Investigations of the role of PAHs in human cancers can be traced back to 1775, when Percivall Pott found an association between exposure to soot and scrotal cancer in chimney sweeps.[28
] More recently, the International Agency for Research on Cancer (IARC) recognized some complex PAH mixtures (e.g. coal tar) and industrial processes (e.g. coke production) as carcinogenic in humans, but individual PAHs were considered only probable or possible carcinogens in humans.[18
] It was only in the most recent IARC review that benzo[a]pyrene exposure in occupational settings was categorized as a definite human carcinogen.[19
An individual’s exposure to PAHs can be estimated in several ways, including measuring PAH metabolites in urine, staining tissues with anti-PAH antibodies, and chemical analysis of target or surrogate tissues (eg. esophageal tissue or blood) for adducted or non-adducted PAHs. Urinary metabolites of PAHs, such as 1-hydroxypyrene glucuronide (1-OHPG), reflect total body exposure in the 24–72 hours prior to collection,[29
] which may be useful for estimating habitual exposures but may not capture episodic exposures. Antibodies raised against PAH immunogens can be used to estimate PAH exposure in specific tissues of interest, such as the esophagus, but they may vary in their specificity and are only semiquantitative. Chemical analysis of adducted or non-adducted PAHs is the most specific and quantitative way to measure PAH exposure, and this analysis can be performed in the target tissue for carcinogenesis (eg. the esophagus) or in surrogates (eg. blood). Since DNA adducts can be repaired, they are usually less permanent than protein adducts.[30
] Studies in experimentally exposed rats using 32
P postlabelling have shown that maximum DNA adduct levels are reached three days after a single dose of BaP, followed by a rapid decay.[27
] Because of the absence of active repair, the stability of protein adducts varies over a longer time scale, which depends on the protein’s stability and the rate of cell turnover in the mucosa.[30
] The strengths and limitations of measuring PAH adducts by immunoassays, 32
P-postlabelling, and mass spectrometry have been extensively discussed.[30
In the current study we tested the association between PAH exposure and ESCC risk in a high-risk population in Golestan Province, Iran. We estimated PAH exposure by immunohistochemical staining of esophageal biopsies. Immunostaining has been previously used successfully to detect PAH-DNA adducts in a pilot study of 5 archival esophageal biospsies in subjects from Linxian, China, another high-incidence area with a pattern of risk factors similar to those identified in Golestan.[31
] Recent studies have also used antisera to BPDE-DNA adducts to evaluate PAH adducts in cancers of the cervix [32
] and prostate.[33
] In our study, we used two monoclonal antibodies, 8E11 and 5D11, to evaluate the PAH-ESCC association. The 8E11 stain showed an appropriate dose-response in the B[a]P-dosed cell lines, and it showed a very strong dose-response association between 8E11 staining and case status, with an adjusted OR (95% CI) for the fifth versus first quintile of staining of 26.6 (5.21–135). The 5D11 staining, on the other hand, did not show the expected dose-response in the dosed cell lines, and did not show a difference in staining between case and control cores on the TMAs. Based on the cell line results, the 5D11 staining patterns observed in the TMAs appear to be non-specific.
Our 8E11 results indicate that PAHs and their metabolites are detectable in epithelial cells of the esophagus (the target of esophageal carcinogenesis), and that the quantity of these compounds in non-tumoral epithelial biopsies is strongly associated with ESCC risk. The large magnitude and clear dose-response pattern of this association argues that this is not a chance finding. The known staining profile of 8E11 suggests that the compound causing this association is most likely a PAH or PAH derivative, however other cross-reactive molecules also cannot be ruled out. There is a need for more specific evaluations to identify the exact compound(s) mediating this association. This association also does not prove that PAHs are causing mutagenesis in these tissues.
As an additional comparison, we examined the 8E11 staining intensity (mean Z score) of current smokers and current non-smokers of tobacco, stratified by case status. Among cases, the mean Z scores were 0.87 in current smokers and 0.61 in non-smokers, a difference of 0.26 (p=0.21). Among controls, the mean Z scores were 0.19 in current smokers and −0.02 in non-smokers, a difference of 0.21 (p=0.41). Thus in both cases and controls, 8E11 staining intensity was greater in those with this known PAH exposure, consistent with a valid measure of this exposure, although the power was too low be sure that this was not due to chance. Additionally, in both cases and controls, the difference in staining intensity (Δ mean Z-score) between smokers and non-smokers was only about one-third of the difference in staining intensity between cases and controls (Δ mean Z-score=0.66, ), implying that the elevated staining in cases was largely due to other exposures. This is consistent with our previous analysis of urinary 1-OHPG in the same area, which showed that 83% of the population was highly exposed to PAHs and only 15% of the variance could be explained by known risk factors.[21
] Thus there is also a need to look for other PAH sources that are significant exposures in this population.
Strengths of this study include the analysis of the target tissue of interest (the esophageal epithelium) rather than a surrogate, the analysis of non-tumoral epithelial tissue (rather than tumor tissue) from both cases and controls in a well designed case-control study, the use of TMAs containing both case and control tissues to minimize differences in how these tissues were processed or evaluated, conducting and averaging triplicate staining measurements, the use of an automated image analysis system to quantify the staining, and the collection of detailed information on environmental factors which allowed adjustment for possible confounders. On the other hand, this study also has limitations. The case-control design of the study leaves open the possibility of reverse causality, that the development of esophageal cancer caused the patients to be more highly exposed to PAHs or to increase their reactivity to 8E11 through some other mechanism. A possible explanation for such an association in this population would be patients smoking opium to relieve the pain from their cancer. In earlier studies we have shown that opium use is relatively common in Golestan [7
] and that people in this area respond truthfully and accurately to questions regarding their opium use.[34
] Such questions were included in the questionnaires filled out by all cases and controls in this study, and adjustment for the answers to these questions did not change the staining associations. But reverse causality due to another unmeasured factor cannot be ruled out.
In conclusion, we found a very strong dose-response relationship between intensity of esophageal tissue staining with 8E11 antibody and ESCC risk in a case-control study in Golestan Province, Iran. This finding strengthens the evidence for a causal role of PAHs in the etiology of esophageal cancer in this high-risk population. It will be important to replicate this study in other high-risk populations, to perform similar studies in prospective cohorts, and to undertake studies with more specific and quantitative chemical analyses to identify the PAH compounds associated with ESCC risk.