Metabolic reduction of chromium by alveolar macrophages and its relationships to cigarette smoke.

Pulmonary alveolar macrophages (PAM), obtained by bronchoalveolar lavage from 47 individuals, reduced hexavalent chromium [Cr(VI)] and decreased its mutagenicity. Their specific activity--mostly mediated by cytosolic, enzyme-catalyzed mechanisms--was significantly higher than in corresponding preparations of mixed-cell populations from human peripheral lung parenchyma or bronchial tree, or from rat lung or liver. At equivalent number of PAM, Cr(VI) reduction, total protein, and some oxidoreductase activities were significantly increased in smokers. No appreciable variation could be detected between lung cancer and noncancer patients. In rats, the Cr(VI)-reducing activity of PAM preparations was induced by Aroclor 1254. Thus, alveolar macrophages provide crucial defense mechanisms not only by phagocytizing metals, but also by metabolically reducing Cr(VI). The epithelial-lining fluid (ELF) also displayed some Cr(VI) reduction. Together with already investigated metabolic processes occurring inside lung cells, these mechanisms are expected to determine thresholds in the pulmonary carcinogenicity of chromium.

Pulmonary metabolism of mutagens and its relationship with lung cancer and smoking habits.

The S-12 fractions of lung peripheral parenchyma obtained from 80 male individuals, aged 17-71 years, were assayed as blind samples for the ability either to convert promutagens into bacterial mutagens or to decrease the potency of direct-acting mutagens in the Ames reversion test. In this system, lung preparations were completely ineffective in activating an N-nitroso compound (i.e., N-nitrosomorpholine) and polycyclic aromatic hydrocarbons [i.e., 3-methylcholanthrene and benzo(a)pyrene] or their metabolites [i.e., 3-hydroxy-benzo(a)pyrene and benzo(a)pyrene-trans-7,8-diol]. They yielded a borderline and sporadic activation of a cigarette smoke condensate, and a weak but frequent activation of an aromatic amine (i.e., 2-aminofluorene), of a heterocyclic amine (i.e., 2-amino-3,4-dimethylimidazo[4,5-f] and of a diamide (i.e., cyclophosphamide). The pulmonary metabolism was more oriented in the sense of detoxification, as shown by the consistent decrease of potency of direct-acting mutagens, including a metal (i.e., sodium dichromate), an acridine and nitrogen mustard derivative (i.e., 2-methoxy-6-chloro-9-[3-(2-chloromethyl)aminopropylamino]acridine or ICR 191), an epoxide (i.e., epichlorohydrin) and an N-oxide (i.e., 4-nitroquinoline-N-oxide). As assessed by means of a numerical score quantifying the variation of mutagenicity, a marked interindividual variability (up to 20-fold) was detected in the ability of lung specimens to affect the mutagenicity of test compounds. Such variability was not significantly related to the protein concentration of S-12 fractions, nor to the age of the patients under scrutiny, who during hospitalization were on normal institutional diets and did not receive any special drug treatment. The only significant difference between 20 noncancer and 60 lung cancer patients, irrespective of the histological type, was a decreased activation of cyclophosphamide in the latter group. Probably due to the high prevalence of smokers among lung cancer patients, a significantly decreased activation of cyclophosphamide was also observed in the group of smokers. Smoking habits were associated with a stimulation of detoxifying mechanisms which, in agreement with the results of a previous study with human alveolar macrophages (F. L. Petrilli et al., J. Clin. Invest., 77:1917-1924, 1986), was significant in the case of sodium dichromate. Such effect was further enhanced by considering only individuals smoking during the last 24 h before collecting lung specimens, and under these conditions it became significant also for ICR 191.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-damaging activity in vivo and bacterial mutagenicity of sixteen hydrazine derivatives as related quantitatively to their carcinogenicity.

