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dissertation entitled

The Effect of Cisplatin on the Adrenal Gland and

Corticosterone Concentration: An In Vivo and In Vitro Study
presented by

Casey Margaret Miller

has been accepted towards fulfillment
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6/01 cJCIFIC/DateOuepGSop. 15

THE EFFECT OF CISPLATIN ON THE ADRENAL GLAND AND
CORTICOSTERONE CONCENTRATION: AN IN VIVO AND IN VITRO STUDY

By

Casey Margaret Miller

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

2000

ABSTRACT
THE EFFECT OF CISPLATIN ON THE ADRENAL GLAND AND
CORTICOSTERONE CONTRATION: AN IN VIVO AND IN VITFIO STUDY

By

Casey Margaret Miller

This study investigated the effect of the anticancer agent cisplatin on the
adrenal gland and corticosterone in an attempt to find out if alterations in adrenal
structure stimulated alterations in the level of corticosterone. Cisplatin (CDDP)
has been shown to alter appetite, weight , water intake, urine output and adrenal
structure in vivo. These effects can alter stress levels in the body and affect the
concentration of corticosterone. After treatment with cisplatin (7mg/kg and
9mg/kg) the animals showed immediate decrease in appetite and extreme weight
loss. Water intake was initially decreased with an increase as post-treatment time
increased. Cisplatin acted as a diuretic initially and then as an antidiuretic in
urine output. Plasma corticosterone concentration showed a trend of increase
that coincided with an increase in adrenal secretion into media for the 9mg/kg
group on day 3 and the 7mg/kg group on day 8. Further studies showed that
there was cellular damage to the mitochondria in disruption of the cristae or total
cavitation of the cristae from the mitochondria. There was enlargement and
degradation of the endothelial lining of the capillaries in both the medulla and the

cortex and an initial decrease in lipid droplets and then increase by day 8

(7mg/kg) post-treatment in the adrenal cortex. Organ culture studies verified
cellular damage but did not confirm the changes in corticosterone levels. The
longer-term cultures exhibited an overall decrease in corticosterone
concentration whereas the shorter-term cultures exhibited no real change from
the control. We feel that cisplatin appears to affect the adrenal gland directly by
endothelial damage, which then causes further damage to the cell and also
indirectly by degranulation of medullary and cortical secretory cells. We can not
verify if this is the primary cause of the alterations in corticosterone concentration

at this time.

Copyright by
CASEY MARGARET MILLER
2000

Dedicated to the memories of my father, mother and brother who encouraged

me, but could not see it to the end.

ACKNOWLEDGEMENTS

I want to express my sincere gratitude to my adviser, Dr. Surinder
Aggarwal for taking me into his laboratory and giving me his support, guidance
and patience. I would also like to thank Drs. R. Neal Band, Ved Gossain for
serving on my committee. I would also like to thank Dr. Karen Klomparens for
serving on my committee and for her mental and financial support. Their
questions and ideas have been crucial in the completion of this thesis.

For their generosity in sharing laboratory resources I would like to thank
Drs. Band, Kopachik and Freidman.

I also want to thank the Zoology staff Tracey, Judy, Lisa, Jan and Chris for
all their help and support through the years and the Zoology faculty for their
support. Past members of our laboratory have also been of immense help and
understanding, Dr. James Fadool and Dr. JoAnn Palma who made class work
and working in the lab and life in general more enjoyable. To all the friends I
meant and shared science and turmoil with at Michigan State, thanks for your

support and encouragement and to my family who helped me attain my goal.

vi

TABLE OF CONTENTS

Page
LIST OF TABLES
LIST OF FIGURES
INTRODUCTION 1
CHAPTER ONE: Cisplatin Induced Physiological Responses and
Alteration of Circulating and Adrenal Explant
Corticosterone Concentrations in the Rat 9
Summary 10
List of References 12
Introduction 1 1
Materials and Methods 13
Results 15
Discussion 23
Conclusion 27
List of References 28
CHAPTER 'IWO: Cisplatin-Induced Structural Changes in the Rat
In Vivo 34
Summary 35
Introduction 36
Materials and Methods 38
Results 40
Discussion 91

Conclusion 95

vii

List of References 96

CHAPTER THREE: Cisplatin-Induced Alterations in Corticosterone
Concentration and Structure of Adrenal Organ

Cultures from the Rat 99
Summary 100
Introduction 101
Materials and Methods 104
Results 107
Discussion 138
Conclusion 140

List of References 141

viii

LIST OF TABLES

CHAPTER THREE:

Table 1.

Table 2.

Group 1 Control and CDDP (7pm/ml) Adrenal
Organ Culture Media Changes and Collection
Times by Post-Treatment and Time from Last
Medial Change.

Group 2 Control and CDDP (7pm/ml) Adrenal
Organ Culture Media Changes and Collection
Times.

Page

105

105

LIST OF FIGURES

Page
CHAPTER ONE:
Figure 1. Food Intake and % Weight Change of Control and
CDDP Treated Rats. 18
Figure 2. Water Intake and Urine Output of Control and
Treated Rats. 20
Figure 3. Plasma Corticosterone Concentration from Control
and CDDP Treated Rats by Radioimmunoassay. 21

Figure 4. Corticosterone Concentration from Control and CDDP
Treated Rat Adrenal Glands. 22

CHAPTER TWO:

Figure 1. Light Micrographs of Sections (2pm) of the Adrenal
Gland of Control and After 7mg/kg CDDP
Treatment. 45

Figure 2. Light Micrographs of Sections (2pm) of the Adrenal
Medulla of Control (A) and After 7mg/kg CDDP
Treatment. 47

Figure 3. Light Micrographs Showing the Magnified View of
Various Cellular Changes Before (A) and After CDDP
(7mg/kg) Treatment. 49

Figure 4. Light Micrographs of Sections (2pm) of the Adrenal
Medulla of Control (A) and After 9mg/kg CDDP
Treatment. 51

Figure 5. Light Micrographs Showing the Magnified View of
Various Cellular Changes Before (A) and
After CDDP (9mg/kg) Treatment. 53

Figure 6. Electron Micrographs of Medullary Region of the
Adrenal Gland of Control (A) and After 7mg/kg
CDDP Treatment. 55

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

Selected Areas from Figure 6 Have Been Enlarged
to Show Changes Before and After CDDP
(7mg/kg) Treatment.

Electron Micrographs of Medullary Region of the
Adrenal Gland of Control and After 7mg/kg
CDDP Treatment.

Selected Areas from Figure 8 Have Been Enlarged

to Show Changes Before and After CDDP Treatment.

Light Micrographs of Sections (2pm) of the Medulla,
Zona Reticularis and Zona Fasciculata of the Adrenal
Gland of Control and 7mg/kg CDDP Treatment.

Light Micrographs of Sections (2pm) of the Medulla
and the Zona Reticularis of the Adrenal Gland of
Control and After 9mg/kg CDDP Treatment.

Electron Micrographs of the Medulla and Zona
Reticularis of the Cortex of the Adrenal Gland
of Control and After 7mg/kg CDDP Treatment.

Selected Areas from Figure 12 Have Been
Enlarged to Show Changes Before and After CDDP
(7mg/kg) Treatment.

Light Micrographs of Sections (2pm) of the Zona
Reticularis and Zona Fasciculata of the Adrenal
Gland of Control (A) and After 7mg/kg CDDP
Treatment.

Light Micrographs Showing the Magnified view
of Various Cellular Changes Before and After
CDDP (7mg/kg) Treatment.

Light Micrographs of Cryo-Sections (10pm) of the
Zona Fasciculata and Zona Glomerulosa of the
Adrenal Gland of Control and After 7mg/kg CDDP
Treatment.

Light Micrographs of Sections (2pm) of the Zona
Reticularis and Zona Fascicularis of the Adrenal
Cortex of Control and After 9mg/kg CDDP
Treatment.

xi

57

59

61

63

65

67

69

71

73

75

77

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

Figure 24.

CHAPTER THREE:

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Light Micrographs Showing the Magnified View
of Various Cellular Changes Before and After
CDDP (9mg/kg) Treatment.

Electron Micrographs of the Zona Fasciculata of
the Adrenal Cortex of Control and After 7mg/kg
CDDP Treatment.

Selected Areas from Figure 19 Have Been Enlarged
to Show Changes Before and After CDDP (7mg/kg)
Treatment.

Electron Micrographs of the Zona Fasciculata of the
Adrenal Cortex of Control and After 9mg/kg CDDP
Treatment.

Selected Areas from Figure 21 Have Been Enlarged

to Show Changes Before and After CDDP Treatment.

Light Micrographs of Sections (2pm) of the Zona
Fasciculata and Zona Glomerulosa of the Cortex
of Control and After 7mg/kg CDDP Treatment.

Light Micrographs of Sections (2pm) of the Zona
Fasciculata and Zona Glomerulosa of the Adrenal

Cortex of Control and After 9mg/kg CDDP Treatment.

Corticosterone Concentration Present in the Media

of Group 1 Adrenal Organ Cultures at Specific Times.

Corticosterone Concentration Present in the Media

of Group 2 Adrenal Organ Cultures at Specific Times.

Light Micrographs of Sections (2pm) of the Medulla
of Group 1 Adrenal Gland Organ Cultures.

Light Micrographs of the Enclosed Areas Enlarged
from Figure 3.

Light Micrographs of Sections (2pm) of the Medulla

and Zona Reticularis (Junction of the Medulla and
Cortex of Group 1 Adrenal Gland Organ Cultures.

xii

79

81

83

85

87

89

91

108

109

115

117

119

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 1 1.

Figure. 12.

Figure 13.

Figure 14.

Light Micrographs of Sections (2pm) of the Zona
Reticularis and Zona Fasciculata of Group 1 Adrenal
Gland Organ Cultures.

Light Micrographs of the Enclosed Areas Enlarged
from Figure 6.

Light Micrographs of Sections (2pm) of the Zona
Fasciculata, Zona Glomerulosa and Capsule Group
1 Adrenal Gland Organ Cultures.

Light Micrographs of Sections(2pm) of the Medulla
of Group 2 Adrenal Gland Organ Cultures.

Light Micrographs of the Medulla Enclosed Areas
Enlarged from Figure 9.

Light Micrographs of Sections (2pm) of the Medulla
and Zona Reticularis of Group 2 Adrenal Gland
Organ Cultures.

Light Micrographs of Sections (2pm) of the Zona
Reticularis and the Zona Fasciculata of Group 2
Adrenal Gland Organ Cultures.

Light Micrographs of the Enclosed Areas Enlarged
from Figure 12.

Light Micrographs of Sections (2pm) of the Zona

Fasciculata, Zona Glomerulosa and Capsule Group
2 Adrenal Gland Organ Cultures.

xiii

121

123

125

127

129

131

133

135

137

INTRODUCTION

Cisplatin (cis-dichlorodiammineplatinum II) is a heavy metal coordination
complex that is now used as a major broad-spectrum anticancer drug in the
treatment of ovarian, testicular, prostate, head and neck, and lung cancers [1].
Its proposed mechanism of action is to inhibit DNA synthesis by inducing
crosslinking of the inter or intrastrands [2-6]. The therapeutic use of cisplatin is
hampered by its severe side effects, which include nausea and vomiting [7-9],
along with kidney damage [10-13]. It is known to cause stomach distention and
ulceration [10, 14, 15], with treatments such as chemical, hormonal and
vagotomy being used to alleviate these effects [16-22]. Steroids and
catecholamines that are synthesized by the adrenal gland have been considered
in the induction of ulcers [23] with stomach mucosa and gastric emptying being
restored in rats after bilateral adrenalectomy [17]. Disease, infection [24],
chemotherapeutic drugs [13, 25, 26], toxic chemicals [27, 28], drugs [29-32],
hormones [33, 34] and peptides [35] can induce stress on the system at the
physical, metabolic and cellular levels. The classic response to stress is for the
system to greatly increase the production of glucocorticoids from the adrenal
gland, which is the key endocrine gland to respond to these conditions [24, 36].
Stress also stimulates the immune system, which then can increase synthesis of
glucocorticoids and the adrenal glands response to stress [24, 37-43]. This
stimulation often overrides the normal feedback inhibition that glucocorticoids
have on pituitary ACTH to allow prolonged production of glucocorticoids from the
adrenal gland [37, 40,44].

