Erythrocytic Nuclear Abnormalities Research Articles

Micronuclei and Nuclear Abnormalities in Erythrocytes from Barbel Barbus peloponnesius Revealing Genotoxic Pollution of the River Bregalnica

Abstract The strong negative anthropogenic pressure on the River Bregalnica resulted in the increase of pollution in the river ecosystem. The aim of this study was to evaluate if aquatic pollutants generate genotoxic effects in barbel (Barbus peloponnesius). Thirty individuals were collected from three localities along the river: one control site and two locations in the middle and lower course of the river with the presence of different pollution sources. Blood smears were stained with May-Grünwald/Giemsa method and the frequency of micronuclei, vacuolated nuclei, binuclei, blebbed nuclei and notched nuclei expressed in per mill was recorded. In fish collected from the control site, the investigated erythrocyte nuclear abnormalities revealed lowest values compared to polluted sites. In general, significantly higher values for erythrocyte deformations, including micronuclei, were noted in the middle and lower part of the river. Significantly higher values of vacuolated and blebbed nuclei were detected in fish from the middle course of the river, while at the lower course the frequencies of occurrence of micronuclei and notched nuclei were significant compared with control site. The middle course of the River Bregalnica has more diverse pollutants compared to the lower course, and consequently the highest frequency of all nuclear deformations was found in barbel from the middle course of the river. Obtained results suggest that pollutants draining into the Bregalnica river ecosystem display a genotoxic effect on the local barbel population.

Acute exposure to a quinalphos containing insecticide (convoy) causes genetic damage and nuclear changes in peripheral erythrocytes of silver barb, Barbonymus gonionotus

The present study was aimed to assess the genotoxic effect in fish caused by convoy, an insecticide commercial formulation containing quinalphos, present in the aquatic waterbody. For this purpose a freshwater teleost, silver barb was exposed to sublethal concentrations (25% and 50% of LC50) of convoy and erythrocytic cellular abnormalities (ECA) and erythrocytic nuclear abnormalities (ENA) tests were performed in addition to the commonly used micronucleus (MN) assay using peripheral erythrocytes and DNA contents in the different tissues after 1, 24, 36, 48, and 72 h of exposures. The obtained results indicated that acute exposure of different sub lethal concentrations of convoy to the fish resulted in significant alterations of erythrocytes as well as significant reduction of DNA contents in blood and vital organs and tissues, such as the brain, liver, kidney and muscle. Compared to each treatment excluding control group, frequencies of ECA, ENA, and MN were found to be elevated with exposure time of the doses. From this study, we conclude that convoy is a hazardous chemical to silver barb. Bioassays can be used as a tool for screening aquatic pollution, especially for insecticides.

Assessment in situ of genotoxicity in tadpoles and adults of frog Hypsiboas cordobae (Barrio 1965) inhabiting aquatic ecosystems associated to fluorite mine

Non-lethal biological techniques such as blood biomarkers have gained attention due to their value as early signals of anthropic effects of contamination representing significant tools to evaluate ecosystems health. We evaluate and characterize in situ genotoxicity of water samples collected from aquatic ecosystems around a fluorite mine using amphibian frogs Hypsiboas cordobae as bioindicator species complemented with 16 physicochemical parameters. Four stations associated with fluorite mine sampling were sampled: a stream running on granitic rock with natural high fluorite content; two streams both running on metamorphic rock with low fluorite content; and an artificial decantation pond containing sediments produced by fluorite flotation process with high variation in physicochemical parameters. We analyses the blood of tadpoles and adults of H. Cordobae, calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, immature and enucleated erythrocytes. Individuals were measured and weighed and body condition was calculated. The results of this study indicate that individuals of decantation pond are exposed to compounds or mixtures which are causing cell damage when compared to those that were collected of stream. Larval stage was more vulnerable than the adult phase and it could be related mainly to the higher exposure time to xenobiotics, which can penetrate easily by skin, mouth and gills; additionally this site offers a reduced availability of food than other sites. Therefore, chronic exposure to pollutants could derive in degenerative and neoplastic diseases in target organs. Moreover these individuals may experience reproductive and behavioral disturbances which could lead to population decline in the long term.

