Effects of dietary retinyl acetate on the promotion of hepatic enzyme-altered foci by polybrominated biphenyls in initiated rats

Vitamin A inhibits the development of some chemically-induced tumours. Since polybrominated biphenyls (PBBs) are hepatic tumour promoters and they affect vitamin A homeostasis in rats, we put forward the hypothesis that dietary levels of vitamin A would influence tumour promotion by PBBs. In the study described here, female Sprague-Dawley rats were initiated on day 1 by ip administration of diethylnitrosamine. On day 7 after initiation, the rats were fed a vitamin A-deficient basal diet that was supplemented with either 2000 IU (low-vitamin A) or 200,000 IU (high-vitamin A) retinyl acetate/kg feed. From day 30 after initiation until the end of the study the following PBBs were added to the diets: Firemaster BP-6 (10 ppm), 2,4,5,2',4',5'-hexabromobiphenyl (10 ppm) or 3,4,5,3',4',5'-hexabromobiphenyl (1 ppm). The control animals received low- or high-vitamin A diets containing no PBBs. On day 180, the rats were necropsied, sections of various tissues were stained for histopathological examination and an evaluation of hepatic enzyme-altered foci was performed. Numbers of gamma-glutamyl transpeptidase-positive foci/cm3 liver and the mean volumes of these foci were lower in the high-vitamin A groups than those in the corresponding low-vitamin A groups, but these differences were not significant. The percentage of the liver volume occupied by foci was significantly greater in the low-vitamin A with 345-HBB group than in the corresponding high-vitamin A group. Thus, high dietary levels of vitamin A had some inhibitory effect on the promotion of hepatic-altered foci by 345-HBB in initiated rats.     

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.     

Effects of polychlorinated biphenyls in rat liver: quantitative analysis of enzyme-altered foci.

The promotional effect of various polychlorinated biphenyls and phenobarbital on enzyme-altered lesions in the rat liver was quantified within the framework of the two-stage carcinogenesis model of Moolgavkar and colleagues. The experiment analyzed here followed an initiation-promotion protocol in which female Wistar rats were initiated with diethylnitrosamine (DEN) at 10 mg/kg body wt for 10 days followed by a 8-week period of promoter treatment with various cytochrome P450 isoenzyme inducing and noninducing compounds. This analysis included 4-monochlorobiphenyl, 2,2',4,5'-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl and 3-methylcholanthrene, all administered at 150 mumol/kg body wt, and phenobarbital which was administered continuously in the diet at 0.05% until termination. Animals were killed either 1 or 9 weeks after the end of treatment and their livers were examined for enzyme histological alterations. Focal transections were classified as falling into three phenotypic categories: ATPase dominant, GGT dominant, or ATPase plus GGT (coextensive). A quantitative method was used to analyze the data consisting of the number and sizes of the focal transections. The number of cells altered by the DEN treatment and cell kinetic parameters measuring the promotional effect of the various compounds were estimated. On the basis of these estimates, we computed the number of nonextinct altered foci and their volume fraction as functions of time. We found that foci exhibiting the coextensive phenotype respond most efficiently to promoter treatment, while GGT dominant foci respond weakly to all the promoters with the exception of 3-MC. For phenobarbital, we observed a significant slowing of focal cell proliferation over time.     

Cardiac and hepatic effects of pre- and postnatal exposure to polybrominated biphenyls in rats

Body weight gain and hepatic concentrations of vitamin A were reduced in Sprague-Dawley rats by pre- and postnatal exposure to 100 ppm polybrominated biphenyls (PBBs). The ratio of liver weight to body weight, activity of hepatic delta-aminolevulinic acid (ALA) synthetase, and urinary excretion of uro- and coproporphyrins were increased by PBBs. Treatment with PBBs also increased the left atrial inotropic response to calcium. However, PBBs had no effect on development of the adrenergic neuronal transport system in heart, left atrial baselike peak tension, or inotropic response to ouabain. Thus PBBs retarded body weight gain and produced a variety of alterations in liver, but had little effect on cardiac contractile function.     

