAL STUDIES 210
I. The Change in the Immature Adrenal Cortex: 212
II. The Change in the Hypothalamic-Hypophysial
Complex: 217,
III. Summary! 223
CHAPTER 6. INVESTIGATION OF A ^-PREGNENOLONE
CONVERSION IN VITRO 225
LIST OF REFERENCES
APPENDIX 261
vii
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LIST OF TABLES
Table Page
1. Description of Neonatal "Imprinting" of the
Hypothaiamus of the Rat 15
2. Standard Curves for Determination of DDT
Analogs "by Electron-Capture Gas Chromatography 79
3. Incubations Performed 83
*K Standard Curves for Determination of Steroids
by Gas Chromatography 9^
5, ^H/ C Ratios: Evidence Supporting Most of the
Steroid Identities Assigned to the Gas
Chromatographic Peaks Obtained During Analysis
of Steroid Metabolite Mixtures 98
6. Effects of Neonatal Injection with o,p'-DDT or
Estradiol on Body and Organ Weights of Intact
and Neonatally Castrated Male Rats 10^
7. Frequency of Increased Reticular Vascularity
in 25 Day Old Rats Injected Neonatally with
o,p'-DDT 142
8. Two-Way Analysis of Variance for Castration
Response Data from Adult, Neonatally Injected
Male Rats: Analysis of Days After Castration
and Before Testosterone Administration 163
9. Effects of Suckling an o,p'-DDT Injected Dam
on Body and Organ Weights of Intact and
Neonatally Castrated Male Rats 185
10. Mnemonics of METFLX 26?
11. Description of the Input Data Format for METFLX 273
viii
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LIST OF FIGURES
Figure
1. Major Steroid Pathways in the Rat 4-
2. Schematic Diagram of the Steroidogenic
Endocrine System of the Male Rat 8
3. Major Metabolic Routes of o,p'-DDT 29
b. Schematic of Approach and Treatments ^0
5. Protocol for Measuring Serum Corticosterone
Levels, Modified from Mattingly and Silber 53
6. Verification of the Sulfuric Acid Fluorescence
Assay for Serum Corticosterone: Parallelism
of the Standard Curve with Serially Diluted
Rat Serum 57
7. The Relationships Between the Response Curves
for Rat Serum and Pituitary Extract and Those
of Radioimmunoassay Standards B-6^0 and B-873 62
8. The Effect of Repeated Tail-Vein Bleeding
Under Ether on the 2^-Hour Periodicity of Serum
LH in Normal Adult Male Rats 68
9. Generalized Protocol for Estimation of DDT
Analog Residues 72
10. Typical Gas Chromatograms Obtained During
Analysis of o,p'-DDT and Its Analogs 77
11. Protocol for the Extraction and Partial
Purification of DDT and Steroid-Metabolites
in in vitro Incubations 87
12. Typical Gas Chromatograms Obtained During
Analyses of Testicular A5_pregnenolone
Metabolites . 91
13. Body Weights of Neonatally Injected Male Rats 11^
1*K Organ Growth in Neonatally Injected Male
Rats: Pituitary 116
15- Organ Growth in Neonatally Injected Male
Rats: Testes 118
16. Organ Growth in Neonatally Injected Male
Rats: Adrenals 121
ix
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LIST OP FIGURES (Cont.)
Figure
17. Organ Growth in Neonatally Injected Male
Rats: Seminal Vesicles
18. Organ Growth in Neonatally Injected Male
Rats: Ventral Prostate 12?
19. Organ Growth in Neonatally Injected Male
Rats: Liver 130
20. Organ Growth in Neonatally Injected Male
Rats: Kidneys 133
21. Normal Adrenal Development in the Male Rat:
Reticular Zone Histology 139
22. Adrenal Reticular Zone Histology at 25 Days
of Age in the Neonatally Injected Male Rat
23. Serum Corticosterone in Neonatally Injected
Male Rats .
2^. Serum Corticosterone in 50 and 75 Day Old
Male Rats Versus Dose of Neonatally Injected
o,p'-DDT
25. Serum LH in Neonatally Injected Male Rats 153
26. Response of Serum LH to Adult Castration in
Neonatally Injected Male Rats 160
27. Response of Serum LH to Adult Castration in
Neonatally Injected Male Rats 162
28. Secretion of DDT Analogs in Rat Milk 173
29. Uptake of DDT Analogs by Suckling Rats 175
30. Uptake of o,p'-DDT by Suckling Rat. Pups:
Total Body Burden Versus Cumulative Dose 181
31. 24-Hour Periodicity of Serum LH in Adult Male
Rats Treated Neonatally Via Their Dams 192
32. Response of Serum LH to Adult Castration in
Male Rats Treated Via Their Dams 199
33. Response of Serum LH to Injection of Large
Doses of LHRH in Adult Male Rats Treated Via
Their Dams 203
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LIST OF FIGURES (Cont.)
Figure page
3^. Flowchart for METFLX 2?0
XI
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LIST OF APPENDICES
Appendix Page
I The Program METFLX 26l
xii
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LIST OF ABBREVIATIONS AND COMMON NAMES
ACTH
Aldrin
A*-
ANOVA
BSA
C
cAMP
Cholesterol
CBG
Ci
CPE
CRH
DDA,DDE,DDD,DDT
o,p'-DDA
p,p'-DBA
o,p'-DDD
p,p'-DDD
o,p'-DDE
adrenocorticotrophic hormone
I,2,3,4,10,10-hexachloro-l,4j4af5,8,8a-
hexahydro-exo-l,^-endo-5,8-dimethano-
naphthalene
k
A -androstenedionej 4-androsten-3,17-
dione
analysis of variance
bovine serum albumin
control
cyclic 3',5'-adenosine monophosphate
5-cholesten-3oC-ol
corticosteroid binding globulin
Curie
5-cholesten-3
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LIST OP ABBREVIATIONS AND COMMON NAMES (Cont.)
p.p'-DDE
p,p'-DDMU
o,p'-DDT
p,p'-DDT
DHA
DMSO
dpm
EST
estradiol
estriol
EV
FID
FSH
GC
GnRH
G-6-P
G-6-PDH
hCG
2,2-dichloro-l,l-bis-(^-chlorophenyl)-
ethylene ; 2 , 2-Ms- ( ^-chlorophenyl )-!,!-
dichloroethylene
2-chloro-l,l-Ms(4~chlorophenyl) -ethyl ene
1,1, 1- trichloro-2- ( 2-chlorophenyl ) -2- (4-
chlorophenyl ) - ethane
1,1, 1- trichloro-2 , 2-bi s- (^-chlorophenyl ) -
ethane
dehydroepiandrosterone; 5-androsten-3°^ -
ol-17-one
dimethyl sulf oxide
disintegrations per minute
Eastern standard time
1? P -estradiol; l,3,5(10)-estratriene-
3,17? -diol
I,3,5(10)-estratriene-3,l6cc,170 -triol
17 ^ -estadiol-17-valerinate
flame ionization detector
follicle stimulating hormone
gas chromatography
gonadotrophin releasing hormone
glucose-6-phosphate
glucose-6-phosphate dehydrogenase
human chorionic gonadotrophin
17 (X. -hydroxyprogesterone; 4-pregnen-
-ol-3, 20-dione
ICSH
i .p.
LD,.rt
interstitial cell stimulating hormone (LH)
intraperitoneal
lethal dose to
xiv
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LIST OF ABBREVIATIONS AND COMMON NAMES (Cont.)
LH luteinizing hormone
LHRH luteinizing hormone releasing hormone
Mirex dodecachlorooctahydro-1,3,4-metheno-2H-
cyclobuta [cd] pentalen-2-one
MTS Michigan Terminal Systems
NADH reduced nicotinamide adenine dinucleotide
NADP nicotinamide adenine dinucleotide
phosphate
OAAD ovarian ascorbic acid depletion
oLH ovine luteinizing hormone
P progesterone? *f- pregnen-3i20-dione
PB phenobarbital
PBS phosphate-buffered-saline
PCB's polychlorinated biphenyls
pg picogram
POPOP l,^-bis-[2-(5-phenyloxazolyl)] -benzene
ppm parts per million
PPO 2,5-diphenyloxazole
psi pounds per square inch
t t*
A -?- A -'-pregnenolone i 5-pregnen-3cC -ol-20-
one
radioimmunoassay
revolutions per minute
subcutaneous
2-diethylaminoethyl-2,2-diphenylpentano-
ate
testosterone? b-androsten-17^ -ol-3-one
p,p'-DDD + 10$ o,p'-DDD
RIA
rpm
s.c.
SKF-525A
T
technical DDD
xv
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LIST OF ABBREVIATIONS AND COMMON NAMES (Cont.)
technical DDT BOfo p,p'-DDT + 15-20$ o,p'-DDT
TLC thin layer chromatography
TP testosterone-17-proprionate
TVBE tail vein-bleeding under ether anesthesia
xvi
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CHAPTER 1
INTRODUCTION
This investigation was undertaken to determine the
feasibility of using the measurement of specific endocrine
functions as a general screen for the effects of foreign
compounds on reproduction and viability. Its primary
purpose was to generate approaches which might be utilized
to shorten current toxicological screening procedures by
augmenting or supplanting the multigenerational tests now
used to measure effects on reproduction. Secondarily, its
goal was to establish whether l,l,l-trichloro-2-(2-chloro-
phenyl)-2-(4-chlorophenyl)-ethane (o,p'-DDT) has any effect
on the steroidogenic endocrine tissues of the developing
male rat and if so, what mechanism was involved in
producing the effect.
The experiments to be described do shed some light on
the secondary questionsand as a result imply the utility
of at least a portion of the approach for use in screening
protocols. Of equal importance, perhaps, they indicate
the potential for utilization of xenobiotics, such as
o,p'-DDT, to produce pathologic states which can provide
information about the normal development and function of
the endocrine system that is not easily obtained by known
procedures. Finally, these experiments indicate both a
direction for future investigations on the mode of action
of o,p'-DDT in the steroidogenic endocrines and a crude
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estimation of how much data of this type must be amassed
to generate conclusions of use in the legal regulation of
synthetic chemicals.
In order to understand the experiments detailed in
Chapters 3-5 and their relation to the objectives an
exposition of some of the background literature and the
resultant experimental approaches and reasoning is neces-
sary. To that end I will briefly discuss the present
models of steroidogenic endocrine function and development
and the known metabolism and biological effects of DDT.
I. Steroidogenic Endocrine Functioni
The physiologic "purpose" of the steroidogenic endo-
crines appears to be the production of the steroid hor-
mones; the major synthetic paths are indicated in Figure
1. The active forms of these hormones typically show
effects on target tissues possessing intracellular steroid
receptor proteins (1,2). Usually these effects are trophic
in nature and lead to growth, proliferation and/or differ-
entiation of the target tissue. However, in the case of
the hypothalamic centers and at least in some of the pitu-
itary cell types (3) the steroids act by suppressing
release of the releasing hormones (-RH, -RF) or the trophic
hormones. In this latter manner they indirectly control
their own production. Likewise, by effecting the func-
tions of the adrenals and/or kidneys and liver (*4-,5), they<>
may ostensibly exert some control over their own inactiva-
tion as well.
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Figure 1. Major Steroid Pathways in the Rat
Shown is a composite of the major pathways found in
the rat testis, adrenal cortex and ovary. It summarizes
the probable sites of action for both trophic hormones
(luteinizing hormone - LH, adrenocorticotrophic hormone -
ACTH) and 3',5'-cyclic adenosine monophosphate (cAMP) as
well as the various enzymes involved in processing the
precursor cholesterol. The enzymes and enzyme complexes
shown ares 0 = cholesterol-acyl esterase; 1 = 20*C -hy-
droxylase + 22R-hydroxylase; 2 = C20-22 lvase; 3 =
A-^-30 -hydroxysteroid dehydrogenase; ^ «=• A-*--* A
isomerase; 5 = 21-hydroxylase; 6 = 11(3 -hydroxylase 5
7 = 17of -hydroxylase; 8 = C.,~ 20 lyase; 9 = aromatizing
enzyme complex; 10 = 17f -hydroxysteroid dehydrogenase.
Substrate specific forms of many of these enzymes occur in
the various tissues.
Those pathways occurring in the testis include the
A - and A^-pathways proceeding from cholesterol and
L
pregnenolone to testosterone and A -androstenedione using
enzymes 1,2,3i^i7t8 and 10* In the adrenals the same path-
ways occur but utilize enzymes 5 and6 to produce cortico-
sterone as the major active product. In the ovary the
major androgen pathways are still present but testosterone
and A -androstenedione undergo aromatization, 9» to form
the major estrogenic products 17fJ -estradiol and estrone.
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FIGURE I
MAJOR STEROID PATHWAYS IN THE RAT
CHOLESTEROL ESTERS
© I
LH OR ACTH
3+4 0"^
PRE6NENOLONE PROGESTERONE
5+6 0'
CORTICOSTERONE
7+8
9 HO
OEHYDROEPt-
ANDROSTERONE
A^ANDROSTENEDIONE
10
ESTRONE
HO'"*^ "**" 3+4
A^ANDROSTENEDIOL TESTOSTERONE
9 HO'
I7P-ESTRADIOL
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Subcellularly, steroids are formed in the adrenals
and gonads from cholesterol or cholesterol esters by the
action of enzymes localized in the lipid droplets (cho-
lesterol-acyl esterase), mitochondria (C_
and 11 f -hydroxylases) , endoplasmic reticulum (3{*- and
t; k,
17 (? -hydroxysteroid dehydrogenases, A^--*A -isomerase,
^17-20 ^yase» 17** ~ an(* 21-hydroxylases and aromatizing
enzymes) and cytosol (20cC -hydroxysteroid de hydro genase)
(6). These multienzyme pathways and the indicated com-
partmentalization allow subcellular modulation of steroid
synthesis. They also impose numerous potential sites for
the action of modulators, e.g., specific enzyme regulation,
membrane modification, blockage or stimulation of cosub-
strate (reduced nicotinamide adenine dinucleotide phos-
phate - NADPH) production, etc. This latter point becomes
particularly important and complex when the precise mode
of action of an extracellular agent is in question.
The rate of synthesis of the steroids appears to be
largely dependent on circulating trophic hormone levels
and the state of activation of intracellular adenylate
cyclase which is proximally associated with the trophic
hormone receptors. Stimulation of the cyclase, or inhibi-
tion of 3'»5'-cyclic nucleotide phosphodiesterase (7),
ultimately leads to protein synthesis generally and sub-
sequently to steroidogenic enzyme synthesis. In addition,
direct activation of cholesterol-acyl esterase and the
cholesterol side-chain cleavage complex occurs acutely
-------
upon stimulation of the cyclase (8). Thus, the rate of
steroidogenesis is both acutely and chronically controlled
by enzyme levels and activation states which are in turn
determined by trophic hormone levels.
Serum trophic hormone levels are controlled by the
rate of synthesis and release of hypophysial trophic hor-
mone stores. In their turn, the stores of these protein
hormones depend on the activation of cellular protein syn-
thesis and on cAMP concentrations which are controlled by
the level of certain peptides, the trophic hormone releas-
ing hormones (factors), in the hypophysial portal blood
(3,9). Further modulation occurs by the direct feedback
actions of the steroids as mentioned above. Once more, as
in the case of the trophic hormones acting on the adrenals
or gonads, an extracellular signal, -RH, controls the
synthesis and release of a hormone.
Finally, the release of the releasing hormones is
controlled by neurons which contain steroid receptors and
which are associated with the neurons'which produce and
release the releasing hormones into the portal vessels.
At this point, however, the modulation by steroid hormones
is inverse, and the negative feedback loop of classic
endocrinology is completed (Figure 2) (3,9,10).
Throughout this complex feedback system the ultimate
effects on steroid target tissues are subject to modulation
by parameters such as receptor levels, Ca + levels, blood
flow, multiple hormone synergisms, etc., which are in turn
-------
Figure 2. Schematic Diagram of the Steroidogenic
Endocrine System of the Male Rat
The products of the hypothalamus, the releasing hor-
mones (luteinizing hormone releasing factor - LHRH, or
gonadotrophin releasing hormone - GnRH, and corticotrophin
releasing hormone - CRH), are shown as being transported
to the anterior pituitary where they induce release (and
synthesis), ©, of the trophic hormones (LH or interstitial
cell stimulating hormone - ICSH, follicle stimulating
hormone - PSH,and ACTH). These hormones are, in turn,
transported via systemic circulation, -», to their respec-
tive target organs, the Steroidogenic glands. These
glands are stimulated, ®, by the trophic hormones to syn-
thesize their steroid products which are then circulated
throughout the body, -».• These steroids produce trophic
effects, ©, on their own respective target organs and pro-
vide feedback controls on the hypothalamus, 0. Simul-
taneously the steroids themselves are catabolized, —-», by
the liver and other tissues to less active'compounds which
may be eliminated.
Sperm production appears to be controlled by an inter-
play of both the pituitary and steroid hormones; FSH, LH
and testosterone all exhibit a stimulatory influence on
the process.
More complex relationships may exist among the
Steroidogenic glands themselves, (|), but these remain to
be clarified.
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FIGURE 2
SCHEMATIC DIAGRAM OF THE STEROIDOGENIC
ENDOCRINE SYSTEM OF THE MALE RAT
HYPOTHALAMUS
LHRH (GflRH). CRN
ANTERIOR
PITUITARY
CORTICOSTERONE
a
TESTOSTERONE
ADRENAL
CORTICOSTERONE
STEROID
METABOLITES a
CONJUGATES
SEMINAL
TESTOSTERONE
PROSTATE
LH (ICSH),
FSH
8
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determined by age, sex, nutrition, health, other physiolog-
ical states and/or the external environment. The feedback
loops, which help maintain organismic homeostajKLs, and the
sensitivity of these loops to large numbers of diverse mod-
ifying influences appear to be quite suitable as indicators
of the unknown effects of exogenous agents on an organism.
Since the initial phenomenological question is always
whether an agent does or does not exhibit a measurable
effect, the fact that exact etiology of an elicited altera-
tion in a hormone feedback loop is unclear is immaterial.
If the elicited change is potentially capable of altering
the viability and/or fertility of the animal involved the
regulatory screening question has been answered. The com-
plexity of endocrine control systems does present problems
in defining exact modes of xenobiotic action but it also
provides experimentally exploitable elements not found in
systems contained within single cells or organs.
In addition to allowing experimental manipulation of
these feedback loops the possibilities of exploiting
temporal changes is alsp available. Studies of the time
course for the maturation of these loops, in both sexes,
and of the cyclical alteration of the loops caused by
pregnancy, menstruation or estrus, provide opportunities
to explore stages of varying sensitivity to environmental
influence. They thus allow identification of the stage(s)
in life during which the exogenous agent being tested pro-
duces the most profound and/or lasting effects.
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Previous work in the general area of toxicity on the
steroidogenie endocrines;has concentrated on the adult rat
and its responses to long term feeding or acute injection
of a variety of drugs, pesticides and other synthetic
/
chemicals. Conney et al. concentrated on the effects of
barbiturates and pesticides on the clearance of steroids
from systemic circulation (11-18) and on the capacity of
target organs (uterus and prostate) to bind steroids in
the presence of the synthetic chemicals (19-23). Their
results have indicated that steroid metabolism and func-
tion is indeed sensitive to agents which induce hepatic
microsomal metabolism? the steroids are metabolized more
quickly and more extensively than normal in treated ani-
mals. Further, this same group along with several others
(2^-27), have shown that some chemicals, e.g., o.p'-DDT
and polychlorinated biphenyls (PGB's) are capable of
specifically blocking the uptake of steroids by their
target organs even in the absence of hepatic induction.
Rybakova et al. (28-30) have shown that the pesticides
DDT and Sevin are capable of increasing pituitary levels of
gonadotrophins in male rats fed contaminated food for long
periods of time. Ottoboni has demonstrated lengthened
reproductive life span in female rats fed DDT (31) while
Ware and Good and Harr et al. have shown a decrease in
fertility and fecundity in mice and rats treated with
Mirex and dieldrin (32,33). Acute blockage of the ovula-
tion surges of both LH and estrogen in the rat have been
10
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shown with barbiturates (3*0 given on the morning of
proestrus. Ethyl ether acutely raises the serum levels of
FSH and LH in rats (35,36). An extensive literature
exists on. the inhibitory action of DDT and its analogs
(especially 2,2-dichloro-l-(2-chlorophenyl)-1-(4-chloro-
phenyl)-ethane (o,p'-DDD)) on the adrenal cortices of man
(37-41), the dog (4-2-47) and guinea pig (48,49) and on the
lack of this effect on the adult rat (50,51). ACTH
release in the rat was increased by dieldrin in a chronic
manner unaccounted for by stress (52). Finally, Heinrichs
and Gellert have demonstrated cystic ovarian development,
persistent vaginal estrus and decreased serum LH levels in
response to adult castration in female rats injected neo-
natally with o,p'-DDT (53,54).
These last investigations (53»54) have a unique and
important character. They demonstrate a permanent altera-
tion of a steroidogenic endocrine control system and,
together with the "imprinting" experiments discussed below,
imply the acute sensitivity of these control systems early
in life, either perinatally in the rat and mouse or
in utero in most other species. Of equal importance, this
sensitive point .occurs coincidentally with complete
maternal dependence and with the time of most effective
transfer of diffusable and lipid soluble chemicals from
mother to offspring (55,56). Since the functioning of
this system after perinatal exposure may be one of the
most sensitive indicators of the potential toxicity
11
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affecting the reproduction and the viability of species
and individuals, the coincidence of these two conditions,
impressionability and dependence, indicate the need to
examine endocrine function subsequent to such exposure.
II. Steroidogenic Endocrine Development; Imprinting
Because the immature steroidogenic endocrine system
appears in the above case (53i5^) "to be so vulnerable to
the presence of an exogenous agent, demonstrating permanent
changes in endocrine status or response, the need to review
steroidogenic development becomes clear. Since most of
the studies have been done on the rat I shall concentrate
there.
Some of the most classical studies concerning the
development of the sex steroid systems relate to the
development of the brain, its sex specificity and control
properties. These studies, reported by Harris (5?)
utilized the technique of ectopic implantation. Ovarian
grafts were made to the anterior chamber of the eye and
vaginal grafts to subcutaneous, sites. ' By surgically and
chemically manipulating the steroid status of the host
animal, e.g., neonatal or adult castration plus steroid
injection, etc., it was possible to determine the basic
outline of hypothalainic sex differentiation. As a result
of these studies and those reported by Barraclough (58-60)
and Gorski (6l) it became established that there are two
hypothalamic centers which control gonadotrophin release
12
-------
and which, in turn, are affected by the presence of
circulating sex steroids.
First, there is a tonic center, anatomically located
in the arcuate-ventromedial nuclear area, which in adult-
hood controls the tonic release of GnRH, and, subsequently,
LH and FSH. This center is subject to negative feedback
inhibition by circulating testosterone (in males) or
1?? -estradiol (in females). Additionally, it remains
largely intact even if doses of exogenous estradiol or
testosterone are given during the perinatal period, 1-10
days of age.
The second center, the cyclic center, is associated
with the preoptic area of the hypothalamus and controls
the gonadotrophin surges seen near the time of ovulation
in the mature female. It differs from the tonic center
both in that it apparently responds positively to circu-
lating estrogen levels above an unknown threshold level by
increasing release of GnRH, LH and FSH and in that it is
effectively eliminated by either perinatal administration
of exogenous steroids or by the testosterone endogenously
produced by the intact neonatal male. It is this elimina-
tion of the cyclic center by exposure to androgens or
estrogen during the critical perinatal period which has
led to the concept of "imprinting". A multitude of manip-
ulations of the castrate and intact state with steroids,
neuronal lesions and surgery now support the imprinting
13
-------
concept (61-63). A summary of some of them is presented
for comparative purposes in Table 1.
Since perinatal steroid status manipulations, such as
those above, alter all those steps controlled by the hypo-
thalamus as well as the hypothalamus itself imprinting has
obvious pleotropic effects. Most prominent of these
effects are phenotypic changes in external genitalia,
growth and sexual behavior (57-61), all of which are
associated with varying degrees of sterility. But
imprinting is not necessarily a clear-cut phenomena; the
degrees of abberation are quite dependent on the steroid
dosages administered and the timing. For example, 100$ of
a group of female rats injected with 1250 ug of testoster-
one propionate on day 5 postpartum showed sterility as
adults while ?0# showed sterility with a dose of 10 ug
(59). Or, as another example, castration of male rats
beyond 5 days of age blocks normal estrus cycles in
implanted ovaries while similar males bearing implants
after castration prior to day 5 do demonstrate normal
estrus cycles. The implications are that endocrine manipu-
lations may alter both the cyclic and tonic centers (63),
the differences in end result being a matter of the
comparative degree of alteration of the two sites.
More subtle changes which may involve sites other
than the two centers defined thus far also take place as
has been shown by Gustafsson et al. in their studies of
specific steroid metabolic enzymes in the liver (6*J—68).
14
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Table 1
•1 2
Description of Neonatal "Imprinting" of the Hypothalamus- of the Rat
Gonadal . Sex Functional Functional Normal
Status g (at birth) Cyclic Center? Tonic Center? Development?
I. Uninjected Pups
Intact 0-15 Days *£*B f + J
Intact 5-15 Days pM^e ; + +
intact Adult p^Je ; + +
H1
Neonatally **„•}+ Male + + -
Castrated HQUXT Female + +
II. Neonatally Injected with Estrogen or High Levels of Testosterone
intact Adult pM^Je I + ? ;
Neonatally .-, , . Male - -
Castrated Female - + -
-------
Table 1 (Cent.)
III. Neonatally Injected with o,p'-DDT
Intact Adult p^e
Neonatally A^I* Male
Castrated HQU.LT; Female
"Imprinting" is defined as fixation of a-biochemical or physiological state or develop-
mental direction; depending on the parameter measured, it may be observed at any time
between 5 and 15-30 days of age.
2
The tonic and cyclic centers of the hypothalamus are those neurons or nuclei which
control tonic or cyclic release of gonadotrophins.
^ Symbols used indicate established normality, = +, or abnormality, <= -, and uncertain func-
tionality » ±. Unknown functionality or conclusions based only on the results in this
thesis are indicated as ?.
The degree of abnormality is dependent on dosage and time of administration after
birth.
-------
Perinatal treatment of females or neonatally castrated
males with high doses of testosterone, 5
-------
necessary for effective specific uptake of androgens into
target tissues and consequently exists as a pseudo-
hermaphrodite. Since changes like these, or others
involving receptors for trophic hormones (73)» may exhibit
temporal and dosage dependencies somewhat different from
those of hypothalamic imprinting per se, they may well be
the biochemical mechanisms ultimately responsible for the
broad spectrum of morphological and physiological changes
seen in adult animals neonatally exposed to androgenizing
or estrogenizing influences at differing times and
dosages.
III. Steroidogenic Endocrine Development; The Normal
Time-Course
Morphologically the testis demonstrates two genera-
tions of testosterone-producing cells (the interstitial
or Leydig cells). These cells, occupying the inter-
stitium between the tubular (sperm-producing) elements
of the testis, exist at birth and are histochemically
demonstrable until roughly 5-7 days of age. They largely
disappear at this time and either regenerate or become
reactivated at approximately 3 weeks of age. Production
of potent spermatozoa in the tubules begins to occur at
roughly 45 days of agej sexual maturity as measured by
sexual potency, peripheral organ enzyme activities and
serum hormone levels is reached by roughly 50-60 days of
age (7^,75).
18
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Steroid production in the first generation of Leydig
cells appears quite similar to that occurring in the adult
and is sensitive to exqgenously administered human
chorionic gonadotrophin (hCG), i.e., LH (?6). The testis
which exists between the first and third week of life
appears steroidogenically inactive and insensitive to exo-
genous LH "because testosterone levels fall even in the
continuous presence of serum LH (77). This may, in fact,
be due to the diminished number of Leydig cells present
(76) although other schemes involving the coincident
absence of the glucocorticoids (see below) have been
advanced in the face of the constitutivity of the testicu-
lar LH receptors (78,79). Full adult steroidogenic
capacity is gradually reached during the period following
weaning (at roughly three weeks of age) and prior to
maturity (80,81), the rise proceeding most rapidly from
about 40 to 60 days of age. Accessory gland development
closely correlates with the increasing serum levels of
active androgens produced during this1 period of maturation
as do the levels of certain enzyme activities found in
peripheral tissues such as the liver (^,5).
Serum hormone levels during the developmental period
have been measured by several groups (77»82,83). Levels
of LH appear highly variable prior to approximately three
weeks.of age after which they stabilize slightly below
adult levels until the onset of maturity at which the
slight rise to adult levels occurs. PSH demonstrates a
19
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stable plateau at roughly adult levels until about three
weeks of age. At this time the levels rise about two-fold
and plateau until ^0-50 days of age after which they fall
back to adult levels. On the hypothalamic level, GnRH con-
centrations begin to slowly rise immediately after birth
and finally reach the adult plateau at about 50-60 days of
age (84). Throughout development the feedback loop remains
sensitive to exogenous LHRH (82) or castration (85-8?)
implying that it is functional and is only modified or
modulated differently during the adult period, the change
taking place during the transitional state, puberty.
Development in the female exhibits a distinctly dif-
ferent pattern. Within about 10 days after birth the
ovaries demonstrate aggregation of several follicular
cells with each egg cell and progression of the initial
meiotic division of the egg cells themselves to the
dictyate stage of prophase I. The oo'cytes appear to ran-
domly begin enlargement, apparently initiating the events
which would eventually lead to ovulation in the adult
ovary. Prior to the cyclical events which occur during the
estrus cycle, however, those oocytes which began develop-
ment and stimulated their associated follicular cells to
divide and form small follicles undergo degeneration long
before reaching the ovulatory state. This atresia seems
to be associated with unfavorable concentrations of estra-
diol, LH and/or FSH (88,89) and may reflect immaturity of
the thecal portion of the ovary.
20
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Slow maturation of the thecal, i.e., interstitial or
stromal, elements of the ovary ensues over the 5-6 weeks
after birth. These steroidogenic elements show sensitivity
to endogenous LH and PSH producing serum estradiol peaks
subsequent to serum LH peaks (77) and supporting uterine
weight gain (90). Near 35-^0 days of age the theca
reaches full maturity and is capable of playing its role
in the cyclic events of normal estrus. Its production of
estradiol, perhaps as a result of the presence of adult
titers of gonadotrophins, becomes sufficient to cause full
differentiation of the .female accessory tissues. This
results in the opening of the vagina, the maturation of
the uterus and the initiation of female sexual behavior.
Steroid synthesis during the immature phase is local-
ized in the thecal tissue, estrogen being the major
product. After the first ovulation, however, the post-
ovulatory follicle forms a temporary gland and site of
progesterone production, the corpus luteum; similar
temporary glands subsequently generate the cyclic changes
in serum progesterone seen during the female cycle.
Trophic hormone production by the female hypophysis
differs from that of the male. FSH is markedly elevated
at birth and remains so until roughly three weeks of age
when it drops to a plateau of 1/3 to l/^ the early level.
It continues there until the periodic rises associated with
ovulation begin to occur at about 5-6 weeks of age (77).
LH, on the other hand, shows nearly the same picture as in
21
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the male; highly variable levels exist until three weeks
of age followed by a plateau near adult diestral (basal)
levels until the onset of cyclicity near *K) days of age
(77). Hypothalamic GnRH rises slowly from birth until 4-6
weeks postpartum when the plateau of maturity is reached
(8*0. The feedback controls are all operative as can be
seen by the abrupt response of serum LH to perinatal
ovariectomy or steroid injection (85»87) or by the halt in
ovarian and uterine growth caused by administration of
anti-gonadotrophin anti-sera (90).
In the female, puberty and final maturity seem closely
associated with ovarian development and/or ovarian
mediated changes in the neural-hypophysial control centers
(91). Conversely, in the male, changes in testicular
sensitivity to LH caused by high FSH levels appear to play
the dominant role (92,93). Though they begin similarly,
the control systems for reaching maturity appear quite
different for the two sexes.
Adrenal cortical development in the rat follows a
similar time course in both sexes for roughly the first
three weeks postpartum. The glomerulosal layer is well
defined at the exterior of the cortex and appears stable
throughout the developmental timecourse. The cortical
tissue within the glomerulosa appears compact and undif-
ferentiated at birth with the reticular and fasicular
zones being morphologically indistinguishable (94). No
definitive perimedullary fetal or x-zone is present, as it
22
-------
is in the mouse, but steroidogenesis does occur as has been
shown by Milkovirf and Milkovic (95). The response to
stress appears blunted for several days following birth
but at no time is it entirely absent (96). Further, this
response is sensitive to hypothalamic control by CRH and
to hypophysial control by ACTH from the 18th fetal day
onward as has been shown in experiments using natural and
experimental anencephally and hypophysectomy (97). In
spite of this functional intactness the adrenal cprtex
exhibits a regression in volume, cell size and steroid
content immediately after birth and does not regain mature
functionality until sometime between 2-3 weeks of age (96).
Beyond this point development in the sexes begins to differ
because estrogen produces a positive trophic action on the
cortex resulting in larger adrenals with higher cortico-
sterone output in the female. This difference has been
implicated in the onset of female puberty through a direct
influence of the adrenal on the ovary (98) although the
exact mechanism is at yet unclear. Daring the period of
3-5 weeks postpartum the fasicular and reticular zones
become differentiated, formation of vascular elements and
tissue cords becoming visible histologically. Finally, by
5-6 weeks of age the adult diurnal fluctuations of corti-
costerone output become established (98) and maturation of
this system is also completed.
23
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IV. Metabolism and Physiological Effects of DDTt
The rat was chosen as the animal model for this work
because of the wealth of information available on its
normal steroidogenic functioning and development and
because of its potential susceptibility to perturbation of
those normal conditions. Choice of the model compound was
based on a review of the known properties of widely used
synthetic chemicals. DDT stands out as one of the few
chemicals which has been extensively examined from essen-
tially all facets: chemical, toxicological, pharmacolog-
ical and ecological. And yet, the extent of its biological
effects, to say little of its mode of action, remain
incompletely defined, as do many of the factors which
cause it to demonstrate specific toxicities, e.g., repro-
ductive toxicity.
In speaking of DDT it must first be borne in mind that
the commercial chemical, technical DDT, is actually a mix-
ture. Most analyses are in general agreement with those
of Haller et al. (99)t finding 70-80#'1,1,1-tri-
chloro-2,2-bis-(^-chlorophenyl) ethane (p,p'-DDT)f 15-20$
o,p'-DDT plus small amounts of 2,2-dichloro-l,l-bis(4-
chlorophenyl) ethane (p,p'-DDD), o.p'-DDD and other syn-
thetic-reaction byproducts. Because of this predominance
of p,p'-DDT in the mixture a large proportion of the
literature on the insecticide has been generated using
pure preparations of p,p'-DDT. Still, it must be said that
24
-------
all the major components have been tested for, and found
to have, biological effects.
Metabolically, in mammals, p,p'-DDT has been found to
undergo either dehydrohalogenation of the side-chain to
form 2,2-dichloro-l,l-bis-(*J-chlorophenyl) ethylene
(p,p'-DDE) or dechlorination of the side-chain to form
p,p'-DDD and subsequently side-chain oxidation to form 2,2-
bis-(4-chlorophenyl) acetic acid (p,p'-DDA) which may be
conjugated (100,101). It has also been shown to be sus-
ceptible to nonenzymatic reductive dechlorination to
p,p'-DDD catalyzed by agents such as lake water, reduced
porphyrins, water-logged soil and boiled pigeon liver
(102-10*0. Yet even though conversion to p,p'-DDD and
then to p,p'-DDA seems to be a ubiquitous possibility the
conversions occur slowly enough for both DDT and its
metabolites to accumulate, sometimes drastically, in
biological tissues. This accumulation, which is respon-
sible for the large majority of the effects seen in
non-target species, is due not only to DDT's slow
metabolic conversion to excretable products, but also to
its high lipid solubility, a property which it shares with
its two major neutral metabolites DDD and DDE.
The proportions and levels of the DDT derived
residues found in mammalian tissues is highly dependent on
factors determined by the species, age, sex, nutritional
status and drug-history of the animal involved (105-112).
These factors not only determine the distribution of body
-------
fat, the major storage tissue for the residues, but also,
more importantly, the activity of the hepatic microsomal
enzymes. These enzymes, which have been demonstrated to
be involved in the active metabolism of p,p'-DDT (100,113,
11*0, are similar to, if not the same as, those involved
in the breakdown of drugs such as phenobarbital or steroids
such as testosterone (12-15, 115-122). Furthermore, in
most mammalian and avian species studied the enzymes are
inducible by drugs (barbiturates) or by DDT itself (11,
13-15,19-21,115-122). Such induction leads to decreases
in the total pesticide load (123,124) and in the DDE/DDD
ratio but it also increases metabolism of normal body
constituents such as steroids and vitamin D (125-129). It
is this latter consideration which is particularly worri-
some in non-target species since it can result in serious
hormonal and nutritional imbalances.
Another major factor influencing the form and levels
of the stored insecticide residues is the presence of gut
flora. McCully et al. established the differences in
DDE/DDD ratios in rats, sheep, chickens, rabbits and
guinea pigs given DDT by several different routes (130).
Their conclusions agree well with other investigations
which have found that DDT metabolism involves extensive
enterohepatic circulation and is highly sensitive to the
presence of gut microflora (105,131). These microbes func-
tion under largely anaerobic conditions and, like those in
anaerobic soils, catalyze the formation of ODD from DDT
26
-------
(132,133). This catalysis overcomes what appears to be a
rate limiting step in DDT breakdown, allowing more rapid
catabolism to DDA and shunting DDT away from the formation
of the inert storage residue, DDE.
Finally, levels of residue retention are, of course,
dependent on the extent of exposure. In the case of low,
1 -50 ppm, exposures in the food of rats, the quantity of
total DDT residues climbs to a plateau over the course .of
17-23 weeks and decreases over the course of about 25-30
weeks after exposure has been discontinued (13*0. This
pattern of residue levels is paralleled, as expected, by
microsomal drug metabolizing activity and is sensitive to
agents which further induce or surpress hepatic microsomal
activity (106,107,129) or which mobilize stores of body
fat (108).
Most of the previous statements concerning metabolism
are also applicable to the other chemicals found in tech-
nical DDT. The normal metabolite p,p'-DDD is broken down
to p,p'-DDA, as mentioned above, and yields no residues of
p,p'-DDE (100,132,133,135,136). On the other hand,
o,p'-DDT, as is shown in Figure 3i is not only subject to
side-chain decomposition but is also degraded by way of
ring hydroxylation reactions (51,137,138). These latter
reactions are predictable on the basis of the open para-
position in the ortho-substituted ring. This extra
metabolic "handle" and the higher polarity of the resultant
hydroxylated metabolites can largely explain the higher
-------
Figure 3. Major Metabolic Routes of o,p'-DDT
The known routes of DDT metabolism are depicted as
separate and parallel pathways. In mammalian systems
pathways I) and II) cross in such ways as.to yield a
variety of hydroxylated intermediates with the side-chain
in various stages of oxidation or conjugation. Pathway
III) appears to be independent for the p,p'-isomer of DDT
producing a major storage form of the pesticides however,
for the o,p'-isomer hydroxylation and other routes seem to
be favored since o,p'-DDE residues are detectible only in
minor quantities,
The specific enzymes involved in the pathways are
largely unknown. The P-450 complex does play some role
since it is induced by DDT and stimulates the elimination
of DDT residues after induction by other agents.
28
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FIGURE 3
MAJOR METABOLIC ROUTES OF o,p'-DDT
I) SIDE CHAIN OXIDATION
Cl
CCI,
DDT
CHCI,
ODD
Cl
Cl
CHCI
DDMU
DDMS
.Cl
CH2
DDNU
.Cl
Cl
CH2OH
DDOH
COOH
DDA
COX
CONJUGATES
II) RING HYDROXYLATION
.Cl .Cl
X= SERINE, GLYCINE,
ASPARTIC ACID
Cl
CH-(O>CI
cci
DDT
(H0),.2 CCI3
Cl
CH"\2/CI
COOH
(H0),_2 CHCI2
III) DEHYDROHALOGENATION
Cl .Cl
^5>CH-^))-CI
CCI3
CCI
2
DDT
DDE
29
-------
metabolic rate of o,p'-DDT and o,p'-DDD in comparison with
the p,p'-isomers. Furthermore, they can also explain the
lack of 2,2-dichloro-l-(2-chlorophenyl)-l-(4-chlorophenyl)
ethylene (o,p'-DDE) in body residues following administra-
tion of either technical DDT or pure o,p'-DDT (131,139).
