An Assessment of the Carcinogenic Potential of Trichloroethylene in Humans

Controversy surrounds the assessments of carcinogenic potential associated with human exposure to trichloroethylene (TCE). The American Conference of Governmental Industrial Hygienists states that TCE is “not suspected to be a human carcinogen.” In contrast, the International Agency for Research on Cancer has classified TCE as a probable human carcinogen, based primarily on the results of animal toxicity studies. Chronic high-dose TCE exposures cause hepatic and pulmonary tumors in mice and renal tumors in rats. Human epidemiology studies, however, do not support a causal association between exposure to TCE at environmentally relevant levels and cancers of the lung, liver, or kidney. The apparent discrepancy between the animal data and the human data can be explained by (1) differences in TCE exposure levels between laboratory animals and humans, (2) species-specific differences in TCE metabolism, and (3) other species-specific mechanisms involved in the development of cancer in rodents. This paper critically assesses the experimental and epidemiological data relevant to the carcinogenic potential of TCE. From the analysis, we conclude that TCE exposure at concentrations likely to be encountered in most environmental media is not likely to cause liver, lung, or kidney cancers in humans.     

Banding carcinogenic risks in developed countries: A procedural basis for qualitative assessment

Readily achieved comparative assessment of carcinogenic risks consequent upon environmental exposures may increase understanding and contribute to cancer prevention. Procedures for hazard identification and quantitative risk assessment are established, but limited when addressing novel exposures to previously known carcinogens or any exposure to agents having only suspected carcinogenic activity. To complement other means of data evaluation, a procedure for qualitative assessment of carcinogenic risk is described. This involves categorizing the relevant carcinogen and circumstances under which exposure occurs. The categories for carcinogens are those used for hazard identification and involve whether the agent is (1) a recognized carcinogen for humans; (2) probably or (3) possibly carcinogenic for humans; (4) characterized by inadequate evidence of carcinogenicity; or (5) lacking carcinogenicity. Exposure is categorized by whether it is one which (1) establishes the agent as a recognized carcinogen; (2) is taken into account in establishing carcinogenicity status; (3) is distinct from those providing clearest evidence of carcinogenicity; (4) is not characterized in relation to carcinogenicity; or (5) involves an exposure in which absence of carcinogenic outcome is observed. These two categories of evidence allow the risk inherent in a situation to be banded as indicative of a proven, likely, inferred, unknown or unlikely carcinogenic outcome, and further characterized using sub-bands. The procedure has been applied to about fifty situations. For recognized carcinogens, including asbestos and polycyclic aromatic hydrocarbons, risks consequent upon occupational exposure, the impact of point source pollution, residence near contaminated sites and general environmental exposure are allocated across the proven band and a likely sub-band. For solvents, pesticides and other compounds having less clearly established carcinogenicity, impact on residents living near a production site, or near earlier related industrial activity is allocated to certain inferred sub-bands. Unknown carcinogenic outcome, which identifies exposure to an agent with inadequate evidence of carcinogenicity rather than being indicative of equivocal or negative data in any context, indicates both the impact of certain pollutants and user-exposure to some consumer products. Situations allocated to the unlikely risk band principally involve certain consumer products. Overall, such risk assessment may be of greatest worth in focusing community attention on proven causes of cancer and associated preventive measures.     

