Activation Of Dimethylnitrosamine Research Articles

Lead acetate does not inhibit dimethylnitrosamine activation and interacts with phenobarbital which is genotoxic in the ST cross of the Drosophila wing spot test

Lead acetate (PbAc) is known to inhibit the synthesis of the heme group, needed for hemeproteins like Cytochromes P450 (CYP450s). Dimethylnitrosamine (DMN) requires metabolic activation by CYP450s. The Drosophila wing spot test was performed to establish whether PbAc inhibits DMN activation in the standard (ST) and high bioactivation (HB) crosses, with different levels of CYP450s. Phenobarbital (PH) was used as an antagonist for its ability to induce CYP450s synthesis. PbAc (0.01, 0.1, 1.0mM) produced significant small spots frequencies in the ST cross, indicating a possible genotoxic activity, however, the total spots frequency was negative at all concentrations. DMN (0.076mM) was genotoxic in both crosses; surprisingly, PH (12mM) was genotoxic and the PH-DMN treatment resulted synergic in the ST cross. Interestingly, the PbAc–PH pre-co-treatments showed a possible interaction in the ST cross. The GC-MS analysis showed a drop in the PH content as the PbAc concentration increased. PbAc also seemed to inhibit the genotoxic activity of PH, except at 0.01mM. It is concluded that PbAc does not inhibit DMN activation by CYP450s in both crosses since it exerted a clear genotoxicity and that PH is genotoxic and interacts with PbAc in the ST but not the HB cross.

Role of hydrocortisone in dimethylnitrosamine-induced suppression of antibody response in the mixed culture of murine hepatocytes and splenocytes

We have previously reported that the hormone-supplemented culture condition for primary hepatocytes is required in dimethylnitrosamine (DMN)-induced suppression of antibody response to sheep erythrocytes in the mixed cultures of murine hepatocytes and splenocytes. In the present investigation, the components of the hormone supplement were screened to identify the component(s) responsible for the increased ability of hepatocytes to activate DMN to its immunosuppressive form. The presence of hydrocortisone in the hepatocyte culture media had the primary role in DMN activation in the co-culture system. Other components of the hormone supplement showed slight or no effects. The effects of hydrocortisone were clearly confirmed through the dose-response study of both DMN and hydrocortisone. To characterize whether the effect of hydrocortisone is glucocorticoid-dependent we tested another potent glucocorticoid, dexamethasone (DEX), and determined if the activity by hydrocortisone could be reversed by RU 486. It was found that hepatocytes cultured in DEX-containing media could also activate DMN to its immunosuppressive form. However, the activity by hydrocortisone to increase DMN-induced immunosuppression was not reversed by RU 486. Furthermore, a possible direct interaction between DMN and hydrocortisone was ruled out. Finally, we transferred DMN-pre-treated culture supernatant from hepatocytes to spleen cell cultures, and found that the metabolite of DMN was very unstable, and that DMN-induced suppression of T-dependent antibody response was hepatocyte-dependent. The present results suggest that glucocorticoids, including hydrocortisone and DEX, in hepatocyte culture media can affect DMN-induced immunosuppression in the hepatocyte/splenocyte co-culture system via a pathway which does not appear to be related to the glucocorticoid receptor.

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).

Dimethylnitrosamine-induced micronucleus formation in mouse bone marrow and spleen

The present study was designed to obtain information on the kinetics of micronucleus (MN) formation following dimethylnitrosamine (DMN) treatment in mice. Male mice were injected once intraperitoneally with 50 or 100 mg/kg DMN. Bone marrow and spleen were obtained at various sacrifice time-points and processed for micronucleus analysis. The vehicle control group had 0.6 and 0.9 MN polychromatic erythrocytes (PCEs)/1000 PCEs in bone marrow and spleen, respectively. DMN, at 50 mg/kg, caused 3.8, 7.8, 8.5 and 10.2 MN PCEs/1000 PCEs in bone marrow and 8.0, 9.2, 19.3 and 32.8 MN PCEs/1000 PCEs in spleen at 12, 24, 36 and 48 h sacrifice times, respectively. A similar time-related elevation of micronucleus frequency was noted for 100 mg/kg DMN. At each sacrifice time-point, spleen PCEs had a higher micronucleus frequency than bone-marrow PCEs. In general, DMN decreased the proportion of PCEs to total erythrocytes, suggesting toxicity. Thus, this study demonstrates the clastogenic activity of DMN in both bone-marrow and spleen PCEs of mice and shows a time-related pattern in elevating DMN-induced MN PCE frequency.

