An aromatase inhibitor and tamoxifen decrease plasma levels of o,p-DDT and its metabolites in Nile tilapia (Oreochromis niloticus)

The objective of this study was to determine if tamoxifen or an aromatase inhibitor (4-hydroxyandrostenedione; 4-OHA) affected plasma concentrations of o,p^′-DDT and its metabolites, o,p^′-DDD and o,p^′-DDE, in mature tilapia. Male and female tilapia were injected 6 times intraperitoneally with o,p^′-DDT (40 mg/kg) alone or in combination with 4-OHA (2 mg/kg) over a 12 day period. An additional group of male fish was injected with tamoxifen (5 mg/kg) plus o,p^′-DDT. At the end of the treatment period, plasma samples were extracted and analyzed by GC/ECD. Females injected only with o,p^′-DDT had significantly higher levels of o,p^′-DDT compared with males. Interestingly, females and males treated concomitantly with o,p^′-DDT and 4-OHA or tamoxifen had significantly lower concentrations of plasma o,p^′-DDT (about 50%) compared with fish treated with only o,p^′-DDT. These initial results suggest that an interaction between endocrine-active compounds occurs in vivo in tilapia and may involve alterations in metabolism of o,p^′-DDT.     

Immunological characterization of metallothionein in marine and freshwater fish

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, metalbinding protein found in all vertebrates. The primary structure of the protein is evolutionary conserved, especially the location of cysteine-residues. There are nonetheless significant differences between MTs from different vertebrate classes, notably in major antigenic epitopes. Previous work has shown that MT from different fish species also differ. The objective of the present work was to characterize MT from temperate, sub-tropical and tropical fish species by their relative reactivities with two anti-fish MT antisera. One or both antisera cross-reacted with MT from all fish species investigated. Surprisingly, the relative reactivities of the two antisera with MT from species belonging to different taxonomic groups appeared to reflect differences at the level of family rather than differences between major phylogenetic lineages.     

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus λGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced ≈7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFκB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3^′-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.     

Quantitation of metallothionein mRNA from the New Zealand common bully (Gobiomorphus cotidianus) and its implications for biomonitoring

Metallothionein (MT) is a protein found in most eukaryotes where its primary role is the regulation of homeostasis of the essential metals copper and zinc. MTs have an additional protective role through their binding of toxic metals such as cadmium and mercury. Increased expression of MT in response to harmful levels of these metals has been demonstrated for several aquatic species. In this study, the expression of MT in the liver tissue of the New Zealand common bully Gobiomorphus cotidianus was measured by quantifying MT mRNA. A statistically significant increase in MT expression was observed in fish exposed to copper for 48 h, but not those exposed to zinc. Field sampling of common bullies showed a wide variation in individual hepatic MT mRNA levels which did not correlate with fish age, sex, or sampling location. A comparison of two populations of common bullies from a polluted and a control site showed a two‐fold higher mean MT mRNA level in fish from the polluted site. Implications for using MT expression in the common bully as a bio‐monitoring tool are discussed.     

Spatial, temporal and diel variations of fish assemblages at two sandy beaches in the Sepetiba Bay, Rio de Janeiro, Brazil

Fish assemblages from two beaches, one in the inner and the other in the outer Sepetiba Bay (latitude: 22°54′–23°04′S; longitude: 43°34′–44°10′W), Southeast Brazil, were sampled by beach seine net, simultaneously, on both seasonal and diel scales, between August 1998 and June 1999. Sites were selected to encompass different environmental conditions which reflect the two bay zones, thus providing a comprehensive assessment of the factors influencing surf zone fish assemblages, and their spatial, seasonal and diel variations. A total of 55 fish species was recorded, mostly young-of-the-year. Anchoa tricolor, Micropogonias furnieri, Gerres aprion, Diapterus rhombeus, Harengula clupeola, Atherinella brasiliensis and Mugil liza were numerically dominant and contributed to 95.2% of the total fish catches. Strong differences in fish assemblages were observed between the two areas, with higher number of species in the outer bay. Increases in fish numbers occurred in winter, while the highest biomass occurred in winter and summer. Transparency, followed by salinity, was responsible for most of the spatial variability and played an important role in structuring fish assemblages. Overall, diel patterns did not reveal any significant trends; however, if we consider each season separately, an increase in fish numbers during the day with peak at sunset was observed in winter, and a higher biomass occurred at night in winter and summer. Species preferences for various combinations of environmental variables are responsible for shifts in the structure and overall abundance of assemblages and dictated some patterns. The sciaenid M. furnieri, the second most abundant species, occurred only in the inner zone, being more abundant in winter. The species of Engraulidae were more abundant in the outer zone in winter/spring during the day. The gerreids G. aprion and D. rhombeus occurred mainly in summer. Overall, temporal fluctuations act more at a specific level than at a structural one, and may be linked to some particular stages of the fish life cycle, but do not significantly influence the spatial organization.     

