Mutagenicity of oxidized microsomal metabolites of 1-nitropyrene in Chinese hamster ovary cells

1-Nitropyrene, a tumorigenic environmental pollutant, is mutagenic in Chinese hamster ovary (CHO) cells in the presence of a liver homogenate 9000 g supernatant fraction (S9). The metabolic pathways involved in this response were studied by comparing the mutagenicities at the hypoxanthine-guanine phosphoribosyl transferase locus of 1-nitropyrene, some oxidized microsomal metabolites of 1-nitropyrene, and related compounds. In the absence of S9, pyrene 4,5-oxide and 6-hydroxy-1-nitropyrene displayed the highest mutagenicities, followed by 1-nitropyrene 9,10-oxide and 1-nitropyrene 4,5-oxide; 3- and 8-hydroxy-1-nitropyrene were weaker mutagens, while pyrene and 1-nitropyrene were essentially without activity. With S9, the order of mutagenic potency was 1-nitropyrene 4,5-oxide greater than 6-hydroxy-1-nitropyrene approximately 1-nitropyrene 9,10-oxide greater than 1-nitropyrene approximately 3-hydroxy-1-nitropyrene approximately 8-hydroxy-1-nitropyrene greater than pyrene approximately pyrene 4,5-oxide, with the latter two compounds being essentially inactive. Inclusion of the epoxide hydrolase inhibitor 1,2-epoxy-3,3,3-trichloropropane during the S9-mediated treatment of CHO cells with 1-nitropyrene increased mutation induction 5-fold. Also, liver microsomes prepared from guinea-pigs treated with Aroclor 1254 mediated a stronger mutagenic response with 1-nitropyrene than microsomes from Aroclor-treated rats. 1-Nitropyrene was essentially non-mutagenic in the presence of microsomes from untreated and phenobarbital-treated rats. Examination of the 1-nitropyrene metabolites produced during the microsomal incubations indicated that Aroclor-induced guinea-pig microsomes yielded substantial amounts of 1-nitropyrene 4,5-dihydrodiol, while Aroclor-induced rat microsomes produced 6-fold more 6- and 8-hydroxy-1-nitropyrene than phenobarbital microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioassay-directed fractionation of 1-nitropyrene metabolites: generation of mutagrams by coupling reverse-phase HPLC with microsuspension mutagenicity assays

We have performed bioassay-directed fractionation of a model complex mixture (rabbit lung S9-generated metabolites of 14C-radiolabeled 1-nitropyrene) by assaying reverse-phase HPLC fractions using two microsuspension mutagenicity assays. A forward-mutation assay measuring mutation at the gpt locus (8-azaguanine resistance) in Salmonella typhimurium TM677 was performed in a total volume of 100 microliters, and a reverse-mutation assay measuring mutation at the hisD3052 allele in S. typhimurium TA98 was performed in a total volume of 200 microliters. HPLC fractions were collected every 30 s for 45 min, resulting in 90 fractions per run. The HPLC chromatogram (absorbance at 280 nm) and the 14C profile were compared to the mutagenicity profiles (mutagrams) and to the mutagenic potencies of pure metabolites studied separately. The results indicate that a fine dissection of the mutagenic fractions can be obtained by coupling HPLC to microsuspension mutagenicity assays. Differences observed between the mutagrams generated by the two bacterial strains were most likely due to metabolic (nitroreductase) differences between the two strains. This method should be generally applicable to the bioassay-directed chemical analysis of complex mixtures.     

