Residual effects of polybrominated biphenyls following perinatal exposure in rats

Rats were exposed to 0.5 ppm of ozone delivered for 23.5 hr per day for up to 180 days. One group of rats was allowed to breathe filtered air for about 2 months after the conclusion of the exposure. Lung collagen and total-protein synthesis rates were quantitated by biochemical analyses performed with lung minces. Other lung lobes from the same rats were used to quantitate collagen and total protein content. Increased levels of lung protein and of lung collagen (hydroxyproline) content were observed at all times sampled during exposure to ozone (3, 30, 50, 88, 180 days); the observed lung hydroxyproline content at 180 days persisted during the 2 months of postexposure recovery. Collagen synthesis rates measured in lung minces were elevated in the exposed rats at all times sampled, consistent with the observed increased lung collagen content seen throughout the study. The observed biochemical changes were consistent with concurrent morphological observations of the occurrence of mild pulmonary fibrosis.     

Action of exogenously administered steroid hormones following perinatal exposure to polybrominated biphenyls.

Polybrominated biphenyls (PBBs) have produced a variety of effects including alterations in the endocrine system and in the activity of microsomal mixed-function oxidases (MFOs). Because steroid hormones are substrates for MFOs, effects of PBBs on the endocrine system may be a consequence of enhanced steroid hormone catabolism. Therefore, as a first step in evaluating the effect of PBBs on steroid hormone catabolism. Therefore, as a first step in evaluating the effect of PBBs on steroid hormone metabolism, it was of interest to determine effects of perinatal exposure to PBBs on the response to exogenously adminstered steroid hormones. Rats were exposed to 0, 10, or 100 ppm PBBs from d 8 of gestation until d 28 postpartum, when experiments were conducted. Responses to labeled steroid hormones were generally modified in a manner directly related to PBB dose and were correlated with changes in serum and target tissue retroactivity. Stimulated metabolism of steroid hormones may account, at least in part, for endocrine-related alterations produced by PBBs. Although the potential for decreased reproductive capacity following PBBs cannot yet be accurately predicted, these experiments suggest that fertility may be reduced following PBBs as a consequence of accelerated steroid hormone metabolism and/or excretion.     

Altered metabolism of progesterone by hepatic microsomes from rats following dietary exposure to polybrominated biphenyls

The local anesthetic mepivacaine and various other drugs known to perturb sarcoplasmic calcium metabolism and/or sarcolemmal sodium conductance were injected into the gastrocnemius or subcutaneously over the gracilis muscles of rats. After 48 hr, the tissues were examined histologically, and muscle damage was rated on a single-blind basis. Following im administration, all of the agents tested which are capable of increasing the intracellular concentration of free calcium—mepivacaine-HCl, quinidine gluconate, A23187, caffeine, and 2,4-dinitrophenol-produced extensive and qualitatively similar myonecrosis. Damage to skeletal muscle was absent or negligible after im injection of control saline solutions or tetrodotoxin, a drug that blocks sodium channels without directly influencing the intracellular calcium distribution. The sc administration of mepivacaine caused extensive damage to fibers approximating the muscle surface, while saline was without effect. To assess the influence of a calcium antagonist on local anesthetic-induced myonecrosis, verapamil-HCl was administered sc, both alone and in combination with mepivacaine. Although verapamil by itself resulted in some minor surface injury, it almost completely blocked the damage produced by mepivacaine. It is concluded that the myotoxicity of local anesthetics is related to a disturbance of intracellular calcium homeostasis rather than to an influence on sarcolemmal sodium conduction.     

Ortho-substituted polybrominated biphenyls activate respiratory burst in granulocytes from humans

The in vivo consequences of exposure to polychlorinated biphenyls (PCB) have been reported to involve reduced phagocytic function, which could be related to increased susceptibility to infections. Though less abundant in the environment, polybrominated biphenyls (PBB) have similar toxicological properties as PCB. In this respect the effect of different PBBs on human granulocytes was elucidated. Ortho-substituted PBBs activated respiratory burst, measured by the chemiluminescence assay, and elevated intracellular calcium. The most active polybrominated congener 2,2',5-TBB increased chemiluminescence in a concentration-dependent manner, and ED(50) was approximately 10 microM. PBBs stimulated elevation of intracellular [Ca(2+)] in human granulocytes. The [Ca(2+)]i was elevated from 50 to 250 nM. The respiratory burst due to stimulation by PBBs was inhibited by U73122, ethanol (1%), wortmannin, and bisindolylmaleimide and by the elimination of extracellular calcium in the same way as shown previously for PCBs, indicating that PBB act by the same mechanisms.     

