Brian Chanas

Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid.

Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (Taxol). It is also the predominant P450 responsible for the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs) in human liver and kidney. In this study, we describe two new CYP2C8 alleles containing coding changes: CYP2C8*2 has an Ile269Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8. CYP2C8*2 was found only in African-Americans, while CYP2C8*3 occurred primarily in Caucasians. Neither occurred in Asians. The frequency of the CYP2C8*2 allele was 0.18 in African-Americans, and that of CYP2C8*3 was 0.13 in Caucasians. CYP2C8*1 (wild-type), CYP2C8*2 and CYP2C8*3 cDNAs were expressed in Escherichia coli, and the ability of these enzymes to metabolize both paclitaxel and arachidonic acid was assessed. Recombinant CYP2C8*3 was defective in the metabolism of both substrates. The turnover number of CYP2C8*3 for paclitaxel was 15% of CYP2C8*1. CYP2C8*2 had a two-fold higher Km and two-fold lower intrinsic clearance for paclitaxel than CYP2C8*1. CYP2C8*3 was also markedly defective in the metabolism of arachidonic acid to 11,12- and 14,15-EET (turnover numbers 35-40% that of CYP2C8*1). Thus, CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel and the physiologically important compound arachidonic acid. This polymorphism has important clinical and physiological implications in individuals homozygous for this allele.

Hemolytic anemia, thrombosis, and infarction in male and female F344 rats following gavage exposure to 2-butoxyethanol

2-butoxyethanol (BE; ethylene glycol monobutyl ether) is used extensively in the manufacture of a wide range of domestic and industrial products which may result in human exposure and toxicity. BE causes severe hemolytic anemia in male and female rats and mice. In a recent report, female F344 rats exposed to 500 ppm BE by inhalation and sacrificed moribund on day 4 of treatment exhibited disseminated thrombosis associated with infarction in several organs. In contrast, no such lesions were observed in male rats similarly exposed to BE. Additional studies were therefore undertaken to compare the effects of BE in rats of both sexes. Rats received 250 mg BE/kg/day by gavage for 1, 2 or 3 days and were sacrificed 24 or 48 hr after the last dose. Control rats received 5 ml/kg water. Progressive time-dependent hemolytic anemia--macrocytic, hypochromic, and regenerative--was observed in both sexes of rats exposed to BE. Additionally, BE caused significant morphological changes in erythrocytes, first observed 24 hr after a single dose, including stomatocytosis, macrocytosis with moderate rouleaux formation, and spherocytosis. These morphological changes became progressively more severe as BE dosing continued and included the occasional occurrence of schistocytes and ghost cells, rouleaux formation in rats of both sexes, and an increased number of red blood cells with micronuclei in female rats. Overall, the progression of hemolytic anemia and morphological changes as a function of the number of days of exposure varied with gender and suggested a faster onset of hemolysis in female rats. The range of BE-related histopathological changes noted in both sexes was comparable; however, while these lesions were observed in female rats following a single dose, similar effects were first observed in males after 3 consecutive days of exposure to BE. Pathological changes involved disseminated thrombosis in the lungs, nasal submucosa, eyes, liver, heart, bones and teeth, with evidence of infarction in the heart, eyes, teeth and bones. Hemoglobinuric nephrosis and splenic extramedullary hematopoiesis were also noted. An apparent correlation between the severity of hemolytic anemia and subsequent disseminated thrombosis in BE-treated rats is proposed. Thrombosis may be related to intravascular hemolysis, which could be triggered by procoagulant release and/or alterations in erythrocyte morphology, as well as increased rigidity.

Effect of methacrylonitrile on cytochrome P-450 2E1 (CYP2E1) expression in male F344 rats.

Tissue-specific induction of cytochrome P-450s (CYP) followed by increased in situ bioactivation may contribute to chemical-induced site-specific toxicity. In rats, methacrylonitrile (MAN) is metabolized by cytochrome P-450 2E1 (CYP2E1) to acetone, which is eliminated along with parent MAN in breath. Gavage administration of MAN to rats causes olfactory epithelial damage and liver enlargement. It was hypothesized that treatment of rats with MAN may result in differential expression of CYP2E1 in tissues leading to tissue-specific toxicity via increased in situ formation of cytotoxic MAN metabolites. In this study, male F344 rats received 60 mg MAN/kg and were sacrificed 6, 12, or 24 h after a single dose, or 24 h after 7 consecutive daily doses. Liver, lung, and nasal tissues were collected. Reverse-transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining were used to assess CYP2E1 expression and localization, and chlorzoxazone hydroxylation was used as a measure of CYP2E1 catalytic activity. Present results showed that CYP2E1 mRNA was increased in lung and nasal tissues with minimal effects in liver of MAN-treated rats. Induction of CYP2E1 protein expression was detected in lung. CYP2E1 activity was higher in liver and lung microsomes from MAN-treated rats when compared to control animals. To compare the effects of MAN and acetone, male F344 rats received a single acetone dose (5 ml/kg) by gavage. After 12 h, acetone treatment resulted in a significant increase in the levels of CYP2E1 mRNA and protein in lung and nasal tissues, with no obvious change noted in the liver. Overall, these data suggest that administration of MAN to rats causes increased expression of CYP2E1 in lung, liver, and nasal tissues. These results also show that acetone induces the expression of CYP2E1 at both the mRNA and protein levels in rat nasal and lung tissues. In conclusion, MAN increased the expression of CYP2E1, and this effect varied as a function of time, length of exposure, and tissue examined. While the damage in the olfactory mucosa due to MAN treatment may not be explained by the observed induction of CYP2E1, it is possible that other CYPs may play a role in the in situ bioactivation of MAN.