Environmental six-ring polycyclic aromatic hydrocarbons are potent inducers of the AhR-dependent signaling in human cells

Highlights

• Effects of environmental PAHs with MW302 on activation of the AhR were studied.

• MW302 PAHs were potent agonists of the AhR in rat and human reporter gene assays.

• MW302 PAHs induced or repressed relevant AhR-responsive genes in human lung cells.

• MW302 PAHs repressed estrogenic responses in human cells in AhR-dependent manner.

• AhR activation could be a major toxic mode of action of environmental MW302 PAHs.

Abstract

The toxicities of many environmental polycyclic aromatic hydrocarbons (PAHs), in particular those of high-molecular-weight PAHs (with MW higher than 300), remain poorly characterized. The objective of this study was to evaluate the ability of selected environmentally relevant PAHs with MW 302 (MW302 PAHs) to activate the aryl hydrocarbon receptor (AhR), since this represents a major toxic mode of action of PAHs. A large number of the evaluated compounds exhibited strong AhR-mediated activities, in particular in human models. The studied MW302 PAHs also significantly contributed to the overall calculated AhR activities of complex environmental mixtures, including both defined standard reference materials and collected diesel exhaust particles. The high AhR-mediated activities of representative MW302 PAHs, e.g. naphtho[1,2-k]fluoranthene, corresponded with the modulation of expression of relevant AhR target genes in a human lung cell model, or with the AhR-dependent suppression of cell cycle progression/proliferation in estrogen-sensitive cells. This was in a marked contrast with the limited genotoxicity of the same compound(s). Given the substantial levels of the AhR-activating MW302 PAHs in combustion particles, it seems important to continue to investigate the toxic modes of action of this large group of PAHs associated with airborne particulate matter.

Introduction

Polycyclic aromatic hydrocarbons (PAHs), derived mostly from various types of combustion processes, are widely distributed in the environment in polluted air, as well as in soil, water, and sediment, as a result of atmospheric transportation, and wet or dry deposition (Zhang et al., 2016). The exposure to airborne PAHs presents a significant threat to human health (Boström et al., 2002; Kim et al., 2013). The airborne carcinogenic PAHs, such as benzo[a]pyrene (BaP), are mostly associated with the fine particulate matter (PM_2.5) (Allen et al., 1998; Hays et al., 2003; Kameda et al., 2005). The carcinogenic or other health risks, associated with exposure to PM_2.5-bound PAHs can be particularly high in locations with high intensities of traffic, coal and biomass combustion or industrial production (Mesquita et al., 2014; Yan et al., 2019; Zhang et al., 2019). Currently, 16 PAHs are included in the priority pollutants list of the U. S. Environmental Protection Agency (US EPA), which includes seven carcinogenic PAHs: BaP, benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), chrysene (Chry), dibenz[a,h]anthracene (DBahA), and indeno[1,2,3-cd]pyrene (IPy) (US EPA, 1993). Although the PAHs compiled in this list contribute significantly to the toxic/carcinogenic effects of complex environmental mixtures, there are numerous other polyaromatic substances that remain poorly characterized. Therefore, using just the set of 16 PAHs can be associated with serious limitations potentially leading to underestimation of the toxicities of complex environmental mixtures, since many toxic PAHs common in polluted air are not included in the current risk assessment schemes (Andersson and Achten, 2015).

The list of priority carcinogenic PAHs has been extended by the European Union authorities, in order to accommodate additional important carcinogenic compounds, in particular dibenzopyrenes (EU, 2005). Dibenzopyrenes belong among PAHs with molecular mass 302 (MW302 PAHs), which have been found to be mutagenic and/or carcinogenic (Bergvall and Westerholm, 2009; Cavalieri et al., 1991; Durant et al., 1996; Durant et al., 1999; Durant et al., 1998; Hannigan et al., 1998). Dibenzo[a,l]pyrene (dibenzo[def,p]chrysene; DBalP) is a particularly strong mutagen (Cavalieri et al., 1991; Durant et al., 1999; Durant et al., 1998), identified in airborne or combustion PM samples (Bergvall and Westerholm, 2007, 2009), which is classified by the International Agency for Research on Cancer as Group 2A carcinogen, i.e. probably carcinogenic to humans. Other isomers of DBalP, such as dibenzo[a,i]pyrene (DBaiP), dibenzo[a,h]pyrene (DBahP) or dibenzo[a,e]pyrene (DBaeP), are often present at higher levels than DBalP, and the first two are also listed as possible (Group 2B) human carcinogens (IARC, 2010). Both dibenzopyrenes and additional mutagenic MW302 PAHs, such as naphtho[2,1-a]pyrene (N21aP), can contribute to the mutagenic activity of PAHs associated with PM_2.5 (Durant et al., 1998). However, there are other MW302 PAHs, which are present in PM_2.5 samples, such as naphtho[1,2-b]fluoranthene (N12bF), naphtho[1,2-k]fluoranthene (N12kF) or dibenzofluoranthenes (Allen et al., 1998; Wei et al., 2011), that have received very limited attention. Large PAHs, including MW302 PAHs, often make a significant contribution to the total mass of PAHs bound to PM_2.5 (Chen et al., 2019). Together with other unsubstituted and halogenated PAHs, MW302 PAHs may contribute to elevated cancer risk in heavily exposed populations (Wang et al., 2012); however, presently, toxicity data for MW302 PAHs are mostly limited to several dibenzopyrenes.

