Inhibition of prostacyclin-like material formation by cadmium.

The activity of cadmium on the platelet and vascular wall prostaglandin system was studied. This metal was found to inhibit the production of prostacyclin (PGI₂) by vascular wall tissue, without affecting the platelet aggregation induced by arachidonic acid or ADP. In view of the vasodilator activity of PGI₂ these data suggest an hypothesis on the origin of cadmium-induced hypertension and on the development of vascular thrombi.     

Inhibition of rat heart mitochondrial respiration by cadmium chloride

Mitochondria were isolated from hearts obtained from adult male Sprague-Dawley rats by two-part differential centrifugation of heart homogenates. Time-dependent (0-120 sec) and concentration-dependent (0-10 microM CdCl2) effects of cadmium on pyruvate-malate-supported state 3 and state 4 respiration were measured in a constant temperature reaction chamber at 37 degrees C, according to established procedures. The ID50 for cadmium chloride on state 3 respiration was determined to be 4.2 microM. The inhibition produced by cadmium chloride in heart mitochondria was compared, using identical procedures, to the effects induced by two compounds, sodium atractyloside and potassium cyanide, which are known to alter mitochondrial respiration at specific sites. The calculated ID50 values for these agents in heart mitochondria were 1.8 and 16 microM, respectively. The concentration-dependent inhibition of mitochondrial respiration induced by either cadmium chloride or potassium cyanide was maintained in the presence of 50 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known uncoupling agent. In contrast, sodium atractyloside did not block the uncoupling effect of 50 microM CCCP. In addition cadmium chloride was also shown to inhibit CCCP-uncoupled mitochondrial respiration. The cadmium-induced inhibition of mitochondrial respiration was reversed partially by cysteine and completely by 2,3-dimercaptopropanol. The results of the present study indicate that, at all concentrations, cadmium chloride acted solely as an inhibitor of rat heart pyruvate-malate-supported mitochondrial respiration. These findings suggest a possible mechanism for the reported disturbances in myocardial metabolism and function that occur in conjunction with acute and chronic cadmium exposure in humans and experimental animals.     

Inhibition of delayed hypersensitivity reaction in mice by cadmium

The effect of a single exposure to Cd on DH was studied through exposure to Cd at different time intervals in relation to antigen administration. Exposure to Cd significantly suppressed the DH reaction, with a dose-response relationship, when administered 2 or 3 days after immunization of BALB/c and DBA/2 mice with sheep red blood cells. A strain difference in sensitivity to Cd was also demonstrated. These results suggest that a single exposure to Cd impairs the cellular immune response in mice.     

Effects of dithiocarbamates and cadmium on the enzymatic activities in liver,kidney and blood of mice

The effects of N-benzyl-d-glucamine dithiocarbamate (BGD), diethyldithiocarbamate (DDTC), and N-p-hydroxymethylbenzyl-d-glucamine dithiocarbamate (HBGD) on the enzymatic activities in mice were studied. The mice were given i.v. injections of these chelating agents (1 mmol/kg) and 3 h later the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ -glutamyltranspeptidase ( γ-GTP), alkaline phosphatase (ALP), leucine aminopeptidase (LAP), and cholinesterase (ChE) in the liver, kidney, and blood were determined. These enzymatic activities were little changed by treatment with these chelating agents. Cadmium (Cd) administration markedly decreased the activities of AST and ALT in the liver and kidney and greatly increased these enzymatic activities in blood. The changes in the enzymatic activities by treatment with Cd were prevented by injection of BGD (1 mmol/kg). These results indicate that BGD, DDTC, and HBGD were not toxic to the liver or kidney of mice and that BGD treatment protected against the acute hepatic and renal toxicity induced by Cd.     

