Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.     

Assessing DNA damage in cnidarians using the Comet assay

The assessment of DNA damage by the Comet assay has been described as a useful non-specific general biomarker of stress in many marine organisms. In field situations it has successfully been employed to distinguish between reference and polluted sites and in the laboratory it has been widely used as a mechanistic tool to determine pollutant effects and mechanisms of DNA damage. To date a wide range of marine vertebrates and invertebrates have been used, however, the usefulness of this assay as a biomarker in cnidarians has not yet been assessed. The aims of this study were to optimize the Comet assay for cnidarian cells and to assess its utility for detecting genotoxic damage in these cells. Cells were isolated from the North American pacific coast temperate sea anemone Anthopleura elegantissima using a non-enzymatic dissociation procedure and viability was determined to be in excess of 90%. Cells were incubated either with (1 h acute exposures) hydrogen peroxide (H(2)O(2)), ethylmethanesulphonate (EMS) or benzo(a)pyrene (B[a]P). In comparison to other marine species, anemone cells exhibited high control or background levels of DNA strand breaks. Despite this, however, we observed dose responses for each of the study chemicals with no reduction in cell viability. This study demonstrates that anemone cells respond to known DNA damaging agents, including B[a]P which requires metabolism to exert its genotoxic effect, and that the Comet assay may prove to be a useful biomarker of stress in cnidarian species.     

DNA strand breaks in grass shrimp embryos exposed to highway runoff sediments and sediments with coal fly ash

Embryo production was reduced in female grass shrimp exposed to sediments with added coal fly ash and to sediments collected from an estuarine station containing high PAH concentrations due to its proximity to a highway storm drain. Grass shrimp embryos exposed to pore water from the high PAH and high metal sediments showed both reduced hatching and increases in DNA strand breaks (comet assay). Sediments with added coal fly ash had high concentrations of vanadium and selenium which may have contributed to effects similar to those observed with sediments with high PAH. The embryo pore water bioassay (hatching/DNA strand breaks) gave results comparable to those observed for reproduction effects (reduced embryo production/embryo hatching) with female grass shrimp exposed to whole sediment.     

Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay

The single-cell microgel electrophoresis assay or the comet assay was used to evaluate DNA damage of dispersed crude oil on sea urchins (Strongylocentrotus droebachiensis) and mussels (Mytilus edulis L.). Sea urchins were exposed to 0.06 and 0.25 mg/L dispersed crude oil in a continuous flow system, while the mussels were exposed to 0.015, 0.06 and 0.25 mg/L dispersed crude oil. Sea urchin coelomocytes and mussel haemocytes were sampled after 4 and 5 weeks exposure, respectively. In the sea urchin coelomocytes, there was a significant concentration-related increase in the percentage of DNA in comet tail. In mussel haemocytes, there was a significantly higher percentage of DNA in comet tail for all treatments compared to the control. The responses were concentration-related up to 0.06 mg/L oil. The two highest exposure concentrations of mussels were not significantly different from each other. These results indicate that the comet assay can be used for biomonitoring of DNA damage in marine invertebrates following oil contamination.     

Interactive effects of UV, benzo[alpha] pyrene, and cadmium on DNA damage and repair in embryos of the grass shrimp Paleomonetes pugio

We examined the link between DNA strand breaks and hatching rates in grass shrimp, (Paleomonetes pugio), embryos exposed to 0.2 microM benzo[alpha] pyrene (BP), 5 microM cadmium (Cd) and 330 kJ/m(2) UV light, either alone or together. After exposure, embryos were transferred to clean seawater with or without 5 microM Cd. Hatching rates and DNA strand breaks (Comet Assay) were determined. DNA lesions caused by exposure to BP, UV light, or BP/cadmium were rapidly repaired and were not associated with any effects on hatching. Exposure to Cd after exposure to BP or UV did not affect embryological development or DNA repair. Exposure to BP/UV resulted in a high level of DNA lesions which were slowly repaired. Exposure to cadmium following BP/UV exposure inhibited hatching and DNA repair. Adducts formed during exposure to BP/UV exposure may be difficult to excise or may saturate the nucleotide excision repair system.     

Histopathological changes in grass shrimp exposed to chromium, pentachlorophenol and dithiocarbamates

