Acute and chronic toxic effects of bisphenol a on Chlorella pyrenoidosa and Scenedesmus obliquus

The acute and chronic toxic effects of Bisphenol A (BPA) on Chlorella pyrenoidosa (C. pyrenoidosa) and Scenedesmus obliquus (S. obliquus) were not well understood. The indoor experiments were carried out to observe and analyze the BPA‐induced changes. Results of the observations showed that in acute tests BPA could significantly inhibit the growth of both algae, whereas chronic exposure hardly displayed similar trend. Superoxide dismutase (SOD) and Catalase (CAT) activities of both algae were promoted in all the treatments. Chlorophyll a synthesis of the two algae exhibited similar inhibitory trend in short‐term treatments, and in chronic tests C. pyrenoidosa hardly resulted in visible influence, whereas in contrast, dose‐dependent inhibitory effects of S. obliquus could be clearly observed. The experimental results indicated that the growth and Chlorophyll a syntheses of S.obliquus were more sensitive in response to BPA than that of C. pyrenoidosa, whereas for SOD andCAT activities, C. pyrenoidosa was more susceptible. This research provides a basic understanding of BPA toxicity to aquatic organisms. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 714–722, 2014.     

Changes in the contractile responses to carbachol and in the inhibitory effects of verapamil and nitrendipine on isolated smooth muscle preparations from rats subchronically exposed to Co2+ and Ni2+

Male Wistar rats were exposed to subtoxic doses of Co²⁺ or Ni²⁺, receiving Co(NO₃)₂ or NiSO₄ with drinking water for 30 days. No significant differences in the body weight and no visible changes in the behaviour of the controls and experimental animals were established. Cumulative concentration-effect curves for carbachol were obtained in ileum and trachea isolated from control and Co²⁺- or Ni²⁺-treated rats. The effect of the Ca²⁺ antagonists on the carbachol-induced contractions was studied by adding increasing concentrations of verapamil or nitrendipine to the bath solution 20 min prior to carbachol. The results showed that exposure of rats to subtoxic doses of Co(NO₃)₂ or NiSO₄ altered the contractile responses to carbachol. The changes in the pD₂ values and the shift to the left of the concentration-effect curves suggest a higher sensitivity to carbachol in preparations from the ileum of Co²⁺- or Ni²⁺-exposed rats. The tracheal strips isolated from control and heavy metal-treated rats showed a less potent sensitiveness to carbachol as compared to the ileal segments. An opposite tendency for decreased cholinergic reactivity was observed in tracheal strips from Co²⁺- and Ni²⁺-treated animals. The inhibitory effect of the Ca²⁺-antagonists on the contractility of ileal preparations from Co²⁺-treated rats increased at all concentrations of verapamil and at the highest concentration of nitrendipine, but decreased at lower concentrations of nitrendipine. The effect of verapamil on the preparations from Ni²⁺-exposed rats was unchanged or even decreased at higher verapamil concentrations. The inhibitory effect of nitrendipine on preparations from Ni²⁺-exposed rats decreased at the lowest concentration but increased at the highest concentration of the blocker. In Co²⁺- or Ni²⁺-treated tracheal preparations verapamil inhibited the contractions induced by low and medium concentrations of carbachol but increased the maximal contractile responses to high concentrations of carbachol.     

Nickel (Ni2+) enhancement of microtubule assembly in vitro is dependent on GTP function

Microtubule (MT) assembly in vitro is accompanied by hydrolysis of tubulin-bound GTP at E-site. Ni2+, a human carcinogen, has been shown to markedly perturb the MT system in cultured cells and enhance MT assembly in vitro. To further probe the mechanisms of such multiple Ni2+ damaging actions on MT, we have focused on dissecting the role of the Ni2+/GTP interaction in influencing MT assembly in vitro as monitored by a turbidity assay at A350 at 27 degrees C using purified bovine brain MT proteins containing 162 microM each of Mg2+ and EGTA. MT assembly was initiated by addition of GTP and progressed in a GTP dose-dependent manner. The minimal and optimal exogenous [GTP] required for MT assembly were 15.6 and 500 microM, respectively. Replacement of GTP (25-87%) with increasing [NiCl2] while keeping the sum of [GTP] and [Ni2+] constant at 500 microM enabled MT assembly to proceed with shortened "lags" but reaching the same maximum plateau levels or elongation rates as with 500 microM GTP only. However, in reactions with Ni2+ replacing >94% of GTP, marked inhibition of MT assembly (lower plateaus) occurred. Electron microscopic (EM) examinations showed that MT formed with high Ni2+ substitutions for GTP appeared shorter, more numerous, and resistant to Ca2+ disruption than those assembled with 500 microM GTP only. Notably, in the presence of 500 microM Ni2+ with no GTP added, no typical MT were observed under EM, despite increases in turbidity of the reaction. In addition, the critical concentration of MT proteins required for assembly was also considerably decreased under conditions of Ni2+ replacements of GTP. These results point to an important role of GTP/Ni2+ interaction in modulating the Ni2+ enhancement of MT assembly in vitro.     

