The effect of dietary glycine on the hepatic tumor promoting activity of polychlorinated biphenyls (PCBs) in rats

Polychlorinated biphenyls (PCBs) are ubiquitious lipophilic environmental pollutants. Some of the PCB congeners and mixtures of congeners have tumor promoting activity in rat liver. The mechanism of their activity is not fully understood and is likely to be multifactorial. The aim of this study was to investigate if the resident liver macrophages, Kupffer cells, are important in the promoting activity of PCBs. The hypothesis of this study was that the inhibition of Kupffer cell activity would inhibit hepatic tumor promotion by PCBs in rats. To test our hypothesis, we studied the effects of Kupffer cell inhibition by dietary glycine (an inhibitor of Kupffer cell secretory activity) in a rat two-stage hepatocarcinogenesis model using 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153, a non-dioxin-like PCB) or 3,3',4,4'-tetrachlorobiphenyl (PCB-77, a dioxin-like PCB) as promoters. Diethylnitrosamine (DEN, 150 mg/kg) was administered to female Sprague-Dawley rats, which were then placed on an unrefined diet containing 5% glycine (or casein as nitrogen control) starting two weeks after DEN administration. On the third day after starting the diets, rats received PCB-77 (300 micromol/kg), PCB-153 (300 micromol/kg), or corn oil by i.p. injection. The rats received a total of 4 PCB injections, administered every 14 days. The rats were euthanized on the 10th day after the last PCB injection, and the formation of altered hepatic foci expressing placental glutathione S-transferase (PGST) and the rate of DNA synthesis in these foci and in the normal liver tissue were determined. Glycine did not significantly affect foci number or volume. PCB-153 did not significantly increase the focal volume, but increased the number of foci per liver, but only in the rats not fed glycine; PCB-77 increased both the foci number and their volume in both glycine-fed and control rats. Glycine did not alter the PCB content of the liver, but did increase the activity of 7-benzyloxyresorufin O-dealkylase (BROD) in liver microsomes from PCB-153 treated rats. However, glycine did not affect the induction of ethoxyresorufin O-dealkylase activity by PCB-77 in liver microsomes. Glycine diminished hepatocyte proliferation in PGST-positive foci, but not in normal tissue. Overall these results do not support the hypothesis that dietary glycine inhibits the promoting activities of PCBs. The observations that PCB-153 increased the number of foci per liver in control rats but not glycine-fed rats and that dietary glycine reduced cell proliferation in PGST-positive foci, however, do not allow us to completely rule out a role for dietary glycine. But the data overall indicate that Kupffer cells likely do not contribute to the tumor promoting activities of PCB-77 and PCB-153.     

Effect of 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl on the induction of hepatic lipid peroxidation and cytochrome P-450 associated enzyme activities in rats

Polychlorinated biphenyls (PCBs) are environmental contaminants that have been widely used for various industrial purposes. In spite of numerous studies on PCBs, however, their mechanism of toxicity remains unknown. The role of cytochrome P-450 in PCBs induced hepatic lipid peroxidation is controversial. Therefore, the present study was undertaken to study the mechanism of action of two PCBs and their role in cytochrome P-450 induction and lipid peroxidation, determined in vivo and during the incubation of subcellular fractions. We also examined whether agonist/antagonist activities between the two PCBs were occurring. Two PCBs were studied: 3,3',4,4'-tetrachlorobiphenyl (PCB-77), a non-ortho-substituted, coplanar PCB; and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153), a di-ortho-substituted, non-planar PCB. Groups of male Sprague-Dawley rats were given a single i.p. injection of one of the two PCBs (at doses of 30, 150, or 300 micromol/kg), both PCBs (at doses of 30 or 150 micromol/kg), or vehicle alone. Rats were sacrificed after 2, 6, or 24 h; or 2, 6, or 10 days. Cytochrome P-450 induction occurred as early as 2 h with PCB-77 and 24 h with PCB-153. Significant increases in thiobarbituric acid reactive substances (TBARS) content in liver tissue occurred 2, 6 and 10 days after treatment with PCB-77 and PCB-153; it was unclear whether these PCBs were synergistic in their induction of TBARS formation. Liver microsomal fractions incubated with NADPH only showed increased TBARS formation at the highest doses of PCB-77 and PCB-153 after 6 days. The results indicate that both PCBs induced cytochrome P-450 enzymes and enhanced lipid peroxidation in liver and subcellular fractions but with different potencies and onsets of action. The results also indicate a larger time difference between cytochrome P-450 induction and lipid peroxidation for PCB-77. Thus, both PCB-77 and PCB-153 are toxic to cells, but may act via different mechanisms to induce their effects.     

