Mutagenicity and clastogenicity evaluation of tacrine by Ames and micronucleus assays

Tacrine was evaluated for its mutagenic and clastogenic activities using the Ames bacterial reverse-mutation assay and the rodent bone marrow micronucleus assay. Tacrine was tested for mutagenic potential at six different concentrations, with 1,250 µg/plate as the highest concentration, followed by five lower concentrations with 2-fold spacing. In clastogenic evaluation, tacrine was administered orally to Wistar rats for 2 days at 5, 10, and 20?mg/kg body weights to assess micronucleus induction in bone marrow erythrocytes. In the Ames assay, tacrine showed nonmutagenicity in four tester strains of Salmonella typhimurium viz. TA98, TA100, TA102, and TA1535, but it was found to be mutagenic in the TA1537 tester strain, both in the presence and absence of a metabolic activation system. Tacrine was found to be nonclastogenic on bone marrow cells of rats at all doses tested and was found to be mutagenic in only the TA1537 strain of Salmonella.     

Mutagenicity and clastogenicity evaluation of metaphenoxy benzyl chloride by ames and micronucleus assays

Metaphenoxy Benzyl Chloride (3-PBC; CAS # 53874-66-1) is a commonly used intermediate in organic synthesis and is a widely used chemical building block. There is likelihood of human contact with 3-PBC in the process of organic or pesticide synthesis and even through as impurity in the product. Further, this chemical has been reported to be found in water reservoirs causing a threat to aqueous flaura and fauna. Genotoxic alert has been suspected for the chemical 3-PBC based on its structure hence, the impetus of present work was to assess the mutagenic potential of 3-PBC using Ames bacterial reverse-mutation assay and in vitro micronucleus assay. 3-PBC was tested for mutagenic potential at six different concentrations, with 0.05 (without metabolic activation) and 0.50 (with metabolic activation) µL/plate as the highest concentrations, followed by five lower concentrations with 2-fold spacing. In clastogenic evaluation, 3-PBC was tested at concentrations ranged from 0.31 to 4.88?µL/mL to assess micronucleus induction in mammalian cells viz. Chinese Hamster Ovarian-K1 cells (CHO-K1 cells). In the Ames assay, 3-PBC did not show mutagenicity in all five tester strains of Salmonella typhimurium viz. TA98, TA100, TA102, TA1535 and TA1537 both in the presence and absence of a metabolic activation system and found to be nonclastogenic when evaluated in “CHO-K1 cells”.     

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON)

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1?mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10?mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.     

N,N-diethylphenylacetamide, an insect repellent: absence of mutagenic response in the in vitro Ames test and in vivo mouse micronucleus test

N,N-diethylphenylacetamide (DEPA), a promising new insect repellent, was tested for mutagenicity in the in vitro Ames Salmonella/microsome mutagenicity test and the in vivo mouse micronucleus test. For the Ames test, DEPA was assayed both in the presence and absence of Aroclor 1254-induced rat-liver S-9 mix (5 and 20% S-9 fraction), using five tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104. For the micronucleus test, mice were exposed to DEPA through ip injection for 2 and 5 days in separate experiments, and bone marrow and peripheral blood were sampled 6 and 48 hr after the final injection, respectively. DEPA did not induce a mutagenic response in the Ames test, and mouse bone marrow and peripheral blood micronucleus tests. DEPA was not considered cytotoxic, as a depression of the percentage PCE was not observed at any dose in the range of 1 to 100 mg/kg body weight with either treatment protocol of the micronucleus test.     

Genotoxicity of quinocetone,cyadox and olaquindox in vitro and in vivo

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His⁺ mutants at 6.9 μg/plate in Salmonella typhimurium TA 97, at 18.2 μg/plate in TA 100, TA 1535, TA 1537, and at 50 μg/plate in TA 98. CYA produced His⁺ mutants at 18.2 μg/plate in TA 97, TA 1535, and at 50 μg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ⩾10 μg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.     