Stress can be expressed by altered body temperature [45], food intake,

weight loss [10, 46-54], water intake, urine output [10, 46, 54-58], or by
stimulating or inhibiting steroid synthesis [13, 26, 29, 34, 59-61].

Cisplatin has been shown to act as a diuretic in Sprage-Dawley [47, 55]
and Long-Evans Rats [46], while Wistar Rats respond to it as an antidiuretic [46].
Cisplatin and other chemotherapeutic drugs have been shown to alter water
intake and urine output [10, 12, 46, 47, 62, 63]. Studies have shown that
cisplatin and other heavy metals can alter the circulating concentration of
corticosterone [27, 64] and testosterone [13, 26, 34, 60]. This can result by a
direct interaction with the steroid or an indirect interaction in the stimulation or
inhibition of other hormones that stimulate glucocorticoid synthesis or by affecting
the structural integrity of the adrenal gland.

The adrenal gland is the major endocrine gland to show noxious
compound induced changes in morphology [65]. Cisplatin [16, 66, 67] and other
noxious chemicals have been shown to disrupt the adrenals ultra-structure by
damaging the mitochondria in the zona fasciculata and their cristae and by
enhancing or depleting lipid droplet formation which can affect both cellular
structure and steroid synthesis [30, 68-77].

To narrow the effects of a compound that may be having on the adrenal
gland and steroid concentration the adrenal gland should be removed from the in
vivo system to stop all influence by uncontrolled hormonal, neurogenic or
hemodynamic factors [78, 79]. Cell and organ culture of adrenal glands have
shown that by treating the culture with toxic agents and/or other hormones that
secretion of glucocorticoids continues, can be monitored and maintained [19, 24,

25,41, 42, 80-87].

10.

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Ribelin, W.E., The effects of drugs and chemicals upon the structure of the
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Francisco Press: San Francisco. p. 1652-3.

de Castro, OR, at al., Ultrastructural effects of nifurtimox on rat adrenal
cortex related to reductive biotransforrnation. Experimental and Molecular
Pathology, 1990. 52: p. 98-108.

de Castro, C.R., E.G.D. de Toranzo, and J.A. Castro, Benznidazo/e-
induced Ultrastructural alterations in rat adrenal cortex. Mechanistic
studies. Toxicology, 1992. 74: p. 223-32.

Magalhaes, M.M., et al., The effects of monensin on golgi complex of
adrenal cortex and steroidogenesis. Tissue and Cell, 1991 . 23(2): p. 209-
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Malendowicz, L.K., et al., Analysis of the preventive action of ACTH on
dexamethasone-induced adrenocortical atrophy in the rat. Cytobios, 1992.
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Malendowicz, L.K., Comparative studies on the effect of 4-APP(4-
aminopyrazolopyrimidine) on hamster and rat adrenal cortex. Exp Toxic
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Mazzocchi, G., et al., Long term effects of the hypocholesterolaemic drug
4-aminopyrazolo- pyrimidine on the zona fasciculata of the rat adrenal
cortex. J Anat, 1986. 149: p. 1-9.

Mazzocchi, G., et al., Effects of the hypolipidemic drug nafenopin on the
zona fasciculata of the rat adrenal cortex: a correlated biochemical and
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Lullmann-Rauch, R. and G.H. Reil, Chlorphentermine-induced Iipidosislike
Ultrastructural alterations in lungs and adrenal glands of several species.
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Lullmann, G. and R. Lullmann-Rauch, Tamoxifen-induced generalized
lipidosis in rats subchronica/ly treated with high doses. Toxicology and
Applied Pharmacology, 1981. 61: p. 138-46.

Robba, C., et al., Opposed effects of chronic prolactin administration on
the zona fasciculata and zona reticularis of the rat adrenal cortex: an
Ultrastructural stereo/ogical study. J Submicrosc Cytol, 1985. 17(2): p.
255-61.

Phelps, J.S., et al., Cisplatin nephrotoxicity: invitro studies with precision-
cut rabbit renal cortical slices. Toxicol Appl Pharmacol, 1987. 90: p. 501 -
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Smith, J.H., The use of renal cortical slices from the Fischer 344 rat as an
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Calogero, A.E., et al., Cocaine stimulates rat hypothalamic corticotropin-
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Mazzocchi, G., et al., Effects of galanin on the secretory activity of the rat
adrenal cortex: in vivo and in vitro studies. Res Exp Med, 1992. 192: p.
373-81 .

Rebuffat, P.m., L. K., et al., Morphology and functional responses of
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Whitcomb, R.W., et al., Monocytes stimulate cortisol production by
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Williams, B.C., et al., Characterization of the steroidogenic
responsiveness and ultrastructure of purified zona fasciculata/reticularis
cells from bovine adrenal cortex before and after primary culture. Journal
of Endocrinology, 1989. 121: p. 317-24.

CHAPTER ONE: CISPLATIN INDUCED PHYSIOLOGICAL RESPONSES
AND ALTERATION OF CIRCULATING AND ADRENAL
EXPLANT CORTICOSTERONE CONCENTRATIONS IN

THE RAT

SUMMARY

Cisplatin has been shown to alter food consumption, body weight, and
water intake and urine output. This study was designed to analyze these effects
of the drug and the effect on corticosterone concentration in plasma and secreted
from the adrenal gland into media. Wistar CRL:(W|)BR male rats were kept in
metabolic cages with food consumption, weight, water intake and urine output
being measured daily. Blood was collected on specific days for corticosterone
analysis. Animals were injected with normal saline, 7mg/kg cisplatin or 9mg/kg
of cisplatin via intraperitoneum. Animals were sacrificed on day three, five and
eight for the 7mg/kg dose and on days three and five for the 9mg/kg dose. At
sacrifice adrenal glands were collected , incubated in media and the medium
collected for steroid analyses. The animal’s appetite was suppressed in both
cisplatin groups immediately after treatment with their weight decreasing 35%
from their initial body weight. Water intake decreased to 0 by day 3 with an
increase in intake by day 5 in the 7mg/kg group, 60% above that of the controls .
Urine output was increased significantly on day 1 and then declined through day
4, with a return to that of the control by day 5. There was a trend of an increase
in corticosterone concentration in plasma though it was not significant from the
control. This increase did coincide with an increase in adrenal secretion of
corticosterone into media for the 9mg/kg group on day 3 and the 7mg/kg group

on day 8.

10

INTRODUCTION

Cisplatin is an important chemotherapeutic agent which has dose limiting
side effects of nausea and vomiting [1, 2] and kidney damage [3-11]. It also
causes ulceration and distention of the stomach [12-16], with many measures
being taken to modify this effect in patients undergoing cisplatin chemotherapy,
protection of the mucosa by both chemical and hormonal means and control of
acid secretion by means of vagotomy [13, 14, 17-24] are two such measures.
Steroids and catecholamines produced by the adrenal gland have been
considered to be a part of the induction of ulcers in rat’s [25]. Ulceration and
gastric emptying are restored when a rat has a bilateral adrenalectomy [17].
Chemotherapeutic drugs [10, 26-29], toxic chemicals [30-32]; drugs [33-38],
hormones [39-46] and peptides [47] are known stressors. Stress can be
expressed by altering food intake and/or weight loss [4, 5, 9, 48-54], water intake
and/or urine output [4-6, 38, 50, 55-57] or by stimulating or inhibiting steroid
production ([10, 26, 33, 36, 37, 43, 44, 47, 58-64]. This stress can arise from the
animals response to the drug by either a direct or an indirect means. The
adrenal gland is the key organ to respond to alarm, adaptation and exhilaration
[65]. The initial response stimulates the release of cortocotropic releasing
hormone (CRH) from the hypothalamus, enhancing the release
adrenocortocotrophic hormone (ACTH) from the pituitary, enhancing the adrenal

to synthesize corticosterone in the rat and cortisol in the human [66, 67]. As the

11

noxious compound induces physical, metabolic and/or cellular alterations to the
system it may also have an affect on the concentration of circulating
corticosterone. The present study was designed to analyze stressors that the
chemotherapeutic agent, cisplatin may initiate physiologically and on steroid
production. This was examined: (i) food consumption and weight loss, (ii) water
intake and urine output and (iii) corticosterone concentration in circulating plasma
(it is the primary glucocorticoid in rats [68]) and adrenal explants production into

media.

12

MATERIALS AND METHODS

Animals and Treatment: Wistar Crl:(Wl)BR male rats weighing 200 to
250 g each from Charles River were placed in metabolic cages, on a 12 hour
light/dark cycle, with food and water adlib. Weight, food and water intake and
urine output was measured daily at 6AM to follow the dinural rhythm of the rats.
Animals were allowed to adapt to their new environment for three days prior to
treatment, and were injected (Day 0) with a single dose via the intraperitoneum
with normal saline (vehicle for cisplatin), 7mg/kg of cisplatin (CDDP) or 9mg/kg of
CDDP. Animals were sacrificed by decapitation at three, five and eight days for
the 7mg per kg treatment, and at days three and five for the 9mg per kg
treatment.

Corticosterone Analysis Plasma: Blood was collected at 10AM from the
tail artery on days -1, 1, 3, 4, 5, 7 and 8 for the 7mg per kg treatment group and
on days -1, 1, and by cardiac puncture on days 3 and 5 for the 9mg per kg
treatment group. Animals were anesthetized by inhalation of Metofane
(PitmanlMoore), a tuberculin syringe coated with Heparin 1000 units/1ml (Elkins-
Senn, Inc.) drew 3 to 4mls of blood. The plasma was collected and kept frozen
at -70 °C until analysis could be conducted. The plasma was analyzed for
corticosterone content using rat corticosterone-3H kit Radioimmunoassay (RIA)
(ICN), read using a Beckman 7500 series scintillation counter.

Corticosterone Secretion from Adrenal Glands: Adrenal glands were

removed from the animal, quartered and incubated for 30 minutes at 37 °C in 1

l3

ml Krebs-Ringer bicarbonate (KRBG) solution with 0.3% glucose (Sigma) . The
medium was discarded and new KRBG with 0.3% bovine serum albumin (fraction
5) (Sigma) was added. After a 30-minute incubation the medium was collected
and stored at -70 °C until corticosterone analysis was conducted according to
Malendowicz, L., et al (1992). The serum was analyzed for corticosterone
content by a rat RIA and read using a Beckman 7500 series scintillation counter.
Statistical Analysis: Statistical analyses were performed using SAS
Institutes Stat View for Windows. Food intake, weight, water intake, urine output,
plasma corticosterone levels and adrenal serum corticosterone levels were
analyzed using Student’s ttest, , ANOVA, or Student’s ttest, Mann-Whitney U
rank-sum test and Kruskal-Wallis H-test as appropriate dependent on the sample
size. The tests were two-tailed and significance was accepted at the P3005

level.

14

RESULTS

Suppression or loss of appetite occurred in both cisplatin groups
immediately after treatment. With food intake decreasing significantly from that
of the controls 20-30mg/day to 0-5mg/day post-treatment by day 4 (Figure 1A).
There also was a significant difference in food consumption between the two
treatment groups on day 2. Both treated groups increased their food intake by
day 5, continuing until the date of sacrifice when intake matched that of the
controls. The initial weight loss correlates with the drop in food consumption
(Figure 1A & B). Both CDDP treated groups had a 35% decrease in weight from
their initial body weight, with little or no increase in weight prior to the date of
sacrifice (Figure 1B). When sacrificed both groups had enlarged and distended
stomachs that were full of food even on days 3 and 5 before food intake
increased. This observation has been made previously and is caused by the lack
of stomach emptying [12-14].

Water intake decreased to zero by day 3 for both treatment groups, and
was significantly different from that of the controls on day 2-3 (Figure 2A). Water
intake in the two treatment groups was significantly different on days 2 and 4,
with water intake increasing for both groups’ days 4-8. The 7mg/kg groups water
intake increased by 60% from that of day 3 and 20% above that of the control
prior to sacrifice. Urine output increased for both treatment groups and was
significantly different from that of the control as of day 1 (Figure 2B). Urine

output continued to decline for both treatment groups through day 4, with the

15

9mg/kg group being significant from the control. On day 5 the urine output of the
treated groups begins approaching the level of the controls, with a significant 2-
fold increase by day 6.

