Inorganic chloramines: a critical review of the toxicological and epidemiological evidence as a basis for occupational exposure limit setting.

Inorganic chloramines are not commercially available, but monochloramine is produced in situ for disinfection or for use in chemical synthesis. Inorganic chloramines are also formed when free chlorine reacts with nitrogen containing substances, e.g. ammonia and urea, present in chlorinated water sources. Occupational exposure may, therefore, occur in e.g. swimming pool facilities and the food processing industry. Monochloramine is soluble and stable in water and the dominating inorganic chloramine in chlorinated water sources. No clinical effects were seen in healthy volunteers given monochloramine in drinking water during 4 or 12 weeks in doses of 0.043 or 0.034 mg/kg bw/day, respectively. Limited data indicate that monochloramine is weakly mutagenic in vitro but not genotoxic in vivo. One drinking water study indicated equivocal evidence of carcinogenicity in female rats but not in male rats and mice. No reproductive or developmental effects were shown in rodents in the few studies located. Dichloramine is soluble but unstable in water. In the only study located, mild histological effects in kidneys, thyroid and gastric cardia were observed in rats administered dichloramine in drinking water for 13 weeks. Trichloramine is immiscible with water and evaporates easily from water into air. Therefore, the primary exposure route of concern in the occupational setting is inhalation. Occupational exposure to trichloramine has been demonstrated in indoor swimming pool facilities and in the food processing industry where chlorinated water is used for disinfection. Exposure-response relationships between airborne levels and self-reported ocular and upper airway irritation have been shown in several studies. Exposure to trichloramine may aggravate asthma symptoms in individuals with existing asthma. The risk of developing asthma following long-term exposure to trichloramine cannot be evaluated at present. No data on genotoxic, carcinogenic, reproductive or developmental effects were located. The toxicological data for mono- and dichloramine are insufficient to recommend health-based occupational exposure limits (OELs).As regard trichloramine, the critical effect is judged to be irritation observed in several studies on pool workers, starting at approximately 0.4 mg/m3 (stationary sampling). Based on these data, a health-based OEL of 0.1 mg/m3 (8-h time-weighted average) is recommended. This corresponds to 0.2 mg/m3 for stationary measurements in swimming pool facilities. No short-term exposure limit (STEL) is recommended.

Evaluation of biocidal efficacy of Chloramine T trihydrate on planktonic and sessile bacteria in a model cooling tower water system.

A model cooling tower system was experimentally seeded with Legionella pneumophila and real industrial cooling tower (CT) water has been run at the closest to full-scale system operating conditions. The water/biofilm samples were taken from the model system monthly, and the effectiveness of the different concentrations of Chloramine T trihydrate biocide was evaluated in terms of its ability to control both planktonic/sessile microbial populations. Although Chloramine T is a recommended commercial formulation for disinfecting CTs, there is a lack of published data on the efficacy of this compound against both planktonic and sessile populations in the cooling tower. Biocide response in both sessile/planktonic bacteria counts varied according to months. Tested biocide concentrations provided the clean tower conditions by reducing the concentration of heterotrophic plate count (HPC) below <104 cfu mL-1, L. pneumophila <10 cfu mL-1 and of adenosine triphosphate (ATP) values <300 relative light units (RLU), after 1, 3 and 24 h of exposure, during a 6-month period. There were no statistically significant differences in efficacy between concentrations in terms of reduction in the number of bacteria, decrease in ATP value and viability. The results revealed that Chloramine T can effectively control biofouling in cooling systems according to the limit values of the successful control program.

Evaluating Evidence for Association of Human Bladder Cancer with Drinking-Water Chlorination Disinfection By-Products.

Exposure to chlorination disinfection by-products (CxDBPs) is prevalent in populations using chlorination-based methods to disinfect public water supplies. Multifaceted research has been directed for decades to identify, characterize, and understand the toxicology of these compounds, control and minimize their formation, and conduct epidemiologic studies related to exposure. Urinary bladder cancer has been the health risk most consistently associated with CxDBPs in epidemiologic studies. An international workshop was held to (1) discuss the qualitative strengths and limitations that inform the association between bladder cancer and CxDBPs in the context of possible causation, (2) identify knowledge gaps for this topic in relation to chlorine/chloramine-based disinfection practice(s) in the United States, and (3) assess the evidence for informing risk management. Epidemiological evidence linking exposures to CxDBPs in drinking water to human bladder cancer risk provides insight into causality. However, because of imprecise, inaccurate, or incomplete estimation of CxDBPs levels in epidemiologic studies, translation from hazard identification directly to risk management and regulatory policy for CxDBPs can be challenging. Quantitative risk estimates derived from toxicological risk assessment for CxDBPs currently cannot be reconciled with those from epidemiologic studies, notwithstanding the complexities involved, making regulatory interpretation difficult. Evidence presented here has both strengths and limitations that require additional studies to resolve and improve the understanding of exposure response relationships. Replication of epidemiologic findings in independent populations with further elaboration of exposure assessment is needed to strengthen the knowledge base needed to better inform effective regulatory approaches.

