Rat liver postmitochondrial supernatant (S9) converted the azo dyes chrysoidine Y and R to products that were mutagenic towards Salmonella typhimurium strain TA100. No such release of mutagens was ...demonstrated using intact rat hepatocytes as an activation system despite the fact that chrysoidine dyes cause unscheduled DNA synthesis in these cells. It appears that genotoxic products produced within hepatocytes either react within the cell or are detoxified prior to release. Following intraperitoneal administration of chrysoidine Y to rats (100 mg/kg i.p.) there was also no evidence of mutagenic or por-mutagenic products excreted in bile or urine. The S9-derived mutagens appear to be largely independent of bacterial acetylation since they were active in the acetylation-deficient strain TA98/1,8-DNP6 in addition to strain TA98. The ultimate mutagenic form(s) are therefore unlikely to be acetoxyarylamines.
Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 (P450) and other enzymes involved in the biotransformation of drugs and other xenobiotics. The list ...of studies made possible with the technology includes discernment of catalytic specificity, elucidation of structure-activity relationships, and various biophysical measurements. There are advantages and disadvantages to each of the vector systems and choices must be made on the basis of needs. Yeast expression systems were used to establish that different P450 2C enzymes are involved in the hydroxylations of tolbutamide and (S)-mephenytion. P450 3A4 was also expressed in yeast and its very broad catalytic specificity was confirmed. Recently, it has been possible to express P450 3A4 as well as other human and animal P450s in bacteria after slight modification of their 5'-coding sequences.
In-vitro studies were conducted to assess the impact of CYP2C9 genotype on the metabolism (methyl hydroxylation) and pharmacokinetics of celecoxib, a novel cyclooxygenase-2 inhibitor and CYP2C9 ...substrate. When compared to cDNA-expressed wild-type CYP2C9 (CYP2C9*1), the Vmax/Km ratio for celecoxib methyl hydroxylation was reduced by 34% and 90% in the presence of recombinant CYP2C9*2 and CYP2C9*3, respectively. These data indicated that the amino acid substitution at position 359 (Ile to Leu) elicited a more pronounced effect on the metabolism of celecoxib than did a substitution at position 144 (Arg to Cys). The Vmax/Km ratio was also decreased in microsomes of livers genotyped CYP2C9*1/*2 (47% decrease, mean of two livers), or CYP2C9*1/*3 (59% decrease, one liver). In all cases, these changes were largely reflective of a decrease in Vmax, with a minimal change in Km. Based on simulations of the in-vitro data obtained with the recombinant CYP2C9 proteins, it was anticipated that the pharmacokinetics of celecoxib (as a much as a five-fold increase in plasma AUC) would be altered (versus CYP2C9*1/*1 subjects) in subjects genotyped heterozygous or homozygous for the CYP2C9*2 (Cys144) or CYP2C9*3 (Leu359) allele. In a subsequent clinical study, the AUC of celecoxib was increased (versus CYP2C9*1/*1 subjects) approximately 2.2-fold (range, 1.6-3-fold) in two CYP2C9*1/*3 subjects and one CYP2C9*3/*3 subject receiving a single oral dose (200 mg) of the drug. In contrast, there was no significant change in celecoxib AUC in two subjects genotyped CYP2C9*1/*2.
