Influence of antimonite, selenite, and mercury on the toxicity of arsenite in primary rat hepatocytes.

The long-term toxicity of arsenic (As) as a result of exposure to contaminated drinking water might be modified by coinciding exposures to elements like selenium, antimony, or mercury. In this study the influence of tetravalent selenite, trivalent antimonite, and divalent mercury was investigated in vitro using cultured primary rat hepatocytes. The cell vitality was assessed in the 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] (MTT), assay with concurrent exposures of the cells to up to 50 microM sodium arsenite(III) and a potential modifier [50 microM sodium(IV) selenite, 10 microM antimony(III) chloride, 25 microM mercuric(II) chloride], which indicated an additive increase in the combined cytotoxicity. Sodium arsenite was tested for genotoxicity in the micronucleus test in a concentration range of 0.25 up to 7.5 microM. In this range, the MTT conversion was at least 80%, indicating high cell viability. Adose-dependent induction of micronuclei was observed. The lowest concentration causing a significantly elevated frequency of micronuclei was 1 microM As (p < 0.05). A significant influence (i.e., reduction of the combined genotoxicity as a result of the presence of a potential modifier) was only observed for 10 and 25 microM antimony chloride (p < 0.05, Fisher's exact test). The metabolic methylation of arsenite was not affected by concurrent incubation with any of the potential modifiers.

Modifications of cellular lipids induce insulin resistance in cultured hepatoma cells.

We altered the cellular lipid composition of an insulin sensitive rat hepatoma cell line through supplementation of the culture medium with linoleic acid (18:2) or 25-hydroxycholesterol, and we studied the effects on insulin stimulation of aminoacid transport system A and glycogen synthesis. The basal rate of sodium-dependent aminoisobutyric acid uptake was slightly reduced in hydroxysterol-treated cells and increased in 18:2-enriched cells. Maximal insulin stimulation of transport was decreased by about 40% in both 18:2 and 25-hydroxycholesterol modified cells, as compared to control cells. In addition to reduced responsiveness, the hydroxysterol-treated cells also showed a diminished sensitivity to insulin, as revealed by a right-shift of the dose-response curve leading to a ED50 of 1.2 X 10(-8) M (P less than 0.02), as compared to 2.45 X 10(-9) M in control cells and 2.13 X 10(-9) M in 18:2 enriched cells. Concerning glycogen synthesis, the basal rate was unaffected by 25-hydroxycholesterol supplementation and slightly reduced in cells enriched in 18:2. Maximal insulin stimulation of glycogen synthesis was reduced by about 40% in both types of lipid modified cells. 25-Hydroxycholesterol again provoked a decrease in sensitivity to insulin: the ED50 was enhanced to 4.9 X 10(-9) M (P less than 0.05), as compared to 1.25 X 10(-9) M in control cells and 1.57 X 10(-9) M in 18:2-supplemented cells. Taken together with the previously reported changes of insulin binding to lipid modified hepatoma cells (Bruneau et al. (1987) Biochim. Biophys. Acta 928, 287-296) our results demonstrate an influence of alterations of the cellular lipid composition on both binding and biological actions of insulin, leading to an insulin-resistant state. Divergences between insulin binding and action were obtained and it was suggested that post-binding events may be responsible for the observed changes. Our findings may be relevant to experimental and clinical states of insulin resistance.

Influence of lipid environment on insulin binding in cultured hepatoma cells.

The influence of alterations of plasma membrane physico-chemical properties on insulin binding have been characterized in an insulin-sensitive rat hepatoma cell line adapted to grow for several generations in culture medium enriched with linoleic acid (18:2) or with 25-hydroxycholesterol. The cells took up 18:2 and 25-hydroxycholesterol added to the culture medium, without exhibiting any sign of intolerance or intoxication. These compounds respectively increased and decreased membrane fluidity at 37 degrees C. The cells demonstrated extensive changes in insulin binding parameters in response to experimental modifications of their membrane lipid composition. When determined at 4 degrees C, insulin receptors were present in the control cells at 136,000 sites/cell but this fell to 111,000 (P less than 0.05) in cells enriched in 18:2, and rose to 176,000 (P less than 0.001) in hydroxysterol-grown cells. According to a two-site model, the main effect of 18:2 was a significant increase of the number of high-affinity sites with a concomitant decrease of low-affinity sites. The hydroxysterol had the opposite effects on these parameters. The high-affinity insulin binding capacity of the hepatoma cells was affected by lipid supplementation in a similar way, whether it was determined at 4 degrees C or at 37 degrees C. Assuming a negative cooperativity model, 18:2 enhanced the degree of negative cooperativity among the sites, while 25-hydroxycholesterol reduced it. The time-course of insulin-induced receptor down-regulation was accelerated in the cells enriched in polyunsaturated fatty acids, but reduced in cells exposed to 25-hydroxycholesterol. These insulin-binding alterations cannot be directly related to modifications of cellular growth rate, receptor internalization or membrane fluidity per se, and are discussed as being more likely due to membrane lipid composition than to overall cell metabolism modifications.

