Experimental and Toxicologic Pathology 68 (2016) 1–13

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Experimental and Toxicologic Pathology

journal homepage: www.elsevier.de/etp

Spontaneous lesions in endocrine glands of experimental Wistar rats

and beagle dogs
G.J. Nataraju, Ramchandra K. Ranvir* , Viren R. Kothule, Shekhar B. Kadam,
B.V. Ravichandra, Upendra Bhatnagar, Mukul R. Jain
Department of Pharmacology and Toxicology, Zydus Research Centre, Cadila Healthcare Ltd., Ahmedabad 382213, Gujarat, India

A R T I C L E I N F O A B S T R A C T

Article history: A retrospective analysis was undertaken at Zydus Research Centre to understand the incidences of
Received 13 January 2015 spontaneous lesions in endocrine glands of Wistar rats and beagle dogs. The data from a total of 841
Received in revised form 25 August 2015 Wistar rats (418 males and 423 females) and 144 beagle dogs (72 males and 72 females) was used from
Accepted 8 September 2015
placebo/vehicle treated control group of different non-clinical toxicity studies. The lesions in various
endocrine glands were classified according to the species and age of the animals at termination of study.
Keywords: Among the endocrine glands, the highest numbers (types) of spontaneous lesions were observed in
Spontaneous lesion
adrenal glands followed in descending order by pituitary, thyroid, endocrine pancreas and parathyroid
Endocrine
Age
glands in Wistar rats. In beagle dogs, highest numbers (types) of spontaneous lesions were seen in
Rat adrenals followed by thyroid, endocrine pancreas, pituitary and parathyroid gland. In adrenal glands of
Dog Wistar rats, the incidences of cortical cell vacuolation, hemorrhages and hemangiectasis/peliosis were
increased with age. Incidence of peliosis at 110 weeks of age was higher in female rats. Among the
proliferative lesions in rats, higher incidences of cortical cell hyperplasia was observed followed by
medullary hyperplasia, complex pheochromocytoma, cortical cell adenoma and cortical adenocarcino-
ma. In beagle dogs, the incidences of hemangiectasis and cortical cell vacuolation in adrenal glands were
higher in 18–21 months aged dogs in both the sexes as compared to 10–12 months of age. In pituitary
gland, the incidences of cystic changes were higher in older rats and dogs and the incidences were more
in beagles as compared to rats. In thyroid glands, C-cell (parafollicular cells) hyperplasia/complex was
observed more frequently in both the species. Few incidences of cystic changes were observed in
parathyroid of 18–21 months aged beagle dogs. In endocrine pancreas, few incidences of islet-cell
vacuolation, atrophy and hyperplasia were observed in both the species. The Islet cell hyperplasia was
found to be more frequent in male rats at 110 weeks of age.
ã 2015 Elsevier GmbH. All rights reserved.

1. Introduction Knowledge of spontaneous histomorphological variations in

endocrine organs of laboratory animals used in routine toxicity
The endocrine system helps to regulate and maintain several studies will assist in accurate and meaningful interpretation of
systemic functions by synthesizing and releasing hormones findings in animal safety/toxicity studies. Keeping in view of the
directly into the bloodstream for chemical signaling of target regulatory requirements for safety assessment of new drugs and
cells. The major areas of control and integration include responses various new chemical entities which are likely to affect the
to stress and injury, growth and development, absorption of endocrine system, it is essential to compile the background
nutrients, energy metabolism, water and electrolyte balance, histopathological data of key endocrine glands such as adrenals,
reproduction, and lactation. Thus, the variation in these physio- pituitary, endocrine pancreas, thyroid and parathyroid glands,
logical functions in response to direct or indirect stimulation which are routinely evaluated in toxicity studies. The review of
including advancement of age largely reflects in the form of literature suggests that chemically induced lesions of the
morphological or structural changes. endocrine organs are most commonly encountered in the adrenal
glands, followed in descending order by the thyroid, pancreas,
pituitary, and parathyroid glands (Frith et al., 2000). Historical
* Corresponding author. control data are especially useful in assessment of increased
E-mail address: [email protected] (R.K. Ranvir). incidence of uncommon neoplasms, when increase in frequency is

http://dx.doi.org/10.1016/j.etp.2015.09.002
0940-2993/ ã 2015 Elsevier GmbH. All rights reserved.
2 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13