They may not, however, be able to explain the differences
in biological actions found with o,p'-DDT and o,p'-DDD in
comparison with p.p'-DDT and p,p'-DDD, respectively.
Though all the compounds are capable of causing
hepatic inductions similar to those seen with p,p'-DDT,
the o,p'-isomers differ in their ability to produce neuro-
logical and steroidal effects. Comparison of the acute
toxicities, reflecting neural toxicity, illustrate these
differences rather dramatically. Oral dosages in rats
required to kill 50$ of the animals dosed (LDer., ) were
^)U S
100-250 mg/kg for p.p'-DDT and 800-2000 mg/kg for o,p'-DDT
(1^0,1^1). Furthermore, the muscular tremors and convul-
sions elicited by a toxic dose of technical DDT have been
shown by Hrinda et al. (1^2,1^3) to be associated with
p,p'-DDTj o,p'-DDT showed no neurotoxicity.
As regards steroidal effects, the situation is nearly
the reverse. As early as 19^9 Nelson and Woodard described
the adrenolytic activity of technical DDD (90$ p,p'-DDD,
10$ o,p'-DDD) in dogs (^2). Within nine years the tech-
nical material had been fractionated and the o,p'-isomer
associated with the observed involution of the fasicular
and reticular cortical zones and with the associated
30
-------
decrease in urinary and cirulating l?-hydroxy- and
17-keto-steroids (^5). After similar activity was shown
in several species including man and the purified material
became available, o,p'-DDD began to be used for the treat-
ment of adrenal hyperfunction as it occurs in Gushing's
syndrome and adrenal carcinoma.
The other outstanding example of steroidal effect is
the innate estrogenicity of o,p'-DDT. In a study of DDT
effects on White Leghorn cockerels published in 1950
Burlington and Lindeman (1^4) demonstrated what appeared
to be an estrogen-like, effect of prolonged exposure to
technical DDT. They found a marked decrease in testicular
growth and in development of secondary sex characteristics
and attributed the effects to a configuration of p,p'-DDT
which superficially resembles the synthetic estrogen
diethylstilbestrol. A conflicting report based on work
with female rats appeared in 1952 (1^5)* but it was not
until 1968 that Bitman et al. (1^-6) and Welch et al. (22)
conclusively demonstrated that only o,'p'-DDT was intrin-
sically estrogenic. These later studies showed that not
only did o,p'-DDT have a trophic effect on the oviducts
of injected chickens and quail (1^6) but it also was
capable of increasing wet weight, glycogen content and
C-glucose uptake into lipid, proteins and RNA in
immature or ovariectomized adult rat uteri (22, 1^7).
Subsequent studies by Singhal et al. (1*1-8) showed that the
uterotropic response in ovariectomized rats also involved
31
-------
enhancement of gluconeogenic and hexosemonophosphate shunt
enzymes; the enzymatic enhancement was inhibited by
actinomycin D or cycloheximide. This report also
indicated that acute administration of o,p'-DDT combined
with 1?@ -estradiol produces a somewhat additive effect.
Notwithstanding this last result, other studies (26,2?)
have shown that o,p'-DDT is capable of effectively com-
peting for uterine cytosolic estradiol receptors in vitro
at levels of 1-10 ppm; such levels are within the
reported tissue levels of DDT under normal environmental
exposure (1^-9). Still later experiments by Gellert et al.
(53,5^,150,151) have demonstrated that o,p'-DDT can
decrease serum LH in ovariectomized adult female rats and
is even capable of causing appearance of persistent-vagi-
nal-estrus in adult rats injected with 3 rag neonatally.
This last effect, perhaps the most striking, has been
demonstrated to be dose-dependent and specific to o,p'-DDT
(if all of its aspects are included). It represents a
starting point for the studies described in this thesis.
It must also be noted that a large body of literature,
coming mostly from the laboratories of A. H. Conney, has
outlined the complicating factor in any in vivo studies
concerning the steroidal actions of DDT and its analogs,
i.e., the induction of hepatic metabolism (11,1^-21,23,39,
118,120,122,127-129,14-3). This series of papers has
demonstrated that chronic doses of pesticides or drugs are
both capable of inducing the hepatic enzymes responsible
32
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for catabolizing circulating steroids of all classes. The
work of Peakall (152), albeit in ring doves, directly
demonstrates reductions in circulating estradiol levels
upon chronic feeding with p,p'-DDT. That such is also the
case in the rat and mouse has been indirectly shown by the
fact that uterine -%-!?£ -estradiol uptake and the utero-
tropic response to estradiol are both blocked by chronic
exposure to halogenated hydrocarbon insecticides (18).
Similar results have also been shown for androgen uptake
and response in rat seminal vesicles (23) or mouse pros-
tate (25). That microsomal induction may occur throughout
the postnatal life of the rat has also been demonstrated
(109,153). All the above observations taken together
require that in vivo experiments on the mode of action of
pesticides, especially in regard to their actions on
steroidogenesis, be interpreted with the possibility in
mind that indirect effects mediated by hepatic steroid
metabolism ere responsible for the effects seen.
The mode of toxic action of DDT and its analogs in
mammals may well be related to its demonstrated actions in
insects. Holan (15*0 briefly described a molecular model
of the membrane site to which DDT binds in insect nerve.
This model was based on molecular-dimension calculations
and on an extensive series of structure-function relation-
ships including at least 57 analogs of p,p'-DDT. The
final picture of the binding of DDT involved insertion of
the trichloroethyl-tail into a pore in the lipid portion
33"
-------
of the membrane with the aromatic rings extending into the
protein layer and forming ?r—bonds to the protein mole-
cules. The pore to which DDT was bound is associated with
Na+ transport? treatment with the insecticide results in
atypical and continuous nerve impulses indicative of per-
turbed Na+ efflux. Other work has indicated coincident
or subsequent changes in K+ and/or Ga++ transport in both
insect and mammalian species and in both nerve and muscle
preparations (155). Obviously, if the compound effects
such universal elements of membrane function as membrane
potential and cation transport it is not surprising that
Hrinda et al. (1^3) have demonstrated increased levels of
cAMP in tissues taken from rats treated with DDT or its
analogs. This is especially so in view of the postula-
tions of cation involvement in the production and actions
of cAMP put forward by Rasmussen (9).
The foregoing discussion has emphasized how much we
know and implied how much we do not know about the DDT
complex (technical DDT), one of the best characterized of
the synthetic chemical mixtures. In spite of the great
humanitarian services DDT performed after its rediscovery
in 1939i a great public outcry arose concerning DDT's
safety after the publication of Rachel Carson's Silent
Spring in 1962 (156). This outcry, combined with the
professional questions which stemmed from the papers
listed in the above discussions and from ecological
studies such as Mickey's Peregrine Falcon Populations.
-------
Their Biology and Decline (157), resulted in the banning
of DDT for use in the United States in 1972. Still, its
environmental persistence (158), global mobility (159.160)
and extensive use outside of the U.S. continue to make it
a material of some ecological importance. Because
o,p'-DDT was implicitly included in the ban and the furor
which brought it about and because it has known effects
which include precisely the type of alterations which
could best be probed by the approaches I have proposed for
screening other materials, I chose to use o,p'-DDT in these
investigations.
Having chosen the endocrine system of the rat and
o,p'-DDT as objects of the experimentation, more specific
questions arose. First, the phenomenological question of
the presence of a measurable effect begged a second ques-
tion which had mechanistic ramifications} what part of
the endocrine system or its control loop is affected?
This question forced an experimental design capable of
answering botn the question of the existence of an effect
and the question of the focus of action of the chemical
under study. Therefore, the object of the experimentation
had to be more fully defined. Being that earlier reports
have generally indicated little reproductive effect of DDT
on the adult rat (161,162) and that several studies had
been published on the effects of neonatal injection with
o,p'-DDT in the female rat (53.5^.150,151), the choice was
made to examine the results of neonatal exposure on the
'SB
-------
function of the endocrine system of the male rat. The
somewhat simpler adult hormone pattern of the male as
compared to the female also made the male system more
attractive than the female. But the question, "On what?"
required even further definitions. It must be understood
that the question of looking for an effect on the organism
as a whole or on a specific subsystem within the organism
required a broad investigation employing a variety of
measurements. Furthermore, the measurements had to probe
both structure and function in order to ascertain if
subtle but still consequential changes had taken place.
In regard to the endocrine system, this emphasis on scope
required that a variety of measurements be made on both
the gonadal-hypothalamo-hypophysial and the adrenal-hypo-
thalamo-hypophysial systems. That is, observations
including morphology, histology and hormone secretions had
to be coupled with function tests, akin to glucose-toler-
ance tests, such as responses to castration or exogenous
LHRH. Obviously, though the investigation was undertaken
to determine the existence of an effect it may. also give
insight into the later question of mechanism of action.
The question of an effect "On what?" was therefore
answered in these studies by noting any differences in the
results of the measurements used in treated and untreated
animals.
It is evident from the previous discussions that the
perinatal period is, at least in the rodent, a period of
"36-
-------
either ambiguity or preparation in the steroidogenic con-
trol systems. It is therefore a period of great sensi-
tivity to outside influences in both the male and female.
Because of this, and because the neonatal detoxification
systems are not as active as they are in the adult (5)» the
potential for exogenous agents such as o,p'-DDT to
influence reproduction and/or viability seems greatest if
exposure occurs perinatally as was proposed in these
experiments. It seemed reasonable that if a model of
interference with the steroidogenic control loops was to
be used in screening for the effects of foreign compounds,
direct introduction of the materials into test groups could
generate the desired answers. If, however, the specific
effects of natural exposure was to be used or questioned,
introduction must occur via the mother, since neonates are
normally completely dependent on her. In these
investigations both routes of exposure were examined.
In the case of either exposure, measurements were
made in both intact and neonatally castrated rats to ascer-
tain if some of the changes found in the female (53»5^il50,
151) could be found in the proportedly neurologically
similar neonatally castrated male (5?-6l). Furthermore,
it was hoped that such parallel experiments might elucidate
the role of the testes in any changes observed in the
intact rat. The measurements performed included body and
organ weights, organ histologies, serum hormone levels
under normal and stressed conditions and pituitary hormone
"17
-------
levels. Studies on in vitro testicular steroid trans-
formations were also initiated and spawned the generalized
techniques and computer program detailed in Chapter 6 and
Appendix I. A general schematic of the approach and treat-
ments used is shown in Figure ^.
The observations described in this thesis are meant
to answer the experimental question posedi "What is the
effect, if any, of neonatal exposure to o,p'-DDT on the
steroidogenic endocrine systems of the male rat?" Some
of the later studies may also shed some light on the locus
of action of the chemical because they were designed to
fix the actual location of the effects of o,p'-DDT more
firmly than the initial studies; they attempted to
answer the question "Exactly where does it act?" Even the
question of when the changes occur was partially addressed
by studies which examined development in conjunction with
function. It is with these questions in mind that the
experiments will be described and related to the primary
objective, i.e., design of a general procedure for
screening foreign compounds for effects on reproduction
and viability.
38"
-------
Figure *K Schematic of Approach and Treatments
Treatment was either via direct s.c. injection into
1-5 day old pups or via the residues carried into the
milk of dams injected daily, i.p., with large quantities
of o,p'-DDT. In both situations growth was measured as
were endocrine tissue development, histology and function.
Tests for subtle alterations of endocrine function were
conducted in surgically modified animals, e.g., neonatal
castrates, (ff, or those stressed by other treatments so
as to exaggerate otherwise masked biochemical or physio-
logical changes.
-------
FIGURE 4
SCHEMATIC OF APPROACH AND TREATMENTS
VIA THE INJECTED DAM
TREATMENTS
DIRECT INJECTION
n n
MEASUREMENTS
PHYSICAL
GROWTH
AND
DEVELOPMENT
TISSUE FUNCTION
NORMAL
STRESSED
40
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CHAPTER 2
MATERIALS AND METHODS
I. Animals«
All rats used in these experiments were Sprague-Dawley
purchased from Spartan Laboratories, Hazlett, Michigan.
They were housed in JQ cm x 30 cm x 15 cm nesting boxes on
wood shavings from birth until weaning and thereafter in
35 cm x 60 cm x 15 cm cages with wire mesh floors. Caging
was in groups of 6-8 dependent on age. All animals were
allowed free access to water and food (Purina Rat Chow).
A light-dark cycle of 12/12 (6.00 - 18.00 EST) was main-
tained. Room temperature and humidity varied somewhat With
the season, but normal values were approximately 25°C and
'). Cages were cleaned every 2-3 days. Medication was
only used when serious outbreaks of respiratory infection
occurred in or near the experimental groups or when
animals were exposed to heavy or repeated ether anesthesia.
In any cases in which medication was used, all test and
control groups were treated equally with Terramycin dis-
solved in the drinking water (50 mg/ml).
When neonates were to be used, the pregnant females
were placed in nesting boxes roughly one week before
delivery. After delivery litter sizes were adjusted to
meet the needs of the experiment, usually 6 or 8-pups per
dam. Litters were maintained, by addition of pups if
necessary, as near to the original number as possible
-------
until weaning. Pups were allowed access to the dams
until 25 days of age by which time natural weaning was
complete in all litters (163).
Neonatal castrations were performed under cold
anesthesia according to the procedure of Pfeiffer (164).
Following the surgery and warming to adult body tempera-
ture, the pups were returned to their dams. Adult castra-
tions were performed under ether anesthesia. A short
midventral-scrotal incision was made and followed by two
small lateral incisions through the tunica vaginalis of
each testis. The testes were expressed through the
incisions and the spermatic artery, vein and the vas
deferens were tied off with sterilized cotton thread. The
testes were severed below the constriction and the wound
closed with wound clips. Adult castrates were placed on
Terramycin for at least 3-5 days after the surgery.
Blood samples were obtained from animals either
after decapitation, in which case trunk blood was col-
lected, or under light ether anesthesia by tail-vein
bleeding (TVBE). The latter procedure was chosen over
heart-puncture, catheterization or tail-vein bleeding with-
out anesthesia for the following reasons. First, I found
heart puncture, during which many animals were killed by
faulty punctures, to be a less predictable process. Since
many of these animals required 2-3 months to obtain I was
conservative in their use. Second, catheterization is
normally only acceptable for bleedings carried out for less
-------
than 3-5 days and requires a moderate amount of surgery
to insert the cather. Because a large number of animals
were to "be bled during periods which exceeded one week
catheterization was discarded. Finally, after several
attempts at bleeding by tail-vein without anesthesia the
ability of the rat to constrict blood flow to the tail
became obvious. Because blood samples of 1.0 ml or more
were needed for the measurements to be made anesthesia
was necessary.
TVBE was carried out by placing the rat on an
ether-moistened cotton pad in a metal can equipped with
a glass cover. After 45-60 seconds the rat was suffi-
ciently anesthetized to be placed in a restraint cage, to
have a small (about 5 mm) fragment of the tail cut off with
a razor blade, and to have 1-3 ml of blood collected in a
test tube prior to regaining full consciousness.
Including the tying off of the tail with a loop of cotton
cord, the time from introduction into the ether chamber
to placing the animal into a holding cage was 3-5 minutes.
Tissue samples were collected after the animals were
weighed, stunned, decapitated and bled. If they were to
be examined histologically they were weighed; divided and
placed into 10$ neutral-buffered formalin (165).
Animal weights were determined on a triple beam
balance to within - 0.5 g. The adrenals and pituitaries
of all of the animals and the testes and accessory glands
of young or steroid-treated rats were rinsed in a modified
-------
Ringer's solution (Buffer I, Table 3), blotted and
weighed on a Sartorious semi-automatic milligram balance
accurate to - 0.03 mg. Kidneys and livers from all the
animals and the testes and accessory glands from older
rats were rinsed and blotted as above and weighed on a
Mettler top-loading balance accurate to - 0.01 g.
II. Chemicals;
All chemicals utilized were obtained from commercial
sources. Miscellaneous solvents, salts, acids and bulk
chemicals were obtained from Mallinckrodt, Inc., Fisher
Scientific Co., Merck Chemical, J. T. Baker Chemical Co.
and Matheson, Coleman and Bell Manufacturing Chemists.
All water used in solutions or washes, etc., was
double-distilled.
DDT analogs were purchased as 99 % pure from Aldrich
Chemical Co., Inc. Subsequent analysis by thin layer
chromatography (TLC) demonstrated single spots for all of
these chemicals? however, gas chromatography (GC) later
showed impurities in the o,p'-DDT, and p,p'-DDD. Bitman
et al. had analyzed Aldrich o,p'-DDT in 1971 as being
98.7f° pure, with l.l£ p,p'-DDT contamination (166).
Analysis of the o,p'-DDT used in these experiments showed
98.5$ o,p'-DDT and 1.5$ p,p'-DDT even after recrystalliza-
tion from several solvent systems (acetone/water, ethanol,
hexane). Contamination of p,p'-DDD was with minor ( <
amounts of o,p'-DDD.
-------
•5 Tk
Labelled steroids, ^H- and C-, were purchased from
Amersham/Searle Corp. Cold steroids were obtained from
Sigma Chemical Co. and Schwartz-Mann. Purity was verified
for all labelled steroids by TLC followed' by radioscanning
at high sensitivity on a Vanguard Model 880 strip scanner.
Three solvent systems were usedi benzene, 8s2-benzene:
acetone, and chloroform. Labelled steroids were used at
the original specific activity determined by the manufac-
turer! 18.6 Ci/mmole, f? n-^HJ- A -pregnenolone; 52
mCi/mmole, k- C-dehydroepiandrosterone; 59.^ mCi/mmole,
k- C-testosterone; 60 mCi/mmole, k- C-A -androstenedi-
one; 53 mCi/mmole, k- C-A^-pregnenolonej 6l.O
mCi/mmole, k- C-progesterone; 61 mCi/mmole k- C-l?ct -
hydroxyprogesterone. They were diluted to appropriate
volumes, i.e., the correct number of .counts per volume,
with l:l-benzene:ethanol or ethanol and stored under
refrigeration. Unlabelled steroids were similarly checked
for purity by TLC and subsequently recrystallized from
ethanol and/or acetone; purity was then checked by TLC,
GC and melting point. Cold steroids were stored at room
temperature in brown glass, and their solutions in ethanol
or benzene-ethanol were refrigerated. Corticosterone solu-
tions were made up in redistilled ethanol and water and
stored for no more than one week under refrigeration.
Cholesterol-3-propyl ether (CPE) was purchased in highly
purified form (verified by GC) from Sigma and was stored
at -20°C. CPE solutions were made up in lil
-------
benzene »ethanol and stored under refrigeration. Testos-
terone-17-propionate and 1? (? -estradiol-l?-valerinate were
also purchased from Sigma and were used as supplied.
Aldrin was obtained from K & K Laboratories, Inc. and
purified by recrystallization from ethanol and hexane after
washing with methanol and acid. Purity was verified by
appearance of a single peak upon GC.
Sodium phenobarbital and 2'-diethylaminoethyl-2,2-di-
phenylpentanoate (SKF-525A) were donated by the labora-
tories of Dr. M. J. Coon and Dr. H. H. Cornish, respec-
tively. The chemicals were dissolved in physiological
saline by addition of acid or base and titrated to pH 7-8
prior to use in injections.
Pyridine nucleotides (NADP* and NADH), glucose-6-phos-
phate (G-6-P), glucose-6-phosphate dehydrogenase from
Baker's or Torula yeast (G-6-P DH) and bovine serum albumin
(BSA) were purchased from Sigma. Synthetic LHRH was
obtained from Calbiochem. All these biologicals were
diluted in modified Ringer's solution'or physiological
saline immediately prior to use. Storage of the crystal-
line materials was at -20°C.
Refined sesame oil and dimethyl sulfoxide (DMSO) were
obtained from Fisher Scientific and used as obtained.
Glass-distilled hexane purchased from Burdick and Jackson
Laboratories, Inc. was also used as received. To remove
fluorescent impurities, diehioromethane from Mallinckrodt
or Fisher Scientific was allowed to stand over
-------
concentrated sulfuric acid for several days, then washed
with sulfuric acid, bicarbonate and water. The wet solvent
was dried over anhydrous sodium sulfate and distilled
through a Vigreaux column (the 39°C fraction was collected).
Similarly, ethanol was distilled after refluxing with
2,^-dinitrophenylhydrazine and hydrochloric acid to remove
fluorescing impurities.
Precoated silica gel G TLC plates were purchased from
Brinkman Instruments, Inc. and thoroughly washed with
methanol prior to use. Gas Chrom Q and silicone GC
coatings were purchased from Applied Science Laboratories.
GC column packings were prepared using filtration and
fluidizer techniques (16?).
Scintillation chemicals, 2,5-diphenyloxazole (PPO)
and l,*J~bis-|2-(5-phenyloxazolyl3-benzene (POPOP), were
obtained from Research Products International, Corp. The
scintillation cocktail used was 6 g PPO plus 0.25 g POPOP
per liter of toluene and was based on the data of Bush and
Hansen (168) for nonquenched systems.1
The ovine-LH (oLH), radioiodinated oLH and anti-rabbit
gamma globulin utilized in the radioimmunoassay (RIA) pro-
cedures were obtained from the Reproductive Endocrinology
Program of the Department of Pathology, The University of
Michigan. The anti-oLH used was a dilution of the
Niswender anti-serum (169) and was also obtained from the
Pathology Department. These biologicals were used as
supplied.
47-
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III. Treatments»
All direct chemical treatments were by injection.
Steroids and o,p'-DDT were dissolved in sesame oil for
subcutaneous (s.c.) injection into neonatal animals. Such
injections were done on the day of birth and repeated on
the following four days; all these injections were volumes
of 0.05 ml. The concentrations used are given in the
experiments. Injections of o.p'-DDT into nursing dams, to
indirectly administer the chemical to the pups, were given
intraperitoneally (i.p.). These injections were begun on
the day of parturition and were continued daily until the
day of weaning (day 25). All injections were either 0.1
ml of 50 mg of o.p'-DDT dissolved in DMSO or 0.1 ml DMSO
alone. The solutions of DDT in DMSO were nearly saturated
and were warmed .prior to injections.
The quantities of steroids given to neonates were
based on the results of imprinting experiments previously
conducted (59) and were chosen to be near the lowest effec-
tive single doses. These doses were divided into five
equal parts, one part of which was given in 0.05 ml of
sesame oil on each of days 1-5 of age; daily doses were
^•0 ug 1? P -estradiol-17-valerinate and 200 ug testoster-
one-17-propionate. The amounts of DDT injected into neo-
nates was meant to cover a range from nearly environmental
exposure levels to levels near those found effective by
Gellert et al. (5*0 in imprinting the neonatal female.
48
-------
The daily dose of o,p'-DDT given the dams was estimated to
•l-V*
be approximately 1/10 of an LD-0 dosage.
In later experiments involving the response of
serum-LH to adult castration, a test of the negative feed-
back loop was performed by injecting testosterone several
hours prior to bleeding. The dose o.f testosterone admin-
istered, s.c., in 0.1 ml of sesame oil was determined, on
the basis of the work of Hutchison and Goldman (170), to
be capable of partially suppressing castrate levels of
serum LH without driving them down to normal intact
levels.
The response to LHRH was measured by injecting a large
(1?1), 1 ug, dose of synthetic LHRH into intact rats. The
injection was in 0.1 ml of physiological saline and was
performed 20-60 minutes prior to bleeding. This period
has been shown to coincide with maximal serum-LH concentra-
tions by previous workers (9,172-17*0 .
The hepatic microsomal inducer, phenobarbital, and the
inhibitor of microsomal induction, SKF-525A, were admin-
istered to neonatal rats i.p. in 0.05 nil of physiological
saline. The doses used were based on the work of Levin
et al. (17) and Harbison (175). Phenobarbital was given
daily in two injections of 100 ug each, spaced 12 hours
apart, for the first five days of life. SKF-525A was
given as a single daily injection of 125 ug over the same
five day period.
49
-------
The amount of DDT actually absorbed by rat pups
suckling injected dams was determined during these
studies and is discussed in Chapter ^.
IV. Measurements;
A. Organ Weights;
Body and organ weights were measured as above and the
percentage of body weight attributable to each organ was
calculated for each animal. Means, standard deviations
and standard errors were then -computed for both absolute
weights and percent of body weight for each treatment
group and each organ. Mean values were then compared
statistically by applying unpaired Student's t-tests.
Significance throughout this investigation was considered
to be attained only if the chance probability of the
observation in question was less than 5$.
B. Organ Histologies!
Tissue fragments fixed in 10$ neutral buffered forma-
lin were prepared for observation by the Histology Labora-
tory of the Department of Pathology, The University of
Michigan. Common histological preparation was followed,
i.e., alcohol/xylene dehydration, paraffin embedding,
sectioning, staining, xylene/alcohol clearing and mounting
(165). The tissues were sectioned at 7-10 mu and stained
with hematoxylin-eosin. Observations were made at 5-100
power using a Zeiss photomicroscope with a polarizing
light source and Nikon objectives. Subjective visual
observations were recorded for each tissue and slide.
50
-------
When differences between treatment groups became apparent
the slides were reexamined to verify the differences. The
adrenals, which showed the most marked changes due to
o,p'-DDT administration, were photographed using the same
microscope and Eastman Kodak Co. color photomicrography
film (ASA 16/13 DIN). The resulting slides were processed
into prints by Kodak. Between-group comparisons of the
frequencies of a given histologic observation were made
2
by using the X test for a 2 x 2 table.
C. Serum Gorticosterone Measurementst
Serum corticosterone was measured by a modification of
the procedures of Mattingly (1?6) and Silber et al. (17?).
Figure 5 shows a schematic diagram of the protocol
utilized.
Upon decapitating an animal a trunk-blood sample was
collected into a 10 or 20 ml beaker. This was allowed to
clot on ice before being transferred to a tube and centri-
fuged for 20 minutes at approximately 5000 rpm (Variac
setting 50) in a Servall tabletop centrifuge. The serum
was decanted and stored at -20°C. Just prior to extrac-
tion the serum was thawed and 1.0 ml was mixed with 2.0 ml
»•
of purified ethanol (all solvents were purified - see II.
Chemicals). The protein precipitate was removed by
centrifugation under the same conditions as previously.
To a measured volume of 0.5-1.0 ml of supernatant in a
screw-capped test tube, 1.0 ml of doubly-distilled water
and 5-0 ml of glass-distilled hexane were added. This
51
-------
Figure 5- Protocol for Measuring Serum Corticosterone
Levels, Modified from Mattingly and Silber
Tests of both the Mattingly (1?6) and Silber (l?7)
procedures for serum corticosterone led to the outlined
protocol. The alkali wash used by Silber was found
unnecessary if the hexane wash was retained and the
reagents were all purified prior to use in the assay.
52
-------
Figure 5
Protocol for Measuring Serum Corticosterone Levels,
Modified from Mattingly and Silber
Fresh Blood Sample
I
Centrifuged 20 min at 5000 rpm in a tabletop centrifuge;
serum decanted
I
Serum
T~
Frozen
i
1.0 ml Thawed Serum
I
2.0 ml EtOH added, mixed then centrifuged as above?
aqueous EtOH decanted
0.5-l.Q ml Aqueous EtOH
T
1.0 ml HpO and 5.0 ml hexane added, mixed, then centrifuged
as above; hexane aspirated off
Washed Aqueous EtOH
5.0 ml CHpClp added, mixed and centrifuged 10 min as above;
aqueous EtOH aspirated off;
CHgClg dried with anhydrous NagSOj, or MgSOj,
Dry CHpClo Extract
I
Stored for 1-2 days under refrigeration before reaction or
reacted immediately by adding 3.0 ml of 7:3~H2SOj, :EtOH,
mixing for 20 sec, centrifuging for 30 sec and
aspirating off the CH^Cl, extract
I * *
Fluorescent Mixture
I
13 min after addition of the HpSO^iEtOH emission read at
535 nm using ^75 nm excitation and a C-70 filter
(cutoff = 510 nm)
53
-------
solution was mixed for at least 20 minutes on a test tube
rotator or a metabolic shaker before being centrifuged as
before. The upper, hexane, layer was carefully aspirated
off and the lower, alcoholic, layer reextracted with 5-°
ml of dichloromethane. Again mixing was for at least 20
minutes on a tube rotator or a metabolic shaker; centri-
fugation was for 1.0 minutes at 5000 rpm. The upper,
alcoholic, layer was carefully aspirated off and the lower,
dichloromethane, layer was dried with anhydrous NagSO^ or
MgSO^. After drying, the tubes could be tightly covered
with Parafilm or teflon and stored under refrigeration
for up to 48 hours prior to assay without altering the
results. Though this short-term storage was possible
assays were completed.as soon as possible after extraction.
The actual measurement was as follows. The dry
dichloromethane extract was added to 3.0 ml of a 7'3 mix-
ture of concentrated sulfuric acid:ethanol in a glass
stoppered conical centrifuge tube. This was Vortexed for
20 seconds and centrifuged for 30 seconds on a Clay Adams
tabletop centrifuge operated at a speed setting of 1-2.
The upper, dichloromethane, layer was aspirated off. At
13 minutes - 30 seconds the fluorescence at 535 nm of the
alcoholic sulfuric acid solution was measured in a 1.0 cm
quartz fluorescence cuvette using ^75 nm excitation light
and a 510 nm cutoff, exit filter (Corning C-?0). The
instrument used was an Aminco-Bowman fluorimeter. The
measured fluorescence was recorded and later corrected for
54
-------
the background fluorescence of blanks run simultaneously.
Calculation of concentration was based on the back-
ground-corrected fluorescence of standards run simultane-
ously with the unknowns.
Initial assays demonstrated that the use of an. alkali
wash of the dichloromethane extract was unnecessary since
similar results were obtained in assays done on the same
sera without the wash. The correctness of the use of
corticosterone standards diluted in water was verified by
showing that a strictly additive relationship occurred in
samples measured with and without the addition of a
standard sample of corticosterone. It was also shown by
the parallelism of a standard curve with a set of serial
dilutions of a serum pool, Figure 6. A water blank was
used in all assays; the stock standard solution was 2 ug
of corticosterone per milliliter in water.
The mean index of precision of the assay, \ , was
-13.^5$; mean sensitivity was 0.969 - 0.027 log units of
fluorescence per log unit of concentration, in ug/ml; the
mean limit of detection, defined as blank plus A, was
0.0062 ug/ml. A measurement of recovery done in pentupli-
cate on extracted and unextraeted standards indicated total
recovery to be 7^4- - 2%. It should be noted that the
parameters given are means and that the values have
improved during the course of experimental repetition so
that the value of A, particularly, may be better reflected
by the value of -9.07$ obtained in the last assay.
-------
Figure 6. Verification of the Sulfuric Acid Fluorescence
Assay for Serum Corticosteronei Parallelism of
the Standard with Serially Diluted Rat Serum
Log emission at 535 nm versus log concentration for
the standard or log relative concentration for serially
diluted serum yield straight lines with slopes of
0.995 - 0.022 and 1.03^ - 0.029, respectively. The
standard curve is plotted as mean log emission - 1 standard
deviation (where errors are covered by the circle they are
not shown); the serum curve shows the individual measure-
ments in a duplicate determination. The serial dilutions
were made starting with a 2.0 ml volume of serum.
Since the standard contained only corticosterone
diluted in water the parallelism with the serially diluted
serum implies that the assay is specific over this con-
centration range (1?8). Any contaminant would have to
demonstrate 2 different partition coefficients equalling
those of corticosterone and have a fluorescence intensity
at 535 nm similar to that of the standard. '
56
-------
FIGURE 6
VERIFICATION OF THE SULFURIC ACID FLUORES-
CENCE ASSAY FOR SERUM CORTICOSTERONE :
PARALLELISM OF THE STANDARD WITH
SERIALLY DILUTED RAT SERUM
o-
10
z
o
«
CO
o
o
-2H
-3-
.4-
STANDARD
SERUM
-2.5 -2.0 -1.5 -1.0 -0.5 0.0
STANDARD CURVE? LOG CONCENTRATION (UG/ML)
SERUM: LOG RELATIVE CONCENTRATION (LOG^VOLUME/Z.O MLJ)
57
-------
The specificity of the assay has been previously
shown by Silber et al. (1??) and is supported by the
parallelism (1?8) of the standards and serially diluted
unknown shown in Figure 6. Interference of DDT metabolites
in treated animals was also eliminated as a source of error
when measurements of fortified blanks showed no fluores-
cence above background.
The ability to store extracted samples for brief
periods of time allowed processing of large numbers of
samples in single assays. By doing this, using four
matched cuvettes and having assistance during the measure-
ment phase of the assay, it was possible to process over
200 samples in four working days or less. This represents
an advantage over the previous manual procedures (176,177)
and an alternative to automated assays which may be of use
in some clinical and laboratory settings.
D. Radioimmunoassays t
1. The Assay
All measurements of LH in serum and pituitary extracts
were made using the double-antibody procedure of Niswender
et al. (169). All assays were performed in the labora-
tories of the Reproductive Endocrinology Program, Depart-
ment of Pathology, The University of Michigan.
The assays utilized the rat pituitary extract, B-6^0,
as the primary intra-laboratory standard for comparison.
Previous bioassays (ovarian ascorbic acid depletion test,
OAAD) and other radioimmunoassays have established that
58
-------
B-64-0 has a potency of 0.03 times that of the National
Institutes of Health LH standard SI (NIH-LH-S1), i.e.,
each nanogram of 6-6^0 is equivalent to 0.03 ng NIH-LH-S1.
Secondary local standards consisting of pooled rat sera
such as B-873 were run in each assay. The potency of
these secondary standards relative to B-640 has been
established by comparisons of the $Q% inhibition points on
12 ei-
plots of percentage of -'I-oLH remaining bound versus log
mass per tube. Even if the remaining portions of the RIA
curves are nonparallel, and the samples used in generating
them are thereby inferred to be dissimilar, the $Q% points
will yield a valid approximation of the relative potencies
of the two samples (179). Such estimates gave B-873 a
value of 212.0 ng B-640/ml or 6.36 ng NIH-LH-Sl/ml.
There have been several (180-182) reports that the LH
values measured in sera under varying physiological condi-
tions show nonparallelism with the pituitary standard in
this RIA. To ascertain that gross errors of measurement
had not been introduced into the LH measurements made for
the experiments to be described later, a check on the
parallelism of the curves obtained with a rat pituitary
extract, B-6^-0, B-873 and a pool of rat serum generated
during the studies for this thesis was performed. The
12^
percentage of JI-oLH remaining bound was plotted versus
the log of the volume of sample taken for assay (which is
proportional to the log of mass per tube). The curves
were displaced on the plot by an arbitrary constant, <* ,
__._..
-------
to prevent their overlap, and were examined visually for
parallelism. The rat pituitary extract and B-6^0 curves
were markedly similar. The curve generated by B-873 was
noticeably nonparallel with B-6^0 but was quite similar to
that given by the pool of rat serum. The curve given by
the pooled serum also showed some similarity to the curve
generated by B-6^0 but, in fact, seemed not to be strictly
parallel throughout its length with either the B-873 or
B-6^0 curves. These results, shown in Figure 7, indicated
the use of B-6^-0 as the standard for pituitary extracts
and B-873 as the standard for serum samples. The latter
guideline was not always followed if a logit plot of
12*5
^I-oLH remaining bound versus volume of unknown serum
added demonstrated better parallelism with B-6^0 or if the
12<
value of the percentage of ^I-oLH bound fell within
^3-57$. In those instances direct comparison with B-6^0
was made. Usually, the curves of serum derived from
castrated male rats appeared to resemble those from B-873
more than those from B-6*fO; serum from intact male rats
yielded curves resembling those given by B-6^0. Whether
the direct comparison was made with B-6*K) or B-873 the
final results for the assay were calculated in units of
mass of B-6*K) per milliliter of unknown and are reported
as such with the potency of B-640, relative to NIH-LH-S1,
noted.
Serum for use in the LH assays was obtained either
from trunk-blood, taken at the time of decapitation and
-------
Figure ?. The Relationships Between the Response Curves
for Rat Serum and Pituitary Extract and Those
of Radioimmunoassay Standards B-64-0 and B-8?3
Four single representative curves - displaced to the
right or left by a constant, cC, for purposes of clarity -
12*5
of percent of -'I-oLH remaining bound versus log of
volume taken, i.e., LH mass added, are plotted. They
illustrate the analytical relationships between the
primary standard preparation B-640 (= 0.03 x NIH-LH-S1 by
OAAD), the secondary standard preparation B-873 and the
serum and pituitary extracts derived from the experimental
animals. Parallelism between B-640 and the pituitary
extracts, and between B-873 and sera prompted the use of
those standards for the respective tissue preparations.
Weighting of all data toward the 50$ binding level did,
however, allow the direct comparison of results from both
tissues to each other even though their radioactivity
displacement curves are nonparallel.
61
-------
FIGURE 7
THE RELATIONSHIPS BETWEEN THE RESPONSE
CURVES FOR RAT SERUM AND PITUITARY EXTRACT
AND THOSE OF RADIOIMMUNOASSAY STANDARDS
B-640 AND B-873
100-
o
z
o
80
GO
UJ
ro
ro
i 40
CD
z
o
f 20
10
M
PITUITARY EXTRACT
B-640
SERUM
r
T
5
T~
10
"1—
100
I 5 10 50
a±LOG OF VOLUME TAKEN FOR ASSAY
500'
62
-------
prepared as discussed previously in regard to corticoster-
one, or from samples obtained by TVBE (see Section I.
Animals). Blood obtained by TVBE was allowed to clot on
ice and then centrifuged for 20 min at 5000 rpm. The
serum from either bleeding procedure was decanted off
after centrifugation and stored at -20°C prior to assay.
Pituitary extracts were prepared from weighed tissues
either immediately after weighing or subsequent to storage
of the whole tissue at -20°C. The whole pituitaries were
individually homogenized in phosphate-buffered saline, pH
7.^ (PBS) (169) using six strokes of the Teflon pestle
(driven by a Tri-R Stir-R Model 5630 at settings;of 5-8)
of a Potter-Elvehjem homogenizer. The final volume of PBS
diluent varied from 10 to 100 ml and was dependent upon
the size of the gland and whether the rat was intact or
castrated,- pituitaries from castrated adults were pre-
pared in 100 ml of PBS while those of weanling intact
animals were homogenized in 10 ml of PBS. Extracts were
stored at -20°C until assayed.
The volume of the duplicate sample aliquots taken for
assay of LH also depended on the previous treatment of the
animal? sera from intact, untreated rats were assayed by
using aliquots of 200 ulj sera from long-term castrated
rats or animals injected with LHRH were assayed by using
aliquots of 10, 20, and/or 50 ul. The number of duplicated
measurements was determined by the volume of the sample
available. Single duplicates were done on sera from intact
63
-------
animals while three duplicates of various sizes were done
on pituitary extracts,
Since the lower limit of reliability for the assay
(the maximum of ^I-oLH remaining bound minus two times
the standard deviation of its estimation) was normally
2-10 ng of B-6^0/ml, values which were computed to lie
below that level, although distinctly above zero, were not
necessarily accurately determined. Such values are
reported as they were calculated} the potential inaccuracy
will be taken into account in the interpretation of experi-
mental results. Samples which generated values which
exceeded the upper limit of the standard curve of a
particular assay were reassayed using smaller aliquots.
The other assay variables for these assays were as follows.
The mean index of precision was -0.0173, i.e., -3.9$; the
12 *•>
mean limit of detection (the maximum 3I-oLH remaining
bound minus the standard error of its estimation) was
215 - 166 pg per assay tube; the mean sensitivity (the
slope of the curve derived for the B-6^0 standard on the
12 *>
plot of percentage of -'I-oLH remaining bound versus log
mass of LH per tube) was -2.55 - 0.15.
2- Effect of Sampling Time and Ether
Several authors have reported 2^-hour variations in
the titers of serum LH in male rats (183,18^). Others
have demonstrated elevations in LH leve.ls in the serum due
to ether anesthesia (36,183,185). Because the blood
samples for several of my experiments were taken under
64
-------
ether (TVBE) and because 2^-hour periodicity was briefly
examined for animals treated via their o,p'-DDT-injected
dams, a short control experiment was conducted. This
experiment examined the effect of serial TVBE on the
2^-hour periodicity of serum LH in normal adult (300-325 g)
male rats.
After allowing the animals to acclimatize to the
day/night cycle for four days the following sampling pro-
cedure was carried out. Beginning at 12.30 (EST) a group
of 5 rats were bled under ether by tail-vein at intervals
of 6 hours for 2^ hours, i.e., 5 times. A second group of
15 rats was divided into 5 subgroups of 3 animals each.