Differential effects of lithium on platelet protein phosphorylation in bipolar patients and healthy subjects

In the present study we have investigated the cAMP-dependent phosphorylation system in platelets of euthymic bipolar patients and healthy volunteers before and after 15 days of lithium treatment. The results showed that 15 days of lithium treatment enhanced the basal and the cAMP-stimulated ³²P incorporation in the 22 and 38 kDa phosphoproteins in bipolar patients, but not in healthy subjects. Moreover, we provided further evidence of increased phosphorylation in the 22 kDa platelet phosphoprotein in untreated euthymic bipolar patients when compared with controls. Overall, these findings suggest an implication of protein phosphorylation in the biochemical action of lithium and in the pathophysiology of bipolar disorder. Received: 9 April 1996/Final version: 1 September 1996     

Differential effects of Ca(2+) on bisphosphonate-induced growth inhibition in breast cancer and mesothelioma cells

Bisphosphonates are widely clinically used inhibitors of bone resorption. Pre-clinical studies indicate that bisphosphonates also inhibit the growth of various cancer cells in vitro, but their in vivo anti-cancer activity varies greatly, depending on the tumor type. We compared the various cellular effects of bisphosphonates in breast cancer and mesothelioma cells, with differences in growth inhibition responses to bisphosphonate-treatment in vivo. We show that the growth inhibitory effects of nitrogen-containing bisphosphonates are significantly affected by excess Ca(2+) in a cell- and bisphosphonate-specific fashion. Furthermore, excess pyrophosphate-resembling bisphosphonates prevent nitrogen-containing-bisphosphonate-induced accumulation of unprenylated Rap1A, p38 phosphorylation and growth inhibition in human MDA-MB-231 breast cancer and mouse AB-12 mesothelioma cells. For some, but not all tested, pyrophosphate-resembling bisphosphonate: nitrogen-containing bisphosphonate combinations these results may be partially explained by the ability of the excess pyrophosphate-resembling bisphosphonates to chelate Ca(2+). In mice, subcutaneous AB-12 and MDA-MB-231 tumors exhibit positive staining for Ca(2+) minerals, as revealed with Von Kossa stainings. We further show that the AB-12 tumors accumulate significantly more of the bone scanning bisphosphonate, Tc99m-medronate, as compared with MDA-MB-231 tumors. In conclusion, our results suggest that Ca(2+) regulates the growth inhibitory effects of bisphosphonates in a target cell and drug-specific fashion. These findings may be of physiological relevance since many tumor types are calcified. They further suggest that bisphosphonates can accumulate in tumors that are growing at the visceral sites and that differences in tumor accumulation of bisphosphonates may regulate their in vivo sensitivity to these drugs.     

Effects of bis(2-ethylhexyl) phthalate and benzo[a]pyrene on the embryos of Japanese medaka (Oryzias latipes)

Effects of two widely found chemical pollutants, bis(2-ethylhexyl) phthalate (DEHP) and benzo[a]pyrene (BaP), on the embryos of Japanese medaka were investigated. The embryos were exposed to different concentrations (0.01, 0.1, 1, and 10 μg/l) of DEHP and BaP. The following were investigated: (1) hatching time and hatching rate in embryos, (2) mortality, sex ratio, body weight and gonadosomatic index (GSI) in adulthood. These two chemicals delayed the hatching time without dose-dependence, but these chemicals had no effect on hatching rate. Mortality was raised and body weight was reduced by DEHP and BaP-treatment; distortion of sex ratio appeared at the lowest concentration of DEHP tested. GSI was decreased because of the BaP-treatment. DEHP and BaP negatively affected Japanease medaka embryos, and the influences of the effects continued into adult stage. Moreover, the effects did not appear to be necessarily dose-dependent.     

