Yolanda Morcillo

Evidence of endocrine disruption in clams - Ruditapes decussata - transplanted to a tributyltin-polluted environment

Clams--Ruditapes decussata--were transplanted to an organotin-polluted marina for a period up to 5 weeks. Tributyltin (TBT) was the major organotin compound detected in clams; TBT accumulation was extremely rapid, it reached a maximum tissue concentration of 290 ng/g wet weight 3 weeks after transplant, which represents a bioaccumulation factor of 12,000. Dibutyltin, monobutyltin and triphenyltin were also detected, although at a lesser extent (4-22 ng/g wet wt). Investigations on endogenous steroid content by radioimmunoassay revealed important changes in TBT-exposed clams. A 33% increase in testosterone titres was observed 5 weeks after transplant. Likewise, a time-dependent decrease in oestradiol levels was evidenced, viz. oestradiol titres decreased 2, 3.5 and 5-fold in clams sampled 1, 3 and 5 weeks after transplant, respectively. These observations together with previous data on TBT-exposure experiments suggest a potential masculinization of clam physiology as a consequence of TBT exposure.

Imposex in the commercial snail Bolinus brandaris in the northwestern Mediterranean

A survey of the degree of imposex in populations of the commercial muricid Bolinus brandaris (L.) was carried out at six locations along the Catalan Coast (NW Mediterranean). Imposex was found to be a widespread phenomenon; the occurrence of a penis was observed in all females studied with the exception of those from St. Carles (South of Catalunya) where only 37% were affected. Other indices such as Relative Penis Length (RPL) and Vas Deferens Sequence (VDS) were applied; RPL varied from 4.5% in St. Carles--the least affected area--to 29.8% in the vicinity of Barcelona; similar results were obtained for VDS. Tissue organotin residues ranged from 6 to 140 ng g(-1) w.w. as Sn. Despite legislation restricting the use of tributyltin (TBT) in antifouling paints, this paper evidences the occurrence of organotin pollution far from the source, with levels of both TBT and triphenyltin (TPhT) high enough to cause environmental concern.

Effects of tributyltin on the Phase I testosterone metabolism and steroid titres of the clam Ruditapes decussata

Abstract The effects of tributyltin (TBT) on the cytochrome P450 monooxygenase system of the clam Ruditapes decussata have been evaluated. Digestive gland microsomes were observed to catalyze the oxidative metabolism of testosterone. The metabolites detected were androstenedione, dihydrotestosterone, dihydroandrostenedione, 6β-, 6α-, 2α- and 2β-hydroxytestosterone, estrone and 17β-estradiol. The effect of 0.1 and 1.0 mM TBT in the incubation mixture lead to a significant reduction in the total metabolism rate (28–39%) and changes in the metabolite profile, viz. a significant decrease in the formation of androstenedione (51–54%) and a stimulation in the formation of hydroxylated metabolites. To fully understand and validate the in vitro data, clams were exposed to different concentrations of TBT in water (0.1, 0.45 and 2.27 μg l−1 of TBT as Sn) for 7 days. When digestive gland microsomes of control and exposed organisms were assayed for their ability to metabolize testosterone, important changes were observed in the pattern of the metabolites formed. There was a significant increase in the formation of 6α-hydroxytestosterone and a dose-dependent decrease in the aromatization of testosterone to estrone and estradiol. Moreover, investigation of steroid concentrations by radioimmunoassay revealed an increase in testosterone in the tissue of TBT-exposed clams. These observations indicate a significant interaction of TBT with androgen metabolism in the clam Ruditapes decussata which may contribute to the masculinization of clam physiology.

Monitoring of organotin compounds and their effects in marine molluscs

Abstract Organotin compounds are ubiquitous contaminants in the environment. The high biological activity of some compounds toward aquatic organisms leads to deleterious impacts in aquatic ecosystems. A comprehensive view is given of the occurrence, biochemical effects and impact of organotin contamination on molluscs along the Catalan coast (Northwestern Mediterranean). Chemical analysis of biota samples revealed that several years after legislation to reduce tributyltin (TBT) inputs, the concentration of this compound in mussels inhabiting Catalan harbours is still elevated. Likewise, levels of organotins outside the harbours are high enough to cause imposex in the commercial muricid Bolinus brandaris . The interaction of TBT with the molluscan cytochrome P450 and the consequences for the organism in terms of hormonal disruptions are discussed.

