Inhalation exposure to multi-walled carbon nanotubes alters the pulmonary allergic response of mice to house dust mite allergen.

Background: Increasing evidence from rodent studies indicates that inhaled multi-walled carbon nanotubes (MWCNTs) have harmful effects on the lungs. In this study, we examined the effects of inhalation exposure to MWCNTs on allergen-induced airway inflammation and fibrosis. We hypothesized that inhalation pre-exposure to MWCNTs would render mice susceptible to developing allergic lung disease induced by house dust mite (HDM) allergen. Methods: Male B6C3F1/N mice were exposed by whole-body inhalation for 6 h a day, 5 d a week, for 30 d to air control or 0.06, 0.2, and 0.6 mg/m3 of MWCNTs. The exposure atmospheres were agglomerates (1.4-1.8 µm) composed of MWCNTs (average diameter 16 nm; average length 2.4 µm; 0.52% Ni). Mice then received 25 µg of HDM extract by intranasal instillation 6 times over 3 weeks. Necropsy was performed at 3 and 30 d after the final HDM dose to collect serum, bronchoalveolar lavage fluid (BALF), and lung tissue for histopathology. Results: MWCNT exposure at the highest dose inhibited HDM-induced serum IgE levels, IL-13 protein levels in BALF, and airway mucus production. However, perivascular and peribronchiolar inflammatory lesions were observed in the lungs of mice at 3 d with MWCNT and HDM, but not MWCNT or HDM alone. Moreover, combined HDM and MWCNT exposure increased airway fibrosis in the lungs of mice. Conclusions: Inhalation pre-exposure to MWCNTs inhibited HDM-induced TH2 immune responses, yet this combined exposure resulted in vascular inflammation and airway fibrosis, indicating that MWCNT pre-exposure alters the immune response to allergens.

DNA Product Formation in Female Sprague-Dawley Rats Following Polyhalogenated Aromatic Hydrocarbon (PHAH) Exposure.

DNA oxidation damage has been regarded as one of the possible mechanisms for the hepatic carcinogenesis of dioxin-like compounds (DLCs). In this study, we evaluated the toxic equivalency factor (TEF) from the standpoint of induced DNA oxidation products and their relationship to toxicity and carcinogenicity. Nine DNA oxidation products were analyzed in the liver of female Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) alone or the tertiary mixture of TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) by gavage for 14, 31, and 53 weeks (5 days/week) by LC-MS/MS: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo); 1,N6-etheno-2'-deoxyadenosine (1,N6-εdAdo); N2,3-ethenoguanine (N2,3-εG); 7-(2-oxoethly)guanine (7-OEG); 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo); malondialdehyde (M1dGuo); acrolein (AcrdGuo); crotonaldehyde (CrdGuo); and 4-hydroxynonenal (HNEdGuo) derived 2'-deoxyguanosine adducts. Exposure to TCDD (100 ng/kg/day) significantly induced 1,N6-εdAdo at 31 and 53 weeks, while no increase of 8-oxo-dGuo was observed. Significant increases were observed for 8-oxo-dGuo and 1,N6-εdAdo at all time points following exposure to the tertiary mixture (TEQ 100 ng/kg/day). Exposure to TCDD for 53 weeks only significantly increased 1,N6-εdAdo, while increases of N2,3-εG and 7-OEG were only found in the highest dose group (100 ng/kg/day). Exposure to the tertiary mixture for 53 weeks had no effect on N2,3-εG in any exposure group (TEQ 0, 22, 46, or 100 ng/kg/day), while significant increases were observed for 1,N6-εdAdo (all dose groups), 8-oxo-dGuo (46 and 100 ng/kg/day), and 7-OEG (100 ng/kg/day). While no significant increase was observed at 53 weeks for 1,N2-εdGuo, M1dGuo, AcrdGuo, or CrdGuo following exposure to TCDD (100 ng/kg/day), all of them were significantly induced in animals exposed to the tertiary mixture (TEQ 100 ng/kg/day). This oxidation DNA product data suggest that the simple TEF methodology cannot be applied to evaluate the diverse patterns of toxic effects induced by DLCs.

Polychlorinated Biphenyls Induce Oxidative DNA Adducts in Female Sprague-Dawley Rats.