One of these subgroups was sacrificed by decapitation each
time the ether-bled test animals were bled. Ether-bleeding
and decapitation were alternated so no time bias would be
introduced into the sampling for either group. All animals
were maintained under normal day/night conditions except
for the two short nighttime sampling periods to which the
ether-bled animals were exposed. The-sera which were
collected at each sampling time were stored at -20°C until
assayed.
The serum LH levels (- 1 standard deviation of the
mean) were calculated for each group and time. These were
plotted versus time of day. The individual means from the
ether-bled rats were tested against the individual means
of the decapitated controls by use of Student's t-tests.
The values at two of the time points differ with
~65
-------
probabilities of more than 95$; at 18.00 the ether-bled
animals show serum levels approximately twice the basal
levels and at midnight the decapitated controls show
levels which are twice those of the ether-bled animals and
twice those of the basal levels seen at any other time of
day. These findings are shown in Figure 8.
The results support those of Dunn et al. (183) and
Hefco and Lackey (184) in regard to the normal 24-hour
periodicity of LH in unetherized male rats. They also
support the findings of Dunn et al. (183), Rowland et al.
(36) and Krulich and Illner (185) in regard to an
ether-induced rise in serum LH during the daytime hours
and the lack of such a rise at night. The plot of the
24-hour periodicity of. the serum LH in ether-bled animals
is nearly superimposable on similar curves generated from
rats suckled by vehicle-injected dams (see intact controls,
Figure 31) and other vehicle injected adult male rats.
The observed pattern is also very similar to the one
Lawton and Smith (186) generated using etherized male rats
and an LH bioassay. Together the reported and present
data indicate a variable sensitivity of the male rat
hypothalamo-hypophysial axis to ether. The peak of normal
LH secretion occurs during the period of peak physical
activity and food intake. This period may represent a
time of passive physiological resistance to stress
resulting in the diminished response to ether at midnight.
This low value may merely represent .a spuriously low mean
66
-------
Figure 8. The Effect of Repeated Tail-Vein Bleeding Under
Ether on the 2*l-Hour Periodicity of Serum LH in
Normal Adult Male Rats
Serum LH measured by the radioimmunoassay of Niswender
et al. (169), as ng of primary standard, B-6^0 (=0.03 x
NIH-LH-S1 by OAAD), is plotted versus time of day. At
each time point indicated the number of animals shown in
parentheses were either bled immediately after brief (^5
sec) exposure to ethyl ether or immediately after decapita-
tion. The same animals were bled under ether throughout
the experiment; missing values were due to technical
problems during sample collection or analysis. The
light-dark schedule was 06.30 to 18.30 as shown. All ani-
mals were fed and watered ad libidum throughout the experi-
ment. Values shown are means - 1 standard deviation;
statistical difference from the decapitated controls was
tested at each time point by Student's t-test (* p< 0.005).
67
-------
FIGURE 8
THE EFFECT OF REPEATED TAIL-VEIN BLEEDING
UNDER ETHER ON THE 24-HOUR PERIODICITY
OF SERUM LH IN NORMAL ADULT MALE RATS
TIME OF DAY
68
-------
(n = 2), however, since the similar data in Figure 31 show
no difference between the serum LH levels of the intact
controls near midnight and the value obtained from the
unetherized control rats in the present experiments
(Figure 8). If the ether-bled and control values at mid-
night are, in fact, similar, the data are consistent with
the claim of Dunn et al. (183) that the serum LH levels
cannot be elevated further at the nighttime peak due to
the normal presence of an optimal LHRH output at that time.
The explanation probably combines facets of both increased
physical activity, respiration, etc., and a normal elevated
LHRH output.
Because of the temporal positions of the serum LH
elevations under normal or ether-stressed conditions,
later experiments were designed to take blood samples
under the most appropriate conditions. If basal levels
were of interest samples were taken by decapitation near
noon; whereas, if the difference between serum LH levels
was to be tested in DDT treated and untreated groups,
samples were taken near 18.00 (EST). In the second
example the time was chosen so as to maximize any differ-
ential effect ether might have on these two groups (see
also intact rats, Figure 31).
E. Estimation of o.p'-DDT Derived Residues in Rat
Pups
The indirect treatment of rat pups, accomplished by
allowing them to suckle o,p'-DDT-injected dams, brought up
69
-------
the question of administered dosage. To answer that
question and to allow analyses of the insecticide used in
these studies the following protocols were adapted.
Neutral hydrophobia compounds similar to DDT form the bulk
of the metabolites normally found in tissues (137,158,187).
Therefore, the "cleanup" protocol was designed to maximize
the recovery of these materials while eliminating the large
bulk of contaminating, nonrelated hydrophobic compounds.
It was suggested in large part by the sulfuric acid
purification of hexane extracts containing PCB detailed
by Widmark (188).
To measure the dam's secretion of neutral DDT
metabolites into her milk and the pup's subsequent uptake
of those metabolites, male pups were removed at various
ages from the litters of both DDT-injected and vehicle-in-
jected dams. The litter sizes were maintained by
replacing the males removed with females of similar age.
Within 10 min of removing the pups from their litters they
were killed by asphyxiation with carbon dioxide. Their
stomachs, containing milk curds, were then removed and the
stomachs and carcasses were frozen and stored separately
until the time of extraction and analysis.
A flowchart for the "cleanup" method is shown in
Figure 9. After weighing the biological sample of interest
it was homogenized with 2-5 weight-volumes of saturated
salt (NaCl) solution and 4-10 volumes of acetone. The
homogenization was done with a Potter-Elvehjem homogenizer
'~70~
-------
Figure 9. Generalized Protocol for Estimation of DDT
Analog Residues
The flowchart outlines the extraction and purification
procedures which were used on each milk curd or rat pup
carcass utilized in the determination of the uptake of DDT
residues by pups suckling o,p'-DDT injected dams. Extrac-
tion and wash volumes used for processing the milk curds
were constant due to the similarity in size of the curds.
However, the large variability in the carcass weights
forced the use of the smallest volume multiples listed in
order to maintain workable total volumes. Even in light
of this problem recovery of the internal standard, aldrin,
remained uniform within each category of samples.
71
-------
Figure 9
Generalized Protocol for Estimation of DDT Analog Residues
Stomach Curd or Carcass
(Parallel Estimations)
I
1. Weighed?
I
2. Homogenized with 2-5 volumes of saturated NaCl
and 4—10 volumes of acetone;
I
3. Internal Standard added;
*K Rehomogenized
Organic Layer
Combined Extracts
|
6. Washed 2 times with
0.05-0.15 volumes
of 80$ MeOH
I
MeOH Wash
Discarded
Acid and Water Washes
(
Discarded
Aqueous Residue
5. Reextracted with 8-20
volumes of benzene and
2-3 times with 0.3-20
volumes of hexane
1
Aqueous Residue
Discarded
Organic Layer
?. Volume measured;
I
8. Washed 1 time with
0.05-0.15 volume of
HgSO/j, (cone.) and 2
times with 0.1 volume
of water
_. -——
Organic Layer
9. Dried with anhydrous
MgSOjj, and evaporated
to appropriate volume
I
Dry Organic Extract
10. Gas chromatography
72
-------
at high speed if the curd was being analyzed or with a
Waring blendor equipped with an explosion-proof chamber if
whole pups were being extracted. After the initial
homogenization a known quantity of the internal chroma-
tography standards used for pesticide analysis, aldrin and
p,p'-DDT, were added (0.5 ug of each were added in curd
extractions; 50 ug of each were added in whole-body
extractions). The solution was then rehomogenized and
placed either into a centrifuge tube or a separatory
funnel. When the organic and aqueous phases had settled
or been separated by centrifugation the organic layer was
removed and saved. The aqueous residue was reextracted
once with 8-20 volumes of benzene and 2 or 3 times with
0.3-20 volumes of hexane. The aqueous residue was dis-
carded. All the organic phases were combined and washed
twice with 0.05-0.15 volume of 80$ aqueous methanol. The
alcoholic washes were discarded and the volume of the
organic layer measured. This solution was then washed
with 0.05-0.15 volume of concentrated1HgSO^. Following
the acid wash the organic layer was washed twice with 0.1
volume of water. Acid and water washes were both dis-
carded. This step, 8, removed the large majority of the
hydrophobia impurities left in the solution. The wet
organic phase was dried over anhydrous MgSOj, and then
evaporated on a steam-cone under nitrogen to near 5 ml
(curd samples) or 50 ml (whole pups). Final volumetric
adjustments were made by adding hexane. Aliquots of 1-8 ul
73
-------
of these dry extracts were injected directly into the gas
chromatograph for qualitative and quantitative analyses.
The precise volumes used in extractions and washes
varied with sample weight. When dealing with whole body
residues a ceiling of 300 ml was used for the addition of
benzene and 600 ml for a total extraction volume. Other
extraction and wash volumes were scaled up or down within
the limits shown to prevent the sample preparation from
becoming unwieldy. For small to moderate size samples,
however, the upper limits of the volumes shown were favored
to minimize problems with emulsions and to maximize clean
recoveries. For curd samples standardized volumes were
used throughout the preparationi 5 ml of saturated salt
solution and 10 ml of acetone, 20 ml of benzene and 2 times
20 ml hexane, 2 times 10 ml of 80$ methanol, 10 ml con-
centrated HrjSQ^, 10 and 5 ml of water. When calibration
standards were processed by this procedure all steps
followed those used for the preparation of stomach curd
samples.
All concentrated extracts were analyzed by a Varian
2100 GC system which incorporated a
-------
were run under a shallow temperature program, 175° to
195 C at 0.5 /min, which had been demonstrated not to
interfere with peak resolution or baseline stability.
Attenuator settings varied slightly but were usually at
16 x 10~ amp/mvj detector standing current was normally
2 x 10"" amp. Chart speed was 0.5 cm/min. The column
packing was suggested by the work of McCully and McKinley
(189) and Abou-Donia and Menzel (190)j the retention times
observed agree fully with their findings. Relative reten-
tion times with respect to aldrin were: 1.45, o,p'-DDEj
1.84, p.p'-DDE; 2.04, o,p'-DDD; 2.39, o.p'-DDT} 2.57,
p,p'-DDD; 3.06, p,p'-DDT. Three typical chromatograms
are shown in Figure 10.
Since the o.p'-DDT was contaminated with 1.5$ p,p'-DDT
(see II. Chemicals) the knowledge that p,p'-DDT is bio-
logically stored preferentially to o,p'-DDT (166) militated
against any reliance on the use of p,p'-DDT as an internal
standard. Therefore, all analyses were performed using
only aldrin as the standard for internal comparison and
deleted quantitation of o,p'-DDT. The sharpness and form
of most of the peaks in the chromatogram indicated that
*•
plots of peak height versus mass, or some transformation
of these measurements, would be suitable for quantitation.
Calibration curves were generated by chromatographing mix-
tures containing various amounts of o,p'-DDE, p,p'-DDE,
o,p'-DDD, o.p'-DDT and p,p'-DDD both before and after
extraction. The linearity of the curves of DDT analog
75
-------
Figure 10. Typical Gas Chromatograms Obtained During
Analyses of o,p'-DDT and Its Analogs
Profiles of detector response versus retention time
are shown for 3 typical samples. Sample A, , is a 1.2
ul injection of an unextracted standard solution containing
1.5 ng/ul of each of o,p'-DDE (II), p,p'-DDE (III),
o,p'-DDD (IV), o,p'-DDT (V), p,p'-DDD (VI) and 1.0 ng/ul
of both aldrin (I) (internal standard) and p,p'-DDT (VII).
Sample B, —->, is a 2.2 ul injection of the residues
extracted from a 15 day old rat pup which had nursed an
o,p'-DDT injected damj 1.0 ng/ul of internal standard has
been added. Sample C, , is a 1.75 ul injection of the
hexane extract of a milk curd from the stomach of a 10 day
old rat pup which had nursed an o.p'-DDT injected dam?
0.1 ng/ul of internal standard has been added.
All samples were run on a 2 m x 2 mm column of 6%/^%
QF-l/SE-30 on 80/100 mesh Gas Chrom Q under a temperature
program of 0.5°/min from 1?5° to 195°C. Injector and
detector temperatures were 2^5° and 295°C,1 respectively.
Gas, N2, flow rates were 63 ml/min under a pressure head
of ?0 psi. An electron capture detector was used at an
attenuator setting of 16 x 10~ amp/mvj chart speed was
0.5 cm/min.
76
-------
FIGURE 10
TYPICAL GAS CHROMATOGRAMS OBTAINED DURING
ANALYSES OF o,p-DDT AND ITS ANALOGS
10
TIME
(MINUTES)
20
30
77
-------
peak height/aldrin peak height versus DDT analog concen-
tration was tested by least-squares regression. Though the
curvature of these plots was not great the best lines were
obtained if a log-lo.g transformation was applied. There-
fore, all unknowns were calculated from standard curves of
log1Q (DDT analog peak height/aldrin peak height) versus
log1Q (concentration of DDT analog). Two overlapping
standard curves were generated using high, 1 ug/ml, and
low, 0.1 ug/ml, final concentrations of aldrin. This was
done to allow more precise measurement of the peak height
ratios and thus to allow more precise measurement of the
residue levels of the unknowns. These two curves were
strictly parallel and their intercepts differed after cor-
rection for aldrin concentration only because the recovery
of aldrin was proportional to the amount added prior to
extraction. The characteristics of each of the high
aldrin concentration curves are shown in Table 2 along
with the extraction recoveries calculated from extracted
and unextracted standards. The somewhat high variability
of the curves most likely reflects both the small number
of replicates, 2-3, done on each sample and departures from
good peak symmetry. Duplicates of unknowns were normally
within - 10-15# and thereby indicate that A is probably a
pessimistic estimate of precision.
78
-------
VO
Table 2
Standard Curves for Determination of DDT Analogs by Electron-Capture Gas Chromatography
Variable
Slope1 b-SD
2
Intercept
atsD
Correlation p
Coefficient K
SEy3
A3
4
Useful Range
(ug/ml)
t
$ Recovery^
o.p'-DDT
0.986^0.032
-0.781-0.020
0.99^
0.065
0.066
0.008-9.0
97.2
o,p'-DDD
0.846-0.024
-0.434^0.017
0.993
0.068
0.080
0.009-9.0
106.3
Analog
o,p'-DDE
0.876-0.021
-0.469-0. 014
0.995
0.061
0.070
0.019-9.0
102.8
p,p'-DDD
0.488^0.029
-0.324^0.017
0.975
0.065
0.133
0.012-9.0
103.0
p,p'-DDE
0.836^0.024
-0.346^0. 017
0.993
0.070
0.084
0.007-9.0
102.7
The standard curve is a plot of Log,Q (Peak Height Analog/Peak Height Aldrin) versus
Log,Q (Analog Concentration) and is of the form Log y = a + b Log x.
2
The intercept is given for the standard curves used at higher analog concentrations
which utilized an aldrin (internal standard) concentration of 1 ug/ml; at lower
analog concentrations an aldrin standard of 0.1 ug/ml was used and the intercept
increased.
-------
oo
o
Table 2 (Cont.)
o
J The error in the predictability of Log y given Log x is the standard error of the
estimate, SEyj the error in the predictability of Log x given Log y is the index of
precision, X » &n& equals SE /b
The useful range is calculated from the linear portion of the parallel curves
generated for both high and low concentrations of analogs.
* Recovery of extracted versus nonextracted standards? aldrin recovery was 66% at
0.1 ug/ml and 8*4$ at 1 ug/ml.
-------
P. Testicular Incubations >
1. Protocol
Measurements were undertaken to investigate the
existence, and possible site, of subcellular interactions
"between o,p'-DDT and the testicular steroids. Incubations
of [? n- H]- A -pregnenolone were conducted in the presence
or absence of o,p'-DDT in testicular homogenates. The
protocol, described below, was so designed that it also
showed the presence or absence of o,p'-DDT metabolism in
the same homogenates.
Testes, obtained during castrations or after animal
sacrifice, were weighed, decapsulated and homogenized at
^°C in 6 weight-volumes of Buffer I. Buffer I is a
modified Ringer's solution suggested by Moldeus et al.
(191) which resembles buffers previously used both in
steroid incubations with testes (182,193) and in drug
incubation with hepatic microsomes (191). Buffer I is
125 mM NaCl, 6 mM KC1, 5 mM MgClg, 15 mM NagHPO^, 10 mM
succinate adjusted to pH 7.^. The homogenization was
achieved with 6 slow strokes of a Potter-Elvehjem homogen-
izer fitted with a Teflon pestle (driven by a Tri-R Stir-R
Model 5^3C at settings of 5-8). The homogenate volume was
measured and poured into 50 ml centrifuge tubes. These
were centrifuged at ^°C at 500 x £ for 10 minutes in a
Lourdes model LRA centrifuge. After centrifugation the
supernatants were decanted into flasks and held on ice
prior to incubation.
81
-------
Incubations were performed in 20 ml beakers to which
200 ul of propylene glycol, measured amounts of ethanol or
ethanolic solutions of A^-pregnenolone and/or o,p'-DDT,
cofactors and 1.90 ml of Buffer I were added. The cofac-
tors included an NADPH-generating system and NADH; their
concentrations in the final incubation volume were 0,825
NADP+, 0.4125 mM G-6-P, 1 unit of G-6-P DH/ml and 0.4125 mM
NADH. .Substrate concentrations were 1 nmole/ml in the final
incubation volume for both A^-pregnenolone (including 5 uCi
of [? n- H] - A -pregnenolone) and o,p'-DDT. Incubations
were initiated by vigorously adding 4.0 ml of supernatant
(40-60 mg of protein) or Buffer I to the chilled cofactor-
substrate solution. Immediately after this addition a 1.0
ml aliquot was removed for a zero-time determination of ste-
roids and/or DDT metabolites. The remaining solution was
quickly placed into a Dubnoff metabolic shaker set at 20
rpm. The incubation proceeded under air at 3?°C. At 5» 10i
30 and 60 minutes of incubation additional 1.0 ml aliquots
were taken. After incubation the residual volume of solu-
tion was retained for determination of protein and to check
the number of tritium counts initially added to the incuba-
tion. Protein was measured by the Biuret method (194) using
BSA as a standard. Counts were determined on a 0.1 ml ali-
quot which was allowed to air dry prior to addition of
scintillation fluid. The protocol is summarized in Table 3,
Each of the incubations, 1-5, was designed to probe a
different facet of the potential differences between
82
-------
Table 3
Incubations Performed
00
Volume of Solutions Added to Each
Addition
Buffer I2 (ml)
3
Cofactors-^
Propylene Glycol (ul)
Supernatant (ml)
[7 n-^HJ-A^-Pregnenolone-5 (ul)
cold A-'-Pregnenolone (ul)
o,p'-DDT7 (ul)
Ethanol (ul)
Total Volume (ml)
Flask
Incubation Number
1
1.90
+
200
IKOO
5
5
8.2
0
6.20
2
1.90
+
200
U-.OO
5
5
0
8.2
6.20
3
1.90
+
200
^.00
0
0
8.2
10.0
6.20
4
1.90
+
200
^. 00
0
0
0
18.2
6.20
5
5.90
+
200
0
5
5
0
8.2
6.20
Flasks were incubated in a Dubnoff metabolic shaker at 20 rpm under air at 37 C and
samples taken at 0, 5» 10, 30 and 60 minutes of incubation. The residual volume of
each flask was retained for determination of protein by the Biuret procedure (BSA
standard) and for determination of the total initial content of ?E counts.
2 Buffer I is 125 mM NaCl, 6 mM KC1, 5 mM MgCl9, 15 mM Na^HPO,,, 10 mM succinicate at
pH ?.*. 2 2 4
-------
Table 3 (Gont.)
Q *l_
^ Final cofactor concentrations in the total incubation volume were 0.825 mM NADP ,
0.^125 mM G-6-P, 0.4125 mM NADH and 1 unit of G-6-P DH/ral.
The 500 x £ supernatant of a 6:1 homogenate of testicular tissue in Buffer I was used.
•^ Concentration of the ethanol solution added was 1 uCi/ul.
Concentration of the ethanol solution added was 1,18 nmoles/ul.
' Concentration of the ethanol solution added was 0.75 nmole/ul.
oo
-------
supernatants derived from treated and untreated animals.
Number 5 serves as a control for thermal or nonenzymatic
steroid transformations and therefore as the baseline for
steroid metabolism in supernatants from both treated and
untreated animals. Number 4 provides a control for the
endogenous presence and/or conversion of o,p'-DDT. Number 3
allows the measurement of the metabolism of DDT in the
absence of exogenously added steroid while number 2 allows
the measurement of the metabolism of A-'-pregnenolone in
the absence of exogenously added o,p'-DDT. Number 1 pro-
vides a direct test of the interactions of steroid metabo-
lism and DDT metabolism by allowing simultaneous measure-
ment of both sets of metabolites.
To allow this simultaneous measurement of both sets
of metabolites an extraction and purification scheme was
devised; it is summarized in Figure 11. The aliquots
taken during incubation were forcefully expressed into 5
ml of ethanol held in a 15 mm x 120 mm ignition tube.
This stopped any metabolic conversions and began the
extraction process. If further processing was not begun
immediately the solutions were stored at -20°C. A recovery
marker (60,000 dpm C-steroid) and carrier steroid (100
ug) were then added for each of the several steroids of
interest. An internal standard, 5 ug of p,p'-DDT, was
also added to monitor the recovery of DDT metabolites.
This solution was Vortexed thoroughly and then centrifuged
for 10-15 minutes at top speed in a Clay-Adams tabletop
85
-------
Figure 11. Protocol for Extraction and Partial Purifica-
tion of DDT and Steroid Metabolites in
in vitro Incubations
The 1.0 ml samples taken from the in vitro incuba-
tions carried out under conditions described in Table 3
were prepared for analysis by this procedure. Steroids
added to aid in recovery and estimation included 100 ug
-ik
cold steroid plus 60,000 dpm 4- C-steroid each for:
A-^-pregnenolone, progesterone, 17cC-hydroxyprogesterone,
dehydroepiandrosterone, A -androstenedione, and tesoster-
one. The p,p'-isomer of DDT was also added (5 ug) as an
internal standard and recovery tracer for the DDT analogs.
The internal standard for the steroids, cholesterol-3-pro-
pyl ether (CPE), was added just prior to gas chroma-
tography in a system which split the column effluent in
approximately a 3*1 ratio between a collection capillary
and the mass detector of the chromatograph. Specifics of
the measurements are discussed in Methods Section III.F.2.
and in Chapter 6.
86
-------
Figure 11
Protocol for the Extraction and Partial Purification of DDT
and Steroid Metabolites in in vitro Incubations
1.0 ml Aliquot
. to 5 ml of ethanol in a 15 mm x 120 mm ignition
; iq>C-steroid plus cold carrier steroid added;
Added
tube? -^C-steroid plus
5 ug of p,p'-DDT added; V.ortexed and centrifuged
Alcoholic Extract
I
Extracted with 2 x 2 ml of hexane
in 60 ml separatory funnel
Alcoholic Solution'
I
Extract with 20 ml
Aspirate
off
alcohol
Wash with 5 ml 1 N
HC1, 5 ml 5% NaHCOv
2 x 5 ml NaCl solu=2
tion; dry over
anhydrous MgSOj,;
evaporate to dryness
under N£ in 10 mm x 75
mm test tube
Store in pyridine at -20°C
Hexane Extract
~~1
Washed with 10 ml cone.
HpSOr and 10 ml HpO; dry
over anhydrous MgSOi,
I ^
Evaporate in vial under
N9 for storage at -20°C
d I
Reconstitute in hexane
I
GasChromatography
1
DDT Data
Evaporate pyridine; add 200 ul
of 1:1-ethanol:benzene and 200
ug CPE
Gas Chromatography-
Area Data
Collected Effluents
- !
Count Data
\
Quantitation Program-
T
Steroid Data
87
-------
centrifuge. The extract was carefully decanted off into a
60 ml separatory funnel. The pellet was reextracted with
another 2 ml of ethanol. The combined ethanol extracts
were extracted twice with 2 ml of hexane. These hexane
extracts were combined and washed with 10 ml of concen-
trated H2SO^ followed by 10 ml of water. The purified
hexane extract was dried over anhydrous MgSOn. Before
storage at -20°C prior to assay, the DDT extract was
evaporated to dryness under nitrogen in a i dram vial.
The hexane-extracted ethanol solution was extracted
further with 20 ml of dichloromethane. The phases were
allowed to separate and the upper alcoholic phase was
aspirated off. Then the dichloromethane, which contained
the majority of the steroids, was washed with 5 ml each of
1.0 N HC1 and 5% NaHCO- and twice with 5 ml of a concen-
trated NaCl solutions each wash was aspirated off. The
dichloromethane extract was then dried over anhydrous
MgSO^ and evaporated to dryness under nitrogen in a
10 mm x 75 mm test tube. Pyridine, 0;i ml, was added and
the tube was sealed with Parafilmj the extract was stored
at -20°C prior to assay.
Just before assay of the DDT extract, 100-1000 ul of
hexane were added. Then a 1-5 ul aliquot of the sample was
quantitated by GC using the conditions mentioned in Methods
Section E., Estimation of o.p'-DDT Derived Residues in Rat
Pups. Before assay of the steroid extract the pyridine was
evaporated off with nitrogen and 200 ul of a 1:1 solution
88
-------
of benzene and ethanol containing 200 ug of the chosen
internal standard for GO of steroids, cholesterol-3-propyl
ether (CPE), was added to make up the assay solution.
2. Estimation of Steroids by Gas Ghromatography
and Scintillation Counting
Aliquots of the final steroid assay solution, 1-5 ul,
were injected onto a 350 cm x 2 mm glass column equipped
with an effluent splitter. The column was packed with
80/120 mesh Gas Chrom Q coated with 1.5$ OV-? and 1.5%
SE-5^. The effluent splitter favored an effluent-cap-
ture-tube over the flame-ionization detector by 3 to 1,
i.e., 75$ of the effluent was captured and 25$ was burned
for qualitative and quantitative analysis. Chromatograms
were run using nitrogen as the carrier gas at a flow rate
of 25 ml/min (head pressure was 70 psi)j hydrogen and
oxygen were used as the flame support gases at flow rates
of 30 and 60 ml/min, respectively. Injector and detector
temperatures were 250° and 295°C, respectively. Column
temperature was programmed on a linear gradient of l°/min
from 1?5° to 275°C. Chart speed was 10 cm/hour. Two
sample Chromatograms are shown in Figure 12.
The use of acetate and methyloxime-silyl derivatives
(195) on this and several other columns had been explored
with varying degrees of success,, in terms of efficient
effluent capture and adequate resolution of the main
steroid peaks of interest. Methyloxime-silyl derivatives
were chromatographically more stable and volatile than the
89
-------
Figure 12. Typical Gas Chromatograms Obtained During
Analysis of Testicular A -Pregnenolone
Metabolites
Two sample chromatograms obtained during analyses of
testicular metabolites of A -pregnenolone are shown.
Plot A was taken from a ?.?5 ul injection of a calibration
standard containing 2.0 ug/ul each of dehydroepiandroster-
one (II), A -androstenedione (III), testosterone (IV),
A^-pregnenolone (V), progesterone (VI), 17oC-hydroxypro-
gesterone (VII) and 200 ng/ul cholesterol-3-propyl ether
(internal standard) (IX). Plot B was obtained from a
6.05 ul injection of an extract prepared via the protocol
of Figure 10 from a sample taken at 30 minutes of incuba-
tion of a testicular homogenate. The homogenate was pre-
pared from the testes of an adult male rat which had been
injected daily on days 1-5 of age with 0.05 ml of sesame
oil containing 4-0 ug estradiol-l?-valerinate. In addition
to the peaks II-VII and IX indicated in the calibration
mixture two unidentified peaks, I and VIII,< are also
evident. It should be noted that in plot B the bulk of
the steroid mass responsible for peaks II-VII is due to
the added carrier steroids; the similarity of the
inter-peak relationships between Plots A and B is a reflec-
tion of the similarity of the inter-steroid mass ratios in
the standard solution and the unknown solution containing
the added carrier steroids. See Materials and Methods
Section IV.F.2. for chromatographic details.
90
-------
FIGURE 12
TYPICAL GAS CHROMATOGRAMS OBTAINED DURING
ANALYSES OF TESTICULAR A5-PREGNENOLONE
METABOLITES
10
30 60
TIME (MINUTES)
i
90
91
-------
parent compounds but their capture efficiency was poor due
to their tendency to form stable aerosols when cooled
(195) rather than condensing onto the capillary capture
tubes. Acetate derivatives worked well also but they had
increased retention times and the peaks for dehydroepi-
androsterone acetate and A -androstenedione overlapped.
Though the peaks for testosterone and A -androstenedione
overlap in the present system their metabolic status as
end-products of both major androgenic pathways (see Figure
1) provides a more logical reason to measure them together
than would be the case for measuring dehydroepiandrosterone
h.
and A -androstenedione concurrently. Although both this ,
peak overlap and the thermal breakdown of 1? cC-hydroxy-
progesterone were recognized problems, the use of parent
compounds obviated the need for derivatization time and the
problems associated with the derivatives.
The components eluted coincidentally with each of the
major peaks of interest, II-VII and IX in Figure 12, were
captured in dry-ice chilled capillaries which were then
flushed into scintillation vials with 10 ml of scintilla-
tion fluid (6 g PPO and 0.25 g POPOP/1 in toluene).
These individual samples were counted 3-*J- times for 10
minutes each time in a Packard Tri-Carb Model 3320 scintil-
lation counter. Tritium was consistently counted at
48-^9$ efficiency in this system while carbon-14 was
counted at 66-69$ efficiency? no quenching was encountered,
o •} h
Counts were converted to -'H and C dpm by using a program
92
-------
written for a Wang 6l4 calculator which was based on the
discriminator-ratio method of Okita et al. (196). Counting
errors were normally less than 1.2$ for ^H and less than
for ^C.
Initial qualitative identification of the peaks was
based on the retention times relative to CPE of known
steroids. Solutions of various known concentrations of
dehydroepiandrosterone, A -androstenedione, testosterone,
A^-pregnenolone, progesterone and 17<£ -hydroxyprogester-
one, each of which contained 1 ug/ul of CPE, were chroma-
tographed to identify the various peaks and to calibrate
the system for the later quantitation of unknowns. The
retention times relative to CPE werei 0.57t dehydroepi-
androsterone; 0.6?, A -androstenedione and testosterone;
0.69, A^-pregnenolone; 0.79, progesterone; and, 0.91,
17oC-hydroxyprogesterone. Peak area data for these
chromatograms were calculated both by triangulation and by
an automatic integration system incorporating an
Adams-Smith interface and an experimental program written
for a Wang 61 *J- calculator; the results from both methods
compared favorably for all but low, broad peaks which the
automatic system failed to detect. The logarithms of peak
areas, normalized by the peak area of CPE, were plotted
versus the log of the concentration of steroid injected.
These standard calibration curves along.with the mean
percentage of steroid captured for counting are described
in Table 4. Overall recoveries for the steroids, including
93
-------
Table 4
Standard Curves for Determination of Steroids "by Gas Chroraatography
Variable
Slope2 b-SD
2
Intercept
a-SD
Correlation R
Coefficient K
SE 3
X3
4
Useful Range
(ug/ml)
% Recovery
DHA
1.005-0.028
0.183-0.018
0.993
0.066
0.066
0.02-25
68.6
A + T
0.988-0.024
0.440^0.013
0.995
0.049
0.050
0.04-50
59.0
Steroid
1.047-0.031
0.153^0.020
0.992
0.073
0.070
0.02-25
88.5
P
1.004-0.031
0.086-0.019
0.992
0.072
0.072
0.02-25
78.1
17
-------
Table *f (Cont.)
The least concentration detectibly greater than zero •§• 2 A to the maximum measured,
5 n It
^ Recovery of C-steroids injected onto the column and trapped with a chilled
capillary tube divided by the actual fraction of chromatographic effluent flowing to
the capillary.
vO
-------
extraction, GC and capillary recovery (which is shown in
Table ty) ranged from 31% for l?oC -hydroxyprogesterone to
66% for A^-pregnenolone. Due to the somewhat high values
of X all measurements on unknowns were done at least in
duplicate.
To further support the identities of the steroids with
Vi k
C ratios were
generated for each GC peak of interest both before and
after each of a series of further purification steps.
Peak effluents were combined from 5 chromatograms of an
assay sample generated from the aliquot taken after 60
minutes of incubation of a testicular supernatant from a
sesame oil-injected rat. The combined effluents for each
peak were eluted from the capillaries with 3 ml of acetone
and evaporated to dryness. They were taken up in 200 ul
of ethyl ether and 10 ul were counted. Fifty micrograms
of each of A -androstenedione and testosterone were added
to the solution containing the effluent of peak III and IV
(Figure 12). This solution was applied to a TLC plate and
run twice in 7:3-hexane:acetone. The spots were located
under UV light, scraped and then eluted with 1.5 ml
l:l-methanol:chloroform. Aliquots of these eluents were
\
counted.
After the addition of 5 mg of the putative steroid
each peak effluent was submitted to derivatization with
either acetic anhydride or methoxylamine hydrochloride.
GC effluents corresponding to peaks II, IV, V and VII were
96
-------
acetylated while the methyloximes were formed for those
corresponding to peaks II and VI. After derivatization
the steroids were recrystallized from ethanol/water. The
crystals were collected and dissolved in acetone; aliquots
were again counted. Recrystallization of the derivatives
was carried out twice more, first from acetone/water and
then from ethyl ether/hexane. In both cases aliquots of
o ~] IL
the collected crystals were counted. The JE/ C ratios
obtained are given in Table 5. Most of the steroids
o
initially lose some -^H counts before the ratio stabilizes,
indicating the inclusion of some other labelled metabo-
lites within the captured effluent or the inclusion of
column-derived background counts for which corrections
have not been made. (Based on effluents corresponding to
the unlabelled CPE peak, column effluents include nonspe-
cific counts amounting to 1-2$ of the total number of
counts injected onto the column for a particular chromato-
gram for both -*H and C.) The peak containing A -andros-
tenedione and testosterone appears reasonably pure as
does l?oC-hydroxyprogesterone. The probable tailing of
testosterone into the A^-pregnenolone peak explains the
•3
initial loss of ^H for this peak. The progesterone peak
obviously contains at least one other product and the
dehydroepiandrosterone peak appears to be grossly con-
taminated by another product or products. Both these last
peaks do, however, show an approach to a stabilized
3 /l^-
•^H/ C ratio by the third crystallization and therefore
97
-------
Table 5
C Ratios s Evidence Supporting Most of the Steroid Identities Assigned to the Gas
Chromatographic Peaks Obtained During Analysis of Steroid Metabolite Mixtures
vO
oo
Treatment
DHA/II
Captured Peak ^.333
TT r
( 7 * 3-Hexane : Ac e tone )
0
Derivatization^ and
Crystallization I 2.763
(EtOH/Water)
Crystallization II 1.790
(Acetone/water)
Crystallization III 1.398
(Ethyl Ether/Hexane)
p
Steroid and Assigned Peak
A^/m T/IV A^/V P/VI 170C/VII
___ Q 7nn - i i?^ i IRQ i i QO
3D'7
-------
Table 5 (Cont.)
o f • • :
Prior to derivatization 5 rag of the putative steroids were added. Acetates of DHA,
T, &-> and 17oC were made by reaction with 2;l-pyridine:acetic anhydride; for A4
and P methyloximes were formed by reaction with a 2% solution of methoxylamine HG1
in pyridine.
-------
support the presence of the putative steroids within the
captured effluents. It should also be noted that these
data were generated by a single set of recrystallizations,
each sampled only once for each step of purification; the
variability of the ratios between crystallizations would
likely have been smaller if duplicates or triplicates had
been run. The results support the identity of the cap-
tured radioactivity with the peak assignments given in
Figure 12 with the possible partial exceptions of dehydro-
epiandrosterone and progesterone. (Other data, reported
in Chapter 6 and the Appendix tend to mollify even these
exceptions.)
Counting and GC data for the metabolism of
A-'-pregnenolone were combined and analyzed by the use
of the procedures discussed in Chapter 6 and the computer
program detailed in the Appendix.
100
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CHAPTER 3
EXPERIMENTS ON THE EFFECTS OF DIRECT INJECTION OF
o.p'-DDT INTO NEONATAL MALE RATS
The approach summarized in Figure 4 implied several
possible divisions of experimental results: by route of
administration, by castration state or by measured vari-
able. The most coherent discussion of the findings seemed
to be based on the route of administration. Therefore,
the next two chapters will describe the results of the
physiological measurements made on animals dosed by direct
(Chapter 3) and indirect (Chapter 4) means. A coordinating
discussion including conclusions and postulations for
future experimental directions comprises Chapter 5. Due
to the different nature and development of the studies
done on testicular incubations they will be discussed
separately in Chapter 6.
I. Organ Weights in Intact Male Rats and in Neonatally
Castrated Adult Male Rats Neonatally Injected with
o.p'-DDT;
The initial experiment in these studies was an
attempt to determine the existence in male rats of a hypo-
thalamic imprinting by o,p'-DDT which was similar to that
which had been reported in females (53). T"t was decided,
on the basis of observations made on animals imprinted with
steroids (57-61,63-67), that alterations of growth and/or
organ weight should be demonstrable if steroidogenic
101
-------
derangement occurred. This would be true if the derange-
ment was caused by either a direct action on the steroido-
genic endocrine glands or by an indirect action via the
hypothalamus or the liver. Neonatally castrated animals
were also included in the experiment to investigate the
possibility that endogenous testosterone was involved in
either the production or the amelioration of any observable
effect of neonatally administered o,p'-DDT. Animals
treated with 170 -estradiol-l?-valerinate (EV) were also
included. They served as positive controls, confirming
the sensitivity of the animals used to imprinting and pro-
viding a comparison of the extremes seen in normal and
imprinted animals. Estradiol was chosen as a control over
testosterone because previous reports supported the idea
that o,p'-DDT acted as a weak estrogen (22,24,26,27,1/1-6-
148,150,162).
Within 6 hours after delivery pups were placed into
litters of 8 pups each and about 1/3 of the males were
castrated. The castrated and intact litters were then
placed into 3 treatment groups and injected on each of
days 1-5 of age. Controls received injections of 0.05 ml
sesame oil; DDT-treated animals received injections of
0.05 ml of sesame oil containing /J-00 ug o,p'-DDT; EV
treated animals received injections of 0.05 ml sesame oil
containing 40 ug 1? f* -estradiol-17-valerinate. Weights
were determined at regular intervals throughout develop-
ment. The rats were sacrificed between 81 and 110 days of
102
-------
age at which time the body and organ weights were recorded
along with any obvious morphological abnormalities.
Body weights measured during development did not
differ noticeably among the groups until after 35-^0 days
of age. At this time the growth of estrogen-treated ani-
mals began to lag behind the other groups; castrates
lagged behind intact animals in all groups after ^5-55
days of age. In neither the intact groups nor the
castrated groups did neonatal treatment with o,p'-DDT
alter the course of weight gain. The effect of estrogen
treatment in both intact and castrate groups was marked,
in accord with the observations of Os'tadalova' and Pari'zek
(197).
The final body weights and organ weights, normalized
by body weight, of all the groups are shown in Table 6.
Body weights for intact control and intact DDT-treated
animals were statistically indistinguishable. Neonatally
castrated control and DDT-treated groups were likewise
similar to each other though the influence of castration
was marked. In both intact and castrated states EV caused
a very significant (p < 0.0005) decrease In body weight,
roughly 35$ in both cases.
The glands directly involved in the production of
trophic and steroid hormones, the pituitary, adrenals and
testes, demonstrated no differences between the control and
DDT-treated groups in either the intact or castrated
states. Estrogen on the other hand, acted on the adrenals
103
-------
Table 6
1 2
Effects of Neonatal Injection with o,p'-DDT or Estradiol on Body and Organ
Weights of Intact and Neonatally Castrated Male Rats
Percent of ~
Body Weight-^
Control (11)
(422±22)
Intact Castrated
DDT(5) EV(ll) Control{5) DDT(?) EV(6)
(404^20) (298±20}$ (240±23) (249-19) (222-11)$
Pituitary x 103
2
Adrenals x 10
Testes
Seminal -i x 1
Vesicles-/x 10-*
Ventral -j x 1
Prostate J x 10-^
Liver
Kidneys
2.75-0.20
1.27-0.10
0.84^0.07
0.28-0.02
3.71-0.30
0.76-0.05
2.59^0.11 2.81-0.18
1.23-0.09 1.99-0.3ot
0.87-0.06 0.25-0.0611
0.30-0.03* 0.04-0.02$
0.11-0.02 0.12-0.01 0.02^0.02$
3.74^0.32 3.39-0.21t
0.73^0.05 0.66^0.05$
3.76-0.18 4.12±0.70 3.42±0.46
1.83-0.29 1.95^0. 15 2.46±o.24#
6.71-2.14 4.84^1.08 32.9-5.7+
3.10^0,71 2.57-0.59 3.06^0.17
3-35-0.25 3.25to.27 3.92-0.10#
0.62^0.06 0.60^0.03 0.68±0.03*
Injected s.c. with 0.05 ml sesame oil, 400 ug o,p'-DDT in 0.05 ml sesame oil or 40 ug
estradiol valerinate in 0.05 ml sesame oil on days 1-5 of age. Reared in litters of
8 pups each and sacrificed at 81 to 110 days of age.
