Turgay Şişman

Toxicologic evaluation of imazalil with particular reference to genotoxic and teratogenic potentials

Imazalil (IMA) is a fungicide that is used extensively in fruit plantations and post-harvest treatments. IMA is suspected to produce craniofacial malformations in vertebrates and scarce data are available about its genotoxicity. Therefore, toxicity tests on embryogenesis of zebrafish (Danio rerio) and genotoxicity biomonitoring assays on human lymphocytes were performed to assess the effects of IMA. For this aim, zebrafish embryos were continually exposed, from 0.5 to 144 h post-fertilization, to a range of concentrations (5, 10, 20, 50 and 100 µM). IMA (0 to 672 µM) were also applied to the whole-blood cultures from two persons. We used chromosomal aberrations (CA) and micronucleus (MN) tests to examine DNA damage by IMA in human peripheral lymphocytes. The fungicide significantly altered zebrafish development even at low concentrations and its effects were dose-dependent. Results of the experiment indicated that IMA concentrations of 10 µM and above negatively affected embryo survival and hatching success. Morphological analysis uncovered a large suite of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, tail defect and cardiac edema. The cytogenetic results clearly showed that IMA caused increases of the frequencies of the structural chromosomal aberrations and the rates of MN as compared to controls in a dose-dependent manner. In conclusion, the present findings are of importance in the assessment of the potential risk of fungicides as IMA on aquatic ecosystems and humans.

Dichlorvos-induced developmental toxicity in Zebrafish

The present study examined effects of the pesticide dichlorvos (O-(2,2-dichlorovinyl)-O,O dimethylphosphate [DDVP]) on embryonic development of zebrafish. In a first set of experiments, early life stages of zebrafish were exposed to five concentrations (5, 10, 25, 50, and 100 mg/L—1) of DDVP for 96 hours post fertilization (hpf). The 24-hpf LC50 value of DDVP in the semistatic test was 39.75 mg/L-1. Developmental abnormalities have been observed in embryos and larvae, such as no blood flow, cardiac edema, delayed hatching, and vertebra malformations. Most of the onserved effects were increased in a concentration-dependent manner. To investigate the behavior of the larvae, zebrafish exposed to 10 and 25 mg/L-1 DDVP were observed on days 6 and 9 after fertilization. 25 mg L-1 DDVP dose caused significant slowing of swimming activity on day 6 and 9 after fertilization. According to present study results, DDVP exposure during early development caused (i) clear behavioral impairments detectable during the posthatching period and (ii) mortality and developmental abnormalities in zebrafish. Dichlorvos is present in the environment with other similar organophosphate compounds. Additive responses to organophosphate compounds may induce lethal or sub-lethal effects in early life stages of fish chronically exposed to this class of chemicals.

Early life stage and genetic toxicity of stannous chloride on zebrafish embryos and adults: Toxic effects of tin on zebrafish

Humans are exposed to stannous chloride (SnCl2), known as tin chloride, present in packaged food, soft drinks, biocides, dentifrices, etc. Health effects in children exposed to tin and tin compounds have not been investigated yet. Therefore, we evaluated the possible teratogenic effects and genotoxic of SnCl2 in zebrafish (Danio rerio) adults and their embryos. In the embryo–larval study, SnCl2 showed embryo toxicity and developmental delay after exposure to the various concentrations of 10–250 μM for 120 h. Teratogenic effects including morphological malformations of the embryos and larvae were observed. The embryos exposed to 100 μM displayed tail deformation at 28 hpf and the larvae exposed to 50 μM showed reduced body growth, smaller head and eyes, bent trunk, mild pericardial edema, and smaller caudal fin at 96 hpf. The results of the teratological study show that SnCl2 induced a significant decrease in the number of living embryos and larvae. Regarding the chromosome analysis, SnCl2 induced a dose‐dependent increase in the micronucleus (MN) frequency in peripheral erythrocytes of adult zebrafish. In blood cells, the 25 μM dose of SnCl2 caused a nonsignificant increase in the total chromosomal aberrations, but the high doses significantly increased the total number of chromosomal aberrations compared with the control groups. Overall, the results clearly indicate that SnCl2 is teratogenic and genotoxic to zebrafish.

