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) superfam ily have been implicated in the metabolic activation of PAH trans-dihydrodi ols because they convert these proximate carcinogens to reactive and redox- active o-quinones. In this study, rat liver 3ahydroxysteroid/dihydrodiol de hydrogenase (AKR1C9) was stably transfected into human breast carcinoma (MC F-7) cells, which represent a null-environment for AKR expression, to detec t the formation of PAH o-quinones in a cellular context and the cellular co nsequences of o-quinone formation. The heterologous transfected cells expre ssed AKR1C9 mRNA and protein. Immunotitration of the enzyme activity indica ted that the expressed protein constituted 1.0% of the soluble protein. The specific activity of the expressed enzyme was also comparable to that obse rved in rat liver cytosol. The transfectants were found to convert (+/-)-tr ans-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 c o-chromatography and by UV-Vis diode-array spectrometry. This conversion wa s not evident in mock-transfected cells. The cytotoxic consequences of BPQ formation was also examined. Transfectants exposed to 1 muM B[a]P-7,8-diol revealed that cytotoxicity, as measured by lactate dehydrogenase (LDH) rele ase, 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 e nzymatically formed BPQ, These data indicate that high stable constitutive expression of AKR1C9 will result in B[a]P-7,8-diol mediated cytotoxicity du e to the formation of unconjugated BPQ.