Corneal topographic evaluation of decentration in photorefractive keratectomy: treatment displacement vs intraoperative drift

Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organophosphorus ester (OP) insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy with paraoxon and plant-activated m-phenylenediamine or with direct-acting 2-acetoxyacetylaminofluorene in Salmonella typhimurium cells [Gichner T et al. (1996): Environ Mol Mutagen 27; 59-66]. In the present study, mammalian-activated m-phenylenediamine, o-phenylenediamine, p-phenylenediamine, benzidine, 2,3-diaminophenazine or 2-aminofluorene, as well as plant-activated benzidine or 2-aminofluorene expressed an elevated mutagenic potency when assayed with S. typhimurium strain YG1024 in the presence of paraoxon. Under non-toxic conditions, paraoxon amplified the S. typhimurium mutant yield induced by these aromatic amines between 1.9-fold and 8.4-fold. Spectrophotometric analysis demonstrated that the rate of degradation of 2-acetoxyacetylaminofluorene was not significantly different in phosphate buffer with or without paraoxon or with S. typhimurium cytosol with or without paraoxon. Also paraoxon-mediated mutagenic synergy does not appear to be due to a direct reaction with aromatic amines. Mutagenic synergy between aromatic amines and OP oxon products may be a cause of concern because people are chronically exposed to environmental and dietary aromatic amines, and a significant segment of the U.S. population tested positive for OP insecticide residues.     

Structural determination of a mutagenic aminophenylnorharman produced by the co-mutagen norharman with aniline

Norharman (9H-pyrido[3,4-b]indole), widely distributed in our environment, including cigarette smoke and cooked foodstuffs, is not mutagenic to Salmonella strains, but becomes mutagenic to S.typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. To elucidate the mechanisms of co-mutagenicity, we tried to isolate the mutagen(s) produced by a reaction between norharman and aniline with S9 mix. By HPLC purification, two mutagenic compounds (I and II), one (I) showing mutagenicity with and the other (II) without S9 mix, were isolated. The structure of compound I was deduced to be a coupled compound of norharman and aniline, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), by a variety of spectrometry techniques and this was confirmed by its chemical synthesis. The mutagenic activity of this novel heterocyclic amine was tested using the pre-incubation method and was found to induce 187,000 revertants in TA98 and 1,783,000 revertants in YG1024 per microg with S9 mix. Compound II was shown to be hydroxyaminophenylnorharman. Formation of the same DNA adducts was observed in YG1024 when aminophenylnorharman or a mixture of norharman plus aniline was incubated with S9 mix. The hydroxyamino derivative also yielded the same DNA adducts in YG1024. Thus, the appearance of mutagenicity by norharman with aniline in the presence of S9 mix suggests that the coupled mutagenic compound, aminophenylnorharman, is formed from norharman and aniline, then converted to the hydroxyamino derivative and forms DNA adducts to induce mutations in TA98 and YG1024.     

Technical report: automated classification of first and second cycle metaphases

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes<2,4-DNT<2,2'-dimethyl-5, 5'-dinitroazoxybenzene (2,2'-DM-5, 5'-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)<4, 4'-dimethyl-3,3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB), and aminonitrotoluenes (2A4AT, 4A2NT)<2,4-DNT<4HA2NT4,4'-dimethyl-3, 3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2, 6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2'-dimethyl-3, 3'-dinitroazoxybenzene (2,2'-DM-3,3'-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

NMDA and AMPA receptors evoke transmitter release from noradrenergic axon terminals in the rat spinal cord