Sixteen hydrazine derivatives (hydrazine, 1,1-dimethylhydrazine, 1,2-dimethylhydrazine, phenylhydrazine, procarbazine, isoniazid, isocarboxazid, nialamide, 2,4-dinitrophenylhydrazine, phenelzine, hydralazine, dihydralazine, carbamylhydrazine, mebanazine, iproniazid, and 1-carbamyl-2-phenylhydrazine) were tested for DNA-damaging activity by the alkaline elution technique and for mutagenic activity in the Salmonella-microsome (Ames) test. The first nine compounds listed (56%) were found to induce a significant DNA fragmentation in the liver and/or in the lung of i.p.-treated male Swiss mice. The DNA-damaging potency varied over an approximately 30-fold range. Thirteen of the first 14 compounds listed (81% of the total), isocarboxazid being inactive, were positive in the Ames test, with a broad range of activity towards the five bacterial strains of Salmonella typhimurium used (TA1535, TA100, TA1537. TA1538, and TA98) and of metabolic behavior in the presence of S-9 mix containing rat liver, mouse liver, or mouse lung postmitochondrial preparations from Aroclor-treated animals. The mutagenic potency varied over an almost 7000-fold range. For 11 of the 16 hydrazine derivatives tested, homogeneous carcinogenicity data (induction of pulmonary tumors in mice chronically treated p.o.) were available from literature. Elaboration of these data showed that carcinogenic potency varied over an approximately 1900-fold range. The five most potent carcinogens were all positive in the DNA damage test. Their carcinogenic potency varied over a 130-fold rage and their DNA-damaging potency varied over a 22-fold range. DNA-damaging potency seemed to vary on a more compressed scale, but regression analysis indicated the existence of a strong positive correlation between in vivo DNA-damaging and carcinogenic potencies, while a lack of correlation was found between mutagenic and carcinogenic potencies. There was no correlation between DNA-damaging and mutagenic potencies.

Specificity and inducibility of the metabolic reduction of chromium(VI) mutagenicity by subcellular fractions of rat tissues.

The mutagenicity of sodium dichromate in the Ames test was decreased as a consequence of chromium(VI) reduction by tissue postmitochondrial (S-9 or S-12) fractions from untreated rats with the following rank of efficiency: liver; kidney; and lung. The effects of lung preparations were significantly enhanced following the intratracheal administration of high doses (0.25 mg/kg) of dichromate itself, 5 times per week for 4 weeks (i.e., 20 fractionated instillations). No changes were conversely detected following single weekly doses of 1.25 mg/kg for the same period (i.e., four cumulative instillations). The local stimulation of chromium(VI) metabolism was also confirmed by testing the mutagenicity of calcium chromate and chromium trioxide, whereas the metabolism of a number of other activatable or deactivatable mutagens was not significantly affected by intratracheal treatment with chromium(VI). Of three enzyme inducers injected i.p. which modified the spectral properties and/or concentration of cytochromes P-450 in liver and lung microsomes, only Aroclor 1254 proved to stimulate chromium(VI) metabolism in lung cells. In liver cells, Aroclor 1254 and to a lower extent phenobarbital induced chromium(VI) reduction, while 3-methylcholanthrene was ineffective. Pretreatment of rats with these three compounds resulted in a selective induction of the metabolic activation of promutagens [benzo(a)pyrene and its trans-7,8-diol, 2-aminofluorene, aflatoxin B1] and of the metabolic deactivation of direct-acting mutagens [2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino] acridine X 2HCl, epichlorohydrin, 4-nitroquinolino-N-oxide] by S-12 and microsomal fractions. These findings indicate that, in addition to already recognized detoxification mechanisms operating outside target cells (26), specific and inducible chromium-reducing pathways, mediating threshold phenomena in chromium carcinogenesis, do also occur in the intracellular environment.

Modulation of biomarkers by chemopreventive agents in smoke-exposed rats.