赤苷脉通注射液的致突变研究

目的探讨中药制剂赤苷脉通注射液的致突变性。方法采用鼠伤寒沙门氏组氨酸营养缺陷型菌株回复突变实验(Ames实验)、中国仓鼠肺成纤维细胞(CHL)染色体畸变实验和小鼠骨髓微核实验来检测赤苷脉通注射液的致突变作用。结果 Ames实验中,赤苷脉通注射液在312.5～5 000μg.皿-1剂量范围内,无论加或不加S9,鼠伤寒沙门氏菌组氨酸缺陷型TA97,TA98,TA100,TA102和TA1535 5株菌的回复突变菌落数均未出现剂量依赖性的增加;染色体畸变实验中,非活化条件或代谢活化条件下,药物质量浓度为1 200,600和300μg.mL-1时,细胞的染色体畸变率均未出现剂量依赖性增加;微核实验中,在1 150,575和287.5mg.kg-1剂量组中均未见骨髓中含微核的嗜多染红细胞数增加。结论在该实验室条件下,Ames实验、CHL细胞染色体畸变实验和小鼠骨髓微核实验结果均为阴性,即中药制剂赤苷脉通注射液无潜在的遗传毒性。     

Tofisopam--evaluation of mutagenic and genotoxic properties

The mutagenic properties of tofisopam, the member of the 2,3-benzodiazepine family, were evaluated on the basis of Ames test with Salmonella typhimurium TA1537, TA97, TA98, TA100 and TA102 strains. The genotoxic properties of tofisopam were estimated on L929 cell line with the cytokinesis-block technique. Under the experimental conditions, no mutagenic activity of tofisopam in tester bacteria strains was found, and no genotoxic activity was observed.     

Non-clastogenicity in mouse bone marrow of fructose/lysine and other sugar/amino acid browning products with in vitro genotoxicity

Heated sugar/amino acid reaction mixtures, known to contain products that are clastogenic and/or mutagenic to cells in vitro, were evaluated for clastogenic activity in mouse bone marrow using the erythrocyte micronucleus assay. Heated (i.e. browned) fructose/lysine reaction mixtures were also evaluated in the Salmonella his-reversion assay and the Chinese hamster ovary (CHO) cell chromosomal aberration assay to confirm and extend previous in vitro observations. Significant mutagenicity of fructose/lysine mixtures was observed in Salmonella typhimurium strains TA100, TA2637, TA98 and TA102, with greater activity in mixtures heated at pH 10 than at pH 7. S-9 decreased the activity in strains TA100, TA2637 and TA98, but increased the activity in strain TA102. Both pH 7 and pH 10 reaction mixtures of the fructose/lysine browning reaction were highly clastogenic in CHO cells. Heated mixtures of fructose and lysine, and of glucose or ribose with lysine, histidine, tryptophan or cysteine, did not increase the frequency of micronucleated erythrocytes in mice when administered by the oral route. This indicates the absence of chromosomal aberrations in erythrocyte precursor cells, and indicates that the genotoxic components of the browned mixtures are not absorbed and distributed to bone marrow cells in amounts sufficient to induce micronuclei when given orally. Because sugar/amino acid browning reactions occur commonly in heated foods, it is important to evaluate further the in vivo genotoxicity of browning products in cell populations other than bone marrow.     

Mutagenic and clastogenic activity of the chloro-nitroimidazole radiosensitizer P40 in vitro and in vivo.

The hypoxic radiosensitizer 1-(2-hydroxy-3-methoxypropyl)-2-chloro-4-nitroimidazole [P40] was investigated for its mutagenic activity in bacterial Ames test as well as for genotoxic activity in micronucleus assay in vivo. This nitroimidazole showed the weak mutagenicity towards TA100 strain (base pair substitution) and towards TA98 strain (frameshift) only in the highest concentration. P40 induced also a significant increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the doses of 0.6 mg/g and 1.2 mg/g. The maximum time response was at 48 h. The decrease of percentage of PCEs suggested the possible cytotoxicity on bone marrow cells after treatment with P40. Positive results in this battery short-term tests provide evidence of clastogenic activity of P40.     

