Tributyltin inhibits the oligomycin-sensitive Mg-ATPase activity in Mytilus galloprovincialis digestive gland mitochondria

Tributyltin (TBT), widely employed in the past in antifouling paints, is one of the most toxic organic pollutants. Although recently banned, it still threatens coastal water ecosystems and accumulates in filter-feeding molluscs. TBT is known to act as a membrane-active toxicant; however data on mussels are scanty and exposure effects on mitochondrial ATPase activities remain hitherto unexplored. TBT effects on the mitochondrial Mg-ATPase activities in the digestive gland of Mytilus galloprovincialis were investigated both in vitro and in TBT-exposed mussels. Both an oligomycin-sensitive Mg-ATPase (OS Mg-ATPase) (70% of total Mg-ATPase activity) and an oligomycin-insensitive ATPase (OI Mg-ATPase) (30%) were found. The OS-Mg-ATPase was as much as 70% in vitro inhibited by 0.7 μM (203 μg/L) TBT, while higher concentrations promoted a partial inhibition release up to 5.0 μM TBT; higher than 10.0 μM TBT concentrations yielded nearly complete enzyme inhibition. Concentrations higher than 1 μM TBT enhanced the OI Mg-ATPase. Mussels exposed to 0.5 and 1.0 μg/L TBT in aquaria showed a 30% depressed OS Mg-ATPase activity, irrespective of TBT dose and exposure time (24 and 120 h). The OI Mg-ATPase activity was apparently refractory to TBT exposure and halved both in control and TBT-exposed mussels after 120 h exposure.     

Intersex Occurrence in Rainbow Trout (Oncorhynchus mykiss) Male Fry Chronically Exposed to Ethynylestradiol

This study aimed to investigate the male-to-female morphological and physiological transdifferentiation process in rainbow trout (Oncorhynchus mykiss) exposed to exogenous estrogens. The first objective was to elucidate whether trout develop intersex gonads under exposure to low levels of estrogen. To this end, the gonads of an all-male population of fry exposed chronically (from 60 to 136 days post fertilization – dpf) to several doses (from environmentally relevant 0.01 µg/L to supra-environmental levels: 0.1, 1 and 10 µg/L) of the potent synthetic estrogen ethynylestradiol (EE2) were examined histologically. The morphological evaluations were underpinned by the analysis of gonad steroid (testosterone, estradiol and 11-ketotestosterone) levels and of brain and gonad gene expression, including estrogen-responsive genes and genes involved in sex differentiation in (gonads: cyp19a1a, ER isoforms, vtg, dmrt1, sox9a2; sdY; cyp11b; brain: cyp19a1b, ER isoforms). Intersex gonads were observed from the first concentration used (0.01 µg EE2/L) and sexual inversion could be detected from 0.1 µg EE2/L. This was accompanied by a linear decrease in 11-KT levels, whereas no effect on E2 and T levels was observed. Q-PCR results from the gonads showed downregulation of testicular markers (dmrt1, sox9a2; sdY; cyp11b) with increasing EE2 exposure concentrations, and upregulation of the female vtg gene. No evidence was found for a direct involvement of aromatase in the sex conversion process. The results from this study provide evidence that gonads of male trout respond to estrogen exposure by intersex formation and, with increasing concentration, by morphological and physiological conversion to phenotypic ovaries. However, supra-environmental estrogen concentrations are needed to induce these changes.     

Modulation of acute steroidogenesis,peroxisome proliferator-activated receptors and CYP3A/PXR in salmon interrenal tissues by tributyltin and the second messenger activator,forskolin

