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The exposome refers to all of the internal and external life-long exposures that an individual experiences. These exposures, either acute or chronic, are associated with changes in metabolism that will positively or negatively influence the health and well-being of individuals. Nutrients and other dietary compounds modulate similar biochemical processes and have the potential in some cases to counteract the negative effects of exposures or enhance their beneficial effects. We present herein the concept of Nutritional Pharmacology/Toxicology which uses high-information metabolomics workflows to identify metabolic targets associated with exposures. Using this information, nutritional interventions can be designed toward those targets to mitigate adverse effects or enhance positive effects. We also discuss the potential for this approach in precision nutrition where nutrients/diet can be used to target gene-environment interactions and other subpopulation characteristics. Deriving these "nutrient cocktails" presents an opportunity to modify the effects of exposures for more beneficial outcomes in public health., Competing Interests: Conflicts of interest: The authors declare no conflicts of interest.

COVID-19 caused by SARS-CoV-2, is an international concern. This infection requires urgent efforts to develop new antiviral compounds. To date, no specific drug in controlling this disease has been identified. Developing the new treatment is usually time consuming, therefore using the repurposing broad-spectrum antiviral drugs could be an effective strategy to respond immediately. In this review, a number of broad-spectrum antivirals with potential efficacy to inhibit the virus replication via targeting the virus spike protein (S protein), RNA-dependent RNA polymerase (RdRp), 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) that are critical in the pathogenesis and life cycle of coronavirus, have been evaluated as possible treatment options against SARS-CoV-2 in COVID-19 patients.

Few investigations have been conducted on the disposition and fate of silver nanoparticles (AgNP) in pregnancy. The distribution of a single dose of polyvinylpyrrolidone (PVP)-stabilized AgNP was investigated in pregnant rats. Two sizes of AgNP, 20 and 110 nm, and silver acetate (AgAc) were used to investigate the role of AgNP diameter and particle dissolution in tissue distribution, internal dose and persistence. Dams were administered AgNP or AgAc intravenously (i.v.) (1 mg kg -1 ) or by gavage (p.o.) (10 mg kg -1 ), or vehicle alone, on gestation day 18 and euthanized at 24 or 48 h post-exposure. The silver concentration in tissues was measured using inductively-coupled plasma mass spectrometry. The distribution of silver in dams was influenced by route of administration and AgNP size. The highest concentration of silver (μg Ag g -1 tissue) at 48 h was found in the spleen for i.v. administered AgNP, and in the lungs for AgAc. At 48 h after p.o. administration of AgNP, the highest concentration was measured in the cecum and large intestine, and for AgAc in the placenta. Silver was detected in placenta and fetuses for all groups. Markers of cardiovascular injury, oxidative stress marker, cytokines and chemokines were not significantly elevated in exposed dams compared to vehicle-dosed control. NMR metabolomics analysis of urine indicated that AgNP and AgAc exposure impact the carbohydrate, and amino acid metabolism. This study demonstrates that silver crosses the placenta and is transferred to the fetus regardless of the form of silver. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

An Adverse Event Following Immunization (AEFI) is an adverse reaction to a vaccination that goes above and beyond the usual side effects associated with vaccinations. One serious AEFI related to the smallpox vaccine is myopericarditis. Metabolomics involves the study of the low molecular weight metabolite profile of cells, tissues, and biological fluids, and provides a functional readout of the phenotype. Metabolomics may help identify a particular metabolic signature in serum of subjects who are predisposed to developing AEFIs. The goal of this study was to identify metabolic markers that may predict the development of adverse events following smallpox vaccination. Serum samples were collected from military personnel prior to and following receipt of smallpox vaccine. The study population included five subjects who were clinically diagnosed with myopericarditis, 30 subjects with asymptomatic elevation of troponins, and 31 subjects with systemic symptoms following immunization, and 34 subjects with no AEFI, serving as controls. Two-hundred pre- and post-smallpox vaccination sera were analyzed by untargeted metabolomics using 1 H nuclear magnetic resonance (NMR) spectroscopy. Baseline (pre-) and post-vaccination samples from individuals who experienced clinically verified myocarditis or asymptomatic elevation of troponins were more metabolically distinguishable pre- and post-vaccination compared to individuals who only experienced systemic symptoms, or controls. Metabolomics profiles pre- and post-receipt of vaccine differed substantially when an AEFI resulted. This study is the first to describe pre- and post-vaccination metabolic profiles of subjects who developed an adverse event following immunization. The study demonstrates the promise of metabolites for determining mechanisms associated with subjects who develop AEFI and the potential to develop predictive biomarkers., (Published by Elsevier Ltd.)

Associations between blood type and disease have been studied since the early 1900s when researchers determined that antibodies and antigens are inherited. In the 1950s, the chemical identification of the carbohydrate structure of surface antigens led to the understanding of biosynthetic pathways. The blood type is defined by oligosaccharide structures, which are specific to the antigens, thus, blood group antigens are secondary gene products, while the primary gene products are various glycosyltransferase enzymes that attach the sugar molecules to the oligosaccharide chain. Blood group antigens are found on red blood cells, platelets, leukocytes, plasma proteins, certain tissues, and various cell surface enzymes, and also exist in soluble form in body secretions such as breast milk, seminal fluid, saliva, sweat, gastric secretions, urine, and amniotic fluid. Recent advances in technology, biochemistry, and genetics have clarified the functional classifications of human blood group antigens, the structure of the A, B, H, and Lewis determinants and the enzymes that produce them, and the association of blood group antigens with disease risks. Further research to identify differences in the biochemical composition of blood group antigens, and the relationship to risks for disease, can be important for the identification of targets for the development of nutritional intervention strategies, or the identification of druggable targets. WIREs Syst Biol Med 2016, 8:517-535. doi: 10.1002/wsbm.1355 For further resources related to this article, please visit the WIREs website., Competing Interests: None., (© 2016 Wiley Periodicals, Inc.)

A comprehensive distribution study was conducted in female rats and mice exposed to a suspension of uniformly carbon-14-labeled C60 ([(14) C(U)]C60 ). Rodents were administered [(14) C(U)]C60 (~0.9 mg kg(-1) body weight) or 5% polyvinylpyrrolidone-saline vehicle alone via a single tail vein injection. Tissues were collected at 1 h and 1, 7, 14 and 30 days after administration. A separate group of rodents received five daily injections of suspensions of either [(14) C(U)]C60 or vehicle with tissue collection 14 days post exposure. Radioactivity was detected in over 20 tissues at all time points. The highest concentration of radioactivity in rodents at each time point was in liver, lungs and spleen. Elimination of [(14) C(U)]C60 was < 2% in urine and feces at any 24 h time points. [(14) C(U)]C60 and [(14) C(U)]C60 -retinol were detected in liver of rats and together accounted for ~99% and ~56% of the total recovered at 1 and 30 days postexposure, respectively. The blood radioactivity at 1 h after [(14) C(U)]C60 exposure was fourfold higher in rats than in mice; blood radioactivity was still in circulation at 30 days post [(14) C(U)]C60 exposure in both species (<1%). Levels of oxidative stress markers increased by 5 days after exposure and remained elevated, while levels of inflammation markers initially increased and then returned to control values. The level of cardiovascular marker von Willebrand factor, increased in rats, but remained at control levels in mice. This study demonstrates that [(14) C(U)]C60 is retained in female rodents with little elimination by 30 days after i.v. exposure, and leads to systemic oxidative stress., (Copyright © 2015 John Wiley & Sons, Ltd.)