Differential effects of tautomycetin and its derivatives on protein phosphatase inhibition, immunosuppressive function and antitumor activity

In the present work, we studied the structure-activity relationship (SAR) of tautomycetin (TMC) and its derivatives. Further, we demonstrated the correlation between the immunosuppressive fuction, anticancer activity and protein phosphatase type 1 (PP1) inhibition of TMC and its derivatives. We have prepared some TMC derivatives via combinatorial biosynthesis, isolation from fermentation broth or chemical degradation of TMC. We found that the immunosuppressive activity was correlated with anticancer activity for TMC and its analog compounds, indicating that TMC may home at the same targets for its immunosuppressive and anticancer activities. Interestingly, TMC-F1, TMC-D1 and TMC-D2 all retained significant, albeit reduced PP1 inhibitory activity compared to TMC. However, only TMC-D2 showed immunosuppressive and anticancer activities in studies carried out in cell lines. Moreover, TMC-Chain did not show any significant inhibitory activity towards PP1 but showed strong growth inhibitory effect. This observation implicates that the maleic anhydride moiety of TMC is critical for its phosphatase inhibitory activity whereas the C1-C18 moiety of TMC is essential for the inhibition of tumor cell proliferation. Furthermore, we measured in vivo phosphatase activities of PP1 in MCF-7 cell extracts treated with TMC and its related compounds, and the results indicate that the cytotoxicity of TMC doesn't correlate with its in vivo PP1 inhibition activity. Taken together, our study suggests that the immunosuppressive and anticancer activities of TMC are not due to the inhibition of PP1. Our results provide a novel insight for the elucidation of the underlying molecular mechanisms of TMC's important biological functions.     

Effect of beta-hydroxy-beta-methylbutyrate (HMB) on protein metabolism in whole body and in selected tissues

Beta-hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite with protein anabolic effect. The aim of the study was to examine the role of exogenous HMB on leucine and protein metabolism in whole body and selected tissues.     

Differential effects of methylmercury on the synthesis of protein species in dorsal root ganglia of the rat

Dorsal root ganglia from control and methylmercury(MeHg)-treated rats were incubated in vitro with ³⁵S-methionine ant the proteins synthesized were analyzed by two-dimensional electrophoresis. The double labelling method, in which proteins of control dorsal root ganglia labelled in vitro with ³H-leucine were added to each of the two samples as an internal standard, was used to minimize unavoidable errors arising from the resolving procedure itself. The results obtained showed that the effect of MeHg on the synthesis of proteins in dorsal root ganglia was not uniform for individual protein species in the latent period of MeHg intoxication. Among 200 protein species investigated, 157 showed inhibition of synthesis close to that of the total proteins in the tissue (68% of the control). Among the remaining protein species, 20 showed real stimulation of synthesis, whereas 7 were moderately inhibited and 16 were inhibited more strongly than the total proteins in the tissue. These results suggest that the effect of MeHg on the synthetic rates for protein species in dorsal root ganglia differs with the species, and that unusual elevation or reduction of the synthesis of some protein species caused by MeHg may lead to impairment of normal nerve functions.This work was supported in part by a grant from the Japanese Environmental Agency     

The action of dl-2,4-diaminobutyric acid on the cataleptogenic effects of pilocarpine and α-flupenthixol in rats

Rats fed diets high in tryptophan, methionine, and tryptophan plus methionine or lysine were subsequently injected with 10 mg/kg d-amphetamine. The amount of amphetamine-induced stereotyped behaviour observed varied as a function of the dietary addition, with methionine showing the greatest difference to controls, and lysine the least. Whole brain catecholamine levels and 5-hydroxytryptamine levels also showed amino acid-specific changes.     