Modelling combined effect of chloramine and copper on ammonia-oxidizing microbial activity using a biostability approach.

Continuous and batch laboratory experiments were used to evaluate the combined effects of copper and chloramine on ammonia oxidizing microbes present in otherwise high nitrifying water samples. The experimental data were analyzed using a biostability concept and quantified with the biostable residual concentratrion (BRC) of monochloramine, or the concentration that prevents the onset of nitrification. In the batch experiments, copper dosing ≥0.25 mg-Cu L(-1) resulted in complete inhibition of nitrification, and a lower copper dosing (0.1 mg-Cu L(-1)) delayed nitrification. The BRC was systematically lowered with the addition of copper. For example, a free-ammonium concentration of 0.1 mg-N L(-1) had a BRC of 0.73 mg-Cl2 L(-1) with no Cu, but addition of 0.1 mg-Cu L(-1) lowered the BRC to 0.16 mg-Cl2 L(-1), while addition of 0.25 mg-Cu L(-1) eliminated the need to add chloramine (BRC = 0). A non-competitive inhibition model fit the experimental data well with a copper threshold of 0.044 mg-Cu L(-1) and can be used to estimate Cu doses needed to prevent nitrification based on the chloramine concentration. Full scale systems applications need further study.

Resveratrol preconditioning increases methionine sulfoxide reductases A expression and enhances resistance of human neuroblastoma cells to neurotoxins.

Methionine sulfoxide reductases A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. Recently, attention has turned to MsrA in coupling with the pathology of Parkinson's disease, which is closely related to neurotoxins that cause dopaminergic neuron degeneration. Here, we firstly provided evidence that pretreatment with a natural polyphenol resveratrol (RSV) up-regulated the expression of MsrA in human neuroblastoma SH-SY5Y cells. It was also observed that the expression and nuclear translocation of forkhead box group O 3a (FOXO3a), a transcription factor that activates the human MsrA promoter, increased after RSV pretreatment. Nicotinamide , an inhibitor of silent information regulator 1 (SIRT1), prevented RSV-induced elevation of FOXO3a and MsrA expression, indicating that the effect of RSV was mediated by a SIRT1-dependent pathway. RSV preconditioning increased methionine sulfoxide(MetO)-reducing activity in SH-SY5Y cells and enhanced their resistance to neurotoxins, including chloramine-T and 1-methyl-4-phenyl-pyridinium. In addition, the enhancement of cell resistance to neurotoxins caused by RSV preconditioning can be largely prevented by MsrA inhibitor dimethyl sulfoxide. Our findings suggest that treatment with polyphenols such as RSV can be used as a potential regulatory strategy for MsrA expression and function.

Novel model to study combined effects of microorganisms and oxidants on development of intestinal necrosis.

BACKGROUND: Although the cause of intestinal necrosis in diseases such as necrotizing enterocolitis (NEC) is multi-factorial, oxidants and bacteria likely are key factors. Many animal models of NEC generate histologic necrosis, but it is rare to see the gross necrosis that is observed in infants. Here, we present a novel model that produces full-thickness intestinal necrosis when chloramine-T, an oxidizing agent, is introduced directly into isolated intestinal loops. The aim of this study was to determine the role of bacteria in this model. METHODS: Three-week-old mice underwent isolated ileal loop construction by suture ligation, and either chloramine-T or saline was injected into the loop. Intestines were then returned to the abdominal cavity, and the incision was closed for 24 h, after which the intestinal loops were analyzed histologically and microbiologically. To determine if bacteria home to the site of injury, some mice had intracecal injection of luminescent Pseudomonas aeruginosa (PA). These mice were sacrificed 2, 6, 8, and 24 h later, and luminescent bacteria were localized via photon camera imaging. RESULTS: No gross necrosis was observed in the saline-treated group, but 59% of the animals in the chloramine-T-treated group had necrosis (p<0.001). Relative bacterial species numbers were calculated for untreated control animals and those with saline-treated loops and chloramine-T-treated loops without and with necrosis. Lactobacillus was detected in 60% of the control animals but was absent in all animals that underwent surgery. Methicillin-sensitive Staphylococcus aureus, PA, and Enterococcus faecium were present only in animals that underwent loop construction; however, bacterial communities did not differ according to loop treatment or the presence or absence of necrosis. After intracecal injection of PA, bacteria homed to the loop and proximal bowel even though the loop was discontinuous with the remaining bowel. CONCLUSIONS: Intraluminal chloramine-T causes full-thickness necrosis in three-week-old mice and is characterized by a loss of probiotic bacteria such as Lactobacillus. Necrotic loops of bowel become colonized rapidly with pathogenic bacteria by unconventional mechanisms. Oxidant stress and colonization by pathogenic bacteria may play important roles in intestinal necrosis across a wide spectrum of pathologic states, including NEC.