Lack of hepatotoxicity or promotion of enzyme-altered liver foci development in rats treated with harman or norharman

The modifying effects of harman or norharman on liver carcinogenesis were investigated in male F344/DuCrj rats initially treated with N-nitrosodiethylamine (DEN). Two weeks after a single dose of DEN (200 mg/kg, intraperitoneally), rats were given harman or norharman at dietary levels of 1000 and 200 parts per million (ppm), or sodium phenobarbital (PB) at 500 ppm as a positive control, for 6 wk. At wk 3 following DEN administration, all animals were subjected to partial hepatectomy. Marked retardation of body weight gain was observed in rats treated with harman or norharman at 1000 ppm, but not at 200 ppm. Increased relative kidney but not liver weights were associated with harman or norharman treatment, especially in the higher dose groups. Although no toxicity-related hepatocyte lesions were found, severe renal toxic tubular lesions and regeneration were evident. Neither harman nor norharman significantly increased the numbers or areas of glutathione S-transferase placental form (GST-P) positive foci observed after DEN initiation, in clear contrast to PB. The results thus demonstrated that harman and norharman are nontoxic for the liver and lack modifying potential for liver carcinogenesis in our medium-term bioassay system.     

Heterogeneity of enzyme-altered foci in rat liver

Enzyme-altered foci (EAF) in liver are assumed to be precursor lesions for tumors in this organ. Results obtained with selected hepatocarcinogens which produce lesions of differing phenotype and growth behavior indicate that not the total number of enzyme-altered cells but rather the proliferation of individual cell clones is of major importance for additional changes leading to malignancy. Analyses including multiple marker enzymes demonstrate a relationship between foci phenotype and proliferation. The inducibility of certain downregulated enzymes in EAF indirectly suggests disturbances in the expression of regulatory genes such as proto-oncogenes. Data on Ha-ras and c-myc proto-oncogene expression in EAF are presented.     

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.     

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.     

Long-term effects of a single oral dose of polybrominated biphenyls on serum and liver lipids in rats

Adult (5 months) male Sherman strain rats received a single dose of either 0 or 500 mg polybrominated biphenyls (PBB) in corn oil/kg body weight by stomach tube. After an 18-month recovery period, serum and liver samples were examined. The primary serum lipid response was an increase in cholesterol (both free and esterified) and in total phospholipids. The percentage of esterified cholesterol was not significantly different from that of the controls, and no significant differences in the cholesterol ester fatty acid composition were observed. Serum triglycerides were also unaffected. In the PBB-dosed animals, the total hepatic fatty acids contained significantly less palmitic acid and more stearic acid, consistent with an increase in palmitic acid chain elongation activity. No significant differences could be detected in the n-3 or n-6 acids except for a slight decline in the content of 22:6 (n-3). Hepatic microsomal phospholipids were slightly higher (per milligram protein) in the PBB-dosed animals, and the cholesterol content was lower. Consequently, the cholesterol-phospholipid ratio was reduced, and microsomes from the latter group appeared to have an altered lipid domain on the basis of steady-state fluorescence anisotrophy measurements. In addition, total hepatic thiobarbituric acid-reactive substances (assayed as malondialdehyde) were significantly increased in the PBB-dosed animals. This observation appeared to reflect an increased susceptibility to peroxidative stress in the latter group, probably resulting from reduced membrane antioxidant concentrations. The PBB-dosed rats had significantly lower serum retinol levels and a reduced content of this vitamin in liver microsomes. Microsomes were also deficient in alpha-tocopherol in the PBB-dosed animals, although serum levels were normal.     

