Enzyme-modified Comet Assay Research Articles

DNA base oxidation in relation to TNM stages and chemotherapy treatment in colorectal cancer patients 2–9 months post-surgery

Accumulation of DNA damage is a critical feature of genomic instability, which is a hallmark of various cancers. The enzyme-modified comet assay is a recognized method to detect specific DNA lesions at the level of individual cells. In this cross-sectional investigation, we explore possible links between clinicopathological and treatment related factors, nutritional status, physical activity and function, and DNA damage in a cohort of colorectal cancer (CRC) patients with non-metastatic disease. Levels of DNA damage in peripheral mononuclear blood cells (PBMCs) assessed 2–9 months post-surgery, were compared across tumour stage (localized (stage I-II) vs. regional (stage III) disease), localization (colon vs. rectosigmoid/rectum cancer), and adjuvant chemotherapy usage, with the last dosage administrated 2–191 days prior to sampling. Associations between DNA damage and indicators of nutritional status, physical activity and function were also explored. In PBMCs, DNA base oxidation was higher in patients diagnosed with regional compared with localized tumours (P = 0.03), but no difference was seen for DNA strand breaks (P > 0.05). Number of days since last chemotherapy dosage was negatively associated with DNA base oxidation (P < 0.01), and patients recently receiving chemotherapy (<15 days before blood collection) had higher levels of DNA base oxidation than those not receiving chemotherapy (P = 0.03). In the chemotherapy group, higher fat mass (in kg and %) as well as lower physical activity were associated with greater DNA base oxidation (P < 0.05). In conclusion, DNA base oxidation measured with the enzyme-modified comet assay varies according to tumour and lifestyle related factors in CRC patients treated for non-metastatic disease.

Further development of CometChip technology to measure DNA damage in vitro and in vivo: Comparison with the 2 gels/slide format of the standard and enzyme-modified comet assay

DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1–2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer’s protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer’s protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO3, 0–4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer’s protocol), using TK6 cells with MMS (100–800 µM, 1 h) and hydrogen peroxide (H2O2, 7.7–122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required.

Unraveling gene expression and genetic instability in dental fluorosis: Investigating the impact of chronic fluoride exposure

Chronic fluoride exposure, even in small quantities, when continuously ingested by the human population, can lead to a significant public health concern known as fluorosis. Our understanding of the effects of fluoride on human health, as well as its potential to impact DNA, is limited. The present study aimed to assess genetic instability in 20 individuals diagnosed with dental fluorosis and 20 individuals without the condition from the state of Rio Grande do Sul, Brazil. The participants' dental fluorosis was evaluated using the Thylstrup-Fejerskov index (TF). To further evaluate genetic instability, several assays were conducted, including the alkaline and modified (+FPG) comet assay (using a visual score, VS), the buccal micronucleus (MN) cytome (BMCyt) assay, the cytokinesis-block MN (CBMN-Cyt) assay, and the measurement of telomere length (TL). In addition, the study utilized tools from Systems Biology to gain insights into the effects of fluoride exposure on humans, which aided in the selection and evaluation of mRNA expression levels of specific genes, namely PPA1 (inorganic pyrophosphatase 1), AQP5 (Aquaporin 5), and MT-ATP6 (Mitochondrially Encoded Adenosine Triphosphate Synthase Membrane Subunit 6). Furthermore, fluoride levels in the blood and urine were assessed using an ion-selective electrode, along with the evaluation of the inflammatory response in serum. The group with dental fluorosis exhibited 2.18 times higher MN frequencies specifically when assessed using the CBMN-Cyt assay, in comparison with individuals without fluorosis. Findings from the enzyme-modified comet assay indicated oxidative damage to purines in DNA. Furthermore, a decrease in TL was observed, along with elevated expression patterns of the PPA1 and AQP5 genes, and significant alterations in cytokine release. Significant correlations were identified between the TF and age, as well as the levels of necrotic cells. Additionally, noteworthy correlations were established between fluoride levels and the levels of MN, VS, and MT-ATP6. Although dental fluorosis results from fluoride exposure, our research highlights the potential influence of this condition on genomic instability and gene expression. Consequently, our findings stress the importance of continuously monitoring populations with a high incidence of dental fluorosis to enhance our comprehension of how genomic instability might correlate with the origins and consequences of health problems in these individuals.