Comparative genotoxicity of 3 procarcinogens in V79 cells as related to glutathione S-transferase activity of hepatocytes from untreated rats and those fed 2% butylated hydroxyanisole

When 7,12-dimethylbenz[ a]anthracene (DMBA) and aflatoxin B 1 (AFB 1) were activated by hepatocytes from Fischer 344 rats fed a diet containing 2% butylated hydroxyanisole (BHA), frequencies of mutation to 6-thioguanine resistance (TG R) at the HGPRTase gene locus and to ouabain resistance (Ou R) at the Na +,K + -ATPase gene locus in V79 cells were 30–70% less than those obtained with hepatocytes from untreated controls. A difference in the mutation frequency did not occur when dimethylnitrosamine (DMN) was activated by BHA induced- rather than control-hepatocytes. Analysis of hepatocytes from rats fed 2% BHA showed a small (1.5-fold), but significant, increase in glutathione levels over that in the controls but no change in activity of cytochrome P450. Cytosolic glutathione S-transferase (GST) activity was increased 2–3-fold in hepatocytes from rats fed the 2% BHA diet. These results suggest that mutagenic response to DMBA and AFB 1 is reduced, at least in part, because of BHA-induction of hepatocyte GST activity; while activation of DMN can occur by pathway(s) unaffected by BHA-induction of these liver enzymes. In contrast to mutation frequencies, significant differences between BHA- and control-activation in the production of sister-chromatid exchange (SCE) and micronucleus formation (MN) were not detected with any of the genotoxins. It was concluded that the mechanism(s) by which SCE and MN occur are likely unrelated to the capacity of BHA to induced activity of hepatic enzymes, e.g. the GSH S-transferases, that directly or indirectly affect mutation end-points.

Dependence on intact cells for the in vitro activation of dimethylnitrosamine to an immunosuppressive form

The in vitro activation of dimethylnitrosamine (DMN) to an immunosuppressive form was studied utilizing liver-enzyme fractions and intact hepatocytes. The N-demethylation of DMN by mouse S9 and microsome preparations was confirmed by determination of formaldehyde generation. S9 fractions from both phenobarbital(PB)- and isopropanol(iso)-pretreated mice displayed significantly greater demethylase activity than uninduced S9 fractions. However, when incubated with spleen cells, neither S9 preparation was capable of activating DMN to a form capable of suppressing antibody responses by recovered spleen cells. In contrast, the positive control, cyclophosphamide, was activated to a markedly immunosuppressive form. S9 fractions failed to activate DMN to an immunosuppressive form regardless of S9 concentration, time of preincubation, or rocking speed. Liver microsomes from PB-pretreated mice displayed significantly greater N-demethylase activity than S9 fractions yet were unable to activate DMN to an immunosuppressive form. In contrast, the addition of DMN to mixed cultures of mouse hepatocytes and mouse spleen cells resulted in activation of DMN and marked suppression of antibody responses. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. Unlike the metabolism of DMN to a mutagenic form, the in vitro activation of DMN to an immunosuppressive form was therefore dependent on intact cells. Furthermore, the activation by intact hepatocytes was shown to be dependent on cell-cell contact or close proximity of activating and target cells.

Alcohol and cancer.

Alcohol and cancer.

Dimethylnitrosamine genotoxicity in rat liver primary cell cultures with low cytochrome P-450 levels.

