A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-mephenytoin. J Pharmacol Exp Ther

ABSTRACT Cytochrome P-450 (CYP) 2C19 is responsible for the metabolism of a number of therapeutic agents such as S-mephenytoin, omeprazole, proguanil, certain barbiturates, diazepam, propranolol, citalopram and imipramine. Genetic polymorphisms in this enzyme are responsible for the poor metabolizers (PM) of mephenytoin, which represent ϳ13-23% of Asians and 3-5% of Caucasians. Several polymorphisms contribute to this phenotype. We have isolated two new allelic variants that contribute to the PM phenotype in Caucasians. CYP2C19*7 contained a single T 3 A nucleotide transversion in the invariant GT at the 5Ј donor splice site of intron 5. The second PM allele, CYP2C19*8, consisted of a T358C nucleotide transition in exon 3 that results in a Trp120Arg substitution. In a bacterial expression system, CYP2C198 protein exhibited a dramatic (ϳ90% and 70%) reduction in the metabolism of S-mephenytoin and tolbutamide, respectively, when compared with the wild-type CYP2C191B protein. Restriction fragment length polymerase chain reaction tests were developed to identify the new allelic variants

Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration

AbstractCyclooxygenases 1 and 2 (COX-1 and COX-2) are key enzymes in prostaglandin biosynthesis and the target enzymes for the widely used nonsteroidal anti-inflammatory drugs. To study the physiological roles of the individual isoforms, we have disrupted the mouse Ptgs1 gene encoding COX-1. Homozygous Ptgs1 mutant mice survive well, have no gastric pathology, and show less indomethacin-induced gastric ulceration than wild-type mice, even though their gastric prostaglandin E2 levels are about 1% of wild type. The homozygous mutant mice have reduced platelet aggregation and a decreased inflammatory response to arachidonic acid, but not to tetradecanoyl phorbol acetate. Ptgs1 homozygous mutant females mated to homozygous mutant males produce few live offspring. COX-1-deficient mice provide a useful model to distinguish the physiological roles of COX-1 and COX-2

Effects of Cigarette Smoke Condensates on Cultured Human Lymphocytes and Separation of Benzo-α-Pyrene Metabolites by High Pressure Liquid Chromatography

Cigarette smoke condensates from all cigarettes tested were found to be potent inducers of AHH enzyme in cultured human lymphocytes and, with the exception of Kent Lights and Carlton CSC's, all were found to be toxic under the experiment conditions. Most of the AHH inducing activity was found in basic and neutral fractions of the lAl standard cigarettes. A radiometric assay of BP metabolites in cultured human lymphocytes was developed in which we were able to separate the primary metabolites and the secondary metabolites from the parent compound (BP) by neutral alumnia HPLC. The primary metabolites were further separated by a selective enzyme hydrolysis and/or reverse phase HPLC

Effect of Dose Volume on the Toxicokinetics of Acrylamide and Its Metabolites and 2-Deoxy-d-glucose

Acrylamide (AA) is a known mutagen and animal carcinogen. Comparison of recent studies revealed significant quantitative differences in AA-induced germ cell mutagenicity. It was hypothesized that despite the administration of AA at similar doses, the discrepancy in the observed effects was most likely due to varying AA concentrations in the administered dosing solution. To test this hypothesis, AA was administered i.p. to mice at 50 mg/kg in a dose volume of 5 or 50 ml/kg, blood was collected at various time points, and AA and its metabolites were quantitated. Changes in dose volume resulted in significant differences in the toxicokinetics of AA and its metabolites and suggested that increased Cmax of AA led to increased metabolism. This theory, in conjunction with the fact that higher levels of AA-derived radioactivity were detected in the testes, may explain the greater toxicity of a 50 mg/kg dose when administered in 5 versus 50 ml/kg. The impact of dose volume on the toxicokinetics of 2-deoxy-d-glucose (DG), a nonreactive, nonmetabolizable substance, was also investigated. The areas under the curve for DG were not different for the two dose volumes; however, Cmax for the more concentrated dose was significantly higher. In conclusion, current studies show that the toxicokinetics of an administered xenobiotic and its metabolites is influenced by the concentration of the parent chemical in the dosing solution. Therefore, it is important to consider the concentration of an administered xenobiotic in the dosing solution because it may affect its toxicokinetics and metabolism and subsequently affect the biological effects of the administered chemical

Kinetics of Trihalogenated Acetic Acid Metabolism and Isoform Specificity in Liver Microsomes

This study determined the metabolism of 3 drinking water disinfection by-products (halogenated acetic acids [HAAs]), bromodichloroacetic acid (BDCAA), chlorodibromoacetic acid (CDBAA), and tribromoacetic acid (TBAA), using rat, mouse, human liver microsomes, and recombinant P450. Metabolism proceeded by reductive debromination forming a di-HAA, the highest under nitrogen \u3e\u3e2% oxygen \u3e atmospheric headspaces. V(max) for the loss of tri-HAA was 4 to 5 times higher under nitrogen than atmospheric headspace. Intrinsic metabolic clearance was TBAA\u3eCDBAA\u3e\u3eBDCAA. At the high substrate concentrations, tri-HAA consumption rate was 2 to 3 times higher than the formation of di-HAA. Liberation of Br(-) from TBAA corresponded to the expected amount produced after DBAA formation, indicating retention of Br(-) by additional metabolite/metabolites. Subsequent experiments with CDBAA detected negligible formation of chlorodibromomethane (CDBM) and failed to account for the missing tri-HAA. Carbon monoxide and especially diphenyleneiodonium ([DPI] P450 reductase inhibitor) blocked CDBAA metabolism. Other chemical inhibitors were only partially able to block CDBAA metabolism. Most effective were inhibitors of CYP 2E1 and CYP 3A4. Immunoinhibition studies using human liver microsomes and anti-human CYP 2E1 antibodies were successful in reducing CDBAA metabolism. However, CDBAA metabolism in wild-type (WT) and CYP 2E1 knockout (KO) mouse liver microsomes was similar, suggesting significant interspecies differences in CYP isoform in tri-HAA metabolism. Additional assessment of CYP isoform involvement was complicated by the finding that recombinantly expressed rat and human P450 reductase was able to metabolize CDBAA, which may be a contributing factor in interspecies differences in tri-HAA metabolism

Effects of dose, age, inhibition of metabolism and elimination on the toxicokinetics of 2-butoxyethanol and its metabolites

Acute exposure to 2-butoxyethanol (BE) causes dose- and age-dependent hemolytic anemia in rats. Recently, we have shown that butoxyacetic acid (BAA) is the proximate hemolytic agent and that inhibition of alcohol or aldehyde dehydrogenases pro-tected rats against BE-induced hemolytic anemia. In the present investigations, the kinetics of 14C-BE metabolism and clearance were studied in control adult (3-4 months old) and old (12-13 months old) male F344 rats and in adult male F344 rats treated with pyrazole, cyanamide or probenecid. Our results showed that the area under the curve (AUC), maximum plasma concen-tration (C) and systemic clearance (Cl5) of BE were dose-dependent. In contrast, there was no effect of dose on half-life (I#{189})or volume of distribution (Vd) of BE. These results also showed that there was no age effect on T#{189},Vd or Cl5 of BE. However, C, and AUC of BE increased as a function of age