GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide.

While redox processes play a vital role in maintaining intracellular homeostasis by regulating critical signaling and metabolic pathways, supra-physiological or sustained oxidative stress can lead to adverse responses or cytotoxicity. Inhalation of ambient air pollutants such as particulate matter and secondary organic aerosols (SOA) induces oxidative stress in the respiratory tract through mechanisms that remain poorly understood. We investigated the effect of isoprene hydroxy hydroperoxide (ISOPOOH), an atmospheric oxidation product of vegetation-derived isoprene and a constituent of SOA, on intracellular redox homeostasis in cultured human airway epithelial cells (HAEC). We used high-resolution live cell imaging of HAEC expressing the genetically encoded ratiometric biosensors Grx1-roGFP2, iNAP1, or HyPer, to assess changes in the cytoplasmic ratio of oxidized glutathione to reduced glutathione (GSSG:GSH), and the flux of NADPH and H2O2, respectively. Non-cytotoxic exposure to ISOPOOH resulted in a dose-dependent increase of GSSG:GSH in HAEC that was markedly potentiated by prior glucose deprivation. ISOPOOH-induced increase in glutathione oxidation were accompanied by concomitant decreases in intracellular NADPH. Following ISOPOOH exposure, the introduction of glucose resulted in a rapid restoration of GSH and NADPH, while the glucose analog 2-deoxyglucose resulted in inefficient restoration of baseline GSH and NADPH. To elucidate bioenergetic adaptations involved in combatting ISOPOOH-induced oxidative stress we investigated the regulatory role of glucose-6-phosphate dehydrogenase (G6PD). A knockout of G6PD markedly impaired glucose-mediated recovery of GSSG:GSH but not NADPH. These findings reveal rapid redox adaptations involved in the cellular response to ISOPOOH and provide a live view of the dynamic regulation of redox homeostasis in human airway cells as they are exposed to environmental oxidants.

Live cell imaging of oxidative stress in human airway epithelial cells exposed to isoprene hydroxyhydroperoxide.

Exposure to respirable air particulate matter (PM2.5) in ambient air is associated with morbidity and premature deaths. A major source of PM2.5 is the photooxidation of volatile plant-produced organic compounds such as isoprene. Photochemical oxidation of isoprene leads to the formation of hydroperoxides, environmental oxidants that lead to inflammatory (IL-8) and adaptive (HMOX1) gene expression in human airway epithelial cells (HAEC). To examine the mechanism through which these oxidants alter intracellular redox balance, we used live-cell imaging to monitor the effects of isoprene hydroxyhydroperoxides (ISOPOOH) in HAEC expressing roGFP2, a sensor of the glutathione redox potential (EGSH). Non-cytotoxic exposure of HAEC to ISOPOOH resulted in a rapid and robust increase in EGSH that was independent of the generation of intracellular or extracellular hydrogen peroxide. Our results point to oxidation of GSH through the redox relay initiated by glutathione peroxidase 4, directly by ISOPOOH or indirectly by ISOPOOH-generated lipid hydroperoxides. We did not find evidence for involvement of peroxiredoxin 6. Supplementation of HAEC with polyunsaturated fatty acids enhanced ISOPOOH-induced glutathione oxidation, providing additional evidence that ISOPOOH initiates lipid peroxidation of cellular membranes. These findings demonstrate that ISOPOOH is a potent environmental airborne hydroperoxide with the potential to contribute to oxidative burden of human airway posed by inhalation of secondary organic aerosols.

An unexpected butadiene diolepoxide-mediated genotoxicity implies alternative mechanism for 1,3-butadiene carcinogenicity.

