Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography

Abstract

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.

The induction of urothelial hyperplasia by methyl methanesulphonate and ethyl methanesulphonate

“…Working on the assumption that differences in carcinogenic activity may be related to the ability of the agent to alkylate the 06 position of the guanine residue in DNA, Frei et al (1978), using a thymic lymphoma model, calculated that the ratio of equipotent doses of EMS and MMS by comparison with MNU would be 21 and 144 respectively. If the induction of bladder cancer by these alkylating agents depends solely on the formation of 06_ methylguanine in urothelial DNA, then the data of Frei et al (1978) predict that, as we confirmed, of the three MNU should be the most powerful carcinogen for the urothelium. However, they also predict that EMS should be considerably more carcinogenic than MMS, which is the reverse of what we found.…”

“…In experiments where the alkylating agents were administered systemically and a range of organs were examined (e.g. brain, liver and thymus), MMS and EMS by comparison with MNU produced little alkylation at the 06 position of guanine and their major alkylation products were 7-methylguanine and 7-ethylguanine respectively (Frei & Lawley, 1976;Frei et al, 1978). Nevertheless, both MMS and EMS are carcinogenic in a variety of organ systems, although they are far less potent than MNU (IARC, 1974).…”

Release of 7-methylguanine residues from alkylated DNA by extracts of Micrococcus luteus and Escherichia coli.

“…In mammalian cells, the m7G is lost at different rates, depending on the biological system studied. A half-life of 24-26 hr has been reported in hamster (9) and in 1OTL1/2 cells (10), and a much longer halflife (60 hr) has been reported in mouse organs in vivo (11,12). This is a faster rate than expected in the case of chemical depurination, which is about 150 hr at pH 7 (13).…”

5-Methylcytosine depletion during tumour development: An extension of the miscoding concept

“…Although N-nitroso and related compounds produce lesions at a variety of sites on DNA, alkylation at the 06-position of guanine is thought to be responsible for the subsequent induction of tumours by these agents (Magee, 1976(Magee, , 1979Pegg & Nicoll, 1976;Lawley, 1980;Frei et al, 1978). The persistence of 06-alkylguanine in DNA of various rat and mouse tissues (Goth & Rajewsky, 1974;Kleihues & Margison, 1974;Margison et al, 1976) correlates with the organ specificity of these compounds, especially when the rate of cell replication of the examined tissue is considered.…”