marginal compared to concurrent controls, and in evaluation of software to analyze and compare the age relation in the incidence
genetic drift within a particular strain (Haseman et al., 1989, 1984). of spontaneous lesions.
As noted previously by Walsh and Poteracki (1994) spontaneous Animals were kept under the species specific identical housing
lesions have been extensively described and quantitated in and husbandry conditions and were maintained and supplied by
commonly used Fischer 344 (F344) rats and in Sprague-Dawley Animal Research Facility of Zydus Research Centre. Wistar rats
rats but most reports in Wistar rats are restricted to specific organ were procured from Harlan laboratory, USA. After receipt of the
systems. animals, they were quarantined and housed in individually
The main objective of this article is to provide a data base of ventilated cages (IVC) made of polysulfonate with autoclaved
spontaneous lesions of endocrine glands in routinely employed corn cob as bedding material. Beagle dogs were procured from
pre-clinical laboratory animal species (Wistar rats & beagle dogs) Beijing Marshall Biotechnology Co., Ltd., and housed individually in
at one place for easy and quick reference to toxicologists and kennels. Individual kennel were provided with animal enrichment
toxicologic-pathologists. This will aid in the interpretation of items and co-mingling was permitted daily for some hours. Both
animal toxicology studies and to determine their significance rats and dogs were kept in environmentally controlled rooms with
further in risk assessment. An attempt was made to compile the 22  3  C temperature and 30–70% relative humidity with 12/12 h
data in accordance to duration of toxicity studies and also to arrive light/dark cycle. Purified water and standard laboratory animal
at a meaningful conclusion about the frequency and pattern of diet was provided. Proximate analysis of feed nutrient content and
occurrence of lesions as related to the age of the animals. microbial contaminant was checked batch wise or periodically.
Quality of water was checked periodically for ensuring acceptable
2. Materials and methods limits of total dissolved solute and microbial contamination as per
Standard Operating Procedures of Zydus Research Centre.
This retrospective analysis was conducted using histopatholog- Necropsy of all the animals was performed and was subjected
ical data from control (vehicle/placebo treated) animals of toxicity for gross examination. The tissues of interest were fixed in 10%
studies conducted between year 2005 and 2014 at Zydus Research neutral buffered formalin, embedded in paraffin, sectioned at 5 mm
Centre, Cadila Healthcare Ltd., Ahmedabad, Gujarat, India. thickness, stained with hematoxylin and eosin and subjected for
Age at termination was in the range of 10 weeks to 2.2 years for microscopic evaluation. All the studies were conducted in AAALAC
Wistar rats and 10 months to 1.9 years for beagle dogs. Percent (Association for Accreditation and Assessment of Laboratory
incidence of lesions in rat and dog at different age was compiled Animal Care) accredited facility in compliance with national and
and tabulated. The data from a total of 841 Wistar rats (418 males international guidelines for animal welfare and all the procedures
and 423 females) and 144 beagle dogs (72 each male and female) used in these studies were reviewed and approved by the
was used for analysis. The data was analyzed and interpreted both Institutional Animal Ethics Committee.
biologically and statistically. Chi-square test for trend followed by
Fisher’s exact test was applied by using Graph Pad Prism ver.6.0

Table 1
Incidences of spontaneous lesions in Wistar rats (%).

Organ/finding Male Female

10–12 weeks 32–34 weeks 110 weeks 10–12 weeks 32–34 weeks (n 110 weeks (n = 108)
(n = 191) (n = 119) (n = 108) (n = 197) = 118)
Adrenal Glands
Cortical cell vacuolation 1 2.5a 17.6b,c 0.5 3.4 13.9b,c
Capsular thickening 0 0 4.6b,c 0 0 1.9
Cystic changes 0 0 0.9 0 0 0
Accessory cortical tissue 2.1 2.5 3.7 2 2.5 4.6
Hemangiectasis/ Peliosis 0 0 2.8 0 3.4a 11.1b,c
Congestion 1 3.4 1.9 1 0.8 12.0b,c
Hemorrhage 0 2.5 0.93 0 1.7 5.6b
Thrombus- venous/ arterial 0 0 0 0 0 1.9
Hemosiderosis 0 0 0.9 0 0 0
Extramedullary hematopoiesis 0 0 0.9 0 0 0.9
Cortical cell hypertrophy, zona 0 0 3.7b,c 0 0 3.7b,c
fasciculata
Cortical cell hyperplasia 0 0 5.6b,c 0 0 5.6b,c
Medullary hyperplasia 0 0 3.7b,c 0 0 2.8b
Pheochromocytoma 0 0 1.9 0 0 0
Complex pheochromocytoma 0 0 0.9 0 0 1.9
Cortical adenoma 0 0 0.9 0 0 0.9
Cortical adenocarcinoma 0 0 0 0 0 0.9

Parathyroid
Parathyroid hyperplasia 0 0 3.7b,c 0 0 5.6b,c
Chief cell hypertrophy 0 0 0 0 0 0.9

Endocrine Pancreas
Islet cell hyperplasia 0 1.7 13.0b,c 0 1.7 0.9
Atrophy of Islets 0 0 3.7b,c 0 0 1.9
Islet cell carcinoma 0 0 0 0 0 0.9
Islet cell vacuolation 0 1.7 0 0 0.8 0
a
Significant at p < 0.05 between 10–12 weeks and 32–34 weeks.
b
Significant at p < 0.05 between 10–12 weeks and 110 weeks.
c
Significant at p < 0.05 between 32–34 weeks and 110 weeks.
 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13 3