Liver primary cell cultures (LPCC) with decreasing concentrations of cytochrome P-450 were used to investigate the genotoxicity of the hepatic carcinogen dimethylnitrosamine (DMN) and the correlation between DMN genotoxicity and cytochrome P-450 levels. Hepatocytes were isolated from partially hepatectomized rats and incubated with [3H]thymidine; single-strand DNA molecular weight was determined by alkaline sucrose sedimentation. The molecular weight of DNA decreased 50% in LPCC plated either 2 or 24 h before being treated for 24 h with 70 micron DMN. Cytochrome P-450 content was 188 pmol per mg protein in freshly isolated hepatocytes, whereas it was 70 and 32 pmol per mg protein in hepatocytes that had been cultured 24 and 48 h, respectively. Incorporation of 14C into acid-insoluble material was the same in LPCC exposed 24 h to [14C]DMN starting either 2 or 24 h after cell plating. At non-toxic concentrations (0.01-1 microM), SKF 525-A, an inhibitor of mixed-function oxidase enzymes, inhibited approximately 20% of the binding of 14C from [14C]DMN to acid-insoluble material in LPCC plated either 2 or 24 h before they were exposed to DMN for 24 h. Hepatocyte cultures exposed to the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (at concentrations ranging between 6.8 X 10(-8) and 6.8 X 10(-5) M) starting 2 and 24 h after plating, exhibited significant unscheduled DNA synthesis. These results indicate that DMN genotoxicity was similar in LPCC differing considerably in cytochrome P-450 levels, and they suggest that DMN genotoxicity in these cultures is due mainly to similar DMN activation than to decreased DNA repair.

Hepatic microsomal enzymes in Schistosoma mansoni-infected mice: I. Effect of duration of infection and lindane administration on dimethylnitrosamine demethylases

The prevalence of schistosomiasis in Egypt, its suspected relationship to carcinoma of the bladder, and the inevitable exposure of schistosomal patients to insecticides prompted the undertaking of this study. Dimethylnitrosamine (DMN) demethylases, responsible for the activation of DMN, were assessed in the hepatic microsomes from albino mice under the influence of Schistosoma mansoni infection and/or lindane administration. DMN-demethylase I responded to infection by an early increase in activity, reaching a peak 30 days after disease induction and declining sharply afterwards. DMN-demethylase II followed the same general pattern, but it reached its peak 20 days after infection and declined at a slower rate than DMN-demethylase I. The effects of lindane, an organochlorine insecticide, were also studied on these two enzymes in uninfected mice and in S. mansoni-infected animals after different durations of disease induction. Given orally, in a dose of 40 mg/kg/day for 3 consecutive days, lindane increased the activity of both enzymes. Since DMN requires metabolic activation for its hepatotoxic and carcinogenic actions, alterations in its activating enzymes, as a consequence to schistosomiasis and/or exposure to lindane, may change susceptibility to its toxic and carcinogenic potentials.

Differential effect of chronic ethanol consumption by the rat on microsomal oxidation of hepatocarcinogens and their activation to mutagens

The effect of chronic ethanol consumption by rats on hepatic microsomal activation of the hepatocarcinogens dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF) was investigated. There was a marked increase in the rate of the oxidative demethylation of DMN and its activation to a mutagen by microsomes foHowing ethanol intake. N- and C-hydroxylation of 2-AAF were measured at substrate concentrations ranging from 2 to 70 μM. The ratio of formation of N-hydroxy-2-acetylaminofluorene to C-hydroxy-2-acetylaminofluorenes increased with decreasing substrate concentration, suggesting enhanced carcinogenic potential of 2-AAF with diminishing levels of carcinogen. Kinetic analysis indicated that N-hydroxylation as well as 7-, 5- and 3-hydroxylation of 2-AAF do not follow Michaelis-Menten kinetics. In contrast to the marked inductive effect of ethanol consumption on the metabolic activation of DMN, only a minimal random effect on the N-hydroxylation of 2-AAF was demonstrable in two separate experiments. Furthermore, N-hydroxylation of 2-AAF by microsomes from control and ethanol-treated rats followed similar kinetics. While ethanol consumption enhanced the mutagenic activation of DMN by hepatic microsomes, no such effect of ethanol consumption on the conversion of 2-AAF to a mutagen was observed. The data indicate that chronic ethanol consumption does not have a general inductive effect on the microsomal activation of hepatocarcinogens.

