Subchronic toxic effects of tributyltin (TBT) and inorganic lead (PbII) in rats

This study evaluated the toxic effects of two doses of inorganic lead (PbII) and tributyltin (TBT), separately and together in different exposure times (30 and 60 days) in rats. After exposure, liver, kidney, brain and blood were sampled for histopathological, hematological and enzymatic analysis. The number of peritoneal cells and lipopolysaccharide (LPS)-induced neutrophil migration after exposure was also analyzed. The liver presented necrotic areas in all exposed individuals while hematological and enzymatic parameters showed no changes. TBT, but not PbII, reduced the number of resident peritoneal macrophages. The combination of both toxicants abolished TBT effects at lower doses and even increased the number of macrophages at higher doses. The neutrophil migration was increased by lead and lead associated with TBT. These results confirm the potential hepatotoxicity of these compounds and they may have antagonistic effects on the immune cells when administered alone. The combination of toxicants induced an increased inflammatory response suggesting that lead effects may prevail over TBT reduction on macrophage number.     

Co-exposure of iron oxide nanoparticles and glyphosate-based herbicide induces DNA damage and mutagenic effects in the guppy (Poecilia reticulata)

Iron oxide nanoparticles (IONPs) have been tested to remediate aquatic environments polluted by chemicals, such as pesticides. However, their interactive effects on aquatic organisms remain unknown. This study aimed to investigate the genotoxicity and mutagenicity of co-exposure of IONPs (γ-Fe₂O₃ NPs) and glyphosate-based herbicide (GBH) in the fish Poecilia reticulata. Thus, fish were exposed to citrate-functionalized γ-Fe₂O₃ NPs (0.3 mg L⁻¹; 5.44 nm) alone or co-exposed to γ-Fe₂O₃ NPs (0.3 mg L⁻¹) and GBH (65 and 130 μg of glyphosate L⁻¹) during 14 and 21 days. The genotoxicity (DNA damage) was analyzed by comet assay, while the mutagenicity evaluated by micronucleus test (MN test) and erythrocyte nuclear abnormalities (ENA) frequency. The co-exposure induced clastogenic (DNA damage) and aneugenic (nuclear alterations) effects on guppies in a time-dependent pattern. Fish co-exposed to NPs and GBH (130 μg glyphosate L⁻¹) showed high DNA damage when compared to NPs alone and control group, indicating synergic effects after 21 days of exposure. However, mutagenic effects (ENA) were observed in the exposure groups after 14 and 21 days. Results showed the potential genotoxic and mutagenic effects of maghemite NPs and GBH co-exposure to freshwater fish. The transformation and interaction of iron oxide nanoparticles with other pollutants, as herbicides, in the aquatic systems are critical factors in the environmental risk assessment of metal-based NPs.     

Ultrastructural studies on the gills of Palaemon serratus (Pennant) in relation to cadmium accumulation

The accumulation of cadmium in the gills of the common shrimp, Palaemon serratus (Pennant) (Crustacea, Decapoda), and the ultrastructural changes in the gill cells were examined after exposure to three cadmium concentrations (5, 25 and 50 ppm). The exposure period was 44 h, which is the LT₅₀ (lethal time for 50% of specimens) in 50 ppm solution at 15°C in 30‰ artificial sea water. Accumulation of cadmium was found to be about 1500% higher in 50 ppm solution compared with the control specimens. The fine structure of the gill cells of P. serratus was mainly affected by cadmium ions at 50 ppm, mitochondria being the most affected organelle. The pinocytotic process and the membrane system involved in osmoregulation were also impaired.     

Tissue-specific genotoxicity and antioxidant imbalance of titanium dioxide nanoparticles (NPTiO2) and inorganic lead (PbII) in a neotropical fish species

The aquatic environment is the major recipient of wastes containing nanoparticles and other contaminants. Titanium dioxide nanoparticles (NPTiO₂) are one of the most produced and used nanoparticle worldwide. This study investigated the toxicity of NPTiO₂, as well as the toxicity interaction between NPTiO₂ and lead (Pb), in response to genetic and biochemical biomarkers using freshwater fish Rhamdia quelen, as an animal model. The results showed genotoxicity in blood and kidney tissues. No effect of NPTiO₂ alone or in co-exposure with Pb on liver genotoxicity were observed. Alterations in the antioxidant hepatic enzymes activities, as well as alterations in glutathione levels indicated that NPTiO₂ alone or in co-exposure with Pb can cause antioxidant imbalance. The lipid peroxidation was also raised after exposure to NPTiO₂. In general, the results of this study indicated that both NPTiO₂ alone and their co-exposure with Pb are capable of producing significant toxic effects in short-term exposure.     

