Superfund Research Program
Predicting the Toxicity of Complex PAH Mixtures
Final Progress Reports
Year: 2019 2012
Studies and Results
The research team has completed microarray based analysis of the significant mRNA expression changes in embryos exposed to the parent PAHs benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at 24 and 48 hours post fertilization (hpf). At both time points, BAA induced expression of a unique set of genes, including several known targets of the AHR, that were not induced by DBT and PYR (Fig. 1). Comparison of the significant DBT vs. PYR gene expression changes indicated that the transcriptional responses to DBT and PYR were very similar at both 24 and 48 hpf suggesting that the relatively small expression differences are due to dose rather than different molecular targets.
The researchers worked with the Analytical Chemistry Research Support Core to conduct uptake studies using GC-MS, and determined the internal body burden of PAHs in embryos following exposure to 0, 1, 5, 10 and 25 uM BAA, DBT and PYR at 24 and 48 hpf. The research group identified striking differences in uptake between the PAHs, with BAA an order of magnitude lower than DBT and PYR. This data was essential for discerning body burden differences from potential mechanistic drivers of differential toxicity induced by the PAHs.
The research team used DAVID functional annotation clustering and Metacore transcription factor prediction to identify significant biological processes and potential transcriptional regulators involved in AHR-dependent (BAA) and AHR-independent (DBT and PYR) mechanisms of developmental toxicity. The investigators found that RELA was an important predicted regulator of both responses, but its targets were different when AHR was activated vs. not activated.
Following a screen of oxygenated PAHs (OPAH) for developmental toxicity and CYP1A induction, the researchers identified three OPAHs, benzanthrone (BEZO), 7,12-benzanthracenedione (7,12-B[a]AQ), and 9,10 phenanthrenequinone (9,10-PHEQ), that induced developmental effects with differential patterns of CYP1A induction and dependence on the AHR. They conducted RNA-Seq analysis of mRNA expression changes induced by these three OPAHs at 48 hpf using Illumina sequencing. Preliminary analysis of differentially expressed transcripts identified 436, 387 and 335 genes significantly differentially expressed in response to BEZO, 7,12-B[a]AQ, and 9,10-PHEQ, respectively. BEZO and 7,12-B[a]AQ had the most overlap, with 156 genes similarly misexpressed, and they identified 46 genes that were common to all three OPAH exposures. Several biomarker genes identified in the microarray with parent PAHs were also identified in the RNA-seq analysis; in addition to cyp1a, the researchers found ctsl.1, sult6b1, gst and several other genes were elevated by the AHR-dependent PAHs. Further analysis of the RNA-seq dataset is in progress to optimize analysis methods, identify novel transcripts, and compare across PAHs to identify molecular mechanisms as well biomarkers of PAH exposure and toxicity that translate across species.
The Seahorse Extracellular Flux Analyzer was used to complete the proposed measurements of in vivo oxidative stress in 24hpf zebrafish embryos and qRT-PCR was conducted at 48hpf using primers for a number of oxidative stress genes in the superoxide dismutase (sod), glutathione transferase (gst), and glutathione peroxidase (gpx) families, important in cellular detoxification and protection from oxidative damage. Several oxidative stress genes were differentially upregulated after developmental exposures to these selected OPAHs. Using the Seahorse assay, mitochondrial function was evaluated by measuring the oxygen consumption rate (ocr). There were decreases in baseline and ATP-linked oxygen consumption rates, and the maximum oxygen capacity in embryos exposed to 9,10-PHEQ, 7,12-B[a]AQ, BEZO, and XAN.
The chemical genetic screen was designed to detect small molecules that could modulate the effects of developmental OPAH exposure. The research team quickly screened approximately 5000 small molecules against the separate effects of BEZO, 7,12-B[a]AQ and phenanthrene quinone and uncovered just over 20 obvious, but putative, hits where OPAH malformation severity was strongly reduced. These results will be pursued via other funding mechanisms. The researchers shifted the focus of Specific Aim 3 to examine PAH effects on early development gene expression changes and cognitive outcomes later in life. The basis for pursuing adult cognitive phenotypes from PAH exposure stems from observations of robust behavioral differences as early as 5 dpf. The researchers are presently analyzing the RNA-Seq data from 6 - 48 hpf exposures of 0.1 and 1ppm B[a]P and 1ppm dibenzo[a,l]pyrene DB[a,l]P. Embryos were exposed in 3 biological replicates of 20 embryos each. The research team is now assessing cognition in adult fish from these same B[a]P and DB[a,l]P exposure cohorts with place preference-shuttle box learning experiments in the laboratory. These experiments will be completed in March 2013 and the investigators are already pursuing a similar approach with the OPAHs BEZO, 7,12-B[a]AQ, 9,10-PHEQ and XAN.
This project pioneered the systematic investigation of the developmental effects of parent and oxygenated PAH (OPAH) exposure. There is growing evidence that OPAHs may be more toxic than parent PAHs. Parent and OPAHs induce different morphological and behavioral effects on development dependent on their structure. Members of each class can be aryl hydrocarbon receptor (AHR) dependent and independent. The gene expression changes, oxidative stress responses and behavioral effects data are becoming a rich body of data from which new insight into the mechanisms of PAH developmental toxicity will continue to emerge. These data will also provide valuable biomarker information for comparison with environmental mixtures containing PAHs. The research team has good reason to anticipate that low micromolar developmental exposures to some parent and oxy PAHs will elicit adverse cognitive outcomes in adults. These studies are important to understanding the mechanisms and long term health impacts of these ubiquitous contaminants.