Lung ADC is a non-small cell lung carcinoma (NSCLC) subtype that is the most prevalent among both smokers and non-smokers (American Cancer Society 2017) with many etiologies besides smoking including a number of environmental and occupational exposures involving PAHs, such as air pollution, diesel exhaust, coal and coke production exposures, all categorized by the IARC as group 1 carcinogens (IARC 2010, 2012a, b, 2013, 2016). In developing and transition countries the use of indoor coal fueled stoves release high levels of PAHs and is strongly linked to high lung cancer mortality, as well as to the development of other respiratory inflammatory diseases, including chronic obstructive pulmonary disease (COPD) (Zhang and Smith 2007). Diesel exposure, particularly in nonsmokers, also significantly associates with increased risk of lung cancer (Silverman et al. 2012). This current study demonstrates the need for an improved understanding of LMW PAHs that are typically far more abundant in these exposures described above than the HMW PAHs and potentially a re-evaluation of their carcinogenicity following additional in vivo studies and/or in vitro studies in human cell lines.
Since the 1990s, B[a]P, a HMW PAH, has been the reference PAH to determine relative potency factors to estimate carcinogenicity and rank toxicity (Nisbet and LaGoy 1992; Environmental Criteria and Assessment; Office 1993), while due to a lack of knowledge, there are many PAHs, particularly those smaller in size, categorized as non-genotoxic, non-carcinogenic, and non-cytotoxic in most models (Ghoshal et al. 1999; Tai et al. 2007; Upham et al. 2008). However, we recently demonstrated that several of these LMW PAHs can elicit numerous adverse lung cell responses in mouse lung cells, namely, induce cytotoxicity, inhibit gap junctions, activate MAP kinases, and induce inflammatory pathways such as cyclooxygenase, Cxcl1 (Kc), and Il6 following acute 30 min–24 h exposures (Osgood et al. 2017). It is also the case that the LMW binary PAH mixtures compared to the individual LMW PAHs did not always respond in an additive manner, but instead in a more synergistic manner, specifically mRNA expression and cytotoxicity (Osgood et al. 2017). This suggests that these PAHs differ in their mechanisms of action (MOA) (Osgood et al. 2017). Importantly, some of these phenotypes are considered key components of the tumor promotion stage of carcinogenicity, specifically, GJIC inhibition, induction of inflammatory mediators, and activation of mitogenic signaling (i.e., MAP kinase pathways), all involved in certain hallmarks of cancer (Hanahan and Weinberg 2011; Klaunig et al. 2000; Trosko and Upham 2010; Zhang and Smith 2007). GJIC is involved in the evasion of growth suppression (Nahta et al. 2015), inflammation in the enabling characteristic of tumor promoting inflammation (Hanahan and Weinberg 2011), and MAP kinase pathways in the self-sufficiency in growth signals (Hanahan and Weinberg 2011). It appears that these LMW PAHs cannot initiate carcinogenesis, however, acting at the promotion stage of cancer or as a co-carcinogen, is still considered as a carcinogen. Thus, in these novel studies we evaluated several established carcinogenic endpoints (DNA adduct formation, GJIC inhibition, and induction of an inflammatory mediator pathway), typically acting during the early stages of carcinogenicity, following exposure of C10 cells to a known classic carcinogen (B[a]P) in combination with the same LMW binary PAH mixture we previously used to provide evidence that these LMW PAHs can act as co-carcinogens in the presence of B[a]P.
B[a]P DNA adducts and cancer development
Our studies revealed that both the B[a]P DNA adducts and gap junctional activity acted in a manner expected for a carcinogen. DNA adducts such as those observed in our studies, are regarded as a critical step in the initiation stage of carcinogenesis. Anti-BPDE adducts can lead to mutations via transversions such as G:C → T:A, although DNA repair mechanisms can remove and replace these adducts (Miller and Ramos 2001). DNA adducts were significantly increased in the presence of the B[a]P and LMW PAH combination compared to B[a]P alone. The fact that these adducts were increased suggests that the LMW PAHs can act as co-carcinogens, however, it does not rule out the potential promoting capability of these LMW PAHs.
