Dimethylnitrosamine Demethylase Activity Research Articles

In vivo potentiation of 1,2-dibromoethane hepatotoxicity by ethanol through inactivation of glutathione- s-transferase

In the rat, a single ethanol (EtOH) pretreatment (2.5 g/kg b.w., per os) was able to strongly enhance the cytotoxicity of 1,2-dibromoethane (DBE) (87 mg/kg b.w., per os). The principal metabolic routes of DBE involve both oxidative and conjugative transformations. Microsomal cytochrome P 450 content and dimethyl nitrosamine demethylase activity were not changed, while a significant loss of cytosolic total GSH-transferase was observed in rats killed 6 h after EtOH pretreatment. Pretreatment with methylpyrazole, an inhibitor of alcoholdehydrogenase prevented the effects provoked by ethanol. The major EtOH metabolite, acetaldehyde, seemed thus to play a fundamental role in the mechanism responsible for the potentiation of DBE toxicity mediated by EtOH. To further support this hypothesis, disulfiram (75 mg/kg b.w.), an inhibitor of aldehyde dehydrogenase, was given i.p. to rats. When DBE was administered to disulfiram- and EtOH-pretreated rats, a marked increase of liver cytolysis was shown and cytosolic GSH-transferase activity was further inhibited if compared to that induced by EtOH treatment alone. The results are consistent with the hypothesis that EtOH given to rats increases DBE liver toxicity because its major metabolite, acetaldehyde, reduces the DBE conjugation to GSH transferase, with consequent shift of DBE metabolism to the oxidative route and accumulation of reactive oxidative intermediates no longer effectively conjugated with GSH.

Effects of dietary restriction on induction of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) in rat liver

The effects of dietary restriction on the induction of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) were studied in the hepatocytes of F344 rats exposed in vivo to dimethylnitrosamine (DMN) or CCl 4. The animals were given food ad libitum, a restricted amount of food (4 g/rat/overnight) or no food. Hepatocytes were isolated 2 h after oral administration of DMN at a dose of 5 mg/kg body weight and 48 h after oral administration of CCl 4 at a dose of 400 mg/kg body weight, and incubated for 4 h in Williams' medium E supplemented with either [ 3H]thymidine for UDS or 5-bromodeoxyuridine for RDS. UDS was determined by autoradiography and RDS was determined by the immunoenzymatic staining method. The background levels of UDS (net grains/nucleus) and RDS (cells in S phase) in control were −12.4 and 0.64% for ad libitum feeding, −6.8 and 0.04% for restricted feeding, and −8.1 and 0% for fasting. UDS induced by DMN and RDS induced by CCl 4 were 19.4 and 3.3% for ad libitum feeding, 34.5 and 10.4% for restricted feeding, and 47.8 and 15.1% for fasting. DMN demethylase activity in rat liver was also found to increase with dietary restriction. These results indicate that dietary restriction modulates the responses of UDS and RDS in the liver of rats.

Importance of hepatocyte culture conditions in dimethylnitrosamine-induced suppression of antibody response in the mixed cultures of murine hepatocytes and splenocytes

The role of the hepatocyte culture media in dimethylnitrosamine (DMN)-induced suppression of antibody responses by splenocytes against sheep erythrocytes (SRBCs) was investigated using the mixed culture system of murine hepatocytes and murine splenocytes. It was observed that hormone-supplemented complete media was required for hepatocyte cultures to optimally activate DMN to its immunosuppressive form(s). In the absence of the hormone supplement, the concentration of DMN required to produce a 50% suppression (IC 50) was increased by over 10-fold (i.e., compare the IC 50 in complete media of <0.5 mM to the IC 50 in basal media of almost 10.0 mM). In contrast, the activation of cyclophosphamide (cytoxan, CTX), which was used in these studies as a comparative control, was not affected by the absence of the hormone supplement. These results indicate that the observed effect on the activation of DMN was not due to a generalized loss of metabolic capability of hepatocytes cultured without hormones. To examine the role of drug metabolizing capabilities of hepatocytes in the differential activation of DMN, we compared phase I and phase II enzyme activities of hepatocytes cultured for 24 h in either basal media or hormone-supplemented complete media. Our results indicated that there was a significant decrease of phase I monooxygenase activities of cultured hepatocytes when compared to freshly isolated hepatocytes. However, our results failed to show any difference in the activities of hepatocytes cultured in the two media. Most notably, there was no difference in the activity of either high-or low-affinity DMN demethylase, as measured by the generation of formaldehyde. We observed a similar profile with phase II conjugative capabilities, specifically sulfotransferase and glucuronyltransferase. These results indicate that the activation of DMN to its immunosuppressive form(s) can be modulated in the co-culture system by culturing hepatocytes under different conditions. Because we failed to show any differences in the metabolic capabilities of hepatocytes cultured under the two media conditions, the results suggest that the modulation of immunosuppressive activity may not be related to a change in the generation of the immunosuppressive metabolite(s).

