Metabolic Activation System Research Articles

Effect of CAT gene polymorphism on sensitivity of human lymphocytes to genotoxic effect of organochlorine pesticide in vitro

Introduction. Over recent decades, toxicogenetic studies have focused on the issues of genome instability under the action of genotoxicants, taking into account biomarkers of sensitivity. The question about the genotoxic potential of chlorpyrifos remains open, since both positive and negative effects have been revealed in various tests. The aim of the study is the investigation of sensitivity of donor peripheral blood lymphocytes to chlorpyrifos in vitro and evaluation of the contribution of polymorphism of antioxidant defense system genes (CAT (rs1001179), SOD2 (rs4880)) to the response of human cells to the action of genotoxicant. Materials and methods. The DNA damaging effect of chlorpyrifos was assessed on lymphocytes from fifty two donors using DNA-comet assay with metabolic activation (+S9) and without it (–S9). The study of cytotoxic effects of chlorpyrifos on human lymphocytes was carried out using an automatic fluorescent cell analyzer ADAMII LS. Results. Chlorpyrifos had a pronounced cytotoxic effect on lymphocytes in most donors in the absence of metabolic activation system. With increasing concentration of the pesticide in the medium and time of cultivation, the viability of lymphocytes decreased, and the proportion of cells in late apoptosis and necrosis increased. Positive genotoxic effects were found on the cells of 33 donors (-S9). In the presence of the S9, mild but statistically significant effects were detected only on cells from 2 donors. % DNA values in the comet tail after exposure to the pesticide varied for cells from different donors. In the absence of metabolic activation, a statistically significant increase in the level of DNA damage was found in cells of individuals with genotype AA (homozygote for the minor allele) for the CAT G262A catalase gene (rs1001179), compared with homozygote for the dominant GG allele. Limitations. The genotoxicity of chlorpyryfos was studied in vitro. Conclusion. The results of the study shown cytotoxic and genotoxic effects of chlorpyrifos. The sensitivity of lymphocytes from different donors to the pesticide was found to be significantly different. The association of the level of DNA damage under exposure of chlorpyrifos in vitro with the G262A polymorphism of the catalase gene was found. The research also confirms the possibility of using a model test-system with peripheral blood lymphocytes to assess the potential genetic risk for humans and to study the contribution of gene polymorphism to individual sensitivity to the action of genotoxicants.

Hazard assessment of fenozan, a released non-intentionally added substance from polyester-based can coating

Since the safety of new-generation polyester-based internal coatings regarding the migration of non-intentionally added substances (NIAS) is poorly documented, studies are needed to identify NIAS originating from these food-contact materials (FCM). The aim of this study was to identify volatile and semi-volatile NIAS from polyester-based coatings in order to assess their hazard and ensure consumers’ safety with regard to exposure from canned food. Extraction and migration tests were carried out on a single polyester-coated tin plate (5 batches) using two solvents: acetonitrile and ethanol 95%, then FCM’s extracts and migrates were analysed by GC-MS. An antioxidant degradation (hydrolysis) product, 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid or fenozan (CAS RN: 20170-32-5), was identified and confirmed by reference standard in all migrates. To assess fenozan’s toxicity, several in vitro bioassays, such as the Ames test (to assess point mutation), the micronucleus assay (to detect chromosomal aberrations), and the iodide uptake assay (to study one mode of action for thyroid disruption) were conducted. Fenozan was negative in the Ames test on three strains of S. typhimurium (TA98, TA100, and TA1535) and on one strain of E.coli (WP2), with and without metabolic activation system (S9 mix) using direct incorporation and pre-incubation methods. The in vitro micronucleus assay conducted on HepG2 cells also exhibited a negative response following a 4-hour treatment with the S9 mix, and a 48-hour treatment without the S9 mix. A weak inhibitory effect was obtained when testing fenozan in the iodide uptake assay using rat thyroid FRTL-5 cells. Significant inhibition started from 800 µM of fenozan, with a maximal inhibition of almost 47% at 1000 µM. The findings indicate that fenozan exhibits an anti-thyroid activity in vitro.