Differential effects of wortmannin on the release of substance P and amino acids from the isolated spinal cord of the neonatal rat

1. Effects of wortmannin, an inhibitor of myosin light chain kinase, on the release of substance P and amino acids, GABA and glutamate, were investigated in the isolated spinal cord preparation of the neonatal rat.
2. Wortmannin at 0.5–10 μM depressed the release of substance P evoked by high-K⁺ (90 mM) medium from the spinal cord (IC₅₀=1.1 μM). Wortmannin also depressed the high-K⁺ (70 mM)-evoked release of substance P from cultured dorsal root ganglion neurons of neonatal rats. In contrast, the high-K⁺ (90 mM)-evoked release of GABA and glutamate from the spinal cord was not affected by wortmannin (0.1–10 μM).
3. Upon stimulation of a dorsal root, a monosynaptic reflex and a subsequent slow ventral root depolarization were evoked in the ipsilateral ventral root of the same segment in the isolated spinal cord preparation. The magnitude of the slow ventral root depolarization was depressed gradually to about 70% of the control during the course of 30 min under wortmannin (1 μM). In contrast, the monosynaptic reflex was unaffected by wortmannin.
4. Immunofluorescent staining revealed that immunoreactivities of substance P and myosin II were colocalized at presynaptic terminals in the dorsal horn of the neonatal rat spinal cord.
5. The present results suggest that myosin phosphorylation by myosin light chain kinase may play a crucial role in the release of substance P, but not in the release of GABA and glutamate in the neonatal rat spinal cord. This may reflect a difference in the exocytic mechanisms of substance P-containing large dense core vesicles and amino acid-containing small clear vesicles.

     

Binding of 63Ni by Cellular Constituents in Some Tissues of Mice after the Administration of 63NiCl2 and 63Ni(CO)4

Abstract One and 24 hours after the administration of ⁶³NiCl₂ and ⁶³Ni(CO)₄ to mice ⁶³Ni was present in association with both particulate and soluble cellular constituents in the lung, liver and kidney. After disruption of the cellular organells by sonication, a considerable part of the ⁶³Ni was still bound to the cellular fragments. Sephadex G-75 chromatography of the cytosol of the lung showed that the largest proportion of ⁶³Ni was eluted in the void volume and a smaller proportion was present in the salt volume. In the kidney, the proportions were reversed. Twentyfour hours after the injection of ⁶³NiCl₂ an intermediate ⁶³Ni-containing peak, with an estimated molecular weight of about 30,000, was found in the lung and the kidney. In the liver of ⁶³NiCl₂-injected mice, most of the nickel was recovered in the void volume, a lesser amount in the salt volume. There was no evidence that ⁶³Ni was bound to metallothionein (induced by Cd-pretreatment) or to superoxide dismutase in the studied tissues. Pretreatments with non-labelled NiCl₂ did not alter the elution profiles. In serum, most ⁶³Ni was present in association with albumin. Gel-chromatograms of red blood-cell hemolysates from ⁶³Ni(CO)₄-injected mice showed ⁶³Ni at an elution volume corresponding to hemoglobin, but ⁶³Ni-binding ligands with higher and lower molecular weights were also present.     

Cerebral dysfunction in type 1 diabetes: effects of insulin, vascular risk factors and blood-glucose levels

Type 1 diabetes can lead to several well-described complications such as retinopathy, nephropathy and peripheral neuropathy. Evidence is accumulating that it is also associated with gradually developing end-organ damage in the central nervous system. This relatively unknown complication can be referred to as ‘diabetic encephalopathy’ and is characterised by electrophysiological and neuroradiological changes, such as delayed latencies of evoked potentials, modest cerebral atrophy and (periventricular) white matter lesions. Furthermore, individuals with type 1 diabetes may show performance deficits in a wide range of cognitive domains. The exact mechanisms underlying this diabetic encephalopathy are only partially known. Chronic metabolic and vascular changes appear to play an important role. Interestingly, the differences in the ‘cognitive profile’ between type 1 and type 2 diabetes also suggest a critical role for disturbances of insulin action in the central nervous system.     