1,3-Butadiene (BD) is abundant in combustion products such as cigarette smoke. While BD has been classified as a known human carcinogen, a long-standing question is the identity of the ultimate carcinogenic metabolite in humans. We hypothesize that 3,4-epoxybutane-1,2-diol (EBD) may play a critical role in human carcinogenesis due to its high bioavailability. We utilized a differential toxicity assay for BD metabolites and newly synthesized EBD analogs in a series of isogenic chicken cells lacking specific DNA repair proteins to address the mode of action of BD genotoxicity and infer a mode of action. Surprisingly, as with the diepoxide 1,2:3,4-diepoxybutane (DEB), the monoepoxide EBD showed remarkable toxicity to cells deficient in Fanconi anemia (FANC) genes. This observation suggests that EBD may be transformed into a bifunctional metabolite and forms interstrand cross-links. EBD and its analog with a hydroxy substituent at C1 were found to be highly toxic to FANCD2-deficient chicken and human cells. The Results suggest that EBD may be transformed to a bifunctional epoxy aldehyde, perhaps by alcohol dehydrogenase, to which the observed FANC sensitivity could be attributed. The implications of this study are very important in considering mechanisms by which EBD may cause leukemia and lymphoma in humans exposed to BD.

Supplementation with omega-3 fatty acids potentiates oxidative stress in human airway epithelial cells exposed to ozone.

BACKGROUND: Dietary intake of the omega-3 family of polyunsaturated fatty acids (ω-3 FA) is associated with anti-inflammatory effects. However, unsaturated fatty acids are susceptible to oxidation, which produces pro-inflammatory mediators. Ozone (O3) is a tropospheric pollutant that reacts rapidly with unsaturated fatty acids to produce electrophilic and oxidative mediators of inflammation. OBJECTIVE: Determine whether supplementation with ω-3 FA alters O3-induced oxidative stress in human airway epithelial cells (HAEC). METHODS: 16-HBE cells expressing a genetically encoded sensor of the reduced to oxidized glutathione ratio (GSH/GSSG, EGSH) were supplemented with saturated, monounsaturated, or ω-3 FA prior to exposure to 0, 0.08, 0.1, or 0.3 ppm O3. Lipid peroxidation was measured in cellular lipid extracts and intact cells following O3 exposure. RESULTS: Relative to cells incubated with the saturated or monounsaturated fatty acids, cells supplemented with ω-3 FA containing 5 or 6 double bonds showed a marked increase in EGSH during exposure to O3 concentrations as low as 0.08 ppm. Consistent with this finding, the concentration of lipid hydroperoxides produced following O3 exposure was significantly elevated in ω-3 FA supplemented cells. DISCUSSION: Supplementation with polyunsaturated ω-3 FA potentiates oxidative responses, as indicated by EGSH, in HAEC exposed to environmentally relevant concentrations of O3. This effect is mediated by the increased formation of lipid hydroperoxides produced by the reaction of O3 with polyunsaturated fatty acids. Given the inflammatory activity of lipid hydroperoxides, these findings have implications for the potential role of ω-3 FA in increasing human susceptibility to the adverse health effects of O3 exposure.

Isoprene-Derived Secondary Organic Aerosol Induces the Expression of MicroRNAs Associated with Inflammatory/Oxidative Stress Response in Lung Cells.

Exposure to fine particulate matter (PM2.5), of which secondary organic aerosol (SOA) is a major constituent, is linked to adverse health outcomes, including cardiovascular disease, lung cancer, and preterm birth. Atmospheric oxidation of isoprene, the most abundant nonmethane hydrocarbon emitted into Earth's atmosphere primarily from vegetation, contributes to SOA formation. Isoprene-derived SOA has previously been found to alter inflammatory/oxidative stress genes. MicroRNAs (miRNAs) are epigenetic regulators that serve as post-transcriptional modifiers and key mediators of gene expression. To assess whether isoprene-derived SOA alters miRNA expression, BEAS-2B lung cells were exposed to laboratory-generated isoprene-derived SOA constituents derived from the acid-driven multiphase chemistry of authentic methacrylic acid epoxide (MAE) or isomeric isoprene epoxydiols (IEPOX) with acidic sulfate aerosol particles. These IEPOX- and MAE-derived SOA constituents have been shown to be measured in large quantities within PM2.5 collected from isoprene-rich areas affected by acidic sulfate aerosol particles derived from human activities. A total of 29 miRNAs were identified as differentially expressed when exposed to IEPOX-derived SOA and 2 when exposed to MAE-derived SOA, a number of which are inflammatory/oxidative stress associated. These results suggest that miRNAs may modulate the inflammatory/oxidative stress response to SOA exposure, thereby advancing the understanding of airway cell epigenetic response to SOA.

