Albino Wistar rats (Rattus norvegius) fed semi-purified diets containing 3.5%, 8%, 27%, and 64% casein, respectively, as the protein source, were poisoned with an intraperitoneal dose of 20mg N-nitrosodimethylamine (NDMA)/kg, following cannulation of the bile duct, in vitro, under urethane anaesthesia. Bile exudates was collected at designated time intervals and analysed for unchanged NDMA using thin layer chromatography and gas liquid chromatography methods. Rats on 64% high protein diet (HPD) were the highest excretors of NDMA, followed by rats on the 3.5% kwashiorkorigenic diet (KWD), 8% low protein diet (LPD) and 27% normal protein diet (NDP) as the least excretors, in that order. The corresponding values for culmulative excretions of NDMA were 4.38%, 2.74%, 2.96% and 4.11%, and for elimination rate contents they were 54.05Kh(-1), 23.01Kh(-1), 23.76Kh(-1) and 48.88Kh(-1), while the respective elimination half-life values were 0.013h, 0.031h, 0.029h and 0.014h. The toxicological and pharmacological implication of the pharmacokinetic findings are discussed.

1. [(14)C]Dimethylnitrosamine was administered to rats, and the formation of free and protein-bound S-methylcysteine was investigated. 2. Very little S-methylcysteine was found in liver acid-soluble fraction, in liver protein or in urine. 3. The methylhistidines formed in liver protein were found to have largely disappeared by 6 days after injection of dimethylnitrosamine. 4. The significance of these results in the carcinogenic action of dimethylnitrosamine is discussed.

Dimethylnitrosamine (DMN) has been characterized as a potent hepatotoxin, carcinogen and mutagen. As described below, immunotoxicity should be added to its profile of activity. Although a broad spectrum of immune parameters is affected by DMN, humoral immunity is particularly sensitive. In order for DMN to produce its traditional profile of toxicity it requires metabolic activation to reactive intermediates which alkylate macromolecules. Similarly, DMN also must be metabolized to produce its immunological effects. However, as this review suggests, the metabolism of DMN to an intermediate capable of suppressing the humoral immune response may be qualitatively and/or quantitatively different from that which mediates hepatotoxicity and genotoxicity.

1. The discoveries that pre-treatment with certain compounds could increase the amounts of drug metabolizing enzymes present in the liver and that metabolism could enhance as well as reduce the toxicity of exogenous molecules were important milestones in toxicology. 2. Some clinically important adverse effects (vitamin D deficiency, reduced efficacy of oral contraceptives, interactions with anticoagulants) were found to be due to enzyme induction by, for example, anticonvulsants. 3. Intestinal enzymes are also inducible and can respond rapidly to individual compounds while the liver enzymes respond more slowly to the diet as a whole. Although promoting hepatic tumours in rats and mice, phenobarbitone does not have this effect in man because there seems to be a threshold for promotion which human use does not exceed. In neither case is there evidence that induction is harmful rather than adaptive in man. 4. As to the future, post-marketing surveillance will continue to be important in assessing the safety of new products, and knowledge of the metabolism and pharmacokinetics of new compounds in experimental animals and in man will assume greater importance. Finally, greater understanding of intracellular processes will pave the way to the study of toxicology at the macromolecular level and thus to critically assess the validity of the animal models currently used in toxicity testing.

Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to alpha-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., alpha-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 X g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous work has shown that induction of a high-affinity NADPH-dependent nitrosodimethylamine demethylase (NDMAd) in liver microsomes occurs in rats due to fasting, ethanol consumption, and streptozotocin-induced diabetes. Several lines of observations suggest that this is due to the induction of specific cytochrome P-450 isozymes. Induction of P-450 species by ethanol has also been observed by other investigators. Since each of the above altered metabolic states has in common elevated levels of ketone bodies, the possible role of acetone, a known inducer of NDMAd, in the induction of the demethylase activity was investigated. Levels of endogenous acetone in fasted rats correlated (r = 0.72) with a three- to fourfold increase in NDMAd activity. However, a dose-response experiment showed endogenous levels of acetone to be capable of causing at most 40% of the induction in fasted rats. This suggests that other ketone bodies or factors may have contributed to the induction. The induction of NDMAd by ethanol was enhanced by alcohol dehydrogenase inhibitors pyrazole and acetaldehyde oxime, suggesting that ethanol, rather than its metabolites, was responsible for the induction.

