DNA Damage In Mammalian Cells Research Articles

Standards for Quantitative Measurement of DNA Damage in Mammalian Cells

As the potential applications of DNA diagnostics continue to expand, there is a need for improved methods and standards for DNA analysis. This report describes several methods that could be considered for the production of reference materials for the quantitative measurement of DNA damage in mammalian cells. With the focus on DNA strand breaks, potentially useful methods for assessing DNA damage in mammalian cells are reviewed. The advantages and limitations of each method, as well as additional concerns with respect to reference material development, are also discussed. In conclusion, we outline strategies for developing candidate DNA damage reference materials that could be adopted by research laboratories in a wide variety of applications.

Detection of Oxidatively Modified Base Lesion(s) in Defined DNA Sequences by FLARE Quantitative PCR.

Assessment of DNA base and strand damage can be determined using a quantitative PCR assay that is based on the concept that damage blocks the progression of a thermostable polymerase thus resulting in decreased amplification. However, some of the mutagenic DNA base lesions cause little or no distortion in Watson-Crick base pairing. One of the most abundant such lesion is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo(d)Gua), although it affects the thermodynamic stability of the DNA, duplex 8-oxo(d)Gua does not inhibit DNA synthesis or arrest most of DNA or RNA polymerases during replication and transcription. When unrepaired, it is a pre-mutagenic base as it pairs with adenine in anti-syn conformation. Recent studies considered 8-oxo(d)Gua as an epigenetic-like mark and along with 8-oxoguanine DNA glycosylase1 (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1) has roles in gene expression via nucleating transcription factor's promoter occupancy. Here, we introduce its identification through fragment length analysis with repair enzyme (FLARE)-coupled quantitative (q)-PCR. One of the strengths of the assay is that 8-oxo(d)Gua can be identified within short stretches of nuclear and mitochondrial DNA in ng quantities. Bellow we describe the benefits and limits of using FLARE qPCR to assess DNA damage in mammalian cells and provide a detailed protocol of the assay.

Elucidating the Implications of Diverse Dynamical Responses in p53 Protein.

p53 is a well-known tumor suppressor gene that acts as a transcription factor to exhibit a variety of dynamical responses by sensing different types and extent of stress conditions causing DNA damage in Mammalian cells. Mathematical modeling has played a crucial role to correlate cell fate decision-making with some of these dynamic p53 regulations. However, it is extremely challenging to explain the various cell-type and stimulus-specific p53 protein dynamics under different stress conditions by using a single mathematical model. In this article, we propose a simple mathematical model of p53 regulation based on a generic p53 regulatory network that elucidates a range of p53 dynamical responses. By employing bifurcation analysis along with deterministic and stochastic simulations, we explain an array of p53 dynamics by correlating it with the corresponding cell fate regulations in a cell type-specific and stimulus-dependent manner. Moreover, our model makes experimentally testable predictions to fine-tune p53 dynamics under various DNA damage conditions and can be systematically used and improved to analyze complex p53 dynamics in the future.

Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells.

During a normal topoisomerase II (TOP2) reaction, the enzyme forms a covalent enzyme DNA intermediate consisting of a 5′ phosphotyrosyl linkage between the enzyme and DNA. While the enzyme typically rejoins the transient breakage after strand passage, a variety of conditions including drugs targeting TOP2 can inhibit DNA resealing, leading to enzyme-mediated DNA damage. A critical aspect of the repair of TOP2-mediated damage is the removal of the TOP2 protein covalently bound to DNA. While proteolysis plays a role in repairing this damage, nucleolytic enzymes must remove the phosphotyrosyl-linked peptide bound to DNA. The MRN complex has been shown to participate in the removal of TOP2 protein from DNA following cellular treatment with TOP2 poisons. In this report we used an optimized ICE (In vivo Complex of Enzyme) assay to measure covalent TOP2/DNA complexes. In agreement with previous independent reports, we find that the absence or inhibition of the MRE11 endonuclease results in elevated levels of both TOP2α and TOP2β covalent complexes. We also examined levels of TOP2 covalent complexes in cells treated with the proteasome inhibitor MG132. Although MRE11 inhibition plus MG132 was not synergistic in etoposide-treated cells, ectopic overexpression of MRE11 resulted in removal of TOP2 even in the presence of MG132. We also found that VCP/p97 inhibition led to elevated TOP2 covalent complexes and prevented the removal of TOP2 covalent complexes by MRE11 overexpression. Our results demonstrate the existence of multiple pathways for proteolytic processing of TOP2 prior to nucleolytic processing, and that MRE11 can process TOP2 covalent complexes even when the proteasome is inhibited. The interactions between VCP/p97 and proteolytic processing of TOP2 covalent complexes merit additional investigation.

