Guidance for the use and interpretation of assays for monitoring anti-genotoxicity.

Since DNA damage can occur spontaneously or be produced by the environmental genotoxins in living cells, it is important to investigate compounds that can reverse or protect DNA damage. An appropriate methodology is essential for the responsive identification of protection offered against DNA damage. This review includes information on the current state of knowledge on prokaryotic cell-based assays (SOS chromotest, umu test, vitotox assay) and cytogenetic techniques (micronucleus assay, chromosome aberration test and sister chromatid exchange assay) with an emphasis on the possibility to explore genoprotective compounds. Throughout the last decade, studies have extrapolated the scientific methodologies utilized for genotoxicity to assess genoprotective compounds. Therefore, shortcomings of genotoxicity studies are also mirrored in antigenotoxicity studies. While regulatory authorities around the world (OECD, US-EPA and ICH) continue to update diverse genotoxic assay strategies, there are still no clear guidelines/approaches for efficient experimental design to screen genoprotective compounds. As a consequence, non-synergetic and inconsistent implementation of the test method by the researchers to execute such simulations has been adopted, which inevitably results in unreliable findings. The review has made the first attempt to collect various facets of experimentally verified approaches for evaluating genoprotective compounds, as well as to acknowledge potential significance and constraints, and further focus on the assessment of end points which are required to validate such action. Henceforth, the review makes an incredible commitment by permitting readers to equate several components of their test arrangement with the provided simplified information, allowing the selection of convenient technique for the predefined compound from a central repository.

Helical chromonema coiling is conserved in eukaryotes.

Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher-order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single-cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super-resolution microscopy, oligo-FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.

Acrylonitrile's genotoxicity profile: mutagenicity in search of an underlying molecular mechanism.

Acrylonitrile (ACN) is a known rodent and possible human carcinogen. There have also been concerns as to it causing adverse reproductive health effects. Numerous genotoxicity studies at the somatic level in a variety of test systems have demonstrated ACN's mutagenicity; its potential to induce mutations in germ cells has also been evaluated. ACN is metabolized to reactive intermediates capable of forming adducts with macromolecules including DNA, a necessary first step in establishing a direct mutagenic mode of action (MOA) for its carcinogenicity. The mutagenicity of ACN has been well demonstrated, however, numerous studies have found no evidence for the capacity of ACN to induce direct DNA lesions that initiate the mutagenic process. Although ACN and its oxidative metabolite (2-cyanoethylene oxide or CNEO) have been shown to bind in vitro with isolated DNA and associated proteins, usually under non-physiological conditions, studies in mammalian cells or in vivo have provided little specification as to an ACN-DNA reaction. Only one early study in rats has shown an ACN/CNEO DNA adduct in liver, a non-target tissue for its carcinogenicity in the rat. By contrast, numerous studies have shown that ACN can act indirectly to induce at least one DNA adduct by forming reactive oxygen species (ROS) in vivo, but it has not been definitively shown that the resulting DNA damage is causative for the induction of mutations. Genotoxicity studies for ACN in somatic and germinal cells are summarized and critically reviewed. Significant data gaps have been identified for bringing together the massive data base that provides the basis of ACN's current genotoxicity profile.

A study on Amygdalin's genotoxicological safety and modulatory activity in human peripheral lymphocytes in vitro.

Amygdalin (AMY), a plant secondary metabolite containing nitrile, is a major component of the seeds of Rosaceae family plants. It is known that this compound has many pharmacological activities such as cancer prevention, antipyretic, and cough suppressant. In this study, the genotoxic and modulatory effects of amygdalin were assessed by chromosomal aberration (CA), sister chromatid exchange (SCE), and cytokinesis-block micronucleus assay (CBMN) assays using human peripheral lymphocytes (HPLs) in the absence and presence of metabolic activator (S9 mix). Lymphocytes were exposed to various concentrations of amygdalin (0.86, 1.72, 3.43, 6.86, and 13.75 µg/mL) alone and in combination with mitomycin-C (MMC, 0.20 µg/mL) or cyclophosphamide (CP, 12 µg/mL). The mitotic index (MI), replication index (RI), cytokinesis-block proliferation index (CBPI), and cytostasis were also evaluated to determine cytotoxicity. Amygdalin alone did not exhibit genotoxic and cytotoxic effects at all the tested concentrations both in the absence and presence of the S9 mix. In contrast, amygdalin significantly reduced the frequencies of CA (especially at 48 h treatments), SCE, and MN (except 0.86 µg/mL in pre- and simultaneous treatment) induced by MMC in all the tested concentrations and treatment protocols. It has also considerably decreased CP-induced CA and SCE frequencies at all the concentrations (except 0.86 µg/mL) in simultaneous treatment. This study demonstrated that amygdalin alone was not genotoxic, on the contrary, it has revealed modulatory effects against chemotherapy agents that induced genomic damage in human lymphocytes, suggesting its chemopreventive potential.