Bacterial mutagenicity,metabolism, and DNA adduct formation by binary mixtures of benzo[a]pyrene and 1-nitropyrene

Air pollutants are a complex mixture containing polycyclic organic compounds. Among these are 1-NP and B[a]P, which are important contributors to the mutagenicity of diesel exhaust and airborne particulate matters. To investigate the interaction of a complex mixture of airborne mutagens, the mutagenicity of 1-NP was examined with S. typhimurium TA98 and TA98NR in the presence and absence of B[a]P. B[a]P exhibited a more antagonistic effect on the mutagenicity of 1-NP in strain TA98 than in strain TA98NR. Also studied were (1) the inhibitory effects of B[a]P on the nitroreductive metabolism of 1-NP and (2) DNA adduct formation by 1-NP. Nitroreductase was associated with the metabolism of 1-NP, and was reduced in a dose-dependent manner in a binary mixture of 1-NP and B[a]P. HPLC analysis showed that the amounts of 1-AP and NAAP, the metabolites of 1-NP, were significantly decreased by the addition of B[a]P in mixtures. The results indicate that the antagonistic effect of B[a]P on the mutagenicity of 1-NP is mediated through altering its nitroreductive metabolism. © 1994 Wiley-Liss, Inc.     

1-nitropyrene as a marker for the mutagenicity of diesel exhaust-derived particulate matter in workplace atmospheres

The use of 1-nitropyrene (1-NP) as a marker for the occupational exposure to diesel exhaust (DE) mutagens was investigated in workplace atmospheres contaminated with DE from a variety of emission sources, such as power supplies, forklifts, trucks, caterpillar vehicles, trains, ships' engines, and vehicles in city traffic. Total suspended particulate matter was collected by area sampling. The 1-NP content of acetone extracts of these samples as determined by gas chromatography-high resolution mass spectrometry varied from 0.080 to 17 μg/g acetone extractable matter, corresponding to air concentrations of 0.012 to 1.2 ng/m³. A sample collected in a rural area contained 0.0017 ng/m³ 1-NP. The mutagenicity of the extracts was tested in the Salmonella typhimurium strains TA98 and TA1538, using the microsuspension assay with and without metabolic activation by an exogeneous metabolizing system (rat liver S9-fraction). In addition, the S. typhimurium strains YG1021 and YG1024 were used because of their high sensitivity towards the mutagenicity of nitro polycyclic aromatic hydrocarbons. When plotting the mutagenic potency of the air sample extracts as determined in the absence of liver S9 versus the particle-associated 1-NP level, a relatively high correlation (r = 0.80–0.91) was observed in all of the S. typhimurium strains. High correlations (r = 0.80–0.93) were also observed when plotting the results of mutagenicity testing after activation by S9 versus the outcome of chemical analysis. These results show that the 1-NP content of workplace air samples is associated with their mutagenic potency, suggesting that 1-NP may be used as a marker for occupational exposure to DE-de-rived particle-associated mutagens © 1995 Wiley-Liss, Inc.     

Suppressive effects of methyl methacrylate on the mutagenicity and DNA adduct formation induced by 1-nitropyrene and benzo[a]pyrene

Methyl methacrylate (MMA) is widely used as a cement in dentistry,orthopaedic surgery and ophthalmology. Studies based on short-termgenotoxicity tests have produced conflicting results in thelast two decades. In the present study, the effects of MMA onthe mutagenicity of 1-nitropyrene (1-NP) and benzo[a]pyrene(B[a]P) were evaluated with the Salmonella typhimurium TA98strain in the absence and presence of S9 mix. The direct-actingmutagenicity of 1-NP was markedly decreased by MMA in a dose-dependentmanner. However, a low inhibitory effect of MMA on the metabolic-actingmutagenicity of B[a]P was observed. MMA did not show mutagenicitywithin the concentrations of 4.7–37.6 µM eitherwith or without S9 mix. The inhibitory effect of MMA was notdue to its cytotoxicity because very low and/or no cytotoxicityof MMA to S.typhimurium TA98 was observed. To confirm the antimutagenicityof MMA against 1-NP and B[a]P, a ³²P-postlabelling method wasused to determine whether MMA modified DNA adduct formationproduced by both compounds in calf thymus DNA. MMA inhibitsthe formation of 1-NP- and B[a]P-DNA adducts in a dose-dependentmanner. The DNA adduct of 1-NP reduced by MMA was greater thanthat of B[a]P. Thus, we suggested that MMA was possibly actingas an inhibitor of chemical carcinogenesis. ¹To whom correspondence should be addressed     

