Metabolism Of Dimethylnitrosamine Research Articles

Enhanced mutagenic activities of N-nitroso compounds in weakly acidic media.

Mutagenesis induced by a number of N-nitrosamines in a liquid phase assay was enhanced 2-8 fold on going from a pH of 7.4 to 6.5, with the degree of enhancement dependent on the compound and the reaction conditions. In general, enhancement was greatest at lower concentrations of mutagen and with compounds of lower molecular weights. With dimethylnitrosamine (DMN) part of this effect was due to an enhanced rate of metabolism at low concentrations of DMN at a pH of 6.5. However, even at higher concentrations of DMN, mutagenesis was more effective at the lower pH despite the fact that metabolism of DMN was twice as rapid at a pH of 7.4 than 6.5. Directly acting mutagens such as N-nitroso-N-methylurea and N-methyl-N'-nitro-N-nitrosoguanidine were 2-10 times more potent at pH values of between 5.5 and 7.0 than at 7.4. No enhancement in the mutagenic activities of methyl methanesulfonate, benzo(a)pyrene or 3-methylcholanthrene was observed on going from a pH of 7.4 to 6.5.

Mutagenicity and metabolism of dimethylnitrosamine and benzo[α]pyrene in tissue homogenates from inbred syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[α]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SsLak, LSH/SsLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. Dimethyl-nitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[α]pyrene as substrate. MC does not induce AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC.

Role of dimethylnitrosamine-demethylase in the metabolic activation of dimethylnitrosamine

In vivo administration to rats of the mixed-function oxidase modifiers 3-methylcholanthrene (MC), pregnenolone-16α-carbonitrile (PCN) or β-naphthoflavone (β-NF) inhibits the hepatic microsome-catalyzed in vitro binding of dimethylnitrosamine (DMN) to DNA. This parallels their effect on DMN-demethylase I, regarded to be the sole activating step in DMN carcinogenesis and fails to account for the previously observed anomaly that MC and PCN inhibit, while β-NF enhances, the hepatocarcinogenic activity of DMN. The in vitro binding of DMN is clearly dependent on microsomes and NADPH, and is strongly enhanced by soluble cytoplasmic proteins; the presence of the latter has no effect. however, on the relative response to pretreatment by the modifiers. In mice β-NF enhances and PCN inhibits DMN-demethylase I; β-NF has no effect on either the cytochrome P-450 level or on the LD 50, while PCN strongly increases the cytochrome P-450 level but without influencing the LD 50. Neither of the two modifiers has any effect in mice on the host-mediated mutagenicity of DMN in a dose-response study, except for the highest dose of DMN (200 mg/kg) where PCN pretreatment significantly enhanced mutagenicity. To account for the anomalous observations, other potential pathways of DMN metabolism have been explored. Whole rat liver nuclei or isolated nuclear membrane fractions contain no DMN-demethylase or diethylnitrosamine-deethylase activity. In a microsomal mixed-function amine-oxidase assay system neither purified enzyme preparations nor whole microsomes catalyze NADPH oxidation in the presence of DMN as substrate. In addition, the purified enzyme does not catalyze formaldehyde production in the DMN-demethylase assay system. Benzylamine, a typical inhibitor of mitochondrial monoamine oxidase (MAO), is a potent inhibitor of DMN-demethylase activity, but microsomes are devoid of MAO activity. Furthermore, purified MAO has no DMN-demethylase activity. The differential effect of modifiers on the carcinogenicity of DMN probably involves pathways other than DMN metabolism.

Methylation of neuronal and glial macromolecules by methylnitrosourea and dimethylnitrosamine in vivo

Rats were injected i.p. with radioactive methylnitrosourea and dimethyl-nitrosamine and neuronal and glial cell fractions separated from brain tissue after 4 h, 72 h and 10 days. DNA, RNA and protein were purified and specific radioactivity was determined. At all labelling times with methylnitrosourea neuronal nucleic acids were methylated to higher (1.5–2.0:1) specific radio-activity than glial nucleic acids. 7-Methylguanine appeared to be the major purine adduct and O 6-methylguanine a minor adduct in the two cell fractions. The proportion of O 6-methylguanine was equal in the two cell fractions. With dimethylnitrosamine, glial macromolecules were methylated to a greater extent than neuronal macromolecules. Neuronal cells appear to be inactive in the metabolism of dimethylnitrosamine. The level of methylation in brain tissue by dimethylnitrosamine was of a lower order than in liver tissue.

