Effects of PBB on cattle. II. Gross pathology and histopathology.

Toxicosis was induced in pregnant Holstein heifers by feeding FireMaster BP-6 (polybrominated biphenyls) in daily oral doses of 25 g/head/day for 33--60 days. The individual heifers were dosed until each became moribund (days 33, 36, 39, 40, 41, or 66), at which time they were necropsied. Gross findings included dehydration, subcutaneous emphysema and hemorrhage, atrophy of the thymus, fetal death with concomitant necrosis of cotyledons, thickened wall of the gallbladder, inspissated bile, edema of abomasal folds, mucoid enteritis, linear hemorrhage and edema of the rectal mucosa, and secondary pneumonia. The livers were enlarged approximately 40%. Kidneys were approximately double the normal size and were pale tan to grey in color. The perirenal lymph nodes were enlarged and edematous. Microscopic changes were the most marked in the kidneys, gallbladder and eyelid. Extreme dilatation of collecting ducts and convoluted tubules with epithelial degenerative changes of cloudy swelling, hydropic degeneration and separation from the basement membrane were principal changes in the kidney. Hyperkeratosis with accumulations of keratin in hair follicles of the epidermis and squamous metaplasia with keratin cysts in the tarsal glands were characteristic findings in sections of eyelids. Moderate to marked hyperplasia and cystic dilatation of the mucous glands in the lamina propria were common changes in the gallbladder. Foci of fatty degeneration and glycogen depletion were observed in liver sections. Necropsy of heifers immediately after 60 days exposure to 0.25 and 250 mg/head/day of PBB showed no gross or histopathological signs indicating toxicosis. Following parturition, at approximately 220 days after the PBB doses, heifers from the 0.25 and 250 mg/head/day groups and their calves were necropsied and displayed no signs of toxicosis. ImagesFIGURE 1.FIGURE 2.FIGURE 3.FIGURE 4.FIGURE 5.FIGURE 6.FIGURE 7.FIGURE 8.FIGURE 9.FIGURE 10.FIGURE 11.FIGURE 12.


Introduction
Following the discovery of accidental contamination of animal feeds with polybrominated biphenyls (PPB) on Michigan farms in 1974, the investigation of the toxicologic effects of these compounds became of primary importance not only to the livestock industry but as a possible health hazard to man. Studies of PBB toxicosis in cattle conducted by our group have included tissue distribution and clearance (1), gross and histologic tissue changes (2), clinicopathologic alterations in blood and urine (3), effects on health and performance (4), renal function (5), and performance of calves from cows exposed to PBB (6). Selected tissues from cattle with acute PBB toxicosis are being processed for examination with the electron microscope. All cattle and calves involved in these studies have been, or will be, subjected to complete necropsy examination upon death or termination of a particular study. The information in this paper will be confined to the description of pathologic changes commonly observed in cattle with experimentally induced PBB toxicosis.

Necropsy Procedure
All cattle that were necropsied were killed by intramuscular injection of 100 mg of succinylcholine chloride followed by exsanguination immediately prior to necropsy. Necropsy dates were established by signs of terminal toxemia in the individual cow.
Representative specimens from organs of all cattle that were necropsied were fixed in neutral buffered formalin. Fixed tissues were processed for paraffin embedding, sectioned at 6 ,um and stained with azure and eosin (7).

Necropsy Findings
Significant gross pathologic changes were not observed in cattle receiving PBB at levels less than 25 g/head/day for a minimum of 25 days (2).
Toxic cattle exhibited signs of dehydration and marked emaciation (Fig. 1). Extensive subcutane-ous emphysema and petechial hemorrhages occurred in the areas of flank, caudal portion of the abdominal wall and medial surface of the hind limbs. Atrophy of the thymus was common. Edematous and hemorrhagic dead fetuses were found in the uterus of pregnant cattle (Fig. 2). Concomitant uterine lesions were hemorrhage and necrosis of cotyledons, with separation of the caruncles and placental degeneration (Fig. 3). The kidneys were greatly enlarged, distended with fluid, pale tan to grey, with little perirenal fat (Fig. 4). The kidneys were approximately double normal size  ( Table 2) with tubules appearing as pale streaks on the cut surface of the cortex (Fig. 4). Perirenal lymph nodes were enlarged and edematous. The liver appeared slightly enlarged and friable but otherwise normal ( Table 2). The gallbladder ap-  peared thickened and filled with inspissated bile.
The changes in the gastrointestinal tract were varied. The most constant obseFvation was the absence of residual feed in the digestive tract. Other changes observed were edema of mucosal folds in the abomasum, occasional mucosal ulcers of the abomasum and colon, mucoid enteritis and linear hemorrhages, and edema of the rectal mucosa (Fig.  5).
Other lesions were pneumonia and petechial hemorrhages of tracheal mucosa, endocardium and myocardium.

