Histotoxic effects of polybrominated biphenyls in Michigan dairy cattle

E~,VIRONMENTAI.

KESE,AKCH 15, 82 -89

Histotoxic H.

Effects of Polybrominated Michigan Dairy Cattle

D.R.

COOK,

(1978)

HELLAKD,

B.H. VAXDEKWEELE,

Biphenyls

AND

R.J.

in

DEJONG

Cattle affected by polybrominated biphenyls (PBB) showed lymphocyte infiltration into liver. kidney. small intestine. and lung. The histology of spleen and lymph nodes and the maintenance of thymus size indicated high immunological activity, while the susceptibility to infections suggested impairment of immunological mechanisms. The liver also showed involvement of hepatocytes. The effects on the small intestine are probably the cause of poor feed efficiency and increased appetite. Some testes samples were devoid of spermatozoa and other cells in various stages of spermatogenesis. Contamination of the original PBB toxin by brominated naphthalenes is possible and could be the cause of some of the symptoms observed.

INTRODUCTION

Toxic effects attributed to polybrominated biphenyls (PBB) occurred after the inadvertant feeding of the compound(s) in the feed concentrate of dairy cattle in central and southern Michigan in the second half of 1973 (Jackson and Halbert, 1974). The circumstances of the feed contamination were described by Jackson and Halbert (1974). Robertson and Chynoweth ( 1975). and Carter (1976). The PBB is sold commercially under the brand name Firemaster BP-6 by the Michigan Chemical Co. It consists of penta-, hexa-, and heptabromobiphenyls and lesser amounts of tetra- and other brominated biphenyls (Kerst. 1974). Sundstrom ct LI/. (1976) have reported 2,1’.4.4’.5.5’-h exabromobiphenyl to be a major component of Firemaster BP-6. Gross symptoms reported by Jackson and Halbert (1974) for Halbert’s cattle include decreased milk production, decreased feed consumption, hematomas in various locations, abnormal growth of hooves, hyperkeratosis, abscesses in the liver and other organs, intra-abdominal adhesions, and severe difficulties associated with reproduction and calving. In one young bull, atrophied testes and aspermia were observed. Toxological studies of the effects of brominated biphenyl on laboratory rodents have also been carried out. Before the Michigan cattle toxicity problems, Aftosmis rt ~1. (1972a,b) reported that, although brominated biphenyls had a low acute oral toxicity, other toxic effects, most notably those on the liver, caused them to conclude that these compounds were not suitable candidates for flame retardants in synthetic fibers. The toxicity of Firemaster BP-6 was also discussed by Kerst (1974). Possible implications of the PBB episode are discussed by Carter (1976). A study by the Food and Drug Administration (1975) comparing dairy cattle exposed and not exposed to low levels of PBB and an investigation by the Michigan Department of Public Health (1975) comparing possible symptoms in people living on quarantined and nonquarantined farms report no consistent differences 82 0013.9351/78/0151-0082$02.00/0 Copyright All

nghrl

0 of

1978 reproductmn

by

Academic in any

Preu,

Inc.

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reserved.

HISTOTOXIC

EFFECTS

OF

PBR

IN

DAIRY

83

CATTLE

or effects attributable to PBB. The disparity of these findings with the clinical effects that are observed on some farms may be due to the fact that control situations are not as prevalent in PBB-effected areas as they were once thought to be. Possible similarities between PBB and PCB (polychlorinated biphenyl) poisoning incidents are discussed by Corbett rt trl. ( 1976). He also mentions the possibility of other toxic contaminants being present in the Firemaster BP-6 mixture. To our knowledge no study on the effects of PBB on the histology of organs of affected dairy cattle has been reported. Because such a study would seem valuable in answering some of the questions raised about the possible toxicity of PBB and because the PBB incident is unique in its scope and nature, it was decided to initiate such a study. MATERIALS AND METHODS Tissues from cattle of five affected herds in the McBain area in central Michigan were collected on two dates. Eye fat samples for PBB determination were also taken. Data on the eight animals, including PBB levels as determined by the Warf Institute,’ are shown in Table 1. Young animals were below the normal size for their age class. Some adult cattle were underweight and showed areas of hyperkeratosis or elongation of the hooves: others appeared generally normal. In all herds from which animals were examined, milk production was below normal, cattle were infertile or subfertile. and feed efficiency was poor. Samples of liver, kidney, lung, spleen, lymph node, small intestine, and abomasum wall were collected from each animal, fixed in buffered formalin, embedded in Paraplast, sectioned, and stained with Harris’ hematoxylin and eosin. Sections known to contain a granular substance, to be described, and control slides were stained according to the method of Gmelin for bilirubin and hematoidin, the method of Perls for ferric iron. and the hematoxylin-lake method for hemosiderin iron. The procedures followed are described in Pearse (1972).