Dietary selenium, glutathione peroxidase activity, and toxicity of 2,3,7,8-tetrachloro-dibenzo-p-dioxin

TCDD has been shown to inhibit selenium-dependent glutathione peroxidase activity. The role of selenium in TCDD toxicity is not known. We have therefore examined the effect of TCDD administration on hepatic glutathione peroxidase, aryl hydrocarbon hydroxylase, glutathione reductase, and glutathione S-transferase activities, glutathione content, and lipid peroxidation in rats fed 0, 0.10, and 2.0 ppm dietary selenium. TCDD treatment significantly inhibited selenium-dependent glutathione peroxidase in animals on diets containing 0.10 and 2.0 ppm selenium. The selenium-dependent glutathione peroxidase activities in rats on 0.10 and 2.0 ppm dietary selenium were 8.3-and 4.7-fold greater than in animals fed a diet containing 0 ppm selenium. TCDD administration enhanced hepatic microsomal lipid peroxidation by factors of 4.0, 4.9, and 9.8 in animals fed diets containing 0, 0.10, and 2.0 ppm selenium, respectively. The administration of a lethal dose of TCDD to rats fed diets containing 0, 0.10, and 2.0 ppm selenium resulted in 0, 46, and 7% survival, respectively, after 66 d. Aryl hydrocarbon hydroxylase, glutathione S-transferase, and glutathione reductase activities were induced by TCDD. The results indicate that optimum dietary selenium provides partial protection from the toxic effects of TCDD.     

Induction of altered hepatic foci by a mixture of dioxin-like compounds with and without 2,2',4,4',5,5'-hexachlorobiphenyl in female Sprague-Dawley rats

The hepatic tumor-promoting activity of a mixture of polyhalogenated aromatic hydrocarbons (PHAHs) was studied in a medium term two-stage initiation/promotion bioassay in female Sprague-Dawley rats. The PHAH mixture contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1, 2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,4,7, 8-pentachlorodibenzofuran (PeCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 2,3,3',4,4', 5-hexachlorobiphenyl (PCB 156), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) and covered >90% of the total toxic equivalents (TEQ) present in Baltic herring. To determine possible interactive effects of di-ortho-substituted PCBs, the PHAH mixture was tested with (PHAH+) and without (PHAH-) PCB 153. Rats were initiated by a diethylnitrosamine injection (30 mg/kg body wt i.p.) 24 h after a partial 23 hepatectomy. Six weeks after initiation, the PHAH mixtures were administered once a week by subcutaneous injections for 20 weeks. Treatment with the PHAH mixtures caused liver enlargement and an increased activity of the hepatic cytochrome P4501A1/2 and P4502B1/2. All PHAH exposure groups exhibited an increased occurrence of hepatic foci positive for the placental form of glutathione-S-transferase. In the PHAH-group dosed 1 microgram TEQ/kg body wt/week, the volume fraction of the liver occupied by foci was significantly lower compared to the TEQ equivalent dosed TCDD group (3.8 vs 8.7%). The volume fraction was significantly increased in the groups treated with 0.5, 1, or 2 micrograms TEQ/kg body wt/week of the PHAH+ mixture (4.5, 5.2, and 6.6%, respectively) compared to the corn oil group (2.0%), but to a lower extent than expected on basis of the TEQ doses. Overall, the TEQ-based administered dose overestimated the observed tumor-promoting effects of this PHAH mixture. The applicability of the toxic equivalency factor concept, the role of differences in toxicokinetic properties and interactive effects of PCB 153 on hepatic deposition of the dioxin-like congeners are discussed.     