Chemoprevention opens new perspectives in the prevention of cancer and other chronic degenerative diseases associated with tobacco smoking, exploitable in current smokers and, even more, in exsmokers and passive smokers. Evaluation of biomarkers in animal models is an essential step for the preclinical assessment of efficacy and safety of potential chemopreventive agents. Groups of Sprague Dawley rats were exposed whole body to a mixture of mainstream and sidestream cigarette smoke for 28 consecutive days. Five chemopreventive agents were given either with drinking water (N-acetyl-L-cysteine, 1 g/kg body weight/day) or with the diet (1,2-dithiole-3-thione, 400 mg; Oltipraz, 400 mg; phenethyl isothiocyanate, 500 mg; and 5,6-benzoflavone, 500 mg/kg diet). The monitored biomarkers included: DNA adducts in bronchoalveolar lavage cells, tracheal epithelium, lung and heart; oxidative damage to pulmonary DNA; hemoglobin adducts of 4-aminobiphenyl and benzo(a)pyrene-7,8-diol-9,10-epoxide; micronucleated and polynucleated alveolar macrophages and micronucleated polychromatic erythrocytes in bone marrow. Exposure of rats to smoke resulted in dramatic alterations of all investigated parameters. N-Acetyl-L-cysteine, phenylethyl isothiocyanate, and 5,6-benzoflavone exerted a significant protective effect on all alterations. 1,2-Dithiole-3-thione was a less effective inhibitor and exhibited both a systemic toxicity and genotoxicity in alveolar macrophages, whereas its substituted analogue Oltipraz showed limited protective effects in this model. Interestingly, combination of N-acetyl-L-cysteine with Oltipraz was the most potent treatment, resulting in an additive or more than additive inhibition of smoke-related DNA adducts in the lung and hemoglobin adducts. These results provide evidence for the differential ability of test agents to modulate smoke-related biomarkers in the respiratory tract and other body compartments and highlight the potential advantages in combining chemopreventive agents working with distinctive mechanisms.

Oltipraz chemoprevention trial in Qidong, People's Republic of China: results of urine genotoxicity assays as related to smoking habits.

A Phase II chemoprevention trial was carried out in Qidong, Jiangsu Province, People's Republic of China. The recruited subjects, all of whom were positive for serum aflatoxin-albumin adducts, were divided into three treatment arms: placebo; oltipraz ([5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione]) given daily at 125 mg p.o.; and oltipraz given once per week at 500 mg p.o. Besides biomarkers related to aflatoxin B(1) exposure, the genotoxicity of blind-coded urine XAD-2 concentrates was evaluated in 201 subjects on the fifth and seventh week of intervention. Genotoxicity was assessed both in the Ames reversion test in strain YG1024 of Salmonella typhimurium, in the presence of an exogenous metabolic system (S9 mix), with or without beta-glucuronidase, and in a DNA repair test in Escherichia coli. Heating of concentrated urine samples or of cigarette smoke condensates was discovered to result in a significant enhancement of their mutagenicity. It was also found that the mutagenicity of condensates from the most extensively used brands of cigarettes in Qidong was much lower than that of Western cigarette brands. Urine mutagenicity was unrelated to treatment with oltipraz, intervention time, gender, and supplement of S9 mix with beta-glucuronidase. Mutagenicity was significantly but variably higher in cigarette smokers than in nonsmokers, which suggests that the urinary excretion of mutagens in the examined population was not exclusively attributable to smoking. Nevertheless, within smokers (28% of the recruited subjects; 67% of all males), the mutagenic potency was significantly correlated with the self-reported number of cigarettes smoked per day and, even more sharply, with the cotinine concentrations in urines. In conclusion, this study demonstrated the validity of urine mutagenicity assays as a biomarker of tobacco smoke exposure that can be investigated on a relatively large scale in chemoprevention trials and provided evidence that oltipraz treatment had no influence on this parameter in the examined population.

Inhibition of urethan-induced lung tumors in mice by dietary N-acetylcysteine.

The thiol N-acetylcysteine (NAC), a precursor of intracellular glutathione (GSH), efficiently prevented the induction of lung tumors in Swiss albino mice, when supplemented to the diet (0.2%) both before and after an i.p. injection of the carcinogen urethan (ethyl carbamate). Irrespective of urethan administration, NAC also significantly enhanced GSH S-transferase activity in liver preparations of the same animals. These data show that, under certain conditions, it is possible to prevent chemically induced cancer by increasing the levels of physiological trapping agents.

Cytosolic activation of aromatic and heterocyclic amines. Inhibition by dicoumarol and enhancement in viral hepatitis B.