The organic extracts of soil collected at parks in residential areas in Osaka and neighboring cities in the Kansai area, Japan, showed mutagenicity in Salmonella typhimurium strain TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). The soil extracts from Ibaraki and two different sites in Osaka, i.e., Sumiyoshi-ku and Minato-ku, were mutagenic in strain TA100 as well as in strain TA98. Direct-acting mutagenicity of soil extracts from Sumiyoshi-ku and Minato-ku toward strain TA98 were 66 or more times higher than that of the other cities. Both extracts exerted stronger mutagenicity in strains YG1021 and YG1024 than TA98 and TA100, and the potency was especially high in strain YG1024: Sumiyoshi-ku, 153 000 revertants/g of soil; and Minato-ku, 246 000 revertants/g of soil. Two mutagenic compounds (I and II) were isolated from the Soxhlet extract of soil from the park in Sumiyoshi-ku by repetitive separation using normal-phase and reversed-phase column chromatography. By comparing the mass and UV spectra and retention times for HPLC on two individual ODS columns of compounds I and II with those of authentic chemicals, we identified these two compounds as 1,6- and 1,8-dinitropyrene (DNPy) isomers. Amounts of DNPy isomers in soil from Sumiyoshi-ku and Minato-ku were 1.7-2.2 ng/g. Forty-three percent and 40% of the mutagenicity of soil from Sumiyoshi-ku and Minato-ku could be attributed to these DNPy isomers, respectively.     

Occupational genotoxicity assessment by mutagenicity assays

Mutagenic activity measured by Ames test and by gene conversion, point mutation and mitochondrial mutability in Saccharomyces cerevisiae D7 strain was determined in the indoor environment of a glass factory. The results suggest that the increase in mutagenicity of air sample collected near the machinery is due to the thermal decomposition of oils. Modified assays were therefore compared for their ability to detect mutagens contained in urinary concentrates of exposed workers. The bacterial tests were performed by microsuspension assay in TA98, TA100 strains and in YG1024, YG1029 strains which overproduce O-acetyltransferase. Significant differences are evidenced both in the eukaryotic and prokaryotic systems.     

Nitroarene concentrations and direct-acting mutagenicity of diesel exhaust particulates fractionated by silica-gel column chromatography

Diesel exhaust particulates were extracted with benzene-ethanol (3:1, v/v) and separated into five fractions by silica-gel column chromatography. Direct-acting mutagenic activity was assayed by the Ames test using the Salmonella typhimurium YG1024 strain. The total activity of five fractions was about four times greater than that of the crude extract, suggesting that the activities in the fractions were suppressed in the crude extract. Strong activity was observed in fraction 4 which was eluted with dichloromethane (61.5% of the total activity) and fraction 5 which was eluted with ethanol (35.3%). Nitropolycyclic aromatic hydrocarbons (NPAHs) were determined by high-performance liquid chromatography with chemiluminescence detection. They were found mainly in fraction 4, although one NPAH was in fraction 3 which was eluted with n-hexane-dichloromethane (3:1, v/v). Based on these results, 53.1% of the activity in fraction 4 was attributed to NPAHs. The contribution of 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrenes was great and that of the other NPAHs was small. The mutagenic compound in fraction 5 was not identified. Fractions 1 and 2, which were eluted with n-hexane, and fraction 3 suppressed the activity of fraction 4. Polycyclic aromatic hydrocarbons in fractions 2 and 3 were considered as possible suppressors of NPAHs.     

Anthracene-9,10-diones as potential anticancer agents: bacterial mutation studies of amido-substituted derivatives reveal an unexpected lack of mutagenicity

Fifteen anthracene-9,10-dione ("anthraquinone") derivatives with (omega-aminoalkyl)carboxamido substituents at the 1-, 2-, 1,4-, or 2, 6-ring positions were tested for bacterial mutagenicity in reverse-mutation assays using Salmonella typhimurium frameshift strains TA1538, TA98, and TA97a, in the presence and absence of a metabolic activation system prepared from the livers of rats treated with Aroclor 1254. Six of the compounds were also tested in S. typhimurium TA100 and Escherichia coli WP2uvrApKM101 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. Two structurally related compounds, mitoxantrone and bisantrene, were tested in parallel as positive controls. Mitoxantrone was mutagenic to S. typhimurium TA1538 and TA98, whereas bisantrene was weakly mutagenic to both these strains but strongly mutagenic toward the TA97a variant. By contrast, although they are also DNA-binding intercalators, none of the amide-functionalized anthracene-9,10-diones of the present study showed significant mutagenic activity in any of the bacterial strains examined. Further, neither substituent position nor systematic alterations in the nature of attached side chains appeared to induce mutagenicity with these agents, although other studies have shown that such structural factors markedly influence their cytotoxic potencies toward mammalian cells in vitro.     

