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Mechanisms of nitrosourea-induced beta-cell damage. Alterations in DNA
Abstract:
The initial step in streptozocin (STZ)-induced beta-cell toxicity has been hypothesized to be the alkylation of specific sites on DNA bases. The enzymatic removal of these lesions results in single-strand breaks that over-activate the nuclear enzyme poly(ADP-ribose) synthetase and critically deplete the cell of NAD. Our studies were performed to quantitatively evaluate the extent of DNA damage in beta-cells and correlate this damage with toxicity. Monolayer cultures of neonatal rat beta-cells were used to determine cytotoxicity and DNA damage after exposure to STZ or the aglycone N-methyl-N-nitrosourea (MNU). Toxicity in beta-cells was determined by correlating morphological alterations observed by phase-contrast microscopy with decrements in immunoreactive insulin release. The extent of DNA damage was determined by alterations in nucleoid density and quantitation of N7-methylguanine formation. Toxicity tests revealed that STZ and MNU were not toxic at equimolar concentrations. Streptozocin was toxic at 10(-3) M, whereas only mild toxicity was observed with MNU at 10(-2) M. Surprisingly, however, at equimolar concentrations the two drugs caused comparable DNA-strand breaks as evidenced by their ability to shift the nucleoid migration ratio in neutral sucrose gradients. Additionally, quantitation of N7-methylguanine formation after exposure to equimolar concentrations of the drugs demonstrated that the two alkylated DNA to the same extent. These findings suggest that factors in addition to the activation of poly(ADP-ribose) synthetase must be responsible for the toxicity seen with STZ, because MNU at a nonlethal concentration is capable of causing comparable DNA damage.
Insights
Streptozocin (STZ) and N-methyl-N-nitrosourea (MNU) cause similar DNA damage in beta-cells. However, STZ is more toxic, suggesting other factors beyond DNA damage activation of poly(ADP-ribose) synthetase contribute to STZ-induced beta-cell toxicity.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Streptozocin (STZ) is known to induce beta-cell toxicity, a process hypothesized to begin with DNA alkylation.
- The repair of these DNA lesions can lead to DNA strand breaks, over-activation of poly(ADP-ribose) synthetase, and NAD depletion.
Purpose of the Study:
- To quantitatively assess DNA damage in beta-cells induced by STZ and N-methyl-N-nitrosourea (MNU).
- To correlate the extent of DNA damage with observed beta-cell toxicity.
Main Methods:
- Neonatal rat beta-cell cultures were exposed to STZ or MNU.
- Cytotoxicity was evaluated through morphological changes and insulin release measurements.
- DNA damage was quantified by analyzing nucleoid density and N7-methylguanine formation.
Main Results:
- STZ induced toxicity at 10(-3) M, while MNU showed only mild toxicity at 10(-2) M.
- Equimolar concentrations of STZ and MNU caused comparable DNA strand breaks.
- Both agents similarly alkylated DNA at equimolar concentrations, indicated by N7-methylguanine formation.
Conclusions:
- DNA damage and subsequent poly(ADP-ribose) synthetase activation alone do not fully explain STZ-induced beta-cell toxicity.
- Additional mechanisms likely contribute to the higher toxicity of STZ compared to MNU, despite similar DNA damage levels.