Increased Oxidation of Dimethylnitrosamine in Pericentral Microsomes after Pyrazole Induction of Cytochrome P‐4502El

The cytochrome P-450IIE1 (CYP2E1) isozyme activates several toxins and procarcinogens. Recent studies employing immunohistochemical and immuno-analysis techniques have shown that this isozyme is predominantly localized in the pericentral zone of the liver acinus. Experiments were conducted to evaluate whether microsomes isolated from the pericentral region of the liver display elevated catalytic activity towards effective substrates for CYP2E1 such as dimethylnitrosamine (DMN) as compared with periportal microsomes. Rats were treated with pyrazole to induce CYP2E1 and hepatocytes prepared from periportal or pericentral zones of the livers by the digitonin-collagenase procedure. Microsomes isolated from these hepatocytes had similar total P-450 contents; however, the microsomes from the pericentral hepatocytes displayed an increased DMSO binding spectrum suggesting an increased content of CYP2E1. Low Km DMN demethylase activity (but not high Km activity) as well as the oxidation of aniline and p-nitrophenol were 2- to 3-fold higher in pericentral compared to periportal microsomes. The oxidation of DMN by both microsomal preparations, as well as the increased rates obtained with the pericentral microsomes, was sensitive to inhibition by carbon monoxide as well as to other CYP2E1 substrates such as ethanol, pyrazole, or 4-methylpyrazole. Anti-CYP2E1 IgG inhibited the oxidation of DMN by both microsomal preparations 75% to 85% and prevented most of the increase found with the pericentral microsomes. Oxidation of aniline and p-nitrophenol was elevated in pericentral hepatocytes compared with periportal hepatocytes to the same extent as in the isolated microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct involvement of human gastric mucosa in the activation of alimentary pro-carcinogens

Exposure to N-nitroso-compounds and aromatic amines, xenobiotics which require an activation in order to exert their genotoxic potential, is causally associated with gastric cancer. We evaluated the capacity of microsome-containing fractions from human gastric mucosa to activate two model carcinogenic compounds. A 9,000 x g supernatant (S9) was obtained from gastric mucosal specimens and, for comparison, from human liver and aroclor-induced and non induced rat liver. The capacity of the S9 to activate N-nitrosopyrrolidine (NPY) and 2-aminofluorene (2AF) to mutagenic metabolites was tested in the Ames/Salmonella reversion assay, while dimethylnitrosamine (DMN) and aminopyrine (AP) demethylation activities were spectrophotometrically evaluated by using an enzymatic assay of the amount of formaldehyde released following the enzymatic demethylation of the corresponding substrates. Results indicate that human gastric S9 fractions may activate 2AF to a genotoxic derivative and are characterized by DMN and AP demethylase activities higher (p < 0.05) than those of human liver, when expressed in mg/protein (p < 0.05). Although the parameters evaluated can only be considered as a partial measure of the general activating capacity toward dietary and environmental procarcinogens, these results suggest that human gastric mucosa may be directly involved in the metabolic activation of these compounds to mutagenic/carcinogenic species.

Immunochemical localization and functional characterization of cytochrome P450IIE1 in rat hepatocyte foci and nodules

P450IIE1 was studied in rat hepatocyte foci and nodules from male Wistar rats, treated for 7 weeks with N-nitroso-morpholine (20, 40 and 80 mg/ml of drinking water). Livers were examined after 15, 23 and 31 weeks. Using specific anti-P450IIE1 IgG, different phenotypes of P450IIE1-altered foci were observed: (i) positive foci, predominant at early times and at the two lower dosages, (ii) negative foci and (iii) mixed-type foci consisting of P450IIE1-positive and -negative hepatocytes which were preponderant at 31 weeks. Immunoblotting of microsomes from livers containing foci and nodules obtained at week 31 of the experiment revealed a decrease in P450IIE1 level, which was correlated to decreased high affinity dimethylnitrosamine demethylase activity. The results suggest phenotypic heterogeneity of P450IIE1-altered foci with predominantly negative foci at later stages.