The relationship between dimethylnitrosamine (DMN) demethylase activity and DMN-induced mutagenesis was investigated in Drosophila melanogaster. The activity of DMN-demethylase was at least 10-fold greater in the Hikone-R strain than in three other Drosophila strains. However, the sex-linked recessive lethal (SLRL) mutations induced by DMN in the four strains differed by less than 2-fold. Several possibilities to explain the lack of correlation between DMN-demethylase activity and DMN-induced mutations were tested and eliminated. They include: (i) the presence of inhibitors of DMN-demethylase in extracts of low-activity strains, (ii) a sex bias in the Hikone-R strain in which the enzyme activity is confined to the females, (iii) the possibility that DMN treatment induces DMN-demethylase activity in the low-activity strains and (iv) the possibility that Hikone-R has a much more efficient DNA repair system than the other strains. The results are discussed in terms of what is known about the role of DMN-demethylase in the metabolic activation of DMN in other systems.

1. The effects of CoCl2 administration to rats on xenobiotic metabolism, dimethylnitrosamine (DMN) metabolism to formaldehyde and methanol, and monoamine oxidase (MAO) enzyme activities in hepatic subcellular fractions have been studied. 2. CoCl2 treatment markedly decreased hepatic mixed-function oxidase enzyme activities and microsomal cytochrome P-450 content. In contrast, the N-oxidation of N, N-dimethylaniline and the activity of microsomal NADPH-cytochrome c reductase was unaffected. 3. The metabolism of DMN to formaldehyde by postmitochondrial supernatant fractions was decreased at substrate concn. of 0 . 5, 5 and 50 mM by CoCl2 treatment but the metabolism of 5 and 50 mM DMN to methanol was affected less. 4. CoCl2 had little effect on MAO activities in whole homogenates, but microsomal MAO activities were markedly inhibited. 5. The inhibition of microsomal MAO indicates that CoCl2 is not a specific inhibitor of cytochrome P-450-dependent biotransformations and consequently the inhibition of DMN metabolism is not evidence of a wholly cytochrome P-450-dependent process.

1. The metabolism of dimethylnitrosamine (DMN) to formaldehyde by rat-liver preparations has been studied at substrate concn. of 0.5, 5 and 50 mM and compared with mixed-function oxidase enzyme activities. 2. The microsomal metabolism of low (0.5 and 5 mM) and high (50 mM) substrate concn. of DMN was differentially affected by acetone addition or KI treatment. 3. A series of heterocyclic compounds related to pyrazole were potent inhibitors of metabolism of 0.5 and 5 mM DMN at concn. which had little effect on mixed-function oxidase activities. In contrast, purine addition slightly stimulated the metabolism of low but not high concn. of DMN. 4. The results are consistent with the suggestion that multiple enzymic pathway(s) are involved in hepatic DMN metabolism and that some of these pathway(s) may be independent of cytochrome P-450.

The effects of 3-methylcholanthrene (MC) pretreatment on metabolism and mutagenic activation of dimethylnitrosamine (DMN) were studied with liver subfractions from two strains of mice differing genetically with respect to aromatic hydrocarbon responsiveness. Both mutagenic activation and DMN N-demethylase activity segregated with aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity as a dominant trait in appropriate crosses between C57BL/6J (Ahb Ahb) and DBA/2J (Ahd Ahd) mice. DMN metabolism and mutagenicity were increased by MC-pretreatment in responsive Ahb Ahb and Ahb Ahd mice, but not in non-responsive Ahd Ahd mice. This indicates the involvement of the Ah locus in the genetic regulation of these activities in mice. Deuteration of DMN reduced mutagenicity and DMN N-demethylase activity by approximately 90 and 50 percent, respectively.