Corticosterone concentration was elevated significantly by day one in the
7mg/kg group (Figure 3). While the 9mg/kg group decreased (not significantly)
from prior to treatment and that of the control on day one. The corticosterone
concentration remained elevated for the remainder of the study in both treatment
groups, though this elevation in concentration was not statistically significant.
Corticosterone standards were unaffected by the addition of CDDP when
analyzed. Secreted corticosterone into media from the adrenal glands of the
9mg/kg group had a significant increase from that of the 7mg/kg group and the
control on day 3 (Figure 4). This coincided with an elevated concentration of
corticosterone in the plasma on this day (Figure 3). There is a decrease in
corticosterone secreted from the adrenal glands from both treatment groups on
day 5, with the controls corticosterone increased though not significantly. The
7mg/kg group increased secretion from the adrenal glands significantly from the
control on day 8, this coincides with an elevation in the concentration of

corticosterone in the plasma.

16

Figure 1: Food Intake and % Weight Change of Control and CDDP Treated Rats.

A)

3)

Food intake (mg) of control and CDDP treated rats. The CDDP treated
groups ceased eating immediately after treatment, with day-1 showing a
significant difference from that of the control (P5005) in the amount of food
consumed. At days 2-4 both treatment groups are significantly different from
the control group (Ps0001) with their food consumption dropping to 0mg, with
a significant difference between the two treatment groups at day-2 (Ps0005)
as well. The 7mg/kg group increased food consumption on days 6-7, with a
significant difference from that of the control at day-6 (P5005) and matching

the controls food consumption by day 8. [Average 4: SD]

The percent weight change of control and CDDP treated rats. The CDDP
treated groups have a weight loss immediately after treatment with days 1-8
showing a significant difference from that of the control group (Ps0001).

[Average : 8.0.]

17

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Figure 2: Water Intake and Urine Output of Control and CDDP Treated Rats.

A)

Water intake of control and CEEP treated rats. Water intake in both
treatment groups decreased significantly (7mg/kg P50005, 9mg/kg
P50001) by day 2 post-treatment and continues to decrease through day 3,
with the two treatment groups also being significantly different (P5005).
Water intake starts to increase in both groups with the 9mg/kg group being
significantly different from the control until sacrifice (P500001) and the
7mg/kg group (P5005). The 7mg/kg group is significant only on day 4
(P50005). By day 7 the 7mg/kg group has had a 4-fold increase in water
intake from day 3. They are in taking a significantly (P50059reater volume

than the controls. [Average :t 8.0.]

Urine output of control and CDDP treated rats. The treated groups had an
increase in urine output by day-1 being significantly different from that of the
control group (7mg/kg P50005, 9mg/kg P500001). On day-2 the 9mg/kg
group is significantly different (P5005) from the control group in urine output
but not the 7mg/kg group. The urine output on day-3 showed no difference
between groups, with a difference appearing again on day-4 between the
9mg/kg treatment group and both the control (P5005) and 7mg/kg group
(P5005). The 7mg/kg groups urine output increased greatly days 6-8 giving

a significant difference from the control (P5005). [Average i 8.0.]

19

 

 

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20

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Control

 

 

 

- 9mg/kg CDDP
- 7mg/kg CDDP

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O

600

 

400

200

Plasma Corticosterone (ng/ml)

 

 

 

 

 

 

 

Figure 3: Plasma Corticosterone Concentration from Control and CDDP Treated
Rats by Radioimmunoassay.

Plasma was collected on specified days and analyzed for the corticosterone

concentration. On day 1 the 7mg/kg group showed a significant difference in the

concentration of corticosterone from that of the control and the 9mg/kg group

(P50005). All other days showed no significant difference in the corticosterone

concentration between groups. [Average i 8.0.]

21

1 000

1:] Control
- 7mg/kg CDDP
- 9mg/kg coup

(n
O
O

*

600

 

 

400

200

Serum Corticosterone (ng/ml)

 

 

 

 

Figure 4: Corticosterone Concentration from Control and CDDP Treated Rats
Adrenal Glands.

The serum was collected from processed from adrenal glands and analyzed for
the level of corticosterone concentration. On day 3 the 9mg/kg group showed a
significant difference in the amount of corticosterone present from the control and
7mg/kg group (P5005). On day-8 the 7mg/kg group showed a significant
difference in corticosterone concentration from that of the control group (P5005).
Day 5 shows no significant difference in the level of corticosterone between the

groups. [Average :I: SO]

22

DISCUSSION

Various chemotherapeutic agents [4, 6, 9, 13, 29, 69-72], toxic chemicals
[30, 32, 51, 73] drugs [33, 36-38, 73] and hormones [43, 50, 74-76] or homnone-
like substances [31, 47, 56, 64] are known to effect food consumption, weight
loss or gain, water intake and urine output of experimental animals. In this
present study, treatment with cisplatin resulted in a decrease in food
consumption and subsequent weight loss in both treatments [4, 9]. It has been
suggested that when noxious compounds are administered, weight loss may be
due to a shift in the regulation level for body [9, 51, 52, 54] and that the weight
reduction is a result of regulation by decreased food intake until such a level of
regulation is reached that is induced by the dose of the drug [51, 54]. Although
the animals have reduced their food consumption to 0mg a day on days 4-5, they
show signs of stomach distention and ulceration at the time of sacrifice [13]. This
is indicative of ulceration of the stomach, possibly related to the levels of

circulating steroids [25].

Water intake and urine outputs were altered suggesting that there is an
effect on hormones that cause dipsia and polyuria. Other groups have found that
cisplatin or other chemotherapeutic drugs [4-7, 10, 11, 71, 72, 77-79], estrogen
[50], pertussis toxin [55] and bradydinin [56] have effects on both water intake
and urine output and cause nephrotoxicity. This pattern in water intake
(decrease through day 3 and then increasing) has been seen previously after

cisplatin administration [4, 6, 9].

23

Cisplatin has been shown to be diuretic in Sprage-Dawley [9, 78] and
Long-Evans Rats [5], while it acts as an antidiuretic in Wistar Rats [5]. This study
shows a diuretic effect on Wistar Rats as they have an increased urine output on
day one, in spite of decreases in water intake. Urine output decreases through
day 4, which parallels decreases in water intake. This suggests that it may not
be the effect of cisplatin acting as a antidiuretic but due to the lower fluid intake.
This is confirmed on days 5-8 when there is an increase in urine output that
parallels an increase in water intake. This pattern has been seen in previous
studies which showed an increase in serum creatinine which is indicative of a
water concentration defect with later tubular changes in the kidney with two

different doses of cisplatin [6, 9].

Concentrations of corticosterone in rats and cortisol in humans are
changed by stress [66, 67, 80, 81] from internal and external sources. Cancer
causes lntemal stress or a direct form, where treatment with chemotherapeutic
drugs and the drugs to reduce the side-affects cause stress from external
sources or an indirect form. Substances such as heavy metals [29, 30, 82], toxic
compounds [31], drugs [33-38, 73, 83], hormones [10, 26, 29, 43, 44, 50, 59, 60,
76, 83] have an affect on the corticosterone levels in rats. Cisplatin is a heavy
metal and chemotherapeutic agent known to effect the structure of the adrenal
gland [13, 84], therefore there is good reason to believe that it could alter

corticosterone concentration.

There was an increase in plasma corticosterone concentration with the

7mg/kg dose of CDDP on all days post-treatment compared with pre-treatment

24

levels. The secretion of corticosterone into media from the adrenal glands on
day 8 confirmed these increases. On day 5 the secretion of corticosterone into
media from the adrenal gland did not show an increase in corticosterone
concentration compared to the control. The concentration in the culture medium
does coincide with the plasma corticosterone levels. This pattern is repeated
with the 9mg/kg group as well on day 5. The differences seen between the two
concentrations could be explained by the stress to the animal when drawing
blood, or stress they experience prior to sacrifice. The animals that received the
9mg/kg dose of cisplatin appeared to be irritable and perhaps more stressed that
the group treated with the 7mg/kg dose. These animals were dehydrated making
the blood draw difficult. Sergejew et al (1996), found that corticosterone
concentration in plasma and that secreted into media from adrenal glands were
increased, although they were not significant from the control. Basal levels in
their studies differed in that they were of a lower concentration from those found
in the present study but this could be attributed to strain and gender differences
[85, 86]. Other studies using male Wistar Rats reported basal levels from 400 to
600 mg/ml that are similar to our findings [33, 35, 47].

The adrenal cortex has a large reserve of precursors for steroid
production, which may cause problems with detecting changes in circulating
corticosterone. This is seen by a study done by Xarli, et al (1978), in human
autopsy material. They found that 1.1% percent of all cases examined had some
type of adrenal hemorrhage with cellular degeneration (not the cause of death).

None of these cases had clinical finding to suggest any adrenocortical

25

dysfunction. Based on this study an increase in corticosterone indicates either
extreme stress, stimulation to steroid synthesis or damage to the adrenal gland

on a cellular level.

Other microscopic studies are required to examine the changes in
structure, at light and EM levels, and explantation of the adrenal gland to organ
culture to allow examination without uncontrolled factors such as changes in

appetite, weight , water intake and urine output.

26

CONCLUSIONS

The physiological affects of alterations in weight and food, water
and urine intake and/or output induce stress on the system and activate the
corticosterone pathway. This gives rise to an increase in the concentration of
corticosterone. To find if the corticosterone concentration increase is due to

cisplatin directly or indirectly further studies will have to be conducted.

27

10.

11.

12.

13.

14.

15.

16.

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33

CHAPTER TWO: CISPLATIN-INDUCED STRUCTURAL CHANGES IN THE

RAT IN VI V0

34

SUMMARY

Wistar CRL:(W|)BR male rats were treated with either normal saline (vehicle for
cisplatin), 7mg/kg or 9mg/kg of cisplatin. Animals were sacrificed at 3, 7 and 8
days for the 7mg/kg group and at 3 and 5 days for the 9mg/kg group. Adrenals
were processed for light and electron microscopy. Both doses showed disruption
of the mitochondria and secretory cells, enlargement and endothelial lining
degradation of the venous channels in both the medulla and cortex of the adrenal
gland and by a decrease in lipid droplets and then increase by day 8 post-
treatment. Cisplatin appears to affect the adrenal gland directly by endothelial
damage, which then causes further damage to the cell, and indirectly by

degranulation of medullary and cortical secretory cells.

35

INTRODUCTION

The structure of the adrenal gland plays a major role in steroidogenesis.
The gland is divided into the medulla and the cortex. The medulla develops from
ectodermal neurocrest cells, and synthesizes nitrogen containing hormones, nor-
epinephrine and epinephrine [1]. The adrenal cortex develops from mesoderrnal
coelemic epithelium and different hormones are synthesized in specific zonae [2].
Therefore the gland is divided into 3 zonae, the innermost being the zona
reticularis which comprises about 7% of the cortex and synthesizes the sex
hormones [3-6]. The middle zone, zona fasciculata comprises 70% of the cortex
and synthesizes the glucocorticoids including cortisol and corticosterone [3-6].
The outermost zone the zona glomerulosa comprises 15% of the cortex and
synthesizes the mineralocorticoid aldosterone [3-6]. The adrenal gland is very
important maintaining the homeostasis of the body because its function is to
respond to alarm, adaptation and exhilaration [7]. The different zonae and the
medulla of the adrenal gland vary in their susceptibility to different noxious
agents’ [8]. The zona glomerulosa receives part of its circulatory supply from the
capsule thus allowing it to be protected from infarctive effects that may damage
the adrenal arterioles in the rest of the cortex or in the medulla [8]. The
mitochondria of the medulla are less prominent and their function is related to the
cellular energy [9]. The mitochondria of the different zonae vary in their design
dependent on the hormone that they are producing [4]. In the zona fasciculata

the mitochondrial membranes play a major role in steroid synthesis. Cytochrome

36

P450 resides on the inner mitochondrial membrane and is the major player in the
first step of steroidogenesis, converting cholesterol to pregnenolone [4, 6, 9-11].
Steroidogenesis then moves to the mitochondrial outer membrane to start the
second phase where it can receive a rapid supply of cholesterol.

Damage to the medulla can alter steroid production by releasing
catecholamines into the medullary venous channel [8, 12]. Damage to the
mitochondria within a specific zona of the cortex influences not only the hormone
synthesis in that zona [11, 13-19], but can also have an effect on the medulla’s
synthesis of catecholamines [20]. Agents that increase lipid concentration, such
as benznidazole, aminoglutethimide, ACTH, prolactin and testosterone in the
adrenal cortex may be activating a degenerative process by destroying cellular
structure and impair function as seen in the liver [17, 21-24]. On the other hand
agents that reduce lipids, such as hypocholesteroloemic drugs, nifurtimox,
monensin, 4-APP, tamoxifen can cause a shutdown in steroid synthesis [13, 25-
28].

