Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis.

The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx-RNA polymerase (Spx-RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx-RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx-RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended -35 box in which nucleotide composition at the -43/-44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

Arsenolysis and thiol-dependent arsenate reduction.

Conversion of arsenate to arsenite is a critical event in the pathway that leads from inorganic arsenic to a variety of methylated metabolites. The formation of methylated metabolites influences distribution and retention of arsenic and affects the reactivity and toxicity of these intermediates. Indeed, some of the toxic and carcinogenic effects associated with exposure to arsenate or arsenite are probably mediated by methylated arsenicals. Recent work has demonstrated a biologically plausible role for phosphorolytic-arsenolytic enzymes in a reaction scheme in which an "activated" arsenate ester is readily reduced by thiols to arsenite. Thiol-dependent reduction of arsenate esters formed by arsenolysis may be one of several functionally reductant processes that control the flux of arsenic into the cellular pathway for arsenic methylation. Integrating these reductive processes into a conceptual model for arsenic metabolism may provide new insights into the cellular machinery for handling this toxic metalloid.

Clastogenic effects of inorganic arsenic salts on human chromosomes in vitro.

Arsenic is well documented as a paradoxical human carcinogen. In West Bengal, several million people were found to be arsenic affected who were exposed to this metalloid principally through drinking water. The arsenic-contaminated drinking water contains both trivalent as well as pentavalent arsenic. In this study, the comparative in vitro cytogenetic effects of two inorganic salts of arsenic, trivalent sodium arsenite (NaAsO(2)) and pentavalent sodium arsenate (Na(2)HAsO4) in three different concentrations, were screened for damage to chromosome and cell division following exposure to human lymphocyte culture. The chromosome-breaking activities in cultured lymphocytes were significantly higher for the compounds with trivalent (NaAsO(2)) than with pentavalent arsenic (Na(2)HAsO(4)), as reflected by the higher chromosomal aberration percentage in the similar doses used. It suggests that sodium arsenite was considerably more clastogenic than sodium arsenate. Moreover, increases in chromosomal aberrations were proportional with the increased dose of exposure for both trivalent and pentavalent forms of arsenic.

Anzer honey prevents N-ethylmaleimide-induced liver damage in rats.

N-ethylmaleimide (NEM) is a sulphydryl blocker which impairs the sulphydryl dependent antioxidant system (mainly glutathione) in the body by alkylating endogenous sulphydryls. This study was designed to investigate the effects of Anzer honey on NEM-induced liver injury in rats. Thirty female Wistar albino rats were divided equally into three groups. Group 1: control; Group 2: NEM; Group 3: Anzer honey+NEM. NEM (0.075mg kg(-1)) was given to both group 2 and 3 administered subcutaneously (s.c.) for 30 days. The animals in the Anzer honey+NEM group were treated with Anzer honey at a dose of 0.275g kg(-1), (p.o.) at 1h prior to every NEM injection. At the end of the 30 day treatment period, liver samples were taken for determination of the glutathione levels and histological examination. NEM treatment alone caused a significant reduction of the liver glutathione levels in group 2. Furthermore, NEM treatment caused congestion and mononuclear cell infiltration in the liver when compared to the control group. In group 3, Anzer honey treatment reversed all the changes in glutathione level, as well as histopathological alterations, normally induced by NEM. The findings imply that depletion of glutathione concentration plays a causal role in NEM-induced liver injury, and that the hepatoprotective effect of Anzer honey may be mediated through sulfhydryl-sensitive processes. They further imply that it may also possess antioxidant properties.

Arsenic salt-induced DNA damage and expression of mutant p53 and COX-2 proteins in SV-40 immortalized human uroepithelial cells.