Quantitative studies on the in vitro metabolic activation of dimethylnitrosamine by rat liver postmitochondrial supernatant.

The metabolic activation of dimethylnitrosamine (DMN) to mutagenic and/or cytotoxic intermediates in vitro has been characterized and the relationship between DMN demethylase and ethoxyresorufin-O-deethylase (EROD) or ethylmorphine-N-demethylase (EMND) has been evaluated. A mammalian assay system which uses the postmitochondrial supernatant (S-15 fraction) prepared from a rat liver homogenate as an enzyme source and V79 Chinese hamster cells as targets for chemically induced damage was used. The enzyme pattern of the S-15 fraction was altered by pretreatment of experimental animals in vivo and/or by the use of enzyme inhibitors in vitro. The results of these studies indicate that the concentration of S-15 fraction in the reaction mixture can markedly influence the degree of DMN-induced cytotoxicity when it is metabolized in vitro and that the degree of DMN-induced cytotoxicity and mutagenicity are linearly related. The degree of cytotoxicity and mutagenicity induced in V79 cells by DMN does not correlate with EROD activity (a measure of 3-methylcholanthrene-inducible mixed-function oxidases) nor with EMND activity (a measure of phenobarbital-inducible mixed function oxidases) in the S-15 fraction.

Studies on the relationship between dimethyl-nitrosamine-demethylase activity and dimethyl-nitrosamine dependent mutagenesis in, drosophila melanogaster

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.

Effect of ethanol on dimethylnitrosamine activation and DNA synthesis in rat liver

Male Wistar rats were treated for 2 weeks with 20% ethanol in the drinking water. Twenty four hours after the termination of treatment animals were injected with different doses of [14C]dimethylnitrosamine and killed 4 h thereafter. The amounts of 7-methylguanine and O6-methylguanine present in liver DNA were determined. There was no significant difference in the levels of either DNA alkylation product between untreated controls and animals pre-treated with ethanol. The O6-methylguanine/7-methylguanine ratio was also unchanged. Specific radioactivity levels in the cellular protein of [14C]dimethylnitrosamine injected animals were slightly lowered after ethanol pre-treatment. In contrast, incorporation of labelled guanine into liver DNA was greatly enhanced at all DMN doses in ethanol pre-treated animals indicating an increase in DNA synthesis. This enhancement of DNA synthesis was confirmed at different ethanol doses, given either in the drinking water or by stomach tube, by the increase in specific radioactivity of liver DNA and the increase in the number of labelled nuclei following [3H]thymidine pulse-labelling.

Ethanol- or acetone-pretreatment of mice strongly enhances the bacterial mutagenicity of dimethylnitrosamine in assays mediated by liver subcellular fraction, but not in host-mediated assays.

The activation of dimethylnitrosamine (DMN) to a bacterial mutagen in liver subcellular fraction and in intrasanguinous host-mediated assays was studied, in particular the effect of pretreatment of the animals with ethanol or acetone. Salmonella typhimurium TA 92 was much more sensitive to DMN mutagenicity than TA 100 and TA 1535 or Escherichia coli WP2uvrA and was used for the main part of the study. Noteworthy, in part already known, features of the in vitro activation are the relatively low pH optimum (pH 6-6.4), the non-linear dose-mutagenic response-relationship and the relatively high doses of DMN required for activation with control preparations. Pretreatment of mice with ethanol or acetone greatly reduced the minimal mutagenically effective concentration of DMN in the in vitro assay. Pretreatment with Aroclor 1254, an inducer frequently used in mutagenicity research, showed little effect when used alone, but reduced the potentiation by acetone. The results of the host-mediated assays substantially differed from those of the in vitro activation assays (a) in the relatively low dose of DMN required for mutagenicity to occur and (b) in the lack of potentiation by acetone-or ethanol-pretreatment. Acetone even led to a marginal decrease in mutagenicity. As a possible explantation for this apparent discrepancy were assume that with the in vitro system the activity of the dilute metabolizing system is limiting for the activation of DMN and induction therefore will increase the mutagenicity, whereas in vivo DMN is quantitatively metabolized in both induced and non-induced animals. The results show that caution has to be taken in the interpretation from in vitro results to the in vivo situation. In particular our in vivo experiments do not support the hypothesis that the induction by ethanol of an activating system with a low Km (which would strongly activate traces of DMN ingested with many foods) is one of the reasons for the increased risk of liver tumors in alcoholics.