Antigenotoxic effect of hyperoside against Mitomycin C and hydrogen peroxide-induced genotoxic damage on human lymphocytes.

Hyperoside is a flavonol glycoside isolated from various plant genera such as Hypericum and Crataegus. It has an important place in the human diet and is used medically to relieve pain and ameliorate cardiovascular functions. However, a comprehensive profile of the genotoxic and antigenotoxic effects of hyperoside is not known. The current study aimed to investigate the genotoxic and antigenotoxic effects of hyperoside against genetic damages induced by two genotoxins (MMC and H2O2) using chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) assays in human peripheral blood lymphocytes in vitro. Blood lymphocytes were incubated with 7.8-62.5 µg/mL concentrations of hyperoside alone and simultaneously with 0.20 µg/mL Mitomycin C (MMC) or 100 µM Hydrogen peroxide (H2O2). Hyperoside did not exhibit genotoxic potential in the CA, SCE, and MN assays. Moreover, it did not cause a decrease in mitotic index (MI) which is an indicator of cytotoxicity. On the other hand, hyperoside significantly decreased CA, SCE, and MN (except for MMC treatment) frequencies induced by MMC and H2O2. Hyperoside, increased mitotic index against both mutagenic agents at 24-h treatment when compared to positive control. Our results demonstrate that hyperoside exhibited antigenotoxic effects rather than genotoxic in vitro human lymphocytes. Therefore, hyperoside may be a potential preventive agent in inhibiting chromosomal and oxidative damage induced by genotoxic chemicals.

Elucidation of the cytogenotoxic potential of vigabatrin and its in silico computer-assisted DNA interaction.

Vigabatrin (VGB) is a gammaaminobutyric acid-ergic (GABA-ergic) antiepileptic drug (AED) and is one of 2 approved drugs available to treat infantile spasms (IS). The aim of this study is to elucidate conflicting data on the toxic effects of VGB and to obtain detailed information about its possible cytogenotoxic effects in human lymphocytes. For this purpose, in vitro Chromosomal Aberration (CA), Sister Chromatid Exchange (SCE), Micronucleus (MN) tests, and Comet Assay were performed to determine possible genotoxic and cytotoxic effects of VGB. In addition, the binding energy level of VGB to DNA was determined in silico by molecular docking. The highest concentration (80 µg/ml) of VGB increased the SCE, CA, MN and micronucleated binuclear cell (BNMN) frequency significantly compared to the control after 24 and 48 hours of treatment. In the tail density and tail length parameters, the dose-dependent increase was found to be statistically significant compared to the control. At the 40 and 80 µg/ml concentrations of VGB for 48 hours caused a statistically significant increase in both CA/Cell and AC percentages, while MI and NDI decreased only significantly at the highest concentration (80 µg/ml) causing. In the Comet Assay head density, tail density and tail length parameters, the dose-dependent increase was found to be statistically significant compared to the control. Also, the in silico molecular docking analysis showed that VGB interacts with B-DNA close to the threshold binding energy. The lowest negative free binding energy (ΔG binding) was found as -5.13 kcal/mol. In conclusion, all results are evaluated together, it has been determined that VGB has cytogenotoxic effects in vitro and binds to DNA in silico with significant free binding energy.

Genotoxicity of nedaplatin in cultured lymphocytes: modulation by vitamin E.

Nedaplatin is a chemotherapeutic agent used widely in cancer therapy. Nedaplatin has been shown to cause DNA damage to cells via the induction of oxidative stress. Vitamin E (Vit E) has an anti-mutagenic activity that can protect cells from DNA damaging agents. The objective of this study is to examine the genotoxic and cytotoxic effects of nedaplatin in human cultured lymphocytes. In addition, modulation of such effects by Vit E was also examined. The frequencies of sister chromatid exchange (SCE) and chromosomal aberrations (CAs) were used as an indicator for genotoxicity. The mitotic and proliferative indices were used to examine the cytotoxic effects of nedaplatin. The results showed that nedaplatin significantly elevated SCE and CA frequencies in human lymphocytes (p Ë‚ 0.01). The increases in the frequencies of SCE and CA caused by nedaplatin were lowered by pretreatment treatment with Vit E (p < 0.05). Nedaplatin significantly lowered mitotic index but Vit E pretreatment did not modulate this effect. These results suggest that Vit E has the potential to ameliorate the genotoxicity of nedaplatin in cultured lymphocytes.

