Plants containing thujone have widespread use and hence have significant human exposure. α-Thujone caused seizures in rodents following gavage administration. We investigated the toxicokinetics of α-thujone in male and female F344/N rats and B6C3F1 mice following intravenous and gavage administration of α-thujone or a mixture of α- and β-thujone (which will be referred to as α,β-thujone). Absorption of α-thujone following gavage administration was rapid without any dose-, species-, sex- or test article-related effect. Absolute bioavailability of α-thujone following administration of α-thujone or α,β-thujone was generally higher in rats than in mice. In rats, females had higher bioavailability than males following administration of either test article although a sex difference was not observed in mice. Cmax and AUC∞ increased greater than proportional to the dose in female rats following administration of α-thujone and in male and female mice following administration of α,β-thujone suggesting possible saturation of elimination kinetics with increasing dose. Dose-adjusted AUC∞ for male and female rats was 5- to 15-fold and 3- to 24-fold higher than mice counterparts following administration of α-thujone and α,β-thujone, respectively (p-value<0.0001 for all comparisons). Following both intravenous and gavage administration, α-thujone was distributed to the brains of rats and mice with females, in general, having higher brain:plasma ratios than males. These data are in support of the observed toxicity of α-thujone and α,β-thujone where females were more sensitive than males of both species to α-thujone-induced neurotoxicity. In general there was no difference in toxicokinetics between test articles when normalized to α-thujone concentration.

1. Methyleugenol (MEG) has been used as a flavouring agent in food, as a fragrance in cosmetic products, and as an insect attractant. MEG was carcinogenic in both rats and mice following gavage administration. In this study we investigated plasma toxicokinetics of MEG in F344 rats and B6C3F1 mice of both sexes following single gavage (37, 75, or 150 mg/kg) and intravenous (IV) (37 mg/kg) administration. 2. Following IV administration, MEG was rapidly distributed and cleared from the systemic circulation in both species and sexes. Absorption of MEG was rapid following gavage administration with secondary peaks in the plasma MEG concentration-versus-time profiles. Cmax and AUCT increased and the clearance decreased greater than proportional to the dose in rats and mice of both sexes. In general, rats had higher internal exposure to MEG than mice. 3. The results for AUCT and clearance suggest that perhaps the metabolism of MEG is saturated at higher doses tested in this study. Absolute bioavailability following gavage administration of 37 mg/kg was low in both rats (~4%) and mice (7–9%) of both sexes indicating extensive first-pass metabolism. There was no sex difference in plasma toxicokinetics of MEG following gavage administration both in rats and mice.

1. Isoeugenol (IEG) has been tested for toxicity and carcinogenicity due to high potential for human exposure and the structural resemblance to known carcinogenic allylbenzenes. In order to support the interpretation of toxicity and carcinogenecity study outcomes, a toxicokinetic study was performed in which both sexes of F344 rats and B6C3F1 mice were given IEG as a single intravenous (IV) or gavage administration. 2. Following IV administration, IEG was rapidly eliminated from systemic circulation in both species and sexes. Gavage administration revealed a rapid absorption of IEG with tmax values ≤20 min for both species and sexes. In rats, AUC increased in a greater than dose-proportional manner and Clapp values decreased with increasing dose in both sexes suggesting saturation of IEG metabolism. On the other hand, Clapp values in male mice increased with increasing dose suggesting induction of IEG metabolism although this was not evident in the females. 3. Absolute bioavailability was greater in female rats (19%) than male rats (10%) (p < 0.0001), but was not different between the sexes for mice (28% males; 31% females) (p = 0.2437). The collective toxicokinetic data supported that low bioavailability following administration of IEG was the result of extensive first-pass metabolism.