Optimalization of rate adaptation using Holter functions in DDD/R pacemakers]

The genotoxicity of the organic peroxide 1,2,3,4-tetrahydronaphthaline-1-hydroperoxide (or tetraline-1-hydroperoxide, THP) was investigated in the Ames assay without a metabolic activating system using Salmonella typhimurium strains TA 98, TA 100, and TA 102. THP served as a model compound for higher organic peroxides, which can arise from autoxidation of hydrocarbons, e.g. in Diesel exhaust. While THP induced no mutagenic response in S. typhimurium TA 98, it was directly mutagenic in strains TA 100 and TA 102. These data, along with findings on mutagenic properties of other alkyl hydroperoxides, suggest that such compounds deserve further investigation regarding their genotoxic potential and occurrence in the environment.     

Genotoxicity of Farbasol and its component: 4-ethyltoluene

A combination of assays for gene mutations in Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains with and without rat liver activation, and for micronucleus and sister chromatid exchange (SCE) in bone marrow cells of Imp:Balb/c mice was used to provide data on the mutagenic and genotoxic properties of the mixture of aromatic solvents, known under the trade name of Farbasol. In addition, 4-ethyltoluene (the main ethylmethylbenzenic component of Farbasol) was also tested for muta- and genotoxicity. The results revealed that neither Farbasol nor 4-ethyltoluene induced an increased reverse mutation in bacterial cells or the formation of micronucleated polychromatic erythrocytes in bone marrow. However, those compounds were found to be active as sister chromatid exchange (SCE) agents.     

Clinical research: any future?

Monochloramine has been suggested as an alternative disinfectant to chlorine to reduce levels of trihalomethanes in treated drinking water, but little is known of the toxicological properties and potential health implications of by-products specific to the chloramination process. Model aqueous fulvic acid solutions (200-400 mg C/liter), serving as surrogates for humic surface waters, were chloraminated over a range of molar Cl:C ratios from 1:40 to 1:2. The resulting by-products were extracted into diethyl ether at pH 2 and investigated with the Ames plate incorporation assay. Extractable mutagenicity increased with increasing chlorine and carbon dose up to about 30,000 revertants/liter at Cl:C ratios of 1:2. Mutagenicity was higher in Salmonella typhimurium strain TA100 than in strain TA98, and was decreased in the presence of S9, indicating that the mutagens formed were direct-acting and induced predominantly base-pair substitutions. Bovine serum albumin decreased slightly, and glutathione reduced greatly, the mutagenic activity detected in extracts. HPLC fractionation of the by-products indicated that most of the mutagenic activity was found in the earliest-eluting (most polar) fraction. The mutagenic by-products appeared to be qualitatively similar to 3-chloro-4-dichloromethyl-5-hydroxy-2-(5H)-furanone (MX) in their chromatographic behavior and responses to glutathione and bovine serum albumin, but were less readily detoxified by S9 than was MX.     

A new mutagenicity assay method for frameshift mutagens based on deleting or inserting a guanosine nucleotide in the beta-lactamase gene

The conventional method of site-directed mutagenesis was used to develop two Salmonella strains, JK-1 and JK-2, for detecting frameshift mutagens. The JK-1 strain was derived from Salmonella typhimurium TA1537 strain transformed by a mutant construct. A guanosine nucleotide was inserted between nucleotide residues 312 and 313 of the beta-lactamase gene. The JK-2 strain was obtained by the same procedure, but a guanosine nucleotide in position 315 of the beta-lactamase gene was deleted. The strains were tested with ten frameshift mutagens and the revertants were selected by ampicillin resistance. Representative mutagens including 2-nitrofluorene (2-NF), 2-acetylaminofluorene (AAF), 9-aminoacridine (9-AA), 2,7-diaminofluorene (2,7-DAF) and 2-methoxy-6-chloro-9-(3-(ethyl-2-chloro-ethyl)-aminopropylamino)acridine (ICR-170) were more potent in the JK-1 strain than the JK-2 strain, and the number of revertant colonies were dose related. Under the same conditions, the ampicillin test was more sensitive than the Ames test. Other types of compounds such as 2-methoxy-6-chloro-9-(2-chloroethylaminopropylamino)acridine (ICR-191), benzo[a]pyrene (BP), 4-nitroquinoline N-oxide (4-NQNO), hycanthone and aflatoxin B1 (AFB1) were not as mutagenic to these new strains. The method is quite promising for studying certain specific frameshift mutagens, but more chemical mutagens should be tested to validate its applicability and reproducibility in general use.     

