Suppression of apoptotic signaling in rat hepatocytes by non-dioxin-like polychlorinated biphenyls depends on the receptors CAR and PXR.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) represent a sub-group of persistent organic pollutants found in food, environmental samples and human and animal tissues. Promotion of pre-neoplastic lesions in rodent liver has been suggested as an indicator for a possible increased risk of liver cancer in humans exposed to NDL-PCBs. In rodent hepatocytes, suppression of DNA damage-triggered apoptosis is a typical mode of action of liver tumor promoters. Here, we report that NDL-PCBs suppress apoptosis in rat hepatocytes treated in culture with an apoptogenic dose of UV light. Suppression became less pronounced when the constitutive androstane receptor (CAR) and/or the pregnane-X-receptor (PXR) where knocked-out using siRNAs, while knocking-out both receptors led to a full reconstitution of apoptosis. In contrast, suppression of apoptosis by the CAR or PXR activators phenobarbital or dexamethasone were CAR- or PXR-specific. Induction and suppression of apoptosis were paralleled by changes in caspase 3/7, 8 and 9 activities. Our findings indicate that NDL-PCBs can suppress UV-induced apoptosis in rat hepatocytes by activating CAR and PXR. It needs further investigation if these mechanisms of action are also of relevance for human liver.

Role of the nuclear xenobiotic receptors CAR and PXR in induction of cytochromes P450 by non-dioxinlike polychlorinated biphenyls in cultured rat hepatocytes.

Polychlorinated biphenyls (PCBs) are among the most ubiquitously detectable 'persistent organic pollutants'. In contrast to 'dioxinlike' (DL) PCBs, less is known about the molecular mode of action of the larger group of the 'non-dioxinlike' (NDL) PCBs. Owing to the life-long exposure of the human population, a carcinogenic, i.e., tumor-promoting potency of NDL-PCBs has to be considered in human risk assessment. A major problem in risk assessment of NDL-PCBs is dioxin-like impurities that can occur in commercially available NDL-PCB standards. In the present study, we analyzed the induction of CYP2B1 and CYP3A1 in primary rat hepatocytes using a number of highly purified NDL-PCBs with various degrees of chlorination and substitution patterns. Induction of these enzymes is mediated by the nuclear xenobiotic receptors CAR (Constitutive androstane receptor) and PXR (Pregnane X receptor). For CYP2B1 induction, concentration-response analysis revealed a very narrow window of EC50 estimates, being in the range of 1-4µM for PCBs 28 and 52, and between 0.4 and 1µM for PCBs 101, 138, 153 and 180. CYP3A1 induction was less sensitive to NDL-PCBs, the most pronounced induction being achieved at 100µM with the higher chlorinated congeners. Using okadaic acid and small interfering RNAs targeting CAR and PXR, we could demonstrate that CAR plays a major role and PXR a minor role in NDL-PCB-driven induction of CYPs, both effects showing no stringent structure-activity relationship. As the only obvious relevant determinant, the degree of chlorination was found to be positively correlated with the inducing potency of the congeners.

Inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin depends on protein biosynthesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic persistent organic pollutant. Most of the toxic effects of TCDD are believed to be mediated by high-affinity binding to the aryl hydrocarbon receptor (AhR) and subsequent effects on gene transcription and protein expression. TCDD causes cancer in multiple tissues in different animal species and is classified as a class 1 human carcinogen. In initiation-promotion studies, TCDD was shown to be a potent liver-tumor promotor. Among other theories it has been hypothesized that TCDD promotes tumor growth by preventing initiated cells from correctly executing apoptosis. In this study, we examined the effects of TCDD on apoptosis induced by UV-C light, ochratoxin A (OTA), and cycloheximide (CHX) in primary rat hepatocytes. Both UV-C light and OTA caused caspase activation and nuclear apoptotic effects. CHX did not activate caspases but nevertheless caused DNA fragmentation and chromatin condensation. TCDD inhibited UV-C light-induced apoptosis and this effect seemed to be dependent on AhR-activation as was shown by employing an AhR antagonist. In contrast to UV-C light-induced apoptosis, TCDD failed to protect primary rat hepatocytes from OTA- or CHX-induced apoptosis. Since both of these compounds inhibit protein biosynthesis as was demonstrated by measuring the incorporation of radiolabeled leucin and protein expression of cytochrome P450 1A1, we propose that the inhibition of apoptosis by TCDD depends on protein biosynthesis. Either TCDD induces some anti-apoptotic protein in an AhR-dependent manner or inhibits pro-apoptotic proteins induced by UV irradiation.