Significant Increase In DNA Damage Research Articles

Aldosterone induces oxidative stress, oxidative DNA damage and NF-κB-activation in kidney tubule cells

An increase of the mineralocorticoid aldosterone is induced by a stimulated renin-angiotensin system in a subgroup of hypertensive patients. Epidemiological studies find higher cancer mortality in hypertensive patients and an increased risk to develop kidney cancer. This work investigated the involvement of oxidants in the genotoxicity of aldosterone and on a potential activation of transcription factor nuclear factor-κB (NF-κB) in kidney tubule cells. Aldosterone, at concentrations as low as 1 nM caused a significant increase of DNA damage, as assessed by comet assay and micronucleus frequency test. Aldosterone also led to a dose-dependent activation of NF-κB. Time courses of DNA damage and NF-κB-activation showed that these effects already occurred after 5 and 3 min of aldosterone exposure, respectively, suggesting non-genomic events of the hormone. Antioxidants prevented aldosterone-induced DNA damage and NF-κB-activation, indicating the involvement of oxidants. In fact, aldosterone caused an increase in intracellular oxidant levels, and in particular of superoxide anions. As a consequence, increased levels of the oxidized DNA modification 7,8-dihydro-8-oxo-guanine were observed in aldosterone-treated kidney cells. Aldosterone-induced DNA damage and NF-κB-activation was dependent on the involvement of the mineralocorticoid receptor. The induction of oxidant-mediated genotoxic effects, and of a long-term activation of the potentially oncogenic cell signal NF-κB by aldosterone could contribute to the increased kidney cancer incidence in hypertensive patients.

The evaluation of the genotoxicity of two food preservatives: Sodium benzoate and potassium benzoate

In this study, the genotoxic effects of sodium benzoate (SB) and potassium benzoate (PB) were investigated in cultured human peripheral lymphocytes using chromosomal aberrations (CA), sister chromatid exchange (SCE), and micronuclei (MN). The level of nuclear DNA damage of SB and PB were also evaluated using the comet assay. The lymphocytes were incubated with different concentrations of SB (6.25, 12.5, 25, 50, and 100 μg/ml) and PB (62.5, 125, 250, 500, and 1000 μg/ml). A significant increase was observed in CA, SCE, and MN, in almost all treatments compared to negative controls. SB and PB significantly decreased the mitotic index (MI) in all the treatments, compared to the negative controls. However, neither of the additives affected the replication index (RI). Although SB significantly increased DNA damage, PB did not cause a significant increase in DNA damage. The present results indicate that SB and PB are clastogenic, mutagenic and cytotoxic to human lymphocytes in vitro.

Determination of genotoxicity of Fenaminosulf by Allium and Comet tests

Genotoxic effects of Fenaminosulf, fungicide and micro-biocide, were examined by using mitotic index (MI), mitotic phase, and Comet assay on the root meristem cells of Allium cepa. In the Allium root growth inhibition test, EC 50 value was firstly determined as 25 ppm and, 12.5 (0.5 × EC 50), 25 (EC 50) and 50 (2 × EC 50) ppm concentrations of Fenaminosulf were introduced to onion tuber roots. Distilled water was used as a negative control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance (ANOVA) were employed and p < 0.05 was accepted as significant value. While MI (except in 24 h at 12.5 and 50 ppm) and prophase index increased, metaphase, anaphase and telophase indexes decreased in all concentrations compared to control at each exposure time. A significant increase in DNA damage was also observed at the concentration of 25 ppm in 24 h, 25 and 50 ppm in 96 h by Comet assay.

Effects of ultraviolet A on the activity of two metabolic enzymes, DNA damage and lipid peroxidation during early developmental stages of the African catfish, Clarias gariepinus (Burchell, 1822).

