The effect of inhibitors of phosphatidylinositol 3-kinase-related kinases on dibenzo[def,p]chrysene genotoxicity measured by γH2AX levels and neutral comet assay in HepG2 human hepatocellular cancer cells

In the study the modulating effect of inhibition of phosphatidylinositol 3-kinase-related kinases (PIKK): ATM (Ataxia Telangiectasia Mutated), ATR (Ataxia Telangiectasia and Rad3 Related) and DNA-PK (DNA-dependent protein kinase) on genotoxicity of dibenzo[def,p]chrysene (DBC) in HepG2 human hepatocellular cancer cells was investigated. The cytotoxicity of DBC was determined, also in combination with PIKK inhibitors, using the MTT reduction assay. The high cytotoxicity of DBC was observed after 72 h incubation (IC₅₀ = 0.06 μM). The PIKK inhibitors applied at non-cytotoxic concentrations: caffeine (1 mM) and KU55933 (2.5 μM) had no significant influence on the DBC cytotoxicity, however NU7026 (5 μM) caused significant increase in the cell viability by about 25%. The combinations of the inhibitors (double or triple) where NU7026 was present also caused increase in the cell viability (i.e. cytoprotective effect) compared to the effect of DBC. The level of damage to the genetic material (DNA double strand breaks, DSB) was assessed by measuring levels of phosphorylated form of H2A histone (γH2AX) and neutral comet assay. DBC induced DSB in a concentration and time-dependent manner. NU7026 considerably reduced the level of DSB level measured by γH2AX and comet assay.The obtained results confirm that DBC is cytotoxic and causes damage to the genetic material including DSB. The DNA-PK inhibitor NU7026 increases cell viability after exposure to DBC and reduces DNA damage, what indicates an important role of the sensor kinase in mediating the effect.     

Carbamate insecticide methomyl confers cytotoxicity through DNA damage induction

Carbamate insecticide methomyl could induce genotoxic effects, including micronuclei, chromosome aberrations and sister-chromatid exchanges. However, methomyl induction of cytotoxicity through DNA damage is largely unknown. Here we identify cytotoxicity and potential genotoxicity of methomyl in vitro. We have employed alkaline comet assay, γH2AX foci formation and DNA ladder assay to detected DNA damage and apoptosis of Drosophila S2, HeLa and HEK293 cells. The alkaline comet assay was used to evaluate total DNA single strand breaks (SSBs) in the target cells exposed in vitro to sublethal concentrations of methomyl. As expected, methomyl induced significant concentration-dependent increases in DNA damage of target cells compared with the negative control, as measured by increases in tail length (μm), tail DNA (percentage of the comet tail) and tail moment (arbitrary units). In agreement with the comet assay data, the percentage of γH2AX positive reaction in HeLa cells also revealed methomyl caused DNA double strand breaks (DSBs) in a time-dependent manner. Moreover, methomyl induced a significant increase of apoptosis in Drosophila S2, HeLa and HEK293 cells in a concentration- and time-dependent manner, as determined by Urea PAGE DNA fragmentation analysis. In conclusion, methomyl is a strongly genotoxic agent that induces cell DNA damage and apoptosis in vitro at these sublethal concentrations.     

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24?h after a single pharyngeal aspiration of MWCNT-S (1–200?μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8?mg/m³) for 4 d, 4?h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200?μg/mouse) and in MNPCEs after inhalation exposure (17.5?mg/m³). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10?μg/cm²), while MWCNT-T increased strand breakage only at 200?μg/cm². Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.     

Evaluation of in vitro cytotoxicity and genotoxicity of copper–zinc alloy nanoparticles in human lung epithelial cells

In the present study, in vitro cytotoxic and genotoxic effect of copper–zinc alloy nanoparticles (Cu–Zn ANPs) on human lung epithelial cells (BEAS-2B) were investigated. XTT test and clonogenic assay were used to determine cytotoxic effects. Cell death mode and intracellular reactive oxygen species formations were analyzed using M30, M65 and ROS Elisa assays. Genotoxic effects were evaluated using micronucleus, comet and γ-H2AX foci assays. Cu–Zn ANPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements. Characterization of Cu–Zn ANPs showed an average size of 200 nm and zeta potential of −22 mV. TEM analyses further revealed the intracellular localization of Cu–Zn ANPs in cytoplasm within 24 h. Analysis of micronucleus, comet and γ-H2AX foci counts showed that exposure to Cu–Zn ANPs significantly induced chromosomal damage as well as single and double stranded DNA damage in BEAS-2B cells. Our results further indicated that exposure to Cu–Zn ANPs significantly induced intracellular ROS formation. Evaluation of M30:M65 ratios suggested that cell death was predominantly due to necrosis.     