Mixed function oxidase activities in lactating rats and their offspring following dietary exposure to polybrominated biphenyls.

Exposure of pregnant rats to diet containing 50 ppm polybrominated biphenyls (PBBs) from day 8 of gestation to day 15 postpartum caused significant increases in hepatic and extrahepatic microsomal mixed function oxidase activity. Hepatic arylhydrocarbon hydroxylase (AHH), epoxide hydratase (EH), hexobarbital hydroxylase (hex-OH), and the 2- and 4-hydroxylation of biphenyl (2-OHBP, 4-OHBP) were increased 10, 3, 3, 23, and 6-fold respectively in animals fed diet containing PBBs. The hex-OH, 2-OHBP, and 4-OHBP activities were not detectable in the S9 fraction from maternal mammary glands of control or PBB-fed rats; however, exposure to PBBs increased mammary AHH 2.5-fold and decreased EH activity 45%. Renal AHH activity was increased 7-fold but renal EH activity was unaltered by feeding PBBs. Pups from control and PBB-exposed mothers were crossfostered at birth to give offspring which received PBB exposure prenatally, postnatally (via mothers milk), or both pre- and postnatally. Each type of exposure produced increases in hepatic AHH and EH activities over those found in pups born to and raised by mothers which received no PBBs. The results demonstrate that PBBs induce hepatic and extrahepatic mixed function oxidase activity in nursing rats and that the extrahepatic effects of the mixture are different from the hepatic effects. Furthermore, PBBs were effective stimulators of hepatic enzymes in 15-day-old rats when the neonates were exposed transplacentally and/or via the mothers' milk. The results suggest potential toxic interaction between PBBs and other agents which are of importance to both mother and young.     

The induction of hepatic microsomal metabolism in rats following acute administration of a mixture of polybrominated biphenyls.

A high frequency of congenital limb defects after maternal treatment with the nitrogen mustard chlorambucil suggested that cytotoxic effects could be analyzed as correlates of teratogenic action of this alkylating agent. Limb buds from $\text{ICR}\text{DUB}$ mouse embryos were exposed in vivo or in vitro to teratogenic doses of chlorambucil. At 0.5- to 72-hr intervals following exposure, the limbs were processed for observation with the light and electron microscopes. Light microscopy of limb buds exposed to the drug in vivo or in vitro showed evidence of cellular involvement within 4 hr following treatment. The effects were manifested in the form of cytoplasmic inclusions which increased in number as the time following exposure increased, and, in vivo, reached a maximum at 24 hr. Increased doses of the drug also produced increased numbers of affected cells. In limb buds exposed to chlorambucil in vitro, the number of affected cells reached a plateau at 48 hr after treatment and decreased significantly by 72 hr, at which time exposure to a second dose of the drug had little or no effect. Affected cells were restricted almost exclusively to regions of undifferentiated mesenchymal cells. Electron microscopic examination of the cytoplasm of affected cells demonstrated the presence of membrane-bound vacuoles containing cellular organelles in various stages of degeneration. The number and size of these vacuoles increased with time following exposure until finally the cells fragmented. Nuclear material was not involved until fragmentation of the cells occurred, at which time the nucleus became pyknotic. Following cellular fragmentation and destruction, macrophages were observed in association with the cellular debris. Acid phosphatase was demonstrated within the vacuoles (by the presence of lead phosphate precipitates) with the electron microscope after incubating limb bud tissue in a buffered solution of β-glycerophosphate/lead nitrate. The amount of demonstrable enzyme was found to increase as the degree of breakdown within vacuoles increased. These results and the histological information provided evidence consistent with a process of autophagocytosis.     

The comparative biologic and toxic potencies of polychlorinated biphenyls and polybrominated biphenyls

