A novel ABC transporter: The first insight into zebrafish (Danio rerio) ABCH1

Abch1 is a novel ATP binding cassette (ABC) transporter found in fish. This study represents an initial characterisation of this transporter using phylogenetic analyses, membrane topology prediction and determination of its tissue expression pattern in adult zebrafish (Danio rerio). Blast search showed that Abch1 orthologs are not present in genomes of other vertebrate taxa and similar genes are found only in invertebrate genomes. Abch1 is most closely related to the ABCG subfamily, although it shares only 12–14% of amino acid sequence identity with ABCG subfamily members. Topology analysis indicated that Abch1 is a half transporter that consists of six transmembrane domains with reverse domain arrangement (NBD-TMD), like ABCG subfamily members, but with differences in loop organization. Tissue distribution pattern revealed the highest Abch1 expression in brain, gills and kidney, followed by lower expression in intestine, gonads, skeletal muscle and liver. Considering moderate similarity in topology and tissue distribution pattern between Abch1 and ABCG subfamily members, we speculate that Abch1 is either involved in sterol transport similar to ABCG1, or is a part of the multidrug/multixenobiotic defence like ABCG2. These hypotheses remain to be addressed in further research.     

The impact of musk ketone on reproduction in zebrafish (Danio rerio).

Nitro musks are a group of nitrated benzenes mainly used by the fragrance industry as a substitute for natural musk. Two of the most common nitro musks, musk xylene and musk ketone, have been detected in water, fish, human adipose tissue, as well as in human breast milk. In this study, female zebrafish were dietary-exposed to musk ketone, prior to spawning with unexposed males. The fish were exposed for 8 weeks at two different dose levels. Accumulation of the compound in the fish as well as reproductive success were studied. Exposed females had reduced body weight and length, as well as reduced liver- and gonad somatic index. The results from the reproduction study showed a dose-dependent reduction in fecundity. Early life-stage mortality was increased and the median survival time was reduced. In addition, an embryo/larvae toxicity test was performed, using newly fertilized zebrafish eggs from unexposed parental fish. Eggs were exposed to a series of different concentrations of musk ketone via the surrounding water. A NOEC value of 10 micrograms/l and a LOEC value of 33 micrograms/l was determined. Conclusively, the present study clearly shows that musk ketone negatively affects reproduction and early life-stage survival in zebrafish.     

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio)

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides.     

Trace elements and polycyclic aromatic hydrocarbons (PAHs) concentrations in deep Gulf of Mexico sediments

The concentrations of polycyclic aromatic hydrocarbons (PAHs) and trace elements were determined for surface (top 2 cm) sediment samples collected during the deep Gulf of Mexico benthos (DGoMB) study .These elements and compounds are known to be toxic to organisms at high concentrations and may affect biological communities. There is no indication of major anthropogenic input of the elements Be, Co, Cr, Fe, Si, Tl, V, K, Mg, Ca, Sr and Zn, based on normalization to Al. The concentrations of these metals in the sediment are a function of the relative amounts of trace-metal-rich Mississippi River-derived silicate material and trace-metal-poor plankton-derived carbonate. This is not true for the elements Ba, Ni, Pb, Cd, As, Cu and Mn, whose concentrations show considerable scatter when normalized to Al and a general enrichment. On a normalized basis, Mn is enriched 5–10 fold, Cu and Ni 2–3 fold and Pb 2 fold over Mississippi River-derived material. These enrichments are likely the result of remobilization of metals from depths in the sediment column where reducing conditions exist. The Ba concentrations at selected sites are higher than those of average clay-rich sediments, but are typical of sediments from near oil well platforms in the northern Gulf of Mexico. In the case of Ba, it seems likely that the enrichments, as high as a factor of 10, are due to disposal of oil well drilling mud. The Ba-enriched samples are from the three shallowest water sites in the Mississippi Trough (sites MT1, 2 and 3) and from site C1 and WC5. All are in an area of intense petroleum exploration and development. PAH concentrations are also elevated at MT1, MT3 and C1. The total PAH concentration ranged from not detected (ND) to 1033 ng/g with a mean of 140 ng/g. Even at the sites most enriched in PAHs and trace elements, the concentrations are not at the levels expected to adversely affect the biota. However, these predicted non-effects are based on research using mostly near-shore estuarine species, not on the indigenous species at the sampling sites.     

