Mammalian epoxide hydrolases in xenobiotic metabolism and signalling

Epoxide hydrolases catalyse the hydrolysis of electrophilic—and therefore potentially genotoxic—epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)—an enzyme prone to detoxification—due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structur

Identification of a CYP3A form (CYP3A126) in fathead minnow ( Pimephales promelas ) and characterisation of putative CYP3A enzyme activity

Cytochrome P450-dependent monooxygenases (CYPs) are involved in the metabolic defence against xenobiotics. Human CYP3A enzymes metabolise about 50% of all pharmaceuticals in use today. Induction of CYPs and associated xenobiotic metabolism occurs also in fish and may serve as a useful tool for biomonitoring of environmental contamination. In this study we report on the cloning of a CYP3A family gene from fathead minnows (Pimephales promelas), which has been designated as CYP3A126 by the P450 nomenclature committee (GenBank no. EU332792). The cDNA was isolated, identified and characterised by extended inverse polymerase chain reaction (PCR), an alternative to the commonly used method of rapid amplification of cDNA ends. In a fathead minnow cell line we identified a full-length cDNA sequence (1,863 base pairs (bp)) consisting of a 1,536bp open reading frame encoding a 512 amino acid protein. Genomic analysis of the identified CYP3A isoenzyme revealed a DNA sequence consisting of 13 exons and 12 introns. CYP3A126 is also expressed in fathead minnow liver as demonstrated by reverse transcription PCR. Exposure of fathead minnow (FHM) cells with the CYP3A inducer rifampicin leads to dose-dependent increase in putative CYP3A enzyme activity. In contrast, inhibitory effects of diazepam treatment were observed on putative CYP3A enzyme activity and additionally on CYP3A126 mRNA expression. This indicates that CYP3A is active in FHM cells and that CYP3A126 is at least in part responsible for this CYP3A activity. Further investigations will show whether CYP3A126 is involved in the metabolism of environmental chemicals. Figure Induction of CYP3A activity by rifampicin and inhibition by diazepam in FHM cell

Ciprofloxacin, enrofloxacin, lincomycin, penicillin G, and penicillin V – Determination of selected antibiotics in urine by LC‐MS/MS. Biomonitoring Method

The working group “Analyses in Biological Materials” of the Permanent Senate Com- mission for the Investigation of Health Hazards of Chemical Compounds in the Work Area developed and verified the presented biomonitoring method. This method allows for the sensitive and precise determination of selected antibiotics (ciprofloxacin, enrofloxacin, lincomycin, penicillin G, and penicillin V) in human urine. Sample preparation includes extracting the analytes by solid-phase extraction on Oasis HLB cartridges, followed by concentrating the eluates under a stream of nitrogen. The analytes are separated from matrix compounds by liquid chromatography and sub- sequently detected with tandem mass spectrometry using electrospray ionisation. Quantitative evaluation is carried out via external calibration in urine. The good precision data and accuracy data show that the method provides reliable and accurate measurement values. Any matrix effects are effectively compensated for by the use of isotope-labelled internal standards. This finding holds similarly true for cipro- floxacin, for which isotope-labelled enrofloxacin was used as internal standard (ISTD). With quantitation limits of 0.1 μg/l for ciprofloxacin, enrofloxacin, and lincomycin as well as 0.3 μg/l for penicillin G and penicillin V, this method is very sensitive and enables the reliable quantitation of occupational exposure to the selected antibiotics