3. Results A higher incidence of cortical cell vacuolation was observed in

both the sexes at 18–21 months (male: 16.7% and female: 22.2%) as
3.1. Adrenal glands compared to 10–12 months (male: 9.3% and female: 5.6%) old dogs.
Hemorrhage and congestion were also observed as other
The spontaneous lesions in adrenals in the present analysis at spontaneous changes in adrenals of dogs (Tables 1–3, Figs. 1–3).
different age were cortical cell vacuolation, vascular congestion,
focal/multifocal hemorrhages, hemangiectasis/peliosis, accessory 3.2. Pituitary gland
cortical tissue, cortical cell hyperplasia, medullary hyperplasia,
cortical cell hypertrophy, extramedullary hematopoiesis, capsular In Wistar rats, non-proliferative changes like, pituitary cysts
thickening, venous/arterial thrombus, complex pheochromocyto- (male: 0.5–1.9%; female: 0.5–0.8%) and vascular congestion (male:
ma, cortical adenoma and cortical adenocarcinoma. 1.9%; female: 0.8%) were observed. Among proliferative changes in
In Wistar rats, cortical cell vacuolation (zona fasciculata and aged Wistar rats (110 weeks), incidences of adenoma of pars
reticularis), focal/multifocal hemorrhages and hemangiectasis distalis (male: 14.8%; female: 25%) and pars intermedialis (male:
were observed as common spontaneous non-proliferative lesions 0.9%) were observed, followed by hyperplasia of pars distalis and
in adrenals. In this analysis, several changes in adrenals of aged rats pars intermedialis in male rats (4.6% and 1.9%, respectively).
(110 weeks) in both the sexes were observed such as, cortical cell Beagle dogs of 18–21 months old revealed incidences of
hypertrophy (3.7% in both sexes), capsular thickening (male: 4.6%; pituitary cysts as 22.2% (male) and 33.3% (female) whereas, these
female: 1.9%) and extra-medullary hematopoiesis (0.9% in both incidences were 9.3% (male and female) in beagle dogs of 10–12
sexes). The other incidences such as cystic changes (male: 0.9%), months old. These cysts were predominantly observed in anterior
hemosiderosis (male: 0.9%), accessory cortical tissue (male: 2.1– lobe (Tables 1–3, Figs. 4 and 5).
3.7%; female: 2–4.6%) and thrombus (female: 1.9%) were also
observed. 3.3. Thyroid gland
Among the proliferative lesions in aged rats (110 weeks),
higher incidence of cortical cell hyperplasia (5.6% in both sexes) In Wistar rats, C-cell (parafollicular cells) hyperplasia, cyst and
were observed followed by medullary hyperplasia (male: 3.7%; ectopic thymus were observed as spontaneous changes in rat up to
female: 2.8%), complex pheochromocytoma (male: 0.9%; female: the age of 32–34 weeks. In aged rats (110 weeks), higher
1.9%), cortical cell adenoma (0.9% in both sexes) and cortical cell incidences of C-cell hyperplasia (23.1% in both the sexes), follicular
adenocarcinoma (0.9% in female). cell hyperplasia (male: 4.6%; female: 0.9%), C-cell adenoma (male:
In beagle dogs, the incidences of hemangiectasis was higher in 0.9%; female: 4.6%) and C-cell adenocarcinoma (male: 2.8%;
18–21 months (male: 11.1% and female: 33.3%) aged dogs in both female: 0.9%) were revealed.
the sexes as compared to 10–12 months (male: 1.9% and female: Beagle dogs of 18–21 months old revealed incidences of C-cell
1.9%) of age. The incidence of accessory adrenocortical tissue was complex as 16.7% (male) and 22.2% (female) whereas, beagle dogs
27.8% in 18–21 months old male dogs.

Table 2
Incidences of spontaneous lesions in Wistar rats (%)

Organ/finding Male Female

10–12 weeks 32–34 weeks 110 weeks 10–12 weeks 32–34 weeks 110 weeks (n = 108)
(n = 191) (n = 119) (n = 108) (n = 197) (n = 118)
Pituitary glands
Cysts 0.5 1.7 1.9 0.5 0.8 0
Hemorrhage 0 0 1.9 0 0 0
Congestion 0 0 1.9 0 0.8 0
Hemangiectasis 0 0 2.8b,c 0 0 0
Mineralization 0 0 0.9 0 0 0
Degeneration-chromophilic 0 0 0.9 0 0 0
cells
Pleomorphic cells 0 0 0.9 0 0 0
Nodular hyperplasia 0 0 1.9 0 0 0
Hyperplasia-pars distalis 0 0 4.6b,c 0 0 0
Hyperplasia-pars 0 0 1.9 0 0 0
intermedialis
Adenoma-Pars distalis 0 0 14.8b,c 0 0 25.0b,c
Adenoma-Pars intermedialis 0 0 0.9 0 0 0
Adenocarcinoma-Pars distalis 0 0 0 0 0 1.9
Ganglioneuroma- Pars nervosa 0 0 0 0 0 0.9

Thyroid gland
Atrophy 0 0 0.9 0 0 0
Cyst 0 3.4 1.9 0.5 0 1.9
C-cell hyperplasia 0 1.7 23.1b,c 0 3.4a 23.1b,c
Follicular cell hyperplasia 0 0 4.6b,c 0 0 0.9
C-cell adenoma 0 0 0.9 0 0 4.6b,c
C-cell adenocarcinoma 0 0 2.8b,c 0 0 0.9
Follicular cell adenoma 0 0 0 0 0 1.9
Ectopic thymus 0 0 0 0.5 0 0
a
Significant at p < 0.05 between 10–12 weeks and 32–34 weeks.
b
Significant at p < 0.05 between 10–12 weeks and 110 weeks.
c
Significant at p < 0.05 between 32–34 weeks and 110 weeks.
4 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13

Table 3
Incidences of spontaneous lesions in beagle dogs (%).

