Active metabolism of thyroid hormone during metamorphosis of amphioxus.

Thyroid hormones (THs), and more precisely the 3,3',5-triiodo-l-thyronine (T(3)) acetic derivative 3,3',5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T(3) and l-thyroxine (T(4)), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T(3), suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage.

Effects of diquat and fomesafen applied alone and in combination with a nonylphenol polyethoxylate adjuvant on Lemna minor in aquatic indoor microcosms.

The influence of tank-mix adjuvants on pesticide toxicity remains largely unknown. Agral 90, a nonylphenol polyethoxylated tank-mix adjuvant, has been used with diquat (bipyridylium herbicide) and fomesafen (diphenyl-ether herbicide) in aquatic indoor microcosms in order to compare the toxicity of the single compounds and of binary herbicide-adjuvant mixtures to Lemna minor. Twenty-four microcosms were used and treatments were performed with substances alone or with herbicide-adjuvant binary mixtures, at two concentrations levels (44.4 and 222.2 microg/L for the herbicides, and 100 and 500 microg/L for Agral 90). Toxicity was assessed weekly for 1 month through growth measurements, as inferred from the relative frond number (RFN) and relative frond area (RFA). Concentrations of diquat and fomesafen in water and sediments were measured weekly. The herbicides showed very different behaviour in microcosms, with a rapid disappearance of diquat from the aqueous phase whereas fomesafen levels remained almost constant over time. Diquat strongly inhibited the growth of L. minor whereas fomesafen had no effect on plant growth. Presence of the adjuvant only slightly reduced the effect of the lowest concentration of diquat, probably as a result of dispersion of the herbicide at the water surface. It is concluded that tank-mix adjuvant designed to improve herbicide efficiency in the terrestrial environment did not have any effect on aquatic plants when applied to the aquatic environment.

Biotransformation of bisphenol F by human and rat liver subcellular fractions.

Bisphenol F [4,4'-dihydroxydiphenyl-methane] (BPF) has a broad range of applications in industry (liners lacquers, adhesives, plastics, coating of drinks and food cans). Free monomers of this bisphenol can be released into the environment and enter the food chain, very likely resulting in the exposure of humans to low doses of BPF. This synthetic compound has been reported to be estrogenic. A study of BPF distribution and metabolism in rats has demonstrated the formation of many metabolites, with multiple biotransformation pathways. In the present work we investigated the in vitro biotransformation of radio-labelled BPF using rat and human liver subcellular fractions. BPF metabolites were separated, isolated by high-performance liquid chromatography (HPLC), and analysed by mass spectrometry (MS), MS(n), and nuclear magnetic resonance (NMR). Many of these metabolites were characterized for the first time in mammals and in humans. BPF is metabolised into the corresponding glucuronide and sulfate (liver S9 fractions). In addition to these phase II biotransformation products, various hydroxylated metabolites are formed, as well as structurally related apolar metabolites. These phase I metabolic pathways are dominant for incubations carried out with liver microsomes and also present for incubations carried out with liver S9 fractions. The formation of the main metabolites, namely meta-hydroxylated BPF and ortho-hydroxylated BPF (catechol BPF) is P450 dependent, as is the formation of the less polar metabolites characterized as BPF dimers. Both the formation of a catechol and of dimeric metabolites correspond to biotransformation pathways shared by BPF, other bisphenols and estradiol.

Effects of the mixture of diquat and a nonylphenol polyethoxylate adjuvant on fecundity and progeny early performances of the pond snail Lymnaea stagnalis in laboratory bioassays and microcosms.