Amino acid chloramine damage to proliferating cell nuclear antigen in mammalian cells.

UNLABELLED: Amino acid chloramines (AACLs) are reactive secondary products of activated neutrophils. To understand AACL damage in cell nuclei, we exploited proliferating cell nuclear antigen (PCNA) as a nuclear protein damage reporter, using western blotting and mass spectrometry. Chloramines of proline, arginine, and glycine caused significant damage to PCNA in cells. Chloramines of taurine and histidine caused slight damage to PCNA in cells. Other AACLs caused no PCNA damage in intact cells. Evidence supports a sulfonamide, sulfinamide, or sulfenamide crosslinking mechanism involving cysteine 148 at the PCNA subunit interface, methionine sulfoxide formation as the basis of electrophoretic mobility shifting, and tyrosine and/or methionine residues as the likely targets of AACL damage to the PCNA antibody epitope. An interstitial fluid model experiment showed that physiological amino acids can mediate HOCl damage to PCNA in the presence of proteins that would otherwise completely quench the HOCl. CONCLUSION: PCNA is a sensitive biomarker of AACL damage in cell nuclei. Arginine chloramine and proline chloramine, or reactive species derived from them, were shown to enter cells and damage PCNA. Amino acids were shown to have at least two different mechanisms for suppressing PCNA damage in cells by their corresponding AACLs. Cysteine 148 was shown to be essential for PCNA subunit crosslinking by AACLs, and a crosslinking mechanism was proposed.

In vitro toxicity and genotoxicity assessment of disinfection by-products, organic N-chloramines.

Disinfection by-products (DBPs) are of concern to both water industries and health authorities. Although several classes of DBPs have been studied, and there are regulated safe levels in disinfected water for some, a large portion of DBPs are not characterized, and need further investigation. Organic N-chloramines are a group of DBPs, which can be formed during common disinfection processes such as chlorination and chloramination, but little is known in terms of their toxicological significance if consumed in drinking water. Only a few in vitro studies using bacterial assays have reported some genotoxic potential of organic N-chloramines, largely in the context of inflammatory processes in the body rather than exposure through drinking water. In this study, we investigated 16 organic N-chloramines produced by chlorination of model amino acids and amines. It was found that within the drinking water-relevant micromolar concentration range, four compounds were both cytotoxic and genotoxic to mammalian cells. A small reduction of cellular GSH was also observed in the treatment with these four compounds, but not of a magnitude to account for the cytotoxicity and genotoxicity. The results presented in this study demonstrate that some organic N-chloramines, at low concentrations that might be present in disinfected water, can be harmful to mammalian cells.

Health effects of disinfection by-products in chlorinated swimming pools.

Increased attendance at swimming pools is correlated with higher input of organic and minerals pollutants introduced by swimmers in the swimming pool water. In most swimming pools, microbiological control is performed by disinfection with the addition of chlorine. Chlorine is now well-known to lead to the formation of many disinfection by-products (DBPs) including trihalomethanes and chloramines. The hypothesis of a link between the presence of eye and skin irritation syndromes in swimmers and contact with swimming pool water treated with chlorine was initially proposed by Mood (1953). During recent decades many epidemiological studies have described the importance of DBPs generated with natural or imported organic matter present in water. Many of these DBPs are suspected to be toxic or even carcinogenic. Trihalomethanes and haloacetic acid families are the most studied but others DBPs, like chloral hydrate, haloacetonitriles, N-nitrosodimethylamine and the bromate ion, are emerging compounds of interest. Epidemiological data about the risk of cancer are still controversial. However, numerous publications highlight a toxic risk especially the risk of allergy and respiratory symptoms for babies and elite swimmers. The few publications dedicated to risk assessment do not suggest increased risk, other than for elite swimmers. These publications are likely to underestimate the risk associated with DBPs because of the lack of data in the literature precludes the calculation of risk associated with certain compounds or certain pathways. Thus for regulations, the need to take into account the risks associated with disinfection by-products is now important without forgetting the need of the control of microbiological hazards in swimming pools.

