Cytotoxicity of chloroacetanilide herbicide alachlor in HepG2 cells independent of CYP3A4 and CYP3A7.
Alachlor is cytotoxic to human hepatoblastoma HepG2s, a cell line that expresses constitutive CYP3A7 and dexamethasone (DEX)-inducible CYP3A4 and CYP3A7. CYP3A4 catalyzes alachlor N-dealkylation to 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA), precursor of 2,6-diethylbenzoquinoneimine, putative reactive metabolite for rat nasal carcinogenicity. We hypothesized that HepG2 alachlor cytotoxicity would be mediated by CYP3A4/7 and increased with DEX. Here, we report time-dependent alachlor cytotoxicity (EC(50) approximately 500 microM and 264+/-17 microM at 6 and 24h, respectively) as assessed by lactate dehydrogenase leakage. DEX pretreatment (25 microM, 48 h) significantly increased CYP3A7-catalyzed luciferin 6' benzylether O-debenzylation, but had no effect on alachlor toxicity. Further, CYP3A4/7 inhibitor triacetyloleandomycin did not prevent, but rather potentiated, alachlor cytotoxicity. In agreement, CDEPA was less toxic than parent alachlor. HepG2 CYP3A4 activity was unaffected by 48 h DEX pretreatment; therefore, studies were done in DPX-2 cells, a HepG2 derivative engineered to overexpress pregnane-X receptor (PXR) that exhibits rifampicin (RIF)-inducible endogenous CYP3A4. Alachlor cytotoxicity in DPX-2 cells occurred over a concentration range equivalent to that in HepG2. CYP3A4 activity of DPX-2 cells treated with RIF (10 microM, 48 h) was twice that of untreated cells, but RIF did not increase alachlor toxicity. These results demonstrate that neither CYP3A4 nor CYP3A7 initiate a pathway leading to a toxic alachlor metabolite.
Miranda
Sonia R
SR
Department of Toxicology, University of Louisiana at Monroe, 700 University Avenue, Sugar Hall, Room #358, Monroe, LA 71209, United States.
Meyer
Sharon A
SA
D000081:Acetamides / Q000378:metabolism / Q000633:toxicity*; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D045744:Cell Line, Tumor; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006540:Herbicides / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008113:Liver Neoplasms / Q000201:enzymology; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid / Q000378:metabolism; D013997:Time Factors
D000081:Acetamides; D006540:Herbicides; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; C000188:alachlor; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase; D001189:Aryl Hydrocarbon Hydroxylases; C103123:CYP3A7 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human