Many ultraviolet-A (UVA)-induced biochemical and physiological changes are valid as biomarkers using aquatic species for detection of the degree of stress. Changes in the concentration and activities of enzymes, such as glucose-6-phosphate dehyderogenase (G6PDH), lactate dehyderogenase (LDH), DNA damage and lipid peroxidation (LPO), can be used as biomarkers to identify possible environmental contamination in fish. This study aimed to investigate the impact of UVA on the activity of the selected enzymes, DNA damage and LPO during early developmental stages of the African catfish Clarias gariepinus. Embryo hemogenates were used for measurements of G6PDH, LDH, DNA damage and LPO concentrations and activities spectrophotometrically at 37°C. The normal ontogenetic variations in enzyme activities, DNA damage and LPO of the early developmental stages (24–168 h-PFS; hours-post fertilization stage) were studied. There was a significant decrease in the activity of G6PDH till 120 h-PFS. Then after 120 h-PFS, the activity of such enzymes insignificantly increased toward higher stages. The LDH activity was recorded with a pattern of decrease till 96 h-PFS, followed by a significant increase toward 168 h-PFS. The polynomial pattern of variations in DNA damage and LPO was also evident. The patterns of the enzyme activities, corresponding DNA damage and LPO of the early ontogenetic stages under the influence of three different UVA doses (15, 30 and 60 min), were recorded. The pattern of variations in G6PDH activity in UVA-induced groups was similar to that of the control group with variation in the magnitude of such activity. In all treated groups, LDH activity decreased till 96 h-PFS, then increased till 168 h-PFS. Within each of the embryonic stages, the increase in UVA led to a significant increase in DNA damage. A significant increase in lipid peroxidation under UVA doses was recorded. The variability in number and molecular weight of proteins under exposure to UVA was evident, reflecting some of the genetic and transcriptional changes during exposure and development.

Proliferation Inhibition, DNA Damage, and Cell-Cycle Arrest of Human Astrocytoma Cells after Acrylamide Exposure

Acrylamide (ACR) has been recognized as a neurological and reproductive toxin in humans and laboratory animals. This study aimed to determine the effects of ACR-induced DNA damage on cell cycle regulation in human astrocytoma cell lines. Treatment of U-1240 MG cells with 2 mM ACR for 48 h resulted in a significant inhibition of cell proliferation as evaluated by Ki-67 protein expression and MTT assay. The analysis of DNA damage with the comet assay showed that treatment of the cells with 0.5, 1, and 2 mM ACR for 48 h caused significant increases in DNA damage by 3.5-, 4-, and 14-fold, respectively. Meanwhile, analysis of cell-cycle arrest with flow cytometry revealed that the ACR treatments resulted in significant increases in the G(0)/G(1)-arrested cells in a time- and dose-dependent manner. Expression of DNA damage-associated/checkpoint-related signaling molecules, including phosphorylated-p53 (pp53), p53, p21, p27, Cdk2, and cyclin D(1), in three human astrocytoma cell lines (U-1240 MG, U-251 MG, and U-87 MG) was also analyzed by immunoblotting. Treatment of the three cell lines with 2 mM ACR for 48 h caused marked increases in pp53 and Cdk2, as well as decreases in cyclin D(1) and p27. Moreover, increases in p53 and p21 were detected in both U-1240 and U-87 MG cells, whereas no marked change in p53 and a decrease in p21 were observed in U-251 MG cells. To address the involvement of ataxia telangiectasia mutated/ATM-Rad3-related (ATM/ATR) kinase in the signaling of ACR-induced G(0)/G(1) arrest, caffeine was used to block the ATM/ATR pathway in U-1240 MG cells. Caffeine significantly attenuated the ACR-induced G(0)/G(1) arrest as well as the expression of DNA damage-associated/checkpoint-related signaling molecules in a dose-dependent manner. This in vitro study clearly demonstrates the critical role of ATM/ATR in the signaling of ACR-induced cell-cycle arrest in astrocytoma cells.

Effects of Dermal Exposure to Nicotiana tabacum (Jean Nicot, 1560) Leaves in Mouse Evaluated by Multiple Methods and Tissues