Animal number I

2 3 4 5 6 7 8 ‘I As determined ’ Wisconsin

Date

of birth

Sex

Date

August 1974 September 1971 October 1974 October 1974 July 1973 July 1972 September 1974 November 1973 by the Warf Alumni

Research

October October October October January January January January

killed 11. 1975 I I, 1975 11. 1975 Il. 1975 7. 1976 7. 1976 7, 1976 7. 1976

Institute. Foundation,

Madison.

Wisconsin.

(ppm

PBB level” in fat basis) 0.13

Not

detected co.01 Not determined 0. IO
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COOK

ET AL

RESULTS

Over half of the cattle killed showed lesions of the liver which were visible on the surface of the organ. In some animals fibrotic areas of the lungs could be similarly observed. Animals 1 and 2 possessed thrombi in one of the ventricles of the heart. Enlargement of lymph nodes was present in most animals. A large thymus gland was present in all animals. Some adhesions of the viscera to the peritoneum of the abdominal wall were present in several animals, particularly No. 8. Animal 7 possessed a small, white spleen which appeared contracted. with prominent white pulp areas. Accumulation of fluid in the peritoneal cavity was observed in several animals. Lil,er. Effects in the liver varied. In many samples lymphocyte infiltration around the triad areas could be observed (Fig. 1). In some of the tissues this infiltration extended around the central veins and interstitial areas. In several animals hepatocytes were also affected, showing fatty degeneration or cloudy swelling and, in more extreme cases, necrotic foci. Proliferation of Kupffer cells was noted in some instances. In one case, aggregates of eosinophilic cells around the triads, with concomitant increases in fibrocytes, were present. In a minority of animals the liver appeared normal. Lymph nodes. Capsules were intact but thin and trabecular attachments were widely spaced because of nodal enlargement. Subcapsular lymphocytes were minimal and possessed compact chromatin material. Many germinal centers were present (Fig. 2), but these were not surrounded by lymphocytes. In the paracortical areas, lymphocytes with dispersed chromatin material were extensively accumulated. Macrophages containing a granular substance, to be further described under Granules in Macrophages, had greatly enlarged the medulla. In some samples, eosinophils were diffusely accumulated throughout the medulla. Spken. White pulp areas within the spleen were abundant and frequently contained rings of intensely dark-staining lymphocytes peripherally (Fig. 3). Red pulp areas contained diffuse accumulations of lymphocytes with widely scattered foci of hematopoiesis. Macrophages, some containing the same granular substance described for the lymph nodes, abounded throughout the red pulp. Kihey. The kidney tissues of one-half of the animals examined were normal in appearance. The other animals showed foci of cortical interstitial lymphocyte

FIG. I. Liver tissue with cloudy swelling of hepatocytes and lymphocyte infiltration (dark cells) around the triad areas. Hematoxylin and eosin. 135x. FIG. 1. Lymph node showing one of many active germinal centers(g) and medulla(m). Hematoxylin and eosin. 80x. FIG. 3. Spleen showing one of the numerous white pulp areas (w) surrounded by red pulp (r). Hematoxylin and eosin. 80x FIG. 4. Small intestine with mucosa (m) invaded by lymphocytes. The submucosa (s) between the mucosa and the inner circular muscular layer (i) is greatly distended by invading lymphocytes. Hematoxylin and eosin. 40x.

