Exogenous Metabolic Activation System Research Articles

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.

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.

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.

Hydromorphone impurity 2,2-bishydromorphone does not exert mutagenic and clastogenic properties via in silico QSAR prediction and in vitro Ames and micronucleus test

The opioid agonist hydromorphone is indicated for the management of severe acute and chronic pain given that alternate treatments are insufficient. While the genotoxicity profile of hydromorphone is well investigated, little is known about the genotoxic potential of its impurities. In this study, 2,2-bishydromorphone was tested in silico and in vitro for both its mutagenic potential in an Ames test performed with Salmonella typhimurium and Escherichia coli tester strains up to a maximum concentration of 5 mg per plate in the absence and presence of metabolic activation. Furthermore, it was tested for its ability to induce micronuclei in TK6 cells in a micronucleus test up to a maximum concentration of 500 µg/mL with or without an exogenous metabolic activation system. 2,2-Bishydromorphone did not reveal any potential for inducing mutagenicity or clastogenicity under the conditions of the respective tests and is therefore considered non-mutagenic and non-clastogenic/aneugenic in vitro. These results are in line with negative in silico quantitative structure-activity relationship (QSAR) prediction for 2,2-bishydromorphone mutagenicity and clastogenicity and provide evidence of good correlation of in silico and in vitro data. Conclusively, these studies add important new clinically relevant information on the safety of hydromorphone as the impurity of 2,2-bishydromorphone is proven to be non-mutagenic and non-clastogenic.

Anthraquinones: Genotoxic until Proven Otherwise? A Study on a Substance-Based Medical Device to Implement Available Data for a Correct Risk Assessment.

A genotoxicological study was carried out on a substance-based medical device (SMD) containing anthraquinones in order to evaluate its potential mutagenic effect. The “In Vitro Mammalian Cell Micronucleus Test” was performed on human TK6 cells by flow cytometry. Cultures were treated with concentrations of SMD tested in the range of 0–2 mg/mL for short treatment time (3 h) both in the absence and presence of an exogenous metabolic activation system, followed by a recovery period in fresh medium (23 h) and for extended treatment time (26 h) without an exogenous metabolic activation system. At the end of both treatment times, cytotoxicity, cytostasis, apoptosis and micronuclei (MNi) frequency were analysed in treated cultures and then compared with those measured in concurrent negative control cultures. The SMD did not induce a statistically significant increase MNi frequency under any of experimental conditions tested. The negative outcome shows that the SMD is non-mutagenic in terms of its ability to induce chromosomal aberrations both in the absence and presence of an exogenous metabolic activation system. The study ended by analyzing intracellular ROS levels to exclude the pro-oxidant ability, typically linked to DNA damage. On the contrary, our results demonstrated the ability the SMD to counteract oxidative stress.

Combining Different In Vitro Bioassays to Evaluate Genotoxicity of Water-Accommodated Fractions from Petroleum Products.

Genotoxicity assessment is of high relevance for crude and refined petroleum products, since oil compounds are known to cause DNA damage with severe consequences for aquatic biota as demonstrated in long-term monitoring studies. This study aimed at the optimization and evaluation of small-scale higher-throughput assays (Ames fluctuation, micronucleus, Nrf2-CALUX®) covering different mechanistic endpoints as first screening tools for genotoxicity assessment of oils. Cells were exposed to native and chemically dispersed water-accommodated fractions (WAFs) of three oil types varying in their processing degree. Independent of an exogenous metabolic activation system, WAF compounds induced neither base exchange nor frame shift mutations in bacterial strains. However, significantly increased chromosomal aberrations in zebrafish liver (ZF-L) cells were observed. Oxidative stress was indicated for some treatments and was not correlated with observed DNA damage. Application of a chemical dispersant increased the genotoxic potential rather by the increased bioavailability of dissolved and particulate oil compounds. Nonetheless, the dispersant induced a clear oxidative stress response, indicating a relevance for general toxic stress. Results showed that the combination of different in vitro assays is important for a reliable genotoxicity assessment. Especially, the ZF-L capable of active metabolism and DNA repair seems to be a promising model for WAF testing.