Aroclor 1254 and fireMaster BP-6, two commercial polychlorinated biphenyl (PCB) and polybrominated biphenyl (PBB) preparations, exhibit comparable biologic and toxic effects. In the present study the commercial PBB was more active than Aroclor 1254 in causing thymic atrophy in male Wistar rats. However, a direct comparison of the relative effects of bromine vs chlorine substituents is not possible with the commercial PBB and PCB mixtures due to their complex congeneric composition. This study reports the synthesis and biologic and toxic effects of a series of laterally substituted 3,3',4,4'-tetrahalobiphenyls which contain the following variable molecular Cl/Br ratios; Br4, Br3Cl, Br2Cl2 (two isomers), BrCl3, and Cl4. 3,3',4,4'-Tetrabromobiphenyl and 3,4,4'-tribromo-3'-chlorobiphenyl (150 mumol/kg)-pretreated animals significantly inhibited the growth rate of and caused thymic atrophy in immature male Wistar rats whereas those isostereomers with reduced Br (and increased Cl) content were either less active or inactive. Pretreatment of male Wistar rats with 10 mumol/kg of the 3,3',4,4'-tetrahalobiphenyls and determination of their effects as inducers of the hepatic microsomal drug-metabolizing enzymes also illustrated the effects of the relative Cl/Br ratios on induction potencies. Both 3,3',4,4'-tetrabromo- and 3,4,4'-tribromo-3'-chlorobiphenyl maximally induced the cytochrome P-448-dependent monooxygenases, benzo[a]pyrene and 4-chlorobiphenyl hydroxylase; the order of potency of the other isostereomers was 4,4'-dibromo-3,3'-dichloro- congruent to 3,4-dibromo-3',4'-dichlorobiphenyl greater than 4-bromo-3,3',4'-trichloro- greater than 3,3',4,4'-tetrachlorobiphenyl. With few exceptions this order of potency was observed for the induction of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase in rat hepatoma cells in culture and for their relative binding affinities to the rat cytosolic receptor protein. The data clearly demonstrate that the biologic activities of this group of isosteric halogenated biphenyls are enhanced with increasing bromine substitution and also support the hypothesis that the activities of this class of chemicals are mediated through the receptor.     

Renal and hepatic microsomal enzyme stimulation and renal function following three months of dietary exposure to polybrominated biphenyls.

Polybrominated biphenyls (PBB) stimulate microsomal enzyme activity and produce a variety of toxic manifestations, including renal and hepatic histopathological changes. Therefore, it was of interest to determine the effect of chronic exposure to PBB on renal and hepatic microsomal enzyme stimulation and renal function. Adult Sprague-Dawley rats were fed diets containing 0 or 100 ppm of PBB for 3 months. Treatment with PBB retarded weight gain and increased the liver to body weight ratio but did not alter kidney to body weight ratio. Biphenyl-4-hydroxylase (BP-4-OH) and biphenyl-2-hydroxylase (BP-2-OH) activities were elevated in the kidney and liver following treatment with PBB. Exposure to PBB increased aryl hydrocarbon hydroxylase (AHH) activity in the kidney and liver. Epoxide hydratase (EH) activity was increased in the liver but decreased in the kidney following exposure to PBB. A three-month exposure to PBB had no effect on blood urea nitrogen, the clearance of inulin, p-aminohippurate (PAH), or fractional sodium excretion. Similarly, the in vitro accumulation of PAH and N-methylnicotinamide (NMN) in thin renal cortical slices and ammoniagenesis and gluconeogenesis in renal cortical slices were not affected by PBB. In conclusion, chronic exposure to PBB resulted in significant alterations in renal and hepatic microsomal enzyme activities but had no detectable effect on renal function. These experiments suggest that alterations in microsomal enzyme activities following PBB do not lead to impairment of renal function; however, this compound may sensitize the kidney to toxicity produced by agents administered subsequent to PBB.     

The human health effects of exposure to polybrominated biphenyls

Polybrominated biphenyls (PBB) were inadvertently introduced into the food chain in Michigan in 1973. Fifty-one people with known exposure to PBB were studied. Twenty-three farmers with disabling health complaints, and 28 chemical workers involved in the PBB manufacturing process were systematically evaluated to determine if these highrisk groups suffered adverse effects from their known exposure to PBB. The farmers had a high frequency of constitutional symptoms, hepatomegaly and skin rashes, findings not commonly noted in the chemical workers. Biochemical and hematologic testing revealed few abnormalities, and electromyograms, nerve conduction velocities, endocrine studies, and lymphocyte transformation studies provided no objective findings that correlated with subjective complaints. There was no relationship between PBB levels and physical or laboratory abnormalities. Present evidence suggests that people exposed to PBB have few objective findings at this time, and reactive depression may be responsible for the high prevalence of constitutional symptoms.     