HISTOTOXIC

EFFECTS

OF

PBB

IN

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CATTLE

85

86

COOK

ET AI.

accumulation. Areas with increased numbers of mesangeal cells within the glomerular tufts were also noted. Grrstroint~~stitlrrl tract. The small intestine was affected in all animals examined. Most notable was lymphocyte invasion of the mucosa and submucosa (Fig. 4). This invasion was pronounced. gave a clubbed appearance to the villi, and caused separation of mucosal gland tissue and a thickened. elevated submucosa. In extreme cases the invasion extended into the muscularis mucosa and the circular and longitudinal muscles. Areas with hyperplastic mucosa or with eroded villi were numerous. Eosinophil invasion of the submucosa was also observed. The abomasum wall was found to be normal in the samples from all four animals that were examined. L~IIIK. Tissue in some samples appeared completely normal: other specimens showed moderate to extreme interlobular fibrosis. Dense parabronchial and parabronchiolar lymphocyte accumulations and alveolar macrophage proliferation were also noted in several samples. Infrequently, involvement of subepithelial tissue and smooth muscle bundles was present. Focally intense interstitial fibrosis and lymphocyte infiltration were also present in the parenchyma; in restricted areas these lesions were advanced to the point of tissue collapse. Testes. While some tissues were essentially normal, in most testes examined mature spermatozoa were reduced in number or absent (Fig. 5). Because other cells in the various phases of spermatogenesis were then also absent, the only cells which were present in the seminiferous tubules were the Sertoli cells. The interstitial cells were normal. Granules it1 mrrcropllnges. The presence of a conspicuous granular substance in macrophages in the spleen and lymph nodes has been mentioned. The substance was also present in small intestine (Fig. 6), kidney, and lung tissue. In all cases it was present in macrophages. The substance stained positively with Perls’ method for ferric iron and negatively with the hematoxylin-lake method for hemosiderin iron and the Gmelin reaction for bilirubin and hematoidin (methods according to Pearse, 1972). DISCUSSION The most notable finding in this study, in our opinion, is the lymphocyte infiltration which was present in many organs. This proliferative response in these cattle was particularly noticeable in organs that are known to originate immunological activity: spleen and lymph nodes. The animals examined also had many lymphocytes in the target organs, where host defenses exist: lung and intestine. It would be logical to consider these animals to possess remarkable immunological capability. However, examination of herd histories and individual animals indicates much vulnerability to common environmental pathogens. The lymphoid hyperplasia. together with the observed maintenance of thymus gland size, causes us to suggest that there is a possibility of immunological incompetence or that there is some block that prevents effective antibody activity. Friend and Trainer (1970) reported increased susceptibility of ducklings to duck hepatitis virus after administration of PCB. Vos and van Genderen (1973) describe impairment of immunologic responses in laboratory animals after administration of chlorinated and brominated

HIS’I’O1‘OXIC

FIG.. 5. Testicular predominate. Frc.. staining

tissue

Hematouylin 6.

Area

material

without and

of the

small

was

contained

EFFECTS

cells

eosin.

intestine

OF

in stage?

PRB

IN

DAIRY

87

CA-I‘1I.E

of bpermatogenesis

or

spermatozc>a.

Se!-toli

cell\

IMY. stained

in macrophages

with

Perls‘ and

was

method

for

present

in several

ferric

iron. types

The

dark.

of tissue.

positively 700r.