Sex differences in biochemical manifestations of selenium deficiency in rat liver with special reference to heme metabolism

In addition to its well-known effect on glutathione peroxidase, selenium deficiency causes: (1) a defect in hepatic heme metabolism characterized by a phenobarbital-mediated increase in microsomal heme oxygenase activity, and (2) an increase in hepatic glutathione S-transferase activity. Since these effects were reported in selenium-deficient male rats, and since female rats have a lower selenium requirement than males, we examined whether these effects were sex-dependent. Weanling male rats, female rats, castrated male rats, and testosterone-treated female rats were fed either a selenium-deficient or a control diet. After 8 weeks, selenium-dependent hepatic glutathione peroxidase activity was 1 per cent of respective control values in each of the selenium-deficient groups. Hepatic glutathione,S-transferase activity was doubled by selenium deficiency in normal, unoperated males but was unaffected in the other groups. In control diet fed rats phenobarbital given as a single injection caused either no significant change or a decrease in the activity of hepatic microsomal heme oxygenase, the rate-limiting enzyme in heme degradation. In contrast, microsomal heme oxygenase activity was stimulated by phenobarbital in all selenium-deficient rats. The stimulation was greatest in males and least in females with intermediate values in castrated males and testosterone-treated females. These results demonstrate a marked effect of sex, castration of males, and testosterone treatment of females on the response of hepatic heme metabolism to phenobarbital and on glutathione S-transferase activity in selenium deficiency even though glutathione peroxidase was reduced to the same extent by selenium deficiency in all groups.     

Polychlorinated biphenyls alter the expression of endothelial nitric oxide synthase mRNA in human umbilical vein endothelial cells

Polychlorinated biphenyls (PCBs) are a group of persistent pollutants that are detected in maternal serum and umbilical cord, suggesting that fetal exposure also needs to be considered. The effects of dioxin-like PCB congeners 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) and a non-dioxin-like compound 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on the expression of endothelial nitric oxide synthase (eNOS), known to maintain blood flow to the fetus, in human umbilical vein endothelial cells (HUVECs) were investigated. The mRNA levels of eNOS, aryl hydrocarbon receptor (AhR) and cytochrome P450 (CYP) 1A1 in cells treated with 5 microM PCBs for 24 hours were analysed by real-time RT-PCR. Cells were also treated with alpha-naphthoflavone (alpha NF), an AhR antagonist or ICI 182780, an estrogen receptor (ER) antagonist, one hour prior to PCB exposure, to observe the effects of these receptors on eNOS modulation. Each PCB increased the eNOS mRNA level by 4.5-fold that was markedly inhibited by alphaNF. ERs were also suspected of altering eNOS levels because ICI 182780 treatment resulted in a decrease in the eNOS level. These results suggest that the eNOS mRNA expression increases due to the action of PCBs related to both AhR and ERs in HUVECs, and that maternal PCB exposure could influence fetal circulation.     

Selected polychlorobiphenyls congeners bind to estrogen receptor alpha in human umbilical vascular endothelial (HUVE) cells modulating angiogenesis

Endocrine disrupting chemicals (EDCs) can behave as agonists or antagonists of several hormone receptors, thus mimicking or antagonizing the physiological activity of endogenous ligands. The involvement of estrogens in the regulation of angiogenesis has convincingly been demonstrated by a large body of experimental studies. Some polychlorobiphenyls (PCBs), considered EDCs, interact with estrogen receptors (ERs), so it is possible that these exogenous compounds affect the angiogenic process. Using fluorescence polarization, we firstly assayed whether PCB 77 (3,3',4,4'-tetrachlorobiphenyl), PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl) and PCB 156 (2,3,3',4,4',5-hexachlorobiphenyl) were able to bind to the alpha isoform of ER, recently found to be involved in angiogenesis. To discriminate the putative agonist or antagonist binding behaviour of these compounds, we tested their ability to activate, similarly to the natural ligand 17-beta-estradiol (17betaE(2)), the extracellular-signal-regulated kinase (Erk) 1/2 in human umbilical vascular endothelial (HUVE) cells. Finally, by using a new angiogenic assay, we evaluated the effect of PCBs treatment on microvessels neoformation. The data obtained in the present study showed that all the PCBs tested were able to bind to ERalpha and to elicit a response which can be agonistic or antagonistic; moreover, PCB 153 and PCB 77 can either positively or negatively modulate the angiogenic process, thus behaving as EDCs in endothelial cells.     