Mutagenicity and genotoxicity assessment of the emerging mycotoxin Versicolorin A, an Aflatoxin B1 precursor

Aflatoxin B1 (AFB1) is the most potent natural carcinogen among mycotoxins. Versicolorin A (VerA) is a precursor of AFB1 biosynthesis and is structurally related to the latter. Although VerA has already been identified as a genotoxin, data on the toxicity of VerA are still scarce, especially at low concentrations. The SOS/umu and miniaturised version of the Ames test in Salmonella Typhimurium strains used in the present study shows that VerA induces point mutations. This effect, like AFB1, depends primarily on metabolic activation of VerA. VerA also induced chromosomal damage in metabolically competent intestinal cells (IPEC-1) detected by the micronucleus assay. Furthermore, results from the standard and enzyme-modified comet assay confirmed the VerA-mediated DNA damage, and we observed that DNA repair pathways were activated upon exposure to VerA, as indicated by the phosphorylation and/or relocation of relevant DNA-repair biomarkers (γH2AX and 53BP1/FANCD2, respectively). In conclusion, VerA induces DNA damage without affecting cell viability at concentrations as low as 0.03 μM, highlighting the danger associated with VerA exposure and calling for more research on the carcinogenicity of this emerging food contaminant.

Exposure to coal mining can lead to imbalanced levels of inorganic elements and DNA damage in individuals living near open-pit mining sites

Coal mining activities are considered harmful to living organisms. These activities release compounds to the environment, such as polycyclic aromatic hydrocarbons (PAHs), metals, and oxides, which can cause oxidative damage to DNA. In this study, we compared the DNA damage and the chemical composition of peripherical blood of 150 individuals exposed to coal mining residues and 120 non-exposed individuals. Analysis of coal particles revealed the presence of elements such as copper (Cu), aluminum (Al), chrome (Cr), silicon (Si) and iron (Fe). The exposed individuals in our study had significant concentrations of Al, sulfur (S), Cr, Fe, and Cu in their blood, as well as hypokalemia. Results from the enzyme-modified comet assay (FPG enzyme) suggest that exposure to coal mining residues caused oxidative DNA damage, particularly purine damage. Furthermore, particles with a diameter of <2.5 μm indicate that direct inhalation could promote these physiological alterations. Finally, a systems biology analysis was performed to investigate the effects of these elements on DNA damage and oxidative stress pathways. Interestingly, Cu, Cr, Fe, and K are key nodes that intensely modulate these pathways. Our results suggest that understanding the imbalance of inorganic elements caused by exposure to coal mining residues is crucial to understanding their effect on human health.

Different Sensitivity of Advanced Bronchial and Alveolar Mono- and Coculture Models for Hazard Assessment of Nanomaterials.

For the next-generation risk assessment (NGRA) of chemicals and nanomaterials, new approach methodologies (NAMs) are needed for hazard assessment in compliance with the 3R's to reduce, replace and refine animal experiments. This study aimed to establish and characterize an advanced respiratory model consisting of human epithelial bronchial BEAS-2B cells cultivated at the air-liquid interface (ALI), both as monocultures and in cocultures with human endothelial EA.hy926 cells. The performance of the bronchial models was compared to a commonly used alveolar model consisting of A549 in monoculture and in coculture with EA.hy926 cells. The cells were exposed at the ALI to nanosilver (NM-300K) in the VITROCELL® Cloud. After 24 h, cellular viability (alamarBlue assay), inflammatory response (enzyme-linked immunosorbent assay), DNA damage (enzyme-modified comet assay), and chromosomal damage (cytokinesis-block micronucleus assay) were measured. Cytotoxicity and genotoxicity induced by NM-300K were dependent on both the cell types and model, where BEAS-2B in monocultures had the highest sensitivity in terms of cell viability and DNA strand breaks. This study indicates that the four ALI lung models have different sensitivities to NM-300K exposure and brings important knowledge for the further development of advanced 3D respiratory in vitro models for the most reliable human hazard assessment based on NAMs.

