Mark J. Wilson

Evaluation of Polycyclic Aromatic Hydrocarbons Using Analytical Methods, Toxicology, and Risk Assessment Research: Seafood Safety after a Petroleum Spill as an Example

Background: Polycyclic aromatic hydrocarbons (PAHs) are abundant and widespread environmental chemicals. They are produced naturally and through man-made processes, and they are common in organic media, including petroleum. Several PAHs are toxic, and a subset exhibit carcinogenic activity. PAHs represent a range of chemical structures based on two or more benzene rings and, depending on their source, can exhibit a variety of side modifications resulting from oxygenation, nitrogenation, and alkylation. Objectives: Here we discuss the increasing ability of contemporary analytical methods to distinguish not only different chemical structures among PAHs but also their concentrations in environmental media. Using seafood contamination following the Deepwater Horizon accident as an example, we identify issues that are emerging in the PAH risk assessment process because of increasing analytical sensitivity for individual PAHs, and we describe the paucity of toxicological literature for many of these compounds. Discussion: PAHs, including the large variety of chemically modified or substituted PAHs, are naturally occurring and may constitute health risks if human populations are exposed to hazardous levels. However, toxicity evaluations have not kept pace with modern analytic methods and their increased ability to detect substituted PAHs. Therefore, although it is possible to measure these compounds in seafood and other media, we do not have sufficient information on the potential toxicity of these compounds to incorporate them into human health risk assessments and characterizations. Conclusions: Future research efforts should strategically attempt to fill this toxicological knowledge gap so human health risk assessments of PAHs in environmental media or food can be better determined. This is especially important in the aftermath of petroleum spills. Citation: Wickliffe J, Overton E, Frickel S, Howard J, Wilson M, Simon B, Echsner S, Nguyen D, Gauthe D, Blake D, Miller C, Elferink C, Ansari S, Fernando H, Trapido E, Kane A. 2014. Evaluation of polycyclic aromatic hydrocarbons using analytical methods, toxicology, and risk assessment research: seafood safety after a petroleum spill as an example. Environ Health Perspect 122:6–9; http://dx.doi.org/10.1289/ehp.1306724

A targeted health risk assessment following the Deepwater Horizon oil spill: polycyclic aromatic hydrocarbon exposure in Vietnamese-American shrimp consumers.

Background: The Deepwater Horizon oil spill of 2010 prompted concern about health risks among seafood consumers exposed to polycyclic aromatic hydrocarbons (PAHs) via consumption of contaminated seafood. Objective: The objective of this study was to conduct population-specific probabilistic health risk assessments based on consumption of locally harvested white shrimp (Litopenaeus setiferus) among Vietnamese Americans in southeast Louisiana. Methods: We conducted a survey of Vietnamese Americans in southeast Louisiana to evaluate shrimp consumption, preparation methods, and body weight among shrimp consumers in the disaster-impacted region. We also collected and chemically analyzed locally harvested white shrimp for 81 individual PAHs. We combined the PAH levels (with accepted reference doses) found in the shrimp with the survey data to conduct Monte Carlo simulations for probabilistic noncancer health risk assessments. We also conducted probabilistic cancer risk assessments using relative potency factors (RPFs) to estimate cancer risks from the intake of PAHs from white shrimp. Results: Monte Carlo simulations were used to generate hazard quotient distributions for noncancer health risks, reported as mean ± SD, for naphthalene (1.8 × 10–4 ± 3.3 × 10–4), fluorene (2.4 × 10–5 ± 3.3 × 10–5), anthracene (3.9 × 10–6 ± 5.4 × 10–6), pyrene (3.2 × 10–5 ± 4.3 × 10–5), and fluoranthene (1.8 × 10–4 ± 3.3 × 10–4). A cancer risk distribution, based on RPF-adjusted PAH intake, was also generated (2.4 × 10–7 ± 3.9 × 10–7). Conclusions: The risk assessment results show no acute health risks or excess cancer risk associated with consumption of shrimp containing the levels of PAHs detected in our study, even among frequent shrimp consumers. Citation: Wilson MJ, Frickel S, Nguyen D, Bui T, Echsner S, Simon BR, Howard JL, Miller K, Wickliffe JK. 2015. A targeted health risk assessment following the Deepwater Horizon Oil Spill: polycyclic aromatic hydrocarbon exposure in Vietnamese-American shrimp consumers. Environ Health Perspect 123:152–159; http://dx.doi.org/10.1289/ehp.1408684