Lead concentration in plasma as a biomarker of exposure and risk,and modification of toxicity by δ-aminolevulinic acid dehydratase gene polymorphism

Blood lead concentration (B-Pb), the main biomarker of lead exposure and risk, is curvi-linearily related to exposure. We assessed plasma lead (P-Pb) as a marker for both lead exposure and toxic effects. We examined claims that δ-aminolevulinic acid dehydratase genotype (ALAD) can modify lead toxicity. In 290 lead-exposed and 91 unexposed Chinese workers, we determined P-Pb, B-Pb, urinary lead (U-Pb), ALAD polymorphism (rs1800435, ALAD1/2; TaqMan assay), and also toxic effects on heme synthesis (blood zinc protoporphyrin and hemoglobin, urinary δ-aminolevulic acid), on the kidneys (urinary albumin, β₂-microglobulin and N-acetyl-β-d-glucosaminidase) and on the peripheral nervous system (sensory and motor conduction velocities).     

Carboxymethyl chitin-glucan (CM-CG) protects human HepG2 and HeLa cells against oxidative DNA lesions and stimulates DNA repair of lesions induced by alkylating agents

A large number of functional foods, including those that contain β-d-glucans, have been shown to prevent human DNA against genotoxic effects and associated development of cancer and other chronic diseases. In this paper, carboxymethyl chitin-glucan (CM-CG) isolated from Aspergillus niger was investigated from two standpoints: (1) DNA-protective effects against oxidative DNA damage induced by H₂O₂ and alkylating DNA damage induced by MMS and MNNG, and (2) a potential effect on rejoining of MMS- and MNNG-induced single strand DNA breaks. The results obtained by the comet assay in human cells cultured in vitro showed that CM-CG reduced significantly the level of oxidative DNA lesions induced by H₂O₂ but did not change the level of alkylating DNA lesions induced by MMS or MNNG. On the other side, the efficiency of DNA-rejoining of single strand DNA breaks induced by MMS and MNNG was significantly higher in HepG2 cells pre-treated with CM-CG. The antioxidative activity of carboxymethyl chitin-glucan was confirmed by the DPPH assay.     

Protective effects of ebselen (Ebs) and para-aminosalicylic acid (PAS) against manganese (Mn)-induced neurotoxicity

Chronic, excessive exposure to manganese (Mn) may induce neurotoxicity and cause an irreversible brain disease, referred to as manganism. Efficacious therapies for the treatment of Mn are lacking, mandating the development of new interventions. The purpose of the present study was to investigate the efficacy of ebselen (Ebs) and para-aminosalicylic acid (PAS) in attenuating the neurotoxic effects of Mn in an in vivo rat model. Exposure biomarkers, inflammatory and oxidative stress biomarkers, as well as behavioral parameters were evaluated. Co-treatment with Mn plus Ebs or Mn plus PAS caused a significant decrease in blood and brain Mn concentrations (compared to rats treated with Mn alone), concomitant with reduced brain E₂ prostaglandin (PGE₂) and enhanced brain glutathione (GSH) levels, decreased serum prolactin (PRL) levels, and increased ambulation and rearing activities. Taken together, these results establish that both PAS and Ebs are efficacious in reducing Mn body burden, neuroinflammation, oxidative stress and locomotor activity impairments in a rat model of Mn-induced toxicity.     