There are uncertainties regarding the role of sex steroids in sexual development and reproduction of gastropods, leading to the recent doubts as to whether organotin compounds do inhibit steroidogenic enzymes in these species. These doubts have led us to suspect that organotin compounds may affect other target molecules, particularly signal transduction molecules or secondary mediators of steroid hormone and lipid synthesis/metabolism. Therefore, we have studied the effects of TBT exposure through food on acute steroidogenesis, PPARs and CYP3A responses in the presence and absence of a cyclic AMP (cAMP) activator, forskolin. Two experiments were performed. Firstly, juvenile salmon were force-fed once with diet containing TBT doses (0.1, 1 and 10 mg/kg fish) dissolved in ethanol and sampled after 72 h. Secondly, fish exposed to solvent control and 10 mg/kg TBT for 72 h were transferred to new tanks and exposed to waterborne forskolin (200 μg/L) for 2 and 4 h. Our data show that juvenile salmon force-fed TBT showed modulations of multiple biological responses in interrenal tissues that include, steroidogenesis (cAMP/PKA activities; StAR and P450scc mRNA, and plasma cortisol), and mRNA for peroxisome proliferator-activated receptor (PPAR) isoforms (α, β, γ), acyl-CoA oxidase-1 (ACOX1) and CYP3A/PXR (pregnan X receptor). In addition, forskolin produced differential effects on these responses both singly and also in combination with TBT. Overall, combined forskolin and TBT exposure produced higher effects compared with TBT exposure alone, for most of the responses (cortisol, PPARβ, ACOX1 and CYP3A). Interestingly, forskolin produced PPAR isoform-specific effects when given singly or in combination with TBT. Several TBT mediated toxicity in fish that includes thymus reduction, decrease in numbers of lymphocytes, inhibition of gonad development and masculinization, including the imposex phenomenon have been reported. When these effects are considered with the present findings, it suggests that studies on mechanisms of action or field studies may reveal endocrine, reproductive or other effects of TBT at lower concentrations than those reported to date from subchronic tests of fishes. Since the metabolic fate of organotin compounds may contribute to the toxicity of these chemicals, the present findings may represent some new aspects of TBT toxicity not previously reported.     

Abdominal-B is essential for proper sexually dimorphic development of the Drosophila gonad

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior-posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. Here, we investigate the role of homeotic genes in the sexually dimorphic development of the gonad in Drosophila. We have found that Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. Our results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species.     

TBT effects on the female genital system of Littorina littorea: a possible indicator of tributyltin pollution

Specimens of the prosobranch Littorina littorea (L., 1758) collected along the East Frisian North Sea coast in summer 1993 exhibited alterations of the pallial oviduct termed as intersex in response to tributyltin (TBT) pollution. The range of TBT body burden was between 150.9 and 1289.5 μg as Sn kg⁻¹ (dry wt.). Five stages of intersex development (0–4) could be distinguished and are documented with scanning electron micrographs. In stages 2–4, which can be found in the direct vicinity of harbours and marinas, the morphological malformations of the oviduct inhibit successful copulation and capsule formation, resulting in sterilization. The intersex index (ISI, calculated as the average intersex stage of a population) and the average prostate length of females were used as parameters for the determination of intersex intensities in the populations. Both indices show significant and positive correlations to the TBT body burden of L. littorea and are promising parameters for TBT biomonitoring. A comparison of TBT bioconcentration factors with populations from England and France indicates that the threshold concentration for intersex development is in the range of 15 ng TBT as Sn/l. Morphometric analyses of the midgut gland revealed no significant differences between sampling stations. In the ovary a retardation and blockage of maturation (atresia) was observed in populations close to harbours. Lytic processes in ovary follicles were observed not only at TBT exposed sites but also at reference stations.     

Apoptotic effects and glucose-6-phosphate dehydrogenase responses in liver and gill tissues of rainbow trout treated with chlorpyrifos

We investigated apoptotic effects and changes in glucose-6-phosphate dehydrogenase (G6PD) enzyme activity in liver and gill tissues of fish exposed to chlorpyrifos. Three different chlorpyrifos doses (2.25, 4.5 and 6.75 μg/L) were administrated to rainbow trout at different time intervals (24, 48, 72 and 96 h). Acute exposure to chlorpyrifos showed time dependent decrease in G6PD enzyme activity at all concentrations (p < 0.05). Immunohistochemical results showed that chlorpyrifos caused mucous cell loss in gill tissue and apoptosis via caspase-3 activation in fish. The present study suggested that chlorpyrifos inhibits G6PD enzyme and causes mucous cell loss in gill and apoptosis in gill and liver tissues.     