Transport and accumulation of organochlorines in the ovaries of Atlantic croaker (Micropogonias undulatus)

The role of vitellogenin in the transport of organochlorines was investigated in Atlantic croaker (Micropogonias undulatus) by exposing them through the diet to o,p′-DDT at a concentration of 10.8 μg/100 g fish/day for 14 days or Aroclor 1254 (PCB) at a concentration of 0.5 mg/100 g fish/day for 30 days during gonadal recrudescence. Tissue samples were taken from the fish at various times after initial exposure, and o,p′-DDT and PCBs were extracted in acetonitrile and analyzed by gas chromatography. Analysis of the ovarian tissue collected 2 and 3 weeks from the start of exposure revealed that the o,p′-DDT concentration increases as the gonadosomatic index (GSI) increases (r² = 0.63), with accumulation ranging from less than 1% to as much as 8% of the total dosage. Interestingly, o,p′-DDT did not accumulate in the testes during the same exposure period. Accumulation of PCBs was found to be 40 times higher in the ovaries than in the testes. Gel filtration of plasma from exposed females showed that o,p′-DDT elutes in the low density lipoprotein and vitellogenin fractions. Control plasma incubated with o,p′-DDT at 4 °C for 16 h followed by chromatography on Sepharose 6B gave similar results with an o,p′-DDT concentration of 0.6μg/mg protein in the vitellogenin fraction. Furthermore, both o,p′-DDT and PCBs were found to bind to purified croaker vitellogenin. These results suggest that lipoproteins, including vitellogenin, are involved in the transport and accumulation of organochlorines in the ovaries of exposed fish.     

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.     

Estradiol-17β and o,p′-DDT stimulate gonadotropin release in Atlantic croaker

Xenobiotic estrogens have the potential to act at a variety of estrogen-responsive target tissues on the hypothalamic-pituitary-gonadal axis. However, to date most studies in fish have focused on stimulation of vitellogenin synthesis by the liver. In the present study the effects of the xenoestrogen o,p′-DDT and estradiol-17β on the neuroendocrine control of gonadotropin secretion were compared. Atlantic croaker (Micropogonias undulatus) were exposed to o,p′-DDT (0.02 and 0.1 μgg⁻¹ body weight day⁻¹) in the diet for 3 and 7 weeks during the gonadal recrudescence phase. The o,p′-DDT exposure elicited a significant increase in plasma gonadotropin levels after both 3 and 7 weeks of exposure. The stimulatory effect of o,p′-DDT on basal (spontaneous) gonadotropin release was accompanied by a slight increase in ovarian growth as evidenced by the increase in gonadosomatic index. It appears that the stimulation of gonadotropin release by o,p′-DDT during early-recrudescence phase results in enhanced ovarian growth. A comparable stimulatory effect was observed with estradiol-17β treatment during early- and late-recrudescence phases of the ovarian cycle using three injections on alternate days and slow release silastic implants (five days). The present study provides the first evidence for an estrogen-like action of o,p′-DDT on gonadotropin release in a teleost model.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Water masses and currents of deep circulation southwest of the Shatsky Rise in the western North Pacific

We conducted full-depth hydrographic observations in the southwestern region of the Northwest Pacific Basin in September 2004 and November 2005. Deep-circulation currents crossed the observation line between the East Mariana Ridge and the Shatsky Rise, carrying Lower Circumpolar Deep Water westward in the lower deep layer (θ<1.2 °C) and Upper Circumpolar Deep Water (UCDW) and North Pacific Deep Water (NPDW) eastward in the upper deep layer (1.3–2.2 °C). In the lower deep layer at depths greater than approximately 3500 m, the eastern branch current of the deep circulation was located south of the Shatsky Rise at 30°24′–30°59′N with volume transport of 3.9 Sv (1 Sv=10⁶ m³ s⁻¹) in 2004 and at 30°06′–31°15′N with 1.6 Sv in 2005. The western branch current of the deep circulation was located north of the Ogasawara Plateau at 26°27′–27°03′N with almost 2.1 Sv in 2004 and at 26°27′–26°45′N with 2.7 Sv in 2005. Integrating past and present results, volume transport southwest of the Shatsky Rise is concluded to be a little less than 4 Sv for the eastern branch current and a little more than 2 Sv for the western branch current. In the upper deep layer at depths of approximately 2000–3500 m, UCDW and NPDW, characterized by high and low dissolved oxygen, respectively, were carried eastward at the observation line by the return flow of the deep circulation composing meridional overturning circulation. UCDW was confined between the East Mariana Ridge and the Ogasawara Plateau (22°03′–25°33′N) in 2004, whereas it extended to 26°45′N north of the Ogasawara Plateau in 2005. NPDW existed over the foot and slope of the Shatsky Rise from 29°48′N in 2004 and 30°06′N in 2005 to at least 32°30′N at the top of the Shatsky Rise. Volume transport of UCDW was estimated to be 4.6 Sv in 2004, whereas that of NPDW was 1.4 Sv in 2004 and 2.6 Sv in 2005, although the values for NPDW may be slightly underestimated, because they do not include the component north of the top of the Shatsky Rise. Volume transport of UCDW and NPDW southwest of the Shatsky Rise is concluded to be approximately 5 and 3 Sv, respectively. The pathways of UCDW and NPDW are new findings and suggest a correction for the past view of the deep circulation in the Pacific Ocean.     