Integrated biomarker response in catfish Hypostomus ancistroides by multivariate analysis in the Pirapó River, southern Brazil

Aquatic pollutants produce multiple consequences in organisms, populations, communities and ecosystems, affecting the function of organs, reproductive state, population size, species survival and even biodiversity. In order to monitor the health of aquatic organisms, biomarkers have been used as effective tools in environmental risk assessment. The aim of this study is to evaluate, through a multivariate and integrative analysis, the response of the native species Hypostomus ancistroides over a pollution gradient in the main water supply body of northwestern Paraná state (Brazil). The condition factor, micronucleus test and erythrocyte nuclear abnormalities (ENA), comet assay, measurement of the cerebral and muscular enzyme acetylcholinesterase (AChE), and histopathological analysis of liver and gill were evaluated in fishes from three sites of the Pirapó River during the dry and rainy seasons. The multivariate general result showed that the interaction between the seasons and the sites was significant: there are variations in the rates of alterations in the biological parameters, depending on the time of year researched at each site. In general, the best results were observed for the site nearest the spring, and alterations in the parameters at the intermediate and downstream sites. In sum, the results of this study showed the necessity of a multivariate analysis, evaluating several biological parameters, to obtain an integrated response to the effects of the environmental pollutants on the organisms.

Impaired growth and erythrocyte nuclear lesions of immature Oreochromis niloticus exposed to waterborne crude oil: Persistent responses

Crude oil impact studies have largely been limited to marine setting and it hampers the understanding and predictability pertaining to freshwater environments following contamination episodes. Growth and erythrocyte nuclear abnormalities (ENA) were followed in freshwater fish Oreochromis niloticus experimentally exposed to crude oil (Arabian light) dispersed in water. Study comprised of a control (no crude oil in water), and two experimental groups with 5 µL/L and 25 µL/L crude oil dispersed in water (5 ppm and 25 ppm respectively), and involved continuous exposure of fish (35 fish/tank in triplicate) with static renewal over a 90-day period. Growth was estimated serially at 18-day intervals (n=40-60). Both exposure groups reduced (p O. niloticus juveniles in freshwater environment.

Genetic and Hematologic Endpoints in Astyanax altiparanae (Characidae) After Exposure and Recovery to Water-Soluble Fraction of Gasoline (WSFG).

The sublethal effects of water-soluble fraction of gasoline (WSFG, 1.5% v/v) were evaluated in the freshwater fish, Astynax altiparanae, after acute exposure (96h) under a semi-static system. In addition, the recovery process was assessed in the fish following contaminant depuration. Recovery treatments were carried out with gradual depuration (GD), consisting of 7days in the WSFG, followed by 8days in clean water; and treatments with total depuration in clean water for 15 (DEP 15) and 30days (DEP 30). The effects were evaluated through the piscine micronucleus test and by differential counting of organic defense cells. Acute exposure increased the frequency of neutrophils. In the GD treatment, the thrombocyte count and erythrocytic nuclear abnormalities (ENA) increased. In the DEP 15 treatment, there was a reduction of ENA; and following 30days of depuration (i.e., DEP 30), the number of lymphocytes increased and the thrombocyte count remained high. These results indicate a long-term response to a condition of stress from WSFG.

Effects of glyphosate on hepatic tissue evaluating melanomacrophages and erythrocytes responses in neotropical anuran Leptodactylus latinasus.