Histopathologic Findings
Pathologic changes were not observed in tissue sections from cattle receiving PBB at levels less than 25 g/head/day for a minimum of 25 days.
The principal lesions observed in kidney specimens from toxic cattle were extreme dilatation of collecting ducts and convoluted tubules, with tubular degenerative changes marked by cloudy swelling, hydropic degeneration and separation from the basement membrane (Figs. 6 and 7). The changes were more prominent in the collecting ducts than in the convoluted tubules. Occasional blood and hyaline casts were found in collecting ducts in the pyramidal area. Vascular changes were not marked and consisted of congestion, with scattered microscopic hemorrhages in the medulla. There were no significant glomerular changes. Interstitial fibrosis was minimal. Hyperkeratosis, with accumulation of keratin in the hair follicles of the epidermis, and squamous metaplasia with keratin cysts in the tarsal glands were common changes in the eyelids (Figs. 8 and 9).
Changes in the gallbladder were characterized by moderate to marked hyperplasia and cystic dilatation of the mucous glands in the lamina propria (Fig.  10). Changes in the liver were not marked and consisted of glycogen depletion of hepatocytes, sinusoidal dilatation, and scattered areas of early centrolobular fatty degeneration (Fig. I 1). Hemorrhage and necrosis of cotyledons, with minimal changes in endometrium and uterine wall, were principal lesions in the uterus from cattle with retained fetuses (Fig. 12). In specimens of uterus from cattle that had aborted, the uterus appeared to be involuting normally.
Changes in the gastrointestinal tract were minimal. Occasional findings were edema of mucosal folds and ulceration in the abomasum, hemorrhage, edema and hyperemia of the mucosa of the terminal colon and rectum. One specimen of pancreas had April 1978 lesions of chronic fibrosing pancreatitis. Moderate to marked involution of the thymus was common to all toxic cattle. Microscopic lesions of early purulent bronchopneumonia were observed in lung specimens from two cows.

Discussion
The pathologic changes observed in PBB toxicosis in cattle are remarkably similar to those described for bovine hyperkeratosis (X disease) caused by highly chlorinated naphthalenes (8)(9)(10)(11). Hyperkeratosis was not as grossly apparent in cattle with PBB toxicosis, as reported for cattle with hyperkeratosis, but was obvious in histologic sec-tions (2). Although the pathologic changes in cattle with PBB toxicosis and bovine hyperkeratosis were quite similar, the concentrations required to elicity toxicosis were markedly different for each of the two chemicals. Cattle have been reported to be relatively sensitive to chlorinated naphthalene intoxication, a single dose of 11 mg to 22 mg/kg producing 100%o mortality (12). In contrast, the cattle in these studies exhibited a wide range of sensitivity to intoxication with PBB, the approximated minimum dose required to produce mortality was 67.2 mg/kg administered daily for 25 to 66 days (Table 1). Based on a 400-kg cow, the approximate total dose of PBB required to produce 100% mortality ranges from 820 to 1500 g ( Table 1). The actual LD50 for PBB in cattle cannot be calculated from the data in these studies, but it does suggest that PBBs are relatively nontoxic.
Commercial synthesis of halogenated hydrocarbons often results in the formation of trace Environmental Health Perspectives     quantities of other hydrocarbon contaminants as well as the expected end-product. Halogenated naphthalenes have been suggested (2) as a possible contaminant of FireMaster BP-6 (PBB); however, published reports of chemical analytical data to date have not verified this fact.
Our studies have shown that PBB toxic cattle have specific, highly characteristic and readily identifiable pathologic changes. This is valuable in that these pathologic changes should serve to clearly identify the problem should future incidents occur. Conversely, it might be used to rule out PBB involvement in incidences of claimed toxicity where specific lesions did not occur. Further, the histopathologic changes identified in the kidneys of PBB toxic cattle suggest the involvement of an extrahepatic target organ not normally associated with halogenated hydrocarbon toxicity. The mechanisms of PBB toxicity in the kidney would certainly merit further study if we are to better understand the pathogenic mechanisms of halogenated hydrocarbons in the environment.