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.

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.

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.

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.

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.

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.

Metabolic myopathy produced by acute inhibition of glyceraldehyde-3-phosphate dehydrogenase with ortho-iodosobenzoic acid.

A previously developed model of exercise-induced muscle contracture using iodoacetate to inhibit glyceraldehyde-3-phosphate dehydrogenase in rat hindlimb muscles produced selective type II myofiber damage. Utilizing a modification of the same model system, rats were given intra-aortic ortho-iodosobenzoic acid (700 nmol/kg body weight), which cleaves tryptophanyl peptides from glyceraldehyde-3-phosphate dehydrogenase. Within 2-4 h, spontaneous electrically-silent contracture developed in the injected musculature resulting in a plantar-flexed position of the hindlimb. After 24 h, the extensor digitorum longus and tibialis anterior muscles appeared grossly swollen (edematous) and discolored. Microscopically, the extensor digitorum longus (composed predominantly of type II myofibers) contained many randomly scattered, damaged myofibers, reduced glycogen content, absent glyceraldehyde-3-phosphate dehydrogenase activity, interstitial edema and focal collections of mononuclear phagocytes. Damaged fibers showed degenerative changes and contained stainable intracellular calcium. On modified trichrome-stained sections, an outer red staining rim of material was identifiable in many fibers. The fibers of the soleus muscle (composed predominantly of type I myofibers) were not damaged, indicating a preferential ortho-iodosobenzoic acid effect on type II myofibers.

Protective effects of methylcobalamin, a vitamin B12 analog, against glutamate-induced neurotoxicity in retinal cell culture.

PURPOSE: To examine the effects of methylcobalamin on glutamate-induced neurotoxicity in the cultured retinal neurons. METHODS: Primary cultures obtained from the fetal rat retina (gestation days 16 to 19) were used for the experiment. The neurotoxicity was assessed quantitatively using the trypan blue exclusion method. RESULTS: Glutamate neurotoxicity was prevented by chronic exposure to methylcobalamin and S-adenosylmethionine (SAM), which is formed in the metabolic pathway of methylcobalamin. Chronic exposure to methylcobalamin and SAM also inhibited the neurotoxicity induced by sodium nitroprusside that release nitric oxide. By contrast, acute exposure to methylcobalamin did not protect retinal neurons against glutamate neurotoxicity. CONCLUSIONS: Chronic administration of methylcobalamin protects cultured retinal neurons against N-methyl-D-aspartate-receptor-mediated glutamate neurotoxicity, probably by altering the membrane properties through SAM-mediated methylation.

Glutathione effects on toxicity and uptake of mercuric chloride and sodium arsenite in rabbit renal cortical slices.

The mechanism of renal uptake of nephrotoxic heavy metals such as HgCl2 and NaAsO2 is not clear. The metals are known to react with endogenous sulfhydryls such as glutathione (GSH), so metal-GSH conjugates may be delivered to the kidney. To study this possibility, renal cortical slices from male New Zealand white rabbits were incubated with 10(-4) M HgCl2 or 10(-3) M NaAsO2 +/- stoichiometric amounts (1-3x) of GSH; or synthetic metal-GSH conjugates [10(-4) M Hg(SG)2 or 10(-3) M As(SG)3]. Incubations were performed at 37 degrees C in DME-F12 buffer (95/5 O2/CO2) for 8 hr. Hg(SG)2 reduced slice K+/DNA content, as an indicator of viability, significantly less than HgCl2. As(SG)3 exhibited a 2-hr delay in K+/DNA content reduction compared to NaAsO2. This delay in toxicity was not correlated to changes in uptake. Arsenic and mercury accumulation, determined by proton-induced X-ray emission, were also identical between the metal salts and the metal-GSH conjugates. Exogenous GSH decreased HgCl2 cytotoxicity and was correlated to a decrease in Hg accumulation in the slice. Exogenous GSH had limited if any protective effects against cytotoxicity by NaAsO2 and a decrease in As accumulation was not observed. Complex metal-GSH interactions appear to exist and impact on the uptake and toxicity of these metals.

The molecular response to reductive stress in LLC-PK1 renal epithelial cells: coordinate transcriptional regulation of gadd153 and grp78 genes by thiols.

Organic thiols are toxic to eukaryotic cells. Treatment of cells with thiols activates expression of grp78, but it is not known if, like other forms of stress, there is a battery of stress response genes that are induced by thiols. In LLC-PK1 renal epithelial cells, mRNAs for both grp78 and gadd153 were induced by thiols with similar time, concentration and structure-activity dependence. Dithiothreitol (DTT) was the most potent reductant and inducer of gene expression among the thiols tested. Nuclear run-on assays demonstrated that DTT activated both grp78 and gadd153 genes transcriptionally. A hamster gadd153 promoter construct which contains enhancer elements necessary for gadd153 activation was stably integrated into the LLC-PK1 cell genome and was activated by DTT. Although auto-oxidation of thiols can generate active oxygen species, transcriptional activation of the gadd153 promoter was not due to formation of hydrogen peroxide or superoxide since neither catalase nor superoxide dismutase prevented activation of the gadd153 promoter by DTT. The concentration dependence for activation of the gadd153 promoter correlated with inhibition of dome formation and protein synthesis, two toxic effects of DTT in LLC-PK1 cells. Thus, both grp78 and gadd153 are members of a gene battery which is responsive to reductive stress. There appears to be considerable, but not complete, overlap between the upstream signaling pathways for activation of both genes.