Rapid decrease of cytochrome P-450IIE1 in primary hepatocyte culture and its maintenance by added 4-methylpyrazole

Studies were conducted to evaluate the possible induction or the maintenance of cytochrome P-450IIE1 in primary hepatocyte cultures by the inducing agent 4-methylpyrazole. Hepatocytes were isolated from control (noninduced) rats and from rats treated in vivo with either pyrazole or 4-methylpyrazole to induce P-450IIE1. The content of P-450IIE1 was determined by Western blots with antipyrazole P-450 IgG, and catalytic activity was assessed by assays of dimethylnitrosamine demethylase activity. The treatment with 4-methylpyrazole in vivo increased the content of P-450IIE1 and dimethylnitrosamine demethylase activity sevenfold and fourfold, respectively. In cultures prepared from noninduced hepatocytes, P-450IIE1 levels fell to values of 76%, 65%, 31% and 1% of freshly isolated hepatocytes after 1, 3, 6 and 9 days in culture. A similar decrease in dimethylnitrosamine demethylase was observed during this time. In cultures prepared from induced hepatocytes, the decline in P-450IIE1 was more rapid as levels fell to 77%, 31%, 3% and 3% of initial values after 1, 3, 6 and 9 days in culture. Again, the fall in dimethylnitrosamine demethylase activity paralleled the decline in content of P-450IIE1 and was more rapid with the induced hepatocytes. With cultures prepared from noninduced or induced hepatocytes, the addition of 4-methylpyrazole in vitro did not increase the content of P-450IIE1 or the activity of dimethylnitrosamine demethylase over the initial values. However, 4-methylpyrazole appeared to stabilize the P-450IIE1 and to decrease its rate of decline in culture. In noninduced cultures, the percent remaining content of P-450IIE1 after 6 days was 31% in the absence of and 52% in the presence of 5 mol/L 4-methylpyrazole. In cultures from 4-methylpyrazole-induced hepatocytes, the percent remaining P-450IIE1 after 3 days was 31% in the absence of inducer and 59% with 4-methylpyrazole added in vitro. Similarly 4-methylpyrazole helped to prevent the rapid decline of dimethylnitrosamine demethylase activity in induced and noninduced cultures. Viability of the induced and noninduced cultures in the absence or presence of added 4-methylpyrazole was similar. Levels of mRNA for P-450IIE1 were similar for livers from control rats and from rats treated in vivo with 4-methylpyrazole. The mRNA levels rapidly declined in induced and noninduced cultures, and this decline, unlike the fall in P-450IIE1 or dimethylnitrosamine demethylase activity, could not be prevented by the addition of 4-methylpyrazole in vitro to the cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Normal and Pathological Human Liver Dimethylnitrosamine Demethylase Activity and Capacity for 2-Aminofluorene and Nitrosopyrrolidine Activation in vitro

Normal and Pathological Human Liver Dimethylnitrosamine Demethylase Activity and Capacity for 2-Aminofluorene and Nitrosopyrrolidine Activation in vitro

Re-evaluation of the effect of ellagic acid on dimethylnitrosamine mutagenicity.

Dimethylnitrosamine (DMN) is activated to mutagenic species in the Ames test (Salmonella typhimurium strain TA100) by hamster hepatic S9 preparation. This S9 activity is induced by administration of ethanol to the animals. The organic solvents dimethylsulphoxide (DMSO) and N-methyl-2-pyrrolidinone (MP) inhibit this mutagenicity, apparently because they inhibit DMN demethylase activity (assayed as formaldehyde production). Ellagic acid, dissolved in DMSO or MP, had no inhibitory effect on DMN mutagenicity, beyond the effect of the solvent vehicle.