Natural background and anthropogenic inputs of polycyclic aromatic hydrocarbons (PAH) in sediments of South-Western Barents Sea

Polycyclic aromatic hydrocarbons (PAH) were measured in sediment cores from 13 locations in South-Western Barents Sea as part of a detailed study of the Norwegian seabed under the MAREANO program. The generally low PAH levels found, an average around 200 ng g⁻¹ dry weight for sum PAH, indicate low inputs of petroleum hydrocarbons to the marine environment in the area. Differences in PAH composition and various PAH ratios indicate a natural, mostly petrogenic origin of PAH in sediments from the open sea locations, while the fjord locations show higher pyrogenic PAH contents with an increase towards upper sediment layers, indicating low inputs from human activities. Petrogenic PAH levels increase in deeper sediments at open sea locations, also when normalised to total organic carbon (TOC) contents, suggesting natural leakages of oil-related hydrocarbons in the area.     

珠江虎门潮汐水道水体中多环芳烃的分布及季节变化

采用GF/F玻璃滤膜对珠江虎门潮汐水道6个表层水样进行过滤分离出多环芳烃的颗粒相和溶解相,并根据美国EPA标准对多环芳烃进行了定量分析.结果显示,洪、枯季水体中16种优先控制多环芳烃总量分别是223～614,6 559～20 031 ng/dm3;洪、枯季多环芳烃总量分别为786～2 098,11 360～34 338 ng/dm3;广州前航道的污染较狮子洋严重.珠江虎门潮汐水道水体中多环芳烃的含量、分布具有明显的季节变化特征,枯季多环芳烃的浓度高于洪季1个数量级以上,且二环多环芳烃的含量占95%以上,主要呈溶解相形式出现;洪季高环多环芳烃的含量明显升高,主要呈颗粒相形式输运.     

综合风化条件下渤海原油中PAHs 变化特征研究

以渤海某原油作为研究对象进行50 d综合模拟风化实验,探讨了原油中五类PAHs组分的分布情况及其风化规律.结果表明:经过50 d风化,原油中PAHs的分布发生了较大的改变:萘系列化合物损失最为严重,相对浓度的损失达到56.78%;菲系列所占的比例有所提高,二苯并噻吩、屈、芴系列则保持相对稳定,其分布特征变化规律为进一步筛选溢油来源鉴别新诊断比值参数提供了一定的导向性;经风化检验,现有常用6种PAHs诊断比值在风化50 d后稳定性良好,可用于风化溢油的鉴别.     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17 beta-estradiol to 19 +/- 11%, 27 +/- 7%, 63 +/- 20% and 61 +/- 12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17 beta-estradiol and 17 alpha-hydroxyprogesterone levels (r = -0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17β-estradiol to 19±11%, 27±7%, 63±20% and 61±12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17β-estradiol and 17α-hydroxyprogesterone levels (r=−0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

Effect of environmental factors on photodegradation of polycyclic aromatic hydrocarbons (PAHs) in the water-soluble fraction of Kuwait crude oil in seawater

Photodegradation of PAHs in the water-soluble fraction of Kuwait crude oil in seawater was investigated under various environmental factors (temperature, light intensity, oxygen levels and presence of a sensitizer) in laboratory conditions. All factors investigated had significant effect on the degradation rates of PAHs. At 15 °C almost all PAHs optimally degraded at an oxygen level of 4 ppm. For lower molecular weight PAHs a light intensity of 500 W/m² in the presence of the sensitizer worked well. Higher molecular weight PAHs degraded at faster rates at a light intensity 750 W/m². At 30 °C, most of the PAHs degraded optimally at an oxygen level of 0 ppm and light intensity of 500 or 750 W/m² in presence of the sensitizer. At 40 °C, most of PAHs degraded optimally at low oxygen concentrations (0 and 4 ppm) and a light intensity of 500 W/m² in the presence of the sensitizer. Linear regression indicated that for most of the compounds, light intensity had the greatest effect on degradation rates.     