Comparative genotoxicity investigation using comet and gammaH2AX assays for screening of genotoxicants in HepG2 human hepatoma cells

As public interest in safety has increased the toxicity evaluation of chemicals become more important. In this study, the DNA-damaging effect of genotoxicants was examined in HepG2 cell line originated from human hepatocellular carcinoma by widely used genotoxicity assays: the comet assay and gammaH2AX immunostaining. Four different direct/indirect genotoxicants were tested in dose-/time-dependent manner. The comet assay and the gammaH2AX immunostaining enables detection of DNA damages in the form of DNA strand breaks with different sensitivity. Therefore, the combination of comet assay and gammaH2AX immunostaining will be complementary tool for evaluation of various forms and degree of DNA damage. Our result also suggested that HepG2 cells could be a suitable model for assessing the genotoxicity of various mutagens and for determining the lowest genotoxic concentration. Further analysis using a larger number of chemicals is warranted to determine the sensitivity and the specificity of HepG2 with in vitro genotoxicity test.     

Evaluation of N-acetyl-cysteine against tetrachlorobenzoquinone-induced genotoxicity and oxidative stress in HepG2 cells

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol (PCP). Although the genotoxic effect of PCP has been comprehensively investigated, there is little known about TCBQ’s genotoxic effects. In the current study, TCBQ was tested for its genotoxicity using HepG2 cells as experimental model. To select the exposure concentration of interest, cell viability was measured and three concentrations were used for further investigation. In single cell gel electrophoresis (SCGE) assay, concentration-dependent increase in tail length, tail DNA percentage and tail moment were detected following TCBQ exposure. Micronucleus (MN) assay indicated TCBQ gradually increased MN frequency and decreased nuclear division index (NDI). Enzyme-linked immunosorbent assay (ELISA) and western blotting analyses both showed TCBQ caused histone H2AX phosphorylation (γ-H2AX). Furthermore, the elevation of 8-hydroxydeoxyguanosine (8-OHdG) and reactive oxygen species (ROS) level indicated TCBQ-induced genotoxicity is associated with oxidative stress. On the other hand, N-acetyl-cysteine (NAC) administration significantly protected cells from the genotoxic effect of TCBQ. Overall, our data suggested TCBQ exerted genotoxic effect possibly via an oxidative damage mechanism in HepG2 cells and this toxicity is prevented by pretreatment with NAC.     

Impact of solvents on the in vitro genotoxicity of TMPTA in human HepG2 cells

Small hydrophobic chemical compounds require solvents to produce suitable solutions for toxicological studies. However, some solvents can modify the biological properties of substances and therefore their toxicity. This specific issue has been raised for PEG-400 as an anti-inflammatory and anti-oxidative compound. Recently, in the context of the REACH Regulation, PEG-400 was used to test the in vivo genotoxicity of trimethylolpropane triacrylate (TMPTA) in the comet assay. TMPTA failed to increase DNA damage whereas it induces genotoxicity in vitro in DMSO. Therefore, we questioned whether PEG-400 could modify the genotoxicity of TMPTA. The aim of this study was to determine the potential impact of PEG-400 on the in vitro genotoxicity of TMPTA, compared to DMSO. TMPTA was dissolved in either PEG-400 or DMSO, and the induction of γH2AX and Caspase-3 was analyzed in HepG2 cells. TMPTA induced γH2AX and Caspase-3 with both PEG-400 and DMSO. However, TMPTA induced effects at 4-fold lower concentrations when PEG-400 is used as the solvent compared to DMSO. While genotoxic effects are observed at much lower concentrations with PEG-400, it does not modify the in vitro genotoxicity of TMPTA. However, further in vitro studies with small hydrophobic compounds should be done to clarify the effect of PEG-400. Moreover, in vivo studies should be performed to confirm that PEG-400 remains suitable for in vivo genotoxicity tests.     