Organ/finding Male Female

10–12 months (n = 54) 18–21 months (n = 18) 10–12 months (n = 54) 18–21 months (n = 18)
Adrenal glands
Cortical cell vacuolation 9.3 16.7 5.6 22.2
Hemangiectasis 1.9 11.1 1.9 33.3*
Congestion 1.9 11.1 3.7 22.2*
Hemorrhage 3.7 5.6 1.9 22.2*
Accessory cortical tissue 0 27.8* 0 0

Thyroid
C-cell complex 1.9 16.7* 1.9 22.2*
Cyst 1.9 16.7 0 16.7*

Parathyroid
Cyst 1.9 11.1 1.9 16.7*

Pituitary
Cyst 9.3 22.2 9.3 33.3*

Endocrine pancreas
Islet cell vacuolation 0 5.6 0 5.6
Islet cell hyperplasia 0 5.6 0 0
*
Significant at p-value < 0.05.

of 10–12 months old showed 1.9% incidences of C-cell complex 3.4. Parathyroid gland
(male and female).
Additionally, we have observed cystic changes, which were In aged rats (110 weeks), higher incidences of parathyroid
lined by cuboidal epithelium and often ciliated in thyroid gland of hyperplasia (male: 3.7%, female: 5.6%) and chief cell hypertrophy
both the sexes (male: 1.9–16.7%; female: 0–16.7%) (Tables 1–3, (female: 0.9%) were observed as spontaneous lesions.
Fig. 7). In beagle dogs, higher incidences of parathyroid cysts were
observed in both the sexes at 18–21 months (male: 11.1%; female:

Fig. 1. Adrenal gland: (a) Normal histological architecture, Wistar rat (H&E 40); (b) hemangiectasis (arrow), Wistar rat (H&E 40); (c) congestion (arrow), Wistar rat (H&E
40); (d) medullary hyperplasia (arrow), Wistar rat (H&E 40).
 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13 5

Fig. 2. Adrenal gland: (a) Normal histological architecture, Wistar rat (H&E 100); (b) accessory cortical tissue (arrow), Wistar rat (H&E 100); (c) cortical cell hypertrophy
(arrow), Wistar rat (H&E 100); (d) cortical adenocarcinoma, Wistar rat (H&E 100).

16.7%) of age as compared to 10–12 months of age (male: 1.9 %; diagnostic criteria can have major impacts on the reliability of
female: 1.9%) (Tables 1–3, Fig. 8). the data when used for comparison purposes. Therefore, to develop
a useful historical control data base, it is essential to periodically
3.5. Endocrine pancreas update and assess the new data which become available and delete
or alter data which may be misleading (Poteracki and Walsh, 1998).
The spontaneous lesions of endocrine pancreas with few In the present analysis, the highest numbers (types) of
incidences were atrophy of islets, islet cell hyperplasia and islet cell spontaneous lesions were observed in adrenal glands followed
carcinoma in aged Wistar rats. The Islet cell hyperplasia was found in descending order by pituitary, thyroid, endocrine pancreas and
to be more frequent in male (13%) when compared to female (0.9%) parathyroid glands in Wistar rats. In beagle dogs, highest numbers
Wistar rats of approximately 110 weeks of age. In aged dogs, (types) of spontaneous lesions were seen in adrenals followed by
incidences of islet cell vacuolation and islet cell hyperplasia were thyroid, endocrine pancreas, pituitary and parathyroid gland.
observed (Tables 1–3, Fig. 6).
4.1. Adrenal glands
4. Discussion
The adrenal cortex of animals is prone to develop degenerative
The endocrine system is an important integrating system of the and proliferative lesions, the etiology of which may be either
body. Finely-regulated hormonal mechanisms make the endocrine spontaneous in nature or experimentally induced (Ribelin, 1984).
system particularly sensitive to toxic effects of exogenous Further, adrenal gland is also one of the most common endocrine
compounds. These effects can interfere with the action, biosyn- organs affected by the chemicals, where the lesions are more
thesis and release of hormones, and result in alterations in frequent in the zona fasciculata and reticularis than in the zona
hormonal target cells. These examples serve to show that the study glomerulosa (Rosol et al., 2001). However, these changes shall be
of endocrine systems in routine toxicity tests is important, not only differentiated from spontaneous lesions.
from a mechanistic point of view, but also to ensure reliable Our analysis revealed that, in Wistar rats, the common
evaluation of the safety of chemicals (Clark and Van Leeuwen, spontaneous non-proliferative lesions like, cortical cell vacuolation
1990). However, stress, housing conditions, diet, age and sampling (zona fasciculata and reticularis), focal/multifocal hemorrhages and
procedures can all influence the results obtained and lead to hemangiectasis were related to the age of rats. Frith et al. (2000)
conflicting results. Therefore, special attention should be given to also reported that these degenerative changes are relatively
the experimental conditions during the study activities as common in aged rats and also may be due to some physiological
functional alteration in endocrine glands as above factors may stress associated with the experimental procedures and husband-
prevail histomorphologically as spontaneous changes in the pre- ry. Severe form of cortical cell vacuolation may progress to cortical
clinical toxicity studies (Everds et al., 2013). cystic degeneration and may be difficult to differentiate from
Variation in spontaneous lesions based on genetic drift within hemangiectasis/peliosis once filled with blood.
the strain/breed, atypically high or low incidence of a specific The three zones of the adrenal cortex normally have some
lesion/neoplasia in a particular study, and modification of degree of vacuolation varying among zones as a state of cellular
6 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13