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.

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.

Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster.

Two arsenic compounds, sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanogaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT) were used. In both tests, a brood scheme of 2-3-3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM for sodium arsenite; and 0.54, 1.08 mM for sodium arsenate) and larval feeding (0.008, 0.01, 0.02 mM for sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). For the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w+/w eye assay as well as in the mwh +/+ flr3 wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds. Sodium arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds.

Effect of arsenic and cadmium on the persistence of mutagen-induced DNA lesions in human cells.

The alkaline single cell gel electrophoresis (SCG test or comet assay) was used to characterize the influence of sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) on the persistence of mutagen-induced DNA lesions. Human blood and SV4O-transformed fibroblasts (MRC5CV1) were treated for 2 hr with methyl methanesulphonate (MMS) or benzo(a)pyrene (BaP). MMS induced concentration-related DNA damage in white Blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage in white blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage by BaP, higher concentrations had to be applied to WBC than to the fibroblast cell line. To study the influence of metal ions on the persistence of DNA lesions, treated cells were further incubated for 2 hr in the absence (postincubation) or presence (posttreatment) of NaAsO2 or CdSO4. After postincubation, MMS and BaP-induced DNA effects were reduced in both cell types, indicating that repair of DNA lesions had taken place. When the cells were posttreated with NaAsO2 or CdSO4, BaP- and MMS-induced DNA lesions persisted in both cell types, indicating an inhibition of DNA repair by these metals. The results suggest a strong interaction of arsenic and cadmium with BaP- and MMS-induced DNA repair processes.

Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts.

Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 microM) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity.

Sodium arsenite and heat shock induce stress proteins in precision-cut rat liver slices.

The present study was undertaken to investigate the usefulness of stress proteins as early, sensitive indicators of hepatotoxicity. Induction of stress protein synthesis in precision-cut rat liver slices was examined following in vitro exposure to sodium arsenite or heat shock. Precision-cut rat liver slices were incubated with 10(-5) or 10(-6) M sodium arsenite for 2, 4 or 8 h in the presence of 35S-methionine or exposed to hyperthermia (42.5 +/- 0.5 degrees C) for 45 min and then incubated with 35S-methionine for 2, 4 or 8 h. Fluorographic analysis indicated an increase in the synthesis of HSP 70 and HSP 90 family of proteins by both treatments. Immunoblot analysis demonstrated that there was a specific induction of HSP 72 and HSP 90. Induction of HSP 70 was greater than that of HSP 90 by both treatments. Stress protein induction occurred at earlier times by concentrations of arsenite which did not alter other viability parameters such as leakage of intracellular K+ or total protein synthesis. The results indicated that induction of stress proteins has the potential usefulness as an early biomarker of arsenite toxicity.

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.

An attempt to explain interindividual variability in 24-h urinary excretion of inorganic arsenic metabolites by C57 BL/6J mice.

Forty C57 BL/6J mice, injected subcutaneously with 0.5 mg/kg arsenic as sodium arsenite, were examined for 24-h urinary excretion of total arsenic metabolites, creatinine and S-adenosylmethionine (SAM) and for 24-h faecal excretion of arsenic and levels of arsenic in the blood, liver, kidneys, lung, skin, spleen and bone at 24-h post-dose. Total urinary arsenic metabolites were calculated by summing up the inorganic (Asi), monomethylated (MMA) and dimethylated (DMA) derivatives directly measured by selective arsine generation-atomic absorption spectrometry (AG-AAS) or were measured by AG-AAS following complete mineralization. Both sets of results showed interindividual differences varying by as much as 7-fold and correlated with the 24-h urinary excretion of both SAM (r = 0.84 and r = 0.86, respectively) and creatinine (r = 0.82 and r = 0.87, respectively). There was interindividual variability of about a 30-fold range in 24-h faecal excretion of arsenic which correlated inversely with 24-h urinary excretion of arsenic metabolites (r = -0.69) and 24-h urinary excretion of both creatinine (r = -0.70) and SAM (r = -0.67). Body tissue levels of arsenic were low and not related to 24-h urinary excretion of arsenic metabolites, SAM and creatinine. Taken together, the results indicate that differences in the profile of urinary arsenic excretion and in the retention of arsenic in a particular organ do not contribute to interindividual variability in 24-h urinary excretion of arsenic metabolites by C57 BL/6J mice, but that variability in faecal excretion does, at least in part. It is speculated that there is most likely a predominant contribution from a diffuse tissue retention of arsenic or from a third route of arsenic elimination, i.e. respiratory, to this phenomenon in view of the small faecal contribution.

Different toxic effects of hydrogen peroxide, nitric oxide, and superoxide on human, pig, and rat islets of Langerhans.

Susceptibility of islet cells to damage by hydrogen peroxide, superoxide, and nitric oxide was determined on islets isolated from humans, pigs, and rats. Islets were incubated for 20 hr at 37 degrees C with different concentrations of hydrogen peroxide, hypoxanthine/xanthine oxidase, or nitroprusside sodium, respectively. Islet cell damage was then measured as trypan blue-uptake. Rat islets showed a higher sensitivity than human or pig islets to damage by reactive oxygen species or nitric oxide. These results indicate that pig islets may be a more suitable model than rat islets to study inflammatory islet cell damage in diabetes and clinical islet transplantation.