2-乙酰氨基芴诱导γH2AX焦点的形成

目的探讨DNA损伤剂2-乙酰氨基芴(2-AAF)是否可诱导γH2AX焦点的形成及γH2AX作为检测DNA损伤的一个新的特异指标的可能性。方法用2-AAF处理中国仓鼠CHL细胞,应用免疫荧光方法检测γH2AX焦点的形成,并通过中性彗星实验验证DNA损伤的程度。结果0.1,1,5和20mg·L-1的2-AAF都可使细胞核内γH2AX焦点数量及有γH2AX焦点生成的细胞数量增加,但只有20mg·L-12-AAF处理的细胞才出现明显的彗尾增长及有彗尾的细胞数量增加。另外,磷脂酰肌醇3-激酶(PI3K)家族抑制物渥曼青霉素可抑制2-AAF诱导的γH2AX焦点形成。结论2-AAF可通过激活PI3K家族成员使H2AX磷酸化,进而诱导γH2AX焦点的形成。γH2AX检测DNA损伤的敏感性优于彗星实验,因此,γH2AX有可能成为衡量DNA损伤程度的良好指标。     

Genotoxicity evaluation of carbon monoxide and 1,3-butadiene using a new joint technology: the in vitro γH2AX HCS assay combined with air–liquid interface system

To investigate the genotoxicity of gaseous toxicants carbon monoxide (CO) and 1,3-butadiene in vitro, a novel combination technology—the in vitro γH2AX high content screening assay combined with air–liquid interface system (ALIS) was established. The results showed that this new technology was available and effective. Based on the joint technology, genotoxicity of CO and 1,3-butadiene was evaluated further in this study. The results showed that treatment concentrations (0, 20%,40%, 80% and 100%, v/v) and exposure time (15, 30, 45, 60 and 90?min) of CO both had no statistically significant effects on the induction of γH2AX (p?>?0.05). However, 1,3-butadiene can induce significant γH2AX (p?in vitro based on the in vitro γH2AX high content screening assay combined with ALIS. Based on the joint technology, CO was not genotoxic in A549 cells, while 1,3-butadiene showed significant genotoxicity in the dose/time-dependency on the induction of γH2AX.     

Genotoxic and apoptotic activities of the food flavourings myristicin and eugenol in AA8 and XRCC1 deficient EM9 cells

Some food flavourings, such as safrole and methyleugenol, are known for their genotoxic and hepatocarcinogenic properties whereas for others, such as myristicin, there is less data. Myristicin and eugenol are both alkenylbenzenes, and we compared their direct genotoxicity in repair proficient (AA8) and repair deficient XRCC⁻ (EM9) Chinese hamster ovary cells. Cell viability was assessed by the MTT assay. The comet assay was used to evaluate DNA breaks, and the γ-H2AX assay to evaluate induction of double strand breaks. We assessed apoptosis by measuring caspases activation, and the TUNEL assay. Reduction of cell viability was similar in AA8 and EM9 cells, for both compounds. After 1 h eugenol produced DNA strand breaks in the comet assay and induced double strand breaks in the γ-H2AX assay in AA8 cells, while myristicin was not genotoxic in both the comet and the γ-H2AX assays. Both flavourings were negative in EM9 cells. After 24 h eugenol and myristicin induced DNA fragmentation detected by TUNEL in both cell lines, but only myristicin activated caspases. Myristicin was more apoptotic than eugenol, in both cell lines. The XRCC1 protein does not influence the apoptotic activity of either compound.     

Sterigmatocystin, 5-Methoxysterigmatocistin,and Their Combinations are Cytotoxic and Genotoxic to A549 and HepG2 Cells and Provoke Phosphorylation of Chk2, but not FANCD2 Checkpoint Proteins

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are structurally related mycotoxins with cytotoxic and genotoxic properties. In the present study, we hypothesized that DNA damage induced by non-cytotoxic concentrations of single and combined mycotoxins could alter the phosphorylation of the checkpoint proteins Chk2 and FANCD2 (ELISA) in HepG2 and A549 cells. The cytotoxic potential (MTT test) of single and combined STC and 5-M-STC, the nature of their interaction (additivity, antagonism, or synergy) and DNA damage level (alkaline comet assay) in HepG2 and A549 cells were also investigated. All experiments were performed after 24 h of mycotoxin treatment. 5-M-STC was 10-folds more cytotoxic than STC to both HepG2 and A549 cells. Both mycotoxins are genotoxic to HepG2 and A549 cells by inducing both double and single DNA strand breaks that activate Chk2 (especially in HepG2 cells) but not the FANCD2 protein. STC exerted higher genotoxic potential than 5-M-STC in HepG2 and A549 cells when both toxins were applied individually at the same concentration. Dual combinations of non-cytotoxic mycotoxin concentrations showed additive to antagonizing cytotoxic and genotoxic effects. The absence and low activation of checkpoint proteins during prolonged exposure to non-cytotoxic concentrations of STC and 5-M-STC could support cell proliferation and carcinogenesis.     