Present is an attempt to study any structural changes after cisplatin treatment

and correlate these changes to alterations in corticosterone levels.

37

MATERIALS AND MEHTODS

Animals: Wistar Crl:(Wl)BR male rats weighing 200 to 250 grams each
from Charles River were placed 3 to a cage. Animals were maintained on a 12-
hour day/night cycle, with food and water ad lib. Animals were allowed to adjust
for three days to the new environment, after which time they were injected (Day
0) with a single dose via the intraperitoneal with normal saline (vehicle for
cisplatin), 7mg per kg or 9mg per kg of cisplatin. Animals were then sacrificed by
decapitation on days 3, 5 and 8 for the 7mg/kg treatment and days 3and 5 for the
9mg/kg treatment, the adrenal glands were processed for light and electron
microscopy.

Light Microscopy: One adrenal was cut in half, and both halves fixed in
2% glutaraldehyde in 0.1M sodium phosphate buffer (PBS), pH 7.4, dehydrated
in graded ethanol series and embedded in JB4 (glycol methacrylate,
Polyscience, lnc.). Sections (2pm) were stained with hematoxylin and eosin for
general histology. The other adrenal was cooled down and frozen in isopentane
(Sigma), further frozen in liquid nitrogen and then stored in liquid nitrogen until
cryosectioned. Sections (10pm) were fixed in 10% normal buffered formalin and
stained with sudan black for lipid localization.

Electron Microscopy: A thin, sagittal slice was cut from each adrenal,
fixed in 2% glutaraldehyde in 0.1 M phophate buffered saline (PBS), and then cut
into smaller sections. Samples were postfixed in 1% 0804, in PBS, dehydrated

in graded acetone series and embedded in Araldite resin. Thick sections were

38

taken and stained with toluidine blue to localize the area for thin sectioning.
Ultra-thin sections were made using an ultratome (LKB- Sweden) and stained
with aqueous 2% uranyl acetate and lead citrate, and examined with a Philips
CM 210 transmission microscope. Fifteen to twenty blocks were embedded for
each of twelve animals in each group. Minimums of five blocks to a maximum of
ten blocks were thick sectioned for each animal to pick areas for thin sectioning

for each animal.

39

RESULTS

The adrenal gland consists of two major areas, the medulla and the
cortex. The medulla synthesizes the hormone norepinephrine and epinephrine.
The cortex is composed of three zones each having specific functions. The zona
glomerulosa is the outer most area it compromises about 15% of the adrenal
gland and synthesizes mineralocorticoids [3-6]. The zona fasciculata is the
middle area and compromises about 70% of the adrenal gland and synthesis the
glucocorticoids one of which is corticosterone [3-6]. The zona reticularis the
innermost layer and borders the medulla, it comprises about 7% of the adrenal
gland and synthesizes the sex hormones [3-6]. Mitochondria are prevalent in
both the medulla and the cortex, although their function is somewhat different in
each. The mitochondria of the medulla are less prominent and their function is
related to the energy process of the cell [9]. The mitochondria of the cortex are
very prevalent and play a major role in steroidogenesis because the
hydroxylases required for steroid synthesis are located at the inner mitochondrial
membranes [10].

The normal structure of the adrenal gland has been well defined and will
not be repeated in this text [3-6, 29].

Medulla

The medullary secretory cell membranes in both of the CDDP treated

groups were ill-defined compared to the controls with an apparent decrease in

the number of nuclei on days 3 and 8 in the 7mg/kg dose (Figure 2-3) and days 3

40

and 5 in the 9mg/kg dose (Figure 4-5). There was an apparent increase in the
number of nuclei on day 5 in the 7mg/kg dose (Figure 1-2). The medullary
capillaries in the 7mg/kg dose were enlarged, have accumulated cellular debris
within the Iumens and the endothelial lining is degenerating (Figure 1-2). The
venous channels of the 9mg/kg dose (Figure 4-5) did not have the
aforementioned stmctural damage. At the EM level the mitochondria of both
groups demonstrated an increase in damage as the days progressed post-
treatment (Figure 6-9). This damage was apparent by the rarefaction and
cavitation of the mitochondria. The granules within the norepinephrine and
epinephrine cells were swollen and reduced in numbers on day 3 with the
7mg/kg dose (Figure 6 -9). By days 5 and 8 there was little distinction between
the norepinephrine and epinephrine cells. Usually they vary in darkness with the
norepinephrine cells appearing more white (the light cells) and the epinephrine
cells appearing darker (the dark cells). Granules were swollen on all days and
appeared to start to increase in number by day 8 with the 7mg/kg dose (Figure
7). The granules in the norepinephrine and epinephrine cells in the 9mg/kg dose
were almost completely depleted.
Cortex

The medullary capillaries extend into and through the zona reticularis on
days 3 and 5 of both treatments (Figure 10 and 11). The zona reticularis of the
9mg/kg dose (Figure 11) and the zona fasciculata (Figure 10) of the 7mg/kg dose
on days 5 and 8 demonstrated clear areas that are indicative of an increase in

the number of lipid compartments and/or an increase in the number of

41

mitochondrial lipid like inclusions. At the EM level (7mg/kg dose only) the
medullary capillaries on days 5 and 8 were enlarged with accumulated cellular
debris and endothelial lining degeneration (Figure 12 and 13). There was
observed a disruption of both the medullary and zona reticularis mitochondria,
increasing with time. The number of lipids in the zona reticularis increased by 2
fold by day 8 in the 7mg/kg dose compared to that of the control. This increase
in lipids verifies what was seen at the light microscopic level.

The zona reticularis cells (Figure 14 and 15) in both treatment groups
showed an apparent decrease in the number of nuclei, with day 5 and 8 (7mg/kg)
showing some increase compared to the earlier treatments. The zona reticularis
(9mg/kg) and the zona fasciculata with both treatment groups and after various
days of post-treatment (Figure 14-18) showed disruption of the secretory cells
with large clear areas which as mentioned previous indicate an increase in the
number of lipid compartments and/or in the number of mitochcondrial lipid like
inclusions. Lipids (7mg/kg dose only) have decreased by day 3 with an increase
(Figure 14 and 15) by day 8. The 9mg/kg dose has decreased lipids on both
days 3 and 5, shown previously [19]. At the EM level (Figure 19-22) there was
observed damage to the mitochondrial cristae with some recovery after day 5
and 8 in the 7mg/kg dose with no recovery with the 9mg/kg dose. The capillaries
were enlarged with cellular debris in the lumen. There was also an increase in
the number lipids with time in the 7mg/kg dose only (Figure 19 and 20), where
both doses of cisplatin showed some lipids cases with increased compartment

size.

42

The zona glomerulosa on day 5 in the 9mg/kg dose appeared to have
decreased cytoplasm and nuclei compared to that of the control. The adrenal
capsules were intact and appear to be unaffected by the treatment of CDDP at

either dose (Figure 23 and 24).

43

Figure 1: Light Micrographs of Sections (2pm) of the Adrenal Gland of Control (A)
and After 7mg/kg CDDP Treatment at (B) 3 Days, (C) 5 Days, (D) 8
Days.

The capillaries (V) in the medulla (M) are enlarged and extending into the zona
reticularis (zr). The zona reticularis has enlarged capillaries on day3, the zona
fasciculata (ZF) by day 5 and the zona glomerulosa (ZG) by day 8. Methylene
blue stain. Bar = 2mm

44

 

Figure 2: Light Micrographs of Sections (2pm) of the Adrenal Medulla of Control
(A) and After 7mg/kg CDDP Treatment at (B) 3 Days, (C) 5 Days, (D) 8
Days.

The secretory cell (arrows) membranes are not as defined, there appears to be
fewer nuclei on days 3 and 8 with an increase on day 5. Note the enlarged
capillaries (V) with red blood cells (R) and cellular debris within the lumen. The
areas enclosed by square enclosures are enlarged in Figure 2. Hematoxylin

and eosin stain. Bar = 0.02mm

46

 

47

Figure 3: Light Micrographs Showing the Magnified View of Various Cellular
Changes Before (A) and After CDDP (7mg/kg) Treatment at (B) 3
Days, (C) 5 Days and (D) 8 Days.

The cell membranes are not as defined and the appearance of fewer nuclei
(arrow) on day 3 and 8. The capillaries (V) are enlarged with red blood cells (R)
and cellular debris with in the lumen. Hematoxylin and eosin stain. Bar =

0.0.006mm

48

 

49

Figure 4: Light Micrographs of Sections (2pm) of the Adrenal Medulla of Control
(A) and After 9mg/kg CDDP Treatment at (B) 3 Days and (C) 5 Days.

The medullary cell membranes are losing definition and there is a decrease in
the number of nuclei (arrows) in the post-treatment. At this magnification there is
no apparent damage to the capillaries (V). The areas enclosed by square
enclosures are enlarged in Figure 5. Hematoxylin and eosin stain.

Bar = 0.02mm

50

,. (Vt.

\

 

'31

Figure 5: Light Micrographs Showing the Magnified View of Various Cellular
Changes Before (A) and After CDDP (9mg/kg) Treatment at (B) 3 Days
and (C) 5 Days.

Day 3 has no membrane distinction between the medullary cells compared to
that of day 5. Both day 3 and 5 appear to have a decrease in the number of
nuclei (arrows). The capillaries (V) show no structural damage. Hematoxylin
and eosin stain. Bar = 0.006mm

52

 

Figure 6: Electron Micrographs of Medullary Region of the Adrenal Gland of
Control (A) and After 7mg/kg CDDP Treatment at (B) 3 Days, (C) 5 Days and (D)
8 Days.

The nuclei (N) and myelinated nerves (n) that are present show no change. The
mitochondria (M) show increasing damage With time. This is evident in the
cristae (arrowhead) or cavitation of the mitochondrial cells (no cristae).
Chromaffin cells both norepinephrine (back arrow) and epinephrine (white arrow)
have granules that appear swollen by day 3. By day 5 there is less differentiation
between the norepinephrine (black arrow) and epinephrine (white arrow) cells,
with fewer granules within the epinephrine cells. The medullary capillaries (V)
are enlarged with red blood cells (R) in the lumen and the endothelial lining
appears degenerated. By day 8 there are fewer granules in the norepinephrine
cells (white arrow) while the epinephrine granules (black arrow) are swollen.

Bars = 400nm

54

 

o)

 

Figure 7: Selected Areas from Figure 6 Have Been Enlarged to Show Changes
Before (A) and After CDDP (7mg/kg) Treatment at 3 Days (B), 5 Days
(C) and 8 Days (D) in the Medulla.

Arrows (black) norepinephrine cell, (white) epinephrine cell, N = nucleus, n =

myelinated nerve, V = capillary, M = mitochondria and arrowhead (black)
cavitated mitochondria. Bars = 150nm

56

 

 

Figure 8: Electron Micrographs of Medullary Region of the Adrenal Gland of
Control (A) and After 9mg/kg CDDP Treatment at (B) 3 Days, (C) 5
Days and (D) 8 Days.

The nuclei (N) show no change. There is progressing disruption of the
mitochondria as shown by the cavitation (arrowhead) of the cell. This disruption
appears to be more prevalent in the norepinephrine cells than in the epinephrine
cells. The norepinephrine (black arrow) and epinephrine (white arrow) granules
appear to be decreased in number as time progresses. Bar = 400pm

58

 

A
O

 

A
d

 

Figure 9: Selected Areas from Figure 8 Have Been Enlarged to Show Changes
Before (A) and After CDDP Treatment at (B) 3 Days and (C) 5 Days, in
the Medulla.

Arrows (black) norepinephrine cell, (white) epinephrine cell, N = nucleus, M =
mitochondria and arrowhead cavitated mitochondria. Bar = 150m

60

 

A)

Figure 10: Light Micrographs of Sections (2pm) of the Medulla, Zona Reticularis
and Zona Fasciculata of the Adrenal Gland of Control (A) and 7mg/kg
CDDP Treatment at (B) 3 Days, (C) 5 Days and (D) 8 Days.

