Organophosphate-resistant forms of acetylcholinesterases in two scallops--the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus.

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.

A possible regulatory role of 17beta-estradiol and tamoxifen on glyoxalase I and glyoxalase II genes expression in MCF7 and BT20 human breast cancer cells.

The glutathione-dependent glyoxalases system, composed of glyoxalase I (GloI) and glyoxalase II (GloII) enzymes, is involved in the detoxification of methylglyoxal, a by-product of cell metabolism. Aberrations in the expression of glyoxalase genes in several human cancers have been reported. Sometimes, these aberrations seem to differ depending on the organs and on the sensitivity of the tumours to estrogens, as we previously detected in the hormone-responsive breast cancer compared to the hormone-independent bladder cancer. To investigate a possible regulatory role of estrogens, as well as antiestrogens, on glyoxalases system, estrogen receptor (ER)-positive MCF7 and ER-negative BT20 human breast cancer cells were cultured in the presence of 17beta-estradiol (E2) and tamoxifen (TAM) performing two independent experiments. After a 24 h or 4 days treatment, we evaluated GloI and GloII mRNA levels, by Ribonuclease Protection Assay (RPA), enzymatic activities spectrophotometrically and cell proliferation by [3H]thymidine incorporation. We found that both steroid molecules affected glyoxalases gene expression and proliferation in a different manner in the cell lines. The modifications in mRNA levels were accompanied by parallel changes in the enzymatic activities. The possibility that modulation of glyoxalase genes by E2 and TAM are due to the presence of estrogen response elements (ERE) or cross-talk mechanisms by proteins of the estrogen signal transduction pathways are discussed. Knowledge regarding the regulation of glyoxalases by E2 and TAM may provide insights into the importance of this enzymes in human breast carcinomas in vivo.

Effects of chlorpyrifos on the catalytic efficiency and expression level of acetylcholinesterases in the bivalve mollusk Scapharca inaequivalvis.

Three acetylcholinesterase (AChE) forms were detected and recovered from foot or gill tissues of the benthonic bivalve mollusk Scapharca inaequivalvis. A study was performed to investigate changes in catalytic and hydrodynamic features of these enzymes, as well as in their expression levels, after a 4-d or a 15-d exposure to a sublethal concentration (0.1 microl/L) of the pesticide chlorpyrifos (CPF). Both considered organs hold, in either CPF-exposed or untreated animals, two nonamphiphilic AChE forms, G2 and G4, which copurified on a procainamide-containing affinity gel and were separated by density gradient centrifugation. A third AChE form, an amphiphilic membrane-anchored G2, was also purified on the same affinity matrix from both organs. All enzymatic forms are true AChEs and are poorly inhibited by CPE They show different increases in the maximum velocity (Vmax) and in the Michaelis constant (Km) values after CPF exposure. Consequently, catalytic efficiency of AChEs, as defined by the ratio Vmax:Km, rises in the gills and drops in the foot. This would produce an overexpression of AChE-specific mRNAs. The effect was longer lasting in the foot. The combined results indicate that overexpression of enzymes in the presence of organophosphate (OP) may be a consequence of OP resistance itself. Again, the resistance of the organism to CPF seem to depend mainly on the resulting increase in AChE content.

Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations.

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.

Different expressions of organophosphate-resistant acetylcholinesterases in the bivalve mollusk Scapharca inaequivalvis living in three different habitats.

The benthic mollusk Scapharca inaequivalvis was collected in spring 1999 from three areas of the northern Adriatic Sea. From the mollusk, molecular forms of acetylcholinesterase (AChE), consisting of two prevailing spontaneously soluble (SS) forms present in the blood, were obtained. These forms are a globular tetramer (SSG4), a dimer (SSG2) of catalytic subunits, and a minor amphiphilic globular dimer (low-salt Triton [LST] G2) phosphatidylinositol tailed. All SS and LST forms, partially purified by affinity chromatography, are AChEs with a marked substrate specificity for acetylthiocholine and poor hydrolysis with butyrylthiocholine. They are poorly inhibited by carbamate eserine and show a different pattern of inhibition by organophosphate diisopropylfluorophosphate (DFP), with totally resistant SS forms from clams collected from the two stations nearest the Po River. Acetylcholinesterase SS and LST forms are expressed at highest, lowest, and middle levels in clams collected from the northern station, closer to the Po delta, and from the two more southern ones, respectively. The possibility that the expression pattern of AChE forms is due to effects of single or mixed classes of chemical pollutants is discussed.