Micronuclei assessment in buccal cells of people environmentally exposed to arsenic in northern Chile.

To determine the genotoxic risk associated to environmental arsenic exposure, the frequency of micronuclei in buccal cells (BCMN) of people drinking arsenic-contaminated water has been evaluated. A group of 105 individuals from the Antofagasta region (north Chile), and 102 individuals from the area of Concepcion, used as reference group, were included in the study. Arsenic concentration in drinking water was high (0.75 mg/L) in the Antofagasta area, 75-fold the maximum recommended level by WHO (0.01 mg/L), while the values obtained in Concepcion were significantly lower (0.002 mg/L). Individual measures of arsenic exposure were also determined in fingernails, which clearly confirm the existence of chronic exposure in the sampled populations from the Antofagasta region (10.15 microg/g versus 3.57 microg/g). The cytogenetic results indicate that, although the BCMN frequency is higher in exposed than in controls, this increase does not attain statistical significance. When the exposure biomarkers were related with the cytogenetic values, no correlations were observed between BCMN and arsenic content in water or in fingernails. In addition, the genotoxicity values do not seem to be related to the ethnic origin from people belonging to the exposed group. As a conclusion it appears that, in the studied population, the chronic ingestion of arsenic-contaminated water does not induce cytogenetic damage, measured as micronuclei, in the cells of the oral mucous in a significant extent.

Insulin receptor processing in hepatoma cells: some steps involved in both basal receptor turnover and insulin-induced down-regulation.

Insulin receptor processing was studied in cultured Zajdela hepatoma cells (ZHC). The basal receptor turnover was estimated in the presence of tunicamycin (TM), which inhibited the insertion into plasma membranes of newly synthesized underglycosylated receptors. After a lag phase of 4 h, the surface receptor number decreased, with a t 1/2 of 7 h, for up to 24 h. This process was markedly slowed down when cells were either briefly preincubated with dansylcadaverine, chloroquine, or cycloheximide or treated for 24 h with TM. The effects of these agents on the insulin-induced receptor down-regulation process was then tested. When cells were treated with chloroquine or dansylcadaverine or placed in calcium-free medium, this process was impeded; similarly, it was inhibited by actinomycin D or cycloheximide, but was not affected by TM after a brief incubation. However, after a 24-h treatment with TM, it disappeared, although receptors remained functional when testing insulin's action upon glycogen synthesis. These results indicate that receptor degradation, both basal and activated by insulin leading to the down-regulated state, was altered under similar experimental conditions. The effects of dansylcadaverine, chloroquine, or the absence of calcium reveal that endocytotic pathways were involved in these processes. The results obtained when mRNA, protein, or glycoprotein synthesis was inhibited indicate that cellular glycoprotein(s) and short-lived protein(s) were necessary for the receptor processing in both cases. These data led us to postulate that basal and insulin-activated receptor degradation may occur in the same way within the cell.

Insulin-induced receptor regulation in cultured Zajdela rat hepatoma cells and relationship to the stimulation of glycogen synthesis.

