Genotoxicity Assessment Research Articles

Are Nano TiO2 Inclusions Improving Biocompatibility of Photocurable Polydimethylsiloxane for Maxillofacial Prosthesis Manufacturing?

(1) Background: The development of a biocompatible material for direct additive manufacturing of maxillofacial extraoral prosthesis is still a challenging task. The aim of the present study was to obtain a photocurable PDMS, with nano TiO2 inclusions, for directly 3D printing of extraoral, maxillofacial prosthesis. The biocompatibility of the newly obtained nanocomposite was also investigated; (2) Methods: 2.5% (m/m) titania nanoparticles (TiO2) oxide anatase and a photoinitiator, benzophenone (BF) 4.5% were added to commercially available PDMS for maxillofacial soft prostheses manufacturing. The three different samples (PDMS, PDMS-BF and PDMS-BF-TiO2) were assessed by dielectric curing analysis (DEA) based on their viscosities and curing times. In vitro micronucleus test (MNvit) was performed for genotoxicity assessment and three concentrations of each compounds (2 mg/L, 4 mg/L and 8 mg/L) were tested in duplicate and compared to a control; (3) Results: The nanocomposite PDMS-BP-TiO2 was fully reticulated within a few minutes under UV radiation, according to the dielectric analysis. PDMS-BF-TiO2 nanocomposite showed the lowest degree of cyto- and genotoxicity; (4) Conclusions: In the limits of the present study, the proposed ex situ preparation of a PDMS-BP-TiO2 offers an easy, simple, and promising technique that could be successfully used for 3D printing medical applications.

Genotoxicity and subchronic general toxicity assessments of Lactobacillus curvatus WiKim 38 using Sprague-Dawley rats

Lactobacillus curvatus WiKim 38 (LCW), isolated from kimchi, has shown novel immunomodulatory and anti-inflammatory properties. In the present study, to obtain data on the safety of LCW, we performed three genotoxicity (bacterial reverse mutation, chromosome aberration, and micronucleus) and two general toxicity (single-dosing and 13-week repeated-dosing) studies. In the genotoxicity assessment, LCW showed no increased reverse mutation for 4 strains of Salmonella typhimurium and a strain of Escherichia coli. In addition, LCW did not induce chromosome aberrations at concentrations up to 5000 μg/mL in cultured Chinese hamster lung (CHL) cells and did not induce an increased frequency of micronuclei in the bone marrow cells of rats at concentrations up to 2000 mg/kg. In the acute toxicity study using Sprague-Dawley (SD) rats, the approximate lethal dose of LCW was determined to be over 5000 mg/kg body weight (b.w.) in both sexes. Finally, in the subchronic toxicity study, no LCW-related adverse effects were observed at concentrations up to 5000 mg/kg b.w./day. Consequently, LCW is considered not to have mutagenic effects, and its no-observed-adverse-effect-level (NOAEL) is 5000 mg/kg b.w., equivalent to approximately 4.71 × 109 CFU/kg b.w., suggesting the LCW could be a potential probiotic for humans based on its safety profile.

Pro-Apoptotic Activity of Artichoke Leaf Extracts in Human HT-29 and RKO Colon Cancer Cells.