Mechanisms of glutamate cysteine ligase (GCL) induction by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end-products of lipid peroxidation and is increased in response to cellular stress and in many chronic and/or inflammatory diseases. HNE can in turn function as a potent signaling molecule to induce the expression of many genes including glutamate cysteine ligase (GCL), the rate-limiting enzyme in de novo glutathione (GSH) biosynthesis. GSH, the most abundant nonprotein thiol in the cell, plays a key role in antioxidant defense. HNE exposure causes an initial depletion of GSH due to formation of conjugates with GSH, followed by a marked increase in GSH resulting from the induction of GCL. GCL is a heterodimeric protein with a catalytic (or heavy, GCLC) subunit and a modulatory (or light, GCLM) subunit. HNE-mediated induction of both GCL subunits and mRNAs has been reported in rat and human cells in vitro; however, the mechanisms or the signaling pathways mediating the induction of Gclc and Gclm mRNAs by HNE differ between rat and human cells. Activation of the ERK pathway is involved in GCL regulation in rat cells while both the ERK and the JNK pathways appear to be involved in human cells. Downstream, MAPK activation leads to increased AP-1 binding, which mediates GCL induction. Some studies suggest a role for the EpRE element as well. As the concentrations of HNE used in all of the studies reviewed are comparable to what may be found in vivo, this makes the findings summarized in this review potentially relevant to GCL regulation in human health and disease.     

Health Risk Assessment of Heavy Metals in Urban Soil of Karachi,Pakistan

The potential health risk due to lifetime exposure to copper, lead, chromium, zinc, and iron in urban soil of Karachi, Pakistan, was evaluated. Mean concentrations of Cu, Pb, Cr, Zn, and Fe in topsoil samples were 33.3 ± 12.8, 42.1 ± 55.8, 9.6 ± 4.2, 99.5 ± 37.3, and 908.4 ± 57.8 mg kg^?1, respectively. A U.S. Environmental Protection Agency model was adopted for the carcinogenic and non-carcinogenic risk assessment from different exposure pathways. Risk assessment indicated that the overall results for the carcinogenic risk were insignificant. However, the carcinogenic risk from Pb due to oral ingestion of soil exceeded the value of 1 × 10^?6, in some areas of the city. It indicates that the exposure to Pb-contaminated soil may cause adverse health effects in humans, especially in children. The Hazard Quotient (HQ) for different metals through ingestion and dermal pathways was also found to be less than 1. The combined Hazard Index (HI) for children through different routes of exposure was 8.9 times greater than for adults. It indicates that the children are more susceptible to non-carcinogenic health effects of trace metals compared to adults. Particularly, non-carcinogenic risk of Pb to children via oral ingestion needs special attention.     

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods.     

Safety testing of monoclonal antibodies in non-human primates: Case studies highlighting their impact on human risk assessment

Monoclonal antibodies (mAbs) are improving the quality of life for patients suffering from serious diseases due to their high specificity for their target and low potential for off-target toxicity. The toxicity of mAbs is primarily driven by their pharmacological activity, and therefore safety testing of these drugs prior to clinical testing is performed in species in which the mAb binds and engages the target to a similar extent to that anticipated in humans. For highly human-specific mAbs, this testing often requires the use of non-human primates (NHPs) as relevant species. It has been argued that the value of these NHP studies is limited because most of the adverse events can be predicted from the knowledge of the target, data from transgenic rodents or target-deficient humans, and other sources. However, many of the mAbs currently in development target novel pathways and may comprise novel scaffolds with multi-functional domains; hence, the pharmacological effects and potential safety risks are less predictable. Here, we present a total of 18 case studies, including some of these novel mAbs, with the aim of interrogating the value of NHP safety studies in human risk assessment. These studies have identified mAb candidate molecules and pharmacological pathways with severe safety risks, leading to candidate or target program termination, as well as highlighting that some pathways with theoretical safety concerns are amenable to safe modulation by mAbs. NHP studies have also informed the rational design of safer drug candidates suitable for human testing and informed human clinical trial design (route, dose and regimen, patient inclusion and exclusion criteria and safety monitoring), further protecting the safety of clinical trial participants.     

