Organ-specific effects of DNA methylation by alkylating agents in the inbred Swiss mouse.

Young adult inbred Swiss mice given single or repeated equitoxic doses of N-methyl-N-nitrosourea (MNUA) or methyl methanesulphonate (MMS) develop thymomas and pulmonary adenomas only following MNUA in spite of nearly identical overall alkylation of DNA of tumour target tissues by both agents due mainly to the biologically ineffective product 7-methylguanine. The main difference in DNA alkylation was the production of O6-methylguinine, a known pre-mutagenic product, by MNUA in amounts 10 or more times as large as following MMS. This supports the possibility that somatic mutations are a part of the process of carcinogenesis.     

Alkylation of cytosine and 5-hydroxymethyl-cytosine by methyl methanesulphonate and N-methyl-N-nitrosourea: its relevance to mutagenesis.

The reaction of cytosine and 5-hydroxymethyl-cytosine (OHMeCyt) with a variety of monofunctional alkylating agents has been investigated to evaluate further the possible role of cytosine alkylation in mutagenesis and the possibility that the immunity of T-even phages to mutation by methyl methanesulphonate (MMS) was due to the unreactivity of OHMeCyt towards this agent. Both cytosine and OHMeCyt reacted equally well with the methylating agents MMS and N-methyl-N-nitrosourea (MNU) affording 6% and less than 1% respectively of the 3-substituted derivative. No product was isolated following subjection of the bases to reaction with ethyl methane-sulphonate (EMS), N-ethyl-N-nitrosourea (ENU) or iso-propyl methane-sulphonate (iPMS).     

The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10⁸ daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl-N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10⁸ daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10⁸ daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O⁶-methylguanine (O⁶-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN₁ character of MNUA would cause a greater formation of phosphotriesters than would MMS.     

Enhancement by caffeine of N-methyl-N-nitrosourea-induced mutations and chromosome aberrations in Chinese hamster cells

N-Methyl-N-nitrosourea (MNU) increased the induction of mutations to 8-azaguanine resistance in Chinese hamster cells in a dose-dependent manner. Mutations were only observed with toxic concentrations of MNU. Since a plot of the fraction of cells surviving alkylation against the extent of methylation of DNA exhibited a shoulder it followed that there was a threshold level of DNA reaction which did not lead to mutations possibly due to efficient repair of DNA damage. Post-alkylation incubation in medium containing caffeine decreased cell survival while at the same time it increased the induced mutation frequency. Mutation frequency was increased whether caffeine was present for 48 h or for a further 12 days in the presence of the selective agent 8-azaguanine. MNU caused chromatid aberrations in Chinese hamster cells and these reached a value of 15% of the treated cells by 48 h after methylation. Post-alkylation incubation in caffeine increased the percentage of cells showing chromosomal damage to a maximum of 86% of treated cells by 40 h after alkylation. A large proportion of cells exhibited completely fragmented or shattered chromosomes. The proportion of cells showing the presence of micronuclei also dramatically increased following incubation of methylated cells in caffeine. These results are discussed in terms of the possibility that damage to DNA is responsible for the lethal, mutagenic and cytological effects of MNU in Chinese hamster cells, and that there is a caffeine sensitive step(s) in the repair of the DNA damage which is responsible for these effects.     

Effect of a single treatment with the alkylating carcinogens dimethynitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. I. Test for induction of liver carcinomas.

A single injection of dimethylnitrosamine (DMN), 12.0-15.6 mg-kg, given to 100 g female rats 24 h after partial hepatectomy, induced hepatocellular carcinoma. No animals receiving DMN without partial hepatectomy developed liver carcinomas. Previous evidence had suggested that the incidence of tumours was highest when DMN was administered during the wave of DNA replication which follows partial hepatectomy. The present experiments made this suggestive evidence statistically significant. A single treatment with diethylnitrosamine (DEN) induced liver cell cancer when given to intact or to partially hepatectomised rats. No tumors developed when another alkylating carcinogen, methyl methanesulphonate (MMS), was administered after partial hepatectomy. The significance of these results in relation to the mechanism of initiation of carcinogenesis is discussed.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. IV. Effect on methylase-mediated methylation of DNA.

The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro.

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.     