Similar to other PAHs, the risk assessment of those MW302 PAHs that are currently included among priority pollutants, is nowadays based on relative potencies or toxic equivalency factors that are linked with their mutagenicity/carcinogenicity (Allen et al., 1998; Bergvall and Westerholm, 2009; Collins et al., 1998; Hannigan et al., 1998; Jiang et al., 2017; Mueller et al., 2019; Nisbet and LaGoy, 1992; Pedersen et al., 2005; Richter-Brockmann and Achten, 2018; Sadiktsis et al., 2012; Wang et al., 2012; Wei et al., 2011). The formation of stable DNA adducts, or induction of oxidative DNA damage, are key steps contributing to tumor initiating properties of many PAHs (Baird et al., 2005; Penning, 2014). However, the mutagenic activity of MW302 PAHs is not the only mechanism contributing to their toxic and carcinogenic properties. The activation of the aryl hydrocarbon receptor (AhR) is a major toxic mode of action of many PAHs (Andrysík et al., 2011; Machala et al., 2001; Pieterse et al., 2013; Vondráček et al., 2017). The AhR activity is important not only for the induction of enzymes producing the genotoxic PAH metabolites, such as cytochrome P450 family 1 (CYP1) enzymes (Nebert and Dalton, 2006), but it is also involved in a wide range of biological processes linked with tumor promotion or progression (Dietrich and Kaina, 2010; Murray et al., 2014; Safe et al., 2013). Importantly, the AhR activation is a major toxic mode of action of various types of complex mixtures of PAHs, which contain many rarely studied environmental PAHs that are not covered in current risk assessment schemes (Huang et al., 2018; Kamelia et al., 2019).

The AhR binding affinities of carcinogenic PAHs have been proposed to correlate with their tumor promoting properties (Boström et al., 2002; Sjögren et al., 1996). When considering the effects of complex mixtures of PAHs, containing many distinct polyaromatic AhR ligands, the AhR activation by such mixtures can be often observed at lower doses than e.g. formation of stable DNA adducts (Andrysík et al., 2011). This could be partly caused by inhibition of CYP1 enzymatic activities by compounds present in such mixtures (Courter et al., 2007; Líbalová et al., 2014b; Mahadevan et al., 2005). Therefore, PAHs may activate various mechanisms contributing to their carcinogenesis, including those initiated via their binding to the AhR, and their relative potency factors based on mutagenicity assays may not always accurately reflect their carcinogenicity (Tilton et al., 2015). The estimation of the relative effective potencies (REPs) of MW302 PAHs acting as AhR agonists may thus provide an important information about their toxicity profiles. Importantly, their AhR-mediated responses may not only include the direct transcriptional effects of this receptor, but also its interactions with additional signaling pathways, such as estrogen receptors (ER). Such effects can be associated with disruption of endocrine signaling or developmental defects induced by environmental PAHs (Kamelia et al., 2019; Zhang et al., 2016). Just recently, two studies addressing developmental toxicity of a broad range of PAHs, which included also 10 PAHs with MW302, have suggested that some of these compounds exert developmental/behavioral defects in zebrafish embryos, as well as stimulate CYP1A expression in some organs. Importantly, based on the results of transcriptomic analysis in zebrafish model, representative MW302 PAHs, such as DBahP and DBaiP clustered together with other known efficient ligands of AhR, such as benzofluoranthenes. Therefore, the authors of those studies have predicted that it will be important to analyze in detail the AhR-inducing potencies of these PAHs (Geier et al., 2018; Shankar et al., 2019). Together, these data indicate that the estimation of the AhR-inducing potencies and determination of regulation of direct endogenous AhR gene targets in human cells, as well as the information about their interactions with additional intracellular signaling modules, such as the ER-mediated cellular responses, may provide an important insight into the toxic modes of action of MW302 PAHs.

In this study, the estimation of the AhR-activating potencies of MW302 PAHs by reporter gene assays was combined with additional in vitro bioassays addressing specific effects of selected non-genotoxic MW302 PAHs on the AhR-mediated cellular responses. Specific objectives were: i) to determine REPs of MW302 PAHs activating rodent and human AhR; ii) to estimate relative contribution of the MW302 PAHs to the total AhR-activity of complex mixtures, as compared with the US EPA priority carcinogenic PAHs (cPAHs); iii) to compare genotoxic (formation of DNA adducts and apoptotic response to DNA damage) and non-genotoxic (endogenous AhR-dependent gene expression) effects of selected MW302 PAHs in human model of alveolar type II cells, adenocarcinoma A549 cell line; and, iv) to determine the ability of MW302 PAHs acting as strong AhR ligands to inhibit estrogenic signaling in cell models represented by human estrogen-sensitive breast carcinoma cell lines. Our overall aim was to characterize in detail the AhR-dependent responses to MW302 PAHs in human cell models.