Genotoxic repercussion of high-intensity radiation (x-rays) on hospital radiographers.

Recent technological advances in the medical field have increased the plausibility of exposing humans to high-intensity wavelength radiations like x-rays and gamma rays while diagnosing or treating specific medical maladies. These radiations induce nucleotide changes and chromosomal alterations in the exposed population, intentionally or accidentally. A radiological investigation is regularly used in identifying the disease, especially by the technicians working in intensive care units. The current study observes the genetic damages like chromosomal abnormalities (CA) in clinicians who are occupationally exposed to high-intensity radiations (x-rays) at their workplaces using universal cytogenetic tools like micronucleus assay (MN), sister chromatid exchange and comet assay. The study was conducted between 100 exposed practitioners from the abdominal scanning, chest scanning, cranial and orthopedic or bone scanning department and age-matched healthy controls. We observed a slightly higher rate of MN and CA (p < .05) in orthopedic and chest department practitioners than in other departments concerning increasing age and duration of exposure at work. Our results emphasize taking extra precautionary measures in clinical and hospital radiation laboratories to protect the practitioners.

In vitro cytogenotoxic evaluation of aripiprazole on human peripheral lymphocytes and computational molecular docking analysis.

Two different drug groups, typical (classic) and atypical (new), are used in the treatment of schizophrenia. Aripiprazole, an atypical antipsychotic chemical, is the active ingredient of the drug Abilify. This study was conducted to determine the possible genotoxic effect of aripiprazole. For this purpose, four different doses of aripiprazole (5; 10; 20, and 40 µg/mL) were examined with Chromosome Abnormality (CA), Sister Chromatid Exchange (SCE), Micronucleus (MN) tests. Based on these tests, Proliferation Index (PI), Percent Abnormal Cells (AC), Mitotic Index (MI), Micronuclear Binuclear Cell (MNBN), and Nuclear Division Index (NDI) levels were determined in human peripheral lymphocytes treated for 24 and 48 hours. Also, to determine possible binding sites of Aripiprazole on B-DNA molecular docking analysis was performed using AutoDock 4.0 (B-DNA dodecamer, PDB code: 1BNA). Aripiprazole binds to B-DNA with a very significant free binding energy (-11.88 Kcal/mol). According to our study, aripiprazole did not significantly change SCE, CA, AC percentage, MN frequencies when compared with control. According to these results, aripiprazole does not have a genotoxic effect. At the same time, no significant change was observed in the PI, MI, and NDI frequencies when compared with the control. In line with these results, it was observed that the use of aripiprazole in the treatment of schizophrenia did not pose any acute genotoxic and cytotoxic risk.

Analysis for Sister Chromatid Exchange.

Sister chromatid exchange (SCE) is the exchange event of genetic material between two identical sister chromatid. Elevation of SCE frequency is considered as a result of replication stress from genetic defects, ROS stress, and genomic damages. SCE staining needs extra processes compared to regular Giemsa staining. Usually two rounds of cell cycle progress are required to observe SCE under microscope. SCE can be visualized with the fluorescence plus Giemsa (FPG) staining method or fluorescence staining methods with immunocytochemistry to BrdU or Click reaction to EdU which provide more clear images of SCE. This chapter will provide the detailed method for the SCE staining and measurement for the traditional FPG staining, BrdU monoclonal antibody staining method, and newly developed EdU Click reaction staining method.

In vitro genotoxic and antigenotoxic effects of an exopolysaccharide isolated from Lactobacillus salivarius KC27L.