This report deals with the histopathological/ultrastructural changes in various tissues of grass shrimp (Palaemonetes pugio) exposed to hexavalent chromium, pentachlorophenol (PCP) and two dithiocarbamate formulations: Aquatreat DNM-30® (15 % sodium dimethyl dithiocarbamate plus 15 % disodium ethylene bis-dithiocarbamate) and Busan-85® (50 % potassium dimethyl dithiocarbamate). The pathological alterations induced by dithiocarbamates and PCP were most severe and first evident in the gills. Of the biocides tested, the dithiocarbamates caused the most extensive branchial damage—the so-called black gill syndrome, involving early melanization and eventual lamellar truncation. Hexavalent chromium, on the other hand, did not induce marked changes in the gills, but it caused invasive melanized cuticular lesions (particularly at the articulations of the pereiopods, pleopods and abdominal segments). Additionally, chromium caused apparent labyrinth hypoactivity in the antennal glands, whereas the dithiocarbamates seemed to induce labyrinth hyperactivity. Hepatopancreatic histopathology was more severe in shrimp exposed to chromium and PCP than in dithiocarbamate-exposed shrimp. The apparent mitotic activity in the hepatopancreas was increased in Aquatreat-exposed shrimp and suppressed in chromium-exposed shrimp; many mitotic figures in the latter case were abnormal. All four compounds caused varying degrees of midgut epithelial hypertrophy, cytoplasmic vacuolization and diminution of basal tubular endoplasmic reticulum, but only PCP caused widespread rupture of midgut epithelial cell apices. Additional notable ultrastructural anomalies included: mitochondrial compartmentalization in PCP-exposed shrimp; mitochondrial fusion in dithiocarbamate-exposed shrimp; and unusual intranuclear inclusions in different tissues of chromium-exposed shrimp. These pathological changes point to differences in the mechanisms of pollutant toxicity and indicate their potential use in the biological monitoring of aquatic pollutants.     

蛋白质精氨酸甲基转移酶1调控DNA损伤修复和细胞凋亡

蛋白质精氨酸甲基转移酶1 (protein arginine methyltransferases 1, PRMT1)是PRMT家族中的重要一员,属于Ⅰ型PRMTs,细胞内超过85%的蛋白质精氨酸的甲基化由其催化。PRMT1通过调节底物的甲基化水平,从而调控蛋白的功能及蛋白参与的细胞活动和生理过程,包括细胞信号转导,DNA损伤修复,转录调控,RNA代谢,蛋白质相互作用等。本文着重讨论PRMT1的结构、底物及其在DNA损伤修复和细胞凋亡中的功能。     

醚菊酯对日本囊对虾肝胰腺细胞DNA损伤效应的研究

为检测中国近海海洋环境中菊酯类农药等污染物对日本囊对虾(Marsupenaeus japonicas)的基因毒性及其危害程度,作者首次使用醚菊酯对日本囊对虾进行毒理实验,将日本囊对虾分为6组进行不同剂量的处理,采用彗星实验分析技术对处理培育25 d的日本囊对虾DNA损伤情况进行检测,并通过Comet Score 1.5分析软件对实验图谱进行拖尾率、尾长、彗尾DNA相对含量、尾距、Olive距等DNA损伤指标的统计分析,在对各指标分别建立一元回归方程的基础上,又通过SPSS软件进行判定分析,筛选影响作用更大的检测指标,建立了多参数的多元回归方程。实验结果发现,与空白对照组相比,日本囊对虾各实验处理组肝胰腺细胞DNA均有不同程度的损伤且各检测指标均有显著性差异(P0.01),各检测指标与处理浓度表现出良好的剂量效应关系,随着处理浓度递增,各检测指标都呈规律性的增长趋势。回归分析和相关系数表明:各检测指标与处理浓度具有高度的相关性,多项式回归方程(P0.05)和多元回归方程(P0.005)均有显著性的统计学意义,通过多元回归方程可以有效地推断醚菊酯处理浓度和处理时间。     

DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.     

0#柴油水溶性成分对僧帽牡蛎DNA损伤的初步研究

在实验条件下,将僧帽牡蛎(Ostrea cucullata)分别置于含有低(0．5mg/L)、中(2mg／L)和高(5mg／L)3种浓度0^#柴油水溶性成分的海水中,利用碱解旋法测定消化腺和鳃INA损伤的程度。结果表明,INA损伤的程度随污染时间的延长而增加,并存在一定的剂量-效应关系;解除污染后,DNA损伤恢复到对照水平。DNA损伤可作为监测海洋石油污染的生物标志物。     

Exposure of grass shrimp to sediments receiving highway runoff: Effects on reproduction and DNA

A grass shrimp bioassay was carried out on sediments from three estuarine stations which were different distances from a highway storm drain. Total polycyclic aromatic hydrocarbon (PAH) concentrations were 29, 1.5 and 0.1 μg/g sediment at stations A (next to drain), B (100 m from drain) and C (500 m from drain), respectively. Lower embryo production and embryo hatching rates and a higher level of DNA strand breaks (comet assay) were observed in grass shrimp exposed to stations A and B sediments. There appeared to be an association between reproduction abnormalities and increased DNA strand breaks as a result of grass shrimp exposure to estuarine sediments receiving highway runoff.     

三种典型POPs对紫贻贝不同组织DNA损伤的比较研究

采用彗星电泳技术,比较研究了Aroclor 1254、BaP、DDT三种典型POPs对紫贻贝(Mytilus edulis)鳃、性腺、消化腺细胞的DNA损伤效应的差异,以期为进一步评价这三种典型POPs遗传毒理机制及其预警监测提供科学依据.结果表明,Tail DNA%是评价DNA损伤的理想指标,另外,在急性毒性试验中,...     