Long chain lipid hydroperoxides increase the glutathione redox potential through glutathione peroxidase 4.

BACKGROUND: Peroxidation of PUFAs by a variety of endogenous and xenobiotic electrophiles is a recognized pathophysiological process that can lead to adverse health effects. Although secondary products generated from peroxidized PUFAs have been relatively well studied, the role of primary lipid hydroperoxides in mediating early intracellular oxidative events is not well understood. METHODS: Live cell imaging was used to monitor changes in glutathione (GSH) oxidation in HAEC expressing the fluorogenic sensor roGFP during exposure to 9-hydroperoxy-10E,12Z-octadecadienoic acid (9-HpODE), a biologically important long chain lipid hydroperoxide, and its secondary product 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE). The role of hydrogen peroxide (H2O2) was examined by direct measurement and through catalase interventions. shRNA-mediated knockdown of glutathione peroxidase 4 (GPx4) was utilized to determine its involvement in the relay through which 9-HpODE initiates the oxidation of GSH. RESULTS: Exposure to 9-HpODE caused a dose-dependent increase in GSH oxidation in HAEC that was independent of intracellular or extracellular H2O2 production and was exacerbated by NADPH depletion. GPx4 was involved in the initiation of GSH oxidation in HAEC by 9-HpODE, but not that induced by exposure to H2O2 or the low molecular weight alkyl tert-butyl hydroperoxide (TBH). CONCLUSIONS: Long chain lipid hydroperoxides can directly alter cytosolic EGSH independent of secondary lipid oxidation products or H2O2 production. NADPH has a protective role against 9-HpODE induced EGSH changes. GPx4 is involved specifically in the reduction of long-chain lipid hydroperoxides, leading to GSH oxidation. SIGNIFICANCE: These results reveal a previously unrecognized consequence of lipid peroxidation, which may provide insight into disease states involving lipid peroxidation in their pathogenesis.

Effect of secondary organic aerosol from isoprene-derived hydroxyhydroperoxides on the expression of oxidative stress response genes in human bronchial epithelial cells.

Isoprene-derived secondary organic aerosol (SOA), which comprise a large portion of atmospheric fine particulate matter (PM2.5), can be formed through various gaseous precursors, including isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene hydroxyhydroperoxides (ISOPOOH). The composition of the isoprene-derived SOA affects its reactive oxygen species (ROS) generation potential and its ability to alter oxidative stress-related gene expression. In this study we assess effects of isoprene SOA derived solely from ISOPOOH oxidation on human bronchial epithelial cells by measuring the gene expression changes in 84 oxidative stress-related genes. In addition, the thiol reactivity of ISOPOOH-derived SOA was measured through the dithiothreitol (DTT) assay. Our findings show that ISOPOOH-derived SOA alter more oxidative-stress related genes than IEPOX-derived SOA but not as many as MAE-derived SOA on a mass basis exposure. More importantly, we found that the different types of SOA derived from the various gaseous precursors (MAE, IEPOX, and ISOPOOH) have unique contributions to changes in oxidative stress-related genes that do not total all gene expression changes seen in exposures to atmospherically relevant compositions of total isoprene-derived SOA mixtures. This study suggests that amongst the different types of known isoprene-derived SOA, MAE-derived SOA are the most potent inducer of oxidative stress-related gene changes but highlights the importance of considering isoprene-derived SOA as a total mixture for pollution controls and exposure studies.

Evidence that endogenous formaldehyde produces immunogenic and atherogenic adduct epitopes.