Near quantitative production of molecular nitrogen from metabolism of dimethylnitrosamine

Molecular nitrogen is produced stoichiometrically from the spontaneous decomposition of N-methyl-N-nitrosourea in phosphate buffer and is readily detected by gas chromatography. It is also generated in high yield from the oxidation of dimethylnitrosamine by mouse or rat liver microsomes. Gas chromatographic analysis for nitrogen generated from dimethylnitrosamine was more difficult because the amount of nitrogen generated was similar to that produced in control tubes which contained no dimethylnitrosamine.

Comparison of the in vitro mutagenicity and metabolism of dimethylnitrosamine and benzo[ a]pyrene in tissues from inbred mice treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls

Homogenates of liver, lung, kidney, stomach, small intestine and colon from 8 strains of mice were compared for their ability to metabolize benzo[ a] rmpyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains CF1, AKR/J, AU/SsJ, DBA/2J, SWR/J, A/J, C3H/HeJ, and C57BL/6J were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. The effects of these drugs on organ weight and on the amounts of DNA, S-10 protein, and microsomal protein per unit weight of tissue are reported. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. For each organ there was an optimal balance between amount of tissue homogenate and concentration of test compound for maximal yield of revertants. A sensitive radiometric assay of DMN demethylase (DMND) is described which permits measurement of the enzyme in liver, lung and kidney. DMN at 1 mM is used as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured in all tissue using BP as substrate. AR and MC are very good inducers of AHH activity in livers of mice classified as aromatic hydrocarbon responsive, but not in those classified as hydrocarbon nonresponsive. Responsiveness is strain-specific and genetically regulated. Metabolism of BP to mutagens by liver homogenates was correlated with extent of AHH induction. This dimorphism of response of AHH to inducers was present, but less pronounced, in non-hepatic tissues. Basal activities of AHH and DMND were correlated in livers and lungs from untreated mice. DMND activities were increased less than 2-fold by PB, MC or AR treatments. Metabolism of DMN to mutagens was not closely correlated with DMND activities. Strain of mouse, type of tissue and test substance are important variables in assessing the potential effect of microsomal enzyme-inducing agents on the metabolism of mutagenic substances.

Inhibition of rat hepatic dimethylnitrosamine demethylase by cyclic and acyclic nitrosamines and secondary amines.

Inhibition of hepatic dimethylnitrosamine (DMN) metabolism by a variety of cyclic and acyclic nitrosamines was demonstrated. Nitrosoproline, a noncarcinogenic nitrosamine, behaved differently from the carcinogenic nitrosamines as an inhibitor for DMN-demethylase. Secondary amines corresponding to the nitrosamines inhibited DMN-demethylase in a manner similar to the nitrosamines.

Mechanism of dimethylnitrosamine metabolism and activation in rats.

Mechanism of dimethylnitrosamine metabolism and activation in rats.

Corrections

In “Deuterium Isotope Effect in the Microsomal Metabolism of Dimethylnitrosamine” by Doron Dagani and Michael C. Archer (57:955–957), a numerical error has been discovered in the data reported on page 956, text-figure 1, which changes three of the kinetic parameters. The correct Km value for DMN-d6 is 110 mm (not 74 mm). The previously reported Km value of 120 mm for DMN remains unchanged. The correct Vmax values for DMN and DMN-d6 are 13.0 and 4.7 nmoles HCHO/mg protein/minute, respectively (not 20 and 5.2 nmoles). KHm/KDm is therefore 1.1 (not 1.6), and VHmax/VDmax is 2.8 (not 3.8). These changes affect neither the conclusions nor the significance of the original paper.

The distribution of [ 14C]dimethylnitrosamine in mice. Autoradiographic studies in mice with inhibited and noninhibited dimethylnitrosamine metabolism and a comparison with the distribution of [ 14C]formaldehyde

The distribution of [ 14C]dimethylnitrosamine was studied by wholebody autoradiography in mice. By applying low temperature autoradiography and autoradiography with dried tape sections in mice with inhibited (by pretreatment with pyrazole, ethanol, or nialamide) and noninhibited dimethylnitrosamine metabolism, a distinction was possible between the distribution of the volatile nonmetabolized dimethylnitrosamine and the nonvolatile metabolites. The results indicated a uniform distribution of the nonmetabolized dimethylnitrosamine in the body. At the short survival intervals, metabolites were trapped primarily in the liver and also to a low degree in the kidneys, probably reflecting the alkylating reactions and other reactions connected with the breakdown of dimethylnitrosamine. At the long survival intervals, metabolites also occurred in tissues with a rapid cell turnover and a high rate of protein synthesis. Since autoradiography of [ 14C]formaldehyde gave this distribution picture, incorporation of radioactivity from [ 14C]dimethylnitrosamine via [ 14C]formaldehyde in the one-carbon pool probably explains the labeling of these tissues. Pyrazole, ethanol, and nialamide strongly depressed the incorporation of radioactivity from [ 14C]dimethylnitrosamine into the acid-insoluble material of the liver. In addition, the pretreatments caused a strong inhibition of the exhalation of 14CO 2 from [ 14C]dimethylnitrosamine.