Differential genotoxicity of Polygoni Multiflori in rat and human: insights from Ames test and S9 metabolic activation system

The Ames test is used worldwide to initially screen the mutagenic potential of new chemicals. In the standard Ames test, S. typhimurium strains (TA100, TA98, TA1535, and TA1537) and Escherichia coli (WP2uvrA) are treated with substances with/without cytochrome P450s (CYPs)-induced rat S9 fractions for identifying mutagens and pro-mutagens. However, many substances show completely different toxicity patterns depending on whether the liver S9 fraction belongs to rats or humans. The natural product Polygoni Multiflori Radix (PMR) can also show bacterial reverse mutation, followed by the rat or human liver S9 fraction. While PMR elicits reverse mutations in the TA1537 strain in rat liver S9 but not in human liver S9, this mechanism has not been verified yet. To explain this, the differences in metabolic enzymes compositions commonly observed between rats and humans have been implicated. This study aimed to explore the key factors that cause differences in the genotoxicity of PMR between rat and human liver S9 metabolic enzymes. The results of next-generation sequencing (NGS) analysis showed that both rat and human metabolic enzymes caused similar mutations in TA1537. However, when the metabolic enzymes in each S9 fraction were analyzed using ion mobility tandem mass spectrometry (IM-MS), rat- and human-specific enzymes were identified among the cytochrome (CYP) family, especially aryl hydrocarbon receptor (AHR)-related CYPs. These findings suggest that CYP1A1 isoforms contribute to the mechanism of PMR in the Ames test. Therefore, an in vitro Ames test might be more reliable in predicting genotoxicity for both rodents and humans. This will also help overcome the limitations of laboratory animal-based toxicity evaluations, which provide unreliable results due to interspecies differences between humans and rodents.

Genotoxicity of selected cannabinoids in human lymphoblastoid TK6 cells

Natural non-psychoactive cannabinoids such as cannabigerol (CBG), cannabidiol (CBD), cannabichromene (CBC), cannabidivarin (CBDV), and cannabinol (CBN) are increasingly consumed as constituents of dietary products because of the health benefits claims. Cannabinoids may reduce certain types of pain, nausea, and anxiety. Anti-inflammatory and even anti-carcinogenic properties have been discussed. However, there are insufficient data available regarding their potential (geno-)toxic effects. Therefore, we tested CBG, CBD, CBC, CBDV, and CBN for their genotoxic potential and effects on mitosis and cell cycle in human lymphoblastoid TK6 cells. The selected cannabinoids (except CBDV) induced increased micronuclei formation, which was reduced with the addition of a metabolic activation system (S9 mix). CBDV induced micronuclei only after metabolic activation. Mitotic disturbances were observed with all tested cannabinoids, while G1 phase accumulation of cells was observed for CBG, CBD and CBDV. The genotoxic effects occurred at about 1000-fold higher concentrations than are reported as blood levels from human consumption. However, the results clearly indicate a need for further research into the genotoxic effects of cannabinoids. The mechanism of the mitotic disturbance, the shape of the dose–response curves and the possible effects of mixtures of cannabinoids are aspects which need clarification.

Identification of Key Toxic Substances Considering Metabolic Activation: A Combination of Transcriptome and Nontarget Analysis.

