TOXICOLOGYANDAPPLIEDPHARMACOLOGY37.433-144

Morphological

Effects of Hexachlorobenzene Female Rhesus Monkeys1

M. J. IATROPOULOS, W. HOBSON, V. KNAUF, International

Center

Received

Toxicity

AND

H. P. ADAMS

of Environmental Safety, Albany Medical Holloman AFB, New Mexico 88330

December

10,1975;

accepted

April

in

College,

I2, I976

Morphological Effects of Hexachlorobenzene Toxicity in Female Rhesus Monkeys. IATKOPOULOS, M.J., HOBSON,W.,KNAUF, V., ANDADAMS, H.P. (1976). Toxicol. Appl. Pharmacol. 37, 433-444. Hexachlorobenzene was administered daily by gavage to five adult female rhesus monkeys for 60 days. Two received 128 mg/kg of HCB, one 64 mg/kg, one 32 mg/kg, and one 8 mg/kg. Complete histopathological examination revealed thymic cortical atrophy; degenerative ovarian changes involving the primary follicles, germinal epithelium, and stroma; degenerative changes in the liver which included alterations compatible with prophyria tarda; and degenerative changes in the kidney. No cutaneous or neuroIogica1 alterations were observed. The mechanism(s) by which HCB and/or its metabolites induce these morphologic effects was not established. Hexachlorobenzene (HCB, C,Cl,) is a water-insoluble, crystalline substance employed in organic synthesis and as a fungicide to control smut disease in cereal grains. This compound is sometimes confused with the insecticide, hexachlorocyclohexane (C,H,CI,), more commonly known as benzene hexachloride. The potential toxicity of HCB was revealed during the years 19551959 when over 3000 cases of toxic porphyria cutanea were reported in southeastern Turkey. The cause of the outbreak was traced to the mass ingestion of HCB-treated wheat intended for planting (Cam and Nigogosyan, 1963). The signs observed were photosensitization, porphyrinuria, and hepatomegaly. This persistent fungicide has a low acute toxicity (Ben-Dyke ef al., 1970) and residues have been detected in mussels, fresh water fish, seed-eating and predatory birds (Koeman et al., 1969a,b). It has also been found in vegetable, animal, and human body fat (Vos et al., 1968; Brady and Siyali, 1972). HCB administered to sheep either orally in capsules for 18 weeks (Avrahami and Steele, 1972) or as pickled wheat for 12 weeks (Craig and Dwyer, 1961) resulted in accumulation of HCB residue in body fat. Chronic toxicity studies conducted on rabbits, guinea pigs, rats, and mice (De Matteis et al., 1961) revealed disturbances in porphyrin metabolism, and neurological rather than cutaneous changes and lesions. Liver changes were reported in the rat (Medline et al., 1973) after the third week of feeding 0.2 % HCB in the diet, which consisted mainly of hepatocyte enlargement with intracytoplasmic inclusions, proliferation 1 This work was supported in part by the Federal Department of Research and Technology of the Federal Republic of Germany, under a coordinated research program entitled “Ecologic-Toxicologic Effects of Foreign Compounds in Non-Human Primates and Other Laboratory Animals.” Copyright C) 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

433

434

IATROPOULOS ET AL.

of smooth endoplasmic reticulum, and formation of large lipid droplets. Ockner and Schmid (1961) induced porphyria in rats by adding 0.2 % HCB in their diet and observed hepatocellular degeneration. However, 39 % of the animals died within the first month of administration after displaying neurological disturbances without any signs of porphyria. In another study involving rats (Kuiper-Goodman et al., 1975), a doserelated accumulation of HCB residues was observed in the tissues, and liver and kidney changes were found. Furthermore, porphyria was observed in the females. Studies with Japanese quail (Vos et al., 1971) have shown extensive liver damage and impaired reproduction with tremor and mortality occurring in the high-dose group when HCB was given orally for 90 days in doses up to 80 ppm. In addition, it has been demonstrated that dietary concentration of HCB of 80 ppm or greater, influences various reproductive parameters in rats (Grant et al., 1975). Little is known of the effects of HCB in nonhuman primates (Rozman et al., 1975). The rhesus monkey was, therefore, selected to assess the effects of chronic ingestion of HCB at the light microscopic level. METHODS