o,p′-DDT induction of vitellogenesis and its inhibition by tamoxifen in Nile tilapia (Oreochromis niloticus)

In order to investigate the mechanism by which o,p′-DDT disrupts endocrine functioning of Nile tilapia in vivo, the estrogenicity of o,p′-DDT was investigated in conjunction with 17β-estradiol (E₂) and tamoxifen. Mature, male tilapia were treated intraperitoneally with o,p′-DDT (60 mg/kg, one dose) or E₂ (5 mg/kg, four doses) in the presence or absence of tamoxifen (5 mg/kg, six doses) for 12 days and then plasma vitellogenin (Vtg) (measured as alkaline-labile phosphorous), E₂, and testosterone (T) were measured. Vtg levels were increased dramatically by E₂ (1744±171 μg/ml) and moderately by o,p′-DDT (82±15 μg/ml) compared with controls (23±3.5 μg/ml). Tamoxifen alone had no effect on Vtg production, but inhibited both E₂ and o,p′-DDT stimulated vitellogenesis. T levels were reduced with E₂ administration (1688±383 pg/ml) and declined further with the combined treatment of E₂ and tamoxifen (281±70 pg/ml), compared with controls (6558±1438 pg/ml). Tamoxifen or o,p′-DDT alone did not affect T levels, but their combined treatment did (2069±647 pg/ml). The results of this study suggest that o,p′-DDT is weakly estrogenic in male tilapia, and that this activity may be mediated through the estrogen receptor.     

Alterations in hepatic metallothionein content in perch, Perca fluviatilis, environmentally exposed to cadmium

During recent years laboratory studies have demonstrated the presence of metallothionein in several species of teleosts.^1–3 The role of metallothionein (MT) in protection against cadmium toxicity has been investigated by exposing fish to high levels of cadmium in the water.^4–6 So far, however, few field studies have focused on the existence of MT and its role in subcellular distribution of cadmium.⁷ In the present study the synthesis of MT as a possible mechanism by which perch adapt to cadmium contamination was investigated in a wild population of perch. An increased MT content of perch liver was found to correlate to an increase in cadmium present in the liver.     

Interaction of CYP1A1 inducers in liver of rainbow trout and eelpout

The hepatic CYP1A1 (ethoxyresorufin-O-deethylase (EROD) and protein level) in rainbow trout and eelpout was induced by isosafrole, β-naphthoflavone, 3,3′,4,4′-tetrachlorobiphenyl and mixtures of two of the compounds. A potentiation effect of the CYP1A1 response was observed when isosafrole was given together with β-naphthoflavone, but not when isosafrole was given with 3,3′,4,4′-tetrachlorobiphenyl.     

Determination of adsorbed and absorbed polychlorinated biphenyls (PCBs) in seawater microorganisms

To distinguish between adsorbed and absorbed PCBs in seawater microorganisms, 2,2′,4,4′,5,5′-hexachlorobiphenyl (IUPAC #153; HCB) was added to a pure culture of Chrysochromulina apheles Moestrup et H.A. Thomsen. After the addition of the HCB, the cells were immediately harvested onto 2 μm polycarbonate filters and rinsed with a gradient of ethanol concentrations. Rinsing with 40% ethanol (v/v) was found to remove 80% of the HCB, which was loosely adsorbed to the cell surfaces, but did not extract the interior of the cells, as tested by chlorophyll a analysis. This method was used in a time course experiment which estimated PCBs adsorption and absorption to different groups of plankton organisms. Three different ¹⁴C-PCBs, 4-chlorobiphenyl (IUPAC #3; MCB), 2,2′,5,5′-tetrachlorobiphenyl (IUPAC #52; TCB), and 2,2′,4,4′,5,5′-hexachlorobiphenyl (IUPAC #153, HCB), were incubated in seawater from the northern Baltic Sea during a spring bloom. Samples were taken every third day and separated by filtration into three fractions; 0.2–2 μm (bacteria), 2–10 μm (flagellates), and >10 μm (microplankton; phytoplankton and protozoa). Two subsamples were retained from each size fraction. One of the subsamples was left untreated, to obtain adsorbed plus absorbed PCB, while the other subsample was rinsed with 40% of ethanol, to obtain the absorbed PCB. The sorption was found to vary depending on the hydrophobicity of the compounds, the structure of the cell membranes, and the lipid content and composition of the cells. The absorption increased for the TCB and the HCB in the largest size fraction over time, which coincided with an increase of the neutral and non-polar lipids.     

