4. Dr. Thomas Rosol

Transcription

4. Dr. Thomas Rosol
Adrenal Glands
Adrenal Cortex and Medulla in
Preclinical Toxicology
Thomas Rosol, DVM, PhD, DACVP
Veterinary Biosciences
Ohio State University, Columbus, Ohio, USA
• Most common endocrine organ with
chemically-induced lesions
• Understanding structure and function is
important for interpreting the mechanisms
and significance of chemically-induced
lesions
01 November 2014
STP-India
Bangalore
Homeostatic Model of
Adrenocortical Growth
HPA Axis
Rat Adrenal Cortex
Zona Glomerulosa (Arcuata) 20%
Zona Fasciculata 65%
Zone Reticularis 15%
Pituitary
ACTH (+)
Adrenal
Capsule cells (Sf1-)
Zona glomerulosa cells (Sf1+)
Zona fasiculata cells (Sf1+)
Zona reticularis cells (Sf1+)
Progenitor cells (Sf1+)
Subcellular Structure/Function
Relationships
Hypothalamus
AVP
CRH
Stem cells (Sf1-, Gli1+)
CORT:
Cortisol (-)
Corticosterone (-)
• Lipid droplets (cholesterol esters)
• Smooth endoplasmic reticulum
• Mitochondria
• Minimal storage of hormone
• Steroids are hydrophobic molecules
diffuse out of cells
Drugs:
Dexamethasone (-)
Copyright: Thomas Rosol
Ohio State University 2014
1
Steroidogenesis
• ACTH (cAMP second messenger)
– ĹStAR protein (steroidogenic acute regulator protein)
• Moves cholesterol to inner mitochondrial membrane
– ĹCyp11A1
• Mitochondria
– Side chain cleavage (Cyp11A1)
– Hydroxylation to Pregnenalone
• SER
– Converted to 11-Deoxycorticosterone
• Mitochondria
– Hydroxylated to Corticosterone or Cortisol
Adrenal Cortex:
Steroid Hormones
Major Glucocorticoids
• Cholesterol is precursor
• Cortisol
• Cytochrome P450 enzymes
– Mitochondria, SER, Shuttling
• Secretion
–
–
–
–
Circadian rhythm
Nocturnal animals (rats, mice, cats): High at night
Daytime animals (dogs, humans): High in morning
Decreased secretion with age
• Bound in serum (90%) to CBG (transcortin)
• Metabolized in liver (hydroxylated and
conjugated)
Acetate
HDL
LDL
ACAT
Cholesterol
HO
nCEH
Pregnenalone
HO
3ß-OH-STEROID DE-OH-ASE' 4,5 ISOMERASE
3ß-OH-STEROID
DE-OH-ASE-
O
' 4,5 ISOMERASE
CYP17
Progesterone O
O
CH 2 OH
O
O HO
CYP21
CH 2OH
O
CYP21
CYP11B2
O
Aldosterone CYP11B1
HO
11-DeoxycorticosteroneO
CH 2 OH
O
CYP11B1
HO
3ß-OH-STEROID DE-OH-ASE' 4,5 ISOMERASE
HO
O
3ß-OH-STEROID
DE-OH-ASE-
O
' 4,5 ISOMERASE
Progesterone O
O
CH 2 OH
O
O HO
CYP21
CH 2OH
O
CYP21
CYP11B2
O
O
Aldosterone CYP11B1
HO
Cortisol
11-DeoxycorticosteroneO
CH 2 OH
O
HO
O
O
CYP17
17 D -OHHO
Pregnenalone
Dehydroepiadrosterone
O
OH CYP17
17 D -OHProgesterone O
CH 2 OH
O
OH
O
Androstenedione
11-Deoxycortisol
CYP11B1
Corticosterone
O
Cholesterol
Esters
O
OH
CYP17
CYP17
17 D -OHProgesterone O
Androstenedione
CH 2 OH
O
OH
CH 2OH
O
OH
nCEH
Pregnenalone
HO
11-Deoxycortisol
Corticosterone
O
O
StAR
O
17D -OHHO
Pregnenalone
Dehydroepiadrosterone
O
OH CYP17
Amphibians
Reptiles
Birds
Rats
Mice
Rabbits (also have
cortisol, increases
during stress)
ACAT
Cholesterol
HO
**CYP11A1**
O
O
OH CYP17
CYP17
–
–
–
–
–
–
ACTH
**CYP11A1**
HO
O
Cholesterol
Esters
cAMP
O
• Corticosterone