Nonadditive hepatic tumor promoting effects by a mixture of two structurally different polychlorinated biphenyls in female rat livers.

This study evaluates and quantifies the interactive hepatic tumor promoting effects of two PCBs, the Ah receptor agonist PCB 126 (3,3',4,4',5-pentachlorobiphenyl) and the constitutive androstane receptor (CAR) agonist PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl). Promotion of altered hepatic foci was evaluated utilizing a medium-term 8-week bioassay for promoters of hepatocarcinogenesis. The assay employs placental glutathione-S-transferase positive (GST-P+) liver cell foci as markers of preneoplasia in female Fischer 344 rats treated with the known initiator diethylnitrosamine followed by partial hepatectomy and by gavage exposure to test chemicals. GST-P+ foci were quantified by histomorphometry and were reported as areas and numbers of GST-P+ foci within the area of liver examined. For PCB 126, the doses were 0.1, 1.0, and 10 microg/kg body weight. For PCB 153, the doses were 10, 100, 1000, 5000, and 10,000 microg/kg body weight. Combined PCB 126 and 153 exposures were 0.1 + 10, 1 + 100, 10 + 1000, 10 + 5000, and 10 + 10,000 microg/kg, respectively. Individual PCB treatment resulted in dose dependent increases in liver and adipose concentrations. Hepatic PCB 153 levels were significantly increased (p < 0.01) after combined exposure. Treatment with PCB 126 or PCB 153 alone resulted in a significant (p < 0.01) dose dependent increase in GST-P+ foci area and number compared with controls. Treatment with the mixture of PCB 126 and 153 resulted in antagonistic GST-P+ focus formation (p < 0.001) for both foci area and number. The less than additive effect was present at all 5 PCB 126/PCB 153 dose combinations, including the low doses of PCB 126 and 153 that did not show significant promotional activity alone.     

Antagonism of aryl hydrocarbon receptor-dependent induction of CYP1A1 and inhibition of IgM expression by di-ortho-substituted polychlorinated biphenyls

Halogenated aromatic hydrocarbons (HAHs) are ubiquitous environment contaminants that produce many of their toxic effects by binding to the aryl hydrocarbon receptor (AhR). However, several investigations have demonstrated that certain polychlorinated biphenyl (PCB) congeners, principally di-ortho-chlorinated PCB congeners, or mixtures containing multiple di-ortho-chlorinated PCBs, inhibit AhR-mediated responses induced by other toxic HAHs. Most relevant to the present study are past reports demonstrating antagonism by these uniquely acting PCB congeners on AhR agonist-mediated inhibition of humoral immune responses. The mechanism responsible for antagonism of AhR agonists by certain PCBs is presently unknown. The present study evaluated the antagonist activity of several di-ortho-substituted PCB congeners [PCB47 (2,2',4,4'), PCB52 (2,2',5,5'), PCB128 (2,2',3,3',4,4'), and PCB153 (2,2',4,4',5,5')] when present in combination with AhR agonists [TCDD (2,3,7,8,-tetrachlorodibenzo-p-dioxin), PCB126 (3,3',4,4',5), and PCB77 (3,3',4,4')] on CYP1A1 induction and inhibition of lipopolysaccharide (LPS)-induced immunoglobulin production in the CH12.LX B cell line. In contrast to non-ortho-substituted PCB (PCB77), which showed additive effects on CYP1A1 induction in combination with TCDD, all of the di-ortho-substituted PCBs examined produced antagonism. Di-ortho-substituted PCB (PCB52) also antagonized TCDD- or PCB126- mediated inhibition of IgM secretion and immunoglobulin heavy chain mRNA expression in the LPS-activated B cells. In addition, PCB52 inhibited TCDD-induced AhR DNA binding to a dioxin-responsive element. Collectively, these results suggest that the mechanism responsible for antagonism by di-ortho-substituted PCB congeners of AhR agonist-mediated CYP1A1 induction and inhibition of antibody responses in B cells occurs through interference with agonist activation of the cytosolic AhR complex.     