Polychlorinated biphenyls (PCBs) are organic chemicals that were traditionally produced and widely used in industry as mixtures and are presently formed as byproducts of pigment and dye manufacturing. They are known to persist and bioaccumulate in the environment. Some have been shown to induce liver cancer in rodents. Although the mechanism of the toxicity of PCBs is unknown, it has been shown that they increase oxidative stress, including lipid peroxidation. We hypothesized that oxidative stress-induced DNA damage could be a contributor for PCB carcinogenesis and analyzed several DNA adducts in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and a binary mixture (PCB 126 + 153) for 14, 31, and 53 wks. Eight adducts were measured to profile oxidative DNA lesions, including 8-oxo-deoxyguanosine (8-oxo-dG), 1,N(6)-ethenodeoxyadenosine (1,N(6)-εdA), N(2),3-ethenoguanine (N(2),3-εG), 1,N(2)-ethenodeoxyguanosine (1,N(2)-εdG), as well as malondialdehyde (M1dG), acrolein (AcrdG), crotonaldehyde (CrdG), and 4-hydroxynonenal-derived dG adducts (HNEdG) by LC-MS/MS analysis. Statistically significant increases were observed for 8-oxo-dG and 1,N(6)-εdA concentrations in hepatic DNA of female rats exposed to the binary mixture (1000 ng/kg/day + 1000 µg/kg/day) but not in rats exposed to PCB 126 (1000 ng/kg/day) or PCB 153 (1000 µg/kg/day) for 14 and 31 wks. However, exposure to PCB 126 (1000 ng/kg/day) for 53 wks significantly increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, and M1dG. Exposure to PCB 153 (1000 µg/kg/day) for 53 wks increased 8-oxo-dG, and 1,N(6)-εdA. Exposure to the binary mixture for 53 wks increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, 1,N(2)-εdG, and N(2),3-εG significantly above control groups. Increased hepatic oxidative DNA adducts following exposure to PCB 126, PCB 153, or the binary mixture shows that an increase in DNA damage may play an important role in hepatic toxicity and carcinogenesis in female Sprague-Dawley rats.

Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Prevalence and sociodemographic correlates of antinuclear antibodies in the United States.

OBJECTIVE: To estimate the prevalence, types, and sociodemographic and biobehavioral correlates of antinuclear antibodies (ANAs) in the US. METHODS: We conducted a cross-sectional analysis of 4,754 individuals from the National Health and Nutrition Examination Survey 1999-2004. ANAs were assessed by indirect immunofluorescence. In ANA-positive individuals, cellular staining patterns were determined, and specific autoantibody reactivities were assessed by immunoprecipitation. RESULTS: The ANA prevalence in the US population of individuals ages 12 years and older was 13.8% (95% confidence interval [95% CI] 12.2-15.5%). ANA prevalence increased with age (P=0.01), and ANAs were more prevalent among females than males (17.8% versus 9.6%; P<0.001), with the female-to-male ratio peaking at 40-49 years of age. ANA prevalence was modestly higher in African Americans compared with whites (age-adjusted prevalence odds ratio [POR] 1.30, 95% CI 1.00-1.70). Remarkably, ANAs were less common in overweight and obese individuals (age-adjusted POR 0.74) than in persons of normal weight. No significant associations of ANA with education, family income, alcohol use, smoking history, serum levels of cotinine, or C-reactive protein were observed. In ANA-positive individuals, nuclear patterns were seen in 84.6%, cytoplasmic patterns were seen in 21.8%, and nucleolar patterns were seen in 6.1%; the most common specific autoantibodies were anti-Ro (3.9%) and anti-Su (2.4%). CONCLUSION: These findings suggest that more than 32 million persons in the US have ANAs, and that the prevalence is higher among females, older individuals, African Americans, and those with a normal body weight. These data will serve as a useful baseline for future investigations of predictors and changes in ANA prevalence over time.

Aloe vera non-decolorized whole leaf extract-induced large intestinal tumors in F344 rats share similar molecular pathways with human sporadic colorectal tumors.