– Fish (teleosts)
– Hamsters
– Dogs (similar to
humans)
– Cats
– Nonhuman primates
– Humans
CH 2OH
O
OH
Cortisol
ACAT: acyl-CoA:cholesterol acyltransferase, nCEH: neutral cholesterol ester hydrolase
Copyright: Thomas Rosol
Ohio State University 2014
2
Acetate
HDL
LDL
Cholesterol
HO
CYP11A1
O
Pregnenalone
HO
HO
3ß-OH-STEROID DE-OH-ASE' 4,5 ISOMERASE
3ß-OH-STEROID
DE-OH-ASE-
O
' 4,5 ISOMERASE
CYP17
Progesterone
O
CH2OH
O HO O
CYP21
CH2OH
O
O
CYP21
CYP11B2
O
O
Aldosterone CYP11B1
HO
HO
Corticosterone
O
HO
Cholesterol
17D -OHProgesterone O
Androstenedione
CH2OH
O
OH
O
OH
CYP17
Pregnenalone
HO
HO
3ß-OH-STEROID DE-OH-ASE' 4,5 ISOMERASE
3ß-OH-STEROID
DE-OH-ASE-
O
' 4,5 ISOMERASE
CYP17
Progesterone O
O
CYP21
CH 2 OH
O
O HO
11-Deoxycortisol
CYP11B1
O
O
O
CH 2OH
O
CYP21
11-DeoxycorticosteroneO
O
O
CH2OH
O
OH
Aldosterone
Angiotensin II
K+
Cortisol
Cholesterol
Esters
**CYP11A1**
17D -OHHO
Pregnenalone
Dehydroepiadrosterone
O
OH CYP17
nCEH
CYP11B2
11-DeoxycorticosteroneO
CH2OH
O
O
O
OH CYP17
CYP17
ACAT
Cholesterol
HO
O
HO
CH 2 OH
O
HO
O
17 D -OHHO
Pregnenalone
Dehydroepiadrosterone
O
OH CYP17
17 D -OHProgesterone O
CH 2 OH
O
OH
O
Androstenedione
11-Deoxycortisol
CYP11B1
Corticosterone
O
CYP17
CH 2OH
O
OH
Cortisol
CYP11A1
O
HO
3ß-OH-STEROID DE-OH-ASE' 4,5 ISOMERASE
3ß-OH-STEROID
DE-OH-ASE-
O
' 4,5 ISOMERASE
CYP17
Progesterone O
O
CH2OH
O HO O
CYP21
CH2OH
O
CYP21
CYP11B2
O
O
Aldosterone CYP11B1
HO
11-DeoxycorticosteroneO
CH2OH
O
HO
O
17D -OHHO
Pregnenalone
Dehydroepiadrosterone
O
OH CYP17
O
17D -OHProgesterone O
Androstenedione
CH2OH
O
OH
11-Deoxycortisol
CYP11B1
Corticosterone
O
O
O
OH CYP17
CYP17
Pregnenalone
HO
CH2OH
O
OH
Cortisol
17E-HSD: 17E-hydroxysteroid dehydrogenase
Cynomolgus Monkey
Copyright: Thomas Rosol
Ohio State University 2014
Cynomolgus Monkey: CYP17A IHC
3
Cynomolgus Monkey: CYP11B2 IHC
Cynomolgus Monkey: Ki-67
Factors Contributing to Chemical
Injury of the Adrenal Cortex
Species Variations
• Rich vascular supply
• Sensitivity varies according to species
• High lipid content (steroidogenesis)
• Variation in pathways of steroid
metabolism
• Bioactivation by cytochrome P450 enzyme
systems to reactive toxic forms
• Limited mechanisms of detoxification
Ultrastructural Lesions
• Variation in xenobiotic metabolism
– Dogs and humans are similar (e.g. o,p’-DDD)
– Rats susceptible to DMBA
Chemical-Induced Injury of the
Adrenal Cortex
• May be more useful than histopathology
– Correlates with mechanism of toxicity
– Organelle-specific enzymes
• Selected Examples of Mechanisms:
– Inhibition of microsomal enzymes
Copyright: Thomas Rosol
Ohio State University 2014
4
Cholesterol
HO
Chemical-Induced Injury of the
Adrenal Cortex
CCl4
CYP11A1
O
O
OH
X
CYP17
Pregnenalone
HO
HO
3ß-OH-STEROID DE-OH-ASE' 4,5 ISOMERASE
3ß-OH-STEROID
DE-OH-ASE-
O
' 4,5 ISOMERASE
X
CYP17
Progesterone O
O
X
CYP21
CH2OH
O HO O
CH2OH
O
X
CYP21
CH2OH
O
OH
CYP11B2
O
O
Aldosterone CYP11B1
HO
11-DeoxycorticosteroneO
CH2OH
O
Lesions:
Swollen SER
and necrosis