(1) Background: Cynara cardunculus L. subsp. scolymus (L.) Hegi, popularly known as artichoke, is an herbaceous plant belonging to the Asteraceae family. Artichoke leaf extracts (ALEs) have been widely used in traditional medicine because of their hepatoprotective, cholagogic, hypoglycaemic, hypolipemic and antibacterial properties. ALEs are also recognized for their antioxidative and anti-inflammatory activities. In this study, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as effect on cell growth of ALEs on human colon cancer HT-29 and RKO cells. HT-29 and RKO cells exhibit a different p53 status: RKO cells express the wild-type protein, whereas HT-29 cells express a p53-R273H contact mutant. (2) Methods: Four different ALEs were obtained by sequential extraction of dried artichoke leaves; ALEs were characterized for their content in chlorogenic acid, cynaropicrin, and caffeoylquinic acids. HT-29 and RKO cells were used for in vitro testing (i.e., cytotoxicity and genotoxicity assessment, cell cycle analysis, apoptosis induction). (3) Results: Two out of the four tested ALEs showed marked effects on cell vitality toward HT-29 and RKO tumour cells. The effect was accompanied by a genotoxic activity exerted at a non-cytotoxic concentrations, by a significant perturbation of cell cycle (i.e., with increase of cells in the sub-G1 phase), and by the induction of apoptosis. (4) Conclusions: ALEs rich in cynaropicrin, caffeoylquinic acids, and chlorogenic acid showed to be capable of affecting HT-29 and RKO colon cancer cells by inducing favourable biological effects: cell cycle perturbation, activation of mitochondrial dependent pathway of apoptosis, and the induction of genotoxic effects probably mediated by the induction of apoptosis. Taken together, these results weigh in favour of a potential cancer chemotherapeutic activity of ALEs.

Application of HepG2/C3A liver spheroids as a model system for genotoxicity studies

HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA), we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA-damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA-damage induced by the direct acting mutagen 4-nitroquinoline N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.

Does storage conditions of whole blood or blood cells effect genotoxicity assessment by comet assay?

Comet assay, applied to in vitro, in vivo and ex vivo systems, is a quick, simple, and sensitive method for the detection of genotoxicity. In general, fresh whole blood or peripheral blood mononuclear cells (PBMCs) are used in the assay for the determination of DNA damage and repair. In this study, the effects of storage conditions on genotoxicity assessed by Comet assay in human whole blood and lymphocyte samples, were evaluated. Whole blood and lymphocyte samples were stored at 4 °C for 1, 2, 3, 4, 5 and 7 days; at −20 °C for 1 month and at −80 °C for 3, 6 and 12 months. 1% DMSO was used as cryoprotectant. No significant differences in DNA damage were demonstrated in all of the storage conditions and durations, and the results were similar according to the median values (p < 0.05). According to Spearman or Pearson correlations, an important correlation was found between the DNA damage of the fresh samples and the samples which were kept at −80 °C for 6 months with temperature and time (p < 0.01 for Pearson and p < 0.05 for Spearman). The results of this study indicated that blood and lymphocyte samples stored in +4 °C, −20 °C and −80 °C up to 12 months can be used instead of fresh samples especially in human biomonitoring studies.

Assessment of reproductive toxicity and genotoxicity of Aconiti Lateralis Radix Praeparata and its processed products in male mice

Ethnopharmacological relevanceAconiti Lateralis Radix Praeparata (Chinese name: Fuzi), the root of Aconitum carmichaelii Debx., is a representative medicine for restoring yang and rescuing patient from collapse. However, less studies had been reported on the reproductive toxicity and genotoxicity of Fuzi. According to the principle of reducing toxicity and preserving efficiency, only processed products of Fuzi are commonly applied in clinic, including Baifupian, Heishunpian and Danfupian. However, whether processing could alleviate the reproductive toxicity and genotoxicity of Fuzi had not been revealed. Aim of the studyTo assess the effect and possible mechanism of Fuzi and its processed products on reproductive toxicity and genotoxicity in male mice. Materials and methodsAqueous extracts of Fuzi and its processed products (Baifupian, Heishunpian and Danfupian, 5.85 g/kg) were administrated by gavage once daily for fourteen consecutive days. The reproductive toxicity was evaluated by testis weight, testis ratio, testis histopathology, sperm count, sperm viability rate and sperm deformity rate. The genotoxicity was evaluated by comet assay and micronucleus test in sperm, peripheral blood cell and bone marrow cell. Possible mechanisms of attenuating toxicity by processing were analyzed by detecting the level of testosterone, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and catalase (CAT). ResultsFuzi significantly caused different degrees of reproductive toxicity and genotoxicity, specifically reducing the weight and testicular coefficient of testis, causing obvious pathological changes in testicular tissue, reducing sperm count and sperm viability rate, increasing sperm deformity rate and DNA damage in sperm/peripheral blood cells/bone marrow cells. Moreover, Fuzi decreased the level of testosterone, SOD, GSH and CAT, while increased the level of MDA in serum. Notably, the reproductive toxicity and genotoxicity induced by the processed products, especially Heishunpian and Danfupian, were significantly lowered compared to Fuzi. Processing could increase the level of testosterone, SOD, GSH, CAT and decrease the level of MDA compared to Fuzi. ConclusionFuzi and its processed products had reproductive toxicity and genotoxicity, but the toxicity of processed products was significantly weakened compared to Fuzi. The protective mechanism of processing to reduce the toxicity of Fuzi might be related to increasing the level of testosterone and decreasing oxidative stress.