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 micro(micro)M) with or without 0.2 micro(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.     

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.     

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.     

miR-430对斑马鱼原始生殖细胞迁移和性腺发育影响的初步研究

为了探究microRNAs(miRNAs)在斑马鱼生殖细胞发育过程中所起的作用,本研究在斑马鱼胚胎期过表达miR-430,结果发现原始生殖细胞(Primordial germ cells,PGCs)迁移出现紊乱,并且通过qRT-PCR发现nanos3、piwil2及tdrd7等生殖质基因的表达水平显著下调.此外,在斑马...     

Elucidating cellular and behavioural effects of contaminant impact (polycyclic aromatic hydrocarbons,PAHs) in both laboratory-exposed and field-collected shore crabs,Carcinus maenas (Crustacea: Decapoda)

Monitoring effects of contaminants at lower levels of biological organisation (e.g. biochemical and cellular) allows for mechanistic evaluation of effects of contaminant exposure through laboratory exposures. However, higher level organism effects (e.g. physiological and behavioural) are deemed more ecologically-relevant. In the present study, cellular (cell viability and immune function), physiological (cardiac activity) and behavioural (foraging behaviour) responses were evaluated in field-collected shore crabs Carcinus maenas from three estuaries [a ‘relatively’ (polycyclic aromatic hydrocarbon) PAH-contaminated site (Plym Estuary) and two ‘comparatively clean’ field sites (Avon and Yealm Estuaries)] and compared with responses of crabs exposed in the laboratory to a model organic contaminant (PAH) pyrene (200 μg l⁻¹ for 28 days). The hypothesis that behavioural indicators may be more sensitive than other more traditional methods was tested. No significant impacts were observed at the cellular or physiological level in Plym-collected crabs (the ‘relatively’ contaminated site), but foraging behaviour was significantly altered (increased prey handling time) compared to individuals collected from the ‘relatively’ uncontaminated sites (Avon and Yealm). When given a cockle as a prey item, both Plym-collected and laboratory-exposed crabs took longer to handle and break into cockle shells. Therefore, ecologically-relevant behavioural observations may serve as valuableindicators of environmental quality.     

Candidate biomarker discovery in plasma of juvenile cod (Gadus morhua) exposed to crude North Sea oil, alkyl phenols and polycyclic aromatic hydrocarbons (PAHs)

In this study we have investigated protein changes in plasma of juvenile Atlantic cod (Gadus morhua) induced by crude North Sea oil and North Sea oil spiked with alkyl phenols and polycyclic aromatic hydrocarbons, a surrogate produced water composition. Using a proteomic approach, we identified 137 differentially expressed proteins at different levels of crude oil exposure. Many of the induced protein changes occurred at low levels of exposure. The results obtained with protein expression profiles after exposure to oil and surrogate produced water indicate effects on fibrinolysis and the complement cascade, the immune system, fertility-linked proteins, bone resorption, fatty acid metabolism as well as increased oxidative stress, impaired cell mobility and increased levels of proteins associated with apoptosis. Although the number of individuals and samples in this study is limited within each treatment group, the protein changes observed in this study represent a first screening for potential biomarker candidates in cod plasma reflecting potential effects of crude oil and produced water exposure on fish.     

In situ sampling reveals rapid uptake and depuration of polycyclic aromatic hydrocarbons by surf clams (Paphies subtriangulata) affected by the Rena oil spill