Structure-activity relationships in the mutagenicity of N-substituted derivatives of phenanthrene-9,10-imine

A series of K-region, N-substituted phenanthrene imines were tested for mutagenicity in Salmonella typhimurium TA100. All chemicals were mutagenic in the absence of an exogenous metabolic activation system. The apparent decay times of the mutagenic species in diffusion plates and their alkylating activities were also measured. The unsubstituted phenanthrene-9,10-imine was approximately 70-fold more mutagenic than the corresponding phenanthrene-9,10-oxide. N-substitution with electron-releasing groups resulted in chemicals that were more mutagenic than those substituted with electron-withdrawing groups. The mutagenic activity of the latter group of chemicals was comparable with that of phenanthrene-9,10-oxide. Except for N-chlorophenanthrene imine, both alkylation of p-nitrothiophenol and apparent decay times in diffusion plates were inversely correlated with mutagenicity. It is hypothesized that reactivity towards p-nitrothiophenol (alkylating activity) and mutagenicity reflect different reactions, in contrast to other chemical mutagens. The results suggest that the high potency of phenanthrene imines as mutagens is possibly due to DNA binding via an aziridinium ion rather than a carbonium ion.     

DNA damage and mutagenicity induced by endosulfan and its metabolites

Endosulfan is a widely used broad-spectrum organochlorine pesticide, which acts as a contact and stomach poison. Nontarget species, such as cattle, fish, birds, and even humans, are also affected. Studies on the genotoxicity and mutagenicity of endosulfan have been inconsistent and nothing is known about the genotoxicity of its metabolites. In the present study, endosulfan (as a commercial isomeric mixture and as the alpha- and beta-isomers), and metabolites of endosulfan (the sulfate, lactone, ether, hydroxyether, and diol derivatives) were assayed for their ability to induce DNA damage in Chinese hamster ovary (CHO) cells and human lymphocytes using the Comet assay and were assayed for their mutagenicity using the Salmonella reversion assay (Ames test with TA98, TA97a, TA102, TA104, and TA100, with and without S9 activation). The compounds produced statistically significant (P < 0.01), concentration-dependent (0.25-10 microM) increases in DNA damage in both CHO cells and human lymphocytes. Endosulfan lactone caused the most DNA damage in CHO cells, while the isomeric mixture of endosulfan produced the greatest response in lymphocytes. The test compounds also were mutagenic in Salmonella strains at concentrations of 1-20 mug/plate (P < 0.05), with TA98 being the most sensitive strain and the diol and hydroxyether metabolites producing the highest responses. The results indicate that exposure to sublethal doses of endosulfan and its metabolites induces DNA damage and mutation. The contribution of the metabolites to the genotoxicity of the parent compound in Salmonella and mammalian cells, however, is unclear, and the pathways leading to bacterial mutation and mammalian cell DNA damage appear to differ.     

Synthesis, molecular docking and PTP1B inhibitory activity of functionalized 4,5-dihydronaphthofurans and dibenzofurans

Protein tyrosine phosphatase 1B (PTP1B) is an enzyme that plays a critical role in down-regulating insulin signaling through dephosphorylation of the insulin receptor. Inhibitors of PTP1B showed increased insulin sensitivity and normalize plasma glucose level and thus are useful therapeutic agents for the treatment of diabetes. A series of functionalized 4,5-dihydronaphthofurans and dibenzofurans were synthesized, studied through molecular docking and evaluated for their PTP1B inhibitory activity.     