Absence of mutagenic activity in Salmonella and of clastogenic activity in CHO cells of Caramel Colours I, II, III and IV.

A total of 15 caramel colours were examined for genotoxic activity using the Salmonella typhimurium plate incorporation assay (Ames test). Five bacterial strains, TA1535, TA1537, TA1538, TA98 and TA100 were used in all the plate incorporation tests. Caramel colours can be divided into four classes, classification depending on the preparative method used. In this study, representatives of all four classes of caramel colour were tested for genotoxic potential in the Ames test, some of the caramel colours being tested both with and without a pre-incubation stage. None of the 15 caramel colours tested exhibited genotoxic potential in any of the five bacterial tester strains. The last two caramel colours tested, in the series of 15 [203-23-4 (Class II) and 311 (Class III)] were also assessed for clastogenic potential. For this test, cultures of CHO cells were exposed to the two caramel colours and metaphase preparations from these cultures examined for evidence of chromosomal aberrations. No evidence of chromosome damaging activity was observed.     

Mutagenicity of recombinant antihemophilic factor (GC-gamma AHF)

This study was carried out to evaluate the mutagenic potential of recombinant antihemophilic factor VIII (GC-gamma AHF). Salmonella typhimurium (S. typhimurium) reversion assay with/without histidine moiety, chromosomal aberration assay on Chinese hamster lung (CHL) fibroblast cells and in vivo micronucleus assay using mouse bone marrow cells and supravital micronucleus assay using peripheral blood were performed. GC-gamma AHF containing histidine did show inconsistent and irregular mutagenic effects on S. typhimurium TA98, TA100, TA1535 and TA1537 both in the absence and presence of the metabolic activation system, however, GC-gamma AHF without histidine showed no mutagenic effects regardless of the metabolic activation system, thus suggesting that the histidine moiety in GC-gamma AHF might cause inconsistent mutagenic effect. Also GC-gamma AHF did not increase the number of cells having structural or numerical chromosome aberration in the cytogenetic test. In classical and supravital micronucleus assay, no significant increases were observed in the occurrence of micronucleated polychromatic erythrocytes and micronucleated peripheral lymphocytes in male ICR mice. These results strongly indicate that GC-gamma AHF has no genetic toxicity under these experimental conditions.     

Methemoglobinogenic potential of primaquine and its mutagenicity in the Ames test

Single doses of primaquine did not produce methemoglobinemia in beagle bitches. Repeated daily administration for 12 days produced a gradually rising level of methemoglobin over that time period, unaccompanied by depletion of erythrocytic reduced glutathione. Primaquine was mutagenic in the Ames test in Salmonella typhimurium strain TA 1537, with or without S9, using a liquid preincubation assay. Primaquine was non-mutagenic in this assay to strains TA 1535, TA 1538, TA 98 and TA 100, regardless of the presence or absence of S9. In the standard overpour Ames test, the drug was non-mutagenic in all 5 Salmonella strains, both with and without S9 metabolic activation.     

Absence of mutagenicity of acid pyrogallol-containing hair gels.