Endogenous formaldehyde is abundantly present in our bodies, at around 100 µM under normal conditions. While such high steady state levels of formaldehyde may be derived by enzymatic reactions including oxidative demethylation/deamination and myeloperoxidation, it is unclear whether endogenous formaldehyde can initiate and/or promote diseases in humans. Here, we show that fluorescent malondialdehyde-formaldehyde (M2FA)-lysine adducts are immunogenic without adjuvants in mice. Natural antibody titers against M2FA are elevated in atherosclerosis-prone mice. Staining with an antibody against M2FA demonstrated that M2FA is present in plaque found on the aortic valve of ApoE -/- mice. To mimic inflammation during atherogenesis, human myeloperoxidase was incubated with glycine, H2O2, malondialdehyde, and a lysine analog in PBS at a physiological temperature, which resulted in M2FA generation. These results strongly suggest that the 1,4-dihydropyridine-type of lysine adducts observed in atherosclerosis lesions are likely produced by endogenous formaldehyde and malondialdehyde with lysine. These highly fluorescent M2FA adducts may play important roles in human inflammatory and degenerative diseases.

Gene Expression Profiling in Human Lung Cells Exposed to Isoprene-Derived Secondary Organic Aerosol.

Secondary organic aerosol (SOA) derived from the photochemical oxidation of isoprene contributes a substantial mass fraction to atmospheric fine particulate matter (PM2.5). The formation of isoprene SOA is influenced largely by anthropogenic emissions through multiphase chemistry of its multigenerational oxidation products. Considering the abundance of isoprene SOA in the troposphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. In this study, we assessed the effects of isoprene SOA on gene expression in human airway epithelial cells (BEAS-2B) through an air-liquid interface exposure. Gene expression profiling of 84 oxidative stress and 249 inflammation-associated human genes was performed. Our results show that the expression levels of 29 genes were significantly altered upon isoprene SOA exposure under noncytotoxic conditions (p < 0.05), with the majority (22/29) of genes passing a false discovery rate threshold of 0.3. The most significantly affected genes belong to the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor network. The Nrf2 function is confirmed through a reporter cell line. Together with detailed characterization of SOA constituents, this study reveals the impact of isoprene SOA exposure on lung responses and highlights the importance of further understanding its potential health outcomes.

Editor's Highlight: Collaborative Cross Mouse Population Enables Refinements to Characterization of the Variability in Toxicokinetics of Trichloroethylene and Provides Genetic Evidence for the Role of PPAR Pathway in Its Oxidative Metabolism.

Background: Trichloroethylene (TCE) is a known carcinogen in humans and rodents. Previous studies of inter-strain variability in TCE metabolism were conducted in multi-strain panels of classical inbred mice with limited genetic diversity to identify gene-environment interactions associated with chemical exposure. Objectives: To evaluate inter-strain variability in TCE metabolism and identify genetic determinants that are associated with TCE metabolism and effects using Collaborative Cross (CC), a large panel of genetically diverse strains of mice. Methods: We administered a single oral dose of 0, 24, 80, 240, or 800 mg/kg of TCE to mice from 50 CC strains, and collected organs 24 h post-dosing. Levels of trichloroacetic acid (TCA), a major oxidative metabolite of TCE were measured in multiple tissues. Protein expression and activity levels of TCE-metabolizing enzymes were evaluated in the liver. Liver transcript levels of known genes perturbed by TCE exposure were also quantified. Genetic association mapping was performed on the acquired phenotypes. Results: TCA levels varied in a dose- and strain-dependent manner in liver, kidney, and serum. The variability in TCA levels among strains did not correlate with expression or activity of a number of enzymes known to be involved in TCE oxidation. Peroxisome proliferator-activated receptor alpha (PPARα)-responsive genes were found to be associated with strain-specific differences in TCE metabolism. Conclusions: This study shows that CC mouse population is a valuable tool to quantitatively evaluate inter-individual variability in chemical metabolism and to identify genes and pathways that may underpin population differences.

DNA Product Formation in Female Sprague-Dawley Rats Following Polyhalogenated Aromatic Hydrocarbon (PHAH) Exposure.

