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Mechanisms of nitrosourea-induced beta-cell damage. Activation of poly (ADP-ribose) synthetase and cellular
G L Wilson1, P C Hartig, N J Patton
1Department of Anatomy, University of South Alabama College of Medicine, Mobile 36688.
Abstract:
It has been hypothesized that the critical step in streptozocin (STZ)-induced beta-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 beta-cell. However, recently it has been shown that a lethal concentration of STZ and a nontoxic concentration of its nitrosoamide moiety methylnitrosourea (MNU) damage beta-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 beta-cell toxicity. Based on this finding, it appeared possible that STZ may be selectively sequestered into some critical site in the beta-cell other than the nucleus. Therefore, studies were initiated with 14C-labeled STZ and MNU to determine whether STZ might be distributed in the beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Streptozocin (STZ) toxicity in beta-cells is not solely due to poly(ADP-ribose) synthetase activation. This study shows STZ and MNU activate the enzyme similarly, suggesting other mechanisms are involved in STZ-induced beta-cell damage.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- The enzyme poly(ADP-ribose) synthetase (PARS) overactivation from DNA strand breaks is hypothesized as critical in streptozocin (STZ)-induced beta-cell toxicity.
- Overactivation of PARS leads to NAD depletion in beta-cells.
- Recent findings show STZ and methylnitrosourea (MNU) cause comparable DNA damage at lethal and non-toxic concentrations, respectively.
Purpose of the Study:
- To investigate whether STZ and MNU activate PARS to the same extent in beta-cells.
- To determine if PARS activation is the sole critical step in STZ-induced beta-cell toxicity.
- To explore differential cellular distribution of STZ and MNU.
Main Methods:
- Exposing neonatal rat islet cell monolayers to varying concentrations of MNU and STZ (10(-3) to 10(-2) M).
- Measuring poly(ADP-ribose) synthetase activation in response to chemical exposure.
- Utilizing 14C-labeled STZ and MNU to assess cellular distribution in RINr and islet cell monolayers.
- Separating cellular DNA and protein fractions using hydroxylapatite columns and quantifying incorporated label via liquid scintillation spectrometry.
Main Results:
- Both STZ and MNU caused comparable activation of poly(ADP-ribose) synthetase across all tested concentrations.
- The data indicate that PARS activation alone is insufficient to explain STZ-induced beta-cell toxicity.
- Preliminary investigations suggest potential differential cellular sequestration of STZ compared to MNU.
Conclusions:
- Poly(ADP-ribose) synthetase activation is not the sole critical factor in STZ-induced beta-cell toxicity.
- Alternative mechanisms, possibly involving selective sequestration of STZ within beta-cells, may contribute to its toxicity.