The medullary (M) cell membranes are less defined as in previous with an

apparent decrease in the number of nuclei. Note the capillaries (V) that are

extending through the zona reticularis (ZR). The zona fasciculata (ZF) has many
clear areas (smaller areas) that are indicative of a higher number of lipid
compartments and/or an increased number of mitochondrial lipid like inclusions
on days 5 and 8 compared to that of day 3 and the control. Hematoxylin and

eosin stain. Bar = 0.02mm

62

 

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. ‘: “7543' . p .g

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v'

 

 

A

Figure 11: Light Micrographs of Sections (2pm) of the Medulla and the Zona

Reticularis of the Adrenal Gland of Control (A) and After 9mg/kg
CDDP Treatment at (B) 3 Days, (C) 5 Days and (D) 8 Days.

The medullary (M) cell membranes have very little to no distinction on day 3, with
day 5 showing an improvement, there appears to be a decrease in the number of
nuclei from the control. The medullary capillaries (V) have extended into the
zona reticularis (ZR) by day 5. The zona reticularis has increased damage as
post-treatment time progresses, with the appearance of small circular clear are as
that are indicative of and increase in the number of lipid compartments and an

increase in the number of mitochondrial lipid like inclusions. Hematoxylin and
eosin stain. Bar = 0.02mm

64

 

 

 

Figure 12: Electron Micrographs of the Medulla and Zona Reticularis of the
Cortex of the Adrenal Gland of Control (A) and After 7mg/kg CDDP
Treatment at (B) 3 Days, (C) 5 Days and (D) 8 Days.

The nuclei (N) in the medulla show no change in structure. The capillaries (\l)
are enlarged, with a red blood cell (R) present in the lumen of the control and an
accumulation of cellular debris at days 5 and 8. The endothelial lining (a) has
increased degeneration as time post-treatment progresses. The mitochondria
(arrowheads) of the medulla have increased damage to their cristae time post-
treatment progresses. The norepinephrine cells appear to have fewer gran ules
(black arrow) at day 3 with an increase in the number of granules at day 5 and 8.
All the granules within the norepinephrine cells from days 3-8 are swollen. The
zona reticularis of the cortex, which consists of many mitochondria (arrowheads)
shows increasing, damage as the time progresses. The number of lipids (L) h as
increased 2 fold by day 8 from the control. Bar = 400pm

66

 

 

0)

Figure 13: Selected Areas from Figure 12 Have Been Enlarged to Show
Changes Before (A) and After CDDP (7mg/kg) Treatment at 3 Days
(B), 5 Days (C) and 8 Days (D) in the Medulla and Zona Reticula ris.

Arrowheads = cavitated mitochondria, arrows = norepinephrine granules,

M =
mitochondria, L = lipids, G = golgi complex, N = nucleus, V = capillaries, R = red
blood cell and e = endothelial lining. Bar = 150um

68

 

H...

I
‘

 

 

Figure 14: Light Micrographs of Sections (2pm) of the Zona Reticularis and Zona
Fasciculata of the Adrenal Gland of Control (A) and After 7mg/kg
CDDP Treatment at (B) 3 Days, (C) 5 Days and (D) 8 Days.

The network like clumps of secretory cells of the zona reticularis (ZR) are less
defined and appear to have fewer nuclei on day 3 compared to days 5, 8 and the
control. Days 5 and 8 appear to have an increase in the number of nuclei but no
apparent improvement in the cellular organization. The narrow chords of
secretory cells of the zona fasciculata (ZF) in all treatment groups show
disruption with clear areas (small areas) (arrows) that indicate an increase in lipid
compartments and an increase in the number of mitochondrial lipid like inclusions
compared to that of the control. The areas enclosed by square enclosures are
enlarged in Figure 15. Hematoxylin and eosin stain. Bar = 0.02mm

70

 

 

Figure 15: Light Micrographs Showing the Magnified view of Various Cellular

Changes Before (A) and After CDDP (7mg/kg) Treatment at (B) 3 days,
(C) 5 Days and (D) 8 Days..

There appears to be a decrease in the number of nuclei (arrows) from day 3-8,
although day 5 and 8 have an increased number of nuclei compared to day 3.
There is an increase in the number of small round clear areas as time post-
treatment progresses. These areas are indicative of an increase in the number
of lipid like compartments and an increase in the number of mitochondrial lipid
like inclusions. Note the larger elongated clear areas (Va) which are capillaries

some with red blood cells (R) in the lumens. Hematoxylin and eosin stain.
Bar = 0.007mm .

72

 

,.
C

 

Figure 16: Light Micrographs of Cryo-Sections (10pm) of the Zona Fasciculata
and Zona Glomerulosa of the Adrenal Gland of Control (A) and After
7mg/kg CDDP Treatment at (B) 3 Days, (C) 5 Days and (D) 8 Days.

Clear areas within the zona fasciculata denote lipid deposition. There is a

depletion of lipids on day 3, with an increasing number on day 5 and 8. Note the
enlarged capillaries (arrow) on day 5 and 8. Sudan black stain. Bar = 0.2mm

74

 

Figure 17: Light Micrographs of Sections (2pm) of the Zona Reticularis and Zone
Fascicularis of the Adrenal Cortex of Control (A) and After 9mg/kg
CDDP Treatment at (B) 3 Days and (C) 5 Days.

The zona reticularis (ZR) of the treated groups appear to have fewer nuclei.
There is an increase in the number of small round clear areas (arrows) in both
the zona reticularis and the zona fasciculata (ZF) of the treated groups with day 5
having a very high percentage of these areas. These areas indicate an increase
in the number of lipid compartments and an increase in the number of
mitochondrial lipid like inclusions. The areas enclosed by square enclosures are
enlarged in Figure 18. Hematoxylin and eosin stain. Bar = 0.02mm

76

 

Figure 18: Light Micrographs Showing the Magnified View of Various Cellular
Changes Before (A) and After CDDP (9mg/kg) Treatment at (B) 3
Days and (C) 5 Days.

The chords of secretory cells that make up the zona fasciculata are not as
defined as the controls, and appear to have a decrease in the number of nuclei.
There is a decrease in the number of small clear areas (arrow) as time
progresses. These areas are indicative of lipid compartments. Note the larger
elongated clear areas (Va) capillaries that are increasing in size, some with red
blood cells within the lumen (R). Hematoxylin and eosin stain. Bar = 0.01 mm

78

 

 

Figure 19: Electron Micrographs of the Zona Fasciculata of the Adrenal Cortex of
Control (A) and After 7mg/kg CDDP Treatment at (B) 3 Days, (C) 5
Days and (D) 8 Days.

The nuclei (N) show no alteration in structure. The mitochondria (M) have
increased disruption to their cristae through day 5 with less apparent disruption
on day 8. There is an increase in the number of lipids (L) as the post-treatment
time progresses, with some showing an increase in the lipid compartment. The
controls capillaries (S) has a red blood cell (R) within its lumen, but does not
have the enlarged size or accumulation of cellular debris within the lumen,
including platelets (p) that is seen in the treated. The endothelial lining (e) of the
sinusoids are degenerate (arrow) in all treated groups. Bar = 400um

80

 

 

 

 

Figure 20: Selected Areas from Figure 19 Have Been Enlarged to Show
Changes Before (A) and After CDDP (7mg/kg) Treatment at 3 Days
(B), 5 Days (C) and 8 Days (D).

N = nucleus, M = mitochondria, arrowhead = cavitated mitochondria, arrow =

mitochondria with damaged cristae, L = lipids, S = capillaries, e = endothelial
lining and p = platelets. Bar = 150um

82

 

n\

Figure 21: Electron Micrographs of the Zona Fasciculata of the Adrenal Cortex of
Control (A) and After 9mg/kg CDDP Treatment at (B) 3 Days and (C)
5 Days.

The nuclei (N) show no apparent difference. The mitochondria (M) have irregular
cristae or no cristae (arrowhead). The disruption of the mitochondria increases
as time post-treatment progresses. There are less lipids (L) with the lipid size
increasing by day 5. Bar = 400um

84

 

Figure 22: Selected Areas from Figure 21 Have Been Enlarged to Show
Changes Before (A) and After CDDP Treatment at 3 Days (B) and 5
Days (C).

N = nucleus, M = mitochondria, arrowhead = cavitated mitochondri, L = lipids.
Bar = 150nm

86

 

 

 

Figure 23: Light Micrographs of Sections (2pm) of the Zona Fasciculata and
Zona Glomerulosa of the Cortex of Control (A) and After 7mg/kg CDDP
Treatment at (B) 3 Days, (C) 5 Days and (D) 8 Days.

As described previously the zona fasciculata (ZF) appears to have a decreased
number of nuclei (arrows) on days 3-8, although days 5 and 8 appear to have
increased nuclei compared to day 3. There is an increased number of elongated
clear areas (arrowheads) enlarged capillaries from the control. There is also an
increased number of small round clear areas on days 5-8 which indicate an
increase in the number of lipid compartments and an increase in the number of
mitochondrial lipid like inclusions. There no apparent difference between groups
in the zona glomerulosa (ZG) or the capsule (Cap). Hematoxylin and eosin stain.
Bar = 0.02mm

88

 

Figure 24: Light Micrographs of Sections (2pm) of the Zona Fasciculata and
Zone Glomerulosa of the Adrenal Cortex of Control (A) and After
9mg/kg CDDP Treatment at (B) 3 Days and (C) 5 Days.

The zona fasciculata (ZF) has increasing disruption to its secretory cells and
appears to have fewer nuclei (arrows) as the time post-treatment progresses.
There are a large number of elongated clear areas (arrowheads) enlarged
capillaries compared to the control, which are increasing as time progresses.
There is a decrease in the number of small round clear areas that are indicative
of a decrease in the number of lipid compartments. The capillaries are starting to
enlarge and also extend into the zona glomerulosa (ZG) by day 5 with an
apparent decrease in the number of nuclei compared to day 3 and the control.
The capsules (Cap) are intact with no apparent change in structure. Hematoxylin
and eosin stain. Bar = 0.0

90

.
.
.
.
.
1 .
..,.
‘-
, r‘ .
. .'

u.
,

7t

5

t

 

DISCUSSION

A number of drugs and noxious chemicals are know to alter the
structural integrity of the adrenal gland by destruction (necrosis and
hemorrhage), degeneration (senile nodular hypertrophy, telangiectasia, focal
fatty changes), adaptive reactions (phospholipid lysomal inclusion) and
neoplastic alterations [8, 12, 30]. The medullary mitochondria cristae are
disrupted with the secretory cells having fewer granules, with the remaining
granules being swollen. The capillaries are enlarged and have signs of
endothelial lining degeneration. It has been suggested that some types of
cortical endothelial injury may be secondary to the medullary damage, which
subsequently releases catecholamine granules into the medullary capillaries and
the cortical capillaries [8, 20, 31]. Cisplatin is known to cause severe
telangiectasia and loss of the lipid vesicles from the zonae fasciculata and
reticularis shortly after treatment and at the9mg/kg dose for the duration of the
experiment, then increasing as time progresses post-treatment [18, 19]. This
occurrence is also seen with other chemotherapeutic drugs [13], and toxic
chemicals [21, 22, 25, 26, 32]. The loss of lipid vesicles could stimulate the
system to retrieve new cholesterol for steroid production, which in rats is mainly
from an exogenous source [33]. Lipids are necessary for the synthesis or steroid
hormones because they carry the cholesterol that will be converted into
pregnenolone [8, 11, 29, 34]. The mitochondria are the most susceptible and are

altered to the point of being abnormal. The mitochondrial compartment is

92

enlarged with the matrix and inner membranes being completely disrupted.
Other studies have reported these same results with chemotherapeutic agents
[13, 21] other toxic chemicals ([16, 22] and hormones such corticotropin
releasing hormone and adrenocorticotropic hormone [11, 15, 24, 35]. Cisplatin
when hydroxylated is known to uncouple oxidative phophorylation in isolated
mitochondria, a necessary part of the mitochondrial function [36]. The
mitochondria of the zona fasciculata are the site of corticosterone synthesis thus
any disruption can effect the concentration of corticosterone [6, 9-11, 20]. The
disruption of the zona fasciculata at first may allow for release of corticosterone
into the system, but then can interfere with steroid production by disrupting the
contact between the smooth endoplasmic reticulum, mitochondria and the lipids.
This contact is very important for the synthesis of steroids by allowing exchange
of enzymes, cholesterol and intermediate products between these areas. We
have previously shown that circulating corticosterone concentration is increased
on day 1 and again at day 5 post-treatment in the lower dose of cisplatin (chapter
1) with corticosterone (ng/ml) output from adrenal explants being relatively the
same. This is most likely due to the release of corticosterone due to an increase
in stress and structural damage caused by CDDP at first and then to the increase
in steroid synthesis seen by the increase of lipids in the cortex. Whereas
corticosterone concentration decreased in the higher dose of CDDP on day 1
with an increase at day 3, this could be do to the difference in dose itself. The

animals treated with the higher dose emulate a much worse overall condition.