There have been numerous studies using effect-directed analysis (EDA) to identify key toxic substances present in source and drinking water, but none of these studies have considered the effects of metabolic activation. This study developed a comprehensive method including a pretreatment process based on an in vitro metabolic activation system, a comprehensive biological effect evaluation based on concentration-dependent transcriptome (CDT), and a chemical feature identification based on nontarget chemical analysis (NTA), to evaluate the changes in the toxic effects and differences in the chemical composition after metabolism. Models for matching metabolites and precursors as well as data-driven identification methods were further constructed to identify toxic metabolites and key toxic precursor substances in drinking water samples from the Yangtze River. After metabolism, the metabolic samples showed a general trend of reduced toxicity in terms of overall biological potency (mean: 3.2-fold). However, metabolic activation led to an increase in some types of toxic effects, including pathways such as excision repair, mismatch repair, protein processing in endoplasmic reticulum, nucleotide excision repair, and DNA replication. Meanwhile, metabolic samples showed a decrease (17.8%) in the number of peaks and average peak area after metabolism, while overall polarity, hydrophilicity, and average molecular weight increased slightly (10.3%). Based on the models for matching of metabolites and precursors and the data-driven identification methods, 32 chemicals were efficiently identified as key toxic substances as main contributors to explain the different transcriptome biological effects such as cellular component, development, and DNA damage related, including 15 industrial compounds, 7 PPCPs, 6 pesticides, and 4 natural products. This study avoids the process of structure elucidation of toxic metabolites and can trace them directly to the precursors based on MS spectra, providing a new idea for the identification of key toxic pollutants of metabolites.

Assessing Genotoxicity of Bovine Pericardium in Guided Tissue Regeneration: Bacterial Reverse Mutation Assay With Exogenous Metabolic Activation.

Introduction Guided tissue regeneration (GTR) is integral to periodontal therapy, facilitating the repair of osseous defects. Due to the widespread use of bovine pericardium (BP) in GTR, a thorough investigation into its genotoxicity is essential for patient safety and treatment efficacy. This study aimed to evaluate the genotoxic effects of local BP in GTR for periodontal osseous defects. Materials and methods The Bacterial Reverse Mutation Assay (Ames test) was used to assess the genotoxic potential of local BP. An exogenous metabolic activation system was employed to evaluate the direct effects of the material on bacterial cells. Results The study investigated the mutagenic effects of local BP across multiple strains of Salmonella typhimurium, utilizing concentrations ranging from 0.3125 mg/plate to 5 mg/plate. While some variability was observed in revertant counts, the generally low SDs suggest a consistent response to the test substance. The maximum revertant count for each strain did not significantly exceed the mean values, indicating the absence of notable outliers or exceptionally high revertant counts at any specific concentration. Based on the data and toxicity assessment criteria, there is insufficient evidence to suggest that the experimental material induces genotoxic effects in the tested bacterial strains under the provided experimental conditions. Conclusion This study assessed the mutagenic potential of local BP membranes used in GTR with the Ames test. Results showed no evidence of mutagenicity, as revertant counts did not exceed twice the negative control in all bacterial strains with exogenous metabolic activation. This suggests that bovine pericardium membranes are safe for medical use under the test conditions. The study highlights the biocompatibility and non-mutagenic nature of BP membranes in GTR for periodontal therapy.

Estrogenic, androgenic, and genotoxic activities of zearalenone and deoxynivalenol in in vitro bioassays including exogenous metabolic activation

Zearalenone (ZEN) and deoxynivalenol (DON) and their derivatives are well-known mycotoxins, which can occur not only in crops but also in water bodies, including drinking water sources. In vitro bioassays can be used to detect biological effects of hazardous compounds in water. To this, when studying biological effects and toxicity in vitro, metabolism is important to consider. In this study, ZEN, α-zearalenol (α-ZEL), DON, 3-acetyl DON, and 15-acetyl DON were evaluated in vitro for hormone receptor–mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and genotoxicity (micronucleus assay) in the presence of an exogenous metabolic activation system (MAS). The ER bioassay proved to be a highly sensitive method to detect low concentrations of the ZEN compounds (EC10 values of 31.4 pM for ZEN, 3.59 pM for α-ZEL) in aqueous solutions. In the presence of the MAS, reduced estrogenic effects were observed for both ZEN compounds (EC10 values of 6.47 × 103 pM for ZEN, 1.55 × 102 pM for α-ZEL). Of the DON compounds, only 3-acetyl DON was estrogenic (EC10 of 0.31 µM), and the effect was removed in the presence of the MAS. Anti-androgenic effects of the ZEN compounds and androgenic effects of the DON compounds were detected in the micromolar range. No induction of genotoxicity was detected for ZEN or DON in the presence of the MAS. Our study highlighted that inclusion of exogenous MAS is a useful tool to detect biological effects of metabolites in in vitro bioassays.