A systematic review of cytogenetic studies conducted in human populations exposed to cadmium compounds

BACKGROUND: Exposure to cadmium fumes or dusts has been associated with an increased risk of lung cancer and the characterisation of the genotoxic potential of cadmium compounds is, among other possible mechanisms, an important element in the assessment of the carcinogenic hazard of the element. While there is some evidence that in experimental systems, cadmium compounds may exert genotoxic effects, the results of the epidemiological studies having examined cytogenetic endpoints in humans exposed to cadmium appear conflicting. Therefore, a systematic review was undertaken to assess whether a cytogenetic effect of cadmium exposure is supported by the studies with the strongest design. METHODS: The relevant literature was identified through several databases and assessed with a check-list by two reviewers. Causes of heterogeneity between studies were looked for. Results were extracted and the strength of the evidence was evaluated with causality criteria. RESULTS: No studies met the criteria for being considered as very convincing. Several factors were identified that could explain contradictory findings (small sample size, selection bias, insufficient characterisation of exposure, lack of consideration of confounders) but their actual impact could not be conclusively assessed with the published information. Importantly, it should be recognised that the absence of a clear mechanism for the cytogenetic action of cadmium compounds did not allow to select the most appropriate endpoint to be examined. CONCLUSIONS: No clear association between cadmium exposure and cytogenetic endpoint appeared but no definite conclusion can be drawn from the existing studies in humans. Future research efforts should mainly focus on experimental studies to understand how cadmium compounds could produce genotoxic/carcinogenic effects, in order to target the most relevant endpoint to be examined in humans.     

Synergy between systemic toxicity and genotoxicity: relevance to human cancer risk

The health risk manager and policy analyst must frequently make recommendations based upon incomplete toxicity data. This is a situation which is encountered in the evaluation of human carcinogenic risks as animal cancer bioassay results are often not available. In this study, in order to assess the relevance of other possible indicators of carcinogenic risks, we used the "chemical diversity approach" to estimate the magnitude of the human carcinogenic risk based upon Salmonella mutagenicity and systemic toxicity data of the "universe of chemicals" to which humans have the potential to be exposed. Analyses of the properties of 10,000 agents representative of the "universe of chemicals" suggest that chemicals that have genotoxic potentials as well as exhibiting greater systemic toxicity are more likely to be carcinogens than non-genotoxicants or agents that exhibit lesser toxicity. Since "genotoxic" carcinogenicity is a hallmark of recognized human carcinogens, these findings are relevant to human cancer risk assessment.     

Mitochondrial glutathione transport: physiological, pathological and toxicological implications

Although most cellular glutathione (GSH) is in the cytoplasm, a distinctly regulated pool is present in mitochondria. Inasmuch as GSH synthesis is primarily restricted to the cytoplasm, the mitochondrial pool must derive from transport of cytoplasmic GSH across the mitochondrial inner membrane. Early studies in liver mitochondria primarily focused on the relationship between GSH status and membrane permeability and energetics. Because GSH is an anion at physiological pH, this suggested that some of the organic anion carriers present in the inner membrane could function in GSH transport. Indeed, studies by Lash and colleagues in isolated mitochondria from rat kidney showed that most of the transport (>80%) in that tissue could be accounted for by function of the dicarboxylate carrier (DIC, Slc25a10) and the oxoglutarate carrier (OGC, Slc25a11), which mediate electroneutral exchange of dicarboxylates for inorganic phosphate and 2-oxoglutarate for other dicarboxylates, respectively. The identity and function of specific carrier proteins in other tissues is less certain, although the OGC is expressed in heart, liver, and brain and the DIC is expressed in liver and kidney. An additional carrier that transports 2-oxoglutarate, the oxodicarboxylate or oxoadipate carrier (ODC; Slc25a21), has been described in rat and human liver and its expression has a wide tissue distribution, although its potential function in GSH transport has not been investigated. Overexpression of the cDNA for the DIC and OGC in a renal proximal tubule-derived cell line, NRK-52E cells, showed that enhanced carrier expression and activity protects against oxidative stress and chemically induced apoptosis. This has implications for development of novel therapeutic approaches for treatment of human diseases and pathological states. Several conditions, such as alcoholic liver disease, cirrhosis or other chronic biliary obstructive diseases, and diabetic nephropathy, are associated with depletion or oxidation of the mitochondrial GSH pool in liver or kidney.     