Aloe vera is one of the most commonly used botanicals for various prophylactic and therapeutic purposes. Recently, NTP/NCTR has demonstrated a dose-dependent increase in large intestinal tumors in F344 rats chronically exposed to Aloe barbadensis Miller (Aloe vera) non-decolorized whole leaf extract (AVNWLE) in drinking water. The morphological and molecular pathways of AVNWLE-induced large intestinal tumors in the F344 rats were compared to human colorectal cancer (hCRC) literature. Defined histological criteria were used to compare AVNWLE-induced large intestinal tumors with hCRC. The commonly mutated genes (Kras, Ctnnb1, and Tp53) and altered signaling pathways (MAPK, WNT, and TGF-ß) important in hCRC were evaluated within AVNWLE-induced large intestinal tumors. Histological evaluation of the large intestinal tumors indicated eight of twelve adenomas (Ads) and four of twelve carcinomas (Cas). Mutation analysis of eight Ads and four Cas identified point mutations in exons 1 and 2 of the Kras gene (two of eight Ads, two of four Cas), and in exon 2 of the Ctnnb1 gene (three of eight Ads, one of four Cas). No Tp53 (exons 5-8) mutations were found in Ads or Cas. Molecular pathways important in hCRC such as MAPK, WNT, and TGF-ß signaling were also altered in AVNWLE-induced Ads and Cas. In conclusion, the AVNWLE-induced large intestinal tumors in F344 rats share several similarities with hCRC at the morphological and molecular levels.

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.

Thyroid follicular lesions induced by oral treatment for 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like compounds in female Harlan Sprague-Dawley rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally-similar dioxin-like compounds affect thyroid function and morphology and thyroid hormone metabolism in animals and humans. The National Toxicology Program conducted eight 2-year gavage studies in female Harlan Sprague-Dawley rats to determine the relative potency of chronic toxicity and carcinogenicity of TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 2,3',4,4',5-pentachlorobiphenyl (PCB118), 2,2',4,4',5,5'-hexachloro-biphenyl (PCB153), a tertiary mixture of TCDD/PCB126/PeCDF, and two binary mixtures (PCB126/PCB153 and PCB126/PCB118). Administration of these compounds was associated with increased incidences of thyroid follicular cell hypertrophy, variably observed in the 14-, 31-, and 53-week interim and 2-year sacrifice groups. In all studies, the incidences of follicular cell adenoma and carcinoma were not increased. Decreased levels of serum thyroxine were primarily noted in the 14-or-later -week interim groups of all chemicals. Serum triiodothyronine (T3) levels were increased in the TCDD, PCB126, PeCDF, TCDD/PCB126/PeCDF, and PCB126/PCB153 studies, while decreased levels were noted in the PCB153 and PCB126/PCB118 studies. TCDD, PCB126, PCB126/PCB153, and PCB126/PCB118 increased levels of serum thyroid-stimulating hormone almost in a dose-dependent manner in the 14-week groups. These data suggest that although dioxin-like compounds alter thyroid hormones and increase follicular cell hyperplasia, there is not an increase in thyroid adenoma or carcinoma in female Sprague-Dawley rats.

Reproductive lesions in female Harlan Sprague-Dawley rats following two-year oral treatment with dioxin and dioxin-like compounds.

Results from previously published animal studies suggest that prenatal and postnatal exposure to dioxin and dioxin-like compounds (DLCs) may profoundly affect the reproductive system of both sexes via endocrine disruption. In the present work, we evaluate the toxicity and carcinogenicity of various DLCs, with an emphasis on their effect on the reproductive organs, induced by chronic exposure of female adult Harlan Sprague-Dawley rats. This investigation represents part of an initiative of the National Toxicology Program to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. For fourteen, thirty-one, or fifty-three weeks or for two years, animals were administered by gavage 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); 2,3',4,4',5-pentachlorobiphenyl (PCB118); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and PCB118. The ranges of treatment-related changes in the reproductive system included chronic active inflammation in the ovary that occurred in the 1,000 and 3,000 microg/kg core groups (two-year exposure) of PCB153 and in the 300 ng/3,000 microg/kg core group of binary mixture of PCB126 and PCB153. Increases in the incidence of acute and/or chronic active inflammation of the uterus were observed in all dosed groups, including the stop-exposure group (withdrawal after thirty-week exposure) of PeCDF and the 1,000 microg/kg and/or higher group dosed with PCB153. The incidence of cystic endometrial hyperplasia was marginally increased in the 92 PeCDF ng/kg group at two years. The incidence of squamous metaplasia was significantly increased in the 44 ng/kg and higher dose group, including the stop-exposure group. The incidence of uterine squamous cell carcinoma was significantly or marginally increased in the 6 ng/kg core and 100 ng/kg stop-exposure groups of TCDD and in the 300 ng/300 microg/kg core group that received the binary mixture of PCB126 and 153. The incidence of uterine carcinoma was marginally increased in the 92 ng/kg PeCDF group at two years and clearly increased in the 1,000 and 4,600 microg/kg PCB118 core group and the 4,600 microg/kg stop group. In the studies of PCB 126, the tertiary mixture, and the binary mixture of PCB126 and PCB118, no increased incidence of any change occurred in the reproductive systems. The range of changes seen with the different compounds suggests that more than one mechanism may have been involved in promoting the female reproductive pathology.