Intersex,a temperature-sensitive mutant of the nematode Caenorhabditis elegans

A temperature-sensitive mutation, isx-1(hc17), is reported in the nematode Caenorhabditis elegans which alters the sexual phenotypes of both genotypic sexes. At the restrictive temperature, XX animals are functionally female rather than hermaphroditic due to the absence of spermatogenesis, and XO animals develop as intersexes. These intersexes have normal male head and tail structures and exhibit some mating behavior, but possess hermaphrodite-like gonads which produce no sperm and usually contain a few oocytes. An abortive vulva is usually present and evidence is presented which suggests that the formation of the vulva by the hypodermis is induced by the underlying gonad. The direct effects of the mutation are confined to the descendants of four primordial gonad cells. Gametogenesis and gonad sheath development do not seem to be tightly coupled and are shown to differ in their responses to X-chromosome dosage. The interaction of the intersex mutation with mutant alleles of two transformer genes tra-1 and tra-2 is discussed and a model for the action of these genes in gonad development and sex determination is proposed.     

Reproductive demographics,intersex, and altered hormone levels in shortnose sturgeon,Acipenser brevirostrum,from Delaware River,USA

Non‐lethal sampling techniques were used to document the reproductive structure of the shortnose sturgeon (Acipenser brevirostrum) population at a fish aggregation site (near Bordentown, New Jersey) in the Delaware River. A total of 68 fish were captured using gill nets (100 × 1.8 m, 12.7 or 15.2 cm mesh) and examined laparoscopically in May–June, and 61 additional fish captured and examined in November during 2006–2011. Six stages of reproductive development were identified in females and five stages were identified in males, encompassing differentiation through maturity in both sexes. Fish captured in the spring were predominantly immature with a higher proportion of females (1 : 1.2 M : F sex ratio), while mature males predominated in the autumn (5.7 : 1 M : F), indicating that the Bordentown area serves as an overwintering/pre‐spawn aggregation site. Three distinct forms of intersex were noted in gonads of 11.6% of fish examined: ovo‐testis, consisting of scattered spermatic cysts in predominantly immature ovary (3.9%); testis‐ova, consisting of ovarian lamellae projecting from a predominantly immature testes (4.7%); and zonal distribution, consisting of multiple, nearly‐homogenous pockets of either testicular or ovarian tissues along the gonad (3%). The hormone profile in fish with ovo‐testis was similar to that of immature males, while the hormone profile in fish with testis‐ova was similar to that of immature females. The relatively high prevalence of intersex raises concerns regarding potential reproductive effects and long‐term impacts on shortnose sturgeon in the Delaware River.     

Gonad development and fatty acid composition of Patella depressa Pennant (Gastropoda: Prosobranchia) populations with different patterns of spatial distribution, in exposed and sheltered sites

The present study examines the effect of shore exposure on the feeding performance (assessed by fatty acid analyses of the whole body) and gonad condition (stage of development and gonad somatic index, GSI) of Patella depressa populations. Male and female limpets were collected at exposed and sheltered sites, during winter and summer. The population at the exposed site was at a more advanced stage of gonad development, with a higher dispersion of gonad stages, both in winter and summer. Additionally, limpets from the exposed site, particularly the males, presented a higher GSI than the corresponding stage in the sheltered site. The quantitatively most important fatty acids were the saturated fatty acids (SFA) 16:0, 14:0, and 18:0, the monounsaturated fatty acids (MUFA) 18:1(n−7), 18:1(n−9), 16:1(n−7) and 20:1(n−9) and the polyunsaturated fatty acids (PUFA) 20:5(n−3) and 20:4(n−6). Females had a significantly higher fatty acid methyl esters (FAME) content (in summer and winter) and higher amounts of SFA and MUFA (in summer), which points to a higher degree of storage of neutral lipids in this sex. Male and female limpets at the exposed site had a significantly higher FAME, SFA, MUFA, PUFA and highly unsaturated fatty acids (HUFA) content than the corresponding sex in the sheltered site in summer. In addition, an inversion in the eicosapentaenoic acid (EPA)/arachidonic acid (ARA) and (n−3)/(n−6) ratios was observed in the sheltered site, as a result of the significantly higher levels of ARA and (n−6) fatty acids and lower amounts of EPA and (n−3) fatty acids found in the sheltered limpets. A high variability among patches in the fatty acid composition in the exposed site was found in winter, possibly related to the aggregation of limpets at this time. The differences found between limpets from the exposed and sheltered sites suggest qualitative and quantitative differences in their diets. Additionally, the results show that the spatial aggregation strategy adopted by limpets in sites of great wave and wind exposure does not affect their feeding and reproductive success, at least in the site examined here. In fact, more developed gonads, a higher GSI and an elevated FAME content was found in the exposed population. Possible factors are suggested and discussed to explain these observations.     

