Project Overview

Overview  Dr. Charles Miller at Tulane University’s School of Public Health & Tropical Medicine, Global Environmental Health Sciences, was awarded an RFP-V grant at $1,574,109 to conduct the RFP-V project titled, “Toxicological Properties of Specific Aromatic Hydrocarbons Isolated from Fresh and Aged Crude Oil from the Deepwater Horizon Spill”. The project consisted of1 institution (Tulane), 1 principal investigator (Miller), 3 co-PIs (Drs. Ed Overton, Jeffrey Wickliffe, Mark Wilson), 2 graduate students (Rebecca Lichtler, Ahmad Alqassim), 1 undergraduate student (Jessica Oswalt), several research technicians and administration personnel, and high school students.  

The scientific goal of this research is to elucidate the highly toxic compounds within fresh and weathered crude oil from the MC252 oil spill. The hypothesis of this research proposal is that a relatively small group of the chemicals in oil accounts for most of the toxicity. Learning the identity of these highly toxic compounds will lead to better predictions of the toxic properties of fresh crude oil and will provide a way to follow these substances as oil weathers in the environment. Oil residues from various sites differ in their composition and toxic activity. Furthermore, oil constituents change dramatically with time and weathering. The ability to identify and quantitate the key toxic compounds in oil will permit predictions of adverse human health effects and ecotoxicity in the future.

 

In human and environmental risk assessment studies, the first steps are hazard identification and dose-response analysis. Oil spills are well recognized for causing toxic effects in people and environmental organisms. However, oil is chemically complex and the specific compounds that contribute to its toxicity are surprisingly poorly defined.

Polycyclic aromatic hydrocarbons (PAHs) represent a large family of toxic chemicals in oil. PAHs have received considerable attention from scientists. However, most of this previous research has focused on the PAHs produced by combustion (pyrogenic products), and these are not well represented in oil. The petrogenic PAHs in oil are distinct in that they are generally alkylated and most have never been evaluated for toxicity. A review article from this research team (Envir. Health Perspect. 122, 6-9, 2014) highlighted the need for toxicological characterization of the PAHs and other toxic chemicals (e.g. benzothiophenes, naphthaenoaromatics, etc.,) in oil. This proposal will help to fill this knowledge gap.

 

The marriage of analytical chemical methodologies with cellular bioassays to identify the highly toxic compounds within fresh and weathered oil samples is proposed here. Dr. Miller has developed an established bioassay that identifies the presence and relative potencies of PAHs and structurally related chemicals that activate the aryl hydrocarbon receptor (AhR). Activation of AhR is the key initial step in a signaling pathway that ultimately results in toxicity, and this bioassay provides the screening system to identify toxic components of oil. Dr. Overton is an expert in using analytical chemical approaches to separate and identify compounds in fresh and weathered crude oils. His group has the ability to fractionate and characterize the many compounds within oil. Drs. Wickliffe and Wilson have developed a novel genotoxicity assay based on normal human cell lines (RPTEC/TERT1 and HepaRG cells). These renal and hepatic cell lines represent toxicologically relevant targets for evaluating the mutagenic action of oil compounds. Dr. Overton and colleagues will prepare fractionated oil samples, Dr. Miller’s lab group will identify which of those fractions have the greatest AhR activity, and the active fractions will be evaluated in the cell lines for genotoxicity. Fractions with the greatest activity will be analyzed for chemical content and then re-fractionated by the Overton group. This fractionate and assay process will be repeated until the individual toxic compounds are identified. The ultimate goal of this research is to use these identified toxic compounds as a gauge of toxicity for any fresh or aged oil sample.

 

Research Highlights

Dr. Miller's research, which included 1 outreach product and activity, resulted to date in 3 peer-reviewed publications, 12 scientific presentations, and 13 datasets being submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are to be made available to the public. Significant outcomes of their research (all related to GoMRI Research Theme 5) are highlighted below. 

 

• Development of methods to fractionate crude oil for bioactivity/toxicity analysis. Drs. Pangeni and Overton have pioneered the development of methods to fractionate oil using silica gel separation methods and then further fractionate oil into sub-fractions using High Performance Liquid Chromatography (HPLC) methodology
  
  

• Identification of oil fractions and sub-fractions with bioactivity/toxicity Drs. Pangeni and Overton have used gas chromatography and mass spectrometry (GC/MS) to identify the chemicals within the fractions and sub- fractions of the oil. They can distinguish between families of polycyclic aromatic hydrocarbons and identify whether they contain one, two, three, or four methyl groups, but in most cases the positions of the methyls are not known. They have developed the HPLC methods (using standards) to resolve the six mono- methylated chrysenes from the parent chrysene molecule in the oil. To our knowledge, this level of isomer resolution has not been described until now. A paper describing the chromatographic fractionation, chemical analysis, and bioassays for toxicity is in progress

 

• Identification of candidate chemical compounds to explain the bioactivity within the oil fractions
  
  The work products of Drs. Pangeni and Overton combined with the bioassays from the Miller lab reveal which compounds in oil may be toxic. Screens through the yeast-based aryl hydrocarbon receptor signaling assay (completed this year) show the later fractions 20 through 24 of the 27-28 fractions from the silica gel column are where the activity resides. There are a number of 4 ringed aromatics that come off the column in fractions 20-24. We tested various pure polycyclic aromatic hydrocarbons (obtained commercially, purity > 98%) and used the process of elimination to discover that dibenzo[a]- and [b]anthracenes as well as triphenylene were ~ 100-fold less potent than the chrysenes that were present in the sub-fractions 20-24. Thus, through a process of elimination, we conclude that the chrysene family of compounds is responsible for the bulk of the bioactivity in MC252 oil.