DNA oxidation damage has been regarded as one of the possible mechanisms for the hepatic carcinogenesis of dioxin-like compounds (DLCs). In this study, we evaluated the toxic equivalency factor (TEF) from the standpoint of induced DNA oxidation products and their relationship to toxicity and carcinogenicity. Nine DNA oxidation products were analyzed in the liver of female Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) alone or the tertiary mixture of TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) by gavage for 14, 31, and 53 weeks (5 days/week) by LC-MS/MS: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo); 1,N6-etheno-2'-deoxyadenosine (1,N6-εdAdo); N2,3-ethenoguanine (N2,3-εG); 7-(2-oxoethly)guanine (7-OEG); 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo); malondialdehyde (M1dGuo); acrolein (AcrdGuo); crotonaldehyde (CrdGuo); and 4-hydroxynonenal (HNEdGuo) derived 2'-deoxyguanosine adducts. Exposure to TCDD (100 ng/kg/day) significantly induced 1,N6-εdAdo at 31 and 53 weeks, while no increase of 8-oxo-dGuo was observed. Significant increases were observed for 8-oxo-dGuo and 1,N6-εdAdo at all time points following exposure to the tertiary mixture (TEQ 100 ng/kg/day). Exposure to TCDD for 53 weeks only significantly increased 1,N6-εdAdo, while increases of N2,3-εG and 7-OEG were only found in the highest dose group (100 ng/kg/day). Exposure to the tertiary mixture for 53 weeks had no effect on N2,3-εG in any exposure group (TEQ 0, 22, 46, or 100 ng/kg/day), while significant increases were observed for 1,N6-εdAdo (all dose groups), 8-oxo-dGuo (46 and 100 ng/kg/day), and 7-OEG (100 ng/kg/day). While no significant increase was observed at 53 weeks for 1,N2-εdGuo, M1dGuo, AcrdGuo, or CrdGuo following exposure to TCDD (100 ng/kg/day), all of them were significantly induced in animals exposed to the tertiary mixture (TEQ 100 ng/kg/day). This oxidation DNA product data suggest that the simple TEF methodology cannot be applied to evaluate the diverse patterns of toxic effects induced by DLCs.

Polychlorinated Biphenyls Induce Oxidative DNA Adducts in Female Sprague-Dawley Rats.

Polychlorinated biphenyls (PCBs) are organic chemicals that were traditionally produced and widely used in industry as mixtures and are presently formed as byproducts of pigment and dye manufacturing. They are known to persist and bioaccumulate in the environment. Some have been shown to induce liver cancer in rodents. Although the mechanism of the toxicity of PCBs is unknown, it has been shown that they increase oxidative stress, including lipid peroxidation. We hypothesized that oxidative stress-induced DNA damage could be a contributor for PCB carcinogenesis and analyzed several DNA adducts in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and a binary mixture (PCB 126 + 153) for 14, 31, and 53 wks. Eight adducts were measured to profile oxidative DNA lesions, including 8-oxo-deoxyguanosine (8-oxo-dG), 1,N(6)-ethenodeoxyadenosine (1,N(6)-εdA), N(2),3-ethenoguanine (N(2),3-εG), 1,N(2)-ethenodeoxyguanosine (1,N(2)-εdG), as well as malondialdehyde (M1dG), acrolein (AcrdG), crotonaldehyde (CrdG), and 4-hydroxynonenal-derived dG adducts (HNEdG) by LC-MS/MS analysis. Statistically significant increases were observed for 8-oxo-dG and 1,N(6)-εdA concentrations in hepatic DNA of female rats exposed to the binary mixture (1000 ng/kg/day + 1000 µg/kg/day) but not in rats exposed to PCB 126 (1000 ng/kg/day) or PCB 153 (1000 µg/kg/day) for 14 and 31 wks. However, exposure to PCB 126 (1000 ng/kg/day) for 53 wks significantly increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, and M1dG. Exposure to PCB 153 (1000 µg/kg/day) for 53 wks increased 8-oxo-dG, and 1,N(6)-εdA. Exposure to the binary mixture for 53 wks increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, 1,N(2)-εdG, and N(2),3-εG significantly above control groups. Increased hepatic oxidative DNA adducts following exposure to PCB 126, PCB 153, or the binary mixture shows that an increase in DNA damage may play an important role in hepatic toxicity and carcinogenesis in female Sprague-Dawley rats.

Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress.

