In vitro Evaluation of Polycyclic Aromatic Hydrocarbon Compound (PAC) Mixtures
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Multiple States have explored the implementation of in vitro new approach methodologies (NAMs) as component of water quality monitoring programs to detect biological activity indicative of potential contaminant and/or contaminant mixture occurrence. Polycyclic aromatic compounds (PACs) have been detected in environmental matrices, and many are known to activate the human Aryl hydrocarbon receptor (hAhR). As part of a larger mixtures study, in collaboration with the Division of Translational Toxicology (NIEHS), we aim to explicate the confounding discrepancies between in vitro and in vivo PAC mixture responses to increase confidence in application of similar NAMs to future monitoring efforts. We used the INDIGO Biosciences hAhR reporter assay, to test 13 PACs (benzo[a]pyrene, benzo[k]fluoranthene, dibenz[a,h]anthracene, chrysene, benz[j]aceanthrylene, indeno[1,2,3-cd]pyrene, phenanthrene, pyrene, dibenzo[a,l]pyrene, dibenzothiophene, acenaphthenequinone, benzo[c]fluorene, and benzo[b]fluoranthene) for hAhR activation. Cells were plated on a 96-well plate and AhR activity (luminescence) and cytotoxicity (absorbance) were quantified. Potency (EC50 values) and efficacy (percent maximal response of MeBio positive control) were determined using the Hill model and dose-response curve fits were developed using the drc package in R. In vitro EC50 values ranged from 1.2x10-8 M (dibenz(a,h)anthracene) to 2.8x10-6 M (benzo(a)pyrene). Dibenzothiophene, dibenzo[a,l]pyrene, pyrene and phenanthrene did not activate the AhR in vitro at exposure concentrations up to 1 mM molar. Benzo[a]pyrene had the highest efficacy (85.9% maximal MeBio response). Three test mixtures were designed based on in vivo responses (immunotoxicity bioassay; antibody formation in B6C3F1/N female mice splenic lymphocytes) and tested in vitro. In general, in vitro EC50 of the test mixtures decreased with increasing molar concentrations of the most potent individual PACs in vitro (e.g. benzo(k)fluoranthene and dibenz(a,h)anthracene), and the Concentration Addition mixtures model over-estimated maximal efficacy of all three test mixtures. Chemical analysis of in vitro exposure media is underway to address relative potency discrepancies between in vitro and in vivo responses of individual compounds. For example, dibenzo[a,l]pyrene, benzo[c]fluorene, and acenaphthenequinone were active in vivo but not in vitro. Overall, our analysis will support future application of this in vitro AhR effects-based approach for detecting PACs and other AhR-activating environmental contaminants likely to cause adverse health outcomes. Abstract does not reflect Agency views or policy.