What's in the pool? A comprehensive identification of disinfection by-products and assessment of mutagenicity of chlorinated and brominated swimming pool water.

BACKGROUND: Swimming pool disinfectants and disinfection by-products (DBPs) have been linked to human health effects, including asthma and bladder cancer, but no studies have provided a comprehensive identification of DBPs in the water and related that to mutagenicity. OBJECTIVES: We performed a comprehensive identification of DBPs and disinfectant species in waters from public swimming pools in Barcelona, Catalonia, Spain, that disinfect with either chlorine or bromine and we determined the mutagenicity of the waters to compare with the analytical results. METHODS: We used gas chromatography/mass spectrometry (GC/MS) to measure trihalomethanes in water, GC with electron capture detection for air, low- and high-resolution GC/MS to comprehensively identify DBPs, photometry to measure disinfectant species (free chlorine, monochloroamine, dichloramine, and trichloramine) in the waters, and an ion chromatography method to measure trichloramine in air. We assessed mutagenicity with the Salmonella mutagenicity assay. RESULTS: We identified > 100 DBPs, including many nitrogen-containing DBPs that were likely formed from nitrogen-containing precursors from human inputs, such as urine, sweat, and skin cells. Many DBPs were new and have not been reported previously in either swimming pool or drinking waters. Bromoform levels were greater in brominated than in chlorinated pool waters, but we also identified many brominated DBPs in the chlorinated waters. The pool waters were mutagenic at levels similar to that of drinking water (approximately 1,200 revertants/L-equivalents in strain TA100-S9 mix). CONCLUSIONS: This study identified many new DBPs not identified previously in swimming pool or drinking water and found that swimming pool waters are as mutagenic as typical drinking waters.

Ability of hypochlorous acid and N-chloramines to chlorinate DNA and its constituents.

Myeloperoxidase is a heme enzyme released by activated phagocytes that is responsible for the generation of the strong oxidant hypochlorous acid (HOCl). Although HOCl has potent bactericidal properties and plays an important role in the human immune system, this oxidant also causes damage to tissues, particularly under inflammatory conditions. There is a strong link between chronic inflammation and the incidence of many cancers, which may be associated with the ability of HOCl and related oxidants such as N-chloramines to damage DNA. However, in contrast to HOCl, little is known about the reactivity of N-chloramines with DNA and its constituents. In this study, we examine the ability of HOCl and various N-chloramines to form chlorinated base products on nucleosides, nucleotides, DNA, and in cellular systems. Experiments were performed with N-chloramines formed on Nalpha-acetyl-histidine (His-C), Nalpha-acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-C), and ammonia (Mono-C). Treatment of DNA and related materials with HOCl and His-C resulted in the formation of 5-chloro-2'-deoxycytidine (5CldC), 8-chloro-2'-deoxyadenosine (8CldA) and 8-chloro-2'-deoxyguanosine (8CldG). With the nucleosides, 8CldG was the favored product in each case, and HOCl was the most efficient chlorinating agent. 5Cl(d)C was the most abundant product on exposure of the nucleotides and DNA to HOCl and His-C, with only low levels of chlorinated products observed with Lys-C, Gly-C, Tau-C, and Mono-C. 5CldC was also formed on exposure of smooth muscle cells to either HOCl or His-C. Cellular RNA was also a target for HOCl and His-C, with evidence for the formation of 5-chloro-cytidine (5ClC). This study shows that HOCl and the model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may play a role in the development of various inflammatory cancers.

Monochloramine produces reactive oxygen species in liver by converting xanthine dehydrogenase into xanthine oxidase.