Fig. 3. Adrenal gland: (a) Normal histological architecture, Wistar rat (H&E 200); (b) extramedullary hematopoiesis (arrow), Wistar rat (H&E 200); (c) cortical cell
vacuolation (arrow), Wistar rat (H&E 200); (d) capsular thickening and cortical cell vacuolation (arrow), Wistar rat (H&E 200); (e) pheochromocytoma, Wistar rat (H&E
200); (f) complex pheochromocytoma, Wistar rat (H&E 200).

activity, predominantly in zona fasciculata. These vacuoles consist degeneration often represents swelling and damage to mitochon-
of large or small, clear, round intracytoplasmic structures dria. An antibiotic, DMNM (a-[1,4-dioxido-3-methylquinoxalin-2-
containing neutral lipids and cholesterol. Physiological cortical yl]-N-methylnitrone) causes impaired steroidogenesis, likely by
vacuolation is generally present in diffuse pattern and may be an blocking the conversion of cholesterol to pregnenolone. The acute
indirect response to a drug or a reflection of some other stress upon exposure of DMNM results in cytoplasmic vacuolation of the zona
the animal (Hamlin and Banas, 1990). Higher degree of cortical fasciculata and reticularis. Chemicals that induce severe vacuolar
vacuolation is pathological and characterized by presence of small degeneration, like DMNM, often lead to loss of adrenocortical cells
spaces or cavities in the cytoplasm of the cortical cells. Spontane- due to necrosis, cell lysis, or apoptosis. Chemicals such as the DDT
ous cortical vacuolar change is a relatively common change in aged derivative, o,p-DDD, which damage mitochondria, can lead to
rats. However, the cortical cell vacuolar degeneration was also severe, acute necrosis and hemorrhage of the adrenal cortex.
shown to be induced by several agents (Yarrington et al., 1996, Ultrastructurally, the smooth endoplasmic reticulum and mito-
1981; Latendresse et al., 1994; Gopinath et al., 1988). chondria will be swollen with disruption of the cristae. In addition,
One of the common toxin-induced changes that can result in there will be an accumulation of lipid bodies due to impaired
excess steroid precursors and cytoplasmic vacuolation is impaired steroidogenesis (Rosol et al., 2001).
steroidogenesis. Triaryl phosphates (group of organophosphates) Similarly, triaryl phosphates which are used in plastics,
results in impaired cholesterol metabolism and increased cyto- hydraulic fluids, and lubricants, block the action of neutral
plasmic lipid vacuoles. Usually the vacuolation begins in the inner cholesterol ester hydrolase with minimal inhibition of acyl
cortex and progresses to the outer zones. If the vacuoles are clear coenzyme A: cholesterol acyltransferase and results in an
by light microscopy, they usually represent lipid, whereas granular accumulation of cholesterol esters in the fat vacuoles in all layers
 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13 7

Fig. 4. Pituitary gland: (a) Normal histological architecture, Wistar rat (H&E 40); (b) hyperplasia (arrow head) and hemangiectasis (arrow)—pars distalis, Wistar rat (H&E
40); (c) hyperplasia-pars intermedialis, Wistar rat (H&E 40); (d) adenoma—pars distalis (arrow), Wistar rat (H&E 40).