Assessment of Genotoxicity and Cytotoxicity of Leached Chemicals from HDPE Plastic Commonly Used by Ambulant Vendors

Plasticizers and additives in plastics like polyethylene terephthalate (PET) bottles have been found to leach – especially when exposed to certain stresses, including temperature. Frequent exposure to these chemicals is associated with a higher risk of developing developmental and reproductive disorders and cancer. In this study, water at varying temperatures (28, 65, 75, and 85 °C) was contained in high-density polyethylene (HDPE) plastic “labo,” a food packaging material common in the Philippines. Gas chromatography–mass spectrometry (GC-MS) analysis showed that the leached chemicals were derivatives of plastic additives and compounds or degradation compounds of these chemicals. Notable leachates include dimethyl sulfoxide, isosorbide, and cyclohexene derivatives. Different compounds leached at different temperatures. The Allium cepa test revealed chromosomal aberrations such as anaphase with bridge, sticky chromosomes, and chromosomal breaks. These signify that the compounds may be genotoxic to Allium cepa root tip cells. The Allium cepa test, brine shrimp lethality assay, and MTT assay on MCF-7 cells were also conducted as preliminary cytotoxicity tests. Results showed an increase in mitotic activity for Allium cepa root tip cells and induced proliferation of MCF-7 cells. Moreover, the mortality rates of Artemia salina were low after 24 h of exposure. These findings provide additional information regarding the leaching from plastic labo – the identity of leached chemicals and the effects of exposure on various cell lines and organisms. These findings may be used as a basis for existing policies regarding the use of plastic as food packaging material.

Novel Leea grandifolia leaves mediated synthesis of ZnO nanorods for photocatalytic and anticancer applications

The main goal of this study was to use a green‐mediated approach for obtaining ZnO nanorods (NRs) using the Leea grandifolia leaf extract. The obtained ZnO NRs were studied using standard methods such as X‐ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy with energy‐dispersive X‐ray analysis, Brunauer–Emmett–Teller, and transmission electron microscopy (TEM), and the results revealed a smaller particle size with a high surface area. The scanning electron microscopy and TEM results showed that the obtained particles were having rod‐like morphology with size ranging from ~20 to ~200 nm. The results of the antioxidant and photocatalytic studies showed excellent photodegradation and free radical scavenging. The results of the in vitro toxicological study revealed that the obtained particles were nontoxic to organisms. The cytotoxicity and genotoxicity assessments concluded that the NR‐treated MCF‐7 had gone through necrosis and apoptosis. The ZnO NRs revealed excellent antibacterial activity towards various pathogens. It was clear that the green‐mediated method could be considered as a better one for the synthesis of ZnO NRs as it imparts excellent anticancer, antibacterial, and photocatalytic properties.

Consumption pattern and genotoxic potential of various smokeless tobacco products in Assam, India: A public health concern

Smokeless tobacco (SLT) consumption is presumed to be one of the major causes of high incidence of oral cancer in India. The present study aimed to document various types of SLT products consumed and their potential impact on the genome instability on the population from Assam state in Northeast India. A cross-sectional study (n = 5000) showed that 60.56 % of the study population consumed at least one of the three forms (sadagura, zarda and khaini) of SLT of which 52.0 % were only sadagura users. Genotoxicity assessment using buccal cytome assay in 240 age and sex matched volunteers revealed that except for zarda, other forms of SLT induced significantly higher incidence micronuclei in the buccal epithelial cells compared to the control individuals. Similar effects were also observed in other cytome parameters related to cell proliferation, cytokinesis defects and cell death. Significantly higher incidence of micronucleus was observed among sadagura and khaini users in lymphocyte cytokinesis-blocked micronucleus assay. The addition of lime in sadagura increased the pH and anion levels which possibly result in higher absorption and may lead to the development of cellular anomalies.

