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.

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Acrylamide (AA) is an animal carcinogen, neurotoxin, and reproductive toxin. AA is formed in baked and fried carbohydrate-rich foods. Metabolism of AA occurs via epoxidation to glycidamide (GA) or direct conjugation with glutathione. Using CYP2E1-null mice, recent studies in this laboratory demonstrated that induction of somatic and germ cell mutagenicity in AA-treated mice is dependent on CYP2E1. We hypothesized that AA metabolism to GA is a prerequisite for the induction of AA-induced mutagenicity. Current studies were undertaken to assess the role of CYP2E1 in the epoxidation of AA to GA and the formation of DNA and hemoglobin (HGB) adducts. AA was administered to CYP2E1-null or wild-type mice at 50 mg/kg ip. Mice were euthanized 6 h later and blood and tissues were collected. Using LC-ES/MS/MS, AA, GA, and DNA- and HGB-adducts were measured. While the plasma levels of AA and GA were 115 +/- 14.0 and 1.7 +/- 0.31 microM in CYP2E1-null mice, they were 0.84 +/- 0.80 and 33.0 +/- 6.3 microM in the plasma of AA-treated wild-type mice. Administration of AA to wild-type mice caused a large increase in N7-GA-Gua and N3-GA-Ade adducts in the liver, lung, and testes. While traces of N7-GA-Gua adducts were measured in the tissues of AA-treated CYP2E1-null mice, these levels were 52- to 66-fold lower than in wild-type mice. Significant elevation of both AA- and GA-HGB adducts was detected in AA-treated wild-type mice. In AA-treated CYP2E1-null mice, levels of AA-HGB adducts were roughly twice as high as those in wild-type mice. In conclusion, current work demonstrated that CYP2E1 is the primary enzyme responsible for the epoxidation of AA to GA, which leads to the formation of GA-DNA and HGB adducts.

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.

Prioritizing the risk posed by thousands of chemicals potentially present in the environment requires exposure, toxicity, and toxicokinetic (TK) data, which are often unavailable. Relatively high throughput, in vitro TK (HTTK) assays and in vitro-to-in vivo extrapolation (IVIVE) methods have been developed to predict TK, but most of the in vivo TK data available to benchmark these methods are from pharmaceuticals. Here we report on new, in vivo rat TK experiments for 26 non-pharmaceutical chemicals with environmental relevance. Both intravenous and oral dosing were used to calculate bioavailability. These chemicals, and an additional 19 chemicals (including some pharmaceuticals) from previously published in vivo rat studies, were systematically analyzed to estimate in vivo TK parameters (e.g., volume of distribution [Vd], elimination rate). For each of the chemicals, rat-specific HTTK data were available and key TK predictions were examined: oral bioavailability, clearance, Vd, and uncertainty. For the non-pharmaceutical chemicals, predictions for bioavailability were not effective. While no pharmaceutical was absorbed at less than 10%, the fraction bioavailable for non-pharmaceutical chemicals was as low as 0.3%. Total clearance was generally more under-estimated for nonpharmaceuticals and Vd methods calibrated to pharmaceuticals may not be appropriate for other chemicals. However, the steady-state, peak, and time-integrated plasma concentrations of nonpharmaceuticals were predicted with reasonable accuracy. The plasma concentration predictions improved when experimental measurements of bioavailability were incorporated. In summary, HTTK and IVIVE methods are adequately robust to be applied to high throughput in vitro toxicity screening data of environmentally relevant chemicals for prioritizing based on human health risks.

Antagonists of cannabinoid receptor 1 (CB1) have potential for the treatment of several diseases such as obesity, liver disease and diabetes. Recently, development of several CB1 antagonists was halted due to adverse central nervous system (CNS) related side effects observed with rimonabant, the first clinically approved CB1 inverse agonist. However, recent studies indicate that regulation of peripherally expressed CB1 with CNS-sparing compounds is a viable strategy to treat several important disorders. Our efforts aimed at rationally designing peripherally restricted CB1 antagonists have resulted in compounds that have limited blood-brain barrier (BBB) permeability and CNS exposure in preclinical in vitro and in vivo models. Typically, compounds with high topological polar surface areas (TPSAs) do not cross the BBB passively. Compounds with TPSAs higher than rimonabant (rimonabant TPSA = 50) and excellent functional activity with limited CNS penetration were identified. These compounds will serve as templates for further optimization.

CUMYL-PICA [1-pentyl-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide] and 5F-CUMYL-PICA [1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide] are recently identified recreationally used/abused synthetic cannabinoids, but have uncharacterized pharmacokinetic profiles and metabolic processes. This study characterized clearance and metabolism of these compounds by human and rat liver microsomes and hepatocytes, and then compared these parameters with in vivo rat plasma and urine sampling. It also evaluated hypothermia, a characteristic cannabimimetic effect. Incubation of CUMYL-PICA and 5F-CUMYL-PICA with rat and human liver microsomes suggested rapid metabolic clearance, but in vivo metabolism was prolonged, such that parent compounds remained detectable in rat plasma 24 h post-dosing. At 3 mg/kg (intraperitoneally), both compounds produced moderate hypothermic effects. Twenty-eight metabolites were tentatively identified for CUMYL-PICA and, coincidentally, 28 metabolites for 5F-CUMYL-PICA, primarily consisting of phase I oxidative transformations and phase II glucuronidation. The primary metabolic pathways for both compounds resulted in the formation of identical metabolites following terminal hydroxylation or dealkylation of the N-pentyl chain for CUMYL-PICA or of the 5-fluoropentyl chain for 5F-CUMYL-PICA. These data provide evidence that in vivo elimination of CUMYL-PICA, 5F-CUMYL-PICA and other synthetic cannabinoids is delayed compared to in vitro modeling, possibly due to sequestration into adipose tissue. Additionally, the present data underscore the need for careful selection of metabolites as analytical targets to distinguish between closely related synthetic cannabinoids in forensic settings.

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.