Single plasmids expressing human steroid hormone receptors and a reporter gene for use in yeast signaling assays.

Single plasmids designed to express the six human type I steroid hormone receptors and detect signaling activity are described in this report. These stably replicating plasmids reported ligand-induced transcriptional activation via lacZ assays in Bakers yeast (Saccharomyces cerevisiae). The ligand concentrations needed to activate signaling in yeast expressing these plasmids spanned five orders of magnitude as based on comparisons of EC(50) values. Radicicol, a direct inhibitor of heat shock protein 90 (Hsp90) and an indirect inhibitor of steroid hormone receptor signaling, was used to determine the functional utility of this yeast reporter system. The inhibitory effect of radicicol was similar on the signaling of all six steroid hormone receptors and was distinguishable from cytotoxic effects that occurred with higher concentrations. These yeast plasmids provide a high throughput system for comparative assessment of steroid hormone receptor signaling and may be useful in screening for pharmacological or xenobiotic activities.

Cadmium alters the formation of benzo[a]pyrene DNA adducts in the RPTEC/TERT1 human renal proximal tubule epithelial cell line.

Previously, we demonstrated the sensitivity of RPTEC/TERT1 cells, an immortalized human renal proximal tubule epithelial cell line, to two common environmental carcinogens, cadmium (Cd) and benzo[a]pyrene (B[a]P). Here, we measured BPDE-DNA adducts using a competitive ELISA method after cells were exposed to 0.01, 0.1, and 1 μM B[a]P to determine if these cells, which appear metabolically competent, produce BPDE metabolites that react with DNA. BPDE-DNA adducts were most significantly elevated at 1 μM B[a]P after 18 and 24 h with 36.34 ± 9.14 (n = 3) and 59.75 ± 17.03 (n = 3) adducts/108 nucleotides respectively. For mixture studies, cells were exposed to a non-cytotoxic concentration of Cd, 1 μM, for 24 h and subsequently exposed to concentrations of B[a]P for 24 h. Under these conditions, adducts detected at 1 μM B[a]P after 24 h were significantly reduced, 17.28 ± 1.30 (n = 3) adducts/108 nucleotides, in comparison to the same concentration at previous time points without Cd pre-treatment. We explored the NRF2 antioxidant pathway and total glutathione levels in cells as possible mechanisms reducing adduct formation under co-exposure. Results showed a significant increase in the expression of NRF2-responsive genes, GCLC, HMOX1, NQO1, after 1 μM Cd × 1 μM B[a]P co-exposure. Additionally, total glutathione levels were significantly increased in cells exposed to 1 μM Cd alone and 1 μM Cd × 1 μM B[a]P. Together, these results suggest that Cd may antagonize the formation of BPDE-DNA adducts in the RPTEC/TERT1 cell line under these conditions. We hypothesize that this occurs through priming of the antioxidant response pathway resulting in an increased capacity to detoxify BPDE prior to BPDE-DNA adduct formation.

compMS2Miner: An Automatable Metabolite Identification, Visualization, and Data-Sharing R Package for High-Resolution LC–MS Data Sets