Mutagenicity and genotoxicity assessments of some industrially processed meat products in Algeria

Processed meat products are presumptive sources of mutagens and genotoxins for consumers. Several epidemiological studies have reported that these products' high intakes have a positive link with cancer incidence. In Algeria, industrially processed meat products are widely consumed. However, there are no earlier studies involving their genotoxic activity. For this end, the current study aimed at evaluating the mutagenicity and the genotoxicity of some representative industrially processed meat products sold in popular supermarkets. All samples were extracted by established method, using both polar and non-polar solvents. The meat extracts mutagenicity was assessed by Ames test, using four strains of Salmonella typhimurium in the presence and absence of metabolic activation, and subsequently by treat and wash assay for extracts showing biologically significant results. The genotoxicity was determined in TK6 human lymphoblastoid cells using the in vitro micronucleus assay in micromethod. The results showed that all extracts analyzed induce no mutagenic activity. However, one of these extracts induced a biologically significant increase in the number of micronucleated cells. Our findings indicate the importance of the genetic damage detection for taking measures to suppress or reduce the exposure to harmful contaminants and encourage further research investigating genotoxic effects of industrially processed meat worldwide.

&lt;i&gt;In sit&lt;/i&gt; assessment of genotoxicity in tadpoles (Amphibia: Anura) in impacted and protected areas of Chapada Diamantina, Brazil

We evaluated the genotoxic potential of surface water bodies in an area impacted by agrochemicals, in the Ibicoara-Mucugê agropole, Bahia and those within protected areas in Chapada Diamantina, Bahia, Brazil, using the micronucleus test as a biomarker in tadpole species native neotropicals. Our hypothesis is that the closer to agricultural farms, the more affected by mutagenic and genotoxic substances will be the environment and resident organisms. The micronucleus frequencies were determined in tadpoles of 11 species collected in 11 surface water bodies in coffee, corn, vegetable, and pumpkin plantations, as well as in adjacent sites within a protection area. Tadpoles of seven species found in agricultural sites exhibited higher frequencies of micronuclei compared to the six species from the protected locations. The species Bokermannohyla oxente and Leptodactylus latrans collected in agricultural areas exhibited the highest frequencies of micronucleated erythrocytes compared to the populations from the protected areas (p &lt;0.05). Our results indicate that compounds present in surface water bodies in agricultural areas of the Ibicoara-Mucugê farm have genotoxic effects on anurans, showing the stress responses caused by agricultural activities associated with the use of complex mixtures of pesticides. Thus, anurans are excellent sentinel species in environmental monitoring, previously indicating the effect of xenobiotics on the environment.

Assessment of aquatic genotoxicity of the Lake Sevan basin, Armenia using natural bioindicators

