Presence Of S9 Mixture Research Articles

In vitro and in vivo evaluation of the genotoxicity of titanium dioxide, GST.

Titanium dioxide (TiO2) was used in various applications in a wide range of products including food, cosmetics and photocatalyst. General toxicity studies of titanium dioxide, GST (Green Sludge Titanium) have been investigated in several reports, whereas studies concerning mutagenicity and genotoxicity have not been elucidated. Herein, we investigated the potential mutagenicity and genotoxicity of GST by genetic toxicology testing. The bacterial reverse mutation test was conducted by the pre-incubation method in the presence and absence of metabolic activation system (S9 mixture). The chromosome aberration test was performed using cultured Chinese hamster lung cell line in the absence and presence of S9 mixture. The micronucleus test was performed by using specific pathogen-free male ICR mice. Genotoxicity tests were conducted following the test guidelines of the Organisation for Economic Cooperation and Development with application of Good Laboratory Practice. No statistically significant increases were found in the bacterial reverse mutation test, in vitro chromosome aberration test, and in vivo micronucleus test when tested for induction of genotoxicity in GST. These results suggest that GST did not induce mutagenicity and genotoxicity in both in vitro and in vivo system.

Co-exposure to deltamethrin and thiacloprid induces cytotoxicity and oxidative stress in human lung cells.

Deltamethrin (DEL) and thiacloprid (THIA) are commonly used synthetic insecticides in agriculture either separately or in combination. There is limited information in human cells for the effects of the mixture of DEL + THIA on oxidative stress. Therefore, the present study was designed to examine the effects of the mixture on cell proliferation and oxidative stress in human lung fibroblast cells. Human telomerase reverse transcriptase (hTERT)-expressing human lung fibroblasts, WTHBF-6 cells, were treated with 2.5 + 37.5, 5 + 75, 12.5 + 187.5, and 25 +375 µM concentrations of DEL + THIA for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and 5 + 75, 12.5 + 187.5, and 25 + 375 µM for lipid peroxidation and reduced glutathione (GSH) assays for 24, 48, and 72 h in the absence and presence of metabolizing fractions of the mammalian liver (S9 mixture). Both the mixture of DEL + THIA and their metabolites significantly reduced cell viability and induced cytotoxicity in WTHBF-6 cells, especially at higher concentrations. The mixture of DEL + THIA significantly decreased GSH levels at the highest concentration for all treatment times and at the highest two concentrations (12.5 + 187.5 and 25 + 375 µM) for 72 h in the presence of S9 mixture. The highest concentration of DEL + THIA mixture caused a significant increase in malondialdehyde (MDA) level at 72 h in the absence of S9 mixture. There were also significant increases in MDA levels at the highest concentration for 48-h and all concentrations of DEL + THIA for 72-h treatment in WTHBF-6 cell cultures with S9. These data showed that the mixture of DEL + THIA and their metabolites can induce cytotoxicity and oxidative stress in human lung fibroblasts.

Antimutagenic and Multi-Biological Activities of Smilax excelsa L. Fruit Extract

In this study, antimutagenic, antimicrobial and antioxidant activities of Smilax excelsa L. fruit extract were evaluated. The antimicrobial effect was investigated by disk diffusion method and the antimutagenic effect was investigated by Ames/Salmonella/microsom test. The antioxidant properties of S. excelsa fruit samples were determined by investigating the total phenolic, flavonoid contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. It was observed that 200 mg plate-1 fruit extract was found to have a mutagenicity inhibition as 81% in the absence of S9 mixture and 67% in the presence of S9 mixture against Salmonella typhimurium TA1535. S. excelsa fruit extract produced an inhibition zone in the range of 11-16 cm against the tested microorganisms. Flavonoid and phenolic contents were found to be 0.7985±0.0124 mgQE 100 mL-1 and 11.9847±0.0041 mgGAE 100 mL-1 at the 200 mg mL-1 extract concentration, respectively. The DPPH removal rate was determined to be 55% at the 200 mg L-1 dose. As a result, it was observed that the S. excelsa fruit tissues exhibited a highly antimutagenic activity and has been determined as a potential natural antimicrobial and antioxidant source.