Insulin receptors were measured in cultured Zajdela rat hepatoma cells (ZHC cells), a stable cell line which presents differentiated hepatic functions. The number of sites was 50,000/cell at 2 C, and the dissociation constant for high affinity binding was 1.6 x 10(-10) M. Down-regulation of receptors occurred rapidly when cells were treated with insulin; this process was related to ambient insulin concentrations and led to a decrease in the number of insulin receptors from 50,000 to 30,000/cell. Cycloheximide prevented part of this regulation. When down-regulated cells were incubated in standard medium devoid of insulin, the number of receptor sites gradually increased and attained control values within 7 h; cyclohexamide inhibited this process. Insulin markedly enhanced glycogen synthesis in ZHC cells, with an ED50 of 1.0 x 10(-9) M, leading to an increase in the total cell glycogen content. In addition, the predicted righthand shift of the dose-response curve was observed for insulin-treated cells. These findings provide evidence of insulin-induced receptor regulation in cultured ZHC cells which is related to the biological effect of the hormone on glycogen synthesis.

Degradation of insulin receptors by hepatoma cells: insulin-induced down-regulation results from an increase in the rate of basal receptor degradation.

The degradation of insulin receptors was studied in cultured Zajdela hepatoma cells (ZHC). Receptor distribution within the cell was evaluated by estimating: i) surface receptor level on entire cells, ii) total cell receptors solubilized by Triton from cell membranes and iii) intracellular receptors solubilized from cells whose surface receptors had been inactivated with trypsin. In the absence of insulin, 80-90% of the insulin binding sites were located on the cell surface. When insulin was added, a rapid decrease of surface receptors was observed. After 2 h, their level was reduced nearly by half; this reduction was accounted for by an actual receptor loss from the cell without an increase in the intracellular pool. These results indicate that insulin enhanced the rate of receptor degradation within the cell. Basal receptor inactivation was studied by using tunicamycin which inhibits new receptor synthesis. The surface receptor number was decreased with a half-life of 7 h, while the level of internal sites remained unchanged. Both basal and insulin-activated receptor degradation were markedly slowed down by chloroquine or dansylcadaverine, indicating the importance of endocytic pathways in this process. Similarly, when de novo protein glycosylation was inhibited for 24 h by tunicamycin, both basal and insulin-activated receptor inactivation were precluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-peptide corticotropin-releasing hormone antagonists: syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines.

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and -imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined the structure-activity relationships of these series and resulted in the identification of a variety of high-affinity agents (Ki's < 10 nM). On the basis of this property and lipophilicity differences, six of these compounds (4d,i,n,x, 8k, 9a) were initially chosen for rat pharmacokinetic (PK) studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds (4d,i,n,x) prompted further PK studies in the dog following both iv and oral routes of administration. Results from this work indicated 4i,x had properties we believe necessary for a potential therapeutic agent, and 4i1 has been selected for further pharmacological studies that will be reported in due course.

Inhibition of IL-2 secretion and IL-2 receptor appearance of activated lymphocytes pretreated with hydroxylated sterols.

When murine BALB/c splenocytes are pretreated for 2 h with different concentrations of 25-hydroxycholesterol (25-HC) or 7 beta-hydroxycholesterol (7-HC), washed and stimulated either with irradiated C57BL/6 splenocytes or with Con A, IL-2 secretion is inhibited in a dose-dependent way as well as the subsequent cell proliferation. Using the same treatment and stimulation conditions, IL-2 receptor appearance on human T lymphocytes, as characterized by anti-Tac antibody binding, is also inhibited in a dose-dependent way. In contrast, the hydrophilic derivative of 7 beta-hydroxycholesterol, the 3.7 bishemisuccinate sodium salt (7-HC BHS), did not influence any of the 3 tested parameters.

Characterization of cytotoxic proteins from mistletoe (Viscum album L.).

Proteins from a laboratory-made oak mistletoe extract and from the commercial mistletoe preparation Iscador Quercus were cytotoxic for leukemia Molt 4 cells in culture. A 50% growth inhibition was obtained with 0.1 microgram/ml proteins for the mistletoe extract and 0.025 microgram/ml for Iscador. On cation exchange chromatography, cytotoxic proteins from the mistletoe extract were mainly eluted at the same positions as purified lectins, while those of Iscador were eluted at the positions of viscotoxins. The data are discussed in relation to the pharmacological activities of the mistletoe protein complexes described in the literature.

Action of insulin on glycogen metabolism in cultured hepatoma cells.

ZHC cells, an established hepatoma cell line characterized by its capacity to synthesize and store glycogen, retain responsiveness to insulin. Sensitivity to insulin is correlated with culture development and is maximal in the confluent monolayer cultures. Insulin induces, within 2-3 h, an increase of glycogen content by stimulating the net synthesis of new glycogen molecules and without affecting their breakdown. Insulin directly acts on glycogen metabolism, and does not modify total cell protein or DNA synthesis. The ZHC cell line can provide a new model for the study of insulin regulation of glycogen metabolism, in the absence of other hormones that modulate the same pathway.

