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.     

Molecular Typing and Genetic Characterization of Pathogenic Variants of Xanthomonas axonopodis pv. citri in Taiwan

Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri (Xac) from Taiwan. These three novel variants of atypical symptom–producing X. axonopodis pv. citri were designated as Xac‐A^f, Xac‐A^p and Xac‐A^r. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine‐responsive regulatory protein (lrp) gene assay and DNA fingerprintings generated by repetitive‐sequence PCR (rep‐PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom–producing strains Xac‐A^f, Xac‐A^p and Xac‐A^r, and additional reference strains from pathotypes Xac‐A, Xac‐A*, Xac‐A^w, X. axonopodis pv. auruantifolii and X. axonopodis pv. citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep‐PCR clearly grouped the atypical symptom–producing variants in types Xac‐ A^f, Xac‐A^r and Xac‐A^p into the same cluster with typical symptom‐producing strains in pathotype Xac‐A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac‐A* and Xac‐A^w in these genotypic analyses. Strains of Xac‐A* and Xac‐A^w were closely related to Xac‐A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv. citrumelo. The results further confirmed the atypical symptom–producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac‐A.     

Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.)

We previously identified a salt and drought stress-responsive TFIIIA-type zinc finger protein gene ZFP252 from rice. Here we report the functional analysis of ZFP252 using gain- and loss-of-function strategies. We found that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes and enhanced rice tolerance to salt and drought stresses, as compared with ZFP252 antisense and non-transgenic plants. Our findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced tolerance to salt and drought stresses.