Effects of the bipyridylium herbicide diquat and tank-mix adjuvant Agral90 were investigated on various life history traits of the freshwater pulmonate snail Lymnaea stagnalis. Trait expression was measured in simple laboratory bioassays on small size groups of snails, and under more complex, indoor microcosm conditions, on larger groups of snails. Microcosms were provided with sediment, plants, and fish, thus allowing a more complex level of intra and inter-specific interactions to develop. Treatments were performed with substances alone or in mixture, at concentrations ranging from 4.4 to 222.2microgl(-1) for diquat, and from 10 to 500microgl(-1) for Agral 90, under a fixed ratio design. Adult growth was negatively affected by diquat and its mixture with Agral 90 both at the highest concentrations (222.2 and 500microgl(-1), respectively). Fecundity expressed differently in bioassays and microcosms, but no effect of the chemicals could be observed on this trait. Progeny development was impaired by 222.2microgl(-1) diquat and its mixture with 500microgl(-1) Agral 90, as reflected by longer development time and reduced hatching rate of clutches laid by the exposed animals, as compared to the controls. Hatching data suggested that diquat bioavailability was lower in microcosms than under bioassay conditions. Consistently, chemical analysis showed that diquat disappeared more rapidly from the water in microcosms than in bioassays. Moreover, the differential expression of several life history traits under bioassays and microcosms conditions was probably also influenced by the level of intraspecific interaction, which differed among the systems. When significant, the effect of diquat was attenuated by the presence of Agral 90, indicating antagonistic interaction between the two substances. Such a deviation from additivity was partly validated statistically.

Comparison of genistein metabolism in rats and humans using liver microsomes and hepatocytes.

Species differences and metabolism are the most crucial factors in considering the effects of genistein. The aim of this study was to have a better knowledge of the metabolic fate of genistein in humans as compared with rats. For this purpose, radiolabeled genistein was incubated with human and rat liver microsomes and with cryopreserved hepatocytes from both species. Incubations were performed using a wide range of genistein concentrations to analyze the kinetics of formation of the metabolites. Metabolite profiling was obtained using an HPLC system connected to a radioactivity detector. Identification of the metabolites was based on their retention times as compared with those of authentic standards and on LC-MS (ESI-MS/MS) or NMR analyses. In both species, liver microsomes produced the same three hydroxylated metabolites (8-OH, 6-OH and 3'-OH-genistein) whereas cryopreserved hepatocytes produced the same glucurono- and sulfo-conjugates (genistein 4'-O-sulfate 7-O-glucuronide, genistein 7-O-glucuronide, genistein 4'-O-glucuronide, genistein 7-O-sulfate and genistein 4'-O-sulfate). The rate of metabolism varied with species. 3'-Hydroxygenistein was the predominant metabolite produced by rat liver microsomes, whereas in humans 3'-hydroxy and 8-hydroxygenistein were produced in the same range. In both human and rat hepatocyte incubations, genistein 7-O-glucuronide represented more than 50% of the incubated dose. Our results on hepatocytes confirmed the predominance of conjugation reaction compared to oxidative reaction observed in vivo.

In vitro aromatic bioactivation of the weak estrogen E(2)alpha and genesis of DNA adducts.

Specific A-ring hydroxylated metabolites of 17beta-estrogens are known to be endogenous pro-carcinogens, more particularly the 4-hydroxylated forms of estrogens produced by cytochrome P4501B1. In this study, we investigated whether estradiol-17alpha, the main hepatic residue of estradiol-17beta in cattle treated for anabolic purposes with estradiol containing implants, could be significantly metabolized by human cells, and whether its aromatic metabolites could induce the formation of DNA adducts as estradiol-17beta and estrone do. First, using a human kidney adenocarcinoma cell line, which expresses specifically the cytochrome P4501B1, we showed that estradiol-17alpha is bioactivated into a mixture of 2- and 4-catechol estrogens leading to the corresponding methoxyestrogens unambiguously identified by LC-APCI-MS/MS. We then demonstrated that the 2- and 4-hydroxylated derivatives of estradiol-17alpha incubated under oxidative conditions with calf thymus DNA gave stable DNA adducts and abasic sites, respectively. From these results, we can consider that human cells expressing CYP1B1-dependent hydroxylation activities metabolize estradiol-17alpha at the same magnitude as estradiol-17beta and estrone, and that in oxidative conditions, the resulting aromatic metabolites can lead to the formation of both stable and unstable DNA adducts.