Five adult female rhesus monkeys (Macaca mulatta) were dosed daily by gastric intubation with hexachlorobenzene’ which was recrystallized twice in benzene. The body weights ranged from 3.72-5.97 kg. Two monkeys received 128 mg of HCB/kg of HCB suspended in 1% methylcellulose daily for 60 days. Three others received 64, 32, and 8 mg/kg/day, respectively, while a fourth received the vehicle only. For statistical purposes, in addition to the one vehicle control, 14 other animals of approximately the same age and body weight from our colony were added for the assessment of ovarian morphology. All animals were individually housed, given water ad libitum, and provided with 250 g of dry monkey chow and half an apple daily. At sacrifice, the animals were exsanguinated under Ketamine3 anesthesia. Complete necropsies were performed on all animals with special attention given to the lips, tongue, oral cavity, external ears, eyes, limbs, vagina, and anus. Each organ was examined in situ and after removal under low power (10x) magnification. Representative samples from salivary glands, mammary tissue, liver, stomach, duodenum, jejunum, ileum, colon, pancreas, adrenals, kidneys, urinary bladder, mesenteric lymph nodes, esophagus, trachea, lungs, heart, thyroid, parathyroids, uterus, and tongue were fixed in 10 % buffered formalin. The eyes were fixed in Zenker’s acetic acid solution, the ovaries in Bouin’s solution (Luna, 1968), and the central nervous system in 37-40% buffered formalin. The femur was fixed for 24 hr in a modified Bouin’s fluid in which the acetic acid content had been reduced by 50 %. Regions of the central nervous system were sampled at the following levels: the precentral gyrus, the superior temporal gyrus, and the lateral calcarine sulcus, the middle peduncle, the anterior third of the cerebellum, the midportion of the pans, and the midlevel of the medulla. The pituitaries were stained with a modified trichrome (Herlant, 1956), periodic acid Schiff (PAS)-orange G (Herlant, 1956), and 1~x01 fast blue-PAS (Kerr, 1965) with or without orange G. The livers were stained with hematoxylin and eosin, von Kossa’s z Eastman Kodak, Rochester, New York. ’ Parke-Davis, Detroit, Michigan.

435

HCB IN MONKEYS

method, the Turnbull blue method (Mallory, 1961), and the A.F.I.P. method for lipofuscin (Luna, 1968). The remaining tissues were routinely stained with hematoxylin and eosin. A semiqualitative method was used to determine the status of the ovarian epitheloid cells. The modified point sampling procedure (Hennig, 1958) was adopted to determine the ratio of corpora lutea to primary follicles from ovaries which were sectioned along their long axis through the mesoovarium. RESULTS In all HCB-exposed animals, microscopic examination of the thymus revealed a reduction or absence of individual lobules. The changes were greater in the monkeys of the 12%mg/kg group, and diminished to normal in the low-dose group. The animals in which the thymic cortex was reduced also manifested hyperplasia of the reticular cells, macrophages, plasma cells, and some lymphocytes in the medulla, along with an increased number of thymic corpuscles. The proximal renal tubules of all experimental groups contained colorless droplets within the cytoplasm, which resulted in a finely granular appearance (Fig. 1). In some of these the tubular cytoplasm was filled with optically empty vacuoles of various sizes (Fig. l), and in some instances perinuclear halos were present. One monkey of the highdose group showed flattening of the epithelia of the proximal tubules, hyperemia of the glomeruli, and thickening of the basement membranes. One animal given 64 mg/kg showed, in addition to the above, fatty degeneration of the tubular epithelium, basal vacuolation of the epithelial cells of the proximal tubules, and scattered foci of chronic inflammatory reaction within some glomeruli and interstitially in the papilla. Liver changes were present in all HCB-treated animals (Table 1) which ranged from cloudy swelling, fatty degeneration (Fig. 2), focal necrosis and fibrosis to centrolobular TABLE INCIDENCE

