Mechanisms of Nitrosourea-Induced β-Cell Damage: Activation of Poly (ADP-Ribose) Synthetase and Cellular Distribution

It has been hypothesized that the critical step in streptozocin (STZ)-induced β-cell toxicity is the overactivation of the nuclear enzyme poly(ADP-ribose) synthetase resulting from DNA strand breaks. Overactivation of this enzyme leads to a lethal depletion of its substrate, NAD, in the β-cell. However, recently it has been shown that a lethal concentration of STZ and a nontoxic concentration of its nitrosoamide moiety methylnitrosourea (MNU) damage β-cell DNA to the same extent and cause comparable amounts of DNA strand breaks. This study was performed to determine whether STZ and MNU activate poly(ADP-ribose) synthetase to the same extent. Monolayer cultures of islet cells from neonatal rats were exposed to concentrations of MNU and STZ of 10⁻³ to 10⁻² M. The results show that both chemicals caused comparable activation of the enzyme at all concentrations tested. These data demonstrate that activation of poly(ADP-ribose) synthetase alone is not the critical step in STZ-induced β-cell toxicity. Based on this finding, it appeared possible that STZ may be selectively sequestered into some critical site in the β-cell other than the nucleus. Therefore, studies were initiated with ¹⁴C-labeled STZ and MNU to determine whether STZ might be distributed in the β-cell differently than MNU. Total cellular DNA and protein from both RINr (clone 38) and islet cell monolayers were separated on hydroxylapatite columns after exposure to ¹⁴C-labeled chemicals. The amount of label incorporated into each fraction was determined by liquid scintillation spectrometry, and the ratio of label incorporated in protein to that in DNA was determined. The results showed that RINr cells, which are resistant to the toxic effects of STZ, have similar protein-to-DNA ratios for MNU (0.32 ± 0.04) and STZ (0.39 ± 0.16) at an equimolar concentration. However, in β-cells the ratio was approximately three times greater for STZ (0.60 ± 0.17) compared with that for MNU (0.21 ± 0.17). These data demonstrate that in β-cells, which are very sensitive to the toxic effects of STZ, many more of the reactive carbonium ions bind to protein. This finding indicates that in β-cells, STZ is sequestered differently than MNU.