Bioavailability of soil-borne polybrominated biphenyls ingested by farm animals

Concentrations of polybrominated biphenyl (PBB) were measured in the fat of livestock on several farms on which soil-borne PBB in confinement areas was the only source of residue. Ratios of concentrations in fat to concentrations in soil were 0.37 for dairy heifers, 0.27 for primaparous dairy cows, 0.10 for multiparous dairy cows, 0.27 for beef cows, 0.39 for beef calves, 0.37 for ewes, and 1.86 for swine. Multiparous dairy cows had lower ratios because of the excretion of PBB in milk during long-term lactation, and swine had higher ratios because they ingest greater amounts of soil than other species. Diets containing 5% PBB-contaminated soil, or 5% contaminated soil amended with activated carbon, were fed to lambs for 56 d. Accumulation of soil-borne PBB in fat, when adjusted for intake, did not differ significantly from accumulation of PBB from a diet in which PBB was added to cornmeal. Amending soil with activated carbon had no effect on residue accumulation. About 70% of PBB in a control diet with PBB added to cornmeal was absorbed, as measured by using titanium as an unabsorbed marker. Absorption of soil-borne PBB was 65% from unamended soil, 57% from soil amended with 0.3% activated carbon, and 56% from soil amended with 0.6% activated carbon. The differences were not great enough to be of practical importance. These results with PBB may be useful in assessing and managing risks of other soil-borne contaminants that have chemical characteristics similar to those of PBB.     

Postoperative disappearance of phenazone from plasma in man

Summary The elimination rate of phenazone after a single oral dose has been studied before and after elective operations. In a group of patients with different illnesses the elimination rate was increased on the fourth to seventh days after operation but was unchanged on the second and third days. The change in elimination rate was highly significant in a standardized group of nine patients with a ligament injury in one knee studied on the fourth or fifth postoperative day. Possible reasons for the changes are discussed.     

Tissue distribution and elimination kinetics of polybrominated biphenyls (PBB) from rat tissue

The concentration of polybrominated biphenyl (PBB) in serum in a large number of organs was determined in a population of rats for 36 weeks a single dose of PBB. Groups were killed at 6, 12, 24 and 36 weeks after exposure to PBB (1 mg/100g body wt, i.p.). Growth, weight gain and appearance of the rats and their internal organs were normal. Complex and varied relationships were found in tissue concentrations with time after PBB administration. Serum and fat had apparent first-order elimination kinetics with calculated half-times of 23.1 and 69.3 weeks, respectively. For five other tissues, apparent t 1/2s ranged from 9.0-63 weeks, while for four others, kinetics could not be determined from these 4 time points. It is likely that a substantial residue of PBB will still remain in the body of the rat at the end of its life span because of the persistence of PBB in lipid-rich tissues (adipose, adrenal, and brain).     

Stimulation of hepatic microsomal metabolism in mice by a mixture of polybrominated biphenyls.

The pattern of stimulation of hepatic microsomal mixed function oxidases has been studied in female NMRI mice following a single ip injection of 150 mg/kg polybrominated biphenyls (PBBs) and compared to the patterns produced by phenobarbital (PB), 3 X 100 mg/kg; 3-methylcholanthrene (MC), 3 X 20 mg/kg; and these two agents administered together at these doses. Cytochrome P450, ethylmorphine N-demethylase, and epoxide hydratase reached maximums of 200, 200, and 350% of control values, respectively, at 48 hr after treatment with PBBs. Ethoxycoumarin O-deethylase and arylhydrocarbon hydroxylase were maximally increased to 400 and 180% of control values, respectively, 96 hr after PBBs. The reduced cytochrome P450 ethylisocyanide difference spectra and the inhibition of ethoxycoumarin O-deethylase and arylhydrocarbon hydroxylase activity by metyrapone and alpha-naphthoflavone indicated that the characteristics of the cytochrome P450 and the cytochrome P450-dependent enzymes changed with time after administration of PBBs. These results indicate that the enzyme-stimulating properties of PBBs alter, changing from PB-like to MC-like, with time after administration. These findings provide an explanation for the effects of PBBs on the toxicity of bromobenzene, indicating that PBBs represent a new and previously unrecognized calss of toxicity-modifying agents sharing properties of both PB and MC.     

Investigation of hyperkeratotic activity of polybrominated biphenyls in Firemaster FF-1

A polybrominated biphenyl (PBB) mixture was fractionated by normal-phase preparative high-performance liquid chromatography. The hexane fractions were concentrated and applied to rabbit ears. Only the most polar fraction produced hyperkeratosis on the rabbit ears. This active fraction was subfractionated by using the same procedure. Again, the extent of hyperkeratotic activity increased with increasing polarity. The PBBs of the largest concentration levels in the active fraction were purified by preparative gas chromatography and tested on rabbit ears. The major compounds did not demonstrate hyperkeratotic activity.     