Increased urinary excretion of pyrimidine and nicotinamide derivatives in rats treated with methyl methanesulphonate

Rats treated with methyl methanesulphonate (MMS) excreted significantly higher quantities of deoxycytidine, thymidine, uracil, 1-methylnicotinamide (1-meNmd) and 1-methyl-6-pyridone-3-carboxylamide (6-pyr-1-meNmd) in their urine 0–24 h after MMS injection (100 $\text{mg}\text{kg}$). Excretion of thymidine, which was not detectable in untreated rats, was dose-dependent. No increase in urinary 7-methylguanine was found, and creatinine excretion was decreased by MMS treatment. Experiments with methyl-¹⁴C-labelled MMS showed transfer of ¹⁴C-label to 7-methylguanine and 1-meNmd. X-Irradiation (500 rad) caused increased excretion of pyrimidines, like MMS, but did not increase excretion of the nicotinamide derivatives.     

Increased urinary excretion of labelled deoxyribonucleosides in rats with stable pre-label led dna treated with methyl methanesulphonate or nitrogen mustard

After the DNA of newborn female rats had been labelled by repeated injections of [¹⁴C]orotate (totalling 36 μCi) during the first 3 weeks of life, approximately 1 000 000 dpm were found in the DNA of the liver, lungs, kidneys, gut, brain, heart and spleen of 8-week-old rats. Methyl methanesulphonate (MMS) (80 mg/kg) and di-(2-chloroethyl)methylamine (HN2) (5 mg/kg) injection increased the amount of ¹⁴C-labelled DNA pyrimidine nucleosides excreted in the urine to 5000 dpm from 350 dpm before injection. The effect on RNA products was much less marked.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. II. Alkylation of DNA and inhibition of DNA replication.

Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.     

Post-treatment with caffeine and the induction of gene mutations by ultraviolet irradiation and ethyl methanesulphonate in V-79 Chinese hamster cells in culture.

The effect of caffeine on V-79 Chinese hamster cells after ultraviolet irradiation or treated with ethyl methanesulphonate was investigated. Caffeine strongly potentiated the killing of both agents, but it had no effect on the induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. The results are consistent with the notion that caffeine slows down an error-prone post-replicative repair mechanism without changing the mutation frequency.     

Caffeine-induced enhancement of chromosome damage in human lymphocytes treated with methyl-methanesulphonate, mitomycin C and X-rays

Two hypothese have been put forward in the literature to explain the synergistic effect of caffeine with several mutagens: (1) binding of caffeine to DNA, and (2) inhibition of DNA repair.Autoradiographic studies with ³H- and ¹⁴C-labelled caffeine did not support the binding hypothesis. Caffeine enchanced in a synergistic way the amount of chromatid breaks and exchanged induced in human lymphocytes with methyl-methanesulphonate (MMS), mitomycin C (MC) and X-rays. The results are best explained if caffiene inhibits a post-replication repair process, particularly the filling-in of gaps in the newly synthesized DNA.     

Repair of DNA damage induced by methylating agents in human uterine cervical cells with or without cancer precursor lesions

Experiments were carried out to study the repair capabilities of normal human cervical fibroblasts and fibroblasts derived from human uterine cervical dysplasia, carcinoma in situ and invasive carcinoma. Sedimentation analysis of DNA in alkaline sucrose density gradient was carried out to monitor the DNA damage induced by a methylating carcinogen, methylnitrosourea (MNU). The results indicate that none of the cell lines, namely, fibroblasts either derived from normal human uterine cervix (T₃₀-11) or from cervical cells of cancer precursor lesions (T₄-3F; T₂₃-3; T₁₈) exhibited any significant repair in 72 h. In contrast fibroblasts derived from normal human skin (GM105) exhibited 38% repair of their DNA damaged by MNU. Epithelial-like cells (T₄-3E) obtained from cervical dysplasia exhibited only 18% repair of MNU-induced DNA damage in 72 h.When the damage was induced by another methylating agent, methyl methanesulfonate (MMS), fibroblasts from normal human skin (GM105) exhibited 40% repair of the damaged DNA whereas fibroblasts from normal human uterine cervix (T₃₀-11) exhibited only a 16% repair, in 72 h.These results suggest that fibroblasts derived from either normal human uterine cervix or from cervix with cancer precursor or cancer lesions exhibit low levels of repair of DNA damged by methylating agents.     

