Suramya Waidyanatha

Comparison of aneuploidies of chromosomes 21, X, and Y in the blood lymphocytes and sperm of workers exposed to benzene†

Benzene is a primary industrial chemical and a ubiquitous environmental pollutant that causes human leukemia and maybe other malignancies. Occupational exposure to benzene has been associated with increased chromosomal aneuploidies in blood lymphocytes and, in separate studies, in sperm. However, aneuploidy detection in somatic and germ cells within the same benzene‐exposed individuals has never been reported. To compare aneuploidies in blood lymphocytes and sperm within the same individuals exposed to benzene, a cross‐sectional study was conducted in 33 benzene‐exposed male workers and 33 unexposed workers from Chinese factories. Air benzene concentrations in the exposed workers ranged from below the detection limit to 24 ppm (median, 2.9 ppm) and were undetectable in the unexposed subjects. Aneuploidies of chromosomes 21, X, and Y in blood lymphocytes were examined by multicolor fluorescence in situ hybridization and were compared to the previously reported aneuploidies in sperm. The results showed that benzene exposure was positively associated with the gain of chromosome 21 but not sex chromosomes in blood lymphocytes. This was in contrast to analysis of sperm, where the gain of sex chromosomes, but not chromosome 21, was significantly increased in the exposed workers. Furthermore, a significant correlation in the gain of sex chromosomes between blood lymphocytes and sperm was observed among the unexposed subjects, but not among the exposed workers. The findings suggest that benzene exposure induces aneuploidies in both blood cells and sperm within the same individuals, but selectively affects chromosome 21 in blood lymphocytes and the sex chromosomes in sperm. Environ. Mol. Mutagen. 2012.

Toxicokinetics of α-thujone following intravenous and gavage administration of α-thujone or α- and β-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 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.

Disposition of bisphenol AF, a bisphenol A analogue, in hepatocytes in vitro and in male and female Harlan Sprague-Dawley rats and B6C3F1/N mice following oral and intravenous administration

Abstract 1.u2002Bisphenol AF (BPAF) is used as a crosslinking agent for polymers and is being considered as a replacement for bisphenol A (BPA). 2.u2002In this study, comparative clearance and metabolism of BPAF and BPA in hepatocytes and the disposition and metabolism of BPAF in rodents following oral administration of 3.4, 34 or 340u2009mg/kg [14C]BPAF were investigated. 3.u2002BPAF was cleared more slowly than BPA in hepatocytes with the rate: ratu2009>u2009mouseu2009>u2009human. 4.u2002[14C]BPAF was excreted primarily in feces by 72u2009h after oral administration to rats (65–80%) and mice (63–72%). Females excreted more in urine (rat, 15%; mouse, 24%) than males (rat, 1–4%; mouse, 10%). Residual tissue radioactivity was <2% of the dose at 72u2009h. Similar results were observed following intravenous administration. 5.u2002In male rats, 52% of a 340u2009mg/kg oral dose was excreted in 24u2009h bile and was mostly comprised of BPAF glucuronide. However, >94% of fecal radioactivity was present as BPAF, suggesting extensive deconjugation in the intestine. 6.u2002Metabolites identified in bile were BPAF-glucuronide, -diglucuronide, -glucuronide sulfate and -sulfate. 7.u2002In conclusion, BPAF was well absorbed following gavage administration and highly metabolized and excreted mostly in the feces as BPAF.

Systemic uptake, albumin and hemoglobin binding of [(14)C]2,3-butanedione administered by intratracheal instillation in male Harlan Sprague Dawley rats and oropharyngeal aspiration in male B6C3F1/N mice.

