Toxicokinetics of α-thujone following intravenous and gavage administration of α-thujone or α- and ß-thujone mixture in male and female F344/N rats and B6C3F1 mice.

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 ß-thujone (which will be referred to as α,ß-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,ß-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,ß-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,ß-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,ß-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.

Toxicokinetics of methyleugenol in F344 rats and B6C3F1 mice.

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. C(max) and AUC(T) 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 AUC(T) 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.

Toxicokinetics of isoeugenol in F344 rats and B6C3F1 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.

Predicting the hepatocarcinogenic potential of alkenylbenzene flavoring agents using toxicogenomics and machine learning.

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.

Compound cytotoxicity profiling using quantitative high-throughput screening.

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.

An interlaboratory study of perfluorinated alkyl compound levels in human plasma.

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.

Comparison of proanthocyanidins in commercial antioxidants: grape seed and pine bark extracts.

The major constituents in grape seed and pine bark extracts are proanthocyanidins. To evaluate material available to consumers, select lots were analyzed using high-performance liquid chromatography, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), gel permeation chromatography (GPC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Atmospheric pressure chemical ionization (APCI) LC/MS was used to identify monomers, dimers, and trimers present. GC/MS analyses led to the identification of ethyl esters of hexadecanoic acid, linoleic acid, and oleic acid, as well as smaller phenolic and terpene components. The GPC molecular weight (MW) distribution indicated components ranging from approximately 162 to approximately 5500 MW (pine bark less than 1180 MW and grape seed approximately 1180 to approximately 5000 MW). MALDI-TOF MS analyses showed that pine bark did not contain oligomers with odd numbers of gallate units and grape seed contained oligomers with both odd and even numbers of gallate. Reflectron MALDI-TOF MS identified oligomers up to a pentamer and heptamer, and linear MALDI-TOF MS showed a mass range nearly double that of reflectron analyses.

Analysis of flavonol aglycones and terpenelactones in Ginkgo biloba extract: A comparison of high-performance thin-layer chromatography and column high-performance liquid chromatography.

Advancements in automated high-performance thin-layer chromatography (HPTLC) have made it feasible to assess its use for the quantitative analysis of marker compounds in botanical preparations. We report here the findings of method comparisons for the terpenelactones and flavonol aglycones by column high-performance liquid chromatography (HPLC) with evaporative light scattering and UV detection, and HPTLC with a scanning densitometer. For the HPTLC assay of terpenelactones, total bilobalide, ginkgolide A, and ginkgolide B consistently achieved <70% of the total determined using HPLC, regardless of variations to postchromatographic derivatization time and temperature. Accuracy testing showed the possibility of a matrix interference. In contrast, a good relationship (95%) was determined between HPTLC and HPLC for determination of total flavonol glycosides (calculated from combined quercetin, kaempferol, and isorhamnetin) from an acid-hydrolyzed Ginkgo biloba L. (GBE) sample. The HPTLC flavonol aglycone method also performed well in terms of accuracy (overall average of 96% recovery for the 3 aglycones) and consecutive plate repeatability (overall percent relative standard deviation of 4.4). It is demonstrated that HPTLC can be a time-saving complement to HPLC for routine analysis of the flavonol glycosides in GBE.

The utility of the guppy (Poecilia reticulata) and medaka (Oryzias latipes) in evaluation of chemicals for carcinogenicity.

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.

Simultaneous quantification of terpenelactones and flavonol aglycones in hydrolyzed ginkgo biloba extract by liquid chromatography with inline ultraviolet and evaporative light scattering detection.

We report here a liquid chromatography (LC) method with inline ultraviolet/evaporative light scattering (UV/ELS) detection for the simultaneous quantification of the terpenelactones and flavonol aglycones in a single sample of hydrolyzed Ginkgo biloba extract (GBE). The sample is hydrolyzed by a rapid and convenient oven heating method for 1 h at 90 degrees C with 10% hydrochloric acid. The 1 h hydrolysis was found to be equivalent to the 2.25 h reflux treatment for dry powder extract, where total flavonol glycosides were 28.4 and 28.1%, respectively. Acceptable precision was achieved for total terpenelactones [relative standard deviation (RSD) = 4.8%] by ELS detection, and total flavonol aglycones (RSD = 2.3%) by UV detection. The analytical range was 1.5 to 7.3% (w/w) for the individual terpenelactones (ELS) and 2.5 to 15.0% (w/w) for the individual glycosides (UV) calculated from the aglycones quercetin, kaempferol, and isorhamnetin. This improved method allows for the first time high throughput sample preparation coupled with the quantification of the predominant compounds generally used for quality control of GBE in a single assay.

