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Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice
R J Graves1, C Coutts, H Eyton-Jones
1Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK.
Abstract:
Methylene chloride (MC) induced DNA damage in freshly isolated hepatocytes from mice and rats, which was detectable as single-strand (ss) breaks by alkaline elution. The lowest in vitro concentration of MC needed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower than for rat hepatocytes (30 mM), and is close to the calculated steady-state concentration of MC in the mouse liver (1.6 mM) at a carcinogenic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectable in hepatocyte DNA from mice which had inhaled 4000 p.p.m. MC for 6 h, but not in hepatocyte DNA from rats similarly exposed. In studies with hepatocytes cultured overnight in the presence of buthionine sulfoximine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjugation of MC with GSH is important in its activation of DNA-damaging species in the liver. The GSH pathway of MC metabolism produces two potential DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH2Cl). Formaldehyde is known to cause DNA ss breaks in cells. However, the lowest concentration of formaldehyde required to induce a significant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikely to be formed following in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That formaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activation system from mouse liver (S9 fraction). Formaldehyde was responsible for the DNA- protein cross-linking effect of MC, but did not cause the DNA damage leading to ss breaks. These DNA ss breaks are likely to be caused by GSCH2Cl. The results suggest a genotoxic mechanism for MC carcinogenicity in the mouse liver, and support the proposal that the observed species differences in liver carcinogenicity result from differences in the amount of MC metabolism via the GSH pathway in the target organ.
Insights
Methylene chloride (MC) causes DNA damage in mouse liver cells, unlike in rat cells, suggesting a genotoxic mechanism for its carcinogenicity. Glutathione conjugation is key to activating MC
Area of Science:
- Toxicology
- Genotoxicity
- Carcinogenesis
Background:
- Methylene chloride (MC) is a widely used industrial solvent.
- Species-specific differences in MC carcinogenicity have been observed, particularly in the liver.
- The underlying mechanisms of MC-induced DNA damage and carcinogenicity require further elucidation.
Purpose of the Study:
- To investigate the mechanism of methylene chloride (MC)-induced DNA damage in hepatocytes.
- To compare the susceptibility of mouse and rat hepatocytes to MC-induced DNA damage.
- To identify the specific metabolites of MC responsible for DNA damage.
Main Methods:
- Alkaline elution assay to detect single-strand DNA breaks in isolated hepatocytes.
- In vitro exposure of hepatocytes to various concentrations of MC.
- In vivo exposure of mice and rats to MC via inhalation.
- Glutathione (GSH) depletion studies using buthionine sulfoximine.
- Analysis of potential DNA-damaging metabolites, including formaldehyde and S-chloromethylglutathione (GSCH2Cl).
Main Results:
- MC induced significant single-strand DNA breaks in mouse hepatocytes at concentrations much lower than in rat hepatocytes.
- In vivo exposure to MC resulted in detectable DNA damage in mouse liver cells but not in rat liver cells.
- Depletion of glutathione (GSH) reduced MC-induced DNA damage, indicating the importance of GSH conjugation.
- Formaldehyde was identified as a DNA-protein cross-linking agent but not the cause of DNA single-strand breaks.
- S-chloromethylglutathione (GSCH2Cl) is proposed as the primary metabolite responsible for MC-induced DNA damage.
Conclusions:
- Methylene chloride (MC) induces genotoxic DNA damage in mouse liver cells, contributing to its carcinogenicity.
- Species differences in MC metabolism via the glutathione (GSH) pathway explain variations in liver carcinogenicity.
- S-chloromethylglutathione (GSCH2Cl) is the likely mediator of MC-induced DNA damage and genotoxicity.