Metabolic activation of aromatic amines by human pancreas

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas. Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens. Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethyl-amine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [3H]N-hydroxy-ABP) of pancreatic cytosols was high, about twothirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using 32P-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.     

Escherichia coli lacZ strains engineered for detection of frameshift mutations induced by aromatic amines and nitroaromatic compounds

Escherichia coli lacZ strains CC107-CC111, which detect specific frameshift mutations, were used to study the mutational specificities of 2-nitro-3-methylimidazo[4,5-f] quinoline (NO2-IQ) and rat hepatic S9-activated 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). New constructs were made in which UvrABC-dependent excision repair was eliminated (strains DJ3107-DJ3111), followed by introduction of plasmid pYG219 conferring acetyl CoA:arylamine N-acetyltransferase/acetyl CoA:arylhydroxylamine O-acetyltransferase (NAT/OAT) activity (strains DJ3207-DJ3211). Sensitivity to mutagens was greatly enhanced. The mutational specificity of NO2-IQ was identical to that of the corresponding amine, IQ. The most prominent mutations caused by the two compounds were -2(CGGC) and 1(CG) frameshifts. +1(AT) Frameshifts play a minor role in the pattern of mutational specificity. Induction of all three mutations was similarly influenced by NAT/OAT activation and UvrABC-dependent excision repair. These new tester strains provide an effective tool for the study of aromatic amine mutational specificity and the influences of excision repair and NAT/OAT activation.     

Inflammatory pulmonary pseudotumor in an adolescent

Among p-phenylenediamine, benzidine and the analogues we previously tested, only the nitro-group containing 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenediamine, 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine caused base-pair reversion in the histidine locus of Salmonella typhimurium TA100. In order to determine the types of mutations involved, such as transversion or transition, these four nitro-group containing compounds were tested with S. typhimurium strains TA100, TA104, TA4001 and TA4006. Dose-mutagenicity relationships occurred with TA100 and TA104. However, the majority of revertants from TA100 and TA104 were insensitive to inhibition by histidine analogue, DL-1,2,4-triazole-3-alanine. These results suggested that the occurrence of histidine revertants was predominantly induced by base-pair (point) mutations and not by suppressor gene mutations. The CG-TA transition and CG-AT transversion are the major types of mutation induced by all these compounds in TA100. The TA-AT transversion also contributed to the mutagenicity of 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine in TA104. These nitro-group containing compounds showed no mutagenicity in TA4001, but induced CG-GC transversion in TA4006.     

Homodimers of the 60 kDa phosphatidylinositol-anchored transforming growth factor-beta 2 binding proteins in FBHEC and MG-63 cells

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His+ revertants to a maximum of 4 times over background at a GTN dose of 5 mumol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 mumol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C-->T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Relation of neuronal endoplasmic reticulum calcium homeostasis to ribosomal aggregation and protein synthesis: implications for stress-induced suppression of protein synthesis

We have been interested in determining the structural and electronic features that may be useful in predicting the mutagenic activity of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs). We have previously found that a correlation between structural and electronic features and direct-acting mutagenicity in Salmonella typhimurium cannot be made using nitro-PAHs with different molecular size. In this study, a series of structurally related nitro-PAHs, the environmental contaminants 1-, 3-, and 6-nitrobenzo[alpha]pyrene (NBaP) and their derivatives, was used to determine structure-activity relationships. It was found that isomeric NBaPs are activated to DNA damaging and mutagenic derivatives by nitroreduction, ring-oxidation, or by a combination of these two pathways. A general finding was that NBaPs and derivatives with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct-acting mutagenicity in S. typhimurium strains TA98 and TA100. In this paper, we also discuss the effect of the location of the nitro group on the metabolism and the mutagenicity of NBaPs and the effect of oxygen-containing functional groups on the mutagenicity of NBaP derivatives. These findings provide a useful molecular basis for interpreting and predicting the direct-acting mutagenicity of nitro-PAHs.     