Regulation of insecticide resistance-related cytochrome P-450 expression in Drosophila melanogaster

Two protein bands, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, account for most of the cytochrome P-450 in Drosophila melanogaster. P-450-A is ubiquitous among strains tested; whereas P-450-B is unique to certain strains. Dimethylnitrosamine demethylase activity is associated with P-450-B. Biochemical and genetic analyses showed that genes located on chromosome II were required for P-450-B expression. These genes were mapped within an interval that includes a major insecticide-resistance locus. Regulatory loci on chromosome III were required for maximum expression of P-450-B. One of these regulatory loci was mapped at another major resistance locus on chromosome III. There was a good correlation between P-450-B expression and resistance to phenylurea among the different strains tested. These results indicate that Drosophila can be a useful model to study the molecular mechanisms of insecticide resistance.

Enzymatic activities of human lung tissue: relationship with smoking habits.

Twenty-two S12 preparations of surgical lung specimens obtained from smoker and non-smoker cancer patients were assayed to detect aryl hydrocarbon hydroxylase (AHH), dimethylnitrosamine demethylase (DMND), and glutathione-S-transferase (GST) activities, in both normal and neoplastic lung tissue from the same patients. Pulmonary fractions were also tested for their ability to activate some precarcinogens into mutagenic metabolites in the Ames test. Statistically significant differences were found for AHH and DMND activities between normal and neoplastic tissue of smoker patients. In addition, higher AHH activity in the neoplastic tissue of the smoker group was observed compared with that found in the non-smoker group. No differences were found for GST activity. All the lung S12 preparations were able to metabolize water-soluble bases and water-insoluble bases, derived from main-stream cigarette smoke condensate, into mutagenic agents in the Salmonella test system. However, S12 preparations from smoker group neoplastic tissues were more effective.

Enzyme induction in rat lung and liver by condensates and fractions from main-stream and side-stream cigarette smoke

Aryl hydrocarbon hydroxylase (AHH) and dimethylnitrosamine demethylase (DMND) activities in pulmonary and hepatic tissues of male Sprague-Dawley rats were assayed following pretreatment with known inducers (benzo[a]pyrene, 3-methylcholanthrene, Aroclor 1254, phenobarbital) and with main-stream (MS) and side-stream (SS) cigarette smoke condensates and their related fractions. Biochemical assays by spectrophotofluorimetry (AHH activity) and spectrophotometry (DMND activity) and by a biological assay (Ames test) were performed to detect AHH and DMND induction. Ames test proved to be much less sensitive than the spectrophotofluorimetric analysis for AHH determination. Both main-stream and side-stream cigarette smoke condensates and some fractions, containing water-soluble bases, water-insoluble bases, and polycyclic aromatic hydrocarbons, were found to induce AHH activity in lung and liver, the lung being induced to the greatest extent. The highest levels of AHH inducibility were found for the SS-smoke condensate and related fractions. In particular, the insoluble bases fractions gave the highest induction. On the contrary, pulmonary DMND activity was not affected by pretreatment with the same materials, while hepatic DMND response was only minimally induced by Aroclor and phenobarbital treatment.

Effects of pretreatment with pyrazole and inducers of mixed function oxidases on DNA repair elicited by dimethylnitrosamine in rat hepatocytes in vivo and in vitro

Experiments were performed to investigate the relationship between the rate of oxidative metabolism of dimethylnitrosamine (DMN) by rat liver microsomes (i.e., DMN demethylase activity, DMNd) and its genotoxicity in liver, as assessed by the in vitro and in vivo/in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assays. Pretreatment of rats with pyrazole (PYR) resulted in a 4-fold increase in DMNd and a 3-fold greater DNA repair response to in vivo administration of 5 mg DMN/kg body weight. Pretreatment with phenobarbital (PB), dichlorodiphenyltrichloroethane (DDT), 3-methylcholanthrene (3-MC), β-naphthoflavone (β-NF) or Aroclor 1254 (ARO) produced a variable degree of inhibition of DMNd and had no significant effects on the response to DMN in the in vivo/in vitro HPC/DR assay. DNA repair elicited by DMN in vitro was decreased in hepatocytes from rats pretreated with 3-MC, while PB, DDT, β-NF and ARO pretreatments had little effect on the response. In contrast, PYR pretreatment produced a 4.5–6.7-fold increase in the in vitro DNA repair response to DMN, and extended detection of positive responses to lower concentrations. Most of the inducers had no effect on DNA repair elicited by the direct acting alkylator, methyl methanesulfonate (MMS). Thus, the pretreatment-related changes in DMN-induced DNA repair were probably due to alterations in DMNd rather than to effects on the DNA repair capacity of the hepatocytes.