A long-standing challenge of untargeted metabolomic profiling by ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) is efficient transition from unknown mass spectral features to confident metabolite annotations. The compMS2Miner (Comprehensive MS2 Miner) package was developed in the R language to facilitate rapid, comprehensive feature annotation using a peak-picker-output and MS2 data files as inputs. The number of MS2 spectra that can be collected during a metabolomic profiling experiment far outweigh the amount of time required for pain-staking manual interpretation; therefore, a degree of software workflow autonomy is required for broad-scale metabolite annotation. CompMS2Miner integrates many useful tools in a single workflow for metabolite annotation and also provides a means to overview the MS2 data with a Web application GUI compMS2Explorer (Comprehensive MS2 Explorer) that also facilitates data-sharing and transparency. The automatable compMS2Miner workflow consists of the following steps: (i) matching unknown MS1 features to precursor MS2 scans, (ii) filtration of spectral noise (dynamic noise filter), (iii) generation of composite mass spectra by multiple similar spectrum signal summation and redundant/contaminant spectra removal, (iv) interpretation of possible fragment ion substructure using an internal database, (v) annotation of unknowns with chemical and spectral databases with prediction of mammalian biotransformation metabolites, wrapper functions for in silico fragmentation software, nearest neighbor chemical similarity scoring, random forest based retention time prediction, text-mining based false positive removal/true positive ranking, chemical taxonomic prediction and differential evolution based global annotation score optimization, and (vi) network graph visualizations, data curation, and sharing are made possible via the compMS2Explorer application. Metabolite identities and comments can also be recorded using an interactive table within compMS2Explorer. The utility of the package is illustrated with a data set of blood serum samples from 7 diet induced obese (DIO) and 7 nonobese (NO) C57BL/6J mice, which were also treated with an antibiotic (streptomycin) to knockdown the gut microbiota. The results of fully autonomous and objective usage of compMS2Miner are presented here. All automatically annotated spectra output by the workflow are provided in the Supporting Information and can alternatively be explored as publically available compMS2Explorer applications for both positive and negative modes ( https://wmbedmands.shinyapps.io/compMS2_mouseSera_POS and https://wmbedmands.shinyapps.io/compMS2_mouseSera_NEG ). The workflow provided rapid annotation of a diversity of endogenous and gut microbially derived metabolites affected by both diet and antibiotic treatment, which conformed to previously published reports. Composite spectra (n = 173) were autonomously matched to entries of the Massbank of North America (MoNA) spectral repository. These experimental and virtual (lipidBlast) spectra corresponded to 29 common endogenous compound classes (e.g., 51 lysophosphatidylcholines spectra) and were then used to calculate the ranking capability of 7 individual scoring metrics. It was found that an average of the 7 individual scoring metrics provided the most effective weighted average ranking ability of 3 for the MoNA matched spectra in spite of potential risk of false positive annotations emerging from automation. Minor structural differences such as relative carbon-carbon double bond positions were found in several cases to affect the correct rank of the MoNA annotated metabolite. The latest release and an example workflow is available in the package vignette ( https://github.com/WMBEdmands/compMS2Miner ) and a version of the published application is available on the shinyapps.io site ( https://wmbedmands.shinyapps.io/compMS2Example ).

The RPTEC/TERT1 cell line models key renal cell responses to the environmental toxicants, benzo(a)pyrene and cadmium

Highlights • RPTEC/TERT1 cells are sensitive to low levels of cadmium and benzo[a]pyrene.• We demonstrate compound specific gene expression responses after co-exposure.• Increased CYP activity was detected but not altered in the presence of cadmium.• RPTEC/TERT1 cells mirror responses expected in the proximal tubule cells in vivo.• The RPTEC/TERT1 cell line provides a biologically relevant in vitro model.

Diet‐induced obesity increases the frequency of Pig‐a mutant erythrocytes in male C57BL/6J mice

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig‐a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30‐week old male mice reared on either a high‐fat diet (60% calories from fat) that exhibit an obese phenotype or a normal‐fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N‐ethyl‐N‐nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig‐a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non‐obese mice with respect to Pig‐a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig‐a mutant frequencies (increased 2.5‐3.7‐fold, p < 0.02) in erythrocytes as compared to non‐obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668–677, 2016.