Urinary mutagenicity as a biomarker in workers exposed to benzidine: correlation with urinary metabolites and urothelial DNA adducts

Urinary mutagenicity has been used in occupational and epidemiological studies for over two decades as a cost-effective, general biomarker of exposure to genotoxic agents. However, few studies have compared urinary mutagenicity to additional biomarkers determined among low- and high-exposed groups. To address this issue, we evaluated the relationship between urinary mutagenicity and other types of biomarkers in a cross-sectional study involving 15 workers exposed to the urinary bladder carcinogen benzidine (BZ, high exposure), 15 workers exposed to BZ-dyes (low exposure), and 13 unexposed controls in Ahmedabad, India. Urinary organics were extracted by C18/methanol and evaluated for mutagenicity in the presence of S9 in the Salmonella strain YG1024, which is a frameshift strain that overproduces acetyltransferase. The results were compared to biomarker data reported recently from the same urine samples (Rothman et al., Proc. Natl Acad. Sci. USA, 93, 5084-5089, 1996) that included a metabolite biomarker (the sum of the urinary levels of BZ + N-acetylbenzidine + N,N'-diacetylbenzidine) and a DNA adduct biomarker [a presumptive N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine (C8dG-ABZ) DNA adduct in exfoliated urothelial cells]. The mean +/- SE urinary mutagenicity (revertants/micromol of creatinine) of the low-exposure (BZ-dye) workers was 8.2 +/- 2.4, which was significantly different from the mean of the controls (2.8 +/- 0.7, P = 0.04) as was that of the mean of the high-exposure (BZ) workers (123.2 +/- 26.1, P < 0.0001). Urinary mutagenicity showed strong, positive correlations with urinary metabolites (r = 0.88, P < 0.0001) and the level of the presumptive C8dG-ABZ urothelial DNA adduct (r = 0.59, P = 0.0006). A strong association was found between tobacco use (bidi smoking) and urinary mutagenicity among the controls (r = 0.68, P = 0.01) but not among the exposed workers (r = 0.18, P = 0.11). This study confirms the ability of a biomarker such as urinary mutagenicity to detect low-dose exposures, identify additional genotoxic exposures among the controls, and correlate strongly with urinary metabolites and DNA adducts in the target tissue (urinary bladder epithelia) in humans.     

Validity of specific subscales of the positive and negative symptom scales in older schizophrenia outpatients

The principal natural food colorants used in modern food manufacture are anthocyanins, betalains, carotenoids, chlorophylls, riboflavin and caramel. Carotenoids (carotenes and xanthophylls) occur naturally in some foods such as carrots, red tomatoes, butter, cheese, paprika, palm oil, corn kernels, marigold petals, annatto, and red salmon. Carotenoids (alpha- or beta-carotene and xanthophylls) are excellent antioxidants and inhibit some types of cancers. In the present study, we used the Salmonella typhimurium tester strain YG1024 in the plate-incorporation test to examine the antimutagenicity of xanthophylls extracted from Aztec Marigold (Tagetes erecta) on 1-nitropyrene (1-NP) mutagenicity. Further, we investigated the effect of lutein on DNA-repair system of tester strain YG1024, using a preincubation test. The possible mechanism of lutein on 1-NP mutagenicity was studied by comparing the absorption spectrum of lutein, 1-NP and lutein plus 1-NP. In a dose-response curve of 1-NP, the mutagenic potency was 4317 revertants/nmol, and the dose of 0.06 microgram of 1-NP/plate was chosen for the antimutagenicity studies. Lutein and xanthophylls from Aztec Marigold (pigments for poultry and human use) inhibited mutagenicity of 1-NP in a dose-dependent manner. Lutein and the pigments were not toxic to the bacteria at the concentrations tested (0.002, 0.02, 0.2, 2.0 and 10 micrograms/plate). The percentages of inhibition of 1-NP mutagenicity were 72%, 92% and 66.2% for lutein (10 micrograms/plate), pigment for poultry use (10 micrograms/plate) and pigment for human use (2 micrograms/plate), respectively. Lutein had no effect on the DNA-repair system of strain YG1024. A new peak was detected at 429 nm when lutein was added at 1-NP, and it was stable throughout the incubation time. The results suggest that the major mechanisms of lutein against 1-NP mutagenicity is the potential formation of a complex between lutein and 1-NP, which could limit the bioavailability of 1-NP.     

