Boston University Superfund Research Program

Background: The ability of a substance to induce a toxicological response is better understood by analyzing the response profile over a broad range of concentrations than at a single concentration. In vitro quantitative high throughput screening (qHTS) assays are multiple-concentration experiments with an important role in the National Toxicology Program’s (NTP) efforts to advance toxicology from a predominantly observational science at the level of disease-specific models to a more predictive science based on broad inclusion of biological observations.

Objective: To develop a systematic approach to classify substances from large scale concentration-response data into statistically supported and toxicologically relevant activity categories.

Methods: The first stage of the approach finds active substances with robust concentration-response profiles within the tested concentration range. The second stage finds substances with activity at the lowest tested concentration not captured in the first stage. The third and final stage separates statistically significant (but not robustly statistically significant) profiles from responses that lack statistically compelling support, or “inactives”. The performance of the proposed algorithm was evaluated with simulated qHTS data sets.

Results: The proposed approach performed well for 14 point concentration-response curves with typical levels of residual error (σ ≤ 25%) or when maximal response (|RMAX|) was greater than 25% of the positive control response. The approach also worked well in most cases for smaller sample sizes when |RMAX| ≥ 50%, even with as few as 4 data points.

Conclusions: The three-stage classification algorithm performed better than one-stage classification approaches based on overall F-tests, t-tests or linear regression.

BACKGROUND: Information on the potential developmental toxicity (DT) of the majority of chemicals is scarce, and test capacities for further animal-based testing are limited. Therefore, new approaches with higher throughput are required. A screening strategy based on the use of relevant human cell types has been proposed by the EPA and others. As impaired neural crest (NC) function is one of the known causes for teratologic effects, testing of toxicant effects on NC is desirable for a DT test battery.

OBJECTIVE: To develop a robust and widely applicable human-relevant NC function assay, allowing sensitive screening of environmental toxicants, and a definition of toxicity pathways.

METHODS: We generated NC cells from human embryonic stem cells, and after establishing a migration assay of NC (MINC), we tested environmental toxicants as well as inhibitors of physiological signal transduction pathways.

RESULTS: Methylmercury (50 nM), valproic acid (> 10 µM) and Lead-acetate (1 µM) affected migration of NC more potently than migration of other cell types. The MINC assay correctly identified the neural crest toxicants triadimefon and triadimenol, additionally it showed different sensitivities to various organic and inorganic mercury compounds. Applying classic pharmacologic inhibitors and large-scale microarray gene expression profiling, we found several signaling pathways that are relevant for the migration of NC in the MINC.

CONCLUSIONS: The MINC assay faithfully models human NC migration, and reveals impairment of this function by developmental toxicants with good sensitivity and specificity.

BACKGROUND: Many bacteria of clinical importance survive and may grow in different environments. Antibiotic pollution may exert on them a selective pressure leading to an increase in the prevalence of resistance.

OBJECTIVES: Determine whether environmental concentrations of antibiotics and concentrations representing action limits used in environmental risk assessment may exert a selective pressure on clinically relevant bacteria in the environment.

METHODS: We used bacterial inhibition as an assessment endpoint to link antibiotic selective pressures to the prevalence of resistance in bacterial populations. Species sensitivity distributions were derived for three antibiotics by fitting log-logistic models to endpoints calculated from minimum inhibitory concentration (MIC) distributions based on worldwide data collated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Bacteria represented in these distributions were placed in a broader context by performing a brief phylogenetic analysis. The potentially affected fraction of bacterial genera at measured environmental concentrations of antibiotics and environmental risk assessment action limits was used as a proxy for antibiotic selective pressure. Measured environmental concentrations and environmental risk assessment action limits were also directly compared to wild-type cut-off values.

RESULTS: The potentially affected fraction of bacterial genera at antibiotic concentrations measured in water environments is estimated not to exceed 7%. Measured environmental concentrations in river sediments, swine feces lagoons, liquid manure and farmed soil are estimated to inhibit wild-type populations in up to 60%, 92%, 100% and 30% of bacterial genera, respectively. At concentrations used as action limits in environmental risk assessment, erythromycin and ciprofloxacin are estimated to inhibit wild-type populations in up to 25% and 76% of bacterial genera.

CONCLUSIONS: Measured environmental concentrations of antibiotics and concentrations representing environmental risk assessment action limits are high enough to exert a selective pressure on clinically relevant bacteria that may lead to an increase in the prevalence of resistance.

BACKGROUND: Inappropriate use of antibiotics in swine feed could cause accelerated emergence of antibiotic resistance genes, and agricultural application of swine waste could spread antibiotic resistance genes to the surrounding environment.