Polychlorinated biphenyls: correlation between in vivo and in vitro quantitative structure-activity relationships (QSARs)

The in vivo quantitative structure-activity relationships (QSARs) for several polychlorinated biphenyls (PCBs) were determined in the immature male Wistar rat. The ED25 and ED50 values for hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction as well as for body weight loss and for thymic atrophy were determined for nine PCB congeners and 4'-bromo-2,3,4,5-tetrachlorobiphenyl. The most active compounds were the coplanar PCB congeners, 3,3',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorobiphenyl; for example, their ED50 values for body weight loss were 3.25 and 15.1 mumol/kg, respectively. The in vivo toxicity of the coplanar PCB, 3,3',4,4'-tetrachlorobiphenyl, was significantly lower (ED50 for body weight loss = 730 mumol/kg) than the values observed for the more highly chlorinated homologs, and this was consistent with the more rapid metabolism of the lower chlorinated congener. The dose-response biologic and toxic effects of several mono-ortho-chloro-substituted analogs of the coplanar PCBs, including 2,3,4,4'5-, 2,3,3',4,4'-, 2',3,4,4',5- and 2,3',4,4',5-penta-, 2,3,3',4,4',5- and 2,3,3',4,4',5'-hexachlorobiphenyl were also determined, and members of this group of compounds were all less toxic than 3,3',4,4',5-penta and 3,3',4,4',5,5'-hexachlorobiphenyl. There was a good rank order correlation between the in vivo QSAR data and the in vitro QSARs for PCBs that were developed from their relative receptor binding affinities and potencies as inducers of AHH and EROD in rat hepatoma H-4-II E cells in culture. These results are consistent with the proposed receptor-mediated mechanism of action for PCBs. In addition, for this series of halogenated biphenyls there was a linear correlation between their in vivo toxicity in rats and their in vitro monooxygenase enzyme induction results. Assuming that the in vivo toxic responses in the rat are representative toxic responses to PCBs, then these results support the predictive utility of the in vitro bioassay with rat hepatoma H-4-II E cells as a short-term test system for the potential toxicity of this class of halogenated aryl hydrocarbons.     

Toxicity of PCB 77 (3,3',4,4'-Tetrachlorobiphenyl) and PCB 118 (2,3',4,4',5-Pentachlorobiphenyl) in the Rat Following Subchronic Dietary Exposure