In the present work, three commercial acid (pH 3.5-4) pyrogallol-containing hair gels, SunSet Alizador Negro (two formulations) and Embelleze Henê Gel, were tested for mutagenicity using two well-established assays. In the Salmonella mutagenicity assay using 648-5000 microg/plate of cosmetic samples, none of the samples reached a 2-fold increase in revertants relative to the controls. Both in the absence and in the presence of S9, the dose-response relation in strains TA98, TA100, TA102, TA1535, and TA1537 was not significant (p>0.01). In the mouse bone marrow micronucleus assay, 10 Swiss male mice were orally administered 2000 mg/kg of sample per body weight/day. The ratio between polychromatic and normochromatic erythrocytes as well as the presence of micronuclei in bone marrow cells were determined. Equal numbers of micronucleated polychromatic erythrocytes were detected between the cells of each treated group and the negative control, using ANOVA and chi-square analyses. Thus, none of the products induced mutagenesis in either assay. Previous studies have shown pyrogallol is mutagenic in various test systems, including Salmonella. However studies have also shown that acidic conditions may repress the reactive-oxygen species (ROS) produced by pyrogallol, and ROS is considered the primary mechanism for the mutagenicity of pyrogallol. Consistent with this are our results, which show that acidic, commercially available pyrogallol-containing hair gels are neither mutagenic in Salmonella nor induce micronuclei in mouse bone marrow in vivo.     

Mutagenicity study of gadobenate dimeglumine formulation (E7155) (1)--Reverse mutation assays in S. typhimurium and E. coli tester strains

The ability of gadobenate dimeglumine formulation (E7155) to cause gene mutations was assessed in five strains of Salmonella typhimurium (TA100, TA1535, TA98, TA1538, and TA1537) and a strain of Escherichia coli (CM891; WP2, uvrA-, pKM101) using the Ames test (agar plate assay). The results suggest that E7155 is non-mutagenic towards these bacterial tester strains.     

Tinospora cordifolia, a safety evaluation

Tinospora cordifolia is one of the indispensable medicinal plants used in veterinary folk medicine/Ayurvedic system of medicine for the treatment of diverse diseases and recommended for improving the immune system by means of body resistance. In the current study, we evaluated the genotoxic risk of the aqueous extract of T. cordifolia (TC) in a battery of four different genotoxicity tests viz., Ames, in vitro chromosome aberration (CA), rodent bone marrow micronucleus (MN), and Comet assay. Experimental results confirmed that in Ames test up to 5000 μg/plate of TC did not exhibit any mutagenic effect in Salmonella typhimurium mutant strains (TA97a, TA98, TA100, TA102, and TA1535). In CA assay, TC was not clastogenic to human peripheral blood lymphocytes up to a concentration of 3000 μg/ml. In MN and Comet assays, TC was pre-treated for 7 days at three dose levels (150, 200 and 250 mg/kg body weight) orally to male Balb/c mice. The results showed that TC treatment did not display clastogenicity and DNA damaging effect in bone marrow erythrocytes and peripheral blood lymphocytes respectively.     

Comparative studies on the mutagenicity of pirenzepine with submammalian/bacterial and mammalian systems

The generic pirenzepine was tested for mutagenicity by bacterial and mammalian procedures. In the Ames test the strains of Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100, and Escherichia coli WP2 (P) were used in the absence and presence of mammalian metabolizing systems. Pirenzepine induced neither base-pair substitutions nor frameshift mutations up to a concentration of 3000 μg/plate. The results of the micronucleus test as well as in vivo cytogenetic analysis of both bone marrow and germ cells in Chinese hamsters were negative after twofold applications of 10, 100, and 1000 mg/kg pirenzepine. No dominant-lethal effects were found after a single treatment of male mice with the same doses.     