• Selected Examples of Mechanisms:
– Inhibition of neutral cholesterol ester
hydrolase
11-Deoxycortisol
CH2OH
O
OH
CYP11B1
HO
Corticosterone
Cortisol
O
O
O
Mechanism:
17D -OHInhibition of
HO
Pregnenalone
Dehydroepiadrosterone
microsomal
O
O
OH CYP17
17D - an 21
17D -OHProgesterone Ohydrolases
Androstenedione
CYP17
Inhibition of Neutral
Cholesterol Hydrolase
TRIARYL PHOSPHATES
Triaryl Phosphate
• Cholesterol lipidosis
• Adrenal cortical and ovarian interstitial
cells
Acetate
HDL
LDL
HO
X
nCEH
**CYP11A1**
O
HO
O
OH
CYP17
Pregnenalone
HO
CORTICAL LIPIDOSIS: 3 WEEKS
Cholesterol
Esters
ACAT
Cholesterol
CYP17
O
17 D -OHHO
Pregnenalone
Dehydroepiadrostero
Triaryl Phosphate
3ß-OH-STEROID
DE-OH-ASE' 4,5 ISOMERASE
3ß-OH-STEROID DE-OH-ASE' 4,5 ISOMERASE
O
O
O
Mechanism: Inhibition
of neutral
cholesterol
OH CYP17
CYP17
17 D -OHester hydrolase
Progesterone
O
O
Progesterone O
Androstenedione
CYP21
CH OH
CH
OH
CH OH
Lesions:
Cytoplasmic
lipid
droplets
in zona
CYP21
O
O
O
O HO
OH
CYP11B2
glomerulosa,
reticularis, and fasciculata
2
2
O
O
Aldosterone CYP11B1
HO
2
11-DeoxycorticosteroneO
CH 2 OH
O
HO
Corticosterone
O
11-Deoxycortisol
CYP11B1
O
Copyright: Thomas Rosol
Ohio State University 2014
CH 2 OH
O
OH
Cortisol
5
Cortical Lipidosis
Chemical-Induced Injury of the
Adrenal Cortex
• Selected Examples of Mechanisms:
– Inhibition of ACAT* Enzyme in Cortical
Cells
L
N
*Acyl Coenzyme: Cholesterol Acyl Transferase
ACAT-Inhibiting Compounds
x Degeneration of zonae fasciculata And
reticularis
– (Ļ) Mitochondria and SER
x Direct cytotoxic effect
x Species sensitivity:
– Dog > Guinea Pig > Rabbit > Monkey > Rat
Chemical-Induced Injury of the
Adrenal Cortex
• Selected Examples of Mechanisms:
Selective Mitochondrial
Degeneration with Vacuolation
ORTHO, PARA, PRIME DDD
(O,P’-DDD)
x Zonae fasciculata and reticularis
–Selective mitochondrial degeneration
x Zona glomerulosa much less sensitive
x Species susceptibility:
– Dog unusually sensitive
Copyright: Thomas Rosol
Ohio State University 2014
6
Vacuolation of Mitochondria
Cessation of O,P’DDD Therapy
Chemical-Induced Injury of the
Adrenal Cortex
Disruption of Organellar
Membrane Turnover
• Examples:
• Selected Examples of Mechanisms:
– Disruption of organellar membrane turnover
– Cationic Amphiphilic Compounds
(Chloroquine, Triparanol,
Chlorophentermine)
– Toxin activation of CYP P450 enzymes
• Lesion: Phospholipidosis of cortical cells
(vacuolation, need EM)
Copyright: Thomas Rosol
Ohio State University 2014
7
Cortical Phospholipidosis
Spontaneous
Degenerative Lesions of
Adrenal Cortex
x Examples in Laboratory Rodents
Adrenal Cortex (Rat)
Cystic Degeneration
Proliferative Lesions of Adrenal
Cortex
Adrenal Cortex
(F344 Rat)
Telangiectasis
Adrenal Cortex (F344 Rat)
Focal (Eosinophilic) Hyperplasia
HYPERPLASIA
x Spindle Cell (Mice)
x Focal (Rats)
x Extracortical Nodular (Dog)
Copyright: Thomas Rosol
Ohio State University 2014
8
BrdU
50
Subcapsular
Spindle Cell Hyperplasia
x Common in mice
x Infrequent in rats and hamsters
