Prediction and validation of apoptosis through cytochrome P450 activation by benzo[a]pyrene

• Benzo[a]pyrene activates by cytochrome P450 was analyzed by in silico approach.
• Benzo[a]pyrene induces apoptosis by activation of intrinsic and extrinsic caspases.
• Caspase inhibitors suppress benzo[a]pyrene mediated apoptotic death.
• CYP1B1 deficiency decreases benzo[a]pyrene induced apoptosis.
• Clotrimazole potent inhibitor of cytochrome P450 playing crucial role in inhibits activity of benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) processed by cytochrome P450 (CYP450) during metabolism is well reported to induce carcinogenesis. The present study has developed a new approach to examine apoptotic activity of a known PAH called benzo[a]pyrene (B[a]P), using protein–ligand and protein–protein interaction through in silico approach, followed by in vitro validation. In silico study showed that the conformational changes and energies involved in the binding of B[a]P to CYP1B1 was crucial with its target proteins. The data showed that activated B[a]P had high affinity to bind with aryl hydrocarbon receptor (AhR) with binding energy of −601.97 kcal/mol. Interestingly, B[a]P–CYP1B1 complex showed strong binding affinity for caspase-8, -9, -3 with binding energy of −625.5, −479.3 and −514.2 kcal/mol respectively. Moreover, the docking of specific caspase inhibitors in the complex showed weak interaction with low binding energy value as compared to B[a]P–CYP1B1 caspase complexes. To validate our in silico work, we showed B[a]P treated HaCaT cells triggered apoptosis with increase in caspase 8, caspase 9 and caspase 3/7 level. Further, in vitro work confirmed that B[a]P induced apoptosis was significantly suppressed in Ac-DEVD-CMK pre-treated cells. In addition, knockdown of CYP1B1 suppressed B[a]P induced apoptosis in HaCaT cells confirming a pivotal role of CYP1B1 in B[a]P induced apoptosis. Interestingly, through in silico modeling, we screened clotrimazole as a potent CYP1B1 inhibitor which completely inhibited B[a]P mediated activation. This hypothesis was validated by MTT assay, caspase activation measurement and showed remarkable inhibition of B[a]P induced cell death; thereby, highlighting a potent therapeutic role for industrial pollution associated diseases.