GJIC and cancer development
The significant inhibition of GJIC observed with B[a]P combined with LMW PAHs at these low doses compared to B[a]P alone further demonstrates that these LMW PAHs act as co-carcinogens. Gap junctions, composed of connexins (Cx), are intercellular channels that allow for molecular communication between neighboring cells that are often inhibited by toxicants, such as tumor promoters (Trosko and Upham 2010). As mentioned above, GJIC is involved in the hallmark of cancer concerning evasion of growth suppression and based on studies in vitro (Cesen-Cummings et al. 1998; Osgood et al. 2017; Tai et al. 2007; Trosko and Upham 2010; Upham et al. 2008) and in vivo (Avanzo et al. 2004), GJIC inhibition is a critical step in the early stages of cancer development. Although controversial, it is hypothesized that connexins act as tumor suppressors that when inhibited, the growth-promoting factors are no longer diluted and intracellular signaling increases which leads to enhanced growth and eventually tumor development (Nahta et al. 2015). For example, Cx43 is significantly reduced in response to multiple toxicants, such as the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in both rodent and human epithelial cell types of the lung (C10 cells), liver (WB cells), and breast (MCF-10A cells) (Osgood et al. 2013; Rakib et al. 2010; Upham et al. 2008). Additionally, mice heterozygous for a Cx43 deficiency developed more lung tumors than their wildtype counterparts (Avanzo et al. 2004) supporting the importance of gap junctions in tumor suppressive activities. Lastly, aberrant GJA1 mRNA expression was observed in NSCLC patients (Chen et al. 2003). However, other mechanisms exist that could be involved in this underlying mechanism (e.g., hemichannel formation) that need to be explored further (Nahta et al. 2015).
Inflammation and cancer
The involvement of inflammation in cancer is not a novel concept and is now considered a critical component of cancer development, elucidated by the classification of inflammation as an enabling characteristic in tumor promotion by Hanahan and Weinberg in 2011. In particular, the pathway evaluated herein, COX-2, leads to the production of prostaglandins, such as PGE2 and PGF2. PGE2 can induce tumor progression through epidermal growth factor receptor (EGFR) signaling and increased proliferative responses in lung ADC (Bazzani et al. 2017). COX-2 is also known to be significantly elevated in NSCLC, although clinical trials for COX-2 inhibitors (e.g., apricoxib) have not proven successful as a therapy for lung ADC (Edelman et al. 2017). Cox-2 was significantly elevated 4–8 h (> 20-fold increase) following treatment with the same LMW binary PAH mixture as used in these studies; however, the doses where these effects were observed were substantially higher (40 µM). The increase observed following 24 h used here at a 8–40-fold lower dose of the LMW binary PAH mixture elicited significant responses with B[a]P or the LMW binary PAH mixture alone and also in all of the combinations of B[a]P with the LMW binary PAH mixture compared to control, further supporting the co-carcinogenic or tumor promoting capabilities of these LMW PAHs. However, the response was significantly increased over the B[a]P or LMW PAH mixture alone when in combination with either B[a]P and 1 or 5 µM PAH mix. Another recent study demonstrated that direct lung application of B[a]P in C57BL/6 mice did not elicit inflammation above that observed in the controls (measured via bronchoalveolar lavage analysis)(Arlt et al. 2015), which supports our in vitro results that B[a]P alone is a weak inflammagen.
PAH exposures and carcinogenicity
Several studies have examined B[a]P carcinogenicity in animal models. For example, a skin cancer study at low doses demonstrated that B[a]P acted as an initiator, and in the presence of promoters (other LMW PAHs), skin tumors developed (Warshawsky et al. 1993), further supporting the tumor promotion potential of LMW PAHs. Additionally, when coal tar was compared to B[a]P alone at the same concentration as observed in the coal tar, B[a]P-induced lung tumor numbers were significantly lower and did not equate to the number of tumors observed in the coal tar exposed group (Fitzgerald et al. 2004). Again, the results suggest that B[a]P does not act alone in PAH-induced lung carcinogenesis.
