José M. Navas

Effects of broodstock dietary lipid on fatty acid compositions of eggs from sea bass (Dicentrarchus labrax)

Samples of sea bass (Dicentrarchus labrax) eggs from broodstock which had been fed either a formulated pelleted feed, containing fish and corn oil, or a local trash fish, bogue (Boops boops) were analysed for lipid class compositions, fatty acid compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) and wax ester and fatty alcohol compositions of wax esters. The pelleted feed contained 1.3 mg g−1 of arachidonic acid (20:4n-6; AA) and an AA/eicosapentaenoic acid (20:5n-3; EPA) ratio of 0.1 while the trash fish contained 4.8 mg g−1 AA and an AAEPA ratio of 0.7. Docosahexaenoic acid (22:6n-3; DHA) concentrations were similar for both diets (about 23 mg g−1). The fatty acid compositions of PC, PE and PI from eggs of fish fed trash fish contained significantly more AA, 22:5n-6 and DHA compared to fish fed the pelleted feed. The AAEPA ratios in these phospholipids were around five-fold higher in the trash fish-fed group compared to those fed the fish and corn oil containing diet. In PI, which contains characteristically high levels of AA, the AAEPA ratios were 1.5 and 8.6 for eggs derived from broodstock fed the pelleted diet and the trash fish, respectively. Determination of lipid class compositions of sea bass eggs revealed the presence of high levels of wax esters which were previously unrecorded in this species. The use of broodstock diets containing blends of corn oil and Northern hemisphere fish oils may be undesirable in that they contain high levels of 18:2n-6 and have low ratios of DHAEPA and of AAEPA. In an effort to improve egg quality and larval viability, efforts should be directed towards establishing the best ratio of DHA/EPA/AA in formulated feeds such that requirements for neural function and visual performance are maximised and that production and efficacy of eicosanoids are adequate to permit physiological functions to operate efficiently.

Antiestrogenicity of β-naphthoflavone and PAHs in cultured rainbow trout hepatocytes : evidence for a role of the arylhydrocarbon receptor

The aims of the present study were to assess, (1) if polyaromatic hydrocarbons (PAHs) are able to inhibit estradiol-regulated vitellogenin synthesis in fish; and (2) if this antiestrogenic activity is mediated through the binding of PAHs to the arylhydrocarbon receptor (AhR). Cultured liver cells of rainbow trout, Oncorhynchus mykiss, were co-exposed to PAHs and 17beta-estradiol (E2), and the resulting effects on induction of AhR-regulated 7-ethoxyresorufin-O-deethylase (EROD) activity and on E2-regulated vitellogenesis were investigated. The following test compounds were compared: the PAH 3-methylcholanthrene (3MC), which is a strong EROD inducer, the PAH anthracene (ANT), which is not an inducer of EROD activity, and the model EROD inducer, beta-naphthoflavone (betaNF). 3MC and betaNF led to significant decreases of E2-triggered hepatocellular VTG synthesis, whereas ANT exerted no antiestrogenic activity. The rank order of the antiestrogenic activity of the test substances agreed with their EROD-inducing potency suggesting that their antiestrogenicity might be mediated through the AhR. Further evidence for this assumption comes from the observation that inhibitors such as alpha-naphthoflavone which interferes with ligand-AhR binding, and 8-methoxypsoralen (8MP), which prevents binding of the occupied AhR to responsive DNA elements, clearly reduced the antiestrogenic effects of the xenobiotics. Furthermore, from the comparison of estradiol concentrations in media of liver cells exposed to the CYP 1A-inducing agents and in media of control cells it is unlikely that the observed antiestrogenic effects were caused by an enhanced E2 catabolism. In conclusion, the results from this study indicate that, (1) AhR-binding PAHs possess an antiestrogenic activity; and (2) that the antiestrogenic activity is mediated through the AhR.

Potencies of estrogenic compounds in in vitro screening assays and in life cycle tests with zebrafish in vivo.