In the National Toxicology Program's toxicity studies, rats were more sensitive than mice to Bis(2-chloroethoxy)methane (CEM) - induced cardiac toxicity following dermal application to male and female F344/N rats and B6C3F1 mice. Thiodiglycolic acid (TOGA) is a major metabolite of CEM in rats. It has been implicated that chemicals metabolized to TOGA cause cardiac toxicity in humans. Therefore, the toxicokinetics of CEM and TOGA were investigated in male and female F344/N rats and B6C3F1 mice following a single intravenous administration or dermal application of CEM to aid in the interpretation of the toxicity data. Absorption of CEM following dermal application was rapid in both species and genders. Bioavailability following dermal application was low but was higher in rats than in mice with females of both species showing higher bioavailability than males. CEM was rapidly distributed to the heart, thymus, and liver following both routes of administration. Plasma CEM C(max) and AUc(infinity) increased proportionally with dose, although at the dermal dose of 400 mg/kg in rats and 600 mg/kg in mice non-linear kinetics were apparent. Following dermal application, dose-normalized plasma CEM C(max) and AUC(infinity) was significantly higher in rats than in mice (p-value <0.0001 for all comparisons except for Cmax in the highest dose groups where p-value = 0.053). In rats, dose-normalized plasma CEM C(max) and AUC(infinity) was higher in females than in males: however, the difference was significant only at the lowest dose (p-value = 0.009 for C(max) and 0.056 for AUC(infinity)). Similar to rats, female mice also showed higher C(max) and AUC(infinity) in females than in male: the difference was significant only for C(max) at the lowest dose (p-value = 0.002). Dose-normalized heart CEM C(max) was higher in rats than in mice and in females than their male counterparts. The liver CEM C(max) was lower compared to that of heart and thymus in both rats and mice following intravenous administration and in rats following dermal application. This is likely due to the rapid metabolism of CEM in the liver as evidenced by the high concentration of TDGA measured in the liver. Dose-normalized plasma and heart TDGA C(max) values were higher in rats compared to mice. In rats, females had higher plasma and heart TDGA C. than males; however, there was no gender difference in plasma or heart TOGA C(max) in mice. These findings support the increased sensitivity of rats compared to mice to CEM-induced cardiac toxicity. Data also suggest that, either CEM C(max) or AUC can be used to predict the CEM-induced cardiac toxicity. Although, both plasma and heart TDGA C(max) was consistent with the observed species difference and the gender difference in rats, the gender difference in mice to cardiac toxicity could not be explained based on the TDGA data. This animal study suggests that toxicologically significant concentrations of CEM and TOGA could possibly be achieved in the systemic circulation and/or target tissues in humans as a result of dermal exposure to CEM. Published by Elsevier Ireland Ltd.

Identification of carcinogenic activity is the primary goal of the 2-year bioassay. The expense of these studies limits the number of chemicals that can be studied and therefore chemicals need to be prioritized based on a variety of parameters. We have developed an ensemble of support vector machine classification models based on male F344 rat liver gene expression following 2, 14 or 90 days of exposure to a collection of hepatocarcinogens (aflatoxin B1, 1-amino-2,4-dibromoanthraquinone, N-nitrosodimethylamine, methyleugenol) and non-hepatocarcinogens (acetaminophen, ascorbic acid, tryptophan). Seven models were generated based on individual exposure durations (2, 14 or 90 days) or a combination of exposures (2+14, 2+90, 14+90 and 2+14+90 days). All sets of data, with the exception of one yielded models with 0% cross-validation error. Independent validation of the models was performed using expression data from the liver of rats exposed at 2 dose levels to a collection of alkenylbenzene flavoring agents. Depending on the model used and the exposure duration of the test data, independent validation error rates ranged from 47% to 10%. The variable with the most notable effect on independent validation accuracy was exposure duration of the alkenylbenzene test data. All models generally exhibited improved performance as the exposure duration of the alkenylbenzene data increased. The models differentiated between hepatocarcinogenic (estragole and safrole) and non-hepatocarcinogenic (anethole, eugenol and isoeugenol) alkenylbenzenes previously studied in a carcinogenicity bioassay. In the case of safrole the models correctly differentiated between carcinogenic and non-carcinogenic dose levels. The models predict that two alkenylbenzenes not previously assessed in a carcinogenicity bioassay, myristicin and isosafrole, would be weakly hepatocarcinogenic if studied at a dose level of 2 mmol/kg bw/day for 2 years in male F344 rats; therefore suggesting that these chemicals should be a higher priority relative to other untested alkenylbenzenes for evaluation in the carcinogenicity bioassay. The results of the study indicate that gene expression-based predictive models are an effective tool for identifying hepatocarcinogens. Furthermore, we find that exposure duration is a critical variable in the success or failure of such an approach, particularly when evaluating chemicals with unknown carcinogenic potency.

