Lethal and mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine potentiated by oxidized glutathione, a seemingly harmless substance in the cellular environment.

Both the lethal and the mutagenic actions of N-methyl-N*-nitro-N-nitrosoguanidine (MNNG) on cells of Streptococcus pneumoniae were greatly potentiated by a component of yeast extract added to the cellular environment. This component was found to be an oxidation product of glutathione, glutathione disulfide (GSSG). At low concentrations in the medium, both GSSG and glutathione potentiated MNNG action, but at high concentrations, glutathione (and other sulfhydryl compounds) abolished the effect. Point mutations in a cellular gene conferred resistance to the potentiating effect, and they blocked uptake of either GSSG or glutathione into the cells as well. This gene apparently encodes a component of the system for glutathione transport in S. pneumoniae. The mechanism by which GSSG, an apparently innocuous substance in the environment, renders low levels of MNNG genotoxic and cytotoxic thus depends on its transport into the cell, whereitisreducedbyglutathionereductaseandthenactivatesintracellularMNNG.Also,itwasobservedthat mutants ofS. pneumoniaedefective in DNA mismatch repair are more resistant to MNNG than are wild-type cells by a factor of 2.5. The Hex DNA mismatch repair system of Streptococcus pneumoniaeacts on heteroduplex DNA formed during genetic transformationandonincorrectlysynthesizedDNAmadeduring chromosomal replication (for a review, see reference 6). The latter activity prevents mutations. Defects in thehexgenes block such repair. The sequences of thehexAgene (29) and of the related mutS gene of Salmonella typhimurium (10) were recently used to detect a homologous human mismatch repair gene, defects of which were shown to cause human colon cancer (8, 22). Some time ago, it was observed that mutants with defective hex genes accumulated in cultures of S. pneumoniae mutagenized by repeated cycles of treatment with Nmethyl-N9-nitro-N-nitrosoguanidine (MNNG) (15). Whereas mutations in other genes ofS. pneumoniaewere found with a frequency of ;0.2%, thehexmutant frequency was as high as 50%. This suggested that the hex mutants might be relatively resistant to MNNG and led to the studies reported here. The carcinogen MNNG is known to exert its mutagenic and lethal effects by methylation of DNA. Although its most prominent products are 7-methylguanine and 3-methyladenine (21), the formation ofO6-methylguanine appears to be responsible for its mutagenic action (7, 24). The Mut mismatch repair system of Escherichia coli, which is related to the Hex system (6, 10, 29), recognizes mismatches ofO6-methylguanine with T or C (7, 12). To exert its action, MNNG must be converted to an active intermediate, methyldiazohydroxide; this occurs slowly in aqueous solution, but the rate of activation can be increased 100-fold by addition of sulfhydryl compounds (21). While investigating the susceptibility of cells of S. pneumoniae to MNNG, we observed a powerful enhancement of mutagenic and lethal action by addition of yeast extract (YE) tothemedium.Aseeminglyinnocuoussubstancewasfoundto be responsible. The potential hazard of such interactions betweenchemicalsubstancesplacedintoourenvironmentshould not be overlooked.