Impairment of lipid storage by cadmium in the European eel (Anguilla anguilla)

Because European silver eels (Anguilla anguilla) fast during their reproductive migration to the Sargasso Sea, the successful completion of their unusual life cycle depends on quantity of lipids stored beforehand. These lipids are mainly accumulated during the growth phase stage of the animals, called yellow eel, as triglycerides in muscle. They are then catabolized to provide sufficient energy to enable migration, gonad maturation and spawning. In the laboratory, we investigated the possible impact of cadmium on the lipid storage efficiency of yellow eels in order to evaluate the possible contribution of this pollutant to the reported decline of European eel populations. Eels were exposed to dissolved cadmium at nominal concentrations of 0 and 5 microgL(-1) for 1 month. Cd toxicity was then examined by studying the activity and expression level of several enzymes involved in liver lipolysis and lipogenesis and by determining lipid content in muscle. Contaminated eels showed a lower body weight growth with a lower efficiency of lipid storage compared to controls. Using two complementary approaches, genetic and enzymatic, it was possible to conclude that this impairment is mainly explained by an increased utilisation of triglycerides since cadmium contamination did not trigger a reduced fatty acid synthesis. These observations suggest an increased fat consumption in presence of cadmium, which could compromise successful reproduction.     

Presynaptic neurotoxins with enzymatic activities

Toxins that alter neurotransmitter release from nerve terminals are of considerable scientific and clinical importance. Many advances were recently made in the understanding of their molecular mechanisms of action and use in human therapy. Here, we focus on presynaptic neurotoxins, which are very potent inhibitors of the neurotransmitter release because they are endowed with specific enzymatic activities: (1) clostridial neurotoxins with a metallo-proteolytic activity and (2) snake presynaptic neurotoxins with a phospholipase A2 activity.     

Inhibition studies of microsomal UDP-glucuronosyltransferase activities by furosemide and salicylamide

Contrarily to cytochrome P-450, a few inhibitors of UDP-glucuronosyltransferase have been described. We verified the nature of the in vitro inhibition due to furosemide, using 4 different aglycones (morphine, p-nitrophenol, borneol and eugenol) presumably belong may to different clusters of UDP-glucuronosyltransferase activities. The variations of these corresponding kinetic parameters (Km, Vmax, specific activities) must correspond to different inhibition mechanisms of furosemide, for example different site(s) of fixation in the area of the active site of UDPGT. Because these variations were not as classically described, we checked the impact of furosemide pretreatment on in vitro levels of different UDPGT activities. We compared these result, with another inhibitor (salicylamide). The apparent induction due to the both molecules enforced the hypothesis of a complexe inhibition mechanism.     

Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.     

Inhibition of oxytocic and hypotensive activities of bradykinin by bradykinin-binding antibodies

The influence of bradykinin-binding antibodies on the hypotensive and the oxytocic responses to bradykinin was investigated. Bradykinin-binding antibodies were obtained from rabbits injected with bradykinin coupled to ovalbumin. Pre- and post-immunization sera were tested for their capacity to bind intrinsically labeled ¹⁴C-bradykinin. Six injected rabbits produced bradykinin-binding antibodies. The binding ofa radio-iodotyrosine analog of bradykinin to antiserum was inhibited by unlabeled bradykinin or kallidin, but was unaffected by oxytocin, vasopressin or angiotensin.Incubation of bradykinin with antiserum resulted in an inhibition of its hypotensive activity in the rat. Bradykinin-induced hypotension was also abolished in rats injected with antiserum. Pre-immunization serum was ineffective in both respects. Bradykinin binding antibodies also inhibited the oxytocic activity of bradykinin on the isolated rat's uterus. The inhibitory effects became evident after dialysis of the antiserum. Undialyzed antisera contained an oxytocic substance which masked the inhibition of bradykinin activity.Inhibition of pharmacological effects of bradykinin by antibradykinin was reversed by heat inactivation of antibodies. Bradykinin-binding antibodies not only inhibited bradykinin activity, but also protected the peptide against kininases.     

