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Simmons S.O.,US Toxicology
Combinatorial Chemistry and High Throughput Screening | Year: 2011

Whole-animal studies have been the mainstay of toxicity testing for decades. These approaches are too expensive and laborious to effectively characterize all of the chemicals currently in commercial use. In addition, there are social and ethical pressures to reduce, refine and replace animal testing in toxicology. The National Research Council (NRC) has outlined a new strategy to transition from animal-based tests to high throughput, cell-based assays and computational modeling approaches to characterize chemical toxicants. Critical to this vision, assays that measure toxicity pathways associated with adverse health effects must be developed. Bioluminescent assays are particularly well suited to the demands of next-generation toxicity testing because they measure a wide range of biological activities in a quantitative and high throughput manner. This review describes the limitations of traditional, animal-based toxicity testing and discusses the current and developing uses of bioluminescent technologies in next-generation testing based on three general assay formats: luciferase-limited assays, ATP-limited assays and luciferin-limited assays. © 2011 Bentham Science Publishers Ltd.

Manjanatha M.G.,US Toxicology | Guo L.-W.,US Toxicology | Shelton S.D.,US Toxicology | Doerge D.R.,US Toxicology
Environmental and Molecular Mutagenesis | Year: 2015

Potential health risks for humans from exposure to acrylamide (AA) and its epoxide metabolite glycidamide (GA) have garnered much attention lately because substantial amounts of AA are present in a variety of fried and baked starchy foods. AA is tumorigenic in rodents, and a large number of in vitro and in vivo studies indicate that AA is genotoxic. A recent cancer bioassay on AA demonstrated that the lung was one of the target organs for tumor induction in mice; however, the mutagenicity of AA in this tissue is unclear. Therefore, to investigate whether or not gene mutation is involved in the etiology of AA- or GA-induced mouse lung carcinogenicity, we screened for cII mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19-111 mg/kg bw/days). Both doses of AA and GA produced significant increases in cII MFs, with the high doses producing responses 2.7-5.6-fold higher than the corresponding controls (P≤0.05; control MFs=17.2±2.2 and 15.8±3.5 × 10-6 in males and females, respectively). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P≤0.01). The predominant types of mutations in the lung cII gene from AA- and GA-treated mice were A:T → T:A, and G:C → C:G transversions, and -1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted. Environ. Mol. Mutagen. 56:446-456, 2015. © 2015 Wiley Periodicals, Inc.

Beger R.D.,National Center for Toxicological Research (NCTR) | Flynn T.J.,US Toxicology
Metabolomics | Year: 2016

Background: Pharmacometabolomics is a relatively new field that measures an individual’s metabolome in biofluids to detect prognostic and diagnostic biomarkers of drug response and to provide an effective means to predict variation in a subject’s response to drug treatment. Pharmacometabolomics has the potential to help clinicians determine the effectiveness and safety of a drug on an individual basis. Aim of Review: To provide information from the current literature in pharmocometabolomics relevant to drug safety including factors besides genetics that can play a role in how a subject responds to a drug treatment. Pharmacometabolomics studies on drug-induced liver toxicity, the use of pharmacometabolomics to detect and predict drug interactions, and future applications of pharmacometabolomics in drug safety are discussed. Key scientific concepts of the review: Pharmacometabolomics can play a role in identifying and/or characterizing toxicity at all stages of drug development. These stages include: pharmacokinetics and ADME; initial toxicity; protective mechanisms; adverse events; late injury; and, injury progression or recovery. Pharmacometabolomics also has the ability to detect endogenous metabolites and markers of other exposure factors including alcohol consumption, impact of the gut microbiome, nutrition, other medications (polypharmacy), dietary supplements, and current individual health-to-disease status, all of which could play a role in patient response to a drug. Pharmacometabolomics alone or in combination with pharmacogenomics can be used to develop customized treatment plans for patients (i.e., personalized medicine) that could significantly reduce adverse events that are sometimes associated with the use of pharmaceuticals. © 2016, Springer Science+Business Media New York (outside the USA).

Jacob C.C.,US Toxicology | Gamboa da Costa G.,US Toxicology
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2011

This paper reports the development and validation of a methodology for the low-level quantification of melamine and cyanuric acid in limited samples of rat serum. The methodology, based upon ion-exchange solid phase extraction (SPE) and ultra-performance liquid chromatography (UPLC) coupled with electrospray tandem mass spectrometry (MS/MS) in multiple reaction monitoring (MRM) mode, relies on the use of stable isotope-labeled internal standards and requires only 15μL samples of serum. The method provides a recovery of 80-110% of melamine with a signal suppression of ca. 55%, and a recovery of 50-90% of cyanuric acid with a signal suppression ca. 40-60%, affording lower limits of quantification (LLOQ) for melamine or cyanuric acid of, respectively, 5. ppb (mean accuracy 109%; CV. =4.9%) and 10. ppb (mean accuracy 96%; CV. =8.6%). The small sample requirements, excellent sensitivity, accuracy and precision, and high-throughput (5. min of instrument run time) make this methodology optimal for toxicokinetic or exposure assessments studies. © 2011.