Genotoxicity evaluation of locally produced nano-hydroxyapatite-silica: An in vitro study using the bacterial reverse mutation test

Background: Nanohydroxyapatite-silica (nanoHA-Silica) has been produced by one-pot sol-gel technique. The material when incorporated into commercial Glass Ionomer Cement (GIC) was found to exhibit higher Vickers hardness, compressive strength, and flexural strength compared to conventional GIC. However, before starting to be used and exposed to the human cell, every material product should undergo for genotoxic evaluation. Thus, the objective of this in vitro study was to evaluate the genotoxicity of locally produced nanoHA-Silica under bacterial reverse mutation assay (Ames test). Materials and Methods: Four Salmonella typhimurium strains TA98, TA102, TA1535, and TA1537 were incubated with nanoHA-Silica in the presence and absence of exogenous metabolic activation system (S9) at five different concentrations (0.3125, 0.625, 1.25, 2.5, and 5 mg/plate) along with appropriate positive and negative controls. The assessment of the results was based on the number of revertant colonies in each plate, and the results were regarded as mutagenic when the number of revertant colonies was more than two-fold of the negative control. Results: There was no significant increase in the number of revertant colonies corresponding to the increase in the concentrations of the test substance for all the five bacterial strains treated with or without S9. Conclusion: NanoHA-Silica-GIC was non-genotoxic and had no mutagenic potential under present test conditions.

Performance of the in vitro transgene mutation assay in MutaMouse FE1 cells: Evaluation of nine misleading ("False") positive chemicals.

The screening of chemicals for the protection of human health and the environment requires the assessment of genetic toxicity. However, existing, internationally-accepted in vitro mammalian genotoxicity tests have been criticized for their low specificity (i.e. high frequency of "false" or "misleading" positive results for compounds that are negative in vivo). An in vitro transgene mutation assay has been established that uses a metabolically competent cell line derived from MutaMouse lung (i.e. FE1 cells). Mutation scoring employs the well-characterized lacZ positive selection system, and the assay is proposed as an alternative in vitro assessment tool. In this study, the performance of the FE1 cell assay was evaluated by examining responses to nine non-DNA-reactive chemicals that previously elicited misleading positive results in other mammalian cell genotoxicity assays. FE1 cells were exposed to concentrations up to approximately 10 mM and/or concentrations that yielded approximately 80-90% cytotoxicity (as measured by relative increase in cell count). The assay demonstrated excellent specificity; exposures to the chemicals examined did not yield any positive responses even when tested in the presence of an exogenous metabolic activation system (i.e. S9) or with an extended sampling time. These results indicate that the FE1 cell mutagenicity assay is an effective and practical alternative to traditional mammalian cell gene mutation assays. The development and validation of effective in vitro tools such as the MutaMouse FE1 cell assay will contribute to international efforts to reduce, refine, and replace experimental animals for toxicity assessment. Environ. Mol. Mutagen. 58:582-591, 2017. © 2017 Wiley Periodicals, Inc.

Complementarity of phosphorylated histones H2AX and H3 quantification in different cell lines for genotoxicity screening.

The in vitro micronucleus assay is broadly used, but is not per se able to discriminate aneugenic from clastogenic compounds, and cytotoxicity can be a confounding factor. In vitro genotoxicity assays generally rely on cell lines with limited metabolic capabilities. Recently, the use of histone H2AX and H3 phosphorylation markers (γH2AX and p-H3) was proposed to discriminate aneugenic from clastogenic chemicals. The aim of the present study was to develop a new genotoxic screening strategy based on the use of the γH2AX and p-H3 biomarkers in combination with cell lines with distinct biotransformation properties. First, we tested a training set of 20 model chemicals comprised of 10 aneugens, five clastogens and five cytotoxics on three human cell lines (HepG2, LS-174T and ACHN). Our data confirm the robustness of these two biomarkers to discriminate efficiently clastogens, aneugens and misleading cytotoxic chemicals in HepG2 cells. Aneugenic compounds induced either an increase or a decrease in p-H3 depending on their mode of action. Clastogens induced γH2AX, and cytotoxic compounds generated a marked decrease in these two biomarkers. Moreover, the use of different cell lines permits to discriminate direct from bioactivated genotoxins without the need of an exogenous metabolic activation system. Finally, we further evaluated this strategy using a test set of 13 chemicals with controversial genotoxic potential. The resulting data demonstrate that the combined analysis of γH2AX and p-H3 is an efficient strategy. Notably, we demonstrated that three compounds (fisetin, hydroquinone and okadaic acid) display both aneugenic and clastogenic properties.

Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.

