Addition Of S9 Mix Research Articles

Sister-chromatid exchanges induced by trihalomethanes in rat erythroblastic cells and their suppression by crude catechin extracted from green tea

An in vitro sister-chromatid exchange (SCE) assay using rat erythroblastic leukemia cells was conducted with four major trihalomethanes (THMs): chloroform, CHCl 3; dichlorobromomethane, CHCl 2Br, dibromochloromethane, CHClBr 2; bromoform, CHBr 3. In the absence of S9 mix, CHBr 3, CHClBr 2 and CHCl 2Br significantly induced SCEs in a clear dose-dependent manner, while CHCl 3 did not significantly induce SCEs. On the other hand, the incidence of CHCl 3-induced SCEs significantly increased, although the incidence of CHBr 3-induced SCEs decreased by the addition of S9 mix. However, there was no difference between the incidence of SCEs induced by CHBr 3, CHClBr 2 or CHCl 2Br in the absence of S9 mix and that in the presence of S9 mix. The addition of crude catechin to the SCE assay system suppressed the ability of CHCl 3 or CHBr 3 to induce SCEs but had no suppressive effect on the other THM-induced SCEs. The suppression of SCEs induced by CHCl 3 or CHBr 3 depended on the crude catechin dose.

Mutagenicity of alkylhydrazine oxalates in Salmonella typhimurium TA100 and TA102 demonstrated by modifying the growth conditions of the bacteria

Alkylhydrazines are important carcinogens. However, they show generally only weak mutagenicity and the activities reported from different laboratories are contradictory. We have developed a sensitive method to detect the mutagenicity of alkylhydrazines. The method is based on a modified preculturing procedures in the Ames test, the emphasis in the modification being a change in the growth period of tester strains. The optimal growth periods were found to be 11 h in Salmonella typhimurium TA100 and 5 h in Salmonella typhimurium TA102. We tested the mutagenic activity of 12 alkylhydrazines; 1,2-dimetehylhydrazine, 1,2-diethylhydrazine, 1,2-dipropylhydrazine. 1,2-dibutylhydrazine, 1,1-dimethylhydrazine, 1,1-diethylhydrazine, 1,1-dipropylhydrazine, 1,1-dibutylhydrazine, methylhydrazine, ethylhydrazine, propylhydrazine, and butylhdyrazine. All 12 alkylhydrazines were clearly mutagenic in Salmonella typhimurium TA102, and 10 hydrazines were mutagenic in Salmonella typhimurium TA100, both in the absence of S9 mix. The mutagenicity was inhibited by the addition of S9 mix or bovine serum albumin. This suggests deactivation of the mutagens by proteins.

The in vitro micronucleus test on isolated human lymphocytes

The in vitro micronucleus test was performed on isolated human lymphocytes using the cytokinesis-block technique with and without a rat liver metabolizing system. Positive control substances were used to evaluate this test: a direct agent (vincristine) requiring no metabolic activation, and three promutagens (cyclophosphamide, benzo[ a]pyrene and dimethylbenz[ a]anthracene). All of them, when compared with controls, caused a significant increase in micronucleus frequency, with a clear dose response. Five compounds were then tested in this in vitro micronucleus test: safrole, azathioprine, procarbazine, diethylstilbestrol and o-toluidine. The chemicals were examined with and without exogenous metabolic activation. Of these five compound, o-toluidine was found to be a marked direct genotoxic agent and azathioprine gave positive results with or without metabolic activation (a better response was noted without the addition of S9 mix). Diethylstilbestrol gave conflicting results and was considered inconclusive. Two chemicals, safrole and procarbazine, were found to be non-genotoxic in this test system, whatever the protocol used.

Four types of induction of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) in rat stomach pyloric mucosa

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.