Development of a Reference Method and Materials for Quantitative Measurement of UV-Induced DNA Damage in Mammalian Cells: Comparison of Comet Assay and Cell Viability

Application of DNA damage diagnostic tests is rapidly growing, in particular for ovarian, prostate, and skin cancers; environmental monitoring; chronic and degenerative diseases; and male infertility. Such tests suffer from significant variability among different laboratories due the lack of standardization, experimental validation, and differences in data interpretation. Reference methods and materials for quantitative measurement of UVA-induced DNA damage in mammalian cells are frequently needed. In this study, we examined the use of the single-cell gel electrophoresis (comet) assay to assess the UVA-induced DNA damage in surface-attached Chinese hamster ovary (CHO) cells treated with a photosensitizer as a candidate cellular oxidative damage reference material. We found that the comet images became diffused and the viability of the cells decreased substantially (>20%) as the UVA dose and benzo [a] pyrene (BaP) concentration exceeded 6.3 J/cm2 and 10−6 mol/L BaP. Maintaining the conditions of exposure within this range can improve DNA damage measurement fidelity, particularly if used as a quantitative reference method and to produce materials considered as an in vitro standard for the comet assay.

Human CYP enzyme-activated genotoxicity of 2,2′,4,4′-tetrabromobiphenyl ether in mammalian cells

Polybrominated biphenyl ethers (PBDEs) are a group of persistent organic pollutants with endocrine-disrupting, neurotoxic, tumorigenic and DNA-damaging activities. They are hydroxylated by human liver microsomal CYP enzymes, however, their mutagenicity remains unknown. In this study, 2,2′,4,4′-tetrabromobiphenyl ether (BDE-47, relatively abundant in human tissues) was investigated for micronuclei induction and DNA damage in mammalian cells. The results indicated that BDE-47 up to 80 μM under a 6 h/18 h (exposure/recovery, covering 2 cell cycles) regime did not induce micronuclei in V79-Mz and V79-derived cell lines expressing human CYP1A1 or 1A2, while it was moderately positive in human CYP2B6-, 2E1-and 3A4-expressing cell lines (V79-hCYP2B6, V79-hCYP2E1-hSULT1A1 and V79-hCYP3A4-hOR, respectively). Following 24 h exposure, BDE-47 induced micronuclei in V79-hCYP2E1-hSULT1A1 and V79-hCYP3A4 cells at increased potencies. In the human hepatoma (HepG2) cells BDE-47 (48 h exposure) was inactive up to 40 μM, however, pretreatment of the cells with ethanol (0.2%, v:v, inducer of CYP2E1) or rifampicin (10 μM, inducer of CYP3A4) led to significant micronuclei formation by BDE-47; pretreatment with bisphenol AF (100 nM) also potentiated BDE-47-induced micronuclei formation (which was blocked by a CYP2E1 inhibitor trans-1,2-dichloroethylene or a CYP3A inhibitor (ketoconazole). Immunofluorescent staining of centromere protein B with the micronuclei formed by BDE-47 in HepG2 cells pretreated with ethanol or rifampicin demonstrated selective formation of centromere-containing micronuclei. The increased phosphorylation of both histones H2AX and H3 in HepG2 by BDE-47 also indicated an aneugenic potential. Therefore, this study suggests that BDE-47 is an aneugen activated by several human CYP enzymes.