-------
Table 6 (Cont.)
Body weights given in grams in parentheses under each heading.
^
All data are given as mean weights or mean % of body weight -1 standard deviation;
numbers in parentheses after the treatment designation are the numbers of animals
examined; comparisons are all versus the appropriate control groups and are made by
unpaired Student's t tests, * p < 0.05, t p < 0.01, # p «£ 0.005, t p <. 0.0005.
-------
of "both intact and castrated animals to cause elevations
of weights to above control group levels. While neonatal
castration itself caused marked hypertrophy to above intact
weights of both the adrenal and pituitary in both control
and DDT-treated groups, the effects of neonatal castration
and EV were nearly additive. This could be seen by com-
paring the intact EV-treated group with both the castrated
control group and the castrated EV-treated group. Estrogen
also markedly diminished testicular weight suppressing it
roughly ?0$ below control values.
The accessory sex tissues, the seminal vesicles and
ventral prostate, should reflect the output of hormonally -
active gonadal androgens (198). DDT only slightly
increased (p < 0.05) "the weight of the seminal vesicles
in intact animals in comparison to controls; it did not
effect the seminal vesicles of castrated rats when com-
pared to similarly castrated controls. Prostate weight in
both intact and castrated animals was unaffected by DDT
administration. Castration alone produced a marked
decrease in sex-accessory weights in both DDT and control
groups. In the estrogen treated groups, however, the con-
sequences of EV treatment and/or castration were more
complex. First, estrogen suppressed intact prostate
weights into the range seen in control castrates; super-
position of castration and EV did not effect prostate
weights more than castration alone. Second, estrogen
decreased (p < 0.0005) the seminal vesicle weights of
106
-------
intact animals into the range seen in EV-treated neonatally
castrated rats. Third, the seminal vesicular weights of
neonatally castrated animals was increased by EV treatment
5-6 fold (p < 0.0005) over the weights observed for
castrated controls. These seminal vesicles (from
EV-treated neonatal castrates) are, however, morphologi-
cally abnormal, being more muscular than those in the
other castrates and retaining an integral association with
what are probably the vestiges of parts of the Miillerian
ductal system. These tissues in the other castrated
groups appear merely immature in comparison to intact
controls.
Liver and kidney weights are also affected by
steroidal status (199) and are, in addition, sensitive to
potential hepatic inducers (5). They were unaffected by
the administration of o,p'-DDT to either intact or
castrated animals. Again castration removed what is an
anabolic influence since the liver and kidneys of castrates
were slightly lighter than those of the intact animals.
Neonatal estrogen again exerted paradoxic effects by
diminishing the organ weights in intact animals into the
control castrate range while increasing the organ weights
in castrated animals toward, or even in excess of, intact
control levels.
Changes due to estrogen treatment were marked, as
anticipated. Inconsistencies of the changes in intact
animals with those seen with castration alone should not,
107
-------
however, allow the misinterpretation that estrogen treat-
ment of intact males is nothing more than chemical castra-
tion (200); body, pituitary and seminal vesicle weights in
intact EV-treated animals do not concur with those found
in control castrates. The issue is confused by the changes
seen in EV-treated neonatal castrates where body, adrenal,
seminal vesicular, liver and kidney weights are increased
rather than suppressed. The continued presence of injected
estrogen at levels sufficient to suppress body weight and
to stimulate adrenal weight in both intact and castrated
animals, to suppress gonadal function in intact animals
and to induce preservation of Mullerian structures serves
as an obvious alternative to imprinting as an explanation
for the results observed.
Clearly, the changes due to neonatal treatment with a
total of 2 mg of o,p'-DDT were minimal. Several interpre-
tations were therefore possible! no effects occurred;
effects were too subtle to be detected by such gross
methodology; or, effects occurred earlier in development
and were not measurable at the time of this experiment.
The first interpretation was presumptuous at this point
and finalistic, in addition; it could lead to no further
experimentation. The second interpretation dictated the
use of more sensitive measurements in subsequent experi-
ments. The third interpretation implied the need for the
examination of some sort of developmental time course.
108
-------
These last two suggestions formed the basis for the next
experiment.
II. Developmental Time Course and Dose-Response to
Neonatally Administered o.p'-DDTi
In addition to probing body and organ weights as a
function of development, this experiment included examina-
tion of organ histology and measurements of serum corti-
costerone, to monitor adrenal output, and serum LH, to
measure pituitary function. It also included several
levels of o,p'-DDT exposure. The inclusion of more than
one dose level was meant to generate dose-response data on
any effects encountered. It was also meant to minimize
the potential effect of multiple antagonistic responses
on the conclusions drawn from the experimental results,
e.g., to prevent hepatic induction from completely elim-
inating a form of DDT which imprints the hypothalamus.
Positive control groups were injected with either EV or
testosterone-17-propionate (TP). Estrogen was included
for the reasons cited previously while testosterone was
included to provide an apparently less drastic though
similar form of imprinting (59) which has been postulated
to be mediated by the estrogen formed by localized hypo-
thalamic aromatization of testosterone (8?). Due to the
size of the proposed experiment neonatal castrates were
not included.
Following parturition, the intact neonatal males were
placed into litters of 8 pups each. Thirty-six litters
109
-------
were divided into 9 groups, 6 vehicle-treated control (C)
litters, 6 EV-treated litters, 6 TP-treated litters and
18 litters treated with o,p'~DDT - 3 litters at each of
6 dose levels. The chemicals were administered s.c. in
5 equal daily doses, each contained in 0.05 ml of sesame
oil, given from the day of birth until 4 days after birth.
Dosages were as follows:
Group
C
I
II
III
IV
V
VI
EV
TP
Chemical
None
o,p'-DDT
o,p'-DDT
o,p'-DDT
o,p'-DDT
o,p'-DDT
o,p'-DDT
Estradiol
Testosterone
Daily Dose (ug)
0
Jj-oo
200
100
20
10
2
40
200
Total Dose (us)
0
2000
1000
' 500
100
50
10
200
1000
After weaning, animals were caged 6 to a cage. Body
weights were recorded throughout development. On days 25,
50, 75 and 110 five to six animals from each DDT-treatment
level and 8-11 animals from each of the control, EV and TP
groups were decapitated between 12.00 and 16.00 (EST).1'2
1
Since the experiment was actually conducted in two
halves started 5 days apart, only 5-6 animals from each
of 3 DDT-treated groups and 5-6 animals from each of the
control, EV and TP groups were processed on any one day.
On day 110 groups C and I and J of groups EV and TP were
110
-------
Animals from all the groups were alternately sacrificed in
order to minimize any time bias between groups. Tissues
were rinsed in Buffer I (see Materials and Methods IV. F.I.),
blotted, weighed, divided and placed into IQfo neutral
formalin.
Data for the halves (see footnote 1, p. 110) of each
group of controls, EV-treated or TP-treated animals were
checked for uniformity by applying Student's t-test
to the two halves. After ascertaining uniformity,
the values from the half-groups were combined. All organ
weights were normalized against total live body weight
before means or errors were calculated. Mean values for
each individual group of treated animals were then tested
by Student's t-tests against the means of the combined
control group, C.
A. -Body and Organ Weights;
Body weight gain in all groups prior to 32-35 days of
age was uniform. By about day 40 estrogen treated animals
began to lag behind in growth and by day 110 were fully 30$
lighter than control rats. Testosterone-treated animals
lagged slightly in growth between days ^0 and 85 but were
nearly normal by day 110. Body weight gain of DDT-treated
rats was -intermediate between that of the controls and
that of the TP group from days ^0 to 80. Beyond 80 days,
groups I-VI showed a somewhat slower growth with groups
sacrificed at 10.00 while the remaining animals were
killed at 19.00 (EST).
Ill
-------
II, III and V demonstrating significantly lower "body
weights by day 110.-* No relationship between DDT dosage
and growth, as measured by body weight was evident. Body
weights for days 25, 50, 75 and 110 are summarized in the
graph in Figure 13.
The mean normalized organ weights of the animals
described in Figure 13 are shown in Figures 1*1—20.
Normalized pituitary weights (Figure 1*0 decreased
uniformly with time. Estrogen treatment caused a signifi-
cant rise, above control levels, in pituitary weight which
was only evident before day 110 of age. Testosterone did f
not alter pituitary weight gain. Although several isolated
changes were seen in the DDT-treated animals those on day
110 may be explained by the lower body weight of the ani-
mals (the mean raw weights of the pituitaries were not
different from controls). The significant value for group
II on day 25 was probably by chance due to the size of the
experiment. No consistent pattern of fluctuation of means
about the control mean was evident for the DDT-treated
animals.
Normalized testicular weights for pairs of testes
(Figure 15) in both control and DDT-treated groups demon-
strated the abrupt hypertrophy associated with puberty,
Group III contracted respiratory infection shortly before
the termination of the experiment; most of the actual
weight loss seen on day 110 in that group and many of the
deviations in normalized organ weights are undoubtedly
due to illness.
112
-------
Figure 13. Body Weights of Neonatally Injected Male Rats
Intact male rats were injected daily, s.c., on days
1-5 of age with 0.05 ml of sesame oil which contained 1/5
of the following total dosagess C = vehicle only, control;
I = 2000ug o,p'-DDT; II = 1000 ug o,p'-DDT; III - 500 ug
o.p'-DDT; IV = 100 ug o.p'-DDT; V = 50 ug o.p'-DDT; VI =
10 ug o.p'-DDT; EV - 200 ug 1?0 -estradiol-17-valerinate;
TP = 1000 ug testosterone-17-propionate. The animals were
raised in litters of 8 pups and had free access to water
and food at all times. Between 12.00 and 16.00 on the days
specified the animals were weighed, decapitated and dis-
sected; fresh organ weights were then determined. (Notej
on day 110 groups C and I and j? of groups EV and TP were
sacrificed at 10.00 while the remaining animals were
killed at 19.00 (EST). This does not alter the interpreta-
tion of the results or the contrasts made except for the
liver which shows marked diurnal weight fluctuations.)
The data shown are means +1 standard deviation;
groups I-VI contained 5-6 rats each while groups C, EV and
TP each contained 8-11 animals. Tests of statistical dif-
ference from the mean of the control group, C, on the given
days were made by unpaired Student's t-tests. Probabil-
ities of the differences seen occurring by chance are
noted for those probabilities of less than 5%: a, p <
0.05; b, p < 0.025; c,~ p < 0.01; d, p < 0.005;
e, p < 0.0005.
113
-------
FIGURE 13
BODY WEIGHTS OF NEONATALLY INJECTED MALE RATS
too
200 300
BODY WEIGHT (G)
400
500
114
-------
Figure 14. Organ Growth in Neonatally Injected Male
Ratsi Pituitary
The pituitary weights, expressed as 1000 x percent
of body weight which are displayed are those of the
animals listed in Figure 13. Organ weights were normal-
ized by body weight for each individual animal prior to
calculating the group means which are shown (+1 standard
deviation). Group sizes were 3-6 for groups I-VI and
8-11 for groups C, EV and TP. Group means were tested
individually by Student's t-test for differences from the
control group, C, mean on each day listed. Statistical
probabilities of less than 0.05 are noted: a, p < 0.05;
b, p < 0.025; c, p < 0.01; d, p -c 0.005; e»
p < 0.0005.
115
-------
FIGURE 14
ORGAN GROWTH IN NEONATALLY INJECTED MALE RATS
(MG/IOO G)
116
-------
Figure 15. Organ Growth in Neonatally Injected Male
Rats: Testes
The testes weights, expressed as percent of body
weight, of the animals listed in Figure 13 are shown.
Organ weights are for pairs of testes and were normalized
by body weight for each individual animal prior to cal-
culating the group means which are given (+1 standard
deviation). Group sizes were 5-6 for groups I-VT and
8-11 for groups C, EV and TP. Group means were tested
individually against the mean of the control group at
each age by Student's t-tests. Statistical differences
with probabilities of less than 0.05 are noteds a, p
< 0.05; b, p < 0.025? c, p < 0.01; d, p < 0.005;
e, p < 0.0005.
117
-------
FIGURE 15
ORGAN GROWTH IN NEONATALLY INJECTED MALE RATS
TESTES
AGE
(DAYS)
25
50
75
e
no
—i
e
0.5
i.o
% BODY
15
WEIGHT
118
-------
activation of steroidogenesis and active spermatogenesis,
between 25 and 50 days of age; thereafter body weight
rose proportionately faster than testicular weight and the
normalized organ weights slowly decreased. As in the case
of the pituitary, several isolated significant deviations
from the control mean were seen in the DDT-treated groups;
all could be explained by differences in body weights.
This was not true, however, for treatment with EV or TP.
Estrogen markedly suppressed testicular weight throughout
the period studied; only a slight alleviation of the
effect was seen between days 50 and 75- Testosterone
yielded marked suppression throughout the experiment.
Still, some sort of recovery, which coincided with the
increased body weight seen after 75 days of age, apparently
took place since normalized testicular weights for this
group slowly rose beyond 50 days of age. This apparent
recovery in TP-treated rats is in concert with similar
observations in mice (59).
Normalized adrenal weights for pairs of adrenals
(Figure 16) declined abruptly for all but the estrogenized
animals between days 25 .and 50; they decreased in propor-
tion to total weight only slowly beyond day 50. Again
DDT appeared ineffective in altering weights although
group IV on day 25 did show an increase above control
levels which was not explicable on the grounds of dif-
ferences in total body weight. The majority of the dif-
ference seen with group III on day 110 was, however,
119
-------
Figure 16. Organ Growth in Neonatally Injected Male
Ratsj Adrenals
The adrenal weights, expressed as 1000 x percent of
body weight, of the animals listed in Figure 13 are
shown. Organ weights are for pairs of glands and were
normalized by body weight for each individual animal prior
to calculating the group means given (+1 standard devia-
tion) . Group sizes were 5-6 for groups I-VI and 8-11 for
groups C, EV and TP. Group means were tested individually
against the mean of the control group at each age by
Student's t-tests. Statistical differences with probabil-
ities of less than 0.05 are noted: a, p < 0.05? b,
p < 0.025; c, p < O.Olj d, p < 0.005; e, p < 0.0005.
120
-------
FIGURE 16
ORGAN GROWTH IN NEONATALLY INJECTED MALE RATS
1000 x % BODY WEIGHT (M6/I006)
50
121
-------
accounted for by a. decreased body weight (see footnote 3»
p. 112). The same explanation for the results in EV and
TP treated animals was insufficient; these steroids
produced an actual hypertrophy of this organ as has been
shown previously in both rats (201) and mice (202).
Interestingly, the hypertrophy was only expressed beyond
day 25, i.e., during puberty (day 50) or maturity (days
75 and 110). Because the expression of this effect has
this time lag and was similar in pattern for both TP and
EV groups, but more marked with estrogen, it bears the
appearance of an imprinting phenomenon. The slight sup-
pressions of the weights in older TP-treated rats probably
is another reflection of the slow recovery of testicular
function in these rats.
The weights of pairs of seminal vesicles, normalized
by total body weight, from groups C and I-VI (Figure 1?)
illustrate the accelerating weight gain between weaning
and maturity seen in organs dependent on circulating
androgens. The abrupt jump between day 50 and 75 and the
plateau thereafter is assuredly a result of the final full
maturation of testicular steroidogenesis. The deviations
from mean control weights shown by the DDT-treated animals
were not - except for group III on day 110 - accounted for
by changes in total body weights. Since the slight devia-
tions (increases) occur only at the -high DDT dose levels
they may be slight (p < 0.05) indications that o,p'-DDT
122
-------
Figure 17. Organ Growth in Neonatally Injected Male
Ratss Seminal Vesicles
The weights of pairs of seminal vesicles, expressed
as percent of body weight, are given for animals listed
in Figure 13. The weights of the blotted pairs of
vesicles with the coagulating glands attached were nor-
malized by body weight for each individual animal prior
to calculating the group means given (+1 standard devia-
tion). Group sizes were b-6 for groups I^VI and 8-11 for
groups C, EV and TP. Group means were tested individually
against the mean of the control group at each age shown by
Student's t-tests. Statistical differences with probabil-
ities of less than 0.05 are noted: a, p •£ 0.05; b,
p < 0.025; c, p < 0.01; d, p < 0.005? e, p < 0,0005.
123
-------
FIGURE 17
ORGAN GROWTH IN NEONATALLY INJECTED MALE RATS
SEMINAL VESICLES
Kltiiiiiiiifiiiiiiiiiiiiiih
AGE
(DAYS)
25
50
75
110
e
0.10 0.20 0.30
% BODY WEIGHT
0.40
124
-------
is a weak imprinting agent which, paradoxically, causes
increased adult gonadal output.
The effect of estrogen was marked (p < 0.0005 on all
days examined). On day 25 an hypertrophy similar to that
seen in estrogen-treated neonatal castrates (experiment l)
and reported by Freud (203) was observed - again, abnormal
morphology may hold the explanation for this result. The
raw weight of the vesicles in these animals continue to
increase slowly until .sometime between 50 and 75 days of
age after which it stabilizes? in no case, however, does
it approach normal levels, indicating a lack of, or
insensitivity to, circulating androgens.
Testosterone also exerts a marked effect on this
tissue beyond 25 days of age (day 25 p < 0.025, days
50-110 p < 0.005). But, as in the cases of the testes
and adrenals, it appears to be mainly a retardation of
development, maturation being completed sometime after
110 days of age in these animals. The results were sug-
gestive of residual titers of injected TP which only fall
below values which suppress LH levels after 75-110 days of
age. If these TP titers were, in addition, insufficient
to support gonadal function (normal function would
antagonize rises in adrenal weight (^,20^)) they could
explain the results discussed thus far for testosterone
treated animals.
The ventral prostate, normalized weights of which are
shown in Figure 18, would be expected to demonstrate the
125
-------
Figure 18i Organ Growth in Neonatally Injected Male
Ratsi Ventral Prostate
The weight of the ventral portion of the prostate
gland, expressed as percent of body weight, is shown for
the animals described in Figure 13. The weight of the
tissue was normalized by body weight for each individual
animal prior to calculating the group means (+1 standard
deviation) depicted. Group sizes were ^-6 for groups I-VI
and 8-11 for groups C, EV and TP. Group means were tested
individually against the mean of the control'group at each
age shown by Student's t-tests. Statistical differences
with probabilities of less than 0.05 are noted? a,
p < 0.05; b, p ^ 0.025; C, p ^ 0.01; d, p < 0.005;
e, p -< 0.0005.
126
-------
FIGURE 18
ORGAN GROWTH IN NEONATALLY INJECTED MALE RATS
VENTRAL PROSTATE
iiiito
0.10
% BODY WEIGHT
0.15
0.20
127
-------
same patterns as the seminal vesicles. This was generally
true for all groups but that treated with EV. The incre-
ments between weaning and immature and between immature
and mature normalized weights were less marked than they
had been for the seminal vesicles of groups C and I-VI.
DDT treatment showed no significant effect on the organ
although variation about the control mean generally agreed
with that found for the seminal vesicles. The pattern of
suppressive effects of TP was the same as that seen with
the seminal vesicles. The same was true of the pattern
seen in EV treated rats beyond day 50. Apparent tissue
proliferation seen at 25 days of age in the seminal
vesicles of estrogen-treated rats was absent here. This
may reflect either the lack of a close association with
Mullerian remanents or an innate difference in the sensi-
tivity of the two sex-accessory tissues to estrogen as has
been discussed by Sufrin and Coffey (205).
Normalized hepatic weights are graphed in Figure 19.
The liver appears largely insensitive- to neonatal treat-
ment with DDT until at least 75 days of age; the unnor-
malized mean weight of the liver in group IV on day 50 was
not different from the control. Even the effects of the
steroids, EV and TP, were minimal through day 75; the
significant deviations that were observed were inconsistent.
The values found for day 110 showed significant devia-
tions for both DDT and steroid treated groups. However,
the explanations for most of these changes lies in the
~ 128
-------
Figure 19. Organ Growth in Neonatally Injected Male
Ratsi Liver
The mean weight of the liver of each of the treatment
groups described in Figure 13 is shown in terms of percent
of body weight at various ages. The organ weights were
normalized to body weight for individual animals prior to
calculation of the means (+1 standard deviation) shown.
Group sizes were 5-6 for groups I-VI and 8-11 for groups
C, EV and TP. Group means were tested individually
against the mean of the control group at each age shown
by Student's t-tests. Statistical differences with
probabilities of less than 0.05 are noted* a, p •£ 0.05s
b, p -*• 0.025; c, p < 0.01; d, p < 0.005; e,
p < 0.005.
The differences seen on day 110 may be more reflec-
tive of differences in time of sacrifice than actual
treatment effects as noted in Figure 13.
129
-------
FIGURE 19
ORGAN GROWTH IN NEONATALLY INJECTED MALE RATS
LIVER
1.5
3.0 4.5
% BODY WEIGHT
6.0
AGE
(DAYS)
25
50
75
110
7.5
130
-------
altered experimental procedure followed on that day and
in the normal diurnal weight fluctuations demonstrated by
the liver (206). In order to allow incubations of the
testes to be done on these rats sacrifice times were
altered from the normal time of 12.00-16.00 (EST) used on
previous days (see footnote 2, p. 110). The data from the
halves of groups EV and TP were compared but failed to
yield significant t values; therefore, the data were
combined. The decreases of the normalized organ weights
to below the control mean in groups II-IV, EV and TP
probably best reflected the 4-9 hour difference in
sampling times rather than actual effects of DDT or steroid
exposures. The magnitude and time course of the change
agree completely with those observed by Potter et al.
(206) in studies of feeding and corticosterone secretion.
Finally, weights for pairs of kidneys, normalized to
body weight, are shown in Figure 20. A consistent decrease
with age in the normalized organ weight was demonstrable
in all treatment groups? the value for group III on day
110 was explained by decreased body weight. In fact, the
greatest portion of each of the deviations from the group
C mean before day 75 are explicable by differences in body
weights since data on raw organ weights indicate minimal
deviations from that of group C. Treatment with TP did
depress kidney weights after day 50 but the effect of
estrogen treatment was significant for both raw and nor-
malized weights only on day 110. Again, as has been the
131
-------
Figure 20. Organ Growth in Neonatally Injected Male
Ratsi Kidneys
The mean weights of pairs of kidneys, expressed as
percent of body weight, are shown for the treatment groups
described in Figure 13 for various ages. Pairs of organs
were blotted and weighed, the weights being normalized by
body weight for each individual animal prior to calcula-
tion of the group means (+1 standard deviation) given.
Group sizes were 5-6 for groups I-VI and 8-11 for groups
C, EV and TP. The individual group means for each age
were tested against the control group means by Student's
t-tests; statistical differences with probabilities of
less than 0.05 are noted: a, p < 0.05; b, p <: 0.025;
c, p <• 0.01; d, p < 0.005; e, p •< 0.0005.
132
-------
FIGURE 20
ORGAN GROWTH IN NEONATALLY INJECTED MALE RATS
o
(T
O
Z
u
UJ
(t
h-
1.5
% BODY WEIGHT
133
-------
case for most of the organs examined, the actions of the
steroids become manifest as alterations in mean normalized
organ weight only after puberty, or full maturity, is
reached.
In summary, neonatal injection with up to 2 mg of
o,p'-DDT (total dose) yielded only minimal, inconsistent
alterations in mean normalized organ weights at 25, 50, 75
and 110 days of age. No consistent correlations could be
drawn between dose and effect on any single organ or com-
bination of organs. Though applications of various forms
of analysis of variance (ANOVA) might have been used in
the data analysis it is doubtful, due to the inconsistency
of the deviations from the control means, whether even
these more powerful statistical methods could have
extracted any further results from the data. In concord-
ance with the results of the first experiment, but
extending it in time, the gross measurement of organ
weights failed to expose any effect of o,p'-DDT on the
male rat.
Treatment with steroids gave results consistent with
those obtained in other studies (59,20?) in regard to
alterations of adult organ weights. But they are also
consistent, in the main, with the continued presence of a
depot of estrogen or testosterone which acts to suppress
gonadotrophin release until sometime between 75 and 110
days of age. The oil droplet which remains after neonatal
treatment, vestiges of which still exist at 110 days of
134
-------
age, can act as a depot (6l). Still, in the absence of
controlled measurements of serum steroids in the mature
animals and in light of the extensive literature on
steroidal imprinting, the present results must be viewed
as being most probably due to neonatal imprinting phenomena
rather than merely steroid feedback inhibition.
B. Organ Histologies:
Having established the lack of any easily located DDT
effect on body weight or organ weights at any age up to
adulthood, measurements for more subtle effects were
undertaken. Organ histologies were done on liver, kidney,
pituitary, testis and adrenal tissues chosen at random
from the groups of treated animals and from the control
group. From each prepared tissue 6-8 sections were
examined; the subjective observations were recorded.
Pituitary, kidney and liver tissues were indistin-
guishable among the groups or ages; they did not differ
overtly from the appearance of the controls under the
general histology conditions used fo^preparation and
staining.
Histology of the testes for groups C and I-VI were
nearly identical at any given age. Sperm production was
absent in animals of 25 days of age but was active at 50t
75 and 110 days. This agreed with the findings of Gellert
et al. (5*0 of motile sperm in adult male rats neonatally
injected with 3 mg of o,p'-DDT. Subjectively, the
DDT-treated groups in my experiment showed some evidence
135
-------
of oligospermia. This was not inconsistent with Gellert's
findings since he did not do sperm counts, It did,
however, reinforce the recent report of Krause et al. (208)
who did histological counts of various testicular cell
types in sections made from the testes of rats injected
with massive doses of technical DDT on day 4 of life.
Estrogen treatment resulted in complete aspermia
through day 75 and only minimal spermatogenesis "by day 110,
results which were in complete accord with a report "by
Maqueo and Kind (209) on histo-morphology of rats injected
with estradiol benzoate on day 5 of age. TP treatment
caused a definite oligospermia until at least day 755
spermatogenesis on day 110, however, appeared normal.
These observations were similar to those reported in mice
by Barraclough (59).
Adrenal histology for all groups on days 50, 75 and
110 seemed qualitatively similar. At all these ages the
glomerulosa layer of the cortex was well defined and
histologically invariate; the medulla was well defined
and highly vascular; the outer (fasicular) layer of the
cortex represented 30-50$ of the cortical area and con-
sisted of fairly large cells containing lipid vacuoles;
the inner (reticular) layer of the cortex appeared highly
vascular with small dense cell-s organized into definite
cords.
On day 25, however, a rather striking picture emerged.
Control and testosterone-treated rats exhibited a lack of
136
-------
the perlmedullary vascular!ty and. cellular organization
typically found later. This observation was in agreement
with the early observations of Howard (9*0. These adrenals
also showed considerably less definition of the reticular
and fasicular zones - cells were uniform throughout the
cortex. In contrast, the glands from animals treated with
o,p'-DDT or estradiol appeared very similar to normal
animals at least 25 days older. The cortex had apparently
aged precociously. Subjectively, the degree of aberration
on the DDT-treated animals was dose-dependent.
Initial observations, summarized in Figures 21 and 22,
were confirmed by examining similar slides prepared from
the remaining control, DDT and EV-treated animal tissues
(Table 7)- Collective DDT and control observations were
?
compared by X analysis using the most vascular 25-day old
control adrenal as a comparative standard. For the slides
examined p was <0.02, implying that DDT treatment with at
least some dosages changed adrenal histology at 25 days of
age. Histology itself cannot imply a'mode of action, but
similarities between EV and DDT-treated adrenals were plain.
Recapitulating, these histological examinations demon-
strate that neonatal DDT treatment did indeed have an
effect on the steroidogenic endocrine tissues, altering
adrenal morphology and possibly sperm production as well.
C. Serum Corticosteronet
In view of the histological findings in the adrenal,
the importance of measuring adrenal function became even
137
-------
Figure 21. Normal Adrenal Development in the Male
Ratt Reticular Zone Histology
These photomicrographs of normal rat adrenal glands
at various ages are representative of the histological
sections obtained from the control animals listed in
Figures 13-20 (intact male rats injected s.c. with 0.05 ml
sesame oil on each of days 1-5 of age). All photographs
were taken at the same magnification (~15X) with a Zeiss
polarizing photomicroscope using Kodak color photo-
micrography film (ASA 16i 13 DIN). The histology slides
were prepared from formalin fixed tissues using paraffin
embedding and hematoxylin and eosin stains; sections were
saggital and 7-10 mu thick.
The medullary region of the adrenals is located at
the left hand or top of the photographs and forms a dis-
tinctive border with the reticular layer of the cortex.
The glomerulosa forms a densely packed region near the
outer edge of the organ, except on day 110 where it is not
pictured. The fasicular and reticular zones are undif-
ferentiated at day 25, but by day 50 the organization of
the vasculature and cords of the reticularis is evident.
This area increases in extent at the expense of the
fasiculata, at least until maturity. This gradual expan-
sion with maturity stands in contrast to the picture seen
in estrogen or o,p'-DDT treated animals (see Figure 22).
138
-------
FIGURE 21
NORMAL ADRENAL DEVELOPMENT IN THE MALE
RAT: RETICULAR ZONE HISTOLOGY
DAY 25
DAY 50
DAY 75
DAY 110
139
-------
Figure 22. Adrenal Reticular Zone Histology at 25 Days
of Age in the Neonatally Injected Male Rat
These photomicrographs of rat adrenal glands at 25
days of age are representative of the histological sec-
tions obtained from the test groups EV, II,. Ill and IV
listed in Figures 13-20 (intact male rats injected daily,
B.C., with 0.05 ml sesame oil containing 1/5 of the total
dosages shown on each of days 1-5 of age). They demon-
strate marked precocious development of the reticular
zone at this age (see Figure 21) and point to a similarity
in the actions of estradiol and o,p'-DDT on this organ.
The histological changes from 25-day old controls
which are evident were not attributed to perimedulary
necrosis in view of the similarity of the histology of the
treated tissue to older control adrenals, of the absence
of polymorphonucleocytes and cell debris, of the apparent
normality of the corticosterone- levels secreted (see
Figure 23), and of the normality of the adrenal weights
obtained (see Figure 16).
Histology slides and photographs were prepared under
the conditions listed in Figure 21.
140
-------
FIGURE 22
ADRENAL RETICULAR ZONE HISTOLOGY AT
25 DAYS OF AGE IN THE NEONATALLY
INJECTED MALE RAT
200U6 EV
1000 UG o,p'-DDT
500 UO o,p'-DDT 100 UG o.p'-DDT
TOTAL DOSE INJECTED BY DAY 5 OF AGE
141
-------
Table 7
Frequency of Increased Reticular Vascularity in
25 Day Old Rats Injected Neonatally with o,p'-DDT
Number of Histological
Preparations with Vascularity
Treatment
Control
DDT
Increased
0
8
Unchanged
10
12
Total
10
20
Total 8 22 30
The standard for comparison was the adrenal from the
control group at 25 days of age showing the most marked
vascularity. The nature of the comparisons do not allow
a clear indication of uncertainty in cases where the dif-
ference from the control were slight. However, these
uncertainties may be partially accomodated when doing
comparisons, by the use of appropriate nonparametrie
statistical procedures such as X »
142
-------
more apparent than when initially planned. Many of the
serum measurements had been done before the histological
investigations were complete but they tended to support
both the histological observations and the organ weights
which had been measured. They are presented in graphical
form in Figure 23.
On days 50 to 110 the mean serum levels, determined
on trunk-blood taken at the time of decapitation, showed
relatively small variation; control means from the rats
killed at 10.00-16.00 (EST) fluctuated from 20-25 ug/100
ml of serum. Likewise, except for an occasional high
value, so did the serum values from the DDT-treated groups.
Testosterone treatment caused a moderate, about 10-20$,
elevation of the levels on day 50 and 75* then fell to, or
slightly below, the control levels on day 110. Estrogen
treatment caused marked elevations above the control
levels observed, ranging from a 100$ elevation on day 50
to approximately a 60$ elevation on day 75. However,
normal levels were evident in the EV group on day 110.
On day 110 the effect of illness was clearly seen in
Group III as was the effect of the diurnal fluctuation of
serum corticosterone in groups EV and TP; the elevated
levels seen in groups II and IV-VI were also interpreted
in terms of the circadian rhythm of corticosterone (210).
Small volumes of serum on day 25 required that
samples be pooled? although multiple determinations were
made on the pooled sera only the single resultant values
143
-------
Figure 23. Serum Corticosterone in Neonatally Injected
Male Rats
Serum corticosterone, in ug per 100 ml, as measured
by sulfuric acid fluorescence is plotted versus age and
treatment group. The serum samples were obtained from
trunk-blood taken at the time of decapitation (12.00-16.00
(EST) except where otherwise indicated) of the animals
listed in Figures 13-20 and a group of intact male rats,
A, injected similarly with 5 daily doses of 2000 ug
o,p'-DDT in 0.05 ml sesame oil (total dose - 10 mg) on
days 1-5 of age.
Serum samples were small on day 25 and were pooled
except for groups C and A. Each serum was assayed in
duplicate; the means for groups were based on the means
of these duplicates. Means +1 standard deviation are
shown, n being 3-6 for groups A-VI and 7-12 for groups C,
EV and TP. Statistically significant differences from
the properly paired control groups were determined by
Student's t-tests (a, p < 0.05; "b. P < 0.025; e,
p 4. 0.0005).
144
-------
FIGURE 23
SERUM CORTICOSTERONE
INJECTED MALE RATS
IN NEONATALLY
C
A
I
II
III
IV
V
VI
EV
TP
AGE
(DAYS)
25
50
75
no
10.00
19.00
10.00
15 30 45 60
SERUM CORTICOSTERONE (U6/IOOML)
145
-------
were available for between-group comparisons. The serum
levels in weanlings in all the treatment groups were about
20-757° higher than those seen at 50 days when normal
maturation of cortical secretion should have occurred
(5-6 weeks) (98). Steroid treatment raised levels fully
^5% higher than even this control mean while DDT treatment
caused less marked, and statistically insignificant,
elevations of 0-20$.
The suggestion of elevated corticosterone levels on
day 25 in DDT-treated animals and the clear rise caused by
estrogen treatment support interpretation of the histo-
logical findings as morphological expressions of increased
steroidogenic activity. However, an altered function need
not, necessarily, be correlated with an altered morphology,
as is shown by the fact that no morphological change was
seen in the testosterone-treated group yet it showed a
marked corticosterone elevation.
The visual suggestion of some correlation between
DDT dose and serum corticosterone level was evident at
both 50 and 75 days of age in groups I-VI. Therefore, a
series of linear regression analyses were done on the
results summarized in Figure 2k. If all the values were
included in the calculations, either linear regression or
LL .
This was true with two exceptions in which samples were
not pooled! l) the control group formed several serum
pools; and 2) a group of males, group A, which was
obtained from a small experiment conducted later but
included in the overall calculations and which was neo-
natally injected with a total of 10 mg of o,p'-DDT.
146
-------
Figure 2k. Serum Corticosterone in 50 and 75 Day Old Male
Rats Versus Dose of Neonatally Injected
o,p•-DDT
The data from groups C, A and I-VI on days 50 and 75
given in Figure 23 are replotted. Data are plotted as
means ±1 standard deviation with n for each point shown
in parentheses. All assays were run in duplicate.
Analysis of variance for either day 50 or 75 leads
to the conclusion that no correlation between corticoster-
one level and o.p'-DDT dose can be found. For doses
between 100 and 2000 ug of o,p'-DDT on day 75* however,
a weak correlation with dose does exist, R = 0.637?
using regression analysis the slope of this line is
non-zero by a t-test with a probability of < 0.01.
147
-------
FIGURE 24
SERUM CORTICOSTERONE IN 50 AND 75
DAY OLD MALE RATS VERSUS DOSE OF
NEONATALLY INJECTED o.p'-DDT
40 -
30 -
20 -
3
2
1 lo"
«w
^1
3 o-
Ul
o
UJ
8 4°-
o
H
K
o 30 -
u
2
=> 20 -
cc
UJ
(O
10 -
0 -
DAY 75
I(3) T4| )
T(I2) i I««j») I(5)i<4)i
DAY 50
r T T (4)
L..I r« T(5) 1
( 1) (3) T « (3)
• • |(6) (5) T »l '
1 | i * r
i i i i i i
0 10 100 1000 10,000
TOTAL DOSE INJECTED (UG)
148
-------
ANOVA led to the conclusion that no alteration from
control levels existed at either 50 or 75 days of age.
However, if low and very high dose levels were eliminated
from the analysis at 75 days of age a weak correlation
with dose appeared, R = 0.637. This regression line
allowed extrapolation of the o,p'-DDT results to the
serum corticosterone levels shown by treatment with
200 ug of EV and, subsequently, to an estimation of
estrogenic potency. The calculations showed that 1 ug
of EV was equivalent to approximately 2,500 ug of o,p'-DDT
on day 75. This estimate was well within an order of
magnitude of the 1/10,000 ratio calculated by Cecil et al.
(1^7) using a uterine glycogen-response bioassay.
The interpretation, that no dose-response effect
existed, was probably true, particularly on day 50.
in view of the large standard deviations which existed
in what was a slightly immature and presumably unstable
system. But the possibility that a multiphasic
dose-response existed and was manifested by a weak
dose-response relationship only in the intermediate dose
range should not be,ignored. DDT generates what may be
antagonistic responses in the liver (microsomal induction
(11-15)) and in estrogen-sensitive tissues (estrogen
mimetic (22,2^,26,27)). The thresholds for expression of
these two effects are different (15i22) so the possibility
that low levels of o,p'-DDT (0-50 ug) stimulated
149
-------
corticosterone breakdown and led to a decrease in serum
corticosterone levels appears reasonable. Because the
balance exists, it also seems reasonable that further
increments (100 ug - 2 mg) of o,p'-DDT dosage could have
favored increased serum corticosterone levels by an estro-
gen-like stimulation of the adrenals and/or an increased
catabolism of circulating androgens. Even further incre-
ments (10 mg) of DDT dosage may have caused breakdown of
the active form(s) of o,p'-DDT by further increasing
microsomal metabolism and/or upset the balance between
adrenal output and hepatic corticosterone catabolism;
either mechanism would result in a drop in serum corti-
costerone levels. It is therefore neither impossible nor
unrealistic to think that the weak correlation between
dose and serum corticosterone which held for intermediate
dosages of o,p'-DDT at 75 days of age might indicate
an action of o,p'-DDT. Obviously, only further
investigations concerning these possibilities will clarify
the meaning of these results.
D. Serum LH;
Earlier studies by Rybakova (28,29) on adult rats
chronically fed low levels of technical DDT uncovered a
stimulatory action on the pituitary gonadotrophs, which
were examined histologically, and on the pituitary content
of LH, which was measured by bioassay. Gellert et al.,
working on the female rat, had also shown that serum LH
could be suppressed by o,p'-DDT in adult rats (150) and
150
-------
that the LH rise in response to adult ovariectomy could
be blunted (5*0* Since the precedents existed for an
effect of o,p'-DDT on hypothalamo-hypophysial function,
determinations of serum LH were conducted concurrently
with my examinations of tissue histology and serum corti-
costerone. A summary of these measurements is given in
Figure 25.
The highly variable LH titers reported for immature
male rats (77,82,83) were evident in control animals on
day 25 and, to a somewhat lesser extent, on day 50. By
day 75 the mean control level had stabilized near the
lower reliable limit of the RIA, 10 ng B-640/ml.
DDT-treated rats also showed highly variable levels on
days 25 and 50. But, in contrast to the controls, only
group VI, the lowest dosage group, had approached stability
on day 50. On days 75 and 110 the levels of all the groups
were statistically indistinguishable from the controls.
On day 110 the high value for group III was probably again
a result of illness (35f36).
Estrogen treatment caused a uniform suppression of
serum LH to essentially nonmeasurable levels until day 75.