Factors influencing the activity of dimethylnitrosamine demethylase in hamster, rat and chicken liver microsomes

In vitro metabolism of dimethylnitrosamine (DMN) by liver microsomal fractions of hamster, rat and chicken revealed that the three species under certain assay conditions, were capable of metabolizing DMN at different rates (hamster > rat > chicken). The magnitude of the demethylase activity was found to be dependent on (a) the nature of the buffer, (b) the concentration of cytochrome P-450 ( P-450) and (c) the concentration of the substrate DMN. Enzyme activity was higher in Hepes buffer than in the phosphate buffer. Concentrations of phosphate higher than 20 mM inhibited the activity of the rat and chicken enzymes. This effect of phosphate was not a consequence of increase in ionic strength since KCl over a wide range of concentration failed to inhibit the activity.

Haloacetonitrile excretion as thiocyanate and inhibition of dimethylnitrosamine demethylase: a proposed metabolic scheme.

Haloacetonitriles, contaminants present in chlorinated drinking water, were administered orally to rats, and the urinary excretion of thiocyanate was measured as an index of cyanide release. The urinary excretion of thiocyanate accounted for 14.2% of the dose of monochloroacetonitrile; 7.7-12.8% of the dose of bromochloro-, dichloro-, and dibromoacetonitrile; and 2.25% of the dose of trichloroacetonitrile. The haloacetonitriles inhibited rat-liver microsomal dimethylnitrosamine (DMN) demethylase in an in vitro assay system. Dibromo- and bromochloroacetonitrile were the most potent inhibitors of DMN demethylase, with Ki = 3-4 X 10(-5) M; dichloro- and trichloroacetonitrile were the next most potent, with Ki = 2 X 10(-4) M; and monochloroacetonitrile was the least potent inhibitor, with Ki = 9 X 10(-2) M. Trichloroacetonitrile, but not dibromoacetonitrile, when administered orally inhibited hepatic DMN demethylase activity. The relative capacity of the haloacetonitriles to inhibit DMN demethylase and to be excreted as thiocyanate did not correlate.

Induction of microsomal dimethylnitrosamine demethylase by pyrazole

Pyrazole, a potent inhibitor of alcohol dehydrogenase, was found to be a potent inducer of the activity of low (iK m) dimethylnitrosamine demethylase (DMN-d). One injection of pyrazole (200 mg/kg body wt) to weanling Wistar rats changed the microsomal DMN demethylase activity by 1.7, 1.9 and 2.5 times the control values at 6, 12 and 24 hr after the injection respectively. Pyrazole administration reduced arylhydrocarbon hydroxylase (AHH) activity. When animals were injected with pyrazole (200 mg/kg body wt) for 1, 2, 3 or 4 consecutive days, the values for DMN-d activity were 277, 297, 306 and 319% of the control values. The corresponding values for AHH were 91, 67, 57 and 45% for l, 2, 3 and 4 injections respectively. Pyrazole-induced DMN-d activity was NADPH dependent and was inhibited by CO; n-butanol gave a 50% inhibition at a concentration of 2 × 10 −3 M. The corresponding value for metyrapone was 1 × 10 −2 M. Cytochrome P-450 was slightly increased by pyrazole and its CO-complex gave an absorption maximum around 451 nm. When the microsomal proteins were separated using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, a large increase in a band at about 51,000 daltons was found in the liver microsomes of pyrazole-treated animals.