Chemical comparison of goldenseal (Hydrastis canadensis L.) root powder from three commercial suppliers.

The characterization of herbal materials is a significant challenge to analytical chemists. Goldenseal (Hydrastis canadensis L.), which has been chosen for toxicity evaluation by NIEHS, is among the top 15 herbal supplements currently on the market and contains a complex mixture of indigenous components ranging from carbohydrates and amino acids to isoquinoline alkaloids. One key component of herbal supplement production is botanical authentication, which is also recommended prior to initiation of efficacy or toxicological studies. To evaluate material available to consumers, goldenseal root powder was obtained from three commercial suppliers and a strategy was developed for characterization and comparison that included Soxhlet extraction, HPLC, GC-MS, and LC-MS analyses. HPLC was used to determine the weight percentages of the goldenseal alkaloids berberine, hydrastine, and canadine in the various extract residues. Palmatine, an isoquinoline alkaloid native to Coptis spp. and other common goldenseal adulterants, was also quantitated using HPLC. GC-MS was used to identify non-alkaloid constituents in goldenseal root powder, whereas LC-MS was used to identify alkaloid components. After review of the characterization data, it was determined that alkaloid content was the best biomarker for goldenseal. A 20-min ambient extraction method for the determination of alkaloid content was also developed and used to analyze the commercial material. All three lots of purchased material contained goldenseal alkaloids hydrastinine, berberastine, tetrahydroberberastine, canadaline, berberine, hydrastine, and canadine. Material from a single supplier also contained palmatine, coptisine, and jatrorrhizine, thus indicating that the material was not pure goldenseal. Comparative data for three commercial sources of goldenseal root powder are presented.

Method validation for determination of alkaloid content in goldenseal root powder.

A fast, practical ambient extraction methodology followed by isocratic liquid chromatography (LC) analysis with UV detection was validated for the determination of berberine, hydrastine, and canadine in goldenseal (Hydrastis canadensis L.) root powder. The method was also validated for palmatine, a major alkaloid present in the possible bioadulterants Coptis, Oregon grape root, and barberry bark. Alkaloid standard solutions were linear over the evaluated concentration ranges. The analytical method was linear for alkaloid extraction using 0.3-2 g goldenseal root powder/100 mL extraction solvent. Precision of the method was demonstrated using 10 replicate extractions of 0.5 g goldenseal root powder, with percent relative standard deviation for all 4 alkaloids < or = 1.6. Alkaloid recovery was determined by spiking each alkaloid into triplicate aliquots of neat goldenseal root powder. Recoveries ranged from 92.3% for palmatine to 101.9% for hydrastine. Ruggedness of the method was evaluated by performing multiple analyses of goldenseal root powder from 3 suppliers over a 2-year period. The method was also used to analyze Coptis root, Oregon grape root, barberry bark, and celandine herb, which are possible goldenseal bioadulterants. The resulting chromatographic profiles of the bioadulterants were significantly different from that of goldenseal. The method was directly transferred to LC with mass spectrometry, which was used to confirm the presence of goldenseal alkaloids tetrahydroberberastine, berberastine, canadaline, berberine, hydrastine, and canadine, as well as alkaloids from the bioadulterants, including palmatine, jatrorrhizine, and coptisine.

Chemical and physical characteristics of cellulose insulation particulates, and evaluation of potential acute pulmonary toxicity.

BACKGROUND: During installation of cellulose insulation (CI) in new and older houses, significant quantities of airborne material are generated. This study characterized the chemical and physical properties, and potential acute pulmonary toxicity of CI. METHODS: CI from four manufacturers was analyzed for inorganic additives and trace element impurities. Aerosols were generated and size fractionated. The number and size of fibrous and nonfibrous particles in the respirable fractions were determined. Respirable CI particulates were intratracheally instilled in rats (5 mg/kg) to evaluate potential pulmonary toxicity. RESULTS: CI samples were similar in composition with small differences due primarily to fire retardants. Less than 0.1% of CI was respirable and contained few fibers. Acute exposure to CI caused transient inflammation in the lungs and increased 4-hydroxyproline. Microscopic evaluation revealed a minimal to mild, non-progressing granulomatous pneumonitis. CONCLUSIONS: Low concentrations of respirable particles were found in CI aerosols. Particles consisted primarily of fire retardants with few fibers, and caused mild pulmonary toxicity in rats.