Toxicity and genotoxicity of wastewater from gasoline stations

The toxicity and genotoxicity of wastewater from eight gasoline stations in Brasília, Brazil's capital city, was studied by assessing chromosomal aberrations, chromosomal malsegregation and the mitotic index in Alliumcepa root cells, and the occurrence of micronucleus and nuclear abnormalities in peripheral erythrocytes of tilapia fish (Oreochromis niloticus). The content of gasoline station effluents was also analyzed based on several physico-chemical parameters. None of the wastewater samples was genotoxic to A. cepa root cells, although cell proliferation was significantly inhibited, especially at the highest concentrations. Likewise, no micronuclei were observed in O. niloticus peripheral erythrocytes, even after exposure to high concentrations, but there was an increase in the number of nuclear abnormalities and fish mortality. These results show that although the effluent from gasoline stations is processed by an oil/water separation system before being discharged into the main sewage system, the wastewater still contains toxic compounds.     

The West African species of Bathygobius (Teleostei: Gobiidae) and their affinities

The circumtropical gobiid fish genus Bathygobius Bleeker, 1878 is discussed and defined in terms of external features, including the head lateral-line system, and osteology. Three West African species are recognized and redescribed: the amphiatlantic B. soporalor (Valenciennes, 1837) and two endemics, B. burtoni (O'Shaughnessy, 1875) and B. casamancus (Rochebrune, 1880). West African B. soporator differs significantly from western Atlantic material only in average values for predorsal scales and some body proportions, so that separation of eastern and western Atlantic populations as morphological species is not warranted. The affinities of the West African Bathygobius species are examined by means of phenetic (presence-only group-average clustering) and cladistic (Camin-Sokal, Dollo and Wagner) numerical processing of a binary database comprising 46 character states in 15 Operational Taxonomic Units (OTUs) formed by species of Bathygobius. Results do not indicate close common ancestry between the West African endemic species and B. soporator , which has probably colonized West Africa from the New World. The implications of various cladistic schemes for the relationships of certain other American species are also noted.     

Mussel micronucleus cytome assay

The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The MN assay is today applied in laboratory and field studies using hemocytes and gill cells from bivalves, mainly from the genera Mytilus. These represent 'sentinel' organisms because of their ability to survive under polluted conditions and to accumulate both organic and inorganic pollutants. Because the mussel MN assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an MN cytome assay. The mussel MN cytome (MUMNcyt) assay protocol we describe here reports the recommended experimental design, sample size, cell preparation, cell fixation and staining methods. The protocol also includes criteria and photomicrographs for identifying different cell types and scoring criteria for micronuclei (MNi) and nuclear buds. The complete procedure requires approximately 10 h for each experimental point/sampling station (ten animals).     

鱼微核试验筛检水体诱变物的应用与研究

对五种试验污染物的遗传毒性采用鱼微核试验技术进行了筛检,试验结果表明鱼微核试验用作筛检诱变物的遗传毒性有效的,对监测水体的质量也具有潜在的价值;同时,对国内外学将各种各样的技术应用到鱼微核式试验进行了总结,概述了微核形成的机理;在此基础上,提出应用鱼微核试验筛检水体诱变物时应注意的问题。     

Biomonitoring of the genotoxic potential (micronucleus and erythrocyte nuclear abnormalities assay) of the Admiralty Bay water surrounding the Brazilian Antarctic Research Station “Comandante Ferraz,” King George Island

The micronucleus (Mn) and other kinds of erythrocyte nuclear abnormality (ENA) assays were used to assess the genotoxic potential of the seawater in front of the Brazilian Antarctic Research Station “Comandante Ferraz” (King George Island). In two consecutive summers, the fish Trematomus newnesi was exposed to the seawater in front of the fuel storage tanks and the sewage discharge outlet of the Station in laboratory bioassays and in situ caging assays. After the exposure, frequencies of ENA and Mn were scored on blood smears. Significantly higher Mn and ENA frequencies in relation to those of the controls were observed in fish exposed to both types of water, in both types of experiment. Seawater in front of the fuel tanks and sewage discharge outlet at the Station was found to induce the formation of micronuclei and other ENAs, indicators of genotoxicity. The need for further investigation and the suitability of the methods for screening mutagenic pollution in polar waters are discussed.     

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.     