OBJECTIVES: This study was conducted to investigate the distribution of plasmid-mediated quinolone resistance (PMQR) genes from swine feedlots and their surrounding environment

METHODS: We used a culture-independent method to identify PMQR genes and estimate their levels in wastewater from seven swine feedlot operations and corresponding wastewater-irrigated farm fields. Concentrations of (fluoro)quinolones in wastewater and soil samples were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry

RESULTS: The predominant PMQR genes in the samples were qnrD, qepA and oqxB, whereas qnrS and oqxA were present only in wastewater samples. Absolute concentrations of all PMQR genes combined ranged from 1.66×10⁷ to 4.06×10⁸ copies/mL in wastewater and 4.06×10⁶ to 9.52×10⁷ copies/g in soil. Concentrations of (fluoro)quinolones ranged from 4.57 to 321 ng/mL in wastewater and below detection limit to 23.4 ng/g in soil. Significant correlations were found between the relative abundance of PMQR genes and (fluoro)quinolone concentrations (r =0.71, p=0.005) and the relative abundance of PMQR genes in paired wastewater and agricultural soil samples (r =0.91, p =0.005)

CONCLUSIONS: Swine feedlot wastewater may be a source of PMQR genes that could facilitate the spread of antibiotic resistance. To our knowledge, this is the first study to examine the occurrence of PMQR genes in animal husbandry environments using a culture-independent method.

Background: Maternal factors are implicated in the onset of childhood asthma. Differentiation of naïve CD4+ T lymphocytes into pro-allergic T helper 2 cells induces interleukin (IL)4 expression and inhibits interferon (IFN)γ expression accompanied by concordant methylation changes in the promoters of these genes. However, it has yet to be established if maternal exposure to polycyclic aromatic hydrocarbons (PAHs) can alter these gene promoters epigenetically during fetal development.

Objectives: This study sought to elucidate the relationship between maternal PAH exposure and promoter methylation status of IFNγ and IL4.

Methods: We assessed the effects of benzo[a]pyrene (BaP), a representative airborne PAH, on the methylation status of the IFNγ and IL4 promoters in Jurkat cells and two lung adenocarcinoma cell lines, and on gene expression. In addition we evaluated methylation status of the IFNγ promoter in cord white blood cells from 53 participants in the Columbia Center for Children's Environmental Health cohort. Maternal PAH exposure was estimated by personal air monitoring during pregnancy.

Results: In vitro exposure of the cell models to low, non-cytotoxic doses (0.1 and 1 nM) of BaP elicited increased promoter hypermethylation and reduced expression of IFNγ, but not IL4. IFNγ promoter methylation in cord white blood cells was associated with maternal PAH exposure in the cohort study subsample.

Conclusion: Consistent with the results for the cell lines, maternal exposure to PAHs was associated with hypermethylation of IFNγ in cord blood DNA from cohort children. These findings support a potential role of epigenetics in fetal reprogramming by PAH-induced environmental diseases.

BACKGROUND: Bisphenol-A (BPA) is the principal constituent of baby bottles, reusable water bottles, metal cans and plastic food containers. BPA exerts estrogen-like activity interacting with the classical estrogen receptors (ERα and ERβ) and through the G protein-coupled receptor named Gpr30/Gper. In this regard, recent studies have shown that Gper was involved in the proliferative effects induced by BPA in both normal and tumor cells.

OBJECTIVES: We studied the transduction signaling pathways through which BPA influences cell proliferation and migration in breast cancer cells and cancer-associated fibroblasts (CAFs).

METHODS AND RESULTS: We used as a model system the SKBR3 breast cancer cells and CAFs that lack the classical ERs. Specific pharmacological inhibitors and gene-silencing procedures were used to show that BPA induces the expression of the Gper target genes c-FOS, EGR-1 and CTGF through the Gper/egfr/erk transduction pathway in SKBR3 breast cancer cells and CAFs. Moreover, we demonstrate that Gper is required for the growth effects and migration stimulated by BPA in both cell types.

CONCLUSIONS: Our results indicate that Gper is involved in the biological action elicited by BPA in breast cancer cells and CAFs. Hence, Gper-mediated signaling should be included among the transduction mechanisms through which BPA may stimulate cancer progression.

Background: Epidemiological studies have assessed T wave alternans (TWA) as a possible mechanism of cardiac arrhythmias related to air pollution in high risk subjects and have reported associations with increased TWA magnitude.

Objective: In this controlled human exposure study we assessed the impact of exposure to concentrated ambient particulate matter (CAP) and ozone (O₃) on T wave alternans in resting volunteers without pre-existing cardiovascular disease.

Methods: 17 participants without pre-existing cardiac disease were randomized to filtered air (FA), CAP (150 μg/m³), O₃ (120 ppb), or combined CAP+O₃ exposures for 2 hours. Continuous electrocardiograms (ECGs) were recorded at rest and T wave alternans (TWA) was computed by modified moving average analysis with QRS alignment for the artifact free intervals of 20 beats along the V2 andV5 leads. Exposure-induced changes in the highest TWA magnitude (TWA_Max) were estimated for the first and last 5 minutes of each exposure (TWA_{Max_}Early and TWA_{Max_}Late respectively). ∆TWA_Max (Late - Early) were compared among exposure groups using analysis of variance (ANOVA).