The aromatic amines 2-aminofluorene (2AF), 2-acetylaminofluorene, and 2-aminoanthracene, and the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, and 3-amino-1-methyl-SH-pyrido[4,3-b]indole (Trp-P-2) were activated by rat liver cytosolic fractions to form mutagenic metabolites in Salmonella typhimurium strains TA98, TA98NR, and TA98/1,8-DNP6. In the case of the Trp-P-2, the cytosolic activation was even more potent than the microsomal activation, which is classically ascribed to N-hydroxylation and subsequent esterification. The cytosolic activation was a) NADPH-dependent, b) induced by pretreatment of rats with 3-methylcholanthrene and especially Aroclor 1254 but not by phenobarbital, and c) inhibited by dicoumarol. The hypothesis is that, following a preliminary oxidative step in the cytosol (pure cytosolic activation) or in microsomes via prostaglandin H synthase (mixed microsomal-cytosolic activation), an oxidized intermediate of amino compounds may serve as substrate for DT diaphorase activity and bielectronically reduced to the corresponding N-hydroxyamino derivative. Purified DT diaphorase, in the presence of either NADPH or NADH as electron donor, produced mutagenic derivatives from IQ and Trp-P-2. An NADPH-dependent activation of Trp-P-2 also occurred in the liver cytosol of woodchucks (Marmota monax), but was not inhibited by dicoumarol. As previously demonstrated with liver S-12 fractions in both humans and woodchucks, the cytosolic activation of Trp-P-2 was enhanced in animals affected by hepatitis B virus infection. This enhanced metabolism, which persisted even after appearance of primary hepatocellular carcinoma in virus carriers, is likely to be ascribed to mechanisms other than DT diaphorase induction, such as glutathione depletion.

Pilot studies evaluating the lung tumor yield in cigarette smoke-exposed mice.

In spite of the major role played by cigarette smoking in the epidemiology of lung cancer, it is very difficult to reproduce the carcinogenicity of this complex mixture in animal models. We implemented a series of pilot experiments in three mouse strains, exposed either to environmental cigarette smoke (ECS) or mainstream cigarette smoke (MCS) or its condensate (MCSC). The whole-body exposure of Aroclor-treated A/J mice to ECS resulted in a rapid and potent induction of micronuclei in peripheral blood erythrocytes. After 6 months of exposure, 6 h a day, followed by 4 months of recovery in filtered air, both lung tumor incidence and multiplicity were significantly increased as compared to sham-exposed mice (77.8% vs. 22.2%, and 1.11+/-0.26 vs. 0.22+/-0.15, means +/- SE). Multiple i.p. injections of butylated hydroxytoluene did not significantly enhance the tumor yield. Another experiment confirmed the responsiveness of A/J mice exposed to ECS for 5 months, followed by 4 months of recovery in air (75.0% vs. 25.0%, and 1.05+/-0.17 vs. 0.25+/-0.10). In contrast, the increase in lung tumor yield after exposure to ECS for 2 months, followed by recovery in air for 7 months, was not significant, and the continuous exposure to ECS for 9 months was totally ineffective. These data, in agreement with previous results of others, show that exposure of A/J mice to ECS for 5-6 months, followed by recovery in air for 4 months, is successful in inducing a weak but significant and reproducible increase in lung tumor yield. Furthermore, the simultaneous exposure to the light emitted by halogen quartz bulbs for 9 months and to ECS for 5 months, followed by 4 months in air, was again weakly tumorigenic (incidence of 55.0% and multiplicity of 0.75+/-0.19), whereas exposure to both ECS and light for 9 months was devoid of effect. The whole-body exposure of A/J mice to MCS, 1 h a day for 5 months, or weekly i.p. injections of MCSC for 5 months, followed in both cases by 4 months of recovery in air, failed to enhance the lung tumor yield. The whole-body exposure of SKH-1 hairless mice to ECS for 6 months, followed by exposure to halogen light for 8 months, resulted in the formation of multiple skin tumors but failed to produce lung tumors. The whole-body exposure of C57BL/6 mice to ECS for 6 months failed to induce any lung tumor but caused alopecia, gray hair, and hair bulb cell apoptosis, which were prevented by the oral administration of N-acetylcysteine.

Rationale and mechanisms of cancer chemoprevention.

