Liver histopathology in Baltic eelpout (Zoarces viviparus) – A baseline study for use in marine environmental monitoring

The use of liver histopathology in various fish species as a biomarker in marine monitoring programmes to assess the biological effect of exposure to anthropogenic contaminants is well established. However, although the eelpout (Zoarces viviparus) has repeatedly been recommended as an appropriate bioindicator for monitoring purposes in the Baltic Sea, information on histopathological liver alterations of this species is scarce. In order to obtain an overview, a total of 1070 eelpout, collected in the period between spring 2001 and winter 2010 from 34 sampling sites distributed over large parts of the Baltic Sea, were histologically analyzed. Liver lesions were recorded and classified according to established liver lesion categories. A high prevalence of non-specific and early toxicopathic non-neoplastic hepatic lesions was recorded. In contrast, pre-neoplastic lesions were observed only in rare cases and neoplastic changes were absent. A comparison with published data on other fish species revealed that the observed alterations may indicate effects of environmental stressors including contaminants. The results of the present study, thus, support the application of eelpout liver histopathology in environmental monitoring.     

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.     

Abnormalities in eelpout Zoarces viviparus upon chemical exposure

Elevated frequencies of abnormal embryos in female eelpout Zoarces viviparus have been demonstrated in Danish, Swedish and German monitoring programmes at certain geographic locations with high levels of anthropogenic input. Pollutants present in areas with high malformation frequencies were selected and tested in a controlled laboratory experiment for their potential to induce abnormalities among eelpout embryos upon injection into pregnant eelpout. Tributyltin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, pyrene, nonylphenol, 2,2′,4,4′-tetrabromophenylether and heptadecafluorooctanesulfonic acid were tested, either individually or combined. Generally, the chemicals were transferred to eggs and/or embryos. Some of the exposures increased the proportion of broods with more than 10% abnormal or 5% malformed embryos, although the average percentages of abnormal development were not affected. Spinal, cranial and eye deformities were evident, similarly to what is seen in nature. Some of the exposures resulted in increased percentages of females with as well a low reproductive capacity as embryos with a low condition index.     

Seasonal variation of hepatic EROD activity in flounder (Platichthys flesus) in the Dutch Wadden Sea

The use of hepatic 7-ethoxyresorufin O-deethylase (EROD) activity in flounder (Platichthys flesus) as a potential biomarker of marine pollution by organic chemicals (as indicated by hepatic PCB-153 concentration) was investigated in the Dutch Wadden Sea in 1989 and 1990. Particular attention was paid to the seasonal variation. The results indicate that EROD activity was reasonably stable during the year, except for a period in January–February when a marked peak occurred. This peak coincided with the absence of mature flounder from the sampling area probably due to the spawning migration. Generally a good relationship existed between hepatic EROD activity and PCB-153 concentration, with one exception. During the period January–April 1990 a peak in PCB-153 concentration occurred immediately after the observed peak in EROD activity. It is concluded that the best period for monitoring EROD activity in flounder is the summer, corresponding to the non-migratory period of the species.     

gamma-Glutamyl transpeptidase as a possible marker of Sertoli cells in fish testes for studies of xenoestrogens.

Estrogenic chemicals are known to have marked effects on the reproductive system of male fish. Finding useful markers of reproductive effects are therefore of great importance and interest. gamma-Glutamyl transpeptidase (gamma-GTP) is a possible marker of Sertoli cells in testes of fish. In the present study we have examined the relationship between the activity of gamma-GTP and the histological structure of the Sertoli cells in testes of five fish species (guppy, Poecilia reticulata; platyfish, Xiphophorus maculatus; eelpout, Zoarces viviparus; rainbow trout, Oncorhynchus mykiss; flounder, Platichthys flesus). In general we found that the more distinct the Sertoli cells the higher the activity of gamma-GTP.     

The viviparous blenny (Zoarces viviparus) as a bioindicator of contaminant exposure: application of biomarkers of apoptosis and DNA damage

The viviparous blenny (Zoarces viviparus), also known as eelpout, is considered a suitable biomonitor for use in European estuarine and coastal waters. We have previously shown that Z. viviparus from the industrialised Tyne estuary (UK) display certain histopathologies (e.g., ovotestis and hepatic nuclear and cellular pleomorphism), which are associated with contaminant exposure [Mar. Environ. Res. 55 (2003) 137]. Furthermore, the prevalence of these pathologies was higher than in fish collected from a less contaminated reference site (the Alde estuary, UK). Here, tissue samples were collected from Z. viviparus from the Tyne and Alde estuaries (in the spring and autumn) and analysed for DNA adducts using the (32)P-postlabelling assay and for histopathology. Z. viviparus caught from the Tyne during the spring (56.4+/-18.5 adducts per 10(8) nucleotides) and autumn (63.1+/-7.5 adducts per 10(8) nucleotides) exhibited patterns of DNA adducts which indicated exposure to a complex mixture of genotoxins. In contrast, levels of DNA adducts in Z. viviparus from Alde estuary were significantly lower during both sampling periods. Histopathological changes to the gonads included intersex (ovotestis) and germ cell apoptosis (detected via the TUNEL assay) in male fish captured from the Tyne estuary. Further studies on the use of germ cell apoptotic rate at different estuarine sites are required if it is to be used as a reproductive biomarker of contaminant exposure in male fish.     