On the hoplonemertean,Kameginemertes parmiornatus (Iwata, 1957) gen. n., comb. n. from Sagami Bay,Japan

Two different tributyltin (TBT)-induced virilisation phenomena in prosobranch snails – intersex in Littorina littorea and imposex in Nucella lapillus – are compared in order to facilitate their simultaneous use in geographical large scale effect monitoring surveys. Imposex in dogwhelks is a more sensitive biomarker and should be used in areas that are only slightly or moderately contaminated with TBT (ambient TBT concentrations < 2.0 ng as Sn l-1). The assessment of intersex intensities in periwinkle populations has considerable advantages in areas with higher TBT concentrations and should be used also wherever dogwhelks are absent irrespective of the TBT exposure level. The intersex index (ISI) and vas deferens sequence (VDS) index are proposed as the most suited parameters for effect monitoring purposes. The geographical uniformity of intersex and imposex is analysed and proven for the coasts of Ireland, France, and Germany. A relative loss of TBT sensitivity in females can be found, but to a varying extent in both species. The implications of this result for biological TBT effect monitoring programmes are discussed in light of the fact that intersex and imposex have both been found to be irreversible. Because it is the objective of these programmes to assess current TBT contaminations and resulting biological effects, only relatively young specimens should be considered in the sampling strategy.     

The effect of inhibitors upon intracellular cyclic AMP levels and chick myoblast differentiation.

The small free-living nematode Caenorhabditis elegans is usually found as a hermaphrodite, but occasionally true males appear in the population. This study provides an account of gonadogenesis in the normal male and in a mutant that is a temperature-sensitive sex transformer.Male and hermaphrodite gonads develop from morphologically identical primordia. The small primordial gonad lies on the ventral side of the worm in the coelomic cavity. The gonadial primordium contains four nuclei at parturition. As this primordium develops in a hermaphrodite, it produces a double-armed, mirror symmetrical gonad that produces first sperm and then eggs. In the male, however, this primordium develops into an asymmetrical structure composed of a ventrally located testis, a loop region, a seminal vesicle, and a vas deferens. The male gonad presents a linear sequence of nuclei in successive stages of spermatogenesis beginning with a mitotic region in the testis, followed by clearly distinguishable stages of meiosis throughout the loop region to the seminal vesicle.A temperature-sensitive sex transformer mutant, tsB202, has been isolated. tsB202 carries an autosomal recessive mutation in linkage group II that at restrictive temperature transforms an XX hermaphrodite into a phenotypic male, complete with a normal male gonad and vestigial external genitalia. These transformed males are classified as pseudomales because they do not exhibit mating behavior. Temperature shift experiments have determined the specific temporal sequences of gonadogenesis, oogenesis, and spermatogenesis. Proper manipulation of the temperature regimen causes the production of intersexes. In one intersex, a male gonad complete with sperm, seminal vesicle, and vas deferens also contains oocytes. In another intersex produced by the complementary temperature shift, a hermaphrodite-shaped gonad develops that produces only sperm and no oocytes.     

Toxicity of Tributyltin in Juvenile Common Carp (Cyprinus Carpio): Physiological Responses,Hepatic Gene Expression,and Stress Protein Profiling

In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na⁺‐K⁺‐ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation–related genes expression and stress‐related proteins profiling in fish liver. Oxidative stress indices and Na⁺‐K⁺‐ATPase showed tissues‐specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF‐I axis and ion‐regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 μg/L) and were inhibited in 20 μg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose‐dependent manner under TBT stress. In short, TBT‐induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long‐term experiments at lower concentrations will be necessary in the future.     

Characterization of abalone Haliotis tuberculata–Vibrio harveyi interactions in gill primary cultures

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.     