2,3-Butanedione (BD) is a reactive diketone in artificial butter flavors that is thought to cause bronchiolitis obliterans in workers in microwave popcorn manufacturing. Bronchiolitis obliterans is generally not diagnosed until irreversible damage has occurred; therefore a biomarker of early exposure is needed. The potential systemic uptake of BD from inhalation exposure has not been evaluated. The objective here was to evaluate the systemic exposure of BD and binding to hemoglobin and albumin. [(14)C]BD was administered to male Harlan Sprague Dawley rats (100 mg/kg, intratracheal instillation) and B6C3F1/N mice (157 mg/kg, oropharyngeal aspiration). Blood and plasma was collected 24 h after administration and analyzed for (14)C content. At 24h, 0.88±0.07% of the administered dose was in rat blood, 0.66±0.06% in rat plasma, 0.38±0.13% in mouse blood and 0.17±0.05% in mouse plasma. Albumin binding in rats was 269±24.2 ng equiv./mg, which accounts for 38% of the radioactivity in plasma. In mice, binding was 85.0±22.3 ng equiv./mg albumin, which accounts for 51% of the radioactivity in plasma. The binding to hemoglobin in rats was 38.2±17.6 ng equiv./mg, and to globin was 29.1±3.96 ng equiv./mg. In mice, the binding to hemoglobin was 16.2±9.0 ng equiv./mg. The site(s) of adduction on hemoglobin and albumin was investigated by mass spectrometry. In rat globin, arginine adducts were detected at R-30 and R-104 of the beta chain in vitro and in vivo. In rat albumin, adducts were detected in vitro on R-219/221, R-360, and R-368, and in vivo on a variety of arginine residues. This study demonstrated that BD enters the systemic circulation and reacts with arginine on hemoglobin and albumin. These results indicate that hemoglobin and albumin adducts may be useful as biomarkers of BD exposure in humans.

Metabolism and disposition of [14C]n-butyl-p-hydroxybenzoate in male and female Harlan Sprague Dawley rats following oral administration and dermal application

n-Butyl-p-hydroxybenzoate (n-butylparaben, BPB) is an antioxidant used in foods, pharmaceuticals and cosmetics. This study investigated the disposition of ring-labelled [14C]BPB in Harlan Sprague Dawley rats, and in rat and human hepatocytes. BPB was rapidly cleared in hepatocytes from rat (t1/2 = 3–4u2009min) and human (t1/2 = 20–30u2009min). The major metabolites detected in rat hepatocytes were hydroxybenzoic acid and in human hepatocytes were hydroxybenzoic acid and hydroxyhippuric acid. [14C]BPB was administered to male rats orally at 10, 100 or 1000u2009mg/kg, intravenously at 10u2009mg/kg and dermally at 10 and 100u2009mg/kg; female rats were administered oral doses at 10u2009mg/kg. Oral doses of BPB were well-absorbed (>83%) and eliminated chiefly in urine (83–84%); ≤1% of the radioactivity remained in tissues at 24u2009h or 72u2009h after dosing. About 4% and 8%, respectively, of 100u2009mg/kg dermal doses were absorbed in 24u2009h and 72u2009h, and about 50% of a 10u2009mg/kg dose was absorbed in 72u2009h. Metabolites detected in urine included those previously reported, BPB-glucuronide, BPB-sulfate, hydroxybenzoic acid and hydroxyhippuric acid, but also novel metabolites arising from ring hydroxylation followed by glucuronidation and sulfation.

From the Cover: Three-Dimensional (3D) HepaRG Spheroid Model With Physiologically Relevant Xenobiotic Metabolism Competence and Hepatocyte Functionality for Liver Toxicity Screening

Effective prediction of human responses to chemical and drug exposure is of critical importance in environmental toxicology research and drug development. While significant progress has been made to address this challenge using invitro liver models, these approaches often fail due to inadequate tissue model functionality. Herein, we describe the development, optimization, and characterization of a novel three-dimensional (3D) spheroid model using differentiated HepaRG cells that achieve and maintain physiologically relevant levels of xenobiotic metabolism (CYP1A2, CYP2B6, and CYP3A4/5). This invitro model maintains a stable phenotype over multiple weeks in both 96- and 384-well formats, supports highly reproducible tissue-like architectures and models pharmacologically- and environmentally important hepatic receptor pathways (ie AhR, CAR, and PXR) analogous to primary human hepatocyte cultures. HepaRG spheroid cultures use 50-100× fewer cells than conventional two dimensional cultures, and enable the identification of metabolically activated toxicants. Spheroid size, time in culture and culture media composition were important factors affecting basal levels of xenobiotic metabolism and liver enzyme inducibility with activators of hepatic receptors AhR, CAR and PXR. Repeated exposure studies showed higher sensitivity than traditional 2D cultures in identifying compounds that cause liver injury and metabolism-dependent toxicity. This platform combines the well-documented impact of 3D culture configuration for improved tissue functionality and longevity with the requisite throughput and repeatability needed for year-over-year toxicology screening.