Adrenal Cortex Adenoma: Rat
Proliferative Lesions of
the Adrenal Cortex
NEOPLASIA
Copyright: Thomas Rosol
Ohio State University 2014
9
Adrenal Cortex Adenoma: Mouse
Subcapsular Type (A and B cells)
Mechanism of Induction of
Cortical Neoplasia
x Hormonal Imbalance: (ĹGonadotropins, esp., ĹLH)
– Gonadectomy
• Species: Mice, Hamsters, Ferrets, Goats
– Transgenic Mice Deficient in Inhibin
– Estrogen Receptor Antagonists
– Ionizing Radiation, Chemicals (DMBA)
• Ovarian Follicle Destruction
• (Ļ)E2, (Ĺ) LH
Development of the adrenal gland and
gonads from the urogenital ridge
Adrenocortical Tumors
(Human Relevance)
• Humans
– Adenomas: 5% of people over 50 years,
incidentalomas, nonfunctional
• Carcinoma is rare (500/yr in USA)
• Dogs: Similar to humans
– Functional tumors more common
• Gonadectomy
– Goats, ferrets, hamsters, and mice
Bielinska M et al. Vet Pathol 2006;43:97-117
The Stress Response and
the Adrenal Cortex
• Physiological response to an environmental
change
• Activation of the HPA axis with increased
glucocorticoids
• Maximizes survivability and suppresses
nonessential functions
• Increased gluconeogenesis; decreased insulin
sensitivity; diabetogenic
• Suppression of reproduction
• Regulation of immune function
• Increases activity and appetite
Copyright: Thomas Rosol
Ohio State University 2014
10
Rat Adrenal Glands
Normal
Chemicals as stressors
• The HPA response is not stressor-specific
• Chemicals can alter homeostatic balance
– Organ effects
– Metabolism effects
Hypertrophy
Increased Adrenal Gland Weight
(Adrenal Hypertrophy)
• Stress
– Known exposure to stress-inducing stimuli
– Histopathology (Morphology)
– Concurrence with other stress-related changes (e.g., thymic
atrophy)
– Clinical chemistry
• Functional challenge
• Differential Diagnosis: Toxic Effect
• High doses of test articles
– Histopathology (Morphology)
– Clinical chemistry
• Functional challenge (functional suppression)
– Mechanistic studies of adrenal cell function (steroidogenic
enzymes)
Adrenal Hypertrophy:
Significance
• Stress-Related
–
–
–
–
Primary and secondary stress-related effects
Less significant finding in toxicology study
Cause should be identified
Functional significance could be tested
Assessment of the HPA Axis
• ACTH challenge assay
– Tests the integrity of the HPA axis through steroid
hormone secretion
• Evaluation of in vitro mechanisms
– H295R human adrenal cortical cells
• Test Compound-Related
–
–
–
–
Potentially serious
Understand mode of action
Human relevance
Species differences
Copyright: Thomas Rosol
Ohio State University 2014
• Enzyme inhibition
– CYP17, not well expressed in rodent adrenal glands
• StAR function (steroidogenic acute regulatory protein; transport
protein for cholesterol in mitochondria and rate limiting step in
steroidogenesis)
• Species differences in susceptibility to toxicants
11
ACTH Stimulation Assay
ACTH STIMULATION TEST
Rodents
16
– Trough of circadian rhythm
• 12 hr light-dark cycle; starting at 6 a.m.