Evaluation of the Mutagenic Properties of Two Lignans from Acanthopanax koreanum Nakai

Acanthopanax koreanum Nakai, a well known traditional herb grown in Jeju Island, South of Korea, has been used as a tonic and sedative agent, as well as in the treatment of diabetes and immune diseases. Mutagenicity of two lignans, syringaresinol and tortoside A isolated from A. koreanum, was assessed using Salmonella/microsome (Ames) test. Tester strains used were Salmonella typhimurium TA98, TA100, TA1535, and Escherichia coli WP2uvrA. The mutagenic activity was determined both in the absence or presence of S9 mixture. As a result, tortoside A did not cause any increase in the number of his+ revertants in S. typhimurium and E. coli WP2uvrA strains in the presence or absence of S9 mix, compared to the controls. Similarly, low concentrations of syringaresinol (750 and 1,500 μg/plate) did not show any mutagenic properties in all bacterial strains, in the presence or absence of S9 mixture. However, in the high concentration of syringaresinol (3,000 μg/plate), the number of revertants were increased in TA1535 strains, in the absence of S9 metabolic activation. Therefore, in vivo experiments such as comet assay are needed to further determine the genotoxic/carciogenic potential of syringaresinol isolated from A. koreanum.

Chemical Structure Determination of DNA Bases Modified by Active Metabolites of Lucidin-3-O-primeveroside

Lucidin-3-O- primeveroside (LuP) is one of the components of madder root (Rubia tinctorum L.; MR) which is reported to be carcinogenic in the kidney and liver of rats. Since metabolism of LuP generates genotoxic compounds such as lucidin (Luc) and rubiadin (Rub), it is likely that LuP plays a key role in MR carcinogenesis. In the present study, the chemical structures of Luc-specific 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts following the reactions of dG and dA with a Luc carbocation or quinone methide intermediate derived from Acetoxy-Luc were determined by liquid chromatography with photodiode array and electron spray ionizaion-mass spectrometry (LC-PDA-ESI/MS). The identification of the two measurable adducts as Luc-N(2)-dG and Luc-N(6)-dA was confirmed by NMR analysis. Subsequently, using a newly developed quantitative analytical method using LC-ESI/MS, the formation of Luc-N(2)-dG and Luc-N(6)-dA from the reaction of calf thymus DNA with Luc in the presence of S9 mixture was observed. The fact that this reaction with Rub also gave rise to the same dG and dA adducts strongly suggests that Rub genotoxicity involves a metabolic conversion to Luc. The precise determination of the modified DNA bases generated by LuP and the method for their analysis may contribute to further comprehension of the mode of action underlying carcinogenesis by MR and related anthraquinones.

Mutagenic activity of river water from a river near textile industrial complex in Korea

The mutagenic activity of XAD-2 adsorbates and water extracts recovered from nine locations of the Kumho River was tested on S. typhimurium TA98 strain to identify the source of the mutagenicity. A sampling site, receiving effluents from the textile industrial complex located in Daegu City, showed extraordinarily high mutagenic activity, especially in the presence of S9 mixture, at all sampling time in both XAD-2 adsorbates and dichloromethane extracts. This indicated the existence of the frame-shift mutagens in the Kumho River, same type of mutagens detected in previous studies by other researchers in the Nakdong River into which the Kumho River discharges. The fractionation study showed that the mutagenic chemicals in the river water are mid-polar. Furthermore, mean tail length obtained by single cell gel electrophoresis assay (Comet assay) showed consistent dose-dependent DNA damage, indicating that the chemicals in the river water not only act as frame-shift mutagens but also break human lymphocytes DNA strain. Chemical identification of the mutagens should be required.

In vitro cytotoxicity assay with selected chemicals using human cells to predict target-organ toxicity of liver and kidney

In order to elucidate the feasibility of predicting liver and kidney target-organ toxicity using in vitro cytotoxicity assay, cytotoxicity of selected chemicals, acetaminophen (AAP), mitomycin (MMC), cupric chloride (CuCl 2), phenacetin, cadmium chloride (CdCl 2) and aristolochic acid (AA), was studied using human hepatoma (Bel-7402) cells and human renal tubular epithelial (HK-2) cells. Cell viability and mitochondrial permeability transition (MPT) were assessed by the neutral red (NR) assay and laser scanning confocal microscope, respectively. The results of the NR assay indicated that cytotoxicity of hepatoxicants, AAP, MMC and CuCl 2 in liver cells was higher than that in kidney cells. Cytotoxicitiy of nephrotoxicant, CdCl 2 was lower in liver cells than that in kidney cells, but nephrotoxicant phenacetin and AA was higher cytotoxicity in liver cells than that in kidney cells. The cytotoxicity of AAP and phenacetin was strengthened in the presence of S9 mixture, indicating that they are metabolism-mediated cytotoxicants. All selected chemicals disrupted MPT in dose-dependent manner. Linear regression analysis revealed a good correlation between the IC 50 values of cytotoxicity and the EC 50 values of MPT in Bel-7402 cells and HK-2 cells ( R 2 = 0.987 and 0.823, respectively). Cytotoxicity assay in vitro using specific cells show good compatibility with target-organ toxicity in vivo. However, limitations of in vitro cytotoxicity assay are due to its incomplete process of ADME and the defect of predicting chronic toxicity effect after long-term exposure to a chemical.

