Effects of vitamin A and [beta]-carotene on respiratory tract carcinogenesis in hamsters : in vivo and in vitro studies

<strong>Summary</strong>Respiratory tract cancer is the leading cause of death by cancer in 'Western' countries. The greater part of lung cancers are caused by smoking. Furthermore, environmental air pollution and occupational exposure contribute to the high incidence of lung cancer. Because it seems to be an almost impossible task to eliminate exposure of man to all these factors, considerable effort has been focused on identifying naturally occurring or synthetic compounds which can prevent the formation of respiratory tract cancer. In this regard, (pro)vitamin A (vitamin A and β-carotene) have been shown very promising. In a large number of epidemiological and experimental studies it has been shown that (pro)vitamin A inhibits the formation of respiratory tract cancer. However, the results of these studies are not always consistent and some studies even showed that (pro)vitamin A increases the incidence of lung cancer. Although the effect of (pro)vitamin A on the formation of respiratory tract cancer has been studied extensively, the mechanisms by which (pro)vitamin A influences the process of respiratory tract carcinogenesis are still not fully understood. In the studies described in this thesis, using both an <em>in vitro</em> and an <em>in</em> vivo approach, the effects of vitamin A and β-carotene on various stages of the process of chemically-induced respiratory tract carcinogenesis were investigated (Figure 1). The emphasis was on the effects of vitamin A and β-carotene on benzo[a]pyrene (B[a]P)-induced DNA-adduct formation, DNA-repair activities, cell proliferation and histomorphological changes in the hamster respiratory tract epithelium. Furthermore, the relationships between DNA-adduct formation, DNA-repair activities, cell proliferation and the expression of the tumour suppressor gene p53 were investigated. <img src="/wda/abstracts/i1924_1.gif" height="830" width="600"/><em>In vitro studies</em>In the first <em>in vitro</em> experiments, the formation and repair of B[a]P-DNA adducts in hamster and rat tracheal epithelial cells was studied (Chapters 3 and 4). It was shown that <em>in vitro</em> the main DNA adduct formed in hamster tracheal epithelial cells was the trans-addition product of deoxyguanosine and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). This is the same DNA adduct as formed <em>in vivo</em> in tracheal epithelial cells of hamsters intratracheally treated with B[a]P. Furthermore, it is the same adduct as has been frequently observed in human respiratory tract cells. In rat tracheal epithelial cells two major DNA adducts were found <em>in vitro</em> : the BPDE-dG adduct and an adduct that is probably derived from interaction of <em>syn</em> -BPDE and deoxyadeno- s-ine. Both the formation of B[a]P-DNA adducts and the B[a]P-induced DNA-repair activities in hamster tracheal epithelial cells were time- and concentration-dependent. In rat tracheal epithelial cells, the formation of B[a]P-DNA adducts was 10 times lower than in hamster tracheas. Furthermore, unlike in hamster tracheal epithelial cells, B[a]P did not induce DNA-repair activities in rat tracheal epithelial cells. In the studies described in Chapter 5, the effect of vitamin A and β-carotene on the formation and repair of B[a]P-DNA adducts in hamster tracheal epithelial cells was investigated. It was shown that both vitamin A and β-carotene slightly inhibited the formation of B[a]P-DNA adducts. In addition, vitamin A and β-carotene increased B[a]P-induced DNA-repair activities. This suggests that the observed decrease in B[a]P-DNA adducts is a positive effect of vitamin A and β-carotene, probably also partly caused by an increase in DNA-repair activities. The effect of vitamin A on DNA-adduct formation and DNA-repair activities depended on the concentration of B[a]P versus the concentration of vitamin A. At a low B[a]P concentration relative to the concentration of vitamin A the formation of B[a]P-DNA adducts was inhibited by vitamin A, whereas at a relatively high concentration of B[a]P the formation of DNA adducts was enhanced by vitamin A.The role of B[a]P and vitamin A in cell proliferation in hamster tracheal epithelium in organ culture is described in Chapter 6. It was shown that the effects of B[a]P and vitamin A on cell proliferation strongly depended on the culture medium used; in tracheas cultured in Ham's F12 medium cell proliferation was decreased by B[a]P treatment compared to control tracheas, while cell proliferation in tracheas treated with vitamin A in combination with B[a]P was increased compared to tracheas treated with B[a]P alone. In tracheas cultured in CMRL-1066 medium, the effects of B[a]P and vitamin A on cell proliferation were opposite to those observed in tracheas cultured in Ham's F12 medium: cell proliferation in tracheas cultured in CMRL-1066 medium and treated with B[a]P was increased compared to control tracheas, while vitamin A decreased B[a]P-induced cell proliferation. To explain these opposite effects of B[a]P and vitamin A on cell proliferation, various medium components and growth factors were investigated. The concentration of CaCl _{<font size="-2">2</font>}<strong>.</strong> 2H _{<font size="-2">2</font>} O revealed to be the most important factor: supplementation of CaCl _{<font size="-2">2</font>}<strong>.