AND GRADING OF LIVER HEXACHLOROBENZENE

1

CHANGES ORALLY

IN RHESUS MONKEYS FOR 60 DAYS

GIVEN

Doseof HCB (mg/kg,/day)

Control

Group Observation Cloudy swelling Fatty degeneration Focal necrosis Centrolobular hepatocellular hypertrophy Cholangioleproliferation Bile casts Granular brown-black hepatocellular pigment = -;

slight

= f;

moderate

= ++;

marked

32

64

128

-

l/+“sb 1/++ 1/++ 1/++

1/+++ 1/+++ -

2/+++ 2/+++

-

l/f -

-

1/++ -

1I+++ 1I+++ 1/++

-

-

1/++

1/++

1/++

nAnimalsper group. b Absent

8

= +++.

-

436

IATROPOULOS

ET AL.

hepatocellular hypertrophy (Fig. 3), cholangiolitic proliferation, bile casts, and granular, dark brown pigment (hemosiderin) within the hepatocytes and Kupffer cells. Proliferation of Kupffer cells was also apparent. The germinal epithelium of the ovaries of all exposed animals was thicker than that of the control group. At the lowest dose level there was a change from the cuboidal,

FIG. 1. Kidney from rhesus monkey given 128 mg/kg/day of HCB for 60 days. Fine and coarse vacuolization of the proximal renal tubules are evident. Hematoxylin and eosin, x290.

unilayer germinal epithelium of the control ovaries to a tall, columnar unilayer form. With increasing dose levels (32 and 64 mg/kg) the germinal epithelium appeared pseudostratified (Fig. 4); however, in the highest dose group the columnar form was observed. The nuclei of the cuboidal layer cells surrounding the primordial follicles were pyknotic and karyorrhectic. At the highest dose these changes were extensive and marked, while in the 32-mg/kg monkey they were slight. Multiple follicular cysts were observed in all HCB-treated animals except in the 32-mg/kg animal. The largest

HCB IN MONKEYS

437

cyst, which was present in the high-dose group, measured 0.5 x 0.2 x 0.7 cm. In all HCB-exposed monkeys, the ratio of corpora lutea to primary follicles was greater than in the controls, and this parameter appeared to be dose-dependent. The ovarian cortices of the animals receiving 128 mg/kg consisted predominantly of dense stroma (Fig. 5) and contained 80% fewer primary follicles (as determined from counts derived from

FIG. 2. Liver from rhesus monkey given 128 mg/kg/day of HCB for 60 days. Fatty metamorphosis and cloudy swelling are evident, involving the periportal area. Hematoxylin and eosin, x290.

sections along the long axis). The cortices of the 64-mg/kg animal (Fig. 4), the 32-mg/kg animal, and the 8-mg/kg animal (Fig. 6) showed varying degrees of degeneration (Table 2) and marked reduction in the number of primary follicles, 71, 17, and 17 %, respectively. The staining methods employed for the pituitaries effectively differentiated the acidophils into lactotropes and somatotropes (Pasteels et al., 1972); however, the basophi1 or mucoid cells were considered collectively as one class. No pathological changes attributable to HCB administration were detected in these classes of cells.

438

IATROPOULOS ET AI,

The remaining organs and systems were either within normal limits or displayed changes which were nonspecific and could be attributed to cadaveric influences, particularly some of the changes observed in the central nervous system, e.g., Purkinje cell changes probably due to hypoxia. The dating of the endometrium (Noyes et al.,

FIG. 3. Liver from rhesus monkey given 8 mg/kg/day of HCB for 60 days. Centrolobular trophy is present. Hematoxylin and eosin, xl 15.

hyper-

1950; and Asdell, 1964) of all animals, including the control, revealed proliferative (follicular phase) endometrium. A benign fibroadenoma of the mammary gland was found in one animal of the highdose group. The other animal of this group showed moderate hyperplasia of the adrenal medulla and the uterus contained a myoma, measuring 3.0 x 2.0 x 1.8 cm, one area of which showed anaplastic change. The animal given 64 mg/kg had slight hyperplasia of the zona fasciculata of the adrenal gland.