Arsenic is widely distributed in the environment, and is a proven toxic and carcinogenic agent that is associated with various human malignancies, including bladder cancer. However, the mechanisms of its carcinogenic action are still not well understood. In addition, over-expression of mutant p53 and cyclooxygenase-2 (COX-2) frequently occurs in a variety of human malignancies. It is therefore of interest to study the genotoxicity of arsenic salts on human uroepithelial cells and the expression of oncoproteins p53 and COX-2. In this study, the relative genotoxicity of sodium arsenite was evaluated in SV-40 immortalized human uroepithelial cells (SV-HUC-1) using the alkaline comet assay. The expression of mutant p53 and COX-2 was also evaluated by immunocytochemistry and western blotting. Our results revealed that sodium arsenite was able to induce DNA damage, and that its genotoxicity is correlated with its concentration. In addition, the expression of mutant p53 increased in parallel with comet scores, and the maximal expression of mutant p53 was observed at 4 microM arsenite. Similarly, sodium arsenite stimulated a concentration-dependent increase in COX-2 expression. In conclusion, this study demonstrated that sodium arsenite is genotoxic to uroepithelial cells in vitro, and that it will induce expression of mutant p53 and COX-2 proteins, indicating a possible key event in carcinogenesis. This study provides us with knowledge of the relationship between p53 and COX-2 over-expression in arsenite-treated urothelial cells and suggests a potential therapeutic role of COX-2 inhibitors in human urothelial malignancies.

Arsenite slows S phase progression via inhibition of cdc25A dual specificity phosphatase gene transcription.

Arsenic acts as a toxicant, a carcinogen, and an effective chemotherapeutic agent, but its mechanisms of action are unclear. We have previously shown that treatment of U937 cells with 5 microM sodium arsenite inhibits cell cycle progression through each cell cycle phase, including S phase. Cdc25A dual specificity phosphatase controls entry into and progression through S phase by dephosphorylating sites of inhibitory phosphorylation on cyclin E-cdk2 (Thr14 and Tyr15). Immunoblotting reveals that a 3-h treatment of U937 cells with 5 microM sodium arsenite results in a dramatic decrease in cdc25A protein levels. Coimmunoprecipitation experiments confirm that cyclin E-cdk2 is more phosphorylated at Thr14 and Tyr15 in the presence of arsenite, and kinase activity assays reveal a decrease in cyclin E-associated cdk2 activity. Therefore, arsenite-dependent cdc25A depletion could contribute to S phase inhibition. There exists an S phase checkpoint known to be mediated by proteasomal cdc25A degradation. However, cycloheximide half-life assay reveals that cdc25A is actually stabilized in arsenite-treated cells. Real-time RT-PCR shows that cdc25A mRNA levels are substantially decreased with arsenite treatment, and actinomycin D half-life assay reveals no change in message stability. Decreased cdc25A message translation is shown by sucrose density gradient polysomal analysis to be an unlikely cause for the profound arsenite-dependent reduction in cdc25A protein levels. Studies are ongoing to establish the mechanism by which 5 microM arsenite decreases cdc25A message abundance, but we surmise that, given the lack of effect on mRNA stability, an inhibition of gene transcription is likely involved.

Environmental and synthetic sulphydryl group inhibitors: effects on bioluminescence and respiration in Vibrio fischeri.

Elemental sulphur (as S0 and S8) is abundant in anaerobic sediments and soil, and is highly toxic in the Vibrio fischeri bioluminescence test. This mode of S0 action remains uncertain. The objective of this research was the analysis of the toxic effects of S0 on bioluminescence and respiration in V. fischeri, in joint action with N-ethylmaleimide (NEM) or 2,4-dithio-DL-threitol (DTT), which are -SH group inhibiting and maintaining synthetic agents, respectively. Non-toxic DTT immediately protected cell bioluminescence against S0 inhibition at low (5.5ppb) and high (55ppb) concentrations of S0, whilst restoration of the inhibitory effect of S0 took up to 30 minutes. NEM (62.5ppb) diminished cell bioluminescence by up to 50% after 5 minutes, but after 60 minutes, the inhibition reached 100%. DTT restored the bioluminescence function inhibited in vivo and in vitro by S0 and NEM. Enhancement of cell respiration by up to 20% and 33% was observed at 2.2ppm of S0 and 36.8ppm of 2,4-dinitrophenol (2,4-DNP; an uncoupler of oxidative phosphorylation), respectively; whilst NEM (3.1ppm) caused a reduction of up to 40%. This comparative analysis confirmed that S0 has multiple modes of action--it acts as both an -SH group inhibitor and an uncoupler of oxidative phosphorylation in V. fischeri cells.

Structure-toxicity analysis of type-2 alkenes: in vitro neurotoxicity.