A comprehensive distribution study was conducted in pregnant and lactating rats exposed to a suspension of uniformly carbon-14 labeled C60 ([(14) C(U)]C60 ). Rats were administered [(14) C(U)]C60 (~0.2 mg [(14) C(U)]C60 kg(-1) body weight) or 5% polyvinylpyrrolidone (PVP)-saline vehicle via a single tail vein injection. Pregnant rats were injected on gestation day (GD) 11 (terminated with fetuses after either 24 h or 8 days), GD15 (terminated after 24 h or 4 days), or GD18 (terminated after 24 h). Lactating rats were injected on postnatal day 8 and terminated after 24 h, 3 or 11 days. The distribution of radioactivity in pregnant dams was influenced by both the state of pregnancy and time of termination after exposure. The percentage of recovered radioactivity in pregnant and lactating rats was highest in the liver and lungs. Radioactivity was quantitated in over 20 tissues. Radioactivity was found in the placenta and in fetuses of pregnant dams, and in the milk of lactating rats and in pups. Elimination of radioactivity was < 2% in urine and feces at each time point. Radioactivity remained in blood circulation up to 11 days after [(14) C(U)]C60 exposure. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impacts of [(14) C(U)]C60 exposure. Oxidative stress was elevated in female pups of exposed dams. Metabolomics analysis of urine showed that [(14) C(U)]C60 exposure to pregnant rats impacted the pathways of vitamin B, regulation of lipid and sugar metabolism and aminoacyl-tRNA biosynthesis. This study demonstrated that [(14) C(U)]C60 crosses the placenta at all stages of pregnancy examined, and is transferred to pups via milk., (Copyright © 2015 John Wiley & Sons, Ltd.)

Obese individuals are at greater risk for hospitalization and death from infection with the 2009 pandemic H1N1 influenza virus (pH1N1). In this study, diet-induced and genetic-induced obese mouse models were used to uncover potential mechanisms by which obesity increases pH1N1 severity. High-fat diet-induced and genetic-induced obese mice exhibited greater pH1N1 mortality, lung inflammatory responses, and excess lung damage despite similar levels of viral burden compared with lean control mice. Furthermore, obese mice had fewer bronchoalveolar macrophages and regulatory T cells during infection. Obesity is inherently a metabolic disease, and metabolic profiling has found widespread usage in metabolic and infectious disease models for identifying biomarkers and enhancing understanding of complex mechanisms of disease. To further characterize the consequences of obesity on pH1N1 infection responses, we performed global liquid chromatography-mass spectrometry metabolic profiling of lung tissue and urine. A number of metabolites were perturbed by obesity both prior to and during infection. Uncovered metabolic signatures were used to identify changes in metabolic pathways that were differentially altered in the lungs of obese mice such as fatty acid, phospholipid, and nucleotide metabolism. Taken together, obesity induces distinct alterations in the lung metabolome, perhaps contributing to aberrant pH1N1 immune responses., (Copyright © 2015 by The American Association of Immunologists, Inc.)

This study was conducted to determine the distribution of [(14)C]C60 in the pregnant rat and fetuses, and in the lactating rat and offspring. Pregnant rats were dosed on gestation day (gd) 15 and lactating rats were dosed on postnatal day (pnd) 8 via tail vein injection with a suspension of approximately 0.3 mg [(14)C]C60 kg(-1) body weight prepared in polyvinylpyrrolidone (PVP), or with PVP alone. Tissues were collected at 24 and 48 h after dosing. The largest portion of the administered dose was detected in the liver (approximately 43%, pregnant dam; approximately 35%, lactating dam) and lung (approximately 25%, lactating dam). Radioactivity (approximately 6%) was distributed to the reproductive tract, placenta and fetuses of the pregnant dam. Lactating rats had radioactivity distributed to the milk (3140 dpm g(-1) tissue, 24 h; 1620 dpm g(-1) tissue, 48 h), and to the pups' GI tract (2.8%, 24 h; 4.4% 48 h) and liver (<1%). Blood radioactivity was significant at 24 h (14-19%) and at 48 h (7%) after dosing; largely accounted for in the plasma fraction. Less that 4% of the dose was recovered in the maternal spleen, heart, brain, urine or feces. Metabolomics analysis of urine indicated that dams exposed to [(14)C]C60 had decreased metabolites derived from the Krebs cycle and increased metabolites derived from the urea cycle or glycolysis, as well as alterations in the levels of some sulfur-containing amino acids and purine/pyrimidine metabolites. This study demonstrated that [(14)C]C60 crosses the placenta and is transmitted to offspring via the dam's milk and subsequently systemically absorbed., (2009 John Wiley & Sons, Ltd.)

This study examines the use of physiologically based pharmacokinetic (PBPK) models for inferring exposure when the number of biomarker observations per individual is limited, as commonly occurs in population exposure surveys. The trade-off between sampling multiple biomarkers at a specific time versus fewer biomarkers at multiple time points was investigated, using a simulation-based approach based on a revised and updated chlorpyrifos PBPK model originally published. Two routes of exposure, oral and dermal, were studied as were varying levels of analytic measurement error. It is found that adding an additional biomarker at a given time point adds substantial additional information to the analysis, although not as much as the addition of another sampling time. Furthermore, the precision of the estimates of exposed dose scaled approximately with the analytic precision of the biomarker measurement. For acute exposure scenarios such as those considered here, the results of this study suggest that the number of biomarkers can be balanced against the number of sampling times to obtain the most efficient estimator after consideration of cost, intrusiveness, and other relevant factors.

1-bromopropane (1-BrP) induces dose- and time-dependent reproductive organ toxicity and reduced sperm motility in rodents. The contribution of cytochrome P4502E1 (CYP2E1) to both 1-BrP metabolism and the induction of male reproductive toxicity was investigated using wild-type (WT) and Cyp2e1-/- mice. In gas uptake inhalation studies, the elimination half-life of [1,2,3-(13)C]-1-BrP was longer in Cyp2e1-/- mice relative to WT (3.2 vs. 1.3 h). Urinary metabolites were identified by 13C nuclear magnetic resonance. The mercapturic acid of 1-bromo-2-hydroxypropane (2OHBrP) was the major urinary metabolite in WT mice, and products of conjugation of 1-BrP with glutathione (GSH) were insignificant. The ratio of GSH conjugation to 2-hydroxylation increased 5-fold in Cyp2e1-/- mice relative to WT. After 1-BrP exposure, hepatic GSH was decreased by 76% in WT mice vs. 47% in Cyp2e1-/- mice. Despite a 170% increase in 1-BrP exposure in Cyp2e1-/- vs. WT mice, sperm motility in exposed Cyp2e1-/- mice did not change relative to unexposed matched controls. This suggests that metabolites produced through CYP2E1-mediated oxidation may be responsible for 1-BrP-induced sperm toxicity. Both 1-BrP and 2OHBrP inhibited the motility of sperm obtained from WT mice in vitro. However, only 2OHBrP reduced the motility of sperm obtained from Cyp2e1-/- mice in vitro, suggesting that conversion of parent compound to 2OHBrP within the spermatozoa may contribute, at least in part, to reduced motility. Overall, these data suggest that metabolism of 1-BrP is mediated in part by CYP2E1, and activation of 1BrP via this enzyme may contribute to the male reproductive toxicity of this chemical.