A recent paper indicated that different carcinogenic PAHs have different MOAs, via studies using transcriptomics (Labib et al. 2016). Carcinogenic HMW PAHs had different transcript profiles and while they all induced DNA adducts, the MOAs for carcinogenicity were not the same, thus cannot be based on B[a]P alone. Therefore, the use of B[a]P as the reference PAH may be overestimating or underestimating the carcinogenicity of these PAHs. For the LMW PAHs, based on our previous report and others (Osgood et al. 2017; Upham et al. 2008), it is critical that other endpoints are evaluated for the PAHs that have no initiating effects, but are acting in a co-carcinogenic or tumor promoting manner, or these important early stage cancer responses could be underestimated and overlooked.
Co-carcinogenic effects of PAHs and toxic equivalency factors
Lastly, to more clearly understand the differences between the complete carcinogenesis endpoints versus co-carcinogenesis and/or promoting endpoints, we assessed the toxic equivalency factors (TEFs) of B[a]P, Flthn and 1-MeA for their co-carcinogenic effects. For this analysis, we used GJIC inhibition as the toxicological endpoint of interest in the C10 cells using the 24 h data generated for these studies and our previous studies (Osgood et al. 2017). We also set B[a]P as our reference standard (TEF = 1.0). Figures 4a and 5a show that 1 µM B[a]P results in approximately 47 and 34% reduction in GJIC (= 53 and 66% fraction of control) which can be averaged to about 40% reduction in GJIC. Our recently published data on the inhibition of GJIC by Flthn and 1-MeA revealed a similar 40% reduction for approximately 10 µM Flthn and 20 µM 1-MeA (Osgood et al. 2017). Therefore, the TEFs for GJIC inhibition in C10 cells following 24 h exposure can be estimated to be approximately 1.0 : 0.1 : 0.05 for B[a]P, Flthn, and 1-MeA with B[a]P showing the highest and 1-MeA showing the lowest GJIC inhibition.
Applying these calculated TEFs to the three mixtures which we have tested (1 µM B[a]P + 1, 5, or 10 µM PAH mix consisting of 1:1 Flthn and 1-MeA) and using an additive model would result in approximately 43, 55, and 70% inhibition of GJIC (= 57, 45, and 30% fraction of control). The actual observed GJIC inhibition in our experiments were 47, 62 and 66% (= 53, 38 and 34% fraction of control, Fig. 5a), respectively, and thus well in agreement with the calculated results.
Overall, our results suggest that the co-carcinogenic effects of PAH mixtures are the sum of the effects caused by the respective individual compounds. In addition, TEFs can be used to assess effects of mixtures such as PAHs. However, the use of TEFs must be endpoint-specific. For example, previously published TEFs which have been established for carcinogenicity [e.g., 1.0 for B[a]P or 0.001 for Flthn; (DFG 2012; Nisbet and LaGoy 1992)] cannot be applied to co-carcinogenic endpoints such as GJIC because they would underestimate these co-carcinogenic effects, in this example by 100-fold. In turn, the TEFs for co-carcinogenic effects cannot be applied to assess the actual carcinogenic outcome of PAH mixtures in animals or humans because they would overestimate carcinogenicity. The molecular endpoints used in our studies reflect those that are known to contribute both to initiating events (DNA adducts) and to co-carcinogenesis or tumor promoting events (GJIC and COX-2) and not meant as endpoints for complete carcinogenesis. This conflation of a carcinogen that is a complete carcinogen versus those that are either co-carcinogens or promoters needs better clarification for future risk assessment. Co-carcinogens and promoters do not result in tumor development unless in the presence of an initiator, such as B[a]P, however, both are still carcinogens.