Similarities between the biochemical actions of cycasin and dimethylnitrosamine

1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [(14)C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of (14)C from l-[U-(14)C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.     

Preferential alkylation of mitochondrial deoxyribonucleic acid by N-methyl-N-nitrosourea

The reaction of the carcinogen N-methyl-N-nitrosourea with mitochondrial DNA from various rat tissues was examined in vivo and in vitro. After incubation of isolated mitochondria or cell nuclei with N[(14)C]-methyl-N-nitrosourea in vitro and subsequent isolation and purification of the DNA the specific radioactivity of the mitochondrial DNA was 3-7 times that of the nuclear DNA. The incorporation of (14)C into embryonic mitochondrial DNA in vitro was about twice that into the liver mitochondrial DNA. Identical incorporation rates, however, were found for the reaction of isolated mitochondrial DNA or nuclear DNA respectively with N[(14)C]-methyl-N-nitrosourea. After intraperitoneal injection of 43.3-58.5mg of N[(14)C]-methyl-N-nitrosourea/kg body wt. to adult rats the labelling of the mitochondrial DNA was on average 5 times that of the nuclear DNA. A smaller specific labelling was observed for the ribosomal RNA, transfer RNA, and mitochondrial RNA as well as for the mitochondrial protein as compared with the mitochondrial DNA. After hydrolysis of the alkylated nucleic acids with hydrochloric acid, fractionation was carried out on Dowex 50 cation-exchange columns. In most experiments 70-80% of the input (14)C radioactivity was eluted in the 7-methylguanine fraction. The preferential alkylation of the mitochondrial DNA by N-methyl-N-nitrosourea in situ is discussed in connexion with the cytoplasmic-mutation hypothesis of carcinogenesis.     

Effect of administration of the carcinogen dimethylnitrosamine on urinary 7-methylguanine

1. Evidence is presented for the excretion of 7-methylguanine in normal rat urine at a rate of approx. 65μg./day. Experiments with animals in which the nucleic acids had been prelabelled by treatment of the neonatal rats with [¹⁴C]-formate gave evidence that the methylated base originated in the nucleic acids of the rat. 2. Injection of [¹⁴C]dimethylnitrosamine leads to an increased excretion of 7-methylguanine, and the base becomes labelled in the methyl group. The disappearance of labelled 7-methylguanine formed in nucleic acids of rats treated with the carcinogen therefore does not take place by an N-demethylation reaction, but by liberation of the intact methylated base.     

Methylation of DNAase-digestible DNA and of RNA in chromatin from rats treated with dimethylnitrosamine.

After injecting rats with di[14C]methylnitrosamine we have prepared liver chromatin and have examined firstly, the methylation level of the DNAase I-degradable fraction of the DNA and secondly, the level of methylation and the stability of methylated sites in chromatin RNA. Our results show that the level of 7-methylguanine in the degradable DNA is about 1.3 times that of whole DNA; therefore in the 20% or so of the DNA which is undegradable by DNAase I, the level must be very low or zero. Experiments using chromatin from rats injected with unlabelled dimethylnitrosamine plus [3H]thymidine show that the specific activity is similar in the DNAase I degradable and undegradable fractions, suggesting that there is no preferential repair in the latter region. In chromatin RNA, the level of 7-methylguanine is higher than that of whole DNA and decreases fairly rapidly within 30 h after dimethylnitrosamine treatment. Our results indicate that this decrease is due to some type of excision or repair process rather than to normal turnover.     

Study of the methylation and lack of deamination of deoxyribonucleic acid by N-methyl-N′-nitro-N-nitrosoguanidine

1. DNA labelled with (14)C in the purine residues was prepared by treating newborn rats with [(14)C]formate and killing them for preparation of nucleic acids at 11-17 months. This DNA was incubated with N-methyl-N'-nitro-N-nitrosoguanidine, and then analysed for products of methylation and deamination reactions. 2. Evidence was found for the formation of 7-methylguanine and a smaller amount of 3-methyladenine, and, after preliminary denaturation of the DNA, 1-methyladenine was detected. The presence of cysteine increased the extent of methylation. No evidence was found for the formation of xanthine or hypoxanthine, even at pH5.5.     