Using Tox21 High-Throughput Screening Assays for the Evaluation of Botanical and Dietary Supplements.

Introduction: Recent nationwide surveys found that natural products, including botanical dietary supplements, are used by ∼18% of adults. In many cases, there is a paucity of toxicological data available for these substances to allow for confident evaluations of product safety. The National Toxicology Program (NTP) has received numerous nominations from the public and federal agencies to study the toxicological effects of botanical dietary supplements. The NTP sought to evaluate the utility of in vitro quantitative high-throughput screening (qHTS) assays for toxicological assessment of botanical and dietary supplements. Materials and Methods: In brief, concentration-response assessments of 90 test substances, including 13 distinct botanical species, and individual purported active constituents were evaluated using a subset of the Tox21 qHTS testing panel. The screen included 20 different endpoints that covered a broad range of biologically relevant signaling pathways to detect test article effects upon endocrine activity, nuclear receptor signaling, stress response signaling, genotoxicity, and cell death signaling. Results and Discussion: Botanical dietary supplement extracts induced measurable and diverse activity. Elevated biological activity profiles were observed following treatments with individual chemical constituents relative to their associated botanical extract. The overall distribution of activity was comparable to activities exhibited by compounds present in the Tox21 10K chemical library. Conclusion: Botanical supplements did not exhibit minimal or idiosyncratic activities that would preclude the use of qHTS platforms as a feasible method to screen this class of compounds. However, there are still many considerations and further development required when attempting to use in vitro qHTS methods to characterize the safety profile of botanical/dietary supplements.

Development of a consensus approach for botanical safety evaluation - A roundtable report.

Botanical safety science continues to evolve as new tools for risk assessment become available alongside continual desire by consumers for "natural" botanical ingredients in consumer products. Focusing on botanical food/dietary supplements a recent international roundtable meeting brought together scientists to discuss the needs, available tools, and ongoing data gaps in the botanical safety risk assessment process. Participants discussed the key elements of botanical safety evaluations. They provided perspective on the use of a decision tree methodology to conduct a robust risk assessment and concluded with alignment on a series of consensus statements. This discussion highlighted the strengths and vulnerabilities in common assumptions, and the participants shared additional perspective to ensure that this end-to-end safety approach is sufficient, actionable and timely. Critical areas and data gaps were identified as opportunities for future focus. These include, better context on history of use, systematic assessment of weight of evidence, use of in silico approaches, inclusion of threshold of toxicological concern considerations, individual substances/matrix interactions of plant constituents, assessing botanical-drug interactions and adaptations needed to apply to in vitro and in vivo pharmacokinetic modelling of botanical constituents.

Characterizing sources of variability in zebrafish embryo screening protocols.

There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.

Screening for Developmental Neurotoxicity at the National Toxicology Program: The Future Is Here.

The National Toxicology Program (NTP) receives requests to evaluate chemicals with potential to cause adverse health effects, including developmental neurotoxicity (DNT). Some recent requests have included classes of chemicals such as flame retardants, polycyclic aromatic compounds, perfluoroalkyl substances, and bisphenol A analogs with approximately 20-50 compounds per class, many of which include commercial mixtures. However, all the compounds within a class cannot be tested using traditional DNT animal testing guideline studies due to resource and time limitations. Hence, a rapid and biologically relevant screening approach is required to prioritize compounds for further in vivo testing. Because neurodevelopment is a complex process involving multiple distinct cellular processes, one assay will unlikely address the complexity. Hence, the NTP sought to characterize a battery of in vitro and alternative animal assays to quantify chemical effects on a variety of neurodevelopmental processes. A culmination of this effort resulted in a NTP-hosted collaborative project with approximately 40 participants spanning across domains of academia, industry, government, and regulatory agencies; collaborators presented data on cell-based assays and alternative animal models that was generated using a targeted set of compounds provided by the NTP. The NTP analyzed the assay results using benchmark concentration (BMC) modeling to be able to compare results across the divergent assays. The results were shared with the contributing researchers on a private web application during the workshop, and are now publicly available. This article highlights the overview and goals of the project, and describes the NTP's approach in creating the chemical library, development of NTPs data analysis strategy, and the structure of the web application. Finally, we discuss key issues with emphasis on the utility of this approach, and knowledge gaps that need to be addressed for its use in regulatory decision making.