DNA adducts in mussels and fish exposed to bulky genotoxic compounds

Biological and procedural factors can influence DNA adduct detection in aquatic organisms. Among them, functional structure and metabolic traits represent major biological determinants for adducts formed by lipophilic pro-mutagenic contaminants. In detecting DNA adducts through the 32P-postlabelling assay, efficiency in DNA purification, digestion, labelling, as well as adduct enrichment and quantification may explain differences between independent studies. Reference DNA adducts have been used to verify some 32P-postlabelling aspects. Data obtained for mussels and fish treated with benzo[a]pyrene (B[a]P) and environmentally exposed to genotoxins confirm the above assertions. Although the 32P-postlabelling assay cannot be proposed for routine biomonitoring it appears a reliable and very sensitive index of exposure to genotoxic pollutants in both fish and mollusks.     

Correlative changes in metabolism and DNA damage in turbot (Scophthalmus maximus) exposed to benzo[a]pyrene

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P < 0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.     

Avoidance behaviour of freshwater fish and shrimp exposed to ammonia and low dissolved oxygen separately and in combination

Abstract

The responses of common smelt (Retropinna retropinna Richardson), inanga (Galaxias maculatus Jenyns), common bully (Gobiomorphus cotidianus McDowall), and shrimp (Paratya curvirostris Heller) to ammonia and low dissolved oxygen (DO), separately and in combination, were measured in a fluvarium. Only common smelt avoided high or low ammonia (c. 8.5 and 2.0 g m^?3 NH3, respectively) and low DO (c. 2 g m^?3) alone and in combination. Shrimp avoided high ammonia and low DO+ammonia but not low DO on its own. Inanga and bullies showed no significant negative response to either contaminant; rather, inanga were strongly attracted to low ammonia and bullies to low DO. These results parallel those from toxicity experiments that show common smelt and shrimp are usually among the most sensitive native species to various contaminants. The consistent and appropriate avoidance behaviour shown by common smelt, in particular, suggests this species would be a good indicator organism for assessing the health of lowland waterways.     

Genetic variability of the blue and red shrimp Aristeus antennatus in the Western Mediterranean Sea inferred by DNA microsatellite loci

Genetic variation at eight microsatellite loci was studied in nine populations of the blue and red shrimp Aristeus antennatus to investigate whether distinct stocks are present in the Western Mediterranean Sea. A high level of gene flow and no evidence of genetic partitioning were discovered. No significant variation was found (F_ST = 0.00673, P-value = 0.067) even when shrimps from exploited and those from deep-water unexploited grounds were compared. No evidence of reduction or expansion of population size in the recent past was found, as indicated by the bottleneck and interlocus g-tests. Our results are consistent with previous studies using mitochondrial gene methods and allozymes, indicating that, for this species, extensive pelagic larval dispersal and adult migration are probably responsible for the genetic homogeneity observed. In particular, due to a different bathymetric distribution of males and females, reported to be associated with different water masses and hence with possible differential dispersal capacity between sexes, the hypothesis of sex-biased dispersal was tested. Mean values of corrected assignment indices and mean relatedness values were higher for males, suggesting that females are the more widely dispersing sex. Molecular assessment of A. antennatus from the Western Mediterranean provides data of biological and evolutionary interest for the successful management of such a highly valuable fishery resource.     

Teratogenesis in Fundulus heteroclitus embryos exposed to a creosote-contaminated sediment extract and CYP1A inhibitors

The goal of these experiments was to explore the relationship between cytochrome P4501A (CYP1A) induction and the teratogenicity of sediments from the Atlantic Wood Industries Superfund site (Elizabeth River, VA) in Fundulus heteroclitus embryos. In these experiments we used embryos spawned from reference site adults to assess CYP1A activity and teratogenicity induced by aqueous Elizabeth River sediment extracts (ERSE). Embryo exposures to ERSE induced CYP1A activity and caused deformities, including pericardial edema, heart elongation and tail shortening. Co-exposures with various CYP1A inhibitors significantly decreased CYP1A activity and increased the teratogenicity of the sediment extract. Potential mechanisms for this increased toxicity are discussed herein.     

苯并(a)芘和芘对梭鱼肝脏DNA损伤的研究

用苯并(a)芘、芘以及它们的等量混和物,分别在浓度为0.1,1,10,20,50μg/dm3浓度下对梭鱼暴污,5d后取梭鱼肝脏和鳃用碱解旋法分别测定其DNA的损伤,结果随着污染物浓度的增加,肝脏DNA损伤程度增加;在相同浓度下,苯并(a)芘和芘的联合毒性大于苯并(a)芘和芘分别作用时的毒性之和。所以苯并(a)芘和芘对DNA损伤的联合作用应为加强作用。