Acrylamide (ACR) is a conjugated type-2 alkene that produces synaptic toxicity presumably by sulfhydryl adduction. The alpha,beta-unsaturated carbonyl of ACR is a soft electrophile and, therefore, adduction of nucleophilic thiol groups could occur through a conjugate (Michael) addition reaction. To address the mechanism of thiol adduct formation and corresponding neurotoxicological importance, we defined structure-toxicity relationships among a series of conjugated type-2 alkenes (1 microM-10mM), which included acrolein and methylvinyl ketone. Results show that exposure of rat striatal synaptosomes to these chemicals produced parallel, concentration-dependent neurotoxic effects that were correlated to loss of free sulfhydryl groups. Although differences in relative potency were evident, all conjugated analogs tested were equiefficacious with respect to maximal neurotoxicity achieved. In contrast, nonconjugated alkene or aldehyde congeners did not cause synaptosomal dysfunction or sulfhydryl loss. Acrolein and other alpha,beta-unsaturated carbonyls are bifunctional (electrophilic reactivity at the C-1 and C-3 positions) and could produce in vitro neurotoxicity by forming protein cross-links rather than thiol monoadducts. Immunoblot analysis detected slower migrating, presumably derivatized, synaptosomal proteins only at very high acrolein concentrations (>or= 25 mM). Exposure of synaptosomes to high concentrations of ACR (1M), N-ethylmaleimide (10mM), and methyl vinyl ketone (MVK) (100mM) did not alter the gel migration of synaptosomal proteins. Furthermore, hydralazine (1mM), which blocks the formation of protein cross-links, did not affect in vitro acrolein neurotoxicity. Thus, type-2-conjugated alkenes produced synaptosomal toxicity that was linked to a loss of thiol content. This is consistent with our hypothesis that the mechanism of ACR neurotoxicity involves formation of Michael adducts with protein sulfhydryl groups.

Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals.

Pentavalent and trivalent organoarsenic compounds belong to the major metabolites of inorganic arsenicals detected in humans. Recently, the question was raised whether the organic arsenicals represent metabolites of a detoxification process or methylated species with deleterious biological effects. In this study, the effects of trivalent arsenite (AsO(3) (3-); iA(III)), the pentavalent organoarsenic compounds monomethylarsonic acid (CH(3)AsO(OH)(2); MMA(V)) and dimethylarsinic acid ((CH(3))(2)AsO(OH); DMA(V)) and the trivalent compounds monomethylarsonous acid (CH(3)As(OH)(2), MMA(III)) and dimethylarsinous acid ((CH(3))(2)As(OH); DMA(III)) were tested on glutamate receptors and on voltage-operated potassium and sodium channels heterologously expressed in Xenopus oocytes. Membrane currents of ion channels were measured by conventional two-electrode voltage-clamp techniques. The effects of arsenite were tested in concentrations of 1-1,000 micromol/l and the organic arsenical compounds were tested in concentrations of 0.1-100 micromol/l. We found no significant effects on voltage-operated ion channels; however, the arsenicals exert different effects on glutamate receptors. While MMA(V) and MMA(III) significantly enhanced ion currents through N-methyl-D: -aspartate (NMDA) receptor ion channels with threshold concentrations <10 micromol/l, DMA(V) and DMA(III) significantly reduced NMDA-receptor mediated responses with threshold concentrations <0.1 micromol/l; iA(III) had no effects on glutamate receptors of the NMDA type. MMA(III) and DMA(V) significantly reduced ion currents through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor ion channels with threshold concentrations <10 micromol/l (MMA(III)) and <1 micromol/l (DMA(V)). MMA(V) and iA(III) had no significant effects on glutamate receptors of the AMPA type. The effects of MMA(V), MMA(III), DMA(V) and DMA(III )on glutamate receptors point to a neurotoxic potential of these substances.

Toxic effects of thiol-reactive compounds on anaerobic biomass.

The objective of this study was to characterize the toxic effects of three well known thiol-reactive electrophilic compounds, N-ethylmaleimide (NEM), pentachlorophenol (PCP) and 1-chloro-2,4-dinitrobenzene (CDNB) on anaerobic biotransformation process. The work was part of a larger investigation on potassium efflux as a possible response mechanism of anaerobic microorganisms to the presence of thiol-reactive organic compounds and the interference of such compounds on the reductive dehalogenation process. Using anaerobic toxicity assay (ATA) and granular anaerobic biomass from a full-scale upflow anaerobic sludge blanket (UASB) reactor, inhibitory concentrations of these compounds that reduced the microbial activity of granular biomass to 50% of a control (IC50) were determined to be 592, 0.97, and 450 mg/l for NEM, PCP, and CDNB, respectively. Toxicity of NEM was also tested on anaerobic biomass from a municipal wastewater treatment plant digester and slightly lower IC50 of 532 mg/l was obtained. The results presented here indicate that anaerobic biomass can acclimate to the three thiol-reactive compounds studied and recover from inhibition as long as the toxicant concentration is below a threshold level. That threshold concentration was found to be 500 mg/l for NEM on biomass from the municipal digester, 1 mg/l for PCP, and 500 mg/l for CDNB, both on granular biomass. Granular anaerobic biomass showed recovery even at NEM concentrations of 1000 mg/l.