Accumulation of M1dG DNA adducts after chronic exposure to PCBs, but not from acute exposure to polychlorinated aromatic hydrocarbons.

Oxidative DNA damage is one of the key events thought to be involved in mutation and cancer. The present study examined the accumulation of M1dG, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, DNA adducts after single dose or 1-year exposure to polyhalogenated aromatic hydrocarbons (PHAH) in order to evaluate the potential role of oxidative DNA damage in PHAH toxicity and carcinogenicity. The effect of PHAH exposure on the number of M1dG adducts was explored initially in female mice exposed to a single dose of either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or a PHAH mixture. This study demonstrated that a single exposure to PHAH had no significant effect on the number of M1dG adducts compared to the corn oil control group. The role of M1dG adducts in polychlorinated biphenyl (PCB)-induced toxicity and carcinogenicity was further investigated in rats exposed for a year to PCB 153, PCB 126, or a mixture of the two. PCB 153, at doses up to 3000 microg/kg/day, had no significant effect on the number of M1dG adducts in liver and brain tissues from the exposed rats compared to controls. However, 1000 ng/kg/day of PCB 126 resulted in M1dG adduct accumulation in the liver. More importantly, coadministration of equal proportions of PCB 153 and PCB 126 resulted in dose-dependent increases in M1dG adduct accumulation in the liver from 300 to 1000 ng/kg/day of PCB 126 with 300-1000 microg/kg/day of PCB 153. Interestingly, the coadministration of different amounts of PCB 153 with fixed amounts of PCB 126 demonstrated more M1dG adduct accumulation with higher doses of PCB 153. These results are consistent with the results from cancer bioassays that demonstrated a synergistic effect between PCB 126 and PCB 153 on toxicity and tumor development. In summary, the results from the present study support the hypothesis that oxidative DNA damage plays a key role in toxicity and carcinogenicity following long-term PCB exposure.

Getting to the Root of the Matter: Challenges and Recommendations for Assessing the Safety of Botanical Dietary Supplements.

The National Toxicology Program's (NTP) mission is "to evaluate agents of public health concern, by developing and applying the tools of modern toxicology and molecular biology." Botanical dietary supplements (BDS) represent agents of public health concern due to widespread exposure to high doses, a lack of safety data for most products, variable quality, and reports of adverse events. This commentary will address lessons learned in NTP testing activities with BDS and recommendations for moving forward.

Naturally complex: Perspectives and challenges associated with Botanical Dietary Supplement Safety assessment.

Due to the extensive use of botanical dietary supplements by consumers in the United States, there is a need for appropriate research and data to support safety assessments. Complexity and variability, both natural and introduced, of botanical dietary supplements make research on these products difficult. Botanical dietary supplements are regulated by the Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act (FD&C Act), as amended by the 1994 Dietary Supplement Health and Education Act (DSHEA). They are regulated as a category of food, which differs from the regulation of pharmaceutical products. Both manufacturers and the FDA are faced with the challenge of determining the best approaches for evaluating and monitoring the safety of botanical products. High quality botanicals research requires accurate identification and characterization of the material being studied. Inconsistent results in efficacy studies of botanical dietary supplements have led to efforts to improve the rigor and reproducibility of research in the field. Addressing the challenges associated with botanical dietary supplement safety is a global effort requiring coordination between numerous stakeholders, including researchers, suppliers, manufacturers, and regulators, all of whom play a role in ensuring that high quality products are available on the market.

Expanding the Concept of Translational Research: Making a Place for Environmental Health Sciences.