93

Cisplatin appears to affect the adrenal gland directly by endothelial
damage, which then causes further damage to the cell, and indirectly by
degranulation of medullary and cortical secretory cells. With the release of
epinephrine and corticosterone into the circulatory system and decrease in the
lipid droplets that store cholesterol esters for steroidsynthesis [37] post-treatment
with lipid recovery as days progress post-treatment. To determine the
mechanism that alters adrenal gland function and corticosterone release, further

studies are needed using adrenal organ culture or adrenal cell culture.

94

CONCLUSIONS

Cisplatin affects the adrenal gland structure by disruption of mitochondria
and secretory cells, enlargement and endothelial lining degradation of the venous
channels in both the medulla and cortex and by decreasing and then increasing
lipid droplets in the cortex. These structural alterations then affect the hormonal
release and synthesis in both the medulla and cortex changing the circulating
concentration and secretion into media by adrenal glands of corticosterone.
Further studies are required using adrenal organ culture and/or adrenal cell

culture to verify these findings.

95

10.

11.

12.

13.

14.

15.

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Simpson, ER. and MR Waterman, Regulation of the syntheisi of
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Boshier, D.P., P. Rebuffat, and G.G. Nussdorfer, Cellular responses of the
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Szabo, S., et al., Thioguanine-lnduced adrenocortical necrosis and its
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de Castro, OR, at al., Ultrastructural effects of nifurtimox on rat adrenal
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Malendowicz, L.K., Sex differences in adrenocortical structure and
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Mazzocchi, G., et al., Investigations on the turnover of adrenocortical
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Durham, SK. and A. Brouwer, 3,4,3',4'-Tetrachlorobiphenyl distribution
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Mazzocchi, G., et al., Effects of testosterone on the zona fasciculata of the
male rat adrenal cortex. A correlated stereological and biochemical study.
J Submicrosc Cytol, 1983. 15(4): p. 991-1005.

Miller, C.M., S.K. Aggarwal, and K.K. Klomparens, Cisplatin-induced
changes in the adrenal of the rat, in Proc Electron Microscope Society of
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Francisco Press: San Francisco. p. 1652-3.

Aggarwal, S.K., et al., Cisplatin-induced peptic ulcers, vagotomy, adrenal
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de Castro, C.R., E.G.D. de Toranzo, and J.A. Castro, Benmidazole-
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Robba, C., G. Mazzocchi, and G.G. Nussdorfer, Effects of a prolonged
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Robba, C., et al., Opposed effects of chronic prolactin administration on
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Mazzocchi, G., et al., Long term effects of the hypocholesterolaemic drug
4-aminopyrazolo- pyrimidine on the zona fasciculata of the rat adrenal
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Magalhaes, M.M., et al., The effects of monensin on golgi complex of
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Malendowicz, L.K., Comparative studies on the effect of 4-APP(4-
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Lullmann, G. and R. Lullmann-Rauch, Tamoxifen-induced generalized
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Szabo, S. and ES. Reynolds, Structure-activity relationships for
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thiols. Environ Haskk Perspect, 1975. 11: p. 135-40.

Robba, C., et al., Effects of prolonged treatment with cyanoketone on the
zona fasciculata of rat adrenal cortex. A combined morphometric and
biochemical study. Cell Tissue Res, 1987. 250(3): p. 599-605.

Boyd, G.S., et al., Cholesterol metabolism in the adrenal cortex. J Steroid
Biochem, 1983. 19: p. 1017-27.

Rapp, j.P., Adrenal steroid biosynthesis and metabolism, in The Adrenal
Gland, P.J. Mulrow, Editor. 1986, Elsevier: New York. p. 65-78.
Malendowicz, L.K., et al., Neuromedin-N increases the levels of circulating
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Aggarwal, S.K., Cisplatin-induced peptic ulcers and calcium modulation.
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Black, V.H., R.l. Brody, and KC. Martin, 3-Hydroxy-3-methylglutaryl
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98

CHAPTER THREE: CISPLATIN — INDUCED ALTERATIONS IN
CORTOCOSTERONE CONCENTRATION AND
STRUCTURE OF ADRENAL GLAND CULTURES FROM
THE RAT

99

SUMMARY

Adrenal glands and a pituitary were removed from Wistar CRL:(W|)BR
rats for two groups, put into organ culture and treated with normal saline (vehicle
for cisplatin) or 70 m/ml for specific times. Medium was collected for group 1 at
24, 0, 8, 20, 32, 56 and 80 hours to analyze for corticosterone concentration and
adrenals were collected and processed for light microscopy at 32, 56 and 80
after treatment with cisplatin. Medium for group 2 was collected at 24, 0, 2, 4, 6
and 8 hours for corticosterone concentration and the adrenals collected and
processed for light microscopy at 2, 4, 6 and 8 hours after treatment. The
corticosterone concentration for group 1 showed a decrease from that of the
control at all times except 32 hours. At this time the control and treated were at
the same concentration. Both the control and treated showed a decrease in
corticosterone concentration over the timeframe of the experiment, possibly due
to a decrease in ACTH. Group 2 showed no real difference in corticosterone
levels throughout the experiment. Microscopically the treated groups showed

increased disruption of the secretory cells compared to that of the controls.

100

INTRODUCTION

Noxious compounds can create trauma and induce stress at the physical,
metabolic and cellular levels in the in vivo system. The initial stress will cause
the system to respond hormonally by the release of cortocotropic releasing
hormone (CRH) from the hypothalamus, stimulating the pituitary to release
adrenocortocotrophic hormone (ACTH) which stimulates the adrenal to
synthesize corticosterone in rats and cortisol in humans [1-4]. Stress caused by
disease, physical, metabolic and or cellular changes can also stimulate the
immune system, which in turn increases the synthesis of glucocorticoids and the
adrenal glands response to this stress [5-12]. This stimulation often overrides
the normal feedback inhibition that glucocorticoids have on pituitary ACTH to
allow prolonged production of glucocorticoids from the adrenal gland [5, 8, 13].

As a noxious compound induces physical, metabolic and/or cellular
alterations to the system it may also have an affect on the concentration of
circulating corticosterone. Is a change in the concentration of corticosterone due
to the stress-related alterations in the body or due to the added noxious
compound itself? To evaluate this problem the adrenal gland can be and
examined at the cellular level or at the organ level. This allows for examination
without the influence of uncontrolled hormonal, neurogenic or hemodynamic

factors [14, 15].

101

Studies of adrenal organ, slice or cell culture have been used to look at
chemotherapeutic agents [14, 16-20], noxious compounds [21, 22], hormones
[20, 23-33], certain peptides [34-38] and interleukins [6-12, 39, 40] influence on
catecholamine and steroid synthesis by secretion into the media and the
structure of the adrenal gland. Other studies have examined specific points in
steroid synthesis to find where stimulation and/or inhibitions take place [25, 26,
31, 32, 41-43]. In some studies (1) ACTH was supplemented to maintain a
basal stimuli and secretion of corticosterone allowing any change in secretion to
be the difference from the basal output [9, 11, 16, 26-28, 31, 35, 44, 45], (2) the
intact pituitary was put into organ culture with the adrenal to achieve stimulus by
a different means, this does not give a basal level that may be constant but
allows more of the natural stimuli, though it can allow for more variability in the
secretion of corticosterone [22, 29, 32], (3) or use no hormonal supplement so
there is only the corticosterone and the effect of what compound you are
supplementing the media with [9, 11, 16, 17, 25, 26, 30, 34, 35, 37, 40, 42, 45].
In cell or organ culture the concentration of the hormone secreted into the media
varies. Sibley et al. [46], found that the variables in the relative amounts of
steroids secreted into the incubation media may be a product not only of a
change in synthesis of the steroid but a change in how it is secreted from the
fissue.

It has been shown that treatment with cisplatin induces physical stress to
the body by lowering body weight and food intake with alterations in water intake

and urine output [47-51], metabolic alterations by the changes in corticosterone

102

concentrations and at the cellular level in the adrenal gland [52-54].To determine
if these alterations are due to cisplatin induced stress on the system or on the
adrenal gland itself, the organ was explanted and put into culture. Its activity was

monitored both structurally and functionally at time intervals.

103

MATERIALS AND METHODS

Organ Culture: Wistar Crl:(Wl)BR male rats weighing 200 to 2509 each
from Charles River were housed 3 to a cage with a light/dark cycle of 12 hours
with food and water ad lib. Animals were allowed to adapt to their new
environment for 3 days prior to adrenal culture. Animals were then decapitated;
the pituitary and two adrenal glands were collected under sterile conditions and
placed in HEPES buffer. Each adrenal gland was cut into 2, and these four
pieces along with a pituitary were placed on a stainless steel mesh support within
a culture dish, such that the medium under the support moistened the adrenal
and pituitary explants for two separate Groups and timeframes were used (see
Table 1 and Table 2). The timeframes for Group 2 were determined after the
analyses of the corticosterone concentration in the culture medium from Group 1.
The adrenal organ cultures were maintained at 37 °C in a humidified
atmosphere of 5% 002 and 95% air. The culture medium was prepared following
the protocol of Szkudlinski, M., eta]. (1989). Media was changed and collected
after a specific time (see Table 1 and Table 2). The cultures were allowed to
adapt to the environment for three days prior to treatment with CDDP, then
treated with physiological saline or 7um/ml media of CDDP for a specific time
(see Table 1 and Table 2). The adrenals were collected and processed for light

and microscopy.

104

Table 1: Group 1 Control and CDDP (7um/ml) Adrenal Organ Culture Media
Changes and Collection Times by Post-Treatment and Time from Last Media
Change. Treatment time is 8 hours. (* = media contains saline or CDDP, bold =

adrenal glands collected and processed for light microscopy)

 

Time
(Hours) =>
Media

 

Chflged -72 -48 -24 0 8* 1 2 1 2 24 24

 

Post-
Treatment 8* 20 32 56 80
Hours

 

 

Collected
(-80°CL -24 0 8* 1 2 1 2 24 24

 

 

 

 

 

 

 

 

 

 

Table 2: Group 2 Control and CDDP (7pm/ml) Adrenal Organ Culture Media
Changes and Collection Times. Treatment time is 2, 4, 6 and 8 hours. (* = media
contains saline or CDDP, bold = adrenal glands collected and processed for light

microscopy)

 

Time
(Hours) :>
Media

 

Chgged -72 -48 -24 0
Collected
(-80°C) -24 0 2* 4* 6* 8*

 

 

 

 

 

 

 

 

 

 

 

 

105

 

Corticosterone Analysis: Corticosterone content in the serum was
analyzed by a Rat Corticosterone-3H Kit (ICN) and read in a Beckman 7500
series scintillation counter as described earlier.

Light Microscopy: The adrenal halves were fixed in 2% glutaraldehyde in
0.1M sodium phosphate buffer (PBS), pH 7.4, dehydrated in graded ethanol
series and embedded in JB4 (glycol methacrylate, Polyscience, lnc.). Sections

(2pm) were stained with hematoxylin and eosin for general histology.

106

RESULTS

Corticosterone Concentration

The corticosterone concentrations in the media from Group 1 (Figure 1)
decreased at 8, 20, 56 and 80 hours post-treatment. There was no significant
difference between the control and CDDP treated cultures prior to treatment. At
32 hours post-treatment the control had a decrease in corticosterone
concentration whereas the treated has an increase in concentration with no
difference between the two. The CDDP treated cultures decrease in
corticosterone revealed a trend towards significance, although the sample size
was insufficient to determine this statistically. The control cultures also had a
decrease in corticosterone concentration at day 0, 32 and 56 this could be due to
a decrease in secreted ACTH from the pituitary gland.