The toxicity of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and 2,3',4,4',5-pentachlorobiphenyl(PCB 118) was investigated in rats following subchronic dietaryexposure. Groups of 10 male and 10 female weanling Sprague-Dawleyrats were administered PCB 77 In the diet at 0, 10, 100, 1000,or 10,000 ppb for 13 weeks. PCB 118 was administered to malesin the diet at 0, 10, 100, 1000, and 10,000 ppb, while the femalegroups received 0, 2, 20, 200, or 2000 ppb of the congener for13 weeks. Growth rate and food consumption were not affectedby treatment. No clinical signs of toxicity were observed. Increasedspleen weight occurred in male rats fed 1000 or 10,000 ppb PCB77. Male rats receiving 10,000 ppb PCB 118 had increased liverweight and hepatic ethoxyresorufin O-deethylase (EROD) activity.Increased hepatic EROD activity but not liver weight was observedin female rats given the 2000-ppb PCB 118 diet. Increased ERODactivity was also noted in male rats given 10,000 ppb and infemale groups receiving 1000 or 10,000 ppb PCB 77. Male ratsexposed to 10,000 ppb PCB 77 had decreased vitamin A in theliver and lung and elevated levels in the kidney. Liver vitaminA of both 1000- and 10,000-ppb PCB 77 female groups was decreased.PCB 118 had no effects on tissue vitamin A at the levels studied.No hematological changes or serum biochemical changes were seenin any of PCB 118- and PCB 77-treated groups, nor were liveruroporphyrin levels altered. A reduction in dopamlne and homovanillinicacid in the substantia nigra region of the brain was observedin female rats fed 2000 ppb PCB 118, while 10,000 ppb PCB 77was associated with an elevation in 3,4-dihydroxyphenylaceticacid in the nucleus accumbens region of male rat brains. Mildto moderate changes were observed in the liver and thyroid ofrats given PCB 77 or PCB 118. PCB 118 accumulated in a dose-dependentmanner in fat and to a much lesser extent in liver. In contrast,very low levels of PCB 77 residue were found in the tissuesexamined. Based on the above data It was concluded that theNOAEL of PCB 77 is 100 ppb in diet or 8.7 sglkg and that ofPCB 118 is 200 ppb in diet or 17 µg/kg body wt/day.     

Gingival carcinogenicity in female Harlan Sprague-Dawley rats following two-year oral treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like compounds.

We evaluated gingival toxicities induced by chronic exposure of female Harlan Sprague-Dawley rats to dioxin and dioxin-like compounds (DLCs) and compared them to similarly induced oral lesions reported in the literature. This investigation represents part of an ongoing initiative of the National Toxicology Program to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. For two years, animals were administered by gavage 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118); control animals received corn oil-acetone vehicle (99:1) alone. A full complement of tissues, including the palate with teeth, was examined microscopically. In the groups treated with TCDD and the mixtures of TCDD, PCB126, and PeCDF; PCB126 and 153; and PCB126 and 118, the incidences of gingival squamous hyperplasia increased significantly. Moreover, in the groups treated with TCDD, PCB126, and the mixture of PCB126 and 153, squamous cell carcinoma (SCC) in the oral cavity increased significantly. This investigation constitutes the first report documenting that chronic administration of dioxin-like PCBs can induce gingival SCC in rats. These results indicate that dioxin and DLCs target the gingiva of the oral cavity, in particular the junctional epithelium of molars.     

Disposition of polychlorinated dibenzo-p-dioxins, dibenzofurans, and non-ortho polychlorinated biphenyls in pregnant long evans rats and the transfer to offspring

Pharmacokinetic properties of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDFs), and non-ortho biphenyls (PCBs) play a critical role in their relative toxicity. The present study examined the transfer of these chemicals to offspring and placenta. Pregnant Long Evans rats received 0.0 (control), 0.05, 0.2, 0.8, and 1.0 microg/kg of dioxin toxic equivalence (TEQ) by oral gavage on the 15th gestational day (GD 15), using a dosing mixture that contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), octachlorodibenzofuran (OCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB 77), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) in ratios approximating that in food. Rats were euthanized on GD 16, GD 21, and postnatal day 4 (PND 4). The chemical concentrations in fetus, pup, placenta, and maternal liver, serum, and adipose tissue were determined using gas chromatography/high-resolution mass spectrometry. A dose-dependent increase in hepatic sequestration was seen with TCDD, PeCDD, 4-PeCDF, OCDF, PCB 126, and PCB 169, and the transfer to offspring was reduced at higher doses. 4-PeCDF, PeCDD and PCB 126 showed higher liver affinity than TCDD. TCDF, 1-PeCDF, and PCB 77 were metabolized rapidly. On GD 16, TCDD and the three PCBs reached equilibration between the fetus and placenta, but this did not occur with PeCDD and 4-PeCDF until GD 21, according to the lipid-based concentrations. Offspring compartments received more of the dosed compounds lactationally than transplacentally (7-28% versus 0.5-3%). The behavior of each congener was dose-dependent; therefore, extrapolation of high-dose experimental data should be used with caution.     