Acrylamide (AM), used in the manufacture of polyacrylamide and grouting agents, is produced during the cooking of foods. Workplace exposure to AM can occur through the dermal and inhalation routes. The objective of this study was to define the kinetics of elimination of AM and its metabolites following oral and dermal administration. This is the second part of a study in which metabolites and hemoglobin adducts of AM were determined in people (Fennell et al., 2005, Toxicol. Sci. 85, 447-459). (1,2,3-(13)C(3))AM was administered in an aqueous solution orally (single dose of 0.5, 1.0, or 3.0 mg/kg) or dermally (three daily doses of 3.0 mg/kg) to sterile male volunteers. Urine samples were collected at 0-2, 2-4, 4-8, 8-16, and 16-24 h following administration orally, or at 0-2, 2-4, 4-8, 8-16, and 16-24 h following each of three daily dermal doses. (13)C(3)-AM and its metabolites in urine, (13)C(3)-glycidamide, (13)C(3)-N-acetyl-S-(3-amino-3-oxopropyl)cysteine and its S-oxide, and (13)C(3)-N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine, were quantitated using liquid chromatography-tandem mass spectrometry. The recovered urinary metabolites accounted for 45.6, 49.9, and 39.9% of a 0.5, 1.0, and 3.0 mg/kg oral dose (0-24 h), respectively, and for 4.5% of the dose after 3 mg/kg was administered daily for 3 days dermally (0-4 days). These results indicate that after oral administration AM is rapidly absorbed and eliminated. The half-life estimated for elimination of AM in urine was 3.1-3.5 h. After dermal administration, AM uptake is slow. This study indicated that skin provides a barrier that slows the absorption of AM, and results in limited systemic availability following dermal exposure to AM.

Workplace exposure to 1-bromopropane (1-BrP) can potentially occur during its use in spray adhesives, fats, waxes, and resins. 1-BrP may be used to replace ozone depleting solvents, resulting in an increase in its annual production in the US, which currently exceeds 1 million pounds. The potential for human exposure to 1-BrP and the reports of adverse effects associated with potential occupational exposure to high levels of 1-BrP have increased the need for the development of biomarkers of exposure and an improved understanding of 1-BrP metabolism and disposition. In this study, the factors influencing the disposition and biotransformation of 1-BrP were examined in male F344 rats and B6C3F1 mice following inhalation exposure (800 ppm) or intravenous administration (5, 20, and 100 mg/kg). [1,2,3-(13)C]1-BrP and [1-(14)C]1-BrP were administered to enable characterization of urinary metabolites using NMR spectroscopy, LC-MS/MS, and HPLC coupled radiochromatography. Exhaled breath volatile organic chemicals (VOC), exhaled CO(2), urine, feces, and tissues were collected for up to 48 h post-administration for determination of radioactivity distribution. Rats and mice exhaled a majority of the administered dose as either VOC (40-72%) or (14)CO(2) (10-30%). For rats, but not mice, the percentage of the dose exhaled as VOC increased between the mid ( approximately 50%) and high ( approximately 71%) dose groups; while the percentage of the dose exhaled as (14)CO(2) decreased (19 to 10%). The molar ratio of exhaled (14)CO(2) to total released bromide, which decreased as dose increased, demonstrated that the proportion of 1-BrP metabolized via oxidation relative to pathways dependent on glutathione conjugation is inversely proportional to dose in the rat. [(14)C]1-BrP equivalents were recovered in urine (13-17%, rats; 14-23% mice), feces (<2%), or retained in the tissues and carcass (<6%) of rats and mice administered i.v. 5 to 100 mg/kg [(14)C]1-BrP. Metabolites characterized in urine of rats and mice include N-acetyl-S-propylcysteine, N-acetyl-3-(propylsulfinyl)alanine, N-acetyl-S-(2-hydroxypropyl)cysteine, 1-bromo-2-hydroxypropane-O-glucuronide, N-acetyl-S-(2-oxopropyl)cysteine, and N-acetyl-3-[(2-oxopropyl)sulfinyl]alanine. These metabolites may be formed following oxidation of 1-bromopropane to 1-bromo-2-propanol and bromoacetone and following subsequent glutathione conjugation with either of these compounds. Rats pretreated with 1-aminobenzotriazole (ABT), a potent inhibitor of P450 excreted less in urine (down 30%), exhaled as (14)CO2 (down 80%), or retained in liver (down 90%), with a concomitant increase in radioactivity expired as VOC (up 52%). Following ABT pretreatment, rat urinary metabolites were reduced in number from 10 to 1, N-acetyl-S-propylcysteine, which accounted for >90% of the total urinary radioactivity in ABT pretreated rats. Together, these data demonstrate a role for cytochrome P450 and glutathione in the dose-dependent metabolism and disposition of 1-BrP in the rat.

Acrylamide (AM), used in the manufacture of polyacrylamide and grouting agents, is produced during the cooking of foods. Workplace exposure to AM can occur through the dermal and inhalation routes. The objectives of this study were to evaluate the metabolism of AM in humans following oral administration, to compare hemoglobin adduct formation on oral and dermal administration, and to measure hormone levels. The health of the people exposed under controlled conditions was continually monitored. Prior to conducting exposures in humans, a low-dose study was conducted in rats administered 3 mg/kg (1,2,3-13C3) AM by gavage. The study protocol was reviewed and approved by Institute Review Boards both at RTI, which performed the sample analysis, and the clinical research center conducting the study. (1,2,3-13C3) AM was administered in an aqueous solution orally (single dose of 0.5, 1.0, or 3.0 mg/kg) or dermally (three daily doses of 3.0 mg/kg) to sterile male volunteers. Urine samples (3 mg/kg oral dose) were analyzed for AM metabolites using 13C NMR spectroscopy. Approximately 86% of the urinary metabolites were derived from GSH conjugation and excreted as N-acetyl-S-(3-amino-3-oxopropyl)cysteine and its S-oxide. Glycidamide, glyceramide, and low levels of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine were detected in urine. On oral administration, a linear dose response was observed for N-(2-carbamoylethyl)valine (AAVal) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal) in hemoglobin. Dermal administration resulted in lower levels of AAVal and GAVal. This study indicated that humans metabolize AM via glycidamide to a lesser extent than rodents, and dermal uptake was approximately 6.6% of that observed with oral uptake.

Dibutylphthalate (DBP) can cause adverse effects on the developing male reproductive tract when administered late in gestation to pregnant rats. The objectives of this study were to evaluate the metabolism of DBP in female rats, and the pharmacokinetics of DBP in pregnant rats on gestational day (g.d.) 20. The identities of DBP metabolites in urine and in maternal and fetal plasma were confirmed by LC-MS/MS, as monobutylphthalate (MBP) and its glucuronide, monohydroxybutylphthalate and its glucuronide, and butanoic acid phthalate and its glucuronide. An LC-MS/MS method was developed for the quantitation of MBP and its glucuronide. MBP and MBP glucuronide were quantitated in maternal and fetal plasma, and in amniotic fluid from pregnant rats administered a single dose of DBP (50, 100, or 250 mg/kg by gavage in corn oil) on g.d. 20. The pharmacokinetics of MBP and MBP glucuronide were determined. MBP was the major metabolite in maternal and fetal plasma. With increasing dose, there was a nonlinear increase in area under the curve (AUC) for MBP, with a ten-fold increase in maternal plasma, and an eight-fold increase in fetal plasma between 50 mg/kg and 250 mg/kg. In amniotic fluid, the major metabolite initially was MBP, but by 24 h after dosing, the major metabolite was MBP glucuronide. Isomers of the MBP glucuronide were detected in amniotic fluid, suggesting acyl group migration, known to occur with acyl glucuronides. This study indicated that MBP, thought to be the active metabolite of DBP, can cross the placenta in late gestation, and that the metabolism of MBP is saturable.