Glyphosate (GLY) is the most used herbicide worldwide and its effects on anurans are well known. Pollutants can cause physiological and morphological effects. Therefore, this study evaluated the effects of GLY on hepatic melanomacrophages as a response to environmental stressors. Three treatments were exposed to different concentrations of pure GLY (100, 1000, and 10,000μgg(-1), respectively), and there was also a control group. After the experimental time, liver and blood were analyzed. Melanomacrophages (MMCs) were located between the hepatocyte cordons, close to sinusoids. GLY increased the melanin area in MMCs of Leptodactylus latinasus exposed since lowest concentration until highest concentration. GLY also changed the occurrence of hepatic catabolism pigments into melanomacrophages and erythrocyte nuclear abnormalities; therefore, it can interfere with the hepatic metabolism. In conclusion, GLY promotes alterations in the hepatic tissue and erythrocyte nuclear abnormalities. Furthermore, MMCs may be useful as morphological responses of GLY effects.

Analysis of nuclear abnormalities in erythrocytes of rainbow trout (Oncorhynchus mykiss) treated with Cu and Zn and after 4-, 8-, and 12-day depuration (post-treatment recovery)

The induction of micronuclei (MN), nuclear buds (NB), bi-nucleated erythrocytes with nucleoplasmic bridge (BNb), vacuolated (VacNuc), blebbed (BL), 8-shaped nuclei, bi-nucleated (BN) and fragmented-apoptotic (FA) erythrocytes was analysed in the peripheral blood, cephalic kidney and liver of rainbow trout Oncorhynchus mykiss after 4-day treatment with copper (Cu) and zinc (Zn) mixture solutions and in 4-, 8- and 12-day depuration process. Fish (three treatment and one control group, N=40) were exposed to 0.0625, 0.125 and 0.25 fractions of 96-h LC50, respectively under semi-static conditions. Exposure of O. mykiss to Cu and Zn induced significant increase of MN (in blood in all test groups; in liver 0.125, 0.25 and in kidney 0.25 groups, respectively), NB and BL (in blood and kidney 0.25 group), 8-shaped (in blood 0.25; in liver 0.125, 0.25 and in kidney all test groups, respectively) and VacNuc (in liver and kidney 0.0625 and 0.125 groups). After 4-day recovery, significantly elevated levels of MN (in blood 0.0625, 0.125; in liver and kidney 0.125 group, respectively) and 8-shaped (in kidney—0.0625 group) were observed in fish. Significant recovery was observed in 0.0625 group after 12-day depuration, estimating the formation of MN in erythrocytes of blood, of 8-shaped nuclei erythrocytes in liver and kidney (after 8-, 12-day and 8-day recovery, respectively). Significant decrease of MN in blood (after 8- and 12-day recovery), in liver (after 8-day recovery), of NB in blood and kidney (after 8-day recovery) and of 8-shaped nuclei erythrocytes in blood (after 8 and 12-day recovery), kidney and liver (after 8-day recovery) was determined in 0.25 group. Changes in gross morphometric indices and biological parameters were observed. The binary metal mixture did not induce FA erythrocytes in any tissue at any test concentration.

Acute and chronic effects of erythromycin exposure on oxidative stress and genotoxicity parameters of Oncorhynchus mykiss

Erythromycin (ERY) is a macrolide antibiotic used in human and veterinary medicine, and has been detected in various aquatic compartments. Recent studies have indicated that this compound can exert biological activity on non-target organisms environmentally exposed. The present study aimed to assess the toxic effects of ERY in Oncorhynchus mykiss after acute and chronic exposures. The here adopted strategy involved exposure to three levels of ERY, the first being similar to concentrations reported to occur in the wild, thus ecologically relevant. Catalase (CAT), total glutathione peroxidase (GPx), glutathione reductase (GRed) activities and lipid peroxidation (TBARS levels) were quantified as oxidative stress biomarkers in gills and liver. Genotoxic endpoints, reflecting different types of genetic damage in blood cells, were also determined, by performing analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and of erythrocytic nuclear abnormalities (ENAs). The results suggest the occurrence of a mild, but significant, oxidative stress scenario in gills. For acutely exposed organisms, significant alterations were observed in CAT and GRed activities, and also in TBARS levels, which however are modifications with uncertain biological interpretation, despite indicating involvement of an oxidative effect and response. After chronic exposure, a significant decrease of CAT activity, increase of GPx activity and TBARS levels in gills was noticed. In liver, significant decrease in TBARS levels were observed in both exposures. Comet and ENAs assays indicated significant increases on genotoxic damage of O. mykiss, after erythromycin exposures. This set of data (acute and chronic) suggests that erythromycin has the potential to induce DNA strand breaks in blood cells, and demonstrate the induction of chromosome breakage and/or segregational abnormalities. Overall results indicate that both DNA damaging effects induced by erythromycin may be related to the oxidative damage observed, shown to occur at environmentally relevant concentrations of erythromycin.

