The mouse skin initiation-promotion model has been used extensively to assess the carcinogenicity of numerous PAHs, singularly and as mixtures (IARC, 2010
) (LaVoie et al., 1993
). The EPA and other agencies use this data, along with results from other animal models, to assess the RPF for PAHs. In this study we employed female FVB/N mice to compare the potency of some complex PAH mixtures found in the environment to the characterized skin carcinogens BaP and DBC. Given the marked increase in tumor incidence, multiplicity and time-to-tumor formation with DBC at 1/100 the molar dose of BaP, we assert that the potency of DBC in this two-stage skin tumor model was more than 100-fold greater than that of BaP and is inconsistent with the current EPA estimate of a BaPeq
of 30 for DBC. ( and 3
). At 100 μg, the RPF of BaP was 100 and DBC 36 μg BaPeq
(1.2 μg × 30). The yield of squamous cell carcinomas was also greater with the 1/100-fold lower DBC dose than with BaP (). Thus, the RPF of DBC in this model is much greater than the currently proposed value of 30 (BaP set at 1). To determine a more accurate RPF for DBC in this model, we would need to conduct a dose-response study.
Examining the results with the environmental PAH mixtures, a similar underestimation of potency can be seen. Based on the published RPFs for the PAHs in the coal tar extract (SRM 1597), the RPF for mix 2 would be 0.34 μg BaPeq
and one would predict a much weaker tumor response compared to BaP alone; however the incidence, latency, multiplicity and tumor type were no different. It is entirely possible that DBC and the mixtures exhibit greater promotional activity than BaP (i.e., are more complete carcinogens). Certainly, with the mixture extracts, there are other components that could be capable of enhancing the TPA promotional activity. These results were somewhat unexpected given previous demonstration that this same PAH mixture inhibits the metabolic activation of BaP and DBC in MCF-7 cells (Mahadevan et al., 2005
) and V79 cells expressing either CYP1A1 or CYP1B1 (Mahadevan et al., 2007b
). The experimental design employed was novel in that we wanted to assess the effect of “mixtures of mixtures”. The lack of response with diesel extract is consistent with the low RPF of 0.004 μg BaPeq
. It would also appear that the increase in RPF (0.47μg, or 0.13 μg above mix 2) with the addition of CSC did not affect carcinogenicity. It is apparent though that the current RPF system for estimation of carcinogenic potency of environmental PAH mixtures or even individual PAHs such as DBC, is inadequate. Our previous observations have shown that individual PAHs exhibit less than additivity and can compete with more potent individual PAHs for the same enzymes (Courter et al., 2006
, Mahadevan et al., 2007a
) It is also unlikely that complex environmental PAH mixtures have been exhaustively characterized with respect to all components and accurate RPFs determined. It would seem a more prudent approach when conducting risk assessments to utilize RPFs of mixtures rather than a summation of individual PAHs. It would also seem prudent to more thoroughly test PAHs such as DBC that exhibit high carcinogenic potency (levels of DBC are rarely reported in environmental samples) using additional models (both in vivo
animal models and in vitro
human cell models). The distinct pattern of gene expression with DBC also raises an important question with respect to whether or not all PAHs are carcinogenic through the same mechanism of action (MOA) which is important with respect to whether or not an RPF approach for carcinogenic risk assessment for mixtures is appropriate.
Another conclusion from this study is that PAH-DNA adduct formation in skin after single administration did not predict the final tumor response (). Based on the total DNA adducts present in skin 12 hours post-initiation, one would predict that BaP would give the most robust tumor response (3-fold greater than DBC). Measuring adducts at one time point may not be sufficient for comparing these particular PAHs; however (Courter et al. 2008
) found similar results at 24 h in Sencar mice. An attempt was made to investigate a time when adduct formation would be at peak levels. The 12 h post initiation time was chosen for measuring DNA adduct formation based on previous work in our lab (Marston et al., 2001
). Sencar mice treated topically with BaP showed peak DNA adduct levels at 12 h. Adduct levels in DBC treated animals peaked at 12 h and were sustained until 24 h post treatment. It has been well documented that PAH structural features (Geacintov et al. 2002
, Wu et al., 2002
), specific types of adducts formed (Dreij et al., 2005
), DNA repair enzyme recognition (Braithwaite et al., 1999
), and replication bypass fidelity (Lagergvist et al., 2011
) all contribute to levels of PAH caused mutagenicity. BaP forms predominantly adducts at the N2
position of dG while DBC forms more at the N6
position of dA. The bay region containing BaP is a planar, less flexible molecule compared to the fjord containing DBC. These two structural features in DNA adduct formation are thought to enable DBC adducts to sit in the large groove of the helix and be unrecognized by repair enzymes (Geacintov et al., 2002
). Initial DNA damage, persistence of the damage, as well as the mutagenic specificity of individual DNA adducts, all contribute to the mutagenic potency and subsequently carcinogenic potency of the tested PAHs. This suggests that one must be cautious in interpretation of DNA adduction as a biomarker of PAH dependent skin tumorigenesis, especially if relying on a single time point or comparing PAHs that may be bioactivated through a variety of pathways.