Identification of key nutrients for gonadal development by comparative analysis of proximate composition and fatty/amino acid profile in tissues and eggs of Chinese sturgeon (Acipenser sinensis Gray, 1835)

The present study was conducted to characterize the proximate composition and fatty/amino acid profile in muscle, liver and gonads obtained from farmed male and female Chinese sturgeon (Acipenser sinensis) with gonad development at stage II, and to discern the key nutrients for gonads by comparing the nutrients between tissues and between fish at different gonad stages. Chinese sturgeon were reared in an outdoor circular cement pool at temperatures ranging from 8°C to 26°C, and fed with 70% artificial diets and 30% frozen forage fish at a feeding rate of 0.2%–1.5% body weight according to the water temperature. Three male and three female A. sinensis were sampled for analyses. Results showed that the lipid content in gonads was significantly higher than in other tissues in both males and females (p < .05). The protein content in male and female gonads was significantly lower than that in the muscle (p < .05). Total content of mono‐unsaturated fatty acids (MUFA) in females was significantly higher than in males (p < .05), while the total content of n‐6 PUFA in females was significantly lower than in males (p < .05). Palmitic acid (C16:0) and oleic acid (C18:1n9) were the major saturated fatty acid (SFA) and MUFA, respectively, in both females and males. The contents of C18:2n6, C20:4n6 (ARA), C18:3n3 and C22:6n3 (DHA) in male gonads were significantly higher than in females (p < .05). The contents of ARA, EPA and DHA in gonads were lower, possibly playing a pivotal role in gonad development. Comparing the nutrients of ovaries in broodstocks at stages III and IV and the eggs at stage IV, the present study suggests that the lipid and fatty acid profile in fish is vital to the gonad development and that proper improvement of lipid nutrition in the broodstock diet to provide sufficient energy and essential fatty acids would be beneficial for the gonad development of Acipenser sinensis.     

Physiological and biochemical response of seaweed Gracilaria lemaneiformis to concentration changes of N and P

This paper presents the studies of nitrogen and phosphorus (N/P) concentrations influence on the antioxidantivity of Gracilaria lemaneiformis and the physio-chemical features in an indoor seawater culture system. The results showed that the specific growth rate, the chemical composition (concentrations of chlorophyll a, phycoerythrin and soluble protein), nitrate reductase activity, antioxidantive defense system (the activities of superoxide dismutase, peroxidase, catalase) and propyldialdehyde content of G. lemaneiformis were affected by changes in concentrations of N and P, and in seaweed culture time. G. lemaneiformis showed increased growth rate when the N/P concentrations increased from 50/3.13 µmol/L to 400/25 µmol/L. But when the N/P concentrations exceeded 400/25 µmol/L, the growth rate dropped significantly. The trend became more obvious once the N/P concentrations reached 600 / 37.5 µmol/L. At these concentrations, the chloroplasts in G. lemaneiformis were damaged as evidences that 1) the number of thylakoids was increased, some of them were swollen up, irregularly aligned, ruptured and partly dissolved; 2) the chloroplast volume was increased; and 3) starch grains in chloroplasts accumulated significantly. On the basis of these results, a remediation critical point for the growth of G. lemaneiformis under high N/P concentrations was proposed.     

中国大鲵生殖腺胚后发育的形态学观察

为探寻中国大鲵(Andrias davidianus)生殖腺胚后发育的特点及规律,采用解剖学与组织学技术对其形态结构变化进行了观察.结果表明,大鲵的原始生殖腺开始出现于出膜28～49 d;出膜133～175 d时一些个体生殖腺内已初步分化出原始卵泡;出膜259 ～343 d时一些个体生殖腺内已初步分化出生精小叶;出膜427 d时,卵巢已明显分化为皮质与髓质,且髓质内出现了卵巢腔,精巢内生精小叶及其内的腔隙、精巢间质等分化已较为明显;出膜511 d时精巢分化为明显的生精小叶和非成熟小叶两个区域.本文认为,大鲵与其他无羊膜类原始生殖腺的分化一般发生在胚后阶段,而且雌性的分化时间早于雄性.     

Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F₁F₀-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F₀, namely oligomycin and N,N′-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F₁, maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F₁ and F₀ domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F₀ proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F₁F₀-ATPase oligomycin sensitivity loss.