The transcription factor NemR is an electrophile-sensing regulator important for the detoxification of reactive electrophiles in Acinetobacter nosocomialis.

NemR is an electrophile-sensing regulator which controls two enzymes required for the detoxification of reactive electrophiles: N-ethylmaleimide (NEM) reductase and glyoxalase I in Escherichia coli. Both enzymes are essential for bacterial survival in the presence of toxic reactive electrophiles, such as N-ethylmaleimide and methyl glyoxal. Here, we report the identification and characterization of NemR from Acinetobacter nosocomialis, a nosocomial pathogen. We confirmed that nemR and the nemA gene which encodes N-ethylmaleimide reductase form a single operon, which is in accordance with the reports from E. coli. Bioinformatic analysis revealed the presence of an NemR binding motif in the promoter regions of nemRA operon and gloA (encoding glyoxalase I) and the binding was confirmed by gel mobility shift assay. The deletion of nemR resulted in increased biofilm/pellicle formation in A. nosocomialis. mRNA expression analysis revealed that NemR acts as a repressor of the nemRA operon and gloA, and that the repressor function is inactivated by the addition of toxic Cys modification agents, contributing to bacterial survival. In addition, it was demonstrated that the nemRA operon is positively regulated by the quorum sensing regulator, AnoR and the operon plays a role in biofilm/pellicle formation in A. nosocomialis.

Increased mitochondrial thioredoxin 2 potentiates N-ethylmaleimide-induced cytotoxicity.

Thioredoxin 2 (Trx2) is a mitochondrially localized antioxidant and antiapoptotic protein, whose functions are mainly dependent on the conserved cysteines at its redox active center. In the current study, we showed by mass spectrometry that a thiol alkylating agent, N-ethylmaleimide (NEM), alkylated a single cysteine residue in the active center of Trx2. The interaction between NEM and Trx2 in intact cells was confirmed by redox Western analysis. Overexpression of Trx2 in cultured 143B osteosarcoma cells caused increased sensitivity to NEM. Covalent modification by NEM resulted in a dominant-negative effect and increased the interaction between Trx2 and peroxiredoxin 3 (Prx3). Our data suggest that the alkylation of the essential thiol(s) of Trx2 has profound impact on the mitochondrial redox circuitry and that such effects are distinct from the responses to agents causing reversible disulfide bond formation between the vicinal dithiols in the active center.

Changes in microbiological metabolism under chemical stress.

Chemical stress may alter microbiological metabolism and this, in turn, may affect the natural and engineered systems where these organisms function. The impact of chemical stress on microbiological metabolism was investigated using model chemicals 2,4-dinitrophenol (DNP), pentachlorophenol (PCP), and N-ethylmaleimide (NEM). Biological activity of Pseudomonas aeruginosa was measured in batch systems, with and without stressors at sub-lethal concentrations. Stressor DNP, between 49 and 140 mg l(-1), and PCP, at 15 and 38 mg l(-1), caused decreases in biomass growth yields, but did not inhibit substrate utilization rates. These effects increased with stressor concentrations, showing as much as a 10% yield reduction at the highest DNP concentration. This suggests that a portion of carbon and energy resources are diverted from growth and used in stress management and protection. Stressor DNP, between 300 and 700 mg l(-1), and PCP at 85 mg l(-1) caused decreases in growth yields and substrate utilization rates. This suggests an inhibition of both anabolism and catabolism. Stressor NEM was the most potent, inhibiting biological activity at concentrations as low as 2.7 mg l(-1). These findings will ultimately be useful in better monitoring and management of biological treatment operations and contaminated natural systems.

Neurotoxic mechanisms of electrophilic type-2 alkenes: soft soft interactions described by quantum mechanical parameters.

Conjugated Type-2 alkenes, such as acrylamide (ACR), are soft electrophiles that produce neurotoxicity by forming adducts with soft nucleophilic sulfhydryl groups on proteins. Soft-soft interactions are governed by frontier molecular orbital characteristics and can be defined by quantum mechanical parameters such as softness (sigma) and chemical potential (mu). The neurotoxic potency of ACR is likely related to the rate of adduct formation, which is reflected in values of sigma. Correspondingly, differences in mu, the ability of a nucleophile to transfer electrons to an electrophile, could determine protein targets of these chemicals. Here, sigma and mu were calculated for a series of structurally similar Type-2 alkenes and their potential sulfhydryl targets. Results show that N-ethylmaleimide, acrolein and methylvinyl ketone were softer electrophiles than methyl acrylate or ACR. Softness (sigma) was closely correlated to corresponding second-order rate constants (k(2)) for electrophile reactions with sulfhydryl groups on N-acetyl-L-cysteine (NAC). The rank order of softness was also directly related to neurotoxic potency as determined by impairment of synaptosomal function and sulfhydryl loss. Calculations of mu showed that the thiolate state of several cysteine analogs was the preferred nucleophilic target of alkene electrophiles. In addition, mu was directly related to the thiolate rate constant (k) for the reaction of the Type-2 alkenes with the cysteine compounds. Finally, in accordance with respective mu values, we found that NAC, but not N-acetyl-L-lysine, protected synaptosomes from toxicity. These findings suggest that the neurotoxicity of ACR and its conjugated alkene analogs is related to electrophilic softness and that the thiolate state of cysteine residues is the corresponding adduct target.