A novel small molecule CFTR inhibitor attenuates HCO3- secretion and duodenal ulcer formation in rats.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) plays a crucial role in mediating duodenal bicarbonate (HCO(3)(-)) secretion (DBS). Although impaired DBS is observed in CF mutant mice and in CF patients, which would predict increased ulcer susceptibility, duodenal injury is rarely observed in CF patients and is reduced in CF mutant mice. To explain this apparent paradox, we hypothesized that CFTR dysfunction increases cellular [HCO(3)(-)] and buffering power. To further test this hypothesis, we examined the effect of a novel, potent, and highly selective CFTR inhibitor, CFTR(inh)-172, on DBS and duodenal ulceration in rats. DBS was measured in situ using a standard loop perfusion model with a pH stat under isoflurane anesthesia. Duodenal ulcers were induced in rats by cysteamine with or without CFTR(inh)-172 pretreatment 1 h before cysteamine. Superfusion of CFTR(inh)-172 (0.1-10 microM) over the duodenal mucosa had no effect on basal DBS but at 10 microM inhibited acid-induced DBS, suggesting that its effect was limited to CFTR activation. Acid-induced DBS was abolished at 1 and 3 h and was reduced 24 h after treatment with CFTR(inh)-172, although basal DBS was increased at 24 h. CFTR(inh)-172 treatment had no effect on gastric acid or HCO(3)(-) secretion. Duodenal ulcers were observed 24 h after cysteamine treatment but were reduced in CFTR(inh)-172-pretreated rats. CFTR(inh)-172 acutely produces CFTR dysfunction in rodents for up to 24 h. CFTR inhibition reduces acid-induced DBS but also prevents duodenal ulcer formation, supporting our hypothesis that intracellular HCO(3)(-) may be an important protective mechanism for duodenal epithelial cells.

Oxidation by thimerosal increases calcium levelsin renal tubular cells.

The effect of thimerosal, a reactive oxidant, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored by using the Ca2+-sensitive dye fura-2. Thimerosal acted in a concentration-dependent manner with an EC50 of 0.5 microM. The Ca2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca2+ reduced 80% of the signal. In Ca2+-free medium, the [Ca2+]i rise induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) was completely inhibited by pretreatment with 5 microM thimerosal. The thimerosal (5 microM)-induced Ca2+ release was not changed by inhibition of phospholipase C with 2 microM U73122. Collectively, this study shows that thimerosal induced [Ca2+]i rises in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity.

Tri-n-butyltin-induced change in cellular level of glutathione in rat thymocytes: a flow cytometric study.

Since some of organotins, accumulated in edible mollusks of aquatic environments, exert a variety of toxic actions on experimental animals, it causes concern for the health of humans. We examined the effects of tri-n-butyltin chloride (TBT) and other organotins (triethyltin chloride, trimethyltin chloride, triphenyltin chloride and tetrabutyltin) on cellular content of glutathione (GSH) in rat thymocytes using a flow cytometer to further characterize the toxicity of TBT. When the cells were incubated with TBT at concentrations of 3 nM or more for 15 min, the cellular content of GSH dose-dependently decreased. However, it completely or partly recovered until 180 min even in the continued presence of TBT. This recovery was temperature-sensitive, suggesting an involvement of metabolic process. The efficacy of TBT to decrease the cellular content of GSH was greater than those of other organotins. Results suggest that TBT and some organotins at environmentally relevant (nanomolar) concentrations significantly reduce the cellular content of GSH, suggesting that they increase the vulnerability to some biological and chemical insults.

Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release.

The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can induce Zn(2+) release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonyl-aspartate-fluoromethylketone. N,N,N', N'-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2- and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn(2+) concentrations. Our studies suggest that under conditions of oxidative stress, Zn(2+) released from intracellular stores may contribute to the initiation of neuronal apoptosis.