Water pollution can cause genomic instability, thereby threatening aquatic wildlife and human health․ For a correct assessment of the impact of the mixture of pollutants, chemical analysis of environmental contaminants should be simultaneously applied with genotoxicity assays. The сomet assay is a sensitive, versatile and extensively used method for genotoxicity assessment in a water environment and was successfully applied in aquatic wild species, including fish and crayfish. Earlier we investigated the genotoxicity of waters of the Lake Sevan basin (Simonyan et al. 2016, Simonyan et al. 2019)․ Lake Sevan is the largest lake of the Caucasus Region, situated in the Republic of Armenia, in the Gegharkunik Province. Water pollution in the lake is a result of both anthropogenic and natural pressures. A combination of comet assay (Tice et al. 2000) and chemical analysis was applied for evaluation of water genotoxicity using gibel carp Carassius auratus gibelio and crayfish Astacus leptodactylus as sentinel organisms. Fish and water samples were collected from the south–southwest of the village Shorzha (40028′33″N and 45014′22″E), Peninsula of Lake Sevan (40033′46″N and 45001′37″E), the estuaries of the Rivers Gavaraget (40025′12″N and 45009′53″E) and Dzknaget (40036′55″N and 44058′13″E). Сrayfish and water samples were collected from the estuary of the River Masrik (40013′25″N and 45038′21″E) and near the villages of Artanish (40027′19″N and 45025′12″E) and Tsapatakh (40024′34″N and 45028′22″E). According to the chemical analysis of waters, the south–southwest of the village Shorzha and the estuary of the River Masrik were chosen as reference sites․ The content of contaminants in water was analyzed according to the standard methods in the Environmental Impact Monitoring Center, Ministry of Environment of the Republic of Armenia. The obtained results showed that the levels of DNA damage were significantly higher in both species in polluted areas than in reference sites. A significant positive correlation between DNA damage in C. auratus gibelio and contents of Al, Fe, Cu and Mn in water was shown. DNA damage in A. leptodactylus correlated with Al, Fe, Cu and Mo. C. auratus gibelio and A. leptodactylus were found to be sensitive mostly to the same pollutants, which indicate that these metals may be of primary concern as contaminants of the studied aquatic environment. Thus, the current study indicates that C. auratus gibelio and A. leptodactylus are sensitive bioindicators for monitoring of water pollution in the Lake Sevan basin.

Therapeutic effects of royal jelly against sodium benzoate-induced toxicity: cytotoxic, genotoxic, and biochemical assessment.

In this study, the protective role of royal jelly (RJ) against the potential toxic effects of sodium benzoate was investigated in Allium cepa L. test material with physiological, genetic, and biochemical parameters. Physiological changes were evaluated by determining weight gain, rooting percentage, root length, and relative injury rate. The genetic evaluations were carried out with chromosomal abnormalities (CAs), micronucleus (MN), tail DNA formation, and mitotic index (MI) ratio parameters. The biochemical evaluations were carried out by determining lipid peroxidation and antioxidant enzyme activities by determining levels of malondialdehyde (MDA), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT). Further, the interaction of sodium benzoate with antioxidant enzymes was evaluated with molecular docking analysis. The antimutagenic effect of RJ was evaluated as the inhibition of chromosomal abnormalities (CAs) and tail DNA formations. A total of six groups were formed in the study. A. cepa L. bulbs in the control group were treated with tap water; the bulbs in the administration groups were treated with sodium benzoate (100 mg/L), RJ (25 mg/L and 50 mg/L doses), and sodium benzoate-RJ combinations with these doses for 72 h. As a result, it was determined that sodium benzoate application caused inhibition of physiological parameters and MI; induced MN, CAs, and DNA damage; and also caused oxidative stress. Depending on the concentration of RJ application, it reduced sodium benzoate toxicity by showing therapeutic effects in all these parameters. Also, the interaction of sodium benzoate with antioxidant enzyme residues was determined by molecular docking analysis. As a result, it has been understood that abandoning the use of sodium benzoate will be beneficial for the environment and human health and concluded that the use of RJ in the daily diet will be effective in reducing the impact of exposed toxic ingredients.

Environmental Genotoxicity Assessment Using Micronucleus (and Nuclear Abnormalities) Test on Intertidal Mussel Perumytilus purpuratus: A Tool for Biomonitoring the Chilean Coast.

The release of pollutants is increasing along the coast of Chile, and the use of biomarkers in biomonitoring programs is essential to assess the early biological effects of marine contamination. The Micronucleus (MN) test was carried out using hemocytes of the mussel Perumytilus purpuratus from two sites in northern-central Chile (La Pampilla and Totoralillo). Nuclear abnormalities were assessed, and high frequencies of micronucleus (10.7-14.4‰) and other abnormalities were found (51.9-76.6‰). These values tended to be higher in La Pampilla, possibly due to the large presence of pollution sources in that site. However, considerably high values were observed in both sites. P. purpuratus is a suitable bioindicator and further monitoring along the Chilean coast using this species should be developed using the MN test and/or other biomarkers to comprehend the effects of human activities on the coastal environment of Chile.