Anti-genotoxic effect of hydrated sodium calcium aluminosilicate on genotoxicity to human lymphocytes induced by aflatoxin B1

The anti-genotoxic potential of hydrated sodium calcium aluminosilicate (HSCAS) was investigated using sister chromatid exchanges (SCEs) induced by aflatoxin B1 (AFB 1) as genotoxic endpoint in human lymphocytes. Whole blood samples from two healthy male donors were used for this experiment and the effects of different concentrations of HSCAS (5 × 10 —6 and 1 × 10—5 M) and AFB1 (1, 5 and 10 μM) were tested. The present results established that the frequencies of SCEs (at doses of 5 and 10 μM except for 1 μM) in lymphocytes were significantly increased by AFB1 compared to controls. When HSCAS was added alone at a molar ratio of 5 × 10—6 and 1 × 10— 5, lymphocytes did not show significant increases in SCE frequencies, but SCE frequencies induced by the various concentrations of AFB1 could be significantly reduced by the presence of HSCAS (at both doses). In fact, HSCAS at 1 × 10—5 M completely inhibited SCE formations caused by AFB1. These results suggest for the first time, that HSCAS can antagonize the ability of AFB1 to cause DNA damage that leads to the formation of SCEs.

Role of Peltigera rufescens (Weis) Humb. (a lichen) on imazalil-induced genotoxicity: analysis of micronucleus and chromosome aberrations in vitro.

Imazalil (IMA), a commonly used fungicide in both agricultural and clinical domains, is suspected to produce very serious toxic effects on vertebrates. On the other hand, in recent years, a number of studies have suggested that lichens might be easily accessible sources of natural drugs that could be used as a possible food supplement. Extensive research is being carried out to explore the importance of lichen species, which are known to contain a variety of pharmacological active compounds. In this context, the anti-genotoxic effects of aqueous Peltigera rufescens (Weis) Humb. extracts (PREs) were studied against the genotoxic damage induced by IMA on cultured human lymphocytes using chromosomal aberrations (CAs) and micronucleus (MN) as cytogenetic parameters. Human peripheral lymphocytes were treated in vitro with varying concentrations of PREs (0, 5, 10, 25, 50 and 100 mg/L), tested in combination with IMA (336 mg/L). PREs alone were not genotoxic and when combined with IMA treatment, reduced the frequency of CAs and the rates of MNs. A clear dose-dependent decrease in the genotoxic damage of IMA was observed, suggesting a genoprotective role of P. rufescens extract. The results of the present study indicate that this plant extract per se do not have genotoxic potential but can minimize the genotoxicity of IMA on human lymphocytes in vitro. In conclusion our findings may have an important application for the protection of human lymphocyte from the genetic damage and side effects induced by agricultural and medical chemicals hazardous in people.

The genoprotective activity of resveratrol on aflatoxin B1-induced DNA damage in human lymphocytes in vitro

Aflatoxin B1 (AFB1) has been consistently shown to be a potent mutagen and carcinogen in humans and animals. On the other hand, resveratrol (RSV), a polyphenol, has several positive biological actions such as protection of cells against DNA damage. In the present study, the antigenotoxic effect of RSV was studied against a genotoxic dose of AFB1 using the damage parameters of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) in cultured human lymphocytes. Whole blood samples from three healthy male donors were used for this experiment and the effects of various concentrations of RSV (0, 10, 15, 25, 40, 75 and 100 µM) and AFB1 (10 µM) were tested. The results revealed that the frequencies of SCEs and CAs in lymphocytes were significantly (p < 0.05) increased by AFB1 as compared to controls. The results also showed that RSV was not genotoxic. Moreover, the number of SCEs and micronuclei induced by AFB1 could be significantly minimized by the presence of RSV. Our results suggest for the first time that RSV can antagonize the ability of AFB1 to cause DNA damage that leads to the formation of SCEs and CAs.

The teratogenic effects of polychlorinated naphthalenes (PCNs) on early development of the zebrafish (Danio rerio)

The present study examines the effects of PCN congeners (PCN50 and PCN66) on zebrafish embryos. The embryos were exposed to different concentrations (20, 30 and 50μg/l) of the respective congeners by dissolving 0.08% acetone. The following endpoints were investigated (mortality, hatching rate, abnormalities and heart rate). No effect on survival was recorded for 20μg/l PCN congeners. On the other hand, embryos exposed to 30 and 50μg/l PCNs showed a significant reduction in survival rate. No developmental defects occurred with any of the congeners at the 20μg/l concentration. However, starting with 30μg/l, weak to very pronounced abnormalities (Lordosis, Kyphosis, disrupted axial body, tail defect, reduced heartbeat, and abnormal hatching) were observed depending on the congener and the concentration used. PCN66 has been shown to be more embryotoxic congener than PCN50. This demonstration of zebrafish responsiveness to PCNs early life stage toxicity coupled with the considerable information on developmental biology and genetics of zebrafish provides a foundation for future investigations into the mechanism of PCNs developmental toxicity.