Chronic degenerative diseases, including cancer, have a multifactorial origin. An intricate network connects each disease with multiple risk factors and also with multiple protective factors. From the point of view of preventive medicine, this implies that removal of a single risk factor will have a beneficial impact on the epidemiology of several diseases. However, in contrast to the situation in infectious diseases, it will never be possible to eradicate any chronic degenerative disease in this way, because each of them is associated with other risk factors at the same time. Similarly, a single protective factor can decrease the risk of contracting different diseases, and the risk of developing a single disease can be attenuated by different protective factors, often in a coordinated fashion. It is thus evident that cancer can be prevented not only by avoiding exposure to recognized risk factors, but also, as a complementary approach referred to as chemoprevention, by favouring the intake of protective factors and by fortifying the physiological defences of the host organism. Chemoprevention can be applied in a primary prevention setting when it is addressed to healthy individuals with the goal of inhibiting occurrence of the disease. Conversely, it is applied in a secondary prevention setting when it is addressed to individuals affected by premalignant tumours, with the goal of reversing the carcinogenesis process. A rational use of chemopreventive agents is based not only on the assessment of their efficacy and safety but also on understanding of their mechanisms of action. A detailed classification is proposed, which covers a variety of mechanisms interfering with different phases of mutagenesis and carcinogenesis. However, this sequence of events does not fit in with a rigid scheme, and several mechanisms, such as inhibition of genotoxic effects, antioxidant activity and scavenging of free radicals, inhibition of cell proliferation, and signal transduction modulation are reiterated several times throughout evolution of these processes. Some of these mechanisms are also involved in advanced stages of tumour progression towards malignancy, invasion and metastasis, and can therefore conveniently be applied for the tertiary prevention of cancer. Most inhibitors work through multiple mechanisms, examples of which are given for 18 chemopreventive agents.

Formation of mutagenic derivatives from nitrite and two primary amines.

Sodium nitrite and two primary aromatic amines, viz. amino antipyrine (AAP) and aniline, were preincubated in vitro with human gastric juice. The resulting derivatives -- presumably diazonium salts -- were directly mutagenic in the Salmonella test. The mutagenic response was more pronounced in the case of AAP, while toxic effects narrowed the range of activity of the aniline derivative. These patterns are consistent with the findings of independent colorimetric analyses, showing that the AAP derivative is more stable at 37 degrees C than the aniline derivative.

Influence of liver S-9 preparations from rats and rainbow trout on the activity of four mutagens.

The mutagenicity of four chemical compounds to strain TA100 of S. typhimurium was affected differently by liver S-9 preparations from untreated Sprague-Dawley rats and from rainbow trout (Salmogairdneri). These two species were equally effective in decreasing the direct mutagenicity of sodium dichromate. Rat preparations were totally inactive and trout preparations were slightly active in producing mutagenic metabolites from benzo(a)pyrene (BP). Conversely, rat homogenates were significantly more efficient in activating aflatoxin B1 (AFB1) and, in particular, 2-aminofluorene (2-AF).

Genotoxicity of mercury compounds. A review.

This article reviews literature data concerning the genotoxicity of 29 mercury-containing agents, including laboratory compounds as well as ingredients of preparations used as fungicides, dyes, disinfectants and drugs. A variety of genetic end-points were investigated in bacteria, yeasts, moulds, plants, insects, cultured cells from fishes, rodents or humans, aquatic organisms, amphibians, mammalia and exposed humans. The overall evaluation is quite complex. Mercury compounds failed to induce point mutations in bacteria but often exerted clastogenic effects in eukaryotes, especially by binding SH groups and acting as spindle inhibitors, thereby causing c-mitosis and consequently aneuploidy and/or polyploidy. Inorganic mercury compounds were also found to induce the generation of reactive oxygen species and glutathione depletion in cultured mammalian cells. Although different mercury compounds tended to produce qualitatively comparable genetic effects, which suggests the involvement of a common toxic entity, methylmercury derivatives and other ionizable organomercury compounds were more active in short-term tests than either non-ionizable mercury compounds (e.g., dimethylmercury) or inorganic mercury salts (e.g., mercuric chloride). The results of cytogenetic monitoring in peripheral blood lymphocytes of individuals exposed to elemental mercury or mercury compounds from accidental, occupational or alimentary sources were either negative or borderline or uncertain as to the actual role played by mercury in some positive findings. Both genotoxic and non-genotoxic mechanisms may contribute to the renal carcinogenicity of mercury, which so far has been convincingly demonstrated only in male rodents treated with methylmercury chloride.

High sensitivity of Salmonella TA102 in detecting hexavalent chromium mutagenicity and its reversal by liver and lung preparations.