Reduced genotoxicity of lignin-derivable replacements to bisphenol A studied using in silico, in vitro, and in vivo methods

Bisguaiacols, lignin‐derivable bisphenols, are considered promising and possibly safer alternatives to bisphenol A (BPA), but comprehensive toxicity investigations are needed to ensure safety. Most toxicity studies of BPA and its analogues have focused on potential estrogenic activity, and only limited toxicological data are available on other toxicity aspects, such as genotoxicity at low exposure levels. In this study, the genotoxicity of six lignin-derivable bisguaiacols with varying regioisomer contents and degrees of methoxy substitution was investigated using a multi-tiered method, consisting of in silico simulations, in vitro Ames tests, and in vivo comet tests. The toxicity estimation software tool, an application that predicts toxicity of chemicals using quantitative structure-activity relationships, calculated that the majority of the lignin-derivable bisguaiacols were non-mutagenic. These results were supported by Ames tests using five tester strains (TA98, TA100, TA102, TA1535, and TA1537) at concentrations ranging from 0.5 pmol/plate to 5 nmol/plate. The potential genotoxicity of bisguaiacols was further evaluated using in vivo comet testing in fetal chicken livers, and in addition to the standard alkaline comet assay, the formamidopyrimidine DNA glycosylase enzyme-modified comet assay was employed to investigate oxidative DNA damage in the liver samples. The oxidative stress analyses indicated that the majority of lignin-derivable analogues showed no signs of mutagenicity (mutagenic index < 1.5) or genotoxicity, in comparison to BPA and bisphenol F, likely due to the methoxy groups on the lignin-derivable aromatics. These findings reinforce the potential of lignin-derivable bisphenols as safer alternatives to BPA.

Biomarkers of DNA Oxidation Products: Links to Exposure and Disease in Public Health Studies.

Environmental exposure can increase the production of reactive oxygen species and deplete cellular antioxidants in humans, resulting in oxidatively generated damage to DNA that is both a useful biomarker of oxidative stress and indicator of carcinogenic hazard. Methods of oxidatively damaged DNA analysis have been developed and used in public health research since the 1990s. Advanced techniques detect specific lesions, but they might not be applicable to complex matrixes (e.g., tissues), small sample volume, and large-scale studies. The most reliable methods are characterized by (1) detecting relevant DNA oxidation products (e.g., premutagenic lesions), (2) not harboring technical problems, (3) being applicable to complex biological mixtures, and (4) having the ability to process a large number of samples in a reasonable period of time. Most effort has been devoted to the measurements of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG), which can be analyzed by chromatographic, enzymic, and antibody-based methods. Results from validation trials have shown that certain chromatographic and enzymic assays (namely the comet assay) are superior techniques. The enzyme-modified comet assay has been popular because it is technically simpler than chromatographic assays. It is widely used in public health studies on environmental exposures such as outdoor air pollution. Validated biomarker assays on oxidatively damaged DNA have been used to fill knowledge gaps between findings in prospective cohort studies and hazards from contemporary sources of air pollution exposures. Results from each of these research fields feed into public health research as approaches to conduct primary prevention of diseases caused by environmental or occupational agents.

Nitrosative stress parameters and the level of oxidized DNA bases in patients with multiple sclerosis.

Multiple sclerosis (MS) is a neurodegenerative disease with various factors affecting its etiology. Overproduction of nitric oxide and subsequent lesions of biopolymers are some of the possible causes of the disease. This study aimed to measure the most relevant nitrosative and oxidative stress biomarkers and the level of modified DNA bases in patients with MS. Each parameter was assayed in 25 patients with MS and 25 healthy controls. This study involved detecting blood plasma and serum nitric oxide metabolites by chemiluminescence detector Sievers NOA-280i, malondialdehyde (MDA) measurements with thiobarbituric acid reactive substance (TBARS) assay, detection of oxidized purines and pyrimidines with the enzyme-modified comet assay. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA) and unpaired t test for the comparison of less than three data sets. DNA single-strand breaks, levels of modified purines and pyrimidines, as well as nitrite and nitrate levels in plasma and serum samples, were significantly higher in patients with MS than in healthy controls. On the contrary, MDA levels appeared to be lower in patients with MS.

Genotoxicity Evaluation of Propyl-Propane-Thiosulfinate (PTS) from Allium genus Essential Oils by a Combination of Micronucleus and Comet Assays in Rats.

