ComparativeP-postlabeling analysis of DNA Adducts in Perch (Perca fluviatilis) from unpolluted and polluted areas in Swedish coastal waters

Perch (Perca fluviatilis), were sampled from unpolluted and polluted areas in Swedish coastal waters. The level of aromatic/hydrophobic DNA adducts in liver tissue was analyzed using the nucleas P1 version of the³²P-post-labeling assay. The level of total adducts measured in the individual fish from polluted areas was between 6 and 22 nmol of adducts/mol of nucleotides, and in the fish from the reference area between 0.2 and 0.6 nmol of adducts/mol of nucleotides.     

Increased toxicity of benzo(a)pyrene-7,8-dihydrodiol in the presence of polychlorobiphenylols

Benzo(a)pyrene (BaP) and polychlorinated biphenyls (PCBs) often co-exist in contaminated environments. Polychlorobiphenylols (OH-PCBs), formed by CYP-dependent monooxygenation of PCBs, are potent inhibitors of the glucuronidation of hydroxylated BaP metabolites. We hypothesized that OH-PCBs could drive the biotransformation of (−)BaP-7,8-dihydrodiol (BaP-7, 8-D) away from detoxication and towards formation of the reactive metabolite. A mixture of five OH-PCBs with 4–6 Cl atoms was infused into isolated, perfused, biliary intact livers (n=3 fish) removed from 3-methylcholanthrene-induced channel catfish. Controls (n=3) were infused with vehicle. Subsequently, [³H]-BaP-7, 8-D was infused into each liver and bile was collected for 1 h. The livers were taken for analysis of metabolites and DNA adducts. Induction status was confirmed by EROD assay. Bile was analyzed for metabolites. It was found that preinfusion of the mixture of OH-PCBs reduced the extent of glucuronidation of BaP-7, 8-D and increased the formation of DNA adducts 5-fold over controls. GSH conjugates, tetrols and triols were increased in the OH-PCB-infused fish, providing further support for our hypothesis that if the glucuronidation were inhibited, CYP-dependent activation would increase. These studies suggest a mechanism for synergy of toxicity of PAH and PCBs.     

Monitoring of the chemical pollution of the river Rhône through measurement of DNA damage and cytochrome P4501a induction in chub (Leuciscus cephalus)

The in vivo response of freshwater fish exposed to pollutants was assessed using two biomarkers, 7-ethoxyresorufin-O-deethylase (EROD) induction and DNA single strand breaks. Chub (Leuciscus cephalus) were caught in spring and in fall at various locations in the river Rhône watershed. EROD activity was measured in the liver while DNA damage was evaluated in chub erythrocytes using the recently developed Comet assay. Chemical contamination was evaluated both in fish muscle (PCBs) and in sediment (PCBs, PAHs, heavy metals) collected at each sampling station. Sex of individuals was shown to influence the level of EROD activity but not the level of DNA damage. The EROD activity as well as the DNA damage were found to be higher in the mostly contaminated stations compared to the reference one. This study shows that multibiomarker-based approach provides complementary informations about early effects in feral fish exposed to complex chemical pollution and highlights the interest of the Comet assay in genotoxicity assessment.     

环雷州半岛海域表层沉积物与鲬鱼汞污染研究

对2018年1月在广东环雷州半岛近海海域采集的海底表层沉积物、鲬鱼(Platycephalus indicus)进行汞含量的测定,分析比较了鲬鱼不同部位汞含量的差异,并采用单因子污染指数法对沉积物和鲬鱼的汞污染状况进行了风险评价。结果表明,环雷州半岛海域表层沉积物中汞含量范围为0.005×10^-6～0.359×10^-6,平均值为0.081×10^-6,雷州半岛东部海域表层沉积物汞含量高于南部和西部海域。鲬鱼的鳃中汞含量范围为0.032×10^-6～0.034×10^-6,平均值为0.033×10^-6;肌肉中的汞含量范围为0.065×10^-6～0.080×10^-6,平均值为0.073×10^-6;肝脏中的汞含量范围为0.228×10^-6～0.270×10^-6,平均值为0.249×10^-6。鲬鱼样品的汞含量都呈现出肝脏＞肌肉＞鳃。单因子污染指数评价结果显示,雷州半岛海域鲬鱼受到汞的轻微污染,雷州半岛东部海域表层沉积物存在汞污染生态风险,而其南部、西部海域尚未受到汞污染的威胁。     