• Pre-test sample usually not collected (to
avoid stress of blood collection)
• Post-ACTH sample at 0.5-2.0 hours
14
PLASMA CORTISOL (ug/dl)
• Most sensitive time to measure
corticosterone in rodents is about 7-10
a.m.
12
10
8
6
4
2
HYPOADRENOCORTICISM
0
BASELINE PLASMA
CORTISOL
POST STIMULATION
PLASMA CORTISOL
ADRENAL MEDULLA
• Less common site of toxic manifestations
• Proliferative lesions
– Important in the rat
– Less common in the mouse
Epinephrine
Copyright: Thomas Rosol
Ohio State University 2014
Norepinephrine
12
Immunohistochemistry
• Tyrosine hydroxylase: All chromaffin cells
– Cytosolic (independent of granules)
– All tumors produce this enzyme
• Chromogranin A: All chromaffin cells
– Secretory Granules (variable in tumors)
• PNMT: Only Epinephrine cells
– Cytoplasmic (variable in tumors)
Tyr Hydroxylase, Chromogranin A
PNMT (Epinephrine Cells)
Focal Medullary Hypeplasia
Focal Medullary Hyperplasia
Copyright: Thomas Rosol
Ohio State University 2014
13
Adrenal Medulla: Neoplasms
Adrenal Medulla: F344 Rat
Pheochromocytoma
• Secretory Cells
– Pheochromocytoma (benign)
•
•
•
•
May start as hyperplasia
Expandsile medullary mass
Well differentiated
Behavior
– Space-occupying mass
– Excess catecholamine secretion
Interspecies Comparison of
Pheochromocytomas
Copyright: Thomas Rosol
Ohio State University 2014
RAT
MOUSE
HUMAN
x Lifetime Frequency
Up to 80%
<5%
<0.09%
x Sex Predilection
MALE
NONE
NONE
x Inducing Agents
NONE
HORMONES
DRUGS, TOXINS
DIETARY FACTORS
NONE
x Cell Type
NE
E+NE
E+NE
14
Factors Influencing Incidence of
Pheochromocytomas in the Rat
• Age
• Strain
– Holtzman
– Wistar
0.5%
67%
• Sectioning technic
– Single vs. serial
– 7.5% of adrenal volume is medulla
• Chronic Stress, Ca2+
– Chronic renal and lung disease
Agents associated with
Pheochromocytomas in Rats
x Phosphodiesterase Inhibitors
– Indolidan
– Isomazole
x Dietary Factors
– Excess Food
– Excess Ca2+, sugars, sugar alcohols
Agents associated with
Pheochromocytomas in Rats
x Hypothalamic-Endocrine
– Growth Hormone
– Estrogen
– Antithyroid Drugs
– Alloxan
– Neuroleptics
x Autonomic Nervous System
– Nicotine
– Reserpine
Agents associated with
Pheochromocytomas in Rats
x Miscellaneous Factors
– Gemfibrozil (Hypolidemic)
– Zomepirone (Anti-inflammatory)
– Diphenylamine (Anti-oxidant)
– Retinol Acetate
– 1,4, Dioxane
– Ethylene Glycol Monoethyl Ether
– P-chloroaniline
– Radiation
– Chronic Respiratory Disease
ADRENAL MEDULLA: NEOPLASMS
• Malignant Pheochromocytomas
– Local invasion
– Invasion into the posterior vena cava
Copyright: Thomas Rosol
Ohio State University 2014
15
Adrenal Medulla: F344 Rat
Malignant Pheochromocytoma
Differential Diagnosis of Adrenal
Medullary and Cortical Tumors
• Chromaffin Reaction
– Dichromate fixative oxidizes catecholeamines
to form brown-black pigment
– Differentiate cortical and medullary neoplasms
Adrenal Medulla: Ganglioneuroma
Copyright: Thomas Rosol
Ohio State University 2014
16
Dual Differentiation (‘Complex’)
Pheochromocytoma & Ganglioneuroma
Copyright: Thomas Rosol
Ohio State University 2014
17

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