Incorporation of biological information in cancer risk assessment: Example — Vinyl chloride

Vinyl chloride (VC) is used as an example to demonstrate how biological information can be incorporated into quantitative risk assessment. The information included is the pharmacokinetics of VC in animals and humans and the data-generated hypothesis that VC primarily affects the initiation stage of the multistage carcinogenesis. The emphasis in this paper is on the improvement of risk assessment methodology rather than the risk assessment of VC per se.Sufficient data are available to construct physiologically-based pharmacokinetic models for both animals and humans. These models are used to calculate the metabolized dose corresponding to exposure scenarios in animals and in humans.On the basis of the data on liver angiosarcomas and carcinomas in rats, the cancer risk per unit of metabolized dose is comparable, irrespective of routes (oral or inhalation) of exposure. The tumor response from an intermittent/partial lifetime exposure is shown to be consistent with that from a lifetime exposure when VC is assumed to affect the first (initiation) stage of the multistage carcinogenic process. Furthermore, the risk estimates calculated on the basis of animal data are shown to be consistent with the human experience.     

Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione

The tripeptide GSH is important in maintenance of renal redox status and defense against reactive electrophiles and oxidants. Previous studies showed that GSH is transported across the basolateral plasma membrane (BLM) into the renal proximal tubule by both sodium-coupled and sodium-independent pathways. Substrate specificity and inhibitor studies suggested the function of several carriers, including organic anion transporter 3 (Oat3). To test the hypothesis that rat Oat3 can function in renal GSH transport, the cDNA for rat Oat3 was expressed as a His6-tagged protein in E. coli, purified from inclusion bodies and by Ni2+-affinity chromatography, and reconstituted into proteoliposomes. cDNA-expressed and reconstituted Oat3 transported both GSH and p-aminohippurate (PAH) in exchange for 2-oxoglutarate (2-OG) and 2-OG and PAH in exchange for GSH, and PAH uptake was inhibited by both probenecid and furosemide, consistent with function of Oat3. mRNA expression of Oat3 and several other potential carriers was detected by RT-PCR in rat kidney cortex but was absent from NRK-52E cells, a rat proximal tubular cell line. Basolateral uptake of GSH in NRK-52E cells showed little PAH- or 2-OG-stimulated uptake. We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers.     

Trapping and identification of the dichloroacetate radical from the reductive dehalogenation of trichloroacetate by mouse and rat liver microsomes

A key question in the risk assessment of trichloroethylene (TRI) is the extent to which its carcinogenic effects might depend on the formation of dichloroacetate (DCA) as a metabolite. One of the metabolic pathways proposed for the formation of DCA from TRI is by the reductive dehalogenation of trichloroacetate (TCA), via a free radical intermediate. Although proof of this radical has been elusive, the detection of fully dechlorinated metabolites in the urine and the formation of lipid peroxidation by-products in microsomal incubations with TCA argue for its existence. We report here the trapping of the dichloroacetate radical with the spin-trapping agent PBN, and its identification by GC/MS. The PBN/dichloroacetate radical adduct was found to undergo an intramolecular rearrangement during its extraction into organic solvent. An internal condensation reaction between the acetate and the nitroxide radical moieties is hypothesized to form a cyclic adduct with the elimination of an OH radical. The PBN/dichloroacetate radical adduct has been identified by GC/MS in both a chemical Fenton system and in rodent microsomal incubations with TCA as substrate.     