Behavior and brain chemistry correlates in mummichogs (Fundulus heteroclitus) from polluted and unpolluted environments

The ability of Fundulus from a polluted environment to capture a prey organism, the grass shrimp (Palaemonetes pugio), was compared to that of Fundulus from an unpolluted reference site. Fish from the unpolluted site took significantly more prey (p < 0.1) during the first 5–10 min of each trial than fish from the polluted site.Analysis of the neurotransmitters dopamine (DA) and serotonin (5-HT) and their metabolites, DOPAC, homovanillic acid (HVA), and 5-hydroxy-indolacetic acid (5-HIAA) in cerebellums and medullas was performed by HPLC. Mummichogs from the polluted site had significantly lower concentrations of 5-HT and its metabolite 5-HIAA in their medullas, but not cerebellums, than fish from the reference site (p < 0.05). Concentrations of DA, DOPAC and HVA were not different between populations in either part of the brain. Neurotransmitter concentrations were shown to be independent of size and therefore, presumably, age.Analysis of Hg in livers and brains of these mummichogs by atomic absorption spectrophotometry showed significant differences between the two populations for both organs. Earlier work showed that the behavioral deficit could be elicited by Hg; thus, this was the putative neurotoxic agent. However, there are other candidates which may contribute to neurotoxicity in the study site.     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

The interplay between mixed function oxygenases and DNA alteration under PAH pollution

Our experience in DNA alteration assessment tells us that aquatic vertebrates, in general, and also some of the invertebrates, by living in polluted areas acquire altered DNA; DNA alterations being defined either as single-strand breaks, and/or alkali sensitive or single-strand specific nuclease-cleavable sites (sse). This can be shown by several methods: (i) Alkaline filter elution;¹ (ii) fluorescence analysis of DNA unwinding;² (iii) alkaline sucrose density gradient centrifugation;³ (iv) alkaline unwinding analysis with hydroxyapatite differentiation;⁴ (v) nucleoid sedimentation analysis;⁵ (vi) supercoiled DNA relaxation detection by electrophoresis;⁶ and (vii) measurement of DNA molecular weight distributions with the electron microscope.⁷ The amount of sse per DNA mass can be a function of either an increased activity of DNA damaging agents or agents that inhibit DNA repair. Among DNA damaging agents are polycyclic aromatic hydrocarbons (PAH), that can cause sse by the ‘fast effect’⁸ or after induction upon uptake, through the action of mixed function oxygenase (MFO). Since MFO induction within certain limits is a function of PAH concentration measurement of this enzyme activity enables at least partial interpretation of the DNA alteration status, which can be a two-phase process.     

Frequencies of liver neoplasia in a feral fish population and associated carcinogens

Previous research had documented prevalent liver neoplasia within feral fish populations from half a dozen polluted waterways of North America.^1–3 A brown bullhead, Ictalurus nebulosus, population in the Black River, Ohio, had a 33% prevalence of grossly observable liver tumors in fish older than 2 years compared to 0 % in 249 bullhead from a reference location (Buckeye Lake).⁴ To approximate the actual prevalence of gross plus microscopic lesions combined in the Black River population, we captured 125 bullhead at least 250 mm in length in 1982. Livers were preserved for histopathology and pectoral fin spines were removed for aging. On average, each liver was sectioned at five levels for diagnosis. Bullheads (whole fish) and Black River sediment were analyzed for polynuclear aromatic hydrocarbons (PAHs) by gas chromatography-mass spectrometry as previously reported.^4,5 Bullheads were found to have a higher incidence of liver tumors than was grossly apparent and to contain elevated levels of polynuclear aromatic hydrocarbons (PAHs).     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3^′,4,4^′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

Nitroaromatic stimulation of superoxide production in three species of freshwater fish

Nitroaromatic compounds frequently contaminate aquatic systems and may, therefore, impact fish. However, a known pathway of nitroaromatic toxicity in mammals, that of nitroaromatic stimulated superoxide (O₂⁻) production, has yet to be addressed in fish. In this study we investigated this pathway in three species of freshwater fish-channel catfish (Ictalurus punctatus), largemouth bass (Micropterus salmoides) and rainbow trout (Salmo gairdneri)-exposed to nitrofurantoin (NF), p-nitrobenzoic acid (PNBA) and m-dinitrobenzene (MDNB). Our results indicate that these nitroaromatics elicit a stimulation of O₂⁻ production by fish liver fractions. Additionally, results suggests a similarity between, fish and mammals in the nitroreductases which mediate nitroaromatic activation and subsequent O₂⁻ production. These findings indicate a potential toxic consequence of fish exposure to nitroaromatic contaminants in aquatic systems.     