Exopolysaccharide isolated from Lactobacillus salivarius (new genus name Ligilactobacillus) KC27L strain (EPSKC27L) exhibits antioxidant properties with 1,1-diphenyl-2-picrylhydrazase (DPPH) radical and superoxide anion radical (O2-.) scavenging effect and iron ion (Fe2+) chelating activity. This study aimed to investigate the in vitro genotoxic effects of EPSKC27L alone (12.50, 25.00, 50.00, and 100.00 µg/mL) and its antigenotoxic activity against DNA damage induced by mitomycin-C (MMC; 0.20 µg/mL), methyl methanesulfonate (MMS; 5.00 µg/mL), and hydrogen peroxide (H2O2; 100 µM). For this purpose, chromosome aberration (CA), sister chromatid exchange (SCE), micronucleus (MN), and comet assays were performed in human peripheral lymphocytes. In addition, the structure of EPSKC27L was investigated in the scanning electron microscope (SEM). EPSKC27L alone did not cause a significant genotoxic effect in CA, SCE, MN, and comet tests. EPSKC27L significantly decreased the frequency of CA, SCE, and MN induced by MMC and MMS. EPSKC27L also significantly reduced DNA damage induced by H2O2. This study showed that the EPSKC27L alone has no genotoxic risk at these concentrations and shows antigenotoxic activity against MMC, MMS, and H2O2. Consequently, EPSKC27L was found to exhibit chemopreventive activity against genotoxic agents. This effect is believed to be due to the antioxidant properties of EPSKC27L.

CNV assessments associated with outcome distinctions for adult and pediatric cancers: Loss of BRCA1 in neuroblastoma associates with a lower survival probability.

DNA copy number variations (CNV) are common in cancer development, however, CNV detection approaches that include assessments of small CNVs, for example, due to locally misaligned sister chromatid exchanges, have not been substantially applied. Using such approaches, CNVs have been detected, in the cancer setting, for regulatory elements common to both proliferation and apoptosis effector genes, but no linkage has yet been made to cancer patient clinical data. Thus, we hypothesized that copy number losses, including local copy number losses, of specific apoptosis effector genes would be associated with reduced survival. Both whole genome and whole exome files were processed for validations and consistency. Results indicated lower late-stage survival for multiple myeloma cases representing reduced BAD and CASP3 copies, as well as for lung adenocarcinoma cases representing reduced BAX and CASP3 copies. Results also indicated that neuroblastoma (NBL) cases representing reduced copies of CASP9 and BRCA1 had reduced overall survival probabilities, with the BRCA1 results being particularly notable due to previous reports of BRCA1 inactivating mutations in NBL. Overall, novel approaches to assessing CNVs offers the promise of establishing patient risk stratifications and of identifying single genes or other small spaces in the genome where a CNV may be linked to specific outcomes.

Dose response to methylating agents in the γH2AX, SCE and colony formation assays: Effect of MGMT and MPG overexpression.

Cells have developed diverse protective mechanisms that enable them to tolerate low doses of genotoxic compounds. DNA repair processes attenuate the mutagenic and carcinogenic effects of alkylating agents, and multiple studies indicate a key role of specific DNA repair factors and pathways in establishing non-linear dose response relationships. Using an overexpression approach, we investigated the impact of O6-methylguanine-DNA-methyltransferase (MGMT), which repairs O6-methylguanine (O6MeG) in a damage reversal reaction, and N-methylpurine-DNA glycosylase (MPG), which acts as an apical enzyme in the BER pathway, on the DNA damage response to the alkylating agents MNNG and MMS. Our data indicate a clear protective effect of MGMT against MNNG-induced nuclear γH2AX foci formation, sister chromatid exchanges (SCE) and cytotoxicity, as determined in the colony formation assay. MGMT protected with similar efficiency against MMS-induced cytotoxicity and γH2AX foci formation, but suppressed SCE induction only weakly, which indicates that recombination events induced by MMS result from other lesions than O6MeG. In contrast, overexpression of MPG had only a very mild protective effect on the cellular defense against MMS and MNNG. Collectively, our data indicate that overexpression of MGMT results in non-linear DNA damage responses to O6MeG inducers. In contrast, MPG overexpression has only minor impact on the DNA damage response to alkylating drugs, indicating that other downstream enzymes in the BER pathway are limiting.

Genotoxic effects of gadobutrol and gadoversetamide active substances used in magnetic resonance imaging in human peripheral lymphocytes in vitro.

Gadobutrol and gadoversetamide are gadolinium-based contrast agents (GBCAs) widely used during magnetic resonance imaging examination. In this study, the genotoxicity of two GBCAs, gadobutrol and gadoversetamide, was investigated by using different endpoints: chromosome aberration (CAs), sister chromatid exchange (SCEs), and micronucleus (MNi). Human peripheral lymphocytes (PBLs) were treated with five concentrations (7 000, 14 000, 28 000, 56 000, and 112 000 µg/mL) of both agents. While a few concentrations of gadobutrol significantly increased abnormal cell frequency and CA/Cell, nearly all the concentrations of gadoversetamide significantly elevated the same aberrations. Similarly, the effect of gadoversetamide on the formation of SCEs was higher than those of gadobutrol. Only one concentration of gadoversetamide significantly increased MN% but no gadobutrol. The comet assay was applied for the only gadobutrol which induced a significant increase in tail intensity at the highest concentration only. On the other hand, significantly decreased mitotic index (MI) was observed following both substances, again gadoversetamide was slightly higher than those of the gadobutrol. The results revealed that both the contrast agents are likely to induce genotoxic risk in PBLs. However, different concentrations and treatment periods should be examined in vitro and specifically in vivo with different test systems for the safer usage of these contrast agents.