The soybean system used for detecting environmental mutagens is analyzed for various types of spots on the leaves of heterozygous y11y11 plants and homozygous y11y11's induced by a nitrosoamine (dimethyl nitrosoamine, DMN) and a nitrosoamide (methyl nitrosourea, MNU). It is shown that the nitrosoamine can be "activated" by the seed (is converted to a true mutagen) without the addition of NADPH or S-9 fraction of the liver homogenate as is necessary in animal tissue culture or bacterial studies. Whereas somatic mosaicism in soybean can be induced with a dose as low as 1.25 ppm of DMN, the upper limit in spot production is reached at around 60 ppm concentration, applied for 0--24 h. Such saturation effect may be due to a limited amount of DMN being converted to true mutagen. MNU, on the other hand, does not show such limitations, perhaps because of its property of being a direct mutagen not necessitating an intermediate step required for converting the promutagen DMN. The frequency of twin spots on Y11y11 leaves increases only slightly by either DMN or MNU, suggesting only a small increase in somatic crossing-over induced by the two chemicals. The yellow spots increase the most, perhaps due to segmental losses carrying Y11 or non-complementary segregation of exchanges involving non-homologous chromosomes. Neither chemical is found capable of mutating y11 to Y11 as seen by the general lack of light green sectors on y11Y11 plants. Usefulness of the soybean system in studying mutagenesis is briefly discussed.

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.

Dimethyl (DMN) and diethyl nitrosamine (DEN) do not give characteristic spectral changes upon interaction with rat liver microsomes, while dipropyl (DPN) and dibutyl (DBN) nitrosamine cause type I spectral changes. The spectral binding constant is 100 mM for DPN and 1.17 mM for DBN. The maximal spectral change is 3.2 X 10(6) and 1.0 X 10(6) absorbance units per milligram protein for DPN and DBN respectively.

The biotransformation of dimethylnitrosamine (DMN) to formaldehyde, generally attributed to the mediation of a demethylase enzyme associated with the microsomal mixed function oxidase system, has been investigated in rat liver preparations. All of the enzyme activity was found in the postmitochondrial fraction and the microsomes contained approximately 50% of this activity. The restoration of total activity resulting from the addition of the cytosol to the microsomal fraction was found to be due to presence of diffusible, heat-labile constituents in the cytosol. Enzyme kinetic studies revealed that DMN was metabolized to formaldehyde by either a multistep or a multicomponent process. DMN demethylase was found to be relatively stable to storage in contrast to cytochrome P-450 and a number of mixed function oxidase enzyme activities. In spectral interaction studies DMN was found to form an atypical interaction spertrum with either control, phenobarbitone-pretreated or phospholipid-depted microsomal preparations. DMN had little effect on Type II spectral interaction of aniline, but noncompetitvely inhibited the Type I spectral interaction of benzphetamine and biphenyl. Whilst the mixed function oxidase enzyme inhibitors SKF 525A and metyrapone markedly reduced the metabolism of ethylmorphine and anailine, DMN demethylase was little affected by the former compound and appreciably enhanced by metyrapone. Moreover, DMN demethylase was strongly inhibited a number of hepatic mixed function oxidases, but significantly reduced anaerobic nitroreductase activity. The results of these studies reveal important differences between the properties of the enzymatic systems which metabolize DMN and mixed function oxidase substrates, and are consistent with the conclusion that the degradation of DMN to formaldehyde by rat liver preparations is a multicomponent system not rate limiting with respect to cytochrome P-450.

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.

1. Aminoacetonitrile, a lathyrogenic agent known to decrease the hepatotoxic action of dimethylnitrosamine, inhibited the metabolism of this compound by rats in vivo and by rat liver slices in vitro. 2. Methylation of nucleic acids in rat liver and kidney by dimethylnitrosamine in vivo was inhibited by treatment of the animals with aminoacetonitrile. 3. These findings are discussed in relation to the hypothesis that dimethylnitrosamine requires metabolism to exert its hepatotoxic and carcinogenic action.