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.     

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.     

Clastogenic activity of sodium fluoride in great ape cells

Nitrobenzimidazole and nitroindole derivatives, related to oxiconazole and characterized by an oxyiminic function, have been synthesized as novel antimycotics and their mutagenic activity tested in Salmonella typhimurium strains TA100 and TA98 with and without an exogenous metabolizing system. TA98NR and TA98/1,8-DNP6 strains were employed to identify a specific metabolic reaction which governs the mutagenic potency. Active compounds are weak direct-acting mutagens. Only derivatives bearing a nitro group on the phenyl ring linked to the oxyiminic function and lacking halogenated substituents show mutagenic activity. Metabolism by bacterial enzyme systems is important to the expression of genotoxicity. The reductive activation of nitrobenzimidazoles and nitroindoles carried out by the 'classical' nitroreductase of Salmonella, which is defective in TA98NR, is required of mutagenicity. Similarly, the O-acetyltransferase defective in TA98/1,8-DNP6 is required for the efficient production of the ultimate electrophilic nitrogen species, which react with DNA. The role of bacterial metabolism in mutation induction needs careful consideration to assess the potential risk to humans from nitrobenzimidazole and nitroindole antimycotics.     

Mutagenicity of mixtures of halogenated aliphatic hydrocarbons and polycyclic aromatic hydrocarbons in the Ames test with TA98 and TA100

Within the framework of the assessment of the genotoxic potential of environment samples the Salmonella-microsome-test (Ames-test) is often used as a screening-test. It is one of the most applied biotest systems and possesses a large scientific acceptance. Because most environment samples are mixtures of various substances, possible effects resulting from the combination should be taken into account with regard to the mutagenic potential. In this context we investigated eight polycyclic aromatic hydrocarbons each combined with six halogenated aliphatic hydrocarbons as to their mutagenicity in the Salmonella-microsome-test with TA98 and TA100. For an exogenous metabolizing system, Arochlor 1254 induced rat liver S9-mix was used. Benz-a-pyrene in combination with bromodichloromethane (Ames neg. in TA98 and TA100 +S9) showed an increase in the number of the revertants up to 25% in TA98 and TA100 (+S9). Carbon tetrachloride (Ames neg. in TA98 and TA100 +S9) showed in TA100 (+S9) an increase in the number of the revertants of 18% at most. In the combination 3-methylcholanthrene with dichloromethane the number of revertants in TA98 (+S9) increased by 25% and in TA100 (+S9) by 18%. Hexachloroethane (weakly mutagenic in TA98 +S9) in combination showed in TA98 (+S9) a slightly increased number of revertants with benz-a-pyrene as well with 3-methylcholanthrene. All the other substances tested (chrysene, phenanthrene, anthanthrene, dibenz-a, i-pyrene, triphenylene, fluoranthene) in combination with either tetrachloroethylene or trichloroethene did not cause an increase in mutagenicity.     

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.     

Synthesis of tools for target identification of the anti-apoptotic compound CGP 3466; Part I

We previously isolated five mutagens, compounds I-V, in blue rayon-adsorbed materials from the Nishitakase River in Kyoto. The chemical structure of compound I, a major mutagen that accounted for 21% of the total mutagenicity, was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). Compound II was also a major mutagen and accounted for 17% of the total mutagenicity. In this study, a large quantity (1.2 mg) of compound II was isolated from adsorbate to 27 kg of blue cotton, and its UV, mass, and 1H NMR spectra were analyzed. On the basis of the spectral data, compound II was deduced to be the PBTA-1 analogue 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). As with PBTA-1, PBTA-2 was synthesized from an azo dye by reduction and chlorination. Since all of the spectra of PBTA-2 coincided with those of compound II obtained from river water, compound II was concluded to be PBTA-2. PBTA-2 is a newly identified potent mutagen, which induces 93 000 and 3 200 000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. Like PBTA-1, PBTA-2 may also be produced from an azo dye during industrial processes in dyeing factories and treatment at sewage plants.     

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.     

Septic thrombosis of the portal vein due to peripancreatic ligamental abscess

Nitro reduction is a critical step in the mutagenic activation of nitroarene. Nitroarene and quinone are known to be reduced by common enzymes, and thus, naphthoquinone (NQ) was studied for its effects on the mutagenicity of nitroarene in the Ames test using Salmonella typhimurium TA98 without S9. The mutagenicity of 1,3-dinitropyrene in TA98 was found to increase 9- and 6-fold as much in the presence of 70 nmol/plate of 2-methyl-1,4-NQ and 2-hydroxy-1,4-NQ, respectively. Mutagenicity also became greater in 1,3,5-trinitronaphthalene, 1-nitropyrene and 3-nitrofluoranthene. Seventy nmol/plate of 2-methyl-1,4-NQ increased the mutagenicity of 1-nitropyrene by 10.5-fold as much.     