Results: Mean values for ∆TWAMax were -2.1 ± 0.4, -2.7 ± 1.1, -1.9 ± 1.5 and -1.2 ± 1.5 in FA, CAP, O₃ and CAP+O₃ exposure groups respectively; with no significant difference between pollutant exposures and FA.

Conclusion: In our study of 17 volunteers without pre-existing cardiovascular disease, we did not observe significant changes in T wave alternans after 2-hour exposures to CAP, O₃, or combined CAP+O₃. This finding, however, does not preclude the possibility of pollution-related effects on TWA at elevated heart rates, such as during exercise, or the possibility of delayed responses.

Background. Specific characteristics of particulate matter (PM) responsible for associations with respiratory health observed in epidemiological studies are not well established. High correlations among and differential measurement errors of individual components contribute to this uncertainty.

Objectives. Investigate which PM characteristics have the most consistent associations with acute changes in respiratory function in healthy volunteers.

Methods. We used a semi-experimental design to accurately assess exposure. We increased exposure contrast and reduced correlations among PM characteristics by exposing volunteers at 5 different locations- an underground train station, two traffic sites, a farm and an urban background site. Each of the 31 volunteers was exposed for 5 hours, while exercising intermittently, 3-7 times at different locations from March-October 2009. We measured PM₁₀, PM_2.5, particle number concentrations (PNC), absorbance, elemental/organic carbon, trace metals, secondary inorganic components, endotoxin content, gaseous pollutants, and PM oxidative potential (OP). Lung function (FEV₁, FVC, FEF_25-75, PEF) and fractional exhaled nitric oxide (FE_NO) were measured before and at three time points after exposure. Data were analyzed with mixed linear regression.

Results. An interquartile increase in PNC (33,000 particles/cm³) was associated with an 11% (95% confidence interval (CI): 5% to 17%) and 12% (95% CI: 6% to 17%) FENO increase over baseline immediately and two hours post-exposure, respectively. A 7% (95% CI: 0.5% to 14%) increase persisted until the following morning. These associations were robust and insensitive to adjustment for other pollutants. Similarly consistent associations were seen between FVC and FEV₁ with PNC, NO₂ and NO_X.

Conclusions. Changes in PNC, NO₂ and NO_X were associated with evidence of acute airway inflammation (FE_NO) and impaired lung function. PM mass concentration and PM₁₀ OP were not predictive of the observed acute responses.

Background: While case-control studies conducted to date have largely affirmed the relationship between polychlorinated biphenyls (PCBs) and non-Hodgkin lymphoma (NHL), occupational cohort studies of PCB-exposed workers have been generally interpreted as negative, thereby raising doubts about a potential causal association. A common theme of immune dysregulation unifies many of NHL’s strongest risk factors, and several authors have posited that sub-clinical immune dysregulation may increase NHL risk by decreasing host resistance, reducing control of cellular proliferation and differentiation, and diminishing tumor surveillance mechanisms.

Objectives: The goals of this review are: I) to evaluate the epidemiological research examining the association between PCB exposure and NHL and discuss the contribution to the weight of evidence of case-control studies and occupational cohort studies; and II) to summarize the evidence for immune dysregulation as a means by which PCBs may cause NHL.

Methods: We performed a literature search using PubMed and 7 additional online biomedical and toxicological referencing libraries to identify literature published through August 2011.

Discussion and Conclusions: Overall, we conclude that the weight of evidence supports a causal role of PCBs in lymphomagenesis. The strongest epidemiological evidence for the relationship between PCBs and NHL comes from case-control studies conducted among the general population. Epidemiological and toxicological data demonstrating immunosuppressive and inflammatory effects of PCBs further contribute to the weight of evidence by providing a plausible explanation for how PCBs can cause NHL through immune dysregulation.

BACKGROUND: Oxidative stress has been implicated in the pathogenesis of a variety of diseases ranging from cancer to neurodegeneration, highlighting the need to identify chemicals that can induce this effect. The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of oxidative stress. Thus, assays that detect the up-regulation of this pathway could be useful for identifying chemicals that induce oxidative stress.

OBJECTIVES: To utilize cell-based reporter methods and informatics tools to efficiently screen a large collection of environmental chemicals and identify compounds that induce oxidative stress.

METHODS: We utilized two cell-based ARE assay reporters, ß-lactamase and luciferase, to screen a U.S. National Toxicology Program 1408 (1340 unique) compound library for their ability to induce oxidative stress in HepG2 cells using quantitative high throughput screening (qHTS).

RESULTS: Roughly 3% (34 of 1340 unique) of compounds demonstrated activity across both cell-based assays. Based on biological activity and structure-activity relationship profiles, we selected 50 compounds for re-testing in the two ARE assays and in an additional follow-up assay that employed a mutated ARE linked to ß-lactamase. Based on this strategy, we identified 30 compounds that demonstrated activity in the ARE-bla and ARE-luc assays and were able to determine structural features conferring compound activity across assays.

CONCLUSIONS: Our results support the robustness of utilizing two different cell-based approaches for identifying compounds that induce ARE signaling. These methods are useful for prioritizing chemicals for further in-depth mechanism-based toxicity testing.