Activation of aminophenylnorharman, aminomethylphenylnorharman and aminophenylharman to genotoxic metabolites by human N-acetyltransferases and cytochrome P450 enzymes expressed in Salmonella typhimurium umu tester strains

Norharman (9H-pyrido[3,4-b]indole) and harman (1-methyl-9H-pyrido[3,4-b]indole) contained in cigarette smoke and cooked foodstuffs, are non-mutagenic to Salmonella strains, but show co-mutagenicity with S9 mixture in the presence of aniline or o-toluidine. The resulting 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (aminomethylphenylnorharman, AMPNH) and 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole (aminophenylharman, APH) are produced by coupling of norharman and aniline, norharman and o-toluidine, and harman and aniline in the presence of S9 mixture, respectively. To clarify the role of human cytochrome P450 (P450) and N-acetyltransferase (NAT) enzymes in the metabolic activation of APNH, AMPNH and APH, we determined the genotoxicity of these coupling chemicals using a variety of umu tester strains established in our laboratories. APNH, AMPNH and APH induced umuC gene expression more strongly in a bacterial O-acetyltransferase-overproducing strain than the parent strain. These chemicals were also found to induce umuC gene expression in NAT2-overexpressing strain at much higher rate than the NAT1-overexpressing strain. Among seven OY strains expressing human P450s and NADPH-P450 reductase used, the genotoxicity of APNH, AMPNH and APH was detected in OY1002/1A2 strain, OY1002/1A1 and OY1002/1A2 strains, and in OY1002/1A2 strain, respectively. From these results, it is concluded that APNH, AMPNH and APH are mainly bioactivated by P450 1A2 and NAT2, followed by NAT1 enzymes. P450 1A1 was also found to activate AMPNH at relatively slower rates.

Mutagenicity of paepalantine dimer and glycoside derivatives from Paepalanthus bromelioides

The first isocoumarin isolated from the methylene chloride extract of Paepalanthus bromelioides, named paepalantine (isocoumarin 1), was found to have antimicrobial activity; but, it is mutagenic clastogenic and cytotoxic. Two other isocoumarins, paepalantine-9- O- β - d -glucopyranoside (isocoumarin 2) and paepalantine-9- O- β - d -allopyranosyl(1→6) glucopyranoside (isocoumarin 3) were isolated from the ethanolic extract. A fourth new isocoumarin, also isolated from the methylene chloride extract of the capitula of P. bromelioides, was characterized as an 8-8′ dimer of paepalantine and denominated isocoumarin 4. The abilities of isocoumarins 2, 3 and 4 to induce mutations in Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 were investigated. Mutagenic activity was observed in strain TA97a treated with isocoumarin 2 in the presence of S9 mixture. The substitution of H at position 9 by glucose or glucose-allose caused reductions in the mutagenic activities of paepalantine, indicating this to be an important site for these properties.

Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677.

Stevioside is natural non-caloric sweetner isolated from Stevia rebaudiana BERTONI, which has been used as a non-caloric sugar substitute in Japan. Pezzuto et al. demonstrated that steviol shows a dose-dependent positive response in forward mutation assay using Salmonella typhimurium TM677 in the presence of metabolic activation system (Aroclor induced rat liver S9 fraction). Our studies were carried out to identify the genuine mutagenic active substance from among the eight steviol derivatives. Steviol indicate almost similar levels of mutagenicity under the presence of S9 mixture, as reported by Pezzuto et al. 15-Oxo-steviol was found to be mutagenic at the one tenth the level of steviol itself under the presence of S9 mixture. Interestingly, specific mutagenicity of the lactone derivative under the presence of S9 mixture was ten times lower than that of the lactone derivative without the addition of S9 mixture.