Insulin receptor-like tyrosine kinase in the tobacco hornworm, Manduca sexta

Urinary bacterial mutagenicity was used as a biomarker of exposure to ambient air pollution in a group of women working outdoors in the city of Teplice (TP; Northern Bohemia) with higher levels of air pollution than a similar group of women in the city of Prachatice (PT; Southern Bohemia). The Salmonella typhimurium plate incorporation assay with the TA98 and YG1041 strains and microsuspension assay with the YG1041 strain were used for testing the urinary mutagenicity. PAH and their metabolites were analyzed by HPLC and GC/MS methods. The significantly higher values of most PAHs/metabolites detected in a TP group confirmed the differences of PAH exposures between both groups. In the plate incorporation assay, the TA98 strain was not able to detect the increase in urinary mutagenicity, but, for the YG1041 strain, the urinary mutagenicity was clearly determined with a significant difference in number of YG1041 + S9 revertants between the TP and PT groups. The microsuspension assay increased the mean response by about 10-fold over the standard plate test; however, no statistical difference between TP and PT groups was found due to high interindividual variability and small sample size. Comparing the urinary PAH/metabolites to urinary mutagenicity, significant correlations were observed between the plate incorporation mutagenicity results with the YG1041 revertants in the presence of metabolic activation and several of the urinary PAH/metabolites. On the contrary, in the microsuspension assay, several urinary PAH/metabolites correlated significantly with the YG1041 revertants only in the absence of metabolic activation. This may indicate the influence of different treatment conditions of assays on the urinary mutagenicity results. The results suggest the insufficient sensitivity of the TA98 tester strain to determinate low urinary level of mutagens. On the contrary, the use of the YG1041 tester strain increases the probability of detecting an effect of environmental exposure and seems to be applicable to biological monitoring. To definitely replace the standard plate incorporation assay with the microsuspension method is not possible without further comparative studies.     

Application of the Salmonella mutagenicity assay and determination of polycyclic aromatic hydrocarbons in workplaces exposed to petroleum pitch and petroleum coke

Workplaces of an Italian carbon electrode factory, exposed to petroleum pitch and petroleum coke, were studied using a coupled chemical and biological approach to evaluate occupational mutagenic/carcinogenic hazards. Analytical procedures for the determination of polycyclic aromatic hydrocarbons (PAH) and Salmonella/microsome mutagenicity tests (with TA98 and TA100 strains) were performed on both industrial ingredients (pitch and coke) and airborne particulate matter of the working environment, after fractionating by sequential Soxhlet extractions with four organic solvents of increasing polarity (benzene, chloroform, methanol and acetone). The results showed: (a) the presence of extraordinarily high PAH (carcinogenic and non-carcinogenic) contents in the benzene extracts of petroleum pitch (3.6 wt% of total PAH) and of airborne particulate samples (up to 0.35 wt% of total PAH), in correlation with very high indirect (after metabolic activation) mutagenic responses of benzene extracts with strain TA98; (b) very high indirect mutagenic responses in the other extracts of the airborne particulate samples (especially with strain TA98); (c) the production during the processing at high temperatures of directly acting mutagens (without metabolic activation) which were absent in the starting materials and their release in the air of workplaces. The comparison of chemical analytical and mutagenicity data has proved to be an interesting approach for better defining the relative health hazards due to occupational exposure to potentially mutagenic/carcinogenic petroleum products.     

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.     

Recent update on the PPAR alpha-null mouse

The most important commercially available nitro- and aminobenzenes and the explosive trinitrobenzene were tested for mutagenicity in the Salmonella typhymurium TA 98 and TA 100 both in the absence and presence of S 9. Ten of the 14 compounds tested (71%) were mutagenic. All the substances showed positive results in TA 98 and 4 substances were also mutagenic in TA 100. The three diaminobenzenes and 4-nitroaniline were mutagenic only with metabolic activation. All other compounds did not require the addition of S 9. Only nitrobenzene, 1,2-dinitrobenzene, aniline and 2-nitroaniline were negative in both strains. In summary, all substances that are derived from nitrobenzene or aniline by addition of a nitro group in the meta- or para-position were mutagenic, whereas nitrobenzene and aniline themselves and their ortho-derivates were nonmutagenic. The possible relationships between the position of the substituents and the mutagenicity are discussed.     