The impact of intrapopulation variability in reproductive traits on population reproductive potential of Grand Bank American plaice (Hippoglossoides platessoides) and yellowtail flounder (Limanda ferruginea)

In geographically extensive fish populations the potential exists for reproductive traits to vary over the population’s range but, the impact that such intrapopulation variability has on overall population reproductive potential has not been formally assessed. Here intrapopulation spatial variability in size at maturity and fecundity are demonstrated for Grand Bank American plaice (Hippoglossoides platessoides) and yellowtail flounder (Limanda ferruginea). Recognition of intrapopulation variability in these reproductive traits coupled with spatial variability in abundance resulted in an increase in estimated population total egg production (TEP) by as much as 10¹⁴ eggs for American plaice and 10¹⁵ eggs for yellowtail flounder as compared to assessment of TEP for the population as a whole. Results highlight the need to explore variability in life history traits not only between but, also within populations and emphasizes the need for sufficient spatial coverage during sampling in order to assess the reproductive potential of fish populations.     

Comparison of microsomal lipid peroxidation in fish and rats

Lipid peroxidation, which is promoted in animal tissues by a variety of toxic agents, is believed to be associated with disruption of cell membranes and loss of activity of membrane-bound enzymes. While the process of lipid peroxidation has been well studied in mammals, only a few non-mammalian species have been examined.¹ In the present study, in vitro assays for lipid peroxidation were performed using microsomes prepared from the livers of rats (Rattus rattus) and a marine fish, mullet (Mugil cephalus). Although lipid peroxidation was observed in microsomes prepared from both mullet and rat tissues, and was stimulated by xenobiotics, the mechanisms of generation appeared to be different. In contrast to rats, NADH was much more effective than NADPH in stimulating lipid peroxidation in mullet liver microsomes. The results suggest that NADH-dependent, as well as NADPH-dependent, lipid peroxidation may be important in vertebrate tissues.     

The effect of supplementary dietary copper on copper and metallothionein levels in the fish, dab, Limanda limanda

Common dab were fed a casein based diet containing copper at a concentration of 1·4 mg copper kg⁻¹ (dry weight) or a similar diet supplemented with 200 mg copper kg⁻¹. Fish were sampled after 10 weeks and 20 weeks and hepatic and renal copper, zinc and metallothionein concentrations were measured. All hepatic cytosolic copper in test and control fish co-eluted from Sephadex G-75 with metallothionein, as did some of the cytosolic zinc. After 20 weeks there was three times as much of this metallothionein-bound copper in the livers of the test fish compared to the control fish, although metallothionein concentration was little elevated. On the basis of these results it is proposed that cytosolic liver copper concentration or heat stable liver copper concentration can be used as an indication of excess dietary copper in the dab Limanda limanda.     

Production of fish in two small streams in the north island of New Zealand

Data are presented on biomass, mortality, growth, and production of fish populations inhabiting two small streams in the Wairarapa district of the North Island of New Zealand. The main water of both streams contained a mixed population of bullies (Philypnodon breviceps Stokell), brown trout (Salmo trutta Linn.), and eels (Anguilla dieffenbachii Gray, and A. australis schmidtii Phillipps). The headwater tributaries of one of the streams contained a large population of Galaxias divergens Stokell and a few eels, but other fish species were absent. All species of fish appeared to be using the same food supply of small benthic invertebrates, mainly ephemeropteran and dipteran larvae. Biomass of benthos per unit area was appreciably higher in the headwater tributary examined than in the main waters downstream. Mean annual stock of benthos in the tributary (Hinau north branch) was 19.75 g/m², in the Hinau main water 10.71 g/m² and in the Hinaki main water 13.47 g/m² Nevertheless, production per unit area from the nearly monospecific fish population in the Hinau north branch (14.28 g/m²) was less than that of the mixed populations in the main waters (24.17 g/m² in the Hinau, 73.54 g/m² in the Hinaki). Possibly fish production in the headwaters might be greater if the species composition of fish was more diverse.     