Evaluation of genetic risks of alkylating agents IV. Quantitative determination of alkylated amino acids in haemoglobin as a measure of the dose after-treatment of mice with methyl methanesulfonate.

The present study explores the possibilities of using specific amino acids in haemoglobin for tissue dosimetry of alkylating agents. The well-known directly alkylating compound methyl methanesulfonate has been used as a model compound.In one experiment ³H-labelled methyl methanesulfonate was given to mice intraperitoneally at three dose levels. The degree of alkylation of haemoglobin exhibited a linear dependence on the quantity of methyl methanesulfonate injected. The degree of alkylation of guanine-N-7 in DNA indicated a slight positive deviation from linearity at high doses.After a single injection the degree of alkylation of cysteine-S and histidine-N-3 in haemoglobin decreased linearly with time reaching the value zero after about 40 days (the life-time of the erythrocytes in the mouse). This demonstrates a stability of these alkylated products, which is fundamental to their use as integral dose monitors.In a second experiment mice were treated with methyl methanesulfonate once a week over a period of 8 weeks. The experiment demonstrated an accumulation of alkylated groups in haemoglobin in agreement with expectation.A method for the quantitative determination of S-methylcysteine in a protein hydrolysate by gas chromatography was developed.     

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.     

Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents.

Rats treated with di(2-chloroethyl)methylamine (HN2), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine 0-24 h after treatment. Ethyl methanesulphonate (EMS) and dimethylnitrosamine (DMN) gave negative results in this respect but all five alkylating agents increased the excretion of 1-methyl-nicotinamide (1-meNmd). In addition, a larger quantity of 7-methylguanine (7MG) and uric acid was excreted after DMN treatment. 1,4-Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5-fluorouracil (5FU), cytosine arabinoside (araC), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz-[a]anthracene (7-BrMBA) gave negative results.     

The synthesis of a DOPA analogue, 1-amino-2-(3, 4-dihydroxyphenyl)-ethylphosphonic acid and distribution studies in mice bearing the Harding-Passey melanoma

The aminophosphonic acid analogue of DOPA, DL-1-amino-2-(3,4-dihydroxy-phenyl) ethylphosphonic acid (ADEP) has been synthesised. The compound was of low toxicity; a single dose of 2 g/kg given to mice subcutaneously was not lethal.[³H]ADEP was injected subcutaneously into mice carrying the established Harding-Passey melanoma, and the distribution of the tritium determined. The highest initial concentration of radioactivity was in the kidneys, adrenal glands and eyes. Isotope content fell to low values in all tissues within 8 days or less but the tumour retained radioactivity for a longer period than did the other tissues examined.ADEP served as a substrate for mushroom tyrosinase.     

Reversions of the two missense and one nonsense trp-mutations induced by UV-light or methyl methanesulfonate in E. coli wild type and its polAi and uvrE502 mutants.

Two missense mutations, trpA58 and trpA78, and one nonsense mutation-trp-ochre, were used to determine the types of base-pair substitution caused by ultra, violet irradiation and methyl methanesulfonate (MMS) in Escherichia coli. UV irradiation of the wild-type bacteria led to the formation of revertants mainly arising as a result of GC yields AT transitions (suppressor revertants of the trpA58 mutant). True revertants of the trp- mutant (arising via transitions of AT pairs) and 5-methyl tryptophan-sensitive (MT-s) Trp+ of the trpA78 mutant (arising via unidentified transversions) occurred at a lower frequency. The polAI mutation did not change the frequency of the UV-induced transitions GC yields AT or that of the substitutions of the AT pairs. The uvrE502 mutation significantly increased the frequency of the UV-induced revertants arising via the transition GC yields AT. Treatment of the wild-type bacteria with MMS resulted in the formation of revertants mainly due to the GC yields AT substitution, and with a lower frequency to the AT yields GC transitions. MMS also induced, with a low frequency, some transversions. The frequency of the MMS-induced GC yields AT transitions was enhanced in the uvrE502 mutant. On the other hand, the uvrE502 mutation eliminated or significantly lowered MMS-induced revertants arising as a result of AT yields GC transitions or transversions.