Multiple biomarker responses in Prochilodus lineatus subjected to short-term in situ exposure to streams from agricultural areas in Southern Brazil

In order to assess the quality of streams susceptible to contamination by pesticides we apply biochemical and genotoxic biomarkers in the Neotropical fish Prochilodus lineatus submitted to in situ tests. Fish were caged, for 96h, in two streams located in areas with intensive use of pesticides, the Apertados (AP) and the Jacutinga (JC), and in a small stream (Godoy stream — GD) found inside a forest fragment adjacent to a State Park. Biochemical parameters, such as biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), non-protein thiols (NPSH), lipoperoxidation (LPO), protein carbonylation (PCO) and acetylcholinesterase (AChE) were evaluated in various fish organs, as well as genotoxic biomarkers (damage to DNA and occurrence of micronuclei and erythrocyte nuclear abnormalities). Samples of water and sediment were collected for analysis of metals (Cu, Cr, Pb, Ni, Mn, Cd and Zn), organochloride pesticides, and triazine and glyphosate herbicides. We observed an increase in liver GST activity in fish at AP and gill GST activity in fish at JC. An increase in liver LPO was also observed in fish exposed to AP and JC. The same animals also exhibited increased DNA damage and erythrocyte nuclear abnormalities (ENAs) compared to the fish kept in GD. A number of compounds showed concentrations higher than the permitted levels, in particular, dichlorodiphenyltrichloroethane (DDT), its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), hexachlorocyclohexanes (HCH), heptachloride, diclofluanid and aldrins. These pesticides were detected at higher concentrations in water and sediment samples from AP, followed by JC and GD. The Integrated Biomarker Response Index (IBR) indicated that AP and JC (AP: 21.7>JC: 18.5>GD: 12.6) have the worst environmental quality. Integrated biomarker analysis revealed that the alterations observed related well with the levels of environmental contaminants, demonstrating the effectiveness of this biomonitoring approach.

Karasu Nehri (Erzurum) Havzasında Yaşayan Pelophylax ridibundus (Anura: Ranidae) Kurbağalarında Kirliliğin Neden Olduğu Eritrositik Çekirdek Anormalliklerinin Tespiti

Karasu River, which is the only river in the Erzurum plain, is the source of the river Euphrates (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 marsh frog, Pelophylax ridibundus collected from two contaminated wetlands environments of the Karasu River Basin, in comparison with frogs from one non-polluted reference site. Heavy metal concentrations in surface water of the river were determined. Genotoxicity assays such as micronucleus (MN) and other nuclear abnormalities (NA) were carried out on the frogs studied. MN and NA (kidney-shaped nucleus, notched nucleus and lobed nucleus) were assessed in peripheral blood erythrocytes of the frog. A significant elevation in MN and NA frequencies was observed in frog collected from the polluted sites compared with those from the reference site. Results of the current study suggest that there is a close realtion between increasing of erythrocytic nuclear abnormalities and environmental contamination. High concentrations of heavy metals have a potential genotoxic effects, and the toxicity is possibly related to industrial, agricultural and domestic activities.

Common toad Rhinella arenarum (Hensel, 1867) and its importance in assessing environmental health: test of micronuclei and nuclear abnormalities in erythrocytes.

Anthropogenic activities may generate significant changes in the integrity of aquatic ecosystems, so long-term monitoring of populations that inhabit them is crucial. Counting micronucleated erythrocytes (MN) and erythrocytic nuclear abnormalities (ENA) in peripheral blood is a widely used method for detecting chromosomal damage due to chemical agents in the water. We analyzed MN and ENA frequency in blood obtained from the common toad Rhinella arenarum populations in sites with different degrees of environmental degradation. The results of this study indicate that there is an association between the frequency of micronuclei and nuclear abnormalities and the degree of environmental alteration recorded for the sites studied.