SUMMARY: The National Institute of Environmental Health Sciences (NIEHS) introduces a new translational research framework that builds upon previous biomedical models to create a more comprehensive and integrated environmental health paradigm. The framework was developed as a graphical construct that illustrates the complexity of designing, implementing, and tracking translational research in environmental health. We conceptualize translational research as a series of concentric rings and nodes, defining "translation" as movement either from one ring to another or between nodes on a ring. A "Fundamental Questions" ring expands upon the research described in other frameworks as "basic" to include three interrelated concepts critical to basic science research: research questions, experimental settings, and organisms. This feature enables us to capture more granularity and thus facilitates an approach for categorizing translational research and its growth over time. We anticipate that the framework will help researchers develop compelling long-term translational research stories and accelerate public health impacts by clearly mapping out opportunities for collaborations. By using this paradigm, researchers everywhere will be better positioned to design research programs, identify research partners based on cross-disciplinary research needs, identify stakeholders who are likely to use the research for environmental decision-making and intervention, and track progress toward common goals. https://doi.org/10.1289/EHP3657.

Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process.

BACKGROUND: Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants. RESULTS: We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/ CONCLUSION: We present proof of the principle that a mechanistically based model can be fit to observations from a single PCR amplification. Initial amounts of amplicon are well estimated without using a standard solution. Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.

The putative tumor suppressor Tsc-22 is downregulated early in chemically induced hepatocarcinogenesis and may be a suppressor of Gadd45b.

Tsc-22 is a novel tumor suppressor gene that represents a new class of transcription factors that has transcriptional repressor activity. We found Tsc-22 downregulation in livers from B6C3F1 mice following treatment for 2 weeks with carcinogenic doses of the antianxiety drug oxazepam (2500 ppm) or the peroxisome proliferator Wyeth-14,643 (500 ppm) but not with two other carcinogens such as o-nitrotoluene or methyleugenol or three noncarcinogens including p-nitrotoluene, eugenol, or acetaminophen. The expression of Tsc-22 was also repressed in B6C3F1 mouse liver tumors that were induced by several chemicals from 2-year carcinogenicity studies as well as in spontaneous liver tumors. To identify potential Tsc-22 target genes in mouse liver, we transfected small interference RNA (SiRNA) designed to inhibit Tsc-22 into murine liver BNL-CL.2 cells. We selected two potential transcriptional targets of Tsc-22, growth arrest and DNA damage-inducible gene 45 beta (Gadd45b) and leucine zipper, putative tumor suppressor 2 (Lzts2) to test based on our previous complementary DNA microarray studies, showing that expression of these cancer-associated genes was increased when Tsc-22 was repressed. SiRNA treatment of BNL-CL.2 cells with Tsc-22 oligonucleotides but not nonspecific oligonucleotides decreased RNA and protein expression of Tsc-22 by 80-90%, while expression of Gadd45b gene, but not Lzts2, was increased over time after an initial decrease. Treatment of these cells with oxazepam for 48 h also resulted in decreased Tsc-22 and increased Gadd45b expression. These data provide evidence that Tsc-22 is a suppressor of Gadd45b expression, which may contribute to an early antiapoptotic response.

Migration of intradermally injected quantum dots to sentinel organs in mice.

Topical exposure to nanoscale materials is likely from a variety of sources including sunscreens and cosmetics. Because the in vivo disposition of nanoscale materials is not well understood, we have evaluated the distribution of quantum dots (QDs) following intradermal injection into female SKH-1 hairless mice as a model system for determining tissue localization following intradermal infiltration. The QD (CdSe core, CdS capped, poly[ethylene glycol] coated, 37 nm diameter, 621 nm fluorescence emission) were injected intradermally (ID) on the right dorsal flank. Within minutes following intradermal injection, the highly UV fluorescent QD could be observed moving from the injection sites apparently through the lymphatic duct system to regional lymph nodes. Residual fluorescent QD remained at the site of injection until necropsy at 24 h. Quantification of cadmium and selenium levels after 0, 4, 8, 12, or 24 h in multiple tissues, using inductively coupled plasma mass spectrometry (ICP-MS), showed a time-dependent loss of cadmium from the injection site, and accumulation in the liver, regional draining lymph nodes, kidney, spleen, and hepatic lymph node. Fluorescence microscopy corroborated the ICP-MS results regarding the tissue distribution of QD. The results indicated that (1) ID injected nanoscale QD remained as a deposit in skin and penetrated the surrounding viable subcutis, (2) QD were distributed to draining lymph nodes through the sc lymphatics and to the liver and other organs, and (3) sentinel organs are effective locations for monitoring transdermal penetration of nanoscale materials into animals.