嗜水气单胞菌(Aeromonas hydrophila)引起斑马鱼肠道生理损伤和肠道菌群失调

研究分析了嗜水气单胞菌(Aeromonas hydrophila)感染斑马鱼后肠道生理健康和肠道微生物的变化。以斑马鱼为研究对象,刮伤皮下真皮,使用105 CFU/mL浓度的嗜水气单胞菌暴露6 h后转入清水,分别在暴露前、暴露后6 h、12 h、24 h取样。使用鱼特异性酶联免疫吸附试验(ELISA)试剂盒,检测肠道中紧密连接蛋白ZO-2 (TJP2)含量和乙氧基异戊二烯-O-脱乙基酶(EROD)酶活性,结果表明,嗜水气单胞菌暴露引起了斑马鱼肠道生理损伤,表现为肠道TJP2含量、EROD酶活性在暴露后显著下调。采用16S rRNA高通量测序检测肠道微生物及其菌群结构变化,嗜水气单胞菌暴露斑马鱼后致病菌不动杆菌属(Acinetobacter)明显增加,OTUs数量明显下降,Alpha多样性降低,结果说明嗜水气单胞菌引起了斑马鱼肠道菌群失调。肠道中微生物的多样性与改善肠道上皮和黏膜屏障功能紧密相关,该研究对从肠道健康的角度评价嗜水气单胞菌毒性,探索嗜水气单胞菌的致病机制具有重要意义。     

GFP标记的迟缓爱德华菌感染诸氏鲻虾虎鱼后组织分布研究

为探讨迟缓爱德华菌(Edwarsiellatarda)入侵途径,建立感染模型,作者通过电转化法构建GFP标记的迟缓爱德华菌EtMc1512(质粒PMDpp-EGFP),实验设立浸泡组、腹腔注射组和肌肉注射组,感染后采集各组实验诸氏鲻虾虎鱼(Mugilogobius chulae)血液、鳃、肝脏、肠、肌肉,培养法统计分析各组织中的荧光细菌数;浸泡组取样时间为0、2、4、6、8、12、24 h,腹腔注射组和肌肉注射组取样时间为6、12、24、48、72、96h。结果显示,构建的EtMc1512-GFP具有较强荧光,GFP标记前后菌株毒力基因(citC、mukF、esrB、katB、fimA、gadB)检测结果均为阳性。浸泡感染后实验鱼各组织内的荧光菌随时间表现为先升后降的趋势,最高菌量出现在肠道(2.51×106CFU/g),其次为鳃(4.19×104CFU/g)、血液(1.65×104CFU/g),肠道荧光菌显著高于其他组织(P0.05);腹腔注射感染后肝脏(4.55×106CFU/g)和血液(4.65×106CFU/g)菌量最高;肌肉注射感染后肌肉在48h首先检出荧光菌,血液(2.93×104 CFU/g)菌量最高。结果表明,肠道、肝脏和肌肉分别是迟缓爱德华菌浸泡感染、腹腔注射感染和肌肉注射感染诸氏鲻虾虎鱼的主要组织器官,在自然条件下迟缓爱德华菌经口感染诸氏鲻虾虎鱼风险较高。     

盐度突变对半滑舌鳎血浆渗透压和鳃丝Na+/K+-ATP酶活性的影响

研究了半滑舌鳎(Cynoglossus semilaevis)由盐度30突变至0、1O、20、35和40盐度后血浆渗透压和鳃丝Na+/K+-ATP酶活性的变化.结果表明,盐度对半滑舌鳎血液渗透压和鳃丝Na+/K+-ATP酶活性均有显著影响(P＜0.05).盐度突变后,各处理组的血液渗透压和鳃丝Na+/K+-ATP酶活性...     

Municipal wastewater contamination in the southern california bight: Part I—metal and organic contaminants in sediments and organisms