Determination of mutagenic potentials of diarylmethylamine based imine compounds by ames test and computational molecular docking

In recent years, studies that investigate the effects of chemical compounds on organisms have increased in direct proportion to their widespread use. In this study, four different bidentate imine ligands and bidentate imine ligands+Cu(II) complexes were synthesized from the bioactive synthetic diarylmethylamine compound. After the ligands and metal complexes obtained were purified using chromatographic and analytical methods, their mutagenic effects were investigated with the Ames/Salmonella test system. In addition, interactions of four different Cu(II) complexes with B-DNA were evaluated with molecular docking analysis. Accordingly, the results indicated a significant increase in the colonies formed in the presence (+S9) and absence (-S9) of the metabolic activation system, meaning a mutagenic effect against strain TA98 and TA100 strains in general.

Synthesis and mutagenic risk of avanafil's potential genotoxic impurities.

In the technical route for the synthesis of avanafil, 1-ethyl-(3-dimethylaminopropyl)carbamyldiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT) are used as reactive acid-amine binding agents. HOBT contains trace amounts of hydrazine residue, and there is a risk of introducing potentially mutagenic impurities with hydrazide-containing structures. The potentially genotoxic impurities E (Imp-E) and F (Imp-F) of avanafil with altering hydrazide-structure were synthesized by chemical method; subsequently, the impurities were evaluated and classified according to ICH M7 guidelines. Two complementary quantitative structure-activity relationship (QSAR) evaluation systems (Derek and Sarah) based on expert rules and statistics were used to preliminarily predict the genotoxicity of Imp-E and Imp-F, and the prediction result of E was suspected to be positive. In the Ames test of Imp-E and Imp-F, in the dose range of 62.5-1000 μg per plate, with or without the presence of metabolic activation system S9, the number of revertant colonies did not exceed 2 times the number of colonies in the solvent control group and did not show a dose-response relationship, and the test results were negative. Imp-E and Imp-F were determined to be negative for genotoxicity, which could be controlled as class 5 in ICH M7, that is, non mutagenic impurity.

Assessment of the genotoxic and mutagenic effects induced by T-2 mycotoxin in HepG2 cells

The T-2 toxin is a mycotoxin produced by molds belonging to Fusarium. Among the Fusarium mycotoxins, trichothecenes are frequently reported in food and feed, being the T-2 toxin (T-2) the mycotoxin which possesses the highest toxicity. According to EFSA, T-2 is found in various cereal grains used in food and feed products, mainly in oats, and it has a high environmental impact due to its mechanisms of toxicity. However, recent information on its genotoxic and mutagenic effects is lacking. This work aimed to evaluate the genotoxic and mutagenic potential of T-2 in vitro. For this purpose, HepG2 cells were exposed to 15, 30, and 60 nM T-2 for 24 h, then the DNA damage was evaluated by the micronucleus and the comet assays. In addition, point mutation analysis was performed by the bacterial reverse mutation test using 0.15–60 nM of T-2 concentrations. The results showed chromosomal damage at 60 nM T-2 since significantly more MN appeared at this concentration than in the control samples. Regarding the comet assay, DNA double helix breaks appeared at all concentrations tested and, in a concentration-dependent manner. However, no mutagenic effects were observed at any of the concentrations tested for the Salmonella typhimurium (S. Typhimurium) strains TA98, TA100, TA1535, TA1537, or the Escherichia coli (E. Coli) WP2 strain in the absence or presence of a metabolic activation system. Therefore, these results showed that T-2 mycotoxin produced genotoxic effects by MN and comet assay, while no mutagenicity was observed. However, further research simulating different metabolic activation pathways and the combined exposure of this mycotoxin with other mutagenic chemicals that could be present in the diet is necessary to discard the mutagenic potential of T-2 fully. These results highlight the carcinogenic potential and danger associated with T-2 exposure and should be considered to prevent associated food risks for the human population.