Toxicological studies with dithranol and its 10-acyl analogues

The oral LD₅₀ values of an antipsoriatic drug, dithranol, were 1542 mg/kg in NMRI mice and 3216 mg/ kg in Wistar rats. Three 10-acyl analogues of dithranol (10-acetyl, 10-propionyl and 10-butyryl dithranol or butantrone) were more toxic both in mice and rats. They were mutagenic only in TA1537 of the five Salmonella typhimurium strains tested. None of them were mutagenic in two Escherichia coli strains. Butantrone was least toxic to test bacteria and had the lowest mutagenic activity on TA1537. In metaphase analysis of in vitro treated human lymphocytes, dithranol, 10-acetyl dithranol and 10-propionyl dithranol produced significant increases in the number of chromosome and chromatid gaps but without a clear dose-response relationship, and without inducing significant breaks. Butantrone did not cause significant increases in gaps or breaks. In the mouse micronucleus test, dithranol and butantrone caused no increases in micronucleated polychromatic or normochromatic erythrocytes, indicating lack of clastogenic activity in vivo at maximum tolerated doses. Hence, dithranol and its 10-acyl analogues have a weak mutagenic activity in vitro. The mutagenic activity of butantrone is lower than that of the other analogues and dithranol.     

Study of mutagenicities of phthalic acid and terephthalic acid using in vitro and in vivo genotoxicity tests

Genotoxicities of phthalic acid (PA) and terephthalic acid (TPA) were examined using three mutagenicity tests: Ames, chromosome aberration (CA), and micronucleus (MN). In the Ames test, these two agents did not produce any mutagenic responses in the absence or presence of S9 mix on the Salmonella typhimurium strains TA98, TA100, TA102, TA1535, or TA1537. The CA test also showed that PA and TPA exerted no significant cytogenetic effect on Chinese hamster ovary (CHO) cells. In the mouse MN test, no significant alteration in occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice ip administered any of these agents at doses of 0, 20, 100, 500, 2500 or 12,500 microM/kg. These results indicate that PA and TPA produced no mutagenic effects using these in vitro and in vivo mutagenic test systems.     

Genotoxic evaluation of the biocomponents of the cricket, Gryllus bimaculatus, using three mutagenicity tests

The mutagenic potential of the extracted components of Gryllus bimaculatus, a species of cricket, was evaluated using short-term genotoxicity tests including the Ames, chromosome aberration, and micronuclei tests. In a Salmonella typhimurium assay, G. bimaculatus extract did not produce any mutagenic response in the absence or presence of S9 mix with TA98, TA100, TA1535, and TA1537. Chromosome aberration testing showed that G. bimaculatus had no significant effect on Chinese hamster ovary (CHO) cells. In the mouse micronucleus test, no significant alteration in occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice intraperitoneally administered with G. bimaculatus extract at doses of 15, 150, or 1500 mg/kg. These results indicate that G. bimaculatus extract exerts no mutagenic effect in these in vitro and in vivo systems.     

Fruit extract of the medicinal plant Crataegus oxyacantha exerts genotoxic and mutagenic effects in cultured cells

Crataegus oxyacantha, a plant of the Rosaceae family also known “English hawthorn, haw, maybush, or whitethorn,” has long been used for medicinal purposes such as digestive disorders, hyperlipidemia, dyspnea, inducing diuresis, and preventing kidney stones. However, the predominant use of this plant has been to treat cardiovascular disorders. Due to a lack of studies on the genotoxicity of C. oxyacantha, this investigation was undertaken to determine whether its fruit extract exerts cytotoxic, genotoxic, or clastogenic/aneugenic effects in leukocytes and HepG2 (liver hepatocellular carcinoma) cultured human cells, or mutagenic effects in TA100 and TA98 strains of Salmonella typhimurium bacterium. Genotoxicity analysis showed that the extract produced no marked genotoxic effects at concentrations of 2.5 or 5 µg/ml in either cell type; however, at concentrations of 10 µg/ml or higher significant DNA damage was detected. The micronucleus test also demonstrated that concentrations of 10 µg/ml or higher produced clastogenic/aneugenic responses. In the Ames test, the extract induced mutagenic effects in TA98 strain of S. typhimurium with metabolic activation at all tested concentrations (2.5 to 500 µg/ml). Data indicate that, under certain experimental conditions, the fruit extract of C. oxyacantha exerts genotoxic and clastogenic/aneugenic effects in cultured human cells, and with metabolism mutagenicity occurs in bacteria cells.