Tertiary amyl methyl ether (TAME) is a fuel additive used to reduce carbon monoxide in automobile emissions. Because of the potential for human exposure, this study was conducted to develop methods for the characterization and quantitation of metabolites in expired air and excreta of rats exposed to a mixture of [13C]- and [14C]TAME ([2,3,4-13C]- and [2-14C]2-methoxy-2-methylbutane). The distribution of TAME in rats was determined following inhalation exposure, and TAME-derived metabolites were characterized in expired air and urine. Male rats were exposed for 6 h via nose-only inhalation to 2500 ppm [14C/13C]TAME, and expired air, urine and feces were collected for up to 7 days. Over 95% of the total recovered radioactivity was excreted by 48 h after exposure. Recovered radioactivity was expired as organic volatiles (44%) and 14CO2 (3%) and excreted in urine (51%) and feces (1%). Both TAME and its metabolite tertiary amyl alcohol (TAA) accounted for > or =90% of the radiolabel in expired air 0-8 h following exposure termination. Three major urinary metabolites of TAME were identified: (1) a direct glucuronide conjugate of TAA; (2) a product of oxidation at the methylene carbon of TAA (2,3-dihydroxy-2-methylbutane); (3) a glucuronide conjugate of metabolite 2. Metabolite 1 accounted for most of the TAME-derived metabolites excreted 0-8 h following exposure termination. Further metabolic products of TAA (metabolites 2 and 3) accounted for most of the excreted TAME-derived metabolites at later time points., (Copyright 2003 John Wiley & Sons, Ltd.)

Tertiary amyl methyl ether (TAME) is a gasoline fuel additive used to reduce emissions. Understanding the metabolism and distribution of TAME is needed to assess potential human health issues. The effect of dose level, duration of exposure and route of administration on the metabolism and distribution of TAME were investigated in male and female F344 rats and CD-1 mice following inhalation or gavage administration. By 48 h after exposure, >96% of the administered radioactivity was expired in air (16-71%) or eliminated in urine and feces (28-72%). Following inhalation exposure, mice had a two- to threefold greater relative uptake of [14C]TAME compared with rats. Metabolites were excreted in urine of rats and mice that are formed by glucuronide conjugation of tertiary amyl alcohol (TAA), oxidation of TAA to 2,3-dihydroxy-2-methylbutane and glucuronide conjugation of 2,3-dihydroxy-2-methylbutane. A saturation in the uptake and metabolism of TAME with increased exposure concentration was indicated by a decreased relative uptake of total [14C]TAME equivalents and an increase in the percentage expired as volatiles. A saturation of P-450 oxidation of TAA was indicated by a disproportional decrease of 2,3-dihydroxy-2-methylbutane and its glucuronide conjugate with increased exposure concentration., (Copyright 2003 John Wiley & Sons, Ltd.)

Interest in understanding the biological behavior of aliphatic ethers has increased owing to their use as gasoline additives. The purpose of this study was to investigate the blood pharmacokinetics of the oxygenate tertiary amyl methyl ether (TAME), its major metabolite tertiary amyl alcohol (TAA) and acetone in rats and mice following inhalation exposure to TAME. Species differences in the area under the curve (AUC) for TAME were significant at each exposure concentration. For rats, the blood TAME AUC increased in proportion with an increase in exposure concentration. For mice, an increase in exposure concentration (100-500 ppm) resulted in a disproportional increase in the TAME AUC. Mice had greater (two- to threefold) blood concentrations of TAA compared with rats following exposure to 2500 or 500 ppm TAME. Mice had a disproportional increase in the TAA AUC with an increase in exposure concentration (100-500 ppm). This difference could result from saturation of a process (e.g. oxidation, glucuronide conjugation) that is involved in the further metabolism of TAA. For each species, gender and exposure concentration, acetone increased during exposure and returned to control values by 16 h following exposure. The source of acetone could be both as a metabolite of TAA or an effect on endogenous metabolism produced by exposure to TAME., (Copyright 2003 John Wiley & Sons, Ltd.)

Acrylamide (AM), which is used to manufacture polymers, is carcinogenic and a reproductive and neurological toxicant. The objective of this study was to compare the metabolism of AM administered orally (po), dermally, intraperitoneally (ip), or by inhalation, and to measure the hemoglobin adducts produced. Rats and mice were exposed to 2.9 ppm [1,2,3-13C] and [2,3-14C]AM for 6 h. [2,3-14C]AM (162 mg/kg) or [1,2,3-13C]AM (13 8 mg/kg) in water was administered dermally to rats for 24 h, and [1,2,3-13C]AM was administered ip (47 mg/kg). Urine and feces were collected for 24 h. Urine was the major elimination route in rats (ip, 62% and po, 53% of the dose; dermal, 44% of the absorbed dose; inhalation, 31% of the recovered radioactivity) and mice (inhalation, 27% of the recovered radioactivity). Signals in the 13C-NMR spectra of urine were assigned to previously identified metabolites derived from AM glutathione conjugation (AM-GSH) and conversion to glycidamide (GA). AM-GSH was a major metabolic route in rats accounting for 69% (ip), 71% (po), 52% (dermal), and 64% (inhalation). In mice, AM-GSH accounted for only 27% (inhalation) of the total urinary metabolites. The remaining urinary metabolites were derived from GA. Valine hemoglobin adducts of AM and GA were characterized using liquid chromatography-mass spectrometry. The ratio of AM to GA adducts paralleled the flux through pathways based on urinary metabolites. This study demonstrates marked species differences in the metabolism and internal dose (Hb-adducts) of AM following inhalation exposure and marked differences in uptake comparing dermal with po and ip administration.

Acrylamide (AM) and N-methylolacrylamide (NMA) are used in the formulation of grouting materials. AM undergoes metabolism to a reactive epoxide, glycidamide (GA). Both AM and GA react with hemoglobin to form adducts that can be related to exposure to AM. The objective of this study was to evaluate the extent to which NMA could form the same adducts as AM. N-(2-carbamoylethyl)valine (AAVal derived from AM) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal derived from GA) were measured following a single oral dose of AM (50 mg/kg) or NMA (71 mg/kg) in male F344 rats. AAVal and GAVal were measured by a modified Edman degradation to produce phenylthiohydantoin derivatives and liquid chromatography/tandem mass spectrometry. In AM-treated rats, AAVal was 21 +/- 1.7-pmol/mg globin (mean +/- SD, n = 4), and GAVal was 7.9 +/- 0.8 pmol/mg. In NMA-treated rats, AAVal was 41 +/- 4.9 pmol/mg, and GAVal was 1.4 +/- 0.1 pmol/mg. Whether AAVal was derived from reaction of NMA with globin followed by loss of the hydroxymethyl group, or loss of the hydroxymethyl group to form AM prior to reaction with globin, is not known. However, the higher ratio of AAVal:GAVal in NMA-treated rats (29 vs. 2.6 in AM-treated rats) suggests that reaction of NMA with globin is the predominant route to AAVal in NMA-treated rats. The detection of GAVal in NMA-treated rats indicates oxidation of NMA, either directly or following conversion to AM. The lower levels of GAVal on NMA administration suggest that a much lower level of epoxide was formed than compared with AM treatment.

Bisphenol A (BPA), which is used in the manufacture of polycarbonates, elicits weak estrogenic activity in in vitro and in vivo test systems. The objectives of this study were to compare the patterns of disposition of radioactivity in adult female F-344 and CD rats after oral administration of (14)C BPA (100 mg/kg), to isolate the glucuronide of BPA and to assess its estrogenic activity in vitro, and to evaluate the transfer of radioactivity to pups from lactating dams administered (14)C BPA. Over 6 days, F-344 rats excreted more radioactivity in urine than CD rats. The major metabolite in urine was identified as bisphenol A glucuronide (BPA gluc) by incubation with beta-glucuronidase and (1)H and (13)C NMR spectroscopy. In lactating CD rats administered (14)C BPA (100 mg/kg) by gavage, only a small fraction of the label was found in milk, with 0.95 +/- 0.66, 0.63 +/- 0.13, and 0.26 +/- 0.10 microg equiv/ml (mean +/- SD) from dams collected 1, 8, and 26 h after dosing, respectively. Radioactivity in pup carcasses indicated exposure in the range of microgram equivalents per kilogram; those values ranged from 44.3 +/- 24.4 for pups separated from their lactating dams at 2 h to 78.4 +/- 10.9 at 24 h. BPA gluc was the prominent metabolite in milk and plasma. In test systems for activation of in vitro estrogen receptors alpha and beta, BPA gluc did not show appreciable efficacy at concentrations up to 0.03 mM, indicating that metabolism via glucuronidation is a detoxication reaction., (Copyright 2000 Academic Press.)