Impact of treated wastewater on organismic biosensors at various levels of biological organization

Relating the treated wastewater quality and its impact on organismic biosensors (Prussian carp, Carassius gibelio and earthworm, Eisenia fetida) was the main objective of the study. The impact on health status of fish living downstream, microbiological contamination and antimicrobial resistance, fish tissue structure, blood biochemistry, oxidative stress, genotoxic effects, as well as multixenobiotic resistance mechanism (MXR) was assessed. Treated wastewater discharged from the WWTP modified the environmental parameters and xenobiotic concentrations of the receiving surface waters. Potential bacterial pathogens from fish and respective waters were found in relatively low numbers, although they comprised aeromonads with a zoonotic potential. High resistance profiles were determined towards the tested antimicrobial compounds, mostly sulfamethoxazole and erythromycin. Histopathology primarily revealed gill lamellar fusion and reduction of interlamellar spaces of effluent fish. A significant increase in plasma values of urea, total proteins, albumins and triglycerides and a significant decrease in the activity of plasma superoxide dismutase were noted in carp from the effluent-receiving canal. Micronucleus test did not reveal significant differences between the examined groups, but a higher frequency of erythrocyte nuclear abnormalities was found in fish sampled from the effluent-receiving canal. Earthworms indicated to the presence of MXR inhibitors in water and sludge samples, thus proving as a sensitive sentinel organism for environmental pollutants. The integrative approach of this study could serve as a guiding principle in conducting evaluations of the aquatic habitat health in complex bio-monitoring studies.

Environmentally Relevant Concentrations of Atrazine and Ametrine Induce Micronuclei Formation and Nuclear Abnormalities in Erythrocytes of Fish.

A rapid and sensitive method using liquid chromatography coupled with mass spectrometry triple quadrupole direct aqueous injection for analysis of atrazine and ametrine herbicides in surface waters was developed. According to the validation method, water samples from six different locations in the Piracicaba River were collected monthly from February 2011 to January 2012 and injected into a liquid chromatographer/dual mass spectrometer without the need for sample extraction. The method was validated and shown to be precise and accurate; limits of detection and quantification were 0.07 and 0.10µg L(-1) for atrazine and 0.09 and 0.14µg L(-1) for ametrine. During the sampling period, concentrations of atrazine ranged from 0.11 to 1.92µg L(-1) and ametrine from 0.25 to 1.44µg L(-1). After analysis of the herbicides, Danio rerio were exposed a range of concentrations found in the river water to check the induction of micronuclei and nuclear abnormalities (NAs) in erythrocytes. Concentrations of atrazine and ametrine >1.0 and 1.5µg L(-1), respectively, induced MN formation in D. rerio. Ametrine was shown to be more genotoxic to D. rerio because a greater incidence of NAs was observed compared with atrazine. Therefore, environmentally relevant concentrations of atrazine and ametrine found in the Piracicaba River are dangerous to the aquatic biota.

Fresh water fish, Channa punctatus, as a model for pendimethalin genotoxicity testing: A new approach toward aquatic environmental contaminants.

Pendimethalin (PND) is one of the common herbicides used worldwide. Fresh water fish, Channa punctatus, was exposed to PND in aquaria wherein its LC50 value was recorded to be 3.6 mg/L. Three sublethal (SL) concentrations, namely, 0.9, 1.8, and 2.7 mg/L were selected for the evaluation of genotoxicity and oxidative stress generated in the fish. In vivo comet assay was carried out in the blood, liver, and gill cells after exposing the fish to aforesaid SL concentrations of PND for 24, 48, 72, and 96 h. The results of the comet assay demonstrated the genotoxicity of PND in all the three tissues. Induction of oxidative stress in the gill cells was affirmed by the increased lipid peroxidation (LPO) and decreased levels of reduced glutathione, superoxide dismutase, and catalase. Frequencies of erythrocytic nuclear abnormalities (ENA) and micronuclei (MN) were also used to assess the genotoxic potential of PND on C. punctatus. MN frequency did not show any enhancement after PND exposure, but the frequency of ENA such as kidney-shaped nuclei, segmented nuclei and lobed nuclei, showed a significant increase after 24-96 h. Thus, ENA seems to be a better biomarker than MN for PND induced genotoxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1520-1529, 2016.