</strong> 2H _{<font size="-2">2</font>} O to the Ham's F12 culture medium mimicted the effects of B[a]P and vitamin A on cell proliferation in CMRL-1066 medium. These results clearly indicate that Ca <sup><font size="-2">2+</font></SUP>is an important regulator of proliferation of hamster tracheal epithelial cells. Furthermore, the results of these experiments showed that the level of B[a]P-DNA adducts was inversely related to cell proliferation in tracheas cultured in Ham's F12 medium. Although these results suggest that the tumour suppressor gene p53 might be involved by inhibiting cell proliferation as a consequence of DNA damage, we were unable to show a direct relationship between the level of B[a]P-DNA adducts, cell proliferation and expression of the p53 tumour suppressor protein in hamster tracheal epithelium in organ culture. <em></em><em>In vivo studies</em>The most widely applied <em>in vivo</em> model to study the aetiology and pathogenesis of respiratory tract cancer in experimental animals is based on repeated intratracheal instillations of a saline suspension of fine crystalline B[a]P particles attached to ferric oxide as a carrier. Various aspects of this method are discussed in Chapter 2, showing that the dose of B[a]P and the size of the B[a]P particles are the most important variables influencing the tumour response. In a first <em>in vivo</em> experiment into the effect of vitamin A and β-carotene on B[a]P-induced (pre)neoplastic changes in the respiratory tract of hamsters, the response of the respiratory tract epithelium was too low. This might be due to an insuficiently high B[a]P dose, possibly in combination with a relatively insensitive strain of hamsters used. The low response hampered studying potential effects of vitamin A or P-carotene on the (pre)neoplastic response (Chapter 7). An interesting observation in this experiment was an exceptionally low mortality of hamsters fed a high-β-carotene diet. Although we were unable to establish the exact cause of death of hamsters not receiving β-carotene, the most conspicuous difference between hamsters in the high-β-carotene group and hamsters in other groups was a decrease in lipid peroxidation in the livers of hamsters in the former group. Probably, this effect was not only due to the high concentration of β-carotene in the diet, but was also related to a high dietary level of α-tocopherol and ascorbyl palmitate (also present in the β-carotene beads and used to protect β-carotene from oxidation). To obtain a higher tumour response, hamsters were treated in a second experiment with a higher total dose of B[a]P (Chapter 8 and 9). In this study, a clear relationship appeared to exist between the extent of B[a]P-DNA adduct formation, the induction of cell proliferation and the immunocytochemically detected expression of the p53 protein in hamster tracheal epithelial cells. However, in this experiment the formation of B[a]P-DNA adducts was not found to be affected by a high dietary level of β-carotene, probably due to the high B[a]P dose. Furthermore, β-carotene did not affect B[a]P-induced cell proliferation and expression of the p53 protein in tracheal epithelial cells. Chapter 9 describes the histomorphological aspects of this hamster study, using conventional histopathology and immunohistochemical techniques for the detection of various cytokeratins and glutathione <em>S</em> -transferase (GST)-isoenzyme Pi. From this study, it appeared that B[a]P influenced both the expression of cytokeratins and the expression of the GST-isoenzym Pi. However, in accordance with the results described in Chapter 8, β-carotene did not inhibit B[a]P-induced lesions in the respiratory tract epithelium of hamsters.<strong>Concluding remarks</strong>Finally, the studies described in this thesis allow the following conclusions:- In vitro, vitamin A and β-carotene decrease slightly but consistently the formation of B[a]P-DNA adducts, probably due to an increase in DNA-repair activities. The effect of vitamin A on the formation of B[a]P-DNA adducts depends on the concentration of B[a]P versus the concentration of vitamin A.- The effects of vitamin A and B[a]P on cell proliferation in hamster tracheal epithelial cells in organ culture strongly depend on the tissue-culture medium used, in particular on the concentration of Ca <sup><font size="-2">2+</font></SUP>in the medium. The effects of B[a]P and vitamin A on cell proliferation observed in tracheas cultured in CMRL-1066 medium are similar to the effects generally observed <em>in vivo</em> .- The hamster tracheal organ culture model is very suitable to study the B[a]P-induced formation of DNA adducts and DNA-repair activities. Both the formation and repair of B[a]P-DNA adducts is dose and time dependent. Furthermore, the main adduct formed <em>in vitro</em> is similar to the adduct formed <em>in vivo</em> after intratracheal instillation of B[a]P, and moreover, this adduct is frequently observed in man.- A high dietary dose of β-carotene, possibly in combination with a high level of et-tocopherol and ascorbyl palmitate, strongly increases the survival of hamsters.- In tracheal epithelia] cells of hamsters treated intratracheally with B[a]P, a relationship between the level of B[a]P-DNA adducts, cell proliferation and p53 expression is observed.- The effect of vitamin A on B[a]P-induced DNA-adduct formation and cell proliferation, as observed in the <em>in vitro</em> experiments, was not found in <em>in vivo</em> experiments, probably due to the high B[a]P dose applied.- β-carotene did not affect the formation of (pre)neoplastic changes in the respiratory tract epithelium of hamsters intratracheally treated with B[a]P as evaluated by conventional histopathology, cytokeratin expression, and glutathione S-transferase isoenzyme Pi expression.- Although intratracheal instillation of B[a]P to Syrian golden hamsters is one of the most widely applied models to study respiratory tract cancer in experimental animals, the tumour response is difficult to control due to a large number of variables affecting the response. The most important variables influencing the tumour response are the dose of B[a]P and the size of the B[a]P particles.In conclusion, although the <em>in vitro</em> experiments described in this thesis show that vitamin A and β-carotene may influence the process of respiratory tract carcinogenesis, <em>in vivo</em> it was not possible to show a modulating effect of vitamin A and β-carotene on B[a]P-induced respiratory tract cancer in hamsters. To explain the inconsistencies in the effect of vitamin A and β-carotene on respiratory tract cancer, further in-depth research should he focused on the molecular mechanisms underlying this effect. The concentration of vitamin A and β-carotene, in particular the concentration of the active metabolite retinoic acid, in target cells should be measured in relation to the action of these molecules on the genomic level.