Oxidative stress is a commonly cited mechanism of toxicity of environmental agents. Ubiquitous environmental chemicals such as the diesel exhaust component 1,2-naphthoquinone (1,2-NQ) induce oxidative stress by redox cycling, which generates hydrogen peroxide (H2O2). Cysteinyl thiolate residues on regulatory proteins are subjected to oxidative modification by H2O2 in physiological contexts and are also toxicological targets of oxidant stress induced by environmental contaminants. We investigated whether exposure to environmentally relevant concentrations of 1,2-NQ can induce H2O2-dependent oxidation of cysteinyl thiols in regulatory proteins as a readout of oxidant stress in human airway epithelial cells. BEAS-2B cells were exposed to 0-1000 µM 1,2-NQ for 0-30 min, and levels of H2O2 were measured by ratiometric spectrofluorometry of HyPer. H2O2-dependent protein sulfenylation was measured using immunohistochemistry, immunoblotting, and isotopic mass spectrometry. Catalase overexpression was used to investigate the relationship between H2O2 generation and protein sulfenylation in cells exposed to 1,2-NQ. Multiple experimental approaches showed that exposure to 1,2-NQ at concentrations as low as 3 µM induces H2O2-dependent protein sulfenylation in BEAS-2B cells. Moreover, the time of onset and duration of 1,2-NQ-induced sulfenylation of the regulatory proteins GAPDH and PTP1B showed significant differences. Oxidative modification of regulatory cysteinyl thiols in human lung cells exposed to relevant concentrations of an ambient air contaminant represents a novel marker of oxidative environmental stress.

Evidence for an unrecognized secondary anthropogenic source of organosulfates and sulfonates: gas-phase oxidation of polycyclic aromatic hydrocarbons in the presence of sulfate aerosol.

In the present study, formation of aromatic organosulfates (OSs) from the photo-oxidation of polycyclic aromatic hydrocarbons (PAHs) was investigated. Naphthalene (NAP) and 2-methylnaphthalene (2-MeNAP), two of the most abundant gas-phase PAHs and thought to represent "missing" sources of urban SOA, were photochemically oxidized in an outdoor smog chamber facility in the presence of nonacidified and acidified sulfate seed aerosol. Effects of seed aerosol composition, acidity and relative humidity on OS formation were examined. Chemical characterization of SOA extracts by ultra performance liquid chromatography coupled to electrospray ionization high-resolution quadrupole time-of-flight mass spectrometry revealed the formation of OSs and sulfonates from photo-oxidation in the presence of sulfate seed aerosol. Many of the organosulfur compounds identified in the smog chamber extracts were also measured in urban fine aerosol collected at Lahore, Pakistan, and Pasadena, USA, demonstrating that PAH photo-oxidation in the presence of sulfate aerosol is a hitherto unrecognized source of anthropogenic secondary organosulfur compounds, and providing new PAH SOA tracers.

Comparative analysis of the relationship between trichloroethylene metabolism and tissue-specific toxicity among inbred mouse strains: liver effects.

Trichloroethylene (TCE) is a widely used organic solvent. Although TCE is classified as carcinogenic to humans, substantial gaps remain in our understanding of interindividual variability in TCE metabolism and toxicity, especially in the liver. A hypothesis was tested that amounts of oxidative metabolites of TCE in mouse liver are associated with hepatic-specific toxicity. Oral dosing with TCE was conducted in subacute (600 mg/kg/d; 5 d; 7 inbred mouse strains) and subchronic (100 or 400 mg/kg/d; 1, 2, or 4 wk; 2 inbred mouse strains) designs. The quantitative relationship was evaluated between strain-, dose-, and time-dependent formation of TCE metabolites from cytochrome P-450-mediated oxidation (trichloroacetic acid [TCA], dichloroacetic acid [DCA], and trichloroethanol) and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione] in serum and liver, and various hepatic toxicity phenotypes. In subacute study, interstrain variability in TCE metabolite amounts was observed in serum and liver. No marked induction of Cyp2e1 protein levels in liver was detected. Serum and hepatic levels of TCA and DCA were correlated with increased transcription of peroxisome proliferator-marker genes Cyp4a10 and Acox1 but not with degree of induction in hepatocellular proliferation. In subchronic study, serum and liver levels of oxidative metabolites gradually decreased over time despite continuous dosing. Hepatic protein levels of CYP2E1, ADH, and ALDH2 were unaffected by treatment with TCE. While the magnitude of induction of peroxisome proliferator-marker genes also declined, hepatocellular proliferation increased. This study offers a unique opportunity to provide a scientific data-driven rationale for some of the major assumptions in human health assessment of TCE.