Interaction of tributyl- and triphenyltin with the microsomal monooxygenase system of molluscs and fish from the Western Mediterranean

Abstract The in vitro interaction of tributyltin (TBT) and triphenyltin (TPhT) with the microsomal cytochrome P450 monooxygenase system of different marine organisms has been evaluated. The selected organisms were two bivalve species, the mussel Mytilus galloprovincialis and the clam Tapes decussata , and the gastropod Thais haemastoma . The fish Mullus barbatus was also studied and the results compared with those obtained for invertebrates. The microsomal fraction isolated from the hepatopancreas or liver of these organisms was incubated in the presence of different concentrations of TBT and TPhT and the interaction with cytochrome P450, cytochrome b 5 , NAD(P)H cytochrome c reductases and 7-ethoxyresorufin O -deethylase (EROD) activity evaluated. Direct effects on spectrally determined P450 and b 5 were only studied in mussels and detected at very high concentrations of TBT or TPhT (1 mM). In general, TBT and TPhT differed in their specificity. TBT led to inhibition of both NADH and NADPH cytochrome c reductase activity in the studied molluscs, whereas TPhT showed little effect. On the contrary, no significant effects on fish hepatic NAD(P)H cytochrome c reductases were detected. EROD activity, however, was significantly inhibited by both TBT and TPhT in Mullus barbatus at a concentration as low as 5–10 μM. The study shows speciesrelated differences and selective effects of TBT and TPhT on different components of the microsomal monooxygenase system.

Interaction of tributyltin with hepatic cytochrome P450 and uridine diphosphate-glucuronosyl transferase systems of fish: In vitro studies

Hepatic microsomes of red mullet (Mullus barbatus) and flounder (Platichthys flesus) were preincubated in the presence of a concentration range of the antifouling agent tributyltin (TBT) chloride, and the interactions of TBT with cytochrome P450 and uridine diphosphate-glucuronyl transferase systems were investigated. The enzyme systems were examined in terms of cytochrome P4501A (CYP1A)-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity and benzo[a]pyrene (BaP) metabolism and in terms of glucuronidation of testosterone and 17beta-estradiol, respectively. Ethoxyresorufin O-deethylase and BaP hydroxylase (BPH) activities of both fish species were progressively inhibited by increasing concentrations of TBT, and the effects were more pronounced for EROD than for BPH (maximal inhibition at 100 microM TBT for EROD and 250-500 microM TBT for BPH). Hydroxylated metabolites of BaP (3-hydroxy-, 7,8-dihydrodiol, and 9,10-dihydrodiol), representing 95% of the total metabolites formed, were reduced up to 75% in the presence of 100 to 500 microM TBT, whereas the formation of other metabolites was less affected. This may alter BaP toxicity and carcinogenicity. Overall, the results were consistent with a specific inhibitory effect of TBT on CYP1A in the two fish species. Additionally, the conjugation of testosterone was significantly inhibited (20%) at low TBT doses (5 microM), with no effect on the glucuronidation of estradiol.

Effects of tributyltin on the cytochrome P450 monooxygenase system and sex steroid metabolism in the clam Ruditapes decussata

Abstract The effects of tributyltin (TBT) on the cytochrome P450 monooxygenase system of the clam Ruditapes decussata have been evaluated. Digestive gland microsomes were observed to catalyze the oxidative metabolism of testosterone. The metabolites detected were androstenedione, dihydrotestosterone, dihydroandrostenedione, 6β-, 6α-, 2α-, and 2β-hydroxytestosterone, estrone and 17β-estradiol. The effect of 0.1 and 1 mM TBT in the incubation mixture led to a significant reduction in the total metabolic rate and changes in the metabolite profile. Clams were also exposed to different concentrations of TBT in water (0.1, 0.45, 2.27 μgl−1 of TBT as Sn) for seven days. A significant increase in testosterone levels was found in the TBT exposed organisms. When digestive gland microsomes of control and exposed organisms were assayed for their ability to metabolize testosterone, a dose-responsive decrease in aromatization of testosterone to estrone and estradiol was observed (p