Primary aromatic amines from side-stream cigarette smoke are common contaminants of indoor air

A very sensitive mass-spectrometry method has been developed for the analysis of aromatic amines in tobacco smoke and in indoor air. Cigarettes were smoked with a smoking machine; the amines from the smoke were trapped in a 5% HCl water solution containing internal standards and detected by gas chromatography/mass spectrometry in the selected-ion-monitoring (SIM) mode. The amines measured were the following: aniline, 2-toluidine, 3-toluidine, 4-toluidine, 2-ethylaniline, 3-ethylaniline, 4-ethylaniline, 2,3-dimethylaniline, 2,4-dimethylaniline, 2,5-dimethylaniline, 2,6-dimethylaniline, 1-naphthylamine, 2-naphthylamine, 2-methyl-1-naphthylamine, 2-aminobiphenyl, 3-aminobiphenyl and 4-aminobiphenyl. We analyzed nine brands of cigarettes sold commercially in Italy (Gauloise, Nazionali, Marlboro, Camel, MS, MS mild and MS lights), with and without filter. Main-stream smoke contained a lower amount of aromatic amines than side-stream smoke: the total level of these amines in main-stream smoke ranged from 200 to 1300 ng/cigarette, whereas the level of aromatic amines in side-stream smoke varied from 20,000 to 30,000 ng/cigarette. The smoke of black-tobacco cigarettes had higher levels of aromatic amines compared to light-tobacco cigarettes and the filters significantly reduced aromatic amines in main-stream smoke. We also determined the levels of aromatic amines in ambient air, offices and houses. Some aromatic amines (aniline and toluidine) were detected in ambient air, as well as in rooms of non-smokers. Most measurements showed a considerable contamination of aromatic amines derived from side-stream smoke, which was detected also in parts of the buildings in which smoking was not allowed.     

Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon ortho-quinones produced by dihydrodiol dehydrogenase

Eight polycyclic aromatic hydrocarbon (PAH) ortho-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II-e (rat hepatoma) cells and for their mutagenicity in the Ames test. Seven of the PAH otrtho-quinones were potent cytotoxins yielding IC50 values for cell survival in the range 1-30 microns. PAH ortho-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained ortho-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz[alpha]anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained ortho-quinones (e.g., benz[alpha]anthracene-3,4-dione and 5-methylchrysene-1,2-dione which reduced cell survival but had no effect on cell viability; and group III contained ortho-quinones (e.g., benzo[alpha]pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival. Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that ortho-quinones underwent preferential enzymatic one-electron redox-cycling and produced superoxide anion radical (O2-.) and/or ortho-semiquinone anion or alternant radicals. ortho-Quinones that reduced cell viability produced O2-. and caused the most total free radical formation, while those that reduced cell survival produced ortho-semiquinone anion or alternant radicals only. PAH ortho-quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104. They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens. The presence of an activating system (Aroclor-induced rat liver S9 plus NADPH) did not increase the mutagenicity of ortho-quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104). These data suggest that PAH ortho-quinones produced by DD are cytotoxic and mutagenic by different mechanisms. The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or ortho-semiquinone anion or alternant radicals. The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH orthooffinones to intercalate and covalently modify DNA.