In order to determine similarities and differences in comparing DBC, BaP and the PAH mixtures with respect to potential mechanisms of action, we examined alterations in the transcriptome of the skin 12 h post-initiation. A total of 922 genes were significantly up or down regulated cumulatively for all treatment groups relative to the toluene control. PAHs are known to exert toxicity, including carcinogenesis, through alteration of Ahr-regulated genes (Andrysik et al., 2011
). As expected BaP and the mixtures containing coal tar extract significantly induced both Cyp1a1
. The response with DBC was unexpected. Although DBC is a much more potent skin carcinogen, dermal Cyp1a1
expression was not induced but was slightly decreased (). PAHs are also known to exhibit toxicity through induction of oxidative stress (Kumar et al., 2012
). A number of genes regulated by the Keap 1- Nrf-2 signaling system (Niestroy et al., 2011
) were up-regulated by BaP and the coal tar-containing mixtures but, again, DBC had no effect or slightly down-regulated expression (). Principal component analysis confirmed that DBC altered a set of genes that did not cluster with BaP or the coal tar-containing mixtures (). In support of the enzyme expression profiles and clustering analysis, we observed that the biological process, response to xenobiotic stimulus (GO:0009410), was significantly (p<0.05) enriched by BaP and not by DBC. In fact, the genes in this category were strongly up-regulated by BaP and either not changed or slightly down-regulated by DBC at 12 hours post-initiation. These data suggest that BAP may be inducing a protective response early during initiation through up-regulation of xenobiotic metabolism, while DBC exposure may result in a less protected cellular environment resulting in a higher tumor incidence. Future studies will examine the regulatory differences between BaP and DBC to understand how early changes during initiation may contribute to tumor outcome.
The principal components analysis also revealed that the coal tar mixtures clustered separately from both DBC and BaP (). Our study shows that the coal tar extract is driving both the tumor incidence and gene expression profiles of the environmental PAH mixtures. Therefore, we focused our bioinformatic analysis of the transcriptional data on genes that are expressed in common between mixtures 2 and 3, but unique from mixture 1, to identify mechanisms associated with skin tumorigenesis. The Venn diagram () shows that over 50% of the genes altered by mix 2 or 3 (270 out of 428 or 521, respectively) were shared between them and only a fraction of these genes (13% or 36 out of 270) were also shared with mix 1. These results call into question whether or not PAHs, in such environmental mixtures, share a common mode of action (MOA), an assumption important in utilizing an RPF approach in risk assessment. Functional analysis of the genes specific to the coal tar PAH mixtures () suggest that down-regulation of DNA repair and cell cycle processes and up-regulation of xenobiotic metabolism are consistent with the enhanced tumorigenicity of these mixtures compared to diesel extract.
In conclusion, the results from this study suggest that an approach to assessing the carcinogenicity of PAH mixtures employing RPFs of individual PAHs has some potential areas of concern. We found DBC and PAH mixtures containing coal tar to have potency as skin carcinogens much greater than would be predicted from the RPFs. A common biomarker to predict carcinogenicity, covalent DNA adducts, did not predict final tumor response. An RPF approach to risk assessment also assumes (as with TEQs) for dioxins/dibenzofurans (Gies et al., 2007
) a common MOA. Our examination of PAH-dependent alterations in the transcriptome from mouse skin calls into question whether or not a common MOA can be assumed. The pattern with DBC was markedly different than BaP and the coal tar-containing mixtures were distinct when compared to either BaP or DBC. Of course, an important caveat to this conclusion is that we sampled a single time-point of 12 hours. The pathways in the , in addition to the well studied Cyp driven diol epoxides, include peroxidase formation of radical cations and AKR formation of o-quinones. Further studies looking at the role of these additional pathways as mechanisms of DBC bioactivation and inhibition of Cyps should be done to broaden our understanding of the complexities and differences among different PAHs and mixtures of PAHs. Further study is needed to determine how the distinct gene clustering in mouse skin at this time point relates to the tumor response in this 25-week two stage initiation-promotion model.