Effect of polybrominated biphenyls on drug metabolizing enzymes in different tissues of C57 mice.

Polybrominated biphenyls (PBBs) are structurally very close to polychlorinated biphenyls (PCBs) which are known to be potent inducers of xenobiotic biotransformation reactions. We have studied the effects of 2 industrial PBB-mixtures, "hexabromobiphenyl" (HBB) and "octabromobiphenyl" (OBB), on enzymes catalyzing drug hydroxylation, epoxide hydration, and conjugation reactions in different tissues of C57 mice. The enzyme activities were measured 10 days after a single i.p. injection of PBBs (75 mg/kg). HBB enhanced the activities of hepatic AHH (1.9-fold), ethoxycoumarin deethylase (5.7-fold), epoxide hydratase (1.5-fold), glutathione S-transferase (1.7-fold) and UDP-glucuronosyltransferase (1.5-fold). In the kidney HBB enhanced the activity of UDP-blucuronosyltransferase 1.5-fold. OBB caused in increase in the activities of liver AHH (1.5-fold), ethoxycoumarin deethylase (2.4-fold) and glutathione S-transferase (1.4-fold). A slight increase was also seen in the activity of UDP-glucuronosyltransferase in digitoninactivated liver microsomes of OBB-treated mice. In the kidney OBB caused a slight but statistically significant decrease in glutathione S-transferase activity. Intraperitoneally injected bromobiphenyls had no effects on these drug metabolizing enzymes in the lung of C57 mice. These results were similar to the effects caused by a mixture of PCBs.     

Effects of polybrominated biphenyls on metabolism of testosterone by rat hepatic microsomes

Polybrominated biphenyls (PBBs) have produced alterations in the male reproductive system of several species. These alterations may be a consequence of modified metabolism of androgens. The purpose of this investigation was to determine the effects of PBBs on hepatic microsomal metabolism of testosterone in vitro. Hepatic microsomes were prepared from male and female rats exposed to 0 or 100 ppm PBBs from Day 8 of gestation until they were killed at 1, 2, or 4 months of age. Testosterone and its metabolites were identified and quantified utilizing high-performance liquid chromatography or gas-liquid chromatography. Steroid identification was confirmed with gas chromatography/mass spectrometry. Hydroxylation of testosterone to 7α- and 6β-hydroxytestosterone in microsomes from both sexes at all ages was increased by pretreatment with PBBs. Microsomal conversion of testosterone to 16 α-hydroxytestosterone and androstenedione was enhanced by PBBs in females at all ages and in 1 month-old males but not from males of 2 and 4 months of age. Reduction of testosterone to dihydrotestosterone and dihydroandrosterone was inhibited in microsomes from both sexes at all ages by pretreatment with PBBs. These results support the contention that the effects of PBBs on the male reproductive system may be due, at least in part, to altered metabolism of testosterone.     

Induction of drug-metabolizing enzymes by fractionated commercial polybrominated biphenyls (PBBs)

A commercial polybrominated biphenyl mixture was separated chromatographically into two fractions on neutral alumina (A_A and A_B) and into three fractions on Florisil (F_A, F_B, and F_C) by sequential elution with solvents of increasing polarity. Using established methods, the activity of each fraction as hepatic microsomal cytochrome P-450- and/or cytochrome P-448-dependent monooxygenase enzyme inducers was examined in the male Wister rat. Like the coadministration of phenobarbitone and 3-methylcholanthrene, commercial PBBs, either unfractionated or reconstituted from its various fractions, induced both cytochromes P-450 and P-448. Both cytochromes were also induced by the less-polar fractions A_A and F_A. In contrast, little or no inductive effects were exhibited by the more polar Florisil fractions, F_B and F_C, indicating that the ability of commercial PBBs to induce cytochrome P-448 is not due to contaminating brominated dibenzofurans or dibenzodioxins. Unlike the polar Florisil fraction, the more polar alumina fraction, A_B, was a potent microsomal enzyme inducer. This fraction was enriched in 2,3,3′,4,4′,5-hexa- and 2,2′,3,3′,4,4′,5-heptabromobiphenyl and also contained unassigned monochloro derivatives of a penta- and hexabromobiphenyl, namely C₁₂H₄Br₅Cl and C₁₂ H₃Br₆Cl, respectively. The data strongly suggest that the biologic effects of the commercial polybrominated biphenyl mixture are due to the various halogenated biphenyls present. These results are discussed in terms of the reported toxic potency of each PBB fraction and with reference to the known biologic activity of individual polybrominated biphenyl congeners or their chloro analogs.