Protective effect of N-acetylcysteine against DNA damage and S-phase arrest induced by ochratoxin A in human embryonic kidney cells (HEK-293)

N-acetylcysteine (NAC) has recently gained particular interest as a beneficial antioxidant. This study investigated the protective effects of NAC against ochratoxin A (OTA)-induced DNA damage and S-phase arrest in human embryonic kidney cells (HEK-293). OTA exposure results in nephrotoxicity, hepatotoxicity as well as immunotoxicity; and, in the present study, the toxicity of OTA toward HEK-293 cells was explored by analyzing the involvement of the oxidative pathway. It was found that OTA treatment led to oxidative damage; meanwhile, OTA treatment induced significant DNA damage and S-phase arrest by down-regulating cyclin A2, cyclin E1, and CDK2 expression. However, NAC pretreatment alleviated OTA-induced ROS overproduction, the loss of mitochondrial membrane potential (ΔΨ_m), and the decrease in superoxide dismutase (SOD) activity. NAC pretreatment was also discovered to attenuate OTA-induced DNA damage using the comet assay and by determining the expression of γ-H2AX. In addition, NAC pretreatment partly ameliorated OTA-induced S-phase arrest by preventing the down-regulation of cyclin A2, cyclin E1 and CDK2 expression in HEK-293 cells. All of these results demonstrated that oxidative damage was involved in OTA-induced DNA damage and cell cycle arrest in HEK-293 cells. Therefore, NAC has the potential to reverse the DNA damage and S-phase arrest induced by OTA.     

Cytotoxic,genotoxic, and neurotoxic effects of Mg,Pb, and Fe on pheochromocytoma (PC‐12) cells

Metals such as lead (Pb), magnesium (Mg), and iron (Fe) are ubiquitous in the environment as a result of natural occurrence and anthropogenic activities. Although Mg, Fe, and others are considered essential elements, high level of exposure has been associated with severe adverse health effects including cardiovascular, hematological, nephrotoxic, hepatotoxic, and neurologic abnormalities in humans. In the present study we hypothesized that Mg, Pb, and Fe are cytotoxic, genotoxic and neurotoxic, and their toxicity is mediated through oxidative stress and alteration in protein expression. To test the hypothesis, we used the pheochromocytoma (PC‐12) cell line as a neuro cell model and performed the LDH assay for cell viability, Comet assay for DNA damage, Western blot for oxidative stress, and HPLC‐MS to assess the concentration levels of neurological biomarkers such as glutamate, dopamine (DA), and 3‐methoxytyramine (3‐MT). The results of this study clearly show that Mg, Pb, and Fe, respectively in the form of MgSO₄, Pb(NO₃)₂, FeCl₂, and FeCl₃ induce cytotoxicity, oxidative stress, and genotoxicity in PC‐12 cells. In addition, exposure to these metallic compounds caused significant changes in the concentration levels of glutamate, dopamine, and 3‐MT in PC‐12 cells. Taken together the findings suggest that MgSO₄, Pb(NO₃)₂, FeCl₂, and FeCl₃ have the potential to induce substantial toxicity to PC‐12 cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1445–1458, 2015.     

Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents

Titanium dioxide nanoparticles (TiO₂-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1–50?μg/mL TiO₂-NPs, 86% anatase/14% rutile, 24?±?6?nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO₂-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO₂-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO₂-NPs.     

Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium

Cytotoxicity and genotoxicity of nitrogen dioxide (NO₂) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO₂ in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract.Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO₂, 0.1 ppm NO₂, 1 ppm NO₂, 10 ppm NO₂ and synthetic air for half an hour. After exposure, genotoxicity was evaluated by the alkaline single-cell microgel electophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively.The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO₂ in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO₂ in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.     

Genotoxicity of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™)

The genotoxic potential of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™) was evaluated in a battery of genotoxicity tests. The results of the bacterial mutation assay (Ames test) were negative. Weak positive results were obtained in 2 separate in vitro chromosomal aberration test in Chinese hamster lung (CHL) fibroblasts. Upon testing in an in vitro chromosomal aberration test in human peripheral blood lymphocytes, no genotoxic activity of PQQ was noted. In the in vivo micronucleus assay in mice, PQQ at doses up to 2000 mg/kg body weight demonstrated that no genotoxic effects are expressed in vivo in bone marrow erythrocytes. The weak responses in the in vitro test CHL cells were considered of little relevance under conditions of likely human exposure. PQQ disodium was concluded to have no genotoxic activity in vivo.     