Effects of beta-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to beta-naphthoflavone (beta-NF, 66 mg/kg, i.p.) elicited a 7-9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to beta-NF, and generally tracked the minimal changes observed in GST-CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by beta-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to beta-NF.     

Regulation of Tilapia metallothionein gene expression by heavy metal ions

Tilapia is a common fish species inhabiting inland waters and estuarine regions in Hong Kong and Southeast Asia, and useful for bio-monitoring of metal pollution. Metallothionein (MT) gene expression in fish tissues has been useful to sub-lethal risk assessment as biomarker of exposure to metal ions in fishes inhabiting metal contaminated area. To investigate metal inductions of Tilapia MT gene expression in vivo, Tilapias were injected with different concentrations of heavy metals and tissues were then removed for quantitative PCR assay using mimic PCR methods. All of the metal ions tested (Cu(2+), Cd(2+), Hg(2+), Ni(2+), Pb(2+) and Zn(2+)) were able to induce hepatic MT mRNA levels. Renal MT mRNA levels of Cd(2+) and Zn(2+) treated fish was not induced with significant fold induction, however MT mRNA levels in gills were sensitive to the administrations of these metal ions. These data indicated that Tilapia MT mRNA levels in gills and liver are sensitive biomarker of exposure to various metal ions.     

Effects of β-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to β-naphthoflavone (β-NF, 66 mg/kg, i.p.) elicited a 7–9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to β-NF, and generally tracked the minimal changes observed in GST–CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by β-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to β-NF.     

Natural variability of hepatic biomarkers in Mediterranean deep-sea organisms

Biomarker assays are widely used as proxies for contaminant-induced effects in aquatic organisms. However, in many cases, their intrinsic natural variability due to exogenous and endogenous factors makes the interpretation of biomarker data difficult. In the present study, we investigated the natural fluctuations of six hepatic biomarkers, namely ethoxyresorufin-O-deethylase (EROD) in fish and pentoxyresorufin-O-deethylase (PROD) in crustacea, catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX) and glutathione reductase (GR) in two deep-sea fish species, namely Alepocephalus rostratus and Lepidion lepidion and the decapod crustacean Aristeus antennatus. The NW Mediterranean deep-sea environment is characterized by very stable temperature and salinity conditions, allowing the exclusion of these two factors as potential sources of interference with biomarker activities. Biomarker results exhibited a clear influence of reproductive processes on enzyme activities, in particular in A. rostratus, which presented a pronounced seasonal pattern linked to variations in the gonadosomatic index (GSI). In addition, other factors such as food availability may also have influenced the observed variability, in particular in specimens of L. lepidion, which did not exhibit variations in reproductive activity throughout the sampling period. Depth-related variability did not exhibit a clear trend and fluctuations across sampling depths were not attributable to any specific factor. Body size had also a significant influence on some biomarkers, although allometric scaling of certain enzyme activities appears to be species-specific. The present work has thus shown that despite the lack of fluctuations of abiotic parameters such as temperature and salinity, biomarker activities in deep-sea organisms still exhibit significant variability, mainly as a result of reproductive processes and food availability.     

Preliminary investigations on vitellogenin m-RNA induction in some bioindicator Mediterranean fish species

This paper describes the development of a RT-PCR method for assaying Vtg gene expression in different marine fish as a potentially valuable and sensitive biomarker of exposure to estrogenic chemicals. The levels of Vtg mRNA have been analyzed using primers specifically designed for the various species and the procedures have been standardized relative to actine mRNA expression levels. Different species were analyzed including organisms with a great potential as bioindicators in the Mediterranean (i.e. the red mullet Mullus barbatus, the striped mullet Mugil cephalus, the European eel Anguilla anguilla) or exposed to biomagnification of halogenated hydrocarbons and with elevated commercial value (the bluefin tuna Thunnus thynnus). The analysis of vitellogenin mRNA levels has been standardized in feral fish providing suitable indications for a future development of this approach.     

Risk of environmental genotoxicity in the Baltic Sea over the period of 2009–2011 assessed by micronuclei frequencies in blood erythrocytes of flounder (Platichthys flesus), herring (Clupea harengus) and eelpout (Zoarces viviparus)

Environmental genotoxicity was investigated at 82 locations encompassing different regions of the Baltic Sea. Micronuclei (MN) analysis was performed in erythrocytes of 1892 specimens of flounder Platichthys flesus, herring Clupea harengus and eelpout Zoarces viviparus, three of the most common native fish species of the Baltic Sea collected in 2009–2011. MN background levels in fish were determined using data obtained in 2001–2011 from 107 Baltic sites. Extremely high genotoxicity risk zones were found for flounder at 11 stations out of 16 in 2009 and 33 stations of 41 in 2010–2011, for herring, at 5 of 18 stations in 2009 and 20 of 43 stations in 2010–2011, in eelpout only at one out of 29 stations. The sampling stations were restricted mainly to the southern and eastern Baltic Sea offshore zones and in most of them, MN frequencies in flounder and herring significantly exceeded the reference and background levels of micronuclei. This is a first attempt to evaluate the background MN responses, as well as low, high and extremely high genotoxicity risk levels for native fish species.     