Stimulation of microsomal dimethylnitrosamine- N-demethylase by pretreatment of mice with acetone

To further investigate the relationship between in vivo microsomal enzyme modifiers and in vitro dimethylnitrosamine (DMN) metabolism, male C57BL/6J mice were pretreated with acetone or Aroclor 1254, two compounds known to influence DMN- N-demethylase activity. Pretreatment with acetone enhanced the in vitro microsomal activity of DMN- N-demethylase, as measured by formaldehyde production from DMN. Accompanying this acetone-enhanced demethylase activity was an increase in the covalent binding of [ 14C] DMN to RNA, protein, and DNA. Four distinct K m values dependent on the substrate concentration were observed for the N-demethylase present in control microsomes. Only one K m value was observed for the demethylase in microsomes from acetone-treated animals, but it was significantly lower than the lowest K m observed in the control microsomes. At DMN concentrations of 1 and 10 mM, acetone significantly increased N-demethylation of DMN as compared to control, but not at 100 mM DMN. Aroclor 1254 pretreatment repressed DMN- N-demethylase at 1 mM DMN but enhanced it at 100 mM. These results suggest that there may be multiple forms of DMN- N-demethylase which are dependent on DMN concentration and respond differently to modifiers of the microsomal drug-metabolizing enzymes.

The influence of DDT on metabolism of dimethylnitrosamine in rats

The rate of in vitro metabolism of dimethylnitrosamine (DMNA) in liver cell preparations from rats given ip injections of DDT (50 mg/kg every second day for two, four, or eight doses) or a single ip injection of 3-methylcholanthrene (100 mg/kg) was compared with the rate of preparations obtained from control rats. The activity of O-demethylase in liver postmitochondrial supernatant was measured as an overall indicator of microsomal enzyme induction. Treatment of rats with DDT two or four times or with 3-methylcholanthrene caused only slight enhancement of the DMNA metabolism rate. Significant differences were observed after eightfold DDT administration. O-Demethylase activity was significantly different among all treated groups, and activity in all treated groups was significantly different from that of the controls. However, there was no correlation between the DMNA metabolism rate and the O-demethylase activity. In vivo studies were carried out to measure the rate of the DMNA disappearance from the blood of rats treated with DDT in the same manner as in the in vitro experiment. DMNA was administered ip to rats at 40 mg/kg 24 hr after the last dose of DDT. No DMNA was detected in the blood of the DDT-induced rats 8 hr after it was injected.

Oxidative metabolism of dimethylnitrosamine: correlation with toxicity.

The relationship between oxidative metabolism and acute toxicity of dimethylnitrosamine (DMN) was examined in neonatal and adult rats to take advantage of developmental changes in activity of the metabolizing enzymes. The objective was to determine the extent to which CO2 production from DMN is correlated with toxicity. Neonatal rat liver and kidney demonstrated the ability to metabolize DMN. This ability progressed to a maximum activity in liver between the 5th and 21st d of age and in kidney between the 15th and 21st d of age. After weaning, the activity of DMN oxidation decreased with age in both tissues. Evidence is also presented to suggest that more than one enzyme may be responsible for the oxidation of DMN to CO2 and that the predominance of individual enzymes varies as the neonate develops. Estimates of LD50 values, used to quantitate the acute toxicity of DMN at various ages, suggest that the rat is most sensitive to DMN toxicity at 5 d of age; however, conversion of DMN to CO2, both in vitro and in vivo, was not well correlated with acute toxicity.

ニトロソアミンの生体内運命と代謝活性化

The biological fate of nitrosamines, known as environmental carcinogens, and their metabolic activation in biological system are reviewed. This article includes the absorption, distribution, metabolism and excretion of dimethylnitrosamine, alkylation of nucleic acid bases with nitrosamines, metabolic activation of dialkylnitrosamines to produce alkyl cations as alkylating agents via α-hydroxylation, and the effects of substituents of α-carbon of dialkylnitrosamines on their biological activities. Emphasis has been placed to α-acetoxyalkylnitrosamines as masked compounds of α-hydroxylated active species. Their chemistry, carcinogenicity, and mutagenicity are compared. Organospecific carcinogenicity of dibutylnitrosamine and N-butyl-N-(4-hydroxybutyl) nitrosamine, urinary bladder carcinogens, are explained in terms of metabolism and metabolic activation.