Cadmium exposure and DNA damage (genotoxicity): a systematic review and meta-analysis.

Existing literature suggests an association between chronic cadmium (Cd) exposure and the induction of DNA damage and genotoxicity. However, observations from individual studies are inconsistent and conflicting. Therefore current systematic review aimed to pool evidence from existing literature to synthesize quantitative and qualitative corroboration on the association between markers of genotoxicity and occupational Cd exposed population. Studies that evaluated markers of DNA damage among occupationally Cd-exposed and unexposed workers were selected after a systematic literature search. The DNA damage markers included were chromosomal aberrations (chromosomal, chromatid, sister chromatid exchange), Micronucleus (MN) frequency in mono and binucleated cells (MN with condensed chromatin, lobed nucleus, nuclear buds, mitotic index, nucleoplasmatic bridges, pyknosis, and karyorrhexis), comet assay (tail intensity, tail length, tail moment, and olive tail moment), and oxidative DNA damage (8-hydroxy-deoxyguanosine). Mean differences or standardized mean differences were pooled using a random-effects model. The Cochran-Q test and I2 statistic were used to monitor heterogeneity among included studies. Twenty-nine studies with 3080 occupationally Cd-exposed and 1807 unexposed workers were included in the review. Cd among the exposed group was higher in blood [4.77 µg/L (-4.94-14.48)] and urine samples [standardized mean difference 0.47 (0.10-0.85)] than in the exposed group. The Cd exposure is positively associated with higher levels of DNA damage characterized by increased frequency of MN [7.35 (-0.32-15.02)], sister chromatid exchange [20.30 (4.34-36.26)], chromosomal aberrations, and oxidative DNA damage (comet assay and 8OHdG [0.41 (0.20-0.63)]) compared to the unexposed. However, with considerable between-study heterogeneity. Chronic Cd exposure is associated with augmented DNA damage. However, more extensive longitudinal studies with adequate sample sizes are necessary to assist the current observations and promote comprehension of the Cd's role in inducing DNA damage.Prospero Registration ID: CRD42022348874.

Cytogenetic effects of antidiabetic drug metformin.

Metformin (MET) is the first-choice antidiabetic drug for type 2 diabetes mellitus treatment. In this study, the genotoxic potential of MET was evaluated by using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) assays in human peripheral lymphocytes as well as comet assay in isolated lymphocytes. Human lymphocytes were treated with different concentrations of MET (12.5, 25, 50, 75, 100, and 125 µg/mL) for 24 h and 48 h. A negative and a positive control (Mitomycin-C-MMC, 0.20 µg/mL, for CA, SCE, and MN tests; hydrogen peroxide-H2O2, 100 µM, for comet assay) were also maintained. MET significantly increased the frequency of CAs at 48 h exposure (except 12.5 µg/mL) compared to the negative control. MET increased SCEs/cells in both treatment periods (except 12.5 µg/mL at 24 h). MET only increased the frequency of MN at 125 µg/mL. While MET significantly increased the comet tail length (CTL) at four concentrations (25, 75, 100, and 125 µg/mL), it did not affect comet tail intensity (CTI) (except 125 µg/mL) and comet tail moment (CTM) at all the treatments. All these data showed that MET had a mild genotoxic effect, especially at a long treatment period and higher concentrations in human lymphocytes in vitro. However, further in vitro and especially in vivo studies should be conducted to understand the detailed genotoxic potential of MET.HighlightsMetformin increased the frequency of CAs and SCEs, especially at 48-h exposure time in human lymphocytes.This antidiabetic drug increased the frequency of MN only at the highest concentration tested (125 µg/mL).Metformin significantly increased the comet tail length in all treatments (except 50 µg/mL).The drug did not significantly affect the comet tail intensity (except 125 µg/mL) and comet tail moment in all treatments.

A SPIDR homozygous nonsense pathogenic variant in isolated primary ovarian insufficiency with chromosomal instability.