In a previous study, we validated an in vitro genotoxicity assay based on γH2AX quantification using the In-Cell Western (ICW) method in HepG2 cells. The assay demonstrated high sensitivity and specificity but failed to detect genotoxicity for few compounds that require specific metabolic bioactivation not sufficiently covered by HepG2 cells. The aim of the present study was to assess γH2AX ICW sensitivity using a broader range of genotoxic molecules with HepG2 cells and three additional human cell lines with distinct biotransformation properties: two cell lines expressing some phase I and II bioactivation capabilities (LS-174T and Hep3B), and one with poor general bioactivation properties (ACHN). We evaluated the four cell lines by testing 24 compounds recommended by European Centre for the Validation of Alternative Methods and a set of 24 additional chemicals with different mode of genotoxic action (MOA) (aneugenicity, DNA adducts formation, induction of oxidative stress), including some known to require specific cytochrome P450 metabolic bioactivation. Results for the 48 compounds tested showed that the γH2AX ICW assay was more sensitive with LS-174T and HepG2 cells than with Hep3B or ACHN cell lines. Among the 38 compounds tested with positive or equivocal carcinogenicity data, 36 (95%) showed a positive genotoxic response with the γH2AX ICW assay compared to only 27 (71%) using the Ames assay. We confirm that the γH2AX ICW assay on HepG2 cells, without an exogenous metabolic activation system, may be a suitable test to predict the in vivo genotoxicity of chemicals with different genotoxic MOA. Moreover, the use of the ACHN cell line in combination with LS-174T and HepG2 cells may permit in many cases to discriminate direct from bioactivated genotoxins. Overall, our results confirm the high sensitivity of the γH2AX ICW assay which, in turn, should reduce the number of animals used for genotoxicity assessment.

Mechanism of hepatotoxicity of BDE-153 involves mitochondrial damage and genotoxicity

To ascertain the in vitro genotoxicity of danthron and its potential mechanism of action, we performed an Ames test, a cytokinesis-block micronucleus assay and a comet assay in Balb/c 3T3 cells. The Ames test revealed that danthron was mutagenic only toward Salmonella typhimurium strain TA102 in the presence of an exogenous metabolic activation system (S9 mix). Danthron (25, 50 and 100 μg/ml) increased the frequencies of micronuclear cells with or without S9 mix, and the comet length, tail length and Olive tail moment in comet assays without S9 mix in a dose-dependent manner. These results demonstrated the in vitro genotoxicity of danthron and that 3T3 cells are capable of activating danthron. When NADP was replaced by NAD in the S9 mix, danthron remained mutagenic toward strain TA102. The addition of dicoumarol, a DT-diaphorase inhibitor, decreased the number of danthron-induced histidine revertants by 35–39%, indicating that DT-diaphorase is involved in the metabolic activation of danthron in the presence of NADH as an electron donor. In 3T3 cells, increases in reactive oxygen species (ROS) formation and 8-hydroxydeoxyguanosine levels as well as a reduction in GSH levels were induced by danthron in a dose-dependent manner, indicating that oxidative stress may be a major contributing pathway in the genotoxicity of danthron.

Terminalia catappa, an anticlastogenic agent against MMS induced genotoxicity in the human lymphocyte culture and in bone marrow cells of Albino mice

BackgroundTerminalia catappa has been used as a folk medicine for treating dermatitis, hepatitis as well as other diseases in India. It possesses anticancer, antioxidant as well as anticlastogenic characteristics. AimThe aim of the present investigation is to highlight the anticarcinogenic and antimutagenic potential of extracts of T. catappa. SubjectsAnticarcinogenic potential of methanolic extract of T. catappa has been tested against the carcinogenicity induced by methyl methanesulfonate in the in vitro and in vivo models. MethodsThe parameters for evaluation included chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and replication indices (RI) both in the presence as well as in the absence of exogenous metabolic activation system (in vitro study) and total aberrant cells and the frequencies of aberrations were used (for in vivo methods). ResultsAlcoholic extracts of T. catappa significantly reduce chromosomal aberration from 34.42%, 70.65% and 82.80% at 24, 48, and 72h produced by methyl methanesulfonate (MMS) to 22.77%, 49.60% and 42.50% levels. Similarly the number of sister chromatid exchanges was reduced from 6.20 per cell to 3.10 per cell at 48h of treatment and replication index was enhanced in vitro for each concentration and duration of treatment. Further their ameliorating potential was dose and duration dependant. Similarly these extracts significantly reduced the number of aberrant cells or frequency of aberrations per cell in vivo. ConclusionExtracts of T. catappa significantly reduced chromosomal aberrations up to 11.65% to 40.30% at different dosages against MMS induced toxicity, similarly sister chromatid exchange was reduced and replication index enhanced in vitro. Similarly in the in vivo experiments, the effective reduction in clastogeny ranges from 19.70% to 40.90%. Their reducing potential was time and dose dependant.