Inhibition of the liberation of arachidonic acid by cadmium ions in rabbit alveolar macrophages

The effects of CdCl₂ on the liberation of arachidonic acid (20∶4) from membrane phospholipids of A23187-stimulated rabbit alveolar macrophages and on the activity of phospholipase A₂ (PLA₂) in a cytosolic fraction were studied. Alveolar macrophages were prelabeled with [³H]arachidonic acid (20∶4) and then treated with A23187. This treatment resulted in a remarkable increase in the liberation of [³H]20∶4 from their phospholipids. Exposure of cells to Cd²⁺ inhibited the liberation of [³H]20∶4 in a dose-dependent manner. Liberation of [³H]20∶4 from cell lipids was calcium dependent and the inhibitory effect of Cd²⁺ competed with the stimulatory effect of Ca²⁺. When Ca²⁺ was removed from the incubation medium, Cd²⁺ did not influence the liberation of [³H]20∶4. Entry of⁴⁵Ca²⁺ into cells was enhanced by treatment of A23187. However, Cd²⁺ did not influence the cellular uptake of⁴⁵Ca²⁺. Treatment with A23187 markedly enhanced entry of¹⁰⁹Cd²⁺ into cells. The effect of Cd²⁺ on the activity of phospholipase A₂ was determined with 1-palmitoyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphocholine as substrate. Calcium-dependent activation of PLA₂ was observed and Cd²⁺ inhibited activation in a dose-dependent manner. These results suggest that exposure of alveolar macrophages to Cd²⁺ causes a reduction in the rate of liberation of 20∶4 from cell lipids, as a possible result of the inhibition of PLA₂ activity by Cd²⁺.     

Inhibition of molting by cadmium in the crab Chasmagnathus granulata (Decapoda Brachyura)

The effect of cadmium on molting of the estuarine crab Chasmagnathus granulata was assayed. Adult males were eyestalk-ablated, in order to synchronically induce molting, and were then chronically exposed to cadmium at 0.25 or 0.50 mg/l. At the highest concentration, a significant mortality was detected at the time of molting, in the few crabs that could reach the E stage. However, most of the crabs exposed from the beginning of the premolt period (D(0) stage) to 0.50 mg/l of cadmium were arrested at the D(1)" stage. This effect was not seen when crabs were exposed to the same cadmium concentration from either D(1)"' or D(3) premolt stages. Crabs arrested by cadmium did not present any difference in the calcium content of carapace, compared to controls, while ecdysteroid levels of those crabs were similar to the ones of control crabs that were in the same premolt stage but could finally molt. These results suggest that cadmium could be preventing the normal peaking of ecdysteroids needed for molting. Since eyestalk-ablated crabs were used, a presumably direct effect of cadmium on Y-organ seems likely, by affecting cytoplasmatic calcium concentration and/or other actions.     

Inhibition of matrix metalloproteinase-2 and -9 activities by selected flavonoids

Matrix metalloproteinases (MMPs) play an important role in physiological and pathological matrix degradation. Here, we report that flavonoids, at physiologically relevant concentrations, inhibit two members of this enzyme family, namely MMP-2 and -9. Eight flavonoids with increasing number of hydroxy groups and other modifications were compared for their capacity to inhibit recombinant catalytic domains of these proteases. EC50 values ranged from 59 and 70 microM (primuletin/5-hydroxyflavone) to 9 and 4 microM (luteolin 7- O-glucoside) for MMP-2 and -9, respectively. Interestingly, the latter glucoside was an equal (MMP-2) or even stronger (MMP-9) inhibitor than its aglycone, luteolin. For luteolin, one of the strongest flavonoids tested, kinetic analysis revealed a non-competitive type of inhibition. Our results add a novel function to the long list of biological effects of these ubiquitous plant constituents that may contribute to and enhance their modulating influence on extracellular matrix degradation and remodelling.     

Inhibition by germanium oxide of the mutagenicity of cadmium chloride in various genotoxicity assays.