A slight rise was evident by day 110. This rise may be
what was reflected in the movement of several of the
normalized organ weights toward control values at this
time. Testosterone treatment similarly suppressed LH
levels on day 25 but it apparently had lost its influence
on tonic LH secretion after day 25 since an essentially
151
-------
Figure 25. Serum LH in Neonatally Injected Male Rats
Levels of serum LH, in ng B-640 per ml, are shown as
functions of age and dose of o,p'-DDT or steroid. Serum
samples are those obtained from trunk-blood at decapitation
of the animals shown in Figures 13-20. (They are the same
samples used to determine serum corticosterone - Figures
23 and 2^). Radioimmunoassays, using the method of
Niswender et al. (169), were done at least in duplicate
for each serum sample. The mean of the duplicates forms
the basis for the group means (+1 standard deviation)
shown; pooled samples were used for groups II-VI on day
25 of age, otherwise, n = 3-6 for groups I-VI and ?-12 for
groups C, EV and TP (.B-6^0 = 0.03 x NIH-LH-S1 by OAAD).
The large group variances at any given age mask any
statistically significant differences in group means.
However, two general trends are evident: 1) the decrease
from generally high levels during immaturity toward low
levels in maturity (75 and 110 days) and 2) the uniformly
low levels of serum LH in the estradiol-treated animals.
152
-------
FIGURE 25
SERUM LH IN NEONATALLY INJECTED MALE RATS
O
oc
O
UJ
2E
UJ
75
10
T~ i ^ r
50 100 150
SERUM LH (N6 B-640/ML)
250
153
-------
normal pattern ensued. Again, as was the case with group
EV, organ weights such as testes, adrenals, seminal
vesicles and ventral prostate seem coordinated with, and
perhaps by, the changes in serum LH.
Although the pituitaries for this study had been
committed to histology, other subsequent small experi-
ments did yield some limited data on hypophysial content
of LHs
Pituitary LH (ug B-640/mg wet tissue)
Day 25 Day 50 Day 75
Control 35-2 (3)* 77 - 10 (3) 36 - 8 (3)
2 mg DDT — — 37 - 11 (3)
10 mg DDT 27 t 8 (3) 69-5 (5)
* Number of animals measured is indicated in parentheses.
The control values agree with those of Yamamoto et al.
(180) and do not differ significantly from those of the
DDT-treated rats.
The pituitary LH values for 81-110 day old neonatally
castrated rats receiving only sesame oil (5 animals)
or a total of 2 mg o,p'-DDT (7 animals) were 6l t 9
and 52-9. respectively, which were similar to the content
in 50 day old males. Neonatal castrates injected with a
total of 200 ug EV (6 animals), however, yielded only
6 - 3 ug B-6^-0/mg wet tissue; their serum levels were
only 50 - 20 ng/ml in contrast to approximately 1.0-1.3 ug
B-64o/ml for the other groups. The extremely low level of
154
-------
pituitary LH in the EV group easily explains the serum
level. If deductive reasoning can be applied to the
absence of serum LH in intact estrogenized animals from
group EV of experiment 2, the result in castrates implies
a similar low pituitary content in the intact rats, a
situation actually found in females by Arai (211) using
OAAD assays. Either of these situations can be explained
by the continued presence of EV until at least 80-100 days
of age. Although such a conclusion is possible on the
basis of Gorski's findings with TP (6l), it would not be
in accord with the findings of Schiavi (207) in the
pituitary. Schiavi found levels only slightly suppressed
in 60 day old .intact rats treated on day 5 with 100 ug of
17 £ -estradiol-17-benzoate and a two-fold elevation of
pituitary LH content above controls at 180 days of age.
Since the same vehicle was used in both Schiavi's experi-
ments and my own the only simple explanations for the dif-
ferences reside in the timing, size and form of the dose
and the total volume in which it was dissolved. Each of
these factors would, from what is known about imprinting
(57-61) tend to favor a less drastic and prolonged effect
in the experiments of Schiavi. Whether the reason is pro-
longed presence or imprinting, neonatal estrogen markedly
affected LH in both the serum and pituitary of neonatally
treated male rats for extremely long periods of time.
In summary, this second .experiment affirmatively
answered the initial experimental query; o,p'-DDT did
155
-------
indeed demonstrate an effect on the steroidogenic
endocrines - it altered the early histological appearance
of the adrenal cortex and possibly its function as well.
Hints of other possible changes involving spermatogenesis
and the production and/or release of LH at 50 days of age
were found. The effects of testosterone propionate and
estradiol valerinate were reexamined in a coordinated
series of measurements not used before. Results stemming
mostly from the estrogen-treated animals shed some doubt
on the idea that estrogen esters given in oil act directly
for only a short period of time after which their effects
are manifestations of steroidal imprinting. This does not
imply, however, that imprinting may, or does, not occur
since too many experiments using other more soluble forms
of the steroid or more easily absorbed vehicles substanti-
ate the initial findings of Pfeiffer (164). Rather, the
question posed is whether or not sesame oil may be a
problematic vehicle in endocrinologic or screening studies.
III. Serum LH Response toAdult Castration;
While some data had been generated on the existence
of an effect on the immature adrenal gland, the total
absence of an effect on the hypothaiamo-hypophysial-gonadal
system of male rats did not seem reasonable in view of the
observations of Rybakova (28,29) , Gellert et al. (53,54,
150,151) and Krause et al. (208). To test the possibility
that such an effect did exist but was only apparent under
156
-------
stressed or competitive situations, a measurement of serum
LH response to adult castration was undertaken.
Six litters of male pups were divided among ^ treat-
ment groups. Two litters received injections of sesame
oil, 0.05 ml s.c. daily, on days 1-5 of ages 4 litters
received 5 daily doses of 2 mg o,p'-DDT in 0.05 ml sesame
oil, a total dose of 10 mg. Two of the DDT-treated litters
also received other drugs; one litter received 10 equal
i.p. doses of 100 ug sodium phenobarbital (PB) in 0.05 ml
saline spaced 12 hours apart over the first 5 days of life
while the other received 5 daily i.p. injections of 125 ug
SKP-525A in 0.05 ml saline. The latter two groups were
included to determine if hepatic activation played any
role in mediating the effects of neonatally administered
o,p'-DDT.
After allowing the animals to mature until 57 days of
age ^ animals from each of the ^ groups were chosen for
the experiment. Two sample bleedings (TVBE) were done on
days 57 and 59 to establish a baseline. Castration was
performed on day 60 and serum samples were taken at
intervals until 14- days after castration, by which time
serum LH levels should have begun to plateau (180). On
day 1^ an s.c. injection of 0.75 mg/kg of testosterone
was given 6 hours prior to bleeding. The dose was small
enough not to drive LH levels to baseline but large enough
to ascertain if the negative feedback loop of the animal
was functional (170). On day 15 after castration the
157
-------
animals were anesthesized lightly with ether and decapi-
tated; trunk-blood samples were collected for LH measure-
ment to determine if steroid clearance of the injected
testosterone and the rebound of serum LH were normal.
After the RIA was completed the group means were calcu-
lated for each day. They were plotted versus time after
castration and submitted to 2-way ANOVA (212,213).
Results for the control and DDT-treated groups are
plotted in Figure 26, those for the PB + DDT and
SKF-525A + DDT groups are plotted in Figure 2?. The
analysis of variance, which covers days 1-11 after castra-
tion (the period after castration but before steroid
treatment), is summarized in Table 8.
The expected hyperbolic rise in serum LH was seen in
all four groups as was a dip of 2$-kQ% subsequent to
testosterone injection and a rise back to pre-testosterone
levels. It was concluded that all groups possessed a
functional feedback system. However, the plateau of the
serum LH levels seen in the control and DDT-treated groups
were distinctly, and statistically, different. The DDT
animals reached a plateau somewhat earlier at a level
fully 30fo below the controls. This corresponded to a p
value of < 0.005 for an ANOVA which included only the
control and DDT groups (this ANOVA is not shown).
Both drug treated groups, SKF-525A + DDT and PB + DDT,
had patterns which fell between the controls and the
DDT-treated group. The pattern of the hepatically induced
158
-------
Figure 26. Response of Serum LH to Adult Castration in
Neonatally Injected Male Rats
Serum levels of LH, given in ng B-6^0 per ml, are
plotted versus time after adult castration for groups of
rats which were injected daily, s.c., on days 1-5 after
birth with either 0.05 ml sesame oil (Control, o-o) or
2 mg o,p'-DDT in 0.05 ml sesame oil (DDT, x-x). Bleedings
were done at 15.00-18.00 (EST) by tail-vein under light
ether anesthesia, the same animals being used throughout
the study. Castration was performed on day 60 of age after
2 baseline bleedings. No treatment other than bleeding
occurred until 1^ days after castration when hypothalamic
feedback control was tested by injecting 0.75 mg/kg
testosterone (T) s.c. in sesame oil 6 hours prior to
bleeding. Bleeding on' day 15 was without further treat-
ment. The serum collected was assayed, at least in dupli-
cate, by the radioimmunoassay procedure of Niswender et al.
(169). The results shown are means, -1 standard deviation,
for groups of ^ animals, except for the controls on days
1^ and 15 in which cases n = 3 and 2, respectively.
Analysis of variance for all of the values indicate a 95$
probability that the control group values were, in fact,
higher throughout the experiment; a similar analysis indi-
cates that, for the days after castration and before treat-
ment with testosterone, the chance occurrence of the differ-
ences seen has a probability of < 0.005. Table 8 shows the
2-way ANOVA for these values and those of Figure 27.
159
-------
FIGURE 26
RESPONSE OF SERUM LH TO ADULT CASTRA-
TION IN NEONATALLY INJECTED MALE RATS
900
800 -
700 -
600-
-------
Figure 27. Response of Serum LH to Adult Castration in
Neonatally Injected Male Rats
Serum levels of LH, given in ng B-^60 per ml, are
plotted versus time after adult castration for groups of
rats which were treated on days 1-5 of age as follows:
group SKF-525A + DDT (V-Y) was injected daily with 2 mg
o,p'-DDT in 0.05 ml sesame oil, s.c., and 125 ug (25 mg/kg)
of SKF-525A in 0.05 ml neutral saline, i.p.j group pheno-
barbital + DDT (•-•) was injected daily with 2 mg o,p'-DDT
in 0.05 ml sesame oil, s.c., and 2 doses - 12 hours apart -
of 100 ug (ifO mg/kg) sodium phenobarbital in neutral
saline, i.p. Bleedings, castration, testosterone treatment
and sample assays were the same as described in Figure 26.
The results shown are means, -1 standard deviation
for groups of 4 animals. All values were examined by
analysis of variance simultaneously with the groups shown
in Figure 26. Comparisons of the SKF-525A + DDT and/or
phenobarbital + DDT groups with the control group (Figure
26) over the entire test period or over the period after
castration and prior to treatment with testosterone indi-
cate no significant differences between these groups (as
determined by Tukey's test for multiple comparisons (214)).
161
-------
FIGURE 27
RESPONSE OF SERUM LH TO ADULT CASTRA-
TION IN NEONATALLY INJECTED MALE RATS
900 -
800 -
700
600 -
-------
Table 8
Two-Way Analysis of Variance for Castration Response Data from Adult, Neonatally
Injected Male Rats: Analysis of Days After Castration and
Before Testosterone Administration
Treatment
Control
o , p ' -DDT
o , p ' -DDT
SKF-525A
o,p'-DDT
PB
*— i
Raw
1
180.72
240.74
233.54
285.25
210 . 98
155.18
172.65
173-00
250.17
226.26
270.06
209.88
260.49
278.75
139-33
292.38
3,579.38
Data from Figures
(LH in ng B-640/ml
Day
3
382.09
323.72
315-40
470.43
343.78
295. 99
336.16
315.30
289 . 34
-330 . 84
367.22
201.95
331.72
392.79
381.01
358.00
5,535.74 7
26 and 27
Serum)
5
543.76
518.11
579.29
511.26
394.95
311 . 08
349.40
402.25
312 . 60
441.90
510.74
390.58
575.83
409.13
327.74
499.39
,078.31
11 2Xi
702.58
435.10 „ ,QO ?2
972.29 f*iyv-f*
496.14
373.95
8i$ 5,382.56
656.87
696.10
484.66 , -,,, ..
788.93 &»^1-33
470.10
489.70
412.20 , n,~ 9<
464.79 6>°63-25
450.00
8,784.43 24,977.86
£x?
3,833,909.77
2,052,224.82
2,919,215.24
2,472,126.38
-------
Table 8 (Cont.}
836,070.53 1,946,633-19 3,260,199.02 5,234,573.47
11,277,476.20
N
b 4
(24,977-86r = 623,893,490.1
= (940.25)2 + (1491.64)2 + (2152.72)2 + (2606.II)2 + (711.8l)2 + (1291.23)2 +
(1457. 68) + (1921. 84)
(956. 37)
(1289. 35)
Day R
(970. 95)2 + (1463. 52)2 + (1812. 09)2 + (1816.69)2 = 43,467,370.57
(7190.72)2 + (53S2.56)2 + (6341.33)2 + (6063.25)2
157,653,873.0
R Day P ? p ? ?
^ (X X)^ = (3579-38T + (5535.74)^ + (7078.31) + (8784.43) - 170,725,061.4
Summary Computations
Source of
Variation
Treatment (R )
A
Day after
Castration
Interaction
Error
Total
Amount
(SS)
105,031.28
- 921,980.55
91,495.03
410.633.56
1,529,140.42
D.P.
3
3
9
48
63
Mean Variance
(MS)
35,010.43
307,326.85
10,166.11
8.554.87
F
versus Error
4.0925
35-9242
1.1883
_ __
Occurrence
by Chance
< 0.025
< 0.0005
>0.05
___
N, = 16 = Number of boxes, i.e. groups of measurements with the same treatment on the
same day.
-------
group, PB + DDT, tended to resemble the DDT hyperbola
more while the SKF-525A + DDT, or hepatically inhibited,
group favored the control pattern. Neither of these
groups differed significantly from the control group or
from each other. A set of pairwise analyses (ANOVA) did
show that the SKF-525A + DDT group was different from the
DDT group (p < 0.05) and that the PB + DDT group
approached statistical difference from both control and
DDT-treated groups (0.05 < p < 0.10).
The analysis of variance including all four treatment
groups, Table 8, demonstrated the anticipated effect of
time after castration by giving it a p value of < 0.0005,
i.e., time after castration contributed significantly to
the overall variance of the results from the overall
(grand) mean of the results. Treatment also showed a
significant effect, p < 0.025. Time after castration and
treatment were shown to be independent when they exhibited
no interaction, p > 0.05. Tukey's (honestly significant
difference) tests (21^) were performed for each of the
possible data contrasts within the data set but only one
reached significance at the 5% level (Control - DDT). The
value of the (Control - (PB + DDT)) contrast approached,
but did not reach significance. A simple estimation of F
for the maximum and minimum cell variances indicated that
the data had fulfilled the requirement for homoscedasticity
and that the use of ANOVA was fully justified^
165
-------
The observed similarity of the SKF-525A + DDT group
and the controls implied that SKF-525A had antagonized
whatever action of o,p'-DDT had resulted in making the
DDT-treated group different from the controls. Because
SKF-525A acts as an inhibitor of microsomal function and
can presumably block DDT breakdown in mammals as it does
in insects (107,215), the amelioration of the DDT effect
on the postcastration rise in LH implied that an. active
metabolite of o,p'-DDT was involved in producing the LH
suppression seen in the DDT-treated group. Further sup-
port, for this conclusion was provided by the position of
the PB + DDT curve. Phenobarbital is a known inducer of
DDT metabolism in the rat (1?5) as is DDT itself (115).
The net result of injection of both PB and DDT could be
either a more rapid formation of an active DDT metabolite
than occurs with DDT treatment alone or a more rapid
inactivation of DDT or its metabolite(s) than occurs with
DDT alone. The first alternative should potentiate any
observed actions of DDT while the second should alleviate
them. Since the PB + DDT curve fell between that of DDT
and SKF-525A + DDT, a balance between the two alternatives
appeared to have occurred.
Other measurements on these animals indicated their
similarities. Variables such as organ weights and pitu-
itary LH content (H(4 - 12 ug B-64o/mg wet weight) did not
differ significantly between the,control, DDT and drug
166
-------
treated groups nor did they differ from published values
>,
(180,20?).
To summarize, a second experiment has indicated the
existence of an effect of o,p'-DDT on the steroidogenic
endocrine system of the male rat. This experiment has
shown the occurrence in male rats neohatally treated with
o,p'-DDT of the same blunting of the LH response to adult
castration which Gellert et al. (5^) demonstrated in the
neonatally injected female rat. At the o,p'-DDT dose used
the decrease of 30$, observed at 11 days postcastration,
agrees well with the 25$ diminution seen in the female at
3 weeks postovariectomy. In either sex the difference
between controls and the DDT-treated animals may be an
hypothalamic imprinting (as proposed by Gellert et al.),
a peripherally mediated change of gonadotrophin or steroid
metabolism, or an alteration of hypothalamic, pituitary or
gonadal hormone sensitivity caused by occupation of
steroid receptors by o,p'-DDT or one of its metabolites.
The indication that a metabolite might be involved is seen
in the present results in the similarity of the responses
of the hepatically-induced, DDT-treated group (PB + DDT)
and the DDT-treated group. It is also seen in the similar-
ity of the responses of the hepatically inhibited,
DDT-treated group (SKP-525A + DDT) and the control group.
These results reinforce the hypotheses of an active
o,p'-DDT metabolite put forward by several authors (22,1^6,
to explain such phenomena as the inhibition of the
167
-------
uterotropic action of o,p'-DDT by pretreatment with the
microsomal poison carbon tetrachloride (22).
168
-------
CHAPTER 4
EXPERIMENTS ON THE EFFECTS OF ADMINISTERING o,p'-DDT TO
NEONATAL MALE RATS VIA THEIR MOTHER'S MILK
The last chapter dealt with experiments in animals
directly injected with doses of o,p'-DDT. This chapter is
a description of the dosage of neonatal rats with
o,p'-DDT via their dams and some of the results of such
dosage. Since most of these experiments were conducted
concurrently with those described in Chapter 3 and some
used similar designs and methodology, they do not repre-
sent advanced experiments "but describe results obtained
by using a different route of exposure.
I. Administration of o.p'-DDT to Neonatal Rats Via the
Dam?
Since the natural route of exposure to o,p'-DDT, or
other synthetic chemicals, during the perinatal period is
via the placenta and/or mother's milk, and since many
materials, including DDT, have been shown to be passed via
those routes (55,56,153,18?), it appeared expedient to
ascertain the effects of o,p'-DDT when administered to the
pup via such a natural route. Therefore, after delivery
and assignment of pups to litters of the needed sizes (6
to 12 per dam) the dams were given daily i.p. injections,
until weaning, of either 0.1 ml of DMSO or the same volume
containing 50 mg of o,p'-DDT dissolved in DMSO. The daily
dose of o,p'-DDT chosen was 1/10 of an acute LD-Q.
169
-------
Body weights for both control and DDT-injected dams
were monitored for one group of dams? growth of the pups
was also measured. Control and DDT-treated dams showed
similar weight profiles through weaning as did their pups.
Growth of the litters was similar to the sesame oil
injected controls used in other experiments and to the
untreated litters of similar size described by Osta'dalova'
et al. (163,216). Other than local discomfort immediately
(0-15 minutes) following injection, the dams did not show
any noticeable untoward effects.
The transfer of o,p'-DDT from dams fed diets con-
taining 20 or 200 ppm of technical DDT to their pups was
implied in a study (187) of the concentration of DDT
metabolites in the milk curds found in the stomachs of
suckling pups. In that study Ottoboni and Ferguson demon-
strated a DDT concentration in milk lipid ranging from 3
to 10 times the concentration in the dams' feed; the
concentration ratio (milk/feed) decreased with increased
DDT concentration in the feed. The study did not, however,
indicate the amount of the DDT dose absorbed by the pups.
Because that study was based on data generated by long
term feeding, because it did not measure DDT uptake by the
pups and because it included feeding during pregnancy it
could only serve as a general model for an investigation
specifically designed to determine what dose of o,p'-DDT
and/or its metabolites were absorbed by pups suckling an
injected dam. The present experiment measured both the
170
-------
secretion of o,p'-DDT and its neutral metabolites and their
absorption by suckling pups.
On the day of parturition pups were sorted into
litters of 6 male pups each and the daily injections of
DMSO or DDT into the dams were begun. The next day, day 1,
one pup from each of 3 DDT-treated and 4 control litters
were removed at random, killed and the stomachs dissected
out for separate storage. The pups were immediately
replaced in the litters by females of similar age. The
same procedure was repeated on days 3* 5» 10, 15 and 20
after birth (only 1-3 samples were collected from each
treatment group after day 10). Collected samples were
processed and measured by the methods described in Figures
9 and 10 (see Chapter 2, Section IV.E.). Only the curded
milk in samples taken at 15 and 20 days was processed to
avoid spuriously low values due to the presence of uncon-
taminated rat chow in the contents of the stomach. No
o,p'-DDE was found in either milk curd or whole body
extracts.
Group means were generated for each compound measured
for each sampling day and these were plotted against time
after birth (which closely approximated time after initial
o,p'-DDT administration). The observed differences in
analog secretion (Figure 28) and uptake (Figure 29)
between control and DDT-treated groups were dramatic.
Concentration of o,p'-DDT in milk (Figure 28) rose
gradually from 1.3 ppm on day 1 to ^ ppm on day 20. The
171
-------
Figure 28. Secretion of DDT Analogs in Rat Milk
The concentration, in ug per g of milk curd, of
various DDT analogs extracted from the curded milk removed
from rat pups of various ages is shown. The pups were
reared in litters of 6 pups each by dams which were
injected daily, i.p., from day 0 (= birth) onward with
either 0.1 ml DMSO (x-x) or 0.1 ml DMSO containing 50 mg
o,p'-DDT (•-•). The curds were extracted according to the
procedure shown in Figure 9 and subjected to gas chroma-
tography under the conditions listed in Figure 10. The
data shown are means, -1 standard error, for both treat-
ment groups; error bars do not appear for those points
where the symbol covers the error interval. Each point
represents 2-k milk curds except for days 10-20 of the
controls where single values are shown.
172
-------
FIGURE 28
SECRETION OF DDT ANALOGS
5 -
M mm
3«
O
oc
32-
o
J 1 -
z
0 0 -
^
o 10 -
n
2
Q.
Q.
~ 5 -
z
o
H 0-
S 2 -
o
O | -
o
<9 _
_^ O **
^ 15 -
10 -
5-
0 -
o.p'-DDT
T
.x^i
| ^^x"^
^ T^r
x 1
o.p'-DDD
\^
-f •
p,p-DDE
^.^-•-
T ^^^^^^ ^
m*-^.\^L~~i.
K "* "" ' 1 ^^**"""» 1* W '^
p,p-DDD
i ,,„ , ..,, 0—**"^
XXX: *
iii '
135 10
IN RAT MILK
i
Lx-^^j
~"[
T T
x^i i
* 1
. ..
•"•^•*"' ' ""^•*^"~~1M^| III m **
I
T
/-I [
1 I
15 20
DAYS
AFTER PARTURITION
173
-------
Figure 29. Uptake of DDT Analogs by Suckling Rats
The tissue concentrations of various DDT analogs, in
ug per g of body weight, is shown for rat pups of various
ages. The carcasses of the pups described in Figure 28
were processed according to the protocol of Figure 9 and
quantitated by gas chromatography under the conditions
described in Figure 10. The data from litters suckling
vehicle-injected dams (x-x) or o,p'-DDT injected dams
(•-•) are shown as means -1 standard error. The number of
pups per point is 2-^ except for days 1 and 20 for the
controls where a single animal is shown. Error bars are
omitted where symbols cover the error intervals.
174
-------
FIGURE 29
UPTAKE OF DDT ANALOGS BY SUCKLING RATS
12.5 -
10 -
~ 7.5 -
x
UJ
o
o
CO
o
o
3
2.5 H
0
3
2 -
I -
0
3
ui
o
2 -
I -
(9 0
3 15
10 H
5
0
o.p'-DDT
o,p'-DDD
p,p-DDE
p.p-DDD
5 10 15
DAYS AFTER BIRTH
T
•
0.
20
175
-------
concentration of o,p'-DDD, the major neutral metabolite
of o,p'-DDT, rose in a fashion parallel to o,p'-DDT from
2 ppm on day 1 to 6 ppm on days 15 and 20. Evidence of
the contamination of the o,p'-DDT, which was injected into
the dams, with the p,p'-isomer, in addition to that pro-
vided in the chemical analysis described in Chapter 2,
Section II, was provided by the appearance of p,p'-DDE and
p,p'-DDD residues. Their concentrations rose somewhat
more slowly above control levels than the o,p'-isomers due
to the smaller amount of p,p'-DDT injected and probably to
the presence of a lag phase of 3-5 days (113,217) for the
hepatic induction by DDT which promoted their formation
in the dam. The metabolites reached amounts of 1 ppm
(p,p'-DDE) and 9 ppm (p,p'-DDD) by days 15 and 20. The
more rapid metabolism of the o,p'-isomers, causing their
loss in the urine and feces, undoubtedly contributed sub-
stantially to the implied unequal retention of the
p,p'-DDT isomer by the dam and to her subsequent secretion
in milk of disproportionately large amounts of p,p'-DDE
and p,p'-DDD.
Whole body o,p'-DDT concentrations in the pups
(Figure 29) rose abruptly from control levels on day 1 to
a plateau of 6-7.5 ppm by day 3. This clearly demonstrated
the absorption of o,p'-DDT. If an estimation of 70$ water
was made for total body weight, and a similar estimation
of 75-80$ water was made for stomach curd weight (218),
the concentrations of o,p'-DDT based on solid weights were
176
-------
18 and 12-15 ppm for the whole body and curd, respectively.
Thus, accumulation was at least an equilibration of the
concentrations in the pups' body tissues with those in the
milk.
Though the body concentrations of o,p'-DDD followed
the temporal pattern of o,p'-DDT accumulation they never
reached the levels found in milk, on either a total or
solid weight basis. A partial answer to this discrepancy
with the results found for o,p'-DDT may lie in the occur-
rence of on-column breakdown of o,p'-DDT to o,p'-DDD.
This breakdown was encountered in an analysis of pure
o,p'-DDT conducted after analysis of the body residues and
the milk curd samples had been completed. A mass of
10-20$ of an injection of o,p'-DDT appeared as o,p'-DDD,
probably as the result of interaction with lipid residues
deposited on the column near the injection port. The same
problem has been described for p,p'-DDT by Burke (219).
It constitutes a needed correction (increase) of less than
20$ for the o,p'-DDT values in milk curd samples (with an
equivalent decrease in measured o,p'-DDD levels).
Analyses of pure pesticides conducted between the body
residue and milk curd analyses indicated no breakdown
problem. Therefore, any adjustments would apply to the
milk curd results only. Possibilities which are more
capable of fully explaining the difference seen in the
milk and body residues of o,p'-DDD in comparison to those
seen with o,p'-DDT may be the occurrences of a more active
177
-------
metabolism or excretion of ingested o,p'-DDD by the pups
or a selective exclusion of o,p'-DDD by the gut.
Body uptake of p,p'~DDE approximates a sigmoidal curve
less steep than the uptake of the o,p'-compounds. The rise
to 1.5 ppm between day 10 and day 15 follows both attain-
ment of the plateau of secretion in milk and the critical
hypothalamic imprinting period. On a solid weight basis
the concentrations of both tissue and milk would be near
b-5 ppm on day 15, a situation adequately described as a
passive accumulation driven by a concentration gradient;
just as might have been expected for a nonmetabolizable,
hydrophobia compound such as p,p'-DDE.
As with body residues of o,p'-DDD, body residues of
p,p'-DDD never approach the levels theoretically possible
on the basis of the residues seen in milk, levels which
might cause neural or reproductive toxicity (see p. 30-31)
or levels approaching those of o,p'-DDT during the critical
imprinting period. A possible on-column breakdown of the
0.1 ng p,p'-DDT per ul, originally added as an internal
standard in milk curd analyses could only account for 1-2$
of the p,p'-DDD mass found in milk and therefore is incon-
sequential in explaining the difference between concentra-
tions seen in milk curds and total body extracts. Time
courses for either p,p'-DDD or o,p'-DDD uptake in pups do,
however, indicate that breakdown after absorption is a very
active process. The abrupt fall to control levels after 15
days is not accompanied by similar falls in o,p'-DDT or
178
-------
p,p'-DDE as would be the case if only decreased uptake, due
to introduction of solid food (163,216), was the cause.
Therefore, active metabolism by pups in the presence of de-
creased intake of all forms of DDT must have caused the ob-
served fall in body residues after day 15 and was probably
responsible at earlier times for maintaining ODD levels be-
low those expected by passive accumulation. Hepatic induc-
tion in pups (112,153) was not clearly indicated but may
have been reflected in the dip in o,p'-DDD and o,p'-DDT
values which occurred between days 3 and 10 since secretion
in milk continued to rise throughout that period.
Total dosage for the period of birth through weaning
was calculated by integrating a set of curves, extrapolated
to 25 days of age, over time. Total administered dose (cumu-
lative dose) was obtained from milk curd concentrations while
total effective or absorbed dose (body burden) was calculated
from body concentrations. Milk production was assumed to be
2^.7 g/day - the mean value obtained by Hanwell and Linzell
(220) for dams suckling litters of 6 pups. Mean body
weights, exclusive of stomach, were used in computation.
When body burden of o,p'-DDT was plotted against
cumulative dose of o,p'-DDT (Figure 30) a curve intermedi-
ate between a straight line and a hyperbola was generated.
Since any curve fitted to the results lay above a line
with a slope of unity and thus forced the conclusion that
more o,p'-DDT was absorbed than was supplied, one of two
conditions must have occurred. Either the assumed
-------
Figure 30. Uptake of o,p'-DDT by Suckling Rat Pupst Total
Body Burden Versus Cumulative Dose
Total body burden, calculated as the product of
o,p'-DDT tissue concentration and average body weight at
the time of sacrifice, is plotted versus cumulative
ingested dose of o,p'-DDT, calculated as the product of
daily milk weight and milk curd concentration integrated
over the age of the animals in days. Milk weight was
taken as 2^.7 g/day based on the findings of Hanwell and
Linzell (220) for litters of 6 pups. The empirical curve
was generated from the data for the DDT exposed pups
described in Figures 28 and 29.
The inset shows a graph of the inverse functions with
a comparison to a line Ax/Ay = 1, describing complete
storage of ingested residues. It should be noted that the
empirical lines describe systems which are storing more
residues than are being ingested. If the errors for both
uptake and storage measurements (see Figures 28 and 29)
are considered, however, the data may be found to include
the approximation of a line with a slope of 1. Thus the
data appear to describe a nonsaturable system, i.e., a
residue sink.
180
-------
FIGURE 30
UPTAKE OF o.p'-DDT BY SUCKLING RAT PUPS*.
TOTAL BODY BURDEN VERSUS CUMULATIVE DOSE
250-
200-
o
Z)
UI
Q
ISO-
00
Q
O
CD
< 100
I-
o
50-
EMPIRICAL
I/X vs I/Y
0.2-
UJ
o
oc
3
m
0.1-
o
o
ID
O.I
0.2
I/ CUMULATIVE DOSE
1
100
1
50 100 ISO
CUMULATIVE DOSE (UG)
200
181
-------
production of milk was too low or the estimates of milk
residues were too low. If allowances were made for a
10-20$ underestimate of o.p'-DDT, caused by on-column
breakdown, and for the variances of "both milk curd and
"body residues, the results adequately approximate a
straight line with a slope of unity. This line (slope
equal to 1.0) indicated that the pups were extremely
efficient in absorbing, and storing, o.p'-DDT administered
in milk; they acted as sinks for o,p'-DDT. Furthermore,
it implied that the total dose absorbed by rats treated in
this manner was between 50 and 100 ug by day 5 or 200-300
ug by day 25. If similar analysis was done for o,p'-DDD,
total doses were 10 and 50 ug for days 5 and 25, respec-
tively. Comparison to cumulative administered dose of
o,p'-DDD indicated that only 10-12$ of that supplied in
the milk was stored in these animals.
Other computations indicated that only 0.35-0-^0$ of
the total dose administered to the dam was eliminated in
her milk and that a total of only 0.19$ of the total dose
administered to the dam was absorbed by the pups. The
absorption rate appeared to be fairly uniform until
self-feeding began at 15 days of age; total retention by
25 days of age was only 0.12$ of the total dose admin-
istered to the dam. It should be noted that the absorption
and retention values given above are probably higher than
the actual exposure experienced by the larger litters used
in other experiments described later since milk production
~182
-------
per pup falls somewhat as the litter size increases (220).
Conversely, because total milk production increases with
larger litters, total dose secreted increases. Therefore,
this experiment only describes an upper limit of exposure
per pup and a lower limit of total secretion by the female.
In summary, this experiment was designed to determine
the dosage of o,p'-DDT administered to rat pups suckled by
DDT-injected dams; it showed that the doses administered
in the milk and absorbed by the pups were well within the
dose-range examined in directly injected rats. It gener-
ated some information on the dynamics of this transfer
from mother to offspring in terms of the forms and
quantities involved. And, it inferred the role of hepatic
induction in transformations in both the dam and pup.
Finally, the experiment indicated that if a "DDT"-saturated"
female were used as a foster dam for a portion of the neo-
natal or preweaning period the efficient absorption of
o,p'-DDT would allow dose response experiments to be con-
ducted by using the natural route of exposure.
II. Organ Weights in Adult Rats Neonatally Treated with
o.p'-DDT Via Their Dams;
A brief experiment similar to the initial .experiment
on directly injected rats was performed using the injected
dam as the means of exposure. Litter size averaged 8 pups.
Autopsies were done at 102-138 days of age; both intact
and neonatally castrated animals were examined. Growth
183
-------
was only examined sporatically but indicated general agree-
ment among the groups prior to 40-50 days of age.
Final body weights (Table 9) were similar in both
castrate groups but the DDT-treated intact animals were
about 10-12$ lighter than their vehicle-exposed controls.
All body weights were indistinguishable from the cor-
responding groups shown in Table 6 and Figure 13.
Organ weights, normalized to body weight, demon-
strated the effects of castration but did not reflect any
overt changes due to treatment with o,p'-DDT. Only the
testes showed a slight change in normalized weight due to
the presence of DDT, but this was explained by the differ-
ence in body weights between the control and DDT-treated
group; raw organ weights did not differ. The other nor-
malized organ weights, with the exception of the pituitary,
were similar to those seen in the directly injected rats
described in Table 6 and in Figures 1^-20.
The weight differences which exist between these
findings and those obtained in the experiments done on
directly injected animals (Table 6 and Figure 1^), in
regard to the pituitaries of both the intact and castrate
groups, were not explicable on the basis of differing body
weights. The difference was also not reflected in a change
in pituitary LH content or serum LH levels in the castrate
groups; pituitary LH content was 55-7 and 55 - 6 ug
B-640/mg Wet tissue for control (3 animals) and DDT-treated
(3 animals) groups, respectively; serum LH levels were
-------
oo
Table 9
-i 2
Effects of Suckling an o,p'-DDT Injected Dam on Body and Organ Weights of
Intact and Neonatally Castrated Male Rats
Percent of -
Body Weight^
Pituitary x 103
2
Adrenals x 10
fpri o 4"pC!
^Jominnn **\ TT 1
Vts c; i i*O o a —J *y TO-*^
Yen"t3?s.] PTO s "l~a "he
T.T VAT*
K" i rifi^irc!
Control
(l±55 t
3-37 - 0
1.36 - o
•4-
0 7^ — 0
o ^ i n
w , j?^ - u
0 OQ - 0
Intact
(6)
.19 3.
.09 1.
07 n
i W f U ,
no n
• ^7 \J •
o? n
DDT
01 ±
51 i
30 t
80 i
-31 +
JJ-i-
11 +
(6)
s #*
29)
0.38
0.06
#
On <
. U J
n no
o n?
Control
(333 ^
^.77 -
2.05 -
fS q^ +
O . _JO —
3Q9 "*"
• 7^ ~
Orjf. "*"
• f O —
Castrated
(9) DDT (1
21) (353 -
0.68 ^.27 - 1
0.28 2.09 - 0
3"^f\ 1 1 T7 "*" "7
. J)O ±±,jf - (
Of.Q r> eJi "•" ft
. DO J . _^f ~ "
0-1 < n "51- n
. J-,? U . f JL — U
^)
35)
,43
06**
. L>O
cn
Ou
T O
• J-3
All pups were suckled prior to weaning at 25 days of age by dams who received daily
i.p. injections of 50 mg o,p'-DDT in 0,1. ml DMSO or 0.1 ml DMSO alone. Litter size
averaged 8 pups per litter and sacrifice was at 102 to 138 days of age.
2
Body weights are given in grams in parentheses under each heading.
-------
Table 9 (Cont.)
„ — -
All data are given as mean weights or mean % of "body weight -1 standard deviation;
numbers in parentheses after the treatment designation are the numbers of animals
examined; comparisons are all versus the appropriate control groups and are made by
unpaired Student's t-tests, * p < 0.05, ** P < 0.025.
GO
O>
-------
1.04 t 0.32 and 0.86 - 0.39 ug B-640/ml for control and
DDT-treated groups, respectively. Nor did a different
group of pituitaries (Section V, this Chapter), taken from
animals treated via their dams, demonstrate weights dif-
fering from those found in directly injected animals for
either control or DDT-exposed groups. Possible explana-
tions of the present results include variability in litter
sizes during the experiment, possible problems with mal-
nutrition due to the presence of primiparous dams and/or
inexperience or inconsistency in dissection and handling
of the pituitary. Whatever the cause, the later deter-
minations, on intact rats, similarly dosed in litters of
6 and 12 pups (Section V, this Chapter), gave values for
normalized pituitary weights of 2.59 - 0.24 for control
animals (21 animals) and 2.69 - 0.25 for DDT-treated ani-
mals (20 animals)} both values agree with those found in
directly injected groups. Similarly, a later determina-
tion of the normalized hypophysial weights of neonatally
castrated, suckled rats raised in 8 pup litters showed
values of 3.?4 ± 0.30 and 3.3? - 0.14 mg/100 g body weight
for control (2 animals) and DDT-treated (3 animals) groups,
respectively; values again in accord with those deter-
mined for directly injected animals.
The above results yielded no clue as to any possible
effect of o,p'-DDT on the male rat. They again, as was
the case with directly injected ra'ts, implied the need for
the use of more sensitive measurements. To that end, and
187
-------
with the data of Rybakova (29,29) and Gellert et al. (53*
54,150,151) in mind, several experiments were done to
probe aspects of pituitary release of LH.
III. Periodicity of LH Release in Adult Rats Neonatally
Exposed to DDT;
Adult castration superimposed on neonatal DDT-treat-
ment was shown to expose a measurable difference between
the responses of sesame oil and DDT-treated LH control
systems (Chapter 3, Section III). Ether had analogously
been shown to alter the 24-hour periodicity of serum LH
levels in untreated animals (Chapter 2, Section IV.D.2.)
(183-186). The following experiment, though originally
performed to measure the direct effect of o,p'-DDT expo-
sure on the 24-hour periodicity of serum LH in both
intact and neonatally castrated rats, is best discussed
in light of the effect of superimposing a second stressor,
ether, on what may be an LH control system which has
already been perturbed by neonatal exposure to o,p'-DDT.
Differences in ether response throughout the day are, in
fact, what was measured.
Two groups of rats were subdivided into 2 subgroups
each, 2 of which were treated with o,p'-DDT via their
dams (DDT-treated) and 2 of which were suckled by vehicle
injected dams (Controls). One subgroup under each treat-
ment was castrated on the day of birth. The pups were
reared in 8 pup litters and were used for the experiment
at 76-89 days of age. After weaning, a pair of subgroups,
188
-------
one DDT-treated and one control, were each split into 3
sets or 2-5 animals each, i.e., 3 sets of 2-5 control ani-
mals were formed and 3 sets of 2-5 DDT-treated animals
were formed. Beginning at 09.00 or 11.00 (EST) individual
animals from a pair of the sets (one DDT-treated set and
one control set) were alternately bled under light ether
anesthesia (TVBE). Animals were returned to their cages
and to the normal light/dark cycle when fully conscious
after bleeding; total elapsed time between removal of an
animal from the cage to its return was about 6-8 minutes.
The serum was separated from the collected blood and
stored at -20°C until analysis by the RIA procedure of
Niswender et al. (169). The bleeding procedure was
repeated at 2 hour intervals, using the sets of animals
pairwise and in rotation so each animal was bled once
every 6 hours, i.e., a total of 4 times over the course of
the test period.
Means for each treatment group and each time of day
were calculated from the results obtained from the RIA.
The means were plotted versus time of day and visually
examined for the existence of any discernable circadian
f-
rhythm(s). Corresponding means for DDT-treated and control
rats were tested for difference by Student's t-tests. Both
the DDT-treated and the control group results for intact
animals were also divided into halves, before 20.00 and
after 20.00 (EST), and examined in a 2-way ANOVA for the
effects of both time, day and night, and exposure to DDT.
