Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice.
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.
Graves
R J
RJ
Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK.
Coutts
C
C
Eyton-Jones
H
H
Green
T
T
D000818:Animals; D001711:Biotransformation; D016466:CHO Cells; D006224:Cricetinae; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000187:drug effects; D005557:Formaldehyde / Q000633:toxicity; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism / Q000633:toxicity; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced*; D008752:Methylene Chloride / Q000493:pharmacokinetics / Q000633:toxicity*; D051379:Mice; D008815:Mice, Inbred Strains; D051381:Rats
D004277:DNA, Single-Stranded; C085664:S-chloromethylglutathione; D005557:Formaldehyde; D008752:Methylene Chloride; D005978:Glutathione