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.

Can the aqueous decoction of mango flowers be used as an antiulcer agent?

This study was designed to determine the effect of Mangifera indica flowers decoction, on the acute and subacute models of induced ulcer in mice and rats. A single oral administration of the aqueous decoction (AD) from M. indica up to a dose of 5 g/kg, p.o. did not produce any signs or symptom of toxicity in the treated animals. The oral pre-treatment with AD (250, 500 and 1000 mg/kg) in rats with gastric lesions induced by ethanol, decreased the gastric lesions from 89.0+/-6.71 (control group) to 9.25+/-2.75, 4.50+/-3.30 and 0, respectively. Pretreatment with AD (250, 500 and 1000 mg/kg) to mice with HCl/ethanol- or stress-induced gastric lesions resulted in a dose-dependent significant decrease of lesion index. In the piroxicam-induced gastric lesions, the gastroprotective effect of AD was reducing with the increase of the AD dose. In the pylorus-ligature, AD (p.o.) significantly decreased the acid output indicating the antisecretory property involved in the gastroprotective effect of M. indica. Treatment with AD during 14 consecutive days significantly accelerated the healing process in subacute gastric ulcer induced by acetic acid in rats. Pretreatment with N-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO-synthase, did not abolish the gastroprotective effects (99% with saline versus 80% with l-NAME) of AD against ethanol-induced gastric lesions. Pretreatment with N-ethylmaleimide (NEM), a blocker of endogenous sulphydryl group, significantly abolished the protective effects of AD against ethanol-induced gastric ulcers (95% with saline versus 47% with NEM). Phytochemical screening showed the presence of steroids, triterpenes, phenolic compounds and flavonoids. Estimation of the global polyphenol content in the AD was performed by Folin-Ciocalteu method and showed approximately 53% of total phenolic on this extract. These findings indicate the potential gastroprotective and ulcer-healing properties of aqueous decoction of M. indica flowers and further support its popular use in gastrointestinal disorders in Caribbean.

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation.

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Differential cytotoxicity of buthionine sulfoximine to "normal" and transformed human lung fibroblast cells.

Glutathione (GSH) depletion has been studied extensively as a possible means to sensitive tumor cells to radiation treatment and chemotherapy. The present study was undertaken to compare the cytotoxicity of GSH depletion in normal and transformed cells. The results showed that specific inhibition of GSH synthesis by L-buthionine sulfoximine (BSO) caused significantly higher cytotoxicity in "normal" human-lung fibroblast cells than in their transformed counterparts. This finding suggests a possibility that depletion of GSH could be more harmful to normal cells than to transformed and/or tumor cells and that the selective cytotoxicity of BSO to normal cells could limit its potential as an effective sensitizer for cancer treatment.

Interactions of metallothionein with murine lymphocytes: plasma membrane binding and proliferation.

Metallothionein (MT) is a thiol rich protein that has been well characterized for its ability to bind and sequester heavy metal cations, free radicals and other reactive toxicants. In addition to induction by these stressors, MT gene expression is upregulated by several cytokines of the acute phase response. In previous work, we have shown that MT can alter aspects of lymphocyte function. MT alone induces modest proliferation of unfractionated splenocytes and acts synergistically with T cell- and B cell-specific mitogens. In contrast, MT inhibits humoral responsiveness in vivo and reduces in vitro T cell responses to processed antigen. In this report, we describe the effects of MT on specific lymphocyte subpopulations in order to further characterize the mechanism of MT-mediated alterations of immune activity. MT binds to the plasma membrane of both T and B lymphocytes, but, in the absence of a costimulatory agent, MT induces lymphoproliferation only in B cells. MT also enhances the capacity of naive B lymphocytes to differentiate into plasma cells. These results demonstrate differential immunomodulatory activities of MT and may explain some of the diverse immunoregulatory effects associated with exposure to environmental toxins.

Four sulfhydryl-modifying compounds cause different structural damage but similar functional damage in murine lymphocytes.