Tobacco farmers are routinely exposed to complex mixtures of the compounds present in tobacco leaves, including organic and inorganic pesticides. Penetration through skin is the most significant route of uptake in occupational exposure to chemicals, including dust and liquids containing toxic and carcinogenic substances. This study evaluates the genotoxic effect of tobacco leaves with and without dermal exposure to flumetralin in Mus musculus, determining cell damage by the micronucleus test and the Comet assay as well as antioxidant enzyme activities and hematologic parameters. Nicotine was used as positive control. Blood samples were collected for 0, 3, 24 and 48 h exposure periods, and DNA damage by Comet assay and micronucleus test was evaluated for all these periods. Bone marrow and liver cells were also evaluated for the 48 h exposure period. Significant differences between Comet assay results in blood cells from animals exposed to tobacco leaves with and without pesticide were found in 24 and 48 h exposure periods in relation to negative control. Bone marrow cells from the group exposed to leaves with pesticide (48 h) also demonstrated significant increase in DNA damage. Concerning the micronucleus test, only animals exposed to tobacco leaves without pesticide (24 h) showed increase in frequency of micronuclei when compared to the negative control. Oxidative stress activities also were demonstrated for different groups. The results demonstrate the injury effect caused by tobacco leaves in different Mus musculus tissues, suggesting that the effects of dermal exposure to tobacco leaves are caused by complex mixtures present in the plant, but mainly by nicotine.

Genotoxic effects of irinotecan combined with the novel platinum(II) complexes in human cancer cells

Combination chemotherapy has become increasingly important as synergistic drugs enable to achieve therapeutic effects at substantially lower doses and limited spectrum of side effects. Irinotecan as a one of the camptothecin analogues has shown a broad spectrum of antitumor activity against various malignancies. It is commonly used in treatment of gastrointestinal and pulmonary cancer. In this study were measured the effect of the novel platinum(II) complexes: cis-[PtCl 2(4-pmOpe) 2] and trans-[PtCl 2(4-pmOpe) 2], with diethyl (pyridine-4-ylmethyl)phosphates (4-pmOpe) as non-leaving ligands, on genotoxicity of irinotecan in human cancer cells. Irinotecan showed genotoxic activity in combination with the new platinum(II) derivatives in cancer cells. Combination of irinotecan with cis-[PtCl 2(4-pmOpe) 2] or trans-[PtCl 2(4-pmOpe) 2] resulted in significant increase in DNA damage in A549 and HT29 cells when compared to effects of irinotecan or platinum(II) complexes used separately. The highest degree of DNA migration in the comet tails was found after the cancer cells were treated with irinotecan and trans-[PtCl 2(4-pmOpe) 2]. In addition, the analysis of DNA repair revealed that irinotecan in combination with trans-[PtCl 2(4-pmOpe) 2] induced unrepairable DNA damage suggesting the poor recognition of the damage by HMG proteins and other repair mechanisms. Thus, genotoxicity of irinotecan was modulated by the structurally different DNA–platinum adducts formed by novel platinum(II) complexes. These data suggest that the use of irinotecan with novel platinum(II) complexes may represent a new strategy for pharmacotherapy in cancer.

Comparative studies on the genotoxicity and cytotoxicity of polymeric gene carriers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer in Jurkat T-cells

A safe alternative to the viral system used in gene therapy is a nonviral gene delivery system. Although polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer are among the most promising gene-carrier candidates for efficient nonviral gene delivery, safety concerns regarding their toxicity remain. The aim of this study was to scrutinize the underlying mechanism of the cytotoxicity and genotoxicity of PEI (25 kDa) and PAMAM (G4). To our knowledge, this is the first study to explore the genotoxic effect of polymeric gene carriers. To evaluate cell death by PEI and PAMAM, we performed propidium-iodide staining and lactate-dehydrogenase release assays. The genotoxicity of the polymers was measured by comet assay and cytokinesis-block micronucleus assay. PEI- and PAMAM-treated groups induced both necrotic and apoptotic cell death. In the comet assay and micronuclei formation, significant increases in DNA damage were observed in both treatments. We conclude that PEI and PAMAM dendrimer can induce not only a relatively weak apoptotic and a strong necrotic effect, but also a moderate genotoxic effect.

Chlorpyrifos induces apoptosis and DNA damage in Drosophila through generation of reactive oxygen species

The present study investigated the apoptosis and DNA damage inducing potential of chlorpyrifos (CP) in Drosophila melanogaster. Third instar larvae of Drosophila were treated with different concentrations of CP (0.015–15.0μg/L) for 2–48h. Reactive oxygen species (ROS) generation, oxidative stress markers, DNA damage and apoptotic cell death end points were measured in them. A significant increase in DNA damage was concomitant with apoptotic mode of cell death in 15.0μg/L CP-treated organisms for 24 and 48h. Depolarization in mitochondrial membrane potential and increased casapase-3 and caspase-9 activities in these organisms indicated both as potential targets of CP. A significant positive correlation was observed among ROS generation, apoptosis and DNA damage. The study suggests that (i) ROS may be involved in inducing apoptosis and DNA damage in the CP-exposed larvae of Drosophila and (ii) D. melanogaster may be used as an alternative in vivo animal model for xenobiotics hazard assessment.

Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells

Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process.

Potential role of melatonin in DNA damage caused by nitrosourea-induced mammary carcinogenesis

Mammary carcinogenesis was induced in female Sprague-Dawley rats by exposure to N-methyl-N-nitrosourea (NMU). Animals were kept under constant light conditions to arrest endogenous melatonin synthesis and were fed the same melatonin dosage, since nitrosourea exposure may also induce cellular injury, especially with extensive proliferative activity. The pro-apoptotic effects of the biogenic amine, melatonin, on rat whole blood leukocytes were assessed by alkaline single cell gel electrophoresis (comet) assay. Potential induction of stress due to animal immobilization and its additional effect on DNA damage was studied. The parameters relevant to the degree of DNA damage in groups with chemocarcinogen treatment demonstrated no significant effects as a result of the immobilization. A significant increase in DNA damage after melatonin treatment in NMU-induced carcinogenesis confirms its involvement in the activation of apoptosis.

The Effects of Arsenic Trioxide on DNA Synthesis and Genotoxicity in Human Colon Cancer Cells

Colon cancer is the third leading cause of cancer-related deaths worldwide. Recent studies in our laboratory have demonstrated that arsenic trioxide is cytotoxic in human colon cancer (HT-29), lung (A549) and breast (MCF-7) carcinoma cells. The purpose of the present study is to investigate the effects of arsenic trioxide on DNA synthesis and the possible genotoxic effects on human colon cancer cells. HT-29 cells were cultured according to standard protocol, followed by exposure to various doses (0, 2, 4, 6, 8, 10, and 12 μg/mL) of arsenic trioxide for 24 h. The proliferative response (DNA synthesis) to arsenic trioxide was assessed by [3H]thymidine incorporation. The genotoxic effects of arsenic-induced DNA damage in a human colon cancer cell line was evaluated by the alkaline single cell gel electrophoresis. Results indicated that arsenic trioxide affected DNA synthesis in HT-29 cells in a biphasic manner; showing a slight but not significant increase in cell proliferation at lower levels of exposure (2, 4 and 6 μg/mL) followed by a significant inhibition of cell proliferation at higher doses (i.e., 8 and 10 μg/mL). The study also confirmed that arsenic trioxide exposure caused genotoxicity as revealed by the significant increase in DNA damage, comet tail-lengths, and tail moment when compared to non-exposed cells. Results of the [3H]thymidine incorporation assay and comet assay revealed that exposure to arsenic trioxide affected DNA synthesis and exhibited genotoxic effects in human colon cancer cells.

Malathion‐induced oxidative stress, cytotoxicity, and genotoxicity in human liver carcinoma (HepG2) cells

Malathion is an organophosphate pesticide that is known for its high toxicity to insects and low to moderate potency to humans and other mammals. Its toxicity has been associated with the inhibition of acetylcholinesterase activity, leading to the interference with the transmission of nerve impulse, accumulation of acetylcholine at synaptic junctions, and subsequent induction of adverse health effects including headache, dizziness, nausea, vomiting, bradycardia, and miosis. Oxidative stress (OS) has been reported as a possible mechanism of malathion toxicity in humans. Hence, the aim of this study was to examine the role of OS in malathion-induced cytotoxicity and genotoxicity. To achieve this goal, MTT, lipid peroxidation, and single cell gel electrophoresis (Comet) assays were performed, respectively, to evaluate the levels of cell viability, malondialdehyde (MDA) production, and DNA damage in human liver carcinoma (HepG(2)) cells. Study results indicated that malathion is mitogenic at lower levels of exposure, and cytotoxic at higher levels of exposure. Upon 48 h of exposure, the average percentages of cell viability were 100% +/- 11%, 117% +/- 15%, 86% +/- 15%, 35% +/- 9%, and 27% +/- 7% for 0, 6, 12, 18, and 24 mM, respectively. In the lipid peroxidation assay, the concentrations of MDA produced were 12.55 +/- 0.16, 20.65 +/- 0.27, 31.1 +/- 0.40, 34.75 +/- 0.45, and 15.1 +/- 0.20 muM in 0, 6, 12, 18, and 24 mM malathion, respectively. The Comet assay showed a significant increase in DNA damage at the 24 mM malathion exposure. Taken together, our results indicate that malathion exposure at higher concentrations induces cytotoxic and genotoxic effects in HepG(2) cells, and its toxicity may be mediated through OS as evidenced by a significant production of MDA, an end product of lipid peroxidation.