Efficacy of Bee Products (Anzer Honey, Pollen and Propolis) in Detection and Healing of Damage Induced by Antidiabetic Drug Vildagliptin/Metformin Hydrochloride in Healthy Human Pancreatic Cells: Cytotoxic, Genotoxic and Biochemical Studies.

Although drugs are powerful therapeutic agents, they have a range of side effects. These side effects are sometimes cellular and not clinically noticeable. Vildagliptin/metformin hydrochloride is one of the most widely used oral antidiabetic drugs with two active ingredients. In this study, we investigated its harmful effects on the metabolic activation system in healthy human pancreatic cells "hTERT-HPNE", and we aimed to improve these harmful effects by natural products. To benefit from the healing effect, we used the unique natural products produced by the bees of the Anzer Plateau in the Eastern Black Sea Region of Turkey. Cytotoxic and genotoxic effects of the drug were investigated by different tests, such as MTT, flow cytometry-apoptosis and comet assays. Anzer honey, pollen and propolis were analyzed by gas chromatography/mass spectrometry (G/C-MS). A total of 19 compounds were detected, constituting 99.9% of the samples. The decrease in cell viability at all drug concentrations was statistically significant compared to the negative control (P<0.05). A statistically significant decrease was detected in the apoptosis caused by vildagliptin/metformin hydrochloride with the supplementation of Anzer honey, pollen and propolis in hTERT-HPNE cells (P<0.05). This study can contribute to other studies testing the healing properties of natural products against the side effects of oral antidiabetics in human cells. In particular, Anzer honey, pollen and propolis can be used as additional foods to maintain cell viability and improve heal damage and can be evaluated against side effects in other drug studies.

Genotoxicity Evaluation of The Novel Psychoactive Substance MTTA.

MTTA, also known as mephtetramine, is a stimulant novel psychoactive substance characterized by a simil-cathinonic structure. To date, little has been studied on its pharmaco-toxicological profile, and its genotoxic potential has never been assessed. In order to fill this gap, the aim of the present work was to evaluate its genotoxicity on TK6 cells in terms of its ability to induce structural and numerical chromosomal aberrations by means of a cytofluorimetric protocol of the "In Vitro Mammalian Cell Micronucleus (MN) test". To consider the in vitro effects of both the parental compound and the related metabolites, TK6 cells were treated with MTTA in the absence or presence of an exogenous metabolic activation system (S9 mix) for a short-term time (3 h) followed by a recovery period (23 h). No statistically significant increase in the MNi frequency was detected. Specifically, in the presence of S9 mix, only a slight increasing trend was observable at all tested concentrations, whereas, without S9 mix, at 75 µM, almost a doubling of the negative control was reached. For the purposes of comprehensive evaluation, a long-term treatment (26 h) was also included. In this case, a statistically significant enhancement in the MNi frequency was observed at 50 µM.