Sediments and organisms were examined for concentrations of organic and metal contaminants from near the Los Angeles County (JWPCP) municipal outfall at Palos Verdes (PV) station 7-3, the Los Angeles City (Hyperion) municipal outfall at Santa Monica Bay (SMB) station 6-4 and reference station SMB 2–3 near Malibu Beach. Flows and mass emission rates of suspended solids, PCBs, Cd and Zn were similar at the two outfalls. Mass emission rate of copper was almost twice as high from Hyperion as from JWPCP, while mass emission rate of DDTs was an order or magnitude higher from JWPCP than from Hyperion.Surficial sediments at PV 7-3 were enriched in most contaminants relative to SMB 6-4 and relative to the mass emission rates of contaminants from the JGVPCP and Hyperion outfalls. Some of this enrichment could be accounted for by the greater accumulation of organic material, measured as total volatile solids, at PV 7-3 relative to SMB 6-4. Some might be accounted for by resurfacing of more contaminated historical deposits buried at PV 7-3. Some of the enrichment of DDTs relative to PCBs could be accounted for by the greater abundance of oxygenated metabolites of PCBs (PCBols) relative to DDTs (DDTols) in sediments.The degree of contamination of organisms by DDTs increased with proximity to PV 7-3 but contamination by PCBs was similar at PV 7-3 and SMB 6-4. DDT concentrations in fish livers ranged from 12 ± 4 ( ) mg/wet kg in longspine combfish from SMB 2–3 to 610 ± 105 (n = 5) mg/wet kg in Pacific sanddab from PV 7-3. DDT concentrations in fish gonads ranged from 0·003 ± 0·003 (n = 5) mg/wet kg in yellowchin sculpin from SMB 6-4 to 1.5 ± 6 (n = 3) mg/wet kg in Pacific sanddabs from PV 7-3. PCB concentrations in fish livers ranged from 1·2 ± 0·4 (n = 4) mg/wet kg in yellowchin sculpin from SMB 2–3 to 16 ± 3 (n = 6) in Pacific Sanddab from SMB 6-4. DDT and PCB concentrations in invertebrate hepatopancreas were only slightly lower than those in fish livers. DDTols and PCBoIs comprised an average of 91 % of the total of parent compounds and oxygenated metabolites in sediments and 66 % in livers and hepatopancreas. Trace metals were frequently decreased in livers and hepatopancreas from near outfalls even though they were highly elevated in sediments.Comparison of sediment and tissue chlorinated hydrocarbon data with that from Elliot and Commencement Bays, Puget Sound, indicated that none of the southern California coastal stations considered in this study were sufficiently lacking contamination to be considered as adequate control sites.     

Health status of red mullets from polluted areas of the Spanish Mediterranean coast,with special reference to Portmán (SE Spain)

A suite of general physiological indicators and hepatic biomarker responses were determined in red mullet (Mullus barbatus) from priority pollution areas of W Mediterranean Sea, including the highly metal polluted area of Portmán (Cartagena, SE Spain). Concentrations of metals and persistent organic pollutants in fish muscle tissue and sediment samples were also analysed. Our results showed that fish from Portmán accumulated the highest concentrations of mercury, lead and arsenic and also of some polycyclic aromatic hydrocarbons and polychlorinated biphenyls congeners. In addition, they had significantly lower condition factor, muscle lipid content and gonadosomatic index, as well as the lowest levels of DNA integrity and the highest ethoxyresorufin-O-deethylase (EROD) activity in liver of the areas investigated. Contaminant body burden in fish only partly corresponded to chemical characteristics of the sediments in the areas in which they were collected. Our findings indicate that red mullets from Portmán had suboptimal health status that warrant further study.     

Biology of the redspotted tonguesole Cynoglossus zanzibarensis (Pleuronectiformes: Cynoglossidae) on the Agulhas Bank,South Africa

The biology of the redspotted tonguesole Cynoglossus zanzibarensis, a common African cynoglossid inhabiting the Agulhas Bank, South Africa, is described. Growth studies based on sectioned sagittal otoliths revealed that C. zanzibarensis is relatively fast-growing and long-lived, attaining ages >8 years. Growth in length was rapid in immature fish, fish attaining 56% of their maximum size within their first year. By sexual maturity, fish had attained 28% of their maximum age and 68% of their maximum length. Total length-at-age was best described by the Von Bertalanffy growth model with combined-sex growth described as L_t = 354.78(1?e^{?0.43 (t+1.17)}) mm TL. Sexually dimorphic growth patterns were evident, females attaining larger lengths, but at a slower growth rate than males. Despite the similar mean size of adult fish, the trawl-sampled adult population was dominated by females, with a sex ratio of 1 male:2.4 females. Female C. zanzibarensis mature in their second year of life (275 mm TL), after which they spawn small, pelagic eggs throughout the year. Approximations of the rates of total, natural and fishing mortality were estimated to be 0.62, 0.48 and 0.14 year^?1 respectively.