Toxic and genotoxic effects of oral administration of furan in mouse liver

In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.

Aldosterone Causes DNA Strand Breaks and Chromosomal Damage in Renal Cells, Which are Prevented by Mineralocorticoid Receptor Antagonists

Epidemiological studies exploring the connection between hypertension and cancer incidence find a higher cancer mortality in hypertensive patients, particularly elevated in hypertension associated with a stimulation of the renin-angiotensin-aldosterone system. Primary aldosteronism, with plasma aldosterone levels between 0.5 and 1 nM (18-36 ng/dL) and local aldosterone levels up to 500 nM (18,000 ng/dL), is now recognised as a more common cause for hypertension. We recently found angiotensin II to be genotoxic due to its induction of oxidative stress. Since aldosterone in higher concentrations also has oxidative effects, its potential genotoxic action in pig LLC-PK1 cells with properties of proximal tubules was analysed. DNA damage was evaluated by two test systems: the comet assay, and the micronucleus frequency test. The results showed that aldosterone concentrations starting from 10 nM (360 ng/dL) caused a significant increase of DNA damage monitored with the comet assay in LLC-PK1, while there was no change in cell vitality and proliferation. The micronucleus frequency test revealed that 10 nM aldosterone also leads to the formation of micronuclei. Furthermore, the formation of superoxide radicals in the cells by this aldosterone concentration could be detected with the superoxide-specific stain dihydroethidium. Further evidence for oxidative stress-induced DNA damage was its reversibility by the antioxidants tempol and catalase. Addition of the steroidal mineralocorticoid receptor antagonist spironolactone or the novel selective nonsteroidal antagonist (R)-BR-4628 reduced the DNA damage and the amount of superoxide radicals indicating a receptor-dependent process.

Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol

The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.

In vivo study of genotoxicity and teratogenicity of silica nanocrystals

The potential genotoxicity of silica nanocrystals (nc-Si) (size 2-7 nm with their agglomerates 5 μm) in mice and influence of nc-Si on the embryonal and the postnatal development of the rats were evaluated. No clastogenic effects were observed in bone marrow cells of mice intraperitoneally exposed to single doses of nc-Si 5, 25 and 50 mg/kg for 24 hr or to doses 5 and 25 mg/kg for seven and 14 days. Comet assay revealed a significant increase in tail DNA percentage in the bone marrow cells of mice treated with nc-Si at a single dose 5 mg/kg for 24 h and both in bone marrow and brain cells when nc-Si was used at a dose 50 mg/kg. There was no significant increase in DNA damage after a 3 h treatment with nc-Si at doses 5 and 25 mg/kg. Also, no elevated DNA damage was observed in mice exposed to nc-Si at 5 mg/kg dose for seven days. Dose of nc-Si at 50 mg/kg caused death of 40-60% of the animals within 2-3 days that made impossible evaluation genotoxicity in this dose at long-term exposure. Nc-Si injected intraperitoneally to rats at a dose of 50 mg/kg on the 1st, 7th and 14th days of pregnancy showed no teratogenicity and had no influence on postnatal development of pups. But negative influence on body weight gain in pregnant rats and newborns at some stages of the experiment was noticed.