Determination of acute and early life stage toxicity of fat-plant effluent using zebrafish (Danio rerio).

The present study examines the effects of an effluent from a fat plant (FP) on zebrafish (Danio rerio) embryos and larvae using the whole effluent toxicity testing methods (WET). The method is based on acute toxicity using 96‐h larval mortality and chronic toxicity using endpoints such as the time to hatch, hatching success, deformity, growth rate, swim‐up failure, accumulative mortality, and sex ratio. On the basis of larval mortality the 96‐h LC50 (the concentration was lethal to 50% of newly hatching zebrafish larvae) was 68.9%. In chronic toxicity test, newly fertilized embryos (<5‐h old) were exposed to 1, 6, 12, 25, 50% effluent concentrations in a 24‐h static renewal system at (27 ± 0.5)°C until 15‐day posthatch. The results showed that all chronic endpoints were significantly different from the control at 50% dilution. Embryos began to show lesions on third day at higher concentrations (12, 25, 50% FP effluent concentrations). Treatment group of 25% dilution showed delayed time to hatch. Morphological abnormalities were observed in newly hatched larvae at 25 and 50% FP effluent concentrations. At 25% dilution, sex ratio of larvae was alternated and there was feminization phenomenon. On the basis of the study, the FP effluent tested here may cause increasing embryotoxicity in the zebrafish embryos. We conclude that the test using zebrafish is feasible to evaluate both acute and chronic toxicities of industrial effluents.

Histopathological changes in gill and liver of Capoeta capoeta living in the Karasu River, Erzurum.

The contamination of surface waters by different pollutants is an important problem worldwide. In this study, the histopathological effects of water pollution were investigated on freshwater fish species Capoeta capoeta caught from the Karasu River. Fish were caught at three different sites in the Karasu River, namely, Aşkale, Dumlu, and Serçeme. The histological changes in gill and liver of fish were detected microscopically and evaluated with quantitative analyses. In addition, heavy metals have also been determined in surface water samples from these sites. Results showed that the Aşkale site was polluted by different kinds of heavy metals. In Aşkale site, some heavy metals such as Cd, Al, As, Pb, and Mn levels were mostly detected at concentrations above than the accepted values by the Turkish Standards Institute. The presence of gill and liver histological alterations were assessed by the degree of tissue change (DTC). In gill filaments, hypertrophy and hyperplasia of the gill epithelium, lamellar epithelial lifting, lamellae shortening, vasodilatation, lamellar disorganization, blood congestion, fusion, and aneurysm were observed. In the liver, the changes included an increase in the number and size of melanomacrophage aggregates, nonhomogenous parenchyma, proliferation of the hepatopancreas, sinusoidal dilatation, vacuolization, hypertrophy of the hepatocytes, congestion and degeneration of central vein, blood congestion, pyknotic nucleus, focal necrosis, and hepatic granuloma. The histological lesions were comparatively most severe in liver. The DTC means were varied from slight to moderate of gill and moderate to severe of liver tissue in the Aşkale site, thus the site is considered to be of low quality. Some pathological alterations were observed in the Serçeme site, although their distribution was lower than sites Dumlu and especially Aşkale. The least DTC means of the Serçeme site demonstrated their good environmental quality. The results suggest that there is a close relationship between amounts of pathological alterations and environmental contamination.

Genotoxic effects of water pollution on two fish species living in Karasu River, Erzurum, Turkey

Karasu River, which is the only river in the Erzurum plain, is the source of the Euphrates River (Eastern Anatolia of Turkey). The river is in a serious environmental situation as a result of pollution by agricultural and industrial sewage and domestic discharges. The present study aims to evaluate genotoxic effects of toxic metals in chub, Leuciscus cephalus, and transcaucasian barb, Capoeta capoeta, collected from contaminated site of the Karasu River, in comparison with fish from an unpolluted reference site. Heavy metal concentrations in surface water of the river were determined. The condition factor (CF) was taken as a general biomarker of the health of the fish, and genotoxicity assays such as micronucleus (MN) and other nuclear abnormalities (NA) were carried out on the fish species studied. MN and NA such as kidney-shaped nucleus, notched nucleus, binucleated, lobed nucleus, and blebbed nucleus were assessed in peripheral blood erythrocytes, gill epithelial cells, and liver cells of the fish. A significant decrease in CF values associated with a significant elevation in MN and NA frequencies was observed in fish collected from the polluted sites compared with those from the reference site. Results of the current study show the significance of integrating a set of biomarkers to identify the effects of anthropogenic pollution. High concentrations of heavy metals have a potential genotoxic effects, and the toxicity is possibly related to industrial, agricultural, and domestic activities.