 

• Oil fraction studies: Silica gel chromatography was used for primary and secondary oil fractionation, and standard and reverse phase high performance liquid chromatography (HPLC) were used for the final fractionation steps. Both gas chromatography (GC-) and HPLC-coupled with mass spectrometry (MS) were used to separate and identify compounds present in the petroleum fractions. Bioactivity of the fractions was followed using a recombinant yeast strain that expressed the human aryl hydrocarbon receptor complex (AhRC) that, when activated by chemical ligand(s), expressed a beta-galactosidase reporter gene. Ligand induced beta-galactosidase enzyme activity was proportional to the amount and potency of the compounds in each fraction. Silica gel separations produced 25-29 initial fractions that were assessed for bioactivity using the AHRC reporter system. Bioactivity peaked with the fractions that contained larger PAHs that included four ring compounds such as the triphenylene, benz-anthracene and chrysene families (MW 228). When tested as individual compounds, the triphenylenes were less potent than the chrysenes, so the latter constituted more of the AHRC signaling activity in the oil fractions. Synthetic mixtures of PAHs were reconstructed based upon the chemical composition of the fraction with the greatest AHRC activity. About one third of the activity in this complex fraction was attributable to the chrysene family of compounds. The chrysenes in this fraction were mixtures of the parent compound along with mono-, di-, tri-, and tetra-methyl derivatives along with other PAHs. The six possible mono-methylchrysenes were obtained and tested for AHRC activity and their composition in oil. Chrysene, 1-, 2-, 3-, and 6-methylchrysene were present, but 4- and 5-methylchrysene were not detected in the bioactive fractions of oil that were resolved by HPLC. When tested individually in the AHRC bioassay, 4-methylchrysene was the most potent activator, and 5-methylchrysene was the least potent. Collectively, these results showed that: 1) the six methylchrysene isomers are similar (within one order of magnitude) to chrysene in their ability to activate the AHRC; 2) although they are a minor group, the chrysene family is toxicologically potent, is not readily weathered away, and constitutes a significant portion of the AHRC activity; and 3) this methodology can be used to identify and characterize the bioactivity of sub-fractions and even individual compounds found in oil.

 

• The Miller lab has used HepG2 cells to examine gene expression of chrysene and its six methylated isomers. We found them all to be approximately the same in their cytotoxic (cell death and/or growth inhibitory actions) and in the ability to induce protein expression of target genes (cytochromes P450 1A1, 1B1, glutathione-S-transferase, and NADH-dependent quinone oxidoreductase).
  
  A preliminary report of this work between the Overton and Miller labs was presented at the “Cellular and Molecular Mechanisms of Toxicity” Gordon Research Conference in Andover, NH, in August 2017 and two posters were presented at the GOMOSES conference in New Orleans in February 2018. This research was published in the journal Environmental Toxicology, 2019 Sep;34(9):992-1000. doi: 10.1002/tox.22770.
  
  An additional publication on these results was submitted for review in the upcoming International Oil Spill Conference in May of 2020.
  
  

• Identification and development of cell lines that are amenable to analysis of oil fractions, gene expression, and genotoxicity studies.   
  
  The Hepa1 cell line was derived several years ago from a mouse liver tumor. We have settled on using this cell line for gene expression (rtPCR) and genotoxicity (PIG-A mutagenesis) assays. This cell line was selected based on its useful gene expression properties of candidate oil-regulated genes such as cytochromes P-450 1A1 and 1B1. A preliminary report of this work was presented at the GOMOSES conference in New Orleans in February 2018.
  
  Hepa1 cells also have good properties for studying oil chemical induced mutations. These properties include clear expression of the phosphatidylinositol glycan, Class A protein (PIG-A) for antibody detection and the ability to make single cell suspensions. These properties are key for using the flow cytometric methodology to identify cells that have lost the PIG- A antigen (indicative of mutation). Dr. Mark Wilson has a new flow cytometer and is conducting the PIG-A assay with oil chemicals. This work will be ready for publication in the fall of 2018. The graduate student (R. Lichtler) is working with Drs. Wilson and Wickliffe on this project.
  
  Recently, we have studied the methylated naphthalenes that are present in the Macondo oil. We found methyl-substituted naphthalenes had appreciable activity in aryl hydrocarbon receptor signaling assays, whereas naphthalene had none. We examined gene and protein expression of these compounds in HepG2 cells. The compounds did not induce marker gene or protein expression in these cells. So despite the ability of some methylated naphthalenes to activate aryl hydrocarbon receptor signaling, they were not able to induce gene expression in a liver cell line. One possibility to explain this result is that the liver cells metabolize the methylnaphthalenes before they can induce aryl hydrocarbon receptor signaling. The significance of this finding is that crude oil is much more abundant in methylated naphthalenes than naphthalene. Methylated naphthalenes are relatively unstudied for toxicological effects and our finding suggests that these compounds deserve more scrutiny.