Modulation of doxorubicin-induced genotoxicity by squalene in Balb/c mice

The present study aims to evaluate the protective effect of squalene against the genotoxicity of the chemotherapeutic agent doxorubicin (Dox) using two genotoxicity assays, the micronucleus assay and the comet assay. Different groups of mice were fed squalene at the doses of 1 and 4 mmol g(-1) body weight (100 or 400 μl as squalene oil) either at 4 h before or 1 h after Dox (20 mg kg(-1)) treatment. 24 h after the Dox treatment, bone marrow erythrocytes were evaluated for the incidence of micronuclei, and the induced DNA strand breaks were examined in heart tissue by the alkaline comet assay. As expected, Dox significantly induced micronuclei in polychromatic (immature) erythrocytes, as well as in total erythrocytes. The frequency of Dox-induced micronucleated erythrocytes was significantly reduced in the mice treated with squalene both before and after Dox administration. Squalene itself obviously did not induce any micronuclei in bone marrow erythrocytes. The comet assay also demonstrated a significant increase in DNA damage, especially DNA single strand breaks in the Dox-treated group of mice as compared to the control. The Dox-induced DNA damage was also effectively reduced by squalene when it was administered either before or after the Dox treatment. Squalene did not induce any significant DNA damage by itself. Compared to the pre-treatment of squalene, post treatment gave rise to more effective prevention against Dox-induced DNA damage. The data suggest that the complimentary use of squalene with Dox will be beneficial to reduce the adverse effect of Dox in cancer chemotherapy, such as the increased incidence of undesirable mutagenic side effects.

180. THE CONSEQUENCES OF ACRYLAMIDE EXPOSURE ON THE MALE GERMLINE

Acrylamide is a common industrial compound that has recently been identified in cooked, carbohydrate-rich foods such as potato chips, breads and cereals. Acrylamide has been found to be a reproductive toxin in rodents, eliciting male infertility and transgenerational toxicity through the male germline; thus dietary exposure to the compound may have consequences for male fertility and reproduction in humans. The aim of this project was to elucidate the mechanisms of acrylamide toxicity in male germ cells of mice. Freshly isolated early male germ cells were exposed to acrylamide and assessed for cell viability and aberrant morphology. DNA damage in these cells was also investigated using a modified version of the Comet Assay, which allows for adduct specificity. Significant increases in cell death or aberrant morphology were not observed following acrylamide exposure (1 µM, 18 hours). However, a significant increase in DNA damage (125% increase in mean tail DNA assessed by Comet) was identified; which may originate from either the metabolic conversion of acrylamide to glycidamide, leading to glycidamide adducts, or from oxidative stress. Additionally, the regulation of cytochrome P450 gene expression was measured using real time PCR and early male germ cells were found to upregulate gene expression of cytochrome P450 enzymes in response to acrylamide exposure. Collectively, these results support a genotoxic mode of action of acrylamide toxicity, in addition to potential oxidative damage in male germ cells. However, the mechanism by which acrylamide elicits toxicity in germ cells requires further investigation. Future outcomes of this research may provide insight into factors necessary for the healthy development of offspring and aid in the risk assessment of dietary acrylamide exposure in humans.

Toxicity and genotoxicity in Astyanax bimaculatus (Characidae) induced by microcystins from a bloom of Microcystis spp

Studies of genotoxicity in fish caused by cyanobacterial microcystins can be useful both in determining the sensitivity of native species, as well as comparing exposure routes. The genotoxicity caused by the microcystins LR and LA from a bloom collected in a eutrophic lake, was revealed in the fish Astyanaxbimaculatus, a native species from South America. LC50 (72 h) was determined as 242.81 μg L (-1) and LD50 (72 h) as 49.19 μg kg (-1) bw. There was a significant increase of DNA damage in peripheral erythrocytes, following intraperitoneal injection (ip) with tested concentrations of 24.58 μg kg (-1) bw and 36.88 μg kg (-1) bw, as well as through body exposure to a concentration of 103.72 μg L (-1) . Micronucleus (MN) induction was observed after ip injections of 24.58 μg kg (-1) bw and 36.88 μg kg (-1) bw for 72 h, as well as following body exposure for 72 at 103.72 μg L (-1) . Thus, both exposure routes resulted in MN induction and DNA damage. Apoptosis-necrosis testing was carried out only by ip injection with concentrations of 24.58 μg kg (-1) bw and 36.88 μg kg- 1 bw. Exposure to microcystins at lower concentrations induced more apoptosis than necrosis in peripheral erythrocytes, whereas exposure at higher concentrations gave rise to both conditions. Thus, Astyanax bimaculatus can be considered as a species sensitive to the genotoxic effects caused by microcystins.