Propyl-propanethiosulfinate (PTS) is a component of Allium essential oils. This organosulfur molecule can be used as a feed additive to decrease the appearance of bacterial resistances caused by the residues of antibiotics. In previous in vitro genotoxicity studies, contradictory results were reported for PTS. In this work, the in vivo genotoxicity of PTS in male and female rats was assessed for the first time, following OECD (Organisation for Economic Co-operation and Development) guidelines. After oral administration (doses: 5.5, 17.4, and 55.0 mg/kg PTS body weight), a combination of the micronucleus (MN) assay (OECD 474) in bone marrow and the standard and enzyme-modified comet assay (OECD 489) was performed. After necropsy, histopathological studies were also carried out. The results did not show the in vivo genotoxicity of PTS at any doses assayed, revealed by the absence of increased MN, and DNA strand breaks or oxidative DNA damage in the standard and enzyme-modified comet assays. The histopathological study revealed that only the highest dose tested (55.0 mg/kg) in the liver and all dose groups in the stomach presented minimal pathological lesions in the organs studied. Consequently, the present work confirms that PTS is not genotoxic at the doses assayed, and it is a promising natural alternative to synthetic preservatives and antibiotics in animal feed.

In vitro genotoxicity assessment of functional ingredients: DHA, rutin and α-tocopherol

The in vitro genotoxicity of three compounds widely used as functional ingredients, docosahexaenoic acid (DHA), rutin and α-tocopherol, was assessed. A miniaturized version of the Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of OECD 471), and the in vitro micronucleus test in TK6 cells (OECD 487) were performed. This strategy is recommended by the European Food Safety Authority for the in vitro genotoxicity assessment of food and feed. In addition, this approach was complemented with the in vitro standard and enzyme-modified comet assay (S9-/S9+) using hOGG1, EndoIII and hAAG in order to assess potential premutagenic lesions in TK6 cells. Rutin showed an equivocal response in the in vitro micronucleus test and also was a potent Salmonella typhimurium revertant inductor in the Ames test. DHA showed equivocal results in the in vitro micronucleus test. In this regard, DHA and rutin seemed to interact with the DNA at a chromosomal level, but rutin is also capable of producing frameshift mutations. No genotoxicity was observed in cells treated with α-tocopherol. This article complements the evidence already available about the genotoxicity of these compounds. However, more studies are needed in order to elucidate the consequences of their use as functional ingredients in human health.

In Vivo Genotoxicity Evaluation of a Stilbene Extract Prior to Its Use as a Natural Additive: A Combination of the Micronucleus Test and the Comet Assay.

Genotoxic data of substances that could be used as food additives are required by the European Food Safety Authority. In this sense, the use of an extract from grapevine shoots containing a stilbene richness of 99% (ST-99), due to its antioxidant and antibacterial activities, has been proposed as an alternative to sulfur dioxide in wine. The aim of this work was to study, for the first time, the in vivo genotoxic effects produced in rats orally exposed to 90, 180, or 360 mg ST-99/kg body weight at 0, 24, and 45 h. The combination of micronucleus assay in bone marrow (OECD 474) and standard (OECD 489) and enzyme-modified comet assay was used to determine the genotoxicity on cells isolated from stomach, liver, and blood of exposed animals. The ST-99 revealed no in vivo genotoxicity. These results were corroborated by analytical studies that confirm the presence of stilbenes and their metabolites in plasma and tissues. Moreover, to complete these findings, a histopathological study was performed under light microscopy in liver and stomach showing only slight modifications in both organs at the highest concentration used. The present work confirms that this extract is not genotoxic presenting a good profile for its potential application as a preservative in the wine industry.

In vitro assessment of the mutagenic and genotoxic potential of a pure stilbene extract

Stilbenes are secondary metabolites of great interest produced by many plant species due to their important bioactive properties. These phytochemicals have become of increasing interest in the wine industry as a natural alternative to sulphur dioxide, which has been associated with human health risks. However, there is still little toxicological information on stilbenes and the results thus far have been contradictory. Considering the key role of genotoxicity in risk assessment and the need to offer safe products in the market, the aim of this study was to assess the mutagenic and genotoxic potential of a stilbene extract with 99% purity (ST-99 extract). A complete series of different in vitro tests (Ames test, micronucleus (MN) test, and standard and enzyme-modified comet assays) was performed before its use as a preservative in wines. The ST-99 extract induces a significant increase of binucleated cells with micronuclei only in presence of the metabolic fraction S9 at the highest concentration assayed. Neither the Ames test nor the comet assay revealed the extract's genotoxic potential. Further studies are necessary, including in vivo assays, to ensure consumer safety before it can be used.