In the present study, we assessed the influence of monochloramine (NH(2)Cl) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, NH(2)Cl (2.5-20 microM) dose-dependently enhanced XO activity concomitant with a decrease in XD activity, implying that NH(2)Cl can convert XD into the reactive oxygen species (ROS) producing form XO. The NH(2)Cl (5 microM)-induced XD/XO interconversion in the rat liver cytosol was completely inhibited when added in combination with an inhibitor of NH(2)Cl methionine (25 microM). A sulfhydryl reducing agent, dithiothreitol at concentrations of 0.1, 1 and 5 mM also dose-dependently reversed the NH(2)Cl (5 microM)-induced XD/XO interconversion. These imply that NH(2)Cl itself acts on the XD/XO interconversion, and that this conversion occurs at the cysteine residues in XD. Furthermore, using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, it was found that NH(2)Cl could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an XO inhibitor, allopurinol. These results suggest that NH(2)Cl has the potential to convert XD into XO in the liver, which in turn may induce the ROS generation in this region.

Changes in blood lead levels associated with use of chloramines in water treatment systems.

BACKGROUND: More municipal water treatment plants are using chloramines as a disinfectant in order to reduce carcinogenic by-products. In some instances, this has coincided with an increase in lead levels in drinking water in those systems. Lead in drinking water can be a significant health risk. OBJECTIVES: We sought to test the potential effect of switching to chloramines for disinfection in water treatment systems on childhood blood lead levels using data from Wayne County, located in the central Coastal Plain of North Carolina. METHODS: We constructed a unified geographic information system (GIS) that links blood lead screening data with age of housing, drinking water source, and census data for 7,270 records. The data were analyzed using both exploratory methods and more formal multivariate techniques. RESULTS: The analysis indicates that the change to chloramine disinfection may lead to an increase in blood lead levels, the impact of which is progressively mitigated in newer housing. CONCLUSIONS: Introducing chloramines to reduce carcinogenic by-products may increase exposure to lead in drinking water. Our research provides guidance on adjustments in the local childhood lead poisoning prevention program that should accompany changes in water treatment. As similar research is conducted in other areas, and the underlying environmental chemistry is clarified, water treatment strategies can be optimized across the multiple objectives that municipalities face in providing high quality drinking water to local residents.

Protective effects of heat shock protein 70 induced by geranylgeranylacetone on oxidative injury in rat intestinal epithelial cells.

OBJECTIVE: Geranylgeranylacetone (GGA), an anti-ulcer agent, has recently been demonstrated to protect a variety of cells and tissues via induction of heat shock protein (HSP)70 against numerous stresses. We investigated whether GGA induces HSP70 and protects against an oxidative stressor, monocrolamine (NH(2)Cl), in a rat intestinal epithelial cell line (IEC-18). MATERIAL AND METHODS: IEC-18 cells pretreated with GGA (0.1-10 microM) were subjected to injury induced by NH(2)Cl. Cell viability was assessed, and endogenous HSP70 levels were determined by enzyme-linked immunosorbent assay in IEC-18 cells. RESULTS: Treatment with GGA (0.1-10 microM) was found rapidly to elevate HSP70 levels and to protect against NH(2)Cl-induced injury in IEC-18 cells. Furthermore, quercetin, an inhibitor of HSP70 synthesis, diminished the protective effects of GGA in IEC-18 cells upon NH(2)Cl-caused injury. CONCLUSIONS: The results of this study suggest that GGA plays an important role in defense mechanisms against oxidative injury in the intestine, primarily via induction of HSP70.

Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption.

Oxidants such as monochloramine (NH(2)Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional proteins. These effects can, in theory, disturb cell polarization and affect critical membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH(2)Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco-2 epithelial monolayers. NH(2)Cl causes a time-dependent decrease in both detergent-insoluble and -soluble zonula occludens (ZO)-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer cytochalasin D resulted in a decreased transepithelial resistance (TER) more quickly than NH(2)Cl but caused a more modest and slower reduction in ZO-1 levels and in occludin redistribution. No changes in the cellular distribution of claudin-1, claudin-5, or ZO-2 were observed after NH(2)Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH(2)Cl. The actin-stabilizing agent phalloidin did not prevent NH(2)Cl-induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol. However, it partially blocked changes in ZO-1 and occludin distribution. Tight junctional fence function was also compromised by NH(2)Cl, observed as a redistribution of the alpha-subunit of basolateral Na(+)-K(+)-ATPase to the apical membrane, an effect not found with the apical membrane protein Na(+)/H(+) exchanger isoform 3. In conclusion, oxidants not only disrupt perijunctional actin but also cause redistribution of tight junctional proteins, resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.

Occupational eosinophilic bronchitis without asthma due to chloramine exposure.