Metal complexes of poly(2-vinylpyridine 1-oxide), poly(4-vinylpyridine 1-oxide) and 2,2′-trimethylenedipyridine 1-oxide

Poly(2-vinylpyridine 1-oxide) ( 3 ), poly(4-vinylpyridine 1-oxide) ( 4 ), and the dimeric analogue 2,2′-trimethylenedipyridine 1-oxide ( 2 ) form metal complexes with chlorides of copper(II), zinc, mercury(II), iron(III), cobalt(II), and nickel(II) salts. Due to steric reasons 4 forms two series (type 6 and 7 ) of complexes by intramolecular or intermolecular crosslinking in which the ratio NO/metal is 2:1 (type 6 ) or 1:1 (type 7 ), whereas 3 and its model compound 2 can form only intramolecular 2:1 complexes of type 5 .     

In vitro and in vivo mutagenicity of the butadiene metabolites butadiene diolepoxide, butadiene monoepoxide and diepoxybutane

Three metabolites of 1,3-butadiene, namely butadiene diolepoxide,butadiene monoepoxide and diepoxybutane, were tested in thebacterial mutation assay using Salmonella typhimurium strainTA100 with and without metabolic activation (S9 mix). All threecompounds showed a mutagenic response. The bifunctional epoxidewas more effective than the diolepoxide which was more effectivethan the monoepoxide. Toxicity appeared to follow the rankingof the chemicals for their mutagenic potency. The monoepoxideand the diolepoxide were also tested for induction of micronucleiin mouse bone marrow erythrocytes and for dominant lethal mutationinduction in postmeiotic male mouse germ cells. The effectsof the diepoxide in both in vivo tests have been published earlier.In the micronucleus assay, the three metabolites gave a positiveresponse whereby the diepoxide was more effective than the monoepoxidewhich was more effective than the diolepoxide. In contrast tothe diepoxide which was positive at a dose as low as 36 mg/kg,the monoepoxide and the diol did not show an induction of dominantlethal effects up to doses of 120 and 240 mg/kg, respectively.It is concluded that the metabolites were mutagenic in bacteriawithout metabolic activation and clastogenic in mouse bone marrow;only the bifunctional diepoxide, however, was active in postmeioticmale mouse germ cells. ⁴To whom correspondence should be addressed     

Synthesis and mutagenicity of 3-halogenated and 3,3', 5,5'-tetrahalogenated benzidines

3-Fluorobenzidine (FBz), 3-chlorobenzidine (CIBz), 3-bromobenzidine(BrBz), 3,3', 5,5'-tetrafluorobenzidine (F₄Bz), and 3,3', 5,5'-tetrachlorobenzidine(Cl₄Bz) were synthesized and tested for their ability to revertSalmonella typhimurium. F₄Bz was the only compound to show direct-actingmutagenicity and was equally potent in strain TA98 and the acetylase-deficientstrain TA98/1,8-DNP₆. In the presence of hamster liver S9, allcompounds except CI₄Bz were mutagenic. The relative mutagenicitiesin TA98 were FBz > CIBz > BrBz > F₄Bz > Bz >Cl₄Bz = 0. In TA98/1,8-DNP₆ the trend was F₄Bz BrBz ClBz >FBz > Bz > CI₄Bz = 0. ¹To whom correspondence should be addressed     

Mutagenicity of (R) and (S) styrene 7,8-oxide and the intermediary mercapturic acid metabolites formed from styrene 7,8-oxide

We have tested the two enantiomers of styrene 7,8-oxide and various thioether metabolites of racemic styrene 7,8-oxide for their direct mutagenicity in Salmonella typhimurium TA100. The mutagenicity data suggests that the (R) enantiomer is more mutagenic than the (S) enantiomer, with the racemic mixture intermediate between the two. The thioether metabolites were not mutagenic. The difference in the mutagenicities of enantiomers probably resulted from a stereoselective process in the Salmonella tester strain. At the present time it is not clear whether the rate-limiting reaction is the interaction of the enantiomers with DNA or some other cellular component.     