Analysis of bases of rat-liver nucleic acids after administration of the carcinogen dimethylnitrosamine

Dimethylnitrosamine is metabolized to form an alkylating intermediate, which may have significance for its carcinogenic action. However, certain other compounds that are known to be highly mutagenic, including nitrous acid and hydroxylamine, might also be formed. Owing to the general reactivity of these compounds, it would be difficult to detect their formation in the intact animal. Instead, the nucleic acids of carcinogen-treated animals were examined for products of reaction with nitrous acid and hydroxylamine, i.e. for deamination of adenine and guanine, and formation of N(6)-hydroxycytosine, respectively. A double-labelling technique was used to detect very small amounts of the abnormal bases. The purine moieties of DNA in adult rat liver were labelled either with (14)C or with (3)H, by treating the neonatal animals with [(14)C]formate or with [(3)H]formate, and then allowing a period for normal growth. During this time, in liver, the labels were largely lost by metabolic turnover from cell components other than DNA. The pyrimidine moieties in DNA were labelled by treating the neonatal animals with [(14)C]orotate. The purine constituents of RNA of adult rat liver were labelled by repeated administration of [(14)C]- or [(3)H]-formate to the adult rats. The [(14)C]nucleic acid-labelled rat could then be treated with the carcinogen, and the [(3)H]nucleic acid-labelled animal could be used as a control. By this means the experimental and control tissues could be homogenized together in a single preparation, and the nucleic acids from the two tissues could be isolated, hydrolysed and analysed in a single sample. It was therefore possible to have an internal control for artifacts due to changes taking place in the nucleic acid bases during the experimental procedures. With this technique, the formation in vivo of 7-methylguanine in rat-liver DNA and RNA after administration of dimethylnitrosamine was confirmed, and no evidence was found for the formation of xanthine, hypoxanthine, N(6)-hydroxycytosine, or any other abnormal base.     

ALKYLATION OF RAT BRAIN NUCLEIC ACIDS BY N-METHYL-N-NITROSOUREA AND METHYL METHANESULPHONATE

Abstract— Alkylation of rat brain nucleic acids in vivo was measured after a single intravenous injection (1 mmol/kg body wt.) of N -[¹⁴C]methyl- N -nitrosourea and [¹⁴C]methyl methanesulphonate. The main product with both compounds was 7-methylguanine, The extents of methylation on this position in DNA and RNA were similar with methylnitrosourea but methyl methanesulphonate produced twice as much 7-methylguanine in DNA as in cytoplasmic RNA. Brain DNA from rats treated with labelled methylnitrosourea contained radioactive O ⁶-methylguanine, accounting for about 12 per cent of the radioactivity present as 7-methylguanine and cytoplasmic RNA contained about half this amount of O ⁶-methylguanine. Neither DNA nor cytoplasmic RNA from methyl methanesulphonatetreated rats contained any detectable O ⁶-methylguanine. Treatment with both compounds resulted in varying small amounts of methylation of other nucleic acid bases including 1-methyladenine, 3-methyladenine and 3-methylcytosine. The possible relevance of alkylation of brain nucleic acids to the induction of brain tumours is discussed.     

The fate of 7[14C]-methylguanine after administration to the rat

1. To assess the significance of the methylation of nucleic acids known to be caused by certain carcinogens, the metabolic fate of 7[¹⁴C]-methylguanine was studied, with special reference to its possible incorporation into RNA and DNA. 2. The major part (approx. 95%) of the dose was excreted unchanged in the urine. A small amount of N-demethylation took place, as evidenced by the formation of radioactive adenine and guanine, and expiration of ¹⁴C-labelled carbon dioxide. No evidence was obtained for the direct incorporation of 7-methylguanine into systems synthesizing nucleic acids, i.e. RNA in liver, DNA in intestine or in the foetus.     

Immunochemical detection of 7-methylguanine residues in nucleic acids

The ability of specific antibodies to react with 7-methylguanine residues in nucleic acids was investigated. Anti-7-methylguanine specific antibodies precipitated polymers of poly-guanylic acid which were methylated to an extent of 35 or 70% at the N-7 position of guanine, indicating that these antibodies could readily detect 7-methylguanine residues in a polynucleotide. This reaction was proportional to the total amount of 7-methylguanine present, suggesting further that quantitation of these residues is possible. To determine the minimal amount required for detection, varying amounts of 7-methylguanine were introduced into calf thymus DNA by alkylation with dimethyl sulfate. While showing no reaction with denatured nonalkylated DNA, the reaction of antibodies with alkylated DNA was proportional to the amount of 7-methylguanine in the preparations. Moreover, the antibodies appeared to detect differences in the distribution of 7-methylguanine residues in extensively methylated DNA. Precipitation was observed with DNA containing as little as one 7-methylguanine residue per 300 nucleotides, suggesting that these antibodies can be used to detect biologically significant levels of 7-methylguanine in viral and cellular nucleic acids.     