Andrographia paniculata a Miracle Herbs for cancer treatment: In vivo and in vitro studies against Aflatoxin B1 Toxicity

BackgroundThe history of natural products used in ancient times and in folk medicine these days, around the world, is the basis for the use of many therapeutic drugs in modern day medicine. Andrographia paniculata belongs to the family Acanthaceae or Kalmegh and is commonly known as ‘king of bitters’. It is extensively used as home remedy for various diseases in Indian traditional system as well as in tribal system in India for multiple clinical applications. AimIn our present work, extracts of these ayurvedic plants were tested for their anticlastogenic, antimutagenic and anticarcinogenic properties against Aflatoxin B1 induced toxicity. Materials and methodsWe used the in vitro method i.e. human lymphocytes culture and in vivo method in bone marrow cells of albino mice, while the parameters studied included chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and cell growth kinetics (RI) both in the presence as well as in the absence of exogenous metabolic activation system for in vitro studies, whereas total aberrant cells and the frequencies of aberrations were used for in vivo methods. ResultsA. paniculata extracts significantly reduced chromosomal aberrations from 35.0%, 62.0% and 69.0% level [at 24, 48, and 72h due to Aflatoxin B1] to 21.72%, 44.0% and 52.0%, similarly sister chromatid exchanges were reduced from 14.60 per cell to 7.50 per cell at 48h of treatments and replication index was enhanced in vitro for each concentration and duration of treatment. ConclusionIn conclusion A. paniculata extracts significantly reduced the number of aberrant cells and frequencies of aberration per cell at each concentration and duration of exposure in vivo; similarly it reduced chromosomal aberrations and sister chromatid exchanges and replication index was enhanced in vitro that was statistically significant at <0.05 level.

In vitro genotoxicity assessment of MTES, GPTES and TEOS, three precursors intended for use in food contact coatings

Organoalkoxysilanes are precursors that are used increasingly in the synthesis of food contact coatings. To comply with the EU regulation, their potential toxicity must be assessed, and very little information is known. The genotoxicity of three common precursors was studied, namely, tetraethylorthosilicate (TEOS), methyltriethoxysilane (MTES) and 3-glycidyloxypropyltriethoxysilane (GPTES). By the Ames test, MTES and TEOS were not mutagenic for bacteria. A significant positive response was observed with GPTES in the TA100 and TA1535 strains. The mutagenic effect was more pronounced in the presence of the exogenous metabolic activation system with an increase of the induction factor (ten-fold higher for the TA1535 strain). In the micronucleus assay performed with a human hepatoma cell line (HepG2 cells), GPTES gave negative results even in the presence of an exogenous activation system. To ascertain the possibility of using this precursor in food contact material, its migration must be monitored according to the coating formulation because migration might result in hazardous human exposure.

Terminalia arjuna, a herbal remedy against environmental carcinogenicity: An in vitro and in vivo study

BackgroundMedicinal plants have been a major source of therapeutic agents from ancient times to cure diseases. The evaluation of rich heritage of traditional medicine is essential. The bark of Terminalia arjuna is rich in polyphenols (60–70%) including flavonoids and tannins. AimThe aim of the present investigation is to highlight the anticarcinogenic and antimutagenic potential of extracts of T. arjuna. Subject and methodsIn this experiment we have used human lymphocyte culture and bone marrow cells of albino mice as assay system. The parameters studied included chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and cell growth kinetics (RI) both in the presence and in the absence of exogenous metabolic activation system for in vitro experiment, whereas total aberrant cells and the total frequencies of aberrations were taken for in vivo study. ResultsThe role of T. arjuna extracts in reducing metaphase aberrations due to aflatoxin B1 is quite significant, the reduction varying from 23.49%, 42.47%, and 59.65% down to 12.32%, 28.00%, and 36.88% respectively at the highest dose (TA4) for the three different durations viz., 24, 48 and 72h. Similarly the number of sister chromatid exchanges got reduced from a higher level of 15.00±1.40 per cell to 7.70±0.50 per cell with S9 mix at 48h of treatment. The replication index was enhanced from 1.33 to 1.55 in vitro. Similar trends were noticed in the in vivo experiments i.e., effective reductions in clastogeny ranging from 15.22% to 54.82% from the mutagen treated positive control and the total frequencies in aberrant cells got reduced from 429 due to AFB1 to 141 due to 5th concentration of Terminalia extracts at 32h of exposure. ConclusionThe ameliorating potential of Terminalia extracts was dose and time dependant.