Mutagenicity of nitro derivatives produced by exposure of dibenzofuran to nitrogen oxides

Dibenzofuran (DF) was reacted with various concentrations of nitrogen oxides (NOx) under light irradiation. The mutagenicities of the reaction mixtures were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP 6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DF-Nox (molar ratios 1:3, 1:6, and 1:18) reaction mixtures exhibited mutagenic potency in strain TA98 without S9 mix, and their direct-acting mutagenicity was reduced in strains TA98NR and TA98/1.8-DNP 6. Four mononitrodibenzofurans and 2 dinitrodiobenzofurans, i.e., 1-nitrodibenzofuran (NDF), 2-NDF, 3-NDF, 4-NDF, 2,7-dinitrodibenzofuran (DNDF) and 2,8-DNDF, were identified with authentic samples in the DF-NOx (1:18) reaction mixture by HPLC cochromatography and a gas The order of mutagenicity of nitrodibenzofurans in strain TA98 without S9 mix was as follows: strains TA98 and TA100 was enhanced by the addition of S9 mix. Since these nitrodibenzofurans were less mutagenic in strains TA98NR and TA98/1,8-DNP 6 than in strain TA98 without S9 mix, it was presumed that their mutagenicity was dependent on their activation by the ‘classical’ bacterial nitroreductase and/pr transacetylase, which are absent in strains TA98NR and TA98/1,8-DNP 6 but present in strain TA98, respectively by 3-NDF and 4-NDF were mutagenic in strain TA100 without S9 mix. 2-NDF and 3-NDF were determined as corresponding amino derivatives in DF-NOx (1:3), (1:6) and (1:18) reaction mixtures. They contributed about 30–65% of the direct-acting mutagenicity of reaction mixtures in strain TA98.

Genotoxic action of an aqueous extract of Heliotropium curassavicum var. argentinum

Heliotropium curassavicum uar. argentinum is widely employed in gout, rheumatism, neuralgias, arteriosclerotic disorders, muscular algias, phlebitis, varix and other illnesses. In order to analyze the genotoxic effect produced in vitro by this medicinal plant, chromosomal aberrations (CA), mitotic index (MI) and anaphase delay (AD) were studied in the CHO cell line, with and without the addition of S9 mix. Prepared according to the Argentine pharmacopeia 0.1, 1, 10 and 100 μg/ml plant decoction (aqueous extract) were assayed. One hundred cells per culture were studied for CA and AD, while MI was calculated for 2000 nuclei. The results revealed a significant increase in the percentage of abnormal metaphases ( p < 0.001) and in total aberrations ( p < 0.001). Both the MI and the AD affected the cell cycle. All results were enhanced by the addition of an S9 fraction. The toxic effect could be associated with pyrrolizidine alkaloids and their N-oxides, which through a process of in vitro metabolism become activated by microsomal oxidation and change into pyrrolic derivatives.

Anticlastogenicity in cultured mammalian cells

Basic and applied research on anticlastogenecity has not only revealed valuable evidence on the mechanisms governing the induction of chromosomal aberrations by environmental mutagens, but also contributed effective ideas on a practical employment of this knowledge for the protection of individuals at risk. Considering the basic role played by chromosomal anomalies in oncogenesis, additional weight must be attributed to studies on anticlastogenicity. The employment of human cells in this kind of study dates back to 1969/70, while classical mammalian cell systems were used only later on. Various modes of application of both clastogens and anticlastogens (AC) were examined, but simultaneous addition to the cultures of both reagents was the most favored way. A wide spectrum of cytogenetic endpoints can be studied, but differences can be demonstrated with regard to efficacy of inhibitors on different types of cytogenetic changes, e.g., open breaks vs. rearrangements, but also vs. SCEs. Depending on their mode of influence on this spectrum, ACs can be arranged in various categories which are of practical importance, for instance, with regard to their oncogenic potential. A wide variety of factors was shown to influence AC action, e.g., time and mode of application of the test substances, physiologic and metabolic features of the cell types studied, type and mechanism of the clastogen used, etc. The addition of S9 mix can drastically change the patterns of efficacy of the ACs. The combined application of two or more ACs, as far as investigated, apparently neither potentiates nor even merely adds their effects.