Increased hepatic acylcarnitines after oral administration of amiodarone in rats

Amiodarone is known to induce hepatic injury in some recipients. We applied an untargeted metabolomics approach to identify endogenous metabolites with potential as biomarkers for amiodarone-induced liver injury. Oral amiodarone administration for 1 week in rats resulted in significant elevation of acylcarnitines and phospholipids in the liver. Hepatic short- and medium-chain acylcarnitines were dramatically increased in a dose-dependent manner, while the serum levels of these acylcarnitines did not change substantially. In addition, glucose levels were significantly increased in both the serum and liver. Gene expression profiling showed that the hepatic mRNA levels of Cpt1, Cpt2, and Acat1 were significantly suppressed, whereas those of Acot1, Acly, Acss2, and Acsl3 were increased. These results suggest that hepatic acylcarnitines and glucose levels might be increased due to disruption of mitochondrial function and suppression of glucose metabolism. Perturbation of energy metabolism might be associated with amiodarone-induced hepatotoxicity.     

Effect of polybrominated biphenyls on hepatic microsomal metabolism of estrogens and uterotropic action of administered estrogen in rats

Perinatal exposure to polybrominated biphenyls (PBBs) increased the hepatic microsomal metabolism of estradiol, estrone, and ethynylestradiol in vitro. Pretreatment with PBBs decreased the effect of estradiol administered exogenously on uterine estrogen cytosolic receptor concentration. The effect of exogenous estradiol on uterine weight and uterine RNA content was also reduced by perinatal exposure to PBBs. Therefore, metabolism of estrogens is altered by PBBs.     

Development of preneoplastic lesions in the liver and nasal epithelium of rats initiated with N-nitrosodimethylamine or N-nitrosopyrrolidine and promoted with polybrominated biphenyls.

In rats, N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR) induce liver tumours and, to a lesser extent, nasal tumours. Polybrominated biphenyls (PBBs) are liver tumour promoters and are highly persistent in tissues of rats. To characterize the development of preneoplastic lesions in the liver and nasal cavity, female Sprague-Dawley rats were initiated with NDMA or NPYR and promoted with Firemaster (FM), a commercial mixture of PBBs. Rats were killed after 30, 120 or 180 days of promotion. Liver and nasal tissues were stained with haematoxylin and eosin and were tested immunohistochemically for glutathione S-transferase placental form (GST-P). Significantly more altered hepatocellular foci (AHF) were evident in rats initiated with NDMA or NPYR and promoted with FM compared with non-promoted groups or rats given only FM. Appreciable numbers of AHF were seen at 120 and 180 days in livers of rats in all other treatment groups, whereas the untreated control rats had no AHF. The percentage volume of the liver occupied by AHF was significantly higher in promoted rats given NDMA than in rats given only NDMA or FM. These results indicate that a single oral dose of PBB can significantly enhance development of AHF in rats initiated with NDMA or NPYR. Preneoplastic lesions in nasal tissues were not detected by staining with GST-P.     

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.     

Enhancing effects of mustard oil on preneoplastic hepatic foci development in Wistar rats

Dietary habits are known to be the major contributory factor in the development of cancer. Mustard oil, which is extensively used in India and elsewhere as a flying and cooking medium, is reported to induce an inflammatory response. The development of altered hepatic foci is an early carcinogenic change in rat liver in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. In the present study, the development of preneoplastic lesions was observed following administration of mustard oil (0.5 mL/day for 8 weeks) in DEN-initiated and partially hepatomized Wistar rats. A significant decrease in the relative and absolute liver weight of mustard oil-exposed rats was recorded. The results revealed a significant increase in the number and area of placental glutathione-S-transferase (GST-P) and gamma-glutamyl transpeptidase (GGT)-positive foci in mustard oil-administered animals. The GST-P- and GGT-positive foci were more prominent in the animals given boiled (up to 300 degrees C for 3 hours) mustard oil in comparison to the animals given fresh mustard oil. These results indicate the possible tumourigenic risk associated with mustard oil consumption.     