Gestational and lactational exposure to TCDD or coplanar PCBs alters adult expression of saccharin preference behavior in female rats

Previous studies have shown that maternal doses of 1 microg/kg or less of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in late gestation can demasculinize and feminize reproductive behavior in male rats. However, it was not known whether coplanar polychlorinated biphenyls (PCBs) had similar effects, or whether non-reproductive sexually dimorphic behaviors such as saccharin preference behavior were also altered. We determined the effects of TCDD or coplanar PCBs on saccharin consumption and saccharin preference in male and female rats. Sprague-Dawley rats were dosed with 3,3',4, 4'-tetrachlorobiphenyl (PCB 77; 2 or 8 mg/kg/day), 3,3',4,4', 5-pentachlorobiphenyl (PCB 126; 0.25 or 1.0 microg/kg/day), TCDD (0. 025 or 0.10 microg/kg/day), or corn oil vehicle on days 10-16 of gestation. Maternal exposure to TCDD or coplanar PCBs did not change saccharin consumption or saccharin preference in male rats. However, TCDD and coplanar PCB-exposed females showed decreased saccharin consumption and saccharin preference. The results indicate that saccharin consumption is masculinized in female rats exposed to TCDD or coplanar PCBs during perinatal development. This effect could be related to the anti-estrogenic actions of these chemicals.     

Differential effects of PCBs on the induction of apoptosis machinery and PKCalpha translocation in rat renal tubular cell cultures

We have demonstrated previously [Pérez-Reyes, P.L., Sánchez-Alonso, J.A., López-Aparicio, P., Recio, M.N., Pérez-Albarsanz, M.A., 2001. Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures. Biosci. Rep. 6, 765-778] that the polychlorinated biphenyls (PCBs) cause loss of cell viability and accelerate apoptosis in cell kidney cultures. Further investigations are necessary to elucidate the mechanism of apoptosis induction. In this way, we have analyzed in the present work the effects of PCBs on protein kinase C (PKC, a protein family intimately involved in the regulation of cell survival) and the expression of two proapoptotic (caspase-3 and Bax) and one antiapoptotic (Bcl-2) proteins. Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, a di-ortho-substituted nonplanar congener) and PCB 77 (3,3',4,4'-tetrachlorobiphenyl, a non-ortho-substituted planar congener), significantly increased PKCalpha activity compared to control cells in the cytosolic and particulate cell fractions, and increased the PKCalpha protein content in the particulate fraction. The nonplanar PCB 153 showed stronger effects than the coplanar congener PCB 77. In addition, Aroclor 1248 decreased both, procaspase-3 levels and the Bcl-2/Bax protein ratio. These findings indicate that PCBs, particularly nonplanar congeners, can induce apoptosis in primary renal tubular cells through the PKCalpha, caspase-3 and Bcl-2/Bax pathway.     

Apoptosis-mediated neurotoxic potential of a planar (PCB 77) and a nonplanar (PCB 153) polychlorinated biphenyl congeners in neuronal cell cultures

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic depending on the chemical structure of the congeners. This study investigated the neurotoxic potential of 3,3',4,4'-tetrachlorobiphenyl (TCB) (PCB 77, a non-ortho-substituted planar congener) and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) (PCB 153, a di-ortho-substituted nonplanar congener) by assessing cell viability and apoptotic cell death in neuronal cell cultures. We have combined morphological and biochemical techniques to establish the relevance of apoptosis in neuronal cell death induced by the two selected PCB congeners. Treatment with both planar and nonplanar congeners caused the loss of cell viability and accelerated apoptosis in a time- and concentration-dependent manner. However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (PCB 77) than for the di-ortho-substituted nonplanar congener (PCB 153). This was correlated with the loss of cell viability since the planar congener was more cytotoxic. Based on our findings, the apoptosis induced by PCBs involves the increase of caspase-3 activity in neuronal cell cultures. It is reasonable to assume that PCB-induced apoptosis may be linked to the neurotoxic effect of these toxicants and that different molecular mechanisms may operate in the induction of apoptosis depending on the planarity or nonplanarity of the PCB congeners.