DNA damage caused by benzo(a)pyrene in MCF-7 cells is increased by verapamil, probenecid and PSC833.
The aim of this study was to clarify whether pharmaceutical drugs capable of inhibiting ABC-transporters affect the toxicity of benzo(a)pyrene (BP). MCF-7 breast adenocarcinoma cells were cultured for 24 and 48 h with benzo(a)pyrene (1 microM) and the transporter inhibitors verapamil (0.125-100 microM), PSC833 (0.05-5 microM) or probenecid (0.05-2 mM). DNA binding of benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) was analyzed by synchronous fluorescence spectrophotometry and p53 protein by immunoblotting. BP metabolism was studied using thin layer chromatography (TLC). MTT assay and ATP quantitation were used for the analysis of cell viability. At 24 h there was no statistically significant increase in the DNA-adduct formation by any of the used inhibitors. However, at 48 h all of the inhibitors, in concentrations known to effectively block ABC transporters, increased the BPDE-DNA adduct formation 1.5 to 2-fold compared to adduct formation with BP only. PSC833 and verapamil also increased p53 protein expression at 48 h (p<0.05). Probenecid decreased glucuronidation of (3)H-BP metabolites. Other inhibitors did not decrease statistically significantly the overall formation of water-soluble metabolites. BP alone slightly decreased viability of cells at 48 h according to ATP quantitation as compared to vehicle treated controls (86.4+/-16.4%). Even though the used inhibitors showed some cytotoxicity, the combination of BP and inhibitors did not decrease cell viability in synergistic manner. According to these results certain pharmaceutical drugs may increase DNA damage caused by benzo(a)pyrene in MCF-7 cells at least partly through the inhibition of transporters. Taking into account the complex metabolism of BP and lack of specificity of the inhibitors used, it is likely that increased DNA damage seen in this study was the result of multiple interactions between the inhibitors, BP metabolism and the efflux of the compounds.
Myllynen
P
P
Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland. paivi.k.myllynen@oulu.fi
Kurttila
T
T
Vaskivuo
L
L
Vähäkangas
K
K
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism; D001564:Benzo(a)pyrene / Q000633:toxicity*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002273:Carcinogens / Q000633:toxicity*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003524:Cyclosporins / Q000494:pharmacology*; D018736:DNA Adducts; D004249:DNA Damage; D004357:Drug Synergism; D005260:Female; D006801:Humans; D011339:Probenecid / Q000494:pharmacology*; D012076:Renal Agents / Q000494:pharmacology*; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors; D014700:Verapamil / Q000494:pharmacology*
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D002121:Calcium Channel Blockers; D002273:Carcinogens; D003524:Cyclosporins; D018736:DNA Adducts; D012076:Renal Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; D001564:Benzo(a)pyrene; D000255:Adenosine Triphosphate; D014700:Verapamil; C022616:thiazolyl blue; D011339:Probenecid; C070272:valspodar