The corticosterone concentration in the media from Group 2 (Figure 2)
decreased 2, 6 and 8 hours into treatment with CDDP. The treated 4-hour
culture exhibited a slight increase in corticosterone concentration, with no
significant difference between the control and CDDP treated cultures prior to
treatment. The sample number was insufficient to determine if the decreases or

increase in corticosterone secretion at the given times is statistically significant.

107

1 400

 

 

 

 

 

 

 

 

 

 

 

1200
$1000
5
g 800
B
:3, 600
O
.2
E 400
O
200
0

-24 0 8 20 32 56 80
Hours

Figure 1: Corticosterone Concentration Present in the Media of Group 1 Adrenal
Organ Cultures at Specific Times.

There is no significant difference in the concentration of corticosterone in the
media between the treated and control groups prior to treatment or at 32 hours
post-treatment. There does appear to be a difference in the corticosterone
concentration present in the media at 8, 20, 56 and 80 hours post-treatment.
The sample number was insufficient to determine whether this is statistically

significant between the two groups. [Average 1: SD]

108

1400 E: Control
- CDDP

1 200

—L
O
O
O

9

 

 

600

400

Corticosterone (ng/ml)

200

 

 

 

 

 

 

 

Hours

Figure 2: Corticosterone Concentration Present in the Media of Group 2 Adrenal
Organ Cultures at Specific Times.

There appears to be no significant difference in corticosterone concentrations in

the media at any treatment hour between CDDP treated and the control groups.

The sample number was insufficient to determine whether there is a significant

difference between the groups. [Average i 8.0.]

109

Group 1 Organ Culture
Medulla

The medullary secretory cells lacked cellular definition (Figure 3-6) in both
the control and cisplatin (CDDP) cultures with membranes becoming more
defined as the time of post-treatment progressed to 80 hours. There was an
apparent decrease in cytoplasm in the CDDP culture at 32 hours post-treatment
and the control and CDDP cultures at 56 hours post-treatment, whereas the 80-
hour post-treatment culture appeared to have increased cytoplasm. There is
apparent nuclear degeneration at 32 hours post-treatment in the CDDP cultures
and the number of nuclei appeared increased as time progresses. The CDDP
cultures capillaries had accumulated cellular debris within the Iumens and were
increased in number to that of the control at 56 hours at 80 hours post-treatment.
Cortex
Zona Reticularis

Both the control and CDDP cultures secretory cells (Figure 5-6) at 32
hours post-treatment demonstrated undefined cell membranes. At 56 hours
post-treatment the control has better membrane definition with both the control
and CDDP cultures having increased cytoplasm compared to the control and
CDDP 32 hour cultures. The 32-hour CDDP culture had a number of intensely
stained condensed nuclei that were not present in the other cultures. All cultures
had enlarged cortical capillaries with the enlargement being more pronounced in

the CDDP cultures as post-treatment time progresses.

110

Zona Fasciculata

The zona fasciculata (Figure 6-8) at 32 hours post-treatment lacked
organization of the cells into the chord-like structure that is seen with intact
adrenal glands in both the control and CDDP cultures. The CDDP culture at 32
hours has no cellular definition and has a number of intensely staining
condensed nuclei. There was an increase in cellularity at 56 hours post-treatment
in both the control and CDDP cultures from that of the 32-hour cultures, although
there was still significant cellular disruption. The 56-hour post-treatment culture
lacked membrane definition compared to its control culture. The zona fasciculata
was more organized at 80 hours as shown by the chord-like seen in the intact
adrenal gland. The control culture had better organization and cellular
membrane definition than the 80 CDDP culture, although there was still
significant cellular separation and disruption in both of the 80-hour post-treatment
cultures.
Zona Glomerulosa and Capsule

The 32 hour controls zona glomerulosa (Figure 8) had better cellular
definition with an apparent increased number of nuclei from the for the CDDP
culture with both having a number of intensely stained condensed nuclei. The
capsule was intact with an apparent increase in the number of nuclei in the
CDDP culture. As post-treatment time progressed the zona glomerulosa in both
the control and CDDP cultures had an apparent decrease in cytoplasm with
increased cellular disruption at both 56 and 80 hours post-treatment with damage

more pronounced in the CDDP cultures. The capsules were intact with an

111

apparent decrease in the number of nuclei as post-treatment time progressed

with only a few visible by 80 hours in both the control and CDDP cultures.

Group 2 Organ Culture
Medulla

The cisplatin (CDDP) culture at 2 hours treatment (Figure 9-11) had
apparent decreased cell membrane definition, cytoplasm and an accumulation of
cellular debris with in the Iumens of the capillaries. Both the control and CDDP
cultures had condensed nuclei that were intensely stained and the control
appeared to have and increased number of nuclei compared to the CDDP
culture. Both the control and CDDP cultures at 4 hours treatment had very little
membrane definition with intensely stained condensed nuclei and some nuclei
that were degenerating. The 4-hour control had accumulated cellular debris
within the lumen of the capillaries. As treatment time progressed the 6 and 8
hour treated cultures had increased membrane degradation. The 6-hour CDDP
had an apparent increase in cytoplasm from the previous 2 and 4-hour cultures
and to that of its control, although the control had increased cellular disruption
and intensely stained condensed nuclei.
Zona Reticularis

The zona reticularis (Figure 11-12) in both the control and CDDP at 2-8
hours treatment had enlarged cortical capillaries going into the zona fascicularis,
with areas of intensely stained nuclei at 2 hours treatment in the CDDP culture.

The 4-hour CDDP treated culture had less membrane definition. At 6 hours

112

treatment the control culture had an apparent decrease in cytoplasm compared
to the CDDP culture. The CDDP cultures at 6 or 8 hours treatment had areas of
condensed nuclei that were intensely stained with the cells having no membrane
definition.
Zona Fasciculata

After 2hrs of treatment (Figure 12-13) the CDDP culture had some
condensed nuclei that were intensely stained. All cultures had an apparent
decrease in cytoplasm, cellular separation and disruption, enlarged capillaries, a
decrease in membrane definition and an increase in the number of nuclei as the
treatment time progressed.
Zona Glomerulosa and Capsule

The zona glomerulosa in both the control and CDDP cultures at 2 and 4
hour treatment had apparent cytoplasmic loss with increased cytoplasm in the
CDDP culture at 2 hours compared to the other cultures. Both the control and
CDDP cultures at the above mentioned times had condensed nuclei that are
intensely stained. There appeared to be an increase in cytoplasm at 6 hours in
both the control and CDDP cultures with condensed intensely stained nuclei in
both the zona glomerulosa and the capsule. At 8-hour treatment both the control
and CDDP cultures appeared to have decreased cytoplasm with condensed

intensely stained nuclei in both the zona glomerulosa and the capsule.

113

Figure 3: Light Micrographs of Sections (2pm) of the Medulla of Group 1 Adrenal
Gland Organ Cultures.

A. Control, B. CDDP 32-Hours Post-Treatment:
The CDDP cultures medullary cell membranes are not well defined, with an
apparent decrease in cytoplasm and fewer obvious nuclei (arrow) that are
degenerating compared to the control. The treated cultures capillaries (V) have
an accumulation of cellular debris within the lumens. The areas enclosed are
magnified in figure 4.

C. Control, D. CDDP 56-Hours Post-Treatment:

Medullary cell number and nuclei (arrow) are relatively equal between the two
cultures, although the CDDP culture appears to have a decrease in cytoplasm
and an increase in the number of capillaries (V). The areas enclosed are
magnified in figure 4.

E. Control, F. CDDP 80-Hours Post-Treatment:

Both the control and CDDP cultures show an increase in cellular membrane
definition, and appear to have an increase in nuclei (arrow). The CDDP culture
appears to have a decrease in cytoplasm, with an increase in and number and
size of the capillaries (V) with an accumulation of cellular debris within their
lumens. The areas enclosed are magnified in figure 4.

All are stained with hematoxylin and eosin. Bar = 0.01

114

 

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115

Figure 4: Light Micrographs of the Enclosed Areas Enlarged from Figure 3.

A. Control, B. CDDP 32-Hours Post-Treatment:
The cellular membranes have no definition with the cells merging together in the
control and CDDP cultures. A number of nuclei (arrow) are condensing, with the
CDDP culture appearing to have a decrease in the number of nuclei (arrow) and
degeneration of those remaining. The CDDP culture also appears to have a
decrease in cytoplasm. The controls capillaries are free of cellular debris unlike
that of the CDDP culture (refer to Figure 3).

C. Control, D. CDDP 56-Hours Post-Treatment:

Both control and CDDP cultures secretory membranes are ill defined with
degenerating nuclei (arrow) and loss of cytoplasm, although the CDDP culture is
in worse condition. The two cultures capillary (V) and endothelial lining is intact
with no apparent difference between the two.

E. Control, F. CDDP 80-Hours Post-Treatment:

The CDDP cultures medullary secretory cell membranes have better definition
and appear to have an increase in the number of nuclei (arrow) compared to the
control culture and previous cultures. The CDDP cultures capillaries (V) are have
increased size with an accumulation of cellular debris within the lumens.

Bar = 0.005mm

116

 

117

Figure 5: Light Micrographs of Sections (2pm) of the Medulla and Zona
Reticularis (Junction of the Medulla and Cortex) of Group 1 Adrenal
Gland Organ Cultures.

A. Control B. CDDP 32-Hours Post-Treatment:

The CDDP cultures medullary (M) cells are less defined; there appear to be
fewer nuclei and a decrease in cytoplasm. The secretory cells of the zona
reticularis (ZR) are undefined in both the control and CDDP cultures, although
the CDDP culture has more enlarged capillaries (clear areas throughout the
fissue)

C. Control D. CDDP 56-Hours Post-Treatment:

Medullary (M) cell number and nuclei are relatively equal between the control
and CDDP cultures, although the CDDP culture appears to have a decrease in
cytoplasm. The ZR secretory cell membranes have better definition in the control
with the CDDP cultures capillaries being of increased size compared to the
control (large clear areas throughout the tissue) and the previous cultures.

E. Control F. CDDP 80=Hours Post-Treatment:

There is improved medullary (M) membrane definition in both the control and
CDDP cultures with the CDDP culture appearing to have a decrease in
cytoplasm and an increase in the number and size of the capillaries (V). The ZR
secretory cells are more defined with better membrane definition in the control
with the CDDP culture having and increase in the size of the capillaries.

All are stained with hematoxylin and eosin. Bar = 0.015mm

118

 

119

Figure 6: Light Micrographs of Sections (2pm) of the Zona Reticularis and Zona
Fasciculata of Group 1 Adrenal Gland Organ Cultures.

A. Control B. CDDP 32-Hours Post-Treatment:

The controls zona reticularis (ZR) cells have better membrane definition with
areas that have condensed nuclei in the CDDP culture. The CDDP culture has
no cell membrane definition in either the ZR or the zona fasciculata (ZF), even
though it appears to have more cytoplasm, although both cultures have large
areas of cellular disruption (arrow). The areas enclosed are magnified in figure 7.

C. Control D. CDDP 56-Hours Post-Treatment:

There appears to be increased cellularity in both the ZR and the ZF from that of
the 32-hour cultures, although the CDDP culture has very little cellular definition.
Again the CDDP culture appears to have more cells with both control and CDDP
cultures still having a significant amount of cellular disruption (arrow). The areas
enclosed are magnified in figure 7.

E. Control F. CDDP 80-Hours Post-Treatment:

The secretory cells of the ZR and the ZF in the control culture have better
organization and membrane definition than those of the CDDP culture and the
previous cultures at 32 and 56 hours, although cellular disruption (arrow) is still
seen in both cultures. The areas enclosed are magnified in figure 7.

All are stained with hematoxylin and eosin. Bar = 0.014mm

120

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Figure 7: Light Micrographs of the Enclosed Areas Enlarged from Figure 6.

A. Control B. CDDP 32-Hours Post-Treatment:
There zona fasciculata has no cellular membrane definition and the nuclei are
condensing in the CDDP culture, although the control appear to have less
cytoplasmic area (arrow).

B. Control D. CDDP 56-Hours Post-Treatment:

There is an increase in cellularity of the ZF in both the control and CDDP cultures
with the control having better cellular definition, although cellular damage (arrow)
is still apparent in both.