浊漳河水体污染物对蚕豆根尖细胞的遗传毒性研究

运用蚕豆根尖细胞微核试验技术对浊漳河南段6个代表性样点水体中有机污染物的遗传毒性进行了研究,为浊漳河流域水体污染状况的有效监测提供理论依据.结果显示:(1)浊漳河南段各监测断面的水体有机污染物对蚕豆根尖细胞均产生了不同程度的损伤,表现出细胞微核、断片、染色体桥等多种异常形态,严重时导致细胞坏死和细胞凋亡.(2)6个样点水样处理的蚕豆根尖细胞微核率在21.47‰～64.77‰,极显著高于对照组(P<0.01).(3)各样点水样对蚕豆根尖细胞的遗传损伤程度依次为:王桥>店上>漳泽水库>北寨>黄碾>五阳;在有丝分裂后期,蚕豆根尖中异常细胞最高达70%,且异常细胞比率有随着污染程度增大而升高的趋势.研究表明,蚕豆根尖细胞微核是水质毒理检测的有效指标,其细胞有丝分裂后期异常细胞比率也可作为水质毒理检测的观察指标.     

Induction of micronuclei in cultured murine splenocytes exposed to elevated levels of ferrous ions, hydrogen peroxide and ultraviolet irradiation

The frequency of micronuclei in cultured mouse splenocytes increased positively and in a dose-related manner to exposure to ferrous ions and ultraviolet irradiation, but not to hydrogen peroxide. Combined treatments, especially when ferrous ions were present with hydrogen peroxide or with ultraviolet irradiation, led to a synergistic enhancement in micronucleus frequency. The results indicate that a significant level of chromosome damage is associated with exposure to ultraviolet light and to general cellular pro-oxidative stress, and indicate that under these conditions the micronucleus assay can provide an effective in vitro model for the study of genotoxicity in relation to oxygen-derived free radicals.     

Formation of micronuclei in erythrocytes of the fathead minnow (Pimephales promelas) after acute treatment with mitomycin C or cyclophosphamide

Despite the widespread use of the fathead minnow in ecotoxicology, there have been relatively few studies on genotoxicity biomarkers in this small, warm-water fish species. Consequently, we investigated the effect of two known genotoxins, mitomycin C and cyclophosphamide, on micronucleus induction in spleen and peripheral blood erythrocytes of this species. Initially, 96-h experiments after intra-peritoneal (i.p.) injections of mitomycin C and cyclophosphamide were undertaken to determine the maximum tolerated dose (MTD). From these studies, MTDs of 10 and 400 mg/kg, respectively, were obtained: doses that were higher than those reported for other fish species. Next, an assessment of micronucleus induction at 1, 2, 4, 8 and 14 days after injection was undertaken for each compound at the MTD. Mitomycin C at 10 mg/kg significantly induced micronuclei in erythrocytes from the spleen, but not from the peripheral blood, at 8 and 14 days. In addition, the overall levels of micronuclei observed were lower than most previously published data from other fish species. In contrast to mitomycin C, treatment with 400 mg/kg cyclophosphamide failed to significantly induce micronuclei in erythrocytes from any of the tissues employed, in contrast to previous reports of significant induction in other species. The reasons for the apparent relative insensitivity of the fathead minnow to these clastogens, with respect to both MTDs and micronucleus induction, are not clear. The fathead minnow, however, has previously been described as relatively insensitive compared to other fish species with respect to selected carcinogens and cytochrome P450 inducers; the latter suggesting that the lack of a significant induction following cyclophosphamide exposure may be due to low metabolic activation in vivo. Consequently, further clarifying work is required to delineate the response shown, considering the extensive use of this species in ecotoxicology research and regulatory testing.     

Histopathological biomarkers and genotoxicity in gill and liver tissues of Nile tilapia Oreochromis niloticus from a polluted part of the Nile River,Egypt

Fish health is affected by water pollution. Oreochromis niloticus collected during summer 2014 from El-Serw, a polluted site on the Nile River, were compared with fish from a reference site, El-Zamalek. Histopathological changes were detected in gill and liver tissue samples using light and electron microscopy. In addition, the degree of DNA damage was measured using the comet assay. To indicate the severity of water pollution at the two sites, physico-chemical properties and heavy metal concentrations were investigated. Gill damage, including lamellar cell hyperplasia and aneurysm, was observed in the fish samples from the polluted site. The livers of fish from the polluted area showed necrosis and an increase in melanomacrophage centres. Histochemical results confirmed a marked rise of gill mucopolysaccharides and a reduction of carbohydrate stored in hepatocytes. Electron microscopy revealed clear alterations in gill and liver tissue of fish from the polluted site. The comet assay showed highly significant DNA damage in tilapia collected from the polluted site, compared to those from the reference site. Histopathological biomarkers and the comet assay may therefore be sensitive indicators of exposure to mixtures of aquatic pollutants in Nile tilapia.     