Evaluation of the cytotoxic and genotoxic effects of Sida planicaulis Cav extract using human neuroblastoma cell line SH-SY5Y

ABSTRACT Sida planicaulis is a weed thought to have originated in Brazil, where it is present in abundant quantities, but also this plant is also found in south-central Florida, Indian Ocean Islands, and the Pacific Islands. Sida planicaulis produces neurotoxicity that adversely affects livestock breeding with heavy animal losses and consequent negative impact on Brazil’s economy. The aim of this study was to determine the chemical profile, cytotoxic and genotoxic effects of ethanolic extracts of S. planicaulis collected in winter (leaf extract) and summer (leaf extract and leaf + flower extract) using an in vitro model of human neuroblastoma cell line SH-SY5Y. Phytochemical screening demonstrated the presence of alkaloids, flavonoids, and apolar compounds. Rutin, quercetin, and swainsonine were detected by HPLC and GC/MS, respectively. Phosphorus, potassium, iron, and zinc were the inorganic elements found. Extracts produced cytotoxicity at all concentrations tested (7–4,000 μg/ml) as evidenced by the colorimetric assay [3-(4,5-dimethyl-thiazol-2-yl) −2,5-diphenyl-tetrazolium bromide (MTT)]. Based upon the alkaline comet assay extracts were found to induce genotoxicity at concentrations ranging from 0.437 to 7 μg/ml. DNA damage produced by extracts was affirmed using a modified comet assay with the enzymes Endo III and FPG in a concentration dependent manner. Further, enzyme-modified comet assay showed both oxidized purines and pyrimidines, and consequently oxidative stress was related to genomic instability and cell death. Data suggest that low concentrations of ethanolic extracts of S. planicaulis (different seasons) induced increased DNA damage related to oxidative stress and chemical composition.

The genotoxic effects in the leukocytes of workers handling nanocomposite materials

The extensive development of nanotechnologies and nanomaterials poses a number of questions to toxicologists about the potential health risks of exposure to nanoparticles (NP). In this study, we analysed DNA damage in the leukocytes of 20 workers who were long-term exposed (18 ± 10 years) to NP in their working environment. Blood samples were collected in September 2016, before and after a shift, to assess (i) the chronic effects of NP on DNA (pre-shift samples) and (ii) the acute effects of exposure during the shift (the difference between pre- and post-shift samples). The samples from matched controls were taken in parallel with workers before the shift. Leukocytes were isolated from heparinised blood on a Ficoll gradient. The enzyme-modified comet assay (DNA formamido-pyrimidine-glycosylase and endonuclease III) demonstrated a considerable increase of both single- and double-strand breaks in DNA (DNA-SB) and oxidised bases when compared with the controls (2.4× and 2×, respectively). Acute exposure induced a further increase of DNA-SB. The welding and smelting of nanocomposites represented a higher genotoxic risk than milling and grinding of nanocomposite surfaces. Obesity appeared to be a factor contributing to an increased risk of oxidative damage to DNA. The data also indicated a higher susceptibility of males vs. females to NP exposure. The study was repeated in September 2017. The results exhibited similar trend, but the levels of DNA damage in the exposed subjects were lower compared to previous year. This was probably associated with lower exposure to NP in consequence of changes in nanomaterial composition and working operations. The further study involving also monitoring of personal exposures to NP is necessary to identify (i) the main aerosol components responsible for genotoxic effects in workers handling nanocomposites and (ii) the primary cause of gender differences in response to NP action.

Novel approach for the detection of alkylated bases using the enzyme-modified comet assay

The enzyme-modified comet assay is widely used for the detection of oxidized DNA lesions. Here we describe for the first time the use of the human alkyladenine DNA glycosylase (hAAG) for the detection of alkylated bases. hAAG was titrated using untreated and methyl methanesulfonate (MMS)-treated TK-6 cells. The hAAG-modified comet assay was compared to the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay, widely used to detect oxidized lesions but that also detects ring-opened purines derived from some alkylated lesions, using cells treated with potassium bromate (oxidizing agent) or MMS. Moreover, neutral and alkaline lysis conditions were used to determine the nature of detected lesions. When alkaline lysis was employed (condition normally used), the level of hAAG-sensitive sites was higher than the Fpg-sensitive sites in MMS-treated cells and hAAG, unlike Fpg, did not detect oxidized bases. After neutral lysis, Fpg did not detect MMS-induced lesions; however, results obtained with hAAG remained unchanged. As expected, Fpg detected oxidized purines and imidazole ring-opened purines, derived from N7-methylguanines under alkaline conditions. It seems that hAAG detected N7-methylguanines, the ring-opened purines derived at high pH, and 3-methlyladenines. Specificity of hAAG towards different DNA lesions was evaluated using a multiplex oligonucleotide-cleavage assay, confirming the ability of hAAG to detect ethenoadenines and hypoxanthine. The hAAG-modified comet assay is a new tool for the detection of alkylated bases.