Does Caesalpinia bonducella ameliorate genotoxicity? An in vitro study in human lymphocyte culture and in vivo study in Albino mice

Most of the world’s populations residing in developing countries depend on alternative medicine and use of plant ingredients. The plant Caesalpinia bonducella belongs to the family of Caesalpiniaceae and it is commonly known as Natakaranja in Hindi. It contains bonducin and two phytosterols namely sitosterol and hepatsane. The twigs and young leaves of C. bonducella are rationally used for curing tumors, inflammation and liver disorders. In our present work alcoholic extracts of this ayurvedic plant were tested for their antimutagenic and anticarcinogenic properties. The aim of the study is to investigate the antimutagenic and antigenotoxic potential of alcoholic extracts of C. bonducella against methyl methane sulfonate (MMS) induced genotoxicity. In this experiment we have used in vitro method i.e., human lymphocyte culture and in vivo method in bone marrow cells of albino mice, while the parameters studied included chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and cell growth kinetics (RI) both in the presence as well as in the absence of exogenous metabolic activation system for in vitro studies whereas total aberrant cells and the frequencies of aberrations were used for in vivo methods. Alcoholic extracts of C. bonducella significantly reduce chromosomal aberration from 42.75%, 44.25%, and 51.75% levels [at 24, 48, and 72h due to methyl methane sulfonate (MMS)] to 28.50%, 30.25%, and 35.10%, respectively similarly sister chromatid exchanges were reduced from 7.70±1.50 to 5.20±1.50 at 48h of treatment and replication index was enhanced in vitro for each concentration and duration of treatment and their ameliorating potential was dose and duration dependent. Similarly these extracts significantly reduced the number of aberrant cells and frequency of aberrations per cell in in vivo.

Spatial and temporal evaluations of estrogenic activity in tap water served by a water plant in Wuhan, China

This study aimed to evaluate the spatial and temporal characteristics of estrogenic activities in tap water served by a water plant in Wuhan, China. Tap water samples were monthly collected from the three sampling sites with different distances of distribution network from the plant during April 2010–March 2011: Min (less than 0.1km), Mid (approximately 4km) and Max (approximately 8km). Estrogenic activities of solid phase-extracted tap waters were measured by using recombinant yeast assay incorporated with and without exogenous metabolic activation system (rat liver S9 fractions) and expressed as 17β-estradiol equivalents (EEQ). Pro-estrogenic and estrogenic activity in tap water ranged from 151.4 to 1395.6pg EEQ/L and 35.2 to 1511pg EEQ/L, respectively. Average pro-estrogenic activity (680.3pg EEQ/L) was significantly higher than estrogenic activity (412.8pg EEQ/L) throughout the whole year. The pro-estrogenic activity significantly increased with the extending of distribution network, and was also statistically correlated with water temperature and pH. However, pro-estrogenic and estrogenic activity was not altered across four seasons. Our results suggest that the pro-estrogenic and estrogenic chemicals are present in tap water served by the water plant.

Antimutagenic Thio Compounds from Sisymbrium officinale

Glucoputranjivin (1) and isopropyl isothiocyanate (2) were isolated from an aqueous dry extract of Sisymbrium officinale and were identified by spectroscopic analysis. The antimutagenic activity of these compounds was evaluated in a bacterial reverse mutation assay using E. coli WP2, WP2uvrA, and WP2uvrA/pKM101 strains, in comparison with the extract. In the absence of the exogenous metabolic activation system S9, the thio compounds exerted antimutagenic activity against the direct-acting mutagen methyl methanesulfonate, in all strains. In the presence of S9, both thio compounds were active against the indirect mutagens 2-aminoanthracene, in WP2uvrA, and 2-aminofluorene, in WP2. The antimutagenicity seems to be due to specific mechanisms, such as the induction of the adaptive response or the excision repair system. Conversely, the inhibition of the CYP450-mediated activation of mutagens was not supported by the present results. An antimutagenic effect was also observed for the S. officinale aqueous extract against the arylamines 2AA and 2AF, but not against MMS. These results suggest that both thio compounds are involved in the antimutagenicity of S. officinale. The antimutagenicity of glucosinolate 1 is reported for the first time.

Development and validation of an in vitro micronucleus assay platform in TK6 cells

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.