Impact of an urban multi-metal contamination gradient: metal bioaccumulation and tolerance of river biofilms collected in different seasons.

The aim of this study was to investigate the repeatability and seasonal variability of the biological response of river biofilms chronically exposed to a multi-metal pressure in an urban contamination gradient. Biofilms were grown on immersed plastic membranes at three sites on the Seine river upstream (site 1) and downstream (sites 2 and 3) from Paris (France). They were collected in four different seasons (autumn, spring, summer and winter). Biofilm tolerance to Cu, Ni, Pb and Zn was measured using a PICT (Pollution-Induced Community Tolerance) approach with a previously developed short-term toxicity test based on ß-glucosidase (heterotrophic) activity. Metal concentrations in the river and also in the biofilm samples (total and non-exchangeable bioaccumulated metals) were also monitored. Biofilm-accumulated metal concentrations reflected the increase of the multi-metal exposure along the urban gradient. These concentrations were strongly correlated with dissolved and particulate organic carbon and with the total metal fraction in the river water, which recalls the significant influence of the environmental parameters on metal uptake processes in river biofilms. Overall, natural biofilms allow monitoring water quality by integrating the variations of a diffuse metal contamination overtime. Tolerance levels globally increased from site 1 to site 3 reflecting the metal pollution gradient measured in the river water collected at the three sites. Cu tolerance tended to increase during warm seasons but no clear seasonal tendency could be found for Ni, Pb and Zn. Furthermore, principal component analysis clearly discriminated samples collected upstream (site 1) from samples collected downstream (sites 2 and 3) along the first principal component which was correlated to the metal gradient. Samples collected in winter were also separated from the others along the second principal component correlated to parameters like water temperature and Total Suspended Solids concentration. This study shows that chronic in situ exposure to environmental metal concentrations has a significant impact on natural biofilms. Biofilm tolerance to metals and biofilm metal bioaccumulation both reflect metal exposure levels although they remain low when compared to Environmental Quality Standards from the European Water Framework Directive. Yet temperature appears as an important environmental variable shaping community structure and response to toxic exposure which shows that the sampling date is an important parameter to consider when using natural river biofilms to assess the impacts of urban pressure.

One-pot and regiospecific synthesis of 2,3-disubstituted indoles from 2-bromoanilides via consecutive palladium-catalyzed Sonogashira coupling, amidopalladation, and reductive elimination.

A practical one-pot and regiospecific three-component process for the synthesis of 2,3-disubstituted indoles from 2-bromoanilides was developed via consecutive palladium-catalyzed Sonogashira coupling, amidopalladation, and reductive elimination.

Pollution-induced community tolerance of freshwater biofilms: measuring heterotrophic tolerance to Pb using an enzymatic toxicity test.

This study aims at investigating the impacts of Pb on freshwater biofilms with a pollution-induced community tolerance (PICT) approach using a recently developed short-term toxicity test based on ß-glucosidase activity to measure biofilms' tolerance to Pb. We first investigated more closely the influence of the total suspended solids (TSS) concentrations of biofilm suspensions used for short-term toxicity tests performed to assess Pb tolerance. The Pb EC(50) values of four dilutions of the same biofilm suspension increased with their TSS concentrations. TSS-normalization allowed to obtain a unique measure of Pb tolerance, thus confirming that TSS-normalization of EC(50) values is a good means to estimate biofilm tolerance to Pb. The experiment was repeated with three different biofilm samples collected at different sites and dates. Second, biofilms were exposed to Pb (0, 1, 10 and 100 µg/L) for 3 weeks in microcosms to assess the impacts of Pb exposure on the communities. An increase in Pb tolerance was observed for the biofilm exposed to 100 µg/L. Automated Ribosomal Intergenic Spacer Analysis revealed modifications of bacterial and eukaryotic community structure with Pb exposure. Moreover, exposure to 100 µg/L Pb also led to an increase in Zn tolerance but not Cu tolerance. This study shows that tolerance acquisition to Pb can be detected after exposure to environmental concentrations of Pb using a PICT methodology and normalized EC(50) values as measures of Pb tolerance.

Indolequinone inhibitors of NRH:quinone oxidoreductase 2. Characterization of the mechanism of inhibition in both cell-free and cellular systems.

We describe a series of indolequinones as efficient mechanism-based inhibitors of NRH:quinone oxidoreductase 2 (NQO2) for use either in cellular or cell-free systems. Compounds were designed to be reduced in the active site of the enzyme leading to loss of a substituted phenol leaving group and generation of a reactive iminium electrophile. Inhibition of NQO2 activity was assessed in both cell-free systems and the human leukemia K562 cell line. Inhibition of recombinant human NQO2 by the indolequinones was NRH-dependent, with kinetic parameters characteristic of mechanism-based inhibition and partition ratios as low as 2.0. Indolequinones inhibited NQO2 activity in K562 cells at nanomolar concentrations that did not inhibit NQO1 and were nontoxic to cells. Computation-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel with the isoalloxazine ring of FAD, and mass spectrometry extended our previous finding of adduction of the FAD in the active site of NQO2 by an indolequinone-derived iminium electrophile to the wider series of indolequinone inhibitors. Modeling combined with biochemical testing identified key structural parameters for effective inhibition, including a 5-aminoalkylamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for the correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least 1 order of magnitude more potent than any previously documented competitive inhibitors of NQO2 and represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems.

Mechanism-based inhibition of quinone reductase 2 (NQO2): selectivity for NQO2 over NQO1 and structural basis for flavoprotein inhibition.

A role for the flavoprotein NRH:quinone oxidoreductase 2 (NQO2, QR2) in human diseases such as malaria, leukemia and neurodegeneration has been proposed. In order to explore the potential of NQO2 as a therapeutic target, we have developed potent and selective mechanism-based inhibitors centered on the indolequinone pharmacophore. The compounds show remarkable selectivity for NQO2 over the closely related flavoprotein NQO1, with small structural changes defining selectivity. Biochemical studies confirmed the mechanism-based inhibition, whereas X-ray crystallography and mass spectrometry revealed the nature of the inhibitor interaction with the protein. These indolequinones represent the first mechanism-based inhibitors of NQO2, and their novel mode of action involving alkylation of the flavin cofactor, provides significant advantages over existing competitive inhibitors in terms of potency and irreversibility, and will open new opportunities to define the role of NQO2 in disease.

A practical mild, one-pot, regiospecific synthesis of 2,3-disubstituted indoles via consecutive Sonogashira and Cacchi reactions.

[Structure: see text] A practical one-pot, regiospecific three-component process for the synthesis of 2,3-disubstituted indoles was developed via consecutive Pd-catalyzed Sonogashira coupling, amidopalladation, and reductive elimination.