Jean-Pierre Girard

Cadmium and copper display different responses towards oxidative stress in the kidney of the sea bass Dicentrarchus labrax

Copper and cadmium were i.p. injected into the fish Dicentrarchus labrax. Cu as Cd-treated fish showed an enlargement of the lysosomal membrane of the kidney (pronephros), Cu being more toxic than Cd. Following injection, metal uptake, measured in muscle, liver and kidney, was much higher with cadmium treatment than with copper, the kidney being the main accumulating organ of cadmium in which metal accumulation is correlated with increased zinc level, suggesting metallothionein induction. In vivo exposure to metal decreases the lysosomal membrane stability of pronephros with a half dose of 127 ng g(-1) Cu and 735 ng g(-1) Cd. Lipid peroxidation, expressed as malondialdehyde equivalents (MDA), and catalase activity were measured in kidney subcellular fractions. When added in vitro, Cu significantly raises the MDA level (365% at 200 µM), Cd having a lower effect (20% at 500 µM). Catalase activity is significantly reduced by Cd whereas Cu does not produce any significant effect at the tested concentrations. Results suggest that although both metals cause in vivo damage to pronephros lysosomal membrane, Cu activates the redox process generating oxyradicals but does not affect in vitro the protective catalase activity unlike Cd which appears to weakly participate in oxyradical generation but alters in vitro protective catalase activity.

Mechanisms regulating intracellular pH in sea urchin eggs

Intracellular pH (pHi) of sea urchin eggs (Paracentrotus lividus) was determined using DMO (dimethyloxazolidinedione) and a rapid filtration technique (P. Payan, J.P. Girard, R. Christen and C. Sardet (1981). Exp. Cell Res. 134, 339-344). Transfer of unfertilized or fertilized eggs from normal sea water into Na+-free artificial sea water leads to a progressive acidification and fall of intracellular Na+ content. A step rise in external Na+ to 10 meq causes a rapid but transient Na+ entry coupled to an excretion of H+, giving rise to a pHi increase. It is shown that the plasma membrane of unfertilized eggs contains a permanent and reversible Na+/H+ exchanger which contributes to the regulation of pHi. This exchange occurs with a 1:1 stoichiometry and is independent of metabolic energy. Proton excretion and sodium entry follow saturable kinetics with respect to external Na+ and are completely inhibited by amiloride. At fertilization, pHi increases from 7.38 to 7.64 and is maintained at this level by two separate mechanisms: (1) a Na+/H+ exchange with the same characteristics as in unfertilized eggs; (2) a H+-excreting system that is dependent on external Na+, amiloride sensitive, and requiring metabolic energy. The relationship between the permanent Na+/H+ exchange involved in pHi regulation and the transient Na+/H+ exchange occurring at fertilization is discussed.

Changes in intracellular cations following fertilization of sea urchin eggs: Na+H+ and Na+K+ exchanges

Sodium and potassium contents, as well as H+ excretion were measured at various times after fertilization in eggs of the sea urchin Paracentrotus, using a rapid filtration technique (Payan, P, Girard, U P, Christen, R & Sardet, C, Exp cell res 134 (1981) 339 [1]). Sodium content changes with a biphasic time course due to the appearance of two exchange mechanisms: 1. A transient Na+/H+ exchange with a l/l stoichiometry causes a rapid rise in intracellular sodium. This exchange lasts about 3 min. and is not inhibited by poisoning the eggs with NaCN. 2. An Na+/K+ exchange, detectable within minutes of fertilization causes sodium content of the egg to fall and settle below unfertilized level. This exchange is energy-dependent, reduced in low K+ (2 mM), totally inhibited by harmaline (10(-4) M) and only partially inhibited by ouabain (10(-2) M). Potassium content rises rapidly after fertilization by an undefined mechanism and is further increased via the Na+/K+ exchange. The Na+/Ka+ exchange increases the cytoplasmic K+/Na+ ratio of the egg from 6 to 12 in the first hour after fertilization. When fertilization occurs in low K+ SW, where the Na+/K+ exchange is reduced, the ratio Na+/K+ decreases and the embryos develop with a preponderance of ectodermal structures (animalization).

Effects of caulerpenyne, the major toxin from Caulerpa taxifolia on mechanisms related to sea urchin egg cleavage

Abstract Caulerpenyne (CYN), the major metabolite synthesized by the alga Caulerpa taxifolia (Vahl), inhibited the first cleavage of sea urchin eggs without affecting fertilization. The effect was dose-dependent with a half maximal dose of 33 μM. Blockage of cleavage was observed when the toxin was added within 40 min of insemination. A preliminary search for the cellular targets of this toxin showed that ionic signals involved in the cell dynamics are altered: caulerpenyne reduced the intracellular ATP-dependent Ca 2+ accumulation in a dose-dependent manner but did not provoke a release of sequestered Ca 2+ . This effect is similar to that of thapsigargin, a specific inhibitor of reticular Ca 2+ -ATPase. CYN had no effect on the incorporation of 35 S-methionine into proteins. 3 H-thymidine incorporation into DNA was inhibited by CYN in a dose-dependent manner: an effect well correlated with cell division kinetics. A CYN concentration of 30 μM, which delayed the first cleavage, inhibited overall protein phosphorylation but did not affect histone kinase phosphorylating activity. Thus, CYN appears to alter the main events of sea urchin egg cleavage, and may therefore constitute an ecological risk for microorganisms and eggs of pluricellular animals living close to these algae. Moreover, this compound is of potential pharmacological interest in view of its antiproliferative properties.

Na+ movements and their oscillations during fertilization and the cell cycle in sea urchin eggs.