We conducted an interlaboratory study which differed from the typical study of this type because of its emphasis on comparing intralaboratory variability in results. We sent specimens to six laboratories experienced in the analysis of perfluorinated alkyl compounds in blood matrices and that use stringent procedures to control and assure accuracy and precision. Each received an identical set of 60 plasma specimens that were analyzed in six completely independent batches. Split specimens were included so that within- and between-batch coefficients of variation could be calculated. All laboratories used liquid chromatography–tandem mass spectrometry (LC–MS/MS). The concentrations of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorohexanesulfonate (PFHxS) measured in the specimens in general showed a high level of agreement, although in some cases the agreement was only moderate. The average within- and between-batch coefficient of variation for PFOS was 9.1% and 9.3%; for PFOA was 14.5% and 14.5%; and for PFHxS was 14.5% and 17.0%. The recent availability of labeled internal standards, among other advances, has facilitated improvement in the accuracy and precision of the assays. Considering the degree of between-subject variation in levels among people in background-exposed populations, the results indicate that biomarker-based epidemiologic studies of associations with health could have reasonable precision.

BACKGROUND: The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects.OBJECTIVE: The National Toxicology Program and the National Institutes of Health Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicologic evaluation.METHODS: A collection of 1,408 compounds previously tested in one or more traditional toxicologic assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics.RESULTS: qHTS of these compounds produced robust and reproducible results, which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited species- or cell type-specific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time dependence in kinetic studies, consistent with known mechanisms of toxicity.CONCLUSIONS: The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicologic evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response.

There has been considerable interest in the use of small fish models for detecting potential environmental carcinogens. In this study, both guppies (Poecilia reticulata) and medaka (Oryzias latipes) were exposed in the aquaria water to three known rodent carcinogens for up to 16 months. Nitromethane, which caused mammary gland tumors by inhalation exposure in female rats, harderian gland and lung tumors in male and female mice, and liver tumors in female mice by inhalation, failed to increase tumors in either guppies or medaka. Propanediol, which when given in the feed was a multisite carcinogen in both sexes of rats and mice, caused increased liver tumors in male guppies and male medaka. There was reduced survival in female guppies and no increased tumors in female medaka. 1,2,3-Trichloropropane, which when administered by oral gavage was a multisite carcinogen in both sexes of rats and mice, caused an increased incidence of tumors in the liver of both male and female guppies and medaka and in the gallbladder of male and female medaka. The results of this study demonstrate that for these three chemicals, under these specific exposure conditions, the fish appear less sensitive and have a narrower spectrum of tissues affected than rodents. These results suggest that fish models are of limited utility in screening unknown chemicals for potential carcinogenicity.