The disposition of styrene was studied in a group of 12 Sprague Dawley rats and two groups of 30 CD1 mice exposed separately to 160 ppm [ring-U-(14)C]styrene of high specific radioactivity of 1.92 TBq x mol(-1) (52 Ci x mol(-1)) for 6 h. A nose-only exposure system was successfully adapted to (1) recirculate a portion of the flow to limit the amount of (14)C-styrene required, and (2) avoid any polymerization of the compound. The mean uptake of styrene in rats was 113 +/- 7 micromol x kg(-1) x h(-1) and stable over time. The mean uptake in mice was higher, 189 +/- 53 and 183 +/- 76 micromol x kg(-1) x h(-1), for the first and second mouse inhalation experiment, but decreased steadily over time. Some of the mice, but none of the rats, showed signs of overt toxicity. The overall excretion of styrene and its metabolites was quantitatively similar in rats and mice. Urinary excretion was the primary route of excretion while fecal excretion accounted for only a very small part of the radioactivity. There was, however, a significant difference between mice and rats in the exhalation of (14)CO(2), which must have resulted from opening and subsequent breakdown of the aromatic ring. In mice the exhalation of (14)CO(2) accounted for 6.4 +/- 1.0 and 8. 0 +/- 0.5% of the styrene retained during the first and second mouse inhalation experiment. In rats, exhalation of (14)CO(2) accounted for only 2.0 +/- 0.7% of the retained styrene. Together with the results from the quantitative whole-body autoradiography (showing significantly higher binding in mouse lung and nasal passages compared to rat) the larger production of (14)CO(2) might be indicative of the formation of reactive ring-opened metabolites in the mouse lung, which, in turn, might be related to the observed development of bronchioalveolar tumors and nasal effects in mice exposed to styrene.

Bronchiolo-alveolar tumors were observed in mice exposed chronically to 160 ppm styrene, whereas no tumors were seen in rats up to concentrations of 1000 ppm. Clara cells, which are predominant in the bronchiolo-alveolar region in mouse lungs but less numerous in rat and human lung, contain various cytochrome P450s, which may oxidize styrene to the rodent carcinogen styrene-7,8-oxide (SO) and other reactive metabolites. Reactive metabolites may form specific DNA adducts and induce the tumors observed in mice. To determine DNA adducts in specific tissues and cell types, rats and mice were exposed to 160 ppm [ring-U-(14)C]styrene by nose-only inhalation for 6 h in a recirculating exposure system. Liver and lungs were isolated 0 and 42 h after exposure. Fractions enriched in Type II cells and Clara cells were isolated from rat and mouse lung, respectively. DNA adduct profiles differed quantitatively and qualitatively in liver, total lung, and enriched lung cell fractions. At 0 and 42 h after exposure, the two isomeric N:7-guanine adducts of SO (measured together, HPEG) were present in liver at 3.0 +/- 0.2 and 1.9 +/- 0.3 (rat) and 1.2 +/- 0.2 and 3.2 +/- 0.5 (mouse) per 10(8) bases. Several other, unidentified adducts were present at two to three times higher concentrations in mouse, but not in rat liver. In both rat and mouse lung, HPEG was the major adduct at approximately 1 per 10(8) bases at 0 h, and these levels halved at 42 h. In both rat Type II and non-Type II cells, HPEG was the major adduct and was about three times higher in Type II cells than in total lung. For mice, DNA adduct levels in Clara cells and non-Clara cells were similar to total lung. The hepatic covalent binding index (CBI) at 0 and 42 h was 0.19 +/- 0.06 and 0.14 +/- 0.03 (rat) and 0. 25 +/- 0.11 and 0.44 +/- 0.23 (mouse), respectively. The pulmonary CBIs, based on tissues combined for 0 and 42 h, were 0.17 +/- 0.04 (rat) and 0.24 +/- 0.04 (mouse). Compared with CBIs for other genotoxicants, these values indicate that styrene has only very weak adduct-forming potency. The overall results of this study indicate that DNA adduct formation does not play an important role in styrene tumorigenicity in chronically exposed mice.

Styrene is used in the manufacture of plastics and polymers and in the boat-building industry. The major metabolic route for styrene in rats, mice, and humans involves conversion to styrene-7,8-oxide (SO). The purpose of this study was to evaluate blood SO, SO-hemoglobin (SO-Hb) adducts, and urinary metabolites in styrene-exposed human volunteers and to compare these results with data previously obtained for rodents. Four healthy male volunteers were exposed for 2 h during light physical exercise to 50 ppm (13)C(8)-styrene vapor via a face mask. Levels and time profiles of styrene in exhaled air, blood, and urine (analyzed by GC) and urinary excretion patterns of mandelic acid and phenylglyoxylic acid in urine (analyzed by HPLC) were comparable to previously published volunteer studies. Maximum levels of SO in blood (measured by GC-MS) of 2.5-12.2 (average 6.7) nM were seen after 2 h, i.e., in the first sample collected after exposure had ended. The styrene blood level in humans was about 1.5 to 2 times higher than in rats and 4 times higher than in mice for equivalent styrene exposures. In contrast the SO levels in human blood was approximately fourfold lower than in mice. The level of hydroxyphenethylvaline (determined by GC-MS-MS) in pooled blood collected after exposure was estimated as 0.3 pmol/g globin corresponding to a SO-Hb adduct increment of about 0.003 pmol/g and ppmh. NMR analyses of urine showed that a major portion (> 95%) of the excreted (13)C-derived metabolites was derived from hydrolysis of SO, while only a small percentage of the excreted metabolites (< 5%) was derived from metabolism via phenylacetaldehyde. Signals consistent with metabolites derived from other pathways of styrene metabolism in rodents (such as glutathione conjugation with SO or ring epoxidation) were not detected., (Copyright 2000 Academic Press.)

Acrylonitrile (AN) and acrylamide (AM) are commonly used in the synthesis of plastics and polymers. In rodents, AM and AN are metabolized to the epoxides glycidamide and cyanoethylene oxide, respectively. The aim of this study was to determine the role of cytochrome P450 in the metabolism of AM and AN in vivo. Wild-type (WT) mice, WT mice pretreated with aminobenzotriazole (ABT, 50 mg/kg ip, 2 h pre-exposure), and mice devoid of cytochrome P450 2E1 (P450 2E1-null) were treated with 50 mg/kg [(13)C]AM po. WT mice and P450 2E1-null mice were treated with 2.5 or 10 mg/kg [(13)C]AN po. Urine was collected for 24 h, and metabolites were characterized using (13)C NMR. WT mice excreted metabolites derived from the epoxides and from direct GSH conjugation with AM or AN. Only metabolites derived from direct GSH conjugation with AM or AN were observed in the urine from ABT-pretreated WT mice and P450 2E1-null mice. On the basis of evaluation of urinary metabolites at these doses, these data suggest that P450 2E1 is possibly the only cytochrome P450 enzyme involved in the metabolism of AM and AN in mice, that inhibiting total P450 activity does not result in new pathways of non-P450 metabolism of AM, and that mice devoid of P450 2E1 do not excrete metabolites of AM or AN that would be produced by oxidation by other cytochrome P450s. P450 2E1-null mice may be an appropriate model for the investigation of the role of oxidative metabolism in the toxicity or carcinogenicity of these compounds.