189
-------
The LH patterns obtained for both neonatally
castrated and intact rats are shown in Figure 31.
significant differences between DDT-treated and control
groups were found at any single time of day for either neo-
natally castrated or intact animals. No rhythm was shown
in either treatment group of castrated rats, The levels
of LH seen in castrates were similar to those reported by
Lawton and Smith (186). The intact animals generated a
picture similar to that seen in Figure 8 which describes
intact, normal, adult males exposed to ether. In intact
males the LH values found during the day were more variable
and higher (p < 0.005) than those obtained at night. A
superficial rise near 2^.00 (EST) may reflect the LH peak
which coincides with peak activity (see Chapter 2, Section
IV.D.2. and Figure 8), however, the values are too near
the reliable limit of the assay to distinguish that point
from any others between 18.00 and 06.00 (EST). Though
there was no significant difference seen in LH values
between the entire set of results from the control and
DDT-treated intact groups (0.05 < p <£ 0.10), the com-
bined values for the results between 15.00 and 19.00 (EST)
did differ for the two groups (t.^ = 2.4-625, p < 0.05),
the controls being higher than the test group.
The possibility that the castrates demonstrated a
larger ether effect than the intact animals was not sup-
ported by the similarity of the present results to the
values of serum LH in long-term castrates bled by
~190~
-------
Figure 31. 24-Hour Periodicity of Serum LH in Adult Male
Rats Treated Neonatally Via Their Dams
Serum LH in ng B-6^0 equivalents per ml is plotted
versus time of day for groups of adult male rats. Both
neonatally castrated and intact rats are shown. The ani-
mals were further grouped by treatment , being suckled in
8 pup litters by dams injected daily, i.p., from day 0 to
25 after parturition, with 0.1 ml DMSO (control, «-•) or
with 0.1 ml DMSO containing 50 mg o,p'-DDT (DDT, x-x).
Bleedings were done by tail-vein under light ether (see
Figure 8) on groups of 2-5 rats. All animals in a given
experiment and treatment group were placed in 3 subgroups
which were bled in rotation every 2 hoursj each individ-
ual subgroup being bled every 6 hours. Animals were
allowed free access to food and water throughout the
experiment. Sera were analyzed for LH by the method of
Niswender et al. (169) using B-6*K) (=0.03 x NIH-LH-S1 by
OAAD) as the primary standard. Values shown are means -1
standard error; r, = 1-5 for each point. No statistically
significant differences between the DDT and control groups
were found.
191
-------
FIGURE 31
24-HOUR PERIODICITY OF SERUM LH IN ADULT MALE
RATS TREATED NEONATALLY VIA THEIR DAMS
2500H
1500-
T
NEONATALLY
I
DDT
CASTRATED J
N
TIME OF DAY
192
-------
decapitation as published "by Yamamoto et al. (180). The
possibility that DDT-treated rats showed an ether response
which compensated for a depression in LH caused by o,p'-DDT
cannot be ruled out. However, the observation that a
response to ether did not eliminate the difference between
DDT-treated and control animals castrated as adults (Chap-
ter 3» Section III) argues against such compensation. The
present finding that no difference was seen due to o,p'-DDT
exposure when a difference did occur after adult castration
in directly injected animals may have been due either to
the longer period which had elapsed after castration -
serum LH continued to rise for at least a month after
castration (180) - or to the difference in the absorbed
dosage (pups treated via the dam absorbed less than 0.5 mg
over a period of 2.5 days while injected pups received 10
mg over 5 days).
The conclusion reached from the experimental results
was that neonatal exposure to o,p'~DDT does not signifi-
cantly alter the 2k-hour periodicity of the response of
serum LH to tail-vein bleeding under ether in either neo-
natally castrated or intact rats. There does appear,
however, to "be a tendency toward lower values in intact
DDT-treated animals at least during the late afternoon.
IV. Serum LH Response to Adult Castration!
Since no significant alteration of the circadian
pattern of LH response to ether was generated by neonatal
DDT exposure in the preceding experiment, an experiment
" 193
-------
was undertaken to measure the effect of neonatal DDT-expo-
sure on the LH response to adult castration. The experi-
ment was later repeated in directly injected animals (the
experiment discussed in Chapter J, Section III). As was
the case in the other experiments dealing with LH release
the empirical bases for the experiment were the observa-
tions of Rybakova (28,29'), showing increased pituitary LH
levels in male and female rats chronically fed technical
DDT, and the observations of Gellert et al. (5*0. showing
the blunting of the response to adult castration in female
rats neonatally injected with 0.001-1.0 mg of o,p'-DDT.
The experiment was initiated with two litters of 8
neonatal pups each. One litter received o,p'-DDT via its -
treated dam (DDT-treated) while the dam of the other
litter received vehicle only (control). Attrition due to
undetermined causes during development and prior to the
beginning of the experiment on day 5? of age, limited the
number of animals to 3 in the control group and ^ in the
DDT-treated group.
To establish a response baseline serum samples were
taken between 15.00 and 18.00 (EST) by TVBE on days 5?, 58
and 59 i the sera were stored for later assay. On day 60
the animals were castrated but no blood samples were taken.
On days 1, 3, 6 and 10 following castration serum was
obtained from all the rats by TVBE at 15.00-18.00 (EST).
Two rats succumbed to ether and a preexisting respiratory
infection leaving 2 control rats and 3 test animals. An
194
-------
injection of testosterone (0.75 mg/kg) was given 6 hours
prior to bleeding on day lb to ascertain if the
gonadal-hypothalamo-hypophysial feedback loop was func-
tioning differently in the DDT-treated and control ani-
mals (1?0). Finally, on day 15 postcastration, the animals
were anesthesized.with ether and decapitated; trunk-blood
was collected to determine if LH had rebounded normally
from the testosterone injection.
Sera were analyzed by RIA using the procedure of
Niswender et al. (169). Means were calculated for each
experimental day and each treatment! a plot of these
results is presented as Figure 32. Two-way ANOVA was also
done on the values obtained between the time of castration
and the time of testosterone administration, i.e., on the
results from days 1-10.
Due to the small number of animals involved the
standard deviations were quite large. At the 5% signifi-
cance level no difference was apparent between the control
and DDT groups. If the acceptance level for significance
was relaxed to 10$, however, the control showed signifi-
cantly higher values than the DDT-treated animals during
the period of 1-10 days postcastration! values for
DDT-treated animals were 15-20$ lower than those for the
controls. The measured LH values for the period following
day 6 were quite similar for both control and DDT-treated
groups to those encountered in the better balanced experi-
ment done on directly injected animals. Controls
~195
-------
Figure 32. Response of Serum LH to Adult Castration in
Male Rats Treated Via Their Dams
Serum LH as ng B-6^0 .(= 0.03 * NIH-LH-S1 by OAAD)
equivalents per ml is plotted versus time after adult
castration for two groups of male rats. The control rats
(•-•) were suckled prior to weaning in a litter of 8 pups
by a dam which received daily i.p. injections of 0.1 ml
DMSO while the DDT-treated rats (x-x) were reared in a
litter of similar size by a dam which received daily i.p.
injections of 0.1 ml DMSO containing 50 mg ofp'-DDT. The
animals were reared with free access to food and water and
were castrated at 60 days of age. Blood samples were taken
from the same animals throughout the experiment by TVBE at
15.00-18.00 (EST) on the days indicated. On day 1^- an
injection of 0.75 mg/kg testosterone (T) was given s.c. in
sesame oil 6 hours prior to bleeding. Sera were assayed
by the method of Niswender et al. (169). Values shown are
means -1 standard deviation for groups of either 2 control
or 3 DDT animals. Two-way ANOVA of the results between
castration and testosterone injection (days 1-10) yielded
a p value between 5 and 10$ for the difference between
control and DDT-treated groups.
196
-------
FIGURE 32
RESPONSE OF SERUM LH TO ADULT CASTRATION
IN MALE RATS TREATED VIA THEIR DAMS
900 -
DAYS
CASTRATION
197
-------
demonstrated a response to testosterone administration
which appeared similar to that observed in directly
injected animals. DDT-exposed animals, on the other hand,
did not demonstrate this response clearly. However, since
the error for the mean on day 14 included the range of
response seen in directly injected rats a total lack of
feedback inhibition could not be inferred.
If the relaxation of the acceptance level for signif-
icance is allowed, the agreement of these results with
those observed in directly injected animals is striking.
Their similarity to the data given by Gellert et al. (5*0
for female rats is equally good. Such correspondence is
particularly important in view of the differences in effec-
tive administered dose. The directly treated rats in my
studies received 10 mg o,p'-DDT while Gellert et al.
observed effects with 0.1-1.0 mg; the calculated total
absorbed dose of all forms of DDT in the suckled animals
was less than 0.5 mg over the full 25 day preweaning period.
If only the first 5 days, the critical period for hypo-
thalamic imprinting in the male, is considered, the
suckled animals absorbed less than 100 ug of all forms of
DDT and only 80-85 ug of o,p'-DDT. Still, in view of the
small numbers of animals used and the resulting large
standard deviations, a final assessment of these experi-
mental results and their importance must await the con-
firmation of future studies.
198
-------
V. Serum LH Response to Exogenous LHRHt
Studies by Rybakova (28,29) indicated a change in the
pituitary content of LH in rats chronically fed low levels
of technical DDT. Other studies by Arai (211) had shown
changes in pituitary stores of LH and hypothalamic stores
of LHRH in response to neonatal estrogen treatment.
Several of the experiments already discussed also implied
a change in the function of the overall feedback system
controlling gonadal steroidogenesis. On the basis of
these observations, and in order to begin answering the
question of the locus of action of neonatal o,p'-DDT, a
measurement of the response of this feedback system to
exogenously administered LHRH was undertaken.
The design of the experiment included the use of two
different litter sizes and two periods of injection of the
dam in order to probe the results of different absorbed
dosages. A 3-level factorial design was generated for 8
litters; a control and a DDT-treated group were con-
structed in each of 2 litter sizes (6-and 12 pups per
litter) and at each of 2 durations of injection of the dam
(5 and 25 days). Growth was monitored by regularly
checking body weights. Beginning at 1^9-15^ days of age
^-6 animals from each litter were subjected to a test of
their serum LH response to injection of synthetic LHRH.
Rats derived from dams injected for 5 days were tested
separately from those reared by dams injected for 25 days.
199
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Treatment Groups
Treatment of the Dam
Litter Size Injected Chemical Number of In.iections
6 DMSO 5
6 DMSO 25
6 o,p'-DDT 5
6 o,p'-DDT 25
12 DMSO 5
12 DMSO 25
12 o,p'-DDT 5
12 o,p'-DDT 25
On the third day of each test1 simple TVBE was done at
17.00-19.00 (EST) to generate a basal level of LH for
reference. The animals from the 6-pup litters in both the
control and DDT-exposed groups were bled in rotation (to
prevent temporal bias) followed by those from the 12-pup
litters. On the fourth day the same rotation was followed
except that, before bleeding each group, each animal
received a 1.0 ug i.p. injection of synthetic LHRH in 0.1
ml of saline. The bleedings were begun 20 minutes after
injection and were completed on each group less than 60
minutes after injection. The same procedure, including
LHRH injection, was repeated on the fifth day of the test.
The test results for the present experiment were plotted
beginning on day 3 to allow comparison of LH levels to
the range of LH values seen in a group of rats which
were neonatally injected with sesame oil and bled as
adults on 8 consecutive days during which they were
injected with saline on days 3-6.
200
-------
On the sixth day no bleeding or injections were performed.
On the seventh day of the test, animals were weighed,
stunned, decapitated, bled and autopsied. Weights and
organ weights were measured as previously described and
assays for LH were done on both sera and pituitary
extracts. Results for the LH assays were plotted versus
day of the test and analyzed for differences by a 3-way
ANOVA as well as by individual t-tests between the means
of each DDT-treated group and its paired control (paired
both in litter size and length of dam injection) on each
day of the experiment. Student's t-tests were likewise
used to compare values for body weights, etc., from each
paired control and DDT-exposed litter.
Growth for litter sizes of 6 and 12 pups diverged as
expected (163,216) between 5 and 10 days of agej o,p'-DDT
injection into the dams for either 5 or 25 days did not
alter weight changes. Pups from 6-pup litters grew
slightly more rapidly than those from 8-pup litters while
pups from 12-pup litters grew somewhat more slowly than
those from 8-pup litters. Final body weights for rats
from 6-pup litters were fully 10% above those of rats from
12-pup litters (significant at p < 0.001). Normalized
organ weights, however, did not differ because of differ-
ence in litter size, DDT treatment or length of DDT-injec-
tion into the dam.
A representative plot of the experimental results
obtained for serum LH is shown in Figure 33 - values
201
-------
Figure 33. Response of Serum LH to Injection of Large
Doses of LHRH in Adult Male Rats Treated Via
Their Dams
The results of a test of the pituitary response to
repeated injection of LHRH in vivo is shown. Serum LH is
plotted versus time for 2 groups of rats raised in 12-pup
litters; Controls (x-x) which were suckled prior to
weaning by a dam which received daily i.p. injections of
0.1 ml DMSO , and DDT treated animals (o-o) which suckled
a dam which received daily i.p. injections of 0.1 ml DMSO
containing 50 mg o,p'-DDT. The 1^9-15^ day old animals,
which had free access to food and water throughout the
experiment, were bled at 17.00-19.00 (EST) on the days
shown by TVBE. Simple, bleedings were performed on days 3
and 7 but 20-60 minutes (unbiased between groups) prior to
the bleedings on days 4 and 5 the animals were injected
i.p. with 1.0 ug of synthetic LHRH in physiological saline.
Sera were assayed by the method of Niswender et al. (169)
using B-6^0 (= 0.03 x NIH-LH-S1) as standard. Values
shown are the means -1 standard deviation for groups of k-6
rats. A Student's t-test for day 5 of the experiment shows
a large (p < 0.005) dependence on DDT treatment for the
observed response to the second daily injection of LHRH.
Simple bleedings were also done on a group of male
rats which received neonatal injections of sesame oil
(S.O.); the limits of the range of their LH levels ("-)
is shown for comparison, with the LHRH treated animals.
202
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FIGURE 33
RESPONSE OF SERUM LH TO INJECTION
OF LARGE DOSES OF LHRH IN ADULT
MALE RATS TREATED VIA THEIR DAMS
500 -
DAY OF EXPERIMENT
8
203
-------
obtained for rats reared in 12-pup litters and suckled by
dams injected for 25 days. Plots for the other litter
sizes and for the other length of dam injection have not
been included.
Serum LH levels did not differ on the third day of the
test due to treatment or length of treatment of the dam.
However, 12-pup litters appeared (p < 0.05) to have higher
basal serum LH values than did 6-pup litters. The result
was probably only apparent because many of the values were
at or below the reliable limit of the RIA.
On the first day of LHRH injection, DDT-treatment,
length of dam injection or litter size did not, themselves,
cause a difference in response to LHRH. Some interaction
between litter size and either the presence of DDT-exposure
or the length of DDT injection into the female was
indicated (p < 0.05), implying some dependency of the
response to injected LHRH on the size of the total
absorbed dose of o,p'-DDT. The absence of any effect of
the major treatment conditions, however, does not allow
clear interpretation of the results.
The second day of LHRH injection yielded a highly
visible difference in the response of those rats in litters
of 6 or 12 pups neonatally exposed to o,p'-DDT by a dam
injected for 25 days and the response of rats exposed only
to DMSO under similar conditions (Figure 33). The
DDT-treated rats gave serum LH responses to injected LHRH
2.5-10 times those given by control rats (p < 0.001).
204
-------
Interaction between treatment and both length of treatment
alone (p < 0.005) and length of treatment and litter size
(p < 0.05) imply that total dosage of o.p'-DDT may have
been the deciding factor in the observed results. Rats
neonatally suckled by females injected for only 5 days did
not differ from their controls.
No differences among LH values from any of the groups
or treatments was evident in the sera collected at the
termination of the experiment. The LH values of the
pituitary extracts (not presented) implied a dependence of
pituitary LH content on the length of treatment of the dam
(p < 0.005), i.e., on the number of injections given the
dam or, possibly, on slightly different amounts of injected
LHRH for the animals in one half of the experiment (reared
by dams injected for 25 days) versus those in the other
half (reared by dams injected for 5 days).
Interpretation of the results of this experiment is
definitely complex. First of all, unintentional experi-
mental differences between the animals derived from dams
injected for 25 days and those derived from dams injected
for 5 days may have arisen because the former group was
tested for LHRH response 5-10 days before the latter.
This may have generated artifactual differences which
would be reflected in significance of the experimental
factor associated with the length of the injection period
for the dam regardless of the presence or absence of
DDT-treatment. If, for example, LHRH degraded measurably
205
-------
during storage in solution, the responses to LHRH for the
animals reared by dams injected for 5 days and injected
with stored LHRH would be less marked than those in animals
reared by dams injected for 25 days and injected with
freshly diluted LHRH. Such a difference would explain
the overall lower serum and pituitary LH values seen in
the second group tested and would also explain the absence
of a measurable potentiation of the response to the second
LHRH injection in rats suckled by dams injected for only 5
days. If, on the other hand, no such problems occurred,
the difference in the results for the two dam injection
periods might be explained on the basis of lower doses
having been received by those animals suckled by dams
injected for only 5 days. Or they might be explained on
the basis of nutritional changes, which were not reflected
in body weight and which potentiated the effect(s) of DDT,
having occurred in rats suckled by dams injected for 25
days.
Whatever the cause of the difference in outcome
related to the 2 different treatment periods, the experi-
ment dealing with animals suckled by dams injected for 25
days with o,p'-DDT did demonstrate a marked increase of
the serum LH response to the second injection of LHRH. In
rats from both litter sizes the potentiation in DDT-exposed
rats was coupled with a decrease in response in control
animals. This may indicate that the two groups differ in
either their rates of LHRH degradation or their rate of
~206
-------
LH synthesis. Though the amount of LHRH injected was
large, calculation of the effective concentration following
injection, based on the half-life of 6.5 minutes given by
Schally et al. (9), indicated that the injected dose would
be cleared in less than 5-3 hours. Even slowing this down
2-3 fold would not explain the results on the basis of
residual LHRH. Potentiation based on the priming effect
of closely spaced injections of LHRH (1?2) would similarly
not be favored due to the length of time between the doses
of LHRH.
The remaining possibilities for explaining the dif-
ferences seen between the control and DDT exposed groups
may involve altered sensitivities of target organs in the
hypothalamo-hypophysial-gonadal system to circulating
hormones as would be caused by neonatal imprinting or they
may involve the continued presence of residual DDT which
interferes with the feedback suppression of pituitary LH
release and/or synthesis. The second mechanism is highly
unlikely since measurements of DDT residues in 120 day old
females injected neonatally with 3 mg of o,p'-DDT demon-
strated no more residues than control rats (53). Since
the animals in the present experiment were even older male
animals, which should have metabolized DDT even faster than
females (105,107), residues should not act directly during
the period used to measure the responses to LHRH. The
alternative mechanism, i.e., imprinted sensitivity changes
which are expressed in the presence of high levels of
207~
-------
circulating LHRH, is in concert with my earlier findings
of probable alterations in the gonadal steroidogenic
control loop. The opposite direction of the changes in
serum LH seen in the DDT-treated rats in the response to
adult castration and the response to repeated LHRH injec-
tions may be manifestations of different DDT effects or
merely the expression of a different facet of the same
effect.
The smaller response of serum LH observed with the
second injection of LHRH, relative to the first, in con-
trol animals may be produced by a depletion of releasable
pituitary LH stores. Initially, this would be due to the
presence of massive amounts of LHRH. Subsequently it
might be due to direct suppression of LH synthesis within
the depleted pituitary by the large quantities of testos-
terone generated as a result of the LH release brought
about by the first injection of LHRH. The precise
mechanism is not clear at this time.
In summary, treatment of neonatal animals with
o,p'-DDT via an injected dam appears quite effective.
Nearly all of the o,p'-DDT which enters the gut in the
milk was absorbed by suckling pups. Such treatment was not
effective in altering body or organ weights, as might have
been anticipated on the basis of both the calculated
absorbed dose and the previous findings in the present
studies. DDT-treatment via the dam was not effective in
altering overall serum LH periodicity. It was, however,
208
-------
effective in modifying the serum LH responses to adult
castration and to repeated large injections of LHRH.
209
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CHAPTER 5
DISCUSSION OP ANIMAL STUDIES
This study began, experimentally, as an
attempt to measure direct interaction between the metabo-
lism of o,p'-DDT and the androgenic steroids in the testes
of adult male rats chronically exposed to o,p'-DDT, i.e.,
it began as a study of interactions in vitro. During the
course of initial experiments, however, technical problems
due to a variety of causes limited the results obtained to
a few very imprecise observations which indicated little
or no effect of DDT exposure. At that juncture (1971) a
growing awareness of the literature made it apparent that
very few effects of DDT or its analogs had been observed -
or tested for - in vivo, much less in vitr<% - in the
steroidogenic tissues of any mammalian speciesi the
effect of o,p'-DDD on the adult adrenal in several species
was the only real exception. Since the available litera-
ture indicated only that o,p'-DDT was a weak estrogen (22,
2^,1^6,1^8,152) the best approach to the problem appeared
to be to conduct a brief but very broad study in vivo
which would indicate which portions of the endocrine system
would be most profitably studied by subsequent studies run
in__y_itro. Body and organ weights seemed to be the most
appropriate measurements for such a screening experiment
since they should indicate changes which might occur in
the the adrenals, the gonads, the liver and the
210
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hypothalamie-pituitary complex. Before the study was
begun, however, a report by Heinrichs et al. (53) became
available. It indicated that some marked changes occurred
in the steroidogenic system of the female rat following
neonatal injection with moderate doses of o,p'-DDT. The
approach of using neonatal injection appeared profitable
both because it yielded quite demonstrable changes and
because it allowed the use of a large body of literature
on neonatal steroid imprinting in the rat (57-63,69-?l) as
a background for comparisons. After several short experi-
ments using the neonatally injected male had failed to
demonstrate any changes in adult organ weights, it was
decided that a longer study incorporating several ages,
several dose levels and examinations of organ histologies,
serum corticosterone and serum LH might prove more
illuminating. This study, described in Chapter 3» Section
II, did yield an indication that development of adrenal
morphology, and possibly function, was effected by neo-
natal injection with a range of concentrations of o,p'-DDT.
The experiment, however, also indicated that if any changes
in parameters dealing directly with gonadal steroidogenesis
had occurred they were rather subtle. Subsequent experi-
ments (Chapter 3, Section III) based on the work of
Rybakova (28,29) and Gellert et al. (5^) concentrated on
measurements of serum LH under conditions which stressed
part of the hypothalamo-hypophysial-gonadal axis. They
were designed to determine "reserve" capacity, i.e., the
211
-------
capacity to respond to stress. Adult castration was found
in such experiments to generate a picture of altered
"reserve" or response capacity in male rats neonatally
injected with o,p'-DDT.
While these experiments on injected rats were being
conducted a parallel series was initiated using the natural
source of environmental dietary contaminants for the pup,
i.e., the mother's milk, as the vehicle for neonatal
exposure to o,p'-DDT. Again, as with directly treated
animals, the results indicated that some changes in the
capacity of the gonad-brain-pituitary axis had occurred
due to DDT exposure but that the changes- were exposed only
by adult castration or exogenous LHRH. However, the
changes occurred at moderately low levels of exposure;
100 ug of DDT in a 5 day old, 10 g pup is a body burden of
10 ppm which is near the levels found in some environmental
exposures. Taken together the results from the two sets
of experiments, in directly injected and suckled rats,
indicated that at least two loci of o,'p'-DDT effects were
demonstrable within the realm of the steroidogenic systems;
l) the immature adrenal cortex, and 2) the hypothalamic-pi-
tuitary complex.
I. The Change in the Immature Adrenal Cortex;
The adrenal has been implicated in the onset of
puberty in the female rat (98) and in the maturation of the
testicular response to hCG (?8,79). If the process of
maturation of the adrenal is interfered with it is
212
-------
conceivable that the maturation of other tissues
influenced by the adrenal will be affected. The coordina-
tion of the rise and fall of several hormone levels can
have marked influence on the normal maturation of tissues,
e.g., only the proper coordination of LH, FSH and E2 will
allow a developing follicle within the ovary to develop to
the stage of ovulation; other conditions result in atresia
(89). Because of this, either adrenal degeneration, or a
precocious adrenal development, as was found in these
studies, may result in pathologic states which could effect
the development of any of a myriad of other tissues which
are known to be effected, by the function of the adrenal
cortex (206).
If precocious development is viewed as accelerated
aging, then the precocious development of the adrenal
cortex seen in animals treated neonatally with estradiol
or o,p'-DDT may result in eventual degeneration similar to
that seen after treatment of the adult adrenal of several
species with o,p'-DDD. Since "the adrenals of
o,p'-DDT-treated rats did not exhibit the specific type of
involuted morphology seen in other species treated with
o,p'-DDD (37-^9) the DDT effects seen may not be the result
of this metabolite. Furthermore, since the adult rat
exhibits a refractoriness to the action of o,p'-DDD it is
improbable, though not impossible, that o,p'-DDD also would
be effective in the neonate.
213
-------
A more likely explanation for the results is that the
estrogenicity of o,p'-DDT may be the cause of its action on
the adrenal. The adrenal is known to be positively
effected both morphologically and functionally by the
presence of circulating estrogen (4). This is true func-
tionally from at least 25 days of age onward and morpho-
logically (normallized weight) from 50 days onward (see
Chapter 3, Section II). Since steroids influence tissues
by way of intracellular mechanisms subsequent to binding a
steroid receptor, it may be assumed that o,p'-DDT may
function by binding to estradiol receptors. DDT has been
shown to bind to uterine estradiol receptors with
affinities of the order of 1-10 x 10~6 M (Kd) - affinity
for estradiol is 4 x 10~ M (26). It has also been shown
to bind to hepatic P-^50 (221) and adrenal P-^50 (222,223)
in such a manner that the substrate difference spectra of
P-^50-steroid and P-450-DDT are similar (Type I).
Furthermore, the estimate of estrogenic potency found
(Chapter 3» Section II.C.) for intermediate dosages of
o,p'-DDT on the basis of corticesterone secretion agrees
with that found using a totally different assay of estrogen
potency, the rat uterine-glycogen bioassay (14?).
Still, there is the element of the time lag between
injection and the observation of an effect which must be
explained before an adequate hypothesis based on estro-
genicity is possible. The potential for the sesame oil
vehicle to act as a depot has already been discussed in
214
-------
regard to the steroids? the same arguments apply to
o,p'-DDT. Another possible mechanism of estradiol and
o,p'-DDT action would be via a peripheral imprinting of
the adrenal cortex analogous to those encountered in the
gonads and/or the liver. Finally, the adrenal precosity
found in animals neonatally injected with estradiol or .
o,p'-DDT may be an indirect effect of hepatic microsomal
induction. In this instance an early compensatory hyper-
trophy of the neonatal adrenal cortex would take place to
maintain serum corticosteroid levels and would not have
fully regressed by 25 days of age. This explanation is,
however, unsatisfactory on two basesi 1) estradiol in
adult rats inhibits microsomal metabolism and induction
(5)i and 2) the serum levels of corticosterone are higher
than normal. Still, unless the levels of free corticos-
terone in serum are measured rather than total corticos-
terone (which includes corticosterone bound to corticos-
teroid-binding-globulin (CBG)) the actual effective level
of the hormone in serum is unknown and the second argument
is weakened.
Obviously, a great deal of research remains to be
done to clarify the meaning and significance of the
observed early development of the adrenal cortex in
treated animals. If the present studies were to be carried
forward a repeat of the first 20-30 days of the direct
injection study using several doses of o,p'-DDT dissolved
in DMSO or ethanol and similar doses dissolved in sesame
215
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oil could be used to verify the initial observations while
eliminating the question of long-term, depot-injection
effects. Such studies would also provide tissues for some
of the following measurements*
1. Measurements of cholesterol conversion into gluco-
corticoids in quartered adrenals in the presence or
absence of ACTH and/or estradiol or o,p'-DDT would deter-
mine the level of functionality of the adrenal at the time
the altered histological appearance was in evidence and
would also test for the altered adrenal's sensitivity to
ACTH, estradiol and o,p'-DDT.
2. Measurements of serum corticosterone and CBG would
establish the amount of free serum corticosterone and could
be used to help differentiate direct effects on the adrenal
from those mediated by the liver.
3. Measurements of hepatic microsomal breakdown of
corticosteroids would also differentiate direct effects on
the adrenal from those mediated by the liver.
^. Measurements of serum ACTH would help to deter-
mine whether the current observation is due to the con-
tinued presence of estradiol or o,p'-DDT or to an imprinted
change in pituitary output of ACTH or hypothalamic produc-
tion and release of CRH.
Other studies involving surgical manipulations, e.g.,
hypophysectomy, of the. dosed animals are, of course,
possible. However, their interpretation would probably be
less precise than the more direct measurements listed above.
-------
11• The Change in the Hypothalamic-Hypophysial Complex»
Because LH (and FSH) are involved in controlling
steroidogenesis and spermatogenesis the observed changes
in the hypothalamic-pituitary axis probably have the
potential for a more direct effect on reproductive
capacity and/or development than those described for the
adrenal. However, the apparent normality of the develop-
ment of the DDT-treated animals argues against such an
effect. On the other hand, it is doubtful whether any
such change would manifest itself in altered reproductive
performance in laboratory rats for whom the stresses of
obtaining and maintaining a mate are in most situations
considerably less than in animals in the wild state.
Therefore, situations in which a somewhat abnormal LH
response capacity might play a role in depressing repro- .
ductive success such as courtship or repulsion of rivals
may not occur in tests of reproductive capacity with
such laboratory rats.
The subjective appearanc'e of at least transient oligo-
spermia in DDT-injected males could result directly in
reduced reproductive success. The observations of Krause
et al. (208) might be used at this point to support the
argument for oligospermia and reduced fecundity, however,
the quantities of DDT which were administered in
that study could well have caused hepatic induction and
testosterone breakdown long after their injection. For
that reason that study is potentially misleading and
217
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cannot be used as clear support for an argument favoring a
hypothalamic or pituitary alteration resulting in impaired
spermatogenesis in DDT-treated rats.
Even if the observed changes in LH response capacity
does not have any effect on reproductive, success it is
still of interest from the viewpoint of understanding the
mechanisms controlling LH synthesis and release, i.e.,
endocrine control. To my knowledge it is the first
observation in the male rat of long term effects caused
by a chemical agent on hypothalamic-pituitary responses to
adult castration or LHRH injection. Therefore, it leads
to some interesting speculations concerning the mechanism
of the response and provides a base for future experimenta-
tion. The apparent differences in the directions of the
two effects (LHRH-response and adult castration response)
may be based on preservation of the cyclic center of the
hypothalamus in DDT-treated male rats but not in the
controls (Chapter 1, .Section II). Such a preservation
might occur by occupation of the center's estrogen recep-
tors by the very weak estrogen, o,p'-DDT, during the
critical phase of hypothalamic development. After the
critical imprinting period had passed and the dosage had
been discontinued the o,p'-DDT (or the actual active
metabolite) would be gradually eliminated by catabolism.
Since endogenous testosterone would thus be unable to act
neonatally, the cyclic center would remain intact. Adult
castration, which removes the source of negative feedback
218
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onto the tonic center (57-61), would not reveal the
presence of such an intact cyclic center. But large doses
of exogenous LHRH, LH, testosterone or, possibly, estro-
gen might stimulate an LH surge of the type seen in the
cycling female rat near ovulation. These agents could
cause the appearance of an LH response superimposed on an
LH surge on the second day of LHRH injection in DDT rats
which would not be possible in control rats. This would be
the case because the endogenous source(s) of LHRH, which
would be necessary for replenishment of pituitary LH stores
after the first injection with LHRH, would be suppressed by
the testosterone produced as a result of the first LHRH
injection in the control but not in the DDT-treated rats.
This mechanism would not explain the lower plateau of the
DDT-treated rats after the adult castration unless some
change in the tonic center had also occurred during its
period of exposure to o,p'-DDT. Still, simultaneous
changes in both cyclic and tonic centers have been
described for neonatal treatment with1androgens and estro-
gens (6l) and thus would not be implausible for o,p'-DDT.
A second more inclusive explanation would involve
either a continued presence of o,p'-DDT which could bind
to hypothalamic steroid (estrogen?) receptors or an
imprinting which caused a modification in the number or
affinity of the testosterone (estrogen?) sensitive recep-
tors within the brain. Either of these conditions would
bring about a state in which a fraction of the total
-------
steroid receptors were, in effect, inactive or occupied by
a weak steroid analog. This fraction would be unavailable
to act in totally suppressing LHRH release but would con-
stantly function at a low level to suppress unstressed LH
levels. This situation would result in an inability of
testosterone, generated perhaps by a burst of LH released
in response to a single LHRH injection, to fully suppress
LHRH release and therefore to prevent stimulation of the
replenishment of releasable LH within the pituitary. It
would also result in an effectively slower fall of serum
testosterone relative to receptor concentration after
castration in DDT-treated as opposed to control animals.
This, in turn, would cause a slower rise in serum LH
following castration. Thus, altered numbers of steroid
receptors could explain both the lower serum LH plateau
after adult castration in the DDT-treated rat and the lack
of a difference between serum LH levels in treated and
untreated animals which were neonatally castrated and
examined as adults (Chapter ^, Section III).
Again, as was the case with the adrenal, additional
postulates for mechanisms cannot be excluded. Only
further studies will fully illuminate the picture. The
initial observations may help guide research on the neo-r
natally dosed male rat by eliminating several approaches
from further consideration and by indicating several
research directions which may be productive '.
220
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1. Measurements of the adult castration response for
longer periods of time after castration should determine if
a final equilibrium state similar to that of controls does
exist.
2. Measurements of LHRH response should be repeated
using more than two injections of LHRH to further define
the magnitude of the response. Combined injections of
LHRH and testosterone or o,p'-DDT might also give clues
as to the functional state of the tonic hypothaiamic
center.
3. Measurements of pituitary release of LH and FSH
in vitro in response to LHRH, testosterone and o,p'-DDT
should be done to provide data on the comparative sensi-
tivities of the pituitaries from DDT-treated and untreated
animals. Such studies could also measure sensitivity to
corticosterone and determine basal ACTH secretion.
4. Histochemical or RIA measurements of brain LHRH
levels could also be done and would probably reveal much
concerning the relative sensitivities-of the brains from
DDT-treated and untreated animals to stimuli such as
exogenous LHRH, testosterone, o,p'-DDT, ether, light, etc.
5. Further studies involving testicular sensitivity
to LH or hCG could also be conducted. They must, however,
utilize cholesterol as substrate (22k ). An initial
measurement using tritiated acetate of very high specific
activity, conducted during these studies, failed to demon-
strate any incorporation of label into the isolated
221
-------
testosterone in either the control tissue, as could have
been predicted (224), or the tissue from rats neonatally
injected with 10 mg of o,p'-DDT. This result could only
imply that the large cholesterol pool (or whatever other
mechanism acts to prevent rats from incorporating labelled
acetate into testosterone) had not been disturbed suf-
ficiently in DDT-treated rats to allow incorporation of
label into testosterone even in the presence of 10 ug/ml
hCG.
6. Studies of specific steroidogenic pathways in
testes taken from DDT-treated and control groups could be
studied. This approach formed the basis for a few
in vitro experiments during this study which will be
discussed in Chapter 6. Briefly stated, they have shown
no conclusive effect of neonatal treatment with o,p'-DDT
on adult testicular steroid production. In view of the
known ability of o,p'-DDT to bind to receptors and the
P-^50 forms found in the adrenal (223) more data should
be collected to test for direct interference of o,p'-DDT
(or its metabolites) with steroidogenesis.
If the male rat continues to be an object of study,
other types of measurements, including assays for serum
androgens, FSH and prolactin, could well be used to moni-
tor hormonal status in much the same manner as has been
attempted in this research.
222
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III. Summary;
Further investigation of the parameters studied or
proposed in this project may well allow more rapid toxico-
logical screening of materials for effects on the functional
status of the steroidogenic endocrine systems. This should
also provide support for, if not an alternative means to,
predictions of effects on reproductive capacity and viability.
Use of the neonatally treated animal is to be recommended
when possible because its susceptibility to permanent damage
is apparent in these and other studies (53,54,57-61,150,151).
The studies demonstrate the type of data which may be
generated in a broad investigation. As a consequence of em-
phasizing scope and of the time consumed in examining it,
depth of study of the positive results has been sacrificed.
The projected experiments were designed to measure the systems
and tissues which have been demonstrated in this study to be
effected by neonatal treatment with o,p'-DDT in more detail.
The whole-animal experiments described demonstrate that
neonatal treatment with o,p'-DDT does effect the steroido-
genic system of the male rat. It causes change in
223
-------
at least two loci, the immature adrenal cortex and the
adult hypothalamo-pituitary axis; such treatment does
not, however, grossly affect development. At least part
of the effects of the treatment may be due to a metabolite
of o,p'-DDT rather than the parent compound. And, the
parent compound and at least some of its metabolites are
effectively transferred to suckling pups who are, in turn,
affected by such naturally administered material.
Though these results seem modest and many other
experiments building on them are possible, let it suffice
that these experiments serve as a basis for more specifically
focused studies which may yield mechanistic information on
the mode of o,p'-DDT action, more insight into the control
and development of the steroidogenic endocrines and, lastly,
more specific rapid and simple testing procedures which
may be of use in clinical and environmental monitoring
situations.
224
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CHAPTER 6
INVESTIGATION OF A^-PREGNENOLONE CONVERSION IN VITRO
The original conception of this project
involved an examination of the mutual interactions of the
metabolism of o,p'-DDT and the metabolism of steroids
within the testes of animals which had been treated with
o,p'-DDT or a vehicle. Since o,p'-DDT had been shown to
act as an estrogen (22,24,53,146-148) and to bind to the
hepatic P-450 involved in steroid hydroxylations (5»12-15,
I8,128,129)i and since the closely related metabolite,
o,p'-DDD, was known to interfere with steroidogenesis in
the adrenal (37,40,42-47), it appeared reasonable to study
the interactive metabolisms of DDT and steroids within the
testes. That is, it seemed reasonable to attempt to
measure the effects of o,p'-DDT and the metabolites which
it formed in vitro on the metabolism of A^-pregnenolone
to androgens in vitro and to concurrently measure the
effects of A^-pregnenolone and the metabolites, e.g.,
androgens, which it formed in vitro on the metabolism of
o,p'-DDT to its metabolites in vitro. To that end incuba-
tion procedures, extraction protocols and metabolite
assay methods were designed to allow measurements to be
done on both sets of metabolites when they were formed in
a single incubation mixture. The attractiveness-of this
combined approach lies in its efficiency in terms of the
time, materials and tissues used to generate a set of
225
-------
results which reflect the multiple forms of interaction
which might occur in vivo. The procedures used in these
studies were developed and improved throughout much of the
study; some of them still need further improve-
ments (present versions were described in Chapter 2, Sec-
tion IV. F.I).
The examination of the metabolisms of two rather
different sets of compounds required that some adjustments
of the incubation conditions commonly used with each be
made to accomodate incubations of both simultaneously.
The discrepant time courses for metabolic incubations pre-
viously done on the steroids (192,225-22?) and DDT analogs
(104,113,133) indicated the necessity of measuring steroid
and DDT metabolites in aliquots of incubations taken over
the course of a reasonable time period (l hour). Because
the potential for DDT interactions with the mitochondrial
P-^50 was unknown, this subcellular fraction was not dis-
carded before incubations were conducted although its
presence for conversions ofA^-pregnenolone to androgens
was unnecessary (Chapter 1, Section I) (6). The
similarity of cofactor requirements for steroid conver-
sions in the testis (6) and for DDT conversions in the
liver (113)f along with the length of the incubation time,
dictated the addition of reduced pyridine nucleotides or
systems capable of generating them. Finally, the protein
concentrations used appeared to be an adequate compromise
226
-------
between those used previously in steroidal studies (192,
226) and in DDT catabolism studies (10^).