Genetic and physical interactions between Pol\u03b7 and Rev1 in response to UV-induced DNA damage in mammalian cells

In response to UV irradiation, translesion DNA synthesis (TLS) utilizes specialized DNA polymerases to bypass replication-blocking lesions. In a well-established polymerase switch model, Polη is thought to be a preferred TLS polymerase to insert correct nucleotides across from the thymine dimer, and Rev1 plays a scaffold role through physical interaction with Polη and the Rev7 subunit of Polζ for continual DNA synthesis. Defective Polη causes a variant form of xeroderma pigmentosum (XPV), a disease with predisposition to sunlight-induced skin cancer. Previous studies revealed that expression of Rev1 alone is sufficient to confer enhanced UV damage tolerance in mammalian cells, which depends on its physical interaction with Polζ but is independent of Polη, a conclusion that appears to contradict current literature on the critical roles of Polη in TLS. To test a hypothesis that the Rev1 catalytic activity is required to backup Polη in TLS, we found that the Rev1 polymerase-dead mutation is synergistic with either Polη mutation or the Polη-interaction mutation in response to UV-induced DNA damage. On the other hand, functional complementation of polH cells by Polη relies on its physical interaction with Rev1. Hence, our studies reveal critical interactions between Rev1 and Polη in response to UV damage.

Protective Effect of Thymidine on DNA Damage Induced by Hydrogen Peroxide in Human Hepatocellular Cancer Cells.

Intracellular ribonucleotide (RN) and deoxyribonucleotide (dRN) pool sizes are critical for the fidelity of DNA synthesis. They are likely to be severely perturbed by many factors which disrupt the integrity and stability of DNA, leading to DNA damage. Exogenously supplied nucleosides are able to increase the deoxynucleoside triphosphate pools, then reverse the DNA damage, and decrease the oncogene-induced transformation dramatically. In this study, the impact of thymidine on the hydrogen peroxide (H2O2)-induced DNA damage was investigated in HepG2 liver cancer cells. From the result of the comet assay, the tail length of cells in the thymidine 600 μM + H2O2 1.0 mM group was dramatically decreased from 42.1 ± 10.8 to 21.9 ± 2.4 μm compared to that exposed with 1.0 mM H2O2 (p < 0.05), suggesting that pretreatment of thymidine reduced the DNA damage of HepG2 cells. Although the RN and dRN contents decreased in the damage group, most of them presented increasing tendency when pretreated with thymidine, especially the key metabolites dCTP, which was mainly related with the decline in the rate of DNA synthesis. The restoration also showed a significant G0/G1 phase arrest of cell cycle progression from 44.6 ± 2.2 to 56.6 ± 0.4% after pretreated with thymidine (p < 0.05). In conclusion, our data demonstrated that the pretreatment with thymidine had a potential protective ability against oxidative damage for DNA in HepG2 cells through the perturbation of RN and dRN pools as well as cell cycle arrest, which should provide new insights into the molecular basis of preventing H2O2-induced oxidative DNA damage in mammalian cells.

Cellular environment controls the dynamics of histone H3 lysine 56 acetylation in response to DNA damage in mammalian cells