Structure-activity relationships in mutagenicity and in nucleophilic ring opening of N-(arylmethyl)phenanthrene 9,10-imines

Ten derivatives of N-benzylphenanthrene 9,10-imine with different substituents on the phenyl ring were synthesized and subjected to mutagenicity tests in Salmonella typhimurium TA100. While electron donating groups were found to enhance the biological activity, electron attracting and bulky substituents lowered the mutagenic potency. A similar dependence on the electronic structure was observed in triethylamine/acetonitrile-promoted interaction of the title imines and 4-nitrothiophenol. This similarity suggests that both biochemical and chemical processes involve mechanisms in which protonation of the aziridine nitrogen is rate controlling, and the attack of the cellular or model nucleophile is a fast step. In contrast to these processes, the reaction of the imines with 4-nitrothiophenol in the presence of 1,5-diazabicyclo[4.3.0] non-5-ene proved to proceed by an SN2 mechanism and to be enhanced by electron attracting substituents.     

Poly(2-vinylquinoline 1-oxide) and some methyl derivatives: Synthesis and viscometric studies

The syntheses of poly[1-(1-oxy-2-quinolyl)ethylene] [poly(2-vinylquinoline 1-oxide)] ( 1a ) and of a number of methyl derivatives ( 1b—f ) are described. Some of the polymers are insoluble in water but soluble in aqueous acetone at several pH values. Those polymers that have a methyl substituent in position 4 are soluble in aqueous acetone below pH < 4 and the viscosity rises steeply with decreasing pH. Polymers with no methyl substituent in position 4 are soluble in aqueous acetone at pH > 6 and the viscosity varies but little with the pH.     

Comparison of microsomal inducer pretreatment on the in vitro α-hydroxylation and mutagenicity of N-nitrosopyrrolidine in rat and hamster liver

The effect of modifiers of liver mixed function oxidase activity on the in vitro α-hydroxylation and mutagenicity of N-nitrosopyrrolidine (NPYR) has been examined in rats and hamsters. Hamster post-mitochondrial supernatants were able to convert NPYR to a mutagen more efficiently than rat preparations under all conditions studied. Aroclor 1254 pretreatment caused the greatest increase in mutagenic activity in both species while phenobarbital and 3-methylcholanthrene pretreatment gave intermediate values when compared to uninduced preparations. Microsomal α-hydroxylation of NPYR was induced by Aroclor 1254 pretreatment in both species. Pretreatment with 3-methylcholanthrene increased α-hydroxylation in hamsters but decreased it in rats. Phenobarbital pretreatment only slightly increased microsomal α-hydroxylation in either species. When microsomal α-hydroxylation rates were expressed per gram wet weight of liver, better agreement between rates of α-hydroxylation and mutagenicity in phenobarbital pretreated animals was obtained since inducer associated changes in total microsomal protein content were taken into account. An example of differential induction of an activation pathway (α-hydroxylation) and a degradative pathway (aldehyde dehydrogenase) is presented to illustrate a potential source of error in the interpretation of metabolic data obtained from post-microsomal supernatants and whole animal studies.     

Genotoxicity of 5-aminolevulinic and 4,5-dioxovaleric acids in the salmonella/microsuspension mutagenicity assay and SOS chromotest

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in some porphyric disorders and in lead poisoning which can undergo metal-catalyzed oxidation producing reactive oxygen species and the keto-aldehyde, 4,5-dioxovaleric acid (DOVA). Evidence in vitro of ALA-induced DNA lesions suggests that ALA and DOVA have mutagenic potential that could possibly contribute to an increased frequency of hepatocellular carcinoma (HCC) in patients with acute intermittent porphyria (AIP). In this study, we evaluated the genotoxic potential of ALA and DOVA. In the absence of exogenous metabolic activation, ALA and DOVA were mutagenic in Salmonella typhimurium tester strain TA104. ALA was also mutagenic in S. typhimurium TA102, but not in TA98, TA100, or TA1535, indicating an oxidative mechanism. Removal of H(2)O(2) with catalase gave only partial protection, suggesting generation of other mutagenic species. Both ALA and DOVA damaged the DNA of Escherichia coli PQ37, inducing the SOS response detected by an increase in beta-galactosidase activity. These results verified the potential mutagenic activity of ALA and DOVA and reinforce the hypothesis that DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.