The possibility that o,p'-DDT might act at any of
several metabolic steps on the pathways from A^-pregnenol-
one to testosterone brought up the question of the value
of measuring intermediates. Since time course data was
already to be generated it was decided that several of the
known intermediates (progesterone, 170C-hydroxyprogester-
one, dehydroepiandrosterone and A -androstenedione, i.e.,
products ^ or D, 5 or E, 1 or A and 2 or B in the
computer program printout of Appendix I) should be examined
at each time point in addition to the substrate and
end-product in order to provide a good picture of the
metabolic flux from the substrate, A-^-pregnenolone (sub-
strate and product 3 or C in the computer printout of
Appendix I), to the end-product, testosterone (product 2
or B in the computer printout of Appendix I). Since the
metabolism of o,p'-DDT in the testis was unknown the
potential production of fairly polar metabolites could not
be excluded. The procedures adopted for extraction, how-
ever, limited examination of the metabolites of DDT to
those which were neutral. Because of this limitation on
the number of DDT metabolites which could be examined,
because of technical limitations on time available for the
entire proposed analysis and because alterations of
steroid metabolism due to treatment with DDT were more
critical to the focus of the project on altered
227
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reproductive capacity, emphasis was placed on examining
steroidal flux within the incubation systems given in
Table 3 (Chapter 2, Section IV.F.I).
Steroid flux of this type appeared to be best studied
by using radioactive substrate (195,228,229). Use of a
second label added after stopping active metabolism could
provide added information by accounting for losses which
occurred during extraction and purification. Similar
information, or information additional to it, could also
be generated by using a sensitive mass detection system.
Since gas chromatography had already been used extensively
for measuring steroids (195,228,229), and because it was a
method of choice for measuring DDT metabolites, it was
chosen as the mass determination system. The GC was also
capable of separating a number of steroids simultaneously
and, thus, could serve as a separation, as well as a
quantitation instrument. Therefore, it seemed reasonable
that if radioactivity could be successfully monitored for
individual chromatographic peaks it should be possible to
generate a series of doubly labelled intermediates and
analyze them simultaneously by combined GC-double label
scintillation techniques. Since the main object of the
overall studies was to analyze the differences between
DDT-treated and untreated animals some decreased precision
in separations and peak identifications was allowable if
samples from incubations of tissues from both types of
animals were analyzed similarly.
228""
-------
With this experimental conception in mind a model for
the analysis of a single compartment metabolic system
emerged.
S.
In this model Y is the endogenous precursor for formation
of substrate S, and subsequently for the formation of P
and Z from S. During the incubation, Y produces amounts
of S and P (SY and Py) which are in addition to the
amounts of endogenous S and P (SE and PQ) which exist prior
to the incubation. SE also forms an amount of product,
!>„, during incubation as does Z, any alternate source of
Ci
product, P7. If tritiated substrate S, is added prior to
L %
incubation it also forms product, P- , during the incuba-
?•
tion. If a sample of an incubation is stepped at time
zero and amounts of carbon-1^ substrate and product, Sn
\j
and Pn, are added, the total concentration of substrate is
u
due to S-, S0 and Sn (two of which are known) (Equation
E 3H C
III, Appendix I). Similarly, the product contained in the
sample would be made up of PQ and PC (one of which is
known) (Equation IV, Appendix I). Because all but one
portion of the mass is known a priori a zero time point
should allow determination of the mass of endogenous
"229"
-------
substrates (SE) (Equation X, Appendix I) and endogenous
products, PQ (Equation XI, Appendix I). At times greater
than zero substrate is made up of Sy, Sp, S^ and S^,
(Equation V, Appendix I) while product is made up of PY»
P0 , Pn, P7 and Pn (Equation VI, Appendix I). In both
JJjT U £l O
cases if mass is corrected for any nonenzymatic breakdown
by measuring an incubated system containing only S« ,
%
which results in substrate and product masses of S^ +
•*H
SG(= SNE) (Equation I, Appendix I) and PC (=PNE) (Equa-
tion II, Appendix I), the conversion from Y to Sy and Py
(Equation XV, Appendix I) and from S and S™ to P,, and
3H ^ JH
Pp (Equation 2, Appendix I) can be computed. This is done
by recalculating the specific activity of the tritiated
substrate to include the initial mass of the cold endogen-
ous substrate SE at time zero or the entire measured mass
of the substrate other than S- .at times greater than zero.
Chromatographic data, Ao and Ap, or other .measurements
of total mass can be combined with the levels of radio-
activity (Cl, 02, C3, Cl', C21, C3') to generate values
for mass of the various subsets of total mass of substrate
or product. By measuring mass and radioactivity at time
zero SE and PQ are determined as stated above. Radio-
activity based on the initial specific activity of S
3H
allows computation of the quantities of S_ and P_ at any
JH • ^H
time beyond zero (Equation 2, Appendix I). If the specific
activity of S is altered to include S,, then P^ can also
3H E E
be determined (Equation 3, Appendix I). If the specific
230
-------
activity is adjusted to include SY as well, PY is also
determined (Equation 4, Appendix I) and by default, PZ is
generated (Equation XVI, Appendix I). Total net conver-
sions for all forms of substrate or for all forms of
product could be measured at any time of incubation by
correcting the measured masses for added SG, (Equation XII,
Appendix I) or P^, (Equation XIII, Appendix I).
The computer program METFLX, presented in Appendix I,
was generated from this model and was subsequently updated
to conform to empirical observations. The program pre-
supposes nothing and generates negative concentration
values for a product if conversions to later products
exceed its formation from endogenous product and all forms
of substrate. It also reflects analytical error in the
radiochemical assays or mass determinations, especially if
too large an amount of cold carrier (added to increase
recovery) is used.
The program uses recovery corrections made possible by
the added C-steroids which were used as internal recovery
standards. It bases chromatographic determination of mass
on the detector response of known products relative to a
chromatographic internal standard and on calibration plots
of Iog10 (relative response) versus Iog10 (concen"tra'tion)
which were generated from mixtures of all of the compounds
of interest and the internal standard. The use of relative
peak areas minimizes errors due to misinjection of samples
or to slight changes in the sensitivity of the mass
231
-------
detector from injection to injection. It corrects
results for individual chromatographic peaks for varia-
tions in sample volume injected, in sample size, in
efficiency of capillary tube capture, in differences in
mass flow to the detector and capture tube, in differences
between internal standard concentration in the samples and
calibration mixtures, etc. Finally, it bases radioactivity
determinations on the specific activity of the added sub-
O T ii,
strate and the computed •'H-dpm and C-dpm for captured
effluent.
Proof of this program was delayed due to problems
involving identification of appropriate GC conditions
which would optimize steroid separations and give the
cleanest results for the proposed approach. Over thirty
single and combined column phases were examined for their
ability to separate the steroids which were chosen for
study. Parent compounds, silyl-methyl-oxime derivatives,
silyl-butyl-oxime derivatives, silyl-benzyl-oxime deriva-
tives, acetate derivatives and benzoate derivatives were
examined on one or several of the phases studied. Choice
of cholesterol-3-pfopyl ether as the chromatographic
internal standard was made after examining its chromato-
graphic properties with respect to those of both the
steroids of interest and 5<£-androstane, 5 cC-pregnane,
estradiol, estriol and cholesterol. The standard had the
desirable characteristics of being stable, fairly similar
in structure to the compounds of interest and of
232
-------
chromatographing in a fashion quite similar to the
steroids of interest. A series of modifications of the
effluent capture procedure led to the final choice of a
simple chilled capillary capture tube. The choice of the
GC column conditions and steroid form were a compromise
of obtaining workable separations and capture efficiencies
without requiring extensive preliminary treatment of the
samples to be measured - the parent compounds were
separated on a 350 cm x 2 mm column containing Gas Chrom
Q coated with 1.5% each of SE-5^ and OV-7 by using a
temperature gradient of l°/minute from 175° to 275°C and
a flow rate of 25 ml/minute for the carrier gas, nitrogen
(Chapter 2, Section IV.F.2). Separations were done using
relatively crude extracts containing amounts of carrier
steroid, sufficient to yield good recoveries of radio-
active steroids throughout isolation and analysis.
By the time these technical matters had been resolved,
the whole animal studies were well under wayi The gen-
erality of the program which had been- written made it
quite desirable to test it on experimental results even if
part of the initial strong impetus to look at direct
interactions of DDT and steroids within the testes had
somewhat waned. Since the testicular steroid conversions
in DDT-treated and untreated animals could still fit into
the scope of the overall project and still might generate
some insight into either any imprinted changes in steroid
metabolism within treated testes or any changes in
233
-------
steroid-DDT interactions within treated tissues, it was
decided to test the program and do the originally pro-
posed measurements on the testes from DDT-treated. and
control animals.
Testes derived from rats exposed neonatally not only
to DDT, but also to EV and TP were incubated under the
protocol given in Table 3. The incubation mixture
aliquots were extracted and stored. Extracts from incu-
bations Type 2 ( A-^-pregnenolone only) and Type 5 (non-
enzymatic incubation) were measured under the conditions
listed in Figures 11 and 12 (Chapter 2, Section IV.P.I and
2). Counting results were normalized to the values which
would have been expected for 1 ul injection volumes to
allow more rapid pre-program comparisons. Relative peak
areas were computed both by means of the fourth revision of
an automatic integration program which linked the GC to an
electronic calculator and by manual triangulation. Exam-
ination of the raw data indicated adequate capture of
o -I Ji
JE- and C-counts (minimally 50-100 dpm/ul above chromato-
graphic background counts for each radioactive label) and
good agreement between automatic and manual integration for
relative areas for all but the l?«c-hydroxyprogesterone
peak, which only manual integration measured. Plots of
o i h
the JE/ C ratio (not shown) for each of the steroid
peaks in the chromatograms from the testicular incubations
of control animals (neonatally injected with sesame oil)
indicated the predicted pattern of -%-labelling.
"234
-------
Tritiated substrate declined to essentially zero levels by
30 minutes of incubation. The products of both the A^-
and A -pathways, testosterone and A -androstenedione,
rose beyond 5-10 minutes of incubation until essentially
3
all the ^H-label was incorporated into them by 30-60
minutes. Progesterone and 17oC -hydroxyprogesterone rose
to a peak by 10 minutes of incubation and declined to base-
line by 30 minutes. Dehydroepiandrosterone (or the label-
led compound associated with that chromat©graphic peak -
Chapter 2, Section IV.F.2) rose slowly after 10-30 minutes
of incubation to a level of about 1/5 that seen for
testosterone plus A -androstenedione. (This agreement of
the radioactivity pattern for dehydroepiandrosterone with
the predicted pattern tends to support the identity of the
label in this chromatographic effluent with the carrier
steroid.) Analogous plots for testicular incubations done
on tissues from rats neonatally injected with EV (200 ug
total), TP (1000 ug total) or o,p'-DDT (500 ug total)
indicated somewhat similar profiles. <
METFLX was run using the results from the incubation
of all the treatment groups. The resultant data are pre-
sented in Appendix I, as an example of the program
printout.
The plots shown at the end of the printout illustrate
o
conversion of the ^H-label and demonstrate patterns
o 1 k
similar to the plots obtained from ^H-/ C- ratios, as
expected. It can be seen in the second plot (Treatment II)
235
-------
that steroid metabolism in EV-treated animals appears more
rapid than in the controls,- this may, however, "be due to
a larger number of Leydig cells per gram of testicular
weight than in the control (62) as may also be the case
in the third plot (Treatment III, TP treatment). Both
sets of results, however, agree with the findings of
Joseph and Kincl (230) in similarly treated rats that
metabolic capability is preserved in the testis of such
animals even though spermatogenesis is largely halted.
The fourth plot (Treatment IV) which describes the situa-
tion for DDT-treated animals appears different in that the
metabolic conversions appear much slower. This may,
however, be an artifact due to inadequate aeration of the
incubation since a larger incubation volume was used for
this tissues it was incubated somewhat earlier than the
steroid and control tissues. Still, it may also indicate
a reduced rate of steroid synthesis in treated animals
and with the results from the whole animal studies imply
an overall sluggishness of response within the hypo-
thalamo-pituitary-testicular axis of DDT-treated rats.
Other data given in the program printout show masses
which reflect the amount of carrier steroids (about 300
nmoles each) which had been added during the isolation
procedure. It is obvious that this mass is too large to
allow good estimation of many of the parameters the program
was designed to calculate. An attempt to measure the
steroids in this system by using a very large incubation
236
-------
"but no carrier steroid demonstrated that GC-measurement
using the FID detection system was possible, "but that the
extract would have to be more rigorously purified prior to
injection onto the column in order to allow the accurate
manual capture of the desired effluents.
The approach at this stage appears usable if a few
modifications are made; the program seems to function
very well, generating accurate results for the desired
parameters and being limited only by the precision of the
values on which it must act. Modifications of this
approach which could make it quite useful in the area of
steroid metabolism and/or comparative steroid metabolism
(as here) would be: 1) to move to the use of electron
capture detection of appropriate derivatives to allow
precise measurements of very small amounts of steroidsj
2) to use minimal amounts of carrier, e.g. 1-10 ng per
steroid, to decrease artifactual change due to deviations
in carrier concentration, 3) to interpose TLC, lipophilic
Sephadex chromatography or some other'purification and/or
steroid-group separation between extraction and GC
analysis to assist in decreasing both chemical and radio-
activity background, and *0 to use capillaries filled with
terphenyl crystals to optimize capture efficiencies so
1 it T
that smaller amounts of C-carrier and/or -'H- could be
accurately detected in the GC effluents (195,228,229).
Potential major modifications of the methodology used in
the present study have evolved elsewhere during the course
-------
of these studies so that now it may be feasible to use
GC-mass spectral equipment in combination with Fourier
transform techniques to measure mass and isotope ratios
simultaneously. Or, RIA might be used for determination
of the masses of most of the steroids involved.
The attractiveness of using an approach such as the
one used in these studies is that when fully developed it
can yield data not only on conversion of labelled sub-
strate but also on conversion of other forms of substrate
and on sizes of metabolic pools. Presently, such data are
generated by doing a series of several experiments, many
times a separate set for each individual product or inter-
mediate. Obviously, for such a limited approach to be
useful in clinical or environmental monitoring the tests
must be quite rapid. The approach which was proposed and
tested in these studies would be perhaps more rapid by
generating all the data for all the compounds of interest
in a somewhat longer single procedure.
The attractiveness of the program lies in its gen-
erality. It may be used to generate solely mass or radio-
activity data or a combination of both? it may be used to
calculate data from any combination of four incubations -
2 test groups and 2 controls, k different buffers, etc.5
and it may be used to compute metabolic flux through a
single compartment system for any of a large variety of
compounds, e.g., amino acids, sugars, drugs, etc. In drug
studies as an example, it can even incorporate measurements
238"
-------
of chemically unidentified intermediates as long as some
sort of standard curve can be generated from which masses
(or absorbance units, etc.) can be computed.
Measurements on the steroids and DDT metabolites from
incubation Types 1, 3 and ^ (Table 3) can still be run on
the samples which were collected during this investigation
though their results must be viewed as preliminary until
they can be repeated using one or more of the suggested
modifications in the isolation, purification and measure-
ment protocols. Measurements of other types of incubation,
•*
e.g., incorporating hCG and -'H-cholesterol in DDT-treated
and control testicular incubations could well extend the
results and spirit of the present investigation.
Contributions of these methodological studies were
twofold: 1) a method of measuring doubly-labelled
steroids in GC effluents, which was proposed earlier as a
desirable objective by Eik-Nes (195) for samples containing
single compounds, and 2) a FORTRAN program, METFLX, which
can analyze results of the type generated in this study
but can also be used in other studies of rather varied
forms.
239
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260
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APPENDIX I
THE PROGRAM METFLX
The program METFLX was designed to reduce data gener-
ated in a comparative metabolism problem. Its purpose is
to work data into a form usable in drawing correlations and
conclusions concerning the metabolic flux of a substrate
through a tissue when incubated in vitro under a variety of
conditions. Specifically, it was written to evaluate a
3 1 k
combination of •'H- C count data and gas chroma to graphic
results which were both to be generated during the course
of experiments on androgen production in rat testes under
the influence of DDT.
The problem was one of taking recovery data, gener-
ated by a C internal standard, and combining it with
total mass data, generated by gas chromatography, and
•a
conversion data, generated by •'H counts derived from
labelled substrate, and converting these to: 1) the values
which actually occurred within the incubation in terms of
total steroid present at given times; 2) the contributions
of the % substrate, the endogenous substrate and all other
substrate sources to the total amount of-a particular
steroid present; 3) "the amount of endogenous steroid
present before the start of the experiment; 4) the net
converions to and from the substrate and products; and
5) the amounts of a particular steroid which are formed
from unlabelled substrates, including the one added,
261
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precursors of the one added and all others which generate
the product by alternate pathways. Graphs of net conver-
sions at given sampling times and under four different
test conditions were plotted to allow graphic comparisons
of flux and kinetics.
The calculations for the program were based on those
generally accepted for either gas chromatographic or
double-label quantitation experiments (195,196,229,231).
They were modified to accommodate the simultaneous use of
both methods in order to measure flux within a system
which contained labelled and unlabelled substrate,
endogenous substrate, endogenous substrate precursors,
endogenous product, alternate product precursors and an
amount of recovery (internal) standard containing a second
label.
The flux model, described in Chapter 5» and some
initial empirical observations dictated the following
equations which yielded the desired pieces of data.
Equation 1) calculates the nanomoles of products or sub-
strate found per milligram of protein at any given time
under any given condition. This equation was calculated
by the function XMOLE.
1) nmoles/mg protein = (G1/C3) x (10 Exp((Log1Q(Al)-SL-
AP)/B)) x (VF/VI) x FF x CE x V x 0.001 /GP
-\L
Cl = number of C counts added
-iL
C3 = number of C counts recovered
262
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Al = chromatographic area of the compound of interest
relative to that of the internal standard (chromato-
graphic) in the same injection of sample
SL = the Log1Q of the ratio of the concentration of the
internal standard (chromatographic) in the calibra-
tion- samples to the concentration of the internal
standard in the sample
AP = the intercept of the calibration plot of I*og10
(relative chromatographic area) versus Log,0 (com-
pound concentration)
VF = final total volume of the sample
VI = initial total volume of the sample
FF = fraction of total gas flow passing through the cap-
ture capillary
CE = efficiency of capillary capture for the compound of
interest
V = volume injected into the chromatograph in ul
GP = concentration of protein in the sample incubation in
mg/ml
This equation was used to generate the following
additional equations and data:
Equation Data
I nmoles/mg-protein of substrate in the
nonenzymatic incubation
II nmoles/mg-protein of product in the
nonenzymatic incubation
263
-------
Equation Data
III nmoles/mg-protein of substrate at time 0 in
a test incubation
IV nmoles/mg-protein of product at time 0 in a
test incubation
V nmoles/mg-protein of substrate at times
greater than 0 in a test incubation
VI nmoles/mg-protein of product at times
greater than 0 in a test incubation
Equation 2) calculated the tritiated product formed during
a test incubation. This equation was calculated by func-
tion H3 and was referred to as Equation 1 in the output.
2) nmoles/mg protein = ((C2 x (C1/C3))/SO)/GP
C2 = number of tritium counts recovered
SO = the initial specific activity of the added tritiated
substrate
Equation 3) calculated the product formed from tritiated
and initially present, endogenous substrate during a test
incubation. This equation was calculated by function H3E
and was referred to as Equation 2 in the output.
3) nmoles/mg protein = ((02 x Cl)/C3)/(CO/((CO/SO) +
(III x GP) - (I))/GP
•a
CO = initial number of -'H counts added
Equation 4) calculated product formed from all forms of
substrate during a test incubation. This equation was cal-
culated by the function ALL and was referred to as Equation
3 in the output.
26 4 ~
-------
*0 nmoles/mg protein = ((02 x 01)/C3/(CO/((CO/SO) +
(V x GP) - (I))/GP
Finally, from the above equations the following equations
and data were obtainedt
Equation Data
X =? III-I nmoles/mg-protein of endogenous substrate
XI = IV-II nmoles/mg-protein of endogenous product
XII = III-V the net number of nmoles of substrate
converted
XIII = VI-IV the net number of nmoles of product
formed
XIV = VIII-VII nmoles/mg-protein of product formed from
endogenous substrate
XV = IX-VIII nmoles/mg-protein of product formed from
substrate precursors
XVI = VI-IV-IX nmoles/mg-protein of product formed from
sources other than the substrate under
study.
Negative values could be obtained from the above
calculations. Such values conveyed the information that
endogenous product or alternative product precursors had
been converted to subsequent products in amounts exceeding
the amount of product produced from the specific substrate
under study.
265
-------
Mnemonics for use in METFLX are summarized in Table
10, while the logical sequence of program steps is out-
lined in the flowchart given in Figure 3^.
A printout of the program and its output were gener-
ated for exemplary purposes from results collected in a
series of control incubations. These incubations were
conducted under the conditions described for incubations
type 2 and 5 in Table 3 (Chapter 2, Section IV.F.l),i.e.,
they were incubations of [7 n- HJ- A^-pregnenolone in the
presence or absence of the 500 x g supernatants at 6:1
testicular homogenates from adult rats. The homogenates
were derived from ^ groups of rats neonatally injected
with 5 consecutive daily s.c. injections on days 1-5 of
age. The injections were each of 0.05 ml of sesame oil
containing: nothing (treatment 1), ^0 ug EV (treatment
2), 200 ug TP (treatment 3) or 500 ug o,p'-DDT (treatment
*0. After the incubations the steroids were extracted
under the protocol listed in Figure 11 (Chapter 2, Section
IV.F.l) and were analyzed under the conditions given in
Figure 12. The steroids were numbered in order of elution
from the GC column: 1 = dehydroepiandrosterone, 2 =
A -androstenedione and testosterone, 3 = A^-pregnenolone,
4 •= progesterone and 5 = I?** -hydroxyprogesterone. All
the steroids were computed as products in the present
program run in order to allow a check on the rate of sub-
strate (product 3) disappearance with time of incubation
as calculated by function H3.
266
-------
Table 10
Mnemonics of METFLX
All Data and calculations were stored in arrays
according to incubation type and time point (0, 5, 10, 30
and 60 minutes). Product data and calculations had the
added dimension of product number.
Symbol Definition
ESDATA Array in which substrate data from the non-
enzymatic incubation were stored
ESUB Vector in which the calculated nmoles/mg-protein
of substrate from the nonenzymatic incubation
were stored
SDATA Array in which the substrate data from the
enzymatic incubation were stored
SUB Array in which the calculated nmoles/mg-protein
of substrate in the enzymatic incubation were
stored
BP The slope of the standard - calibration - curve
of Log1Q(Al) versus Log ,Q(concentration)
AP The intercept of the standard - calibration -
curve of Log,Q(Al) versus Log,Q(concentration)
PDATA Array in which the product data from the non-
enzymatic incubation were stored
EPRO Array in which the calculated nmoles/mg-protein
of product in the nonenzymatic incubation were
stored
PRO Array in which the data for the product from the
enzymatic incubation were stored
PROD Array in which the calculated nmoles/mg-protein
of product in the enzymatic incubation were
stored
XPROD Array where the calculated values for H3 were
stored
YPROD Array where the calculated values for H3E were
stored
267
-------
Table 10 (Cont.)
Symbol
ZPROD
X to XVI
T
NSGALE
CHAR
POINT
GP
VI
CE
FF
SL
VF
N
B
SO
CO
Definition
Array where the calculated values for ALL were
stored
Arrays in which the calculated values for equa-
tions X to XVI were stored
Vector in which the sampling times were stored
Vector which held scaling information for use in
PLOT1
Vector which held the character labels for use
in PLOT 4
The value of the ordinate for points plotted by
PLOTl-iJ-
Vector which held the values of mg-protein per
ml for all the incubation conditions
Vector which held the values of the initial sample
volumes for all the incubation conditions
Vector which held the values of the capture
efficiency of capillary capture for each of the
chromatographic peaks examined
The value which defined the flow fraction of gas
which passed through the capillary capture tube
The .value which defined the Log10 of the ratio of
the concentration of the internal chromatographic
standard in the calibration' mixture over the
concentration of the internal chromatographic
standard in the measured sample
The value which defined the final total volume of
the sample
The value which defined the number of products
measured
BP for the substrate
Initial specific activity of the added tritiated
substrate in dpm/nmole
Initial number of tritium counts added in dpm
268
-------
Table 10 (Cqnt.)
Symbol Definition
XMOLE The function which calculated the total number
of nmoles/mg-protein in the sample
PROCAL The subroutine which calculated PROD, XPROD,
YPROD, ZPROD, XI, XIII, XIV, XV and XVI
Cl The initial number of carbon-1^ dpm added
C2 The number of tritium dpm recovered
C3 The number of carbon-1^ dpm recovered
Al The area of the chromatographic peak which cor-
responded to a given compound relative to the
area of the internal standard peak in the same
injection
V The volume of the sample injected for measure-
ment
YMIN The minimum value for the ordinate for a given
data plot, used in PLOT1-4
YMAX The maximum value for the ordinate of a given
data plot, used in PLOT1-4
PLOT1-A- A series of computer printer plot subroutines
located within the *LIBRARY of the MTS computer
system of The University of Michigan
H3 The function which calculated the conversion of
tritium dpm from substrate to product for only
tritiated substrate
H3E The function which calculated the conversion of
endogenous plus tritiated substrate to product
ALL The function which calculated -the formation of
product from all sources of substrate
ALOG10 The single precision estimate of the logarithm
to the base 10
SUBO SUB at time zero
269
-------
(start J
Read and Writes.
Calculation
Variablesi
CE(I), FF, SL,
VF, N, GP(I),
VI(I), B, SO,
CO, AP(3),
CE(6)
Read and Writes
Nonenzymatic
Substrate
Data
XMOLE
Store in ESUB(I)
Read and Write""'
Enzymatic
Substrate
Data
XMOLE
Store in SUB(I,J)1
Calculate Equa-
tions 5 X and
Store in X(I)
XII and Store in
XII(I,J)
(Do 200 M = l.N>
"^
Read and
BP(M) and AP(M)
Read and Write^
Nonenzymatic
Product Data_
( XMOLE J
Figure 3^
Flowchart for METFLX
Store in EPRO(M,ll
J_
Read and Write1^
Enzymatic
Product Data
VPROCAL J
yes
Writes Definitions
B, SO, CO, AP(3),
CE(6)
Nonenzymatic
Substrate Data =
BSDATA(I,J)
Enzymatic Substrate
Data = SDATAJI.J.K)
(Do 400'M - 1,N>"
*
Writes BP(M), AP(M),
CE(M),
Nonenzymatic
Product Data =
PDATA(M,I,J)
Enzymatic Product
Data = PROdVl.I.J.K)
yes
Writes Explanations
of Equations and
Incubations or
Treatments,
ESUB(I)
SUB(I,J)
X(I)
XII(IJ)
-------
Figure 3^ (Cont.)
Write: EPRO(M.I),
PROD(M,I,J),
XPROD(M,I,J),
YPROD(M,I,J),
ZPROD(M,I,J),
XIV(M,I,J),
XV(M,I,J),
yes
Determine YMIN
and YMAX
I
(Do 300 I =
(call Plotl)
/Call Plot^)
(Do 601 M = 1,N>
_
(Do 602 K = 1,5)
XPROD(M,I,K)
POINT(K)
( XMOLE J
ICalculate Function XMOLE)
CReturn)
PROGAL
f
J
XMOLE
Store in PROD(M,I.J)j
in XPROD(M,I,J)
[Store in YPROD(M,I,J)
[Store in ZPROD(M,I.J)
E
Calculate ?
XI and Store in XI(M,I)
XIII and Store in XIII(MfI,J)
XIV and Store in XIV(M,I,J)
XV and Store in XV(M,I,J)
XVI and Store in SVI(M,I)
(Return)
Calculate Equation H3
(Return)
Calculate~Equation H3E|
(Return)
Calculate~Equation ALL]
(Return)
271
-------
Chromatographic area data and radioactivity levels
C1 C-dpm and 3H dpm) were normalized to 1 ul of GC injec-
tion volume prior to the computer run in order to facili-
tate comparisons of the raw data. Values-for samples
were entered in order of incubation number, 1-^, and time
of incubation - 0, 5, 10, 30 or 60 minutes. These nor-
malized results were entered according to the format
shown in Table 11 along with the calculated values for
such items as the initial specific activity of the sub-
strate (SO), slopes and intercepts for the GC calibration
curves for the various steroid peaks (BP and AP), capil-
lary tube capture efficiencies for each of the steroid
chromatographic peaks (CE), etc.
The program output is given in 5 sections. First, a
program listing of the steps used in METFLX is given on
pages 277-282. Second, a printout echoing the values
entered on cards is given on pages 283-285; this serves
as a visual check on the correctness and order of the
values entered. Third, the same data is printed in organ-
ized form according to substrate and product number,
according to variable and according to the presence or
absence of tissue within the steroid incubation (pages
286-291). The order of values, proceeding from top to
bottom in each list, is by time of incubation sample and
treatment number, i.e., values for treatment 1, zero
minutes, treatment 1, five minutes, ... treatment 4, 60
minutes. Fourth, the data output of the computations is
272
-------
Table 11
Description of the Input Data Format for METFLX
There are 10 types of data cards which were entered
in the following orders
1. One card
Constants CE(1), CE(2) CE(6)
2. One card
Constants FP, SL, VF
3. One card
Constants N, GP(1), GP(2), GP(3), GP(^)
kr. One card
Constants VI(1), VI(2), VI(3) and VI(^)
5. One card
Constants B, SO, CO, AP(3), CE(6)
6. Five cards
Data cards for the substrate from the
enzymatic incubation
Each card contained ^ values« Cl, C3, Al and V
Cards were read according to increasing timei
0, 5. 10» 30, 60 minutes
7. Twenty cards
Data cards for the substrate from the enzymatic
incubations
Each card contained values for: Cl, C3, Al
and V
Each card represented one time point $ they were
read in the same order as in 6.
Each set of 5 cards represented one incubation;
there were a total of M- incubations
8. One card
Constants BP and AP
9. Five cards
Data cards for the product from the nonenzymatic
incubation
Each card contained ^ values5 Cl, C3, Al and V
Cards were read according to increasing timei
0, 51 10, 30 and 60 minutes
273
-------
Table n (Cont.)
10. Twenty cards
Data cards for the product from the
enzymatic incubation
Each card contained 5 values: 01, 02, 03,
Al and V
Each card represented one time points they
were read in the same order as in 9.
Each set of 5 cards represented one incuba-
tion; there were a total of ^ incubations
Formats 8., 9. and 10. were repeated as many times as
there were products.
274
-------
printed (pages 292 to 302). The substrate is listed first
followed by each of the products (product 3 = substrate).
The values for the various incubation conditions, times
and equations are printed in clearly labelled order.
o
Fifth, plots of the content of ^H-labelled substrate and
products versus time of incubation are given for each
animal treatment group. These allow easy comparison of
rates and levels of conversion .
The results generated demonstrate the need for
several modifications in the experimental procedures.
First, the total nanbmoles of substrate and products
closely resemble the amounts added as carrier. This
destroys the utility of the program for computing endogen-
ous levels of substrate and/or product or quantities
based on those levels. Therefore, large reductions, or
elimination of carrier is suggested. This might necessi-
tate the use of electron capture detection of appropriate
steroid derivatives but that possibility is presently
used in a number of laboratories (195»229). Second, the
need to use uniform sample sizes is illustrated by the
differences seen between treatment groups 1-3» which used
1 ml sampling aliquots and those in group 4 which used
5 ml sampling aliquots.
In the program printout the following numbers and letters
are used to identify specific steroid chromatographic
peaksi l=A=dehydroepiandrosterone, 2=B=A -androstenedi-
one + testosterone, 3s=C=A5_pregnenolone, ^-D=progester-
one, 5=E=17°C-hydroxyprogesterone.
27 5
-------
Modifications of the program are indeed possible.
Extensions of printout to include plots of all the equa-
tion results and inclusion of the protein concentrations
in each incubation could easily be made. Similarly, addi-
tional correction or dilution factors may be included as
well as extensions to different numbers of sampling times
or incubation or treatment conditions.
Finally, utility of the program is not limited to the
coupled GG-scintillation counting system described here.
i
Any mass determination which does not interfere with the
o n h,
simultaneous quantitation of -'H- and C- may just as
easily be used in the context of this program. Similarly,
the program is not limited to use with steroids but is
easily extended to numerous other metabolic problems. It
is hoped that the generality of its inception may serve to
make the program useful in many studies both in and out of
the area of steroid biochemistry.
276
-------
C METFLX CALCULATES THE METABOLIC FLUX OF COMPOUNDS IH A TISSUE
C INCUBATION BASED OH DATA GENERATED BY CC AND DOUBLE-LABEL SCINTILLA-
C TION COUNTING. INITIALLY APPLIED TO ANDROGEN METABOLISM.
DIMENSION ESDATA<5,5),ESUBI5),SDATA<4,5,5),SUB(4»5),BP(6»,APJ6)
DIMENSION PDATA(6,5»5l,EPRO»6,5),PRO(6.4»5,6)
DIMENSION PROD<6»4,5>,XPROD(6,4,5»»YPROD(6,4,5),ZPROD(6.4,5I
DIMENSION X«4),XI<6,4I,XII<4,5),XIII<6»4,5),XIV<6,4,5),
1XY<6.4,5),XYI(6,4,5I
DIMENSION T(5»,NSCALE(5),CHAR<7)
DIMENSION POINT(5),GP(4),VI(4)
DIMENSION CEI6)
410 FORMATC6F10.8)
READ(5,410HCEU),I=1,6>
WRITE(6,411MCEm»I=l»6I
411 FORMATC ',6F10.8»
READ(5,420)FF,SL»YF
WRITE<6»421)FF»SL»VF
420 FORMAT(3F10.8»
421 FORMATC ' ,2F10.8,F10«3)
READ(5,201)N,(GP(I),I«1,4)
WRITE(6,202)N,«GP(I),I=1,4>
201 FORMAT(I1.4F10«8)
202 FORMATC ',I3,4F10.4I
READ(5,207)(VIU),I»1,4)
207 FORMAK4F10.3)
WR!TE{6,208)(TI(I),I«1,4)
208 FORMATC '.4F10.3)
READ(5«1)BtSO,CO*AP(3),CE(6l
WRITE(6,203)B,SO,CO,AP(3)*CE(6)
1 FORMATJ5F10.8)
203 FORMATC • ,F10.6,2F10.2,F10.6,F10.8)
READJ5.2K (ESDATAU»J),J»1»4),I=1.5)
WRITE(6,204M(ESDATA(K,L),L»1,4),K-1,5)
2 FORMAT(F10>4,10X,3F10.4)
204 FORMATC ' ,F10.2,10X,F10.2,F10.4,F10.2)
DO 100 1*1.5
100 SSUB(I)»XMOLE(ESDATA(I,1),ESDATA(I,2),ESDATA(I,3),ESDATA(I,4),
1B,1.0.AP(3),FF,SL,CE(6),VI(1),VF)
READ(5,2)(((SDATA(I,J.K).K=li4),J=l,5).I=l,4)
WRITE!6,204 »«(SDATA(L.M.K),K=1,4) .H=l,5),L*1*41
DO 101 1=1,4
DO 101 J«1.5
101 SUB«I,JNXMOLE(SDATAfI,J,l),SDATA(I,J»2>,SDATA
23 FORMAT(2F10.6»
'205 FORMATC S2F10.6)
277
-------
READ«5»2H{PDATAfM.I»J»»J*l,4),I<=4,5)
WRITE(6,204MCPDATA{M,K.L).L=1.4).K=1.5)
DO 102 1=1»5
102 EPRO(M.I)=XMOLB
READi5,3)mpRO(M,I,J,K),K=l,5),J«l,5).Ial.4)
VRITE(6,206MUPRO
881 FORMATC '.4X.'AP - INTERCEPT OF STANDARD PLOT OF RELATIVE AREA ?E
3RSUS CONCENTRATION OF X')
WRITEI6.680)
680 FORMATC ',4X.'SO - INITIAL SPECIFIC ACTIflTTi DPM/HM. OF 3H SOBST
IRATE ADDED')
WRITEI6.690)
690 FORMATC ',4Xi'CO - INITIAL NUMBER OF 3H-SUBSTRATE COUNTS ADDED TO
1 SAMPLE')
WRITE(6f700)
700 FORMATC S4Xf'Cl - INITIAL NUMBER OF 14C DPM ADDED TO SAMPLE AS X
1M
WRITE(6,710>
710 FORMATC f.4Xt'C2 - NUMBER OF 3H DPM RECOVERED FROM SAMPLE AS XM
WRITE(6i720)
720 FORMATC 't4Xt'C3 - NUMBER OF 14C DPM RECOVERED FROM SAMPLE AS X* I
WRITE<6.730)
730 FORMATC 'i4X.'Al - RELATIVE AREA OF PEAK FROM COMPONENT X IN ALIQ
1UOT OF SAMPLE'J
WRITE(6t750»
750 FORMATC S4X.'V - VOLUME INJECTED FOR MEASUREMENT1 I
WRITE(6,880)
880 FORMATC '»4Xi'CE - EFFICIENCY OF CAPILLARY CAPTURE FOR THIS COMPOWBUT
WRITEC6.61)B,SO,CO,AP(3),CE(6)
61 FORMATC-t»lB='tF10.6,3X»'SO=',F10.2f3X.'CO»',F10.2.3XtlAP«ttF10.6'C6
3*',F8.6)
WRITE(6t62>
62 FORMATC-'.'NONENZYMATIC SUBSTRATE DATA')
WRITE(6»63>
63 FORMAT ('-'.4X.'Cl' .10X.'C3'.9X.' Al' .9Xt * VI
WRITEJ6.64)1(ESDATA(I»J).J=l.4 I»1=1,51
64 FORMATC ' »F10.2.F10.2.F10.4.F10.2J
WRITE(6.65>
65 FORMAT('-'.'ENZYMATIC SUBSTRATE DATA')
WRITEJ6.63)
WRITE(6,64>mSDATA(I»JtK>»K»l»4»»Jsl«5),I°*l94r
DO 400 M=1»N
278
-------
WRITE!6.69)8P!M).AP(M).CE!M)
69 FORMAT!'-'»'BP=',F10.6i5Xf'AP-'iF10.6t5X.'CE«'.F10.6)
L»M
WRITE(6,66)L
66 FORMAT !'-','NONENZYMATIC PRODUCTMX.I1, IX, 'DATA')
WRITE!6.63)
WRITE!6t64H!PDATA(M.I«J)tJ*l,4)tI»1.5)
URITE!6t67)t
67 FORMAT!'-'t'ENZYMATIC PRODUCTMX.il. IXt'DATA' )
WRITE16.68)
68 FORMAT{4X.lClttlOX.tC2(,8X»'C3't8X,lAl'.9X»'V1I
WRITE16.70M !
-------
7 FORMATC-1, 'NMOLES/MG-PROTBIN, SUBSTRATE* ENZYMATIC INCUBATION ')
V/RITE<6,5)
WRITE(6,8HI,(SUB(I»J),J=1,5),I=1,4I
8 FORMATC ' , II ,3X,5F10.4 )
WRITEC6.15)
15 FORMATC-', 'EQUATION X* )
WRITE(6,16)U»X(I),I=1,4)
16 FORMATC ' , II ,3X,F10.4 )
WRITE(6,18)
18 FORMATC-', 'EQUATION XII')
WRITE(6,5)
WRITE(6,8)(I,(XII(I.J),J=1,5>,I=1,4)
DO 500 M=1.N
L=M
WRTTE(6,31)L
31 FOTRWATt 'I1 »21Xi 'PRODUCT* .1X.I1 )
WRITE(6t9)
9 FORMAT* '-'.'NMOLES PRODUCT: HONENZYMATIC IKCUBATION SAMPLE' I
WRITE(6,5J
WRITE(6»6XEPRO(MtI).I=lf5)
WRITE(6tlO)
10 FORMAT! '-' . 'NMOLES/MG-PROTEIN » PRODUCT: ENZYMATIC INCUBATION')
WRITEJ6.5I
WRITE (6tai( It (PROD(M.IiJ)iJ=1.5UI=lt4)
WRITE(6fl2)
12 FORMATC-', 'EQUATION 1')
WRITE<6,5)
WRITE(6,8HI»«XPROD(MtI,J),J=l,5),I=l,4)
WRITE(6,13)
13 FORMATC-', 'EQUATION 2')
WRITE(6,8»(I,(YPROD(M,I,J),J=1,5).I=1,4)
WRITE(6,14)
14 FORMATC-' .'EQUATION 3')
WRITE16.5)
WRITE(6,8)(I,(ZPROD(M,I,J»,J=1,5),I=1,4)
WRITE(6,17)
17 FORMATC-', 'EQUATION XI1 »
WRITE(6,16)(I,XHM,I),I=1»4)
WRITEC6,19)
19 FORMATC-', 'EQUATION XIII')
WRITE16.5)
WRITE(6,8)(I,(XIII(M,I,J),J=1,5),I=1,4)
KRITBI6.20)
20 FORMATC-', 'EQUATION XIV)
WRITE(6.5)
WRITS(6,8)(I»(XIV(M,I,J),J=1,5),I=1,4)
WRITE(6,21)
21 FORMATC-', 'EQUATION XV)
WRITE(6,5)
HRITE(6»8)(It(X7CN*ItJ)tJ«lt5)tI«lff4)
WRITE(6.22)
22 FORMATC-', 'EQUATION XVI')
WRITE (6, 5)
280
-------
WRITE!6,8)11,1XVIIM.I,J),J=1,51,1=1,4 I
500 CONTINUE
T(l)»0.