Cadmium carcinogenesis

Cadmium is a heavy metal of considerable environmental and occupational concern. Cadmium compounds are classified as human carcinogens by several regulatory agencies. The most convincing data that cadmium is carcinogenic in humans comes from studies indicating occupational cadmium exposure is associated with lung cancer. Cadmium exposure has also been linked to human prostate and renal cancer, although this linkage is weaker than for lung cancer. Other target sites of cadmium carcinogenesis in humans, such as liver, pancreas and stomach, are considered equivocal. In animals, cadmium effectively induces cancers at multiple sites and by various routes. Cadmium inhalation in rats induces pulmonary adenocarcinomas, in accord with its role in human lung cancer. Cadmium can induce tumors and/or preneoplastic lesions within the rat prostate after ingestion or injection. At relatively high doses, cadmium induces benign testicular tumors in rats, but these appear to be due to early toxic lesions and loss of testicular function, rather than from a specific carcinogenic effect of cadmium. Like many other metals, cadmium salts will induce mesenchymal tumors at the site of subcutaneous (s.c.) or intramuscular (i.m.) injections, but the human relevance of these is dubious. Other targets of cadmium in rodents include the liver, adrenal, pancreas, pituitary, and hematopoietic system. With the exception of testicular tumors in rodents, the mechanisms of cadmium carcinogenesis are poorly defined. Cadmium can cause any number of molecular lesions that would be relevant to oncogenesis in various cellular model systems. Most studies indicate cadmium is poorly mutagenic and probably acts through indirect or epigenetic mechanisms, potentially including aberrant activation of oncogenes and suppression of apoptosis.     

Heavy metal poisoning: the effects of cadmium on the kidney

The heavy metal cadmium (Cd) is known to be a widespread environmental contaminant and a potential toxin that may adversely affect human health. Exposure is largely via the respiratory or gastrointestinal tracts; important non-industrial sources of exposure are cigarette smoke and food (from contaminated soil and water). The kidney is the main organ affected by chronic Cd exposure and toxicity. Cd accumulates in the kidney as a result of its preferential uptake by receptor-mediated endocytosis of freely filtered and metallothionein bound Cd (Cd-MT) in the renal proximal tubule. Internalised Cd-MT is degraded in endosomes and lysosomes, releasing free Cd²⁺ into the cytosol, where it can generate reactive oxygen species (ROS) and activate cell death pathways. An early and sensitive manifestation of chronic Cd renal toxicity, which can be useful in individual and population screening, is impaired reabsorption of low molecular weight proteins (LMWP) (also a receptor-mediated process in the proximal tubule) such as retinol binding protein (RBP). This so-called ‘tubular proteinuria’ is a good index of proximal tubular damage, but it is not usually detected by routine clinical dipstick testing for proteinuria. Continued and heavy Cd exposure can progress to the clinical renal Fanconi syndrome, and ultimately to renal failure. Environmental Cd exposure may be a significant contributory factor to the development of chronic kidney disease, especially in the presence of other co-morbidities such as diabetes or hypertension; therefore, the sources and environmental impact of Cd, and efforts to limit Cd exposure, justify more attention.     

Evaluation and Spatial Diffusion of Health Risk of Persistent Organic Pollutants (POPs) in Soils Surrounding Chemical Industrial Parks in China

A case study of the cancer risk to humans posed by persistent organic pollutants (POPs) in an industrial area of China, which has a long history of contamination from many sources, is presented. Relatively great concentrations of POPs around the chemical industrial parks have the potential to be chronically carcinogenic to local people. Sixteen individual PAHs listed for priory control by the U.S. Environmental Protection Agency (USEPA), metabolites of DDTs, and isomers of HCHs were measured in soils and a human health risk assessment was conducted by use of USEPA exposure models for children and adults, respectively. Geostatistical methods were used to simulate the spatial diffusion of potential carcinogenic risk, and non-parametric Mann-Whitney U and Kruskal-Wallis tests were employed to analyze the impact of point sources on the surrounding area. The mean value of the sum of Excess Lifetime Cancer Risk (∑ELCR) exceeded the generally acceptable risk level of 1.0E-06 recommended by the USEPA for carcinogenic chemicals. The maximum ∑ELCR was 2.9E-04 for children, which was observed inside the chemical industrial parks. Contamination at the chemical industrial parks caused significant spatial diffusion of ELCR values caused by PAHs, DDT, and HCH.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.