Evaluation of micronucleus induction in a Chilean population environmentally exposed to arsenic.

In the present study we have evaluated whether or not environmental exposure to arsenic in ground drinking-water results in a significant increase in the frequency of micronuclei (MN) in peripheral blood lymphocytes. Thus, 106 individuals from the Antofagasta region (North Chile), together with 111 individuals from the area of Concepción, were used in this investigation. In the Antofagasta area, arsenic levels in drinking-water as high as 0.750 mg/L were measured. In Concepción, located about 2500 km towards the south and used as reference area, arsenic levels in tap water were as low as 0.002 mg/L. The total content of arsenic in fingernails was determined as a biomarker of individual exposure. The cytogenetic results obtained in this study indicate that in the exposed group the overall frequency of binucleated micronucleated cells (BNMN) is higher than in the reference group, the difference being statistically significant. In addition, no differences were found between the exposed and the reference groups, regarding the cytokinesis-block proliferation index (CBPI). No association was observed between BNMN and arsenic content in water or arsenic in fingernails. On the other hand, when the exposed group was divided according to their Atacameno or Caucasian ethnicity, no significant differences were observed between them. In addition, as usually found in other human biomonitoring studies, sex and age are factors that modulate the frequency of MN in both exposed and reference populations.

Pharmacognosy of Mimosa tenuiflora (Willd.) Poiret.

A chemical investigation of the bark of Mimosa tenuiflora (Willd.) Poiret, performed in our laboratory, allowed the isolation and identification of three new triterpenoid saponins (mimonosides A, B and C), three steroid saponins (3-O-beta-D-glucopyranosyl campesterol, 3-O-beta-D-glucopyranosyl stigmasterol and 3-O-beta-D-glucopyranosyl beta-sitosterol) together with lupeol, campesterol, stigmasterol and beta-sitosterol. The three new triterpenoid saponins were subjected to in vitro biological tests (immunomodulation and proliferation) using different animal and human cells in culture. The results of these tests contribute to explain the traditional use of this plant material.

In vitro cytotoxicity of polyindolenine alkaloids on rat hepatoma cell lines. Structure activity relationships.

New groups of polyindolenine alkaloids have been isolated from new species of the tribe Psychotriae (Rubiaceae). The cyto-inhibitory effects of these compounds on tumoral cell lines have been thoroughly investigated and thus allow the statement of relationships between some original structural patterns and the observed biological effects.

Altered pharmacology of native rodent spinal cord TRPV1 after phosphorylation.

BACKGROUND AND PURPOSE: Evidence suggests that phosphorylation of TRPV1 is an important component underlying its aberrant activation in pathological pain states. To date, the detailed pharmacology of diverse TRPV1 receptor agonists and antagonists has yet to be reported for native TRPV1 under phosphorylating conditions. Our goal was to optimize a relatively high-throughput methodology to allow pharmacological characterization of the native TRPV1 receptor using a spinal cord neuropeptide release assay under naive and phosphorylating states. EXPERIMENTAL APPROACH: Herein, we describe characterization of rodent TRPV1 by measurement of CGRP release from acutely isolated lumbar (L1-L6) spinal cord using a 96-well technique that combines use of native, adult tissue with quantitation of CGRP release by ELISA. KEY RESULTS: We have studied a diverse panel of TRPV1 agonists and antagonists under basal and phosphorylating conditions. We show that TRPV1-mediated CGRP release is evoked, in a temperature-dependent manner, by a PKC activator, phorbol 12,13-dibutyrate (PDBu); and that treatment with PDBu increases the potency and efficacy of known TRPV1 chemical agonists, in an agonist-specific manner. We also show that the pharmacological profile of diverse TRPV1 antagonists is dependent on whether the stimulus is PDBu or capsaicin. Of note, HPPB was identified as an antagonist of capsaicin-evoked, but a potentiator of PDBu-evoked, CGRP release. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that both TRPV1 agonist and antagonist profiles can be differentially altered by PKC activation. These findings may offer new insights for targeting TRPV1 in pain states.