In Vitro Genotoxicity Assessment of Functional Ingredients: Betaine, Choline, and Taurine.

This article focuses on a complete in vitro genotoxicity assessment of three nutrients widely used as functional ingredients in the European market: betaine, choline, and taurine. The European Food Safety Authority (EFSA) tiered approach for food additives in concordance with the safety assessment of chemicals in food developed by Food and Agriculture Organization/World Health Organization (FAO/WHO) was followed; the miniaturized Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of Organization for Economic Co-operation and Development (OECD) 471), and the micronucleus test (OECD 487) in TK6 cells were performed. In addition, the in vitro standard and enzyme-modified (human 8-oxoguanine DNA glycosylase 1 (hOGG), endonuclease III (EndoIII), human alkyladenine DNA glycosylase (hAAG)) comet assay (S9−/S9+) was conducted in order to assess the potential premutagenic lesions in TK6 cells. None of the compounds produced any signs of genotoxicity in any of the conditions tested. This article increases the limited evidence available and complements the EFSA recommendations for the in vitro genotoxicity testing of nutrients.

‘Site of contact genotoxicity’ assessment for implants - Potential use of single cell gel electrophoresis in biocompatibility testing of medical devices

Toxicological risk assessment of medical devices requires genotoxicity assessment as per ISO 10993, Part 3, which is designed to address gene mutations, clastogenicity and/or aneugenicity endpoints. ‘Site of contact genotoxicity’ is a potential genotoxic risk especially for medical implants, that is currently not addressed in biocompatibility standards. We therefore performed initial validation study on the use of alkaline single cell gel electrophoresis (comet assay) for detecting ‘site of contact genotoxicity’ of medical devices, using test items made of acrylic implants impregnated with ethyl methanesulphonate (EMS). Comet assay detected increased DNA migration at the site of implantation, but not in the liver. The same implants also failed to show any genotoxicity potentials, when tested on the standard test battery using Salmonella/microsome and chromosome aberration assays. The study suggested that some medical implants can cause ‘site of contact genotoxicity’, without producing systemic genotoxicity. In conclusion, comet assay will add new dimension to safety assessment of medical devices, and this assay can be added to the battery of genetic toxicology tests for evaluating biocompatibility of medical implants.

Transforming early pharmaceutical assessment of genotoxicity: applying statistical learning to a high throughput, multi end point in vitro micronucleus assay

To provide a comprehensive analysis of small molecule genotoxic potential we have developed and validated an automated, high-content, high throughput, image-based in vitro Micronucleus (IVM) assay. This assay simultaneously assesses micronuclei and multiple additional cellular markers associated with genotoxicity. Acoustic dosing (≤ 2 mg) of compound is followed by a 24-h treatment and a 24-h recovery period. Confocal images are captured [Cell Voyager CV7000 (Yokogawa, Japan)] and analysed using Columbus software (PerkinElmer). As standard the assay detects micronuclei (MN), cytotoxicity and cell-cycle profiles from Hoechst phenotypes. Mode of action information is primarily determined by kinetochore labelling in MN (aneugencity) and γH2AX foci analysis (a marker of DNA damage). Applying computational approaches and implementing machine learning models alongside Bayesian classifiers allows the identification of, with 95% accuracy, the aneugenic, clastogenic and negative compounds within the data set (Matthews correlation coefficient: 0.9), reducing analysis time by 80% whilst concurrently minimising human bias. Combining high throughput screening, multiparametric image analysis and machine learning approaches has provided the opportunity to revolutionise early Genetic Toxicology assessment within AstraZeneca. By multiplexing assay endpoints and minimising data generation and analysis time this assay enables complex genotoxicity safety assessments to be made sooner aiding the development of safer drug candidates.

Biomarker-based evaluation of cytogenotoxic potential of glyphosate in Vigna mungo (L.) Hepper genotypes.