Comparative analysis of the relationship between trichloroethylene metabolism and tissue-specific toxicity among inbred mouse strains: kidney effects.

Trichloroethylene (TCE) is a well-known environmental and occupational toxicant that is classified as carcinogenic to humans based on the epidemiological evidence of an association with higher risk of renal-cell carcinoma. A number of scientific issues critical for assessing human health risks from TCE remain unresolved, such as the amount of kidney-toxic glutathione conjugation metabolites formed, interspecies and interindividual differences, and the mode of action for kidney carcinogenicity. It was postulated that TCE renal metabolite levels are associated with kidney-specific toxicity. Oral dosing with TCE was conducted in subacute (600 mg/kg/d; 5 d; 7 inbred mouse strains) and subchronic (100 or 400 mg/kg/d; 1, 2, or 4 wk; 2 inbred mouse strains) designs. The quantitative relationship was evaluated between strain-, dose, and time-dependent formation of TCE metabolites from cytochrome P-450-mediated oxidation (trichloroacetic acid [TCA], dichloroacetic acid [DCA], and trichloroethanol) and glutathione conjugation [S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione], and various kidney toxicity phenotypes. In subacute study, interstrain differences in renal TCE metabolite levels were observed. In addition, data showed that in several strains kidney-specific effects of TCE included induction of peroxisome proliferator-marker genes Cyp4a10 and Acox1, increased cell proliferation, and expression of KIM-1, a marker of tubular damage and regeneration. In subchronic study, peroxisome proliferator-marker gene induction and renal toxicity diminished while cell proliferative response was elevated in a dose-dependent manner in NZW/LacJ but not C57BL/6J mice. Overall, data demonstrated that renal TCE metabolite levels are associated with kidney-specific toxicity and that these effects are strain dependent.

Identification and characterization of 2'-deoxyadenosine adducts formed by isoprene monoepoxides in vitro.

Isoprene, the 2-methyl analogue of 1,3-butadiene, is ubiquitous in the environment, with major contributions to total isoprene emissions stemming from natural processes despite the compound being a bulk industrial chemical. Additionally, isoprene is a combustion product and a major component in cigarette smoke. Isoprene has been classified as possibly carcinogenic to humans (group 2B) by IARC and as reasonably anticipated to be a human carcinogen by the National Toxicology Program. Isoprene, like butadiene, requires metabolic activation to reactive epoxides to exhibit its carcinogenic properties. The mode of action has been postulated to be that of a genotoxic carcinogen, with the formation of promutagenic DNA adducts being essential for mutagenesis and carcinogenesis. In rodents, isoprene-induced tumors show unique point mutations (A→T transversions) in the K-ras protooncogene at codon 61. Therefore, we investigated adducts formed after the reaction of 2'-deoxyadenosine (dAdo ) with the two monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (IP-1,2-O) and propen-2-yloxirane (IP-3,4-O), under physiological conditions. The formation of N1-2'-deoxyinosine (N1-dIno) due to the deamination of N1-dAdo adducts was of particular interest, since N1-dIno adducts are suspected to have high mutagenic potential based on in vitro experiments. Major stable adducts were identified by HPLC, UV-spectroscopy, and LC-MS/MS and characterized by (1)H NMR and (1)H,(13)C HSQC and HMBC NMR experiments. Adducts of IP-1,2-O that were fully identified are R,S-C1-N(6)-dAdo, R-C2-N(6)-dAdo, and S-C2-N(6)-dAdo; adducts of IP-3,4-O are S-C3-N(6)-dAdo, R-C3-N(6)-dAdo, R,S-C4-N(6)-dAdo, S-C4-N1-dIno, R-C4-N1-dIno, R-C3-N1-dIno, S-C3-N1-dIno, and C3-N7-Ade. Both monoepoxides formed adducts on the terminal and internal oxirane carbons. This is the first study to describe adducts of isoprene monoepoxides with dAdo. Characterization of adducts formed by isoprene monoepoxides with deoxynucleosides and subsequently with DNA represent the first step toward evaluating their potential for being converted into a mutation or as biomarkers of isoprene metabolism and exposure.

