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, Fig 3. Formation of B[a]P metabolites was altered by steatosis and upon ethanol co-exposure

, alone or in combination with 5 mM ethanol for 5 days. B[a]P metabolites in culture media were analyzed by gas chromatography tandem mass-spectrometry. Results are quoted as proportion of B[a]P relatively to total OH-and total diOH-B[a]P metabolites detected under the different conditions (cf. Supplementary supplementary Table S2 for values), Non-steatotic or steatotic hepatocytes were treated with 10 nM B

, Non-steatotic or steatotic hepatocytes were treated or not (C; treated with DMSO) with 5 mM ethanol (E), 10 nM B[a]P (B) or a combination of both toxicants (BE) for 5 days (A-C, F) or 3 h (D,E), in presence or not of inhibitor. The involvement of ethanol metabolism was analyzed by testing the effect of the, steatotic WIF-B9 cells

, UV detection) of the formation of OCZX. ADH activity was evaluated by measuring the NADH production by spectrophotometry in the absence (D) or presence (E) of the AhR inhibitor CH-223191 (CH; 3 µM). This activity was given relative to control cells. (F) Involvement of AhR in apoptosis was evaluated after co-treatment with CH. Results are means ± SD for at least three independent cultures. *: Significantly different from condition without inhibitor (4-MP or CH). a: Significantly different from corresponding control (with or without steatosis), Hoechst 33342 staining, and (B) DNA damage evaluated by counting cells positive for ?H2AX staining. (C) CYP2E1 activity was assessed by HPLC analyses

, Non-steatotic or steatotic hepatocytes were treated or not (C; treated with DMSO) with 5 mM ethanol (E), 10 nM B[a]P (B) or a combination of both toxicants (BE) for 5 days, in presence or not of antioxidant. The involvement of oxidative stress in toxicity was evaluated by testing the effects of the antioxidant molecule thiourea (6.25 mM) on (A) apoptosis after Hoechst 33342 staining, and (C) DNA damage evaluated by counting cells positive for ?H2AX staining. (B) Lipid peroxidation was assessed by measuring the production of malondialdehyde (MDA) by HPLC. (D) The superoxide anion production was assessed by the measurement in fluorescence of 2-OH ethidium using DHE probe. Results are means ± SD for at least three independent cultures. *: Significantly different from condition without thiourea. a: Significantly different from corresponding HIGHLIGHTS ? 1-Death of steatotic hepatocytes co-exposed to, Fig 5. Involvement of oxidative stress in the cell death induced by

, ? 3-AhR and NF?B would be involved in iNOS induction, thus leading to NO production

, ? 5-Death of co-exposed steatotic cells involves a peroxynitrite-dependent DNA damage