Regulation of Tilapia metallothionein gene expression by heavy metal ions

Tilapia is a common fish species inhabiting inland waters and estuarine regions in Hong Kong and Southeast Asia, and useful for bio-monitoring of metal pollution. Metallothionein (MT) gene expression in fish tissues has been useful to sub-lethal risk assessment as biomarker of exposure to metal ions in fishes inhabiting metal contaminated area. To investigate metal inductions of Tilapia MT gene expression in vivo, Tilapias were injected with different concentrations of heavy metals and tissues were then removed for quantitative PCR assay using mimic PCR methods. All of the metal ions tested (Cu²⁺, Cd²⁺, Hg²⁺, Ni²⁺, Pb²⁺ and Zn²⁺) were able to induce hepatic MT mRNA levels. Renal MT mRNA levels of Cd²⁺ and Zn²⁺ treated fish was not induced with significant fold induction, however MT mRNA levels in gills were sensitive to the administrations of these metal ions. These data indicated that Tilapia MT mRNA levels in gills and liver are sensitive biomarker of exposure to various metal ions.     

Parasites of the deep-sea fish Mora moro () from the NW Mediterranean Sea and relationship with fish diet and enzymatic biomarkers

Specimens of Mora moro were collected in two seasons and three localities of the Balearic Sea (NW Mediterranean Sea) and parasitological, dietary (to prey-species level), enzymatic and histological data were obtained, alongside with environmental information (T, S, O₂). The relationships among fish parasite load, condition indices, diet, enzymatic activity of muscular acetylcholinesterase (AChE) and lactate dehydrogenase (LDH), intensity of splenic melano-macrophage centres (MMC) and hepatic granulomas were tested. M. moro showed a rich and abundant parasite fauna, and was a new host record for 17 out of the 18 different endoparasite taxa found. Significant differences were detected among locality-season groups, in turn related to different environmental variables, for Anisakidae gen. sp., Anisakis Type II and Tetraphyllidea fam. gen. sp.; thus, they are proposed as potentially useful as biological tags for geographical discrimination of M. moro in the NW Mediterranean Sea. Detailed relationships were found between parasite taxa and prey ingested (e.g. Anisakidae gen. sp. related with meso-bathypelagic crustaceans; Anisakis Type I with benthopelagic squids). Most parasites were linked to samples with highest levels of near-bottom O₂, which is consistent with direct relationships found between near-bottom O₂ and zooplankton biomass in the Balearic Basin. Total parasite abundance and the abundance of Tetraphyllidea fam. gen. sp. showed a significant relationship with the activity of AChE and the abundance of Anisakis Type II with LDH. AChE was associated with hepatosomatic index (HSI) and condition factor (K); LDH with gonadosomatic index (GSI), K and fish total length (TL). LDH activity showed differences among sampling groups. Splenic MMC and hepatic granulomas were not associated with fish parasite load. A positive relationship was found between MMC area and fish TL and LDH activity.     

Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in the liver and gill of brown trout (Salmo trutta)

In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole [(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole] and fenpropimorph [(+/-)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc] were administrated in the water separately and together in a static system (80 microg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole[2-(thiazol-4'-yl)benzimidazole] in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment.     

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.     

Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in the liver and gill of brown trout (Salmo trutta)

In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole {(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole} and fenpropimorph {(±)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc} were administrated in the water separately and together in a static system (80 μg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole{ 2-(thiazol-4′-yl)benzimidazole} in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment.     

原油WSF对三种海洋鱼类肝微粒体EROD活性的诱导和恢复的比较

研究了实验室条件下原油水溶性组分(WSF)暴露对黑鲷、黄鳍鲷和褐菖鲉肝微粒体EROD活性的剂量-效应,时间-效应和恢复过程。实验结果表明,在剂量诱导实验中,褐菖鲉肝EROD活性在原油WSF浓度为50μg/dm3时呈现生物统计学上的显著差异,而黑鲷和黄鳍鲷肝EROD活性在75μg/dm3时才呈现生物统计学上的显著差异;褐菖鲉肝EROD活性诱导倍数最高,但黑鲷的诱导浓度范围较广。在时间诱导实验中,在40μg/dm3原油WSF暴露下黄鳍鲷肝EROD活性在2 d时首先呈现显著差异;三种鱼肝EROD活性均在第4天达到最高,并呈现显著性变化,此后随着暴露时间的延长而逐渐下降并接近对照组水平。在恢复实验中三种鱼肝EROD活性下降并恢复到对照组水平。研究结果表明:对于石油污染物,黑鲷、黄鳍鲷和褐菖鲉肝EROD活性都可以作为污染生化效应监测指标,然而就三种鱼类比较而言,褐菖鲉最敏感,更适合于作为石油类污染及其生化效应,尤其是低剂量效应的监测生物。