Structural characterization of formaldehyde-induced cross-links between amino acids and deoxynucleosides and their oligomers.

Exposure to formaldehyde results in the formation of DNA-protein cross-links (DPCs) as a primary genotoxic effect. Although DPCs are biologically important and eight amino acids have been reported to form stable adducts with formaldehyde, the structures of these cross-links have not yet been elucidated. We have characterized formaldehyde-induced cross-links of Lys, Cys, His, and Trp with dG, dA, and dC. dT formed no cross-links, nor did Arg, Gln, Tyr, or Asn. Reaction of formaldehyde with Lys and dG gave the highest yield of cross-linked products, followed by reaction with Cys and dG. Yields from the other coupling reactions were lower by a factor of 10 or more. Detailed structural examination by NMR and mass spectrometry established that the cross-links between amino acids and single nucleosides involve a formaldehyde-derived methylene bridge. Lys yielded two additional products with dG in which the linking structure is a 1,N(2)-fused triazino ring. The Lys cross-linked products were unstable at ambient temperature. Reactions between the reactive N(alpha)-Boc-protected amino acids and the trinucleotides d(T(1)B(2)T(3)) where B(2) is the target base G, A, or C and reactions between dG, dA and dC and 8-mer peptides containing a single reactive target residue at position 5 yielded cross-linked products with structures inferred from high resolution mass spectrometry and fragmentation patterns that are consistent with those between N(alpha)-Boc-protected amino acids and single nucleotides rigorously determined by NMR studies. These structures will provide a basis for investigation of the characteristics and properties of DPCs formed in vivo and will be helpful in identifying biomarkers for the evaluation of formaldehyde exposure both at the site of contact and at distant sites.

Liquid chromatography electrospray ionization tandem mass spectrometry analysis method for simultaneous detection of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine.

Trichloroethylene (TCE, CAS 79-01-6) is a widely used industrial chemical, and a common environmental pollutant. TCE is a well-known carcinogen in rodents and is classified as "probably carcinogenic to humans". Several analytical methods have been proposed for detection of TCE metabolites in biological media utilizing derivatization-free techniques; however, none of them is suitable for simultaneous detection of both oxidative metabolites and glutathione conjugates of TCE in small volume biological samples. Here, we report a new combination of methods for assessment of major TCE metabolites: dichloroacetic acid (DCA), trichloroacetic acid (TCA), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG). First, DCA and TCA were extracted with ether. Second, the remaining aqueous fraction underwent solid phase extraction for DCVC and DCVG. Then, DCA and TCA were measured by hydrophilic interaction liquid chromatography ion exchange negative electrospray ionization tandem mass spectrometry, while DCVC and DCVG were measured by reverse phase positive electrospray ionization tandem mass spectrometry. This method was applied successfully to measure all 4 TCE metabolites in as little as 50 microl of serum from mice orally exposed to TCE (2100 mg/kg, 2h). Serum concentrations (mean+/-standard deviation) of the TCE metabolites obtained with this method are comparable or equivalent to those previously reported in the literature: DCA, 0.122+/-0.014 nmol/ml (limit of detection: 0.01 nmol/ml); TCA, 256+/-30 nmol/ml (0.4 nmol/ml); DCVG, 0.037+/-0.015 nmol/ml (0.001 nmol/ml); DCVC, 0.0024+/-0.0009 nmol/ml (0.001 nmol/ml). This method opens new opportunities to increase throughput and decrease number of animals required for mechanistic studies on TCE in rodents.