Y Chromosome aneuploidy,micronuclei, kinetochores and aging in men

This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant mcrease in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.     

Assessment of the genotoxicity of olive mill waste water (OMWW) with the Vicia faba micronucleus test

The present study concerns the genotoxicity of olive mill waste water (OMWW) generated in mills producing olive oil in Morocco. The Vicia faba micronucleus test was used to evaluate the genotoxicity of OMWW and the six major phenolic compounds identified by HPLC in this effluent. Five dilutions of OMWW were tested: 0.1, 1, 5, 10 and 20%. Maleic hydrazide was used as a positive control. The results showed that OMWW was genotoxic at 10% dilution. In order to investigate the components involved in this genotoxicity, the six major phenols present in this effluent, oleuropein, gallic acid, 4-hydroxyphenyl acetic acid, caffeic acid, paracoumaric acid and veratric acid, were studied at concentrations corresponding to the genotoxic concentration of the OMWW itself. Two phenols, gallic acid and oleuropein induced a significant increase in micronucleus frequency in Vicia faba; the four other phenols had no significant genotoxic effect. These results suggest that under the experimental conditions of our assay, OMWW genotoxicity was associated with gallic acid and oleuropein.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Genotoxicity of wastewater samples from sewage and industrial effluent detected by the Allium root anaphase aberration and micronucleus assays.

The genotoxicity of wastewater samples from sewage, and industrial effluent from the Amritsar, India, area were investigated using the Allium micronucleus and anaphase aberration assays. Raw sewage samples and acetone extracts of the dehydrated sewage were use for treatment of the Allium roots. Industrial effluents were collected and stored in the form of sludge (semi-dried matter). The acetone extracts of the sludge samples were also used for treatment of the Allium roots. From the Allium root micronuclei tests on the sewage extracts, no significant increase in the number of micronuclei was found in comparison with negative controls. All the other extracts from industrial effluent showed positive responses both in the micronucleus and anaphase aberration assays.     

The genotoxic risk of hospital, pharmacy and medical personnel occupationally exposed to cytostatic drugs--evaluation by the micronucleus assay

The aim of this study was to evaluate the genotoxicity of cytostatic drugs in hospital and pharmacy employees (n=100), occupationally exposed. The micronucleus assay was used to study lymphocytes in 247 peripheral blood samples. Samples were collected at "baseline level" without any cytostatic drugs exposure before recruiting or after at least 3 weeks without cytostatic drugs contact and at three times (cycle 1-3) post-exposure. Samples from 60 office employees served as controls. Furthermore, our results were compared to urinary analyses of cytostatic drugs (oxazaphosporines, anthracyclines, platinum) which were collected in parallel to the cytogenetic investigation. Statistical analyses were performed under consideration of age, gender and X-ray exposure. The frequency of micronuclei was significantly related to the age of the subjects (r(Spearman)=0.16; P<0.05). However, there were no significant differences in micronucleus rates between controls and exposed hospital workers. Similarly, micronucleus rates were not significantly different at the various sampling time points and there was no correlation between duration of employment and micronucleus rates. Furthermore, no correlation between current biomonitoring data of exposure (urine tests) and micronuclei frequency was found. Therefore, significantly increased genotoxic damage of the lymphocytes investigated in this study could not be demonstrated.     

Genotoxic effects of deltamethrin in the mouse bone marrow micronucleus assay

The genotoxicity of deltamethrin was studied in Swiss albino male mice (five animals/group) using the bone marrow micronucleus assay. Deltamethrin (two i.p. injections, 30 h and 6 h before sample collection) was found to induce micronuclei at 162.5 and 300.0 mg/kg body weight. A lower dose (32.5 mg/kg body weight) failed to induce a significant increase in micronuclei over the control level.     