Regulation of dimethylnitrosamine metabolism by androgenic hormones.

Hormonal regulation of dimethylnitrosamine (DMN) metabolism by mouse kidney microsomes was investigated using an in vitro mutagenesis system that detects bioactive metabolites of this procarcinogen by measuring reverse mutation in Salmonella typhimurium his- auxotrophs. Induction of microsomal DMN metabolizing enzymes was androgen-specific. Testosterone and medroxyprogesterone acetate were active inducers, d/1 norgestrel was less active, while epitestosterone, estradiol, and progesterone were ineffective. THe response to testosterone and medroxyprogesterone acetate was time-and dose-dependent. Eleven strains of inbred mice were screened for their response to exogenous testosterone. DMN metabolism was stimulated in all mouse strains except for RF/J mice. Other androgenic end points were responsive in the latter strain, however. These observations suggest that induction of renal microsomal DMN metabolising enzymes is androgen-specific and probably mediated via the androgen receptor. The insensitivity of RF/J mice may be due to a mutation affecting a key step in the enzymatic activation of DMN.

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.

Mutagenicity testing on Chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[ a]pyrene

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[ a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN — both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasised that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.

Use of human-liver microsomes from kidney-transplant donors for the induction of chromatid aberrations and sister-chromatid exchanges by means of pre-carcinogens in Chinese hamster cells in vitro

Samples of two human livers taken during operation of kidney donor patients were processed for microsome fractions and used for metabolization of cyclophosphamide (CP) and dimethylnitrosamine (DMN) in combination with the NADPH-generating system. Rat-liver microsomes were checked for comparison. Induction of chromatid aberrations and sister-chromatid exchanges in a newly isolated clone of Chinese hamster fibroblasts served as indicators of activity. Human S-9 fractions standardized on protein content showed strong variations of CP and DMN activation. Whereas liver microsomes of one patient (who also suffered from Gaucher's disease) were highly active for both pre-carcinogens and metabolized DMN at the same level as the uninduced rat-liver microsomes, the S-9 fraction from the second patient failed to activate CP, but was distinctly positive for DMN. It is suggested that samples of liver and other organs of renal transplant donors might be a practicable source of freshly prepared human microsome fractions usable in biochemical, genetic and carcinogenetic studies. Problems concerning the extrapolation of results are discussed.

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 use of short-term tests to measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds

The effect of reducing agents on the nitrosation of methylguanidine (MG) and on the in vitro activation of dimethylnitrosamine (DMN) was examined by measuring DNA-repair synthesis (unscheduled incorporation of [ 3H]TdR). shifts in alkaline sucrose gradients, frequency of chromosome aberrations, and clone-forming capacity of cultured human fibroblasts. The reducing agents examined were sodium ascorbate, cysteine, cysteamine, and propyl gallate. Since the short-term bioassays used can be quantitated, it has become relatively easy to detect the inhibitory action of reducing compounds on the nitrosation reaction of MG and metabolic activation (with S-9 preparation) of the precarcinogen DMN, to measure their effective dose range, and to establish the most effective ratios between inhibitory agent and reactant. The results indicate that DNA-repair synthesis is a suitable short-term test for studying the numerous combinations and permutations between several carcinogenic or non-carcinogenic agents, and for estimating the capacity of inhibitory agents to affect formation and activation of chemical carcinogens.