Trichloroethylene effects on the formation of enzyme-altered foci in rat liver

The initiating and promoting effects of trichloroethylene in rat liver were investigated using the enzyme-altered foci bioassay. The incidence of gamma-glutamyl transpeptidase (GGT)-positive foci was used as an early histochemical marker of putative preneoplastic hepatocytes. A single PO dose of trichloroethylene (490 mg/kg) was administered in corn oil to rats which had been partially hepatectomized 24 h previously. Three days following gavage with the chlorinated hydrocarbon the rats were promoted with an 8-week regimen of 500 ppm phenobarbital in drinking water. This protocol is known to induce enzyme-altered foci in the livers of animals which have received an initiating dose of a genotoxic carcinogen. Trichloroethylene was not found to induce GGT-positive foci under these conditions. Additionally, groups of rats were partially hepatectomized, initiated with N-nitrosodiethylamine (30 mg/kg; PO) and administered five times weekly doses of 200 mg trichloroethylene per rat in order to investigate the promoting activity of the chlorinated hydrocarbon in rat liver. No significant promoter effects were observed with trichloroethylene, although the results in this case were somewhat equivocal. The findings of these investigations are taken as partially supportive of an epigenetic, cytotoxic mechanism of tumorigenic action of trichloroethylene.     

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.     

Differential tissue distribution of various polybrominated biphenyls of Firemaster FF-1 in male rats

Gas chromatography with an electron-capture detector was used to identify the major components of the polybrominated biphenyls (PBB's) in Firemaster FF-1. The distribution and clearance of the PBB's in rat whole blood, grey and white matter, cerebellum, heart, kidney, liver, spleen, lung, jejunum, testes, and subcutaneous fat from the inguinal region were determined at various times after a single dose of 10 mg/kg of Firemaster FF-1 given orally. Peak blood levels of PBB occurred within 4 hr whereas levels in inguinal subcutaneous fat peaked 7--14 days after the single oral dose. Concentrations in other tissues peaked usually within 12 hr and showed complex logarithmic decline over time. 2,2',4,5,5'-Pentabromobiphenyl (PnBBa) was cleared much more rapidly from the rat than 2,3',4,4',5-pentabromobiphenyl (PnBBb) or any of the higher molecular weight analogs. Furthermore, PnBBa penetrated the brain more rapidly. Each of the PBB analogs was found in all tissues examined but varied in both rate of absorption and clearance.     

Nicotine administration enhances conditioned inhibition in rats

The effect of nicotine on conditioned inhibition was examined using a serial feature negative discrimination task. Nicotine (0.35 mg/kg) or vehicle was administered before each of the 16 training sessions. On some trials in each session, a tone was presented and followed by food reward. On other trials, the tone was preceded by a visual stimulus and not reinforced. Nicotine-treated rats exhibited greater discrimination between the two trial types as evidenced by less frequent responding during non-reinforced trials, and learned the discrimination in fewer sessions than vehicle-treated rats. In contrast, there were no group differences in responding during the reinforced trials.     

Endocrinological, neurochemical, and anabolic effects of polybrominated biphenyls in male and female rats

Pregnant rats were placed on a control diet or on a diet containing 100 ppm polybrominated biphenyls (PBBs, Firemaster PB-6) on Day 8 of gestation. On Day 28 postpartum the pups were weaned onto the same diet as their mothers, and maintained on this diet until sacrificed during the ninth week. Body weight gain was reduced in both the male and female rats exposed to PBBs. The absolute weights of some organs (ventral prostate and seminal vesicles in males, and adrenal and pituitary glands in females) were reduced in animals maintained on the PBB diet, but when expressed as a percentage of body weight only the ventral prostate weight remained significantly reduced. Exposure to PBBs did not affect plasma concentrations of luteinizing hormone, prolactin, or corticosterone; nor did it affect the increase in the plasma concentration of the latter two hormones in response to a mild environmental stress. Exposure to PBBs did not alter the steady-state concentrations of norepinephrine or dopamine in the posterior pituitary or selected brain regions. Furthermore, treatment with PBBs did not alter the rates of synthesis of these catecholamines (as estimated by the rate of accumulation of dihydroxyphenylalanine following inhibition of dihydroxyphenylalanine decarboxylase) in the brains of female rats on the day of diestrus or estrus. On the basis of vaginal cytology, exposure to PBBs significantly lengthened the estrous cycle of female rats.