Micronuclei and other nuclear anomalies in buccal smears: methods development

Laboratory work aimed at improving the epidemiologic utility of an innovative genotoxicity assay is described. The exfoliated cell micronucleus assay involves microscopic analysis of epithelial smears to determine the prevalence of micronucleation, an indicator of structural or numerical chromosome aberrations. While the assay holds promise for the study of epithelial carcinogens, it is hampered by the fact that exfoliated cells are moribund and undergo degenerative phenomena that can produce extranuclear objects difficult to distinguish from classical micronuclei. Modifications in the protocol were assessed in sample buccal smears from several study populations: radiotherapy patients, nonusers of tobacco, and snuff users. Refinements in micronucleus scoring criteria and the inclusion of other nuclear anomalies in the scoring system are proposed. We demonstrate that our criteria are successful in detecting excess micronucleation in positive controls. We also provide evidence that other nuclear anomalies are at least as common as micronucleation and that therefore there is the potential for extensive misclassification. Reliability was assessed in duplicate readings.     

Micronuclei and other nuclear anomalies in buccal smears: methods development.

Laboratory work aimed at improving the epidemiologic utility of an innovative genotoxicity assay is described. The exfoliated cell micronucleus assay involves microscopic analysis of epithelial smears to determine the prevalence of micronucleation, an indicator of structural or numerical chromosome aberrations. While the assay holds promise for the study of epithelial carcinogens, it is hampered by the fact that exfoliated cells are moribund and undergo degenerative phenomena that can produce extranuclear objects difficult to distinguish from classical micronuclei. Modifications in the protocol were assessed in sample buccal smears from several study populations: radiotherapy patients, nonusers of tobacco, and snuff users. Refinements in micronucleus scoring criteria and the inclusion of other nuclear anomalies in the scoring system are proposed. We demonstrate that our criteria are successful in detecting excess micronucleation in positive controls. We also provide evidence that other nuclear anomalies are at least as common as micronucleation and that therefore there is the potential for extensive misclassification. Reliability was assessed in duplicate readings.     

Ecogenotoxicological studies for an early toxicity screening and monitoring in Epinephalus chlorostigma and Scamberomorus commerson

The study was planned to investigate DNA fragmentation in fish to screen aquatic toxicity and in Epinephalus chlorostigma and Scamberomorus commerson collected from Red sea near Jizan, Saudi Arabia from three locations “(Corniche North park: “16.92161, 42.54631; Jizan Port: 16.874, 42.54952” N and Jizan Economic City: 17.26589, 42.34738“ ”)“ were used as a case study for the application of comet assay. The study area of the Red Sea is polluted due to anthropogenic activities and the disposal of wastes from multiple sources. Comet and micronucleus assays were used to detect genotoxicity in these fish species harvested from three sites. The concentration of Pb, Cr, Zn, Mn, Cu, Cd, Sn, and Hg was higher in the water samples collected from the polluted site compared to the non-polluted site of the Red sea. Comet assay for S. commerson showed significant (p < 0.05) genetic damage about 44.33 ± 3.03% DNA in comet tail at site S1. It was subsequently reduced to 31.71 ± 3.52% and 22.11 ± 2.52% at sites S2 and S3. E. chlorostigma also showed significant DNA in comet tail as 17.34 ± 2.19%, 11.87 ± 3.01%, and 36.41 ± 3.98% at site S1-S3, respectively. Significant (p < 0.05) DNA damage was observed in the fishes procured from non-polluted locations and upstream locations. The micronucleus induction in E. chlorostigma was recorded as 23.20 ± 4.19 and 2.20 ± 0.58%, respectively, non-polluted and polluted sites. S. commerson exhibited significant differences between polluted and non-polluted sites (44.80 ± 3.73 and 8.20 ± 2.20‰) polluted and upstream (44.80 ± 3.73 and 20.60 ± 4.02‰), respectively. A significant difference was obtained between E. chlorostigma and S. commerson for nuclear abnormalities S. commerson showed higher frequencies for nuclear deformities than E. chlorostigma. S. commerson showed substantial micronucleus induction frequencies collected from an area of low pollution intensity (upstream). This study showed that E. clorostigma and S. commerson could be successfully used as a bioindicator to determine the health of the Red Sea through the most specific assays such as comet and micronucleus tests as an early warning and to devise the monitoring strategies to ensure a safe supply of fish for human consumption.