Herbicides have proven to be a boon for agricultural fields. Their inherent property to kill weeds and unwanted vegetation makes them an essential biological tool for farmers and agricultural systems. Besides being capable of destroying weeds, they also exhibit certain effects on non-target crop plants. In the present study, a laboratory experiment was performed to assess the effect of glyphosate on Vigna mungo root meristem cells. Seeds of five different genotypes of V. mungo were treated with a series of concentrations of glyphosate ranging from 1 to 10mM, and their effects on mitotic cell division were studied. Healthy and uniform-sized seeds were selected and were allowed to grow in Petri plates for 3days, and all the doses were maintained in triplicates. Roots were fixed at day 3 after treatment (DAT) for cytological microscopic slide preparation. The results obtained indicate the dose-dependent reduction in the mitotic index in all the genotypes and an increase in the percentage of chromosomal aberrations (CAs) and relative abnormality rate (RAR). Most commonly observed chromosome aberrations at lower doses (< 6mM) were fragments, stickiness, and disoriented metaphase, while at higher doses (6 to 10mM) bridges, laggards, spindle disorientation, and clumping were obvious. The increase in the percentage of CAs and RAR indicates the inhibitory effect of glyphosate on cell cycle progression at various stages in root tip cells. The present study is a fine example of a biomarker-based genotoxic assessment of mitotic damage caused by glyphosate.

Genotoxic risk assessment and mechanism of DNA damage induced by phthalates and their metabolites in human peripheral blood mononuclear cells

The human genome is persistently exposed to damage caused by xenobiotics, therefore the assessment of genotoxicity of substances having a direct contact with humans is of importance. Phthalates are commonly used in industrial applications. Widespread exposure to phthalates has been evidenced by their presence in human body fluids. We have assessed the genotoxic potential of selected phthalates and mechanism of their action in human peripheral blood mononuclear cells (PBMCs). Studied cells were incubated with di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP) and their metabolites: mono-n-butylphthalate (MBP), mono-benzylphthalate (MBzP) in the concentrations range of 0.1–10 µg/mL for 24 h. Analyzed compounds induced DNA single and double strand-breaks (DBP and BBP ≥ 0.5 µg/mL, MBP and MBzP ≥ 1 µg/mL) and more strongly oxidized purines than pyrimidines. None of the compounds examined was capable of creating adducts with DNA. All studied phthalates caused an increase of total ROS level, while hydroxyl radical was generated mostly by DBP and BBP. PBMCs exposed to DBP and BBP could not completely repair DNA strand-breaks during 120 min of postincubation, in opposite to damage caused by their metabolites, MBP and MBzP. We have concluded that parent phthalates: DBP and BBP caused more pronounced DNA damage compared to their metabolites.

Assessment of the in vivo genotoxicity of pendimethalin via mitochondrial bioenergetics and transcriptional profiles during embryogenesis in zebrafish: Implication of electron transport chain activity and developmental defects

Pendimethalin, an herbicide used to control weeds, acts by inhibiting plant cell division and mitosis. Several studies have reported the detrimental effects of pendimethalin on non-target organisms. It has been found to be especially toxic to aquatic life. Additionally, there is some evidence that pendimethalin induces mitochondrial stress. However, none of the studies have provided information about the functional defects in mitochondria and toxicity during embryogenesis. In this study, we evaluated the impact of pendimethalin on the electron transport chain (ETC) activity and mitochondrial complexes via in vivo screening of oxidative phosphorylation and transcriptional profiles in zebrafish embryos. The results showed that pendimethalin interferes with mitochondrial complexes I and V, which inhibit embryo energy metabolism, thereby leading to developmental defects. Transgenic zebrafish, fli1:eGFP and olig2:dsRed, were used to confirm pendimethalin-induced functional depletion in neurogenesis and vasculogenesis during embryo development. This study provides new insights into the methodology of environmental assessment of biohazard chemicals that target ETC activity in mitochondria. Additionally, the results suggest that real-time respiratory and metabolic monitoring in zebrafish will be useful for the genotoxicity assessment of environmentally hazardous substances and may be used as an alternative model for the control of aquatic environmental pollutants.