Protection from cytotoxic effects induced by the nitrogen mustard mechlorethamine on human bronchial epithelial cells in vitro.

The present study was undertaken to find potent molecules against the toxicity of nitrogen mustard mechlorethamine (HN2) on respiratory epithelial cells, using a human bronchial epithelial cell line (16HBE14o-) as an in vitro model. The compounds examined included inhibitors of poly(ADP-ribose) polymerase (PARP), sulfhydryl-group donors as nucleophiles, and iron chelators and inhibitors of lipid peroxidation as antioxidants. Their effectiveness was determined upon observance of metabolic dysfunction induced by HN2 following a 4-h exposure, using (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and ATP-level assays as indicators. Moreover, the fluorescent probe, monobromobimane (mBBr), and 2',7'-dichlorofluorescin-diacetate (H2DCF-DA) were used to assess intracellular sulfhydryl and peroxide level modifications by flow cytometry, respectively, following a 3-h exposure. At last, cell death was assessed by flow cytometry using the propidium iodide (PI)-dye-exclusion assay following 24-h exposure. PARP inhibitors (niacinamide, 3-aminobenzamide, 6(5H)-phenanthridinone), and two sulfhydryl-group donors (N-acetylcysteine, WR-1065) were found to be effective in preventing HN2-induced metabolic dysfunction when added in immediate or delayed treatment with HN2. Only N-acetylcysteine, however, was found to prevent cell death induced by HN2, though it must be present at the time of the HN2 challenge. Flow cytometric measurements of intracellular sulfhydryl levels strongly suggested that N-acetylcysteine and WR-1065 are preventive in alkylation of cellular compounds, mainly by direct extracellular interaction with HN2. PARP inhibitors prevent secondary deleterious effects induced by HN2, considering metabolism dysfunction as the endpoint. Elsewhere, the oxidative stress appears to be a side effect in HN2 toxicity only upon considering the inefficiency of several antioxidants.

Dehydroepiandrosterone (DHEA) protects hippocampal cells from oxidative stress-induced damage.

It has been postulated that decreases in plasma levels of dehydroepiandrosterone (DHEA) may contribute to the development of some age-related disorders. Along with neuroprotective and memory enhancing effects, DHEA has been shown to display antioxidant properties. Moreover, oxidative stress is known to cause lipid peroxidation and degenerative changes in the hippocampus, an area involved in memory processes and especially afflicted in Alzheimer's disease (AD). Accordingly, we investigated the antioxidant effects of DHEA in models of oxidative stress using rat primary hippocampal cells and human hippocampal tissue from AD patients and age-matched controls. A pre-treatment of rat primary mixed hippocampal cell cultures with DHEA (10-100 microM) protected against the toxicity induced by H2O2 and sodium nitroprusside. Moreover, DHEA (10-100 microM) was also able to prevent H2O2/FeSO4-stimulated lipid oxidation in both control and AD hippocampal tissues. Taken together, these data suggest that DHEA may be useful in treating age-related central nervous system diseases based on its protective effects in the hippocampus.

Characterization of antioxidant properties of natural killer-enhancing factor-B and induction of its expression by hydrogen peroxide.

Natural killer-enhancing factor B (NKEF-B) belongs to a highly conserved family of recently discovered antioxidants. The role of NKEF-B as an antioxidant was demonstrated by its protection of transfected cells to oxidative damage by hydrogen peroxide. To further characterize the antioxidant properties of NKEF-B, we compared the sensitivity of a human endothelial cell line ECV304 and its transfectant, B/1 that hyperexpresses NKEF-B, to various oxidants. In addition, we investigated the changes in the expression of NKEF-B mRNA upon oxidative stress. We found that B/1 was significantly more resistant than the control cells to the oxidative stresses caused by t-butyl hydroperoxide (t-BHP) and methyl mercury (MeHg). In contrast, there was no difference in the sensitivity of B/1 and the control cells to sulfhydryl reactive agents, diethyl maleate and diamide. B/1 was also as sensitive as the control cells to buthionine sulfoximine. The expression of NKEF-B mRNA was induced when the parental cell line ECV304 was treated with 2 mm HP. The induction reached a maximum level around 2 hr and decreased to the basal level around 4 hr. NKEF-A mRNA was not induced by HP. These results demonstrate antioxidant activities of NKEF-B toward prooxidants such as alkyl hydroperoxide and MeHg. Together with its antioxidant activity, the induction of NKEF-B by HP indicates that NKEF-B is an important oxidative stress protein providing protection against a variety of xenobiotic toxic agents.