Pyrazole-induced cytochrome P-450 in rat liver microsomes: An isozyme with high affinity for dimethylnitrosamine

Treatment of rats with pyrazole elevated the hepatic microsomal dimethylnitrosamine demethylase activity (DMNd) by several fold. Methylethylnitrosamine demethylase activity was also increased by pyrazole, but some classical monooxygenase activities were not induced. The treatment induced a new protein species which has an apparent molecular weight of 52,000 dal and is believed to be a cytochrome P-450 isozyme. The involvement of a hemoprotein in the pyrazole-induced DMNd was demonstrated in an experiment with CoCl 2 which decreased both the microsomal cytochrome P-450 content and DMNd. The induced enzyme with a single K m value of 0.061 mM and V max of 12.1 nmol/min/mg is probably the most efficient enzyme known to metabolize nitrosamines. NADPH-cytochrome P-450 reductase was also demonstrated to be an essential component enzyme of the DMNd. These results further substantiate the idea that the P-450-containing monooxygenase is responsible for the metabolism of dimethylnitrosamine in both the control and pyrazole induced microsomes.

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Oxidative demethylation of dimethylnitrosamine (DMN) was detectable with liver homogenate preparations from fetal mice (noninbred Swiss) by day 16 of gestation; this activity increased to adult levels by postnatal day 7. Treatment of pregnant mice with a mixture of polychlorinated biphenyls (PCB's), 500 mg Aroclor 1254/kg, on the last day of gestation caused a large elevation in DMN demethylase activity in the livers of their sucklings, relative to the normal ontogenic increase, reaching a maximum at postnatal day 21. Foster nursing experiments showed that this inductive effect was due primarily to lactational delivery of the PCB]s. DMN demethylase activity in the maternal livers was significantly lower than normal during pregnancy. The mothers' livers were also less responsive to DMN demethylase induction by PCB's during the last 5 days of gestation in comparison with the response earlier in pregnancy or just after delivery. In the livers of the mothers, induced activity resulting from treatment with PCB's on day 19 of gestation reached a maximum on postnatal days 4-7 and declined to normal levels by the time of weaning.

Effects of indole and tryptophan on cytochrome P-450, dimethylnitrosamine demethylase, and arylhydrocarbon hydroxylase activities

The effects of indole and l-tryptophan feeding on the activities of two liver microsomal enzymes, dimethylnitrosamine demethylase (DMN-d) and arylhydrocarbon hydroxylase (AHH), were studied in rats and hamsters. Both indole and tryptophan increased cytochrome P-450 concentration and DMN-d activity in rats, but only indole was effective in hamsters. AHH activity in both species was induced by indole. Both indole and tryptophan increased the ratio A 392–500nm/ A 410–500nm obtained by n-octylamine difference spectroscopy of rat liver microsomes but not of hamster liver microsomes. Indole increased the absorbance difference ratio A 455–500nm/ A 430–500nm of rat liver cytochrome P-450 obtained by ethyl isocyanide. The opposite was true for hamsters, which showed a low band at 455 nm. Indole gave a type II binding spectrum with rat liver microsomes, but the values for absorption minimum and maximum for tryptophan were somewhat outside the reported values for type II binders. Indole which induced liver AHH activity, protected against 7,12-dimethylbenz[a]anthracene (DMBA)-produced mammary gland carcinogenicity, whereas tryptophan was without effect.

Differential effects of acetone or aroclor 1254 pretreatment on the microsomal activation of dimethylnitrosamine to a mutagen

Pretreatment of mice with acetone enhances the microsomal N-demethylation of dimethylnitrosamine (DMN) at low substrate concentrations (<5 mM), while pretreatment with Aroclor 1254 represses this activity at low, but enhances it at high (> 35 mM) DMN concentrations. To relate the activity of DMN demethylase with the mutagenicity of DMN, liver microsomes were isolated aseptically from mice 18 h after acetone (3 ml/kg, ip), 5 days after Aroclor 1254 treatment (500 mg/kg, ip), or after treatment with the appropriate injection vehicles, and incubated with S. typhimurium (TA 1535), NADPH and DMN (1, 3 or 70 mM) for 5 to 60 min. After a 48-h incubation on minimal media, revertants per plate were determined. Microsomes from acetone pretreated mice bioactivated DMN to a mutagen at significantly higher ( p < 0.001) levels when incubations were performed at 1 mM DMN. Aroclor-1254 microsomes exhibited a decreased ability to convert DMN to a mutagen at both 1 and 3 mM DMN ( p < 0.05) and a significantly higher ( p < 0.05) ability at 70 mM DMN. These data and published reports suggest multiple microsomal enzymes for DMN bioactivation and that acetone may enhance the enzyme that operates at environmentally important levels of DMN.