Characterization of aqueous formulations of tetra- and pentavalent forms of vanadium in support of test article selection in toxicology studies

Tetravalent (VIV) and pentavalent (VV) forms of vanadium were selected for testing by the National Toxicology Program via drinking water exposure due to potential human exposure. To aid in the test article selection, drinking water formulations (125–2000xa0mg/L) of vanadyl sulfate (VIV), sodium orthovanadate, and sodium metavanadate (VV) were characterized by ultraviolet/visible (UV/VIS) spectroscopy, mass spectrometry (MS), or 51V nuclear magnetic resonance (NMR) spectroscopy. Aqueous formulations of orthovanadate, metavanadate, and vanadyl sulfate in general were basic, neutral, and acidic, respectively. Changes in vanadium speciation were investigated by adjusting formulation pH to acidic, neutral, or basic. There was no visible difference in UV/VIS spectra of pentavalent forms. NMR and MS analyses showed that the predominant oxidovanadate species in both ortho- and metavanadate formulations at basic and acidic pH, respectively, were the monomer and decamer, while, a mixture of oxidovanadates were present at neutral pH. Oxidovanadate species were not observed in vanadyl sulfate formulations at acidic pH but were observed at basic pH suggesting conversion of VIV to VV. These data suggest that formulations of both ortho- and metavanadate form similar oxidovanadate species in acidic, neutral and basic pH and exist mainly in the VV form while vanadyl sulfate exists mainly as VIV in acidic pH. Therefore, the formulation stability overtime was investigated only for sodium metavanadate and vanadyl sulfate. Drinking water formulations (50 and 2000xa0mg/L) of metavanadate (~pH 7) and vanadyl sulfate (~pH 3.5) were ≥92xa0% of target concentration up to 42xa0days at ~5xa0°C and ambient temperature demonstrating the utility in toxicology studies.

Toxicokinetics of Isoeugenol in F344 rats and B6C3F1 mice

Abstract 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 ≤20u2009min 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%) (pu2009<u20090.0001), but was not different between the sexes for mice (28% males; 31% females) (pu2009=u20090.2437). The collective toxicokinetic data supported that low bioavailability following administration of IEG was the result of extensive first-pass metabolism.

How similar is similar enough? A sufficient similarity case study with Ginkgo biloba extract

Botanical dietary supplements are complex mixtures that can be highly variable in composition and quality, making safety evaluation difficult. A key challenge is determining how diverse products in the marketplace relate to chemically and toxicologically characterized reference samples (i.e., how similar must a product be in order to be well-represented by the tested reference sample?). Ginkgo biloba extract (GBE) was used as a case study to develop and evaluate approaches for determining sufficient similarity. Multiple GBE extracts were evaluated for chemical and biological-response similarity. Chemical similarity was assessed using untargeted and targeted chemistry approaches. Biological similarity was evaluated using in vitro liver models and short-term rodent studies. Statistical and data visualization methods were then used to make decisions about the similarity of products to the reference sample. A majority of the 26 GBE samples tested (62%) were consistently determined to be sufficiently similar to the reference sample, while 27% were different from the reference GBE, and 12% were either similar or different depending on the method used. This case study demonstrated that approaches to evaluate sufficient similarity allow for critical evaluation of complex mixtures so that safety data from the tested reference can be applied to untested materials.

Toxicokinetics of methyleugenol in F344 rats and B6C3F1 mice

1.u2002Methyleugenol (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 150u2009mg/kg) and intravenous (IV) (37u2009mg/kg) administration. 2.u2002Following 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.u2002The 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 37u2009mg/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.