Study on Mutagenic Properties of the Nakatsu River Sediment.

We have reported previously the presence of mutagenicity in the extract from the sediment collected from some points of the Nakatsu river. In order to characterize mutagens in the river sediment, the extract from the Nakatsu river sediment was fractionated into 9 fractions by HPLC and tested using Salmonella typhimurium TA98, YG1021 and YG1024. The middle-high polar fractions in the extract from the sediment showed mutagenicity to YG1024 by the addition of S9 mix. Then, some aminoarenes were investigated in these fractions by NPD-GC. More polar two fractions revealed mutagenicity to TA98 without S9 mix. In these fractions some mutagenic substances were surveyed and identified by GC-MS, but unknown peaks were present. Mutagenicity, PAH concentration, amount of extract and ignition loss of the sediments from the Nakatsu river were compared with those of the Sagami river. As the result it is comfirmed that the Nakatsu river is specific on mutagenic contamination.

Diurnal mutagenicity of airborne particulate matter in a cold district

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2–30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without β-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of β-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N,O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.

Mutagen formation on nitrite treatment of indole compounds derived from indole-glucosinolate

The mutagenicities of 8 indole compounds (indole-3-acetonitrile, indole-3-carbinol, indole-3-acetamide, indole-3-acetic acid, 3-methylindole, indole-3-aldehyde, indole-3-carboxylic acid and indole) derived from indole glucosinolate were studied by mutation tests on Salmonella typhimurium TA98 and TA100 and Escherichia coli WP2 uvrA/pKM101 with and without S9 mix. None of the 8 indole compounds were mutagenic, but they became mutagenic on these 3 tester strains when treated with nitrite at pH 3. The nitrite-treated indole compounds were mutagenic without metabolic activation system (S9 mix), and their mutagenicities were decreased by the addition of S9 mix.

Mutagenic properties of 2-amino- N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino- N 6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/μg. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 μl S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 μl S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1 1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DT r) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DT r mutants increased dose-dependently at concentrations of 2.5–15 μg/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DT r mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.

Mutagenicity of urine from rats after 1-nitropyrene and 2-nitrofluorene administration using new sensitive Salmonella typhimurium strains YG1012 and YG1024

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2–30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without β-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of β-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N, O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.

Sister-chromatid exchange induced by mitomycin C in early developmental mouse embryo in utero

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2–30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without β-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of β-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N,O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.

Mutagenic evaluation of trichlorfon using different assay methods with Salmonella typhimurium

Evaluation of the mutagenicity of trichlorfon pesticide was carried out with strains TA1535, TA100, TA97, TA98 and TA104 of Salmonella typhimurium by means of several assay methods: (i) spot test; (ii) standard plate incorporation test; (iii) plate incorporation test with preincubation; (iv) fluctuation test and (v) fluctuation test with preincubation, with and without post-mitochondrial liver fraction (S9) from Wistar rats pretreated with phenobarbital and 5,6-benzoflavone as a metabolic activation system. Trichlorfon induced base-pair substitution mutations, and its mutagenic activity was decreased by the addition of S9 mix. The fluctuation test and fluctuation test with preincubation were the most sensitive assay methods for detecting the mutagenicity of trichlorfon.

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP 6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.

Effect of vitamin C depletion and repletion on antioxidant defense indices in human subjects