Epigenetic changes play a crucial role in sensing signals and responding to fluctuations in the extracellular environment. How the cellular micro-environment affects DNA damage response signalling in chromatin context is not extensively studied. Histone acetylation is dynamic and very sensitive to changes in the extracellular environment. Existing literature on H3 lysine 56 acetylation (H3K56ac) levels upon DNA damage in mammals presents a conflicting picture. The occurrence of both increased and decreased H3K56ac upon DNA damage in our experiments led us to investigate the role of the micro-environment on H3K56ac. Here, we show that the global levels of H3K56ac increase as cells grow from low density to high density while SIRT1 and SIRT6 expression decrease. Additionally, rising lactic acid levels increase H3K56ac. Our results show that cell density and accumulation of metabolites affect dynamics of H3K56ac in response to DNA damage. Upon DNA damage, H3K56ac increases in low density cells with low initial acetylation, while acetylation decreases in high cell density cells. These results highlight that H3K56ac levels upon DNA damage are dependent on the metabolites in the extracellular milieu which impact chromatin structure by regulating chromatin modifying enzymes. Accumulation of lactic acid at high cell density reflects conditions similar to the tumour micro-environment. As H3K56ac increases in tumours, lactic acid and low pH might alter H3K56ac in tumours, leading to deregulated gene expression, contributing to tumour progression.

The Use of Single-Cell Comet Assay on Oral Cells: A Critical Review.

Genotoxicity is the capacity of an agent to induce damage to DNA. Given the close relationship between genotoxicity and carcinogenesis, several assays have been developed for detecting genetic damage. Among them, the single-cell gel (comet) assay plays an important role for evaluating DNA damage in mammalian cells, including those of the oral cavity. The purpose of this article was to provide a critical review of the application of single-cell gel comet assay to buccal cells. A search of the scientific literature was conducted of published studies available on single-cell gel comet assay and oral cells. The results showed that the majority of studies were conducted on humans, whereas few were designed for use in rodents and in vitro. Further studies within the field are relevant for better understanding the underlying mechanisms of genotoxicity in oral cells, especially since the use of humans is quite complicated due to issues of ethics.

Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains.

The overproduction of riboflavin (vitamin B2) by Ashbya gossypii, one of the most distinctive traits of this filamentous hemiascomycete, has been proposed to act as an ecological defense mechanism, since it is triggered by environmental stress. The interaction of endogenous riboflavin with light generates reactive oxygen species (ROS) and induces oxidative DNA damage in mammalian cells, but exogenous riboflavin was shown to protect A. gossypii spores against ultraviolet light. Envisioning a better understanding of this biotechnologically relevant trait, here we investigated the putative genotoxic effects associated with the overproduction of riboflavin by A. gossypii. For assessing that we developed the Ashbya Comet Assay, which was able to reproducibly measure oxidative (H2O2/menadione-mediated) and non-oxidative (camptothecin-mediated) DNA damage in A. gossypii. Using this protocol, we determined that exposure to sunlight-mimicking light during growth significantly increased the DNA damage accumulation in riboflavin-overproducing cells, but not in non-overproducing ones. The exposure of overproducing cells to light induced the intracellular accumulation of ROS and increased the production of riboflavin 1.5-fold. These results show that riboflavin-overproducing strains are highly susceptible to photo-induced oxidative DNA damage and draw attention for the importance of controlling the exposure to light of biotechnological riboflavin production processes with A. gossypii.

Cell damaging by irradiating non-thermal plasma to the water: Mathematical modeling of chemical processes.

Recently non-thermal plasma (NTP) is applied for many therapeutic applications. By NTP irradiating to the tissues or cell-lines, the water molecules (H2O) would be also activated leading to generate hydrogen peroxide (H2O2). By irradiating plasma to bio-solution, its main output including vacuum UV to UV causes the photolysis of H2O leading to generate hydroxyl (OH) molecules in couple forms with ability to convert to H2O2. Additionally, other plasma's output the oxygen atoms could also penetrate under the liquid's surface and react with H2O to generate H2O2. In NTP applications for killing unwanted-cells of microorganisms (e.g. sterilization) or cancerous tissues, the H2O2 molecule is the main reactive species for cell death via inducing DNA damage in mammalian cells. In this paper we proposed a mathematical model for NTP application describing the formation of hydroxyls in the bio solution and other subsequent reactions leading to DNA damage in vitro. The instant concentrations of the OH and H2O2, the main species for DNA oxidation were obtained and investigated in this simulation. In order to validate the model, the cellular response to NTP stimulation was compared with some experimental findings from viewpoint of DNA damage to show the significant consistency.