The recently developed strain TA102, particularly suited to the detection of oxidative mutagens (Levin et al., 1983), was the most sensitive out of 9 strains of S. typhimurium his- in revealing the mutagenicity of Cr(VI) compounds (sodium dichromate, calcium chromate and chromium trioxide). The rank of sensitivity was the following: TA102, TA100, TA97, TA92, TA1978, TA98, TA1538 and TA1537, TA1535 being the only insensitive strain. Cr(III) compounds (chromic acetate, chromic nitrate and chromic potassium sulfate) were totally inactive with all strains. The direct mutagenicity of Cr(VI) was markedly decreased, through NADPH-requiring mechanisms, by rat-liver S9 fractions and, to a lower extent, by human lung S12 fractions, which supports the hypothesis of a metabolically regulated threshold in chromium pulmonary carcinogenicity.

Inhibition of the 'spontaneous' mutagenicity in Salmonella typhimurium TA102 and TA104.

Thirty-four compounds belonging to various chemical classes were assayed for the ability to modulate the 'spontaneous' mutagenicity in strain TA104 of S. typhimurium, and 17 of them were also assayed in TA102. All test agents, many of which were already known or suspected to act as inhibitors of induced mutagenicity, had been previously monitored in our laboratory for antimutagenicity towards either 4-nitroquinoline 1-oxide in TA100 and/or cigarette smoke in TA98 with S9 mix. A considerable proportion of test compounds decreased the number of spontaneous revertants in TA104 (44.1%) and/or TA102 (41.2%) to a significant extent, with dose-related and reproducible effects. In almost all cases the antimutagenic effect was genuine and not related to bacterial killing or growth inhibition. The results obtained suggest that the DNA repair background plays a prominent role in the genesis of spontaneous mutations in these strains, containing the hisG428 mutation which is typically reverted by oxidative mutagens. Due to its theoretical and practical implications, the finding that several chemopreventive agents can attenuate the rate of spontaneous reversion deserves attention.

Genotoxic effects in bacteria of the light emitted by halogen tungsten lamps having treated quartz bulbs.

Traditional halogen tungsten lamps, which are extensively used worldwide for the illumination of indoor environments, have a quartz bulb which transmits not only visible light but also ultraviolet (UV) light. Due to the output of far-UV wavelengths, halogen lamps were found in previous studies to be potently genotoxic in bacteria, clastogenic in cultured human cells, and carcinogenic in hairless mice. This discovery prompted the launching of new halogen lamps, known as UV-Stop, UV-Block, or similar trade names, which have the quartz glass treated in such a way to reduce its permeability to UV radiation. Surprisingly, these lamps are advertised for attenuating discolouration of UV-sensitive materials, such as fabrics, paintings, works of art and furniture, whereas protection of the human skin from potential carcinogenic risks is overlooked. We tested forty-seven 12 V-powered lamps with treated quartz bulb, which were made available by five producers as blind-coded samples. After exposure to either 1000 lx for 30 min or 2500 lx for 60 min, the 50 W lamps from two producers were borderline mutagenic in strains TA100 and TA104 of S. typhimurium, and induced an evident and dose-related DNA damage in the E. coli strain CM871 (uvrA- recA- lexA-), as compared to its isogenic, DNA repair-proficient counterpart WP2. The 50 W lamps supplied by the other three producers also induced a significant genotoxic damage, but only after exposure for 60 min at illuminance levels of 2500 lx or higher. In calibration experiments, one of these three lamp brands was found to induce in 60 min a genotoxic damage which was equivalent to the one induced in just 55 s by a traditional halogen lamp. Therefore, the new types of lamps with treated quartz bulbs provide an appreciable step forward in the safety of halogen lamps, but some output of genotoxic UV radiations does still occur. Moreover, the lamps manufactured by different producers are not equally effective to this respect. By comparison, the simple application of a glass cover to a traditional halogen lamp completely prevented genotoxic effects, even after 60 min of exposure at an illuminance of 10,000 lx. Suitable regulations are urgently needed for controlling the biological safety or artificial illumination systems.

Reversal of sodium-azide mutagenicity by liver preparations and by gastric juice.

Sodium azide was found to be mutagenic for Salmonella typhimurium by inducing base-pair substitutions that were not enhanced by pKM101 plasmid (R factor). However, the mutagenicity of sodium azide was decreased by enzyme proteins contained in rat-liver post-mitochondrial fractions, depending on the NADPH-generating system. Pre-incubation with human gastric juice also decreased azide mutagenicity. These metabolic effects might explain the conflicting nature of the mutagenicity and carcinogenicity tests reported in the literature. Laboratory reagents containing 0.1% sodium azide as a preservative showed the expected patterns of mutagenicity and of metabolic deactivation, and no aspecific interaction could be detected between azide and the various components, including proteins, of the reagents tested.