The evidence of clonal evolution with monosomy 7 in aplastic anemia following granulocyte colony-stimulating factor using the polymerase chain reaction

The polycyclic aromatic hydrocarbons (PAH) containing fractions of smoked and charcoal-broiled foods, namely, Sheat fish (Kytopterus apogon), Mimrow (Crossocheilus reba), Freshwater catfish (Clarias batrachus), chicken wings, rice pork sausage and pork, in addition to naphthalene, acenaphthene, anthracene, phenanthrene, fluoranthene, pyrene, benz[a]anthracene, naphthacene, benzo[a]pyrene, benzo[e]pyrene, 9,10-dimethyl-1,2-benzanthracene, dibenz[ah]anthracene, benzo[ghi]perylene and coronene, were evaluated for their mutagenic potential using Salmonella typhimurium strains TA98 and TA100 in the absence of metabolic activation after being treated with nitrite (500 mM) for 4 hr at 37 degrees C and in acid solution pH 3.0-3.5. The presence of N-nitroso compounds was also determined. Results showed that nitrite could convert most samples to direct-acting mutagens towards both strains except for fluoranthene and benzo[ghi]perylene, which exhibit mutagenicity only with TA98. It was demonstrated that treatment of PAHs with nitrite in acid solution produced some non-N-nitroso direct-acting mutagens, suggesting that they might belong to nitro-PAHs. Therefore, the consumption of charcoal-broiled and smoked foods simultaneously with nitrite is not recommended.     

The genotoxicity and carcinogenic potential of gastrofenzin

Genotoxicity of the Bulgarian drug gastrophensin was studied by using a battery of two genotoxicity assays "in vitro" - Salmonella/mutation assay and "in vivo" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" was tested in five mutant, histidine auxotrophic strains - TA 1535, TA 1537, TA 1538, TA 98 and TA 100 without and in the presence of metabolic activation (+/- S9) at concentration of 0.4, 2 and 10 mg center dot ml-1. Gastrophensin did not induce mutagenic response in the Salmonella/mutation assay in a range of tested concentrations in both series of assays (+/- S9). Gastrophensin did not induce micronuclei in bone marrow cells of male C57Bl6 mice at 24, 48 and 72 hours after single oral treatment with 236 mg center dot kg-1 (80% DL50 oral, mice) and 118 mg center dot kg-1 (40% DL50 oral, mice). Based on the present data a conclusion of the lack of mutagenicity and of carcinogenic potency of gastrophensin was made.     

Comparison of CoMFA models for Salmonella typhimurium TA98, TA100, TA98 + S9 and TA100 + S9 mutagenicity of nitroaromatics

Comparative Molecular Field Analysis (CoMFA) was applied to a comprehensive data set of heterogeneous nitroaromatics tested in Salmonella typhimurium TA98 and TA100 with and without S9 microsomal activation. The four CoMFA models developed agree with postulated mechanisms of mutagenicity, and explain over 70% of the corresponding mutagenic variance The standard deviation coefficient contours common in the four models included high electronic density regions equivalent to C4-C5 in the pyrene ring, and an electron deficient site equivalent to C6. These areas are associated with high mutagenicity. Electron deficient areas may be related with the nitroreductive bioactivation of nitroaromatics. Electron rich sites may be involved with oxidative mechanisms analogous to the bioactivation pathway of polycyclic aromatic hydrocarbons. The contribution of steric factors to mutagenicity follows the order TA98 + S9 > TA98 > TA100 + S9 > TA100. The models indicated that increasing bulk perpendicular to the aromatic plane would decrease mutagenicity, but increasing the aromatic ring system along a region corresponding to C6-C7 in 1-nitropyrene would increase mutagenicity.     

The role of the peripherin/RDS gene in retinal dystrophies

The effect of a potent endogenous antioxidant, the pineal gland indole melatonin (MLT) on the mutagenicity of twelve well-known mutagens and carcinogens has been investigated using two in vitro tests the Ames test and the single cell gel electrophoresis assay (SCGE assay or COMET assay). The 12 mutagens used were 7, 12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP), 2-aminofluorene (AF), 1,2-dimethylhydrazine (DMH), bleomycin, cyclophosphamide (CP), 4-nitroquinoline-N-oxide (NQO), 2,4, 7-trinitro-9-fluorenone (TNF), 9-aminoacridine (AA), N-nitrosomethylurea (NMU), mitomycin C and sodium azide tested in the absence or in the presence of S9 mix. MLT alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested (100 microM) in the SCGE assay. In four Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 MLT significantly reduced the mutagenicity of chemicals which require S9 activation. In the SCGE assay performed on CHO cells, preincubation with MLT led to a strong inhibition of clastogenic activities of DMBA and CP, and in a lesser extent with BP and NMU. With mitomycin C, MLT exacerbated responses in both tests. The possible mechanisms of MLT's inhibitory action are discussed.     

