Relationship of cellular topoisomerase IIα inhibition to cytotoxicity and published genotoxicity of fluoroquinolone antibiotics in V79 cells.

Fluoroquinolone (FQ) antibiotics are bacteriocidal through inhibition of the bacterial gyrase and at sufficient concentrations in vitro, they can inhibit the homologous eukaryotic topoisomerase (TOPO) II enzyme. FQ exert a variety of genotoxic effects in mammalian systems through mechanisms not yet established, but which are postulated to involve inhibition of TOPO II enzymes. To assess the relationship of inhibition of cell nuclear TOPO II to cytotoxicity and reported genotoxicity, two FQ, clinafloxacin (CLFX) and lomefloxacin (LOFX), having available genotoxicity data showing substantial differences with CLFX being more potent than LOFX, were selected for study. The relative inhibitory activities of these FQ on nuclear TOPO IIα in cultured Chinese hamster lung fibroblasts (V79 cells) over dose ranges and at equimolar concentrations were assessed by measuring nuclear stabilized cleavage complexes of TOPO IIα-DNA. Cytotoxicity was measured by relative cell counts. Both FQ inhibited V79 cell nuclear TOPO IIα. The lowest-observed-adverse-effect levels for TOPO IIα inhibition were 55 µM for CLFX, and 516 µM for LOFX. The no-observed-adverse-effect-levels were 41 µM for CLFX, and 258 µM for LOFX. At equimolar concentrations (175 µM), CLFX was more potent than LOFX. Likewise, CLFX was more cytotoxic than LOFX. Thus, the two FQ, inhibited TOPO IIα in intact V79 cells, differed in their potencies and exhibited no-observed-adverse-effect levels. These findings are in concordance with published genotoxicity data and observed cytotoxicity.

CYP2D6 increases toxicity of the designer drug 4-methylthioamphetamine (4-MTA).

4-Methylthioamphetamine (4-MTA) belongs to a group of new amphetamine derivatives that is usually sold as "ecstasy" or "flatliners" on the illicit drug market. Large interindividual differences in 4-MTA mediated toxicity have been reported in humans. Therefore, we tested whether CYP2D6 or its variant alleles as well as CYP3A4 influence the susceptibility to 4-MTA. For this purpose, we used the colony formation assay with Chinese hamster lung fibroblast V79 cells expressing human wild-type CYP2D6 (CYP2D6*1), the low activity alleles CYP2D6*2, CYP2D6*9, as well as human CYP3A4. The obtained results showed that the expression of wild type CYP2D6*1 clearly enhanced the susceptibility to the cytotoxic effects of 4-MTA compared with the parental cells devoid of CYP-dependent enzymatic activity. Toxicity in V79 CYP2D6*1 was also higher compared to the V79 cell lines expressing the low activity alleles CYP2D6*2 and CYP2D6*9. In contrast to CYP2D6, the CYP3A4 isoenzyme did not enhance 4-MTA toxicity. In conclusion, our results suggest that CYP2D6 rapid metabolizers may be more susceptible to 4-MTA toxicity than CYP2D6 poor metabolizers.

Influence of CYP2D6 polymorphism on 3,4-methylenedioxymethamphetamine ('Ecstasy') cytotoxicity.

OBJECTIVES: Remarkable interindividual differences in 3,4-methylenedioxymethamphetamine ('Ecstasy')-mediated toxicity have been reported in humans. Therefore, we tested whether CYP2D6 or its variant alleles as well as CYP3A4 influence the susceptibility to 3,4-methylenedioxymethamphetamine. METHODS: 3,4-Methylenedioxymethamphetamine cytotoxicity was determined in V79 cells expressing human wild-type CYP2D6 (CYP2D6*1), the low-activity alleles CYP2D6*2, *9, *10, and *17, as well as human CYP3A4. Metabolites of 3,4-methylenedioxymethamphetamine formed by the different cell lines were quantified by high-performance liquid chromatography/electrochemical detector. RESULTS: Toxicity of 3,4-methylenedioxymethamphetamine was clearly increased in cells expressing CYP2D6*1 compared with the parental cells devoid of CYP-dependent enzymatic activity. Toxicity in V79 CYP2D6*1 cells was also higher than in V79 cell lines expressing the low-activity alleles CYP2D6*2, *9, *10, or *17. In contrast to CYP2D6, the CYP3A4 isoenzyme did not enhance 3,4-methylenedioxymethamphetamine toxicity. Formation of the oxidative 3,4-methylenedioxymethamphetamine metabolite N-methyl-alpha-methyldopamine was greatly enhanced in V79 cell line transfected with CYP2D6*1 compared to all other cell lines. The increase in the cytotoxic effects of 3,4-methylenedioxymethamphetamine observed in this cell line was therefore suspected to be a consequence of the production of this metabolite. This was further investigated by testing the cytotoxicity of N-methyl-alpha-methyldopamine to the control cell line. The results confirmed our hypothesis as the metabolite proved to be more than 100-fold more toxic than the parent compound 3,4-methylenedioxymethamphetamine. CONCLUSIONS: CYP2D6*1 mediates 3,4-methylenedioxymethamphetamine toxicity via formation of N-methyl-alpha-methyldopamine. Therefore, it will be important to investigate whether CYP2D6 ultrarapid metabolizers are overrepresented in the cases of 3,4-methylenedioxymethamphetamine intoxications.