Serine is the major residue for ADP-ribosylation upon DNA damage

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that synthesise ADP-ribosylation (ADPr), a reversible modification of proteins that regulates many different cellular processes. Several mammalian PARPs are known to regulate the DNA damage response, but it is not clear which amino acids in proteins are the primary ADPr targets. Previously, we reported that ARH3 reverses the newly discovered type of ADPr (ADPr on serine residues; Ser-ADPr) and developed tools to analyse this modification (Fontana et al., 2017). Here, we show that Ser-ADPr represents the major fraction of ADPr synthesised after DNA damage in mammalian cells and that globally Ser-ADPr is dependent on HPF1, PARP1 and ARH3. In the absence of HPF1, glutamate/aspartate becomes the main target residues for ADPr. Furthermore, we describe a method for site-specific validation of serine ADP-ribosylated substrates in cells. Our study establishes serine as the primary form of ADPr in DNA damage signalling.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Induction of DNA damage in mammalian cells by hydrogen peroxide generated by glucose oxidase immobilized in agarose slides

A new model system has been developed to study the influence of reactive oxygen species on isolated mammalian cells in conjunction with the comet assay. The glucose-glucose oxidase system was used as a hydrogen peroxide generating source. The level of DNA damage was assessed in the splenocytes and the cells of bone marrow of mouse and in human leukocytes both in untreated cells and in cells treated with hydrogen peroxide generated by glucose oxidase using the alkaline comet assay in vitro. Various options for the location of the enzyme in the slides have been studied: in the layer with the cells, in the layer above the cells, or in solution on the surface of the slides. The option where glucose oxidase was in the upper layer of 0.5% agarose over the layer of the cells was optimal. It provided separation of the enzyme from the cells and avoided obstruction to the hydrogen peroxide exposure. For the whole blood study, the content of endogenous glucose must be taken into account. This approach can be used to study the level of DNA damage induced in vitro and for the detection of DNA repair, thereby expanding the possibilities of the method, while the experiments are conducted under controlled conditions.

Meglumine Antimoniate (Glucantime) Causes Oxidative Stress-Derived DNA Damage in BALB/c Mice Infected by Leishmania (Leishmania) infantum.

Leishmaniasis is a neglected tropical disease caused by >20 species of the protozoan parasite Leishmania Meglumine antimoniate (Glucantime) is the first-choice drug recommended by the World Health Organization for the treatment of all types of leishmaniasis. However, the mechanisms of action and toxicity of pentavalent antimonials, including genotoxic effects, remain unclear. Therefore, the mechanism by which meglumine antimoniate causes DNA damage was investigated for BALB/c mice infected by Leishmania (Leishmania) infantum and treated with meglumine antimoniate (20 mg/kg for 20 days). DNA damage was analyzed by a comet assay using mouse leukocytes. Furthermore, comet assays were followed by treatment with formamidopyrimidine-DNA glycosylase and endonuclease III, which remove oxidized DNA bases. In addition, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the animals' sera were assessed. To investigate mutagenicity, we carried out a micronucleus test. Our data demonstrate that meglumine antimoniate, as well as L. infantum infection, induces DNA damage in mammalian cells by the oxidation of nitrogenous bases. Additionally, the antileishmanial increased the frequency of micronucleated cells, confirming its mutagenic potential. According to our data, both meglumine antimoniate treatment and L. infantum infection promote oxidative stress-derived DNA damage, which promotes overactivation of the SOD-CAT axis, whereas the SOD-GPx axis is inhibited as a probable consequence of glutathione (GSH) depletion. Finally, our data enable us to suggest that a meglumine antimoniate regimen, as recommended by the World Health Organization, would compromise GPx activity, leading to the saturation of antioxidant defense systems that use thiol groups, and might be harmful to patients under treatment.