In vivo formation of mutagens by intraperitoneal administration of polycyclic aromatic hydrocarbons in animals during exposure to nitrogen dioxide

Consumption of fossil fuels has increased indoor and outdoor concentrations of polycyclic aromatic hydrocarbons (PAHs) and nitrogen dioxide (NO2). To study the combined effect of PAH administration and NO2 exposure on mutagenicity of urine from animals we injected 400 mg/kg body wt i.p. one of five kinds of PAH (pyrene, fluoranthene, fluorene, anthracene and chrysene) into ICR mice, Wistar rats, Syrian golden hamsters or Hartley guinea pigs after exposure to 20 p.p.m. NO2 gas for 24 h and then exposed the animals to NO2 gas for an additional 24 h. During the latter 24 h we collected the urine and assayed its mutagenicity with the Ames Salmonella strains after treatment with beta-glucuronidase and arylsulfatase and extraction with dichloromethane. The urine from mice treated with both PAH and NO2 showed high mutagenicity for Salmonella typhimurium strains TA98 and TA100, whereas the urine from mice treated with PAH and air showed almost no mutagenic activity. The mutagenicity was decreased in nitroreductase- and acetyltransferase-deficient strains TA98NR and TA98/1,8-DNP6 respectively. Treatment with a mixture of 20% of each of the five kinds of PAH and NO2 augmented the urinary mutagenicity of mice 1.5-fold. The urine from hamsters treated with pyrene or fluoranthene and NO2 was also highly mutagenic, but that from rats or guinea pigs was not very mutagenic. The mutagenicity was also decreased in strains TA98NR and TA98/1,8-DNP6. These results suggest that the urine contains nitro compounds and that the nitration of PAHs occurs in the body of animals under exposure to NO2 gas. Actually, the nitrated metabolites of pyrene, 1-nitro-6/8-hydroxypyrene and 1-nitro-3-hydroxypyrene, were detected in the urine from mice treated with pyrene under exposure to NO2 gas. To elucidate the mechanism of in vivo nitration, NO2 (20 p.p.m.) was bubbled through 50 mM Tris-HCl buffer (pH 7.4) or dichloromethane solution containing pyrene or 1-hydroxypyrene (10 microg/ml). Pyrene was not nitrated by NO2 in either aqueous or organic solutions. However, 1-hydroxypyrene was changed to nitrohydroxypyrenes by NO2 in the Tris-HCl buffer, but not in the organic solution. Ascorbic acid, alpha-tocopherol, glutathione oleic acid and hemoglobin were found to inhibit the nitration of 1-hydroxypyrene in aqueous solution. The urinary mutagenicity of mice treated with both pyrene and NO2 was also decreased by oral administration of ascorbic acid and alpha-tocopherol. These results suggest that 1-hydroxypyrene is nitrated by an ionic reaction in the animal body after hydroxylation of pyrene in the liver.     

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.     

Independent responsiveness of frog liver low-density lipoprotein receptor and HMGCoA reductase to estrogen treatment

We examined the mutagenicity of cigarette smoker's urine in 32 healthy male cigarette smoker and 37 healthy male non-smoker. Twenty-four-hour urine specimens were subjected to blue rayon extraction which selectively adsorb polycyclic compounds, after which the elutions were fractionated by carboxymethyl cellulose column chromatography for removing antimutagenic compounds. The mutagens were measured by using an S9-mediated Salmonella mutagenicity test on strain TA98. Compared with those with non-smokers, smokers' urine showed a significantly higher urinary level of mutagens in the acid-elutable and in the sum of all chromatography fractions. A similar tendency was also seen in the alkali-elutable fraction. The subjects were classified into three groups according to the number of smoked cigarettes. Heavy smokers, who smoked more than 20 cigarettes per day, showed a significantly higher urinary level of mutagens than both non-smokers and light smokers especially in the acid-elutable and in the sum of all chromatography fractions. Our findings suggest that smokers are exposed to a great amount of polycyclic carcinogens and mutagens by cigarette smoking. These results also suggest that urinary level of mutagens measured by using blue rayon extraction combined with carboxymethyl cellulose chromatography could be a good index for estimating the exposure to carcinogens and mutagens such as polycyclic compounds.     

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.