biphenyls. The sparse literature on the effects of PCB and PBB on the immunologphenomena in ical system is reviewed by Kolbye et rrl. (1976). Immunological cattle affected by PBB are thus of interest and need further study. Special mention should be made of the small intestine. The chronic inflammatory changes in all layers of the intestine possibly compromised the absorption capability of the gastrointestinal tract. This would explain the poor feed efficiency and ravenous appetite described for PBB-affected cattle. Jackson and Halbert ( 1974) described poor appetites of cattle being fed a PBB-containing diet. However. it is widely observed that feed efficiency is decreased and appetite increased in PBB-affected cattle after the initial ingestion of the toxin. The granular substance observed in several tissues, especially spleen, lymph nodes, intestine. and lung. was not chemically identified. Kolbye et rrl. (1976). in their literature review, suggest that polyhalogenated biphenyls can be the cause of porphyria. especially hepatic porphyria. Strik (1973a,b,c) has described porphyrias caused by brominated and chlorinated biyhenyls. A porphyria due to overproduction of cytochrome enzyme after administration of PCB has been reported by Goldstein et LI/. (1974). Wit ( 1972) has reported on porphyria caused by brominated biphenyl. The possibility that the substance observed in our experi-

88

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ET AL

merit, which stained positively for ferric iron, is derived from a cytochrome enzyme needs further study. Infertility has been reported as one of the major deleterious effects of PBB (Jackson and Halbert, 1974). Tissues from the female reproductive system are not reported in this paper. Testes examined were subfertile or infertile, and it would seem that male infertility may also be one of the causes of the failure of cows to breed. The amounts of PBB in the fat of the cattle studied in this investigation (Table 1) were determined from fat samples collected at the time the cattle were killed. PBB levels were probably higher shortly after the toxin was ingested in the feed. Many animals in the herds which were sampled died or were killed in 1973 and 1974. It is possible that a different pathogenesis existed in these earlier mortalities. Since the animals sampled differed in age, sex, parity, and other characteristics, no conclusions from the PBB levels were drawn. The hyperkeratosis over the withers, observed in several of the animals, is intriguing. Sikes et nf. (19.521, Gregory et ctl. ( 1954), and Bell (1954) have described the hyperkeratosis, “X disease,” caused by administration of chlorinated naphthalenes. It is known that chlorinated naphthalenes are common contaminants of PCB (for a review, see Kolbye or cl/., 1976). It has been found that PBB contains detectable amounts of brominated naphthalenes (P. W. O’Keefe. personal communication). it would seem possible that the hyperkeratosis observed in PBB-affected cattle is due to the presence of brominated naphthalenes in this PBB. Thus, the presence and effects of possible contaminants in PBB, such as brominated naphthalenes and brominated dibenzofurans, need further study. ACKNOWLEDGMENTS We thank Zuiderveen.

Drs. A. S. Clark and G. Zuiderveen

and L. J. Witt and Messrs. for their help in obtaining

C. Becker, R. Benson, E. Prehn. the tissues used in this study.

K. L.

REFERENCES Aftosmis. J. G.. Culik. R., Lee. K. P., Sherman. H.. and Waritz. R. S. (1972a). The toxicology of brominated biphenyls. I. Oral toxicicity and embryotoxicity. /!I “Report of Haskell Laboratory E. I. du Pont de Nemours and Co.. Inc., Wilmington. for Toxicology and Industrial Medicine.” Del. Aftosmis, J. G.. Dashiell. 0. L.. Griffith. F. D.. Hornberger. C. S.. McDonnell. M. E.. Sherman. H., Tayfun. F. 0.. and Waritz, R. S. t 1972b). The toxicology of brominated biphenyls. II. Skin. eye, and inhalation toxicity and an acute test method for evaluating hepatotoxicityand accumulation in body fat. III “Report of Haskell Laboratory for Toxicology and Industrial Medicine.” E. I. du Pont de Nemours and Co., Inc.. Wilmington, Del. Bell. W. B. (1954). The production of hyperkeratosis (X disease) by a single administration of chlorinated naphthalenes. J. Amrr. C’~T. hfed. A.~rrr. 124, 289-290. Carter, L. J. (1976). Michigan’s PBB incident. Chemical mixup leads to disaster. Science 192, 240-243. Corbett. T. H.. Beaudoin. A. R.. Cornell. R. G.. Anver, M. R., Schumacher. R., Endres. J.. and Szwabowska, M. (1975). Toxicity of polybrominated biphenyls (Firemaster BP-61 in rodents. E~,\~ii-on. R~J. 10, 390-396. Food and Drug Administration. Bureau of Veterinary Medicine, Veterinary Medical Research (1975). “Michigan State Dairy Herd Survey. A Report on Herd Health. Status of Animals Exposed to Polybrominated Biphenyls (PBB),” April ‘25. Beltsville, Md. Friend. M.. and Trainer. D. 0. (1970). Polychlorinated biphenyl: Interaction with duck hepatitis virus. Scic~nc~c 170. 13141316.