Toxic metals occur naturally at low concentrations throughout the environment, but are found in higher concentrations at many of the hazardous waste sites on the EPA Superfund list. As part of the Agency for Toxic Substances and Disease Registry (ATSDR) mandate to evaluate the toxicity of metals and mixtures, we chose four of the high-priority metal pollutants from ATSDR's HAZDAT list, including arsenic, cadmium, chromium, and lead, to test in a commercially developed assay system, CAT-Tox(L) (Xenometrix). This assay employs a battery of recombinant HepG2 cell lines to test the transcriptional activation capacity of xenobiotics in any of 13 different signal transduction pathways. Our specific aims were to identify metal-responsive promoters and determine whether the pattern of gene expression changed with a mixture of metals. Humic acid was used in all assays as a carrier to help solubilize the metals and, in all cases, the cells were exposed to the humic acid–metal mixture for 48 h. Humic acid alone, at 50–100 μM, showed moderate activation of the XRE promoter, but little other notable activity. As(V), at doses of 50–250 μM, produced a complex profile of activity showing significant dose-dependent induction of the hMTIIA, GST Ya, HSP70, FOS, XRE, NFκBRE, GADD153, p53RE, and CRE promoters. Pb(II) showed dose-related induction of the GST Ya, XRE, hMTIIA, GRP78, and CYP IA1 promoters at doses in the range of 12–100 μM. Cd(II), at 1.25–15 μM, yielded significant dose-dependent induction of hMTIIA, XRE, CYP IA1, GST Ya, HSP70, NFκBRE, and FOS. Whereas Cr(III) yielded small, though significant inductions of the CRE, FOS, GADD153, and XRE promoters only at the highest dose (750 μM), Cr(VI) produced significant dose-related inductions of the p53RE, FOS, NFκBRE, XRE, GADD45, HSP70, and CRE promoters at much lower doses, in the range of 5–10 μM. Assays testing serial dilutions of a mixture comprising 7.5 μM Cd(II), 750 μM Cr(III), and 100 μM Pb(II) (the combination of metals most frequently found at National Priority List sites) showed significant dose-dependent induction of the hMTIIA promoter, but failed to show dose-related induction of any other promoter and showed no evidence of synergistic activation of gene expression by the metals in this mixture. Our results thus show metal activation of gene expression through several previously unreported signal transduction pathways, including As(V) induction of GST Ya, FOS, XRE, NFkBRE, GADD153, p53RE, and CRE; Pb(II) induction of GST Ya, XRE, Cyp IA1, and GADD153; Cd(II) induction of NFkBRE, Cyp IA1, XRE, and GST Ya; and Cr(VI) induction of p53RE, XRE, GADD45, HSP70, and CRE promoters, and thus suggest new insights into the biochemical mechanisms of toxicity and carcinogenicity of metals. It is also an important finding that no evidence of synergistic activity was detected with the mixture of Cd(II), Cr(III), and Pb(II) tested in these assays.

A quantitative method was developed for determination of alpha(2u)-globulin in urine and kidney samples collected from male rats using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS). Samples prepared from urine and kidney homogenates using size exclusion filters were subject to reversed-phase liquid chromatography and the effluent passed into an electrospray ionization source. Quantitative analysis using external standard calibration was based upon selected ion monitoring of protonated molecular ions by the mass spectrometer. Linear calibration curves were developed over the range of similar to 4.6-370 mu g of alpha(2u)-globulin/mu l for spiked urine standards and over the range of similar to 4.6-550 mu g of alpha(2u)-globulin/mu L for spiked kidney standards. The precision (relative standard deviation) for repeated injection (using urine samples) and intra-assay precision (using both urine and kidney samples) were within +/-10.4% and +/-13.2%, respectively. Using spiked urine standards, inter-assay precision, intra-assay accuracy, and inter-assay accuracy were within +/-20%, +/-20%, and +/-15%, respectively. Using spiked kidney standards, intra-assay accuracy was within +/-15%. The limits of detection (LOD) for the determination of alpha(2u)-globulin in urine and kidney samples were similar to 0.41 pg/nL (1.0 fmol injected) and 25 pg/nL (similar to 13 fmol injected), respectively. The limits of quantitation (LOQ) for determination of alpha(2u)-globulin in urine and kidney samples were calculated as 1.4 pg/nL (3.7 fmol injected) and 83 pg/nL (45 fmol injected), respectively. Applicability of the LC-ESI/MS method was demonstrated by determination of alpha(2u)-globulin in both urine and kidney samples collected from male Fischer 344/N rats dosed intravenously with cis-Decalin at concentrations of 0, 2.5, 5.0, 10, and 20 mg/kg. A dose-dependent relationship was found between the amount of cis-Decalin administered and alpha(2u)-globulin accumulation in kidney samples, whereas no significant change in the urinary levels of alpha(2u)-globulin occurred. These observations are consistent with excessive accumulation of alpha(2u)-globulin occurring in protein droplets in renal proximal tubule epithelial cells as a result of decreased catabolic activity due to formation of ligand-protein complexs with Decalin and its metabolite(s). This report demonstrates that LC-ESI/MS may be routinely applied for quantitative analysis of alpha(2u)-globulin in rat urine and kidney samples to address alpha(2u)-globulin accumulation and its role in the development of nephrotoxicity associated with chemical exposures.