Long-term exposures to low doses of titanium dioxide nanoparticles induce cell transformation,but not genotoxic damage in BEAS-2B cells

Abstract

There is a great interest in a better knowledge of the health effects caused by nanomaterials exposures and, in particular to those induced by titanium dioxide nanoparticles (nano-TiO₂) due to its high use and increasing presence in the environment. To add new information on its potential genotoxic/carcinogenic risk, we have carried out experiments using chronic exposures (up to 4 weeks), low doses, and the BEAS-2B cell line that, as a human bronchial epithelium cells, can be considered a good cell target. Cell uptake has been assessed by transmission electron microscopy (TEM) and flow cytometry (FC); genotoxicity was evaluated using the comet and the micronucleus (MN) assays; and cell-transforming ability was evaluated using the soft-agar assay to detect anchorage-independent cell growth. Results show an important cell uptake at all the tested doses and sampling times used (except for 1?µg/mL and 24-h exposure). Nevertheless, no genotoxic effects were observed in the comet and in the MN assays. This lack of genotoxic effect agrees with the FC results showing no induction of intracellular reactive oxygen species (ROS), the data from the comet assay with formamidopyrimidine DNA glycosylase (FPG) enzyme showing no induction of oxidized bases, and the lack of induction of expression of heme-oxygenase (HO-1) gene both at the RNA and protein level. On the contrary, significant increases in the number of clones growing in an anchorage-independent way were observed. This study would indicate a potential carcinogenic risk associated to nano-TiO₂ exposure, not mediated by a genotoxic mechanism.     

4β‐Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells

Reactive oxygen species (ROS) induction had been previously reported in 4β‐hydroxywithanolide (4βHWE)‐induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8‐oxo‐2′deoxyguanosine [8‐oxodG]) were detected in 4βHWE‐treated oral cancer (Ca9‐22) and/or normal (HGF‐1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N‐acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE‐treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine‐DNA glycolyase (Fpg)‐based comet assay and 8‐oxodG‐based flow cytometry, the 8‐oxodG expressions were higher in 4βHWE‐treated oral cancer cells than in oral normal cells. All the 4βHWE‐induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS‐mediated selective killing of oral cancer cells.     

In vitro investigation of the genotoxicity of portimine,a cyclic imine toxin produced by the dinoflagellate Vulcanodinium rugosum,on human hepatic HepaRG cells

Portimine, a recently identified cyclic imine produced by the dinoflagellate Vulcanodinium rugosum, has been described as a potent apoptotic agent in contrast to most of the cyclic imines that are well-known to be neurological toxins. As apoptosis can be a consequence of a high level of DNA lesions, we investigated the responses of portimine on several endpoints aimed at detecting DNA damage in the hepatic cell line HepaRG. Portimine induced phosphorylation of H2AX, which could possibly be consistent with the previously published induction of apoptosis with this toxin. In addition, detection of apoptosis through the activation of caspase-3, the induction of strand breaks detected by the comet assay as well as chromosome and genome mutations using the micronucleus assay were addressed. Surprisingly, portimine treatment resulted in increases in only γH2AX in differentiated HepaRG cells whereas no effects on the other endpoints were detected. These increases in γH2AX in the absence of genotoxic effects in the other tests could indicate that portimine could possibly induce a DNA replication stress and/or that the compound can be detoxified by the HepaRG cells.     

Assessment of the chemopreventive effect of casearin B,a clerodane diterpene extracted from Casearia sylvestris (Salicaceae)

