Previous data from our laboratory showed that exposure to foreign particles, such as CD and silica suppresses the PAH-mediated induction of CYP1A1 in rat lung [3
]. By histopathologic assessment, particle exposure produces AT-II cell hyperplasia and hypertrophy in the lungs of rats exposed to either respirable silica or respirable coal dust [3
]. Previous studies indicated that AT-II cell hypertrophy and hyperplasia were related processes and a response to alveolar type I cell injury in the particle-exposed lung [24
]. Using immunofluorescent co-localization of CYP1A1 and the AT-II cell markers cytokeratins 8/18 in CD-exposed rats, studies from our laboratory showed that these hyperplastic and hypertrophic cells expressed decreased amounts of PAH-induced CYP1A1 protein. This suggested that CD exposure resulted in appearance of a new population of AT-II cells with diminished capacity for CYP1A1 induction [3
]. Since CYP1A1 is induced by ligand activation of the AhR, which is localized in the cytoplasm in the inactive state, we isolated AT-II cells from in vivo
CD-exposed rats to localize and quantify AhR in the cytoplasm and nucleus after in vivo
treatment with the specific CYP1A1 inducer, BNF.
AhR translocation is essential for CYP1A1 induction by PAHs. After being stimulated by an inducer, the AhR is translocated to nucleus and binds to another protein called AhR nuclear translocator (ARNT) forming a heterodimeric protein complex [25
]. This complex binds to the xenobiotic responsive element (XRE) located at the enhancer region of CYP1A1 gene producing conformational changes in chromatin structure and initiates CYP1A1 transcription [26
Morphometric analysis of immunofluorescence staining in this study showed that the proportional AhR expression in AT-II cell nuclei, an indicator of AhR localization in the nucleus, was significantly increased in BNF-exposed rats compared with vehicle control rats. Moreover, the total area of AhR expression per AT-II cell showed a significant increase in BNF-exposed rats compared to vehicle controls. This finding is consistent with many studies involving BNF as a specific potent inducer for CYP1A1 through activation of AhR and served as a positive control for the immunofluorescent procedure used in our study [27
Conversely, in CD exposed rats, the localization of AhR in AT-II cell nuclei, measured as a proportional AhR expression in the cell nucleus, was not significantly affected by BNF. In addition, in CD-exposed rats, the percentage of AT-II cells with nuclear AhR expression was not significantly affected by BNF. These findings suggest that the mechanisms of CD-induced inhibition of CYP1A1 induction may involve failure of the CD-exposed cells to translocate AhR after PAH exposure. One possible explanation for the alteration of AhR localization in AT-II cell nucleus of CD-exposed rats after being activated by BNF is AhR proteolysis. Since CD contains small amounts of PAHs and ligand-activated AhR is rapidly proteolyzed resulting in an immediate reduction of the AhR number [33
], it is possible that the prior exposure to low levels of PAH decreased the AhR pool in the cell. Gu and co-workers suggested that this ligand-dependant degradation of AhR aims to ameliorate the response of the cell to environmental changes, thereby protecting the cell from prolonged exposure to excessive concentrations of agonists as an adaptive mechanism [33
]. However, the amount of PAHs quantified in CD was 500 times less than the dose of BNF injected. Furthermore, the BNF used in this study was injected daily for three days to repeatedly activate AhR. Thus, increased AhR proteolysis due to prior PAH-activation in the CD-exposed rats seems unlikely.
The decreased AhR nuclear translocation in alveolar type II cells after CD exposure may well be a general response of alveolar type II cells to inflammation associated with lung-deposited particles, since CYP1A1 induction and its dependent activity (EROD) were inhibited by exposure to another type of occupational dust, crystalline silica, which does not contain PAH [23
]. Histopathological and bronchoalveolar lavage examinations showed that both CD and silica were associated with pulmonary inflammation [3
]. Inflammation can downregulate CYP-dependent activity in the liver [40
]. In the liver, a number of different CYPs are suppressed by inflammation and reduced CYP activity involves both transcriptional and post-transcriptional mechanisms [43
]. Because exposure to CD is associated with pulmonary inflammation [3
], the mechanism of suppressed PAH-induced CYP1A1 activity in the particle-exposed lung is likely to involve proinflammatory mediators known to influence CYP1A1 activity. In particular, in vitro
NF-κB suppresses PAH-induced CYP1A1 activity by preventing acetylation of the CYP1A1 promoter and thereby reducing transcription [8
]. In addition, nitric oxide binds to the catalytic heme moiety of CYP1A1, leading to post-transcriptional downregulation of CYP1A1 activity [46
]. Indeed, our previous studies showed, that PAH induced CYP1A1 activity was inversely related to CD-induced pulmonary inflammation [3
However, in both the silica and coal dust-exposed rat lungs, alveolar type II cells proliferate following pulmonary inflammation and are often refractory to CYP1A1 induction [3
]. A previous in vitro
study with cells of apparent alveolar type II cell origin indicated that proliferating cells had less constituitive EROD activity than confluent (quiescent) cultures [47
]. In a keratinocyte cell line, differentiated cultures had more AhR mRNA and more TCDD-inducible CYP1A1 than did proliferating cultures [48
]. Similarly, in vivo
rat skin cells that remain refractory to CYP1A1 induction are predominantly basal cells, the progenitor population of the skin [49
]. In this study, we have investigated the hypothesis that like proliferating keratinocytes, alveolar type II cells from CD-exposed rats have decreased AhR translocation to the nucleus following in vivo
PAH exposure. Our findings are consistent with these previous findings for other proliferating cells and indicate that the alveolar type II cells of the CD-exposed rat lungs have decreased capacity for BNF-induced AhR nuclear translocation. Thus, suppression of PAH-induced CYP1A1 activity in the CD-exposed lung is associated with both inflammation and the expansion of an alveolar type II cell population with reduced capacity for BNF-induced AhR nuclear translocation.
Since CYP1A1 induction is dependent upon AhR nuclear translocation [9
], the alveolar type II cells of the particle-exposed lung would be expected to produce fewer CYP1A1-dependent metabolites of PAH carcinogens. It is these PAH metabolites which are believed to cause the DNA adducts which may initiate lung cancer and AT-II cells are potential progenitor cells for lung cancer [51
]. Thus, our findings indicate CD exposure prior to PAH exposure decreases the inducibility of PAH metabolism in the lung, at least in part because there is diminished capacity for AhR nuclear translocation in the type II cells. However, if DNA adducts have already caused DNA mutations in alveolar type II cells, expansion of initiated cells is integral to the process of cancer promotion [53
]. Studies are still needed to determine the effect of CD upon the lung that has been previously exposed to PAH carcinogens. It may well be that CD is actually a complex modifier of PAH carcinogenesis with both the negative modification of PAH-induced metabolism indicated in this and previous studies [3
] and the capacity for promotion through expansion of initiated cells.
Taken together, our results demonstrate that exposure of rats to CD modifies nuclear translocation of AhR in AT-II cells after subsequent BNF exposure. This provides an explanation for at least some of the diminished CYP1A1 induction observed in the particle-exposed lung upon subsequent BNF exposure.