of the adrenal cortex. Administration of excess glucocorticoids vascular changes in addition to cytoplasmic alterations (Pudney
leads to increased negative feedback in the pituitary gland and a et al., 1984).
reduction in ACTH secretion. This will inhibit the action of CYP11A1 The hyperemia is followed by extravasation of erythrocytes into
and lead to reduced cholesterol metabolism, accumulation of fat intercellular spaces and disruption of the cord like organization of
vacuoles in the cortical cells, and eventual atrophy of the zona the cells. This condition may progress to vascular pooling
fasciculata and reticularis (Rosol et al., 2001). (angiectasis) and hemorrhage due to cell deletion and hemorrhagic
In order to differentiate the spontaneous cortical cell vacuola- infarction. These ACTH induced changes probably represent
tion with that of chemically induced, one needs to compare with pathological reaction to over-stimulation rather than physiological
concurrent control group of animals for the pattern and severity of responses Frith et al. (2000).
lesion, the nature of the test compound, whether it has any effect As reported by Frith et al. (2000), the accessory adrenal cortical
on mechanism of steroidogenesis which may contribute to the tissue is characterized by presence of concomitant adrenocortical
development of vacuolation as discussed earlier. tissue outside the adrenal capsule or in the periadrenal tissue. It is
Focal/multifocal hemorrhages are characterized by accumula- most commonly found in the retroperitoneal fat adjacent to the
tion of free erythrocytes outside of vascular channels. However, if adrenal gland or kidney. Accessory cortical tissue shall be
the condition is longstanding, hemosiderin-laden macrophages, differentiated from cortical neoplasms wherein, accessory cortical
cholesterol clefts, fibrosis and sometimes infarction may be tissue lacks typical zonal arrangement of the adrenal cortex but the
noticed. Hemorrhage may occur secondary to neoplasia and is central portion of the nodule may contain dilated capillaries and an
sometimes seen in animals with mononuclear cell leukemia incomplete capsule may be present. This makes the differentiation
(MacKenzie and Boorman, 1990). from a cortical neoplasm difficult as the cortical tissue may extend
Hemangiectasis observed in the cortex or medulla is character- past the capsular borders. However, features of cellular atypia and
ized by blood filled spaces lined by endothelium and it may be vascular or capsular invasion are not present in accessory cortical
associated with inflammatory, degenerative and or neoplastic tissue which will aid in differentiating with that of neoplasm.
disease processes. Hyperemia with hemangiectasis and dilatation Among the proliferative lesions in aged rats (110 weeks),
of intercellular spaces may be related to stress and/or exogenous higher incidence of cortical cell hyperplasia were observed
administration of adrenocorticotropic hormone (Frith et al., 2000). followed by medullary hyperplasia, complex pheochromocytoma,
Further results of analysis revealed that the incidence of peliosis cortical cell adenoma and cortical cell adenocarcinoma. Other
in 110 weeks old female rats was higher (11.1%) than male rats authors also reported similar findings however with slightly higher
(2.8%). This was consistent with the other reported findings (Dhom incidence of benign pheochromocytoma (male: 9.7%; female:
et al., 1981; Frith et al., 2000) and considered to be associated with 1.3%), malignant pheochromocytoma (male: 1.7%; female: 0.4%),
constant estrus in aging female rats as probable reason for higher cortical cell adenomas (males: 3.4%; females: 4.1%) and less
incidence (Dhom et al., 1981). Although the adrenals are amongst common cortical cell adenocarcinomas (<0.9%) (Poteracki and
the most highly vascularized organs this is not always appreciated Walsh, 1998). The cortical cell hyperplasia which is of higher
due to collapse of the vasculature in conventional histological incidence in our studies should be differentiated from chemically
preparation (Rhodin, 1971). Administration of ACTH to rats causes induced hyperplasia and one has to look for the type and
8 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13

Fig. 5. Pituitary gland: (a) Normal histological architecture, Wistar rat (H&E 100); (b) cyst (arrow), beagle dog (H&E 100); (c) congestion (arrow), Wistar rat (H&E 100);
(d) adenocarcinoma—pars distalis, Wistar rat (H&E 100); (e) normal histological architecture, Wistar rat (H&E 200); (f) ganglioneuroma—pars nervosa, Wistar rat (H&E
200).

distribution of the lesion and historical control data before spontaneous (age related) or can result from dietary modifications
concluding it as treatment related effect. Usually the chemically and the administration of exogenous chemicals (Rosol et al., 2001).
induced cortical cell hyperplasia will be of nodular in type, often The compounds which induce medullary hyperplasia include
multiple in distributions, whereas excess ACTH stimulation nicotine, reserpine, synthetic retinoids, thiouracil, neuroleptics,
induces diffuse cortical cell hyperplasia (Klaassen et al., 2013). growth hormone, 4-chloro-m-phenylenediamine, 11, 2-trichloro-
The cortical adenomas are usually well-defined nodular ethane (Zomepirac), an analgesic, a b-adrenergic blocker (Bloca-
proliferations that arise from cells of the zona fasciculata and dren), and lactose & sugar alcohols, such as mannitol, sorbitol, and
zona reticularis. The expansive growth of the tumor causes definite xylitol and lactitol. These changes are widely reported in rats but
compression around the periphery. Adenomas appear to be far there are few reports in other species (Gopinath et al., 1988).
more prevalent than carcinomas. Cortical carcinomas are usually The relatively high incidence of pheochromocytoma, especially
large with significant distortion of the cortex. Adrenal cortical in male rats of certain strains, makes it difficult to differentiate
neoplasms have been induced experimentally in rats with a wide between spontaneous and xenobiotic induced pheochromocyto-
variety of agents including estrogens, irradiation, certain chlori- mas. There is a strong genetic component to the pathogenesis of
nated hydrocarbons and benzenes. The spontaneously developing pheochromocytomas in rats, exemplified by the different inci-
adrenal cortical tumors occur at a relatively low frequency in most dence of pheochromocytomas between strains of rats. Studies
rat stocks and strains (Brown et al., 1995). from the NTP historical database of 2-year-old F344 rats have
The adrenal medulla has been regarded as a less common site of shown that the incidence of pheochromocytomas was 17.0 and
chemically induced degenerative lesions. However, proliferative 3.5% for males and females, respectively.
lesions of the medulla in rats occur frequently and can be
 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13 9

Fig. 6. Endocrine pancreas: (a) Normal histological architecture, Wistar rat (H&E 100); (b) islet cell hyperplasia (arrow), Wistar rat (H&E 100); (c) normal histological
architecture, Wistar rat (H&E 200); (d) islet cell hyperplasia (arrow), Wistar rat (H&E 200).