Evaluation of the genotoxic effect of tartrazine using a metabolic activation system in human lymphocyte culture under cytokinetic block conditions

The expansion of the spectrum of use of food additives and, in particular, food dyes (FD), increases the risk of increasing human exposure to genotoxicants. Since in real life, not pure substances with proven genetic safety are in contact with a person, but complex mixtures of unknown composition, even minor impurities in which can become an additional source or modifier of genome instability effects. Of particular concern in this aspect are synthetic FD azo and diazo compounds which can be transformed by human intestinal microflora to some forms of genotoxicants. The purpose of the work is to evaluate the genotoxic effects of 02 mg/mL of Tartrazine FD (E102) purchased in a retail network in a micronucleus test on human blood cells cultured under cytokinetic block conditions in parallel in presence and without rat S9 hepatocyte metabolic activation system.&#x0D; Genotoxic effects were found in cultures without metabolic activation at 0.00002560,00064 mg/mL and 0.4 mg/mL of tartrazine, and in the presence of S9 at 0.0000256 mg/mL, 0,000128 mg/mL and 0.16 mg/mL of tartrazine. For the first time, a dose-dependent suppression of mitotic and proliferative activity of lymphocytes induced by the tested tartrazine sample was revealed, as well as a dose-dependent U-shaped curve in the frequency of apoptosis. The data obtained indicate the presence of genotoxic activity of the studied sample.&#x0D; We discuss the necessity to create the system for evaluation the genotoxic safety of FD real mixtures from a retail network.

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.

Abstract 5338: A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy

Abstract It has been well-established that hepatic drug metabolism may have significant impact on the efficacy and off-target toxicity of anticancer drugs. We present here an in vitro assay, the metabolism-dependent cytotoxicity assay (MDCA), for the evaluation of the roles of specific pathways on the efficacy of anticancer drugs. The assay employs a novel in vitro hepatic metabolic system, the permeabilized cofactor-supplemented human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system for the evaluation of the in vitro cytotoxicity of anticancer agents towards a designated tumor and nontumor cells. MMHH are derived from cryopreserved human hepatocytes isolated from human livers procured for but not used for transplantation provided to our laboratory from the International Institute for the Advancement of Medicine. We have previously demonstrated that MMHH retained all drug metabolizing enzyme pathways present in human hepatocytes. The advantages of MMHH include the following: 1. Drug metabolizing enzyme activities are not affected by cytotoxicity of the drug substrate, a major limitation of intact human hepatocytes; and 2. Drug metabolism pathways can be selected by cofactor specification (e.g. NADPH/NAD+ for phase 1 oxidation; Uridine 5′-diphosphoglucuronic acid (UDPGA) for glucuronidation; 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for sulfation; N-acetyl coenzyme A for N-acetylation). We present here a proof-of-concept study to evaluate the role of various hepatic metabolic pathways towards the cytotoxicity of cyclophosphamide, a commonly used drug for the treatment of renal carcinoma, towards a renal cancer cell line, HEK293 cells. We observed that NADPH enhanced the cytotoxicity, consistent with the known metabolic activation of cyclophosphamide by P450 isoforms (e.g. CYP2B6; CYP3A4) to the known metabolic activation of cyclophosphamide to 4-hydroxycloophosphamide, followed by the formation of the ultimate cytotoxic metabolites phosphoramide mustard and acrolein, which are responsible for its anticancer properties. In addition, we found that the addition of L-glutathione attenuated cyclophosphamide cytotoxicity towards the HEK293 cells, an observation consistent with the roles of this cellular detoxification system in the resistance of tumor cells to chemotherapeutic agents. Minimal effects were observed for UDPGA and PAPS, consistent with the known noninvolvement of these phase 2 conjugating pathways in cyclophosphamide metabolism. MDCA represents an in vitro experimental system that can be routinely applied towards the discovery and development of novel anticancer drugs to evaluate the roles of drug metabolism in efficacy and safety, with various cancer cells (e.g. prostate carcinoma, neuroblastoma) and cells from normal tissues (e.g. cardiomyocytes, intestinal mucosal cells), respectively, as target cells. Citation Format: Albert P. Li, Hong Wei. A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5338.