Oxidative DNA damage is involved in ochratoxin A-induced G2 arrest through ataxia telangiectasia-mutated (ATM) pathways in human gastric epithelium GES-1 cells in vitro

Ochratoxin A (OTA), one of the most abundant mycotoxin food contaminants, is classified as “possibly carcinogenic to humans.” Our previous study showed that OTA could induce a G₂ arrest in immortalized human gastric epithelium cells (GES-1). To explore the putative roles of oxidative DNA damage and the ataxia telangiectasia-mutated (ATM) pathways on the OTA-induced G₂ arrest, the current study systematically evaluated the roles of reactive oxygen species (ROS) production, DNA damage, and ATM-dependent pathway activation on the OTA-induced G₂ phase arrest in GES-1 cells. The results showed that OTA exposure elevated intracellular ROS production, which directly induced DNA damage and increased the levels of 8-OHdG and DNA double-strand breaks (DSBs). In addition, it was found that OTA treatment induced the phosphorylation of the ATM protein, as well as its downstream molecules Chk2 and p53, in response to DNA DSBs. Inhibition of ATM by the pharmacological inhibitor caffeine or siRNA effectively prevented the activation of ATM-dependent pathways and rescued the G₂ arrest elicited by OTA. Finally, pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) reduced the OTA-induced DNA DSBs, ATM phosphorylation, and G₂ arrest. In conclusion, the results of this study suggested that OTA-induced oxidative DNA damage triggered the ATM-dependent pathways, which ultimately elicited a G₂ arrest in GES-1 cells.     

Ex vivo toxicity of nitrogen dioxide in human nasal epithelium at the WHO defined 1-h limit value

Current pollution limits indicating potential harm to human health caused by nitrogen dioxide have prompted a variety of studies on the cytotoxicity and genotoxicity of nitrogen dioxide (NO₂) in vitro. The present study focuses on toxic effects of NO₂ at the WHO defined 1-h limit value of 200 μg NO₂/m³ air, equivalent to 0.1 ppm NO₂.Nasal epithelial mucosa cells of 10 patients were cultured as an air-liquid interface and exposed to 0.1 ppm NO₂ for 0.5 h, 1 h, 2 h and 3 h and synthetic air as negative control. After exposure, analysis of genotoxicity was performed by the alkaline single cell microgel electrophoresis (comet) assay and by the micronucleus test. Depression of proliferation and cytotoxic effects were checked by the micronucleus assay and the trypan blue exclusion assay.The experiments demonstrated significant DNA fragmentation even at the shortest exposure duration of half an hour in the comet assay. The amount of DNA fragmentation significantly increased with extended NO₂ exposure durations. The amount of DNA fragmentation increased with extended exposure durations to synthetic air at a significantly lower level as compared to NO₂ exposure. Micronucleus inductions were seen only at the longest exposure duration of 3 h. There were no changes in proliferation seen in the micronucleus assay under any experimental setup. Moreover, no signs of necrosis, apoptosis or changes in viability were detected. Data demonstrate genotoxicity of NO₂ at concentrations found in the urban atmosphere during short exposure durations. DNA alterations in the micronucleus assay at an exposure time of 3 h indicate a significant DNA alteration possibly being hazardous to humans.     

Genotoxic effects of the water-soluble fraction of heavy oil in the brackish/freshwater amphipod Quadrivisio aff. lutzi (Gammaridea) as assessed using the comet assay

Amphipod crustaceans have been widely used as invertebrate models in ecotoxicology due to their importance in the food chain. However, few studies have evaluated the genotoxic effects of pollutants in this model using the comet assay. The main obstacle to using amphipods in the comet assay is the difficulty in obtaining enough blood cells from a single individual. In this study, we evaluated the genotoxic effects of the water-soluble fraction (WSF) of heavy oil on the brackish/freshwater amphipod Quadrivisio aff. lutzi, which is common in the coastal lagoons of southeastern Brazil, using hemocytes obtained from single amphipods (without pooling) after optimizing hemolymph extraction. The comet assay revealed significantly higher DNA damage levels (2- to 6-fold higher) in treated amphipods compared to untreated ones with a sublethal concentration of 17.6 % of the WSF within 72 h of treatment. Two independent experiments confirmed an “up and down” pattern of DNA damage, measured as the % of DNA contained in the tail of the comets. Elevations in DNA damage levels were observed at the 6 and 48 h time points, while very low levels of DNA damage were observed at the 24 and 72 h time points. Furthermore, the comet assay revealed gender variability in the levels of DNA damage after short-term exposure.     