Studies have shown that Casearia sylvestris compounds protect DNA from damage both in vitro and in vivo. Complementarily, the aim of the present study was to assess the chemopreventive effect of casearin B (CASB) against DNA damage using the Ames test, the comet assay and the DCFDA antioxidant assay. The genotoxicity was assessed by the comet assay in HepG2 cells. CASB was genotoxic at concentrations higher than 0.30 μM when incubated with the FPG (formamidopyrimidine-DNA glycosylase) enzyme. For the antigenotoxicity comet assay, CASB protected the DNA from damage caused by H₂O₂ in the HepG2 cell line in concentrations above 0.04 μM with post-treatment, and above 0.08 μM with pre-treatment. CASB was not mutagenic (Ames test) in TA 98 and TA 102. In the antimutagenicity assays, the compound was a strong inhibitor against aflatoxin B1 (AFB) in TA 98 (>88.8%), whereas it was moderate (42.7–59.4%) inhibitor against mytomicin C (MMC) in TA 102. Additionally, in the antioxidant assay using DCFDA, CASB reduced reactive oxygen species (ROS) generated by H₂O₂. In conclusion, CASB was genotoxic to HepG2 cells at high concentrations; was protective of DNA at low concentrations, as shown by the Ames test and comet assay; and was also antioxidant.     

Molecular model of naphthalene-induced DNA damage in the murine lung

Airway epithelial damage and repair represents a novel therapeutic target in asthma and chronic obstructive pulmonary disease. An established mouse model of airway epithelial damage involves the Clara cell cytotoxicity of parenterally administered naphthalene, an important environmental toxicant with genotoxic and carcinogenic potential. The objective of the current study was to investigate naphthalene-induced toxicity and to identify and quantify DNA double-strand breaks in a murine naphthalene model of airway epithelial damage. Male C57/BL6 mice were injected with 200 mg/kg naphthalene and culled at 12-, 24-, 48- and 72-h time points. Lung function and bronchoalveolar lavage was performed and the lungs were dissected for histological analysis and for quantitation of DNA double-strand breaks using γH2AX as a molecular marker. Mice injected with naphthalene had increased epithelial denudation, bronchoalveolar lavage fluid cellularity and reactivity to nebulized methacholine chloride as compared to corn oil vehicle controls. Histological changes were most pronounced at the 12- and 24-h time points. DNA double-strand breaks, quantitated as the number of γH2AX foci per cell, were highest at the 24- and 48-h time points. All parameters had decreased at the 72-h time point, consistent with airway re-epithelization and cellular repair. Our findings indicate a time-dependent accumulation of γH2AX foci in mouse airway epithelial cells following administration of naphthalene. Naphthalene airway epithelial injury constitutes a model of DNA double-strand breaks in mice, which can be adapted as a suitable model for further investigation of genotoxic damage for evaluating the efficacy of potential therapeutics.     

Combined cytotoxic and genotoxic effects of ochratoxin A and fumonisin B1 in human kidney and liver cell models

Food products can be contaminated by several fungi species and each species may produce different mycotoxins, leading to human combined exposure. Although predictions about the joint toxic effects of mycotoxins can be made from their individual toxicities, experimental data is still limited to allow a reliable hazard assessment. Thus, this study aimed to characterize the combined cytotoxic and genotoxic effects of ochratoxin A (OTA) and fumonisin B1 (FB₁) in cell lines representative of their target organs, kidney and liver. Interactions were ascertained using mathematical extensions to the reference models of concentration addition and independent action. Cytotoxicity (MTT assay) data modeling revealed a synergistic pattern for low doses of both FB₁ and OTA shifting to antagonism at higher concentration levels, irrespectively of the reference model applied. Concerning genotoxicity assessment, neither OTA nor FB₁, individually or in combination, induced a prominent increase in DNA damage (comet assay) or oxidative DNA damage (FPG-comet assay). In conclusion, this study revealed a synergistic cytotoxic effect for OTA and FB₁ at low concentration levels. Given that human co-exposure to these two mycotoxins is probable to occur at low doses, these results raise concerns regarding their potential health outcomes that seem to differ from those predicted by an additive model.     

In vitro cytotoxicity,genotoxicity and antigenotoxicity assessment of Solanum lycocarpum hydroalcoholic extract

Context: Solanum lycocarpum A. St.-Hil. (Solanaceae), popularly known as ‘fruta-do-lobo’ (wolf fruit), ‘lobeira’ and ‘jurubebão’, is commonly used by native people of Central Brazil in powder form or as a hydroalcoholic extract for the management of diabetes and obesity and to decrease cholesterol levels. Objective: The present study determines the possible cytotoxic, genotoxic and antigenotoxic activities of hydroalcoholic extract of the S. lycocarpum fruits (SL).