A case is discussed of eosinophilic bronchial inflammation without asthma due to chloramine T (CLT) exposure in a nurse. She reported a non-productive chronic cough on contact with CLT during workshifts. She had negative results of skin prick testing to CLT. However, sensitisation to CLT was confirmed by the presence of specific anti-chloramine IgE. Airway responsiveness to histamine was normal before and after CLT challenge. Eosinophil proportion in sputum was increased at 6 and 24 h after CLT challenge.

Monochloramine induces acute and protracted colitis in the rat: response to pharmacological treatment.

Monochloramine is a powerful oxidative molecule that is produced in inflammatory sites. We investigated the effect of intrarectally administered monochloramine (3.2 mg) in the rat. A single enema induced after 24 h an intense inflammatory reaction characterized by mucosal necrosis, submucosal edema, hemorrhage and colonic thickening, as well as induction of nitric oxide synthase and tumor necrosis factor and an increase in the interferon gamma/interleukin 4 ratio. The inflammatory response peaked 3-5 days after monochloramine administration and then followed a extended recovery phase. At 1 week there was substantial but incomplete mucosal repair, submucosal edema, neutrophil/macrophage infiltration and increased myeloperoxydase and alkaline phosphatase activities. Oxidative stress, as determined by malonyldialdehyde levels, was prominent only in the acute phase (3-5 days). Monochloramine colitis was amenable to pharmacological treatment with sulphasalazine or prednisolone, suggesting that it may be used as an experimental model of inflammatory bowel disease. In conclusion, monochloramine induces acute and protracted colonic inflammation in the rat. Locally produced monochloramine might contribute to the perpetuation of inflammatory bowel disease.

Bacterial superantigen-treated intestinal epithelial cells upregulate heat shock proteins 25 and 72 and are resistant to oxidant cytotoxicity.

While the pathological events evoked by infection are commonly described, effective host responses to bacteria and their products should primarily be protective. Heat shock protein (Hsp) expression is upregulated by many stimuli and serves to maintain intracellular protein integrity. The ability of the prototypic superantigen, Staphylococcus aureus enterotoxin B (SEB) to induce Hsps was investigated with BALB/c mice and by in vitro addition to the murine small intestinal epithelial cell line MSIE. SEB-treated (5 or 100 microg intraperitoneally) mice revealed increased Hsp25 and Hsp72, but not Hsc73, in jejunal lymphocytes and epithelial cells. A similar Hsp response to SEB occurred in MSIE cells and was preceded by activation of the ERK1/2 and p38 mitogen-activated protein kinases but not the SAPK/JNK pathway; pharmacological inhibition of ERK1/2, but not p38, significantly reduced SEB-induced Hsps. Moreover, SEB-treated MSIE cells were protected against oxidant-induced cytotoxicity (measured by 51Cr release) and F-actin depolymerization. Thus, SEB exposure results in a rapid induction of the Hsp25 and Hsp72 in intestinal epithelial cells, both directly and through lymphocyte activation, and we suggest that this event is important in protecting the gut from damage by Staphylococcus infection or in the reparatory process and may be a generalized response to lumen-derived bacterial toxins.

HOCl-mediated cell death and metabolic dysfunction in the yeast Saccharomyces cerevisiae.

The nature of oxidative damage to Saccharomyces cerevisiae caused by levels of HOCl that inhibit cell replication was explored with the intent of identifying the loci of lethal lesions. Functions of cytosolic enzymes and organelles that are highly sensitive to inactivation by HOCl, including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the mitochondrion, were only marginally affected by exposure of the yeast to levels of HOCl that completely inhibited colony formation. Loss of function in membrane-localized proteins, including the hexose transporters and PMA1 H(+)-ATPase, which is the primary proton pump located within the S. cerevisiae plasma membrane, was also marginal and K(+) leak rates to the extracellular medium increased only slowly with exposure to increasing amounts of HOCl, indicating that the plasma membrane retained its intrinsic impermeability to ions and metabolites. Adenylate phosphorylation levels in fermenting yeast declined in parallel with viability; however, yeast grown on respiratory substrates maintained near-normal phosphorylation levels at HOCl doses several-fold greater than that required for killing. This overall pattern of cellular response to HOCl differs markedly from that previously reported for bacteria, which appear to be killed by inhibition of plasma membrane proteins involved in energy transduction. The absence of significant loss of function in critical oxidant-sensitive cellular components and retention of ATP-synthesizing capabilities in respiring yeast cells exposed to lethal levels of HOCl suggests that toxicity in this case may arise by programmed cell death.