In vivo alkylation of foetal, maternal and normal rat tissue nucleic acids by 3-methyl-1-phenyltriazene.

The carcinogen 3-methyl-1-phenyltriazene (MPT) was administered subcutaneously to normal or pregnant BD VI rats and DNA and RNA were isolated from various tissues after 8 h or 15 h, respectively. Sephadex G-10 chromatography of DNA hydrolysates showed the presence of 7-methylguanine in all tissues examined including that of the brain, one of the target organs for tumour induction. The amounts of the minor product, O6-methylguanine, were characteristic of an SN1 reaction mechanism. Dowex-50 chromatography of RNA hydrolysates showed the presence of 7-methylguanine and of the minor product, 3-methylcytosine. The relative amounts, both of the methylated bases in the individual nucleic acids and of 7-methylguanine in DNA and RNA, were similar to those found previously after administration of 3,3-dimethyl-1-phenyltriazene (DMPT). This suggests the involvment of a common alkylating intermediate. De novo incorporation of radioactivity into purine bases was detected in both DNA and RNA although the levels were not related to the amounts of methylation. The results show that MPT is sufficiently stable to alkylate nucleic acids in vivo and are consistent with the hypothesis that this reaction is a prerequisite for tumour induction. Futhermore, they support the proposal that MPT is the active intermediate in the induction of tumours by DMPT.     

Methylation of DNA guanine via the 1-carbon pool in dimethylnitrosamine-treated rats

Treatment of rats with radioactive methionine and nonradioactive dimethylnitrosamine resulted in the formation of radioactive 7-methylguanine in rat-liver DNA. By comparing the specific activity of administered [14C-Me]-dimethylnitrosamine to the specific activity of isolated 7-methylguanine it was determined that following 20 mg/kg dimethylnitrosamine DNA methylation via the 1-carbon pool may account for up to 30% of the total 7-methylguanine formed.     

Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney

1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [(14)C]leucine and [(14)C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [(14)C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [(14)C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [(14)C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [(3)H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.     

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2-3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[(14)C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.     

In vivo alkylation studies with dichlorvos at practical use concentrations

Twenty male CFE rats were exposed to atmospheres containing 0.064 μg/l of [Me-¹⁴C]dichlorvos (113 Ci/mol) for 12 h. Analysis of the DNA and RNA from the total soft tissues of these rats revealed no methylation of the N₇ atom of guanine moieties. The limits of detection of methylation were one methyl group per 6.0 × 10¹¹ and per 2 × 10⁹ nucleotide units for DNA and RNA, respectively. Only 0.000001% of the administered dose would have needed to react with DNA in order to produce detectable methylation of this macromolecule.The exposure period employed in this study (12 h) constituted a significant fraction of the half-life of 7-methylguanine moieties in DNA (3 days). On the basis of this information and the extremely rapid metabolism of dichlorvos in a wide range of mammalian tissues and species it was concluded that dichlorvos does not methylate the nucleic acids of mammalian tissues when it is inhaled continuously at practical use concentrations.     

Cellular injury and carcinogenesis. The effect of a protein-free high-carbohydrate diet on the metabolism of dimethylnitrosamine in the rat

1. Rats fed on a protein-free high-carbohydrate diet for 7 days metabolized dimethylnitrosamine at only 55% the rate of rats fed on a commercial diet. 2. Dimethylnitrosamine was metabolized by liver slices from rats fed on the protein-free diet at less than half the rate attained by slices from rats fed on a commercial diet. But kidney slices from these rats metabolized dimethylnitrosamine at the same rate as kidney slices from rats on a commercial diet. 3. Methylation by dimethylnitrosamine (70mg/kg body wt.) of N-7 of guanine of the liver RNA and DNA of rats fed on a protein-free diet was only slightly higher than in rats fed on a normal diet given 27mg/kg body wt. In contrast, the methylation by dimethylnitrosamine of guanine in kidney nucleic acids of these rats was three times that in the rats fed on a normal diet. 4. In rats fed on a protein-free diet the incidence of kidney tumours produced by a single dose of dimethylnitrosamine is increased.     