Genotoxic responses of juvenile tilapia (Oreochromis niloticus) exposed to florfenicol and oxytetracycline

Florfenicol (FLC) and oxytetracycline (OTC) are the two most commonly used antibiotics for bacterial treatment in fish farming in Brazil, and because of their intensive use, the potential harmful effects on aquatic organisms are of great concern. This study evaluated the effects of environmental concentrations of FLC and OTC on the genetic material of juvenile tilapia (Oreochromis niloticus) erythrocytes by using the comet assay and the occurrence of micronuclei (MN) and other erythrocytic nuclear abnormalities (ENAs) after exposure to 96hour. The comet assay showed that fish erythrocytes exhibited significantly higher DNA damage after exposure to environmental concentrations of FLC and OTC. Although MN was not observed, ENAs were significantly higher after exposure to FLC, indicating that ENAs are a better biomarker for FLC than MN. The results showed that environmental concentrations of FLC and OTC were genotoxic to erythrocytes of O. niloticus; however, future studies on DNA damage recovery are needed.

Erythrocytic abnormalities in three Antarctic penguin species along the Antarctic Peninsula: biomonitoring of genomic damage

Pollutants and toxic contaminants produced in all parts of the world are transported to remote regions including Antarctica. Tourism, research, and fishing activities on this continent are another source of contamination. Toxic substances affect Antarctic species, and some produced genomic damage to the fauna. The genetic damage can be detected by microscopic observation of erythrocytic nuclear abnormalities (ENAs). We counted the number of ENAs in seven populations of three Pygoscelid penguin species, Adélie (Pygoscelis adeliae), Chinstrap (Pygoscelis antarctica), and Gentoo (Pygoscelis papua), and found important differences among species exposed to the same conditions. ENAs were more frequent in Adélie penguins than in the other two species. Inter-population comparisons within species showed remarkable differences in Adélie and Chinstrap penguins but not in Gentoo penguin. Frequency of ENAs in Adélie penguins was the highest in Yalour Island population, intermediate in King George Island population, and the lowest in Torgersen Island and Avian Island populations. In Chinstrap penguins, the highest number of ENAs was found on Deception Island, and significant differences were found only between Deception Island and King George Island populations. This information will provide baseline data to be used for assessing the evolution of genomic damage of penguins along the Antarctic Peninsula in the future.

Comparing the genotoxicity of a potentially carcinogenic and a noncarcinogenic PAH, singly, and in binary combination, on peripheral blood cells of the European sea bass.

Research on the toxicological mechanisms of polycyclic aromatic hydrocarbons (PAHs) deemed carcinogenic and noncarcinogenic has mostly been developed for individual compounds even though, in the environment, PAHs invariably occur in mixtures. The present work aimed at understanding the interaction effects of two model PAHs, the potentially carcinogenic benzo[b]fluoranthene (B[b]F) and the noncarcinogenic phenanthrene (Phe) to a marine fish (the sea bass Dicentrarchus labrax). The study endeavoured an ecologically-relevant scenario with respect to concentrations and contaminant matrix, sediments, which are the main reservoirs of these substances in the environment, due to their hydrophobic nature. For the purpose, 28-day laboratorial bioassays with spiked sediments (with individual and combined PAHs at equitoxic concentrations) were conducted. Genotoxicity was determined in peripheral blood through the "Comet" assay and by scoring erythrocytic nuclear abnormalities (ENA). The results showed that exposure to either PAHs induced similar levels of DNA strand breaks, although without a clear dose- or time-response, likely due to the low concentrations of exposure and potential shits in PAH bioavailability during the assays. However, clastogenic/aneugenic lesions were only observed in fish exposed to B[b]F-spiked sediments. Conversely, the combination assays revealed a supra-additive effect especially at chromosome level, linked to concentrations of PAHs in water. A decrease in DNA-strand breakage was observed over time during all assays, revealing some ability of fish to cope with this DNA lesion. Overall, the findings show that low-moderate concentrations of sediment-bound mixed PAHs may significantly increase the hazard of mutagenesis even when the individual concentrations indicate low risk, especially considering that chromosome-level damage is unlikely to be repaired, leading to the fixation of DNA lesions upon prolonged exposures. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1307-1318, 2016.