TABLE OF RHESUS

Interstitial epitheloid cells” Condition of primary follicles and degreeof degeneration“ Cysts” .~

Germinal epithelium

IN THE OVARIES

2 ORALLY

14.0 0.05 12.0

3.6 0.011 7.7

I/+

-I280.0

WNL

WNLb

339.0

1/+

Unilayered with flat columnar cells

Unilayered with flat cuboidal cells 7/+

1

15

16.0

0.25

0.1

80.0

1/++ 1/+

1/+

20.0

14.5

1

128

60 DAYS

21.7

0.44

30.0

68.0

PseudoUnilayered stratified with with tall squamous columnar cells cells

1

64

FOR

30.0

280.0

1/+ -I-

1/+

Pseudostratified with squamous cells

1

32

Dose of HCB (mg/kg/day)

HEXACHLOROBENZENE

8

GIVEN

Control

MONKEYS

n Incidence and grading: incidence expressed in absolute number of animals. Grading is defined as follows: - = absent; slight = +; moderate = ++; marked = +++. b WNL = within normal limits.

Semiquantitativemicroscopicfindings Average number of primary follicles Average number of corpora lutea Ratio of corpora lutea to primary follicles Thickness(pm) of the ovarian germinal epithelium

Semiqualitativemicroscopicfindings

No. of animals

Group

SUMMARY OF MORPHOLOGICAL FINDINGS

440

IATROPOULOSETAL.

FIG. 4. Ovary from rhesus monkey given 64 mg/kg/day of HCB for 60 days. A pseudostratified, germinal epithelium is evident. Scattered, occasional follicles are visible within the cortex, ail of which have undergone some degree of degenerative change. Hematoxylin and eosin, x290.

DISCUSSION Little information is available concerning the dynamic equilibrium, steady state of storage, metabolic behaviour, enzyme induction potential, and pathological alterations in rhesus monkeys given HCB. Originally the compound was believed to be very stable with respect to metabolic conversion or change; however, Rozman et al. (1975) have recently demonstrated that approximately 3 ‘A of ingested HCB is metabolized primarily to pentachlorobenzene and in trace amounts to tetrachlorobenzene. In the rat (Iatropoulos et al., 1975), the initial absorption of this compound proceeds very slowly. Ingested HCB is primarily absorbed and distributed by the lymphatic system and eventually accumulates in the adipose tissue, bypassing the systemic circulation and excretory organs. In the rhesus monkey, Rozman et al. (1975) have demonstrated

HCBINMONKEYS

441

FIG. 5. Ovary from rhesus monkey given I28 mg/kg/day of KCB for 60 days. Note depletion of the ovarian cortex of follicles and its substitution by dense stroma. Hematoxylin and eosin, x290.

that, in spite of its extremely slow metabolic conversion, HCB apparently never accumulates to high concentrations in the liver, and the bulk of the body distribution occurs in adipose tissue, thymus, bone marrow, and mesenteric lymph nodes, in that order. Since HCB is primarily distributed via the lymphatic system, it did not seem unusual to find dose-dependent atrophy of the thymic cortex and reduction in the number of lymphocytes, especially the small lymphocytes. In the ovaries, although the distributional pattern of HCB concentration was demonstrated to be relatively small (Rozman et al., 1975), there were significant and characteristic dose-dependent degenerative changes. These changes involved all ovarian elements, including the primary follicles, germinal epithelium, and stroma, and were morphologically similar to postmenopausal changes. Recently, Grant et al. (1975) studied the effect of HCB on rat reproduction. It was found that HCB did depress

442

IATROPOULOS

ET AL.

FIG. 6. Ovary from rhesus monkey given 8 mg/kg/day of HCB for 60 days. Follicles at different stages of development are present, some undergoing degenerative changes. Note thin, small germinal epithelium. Hematoxylin and eosin, x290.

reproduction in that species, although no gross abnormalities were present in the pups. The fact that the endometrium of all animals was in the follicular and not the luteal phase suggests the presence in all treated animals of a disproportionate number of nonfunctional corpora lutea or corpora aberrantia (Table 2). This suggests that the corpora lutea are either not receptive to gonadotropin stimulation or incapable of steroidogenesis; a third alternative would be a lack of uterine response to luteal transition. Since the activity state of the gonadotropes (mucoid cells) was not clear, it remains to be elucidated at which level(s) HCB exerts its effect on the reproductive system. The presence of uniformly distributed coarse and fine vacuoles together with the deposition of granular hemosiderin within the hepatocytes and Kupffer cells is characteristic of porphyria tarda (Creutzfeldt et al., 1966). The term “cutanea” has been purposefully deleted, because no skin lesions were observed. Other hepatic changes,