Mutagenicity testing with TA97 and TA102 of 30 DNA-damaging compounds, negative with other Salmonella strains.

In a comparative study on 135 compounds of various chemical classes, 30 agents inducing direct nonreparable DNA damage in repair-deficient E. coli failed in reverting strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium (De Flora et al., 1984b). These compounds were re-assayed in the Ames test using strains TA97 and TA102. A dose-dependent mutagenic response was detected with aminoantipyrine and p-rosaniline in TA97 and with streptomycin and formaldehyde in TA102. p-Rosaniline was the only mutagen requiring metabolic activation. 5 compounds, i.e. o-aminophenol in TA97 and methanol, ethanol, cadmium chloride and cadmium sulfate in TA102, induced a reproducible increase in revertants over controls, but this was less than 2-fold. The remaining 21 chemicals--including amino compounds, aliphatics, aromatics, heterocycles, hydrazine derivatives and inorganics--confirmed their inactivity in the Ames test. Overall data for 135 compounds, comparing the Ames test (7 strains) and the DNA-repair test (3 strains), are re-assessed on the basis of these findings.

Mutagenicity of active oxygen species in bacteria and its enzymatic or chemical inhibition.

The mono-electronic reduction of oxygen in the hypoxanthine-xanthine oxidase system led to the formation of active species eliciting an evident and highly reproducible mutagenic response in strain TA104 of S. typhimurium. Similar effects were observed by generating oxy radicals either extracellularly or inside bacterial cells. Mutagenicity was selectively detected in TA104 and not in other Salmonella strains, which points out the importance of the hisG428 mutation and of the deletion excising the uvrB gene, as far as sensitivity to oxy radicals is concerned. The mutagenicity of the system was further enhanced in the presence of superoxide dismutase. Catalase did not affect the mutagenicity of hypoxanthine plus xanthine oxidase, whereas it inhibited the mutagenicity induced by the mixture of hypoxanthine with xanthine oxidase and superoxide dismutase. This demonstrates that not only hydrogen peroxide but also the superoxide radical anion is positive in this system. Glutathione and 2 synthetic thiols, i.e., N-acetylcysteine and alpha-mercaptopropionylglycine, besides decreasing the high spontaneous mutagenicity of TA104, efficiently prevented the mutagenicity of active oxygen species.

Experimental databases on inhibition of the bacterial mutagenicity of 4-nitroquinoline 1-oxide and cigarette smoke.

Two antimutagenicity databases were prepared by applying a co-treatment procedure to the Salmonella reversion assay. Ninety compounds belonging to various chemical classes were quantitatively tested for antimutagenicity towards the direct-acting mutagen 4-nitroquinoline 1-oxide (4NQO) in strain TA100 of S. typhimurium and 63 of them were additionally tested for antimutagenicity towards unfractionated mainstream cigarette smoke (CS) in strain TA98, in the presence of S9 mix. Twelve compounds (13.3%) inhibited 4NQO mutagenicity by at least 50%, with a MID50 (dose inhibiting 50% of mutagenicity) varying over a 1226-fold range. Twenty-six compounds (41.3%) inhibited CS mutagenicity, with a MID50 varying over a 520-fold range. Three compounds only, i.e., bilirubin, curcumin and myricetin, were capable of inhibiting the mutagenicities of both 4NQO and CS. However, myricetin and the other flavonoid rutin were at the same time mutagenic by inducing frameshift mutations following metabolic activation. There was a rather rigorous selectivity of antimutagenicity data depending on the chemical class of inhibitors and it was possible to discriminate protective effects within several pairs or series of structurally related compounds. For instance, all eight thiols and aminothiols inhibited 4NQO mutagenicity, which contrasted with the inactivity of the remaining 17 sulfur compounds tested, all of them lacking a free sulfhydryl group. The mutagenicity of CS was consistently inhibited by the majority of phenols (eight out of 10 tested) and by all two isothiocyanates, two dithiocarbamates, three indole derivatives, three tetrapyrrole compounds and three flavonoids tested. Although the results obtained cannot be extrapolated to other mutagens or test systems, they may provide a useful source of information for research in the area of antimutagenesis and for the development of chemopreventive agents.