The strains of rats with high incidences of pheochromocytoma been diagnosed as pheochromocytomas and ganglioneuromas.
include Wistar, NEDH (New England Deaconess Hospital), Long– Complex pheochromocytomas often are observed in conjunction
Evans, and Sprague-Dawley. Pheochromocytomas are considerably with proliferative lesions of pituitary, thyroid C-cells and/or
less common in Osborne-Mendel, Charles River, Holtzman, and pancreatic islet cells. This apparent relationship bears some
WAG/Rij rats. Most studies have reported a higher incidence in similarities to Mixed Endocrine Neoplasm Syndrome of humans
males than in females. Cross breeding of animals with high and low (Brown et al., 1995).
frequencies of adrenal medullary proliferative lesions results in F1 Beagle dogs also exhibited lesions similar to Wistar rat with
animals with an intermediate tumor frequency. Multiple factors incidence of cortical cell vacuolation, hemangiectasis, hemorrhage
are involved in the pathogenesis of pheochromocytomas in rats and congestion. Definite age relation was observed with the
which include the genetic background, chronic high levels of occurrence of spontaneous lesions in adrenals of dogs where the
growth hormone or prolactin associated with pituitary tumors, incidence rate was higher by 2–15 folds at 18–21 months as
dietary factors, and stimulation of the autonomic nervous system. compared to the findings at 10–12 months of age.
Chronic administration of reserpine results in decreased blood Our analysis revealed that the incidences of hemangiectasis
pressure, neural stimulation of the adrenal medulla and develop- were higher at 18–21 months aged beagles as compared to 10–12
ment of hyperplasia and pheochromocytoma in the rat. Several months of age in both the sexes. The incidence of accessory
sugars and sugar alcohols have produced adrenal medullary adrenocortical tissue was 27.8% in 18–21 months old male dogs.
tumors at high dosages, including xylitol, sorbitol, lacitol, and The accessory adrenocortical tissue is the aberration of adreno-
lactose at concentrations of 10–20% in the diet. Although the cortical tissue separated from the original body and surrounding
mechanism involved is not completely understood, a role for capsule and is common inside and outside of the adrenal capsule
calcium has been postulated. High doses of slowly absorbed sugars (Sato et. al., 2012). Higher (1.5–2 fold) incidences of cortical cell
and starches increase the absorption and urinary excretion of vacuolation at the age of 10–12 months old dogs is supported by
calcium. In addition, vitamin D3 induces proliferative lesions the report of Sato et al. (2012) wherein he reported that
(hyperplastic nodules and pheochromocytomas) in the rat (Rosol vacuolation of the cells of the zona glomerulosa and various
et al., 2001). Administration of growth hormone, thiouracil, degrees of zonal vacuolation in the cells of the zona fasciculata
estrogens, radiation, retinol acetate, neuroleptics, vitamin A occur occasionally, especially near the zona reticularis, where
analogues, nicotine, reserpine, zomepirac sodium and 4-chloro- accumulation of large lipid droplets containing cells is frequently
m-phynelenediamine have been associated with induction of seen in the zona glomerulosain beagle dogs.
pheochromocytomas in rats (Brown et al., 1995). The lesions observed in adrenal glands might be attributed to
Complex pheochromocytomas are usually solitary masses various factors contributing to stress (Frith et al., 2000) and
within the medulla and the tumor is composed of a mixture of physiological alterations (e.g. estrus in females) (Dhom et al.,
pheochromocytes, ganglion cells, Schwann cells, and neurofibrils. 1981). Factors that induce stress responses in laboratory animals
Hence it has the morphological components of both pheochromo- during the pretest and study periods include transportation,
cytoma and ganglioneuroma. These tumors are rare and actual housing conditions, handling, restraint, diet, diagnostic proce-
incidence is difficult to determine as many of these lesions have dures, sample collection, and dosing (Conour et al., 2006). The
10 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13

Fig. 7. Thyroid and parathyroid gland: (a) Normal histological architecture, Wistar rat (H&E 40); (b) C-cell adenoma, Wistar rat (H&E 40); (c) normal histological
architecture, Wistar rat (H&E 100); (d) cyst (arrow), Wistar rat (H&E 100); (e) C-cell complex, beagle dog (H&E 100); (f) C-cell adenocarcinoma, Wistar rat (H&E 100).

