Stable expression of rat dihydrodiol dehydrogenase (AKR1C9) in human breast MCF-7 cells results in the formation of PAH-o-quinones and enzyme mediated cell death.
Dihydrodiol dehydrogenase members of the aldo-keto reductase (AKR) superfamily have been implicated in the metabolic activation of PAH trans-dihydrodiols because they convert these proximate carcinogens to reactive and redox-active o-quinones. In this study, rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (AKR1C9) was stably transfected into human breast carcinoma (MCF-7) cells, which represent a null-environment for AKR expression, to detect the formation of PAH o-quinones in a cellular context and the cellular consequences of o-quinone formation. The heterologous transfected cells expressed AKR1C9 mRNA and protein. Immunotitration of the enzyme activity indicated that the expressed protein constituted 1.0% of the soluble protein. The specific activity of the expressed enzyme was also comparable to that observed in rat liver cytosol. The transfectants were found to convert (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) to benzo[a]pyrene-7,8-dione (BPQ). The identity of this metabolite was confirmed by co-chromatography and by UV-Vis diode-array spectrometry. This conversion was not evident in mock-transfected cells. The cytotoxic consequences of BPQ formation was also examined. Transfectants exposed to 1 microM B[a]P-7,8-diol revealed that cytotoxicity, as measured by lactate dehydrogenase (LDH) release, occurred over the time course of o-quinone formation leading to 77% of the cellular LDH being released by 16 h. AKR1C9 inhibitors blocked the B[a]P-7,8-diol dependent cytotoxicity indicating that it was mediated by the enzymatically formed BPQ. These data indicate that high stable constitutive expression of AKR1C9 will result in B[a]P-7,8-diol mediated cytotoxicity due to the formation of unconjugated BPQ.
Tsuruda
L
L
Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6084, USA.
Hou Yt
Penning
T M
TM
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism / Q000633:toxicity; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms; D002273:Carcinogens / Q000378:metabolism / Q000633:toxicity; D016923:Cell Death; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism* / Q000633:toxicity; D011809:Quinones / Q000378:metabolism*; D012333:RNA, Messenger; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D001580:Benzopyrenes; D014408:Biomarkers, Tumor; D002273:Carcinogens; D011084:Polycyclic Aromatic Hydrocarbons; D011809:Quinones; D012333:RNA, Messenger; C076588:benzo(a)pyrene-7,8-dione; C403192:3-hydroxysteroid dihydrodiol dehydrogenase; D000429:Alcohol Oxidoreductases; D006913:Hydroxysteroid Dehydrogenases; C023125:dihydrodiol dehydrogenases; D007770:L-Lactate Dehydrogenase