Materials and methods: The clonogenic efficiency assay was used to determine the cytotoxicity. Three concentrations of SL (16, 32 and 64?μg/mL) were used for the evaluation of its genotoxic and antigenotoxic potential on V79 cells using the micronucleus and comet assays. In the antigenotoxicity assays, the cells were treated simultaneously with SL and the alkylating agent methyl methanesulphonate (MMS, 44?μg/mL for the micronucleus assay and 22?μg/mL for the comet assay) as an inducer of micronuclei and DNA damage.

Results: The results showed that SL was cytotoxic at concentrations up to 64?μg/mL. No significant differences in the rate of chromosome or DNA damage were observed between cultures treated with SL and the control group. In addition, the frequencies of micronuclei and DNA damage induced by MMS were significantly reduced after treatment with SL. The damage reduction percentage ranged from 68.1% to 79.2% and 12.1% to 16.5% for micronucleus and comet assays, respectively.

Discussion and conclusion: SL exerted no genotoxic effect and exhibited chemopreventive activity against both genomic and chromosome damage induced by MMS.     

Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells

Abstract

Quinocetone (QCT), a new quinoxaline 1,4-dioxides, has been used as antimicrobial feed additive in China. Potential genotoxicity of QCT was concerned as a public health problem. This study aimed to investigate the protective effect of curcumin on QCT-induced oxidative stress and genotoxicity in human hepatocyte L02 cells. Cell viability and intracellular reactive oxygen species (ROS), biomarkers of oxidative stress including superoxide dismutase (SOD) activity and glutathione (GSH) level were measured. Meanwhile, comet assay and micronucleus assay were carried out to evaluate genotoxicity. The results showed that, compared to the control group, QCT at the concentration ranges of 2–16?μg/mL significantly decreased L02 cell viability, which was significantly attenuated with curcumin pretreatment (2.5 and 5?μM). In addition, QCT significantly increased cell oxidative stress, characterized by increases of intracellular ROS level, while decreased endogenous antioxidant biomarkers GSH level and SOD activity (all p?<?0.05 or 0.01). Curcumin pretreatment significantly attenuated ROS formation, inhibited the decreases of SOD activity and GSH level. Furthermore, curcumin significantly reduced QCT-induced DNA fragments and micronuclei formation. These data suggest that curcumin could attenuate QCT-induced cytotoxicity and genotoxicity in L02 cells, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, consumption of curcumin may be a plausible way to prevent quinoxaline 1,4-dioxides-mediated oxidative stress and genotoxicity in human or animals.     

Cytotoxicity and genotoxicity of versicolorins and 5-methoxysterigmatocystin in A549 cells

Aspergillus versicolor and A. flavus are primary colonizers in damp dwellings, and they produce sterigmatocystin (ST) and aflatoxin B1 (AFB(1)), respectively. These hepatotoxic and carcinogenic mycotoxins and their precursors and derivates possess a furofuran ring, which has proven responsible for their toxicity. The aim of this study was to investigate the cytotoxicity and genotoxicity of versicolorin A (VER A) and versicolorin B (VER B), as the furofuran precursors of aflatoxins and ST, and of 5-methoxysterigmatocystin (5-MET-ST), a methoxy derivative of ST, in human adenocarcinoma lung cells A549. The IC(50) values of the tested compounds were obtained by the cell proliferation MTT test as follows: 109?±?3.5?μM (VER A), 172?±?4?μM (VER B) and 181?±?2.6?μM (5-MET-ST). The comet assay and micronucleus test were used to assess their genotoxic potential after 24?h of treatment with concentrations corresponding to ? and ? IC(50) in comparison with AFB(1) and ST, applied in concentrations corresponding to ? IC(50), as previously determined in A549 cells. DNA damage parameters assessed by the comet assay were tail length, tail intensity and tail moment, while the level of DNA damage in the micronucleus test was evaluated by the number of formed micronuclei (MN), nuclear buds (NB) and nucleoplasmic bridges (NPB) in 1,000 binucleated cells. Considering the three comet parameters, all applied toxins exerted significant DNA damage compared to the control, while ST and VER B produced the highest DNA damage. All toxins provoked a statistically significant increase in MN, and a slightly decreased formation of NB and NPB. AFB(1), ST and 20?μM VER A showed a statistically significant increase in all three micronucleus parameters compared to the control, and the highest increase in the number of MN occurred in cells treated with 50?μM VER A. The differences between results obtained by the micronucleus test and comet assay could be explained by the fact that the micronucleus detects irreversible DNA damage, which is usually correlated with the previously determined cytotoxic potential of the AFB(1) precursors.