Effects of thiamine deficiency on the metabolism and acute toxicity of dimethylnitrosamine in the rat

Effects of thiamine deficiency on the metabolism and acute toxicity of dimethylnitrosamine in the rat

Further studies on the metabolism of dimethylnitrosamine by rat liver in vitro [proceedings

Further studies on the metabolism of dimethylnitrosamine by rat liver in vitro [proceedings

Effects of pyrazole and 3-amino-1,2,4-triazole on the metabolism and toxicity of dimethylnitrosamine in the rat.

Pretreatment of rats with pyrazole or 3-amino-1,2,4-triazole (3-AT) known inhibitors of alcohol metabolism, profoundly inhibited the metabolism of dimethylnitrosamine (DMN), both in terms of [14C]CO2 excretion and of the decline in the blood concentration. Additionally, 4-methylpyrazole, tetraethylthiuram disulfide (disulfiram), methanol, and ethanol inhibited the metabolism of DMN in the whole animal. In parallel experiments with [14C]aminopyrine, no substantial inhibitory effect was found with pyrazole, 3-AT, or disulfiram pretreatment. Investigations into the effects of pyrazole and 3-AT pretreatment on the acute toxicity and hepatotoxicity of DMN showed that pyrazole significantly increased the median lethal dose (LD50) of DMN and provided substantial protection against the hepatotoxicity of DMN, in that centriblobular necrosis was not seen at dose levels of DMN up to 25 mg/kg and early histochemical changes indicative of liver injury were not observed at a dose level of 15 mg DMN/kg. In contrast, 3-AT pretreatment did not affect the LD50 of DMN or provide any protection against the hepatotoxicity of DMN. Further, although both inhibitors delayed the incorporation of radioactivity from [14C]DMN into hepatic subcellular organelles, pyrazole was significantly more effective than was 3-AT.

Inhibition of Dimethylnitrosamine-Induced Strand Breaks in Liver DNA and Liver Cell Necrosis by Diethyldithiocarbamate 2

Diethyldithiocarbamate (DEDTC) prevented dimethylnitrosamine (DMN)-induced strand breaks in liver DNA and liver cell necrosis in male Wistar rats. In contrast, DEDTC did not inhibit the fragmentation of liver DNA caused by several other chemical carcinogens (N-hydroxy-2-acetylaminofluorene, 3-hydroxyxanthine, aflatoxin B1, N-acetoxy-2-acetylaminofluorene, methyl methanesulfonate, methylnitrosourea, and methylazoxy-methanol acetate), whether or not they required metabolic activation. Aminoacetonitrile exerted an action similar to that of DEDTC. The inhibitory effect was transitory, lasting at least for 4 hours, and protection for longer than 4 hours required multiple administrations of DEDTC. DEDTC also inhibited the serum clearance of DMN, methylation of liver DNA, and oxidative demethylation of DMN in the in vitro hepatic microsomal system prepared from either male Wistar rats or from hamsters. Interference of the metabolism of DMN appeared to be the mechanism by which DEDTC arrested DMN-induced biochemical and biologic effects.

Influence of lead nitrate oń dimethylnitrosamine intoxication

The effect of lead nitrate, an inhibitor of the hepatic drug-metabolizing enzyme system upon the acute hepatotoxicity of dimethylnitrosamine (DMN) was studied. Lead pretreatment significantly prevented polysomal disaggregation induced by the nitrosamine. Cell necrosis, evaluated morphologically and by the release of serum glutamic-pyruvic transaminase (GPT), was also diminised. The metabolism of DMN in rats pretreated with lead nitrate was investigated by following its clearance from blood and by determining, in vitro the demethylation of the nitrosamine. Lead increased, although not significantly, the clearance of DMN from blood, but it lowered the activity of DMN-demethylase 24 h after its administration. Finally, lead lowered the lethal effects of DMN. The mechanism by which lead influenced DMN toxicity is discussed.

Effect of pregnenolone-16α-carbonitrile on the metabolism of dimethylnitrosamine and binding to rat liver macromolecules

Effect of pregnenolone-16α-carbonitrile on the metabolism of dimethylnitrosamine and binding to rat liver macromolecules