Hematological and biochemical investigations on the effect of vitamin E and C on Oreochromis niloticus exposed to zinc oxide nanoparticles

This study was carried out to determine the LC50 of zinc oxide nanoparticles (ZnONPs) on Oreochromis niloticus and to investigate the effect of vitamin E and C on hematological and biochemical alterations induced by two sublethal concentrations (1 and 2mg/L) of ZnONPs. One hundred and eighty fish were used for studying the lethal concentrations of ZnONPs. For sublethal study two hundred and twenty-five males of O. niloticus were equally divided into five groups, control, the second and the third were treated with 1 and 2mg/L ZnONPs respectively. The fourth and fifth were exposed to the same concentrations of ZnONPs plus vitamins E and C. The results revealed that the 96h LC50 of ZnONPs was 3.1±0.4mg/L. The sublethal study revealed the presence of normocytic normochromic anemia in groups (2, 3 and 5) along the experiment period. The 4th group showed normocytic normochromic anemia at the 7th day and microcytic hypochromic anemia at the 15th day. Leukocytosis, heterophilia, lymphopenia and monocytopenia were recorded at the 7th day in all treated groups compared with the normal control. At the 15th day heteropenia, lymphopenia and monocytopenia were reported in all treated groups. A significant increase in the serum levels of alkaline phosphatase, aminotransferases, urea, creatinine and erythrocytic nuclear and morphological abnormalities along the experimental periods in all treated groups compared with the normal control. Serum total protein and albumin levels were significantly decreased at the same period in the same groups. Addition of vitamin E and C to the diet (groups 4 and 5) significantly improved all measured parameters compared with groups (2 and 3) which treated with ZnONPs only.

Genotoxicity, potential cytotoxicity and cell uptake of titanium dioxide nanoparticles in the marine fish Trachinotus carolinus (Linnaeus, 1766)

Nanoparticles have physicochemical characteristics that make them useful in areas such as science, technology, medicine and in products of everyday use. Recently the manufacture and variety of these products has grown rapidly, raising concerns about their impact on human health and the environment. Adverse effects of exposure to nanoparticles have been reported for both terrestrial and aquatic organisms, but the toxic effects of the substances on marine organisms remain poorly understood. The main aim of this study was to evaluate the genotoxicity of TiO2–NP in the marine fish Trachinotus carolinus, through cytogenotoxic methods. The fish received two different doses of 1.5μg and 3.0μg–TiO2–NPg−1 by intraperitoneal injection. Blood samples were collected to analyze erythrocyte viability using the Trypan Blue exclusion test, comet assay (pH>13), micronucleus (MN) and other erythrocyte nuclear abnormalities (ENA) 24, 48 and 72h after injection. The possible cell uptake of TiO2–NP in fish injected with the higher dose was investigated after 72h using transmission electron microscopy (TEM). The results showed that TiO2–NP is genotoxic and potentially cytotoxic for this species, causing DNA damage, inducing the formation of MN and other ENA, and decreasing erythrocyte viability. TEM examination revealed that cell uptake of TiO2–NP was mainly in the kidney, liver, gills and to a lesser degree in muscle. To the extent of the authors’ knowledge, this is the first in vivo study of genotoxicity and other effects of TiO2–NP in a marine fish.