Study of in vitro cytotoxicity of a water soluble organic arsenic compound, arsenosugar, in seaweed.

In the present study, we demonstrated the cytotoxic effect of a dimethylarsenic compound in seaweed, (R)-(2',3'-dihydroxypropyl) 5- deoxy-5-dimethylarsinoyl-beta-D-riboside, namely arsenosugar (AsSug), on mammalian cells, murine macrophages, in comparison with that of an inorganic arsenical, arsenite, in vitro. More than 99.5% pure AsSug was synthesized. Arsenite was strongly and equally toxic to both peritoneal macrophages (PMs) and alveolar macrophages (AMs), and the concentration of arsenite that inhibited the viability of cells by 50% compared to the viability of control cells (50% inhibitory concentration; IC50) was 5 microM. In contrast, AsSug showed no cytotoxicity to both PMs and AMs at the microM concentration level; however, it induced different and interesting cellular responses in both macrophages at high concentrations, 1-10 mM. AsSug enhanced the viability of PMs at an optimal dose of 5 mM; conversely, it showed weak but significant cytotoxicity to AMs (IC50 = 8 mM).

Glutathione peroxidase and catalase modulate the genotoxicity of arsenite.

The X-ray hypersensitive Chinese hamster ovary (CHO) cells, xrs-5, are also more sensitive to sodium arsenite in terms of cell growth and micronucleus induction than CHO-K1 cells. Since reactive oxygen species are suggested to be involved in arsenic toxicity, we have measured antioxidant mechanisms in xrs-5 as well as CHO-K1 cells. There were no apparent differences in the activities of superoxide dismutase, glutathione S-transferase, glutathione reductase, and the levels of glutathione between xrs-5 and CHO-K1 cells. However, the activities of glutathione peroxidase and catalase were 5.4- and 5.8-fold lower, respectively, in xrs-5 cells. The addition of catalase or glutathione peroxidase to cultures reduced the arsenite-induced micronuclei in xrs-5 cells. Whereas, simultaneous treatment with mercaptosuccinate, an inhibitor of glutathione peroxidase, and 3-aminotriazole, an inhibitor of catalase, synergistically increased the arsenite-induced micronuclei. These results suggest that both catalase and glutathione peroxidase are involved in defense against arsenite genotoxicity. The xrs-6 cells, another line of x-ray hypersensitive CHO cells, which had 1.6-fold higher catalase activity and 2.5-fold higher glutathione peroxidase activity than xrs-5 cells, were also more sensitive than CHO-K1 cells but were less sensitive than xrs-5 cells to cell growth inhibition of arsenite. Moreover, a 1.6-fold increase of glutathione peroxidase activity by selenite adaptation effectively removed the arsenite-induced micronuclei in CHO-K1 cells. These results suggest that glutathione peroxidase is more important than catalase in defending against arsenite toxicity. Our results also suggest that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic poisoning.

New ulcerative colitis model induced by sulfhydryl blockers in rats and the effects of antiinflammatory drugs on the colitis.

We tried to produce a new ulcerative colitis model in rats by topical administration of sulfhydryl blockers. After male SD rats were fasted for 24 hr, 100 microliters of 3% N-ethylmaleimide (NEM) or iodoacetamide (IA) was introduced into the colon via a Nelaton's catheter. Both NEM and IA caused severe diarrhea with rectal bleeding and decreased body weight for about 7 days. At autopsy, adhesions and dilatation of the colon and severe mucosal lesions were observed. Both the weight and myeloperoxidase activity of the colon increased markedly. Maximum changes were observed within 1-3 days followed by gradual recovery, but even on day 21, some abnormalities were still observed. The ulceration and inflammation of the colon were confirmed by histological studies. Antiinflammatory drugs such as indomethacin inhibited the inflammation of the colon by NEM, but aggravated the ulceration. These results revealed that sulfhydryl blockers instilled into the colon caused ulcerative colitis in the rat. This model may be useful in studies on the pathogenesis of ulcerative colitis and the evaluation of drugs for therapy. Furthermore, it was suggested that antiinflammatory drugs may delay the healing of colonic ulcers.