Different effects of Pb(2+) and Cu(2+) on immune and antioxidant enzyme activities in the mantle of Pinctada fucata

The aim of this study was to investigate the natural role of the mantle in pearl oyster, Pinctada fucata. The mantle is believed to be the tissue responsible for shell and pearl formation. However, our current study on lead and copper accumulation in tissues of the oyster showed that the secondary tissue for lead accumulation was not the digestive gland but the mantle. In view of high lead concentrations in the mantle, its general metabolic condition (including immune and antioxidant defense systems) as affected by the two metals was studied. The results indicated that activities of antioxidant enzymes (superoxide dismutase, SOD; Se-dependent glutathione peroxidase, Se-GPx) were altered by lead and copper in the similar way. However, the immune enzyme activities (acid phosphatase, AcPase; phenoloxidase, PO) were perturbed differently by two metals. Therefore, the mantle of P. fucata was predicted to participate in immune processes and accumulation or detoxification of lead besides shell formation. Our observations described here may also provide important clues to further understanding of the biomarker responses of bivalves.     

A comparative study of histamine and K+ effects on (Ca(2+)-Mg2+)-ATPase activity in synaptosomes.

Histamine (10(-4) M) and 60 mM K+, but not 60 mM Na+ or 60 mM choline+, increased the maximal synaptosomal (Ca(2+)-Mg2+)-ATPase activity by 15 and 36% respectively and decreased the extrasynaptosomal Ca2+ concentration necessary to reach it. Histamine and K+ enhanced the synaptosomal (Ca(2+)-Mg2+)-ATPase activity in a concentration-dependent manner. In synaptic plasma membranes histamine (10(-4) M) and 60 mM choline+ were not able to alter the enzymatic activity, however 60 mM K+ and 60 mM Na+ elevated (Ca(2+)-Mg2+)-ATPase activity by 20 and 15%, respectively, without altering the affinity for Ca2+. Histamine effects in synaptosomes were mediated by H2 receptor stimulation. 3-Isobutyl-1-methyl-xanthine (10(-4) M) potentiated (15%) the maximal histamine effect. The slow Ca2+ channel antagonists verapamil and diltiazem, both at 10(-6) M, completely inhibited K+ effects in synaptosomes, however histamine effects were only blocked by verapamil. The data suggest that K+ and histamine effects on synaptosomal (Ca(2+)-Mg2+)-ATPase activity are mediated by increases of intrasynaptosomal Ca2+ levels. Moreover, histamine effects on synaptosomal enzyme activity were mediated by cAMP.     

Differential effects of arsenic on folate binding protein 2 (Folbp2) null and wild type fibroblasts

Exposure to arsenic results in a wide variety of adverse effects. It has been postulated that one mechanism of arsenic toxicity is disruption of cellular methyl biochemistry. Because dietary folate is required to generate the methyl donor S-adenosyl methionine, we hypothesized that loss of folate binding protein 2 (Folbp2) results in increased susceptibility to arsenic-induced cytotoxicity. Using Folbp2 +/+ and -/- fibroblasts, we determined that Folbp2 null cells display increased sensitivity to arsenic exposure. Folic acid supplementation partially rescues wild type cells from arsenic toxicity, but Folbp2 null cells are not protected. Arsenic inhibits folic acid uptake in Folbp2 null fibroblasts, but not wild type cells; baseline uptake is similar in both cell types. These results support the possibility that arsenic toxicity occurs, in part, by perturbing cellular methyl biochemistry. Furthermore, identification of Folbp2 as a protective protein presents an opportunity to identify populations at increased risk for serious effects of arsenic exposure.     

The inhibitors of protein acylation, cerulenin and tunicamycin, increase voltage-dependent Ca(2+) currents in the insulin-secreting INS 832/13 cell

As it has been suggested that protein acylation plays a role in nutrient stimulus-secretion coupling in the pancreatic beta-cell, we examined the insulin-secreting INS 832/13 beta-cell line for evidence that protein acylation was involved. The perforated whole-cell configuration was employed to voltage-clamp INS 832/13 cells. Voltage pulses were applied and Ca(2+) currents measured in the presence and absence of the protein acylation inhibitors cerulenin and tunicamycin. Both inhibitors enhanced the peak amplitude of I(Ca,L). Both increased the peak inward current in the range between -40 and +30mV and shifted the apparent maximum current by 10mV in the hyperpolarizing direction without affecting the activation threshold of -40mV. The two drugs had qualitatively and quantitatively similar effects. Steady-state activation curves revealed that cerulenin and tunicamycin shifted the activation curves in the hyperpolarization direction. Activation time constants were significantly reduced in the presence of both drugs. The Ca(2+) charge influx was increased by the drugs at all potentials tested. In contrast to these effects on the L-type Ca(2+) channel, the two inhibitors of protein acylation had no effect on the ATP-sensitive K(+) channel. The results suggest that protein acylation exerts a tonic inhibitory effect on L-type Ca(2+) channel function in the insulin-secreting beta-cell.     