IN VITRO CHROMOSOME ABERRATION ASSAY USING HUMAN BRONCHIAL EPITHELIAL CELLS

The determination of chromosome aberrations (CA) in Chinese hamster lung fibroblast (CHL) cells was compared with that in normal human bronchial epithelial (BEAS-2B) cells, which upon adenovirus infection were reported to possess carcinogen metabolizing capacities similar to polycyclic aromatic hydrocarbons. CHL and BEAS-2B cells were treated with increasing concentrations of benzo\\[a]pyrene ( BaP) or N -nitrosodiethylamine (NDEA) . In BEAS-2B cells, BaP, at a concentration of 50 mug/ml, produced a significant increase in the CA frequency, while NDEA did not markedly alter the number of aberrations in the absence of S9 mixture. The CHL cells exposed to BaP and NDEA in the presence of S9 mixture responded as anticipated with a 30% and 14% frequency of CA observed in the BaP (50 mug/ ml) and NDEA (1000 mug/ ml) treated cells, respectively. The results of this study show that the CA assay using human cell line with intrinsic metabolic activation system, such as BEAS-2B cells, may be a useful model for predicting human clastogens and carcinogens.

Antimutagenic activity of extracts from Japanese eggplant

Using the Salmonella/microsome assay, the antimutagenic effects of specific components of the extracts from eggplant fruits were investigated. The eggplant fruit juice exhibited an antimutagenic activity against 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2) induced mutagenicity. In some of the fractions extracted with several organic solvents (acetone, petroleum ether, ethyl acetate, and methanol), the activity was recognized. No mutagenicity or toxicity for Salmonella typhimurium TA98 in the presence of S9 mixture was observed with any of the extracts. It is suggested that there are multiple components of the activities that exist in the eggplant fruit. We isolated lutein from the 84% methanol (methanol/water, v/v) layer, pheophorbide or chlorophyllide from the 70% methanol layer and tannins containing sugar-moieties from the water layer. Pheophytin a and b, Mg-free derivatives of chlorophyll a and b, were isolated from the petroleum ether layer as possible antimutagens. The pheophytin a with S9 mix inhibited by 30–40% the mutagenicity of Trp-P-2.

Bacterial mutagens in the urine of patients under tinidazole treatment

Tinidazole is an antiparasitic drug belonging to the 5-nitroimidazole family. It is prescribed against protozoal infestations and is widely used in Mexico as well as other underdeveloped countries where infectious diseases are the first cause of children mortality. The drug is a direct mutagen in Salmonella typhimurium TA100 strain and the presence of S9 mixture did not modify its mutagenic effect. At low doses no mutagenicity was detected with strains TA100NR, TA98 or UTH8414 (Uvr + derivative of TA100). Urine from four patients under tinidazole treatment exhibited a mutagenic activity on strain TA100, greater than expected from the tinidazole concentrations determined by high-performance liquid chromatography (HPLC). Components from the urine samples were separated on thin-layer chromatography (TLC) plates, and their mutagenic effects tested by direct application of the Salmonella assay onto sections of the developed chromatoplate. The R f of one component (0.62) corresponded to the one obtained for a tinidazole standard and showed the expected mutagenicity, while a second component with an R f = 0.39 , exhibited a mutagenic potency slightly greater than the observed for tinidazole; however, as in the case of the drug itself, reduction of the nitro group was necessary for a mutagenic activity.

Comparison of Antimutagenic Effect of Various Tea Extracts (Green, Oolong, Pouchong, and Black Tea)

The antimutagenic effects of various tea extracts prepared from nonfermented tea (green tea), semifermented tea (oolong tea and pouchong tea), and fermented tea (black tea) were investigated by Salmonella/microsome assay. No mutagenicity or toxicity in Salmonella typhimurium TA98 and TA100 was observed with any tea extract. The tea extracts markedly inhibited the mutagenicity of 2-amino-3-methylimidazo(4,5-f)quinoline, 3-amino-1,4-dimethyl-5H-pyridol(4,3-b)indole,2-amino-6-methyldipyrido(l,2-a:3′,2′-d)imidazole, benzo[a]pyrene, and aflatoxin B1 toward S. typhimurium TA98 and TA100 in the presence of S9 mixture, especially those of oolong and pouchong teas inhibited over 90% mutagenicity of these five mutagens at the dosage of 1 mg per plate. Among four tea extracts, black tea exhibited the weakest inhibitory effect on mutagenicity of these five mutagens. The mutagenicity of 4-nitroquinoline-N-oxide, a direct mutagen, was not inhibited by black and oolong tea extracts to S. typhimurium TA98 in the absence of S9 mixture but was increased by the tea extracts at the dose of 1 mg per plate to S. typhimurium TA100. As the antimutagenic effect of semifermented tea was stronger than nonfermented and fermented teas, some antimutagenic substances might be formed during manufacturing processes of tea.