E. Control F. CDDP 80-Hours Post-Treatment:

The secretory cells of the ZF are more organized in both the control and CDDP
cultures compared to the previous cultures at 32 and 56 hours, this is apparent
by the chord-like structure of the cells as is seen in the intact adrenal gland. The
CDDP culture has little cellular membrane definition with both the control and
CDDP cultures having separation of the cells and disruption (arrow).

Bar = 0.008mm

122

 

123

Figure 8: Light Micrographs of Sections (2pm) of the Zona Fasciculata, Zona
Glomerulosa and Capsule Group 1 Adrenal Gland Organ Cultures.

A. Control B. CDDP 32-Hours Post-Treatment:

The secretory cells (arrow) of the upper regions of the zona fasciculata (ZF)
closer to the zona glomerulosa (ZG) in both the control and CDDP cultures
appear to have less cellular damage compared to lower regions of the ZF in
previous figures. The cells are lacking the organization needed to form the
chord-like structure that the ZF displays in the intact adrenal gland. There
appears to be an increase in the number of nuclei in the 2G in the CDDP culture
compared to the control with the nuclear condensation apparent in both the
control and CDDP cultures. The adrenal capsule (Cap) appears to be intact with
a number of intensely stained condensed nuclei in the CDDP culture.

C. Control D. CDDP 56-Hours Post-Treatment:

The control and CDDP culture appear to have a decrease in cytoplasm (arrow)
with the CDDP culture having little cellular definition (arrow) in the ZF. There is
increased disruption of the secretory cells of the ZG in both the control and
CDDP cultures from that of the 32 hour cultures with some intensely stained
condensed. The adrenal capsules (Cap) appear intact with a decrease in the
number of nuclei from the 32-hour cultures.

E. Control F. CDDP 80-Hours Post-Treatment:

The secretory cells (arrow) of the ZF in both the control and CDDP cultures
appear to be trying to organize into the chord-like structure apparent in an intact
adrenal gland, although they are lacking cellular membrane definition. There
appears to be a decrease in the number of secretory cells, organization and in
the number of nuclei in ZG of both the control and CDDP cultures. The adrenal
capsules (Cap) are intact but devoid of nuclei in both the control and CDDP
cultures.

All are stained with hematoxylin and eosin. Bar = 0.01 mm

124

 

Figure 9: Light Micrographs of Sections(2pm) of the Medulla of Group 2 Adrenal
Gland Organ Cultures.

A. Control B. CDDP 2-Hours Treatment:

The CDDP culture appears to have a loss of cytoplasm and membrane definition
with both the control and CDDP cultures having condensed nuclei. The CDDP
cultures capillaries (V) have accumulated cellular debris within the lumens. The
areas enclosed are magnified in figure 10.

C. Control D. CDDP 4-Hours Treatment:

The CDDP culture has little membrane definition with the capillaries (V) having
no apparent damage in either control or CDDP cultures. The areas enclosed are
magnified in figure 10.

E. Control F. CDDP 6-Hours Treatment

The control appears to have an increased number of nuclei and cellular
disruption with a decrease in the cytoplasm compared to the 2-hour cultures and
its CDDP partner. The CDDP culture has an increase in cytoplasm. The
capillaries (V) are intact with no apparent damage to either the control or CDDP
cultures. The areas enclosed are magnified in figure 10.

G. Control H. CDDP 8-Hours Treatment:

Both the control and CDDP cultures have a loss of membrane definition,
decreased nuclei with the CDDP culture having a decrease in cytoplasm. The
venous channels (V) appear to be intact with no apparent damage in either
control or CDDP cultures. The areas enclosed are magnified in figure 10.

All are stained with hematoxylin and eosin. Bar = 0.02mm

126

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Figure 10: Light Micrographs of the Medulla Enclosed Areas Enlarged from
Figure 9.

A. Control B. CDDP 2-Hours Treatment:

There is no membrane (arrow) definition, with condensed and degenerating
nuclei, an apparent loss of cytoplasm and accumulated debris in the lumen of the
capillaries (V) of the CDDP culture with the control having better membrane
definition and increased pronounced nuclei.

C. Control D. CDDP 4-Hours Treatment:

There is no membrane (arrow) definition with condensed and degenerating nuclei
in the CDDP culture, with both the control and CDDP cultures having an
apparent loss of cytoplasm. The capillaries (V) of the control have accumulated
some cellular debris.

E. Control F. CDDP 6-Hour Treatment:

Neither the control nor CDDP cultures have membrane (arrow) definition. The
nuclei in the control are condensed, with an apparent loss of cytoplasm from the
cells and the CDDP culture has nuclear degeneration.

G. Control H. CDDP 8-Hours Treatment:
Both the control and CDDP cultures lack membrane definition (arrow), have an
apparent loss of cytoplasm and a decrease in the number of nuclei.

Bar = 0.007mm

I28

 

Figure 11: Light Micrographs of Sections (20m) of the Medulla and Zona
Reticularis of Group 2 Adrenal Gland Organ Cultures.

A. Control B. CDDP 2- Hours Treatment:

The CDDP cultures medullary (M) cells appear to have decreased cytoplasm and
membrane definition. The zona reticularis (ZR) in both the control and CDDP
cultures have enlarged capillaries (clear areas) with the treated cultures being
more pronounced and having a number of nuclei that are condensed.

C. Control D. CDDP 4-Hours Treatment:

Both the control and CDDP cultures have an apparent loss of cytoplasm in the
medulla (M) with the ZR having enlarged capillaries (clear areas) with the CDDP
culture having no cellular membrane definition. The medullary capillaries (V) are
intact with no apparent damage in either the control or CDDP cultures.

E. Control F. CDDP 6-Hours Treatment:

The controls medulla (M) and areas of the ZR appear to have decreased
cytoplasm and a number of condensed nuclei with both the control and CDDP
cultures having enlarged capillaries (clear areas). The medullary capillaries (V) in
the treated culture have no apparent damage to the endothelial lining.

G. Control H. CDDP 8-Hours Treatment:

The control has an apparent decrease in cytoplasm in the medulla (M) and the
ZR has areas of intensely stained nuclei that are condensed. The CDDP culture
appear to have an increase in cytoplasm with no cellular membrane definition
and the medullary capillaries (V) have no apparent damage to the endothelial
lining.

All are stained with hematoxylin and eosin. Bar = 0.02mm

130

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Figure 12: Light Micrographs of Sections (2pm) of the Zona Reticularis and the
Zona Fasciculata of Group 2 Adrenal Gland Organ Cultures.

A. Control B. CDDP 2-Hours Treatment:

The zona reticularis (ZR) in both the control and CDDP cultures have enlarged
capillaries and cellular separation (clear areas) that go into the zona fasciculata
(ZF). The CDDP culture has no cellular definition and areas with condensed
nuclei. The areas enclosed are magnified in figure 13.

C. Control D. CDDP 4-Hours Treatment:

The ZR in both the control and CDDP cultures has enlarged capillaries (clear
areas) that go into the ZF, being more pronounced in the control. The CDDP
culture to have an increase in cytoplasm but the cells have no membrane
definition. The areas enclosed are magnified in figure 13.

E. Control F. CDDP 6-Hours Treatment:

Both the control and CDDP cultures have enlarged capillaries and cellular
separation (clear areas), that are more pronounced in the ZR of the control and
the ZF of the treated. The cell membranes have no apparent definition in either
the control or CDDP cultures. The areas enclosed are magnified in figure 13.

G. Control H. CDDP 8-Hours Treatment:
The cells of the ZR and the ZF in both the control and CDDP cultures have no

membrane definition, with the enlarged capillaries being more pronounced in the
control culture. The areas enclosed are magnified in figure 13.

All are stained with hematoxylin and eosin. Bar = 0.02mm

132

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Figure 13: Light Micrographs of the Enclosed Areas Enlarged from Figure 12.

A. Control B. CDDP 2-Hours Treatment:

C. Control D. CDDP 4-Hours Treatment:

E. Control F. CDDP 6-Hours Treatment:

G. Control H. CDDP 8-Hours Treatment:

The ZF appears to have a decrease in cytoplasm, enlarged capillaries (arrow)
with separation of cells and a decrease in membrane definition and nuclei as

time in culture progresses in both the control and CDDP cultures.

Bar = 0.007mm

134

 

Figure 14: Light Micrographs of Sections (2pm) of the Zona Fasciculata, Zona
Glomerulosa and Capsule Group 2 Adrenal Gland Organ Cultures.

A. Control, B. CDDP 2-Hours Treatment:

Both the control and CDDP cultures have enlarged capillaries and an apparent
decrease in cytoplasm through the zona fasciculata (ZF). The control and CDDP
cultures have areas of condensed nuclei in the ZF, zona glomerulosa (ZG) and
the capsule (Cap) with the control appearing to have a greater loss of cytoplasm
in the ZG, but allover better membrane definition than the CDDP culture.

C. Control, D. CDDP 4-Hours Treatment:

There are enlarged capillaries that go from the ZF into the ZG, with cytoplasmic
loss and condensed nuclei in the upper ZF, ZG and the Capsule (Cap), in both
the control and CDDP cultures with the CDDDP culture having no membrane
definition.

E. Control, F. CDDP 6-Hours Treatment:

There appears to be a decrease in cytoplasm, enlarged capillaries, cellular
separation and condensed nuclei in the ZF, with increased cytoplasm and a
number of condensed nuclei in the ZG in both the control and CDDP cultures.
The Capsule (Cap) is intact with a number of intensely stained condensed nuclei
in both the control and CDDP cultures.

G. Control, H. CDDP 8-Hours Treatment:

There is an apparent increase in cytoplasm with no cellular membrane definition;
the cells are merged together in the ZF in both the control and CDDP cultures.
The ZG and capsule (Cap) have a loss of cytoplasm with an increase in the
number of intensely stained condensed nuclei in both the control and CDDP
culture.

All are stained with hematoxylin and eosin. Bar = 0.02

136

 

137

DISCUSSION

Corticosterone concentration for the Group 1 cisplatin treated decreased
throughout the experiment except for at 32 hours where it was equal to the
control. In the controls, corticosterone concentrations also declined throughout
the experiment. This may be indicative of the drop in ACTH synthesis from the
pituitary in culture with the adrenal gland and/or the length of time in culture.
Some studies have shown that a constant concentration of ACTH is necessary to
maintain the adrenal glands cellular structure and that without it apoptosis will
become activated [25, 26]. Group 2, which was in culture for a shorter time did
not show this decline in corticosterone concentration in either the control or the
cisplatin treated. There were differences in corticosterone concentration
between the treated and the control, but they do not appear to be significant.

Cellular damage was apparent in all the organ cultures, with a trend of
increasing damage as time in culture progressed. The zona glomerulosa is
usually spared the infarctive effects of damage that the arterioles have in the
other zonae [55]. This difference is due to the zona glomerulosa receiving part of
its vascular supply from the capsule [55] thus sparing it from catecholamines and
cellular debris being dumped into the arterioles. There is apparent cytoplasmic
loss and an increased number of condensed nuclei in the zona glomerulosa of
the organ cultures. Thus it has lost its protection in culture and has induced
apoptosis. The morphology of the organ cultures does not show the fine

structure that is in the intact gland.

138

Our in vivo studies showed an overall increase in corticosterone
concentration which conflict with the results from the in vitro studies. Conflicting
results in corticosterone concentrations between in vivo and in vitro studies have
been seen before [36, 37]. The main thought for this occurrence is the alteration
in the hypothalamic-pituitary-adrenal axis, which is non-existent in organ culture.
Interleukin 18 and interleukin 6 have been found to stimulate corticosterone
concentration in vivo [6, 7, 10-12, 39], with variable results in vitro [9].

Further studies are needed to verify the results found in this study. Organ
cultures should be repeated with addition of ACTH to stimulate corticosterone
synthesis and the cellular integrity of the culture. Additional in vivo studies
should be done with rats that are on a constant level of dexamethasone, a
synthetic glucocorticoid agonist to analyze if cisplatin effects it. Studies of the
immune system’s interaction with the Hypothalamus-Pituitary—Adrenal axis and
corticosterone concentration would be beneficial to how the system relates to

stress and the toxic effects of the cisplatin.

139

CONCLUSIONS

This study has left more unanswered questions than answers. The
immune systems interaction with the hypothalamus-pituitary—adrenal axis (HPA)
and corticosterone concentrations in the induction of prolonged stress would be
of great importance in contributing to the knowledge of how the system deals

with disease and the toxic effects of treating that disease.

140

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

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