Differential effects of amphetamine and fenfluramine on dietary self-selection in rats

Daily caloric intakes and dietary self-selection of the three macronutrients, protein, fat and carbohydrate were examined in female rats following administration of d-amphetamine sulfate (0.0, 0.5, 1.0 and 2.0 mg/kg, IP) or fenfluramine hydrochloride (0.0, 1.5, 3.0 and 6.0 mg/kg, IP). Animals were maintained on ground Purina Chow or one of two self-selection regimes, one with a high-caloric fat ration (7.85 kcal/g) and the other with a fat ration isocaloric to the carbohydrate and protein rations (3.76 kcal/g). Animals received drug injections at the beginning of a daily 8-hour feeding period with nutrient intakes measured at 2, 4 and 8 hrs following injections. While both amphetamine and fenfluramine led to dose-related decreases in total caloric intakes, the two drugs resulted in different temporal patterns of feeding. Amphetamine produced its greatest effect on caloric intake during the first 2 hours of the feeding period, whereas fenfluramine suppressed caloric intake equivalently across the 8-hour feeding period. The two anorectic drugs also led to different patterns of nutrient choice. When animals were given the high-caloric fat ration, amphetamine selectively decreased fat intake while fenfluramine produced decreases in both protein and fat intakes, sparing carbohydrate intake. In contrast, when animals were given the isocaloric fat ration, amphetamine resulted in a general suppression of nutrient intakes while fenfluramine led to a sustained decrease in fat intake with a relative sparing of protein and carbohydrate consumption.     

Inhibitory effects of Polygonum cuspidatum water extract (PCWE) and its component resveratrol [correction of rasveratrol] on acyl-coenzyme A-cholesterol acyltransferase activity for cholesteryl ester synthesis in HepG2 cells

The pharmacological effects of Polygonum cuspidatum water extract (PCWE) on lipid biosynthesis were investigated in cultured human hepatocyte HepG2 cells. The addition of PCWE (5 and 20 microg/ml), which had no effect on cell proliferation and cellular protein content, caused a marked decrease in the cellular cholesterol content, particularly, the cholesteryl ester content following 24 h of incubation. The incorporation of (14)C-oleate into the cellular cholesteryl ester fraction was also reduced remarkably during incubation for 6 and 24 h. The effect of PCWE on acyl-coenzyme A-cholesterol acyltransferase (ACAT) activity were studied in vitro to explore the mechanism by which PCWE inhibits cholesterol ester formation. The data confirmed that PCWE, in a dose dependent manner, remarkably inhibits ACAT activity. Among the main active chemicals of P. cuspidatum, resveratrol, a kind of flavonoid, decreased ACAT activity in a dose-dependent manner from the level of 10(-3) M. Theses results strongly suggest that PCWE reduces the cholesteryl ester formation in human hepatocytes by inhibiting ACAT.     

HPLC with programmed wavelength fluorescence detection for the simultaneous determination of marker compounds of integrity and P-gp functionality in the Caco-2 intestinal absorption model

A high sensitivity reversed-phase HPLC method is presented for the simultaneous determination of marker compounds of paracellular transport (atenolol), transcellular transport (propranolol) and P-gp functionality (talinolol) in the Caco-2 system. The Caco-2 system is presently commonly accepted as an in vitro cell culture model of the intestinal mucosa. A programmed wavelength fluorescence detection method was used to optimise the response of the marker compounds. This marker compound mixture and the corresponding HPLC assay can be used for in house validation of the Caco-2 system or to evaluate simultaneously the effect of test compounds or absorption enhancing strategies on monolayer integrity and P-gp functionality. The method can easily be adapted to determine the concentration of atenolol, propranolol and talinolol in blood, thus allowing to use the same compounds in the in situ rat perfusion system with blood sampling from the mesenteric vein.