DNA damage and cytotoxicity in pancreatic β-cells expressing human CYP2E1

Abstract Epidemiological studies have identified nitrosamines as a risk factor for Type I (insulin dependent) diabetes mellitus. These compounds require bioactivation by cytochrome P450 2E1 (CYP2E1) for exertion of their toxic effects. Two mammalian insulin secreting pancreatic β-cell lines BRIN BD11h2E1 and INS-1h2E1, which express human full length CYP2E1 cDNA, were used to elucidate the role of CYP2E1-mediated nitrosamine bioactivation in pancreatic β-cell dysfunction and destruction. These cell lines were shown to metabolise dimethylnitrosamine to produce formaldehyde at rates of 3.41 ± 0.24 and 3.65 ± 0.26 nmol/min mg microsomal protein, respectively. Following incubation with various concentrations of the nitrosamines dimethylnitrosamine, N -nitrosopyrrolidine and 1-nitrospiperidine, all of which are bioactivated by CYP2E1, cytotoxicity and DNA damage were assessed using either the neutral red assay or comet assay respectively. Exposure of CYP2E1 expressing cells to nitrosamines resulted in significant dose-dependent decreases in cell viability, which were not seen in cells which did not express CYP2E1. Following culture with nitrosamine concentrations as low as 2.5 mM 1-nitrosopiperidine, cell viability was significantly lower in BRIN BD11h2E1 and INS-1h2E1 cell lines in comparison to the BRIN BD11 and INS-1 parental cell lines (72.5 ± 4.96 and 66.4 ± 3.09% in BRIN BD11h2E1 and INS-1h2E1 versus 109.0 ± 3.40 and 100.0 ± 3.25% in BRIN BD11 and INS-1 respectively, P N -nitrosopyrrolidine, respectively ( P