HIS-I‘OI‘OSI(.

t:FFE(‘TS

()I;

PRR

IS

DAIRY

(.ATTI.E

89

Goldstein. J. A.. Hickman, P., and Jue. D. L. (1974). Experimental hepatic porphyria induced by polychlorinated biphenyls. To.ricol. A/>p/. Pl~trrrrrc~c,o/. 27. 437-448. Gregory. R. P.. Wise, J. C.. and Sikes. D. (1954). Experimental production of bovine hyperkeratosis with a feed concentrate exposed to vapors of a highly chlorinated naphthalene. J. Amcdr. 1’~1. Mcti. A.F.\lU 125, 744-1-46. Jackson. T. F.. and Halbert. F. L. (1974). A toxic syndrome associated with the feeding of polybromlnated biphenyl-contaminated protein concentrate to dairy cattle. .I. Amrr. \‘(,I. Med. Astcx.. 16.5. 437-439. Kerst. A. F. (1974). “Report to the Michigan Environmental Review Board by the Michigan Chemical Corporation.” Sept. ‘3. Lansing. Mich. Kolbbe, A. C. (Ed.) and the Subcommittee on the Health Effects of Polychlorinated Biphenyls and Polybrominated Biphenyls (1976). “Final Report.” Department of Health. Education. and Welfare. Wash., D. C. Michigan Department of Public Health ( 19751. “The Short Term Effects of PBB on Health.” May I. Lansing. Mich. Pearse. A. G. E. (1972). “Histochemistry. Theoretical and Applied.” 3rd ed.. Vol. 2. Williams & Wilkins. Baltimore. Robertson. L. W.. and Chynoweth. D. P. (1975). Another halogenated hydrocarbon. E/~~,iron/r~c~/~f 17, 15-27. Sikes. D., Wise. J. C., and Bridges. M. E. (1952). The experimental production of “X disease” (hyperkeratosist in cattle with chlorinated naphfhalenes and petroleum products. .I. Amc,r. I,‘P~ ,2lctl. A.\soi-. 121, 337-344. Strik. J. J. T. W. A. (1973a). Chemical porphyria in Japanese quail. ,!%:yr~(, 16. ?I l-233. Strik. J. J. T. W. A. ( l973b). Species differences in experimental porphyria caused by polyhalogenated aromatic compounds. G~:~rnca 16. 114-230. Strik. J. J. T. W. A. (1973~). Toxicity of hexachlorobenzene (HCB) and hexabromobiphenyl (HBB). ,Zlrc/cJd. HijX rftrc-. Ltr,rdh~)rru,11~r/r~t\(~/~, Grut. 38. 709- 7 16. Sundstrom. G.. Hutzinger. 0.. and Safe, S. (1976). identification of 7 . -1’.4.4’.5.5’-hexabromobiphenyl as the major component of flame retardant Firemaster BP-6. C/~f,~,lr,.~p/lr,~, I, I I - 14. Vos. J. G.. and van Genderen (19731. Toxological aspects of immunosuppression. 1~ “Pesticides and the Environment: A Continuing Controversy” (W. B. Deichman, Ed.). pp. S1,7-545. Stratton Intercontinental Medical Book. New York. Wit. J. G. (19711. “Enzyme Induction with Specific Reference to Chemical Porphyria,” Paper presented at the Third Technical Meeting of Occurrence and Significance of Chemicals in the Environment. Berlin. Germany. Organization for Economic Co-operation and Development, Paris.