The objective of this study was to compare the estrogenic potency of environmental estrogens at two testing tiers: at the initial level of in vitro screening assays, and at the level of definitive fish reproduction tests in vivo. The in vitro tests included a recombinant yeast estrogen receptor (ER) assay, a competitive radioreceptor assay using the hepatic ER of carp (Cyprinus carpio), and assays on vitellogenin induction in cultured hepatocytes of rainbow trout (Oncorhynchus mykiss) and carp. In vivo, full life cycle tests with zebrafish (Danio rerio) were performed, using fertilization success as estrogen-sensitive reproductive endpoint. The test compounds included the natural estrogen 17beta-estradiol (E2) (only applied in the in vitro assays); the synthetic estrogen ethynylestradiol (EE2); and two xenoestrogens, 4-tert-octylphenol (OP) and bisphenol A (BPA). Among the in vitro assays, differences were observed in the relative ranking of the test substances, and in the absolute sensitivity (EC50 values), although the interassay differences of EC50 values were within one order of magnitude. The in vivo activity of the test compounds was not accurately predicted by the in vitro assays, with respect to neither sensitivity nor ranking. The in vitro assays tended to overestimate the relative potency of the xenoestrogens; i.e. the ratio between the activity of the reference compound, EE2, and that of the test compound. The best prediction of the in vivo fish test results was obtained from the recombinant yeast assay.

Estrogen-mediated suppression of cytochrome P4501A (CYP1A) expression in rainbow trout hepatocytes: role of estrogen receptor

Hepatic CYP1A expression in fish can be modulated by the female sex hormone, 17beta-estradiol (E2), however neither the mechanism of E2 suppression of CYP1A nor the capacity for hormonal regulation to overcome CYP1A induction by xenobiotics are known. The present study investigates for the first time in fish if the estrogen receptor (ER) is involved in the suppressive action of E2 on CYP1A gene expression. The study further examines, if the E2 effect is able to overcome xenobiotic induction of CYP1A. As experimental model, in vitro cultures of rainbow trout, Oncorhynchus mykiss, hepatocytes were used. The effect of E2 on CYP1A was assessed by measuring the CYP1A-associated 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity, and CYP1A mRNA contents. E2 at non-cytotoxic concentrations caused a significant time- and concentration-dependent decline of basal but not of induced hepatic EROD activities. The inhibitory action of E2 on basal CYP1A was also evident at the mRNA level. The presence of the ER antagonist tamoxifen abolished the inhibitory action of E2 on CYP1A expression. The results from these in vitro experiments provide evidence (a) that the ER is involved in the suppressive action of E2 on CYP1A, and (b) that E2 inhibitory action does not overcome xenobiotic induction of CYP1A.

Estrogen Receptors Are Expressed in a Subset of Tyrosine Hydroxylase-Positive Neurons of the Anterior Preoptic Region in the Rainbow Trout

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distribution of estrogen receptor-immunoreactive cells and tyrosine hydroxylase-immunoreactive neurons on the same sections of the preoptic region of adult female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the anterior preoptic region surrounding the preoptic recess and its large lateral extensions. Tyrosine hydroxylase-immunoreactive cells were consistently detected in the ventral and ventrolateral walls of the preoptic recess, in an area that was named nucleus preopticus pars anteroventralis. Dopamine immunohistochemistry and Dil retrograde transport studies indicated that part of these catecholaminergic neurons are dopaminergic and could project to the pituitary. Double staining studies showed consistently that most estrogen receptor-positive cells located ventral to the large extensions of the preoptic recess are also tyrosine hydroxylase-positive, indicating that this region is a major target for estradiol feedback. The results are discussed in relation to the role of the nucleus preopticus pars anteroventralis in mediating the negative feedback actions of estradiol on the secretion of gonadotrophin (GTH2) secretion. A hypothesis is drawn in order to explain the synchronizing role of estradiol at the time of ovulation in rainbow trout.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Antiestrogenic activity of anthropogenic and natural chemicals

A number of natural and man-made chemicals possess antiestrogenic activity, i.e. they antagonize a broad spectrum of estrogen-induced responses in vertebrates. Examples of antiestrogens include dioxin, furan and PCB congeners, certain PAHs, pesticides and indol-3-carbinol derivatives. Major mechanisms of antiestrogenicity are antagonistic action of chemicals at the estrogen receptor, or binding of chemicals to the arylhydrocarbon (Ah) receptor and subsequent interaction with estrogen-responsive genes. Toxicological consequences resulting from antiestrogenic activity have not been conclusively demonstrated to date, although antiestrogenic compounds could critically affect sensitive reproductive and developmental processes.