The effects of germanium oxide on the genotoxicity of cadmium chloride were investigated. The incorporation of [3H]thymidine into testicular DNA was inhibited in mice injected ip with 1.35, 1.80 or 2.70 mg cadmium chloride/kg body weight. Germanium oxide (0.05 or 0.1 mg/kg body weight, sc) alone did not affect [3H]thymidine incorporation into testicular DNA but 0.05 mg germanium oxide/kg antagonized the inhibitory effect of 1.35 mg cadmium chloride/kg. However, combinations of the other doses of the two compounds did not show statistically significant antagonistic effects. Cadmium chloride significantly increased the frequencies of micronucleus formation in polychromatic erythrocytes, and of chromosome aberrations in the bone marrow of mice treated with 0.7, 1.4 or 2.7 mg/kg body weight, in a dose-related manner. These effects were inhibited by germanium oxide at doses of 0.1 or 0.5 mg/kg body weight, although germanium oxide alone did not affect micronucleus formation or the chromosome aberration rate. Cadmium chloride produced a dose-related increase in the frequency of sister chromatid exchanges in cultured human lymphocytes at concentrations of 5, 10 or 50 mumol. This effect was also inhibited by germanium oxide (0.05 or 0.1 mumol), although germanium oxide alone had no effect. There was a dose-related increase in the frequency of sperms with abnormal head morphologies from mice treated with 0.6, 1.1 or 2.2 mg cadmium chloride/kg body weight and this too was antagonized by the injection of germanium oxide (0.1 or 0.5 mg/kg body weight). Germanium oxide alone did not affect the frequency of sperm-head abnormalities.     

Antioxidant and glutathione-related enzymatic activities in rat sciatic nerve

The present work tries to establish the antioxidant capacity of the peripheral nervous tissue of the rat, in terms of the enzymatic activities present in this tissue that either prevent the formation of activated species as the semiquinone radical (DT-diaphorase), protect against activated oxygen species (superoxide dismutase, glutathione peroxidase), conjugate natural toxic products or xenobiotics (glutathione S-transferases, especially the activity conjugating 4-hydroxy-nonenal), or complete the glutathione system metabolism (glutathione disulfide reductase, γ-glutamyl transpeptidase). All the activities studied are lower in this tissue than they are in liver, except for γ-glutamyl transpeptidase. The relevance of the results obtained and its possible relationship with different neuropathies is discussed. It is concluded that the peripheral nervous tissue is by far less protected than the liver against oxidative damage.     

Effects of crocetin on antioxidant enzymatic activities in cardiac hypertrophy induced by norepinephrine in rats

Crocetin, a carotenoid isolated from the Chinese herbal medicine Crocus sativus L. (Saffron), has been shown to have cardiovascular protective effects. The present study investigated the protective action of the antioxidant crocetin against cardiac hypertrophy induced by norepinephrine (NE). This was evaluated by assaying for pathological histological changes with an optical microscope and cell image analysis system. Lipid peroxidation was quantified using thiobarbituric acid-reactive substances (TBARS). Myocardial superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and myocardial catalase (CAT) activities were assayed to evaluate the antioxidant capacity. After long term treatment with NE, antioxidant enzymatic activities were significantly decreased, while products of lipid peroxidation increased. Crocetin markedly reduced the content of lipid peroxidation (LPO), increased the GSH-Px and SOD activity in cardiac hypertrophy, and significantly improved the myocardial pathological histological changes induced by NE. These results suggest that the cardioprotective effects of crocetin are related to modulation of endogenous antioxidant enzymatic activities. Comparing crocetin with captopril, our results indicated that antioxidant activity is an important factor in the therapy of cardiac hypertrophy, but as an antioxidant only, its effects may be limited.     

Tracing some enzymatic activities in three virulent pathogenic Erwiniae

The absence, in the biomass, of glutamic-oxalacetic and glutamic-pyruvic transaminases and the prevalance of gamma-glutamic transpeptidase were taken as basic criteria to differentiate betweenErwinia carotovora var.carotovora fromE. carotovora var.citrullis andE. toxica. For further identification, detection of cholesterol in the biomass confirmed thetoxica species whereas the absence of glucose confirmed thecitrullis variety.