Repair studies on methylated DNA of CHO cells

DNA methylation pattern was investigated on Chinese hamster ovary (CHO) cells after treatment with N-(14C)-methyl-N-nitrosourea (14C-MNU). The main target was the N-7 position of guanine, exceeding the methylation in the O6 position of guanine by a factor of 8 and that in the N-3 position of guanine and adenine by a factor of 20. No DNA repair could be observed within 2 hours after methylation. Pretreatment of cells with gamma irradiation (7 rad) before application of MNU induced repair of N-7-methylguanine. This methylation product was decreased to about 50% within two hours, whereas the repair of the other methylated bases was not influenced. The analysis was carried out by high performance liquid chromatography after acid hydrolysis of isolated DNA. 14C-methylated products were determined by liquid scintillation counting.     

Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in vivo with methyl methanesulphonate and NN-dimethylnitrosamine

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection of [(14)C]methyl methanesulphonate (50mg/kg) or NN-di[(14)C]methylnitrosamine (2mg/kg). These doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [(14)C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (H(+) form) and Dowex-50 (NH(4) (+) form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the O-6 position of guanine was a significant feature of rRNA obtained from the NN-di[(14)C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [(14)C]methyl methanesulphonate.     

The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine

1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [¹⁴C]methyl methanesulphonate (50mg/kg) or [¹⁴C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [³H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.     

Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N′-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation

1. In neutral aqueous solution N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) yields salts of nitrocyanamide as u.v.-absorbing products. With cysteine, as found independently by Schulz & McCalla (1969), the principal product is 2-nitràminothiazoline-4-carboxylic acid. Both these reactions liberate the methylating species; thiols enhance the rate markedly at neutral pH values. An alternative reaction with thiols gives cystine, presumably via the unstable S-nitrosocysteine. 2. Thiols (glutathione or N-acetylcysteine) in vitro at about the concentration found in mammalian cells enhance the rate of methylation of DNA markedly over that in neutral solution. 3. Treatment of cultured mammalian cells with MNNG results in rapid methylation of nucleic acids, the extent being greater the higher the thiol content of the cells. Rodent embryo cells are more extensively methylated than mouse L-cells of the same thiol content. Cellular thiol concentrations are decreased by MNNG. Proteins are less methylated by MNNG than are nucleic acids. 4. Methylation of cells by dimethyl sulphate does not depend on cellular thiol content and protein is not less methylated than nucleic acids. Methylation by MNNG may therefore be thiol-stimulated in cells. 5. Both in vitro and in cells about 7% of the methylation of DNA by MNNG occurs at the 6-oxygen atom of guanine. The major products 7-methylguanine and 3-methyladenine are given by both MNNG and dimethyl sulphate, but dimethyl sulphate does not yield O(6)-methylguanine. Possible reaction mechanisms to account for this difference between these methylating agents and its possible significance as a determinant of their biological effects are discussed.     

Origin and formation of 1,7-dimethylguanosine in tRNA chemical and enzymatic methylation

tRNA chemical methylation: 1. 1,7-Dimethylguanosine was found in in vivo methylated tRNA from liver and kidney of rat after exposure to a low dose of dimethylnitrosamine (4 mg/kg body weight). 2. At 4 h after dimethylnitrosamine administration, the 1,7-dimethylguanosine:7-methylguanine ratio (product ratio) for liver and kidney tRNA was 0.017 and 0.091, respectively. At 24 h after dimethylnitrosamine administration, the product ratio was lower in both hepatic and renal tRNA. 3. When dimethylnitrosamine was given in four separate daily injections, the product ratio in hepatic tRNA 4 h after the last dose was the same as for the same total dose given by a single injection, but in renal tRNA it was lower. No dialkyl compound was found in liver and kidney tRNA 24 h after the last multiple injection. tRNA enzymatic methylation: 1. Base analyses of Escherichia coli B tRNA methylated in vitro, by using S-adenosylmethionine as physiological methyl donor and enzyme preparations from liver and kidney of normal rat, indicated that 1,7-dimethylguanosine was also a product of enzymatic methylation. 2. The amount of 1,7-dimethylguanosine formed by kidney enzyme preparation was 3-times that produced by the liver extract. 3. A second type of enzymatic methylation assay where chemically methylated tRNA was used as substrate indicated that the 7-methylguanosine residues in the nucleic acid are not the substrate of the methylase activity forming the 1,7-dimethylguanosine moieties. Analogous data were obtained for the origin of 1,7-dimethylguanosine residues in tRNA chemical methylation by dimethyl sulphate.