The RPTEC/TERT1 Cell Line as an Improved Tool for In Vitro Nephrotoxicity Assessments

In earlier studies, we have characterized a newly developed cell line derived from the renal proximal tubule epithelial cells (RPTEC) of a healthy human male donor in order to provide an improved in vitro model with which to investigate human diseases, such as cancer, that may be promoted by toxicant exposure. The RPTEC/TERT1 cell line has been immortalized using the human telomerase reverse transcriptase (hTERT) catalytic subunit and does not exhibit chromosomal abnormalities (Evercyte Laboratories). We have previously conducted single-compound and binary mixture experiments with the common environmental carcinogens, cadmium (Cd), and benzo[a]pyrene (B[a]P). Cells exhibited cytotoxic and compound-specific responses to low concentrations of B[a]P and Cd. We detected responses after exposure consistent with what is known regarding these cells in a normal, healthy kidney including significant gene expression changes, BPDE-DNA adducts in the presence of B[a]P, and indications of oxidative stress in the presence of Cd. The RPTEC/TERT1 cell line was also amenable to co-exposure studies due to its sensitivity and compound-specific properties. Here, we review our earlier work, compare our findings with commonly used renal cell lines, and suggest directions for future experiments. We conclude that the RPTEC/TERT1 cell line can provide a useful tool for future toxicological and mixture studies.

Consumption of Fish and Shrimp from Southeast Louisiana Poses No Unacceptable Lifetime Cancer Risks Attributable to High‐Priority Polycyclic Aromatic Hydrocarbons

Following oil spills such as the Deepwater Horizon accident (DWH), contamination of seafood resources and possible increased health risks attributable to consumption of seafood in spill areas are major concerns. In this study, locally harvested finfish and shrimp were collected from research participants in southeast Louisiana and analyzed for polycyclic aromatic hydrocarbons (PAHs). PAHs are some of the most important chemicals of concern regarding oil-spill-contaminated seafood resources during and following oil spills. Some PAHs are considered carcinogens for risk assessment purposes, and currently, seven of these can be combined in lifetime cancer risk assessments using EPA approaches. Most PAHs were not detected in these samples (minimum detection limits ranged from 1.2 to 2.1 PPB) and of those that were detected, they were generally below 10 PPB. The pattern of detected PAHs suggested that the source of these chemicals in these seafood samples was not a result of direct contact with crude oil. Lifetime cancer risks were assessed using conservative assumptions and models in a probabilistic framework for the seven carcinogenic PAHs. Lifetime health risks modeled using this framework did not exceed a 1/10,000 cancer risk threshold. Conservative, health-protective deterministic estimates of the levels of concern for PAH chemical concentration and seafood intake rates were above the concentrations and intake rates modeled under this probabilistic framework. Taken together, consumption of finfish and shrimp harvested from southeast Louisiana following the DWH does not pose unacceptable lifetime cancer risks from these seven carcinogenic PAHs even for the heaviest possible consumers.

Assessment of an irritant gas plume model for epidemiologic study

Abstract Previously, we reported the development of a Hazard Prediction and Assessment Capability plume dispersion model of the 2005 Graniteville, South Carolina, USA accidental release of chlorine. Here, we assess this model by spatial and statistical comparison with post-incident observed environmental indicators of exposure and other types of observations. Spatial agreement was found when the model was compared to phytotoxic bleaching and corrosion events observed in 2 km radius around the release site. When spatially compared to locations of injured or killed animals, model predictions of the plume footprint were in relatively good agreement. Model-predicted human casualties differed from observed casualty counts primarily due to the shielding effect of buildings. A statistical comparison of observed dog health outcome-derived exposure vs. model predicted exposure showed relatively good agreement, particularly when a subcohort of indoor dogs was excluded. Evaluation and assessment of the building infiltration effect would further improve the model prior to application in epidemiologic study.