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NAD+ depletion and cytotoxicity in isolated hepatocytes
1Department of Pharmacology, School of Pharmacy, University of London, U.K.
Abstract:
Activation of poly(ADP-ribose)polymerase by DNA damaging agents causes a depletion of intracellular NAD+ and subsequent lowering of ATP pools, which if extensive may lead to cell death. We have studied the cytotoxicity to isolated hepatocytes of dimethyl sulphate, a direct-acting carcinogen and mutagen, hydrogen peroxide, generated by glucose/glucose oxidase, and menadione (2-methyl-1,4-naphthoquinone) in relation to their effects on intracellular NAD+ and ATP levels. Both dimethyl sulphate and glucose/glucose oxidase caused a depletion of NAD+, which was apparently due to an activation of poly(ADP-ribose)polymerase as it was prevented by inhibitors of the polymerase, i.e. 3-aminobenzamide and nicotinamide. This protection of intracellular NAD+ was accompanied by a prevention of the cytotoxicity of both dimethyl sulphate and glucose/glucose oxidase, while it did not alter the decrease in intracellular ATP they induced. This apparent dissociation of effects on ATP from NAD+ does not support the suggestion that activation of poly(ADP-ribose)polymerase leads to a decrease in cellular ATP as a consequence of NAD+ depletion. Menadione also caused a depletion of NAD+ which preceded cytotoxicity, but in contrast to dimethyl sulphate and H2O2 this depletion did not involve poly(ADP-ribose)polymerase as it was not prevented by inhibitors of the enzyme. Our results also indicate that the cytotoxicity of menadione is not mediated by H2O2 alone. Marked depletion of intracellular NAD+ prior to toxicity and a protection against toxicity associated with maintenance of NAD+ suggest a possible role for the maintenance of intracellular NAD+ in cellular integrity.
Insights
DNA damaging agents activate poly(ADP-ribose)polymerase, depleting NAD+ and potentially causing cell death. Maintaining NAD+ levels protected hepatocytes from toxicity, suggesting a role in cellular integrity.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- DNA damaging agents can activate poly(ADP-ribose)polymerase (PARP).
- PARP activation depletes intracellular nicotinamide adenine dinucleotide (NAD+) and can lower adenosine triphosphate (ATP) pools.
- Extensive depletion of these energy molecules may lead to cell death.
Purpose of the Study:
- To investigate the cytotoxicity of dimethyl sulphate, hydrogen peroxide, and menadione on isolated hepatocytes.
- To examine the relationship between the cytotoxicity of these agents and their effects on intracellular NAD+ and ATP levels.
- To determine the role of poly(ADP-ribose)polymerase activation in the observed cytotoxicity.
Main Methods:
- Isolated hepatocytes were exposed to dimethyl sulphate, glucose/glucose oxidase (for H2O2 generation), and menadione.
- Intracellular NAD+ and ATP levels were measured.
- The effects of poly(ADP-ribose)polymerase inhibitors (3-aminobenzamide, nicotinamide) on cytotoxicity and NAD+ levels were assessed.
Main Results:
- Dimethyl sulphate and glucose/glucose oxidase induced NAD+ depletion via PARP activation, which was prevented by PARP inhibitors.
- PARP inhibition protected hepatocytes from dimethyl sulphate and glucose/glucose oxidase cytotoxicity, without affecting ATP levels.
- Menadione also caused NAD+ depletion preceding cytotoxicity, but this was independent of PARP activation and H2O2.
Conclusions:
- The study suggests that maintaining intracellular NAD+ levels is crucial for cellular integrity against certain toxins.
- The findings challenge the notion that NAD+ depletion directly causes a decrease in cellular ATP following PARP activation.
- Menadione-induced cytotoxicity is not solely mediated by hydrogen peroxide and involves a PARP-independent mechanism.