T(2»=5.
T<3)=10.
T<4)=30«
T(5)=60»
DATA CHARm,CHAR<2),CHAR(3),CHAR(4),CHAR<5),CHAR(6)/'A','B'»
4IC'»'D',IEI.'FV
YMAX=0
YMIN=0
DO 900 M=1,N
DO 900 1=1,4
DO 900 J=l,5
IFJXPROD(M*I,J).GT.YMAX) GO TO 901
IF(XPRODfM,I,J>.LT.YMIN) GO TO 902
GO TO 903
902 YMIN=XPROD«M,I,J)
GO TO 903
901 YMAX=XPROD«M,I.J,J
903 CONTINUE
900 CONTINUE
DO 300 1=1,4
CALL PLOT1<0»6.9,7,9)
CALL PLOT2(0,T(5),T(U»YMAX,YMIN)
DO 601 M=1,N
DO 602 K=l,5
602 POINT(K)=XPROD
87 FORMAT('1',26X,'CONVERSION OF 3H1 TREATMENT HIM
GO TO 89
84 WRITE(6,88)
88 FORMAT('1',26X»'CONVERSION OF 3HI TREATMENT IV)
89 CALL PLOT4U7»'NMOLES/MG PROTEIN1)
300 WRITE<6,303)
303 FORMAT!' ',34X,'TIME IN MINUTES')
STOP
END
SUBROUTINE PROCAL(PRO»SUB,ESUB»EPRO,BP,SO,CO,PROD»XPROD,YPROD,
5ZPROD,XI,Xni,XIV,XVtXVI»GP,M»AP»FF»SL»CE«VI,VF>
DIMENSION PRO(6,4,5,6),SUB(4,6),ESUBI5),EPRO(6,5)
DIMENSION PROD(6,4»5),XPROD(6,4»5),YPROD(6,4,5),ZPROD(6,4t5)
DIMENSION XI(6,4)",XIII(6t4t5)»XIV(6,4,5),BP(6)
DIMENSION XV(6,4t51,XVIC6,4,5),GPI4)»VI(4)
DIMENSION AP(6),CE(6)
DO 99 I»l,4
281"
-------
DO 99 J=l»5
PROD(M,I.J^XMOLE(PROIM,I,J,lJ,PRO(MtI»J,3}.PROIM.I.J»4).
7PRO(M»I»J»5»»BPmtGPm.AP
XPROD(M.I.J)»H3«PRO!M,I.J,1>,PRO,PRO(M»I»J,2>rPRO(M.I»J»3),SO.
8SUBU»l)»£SUBm,GPIIH
99 ZPROD(M,I,J)=ALl(CO,PRO{M.I.J,l).PRO(M,I,J.2).PRO-SPRO
96 XVHM,I.J)=XIII(M.I.J)-ZPROD(M,I.J>
RETURN
END
FUNCTION XMOLE(C1,C3.A1.V.B.GP,AP,FF.SL.CS;VI,VF)
XMOLE«=(CCl/C3)*(10**U(ALOG10(A-.5-SL»AP>/B)»* I >/GP
RETURN
END
FUNCTION AIL(CO.C1.C2.C3»SO.SUB.ESUB,GP J
ALL=< (C2*JC1/C3)»/ (CO/«(CO/SO )+(SUB*GP)-«SSUB)) 1
RETURN
END
182
-------
0.685827970•589727340• 884790600.780579860.491454540•88479060
0-74367738-.25467902 Q.200
5 9.1730 7.7820 7.7030 29.4550
1.000 1.000 1*000
1.0469241898380.001846948.00
5.000
•3.5120810.88479060
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
1.005030
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734-00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
-3.375531
14.52
73.67
94*48
151.77
144.60
18*45
495.79
224.79
435.25
236.25
41.51
155.66
83.03
, 270.20
249.77
153.70
194*15
361 . 49
90.88
90.88
90*88
93.60
93.60
33*93
80*09
74*55
76*88
39.62
51*21
90*99
92*45
92.75
45.64
54.05
95.06
83*07
91*89
49*49
53*78
50.78
61.51
48*10
37.27
76.27
76.27
76.27
79.78
79.78
22.86
75.35
64*60
80*27
26*40
32*73
87.27
72*11
83*53
29.53
50*51
96*96
78*33
88*96
42.66
37.56
50*26
53.23
0.8440
0.8440
0.8440
0*7870
0*7870
0.4520
0.5480
0.5210
0.8005
0*6965
0*7480
0*7880
0.7345
0.7635
0*8580
0.8295
0.9090
0.7530
0*8105
0*6805
0*5190
0*3375
0*3970
0*3240
0*3840
0*8280
0.8280
0*8280
0*7940
0*7940
0*4105
0.4670
0.4235
0.7795
0.5770
0.6320
0.7210
0.6375
0*7075
0.7395
0.7130
0.7325
0.7210
0.8045
0.5830
0.4960
0.3860
0.4340
1.00
1.00
1.00
1.00
1*00
1.00
1.00
1.00
1.00
1.00
1*00
1.00
1.00
1.00
1.00
1*00
1.00
1.00
1*00
1*00
1*00
1*00
1*00
LOO
1.00
1.00
1.00
1*00
1.00
1.00
1.00
1*00
1.00
1*00
1.00
1*00
1.00
1.00
1.00
1.00
1*00
1*00
LOO
1.00
1.00
1*00
1.00
1.00
-------
61734.00
61734.00
0 ' 9R8246
12809'. 00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
1.046924
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
631.14
531.94
-3.357901
35.54
58.90
158.72
1396.45
635.75
42.07
274.97
1445.56
1334.55
655.33
48*09
131.50
453.02
1508.75
876.23
268.61
208.53
273.08
2432.41
3920.06
-3.512081
588. 77
1790.69
814.44
111.15
182.80
1141.55
193.14
117.34
122.13
151.03
1461.38
1176.97
319.97
121.10
171.91
6207.46
6568.69
5871.14
1172.33
582.00
43.67
30.51
163.41
163.41
163.41
163.38
163.38
54.86
150.90
127.57
160.67
60>34
65.77
173.57
184.33
169.50
70.31
93.40
182.33
169.02
183.81
95.52
99*22
109.81
117.28
98.68
79.98
90.88
90.88
90*88
93.60
93.60
33.93
80.09
74.55
76.88
39.62
51.21
90.99
92.45
92.75
45.64
54.05
95.06
83.07
91.89
49.49
53.78
50.78
61.51
48.10
37.27
0*3850
0.3315
1.8700
1.8700
1.8700
1.6890
1 . 6890
0.9385
1.2230
1.0495
1.5425
1.2985
1.3160
1.4615
1.6740
1.5400
1.5250
1.2285
1.5965
1.6350
1.6060
1.3680
1.1020
0.8220
0.8500
0.8140
0*7620
0.8440
0.8440
0*8440
0.7870
0.7870
0.4520
0*5480
0*5210
0*8005
0*6965
0.7480
0.7880
0.7345
0.7635
0*8580
0.8295
0.9090
0.7530
0.8105
0*6805
0.5190
0.3375
0.3970
0.3240
0.3840
1*00
1.00
1.00
1.00
1.00
1.00
1.00
1*00
1*00
1.00
1.00
1.00
1*00
1*00
1*00
1.00
1.00
1.00
1.00
LOO
1.00
1.00
1.00
1.00
1*00
1*00
1*00
1*00
1*00
1.00
1.00
LOO
1*00
1*00
1*00
1.00
1.00
1*00
1*00
1.00
1.00
1.00
1*00
1*00
1*00
1.00
1.00
1*00
1.00
1*00
1.00
1.00
-------
1.003495
63606.00
63686*00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
0.993589
62367. 00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
-3.329786
64.46
191.31
444.83
193.24
95.74
130.17
840.3?
281.70
93.57
101.39
166.70
690 . 68
521.33
146.58
109.77
553.61
584.10
932.11
1230.05
526.88
-3.482027
36.54
79.51
281 . 96
167.03
60.93
42.98
284.47
189.74
92.43
64.06
80.54
261.73
542.09
127.47
96.72
237.39
257.80
413.96
1042.03
391.59
97.01
97.01
97.01
100.92
100.92
33.20
89.29
84.81
93.80
42.22
51.89
104.41
121.07
102.12
44.25
64.40
112.74
104.72
109.41
56.01
50.75
54.73
59.06
50.47
41.36
68.38
68.38
68.38
73.06
73.06
23.31
75.36
65.62
78.07
25.41
35.75
89*69
92.73
86.71
31.37
43.37
97.92
83.00
93.92
40.79
49.90
57.97
56.57
50.18
35.03
0*8530
0*8530
0.8530
0.7730
0.7730
0.4570
0*6615
0.5275
0.7690
0.6670
0.7690
0.7395
0.7785
0.8100
0.7435
0.7065
0.8665
0.8155
0.8020
0.6695
0.4220
0.3345
0.3405
0.2980
0.3170
0.4530
0*4530
0.4530
0*3220
0.3220
0.2780
0.4565
0.3520
0.4650
0.3970
0.3710
0.4545
0.3825
0.4805
0*4645
0.4365
0.5200
0.4160
0.4790
0.3870
0*4550
0*3310
0.2205
0.1615
0.1650
1*00
1*00
1*00
1*00
1*00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
LOO
1.00
1*00
1*00
1*00
1*00
1.00
1.00
1.00
1*00
1*00
1*00
1*00
,1*00
1*00
1.00
1*00
1*00
1.00
1*00
1*00
1.00
1.00
1.00
1.00
1*00
1*00
1*00
1*00
1*00
1*00
1*00
1*00
1-00
1*00
1*00
ZB5
-------
DEFINITION OF VARIABLES:
BCP) - SLC" C OF STANDARD PLOT OF RELATIVE AREA VERSOS CONCEHTRATION
AP - INTERCEPT OF STANDARD PLOT OF RELATIVE ARBA VERSUS CONCENTRATION
SO - INITIAL SPECIFIC ACTIVITY. DPM/NM. OF 3H SUBSTRATE ADDED
CO - INITIAL NUMBER OF 3H-SUBSTRATB COUNTS ADDED TO SAMPLE
Cl - INITIAL NUMBER OP 14G DPM ADDED TO SAMPLE AS K
C2 - NUMBER OF 3H DPM RECOVERED FROM SAMPLE AS X
C3 - NUMBER OF 14C DPM RECOVERED FROM SAMPLE AS X
Al - RELATIVE AREA OF PEAK FROM COMPONENT X IN ALIQUOT OF SAMPLE
V - VOLUME INJECTED FOR MEASUREMENT
CE - EFFICIENCY OF CAPILLARY CAPTURE FOR THIS COMPONENT
B= 1.046924 50=1898380.00 CO-1846948-00 AP= -3.5120B1
NONENZYMATIC SUBSTRATE DATA
Cl
62016.00
62016.00
62016.00
62016.00
62016.00
C3
90.88
90*88
90.88
93-60
93.60
Al
0.8440
0.8440
0.8440
0.7870
0.7870
V
1.00
1*00
1.00
1.00
1*00
ENZYMATIC SUBSTRATE DATA
Cl
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
C3
33.93
80.09
74.55
76.88
39.62
51.21
90.99
92.45
92.75
45.64
54.05
95.06
83.07
91.89
49*49
53.78
50.78
61.51
48.10
37.27
Al
0.4520
0.5480
0.5210
0.8005
0.6965
0.7480
0.7880
0.7345
0.7635
0.8580
0-8295
0.9090
0.7530
0.8105
0.6805
0.5190
0.3375
0.3970
0.3240
0.3840
V
• 00
.00
>00
>00
.00
.00
.00
.00
.00
.00
.00
.00
1*00
00
00
00
00
00
00
00
286
-------
BP» 1-005030
AP= -3.375531
CE» 0.685828
NOHENZYMATIC PRODUCT 1 DATA
Cl
61734.00
61734.00
61734.00
61734.00
61734.00
C3
76.2? 0
76.27 0
76.27 0
79. 78 0
79. 78 0
Al
.8280
.8280
.8280
.7940
.7940
¥
1.00
1.00
1.00
1.00
1.00
ENZYMATIC PRODUCT 1 DATA
Cl
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
61734.00
C2
14.52
73.67
94.48
151.77
144.60
18.45
495.79
224.79
435.25
236.25
41.51
155*66
83.03
270.20
249.77
153.70
194.15
361.49
631.14
531.94
C3
22.86
75.35
64.60
80.27
26.40
32.73
87.27
72.11
83.53
29.53
50.51
96.96
78.33
88.96
42.66
37.56
50.26
53.23
43.67
30*51
Al
0.4105
0.4670
0.4235
0.7795
0.5770
0.6320
0.7210
0.6375
0.7075
0.7395
0.7130
0.7325
0.7210
0.8045
0.5830
0.4960
0.3860
0.4340
0.3850
0.3315
1-00
1.00
1*00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1*00
1.00
1.00
1.00
1.00
1*00
1.00
1.00
1.00
287
-------
BP= 0.988246
AP= -3.357901
CE« 0.589727
NONmYMATIC PRODUCT 2 DATA
Cl
128091.00
128091.00
128091.00
128091.00
128091.00
C3
163.41
163.41
163.41
163.38
163.38
Al
1.8700
1.8700
1 . 8700
1.6890
1.6890
¥
1*00
1.00
1.00
1.00
1.00
ENZYMATIC PRODUCT 2 DATA
Cl
128091.00
128091.00
128091*00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091*00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
128091.00
C2
35.54
58*90
158.72
1396.45
635.75
42.07
274.97
1445.56
1334.55
655.33
48.09
131.50
453.02
1508.75
876.23
268.61
208*53
273.08
2432.41
3920*06
C3
54.86
150.90
127.67
160.67
60.34
65.77
173.57
184.33
169.50
70.31
93*40
182*33
169*02
183*81
95*52
99.22
109.81
117.28
98.68
79.98
Al
0*9385
1.2230
1.0495
1.5425
1.2985
1.3160
1.4615
1.6740
1.5400
1.5250
1.2285
1.5965
1.6350
1.6060
1.3680
1.1020
0*8220
0.8500
0.8140
0.7620
00
00
00
00
00
00
1*00
1.00
1*00
1*00
1*00
1*00
1*00
1*00
1*00
1*00
1*00
1*00
1*00
1.00
288
-------
BP= 1.046924
AP= -3.512081
CE= 0.884791
HONEHZYMATIC PRODUCT 3 DATA
Cl
62016.00
62016.00
62016.00
62016.00
62016.00
C3
90*88 0
90.88 0
90.88 0
93.60 0
93.60 0
Al
.8440
.8440
• 8440
.7870
.7870
V
1*00
1.00
1.00
1*00
1*00
ENZYMATIC PRODUCT 3 DATA
Cl
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
62016.00
620 16. Op
62016.00
62016.00
62016.00
62016.00
C2
588.77
1790.69
814.44
111.15
182.80
1141.55
193.14
117.34
122.13
151.03
1461.38
1176.97
319.97
121.10
171.91
6207.46
6568.69
5871.14
1172.33
582.00
C3
33.93
80.09
74.55
76.88
39.62
51.21
90.99
92.45
92.75
45.64
54.05
95.06
83.07
91.89
49*49
53.78
50.78
61.51
48.10
37.27
Al
0.4520
0*5480
0.5210
0*8005
0*6965
0*7480
0.7880
0.7345
0.7635
0.8580
0.8295
0*9090
0*7530
0*8105
0.6805
0.5190
0.3375
0.3970
0.3240
0*3840
00
00
00
00
00
1.00
1.00
1*00
1.00
1*00
1*00
1.
1.
1.
1.
1.
1.
1
1.
00
00
00
00
00
00
00
.00
>00
289
-------
BP= 1.003495
AP« -3.329786
CE* 0.780580
HONENZYMATIC PRODUCT 4 DATA
Cl
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
63686.00
C3
97.01
97.01
97.01
100.92
100.92
Al
0.8530
0.8530
0.8530
0.7730
0.7730
V
1.00
1.00
1-00
1.00
1.00
DUCT 4 DATA
C2
64.46
191.31
444.83
193.24
95.74
130.17
840.37
281 . 70
93.57
101.39
166.70
690.68
521.33
146.58
109.77
553.61
584.10
932.11
1230.05
526.88
C3
33.20
89.29
84.81
93.80
42.22
51.89
104.41
121.07
102.12
44.25
64.40
112.74
104.7.2
109.41
56.01
50.75
54.73
59.06
50.47
41.36
Al
0.4570
0.6615
0.5275
0.7690
0.6670
0.7690
0.7395
0.7785
0.8100
0.7435
0.7065
0*8665
0.8155
0.8020
0.6695
0.4220
0.3345
0.3405
0.2980
0.3170
00
00
00
00
1*00
1.00
1.00
• 00
.00
1.00
1.
1.
1.
1.
1.
1.
1.
1.
1
1
00
00
00
00
00
00
00
00
• 00
• 00
290
-------
BP* 0.993589
AP» -3.482027
CE= 0.491455
NONENZYMATIC PRODUCT 5 DATA
Cl
62367.00
62367.00
62367.00
62367.00
62367.00
C3
68.38 0
68*38 0
68.38 0
73.06 0
73.06 0
Al
.4530
• 4530
• 4530
.3220
• 3220
?
1.00
1*00
1.00
1.00
1.00
ENZYMATIC PRODUCT 5 DATA
Cl
62367.00
62367.00
62367.00
62367.00
62367. 00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367.00
62367. 00
62367.00
62367.00
62367.00"
62367.00
62367.00
C2
36.54
79.51
281.96
167.03
60.93
42.98
284.47
189.74
92.43
64.06
80*54
261.73
542*09
127.47
96.72
237.39
257. 80
413.96
1042*03
391*59
C3
23.31
75.36
65*62
78.07
25.41
35.75
89.69
92.73
86.71
31.37
43.37
97.92
83.00
93.92
40.79
49*90
57.97
56.57
50.18
35.03
Al
0*2780
0.4565
0.3520
0*4650
0.3970
0.3710
0*4545
0*3825
0*4805
0.4645
0.4365
0.5200
0.4160
0*4790
0.3870
0.4550
0.3310
0.2205
0.1615
0.1650
00
00
• 00
,00
>00
.00
• 00
• 00
• 00
.00
• 00
1*00
1.00
1.
1.
1.
1.
1.
1.
1.
00
00
00
00
00
00
00
291
-------
EQUATIONS:
1 - 3H PRODUCT FORMED
2 - PRODUCT FORMED FROM 3H AMD ENDOGENOUS SUBSTRATE
3 - PRODUCT FORMED FROM ALL SOURCES
X - ENDOGENOUS SUBSTRATE
XI - ENDOGENOUS PRODUCT
XII - NET CONVERSION OF SUBSTRATE
XIII - NET FORMATION OF PRODUCT
XIV - PRODUCT FORMED FROM ENDOGENOUS SUBSTRATE
XV - PRODUCT FORMED FROM SUBSTRATE PRECURSORS
XVI - PRODUCT FORMED FROM OTHER SUBSTRATES
ANIMAL TREATMENTS:
HI) - CONTROL. SESAME OIL INJECTED
2(11) - ESTRADIOL INJECTED
3UII) - TESTOSTERONE INJECTED
41IV) - DDT INJECTED
NMOLES SUBSTRATE: NONENZYMATIC INCUBATION SAMPLE
0 MIN
302.5964
5 MIN
302.5964
10 MIN
302.5964
30 MIN
274.8206
60 MIN
274.8206
NMOLES/MG-PROTEIN, SUBSTRATE: ENZYMATIC INCUBATION
1
2
3
4
0 MIN
48-6618
61.4888
64.9663
2.1821
5 MIN
24.7792
36.3720
40.3137
1.5321
10 MIN
25.3664
33.4725
38.5391
1.4770
30 MIN
37.0729
34.6214
37.3770
1.5556
60 MIN
62.9832
78.6538
58.7261
2.3614
EQUATION X
1 15.6741
2 22.6047
3 25.6834
4 -7.1481
EQUATION XII
1
2
3
4
0 MIN
0-0
o.o
o.o
o.o
5 MIN
23.8826
25.1168
24.6527
0.6500
10 MIN
23.2954
28.0163
26.4272
0.7051
30 MIN
11.5889
26.8674
27.5894
0.6265
60 MIN
-14.3214
-17.1650
6.2402
-0.1793
292
-------
PRODUCT 1
NMOLES PRODUCT: NONENZYMATIC INCUBATION SAMPLE
0 KIN
280*0796
5 MIN
280.0796
10 MIN
280*0796
30 MIN
256.8162
60 MIN
256*8162
NMOLES/MG-PROTEIN, PRODUCT! ENZYMATIC INCUBATION
1
2
3
4
0 MIN
50.6822
64.1021
47*3132
2.3192
5 MIN
17.4812
27.4085
25.3178
1.3505
10 MIN
18*5000
29.3472
30.8498
1.4329
30 MIN
27.3204
28*1022
30*2928
1*5503
60 MIN
61.5816
83.0680
45.8517
1.9121
EQUATION 1
1
2
3
4
0 MIN
0.0023
0.0024
0.0035
0.0045
5 MIN
0.0035
0.0237
0*0068
0*0043
10 MIN
0.0052
0-0130
0*0045
0.0075
30 MIN
0*0067
0*0218
0*0128
0*0160
60 MIN
0.0194
0.0334
0.0247
0*0192
EQUATION 2
1
2
3
4
0 MIN
0.3350
0-4283
0*7090
-1*1022
5 MIN
0*5157
4*3161
1*3850
-1*0404
10 MIN
0.7714
2.3683
0.9144
-1.8291
30 MIN
1*1886
4.5804
2.9863
-3.4371
60 MIN
3.4433
7.0326
5.7566
-4.1464
EQUATION 3
1
2
3
4
0 MIN
0*3350
0*4283
0.7090
-1.1022
5 MIN
-0.2648
-0.4533
0*0621
-1*1244
10 MIN
-0.3674
-0.5508
-0.0219
-1.9892
30 Mir
0.4562
-0.0990
0*1854
-3.7397
60 MIN
6.0653
11.6227
4.5354
-4.0419
EQUATION XI
1 20.1492
2 28*1114
3 10*9534
4 -7.1895
293
-------
EQUATION XIII
1
2
3
4
0 MIN
0.0
o.o
o.o
0.0
5 MIN
-33.2010
-36.6936
-21.9954
-0.9687
10 MIN
-32.1822
-34.7549
-16.4634
-0.8863
30 MIN
-23.3618
-35.9999
-17.0205
-0.7689
60 MIN
10.8994
18*9660
-1.4615
-0.4072
EQUATION XIV
1
2
3
4
0 MIN
-50.3472
-63.6738
-46. 6043
-3.4214
5 MIN
-50.1665
-59.7859
-45.9283
-3.3597
10 MIN
-49.9108
-61.7337
-46.3988
-4.1483
30 MIN
-49.4936
-59.5217
-44.3269
-5.7563
60 MIN
-47.2389
-57-0695
-41.5566
-6.4656
EQUATION XV
1
2
3
4
0 MIN
0*0
o.o
o.o
o.o
5 MIN
-0.7805
-4.7694
-1.3229
-0.0839
10 MIN
-1.1388
-2.9192
-0.9363
-0.1600
30 MIN
-0.7324
-4.6794
-2.8009
-0.3026
60 MIN
2.6219
4.5901
-1.2212
0.1045
EQUATION XVI
1
2
3
4
0 MIN '
-0.3350
-0.4283
-0.7090
1.1022
5 MIN
-32.9362
-36.2403
-22.0575
0.1556
10 MIN
-31.8148
-34.2040
-16.4415
1.1028
30 MIN
-23.8180
-35.9009
-17.2059
2.9708
60 MIN
4.8341
7.3433
-5.9969
3.6348
294
-------
PRODUCT 2
WHOLES PRODUCT! NONBN2YMATIC INCUBATION SAMPLE
0 MIN
586.0391
5 MIN
586.0391
10 MIN
586.0391
30 MIN
528*7720
60 MIN
528*7720
NMOLES/MG-PROTEIN. PRODUCT: ENZYMATIC INCUBATION
1
2
3
4
0 MIN
94. 7259
131.1253
87.0086
3.8378
5 MIN
45.0190
55.2489
58.1029
2.5776
10 MIN
45*6145
59*6842
64*2080
2*4966
30 MIN
53.4746
59*6514
57.9821
2.8401
60 MIN
119*6202
142*3873
94*8595
3*2777
EQUATION 1
1
2
Z
4
0 MIN
0.0048
0.0055
0.0045
0.0062
5 MIN
0*0029
0.0137
0*0063
0*0044
10 MIN
0*0092
0*0680
0*0235
0*0053
30 MIN
0.0639
0.0683
0.0719
0*0565
60 MIN
0*0775
0*0808
0*0804
0*1123
EQUATION 2
1
2
Z
4
0 MIN
0.7090
1.0083
0.9216
-1.5129
5 MIN
0*4272
2*4973
1*2910
-1*0613
10 MIN
1*3616
12*3622
4*7976
-1*3012
30 MIN
11.3371
14.3604
16*7451
-12.1633
60 MIN
13*7433
16.9998
18*7139
-24*1854
EQUATION Z
1
2
Z
4
0 MIN
0.7090
1.0083
0.9216
-1.5129
5 MIN
-0.2193
-0.2623
0.0579
-1.1469
10 MIN
-0*6485
-2*8753
-0*1148
-1*4151
30 MIN
4*3515
-0*3104
1*0396
-13*2343
60 MIN
24*2081
28*0954
14*7439
-23*5761
EQUATION XI
1 30*8385
2 55.8183
3 10.9293
4 -16.0582
'295
-------
EQUATION XIII
1
2
3
4
0 MIN
0*0
o.o
O'O
0.0
5 MIN
-49. 7069
-75.8764
-28.9057
-1.2602
10 MIN
-49.1115
-71.4410
-22.8006
-1.3412
30 MIN
-41.2513
-71.4739
-29.0265
-0.9977
60 MIN
24.8942
11.2620
7.8509
-0.5601
EQUATION XIV
1
2
3
4
0 MIN
-94.0170
-130.1169
-06. 0870
-5.3508
5 MIN
-94.2988
-128.6280
-85.7176
-4.8991
10 MIN
-93.3643
-118.7630
-82.2110
-5.1391
30 MIN
-83.3889
-116.7649
-70.2635
-16.0011
60 MIN
-80.9826
-114.1255
-68.2947
-28.0233
EQUATION XV
1
2
3
4
0 MIN
0.0
0.0
0*0
0.0
5 MIN
-0.6465
-2.7595
-1.2331
-0-0856
10 MIN
-2.0101
-15.2375
-4.9124
-0.1139
30 MIN
-6.9855
-14.6708
-15.7056
-1.0710
60 MIN
10.4648
11.0956
-3.9700
0.6094
EQUATION XVI
1
2
3
4
0 MIN
-0.7090
-1 . 0083
-0.9216
1.5129
5 MIN
-49.4876
-75.6141
-28. 9636
-0.1133
10 MIN
-48.4630
-68.5658
-22.6858
0.0739
30 MIN
-45.6028
-71.1635
-30.0660
12.2365
60 MIN
0.6862
-16.8334
-6.8930
23.0159
296
-------
PRODUCT 3
NHOLES PRODUCT! NONENZYMATIC INCUBATION SAMPLE
0 MIN
302.5964
5 MIN
302.5964
NMOLES/MG-PROTEINt PRODUCT
1
2
3
4
0 MIN
48.6618
61.4888
64.9663
2.1821
5 MIN
24.7792
36.3720
40.3137
1.5321
10 MIN
302.5964
30 MIN
274.8206
60 MIN
274.8206
: ENZYMATIC INCUBATION
10 MIN
25.3664
33.4725
38.5391
1.4770
30 MIN
37.0729
34.6214
37.3770
1.5556
60 MIN
62.9832
78.6538
58.7261
2.3614
EQUATION 1
1
2
3
4
0 MIN
0.0618
0.0936
0.1147
0.1280
5 MIN
0.0796
0.0089
0-0525
0.1435
10 MIN
0.0389
0.0053
0.0163
0.1059
30 MIN
0.0051
0.0055
0.0056
0.0270
60 MIN
0*0164
0*0139
0*0147
0.0173
EQUATION 2
1
2
3
4
0 MIN
9.1944
17.0130
23.4314
-31.2300
5 MIN
11.8468
1.6200
10.7300
-34.9998
10 MIN
5.7886
0.9687
3.3381
-25.8259
30 MIN
0.9130
1.1628
1.3017
-5.8228
60 MIN
2.9138
2*9222
3.4309
-3.7307
EQUATION 3
1
2
3
4
0 MIN
9.1944
17.0130
23.4314
-31.2300
5 MIN
-6.0829
-0.1701
0.4810
-37. 8230
10 MIN
-2.7568
-0.2253
-0.0799
-28.0857
30 MIN
0.3505
-0.0251
0*0808
-6.3355
60 MIN
5*1325
4*8294
2*7031
-3*6367
EQUATION XI
1 15.6741
2 22.6047
3 25.6834
4 -8.0911
297
-------
EQUATION XIII
1
2
3
4
0 MIN
0.0
o.o
0.0
0.0
5 MIN
-23.8826
-25.1168
-24.6527
-0.6500
10 MIN
-23.2954
-28.0163
-26.4272
-0.7051
30 MIN
-11.5889
-26.8674
-27.5894
-0.6265
60 MIN
14.3214
17.1650
-6.2402
0.1793
EQUATION XIV
1
2
3
4
0 MIN
-39.4675
-44.4758
-41.5349
-33.4121
5 MIN
-36.8150
-59.8688
-54.2363
-37.1819
10 MIN
-42.8732
-60.5201
-61.6283
-28.0080
30 MIN
-47.7488
-60.3260
-63.6647
-8.0049
60 MIN
-45.7480
-58.5666
-61.5354
-5.9128
EQUATION XV
1
2
3
4
0 MIN
o.o
0.0
o.o
0.0
5 MIN
-17.9297
-1.7902
-10.2490
-2.8232
10 MIN
-8.5454
-1.1940
-3.4180
-2.2597
30 HIN
-0.5626
-1.1879
-1.2209
-0.5127
60 MIN
2.2187
1.9073
-0.7278
0.0940
EQUATION XVI
1
2
3
4
0 MIN
-9.1944
-17.0130
-23.4314
31.2300
5 MIN .
-17.7998
-24.9466
-25.1337
37-1730
10 MIN
-20.5386
-27.7909
-26.3474
27.3806
30 MIN
-11.9394
-26.8423
-27.6702
5.7090
60 MIN
9.1889
12.3356
-8.9433
3.8160
298
-------
PRODUCT 4
MMOLES PRODUCT: NONENZYMATIC INCUBATION SAMPLE
0 MIN
242. 7879
5 MIN
242.7879
10 MIN
242.7879
30 MIN
211*5665
60 MIN
211.5665
NMOLES/MG-PHOTEIN, PRODUCT: ENZYMATIC INCUBATION
1
2
3
4
0 MIN
41.5246
52.6021
39.3501
1.5628
5 MIN
22.3201
25.1429
27.5486
1 . 1496
10 MIN
18.7537
22.8225
27.9188
1.0844
30 MIN
24.6867
28.1485
26.2812
1.1111
60 MIN
47.5952
59.6456
42.8831
1*4419
EQUATION 1
1
2
3
4
0 MIN
0.0071
0*0108
0.0113
0.0124
5 MIN
0.0078
0.0347
0.0267
0.0122
10 MIN
0.0192
0.0100
0.0217
0*0180
30 MIN
0.0075
0*0039
0.0058
0.0278
60 MIN
0.0083
0.0099
0.0085
0.0145
EQUATION 2
1
2
3
4
0 MIN
1.0565
1*9661
2.3037
-3.0310
5 MIN
1.1658
6.3083
5.4522
-2.9654
10 MIN
2*8540
1.8236
4.4305
-4.3852
30 MIN
1.3361
0.8309
1.3589
-5.9794
60 MIN
1 .4707
2*0778
1.9879
-3.1254
EQUATION 3
1
2
3
4
0 MIN
1.0565
1.9661
2.3037
-3.0310
5 MIN
-0.5986
-0.6625
0.2444
-3.2046
10 MIN
-1*3592
-0*4241
-0.1060
-4*7689
30 MIN
0.5128
-0.0180
0.0844
-6.5059
60 MIN
2*5905
3*4340
1*5661
-3*0466
EQUATION XI
1 15.0569
2 21.4034
3 7.8314
4 -6.6799
299
-------
EQUATION XIII
1
2
3
4
0 MIN
0>0
0.0
0*0
0*0
5 MIN
-19.2045
-27.4592
-11.8014
-0.4132
10 MIN
-22.7709
-29.7796
-11.4312
-0.4784
30 MIN
-16.8379
-24.4536
-13.0689
-0.4517
60 MIN
6.0706
7.0435
3.5331
-0.1209
EQUATION XIV
1
2
3
4
0 MIN
-40.4681
-50.6360
-37.0464
-4.5938
5 MIN
-40.3587
-46. 2938
-33. 8979
-4.5282
10 MIN
-38.6706
-50.7785
-34.9195
-5.9481
30 MIN
-40.1885
-51.7712
-37.9912
-7.5422
60 MIN
-40.0539
-50.5243
-37.3622
-4.6882
EQUATION XV
1
2
3
4
0 MIN
0.0
o.o
o.o
0*0
5 MIN
-1.7644
-6.9708
-5.2078
-0.2392
10 MIN
-4.2131
-2.2478
-4.5365
-0.3837
30 MIN
-0.8232
-0.8489
-1.2745
-0.5265
60 MIN
1.1198
1.3562
-0.4217
0.0787
EQUATION XVI
1
2
3
4
0 MIN
-1.0565
-1.9661
-2.3037
3.0310
5 MIN
-18.6059
-26.7966
-12.0458
2.7914
10 MIN
-21.4117
-29.3555
-11.3252
4.2905
30 MIN
-17.3507
-24.4356
-13.1532
6.0541
60 MIN
3.4801
3.6095
1.9669
2.9257
300
-------
PRODUCT 5
NMOLES PRODUCTl NONENZYMATIC IKCUBAflOH SAMPLE
0 MIN
173.2276
5 MIN
173.2276
NMOLES/MG-PROTEIN* PBODOCT
1
2
3
4
0 MIN
33.8900
34.8253
34.1568
1.6190
5 MIN
17.2686
17.0276
18.0429
1.0117
10 MIN
173.2276
30 MIN
114.9919
60 MIN
114.9919
J ENZYMATIC INCUBATION
10 MIN
15.2664
13.8450
17.0045
0.6888
30 MIN
16.9816
18.6271
17.3189
0.5676
60 MIN
44.4992
49.7619
32.1739
0.8309
EQUATION 1
1
2
3
4
0 MIN
0.0056
0.0051
0.0079
0.0053
5 MIN
0.0038
0.0134
0.0114
0.0050
10 MIN
0.0154
0*0086
0.0279
0.0082
30 MIN
0.0077
0*0045
0.0058
0*0232
60 MIN
0*0086
0*0086
0*0101
0*0125
EQUATION 2
1
2
3
4
0 MIN
0-8353
0-9227
1.6185
-1.2945
5 MIN
0.5622
2.4344
2.3295
-1.2101
10 MIN
2.2896
1.5705
5.6921
-1.9911
30 MIN
1.3588
0.9466
1.3481
-4.9892
60 MIN
1.5229
1*8135
2.3553
-2.6858
EQUATION 3
1
2
3
4
0 MIN
0.8353
0.9227
1.6185
-1.2945
5 MIN
-0.2887
-0.2557
0.1044
-1.3077
10 MIN
-1.0904
-0.3653
-0.1362
-2.1654
30 MIN
0.5216
-0*0205
0.0837
-5.4285
60 MIN
2*6825
2.9971
1*8556
-2*6181
EQUATION XI
1 15.0054
2 12.5653
3 11.6685
4 -4.2621
'301
-------
EQUATION XIII
1
2
3
4
0 MIN
o.o
0*0
0.0
0*0
5 MIN
-16.6214
-17.7977
-16.1139
-0.6073
10 MIN
-18.6236
-20.9803
-17.1524
-0.9301
30 MIN
-16.9084
-16.1982
-16.8380
-1.0513
60 MIN
10.6092
14.9366
-1.9829
-0.7881
EQUATION XIV
1
2
3
4
0 MIN
-33. 0547
-33.9025
-32.5383
-2.9134
5 MIN
-33.3277
-32.3909
-31 . 8273
-2.8290
10 MIN
-31.6003
-33.2548
-28.4647
-3.6101
30 MIN
-32.5311
-33.8787
-32.8087
-6.6082
60 MIN
-32.3671
-33.0118
-31.8016
-4.3048
EQUATION XY
1
2
3
4
0 MIN
0.0
0.0
0.0
0.0
5 MIN
-0.8509
-2.6900
-2.2251
-0.0976
10 MIN
-3.3800
-1.9357
"5.8283
-0.1742
30 MIN
-0.8372
-0.9671
-1.2644
-0.4393
60 MIN
1.1596
1.1836
-0.4996
0.0677
EQUATION XVI
1
2
3
4
0 MIN
-0.8353
-0.9227
-1.6185
1.2945
5 MIN
-16.3327
-17.5420
-16.2184
0.7004
10 MIN
-17.5331
-20.6150
-17.0162
1.2352
30 MIN
-17.4299
-16.1777
-16.9216
4.3771
60 MIN
7.9267
11.9395
-3.8386
1.8300
302
-------
0.143
CONVERSION OF iH; TREATMENT
0.115
s
H
0
I
£
S
/
H
G
P
R
0
T
t
I
N
12.036
-0.008 + --
0.0
10.000 20.000 30.060 40.000
TIME IK MI MUTES
50.000
60.000
303
-------
CONVERSION 0? 3H; TREATMENT II
K
H
0
L
I
S
I
H
C
P
H
0
T
E
I
N
0.115
0.086
0.029
-0.000
i
I
1
I
I
1
I
I
I
I
I
I
I
1
I
C
lH
1\
!\
j\
1 \
1 I
i5
fi
i i
!
i 1'
' \
1*1
1 / )
I • /I
"U /
o /;»*'
ly
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-60Q/1-77-Q42
3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE
METABOLIC INTERACTIONS OF HORMONAL STEROIDS AND CHLORINAtED
HYDROCARBONS - Effects of Neonatal Treatment with o,p'
DDT on the Development of the Steroidogenic Endocrine
System nf the Male Rat
5. REPORT DATE
September 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Kenneth Lyle Campbell
B. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
The University of Michigan Medical School
Ann Arbor, Michigan 48104
10. PROGRAM ELEMENT NO.
5
11. CONTRACT/GRANT NO.
R-800637
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP.NC
14. SPONSORING AGENCY CODE
EPA-600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This study was an attempt to generate approaches to the measurements of the potential
of xenobiotics to alter reproductive capacity and viability which may allow improvement
of those currently in use. More specifically, it was an attempt to identify the effects
of neonatal exposure to the model compound, o,p'-DDT (l,l,-trichloro-2-(2-chlorophenyl)
-2-(4-chlorophenyl)-ethane), on the developing Steroidogenic endocrine system of the
model animal, the male rat. The effect of exposures by direct injections before day
5 of age and by indirect treatment via an o,p'-DDT injected dam were examined initially
by measurement of growth, organ weights, organ histology, and of serum corticosterone
and luteinizing hormone (LH). Later experiments included determinations of the effects
of o,p'-DDT on the serum LH responses to challenges of the hypothalamo-pituitary axis
with adult castration and repeated injections of luteinizing hormone releasing hormone
(LHRH). The observed effects indicated a precocious development of the adrenal cortex
in immature animals treated neonatally with o,p'-DDT similar to that seen in rats neo-
natally injected with 17 -estradiol-17-valerinate. They also demonstrated altered
serum LH responses in treated rats to challenges with adult castration or repeated
injections of LHRH. These results are discussed in relation to previous work on
steroids and o,p'-DDT in several animal systems, to possible mechanisms, to prospectiv*
experiments and to the original goal of the research.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Chlorohydrocarbons
Steroids
Endocrine glands
Metabolism
Biochemistry
Rats
DDT
06 A, F
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
322
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
307
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