The mutagenicity of three isomers of nitro-6H-dibenzo[b,d]pyran-6-one (NDBP), 2-NDBP, 3-NDBP and 4-NDBP, was characterized in the plate-incorporation (PI) and microsuspension (MS) assay using Salmonella typhimurium tester strains in the presence or absence of S9 mix. In both assays, all of the NDBPs showed mutagenicity in every strain. In the absence of S9 mix, TA98 was the strain most sensitive to the mutagenicity of NDBPs. The activity of NDBPs was reduced in TA98NR and TA98/1,8-DNP6 strains relative to TA98, suggesting that NDBPs cause frameshift mutation and that nitroreduction by ‘classical’ nitroreductase and acetylation are significant steps for their metabolic activation. Mutagenic potency of NDBPs in TA98 without S9 mix in the MS assay (2-NDBP 104 300 rev./μg, 3-NDBP 23 500 rev./μg, and 4-NDBP 15 300 rev./μg) was much higher than that in the PI assay (2-NDBP 38 rev./μg, 3-NDBP 162 rev./μg, and 4-NDBP 7 rev./μg). Although additional S9 mix increased the mutagenicity of NDBPs in the PI assay, the mutagenic potency of NDBPs in the MS assay using strains TA98 and TA100 was decreased by the addition of S9 mix. In the PI assay, frameshift and base-substitution activities of both isomers were enhanced by the addition of the pKM101 plasmid, suggesting the induction by these isomers of complex frameshifts (frameshifts with associated base substitutions) in strain TA98. In the PI assay, 2-NDBP generally exhibited more base-substitution than frameshift activity; however, the reverse was true for 3-NDBP. In the MS assay, both isomers exhibited more frameshift than base-substitution activity.

Genotoxicity of quinoxaline 1,4-dioxide derivatives in Escherichia coli and Salmonella typhimurium

The mutagenicities of 5 quinoxaline 1,4-dioxide (QdO) derivatives were tested by 2 bacterial assays using forward mutation with Escherichia coli WP2uvrA/pKM101 and reverse mutation with Salmonella typhimurium TA100 and TA98. Potent mutagenic activities of all QdOs tested were observed in both mutation assays. Mutagenicities of these compounds were varied by addition of S9 mix. Their SOS-inducing activities were examined with a ‘Rec-lac test’ that has been newly developed by us for detecting genotoxins. A high level of SOS-inducing activity was observed in all samples tested. These results suggest that the mutagenicity of QdOs results from the error-prone repair involved in SOS responses.

Mutagenicity studies on fenitrothion in bacteria and mammalian cells

The mutagenicity of fenitrothion was determined in strains of Salmonella typhimurium and Escherichia coli. Fenitrothion was found to be non-mutagenic in Salmonella typhimurium strains of TA98, TA1535 and TA1537 and in Escherichia coli WP2 uvrA both with and without S9 mix, while weak mutagenicity was observed only in Salmonella typhimurium TA100 and enhanced by the addition of S9 mix. The mutagenicity observed in the TA100 strain was not expressed in a nitroreductase-deficient strain, TA100 NR, and decreased in a transacetylase-deficient strain, TA100 1,8-DNP 6. The mutagenicity of fenitrothion was also examined by a gene mutation assay using the gene for hypoxanthine-guanine phosphoribosyltransferase ( hgprt) in V79 Chinese hamster lung cells. Fenitrothion did not induce any increment of 6-thioguanine-resistant mutant cells at doses ranging from 0.01 to 0.3 mM regardless of the presence or absence of S9 mix. These results suggest that reduction of fenitrothion by a bacterial nitroreductase of TA100 to an active form is essential for the expression of the mutagenicity of fenitrothion in TA100 and that a bacterial transacetylase of TA100 also has an important role in the process of mutagenic activation.

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella strains showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.

2-( N-nitroso- N-methylamino)propiophenone, a direct acting bacterial mutagen found in nitrosated Ephedra altissima tea

A new f-nitroso compound identified in a nitrosated tea extract made from the plant Ephedra altissima and shown to be formed under in vivo conditions was identified as 2-( N-nitroso- N-methylamino)propiophenone (NMAP). N-Nitrosoephedrine (NEP), another N-nitroso compound detected in nitrosated Ephedra altissima tea and NMAP are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Base-pair substitution mutation-detecting strains (TA100 and TA1535) showed both compounds to be weak direct-acting mutagens without the addition of S9-mix. The identification, synthesis and mutagenicity of NMAP are discussed.