title: DNA damage and cytotoxicity in pancreatic beta-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 beta-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 beta-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/minmg 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 2.5mM 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 < 0.001). The highest dose of any of the nitrosamines tested failed to significantly reduce cell viability in the cells which lacked CYP2E1. Expression of CYP2E1 did not cause any change in the basal level of DNA damage in any of the cell lines. However, 16 h exposure to various nitrosamines resulted in significant dose-dependent DNA damage in the BRIN BD11h2E1 and INS-1h2E1 cells compared to their respective non CYP2E1-expressing parental controls, e.g. DNA damage increased from 34.38 +/- 1.25 to 44.01 +/- 1.56% DNA in comet tail in BRIN BD11h2E1 cells incubated with 10 or 40 mM N-nitrosopyrrolidine, respectively (P < 0.001). Similar treatment of the BRIN BD11 and INS-1 cell lines did not result in a significant increase in DNA damage (20.33 +/- 1.0 and 22.4 +/- 0.98% DNA in comet tail). The pancreatic beta-cell is richly vascularised and expresses CYP2E1. This study suggests that expression of human CYP2E1 in pancreatic beta-cells make them highly susceptible to cytotoxicity and DNA damage by nitrosamines and other agents bioactivated by CYP2E1.
authors.affiliation: School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry BT52 1SA, N. Ireland, UK. djlees@ulster.ac.uk
authors.forename: Diane J
authors.initials: DJ
authors.lastname: Lees Murdock
authors.forename: Yvonne A
authors.initials: YA
authors.lastname: Barnett
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Barnett
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects*; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D009499:Neutral Red; D009602:Nitrosamines / Q000494:pharmacology*; D051381:Rats
chemical_list: D009602:Nitrosamines; D009499:Neutral Red; D019392:Cytochrome P-450 CYP2E1