After exposure to methyl tert-butyl ether (MTBE), a gasoline additive, only one metabolite [tert-butyl alcohol (TBA), <1% of dose] has been identified in human urine [Nihlén, A., et al. (1998) Toxicol. Appl. Pharmacol. 148, 274-280]. In the study presented here, metabolites of MTBE were characterized by (1)H-decoupled (13)C NMR spectroscopy in urine obtained from four volunteers experimentally exposed to 50 ppm (13)C-labeled MTBE ([1,2-(13)C(2)]MTBE) vapor (facemask) for 2 h during a light physical work load (50 W). Chemical shifts for the two adjacent (13)C-labeled carbons in [1, 2-(13)C(2)]MTBE-derived metabolites were consistent with the shifts obtained for spiked standards of alpha-hydroxyisobutyric acid (HBA) and 2-methyl-1,2-propanediol (MPD). NMR signals were not detected for labeled MTBE, TBA, or possible MTBE-derived conjugates. Quantification of HBA and MPD was performed by NMR for two urine samples (collected 20 h after exposure). One subject had 11% HBA and 1% MPD, and the other individual had 3% HBA and 1% MPD in the urine, expressed as a percentage of MTBE uptake. This indicates that HBA and MPD occur at significantly higher levels in the urine (detected by NMR) than MTBE and TBA (detected by GC). To our knowledge, this is the first characterization of MTBE metabolites, other than TBA, in humans. Further urine, blood, and expired air were collected up to 22 h after exposure, and the toxicokinetics of MTBE, TBA, and acetone were determined by GC. Low relative uptake (39%), a low level of postexposure exhalation of MTBE (17%), and low recovery of TBA in the urine (<1%) were observed. The same subjects had previously been exposed to unlabeled MTBE in a whole-body exposure study [Nihlén, A., et al. (1998) Toxicol. Appl. Pharmacol. 148, 274-280], and the toxicokinetics of MTBE and TBA in this facemask exposure did not differ from the previous whole-body chamber exposure.

The oxygenate tert-amyl methyl ether (TAME) is a gasoline fuel additive used to reduce carbon monoxide in automobile emissions. To evaluate the relative health risk of TAME as a gasoline additive, information is needed on its pharmacokinetics and toxicity. The objective of this study was to use a physiologically-based pharmacokinetic (PBPK) model to describe the disposition of TAME and its major metabolite, tert-amyl alcohol (TAA), in male Fischer-344 rats. The model compartments for TAME and TAA were flow-limited. The TAME physiological model had 6 compartments: lung, liver, rapidly perfused tissues, slowly perfused tissues, fat, and kidney. The TAA model had 3 compartments: lung, liver, and total-body water. The 2 models were linked through metabolism of TAME to TAA in the liver. Model simulations were compared with data on blood concentrations of TAME and TAA taken from male Fischer-344 rats during and after a 6-hour inhalation exposure to 2500, 500, or 100 ppm TAME. The PBPK model predicted TAME pharmacokinetics when 2 saturable pathways for TAME oxidation were included. The TAA model, which included pathways for oxidation and glucuronide conjugation of TAA, underpredicted the experimental data collected at later times postexposure. To account for biological processes occurring during this time, three hypotheses were developed: nonspecific binding of TAA, diffusion-limited transport of TAA, and enterohepatic circulation of TAA glucuronide. These hypotheses were tested using three different model structures. Visual inspection and statistical evaluation involving maximum likelihood techniques indicated that the model incorporating nonspecific binding of TAA provided the best fit to the data. A correct model structure, based upon experimental data, statistical analyses, and biological interpretation, will allow a more accurate extrapolation to humans and, consequently, a greater understanding of human risk from exposure to TAME.

The purpose of this study was to examine the feasibility of using 13C NMR spectroscopy to analyze urinary metabolites produced following coadministration of two structurally similar carbon-13-labeled compounds to rodents. Acrylonitrile (AN) and acrylamide (AM) are used in the chemical industry to manufacture plastics and polymers. These compounds are known to produce carcinogenic, reproductive, or neurotoxic effects in laboratory animals. The potential for human exposure to AN and AM occurs in manufacturing facilities and environmentally. Male F344 rats and B6C3F1 mice were coadministered po [1,2,3-13C]AN (16-17 mg/kg) and [1,2,3-13C]AM (21-22 mg/kg) after 0 or 4 days of administration of unlabeled AN or AM. Urine was collected for 24 h following administration of the 13C-labeled compounds and analyzed by 13C NMR spectroscopy. Rats and mice excreted metabolites derived from glutathione (GSH) conjugation with AM or AN or derived from GSH conjugation with the epoxides cyanoethylene oxide (CEO) or glycidamide (GA). GA and its hydrolysis product were also detected in the urine of rats and mice. For mice, an increased urinary excretion of total AN- and total AM-derived metabolites (p < 0.05) on repeated coadministration suggested a possible increase in metabolism via oxidation. In addition, mice had an increased (p < 0.05) percentage of dose excreted as metabolites derived from GSH conjugation with AM, AN, CEO, or GA after five exposures as compared with one exposure that may be related to a significant increase in the synthesis of GSH or an increase in glutathione transferase activity. The only significant (p < 0.05) increase between one and five exposures for the rat was in the percentage of metabolites produced following conversion of AM to GA. The use of 13C NMR spectroscopy has provided a powerful methodology for elucidation of the metabolism of two 13C-labeled chemicals administered simultaneously.

Styrene is used for the manufacture of plastics and polymers. The metabolism and hepatotoxicity (mice only) of styrene was compared in male B6C3F1 mice, CD-1 mice, and F344 rats to evaluate biochemical mechanisms of toxicity. Rats and mice were exposed to 250 ppm styrene for 6 h/day for 1 to 5 days, and liver (mice only) and blood were collected following each day of exposure. Mortality and increased serum alanine aminotransferase (ALT) activity were observed in mice but not in rats. Hepatotoxicity in B6C3F1 mice was characterized by severe centrilobular congestion after one exposure followed by acute centrilobular necrosis. Hepatotoxicity was delayed by 1 day in CD-1 mice, and the increase in ALT and degree of necrosis was less than observed for B6C3F1 mice. Following exposure to unlabeled styrene for 0, 2, or 4 days, rats and mice were exposed to [7-14C]-styrene (60 microCi/mmol) for 6 h. Urine, feces, and expired air were collected for up to 48 h. Most styrene-derived radioactivity was excreted in urine. The time-course of urinary excretion indicates that rats and CD-1 mice eliminated radioactivity at a faster rate than B6C3F1 mice following a single 250 ppm exposure, consistent with a greater extent of liver injury for B6C3F1 mice. The elimination rate following 3 or 5 days of exposure was similar for rats and both mouse strains. Following three exposures, the total radioactivity eliminated in excreta was elevated over that measured for one exposure for both mouse strains. An increased excretion of metabolites on multiple exposure is consistent with the absence of ongoing acute necrosis following 4 to 5 daily exposures. These data indicate that an induction in styrene metabolism occurs after multiple exposures, resulting in an increased uptake and/or clearance for styrene.

1,3-Butadiene (BD) is a carcinogen in rats and mice. Previous in vitro studies showed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. Blood and tissue levels of BDE were significantly lower in rats than in mice following exposure to BD. Since mice are much more susceptible to cancer induced by BD than rats, these findings suggest a key role for BDE in BD-induced carcinogenicity. The aim of this study was to characterize the glutathione (GSH) conjugation of BDE by cytosol from human liver and mouse and rat liver and lung in vitro. BDE and radiolabeled GSH were incubated with cytosol. Conjugates were identified by 13C-NMR and FAB mass spectroscopy and quantitated by HPLC. The enzyme kinetics for the conjugation of BDE with GSH suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in GSH conjugation of BDE.

