Related Experiment Videos
Analysis of oxygen radical toxicity in pancreatic islets at the single cell level
Abstract:
Despite extensive studies on streptozotocin, alloxan and nitric oxide toxicity in pancreatic islets the mechanism of oxygen radical induced islet cell death has not been determined. The present study shows at the level of single cells that following exposure to oxygen radicals generated from xanthine oxidase DNA strand breaks occur in cell nuclei within 5-60 min and precede cell death by several hours. Similar kinetics were seen when treating islet cells with the alkylating agent streptozotocin. Immunofluorescence studies demonstrated the endogenous formation of ADP-ribose polymers in nearly all islet cell nuclei within minutes of treatment with xanthine oxidase, indicating activation of the enzyme poly(ADP-ribose) polymerase (PARP). Concomitantly, cellular NAD+ depletion was noted. Nicotinamide largely prevented NAD+ depletion and in parallel resulted in islet cell survival. These findings identify islet cell nuclear DNA as a primary target of oxygen radical toxicity and suggest related pathways of oxygen radical, nitric oxide and streptozotocin toxicity.
Insights
Oxygen radicals cause DNA strand breaks in pancreatic islet cells, leading to cell death. Nicotinamide protects islet cells by preventing NAD+ depletion, suggesting a common toxicity pathway.
Area of Science:
- Cell biology
- Toxicology
- Endocrinology
Background:
- The precise mechanism of oxygen radical-induced pancreatic islet cell death remains unclear.
- Previous research focused on streptozotocin, alloxan, and nitric oxide toxicity.
Purpose of the Study:
- To elucidate the mechanism of oxygen radical-induced islet cell death.
- To investigate the role of DNA damage and poly(ADP-ribose) polymerase (PARP) activation.
Main Methods:
- Exposure of single islet cells to oxygen radicals generated by xanthine oxidase.
- Analysis of DNA strand breaks using immunofluorescence.
- Measurement of ADP-ribose polymer formation and cellular NAD+ levels.
- Assessment of nicotinamide's protective effects.
Main Results:
- Oxygen radicals induced DNA strand breaks in islet cell nuclei within 5-60 minutes, preceding cell death.
- Xanthine oxidase treatment activated PARP, leading to endogenous ADP-ribose polymer formation.
- Cellular NAD+ depletion occurred concurrently with PARP activation.
- Nicotinamide prevented NAD+ depletion and significantly improved islet cell survival.
Conclusions:
- Islet cell nuclear DNA is a primary target for oxygen radical toxicity.
- PARP activation and subsequent NAD+ depletion are key events in oxygen radical-induced islet cell death.
- This pathway may be shared with nitric oxide and streptozotocin toxicity.