EM changes and other toxic effects of firemaster BP-6 (polybrominated biphenyls) in the mouse.

Groups of Swiss ICR mice were fed 1000 ppm polybrominated biphenyls (FireMaster BP-6) in rodent chow for 4, 8, 11, and 14 days. Control groups were fed standard rodent chow without FireMaster BP-6. Animals were killed at the end of each feeding period and the livers examined by electron microscopy. EM changes noted were progressive increase in size of hepatocytes, a decrease in rough endoplasmic reticulum, a marked increase in smooth endoplasmic reticulum, mitochondrial degeneration, increased lysosomes, and a decrease in glycogen. In addition, there was increasing proliferation of microvilli in bile canaliculi with increasing feeding times. A group of mice fed 1000 ppm FireMaster BP-6 in rodent chow for 11 days had livers with a mean of 13.93% of total body weight as compared with 6.49% for the control group (P=0.02). Tissue distribution following ingestion of 100 ppm FireMaster BP-6 for 14 days was studied. Twelve weeks post-feeding, the tissue concentrations of hexabromobiphenyl in order of highest concentration to lowest were as follows: perithymic fat, perirenal fat, adrenal glands, thymus gland, liver and stomach.


Introduction
In 1975, we reported that the commercial mixture of FireMaster BP-6 was weakly teratogenic in mice, produced decreasing birth weights with increasing dosage during pregnancy in both mice and rats, and produced a marked increase in liver size and weight with a concomitant increase in P450 activity (1). The dietary doses studied ranged from 50 to 1000 ppm. In the teratogenicity studies, rodents were fed FireMaster BP-6 in the diet from day 7 to day 18 of the pregnancy for mice and from day 7 to day 20 of the pregnancy for rats. The birth defects produced in mice included exencephaly and cleft palate. In the liver studies, the only dose tested was 1000 ppm in the diet for 11 days.
In addition, we performed a pilot study feeding 100 ppm FireMaster BP-6 to six timed-pregnant Swiss/ICR mice on days 7-18 of the pregnancy, with six identical mice on a normal diet as controls. The offspring of both groups were born spontaneously and nursed by their mothers and maintained for 15 months. Five of the six FireMaster BP-6-fed mothers died prior to termination of the experiment and unfortunately were not examined. At autopsy, the one remaining mother had multiple hyperplastic nodules throughout the liver which weighed 7.2 g. The hexabromobiphenyl concentration in the liver was 2.4 ppm. One of 22 male offspring had a solitary liver nodule while 0/20 female offspring had nodules. None of the control mice had nodules (2).
These findings prompted us to perform electron microscopic studies of liver from mice fed 1000 ppm Firemaster BP-6, perform tissue distribution studies of polybrominated biphenyls, and undertake a carcinogenicity bioassay of the chemical mixture in mice which is still in progress.

Materials and Methods
A group of 8-week-old male Swiss/IRC mice was fed a diet of granular rodent chow containing 1000 April 1978 ppm Firemaster BP-6 (polybrominated biphenyls) up to 14 days. During the feeding period, the animals were maintained in an environmental exposure chamber designed for use with toxic substances. A control group of similar mice was maintained in a second chamber and fed noncontaminated granular rodent chow. All animals were weighed on day 0, 4,8,11, and 14 of the study. . Three animals from each group were killed and the livers weighed and prepared for histologic examination on days 0, 4, 8, 11, and 14 of the feeding period. Liver specimens were minced into approximately 1-1.5 mm3 pieces and fixed in 2.5% cacodylate-buffered gluteraldehyde at pH 7.4 for several days, after which tissues were rinsed in cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon 812. (3,4). Sections were obtained with a Sorvall MT-2 microtome. Survey sections prepared for light microscopy, 1 ,u thick, were made at least from seven blocks in each animal and stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with Hitachi HU-11 C electron microscope.
Testicular weights of each animal were also recorded and the tissues prepared for light microscopy.
Two groups of 8-week-old Swiss ICR mice were maintained in separate environmental exposure chambers designed for use with toxic substances. One group was fed granular rodent chow containing 100 ppm FireMaster BP-6 (polybrominated biphenyls) while the other was fed with normal granular rodent chow. The experimental feeding period lasted 14 days.
At both 6 hr and 14 weeks post-feeding, three animals from each group were killed and various tissues or organs were isolated, weighed, and combined within each group for hexabromobiphenyl (HBB) analysis. Portions of tissues or organs were preserved in 10% formalin and prepared for histologic examination. The hexabromobiphenyl concentrations were determined by a commercial laboratory, Anatech Services, Ann Arbor, Michigan, using micro-Soxhlet extraction with diethyl ether and Florisil clean-up, and gas liquid chromatography (5,6).
Once the results of the first feeding were examined, a second study was initiated. Groups of 8-week-old Swiss ICR mice were again fed and maintained in a similar manner and then examined twelve weeks post-feeding.
Transplacental transfer of hexabromobiphenyl was studied in a pregnant dam fed 100 ppm FireMaster BP-6 from day 7 to day 18 of the pregnancy. The pregnancy was terminated on day 18 and the following tissues or organs were analyzed for hexabromobiphenyl: maternal fat, maternal liver, whole fetus, and fetal liver. A control group of pregnant dams was fed normal granular rodent chow, maintained and examined in a similar manner.
In addition, fat and thymus tissues from two 3day-old human infants who had expired were examined for hexabromobiphenyl concentration.