Teeguarden J.,Pacific Northwest National Laboratory | Hanson-Drury S.,Pacific Northwest National Laboratory | Fisher J.W.,US Toxicology | Doerge D.R.,US Toxicology
Food and Chemical Toxicology | Year: 2013

Mammalian estrogen receptors modulate many physiological processes. Chemicals with structural features similar to estrogens can interact with estrogen receptors to produce biological effects similar to those caused by endogenous estrogens in the body. Bisphenol A (BPA) is a structural analogue of estrogen that binds to estrogen receptors. Exposure to BPA in humans is virtually ubiquitous in industrialized societies, but BPA is rapidly detoxified by metabolism and does not accumulate in the body. Whether or not serum concentrations of BPA in humans are sufficiently high to disrupt normal estrogen-related biology is the subject of intense political and scientific debate. Here we show a convergence of robust methods for measuring or calculating serum concentrations of BPA in humans from 93 published studies of more than 30,000 individuals in 19 countries across all life stages. Typical serum BPA concentrations are orders of magnitude lower than levels measurable by modern analytical methods and below concentrations required to occupy more than 0.0009% of Type II Estrogen Binding Sites, GPR30, ERα or ERβ receptors. Occupancies would be higher, but ≤0.04%, for the highest affinity receptor, ERRγ. Our results show limited or no potential for estrogenicity in humans, and question reports of measurable BPA in human serum. © 2013 Elsevier Ltd.

Dobrovolsky V.N.,US Toxicology | Elespuru R.K.,Health-U | Bigger C.A.H.,White Oak Research Center | Robison T.W.,White Oak Research Center | Heflich R.H.,US Toxicology
Environmental and Molecular Mutagenesis | Year: 2011

The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10 -6; median of 3.8 × 10 -6 and mutant frequency less than 8 × 10 -6 for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ∼300 × 10 -6 and ∼100 × 10 -6. We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ∼6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine the assay's sensitivity in detecting mutations induced by genotoxic agents acting via different mechanisms. © 2011 Wiley-Liss, Inc.

Llorens A.,CSIC - Institute of Agricultural Chemistry and Food Technology | Lloret E.,IRTA - Institute of Agricultural-Alimentary Research and Technology | Picouet P.A.,IRTA - Institute of Agricultural-Alimentary Research and Technology | Trbojevich R.,US Toxicology | Fernandez A.,CSIC - Institute of Agricultural Chemistry and Food Technology
Trends in Food Science and Technology | Year: 2012

Metallic-based micro and nano-structured materials are incorporated into food contact polymers to enhance mechanical and barrier properties, and to prevent the photodegradation of plastics. Additionally heavy metals are effective antimicrobials in the form of salts, oxides, and colloids, complexes such as silver zeolites, or as elemental nanoparticles. They are incorporated for food preservation purposes and to decontaminate surfaces in industrial environments. Other relevant properties in active food packaging, such as the capability for ethylene oxidation or oxygen scavenging, can be used to extend food shelf-life. Silver based nano-engineered materials are currently the most commonly used in commodities due to their antimicrobial capacity. Copper, zinc and titanium nanostructures are also showing promise in food safety and technology. The antimicrobial properties of zinc oxide at the nanoscale will provide affordable and safe innovative strategies. Copper has been shown to be an efficient sensor for humidity, and titanium oxide has resistance to abrasion and UV-blocking performance. The migration of cations from the polymer matrices is the key point to determine their antimicrobial effectiveness; however, this cation migration may affect legal status of the polymer as a food-contact material. © 2011 Elsevier Ltd.

Gibbs-Flournoy E.A.,University of North Carolina at Chapel Hill | Bromberg P.A.,University of North Carolina at Chapel Hill | Hofer T.P.J.,Helmholtz Center for Environmental Research | Samet J.M.,Health-U | Zucker R.M.,US Toxicology
Particle and Fibre Toxicology | Year: 2011

Background: Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane.Results: Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependant internalization process.Conclusions: DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels. © 2011 Gibbs-Flournoy et al; licensee BioMed Central Ltd.

Cheng Y.,Texas A&M University | Jutooru I.,Texas A&M University | Jutooru I.,Covance | Chadalapaka G.,Texas A&M University | And 2 more authors.
Oncotarget | Year: 2015

HOTTIP is a long non-coding RNA (lncRNA) transcribed from the 5′ tip of the HOXA locus and is associated with the polycomb repressor complex 2 (PRC2) and WD repeat containing protein 5 (WDR5)/mixed lineage leukemia 1 (MLL1) chromatin modifying complexes. HOTTIP is expressed in pancreatic cancer cell lines and knockdown of HOTTIP by RNA interference (siHOTTIP) in Panc1 pancreatic cancer cells decreased proliferation, induced apoptosis and decreased migration. In Panc1 cells transfected with siHOTTIP, there was a decrease in expression of 757 genes and increased expression of 514 genes, and a limited gene analysis indicated that HOTTIP regulation of genes is complex. For example, Aurora kinase A, an important regulator of cell growth, is coregulated by MLL and not WDR5 and, in contrast to previous studies in liver cancer cells, HOTTIP does not regulate HOXA13 but plays a role in regulation of several other HOX genes including HOXA10, HOXB2, HOXA11, HOXA9 and HOXA1. Although HOTTIP and the HOX-associated lncRNA HOTAIR have similar pro-oncogenic functions, they regulate strikingly different sets of genes in Panc1 cells and in pancreatic tumors.

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