1,3-Butadiene (BD) is used in the production of synthetic rubber and other resins. Carcinogenic effects have been observed in laboratory animals exposed to BD, with mice being more sensitive than rats. Metabolic oxidation of butadiene to epoxides is believed to be a crucial step in the initiation of tumors by BD. However, limited information is available that describes the in vivo metabolism of BD. Male Sprague-Dawley rats and B6C3F1 mice were exposed to 800 ppm [1,2 3,4-13C]butadiene for 5 h, and urine was collected during and for 20 h following exposure. Urinary metabolites were characterized using 1- and 2-dimensional methods of NMR spectroscopy. Three metabolites previously detected in vivo, N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine, N-acetyl-S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine, and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine, were present in both rat and mouse urine, accounting for 87% and 73% of the total metabolites excreted, respectively. A fourth metabolite, previously detected in vitro, 3-butene-1,2-diol, was also present in both rat and mouse urine and comprised 5% and 3% of the total metabolites excreted, respectively. An additional metabolite detected only in mouse urine that is derived from glutathione conjugation with epoxybutene was identified as S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine (4%). N-Acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine (4%), detected in mouse urine, is a thiohemiacetal product of 3-butenal. Additionally, mice excreted N-acetyl-S-(3-hydroxypropyl)-L-cysteine (5%) and N-acetyl-S-(2-carboxyethyl)-L-cysteine (5%), which could be derived from further metabolism of N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine or from glutathione conjugation with acrolein. Mice excreted N-acetyl-S-(1-(hydroxymethyl)-3,4-dihydroxypropyl)-L-cysteine (5%), which could be derived from glutathione conjugation with diepoxybutane (BDE), while rats excreted 1,3-dihydroxypropanone (5%), which may be derived from hydrolysis of BDE. These studies indicate that reactive aldehydes are produced as metabolites of BD in vivo, in addition to the reactive monoepoxide and diepoxide of BD. The greater toxicity of BD in mice compared with rats may be attributed to the greater ability of rats to detoxify BDE via hydrolysis, and/or to the production of reactive aldehydes.

1,3-Butadiene (BD) has been classified as a probable human carcinogen based on sufficient evidence of a carcinogenic response in B6C3F1 mice and Sprague-Dawley rats and limited human evidence of carcinogenicity. Mice are much more susceptible to BD-induced carcinogenicity than rats. Previous in vitro studies revealed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. BDE was also readily quantitated in blood and tissues of mice exposed to BD but could not be detected in rats similarly exposed. These findings suggest a key role for BDE in BD-induced carcinogenicity. The purpose of this study was to characterize the glutathione (GSH) conjugation of BDE by liver and lung cytosol from B6C3F1 mice and Sprague-Dawley rats and human liver cytosol from six different individuals in vitro. BDE and glycine-[2-3H]GSH were incubated, at pH 7.4, with cytosol. 13C NMR and mass spectral analysis indicated formation of two isomeric conjugates, S-(1-(hydroxy-methyl)-2,3-epoxypropyl)glutathione and S-(2-hydroxy-3,4-epoxy--butyl)glutathione, which were rapidly hydrolyzed in cytosol to the corresponding trihydroxy conjugates. Total conjugates were quantitated by HPLC. Conjugation of BDE with GSH followed Michaelis-Menten kinetics in human as well as rat and mouse cytosolic fractions. The conjugation rates in mouse and rat liver cytosol were similar (Vmax 162 +/- 16 and 186 +/- 37 nmol/mg protein/min, respectively) and an order of magnitude higher than in human liver cytosol (Vmax 6.4 +/- 1.9 nmol/mg protein/min). the apparent KM values were lower in human (2.1 +/- 1.4 mM) than mouse (6.4 +/- 1.6 mM) or rat (24 +/- 6 mM) liver. Mouse lung cytosol (Vmax 38.5 +/- 2.5 nmol/mg protein/min, KM 1.70 +/- 0.37mM) is also more efficient in GSH conjugation than rat lung cytosol (Vmax 17.1 +/- 3.0 nmol/mg protein/min, KM +/- 1.7 mM). These results suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in hepatic or pulmonary GSH conjugation of BDE. Also, considering the low oxidation rates of BD to BMO and of BMO to BDE in humans as compared to mice, the relatively low capacity of GSH conjugation of BDE in human liver will not necessarily lead to increased BDE blood levels in humans potentially exposed to BD.

Cyanoethylene oxide (CEO), a putative toxic and carcinogenic metabolite of acrylonitrile, is a direct-acting mutagen. The focus of this study was to elucidate potential adducts responsible for the mutagenic effect of CEO by characterizing products from the reaction of CEO with nucleotides. The reaction of CEO with the 5'-monophosphates of deoxyguanosine, deoxyadenosine, deoxycytidine or deoxythymidine resulted in the formation of at least one adduct for each nucleotide. Using two-dimensional NMR spectroscopy and fast atom bombardment mass spectrometry, CEO-nucleotide adducts (approximately 25% modification) were characterized as 2-cyano-2-hydroxyethyl phosphodiesters. The isolate from the reaction of deoxyguanosine-5'-monophosphate (dGMP) with CEO contained a second adduct, identified as N7-(2-cyano-2-hydroxyethyl)-dGMP. Single and double strand breaks, which were observed in supercoiled pBR322 plasmid DNA exposed to CEO (> 50 mM), may arise following formation of cyanohydroxyethyl phosphotriester adducts. The characterization of these phosphodiester adducts in vitro may provide insight into the intermediates responsible for the genotoxic effect of CEO in vivo.

The dose dependence of the urinary excretion of acrylonitrile (ACN) metabolites was studied after oral administration of [2,3-14C]ACN to male F-344 rats (0.09 to 28.8 mg/kg) and male B6C3F1 mice (0.09 to 10.0 mg/kg). Urine was the major route of excretion of ACN metabolites (77 to 104% of the dose), with less than 8% of the dose excreted in the feces. Reverse-phase HPLC analysis of urine from treated animals indicated five major components (1 through 5 in order of elution) that accounted for 75 to 100% of the total urinary radioactivity. Component 4 was observed in the urine of ACN-treated mice but was only present in trace amounts in the urine of ACN-treated rats. Components 1, 2, and 3 were present in the urine of animals administered [2,3-14C]cyanoethylene oxide (CEO), indicating that these components were derived from the epoxide metabolite of ACN. The ACN urinary metabolites were isolated by HPLC and identified by chromatographic and mass spectral analysis. Component 5 was N-acetyl-S-(2-cyanoethyl)cysteine and component 4 was S-(2-cyanoethyl)thioacetic acid, both derived from the glutathione (GSH) conjugate of ACN. Component 3 contained N-acetyl-S-(2-hydroxyethyl)cysteine, N-acetyl-S-(carboxymethyl)cysteine, and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine. Component 2 was thiodiglycolic acid. These urinary metabolites are derived from catabolism of the GSH conjugates of CEO. The polar component 1 was not identified. These results demonstrate that GSH conjugation is the major disposition pathway of ACN. The excretion of metabolites derived from CEO was an approximately linear function of dose in both species, whereas the excretion of N-acetyl-S-(2-cyanoethyl)cysteine increased nonlinearly with dose. This nonlinearity indicates the presence of a saturable pathway competing with glutathione for ACN, most likely the cytochrome P450-dependent oxidation of ACN. Thiodiglycolic acid was formed 10-fold more in mice than in rats, but this species difference in the oxidative processing of GSH conjugates is probably not of toxicological significance. The ratio of ACN epoxidation to GSH conjugation was 0.50 in rats and 0.67 in mice. This species difference in ACN oxidation could have important toxicological implications, since CEO is believed to mediate the carcinogenic effects of ACN.