Results
The results of the feeding study during which mice were fed 1000 ppm FireMaster BP-6 and examined after 0, 4, 8, 11, and 14 days are depicted +40%control mice 4th 8th 11th 14th day day day day   Figures 1 and 2. Figure 1 shows that, while the control animals continually gained weight during the course of the experiment, the PBB-fed mice lost weight until day 14, when they returned to their starting weights. Figure 2 shows the liver/body weight ratios increased progressively with the length of feeding for the PBB-fed mice only. Testicular/body weight ratios did not vary significantly between the experimental and control mice.
The results of the feeding study during which the experimental group was fed 100 ppm FireMaster BP-6 for 14 days and animal tissues examined for hexabromobiphenyl concentrations both 6 hr and 14 weeks after feeding are shown in Table 3. In this series, the thymus gland had the highest concentrations at both examination periods. At the 6 hr examination, the hexabromobiphenyl concentrations, in decreasing order, were thymus gland, perirenal fat, liver, brain, spleen, pancreas, and testicles. The adrenal glands were not examined. At the 14-week examination, the hexabromobiphenyl concentrations, in decreasing order, were thymus, adrenal glands, perirenal fat, liver, testicles, spleen, brain, and pancreas. The ratio of hexabromobiphenyl concentrations for each organ or tissue/perirenal fat, expressed as per cent, is noted in parentheses in Table 3.
Because we suspected that the thymus gland had abnormally high concentrations of HBB due to adherent fat tags, additional groups of mice were fed and maintained in a similar manner and examined 12 weeks post-feeding. The thymus x 13,082.   glands and adrenal glands were carefully stripped of adherent fat under a dissection microscope before being sent to the laboratory for analysis. The results of this experiment are shown in Table 4. The concentrations of hexabromobiphenyl found in this experiment, decreased in the order, perithymic fat, perirenal fat, adrenal glands, thymus gland, liver, and stomach.
Results of the study of the transplacental transfer of hexabromobiphenyl are shown in Table 5. The maternal fat concentration after 100 ppm FireMaster BP-6 in the diet on days 7-18 of the pregnancy was 112.74 ppm, while the maternal liver had 12.02 ppm. The maternal liver/maternal fat ratio, expressed as per cent, was 11%. The whole fetus concentration was 0.95 ppm while the fetal liver concentration was 5.86 ppm. The whole fetus/maternal fat and fetal liver/maternal fat ratios, expressed as per cent, were 0.8% and 5.2%, respectively.
The results of analysis of fat and thymus specimens from two human infants, taken at autopsy, are shown in Table 6. The ratios of thymus/fat HBB concentrations, expressed as per cent, were 13% and 37%.

Discussion
We have demonstrated a number of cellular changes in livers of mice fed 1000 ppm FireMaster BP-6 for periods up to 14 days. On the basis of our previous studies showing marked increase in P450 activity in livers of mice fed 1000 ppm FireMaster BP-6 for 11 days, the finding of marked increase in smooth endoplasmic reticulum was expected. The additional findings of marked bile canalicular microvilli proliferation and mitochondrial damage was unexpected. The long-term significance of these findings is unknown. Using the concentration of hexabromobiphenyl in perirenal fat as standard reference of 100%, we have attempted to demonstrate, in a small series, the relative tissue distribution of this chemical in the mouse after several periods of time post-feeding. Liver and brain concentrations were relatively high initially after the feeding period; however, liver appears to have relatively concentrated the chemical Environmental Health Perspectives with passing time, while the brain concentrations appear to have dropped considerably. The concentration in the testicles started relatively low compared to perirenal fat but dropped more slowly, so that the relative concentration rose after 14 weeks. Adrenal glands appear to concentrate hexabromobiphenyl to the same degree as perirenal fat. If we disregard the first thymus studies as erroneous due to extraneous fat, the thymus gland had a relative concentration of 9.8% at 12 weeks post-feeding. This compares favorably to relative concentrations of 13% and 37% in two newborn human children. In light of the findings of Allen et al. (7) demonstrating hyperplasia of the gastric mucosa in monkeys following ingestion of the related polychlorinated biphenyls, the finding of persistent concentrations of hexabromobiphenyl in the stomach (relative concentrations of 6.1% 12 weeks after feeding) warrants investigation of possible similar effects in that organ with polybrominated biphenyls.
The persistence of hexabromobiphenyl in tissues up to 14 weeks after feeding suggests that delayed onset disease may occur in these organ systems. Further studies of the tissue distribution and effects of FireMaster BP-6 in rodents for longer time periods are in progress. Unpublished data from human autopsy fat specimens (8) have indicated that a majority of Michigan nonfarm residents have measurable levels of HBB with levels as high as 0.75 ppm.
Further studies of tissue distribution and effects of HBB and other brominated biphenyls and contaminants in both animals and humans are indicated.