The role of the enzyme-modified comet assay in in vivo studies

The in vivo comet assay is an established genotoxicity test, with an OECD test guideline, but in its standard form it measures only DNA strand breaks. Including in the assay an additional step, in which the DNA is incubated with a lesion-specific enzyme, can provide important information about the nature of the DNA damage. Formamidopyrimidine DNA glycosylase, 8-oxoguanine DNA glycosylase or endonuclease III are commonly used in the in vitro genotoxicity test and in human biomonitoring to detect oxidised bases, but in vivo applications are rarer. A systematic literature search has identified a total of 60 papers that report such in vivo experiments, testing a variety of agents. In many cases, strand breaks were not seen, but significant levels of enzyme-sensitive sites were induced - indicating a mechanism of action involving oxidative stress. Compounds such as methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS) could be used as positive controls in both the standard and the enzyme-modified in vivo comet assays.

Cytotoxic and genotoxic evaluation of cotinine using human neuroblastoma cells (SH-SY5Y).

Cotinine is the main metabolite of nicotine, which is metabolized in the liver through a cytochrome P450 enzyme. Different studies point to genetic instability caused by nicotine, such as single and double DNA strand breaks and micronuclei formation, but little is known about the effect of cotinine. Therefore, the present in vitro study assessed the effects of cotinine on cell viability and DNA damage in SH-SY5Y neuroblastoma cells, as well as genotoxicity related to oxidative stress mechanisms. Comparisons with nicotine were also performed. An alkaline comet assay modified by repair endonucleases (FPG, OGG1, and Endo III) was used to detect oxidized nucleobases. SH-SY5Y neuronal cells were cultured under standard conditions and exposed for 3 h to different concentrations of cotinine and nicotine. Cytotoxicity was observed at higher doses of cotinine and nicotine in the MTT assay. In the trypan blue assay, cells showed viability above 80% for both compounds. Alkaline comet assay results demonstrated a significant increase in damage index and frequency for cells treated with cotinine and nicotine, presenting genotoxicity. The results of the enzyme-modified comet assay suggest a DNA oxidative damage induced by nicotine. Unlike other studies, our results demonstrated genotoxicity induced by both cotinine and nicotine. The similar effects observed for these two pyridine alkaloids may be due to the similarity of their structures.

Measurement of DNA damage with the comet assay in high-prevalence diseases: current status and future directions.

The comet assay is widely used in studies on genotoxicity testing, human biomonitoring and clinical studies. The simple version of the assay detects a mixture of DNA strand breaks and alkali-labile sites; these lesions are typically described as DNA strand breaks to distinguish them from oxidatively damaged DNA that are measured with the enzyme-modified comet assay. This review assesses the association between high-prevalence diseases in high-income countries and DNA damage measured with the comet assay in humans. The majority of case-control studies have assessed genotoxicity in white blood cells. Patients with coronary artery disease, diabetes, kidney disease, chronic obstructive pulmonary disease and Alzheimer's disease have on average 2-fold higher levels of DNA strand breaks compared with healthy controls. Patients with coronary artery disease, diabetes, kidney disease and chronic obstructive pulmonary disease also have 2- to 3-fold higher levels of oxidatively damaged DNA in white blood cells than controls, although there is not a clear difference in DNA damage levels between the different diseases. Case-control studies have shown elevated levels of DNA strand breaks in patients with breast cancer, whereas there are only few studies on colorectal and lung cancers. At present, it is not possible to assess if these neoplastic diseases are associated with a different level of DNA damage compared with non-neoplastic diseases.

In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin.

The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04–2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays. The exposure periods ranged between 4 and 72 h depending on the assay. The genotoxicity of the mixture was observed only in the MN test with S9 metabolic fraction, similar to the results previously reported for CYN individually. These results indicate that cyanobacterial mixtures require a specific (geno)toxicity evaluation as their effects cannot be extrapolated from those of the individual cyanotoxins.