Acute toxicity and genotoxicity studies on new melatonergic antidepressant GW117

GW117, a novel derivate compound of agomelatine that acts as both a 5-HT2C receptor antagonist and a MT1/MT2 receptor agonist, likely underlines the potent antidepressant action with less hepatotoxicity than agomelatine. We evaluated the acute toxicity of GW117, and the genotoxicity of GW117 using bacterial reverse mutation test, mammalian chromosomal aberration test in Chinese hamster lung cells (CHL) and mouse bone marrow micronucleus test. The acute toxicity test results showed that maximum tolerated dose (MTD) of GW117 was 2000 mg/kg, under which mean Cmax and AUC0→t was 10,782 ng/mL and 81,046 ng/mL × h, respectively. The result of bacterial reverse mutation test showed that the number of bacterial colonies in each dose group of GW117 did not increase significantly compared with that in the solvent control group with or without S9 metabolic activation system. In vitro chromosome aberration test of CHL cells, the chromosome aberration rate of each dose group of GW117 did not increase with or without S9 metabolic activation system. In mouse micronucleus test, the highest dose was 2000 mg/kg, the micronucleus rate did not increase significantly. Under the conditions of this study, the MTD of a single GW117 administration was 2000 mg/kg, there was no genotoxicity effect of GW117.

Genotoxicity assessment of root extracts of Paeonia lactiflora Pall

The roots of Paeonia lactiflora Pall., (Paeoniae Radix, PL) are a well-known herbal remedy used to treat fever, rheumatoid arthritis, systemic lupus erythematosus, hepatitis, and gynecological disorders in East Asia. Here we evaluated the genetic toxicity of PL extracts (as a powder [PL-P] and hot-water extract [PL-W]) in accordance with the Organization for Economic Co-operation and Development guidelines. The Ames test revealed that PL-W was not toxic to S. typhimurium strains and E. coli in absence and presence of the S9 metabolic activation system at concentrations up to 5000μg/plate, but PL-P produced a mutagenic response to TA100 in the absence of S9 mix. PL-P was cytotoxic in in vitro chromosomal aberrations (more than a 50% decrease in cell population doubling time), and it increased the frequency of structural and numerical aberrations in absence and presence of S9 mix in a concentration-dependent manner. PL-W was cytotoxic in the in vitro chromosomal aberration tests (more than a 50% decrease in cell population doubling time) only in the absence of S9 mix, and it induced structural aberrations only in the presence of S9 mix. PL-P and PL-W did not produce toxic response during the in vivo micronucleus test after oral administration to ICR mice and did not induce positive results in the in vivo Pig-a gene mutation and comet assays after oral administration to SD rats. Although PL-P showed genotoxic in two in vitro tests, the results from physiologically relevant in vivo Pig-a gene mutation and comet assays illustrated that PL-P and PL-W does not cause genotoxic effects in rodents.

Application of OECD QSAR Toolbox software for predicting the mutagenic effects of chemicals

Introduction. The introduction of new chemical products to the market requires a preliminary assessment of their danger to human health and the environment, including specific and long-term effects. Taking into account the medical and social significance of the consequences of exposure to chemicals with a mutagenic effect on the body, the study of genotoxicity / mutagenicity seems to be extremely relevant. In order to reduce the number of experiments performed on animals, calculation methods are being actively introduced into toxicological practice, in particular OECD QSAR Toolbox software. Therefore, the aim of our research was to study the possibility of using the OECD QSAR Toolbox software to predict the mutagenic effects of chemicals. Material and methods. OECD QSAR Toolbox software version 4.4.1., OECD guidelines on the assessment of the mutagenic effect. Results and discussion. We have developed the “Guidelines for predicting the mutagenic effects of chemicals”, which includes a detailed description and work with the program when predicting the mutagenic effects of chemicals. Based on the analysis of OECD documents on the assessment of the mutagenic potential of chemicals, seven most widely used tests were selected, which allow evaluating the effect on various test objects in the presence / absence of metabolic activation systems. For methods, indicators were selected that must be set to start the calculation in the QSAR Toolbox software. The most suitable databases and profilers were also indentified. Conclusion. Forecasting using the OECD QSAR Toolbox software is a screening method that involves performing several calculations using the results of various experimental studies for analogs, and subsequent expert interpretation of the data obtained.