ABSTRACT

To investigate the uptake and depuration of polycyclic aromatic hydrocarbons associated with the Rena oil spill we sampled the surf clam Paphies subtriangulata at two open coast locations (6?km apart) just prior to oil coming ashore (7 October 2011), then at 1–3 week intervals for the next 4 months. Total polycyclic aromatic hydrocarbons (tPAH) increased at both sites from 1 to 96–124?µg?kg^?1 (wet weight) by 18 October before declining to low levels (<4?µg?kg^?1) by February 2012. Ongoing sampling throughout 2012–2014 included three additional sites to the north east (up to 30?km away) and a site 5?km to the south east revealing tPAH levels generally <10?µg?kg^?1 except in October 2013 where levels ranged between 39–45?µg?kg^?1 at all sites. A comparison of PAH component profiles with oil-contaminated beach sediment indicated that the high levels observed in surf clams between October–December 2011 were clearly associated with the Rena spill. However, the October 2013 peak had a PAH profile inconsistent with weathered Rena oil, suggesting an alternative source of contamination. Our results highlight the potential for P. subtriangulata as a PAH monitoring tool but recognise more study is needed to better quantify baseline levels and uptake and depuration dynamics.     

(Laser-excited) Shpol'skii fluorometry: applications in marine environmental analysis of polynuclear aromatic hydrocarbons

Shpol'skii spectroscopy is a technique that can be employed to obtain highly resolved spectral signatures of molecules. The high-resolution spectra are obtained by using n-alkanes as solvents that form neat crystalline matrices at low temperatures. The specificity and sensitivity of the Shpol'skii method can be further improved by application of selective laser excitation. Summarizing, Shpol'skii spectra show the highly specific fingerprinting properties of IR spectra, but at the same time have the inherently high sensitivity of fluorescence techniques.In this paper attention will be given to principles and general experimental aspects of the Shpol'skii technique. Furthermore, some applications in environmental analysis will be discussed in more detail: (1) the qualitative determination of polycyclic aromatic hydrocarbons (PAH) in suspended matter collected in estuarine water; (2) the quantitative determination of PAH in NRCC sediment reference material SES-1; and (3) the application of laser-excited Shpol'skii spectroscopy for the qualitative determination of PAH in crude extracts of biotic samples. It is shown that the Shpol'skii technique can be used successfully as an independent reference method for both qualitative and quantitative checks of conventionally used determination methods. Because of the high selectivity of the low-temperature method, PAH can be quantitatively determined without any sample clean-up, even in extremely complex biotic samples.     

Measurement of a time-weighted average concentration of polycyclic aromatic hydrocarbons in seawater: An improved procedure of blue rayon hanging technique for monitoring benzo(a)pyrene

We have proposed a time-weighted measurement using blue rayon that selectively adsorbs and concentrates polycyclic aromatic hydrocarbons (PAH), e.g. benzo(a)pyrene (BaP). Though the method was demonstrated to be a convenient way to monitor PAH, the amount adsorbed to the blue rayon depends on the intensity of water stream and the level of PAH. The intensity of water stream was measured by ‘plaster ball’ method, while TWA of PAH in water was measured by a portable sampler using solid phase extraction cartridge. A level of BaP measured by the original blue rayon technique was corrected in this way by the water stream intensity, which correlated well with the TWA of BaP measured by the portable sampler. The improved blue rayon hanging method was applied to several field sites in the Seto Inland Sea of Japan. TWA of BaP ranged from 0.08 to 3.78 ngl⁻¹. These results showed the possibility that the method could be used to evaluate pollution in aquatic environment.     

Phase I and II biotransformation enzymes and polycyclic aromatic hydrocarbons in the Mediterranean mussel (Mytilus galloprovincialis,Lamarck, 1819) collected in front of an oil refinery

The aim of the present study was to investigate the responses of phase I and II biotransformation enzymes and levels of PAHs in the Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) collected from three sites at different distance from an oil refinery. Phase I enzyme activities as NAD(P)H-cyt c red, NADH ferry red, B(a)PMO and phase II as UDPGT, GST were measured in digestive gland while 16 PAHs (US-EPA) in whole soft tissue. An added value to the data obtained in the present study rely on the RDA analysis which showed close correlations between PAHs levels and phase I enzyme activities in mussels collected in front of the refinery. And again a significant spatial correlation between B(a)P levels and NADPH-cyt c red activities was observed using linear models. No differences among sites for B(a)PMO and phase II GST activities were observed, while the application of UDPGT as biomarkers requires further investigation.