Four thiol-modifying compounds were used to inhibit murine lymphocyte mitogenesis. The compounds were a copper sulfate/O-phenanthroline complex (CuP) to oxidize surface thiols, N-ethyl maleimide (NEM) to alkylate surface and intracellular thiols, D,L-buthionine-S,R-sulfoximine (BSO) to prevent synthesis of glutathione, and hydrogen peroxide, which reacts with various cellular constituents, including sulfhydryls. Splenic lymphocytes were incubated with one of the four compounds, washed, and then stimulated with the B cell mitogen, LPS, or the T cell mitogen, Con A. In spite of their differing chemical reactivities and differing effects on cell viability, lipids, and total, protein, and non-protein thiols, the four sulfhydryl-modifying compounds had very similar effects on the kinetics and inhibition of lymphocyte growth. All compounds had complex effects on mitogenesis, causing enhanced, delayed, or inhibited tritiated thymidine incorporation. Although the total thiol contents of untreated T cells and B cells were found to be equivalent, the LPS response consistently was inhibited by lower concentrations than the Con A response, suggesting that B cells were more sensitive than T cells to thiol modification. To compare compounds the efficiency of inhibition was determined by functionally relating reductions in mitogenesis with reductions in thiol content of the cells. The compounds differed in inhibitory efficiency; thus, damage to some thiols must be more important than damage to others. CuP ablated mitogenesis with the least change in thiol content. Therefore, surface sulfhydryls appear critical in lymphocyte mitogenesis. With all compounds inhibition of mitogenesis occurred over a very narrow range of thiol content, suggesting that the thiols important in inhibition were few in number relative to the total thiol content of the cell.

H(2)O(2) mediates oxidative stress-induced epidermal growth factor receptor phosphorylation.

We used a well-established thiol-alkylating agent, N-ethylmaleimide (NEM), to oxidatively stress human keratinocytes. Time course studies revealed that NEM rapidly depleted keratinocytes of reduced glutathione (GSH), which was followed by rapidly increasing levels of intracellular reactive oxygen species (ROS) and subsequently by phosphorylation of epidermal growth factor receptor (EGFR). Pretreatment with antioxidants or enhanced catalase activity in keratinocytes inhibited ROS/H(2)O(2) accumulation and EGFR phosphorylation, demonstrating that H(2)O(2) production is a mediator required for EGFR phosphorylation. Collectively, these results suggest a sequence of events leading to EGFR phosphorylation which is likely shared by oxidative stress-inducing agents, namely: (1) GSH depletion; (2) H(2)O(2) accumulation; and (3) EGFR phosphorylation. We propose that depletion of GSH and accumulation of H(2)O(2) are upstream events and critical mediators required for ligand-independent phosphorylation of growth factor receptors in response to oxidative stress.

The role of gastric mucosal sulphydryls in the ulcer-protecting effects of cisapride.

The present study was designed to examine the role of endogenous sulphydryls (SHs) in the gastro-protection induced by cisapride (CIS) (10, 25 and 50 mg kg-1 i.p.), a potent benzamide stimulating gastrointestinal motility in mucosal injury induced by 50% v/v ethanol. Results were compared with those of 5-hydroxytryptamine (5-HT) (10mg kg-1). Ethanol mucosal damage was significantly reduced by treatment with CIS and 5-HT. On the contrary, administration of n-ethylmaleimide (NEM) (10 mg kg-1) an SH alkylator, markedly worsened lesion formation and counteracted the protective effect of CIS. Rats pretreated with CIS significantly increased the total sulphydryls as reflected in the non-protein and protein fractions however, 5-HT treatment showed a fall in the non-protein level. The present results suggest that 5-HT-ergic dependent mechanisms have no relation to the gastro-protection afforded by CIS in this experimental model. It is possible that mucosal SHs could be involved.

Knockdown of the gene for homeobox protein HOXB13 reduces toxicity of oxidative-stress inducers in HEK293 cells.

HEK293 cells transfected with a double-stranded siRNA to suppress expression of the homeobox gene HOXB13 were highly resistant to oxidative stress-inducing agents, such as hydrogen peroxide, N-ethylmaleimide (NEM), and paraquat. This finding suggests that HOXB13 might enhance cell injury caused by oxidative stress.

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.

Response of hydroperoxidase and superoxide dismutase deficient mutants of Escherichia coli K-12 to oxidative stress.

