Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes

Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For fipronil sulfone, cytotoxic effects increased throughout the dose range. The trypan blue assay indicated that cytotoxic effects contributing to an increase of greater than 10% of control values was indicated at doses above 12.5 microM. However, fipronil sulfone induced cytotoxic effects at lower doses. The possibility that cytotoxic effects were due to apoptosis was indicated by significant time- and dose-dependent induction of caspase-3/7 activity in both HepG2 cells and human hepatocytes. Fipronil mediated activation of caspase-3/7 in concurrence with compromised ATP production and viability are attributed to apoptotic cell death.     

4S polycyclic aromatic hydrocarbon receptor (glycine N‐methyltransferase) and the aryl hydrocarbon receptor nuclear translocator (hypoxia inducible factor‐1β) interaction in Chinese hamster ovary and rat hepatoma cells: 4S PAH‐R/ARNT hetero‐oligomers?

Rat CYP1A1 promoter‐luciferase, transiently transfected wild‐type and 4S PAH receptor (glycine N‐methyl transferase, GNMT)‐transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild‐type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT‐transformed CHO cells and wild‐type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno‐precipitated with anti‐GNMT antibodies, separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti‐aryl hydrocarbon receptor nuclear translocator (ARNT, HIF‐1β) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero‐oligomer (dimer?) with ARNT/HIF‐1β which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor‐1 function with respect to the HIF‐1β subunit (ARNT). J. Cell. Biochem. 112: 2015–2018, 2011. © 2011 Wiley‐Liss, Inc.     

Eugenol inhibit 7,12-dimethylbenz[a]anthracene-induced genotoxicity in MCF-7 cells: Bifunctional effects on CYP1 and NAD(P)H:quinone oxidoreductase

Typically, chemopreventive agents either inhibit the cytochrome P450s (CYPs) that are essential for the metabolism of carcinogens or induce phase II detoxifying enzymes. This study examined the chemopreventive effect of eugenol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells. Eugenol inhibited the formation of the DMBA-DNA adduct in a dose dependent manner. CYP1A1 and CYP1B1 activity, which catalyze the biotransformation of DMBA, were strongly inhibited by eugenol. Eugenol also suppressed the CYP1A induction by DMBA through decreased aryl hydrocarbon receptor activation and subsequent DNA binding. Furthermore, eugenol increased the expression and activity of NAD(P)H:quinone oxidoreductase (QR), a major detoxifying enzyme for DMBA, through NF-E2 related factor2 binding to antioxidant response element in QR gene. Therefore, eugenol has a potent protective effect against DMBA-induced genotoxicity, presumably through the suppression of the DMBA activation and the induction of its detoxification. These results suggest that eugenol has potential as a chemopreventive.     

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic” quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.