HCB IN MONKEYS

443

e.g., the increased size and number of Kupffer cells, centrolobular hepatocellular degeneration, are similar and comparable to the HCB-related changes observed in rats (Ockner and Schmid, 1961) and humans (Cam and Nigogosyan, 1963). The degenerative changes in the kidney appear to be HCB-specific and are in agreement with the findings of Kuiper-Goodman et af. (1975). Despite the extensive changes which were observed in the thymus, ovaries, liver, and kidneys, the mechanism by which HCB or its metabolites induce these changes is not understood. The means by which HCB interferes with porphyrin synthesis and ovarian function is currently under investigation. REFERENCES ASDELL, S. A. (1964). Patterns of Mammalian

Reproduction.

2nd ed., pp. 143-151. Cornell

University Press, Ithaca, New York. AVRAHAMI, M., AND STEELE, R. T. (1972). Hexachlorobenzene.1. Accumulation and elimination of HCB in sheepafter oral dosing.N.Z. J. Agr. Res. l&476-481. BEN-DYKE, R., SANDERSON, D. M., AND NOAKES, D. M. (1970).Acute toxicity data for pesticides. World Rev. P. 9, 119-127. BRADY, M. N., AND SIYALI, D. S. (1971). Hexachlorobenzenein human body fat. Med. J. Aust. 1, 158-161. CAM, C., AND NIGOGOSYAN, G. (1963).Acquired toxic porphyria cutaneatarda due to hexachlorobenzene.J. Amer. Med. Assoc. 183,88-91. CRAIG, J., AND DWYER, H. P. (1961).Feedingpickled wheatto sheep.J. Dep. Agr. West. Aast.

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biphenylsin fish, musselsand birds from the river Rhine and the Netherlandscoastalarea. Nature (London) 221, 1126-l 128. LUNA, L. G. (1968).Manualof Histologic Staining Methods. 3rd ed. McGraw-Hill, New York. MALLORY, F. B. (1961).Pathological Technique. Hafner PublishingCompany, New York, MEDLINE, A., BAIN, E., MENON, A., AND HABERMAN, H. (1973).Hexachlorobenzeneand rat liver. Arch. Pathol. 96, 61-65. NOYES, R. W., HERTIG, A. T., AND ROCK, J. (1950).Dating the endometrialbiopsy. Fertil. Steril. 1. 3-21.

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OCKNER, R. K., AND SCHMID, R. (1961).Acquired prophyria in man and rat due to hexachlorobenzeneintoxication. Nature (London) 189,499. PASTEELS, J. L., GANSSET, P., DANGUY, A., ECTORS, F., NICHOLL, C. S., AND VARAVUDHI, P.

(1972). Morphology of the lactotropes and somatropesof man and rhesusmonkeys.J. Clin. Endocrinol. Metab. 34, 959-967. ROZMAN, K., MUELLER, W., IATROPOULOS, M., COULSTON, F., AND KORTE, F. (1975).Auss-

cheidung, Koerperverteilung and Metabolisierung von Hexachlorobenzol nach oraler Einzeldosisin Ratten und Rhesusaffen.Chemosphere 5,289-298. Vos, J. G., BREEMAN, H. A., AND BENSCHOP, H. (1968).The occurrenceof the fungicidehexachlorobenzenein wild birdsandits toxicological importance.A preliminary communication. Meded Rijksfac. Landbouwetensch. Gent. 33, 126331269. Vos, J. G., VANDER MAAS, H. L., MUSCH, A., ANDRAM, E. (1971). Toxicity of hexachlorobenzenein Japanesequail with specialreferenceto porphyria, liver damage,reproduction and tissueresidues.Toxicol. Appl. Pharmacol. 18, 944-957.