Abstract We have analysed in detail the Na + content and Na + influx during fertilization and first divisions of the sea urchin egg ( Paracentrotus lividus ) using a filtration technique devised to eliminate rapidly contamination by the Na + of external sea water. In the first 5 min following fertilization the egg fills up with Na + (+ 30%). Thereafter Na + is extruded and the Na + content stabilizes at about 60% of the unfertilized egg level by the second cleavage (2 h). The initial increase in Na + content is due to a large increase in Na + influx already detected at 20 sec. The Na + influx reaches its maximum at 1 min and its minimum at 5 min. H + excretion follows the same kinetics. A second increase in Na + influx is noted 5–10 min after fertilization; it reaches its maximum at prophase metaphase (30 min) and its minimum during cleavage (60 min). These oscillations in Na + influx were observed for the first three divisions. Fertilization also immediately stimulates the Na + efflux which remains elevated throughout the cell cycle and is responsible for the depletion of the Na + content of the embryos. Activation of the eggs by weak amine bases (5 mM NH 4 Cl) which bypasses the early cortical reaction produces only a depletion in the Na + content of the egg similar to that produced by fertilization. NH 4 Cl also increases the Na + influx soon after fertilization, although no transient variations are noted.

Toxic effects of Gymnodinium cf. mikimotoi unsaturated fatty acids to gametes and embryos of the sea urchin Paracentrotus lividus

Abstract The toxicity of the main Gymnodinium cf. mikimotoi polyunsaturated fatty acid, has been investigated using the sea urchin gamete and embryo bioassays. The 18:5n3 fatty acid delays or inhibits first cleavage of Paracentrotus lividus eggs and provokes abnormalities in the embryonic development. These effects were compared with those of other polyunsaturated fatty acids, 18:4n3, 20:5n3 and 22:6n3, which are also present in this alga. A classification of the different fatty acids, based on their effects on sea urchin egg cleavage and the determination of the half inhibiting concentrations (IC 50 ) is proposed.

The nitroxide stable radical tempo prevents metal-induced inhibition of CYP1A1 expression and induction.

Heavy metals are known to provoke oxidative stress in fish liver cells. Because H2O2, OH*- and intracellular superoxide are involved in this oxidation, we investigated the effect of nitroxide radical, 2,2,6,6-tetramethylpiperidinyl-N-oxyl (abbreviated as TEMPO), a cell-permeable agent possessing antioxidant properties, on CYP1A expression in trout (Oncorhynchus mykiss) hepatocytes. 3-methylcholanthrene (3-MC) induced the CYP1A-related EROD activity. This induction was inhibited by concomitant exposure to Cd (II), Cu (II), Pb (II) or Zn (II). CYP1A mRNA levels were also reduced. Simultaneous treatment with 3-MC, a heavy metal and TEMPO suppressed both the inhibition of EROD activity and the decrease of CYP1A mRNA expression. These results suggest a working hypothesis that heavy metals produce multiple oxidative effects, including generation of hydroxyl radicals, which could down-regulate CYP1A1 expression. This metal-induced inhibition was prevented by TEMPO, which might protect trout hepatocytes by scavenging free radicals and thus preventing their inhibitory effects on CYP1A induction and expression.

Characterization of valine transport in sea urchin eggs

In unfertilized eggs, the mechanism of valine uptake can be summarized as follows. It is saturable over the external concentration of valine and insensitive to the presence of external sodium, depletion of cellular energy supplies and intracellular acidosis. The activation energy for the transport reaction (16.3 kcal/mol) is within the range of values reported for active transport of small molecules. In fertilized eggs, the total rate of valine uptake can be divided into two components: (i) a Na+-insensitive uptake which accounts for about 7% of total absorption as shown by studies in Na+-free medium seems to possess the same characteristics as in unfertilized eggs, (ii) a Na+-dependent transport of valine which constitutes the main entry is formed about 5 min after fertilization. It follows Michaelis-Menten kinetics characterized by 15-fold increase in Vmax with no change in Km. These two mechanisms have characteristics in common, such as their insensitivity to metabolic energy supply, their energy of activation and their ability to concentrate valine. The relationship between the establishment of the Na+-dependent valine uptake and the ionic events triggered by fertilization is discussed.

Effects of tributyltin on Ca2+ homeostasis and mechanisms controlling cell cycling in sea urchin eggs

Abstract Tributyltin (TBT) is one of the widespread organotins in the marine environment: we have investigated its cellular targets in the eggs of the marine invertebrate sea urchin Paracentrotus lividus . TBT was used at concentrations ranging from 10 −9 to 10 −4 M. The lipophilic character of TBT makes that the magnitude of the effects varied according to the concentration of egg used. Concentrations of TBT being without effect on egg fertilisation inhibited first and second cleavage in a dose-dependent manner. Concentrations of TBT which block egg cleavage did not alter egg membrane permeabilities to Ca 2+ and Na + . TBT inhibited intracellular ATP-driven sequestration of Ca 2+ into reticular compartment but did not provoke Ca 2+ leakage from these compartment. Blockade of eggs cleavage by TBT is accompanied by an inhibition of protein synthesis ( 35 S-methionine incorporation) and DNA synthesis ( 3 H-thymidine incorporation). Protein phosphorylation ( 32 P incorporation) and histone H1 kinase activity were inhibited when cell cycling was arrested by organotin. Results are discussed in terms of concentration of TBT per egg protein in order to compare the particular sensitivity of the numerous cellular process affected by TBT.

Ryanodine Activates Sea Urchin Eggs

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP3, produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner 45Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP3 stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg++, ruthenium red and heparin. Our results suggest that ryanodine‐sensitive intracellular calcium channels exist in the sea urchin egg.