Mutagenicity evaluation of riot control agent o-chlorobenzylidene malononitrile (CS) in the Ames Salmonella/microsome test.

o-Chlorobenzylidene malononitrile (CS), a riot control agent, was evaluated for its possible mutagenic activity in the Ames Salmonella/mammalian microsome mutagenicity test. Five histidine-deficient (His-) mutant tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104--were used. The liquid preincubation procedure was used with metabolic activation (presence of S9 mixture) and without metabolic activation (absence of S9 mixture). For the experiments with metabolic activation, three different concentrations of S9 fraction (supernatant of Aroclor 1254-induced rat liver homogenate at 9000 g)--5%, 15% and 30% in S9 mixture--were used. Along with mutagenic activity, CS was also evaluated for cytotoxic activity in all the five tester strains of Salmonella typhimurium, both in the presence and absence of S9 mixture. The mutagenic and cytotoxic activities of CS were assessed by counting the His+ revertant colonies and by counting the microcolonies (His-, auxotrophs in the background lawn), respectively, and the respective mean values were compared with the relative negative (solvent) control. A dose range of 12.5-800 micrograms plate-1 for CS did not induce a mutagenic response either in the presence or absence of S9 mix. No change in the negative mutagenic response of CS has been observed even in the presence of an elevated level of S9 fraction in the S9 mix. A dose of 200 micrograms plate-1 for CS was found to be cytotoxic by decreasing the surviving cells as well as His+ revertant colonies; however, the effect was reduced in the presence of an elevated level of S9 fraction in the S9 mix.

DNA repair synthesis in primary culture of bovine bile duct epithelial cells induced by chemical agents in relation to bile duct cancer

Unscheduled DNA synthesis (UDS) was measured autoradiographically in a primary culture of extrahepatic bile duct epithelial cells of Holstein cows following exposure to chemicals known to be capable of developing bile duct cancers in experimental animals, i.e., N-methyl- N′-nitro- N-nitrosoguanidine (MNNG, CAS No. 70-25-7), N-ethyl- N′-nitro- N-nitrosoguanidine (ENNG, CAS No. 4255-77-6), 20-methylcholanthrene (MCA, CAS No. 56-49-5), N-nitrosodimethylamine (DMN, CAS No. 62-75-9) and aflatoxin B 1 (AFB 1, CAS No. 1162-65-8). MNNG and ENNG induced UDS without addition of S9 mixture. MCA elicited UDS only if S9 mixture was added. Regardless of the presence or absence of S9 mixture, DMN failed to induce UDS. DNA repair by AFB 1 was enhanced by the presence of S9 mixture. Therefore, for MNNG, ENNG and high doses of AFB 1 activation by the liver is not necessary to exert genotoxic effects and they seem to be capable of direct action on bile duct epithelial cells in the presence of a bilioenteric fistula or anastomosis.

Mutagenicity of smoke condensate of bidi--an indigenous cigarette of India.

The possible mutagenicity of bidi smoke condensate (BSC), an indigenous form of an Indian cigarette has been studied using three short-term test systems. It was seen that BSC caused frame-shift mutations in Salmonella typhimurium strains TA 98 and TA 1538 and required metabolic activation. In the mammalian test systems, BSC induced 8 azaguanine resistant mutations in V79 Chinese hamster cells in the presence of S9 mixture and induced elevated frequencies of micronucleated erythrocytes in the bone marrow of Swiss mice.

Induction of sister chromatid exchange by various selenium compounds in Chinese hamster cells in the presence and absence of S9 mixture

This study was designed to investigate the effect of 3 selenium compounds, namely, sodium selenite, sodium selenide and sodium selenate on induction of sister chromatid exchange (SCE) in the Chinese hamster V79 cell line in the presence and absence of S9 mixture. The results indicated that the most potent SCE inducer in the presence of S9 mixture was sodium selenite and this was followed by sodium selenide, while in the absence of S9 mixture the most effective SCE inducer was sodium selenide which was then followed by sodium selenite. For sodium selenate, the data indicated no increase in SCE rate as compared to the control values both in the presence and absence of S9 mixture. In addition, it was observed that growth inhibition as measured by no sister chromatid differentiation or no metaphases was produced by certain doses of the compounds tested. Based on this, the compound that induced the most growth inhibition both in the presence and absence of S9 mixture was sodium selenide and this was followed by sodium selenite. For sodium selenate no growth inhibition was observed. The different capabilities of the selenium compounds tested to induce SCE were clearly demonstrated in the system employed in this study. It is felt that this effect is an important cytogenetic characteristic of these compounds, yet how this activity relates to the antimutagenic and anticarcinogenic properties of these agents is difficult to discern.