Small amounts (6-12%) of radioactivity administered by gavage as 14C-labeled 2-methoxyethanol (2-ME) or 2-methoxyacetic acid (2-MAA) to pregnant mice are exhaled as 14CO2 as well as accumulated in tissues that are highly active in the synthesis of macromolecules (Sleet et al., Toxicol. Appl. Pharmacol. 84, 25-35, 1986; Mebus et al., Toxicol. Appl. Pharmacol. 112, 87-94, 1992). In addition, pregnant CD-1 mice similarly administered 13C-labeled 2-ME excrete urinary metabolites that may arise from incorporation of a coenzyme A thioester of 2-MAA into the Krebs cycle, forming methoxycitrate (Sumner et al., Chem. Res. Toxicol. 5, 553-560, 1992). Based on these previously published observations, we propose a mechanism for the further metabolism of methoxycitrate that is consistent with the detection of 14CO2 after administering either [1-14C]2-MAA, [2-14C]2-ME, or [methoxy-14C]2-ME to mice. This postulated pathway may also explain the tissue-specific accumulation of radioactivity arising from [14C]2-ME.

The conformational behavior in solution of two receptor selective tachykinin agonists, senktide (succinyl-D-F-MeF-G-L-M-NH2) and septide (pQ-F-F-P-L-M-NH2) is described. Two dimensional cross relaxation NMR spectroscopy is used together with coupling constant data to obtain interproton distance constraints. These results are used in conjunction with semi-empirical energy computations to indicate favorable conformations. Senktide is found to have a high degree of conformational order which is attributed to rotational restriction associated with the N-methylation of phenylalanine. The lowest energy conformation in accord with the experimental interproton distances contains a beta-turn. Interproton distances indicate that septide exists as a random coil or in an extended chain conformation. Energy computations suggest that septide is primarily an extended chain with internal reorientation restricted by the proline residue. These results may be related to the selectivity of these peptides for different receptors, in that the analogs, with conformations more stable than tachykinins, are more receptor selective.

2-Methoxyethanol (2-ME) is an industrial solvent that induces developmental and testicular toxicity in laboratory animals. Oxidation of 2-ME to 2-methoxyacetic acid (2-MAA) is required for the generation of these adverse effects. The urinary metabolites of 2-ME were investigated to characterize the fate of 2-ME and 2-MAA. 13C NMR spectroscopy was used to detect and assign metabolites in the urine of pregnant CD-1 mice following administration of 250 mg/kg of [1,2,methoxy-13C]-2-ME. Two-dimensional NMR methods were used to correlate signals from the labeled carbons in each 2-ME metabolite and to determine the number of hydrogens attached to each carbon. Structures were assigned from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison to standards. Pathways involved in forming metabolites assigned in this study include transformation of 2-ME via ethylene glycol, conjugation with glucuronide or sulfate, and oxidation to 2-MAA. Additional metabolites were assigned that can be formed from further conversion of 2-MAA to glycine and glucuronide conjugates, as well as metabolites derived from the incorporation of 2-methoxyacetyl CoA derivatives into intermediary metabolism. Elucidation of the further metabolism of 2-MAA may be important for understanding the mechanisms by which 2-ME induces adverse effects.

Hemoglobin adducts formed by chemical carcinogens can be used as biomarkers of exposure. The kinetics of adduct formation and removal is complex and depends on the processes involved in erythrocyte removal, adduct stability, and the duration and extent of exposure. In order to relate the formation of adducts to the extent of exposure in complex exposure scenarios, a model has been developed to describe the kinetics of accumulation and removal of adducts formed in vivo. The exposure scenario, lifetime of erythrocytes, and extent of adduct formation following a single exposure are required input parameters. Predictions of adduct accumulation have been generated for a wide variety of exposure scenarios and compared with both the solutions to equations derived for adduct formation and removal and experimental observations. Loss of adduct by removal of erythrocytes from circulation, both by senescence and random removal and as a result of chemical instability, has been simulated. Equations have been derived to describe the removal of hemoglobin adducts under conditions of exposure for less than the lifetime of the erythrocyte, when removal is initially a linear function of time. This model makes possible the comparison of data obtained from different exposure scenarios and in different species.

Acrylamide, widely used for the production of polymers and as a grouting agent, causes neurotoxic effects in humans and neurotoxic, genotoxic, reproductive, and carcinogenic effects in laboratory animals. In this study, 13C NMR spectroscopy was used to detect metabolites of acrylamide directly in the urine of rats and mice following administration of [1,2,3-13C]acrylamide (50 mg/kg po). Two-dimensional NMR experiments were used to correlate carbon signals for each metabolite in the urine samples and to determine the number of hydrogens attached to each carbon. Metabolite structures were identified from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison with standards. The metabolites assigned in rat and mouse urine are N-acetyl-S-(3-amino-3-oxopropyl)cysteine, N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine, N-acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)cysteine, glycidamide, and 2,3-dihydroxypropionamide. These metabolites arise from direct conjugation of acrylamide with glutathione or from oxidation to the epoxide, glycidamide, and further metabolism. Acrylamide was also detected in the urine. Quantitation was carried out by integrating the metabolite carbon signals with respect to that of dioxane added at a known concentration. The major metabolite for both the rat (70% of total metabolites excreted) and the mouse (40%) was formed from direct conjugation of acrylamide with glutathione. The remaining metabolites for the rat (30%) and mouse (60%) are derived from glycidamide. The species differences in extent of metabolism through glycidamide may have important consequences for the toxic and carcinogenic effects of acrylamide.

Acrylonitrile, a carcinogen in rats, undergoes extensive metabolism via two routes: direct glutathione conjugation or epoxidation. Metabolism to cyanoethylene oxide may mediate the carcinogenic and toxic activity of acrylonitrile. To characterize comprehensively the metabolism in vivo of acrylonitrile, the detection and identification of metabolites in urine of rodents dosed with acrylonitrile have been carried out using NMR spectroscopy. Following administration of [1,2,3-13C]acrylonitrile to male Fisher 344 rats (10 or 30 mg/kg, po) or B6C3F1 mice (10 mg/kg, po), urine samples were collected for 24 h. Carbon-13 NMR spectra were acquired directly on the urine samples after centrifugation and addition of 10-25% D2O. Resonances were assigned to carbons of acrylonitrile metabolites on the basis of chemical shift, proton multiplicity, carbon-carbon coupling, and calculated values of shift, and by comparison with standards. The proton multiplicity of each carbon was determined by heteronuclear 2D J-resolved spectroscopy (HET2DJ), and the carbon-carbon connectivities of resonances were determined using incredible natural abundance double quantum transfer spectroscopy (INADEQUATE). The metabolites identified in rat urine were thiocyanate, N-acetyl-S-(2-cyanoethyl)cysteine, N-acetyl-S-(2-hydroxyethyl)cysteine, N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine, thiodiglycolic acid, thionyldiacetic acid, and S-(carboxymethyl)cysteine or its N-acetyl derivative. These metabolites were also identified in mouse urine. Metabolites were quantitated by integrating metabolite carbon resonances with respect to that of dioxane added at a known concentration. Thiodiglycolic acid and (carboxymethyl)cysteine (or its N-acetyl derivative) were the major metabolites in the mouse, while N-acetyl-S-(2-cyanoethyl)cysteine and N-acetyl-S-(2-hydroxyethyl)cysteine were the major metabolites in the rat. Metabolites derived from cyanoethylene oxide (CEO) accounted for approximately 60% of the products excreted in rat urine, compared with 80% in the urine from mice. Differences between rat and mouse in the further metabolism of CEO were also observed. The proportion of the dose metabolized via CEO may be an important determinant of the toxicity and carcinogenicity of acrylonitrile.

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.

A receptor selective linear hexapeptide tachykinin analog, senktide, is shown to be highly ordered in solution. The conformational restriction is attributed to steric and electrostatic interactions produced by N-methylation of the third amino acid residue in the sequence and the negatively charged N-terminus. The structure of senktide is described as a dynamic mixture of similar conformations where the predominant one is a distorted antiparallel hydrogen bonded beta-pleated sheet. The observed senktide-receptor specificity is suggested to result from a selection of this or a closely related conformation.