Effect of dietary lipid composition on vitellogenin, 17β-estradiol and gonadotropin plasma levels and spawning performance in captive sea bass (Dicentrarchus labrax L.)

Abstract The influence of dietary lipid composition on the reproductive performance of the sea bass ( Dicentrarchus labrax ) and on vitellogenin (VTG), 17 β -estradiol (E2) and gonadotropin II (GtH II) plasma levels has been investigated. The control group was fed with a natural diet consisting of trash fish ( Boops boops ). Two experimental groups were fed with pelleted diets containing different amounts of lipids: Group A was fed with a commercially available diet with a 10% lipid content and Group B fed the base diet enriched to a 22% lipid content using refined fish oil enriched with n −3 fatty acids. The control group exhibited higher egg viability and hatching rate than the experimental groups. The better spawning performance of the control group, with respect to the experimental groups, was associated with differences in endocrine profiles during the reproductive cycle. In the experimental groups, E2 levels were higher than in controls during the period of vitellogenesis. In the profiles of VTG levels, groups A and B exhibited a greater decrease of plasma VTG during the mid spawning time as compared to the control group. Profiles of plasma GtH II levels were determined for the first time in the sea bass and showed a single annual peak during the spawning period. At that time, GtH II levels from groups A and B were higher than in the controls. The present data suggest that dietary lipid composition significantly affects the reproductive performance of the sea bass.

Species-specific toxicity of copper nanoparticles among mammalian and piscine cell lines.

Abstract The four copper nanoparticles (CuNPs) with the size of 25, 50, 78 and 100 nm and one type of micron-sized particles (MPs) (∼500 nm) were exposed to two mammalian (H4IIE and HepG2) and two piscine (PLHC-1 and RTH-149) cell lines to test the species-specific toxicities of CuNPs. The results showed that the morphologies, ion release and size of the particles all played an important role when investigating the toxicity. Furthermore, the authors found that the particle forms of CuNPs in suspensions highly contribute to the toxicity in all exposed cell lines whereas copper ions (Cu2+) only caused significant responses in mammalian cell lines, indicating the species-specific toxicity of CuNPs. This study revealed that the morphologies, ion release rate of NPs as well as the species-specific vulnerabilities of cells should all be considered when explaining and extrapolating toxicity test results among particles and among species.

Toxic effects of an oil spill on fish early life stages may not be exclusively associated to PAHs: studies with Prestige oil and medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs) are assumed to be the primary determinant of oil petroleum toxicity. Since the PAH content in Prestige oil was relatively high, we investigated the effects of different oil fractions (crude or weathered oil -0.05 to 50 g/L, and shaken or sonicated water accommodated fractions, WAFs, 25-100%, v/v) on the embryo-larval development of medaka (Oryzias latipes). Concentrations of summation operator16PAHs analyzed in the incubation medium were highest in the shaken WAF followed by the crude oil, the sonicated WAF and the weathered oil. Both oils (> or =0.25 g/L) induced developmental abnormalities whereas no significant effects were seen in the WAF exposures. In vivo morphometric analysis of the surface of the gallbladder during advanced embryo organogenesis (192 h post-fertilization, hpf) revealed significant dilation in both WAF exposures (>3 x 10(4) microm(2) at > or =25%, v/v, compared to <1.7 x 10(4) microm(2) at 0%, v/v) followed by the crude oil (>2.2 x 10(4) microm(2) at > or =0.05 g/L). Fluorescent aromatic compounds were observed in the gallbladder and the yolk sac of 168-hpf embryos exposed to all oil fractions. Results suggest the presence of components in both oils capable of penetrating the chorion and inducing a toxicity not observed in the WAFs. Hence, the hazard and risk assessment of Prestige oil should not be based solely on the presence of PAHs since proximity or direct contact may induce toxicity not associated exclusively to these compounds. This research offers a new hypothesis for explaining the reported biological observations, which could be correlated to direct oil exposure rather than the traditional mechanism of waterborne PAH exposure. Further research is needed to identify those oil components responsible for toxicity.