Characterization of Lantana Camara Roots (Pentacyclic Triterpenoid) and Mutagenicity Testing of Extracted Oleanolic Acid Using Salmonella Typhimurium

The objective of the present study was to isolate oleanolic acid from the roots of Lantana camara, and characterize it chemically using DSC, HPLC, and FTIR methods, and additionally carrying out a short term assay for assessment of its mutagenic potential by conducting bacterial reverse mutation test to evaluate the ability of the “Oleanolic acid (Pentacyclic Triterpenoid)” to induce point mutations in tester strains of Salmonella typhimurium in both presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes. Differential scanning calorimetry (DSC), High Performance Liquid chromatography (HPLC), and Fourier transform infrared (FTIR) spectrometers were used to chemically analyze the isolated molecule. Oleanolic acid was utilized to carry out Preliminary Cytotoxicity and mutagenicity study. According to the results of spectrophotometric research, oleanolic acid extracted from Lantana camara roots exhibits identical spectrum characteristics to standard oleanolic acid also the mutagenic potential of Oleanolic acid (Pentacyclic Triterpenoid). Oleanolic acid was found to be non-mutagenic in all five test strains of Salmonella typhimurium—TA98, TA100, TA102, TA1535, and TA1537 employing plate incorporation assays. It may be determined that oleanolic acid isolated from Lantana camara roots gives identical, identifiable signals and absorbance like previously reported reference standard based on the results of DLC, HPLC, and FTIR spectra and their interpretation and was determined that oleanolic acid purified from Lantana camara roots is non-mutagenic in Salmonella typhimurium.

Comparative evaluation of the genotoxicity of carbendazim technical grade active ingredients in the Ames test and micronucleus in vivo test

Introduction. Carbendazim is a systemic benzimidazole fungicide used against a wide range of crop diseases. The ability of carbendazim to induce the incidence of chromosomal aberrations and micronuclei in mammalian cells by influencing the processes of mitotic spindle formation in the cell cycle have been shown in various in vitro and in vivo tests. Contradictory data were obtained in the bacterial test system Salmonella/microsomes, indicating both the absence and the presence of mutagenic activity of carbendazim. The discrepancy in the results may stem from the presence of impurities. The aim of the study was a comparative evaluation of the genotoxicity of various technical products of carbendazim. Materials and methods. The genotoxicity of carbendazim was studied using the plate incorporation version of the Ames test on 5 strains of Salmonella typhimurium in the presence and the absence of metabolic activation system (+S9/-S9) and in a micronucleus test in CD-1 mice. Two technical grade active ingredients (TGAI) and an analytical standard for carbendazim were tested. Results. In the Ames test, the analytical standard of carbendazim possessed no mutagenic activity (±S9) on any of the strains. The most pronounced mutagenic effect was observed for the TGAI I in TA98 strain, the number of revertants at the maximum concentration was 5-7 times higher than that in the negative control. The positive effects of carbendazim TGAIs in the Ames test are likely mediated by the presence of impurities. Under in vivo conditions, all tested TGAIs of carbendazim induced a statistically significant and dose-dependent formation of micronuclei in polychromatic erythrocytes (PCE) of mouse bone marrow. The mean frequency of PCE with micronuclei at the maximum dose exceeded this rate in the negative control by 21-24 times. Research limitations. The study is limited to testing the mutagenic activity of two samples of carbendazim technical products and one sample of its analytical standard in both in vivo and in vitro tests. Conclusion. Taking into account the high content of the active substance in the tested TGAIs, the bacterial reverse mutation test is a highly sensitive method for assessment of the equivalence of carbendazim generic products. The use of a micronucleus test for evaluating of the equivalence of carbendazim TGAIs to the original substance is inappropriate due to the pronounced aneugenic effect.