Mitophagy induction through mito CYP1B1/MnSOD axis facilitates Benzo[a]pyrene-mediated cell death

Abstract

Background: Mitophagy, a special type of autophagy plays an important role in mitochondrial dynamics and mitochondria associated cell death. In this study, we examined the molecular mechanism of Benzo[a]pyrene (B[a]P) facilitated mitophagy-dependent cell death. Method: Autophagic flux and mitophagy in HaCaT cells exposed to B[a]P were monitored by measuring levels and location of autophagy markers through Western blot, immunostaining and confocal microscopy. The mitochondrial energy status was quantified by oxygen consumption and flow cytometry. The involvement of MnSOD was evaluated by biochemical assay, docking and colocalization study. Results: B[a]P found to display cytotoxicity in Beclin-1 deficient HaCaT cells indicating potential existence of non-apoptotic cell death with B[a]P. We showed that B[a]P triggered Beclin-1 dependent mitophagy through the mTOR-AMPK pathway. Intriguingly, mitophagy by B[a]P was suppressed in CYP1B1 and AhR knockdown HaCaT cells indicating the crucial role of B[a]P activation in mitophagy-regulated cell death. B[a]P was shown to accumulate dysfunction mitochondria resulting fall in ATP level along with abrogation of oxygen consumption rate in HaCaT cells. Importantly, supplementation of methyl pyruvate compensated B[a]P-induced fall in ATP level, mitigated reactive oxygen species burden and autophagy. Mechanistically, B[a]P inhibited MnSOD activity and further we found that activated mitochondrial CYP1B1 interact with MnSOD, inflicting mitophagy that leads to cell death.