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.     

Isochaihulactone protects PC12 cell against H(2)O(2) induced oxidative stress and exerts the potent anti-aging effects in D-galactose aging mouse model

Aim:

To investigate the effect of isochaihulactone (also known as K8), a lignan compound of Bupleurum scorzonerifolium, on H₂O₂-induced cytotoxicity in neuronally differentiated PC12 cells (nPC12).

Methods:

Viability of neuronal PC12 cells was measured using MTT assay. Protein expression was determined by Western blot. Apoptotic cells was determined using TUNEL assay. D-galactose aging mice were used as a model system to study the anti-oxidant effects of isochaihulactone in vivo.

Results:

Pretreatment with isochaihulactone (5–10 μmol/L) increased cell viability and decreased membrane damage, generation of reactive oxygen species and degradation of poly (ADP-ribose) polymerase in H₂O₂-treated nPC12 cells and also decreased the expression of cyclooxygenase-2, via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. The results suggest that isochaihulactone is a potential antioxidant agent. In a murine aging model, in which chronic systemic exposure to D-galactose (D-gal) causes the acceleration of senescence, administration of isochaihulactone (10 mg·kg^-1·d^-1, sc) for 7 weeks concomitant with D-gal injection significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, H&E staining to quantify cell death within hippocampus showed that percentage of pyknotic nuclei in the D-gal-treated mice were much higher than in control.

Conclusion:

The results suggest that isochaihulactone exerts potent anti-aging effects against D-gal in mice possibly via antioxidative mechanisms.     

DETECTION OF POLY(ADP-RIBOSE) SYNTHETASE ACTIVITY IN ALVEOLAR MACROPHAGES OF RATS EXPOSED TO NITROGEN DIOXIDE AND OZONE

The toxic effects of nitrogen dioxide (NO₂) and ozone (O₃) are mediated through the formation of free radicals, which can cause DNA strand breaks. The present study demonstrates that exposure to NO₂ and O₃ causes a stimulation of poly (ADP-ribose) (poly ADPR) synthetase in alveolar macrophages of rats. Three-month-old male Sprague-Dawley rats, specific pathogen free, were exposed to either 1.2 ppm NO₂ or 0.3 ppm O₃ alone or a combination of these 2 oxidants continuously for 3 days. The control group was exposed to filtered room air. To evaluate whether exposure to these two oxidants (NO₂ and O₃) caused DNA damage to lung cells, the activity of poly ADPR synthetase was measured. Cellular DNA repair is dependent upon the formation of poly (ADP-ribose) polymerase, which is catalyzed by poly ADPR synthetase. Poly ADPR synthetase is known to be activated in response of DNA damage. The results showed that the enzyme activity was stimulated after exposure to O₃ or exposure to NO₂ + O₃. Ozone exposure caused a 25% increase in the enzyme activity as compared to the control. Combined exposure to NO₂ + O₃ showed a 53% increase in the enzyme activity. These results were statistically significant as compared to the control and NO₂ exposure groups. Other parameters such as total cell count, cell viability, and differential cell count were also determined. The stimulation of poly ADPR synthetase activity after O₃ exposure or NO₂ + O₃ exposure reflects a response to lung cellular DNA repair, which may be used as an indicator for assessing DNA damage caused by oxidant injury.     

Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using comet assay and alkaline unwinding assay

This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl₂) on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 µg/L) of CdCl₂. In vitro exposure of hydrogen peroxide (H₂O₂; 1, 10, 25, and 50 µM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl₂ exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 µg/L of CdCl₂ produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl₂ (10, 25, 50, and 75 µg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 177–187, 2015.