adrenal gland is an essential stress-responsive organ that is part of hemangiectasis, mineralization and degeneration of chromophilic
both the hypothalamic-pituitary-adrenal axis and the sympatho- cells were the other observed spontaneous changes in rats. The
adrenomedullary system. Animals respond to these stressors by anterior pituitary of aged rats frequently has hyperplastic and
releasing a number of mediators such as catecholamines and neoplastic lesions that consist of one or more of the hormone-
glucocorticoids and their effects tend to be transient and result in producing cell types that are normally present in this gland (Van
habituation within 1–4 days (Prager et al., 2011). Prolonged Putten and Van Zwieten, 1988; Van Putten et al., 1988; Sandusky
exposure to stress commonly increases adrenal weight and is et al., 1988). The incidence of pituitary cysts was also found to be
associated with spontaneous occurrence of changes such as higher at 110 weeks old rats.
cellular hypertrophy and/or hyperplasia (Ulrich-Lai et al., 2006). Results of analysis evoked higher incidences of pituitary cysts in
beagle dogs. Similar findings were reported by Morishima et al.
4.2. Pituitary gland (1990). In consistent with our findings, Sato et al. (2012) also
reported that pituitary cysts were often found in the anterior lobe.
In Wistar rats, the higher incidence of pituitary adenomas in The pituitary cysts are considered to develop from remnants of the
females suggests the role of estrogen, either by direct effect on distal cranio-pharyngeal duct (Capen, 1985) and were thought to
pituitary cells or inhibition of dopamine. The effect of anti- be of congenital origin (Morishima et al., 1990). Further, the higher
estrogenic compound, tamoxifen was demonstrated by adminis- incidences of cystic changes were noticed in beagles as compared
tering it for 2 years which resulted in reduction in incidences of to Wistar rats in our present analysis.
pituitary adenomas and adenomas related deaths in treatment The pituitary gland neoplasms are among the most commonly
group animals (Greaves et al., 1993). Adenoma and adenocarcino- encountered in most of the rat strains during routine long term
ma of pars distalis, ganglioneuroma of pars nervosa, toxicological studies. The majority of these neoplasms occur in the
 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13 11

Fig. 8. Parathyroid gland: (a) Normal histological architecture, Wistar rat (H&E 100); (b) cyst (arrow), beagle dog (H&E 100); (c) normal histological architecture, Wistar rat
(H&E 200); (d) chief cell hypertrophy (arrow), Wistar rat (H&E 200); (e) hyperplasia, Wistar rat (H&E 200).

pars distalis. Only few chemical compounds have been shown to 4.3. Thyroid gland
induce increase in the incidences of pituitary neoplasms in the rats.
The test compounds which alter the normal pituitary-target organ Similar to our findings, many authors have reported spontane-
axis would be suspected to secondarily induce pituitary neoplasms ous proliferative changes in thyroid glands with varying inciden-
by hyper stimulatory mechanisms due to altered feedback ces. Haschek et al. (2013) reported that the C-cell proliferative
regulation. Some estrogenic agents and/or irradiation have been lesions occur commonly in many rat strains such as, Sprague-
reported to increase the incidence of pituitary neoplasms. High Dawley, Fischer and Wistar and the incidence varies from 19% to
caloric and/or high protein levels in the diet also known to be cause 33% with no sex differences.
for high incidences of pituitary neoplasms in rats (Majka et al., Burek (1978) reported that crossbreeding of high (WAG/Rij) and
1990). As reported by Uchida et al. (1981), all the rats with pituitary low (BN/Bi) incidence strains results in F1 animals with
tumors had hyperplastic mammary glands, associated mostly with intermediate tumor incidences in thyroid gland. As with prolifer-
lactation which suggests that spontaneous pituitary tumors have a ative lesions of other endocrine organs there is a correlation
hormonal effect on mammary glands. between the age of the animal and the presence of the C-cell
While differentiating the spontaneously occurring neoplasms proliferative lesions. Rats which were between 12 and 24 months
with that of chemically induced, one needs to compare with of age had a 10% incidence whereas approximately 20% of 2–3 year-
concurrent control group of animals for the rate of incidence and old rats had C-cell tumors. Similarly, the frequency of focal and
the nature of the test compound, whether it has any estrogen like diffuse C-cell hyperplasia increased progressively with age.
effects and/or effect on pituitary-target organ axis etc. The Although stimulus for C-cell proliferation remains unknown, the
historical data of pituitary tumors will be of immense use while results by Delellies et al. (1979) in rats showed that there is a
deciding the effects of test compounds. prolonged phase of diffuse and nodular hyperplasia of C-cells
12 G.J. Nataraju et al. / Experimental and Toxicologic Pathology 68 (2016) 1–13

which is accompanied by elevation of serum calcitonin. According induction of toxicity of diabetogenics like alloxan and streptozo-
to the National Toxicology Program historical database of 2-year- tocin.
old F344 rats, the incidence of C-cell neoplasms was 8.9% in males
and 8.5% in females. (Klaassen et al., 2013). Acknowledgement
In beagle dogs, similar higher incidences of C-cell complex were
reported by Morishima et al. (1990). C-cell complex which is We thank Mr. Rajesh J. Patel for statistical analysis of data and
characterized by solid islands of C-cell-like clear cells are histo technicians of our laboratory for their skillful technical
frequently seen in the thyroid tissue and are considered to be assistance.
remnants of ultimobranchial bodies formed before differentiation
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