Anomalously low alkenone temperatures caused by lateral particle and sediment transport in the Malvinas Current region, western Argentine Basin

We analysed the alkenone unsaturation ratio (U^K′₃₇) in 87 surface sediment samples from the western South Atlantic (5°N–50°S) in order to evaluate its applicability as a paleotemperature tool for this part of the ocean. The measured U^K′₃₇ ratios were converted into temperature using the global core-top calibration of Müller et al. (1998) and compared with annual mean atlas sea-surface temperatures (SSTs) of overlying surface waters. The results reveal a close correspondence (<1.5°C) between atlas and alkenone temperatures for the Western Tropical Atlantic and the Brazil Current region north of 32°S, but deviating low alkenone temperatures by −2° to −6°C are found in the regions of the Brazil–Malvinas Confluence (35–39°S) and the Malvinas Current (41–48°S). From the oceanographic evidence these low U^K′₃₇ values cannot be explained by preferential alkenone production below the mixed layer or during the cold season. Higher nutrient availability and algal growth rates are also unlikely causes. Instead, our results imply that lateral displacement of suspended particles and sediments, caused by strong surface and bottom currents, benthic storms, and downslope processes is responsible for the deviating U^K′₃₇ temperatures. In this way, particles and sediments carrying a cold water U^K′₃₇ signal of coastal or southern origin are transported northward and offshore into areas with warmer surface waters. In the northern Argentine Basin the depth between displaced and unaffected sediments appears to coincide with the boundary between the northward flowing Lower Circumpolar Deep Water (LCDW) and the southward flowing North Atlantic Deep Water (NADW) at about 4000 m.     

Polychlorinated biphenyls in fish-eating sea birds—molecular features and metabolic interpretations

Polychlorinated biphenyls (PCBs) are an important group of environmental pollutants, there being a total of 209 theoretical congeners. Residue analysis of the adipose tissue of five species of fish-eating sea birds from British and Irish coastal waters revealed the presence of up to 60 different congeners. By GC-MS, GC-MSD and high resolution capillary GC-ECD using authentic standards, it was possible to identify and quantify 40 different congeners. Despite the large number of PCB congeners identified only 10 accounted for > 80% of the total PCBs. A PCB congener was identified accounting for 5% of the total PCBs (2,3′,4,4′,5′,6-hexachlorobiphenyl) [168] not usually reported in biological samples. Comparison of the molecular structure for the persistent PCB congeners revealed the lack of meta-para unsubstituted adjacent carbon atoms. It has been shown that meta-para unsubstituted adjacent carbon atoms facilitate the metabolism of PCBs and it is hypothesised that the formation of hydroxy derivatives may depend upon such a requirement.     

Mycobacteria, but not mercury, induces metallothionein (MT) protein in striped bass, Morone saxitilis, phagocytes, while both stimuli induce MT in channel catfish, Ictalurus punctatus, phagocytes

Recent advances in molecular immunology indicate that the expression of inducible pro-inflammatory proteins is increased in vertebrates in response to both infectious disease agents and various xenobiotics. For example, iNOS, COX-2, and CYP1A are induced by both inflammation and AhR ligands. Moreover, the expression of these proteins in response to stimuli varies among individuals within populations. Little is known of the differences among fish in the inducibility of proinflammatory proteins in response to both infectious agents and xenobiotics. Through random screening of a striped bass, Morone saxitilis, peritoneal macrophage cDNA library, a full length metallothionein (MT) gene was cloned and sequenced. MT is a low-molecular weight (6–8 kDa), cysteine-rich metal binding protein. Metals are required by pathogenic bacteria for growth, and by the host defense system by serving as a catalyst for the generation of reactive oxygen intermediates (ROIs) by phagocytes. A recombinant striped bass MT (rMT) was expressed and purified, then used to generate a specific mAb (MT-16). MT protein expression was followed in freshly isolated striped bass and channel catfish, Ictalurus punctatus, phagocytes after in vitro exposure to the naturally occurring intracellular pathogen Mycobacteria fortuitum or to 0.1 and 1 μM mercury (Hg), as HgCl₂. MT expression was increased by 24 h in both channel catfish and striped bass phagocytes as a result of exposure to M. fortuitum cells. On the other hand, MT was induced by Hg in channel catfish cells, but not those of striped bass. These results indicate that metal homeostasis in phagocytes is different between catfish and striped bass. In addition, these data suggest that care should be taken to distinguish between inflammation-induced vs. metal-induced MT when using MT expression as a biomarker of metal exposure.