Primary ovarian insufficiency (POI), affecting 1% of women under 40 years is a public health problem. Genes involved in meiosis/DNA repair were recently shown to be the leading family of associated causal genes, some of them also cause tumors/cancers. Here, using targeted next-generation sequencing in an Indian POI patient with primary amenorrhea and streak ovaries, we identified a novel homozygous nonsense variant in exon 7 of SPIDR (KIAA0146) c.814C > T, R272*, predicted to lead a nonsense-mediated mRNA decay. SPIDR was recently identified by in vitro assays as an auxiliary protein interacting with RAD51 and BLM, two major proteins involved in genome stability. Consistent with alteration of the RAD51 pathway, we observed a strong increase in mitomycin C-induced DNA breaks and aberrant metaphases in the patient's cells compared to a control. However, sister chromatid exchanges were normal in contrast to the sharp increase characteristic of the BLM pathway. This is the first evidence of chromosomal instability associated with a SPIDR molecular defect, which supports the role of SPIDR in double-stranded DNA damage repair in vivo in humans and its causal role in POI. Our study increases knowledge on the SPIDR function and has broad implications in the management of such patients.

Genotoxicity of Mercury and Its Derivatives Demonstrated In Vitro and In Vivo in Human Populations Studies. Systematic Review.

Beside partial coverage in three reviews so far (1994, 2009, 2019), there is no review on genotoxic studies dealing with mercury (Hg) and human exposure using the most usual genotoxic assays: sister chromatid exchanges (SCE), chromosomal aberrations (CA), cytochalasin B blocked micronucleus assay (CBMN), and single-cell gel electrophoresis (SCGE or alkaline comet assay). Fifty years from the first Hg genotoxicity study and with the Minamata Convention in force, the genotoxic potential of Hg and its derivatives is still controversial. Considering these antecedents, we present this first systematic literature overview of genotoxic studies dealing with Hg and human exposure that used the standard genotoxic assays. To date, there is not sufficient evidence for Hg human carcinogen classification, so the new data collections can be of great help. A review was made of the studies available (those published before the end of October 2021 on PubMed or Web of Science in English or Spanish language) in the scientific literature dealing with genotoxic assays and human sample exposure ex vivo, in vivo, and in vitro. Results from a total of 66 articles selected are presented. Organic (o)Hg compounds were more toxic than inorganic and/or elemental ones, without ruling out that all represent a risk. The most studied inorganic (i)Hg compounds in populations exposed accidentally, occupationally, or iatrogenically, and/or in human cells, were Hg chloride and Hg nitrate and of the organic compounds, were methylmercury, thimerosal, methylmercury chloride, phenylmercuric acetate, and methylmercury hydroxide.

Analysis of repair of replication-born double-strand breaks by sister chromatid recombination in yeast.

The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.

Assessment of the genotoxic potential of tetrachlorvinphos insecticide by cytokinesis-block micronucleus and sister chromatid exchange assays.

Tetrachlorvinphos is an organophosphate that is classified as a carcinogen in humans by several authorities. Due to very limited data regarding the genotoxic potential, we aimed to comprehensively investigate in vitro genotoxic potential of tetrachlorvinphos. We performed our study by applying the cytokinesis-block micronucleus cytome and sister chromatid exchange (SCE) assays to human peripheral blood lymphocytes. We evaluated micronucleus (MN) and SCE frequencies and cytokinesis-block proliferation index in both exposed and non-exposed lymphocytes. We also calculated the chromosomal instability level in response to exposure by combining the results of MN and SCE. We found that MN frequency did not increase with exposure to tetrachlorvinphos (0-50 µg/ml). In contrast, we observed that SCE frequencies significantly increased with exposure to ≥5 µg/ml tetrachlorvinphos. Furthermore, exposure to tetrachlorvinphos at concentrations of 50 µg/ml induced a significant increase in chromosomal instability level (p < 0.05). Cytokinesis-block proliferation index level did not significantly decrease in response to tetrachlorvinphos exposure. Our findings reveal that tetrachlorvinphos resulted in different DNA damages that were measured by two assays. Furthermore, our findings suggested that exposure to tetrachlorvinphos increased chromosomal instability that is a hallmark of many malignancies. We conclude that although tetrachlorvinphos does not significantly increase the MN level, the significant increase of both SCE and CIN frequencies indicates the genotoxic potential of tetrachlorvinphos in human peripheral lymphocytes. Additionally, tetrachlorvinphos is not cytotoxic in the range of tested concentrations.