In Escherichia coli, the coordinate action of two antioxidant enzymes, superoxide dismutase and hydroperoxidase (catalase), protect the cell from the deleterious effects of oxyradicals generated during normal aerobic respiration. To evaluate the relative importance of these two classes of enzymes, strains of E. coli deficient in superoxide dismutase and (or) hydroperoxidase were constructed by generalized transduction and their physiological responses to oxygen and oxidant stress examined. Superoxide dismutase was found to be more important than hydroperoxidase in preventing oxygen-dependent growth inhibition and mutagenesis, and in reducing sensitivity to redox-active compounds known to generate the superoxide anion. However, both types of enzymes were required for an effective defense against chemical oxidants that generate superoxide radicals and hydrogen peroxide.

The role of non-protein sulfhydryl compounds in gastric adaptive cytoprotection against ethanol-induced mucosal damage in rats.

The contribution of the endogenous nonprotein sulfhydryl compounds (SH) in gastric adaptive cytoprotection was investigated in rats. N-ethylmaleimide (NEM) treatment significantly reduced mucosal SH level, and aggravated the mucosal injury induced by absolute ethanol. Oral administration of the mild irritants, 20% ethanol, 5% NaCl or 0.3 M HCl, significantly increased the basal mucosal SH level. These agents also showed a cytoprotective action against the necrotizing effect of absolute ethanol. Administration of NEM did not alleviate this cytoprotective potential, although it abolished the increased SH level evoked by these mild irritants. Thus, it is concluded that modulation of endogenous SH by mild irritants perhaps only plays a minor role in the gastric adaptive cytoprotection.

Interactive cytotoxicities of selected organic and inorganic substances to brown cells of Mercenaria mercenaria.

Toxicities of binary mixtures of Cu2+, Cd2+, benzo(a)pyrene [B(a)P] and N-ethylmaleimide (NEM) were screened using the in vitro neutral red (NR) assay to test the hypothesis that combined toxicity is more than or less than additive relative to the influence of each mixture constituent on toxicant uptake and brown cell lysosomal membrane stability. Significant cytotoxicity was observed at 25 mumol/L Cu2+, 500 mumol/L Cd2+ and 25 mumol/L NEM. B(a)P at 12 mumol/L exerted no toxicity under the conditions of the assay. Interactions between Cu2+ and NEM, between Cd2+ and NEM, and between Cd2+ and B(a)P significantly influenced brown cell survival. Comparison of observed joint toxicity with estimates made using a model of independent joint action indicates that interactive effects are less than additive in character. The 3-way interaction involving Cu2+, B(a)P, and NEM also affected brown cell survival to a statistically significant degree. However, the interactive cytotoxicity of this mixture is attributable mainly to the combined effect of Cu2+ and NEM. Results also indicate that new hypothesis and additional experimentation are needed to understand the interactive toxicity of mixture constituents.

Formation of protein-glutathione mixed disulfides in the developing rat conceptus following diamide treatment in vitro.

Protein-glutathione mixed disulfide (protein-S-SG) formation was investigated in developing rat conceptuses during early organogenesis (gestational day 10, GD 10) using the whole embryo culture system. Low levels of protein-S-SG (25.0 +/- 6.6 pmoles resolved GSH/conceptus) were found in conceptuses under normal culture conditions. Incubation of the conceptuses with 75-500 microM diamide (a thiol oxidant) resulted in rapid increases in protein-S-SG (to 2- to 16-fold that of control values) in a dose-dependent manner during 30 min of the culture period. Approximately 20% of the observed cytosolic glutathione (GSH) depletion following diamide (500 microM) could be accounted for as mixed disulfides of protein sulfhydryls, when determined in whole conceptual tissues after 15 min. The most extensive S-thiolation of protein sulfhydryls by GSH was observed in visceral yolk sac (VYS) when compared to embryo proper and ectoplacental cone. This result indicates that the most abundant, sensitive, or accessible protein sulfhydryls were found in the VYS. Inhibition of glutathione disulfide reductase activity by pretreatment of the conceptuses with 25 microM BCNU for 2 hr potentiated protein-S-SG formation elicited by 75 microM diamide. Reincubation of the conceptuses in fresh media, following the 15-min treatment with 500 microM diamide, reversed both the GSH depletion and the protein-S-SG formation in conceptal tissues. The reduction of the protein-S-SG was dependent on adequate intracellular GSH levels and was inhibited when GSH was rapidly depleted by subsequent addition of N-ethylmaleimide (NEM, 100 microM). Under the same experimental conditions, addition of 1 mM dithiothreitol (DTT) did not significantly enhance the GSH restoration rate nor the protein-S-SG reduction rate. The results also indicated that low levels of intracellular cysteine do not play an important role in the reduction of protein-S-SG. Protein-S-SG formation may be important for cellular regulation and in mediating the embryotoxicity elicited by diamide or other oxidative stresses.