Large interindividual variability in the in vitro formation of tamoxifen metabolites related to the development of genotoxicity.

AIMS: To characterize the interindividual variability and the individual CYP involved in the formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen from tamoxifen. METHODS: Microsomes from 50 human livers were used to characterize the interindividual variability in the alpha-hydroxylation, N-desmethylation and N-didesmethylation of tamoxifen. Selective inhibitors and recombinant enzymes were used to identify the forms of CYP catalysing these reactions. RESULTS: The rates of formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen were highly variable, and correlated with each other (P < 0.0001). The respective ranges were 0.7-11.4, 25.7-411, and below the limit of quantification--4.4 pmol mg(-1) protein min(-1). Formation of all metabolites was observed with expressed recombinant CYP3A4, inhibited by troleandomycin (65, 77 and 35%, respectively, P < 0.05) and associated with CYP3A4 expression (rs = 0.612, rs = 0.585 and rs = 0.430, P < 0.01, respectively). CONCLUSIONS: Formation of alpha-hydroxy-, N-desmethyl- and N-didesmethyl-tamoxifen in vitro is highly variable and mediated predominantly by CYP3A4.

V79 Chinese hamster cells genetically engineered for polymorphic cytochrome P450 2D6 and their predictive value for humans.

With more than 30 genetic variants human cytochrome P450 2D6 (CYP2D6) presents the most extensive variation among all cytochromes P450. At the same time, roughly 30% of all drugs are metabolised by CYP2D6. Therefore, V79 Chinese hamster cells were genetically engineered for the genetic variants *1, *2, *9, *10, and *17 encoding active enzymes. These cells are to be used to understand and to predict variant-dependent metabolism of drugs and drug candidates. The V79-derived cell lines were extensively characterised for stable expression of mRNA, for enzyme activity using bufuralol hydroxylation, for CYP content by CO difference spectra, and for protein distribution and cellular location by in situ immunofluorescence. Based on these results, CYP-mediated metabolism of tamoxifen was investigated.

The influence of CYP2B6, CYP2C9 and CYP2D6 genotypes on the formation of the potent antioestrogen Z-4-hydroxy-tamoxifen in human liver.

AIMS: To investigate in a large panel of 50 human liver samples the contribution of CYP2C9, CYP2D6, and CYP3A4 to the overall formation of the potent antioestrogen Z-4-hydroxy-tamoxifen, and how various genotypes affect its formation from tamoxifen. METHODS: The formation of Z-4-hydroxy-tamoxifen from 10 microm tamoxifen was studied in human liver microsomes (n=50), characterized for CYP2B6, CYP2C9, CYP2D6 and CYP3A4 expression, and CYP2B6, CYP2C9 and CYP2D6 genotype. The effect of chemical and monoclonal antibody inhibitors, and the formation in supersomes expressing recombinant CYP isoforms was also investigated. Z-4-hydroxy-tamoxifen was quantified using LC-MS analysis. RESULTS: Z-4-hydroxy-tamoxifen was formed by supersomes expressing CYP2B6, CYP2C9, CYP2C19 and CYP2D6, but not CYP3A4. In agreement with these data, the mean formation of Z-4-hydroxy-tamoxifen was inhibited 49% by sulphaphenazole (P=0.001), 38% by quinidine (P<0.05) and 13% by monoclonal antibody against CYP2B6 (MAB-2B6, P<0.05). Furthermore, Z-4-hydroxy-tamoxifen formation significantly correlated with both CYP2C9 expression (r(s)=0.256, P<0.05) and CYP2D6 expression (r(s)=0.309, P<0.05). Genotypes of CYP2D6, CYP2B6 and CYP2C9 had an effect on metabolite formation in such a way that samples with two nonfunctional CYP2D6, or two variant CYP2C9 or CYP2B6 alleles, showed lower enzyme activity compared with those with two functional or wild-type alleles, (5.0 vs 9.9 pmol mg(-1) protein min(-1), P=0.046, 5.1 vs 9.9 pmol mg(-1) protein min(-1), P=0.053, and 6.8 vs 9.4 pmol mg(-1) protein min(-1), P=0.054, respectively). CYP2D6 and CYP2C9 contribute on average 45 and 46%, respectively, to the overall formation of Z-4-hydroxy-tamoxifen. CONCLUSIONS: CYP2B6, CYP2C9 and CYP2D6 genotypes all affected Z-4-hydroxy-tamoxifen formation and can predict individual ability to catalyse this reaction.