Cyclo(phenylalanine-proline) induces DNA damage in mammalian cells via reactive oxygen species.

Cyclo(phenylalanine-proline) is produced by various organisms such as animals, plants, bacteria and fungi. It has diverse biological functions including anti-fungal activity, anti-bacterial activity and molecular signalling. However, a few studies have demonstrated the effect of cyclo(phenylalanine-proline) on the mammalian cellular processes, such as cell growth and apoptosis. In this study, we investigated whether cyclo(phenylalanine-proline) affects cellular responses associated with DNA damage in mammalian cells. We found that treatment of 1 mM cyclo(phenylalanine-proline) induces phosphorylation of H2AX (S139) through ATM-CHK2 activation as well as DNA double strand breaks. Gene expression analysis revealed that a subset of genes related to regulation of reactive oxygen species (ROS) scavenging and production is suppressed by the cyclo(phenylalanine-proline) treatment. We also found that cyclo(phenylalanine-proline) treatment induces perturbation of the mitochondrial membrane, resulting in increased ROS, especially superoxide, production. Collectively, our study suggests that cyclo(phenylalanine-proline) treatment induces DNA damage via elevation of ROS in mammalian cells. Our findings may help explain the mechanism underlying the bacterial infection-induced activation of DNA damage response in host mammalian cells.

New investigations into the genotoxicity of cobalt compounds and their impact on overall assessment of genotoxic risk

The genotoxicity of cobalt metal and cobalt compounds has been widely studied. Several publications show induction of chromosomal aberrations, micronuclei or DNA damage in mammalian cells in vitro in the absence of S9. Mixed results were seen in gene mutation studies in bacteria and mammalian cells in vitro, and in chromosomal aberration or micronucleus assays in vivo. To resolve these inconsistencies, new studies were performed with soluble and poorly soluble cobalt compounds according to OECD-recommended protocols. Induction of chromosomal damage was confirmed in vitro, but data suggest this may be due to oxidative stress. No biologically significant mutagenic responses were obtained in bacteria, Tk+/− or Hprt mutation tests. Negative results were also obtained for chromosomal aberrations (in bone marrow and spermatogonia) and micronuclei at maximum tolerated doses in vivo. Poorly soluble cobalt compounds do not appear to be genotoxic. Soluble compounds do induce some DNA and chromosomal damage in vitro, probably due to reactive oxygen. The absence of chromosome damage in robust GLP studies in vivo suggests that effective protective processes are sufficient to prevent oxidative DNA damage in whole mammals. Overall, there is no evidence of genetic toxicity with relevance for humans of cobalt substances and cobalt metal.

Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

Dichlorvos-induced toxicity in HCT116 cells: Involvement of oxidative stress and apoptosis

Organophosphorous (OP) pesticides are widely used in the agriculture and home. Among those pesticides, Dichlorvos (DDVP) is a worldwide used insecticide for pest control. DDVP is commonly used as an insecticide for maintenance and growth of agricultural products, to control the internal and external parasites of farm animals, and to eradicate insects threatening the household, public health, and stored products. Although substantial information is available regarding the environmental and ecological impact of DDVP, not much is known in regard to its toxicity in the mammalian system. Therefore a study was conducted for the assessment of cytotoxic and genotoxic effects of DDVP in human colon carcinoma (HCT116) cell line. We demonstrated that DDVP significantly decreased cell viability as assessed by the MTT assay. The increase in cell death was accompanied by a reduction in the mitochondrial membrane potential. Besides, pretreatment with Z-VAD-FMK, a general caspases inhibitor, decreased significantly the DDVP-induced cell death. We also shown that DDVP induced reactive oxygen species (ROS) generation followed by lipid peroxidation as evidenced by an increase in the MDA levels. Our results also indicate that DDVP induced a concentration-dependent increase in DNA damage as evident by the comet assay. These data indicate that DDVP produces cytotoxicity and DNA damage in mammalian cells and should be used with caution.