Environmental Health Perspectives 102, Supplement 10, December 1994
The Role of the Cellular Antioxidant Defense in Oxidant Carcinogenesis
Peter Cerutti, Rita Ghosh, Yukiko Oya, and Paul Amstad
Department of Carcinogenesis, Swiss Institute for Experimental Cancer Research, Epalinges/Lausanne, Switzerland
Abstract
Oxidant carcinogens interact with multiple cellular targets including membranes, proteins, and nucleic acids. They cause structural damage to DNA and have the potential to mutate cancer-related genes. At the same time, oxidants activate signal transduction pathways and alter the expression of growth- and differentiation-related genes. Indeed, the carcinogenic action of oxidants results from the superposition of these genetic and epigenetic effects. All cells possess elaborate antioxidant defense systems that consist of interacting low and high molecular weight components. Among them, superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalase (CAT) play a central role. Our studies with mouse epidermal cells demonstrate that the balance between several antioxidant enzymes rather than the activity of a single component determines the degree of protection. Unexpectedly, increased levels of Cu,Zn-SOD alone in stable transfectants resulted in sensitization to oxidative chromosomal aberrations and DNA strand breaks. However, a concomitant increase in CAT or GPx in double transfectants corrected or overcorrected the hypersensitivity of the SOD clones depending on the ratios of activities CAT/SOD or GPx/SOD. The cellular antioxidant capacity also affected oxidant induction of the growth-related immediate early protooncogene c-
fos. Increases in CAT or SOD reduced the accumulation of c-
fos message, albeit for different reasons. The cellular antioxidant defense also affects the action of UVB light (290-320 nm) that represents the most potent carcinogenic wavelength range of the solar spectrum. UVB light is known to exert its action in part through oxidative mechanisms. Increases in CAT and GPx protected mouse epidermal cells from UVB-induced DNA breakage. An increase in GPx enhanced the induction of c-
fos by UVB probably because it diminished DNA breaks. DNA breaks appear to exert a long-range effect on chromatin confirmation, which is incompatible with efficient transcription. --
Environ Health Perspect 102(Suppl 10):123-130 (1994)
Key words: superoxide dismutase, catalase, glutathione peroxidase, DNA strand breaks, fos protooncogene, jun protooncogene, ultraviolet light, hydrogen peroxide, superoxide, antioxidant defense, carcinogenesis, xanthine oxidase, chromosomal aberrations, polyADP-ribosylation
This paper was presented at the Conference on Oxygen Radicals and Lung Injury held 30 August-2 September 1993 in Morgantown, West Virginia.
This work was supported by the Swiss National Science Foundation, the Swiss Association of Cigarette Manufacturers, and the Association for International Cancer Research.
Address correspondence to Peter Cerutti, Department of Carcinogenesis, Swiss Institute for Experimental Cancer Research, Ch. Boveresses 155 1066 Epalinges/Lausanne, Switzerland. Telephone 41 21 316 5858. Fax 41 21 652 6933.
Introduction
It is well recognized that oxidants play a role in several stages of carcinogenesis. They are ubiquitous in our natural environment but they are also formed in the tissue by endogenous cellular mechanisms (1-3). Oxidants can introduce structural changes in cancer-related genes in the form of chromosomal aberrations and point mutations. Indeed, point mutations in the ras-family protooncogenes (4) and in the p53 tumor suppressor gene (5,6) represent the most frequent genetic changes in human malignancies, and at least some of them may be caused by oxidants. In addition to these genotoxic effects, oxidants activate signal transduction pathways that lead to the modulation of the expression of entire families of growth- and differentiation-related genes (7-9). However, unlike polypeptide growth factors, oxidants always induce macromolecular damage, cytotoxicity, and cell killing. All the effects of oxidants are influenced by the cellular antioxidant defenses (10,11). This multilayer system consists of low molecular weight components and several antioxidant enzymes. It is evident that the biological consequences of the exposure to an oxidant carcinogen, e.g., growth stimulation, growth inhibition, differentiation, or cell death is not readily predictable. It may vary with the dose, the type of the oxidant, and the tissue because it is the result of the superposition of effects on the multiple cellular targets mentioned above.
Oxidants are likely to play an important role in lung carcinogenesis. Chronic tissue injury by physical and chemical irritants frequently results in inflammation accompanied by the infiltration of phagocytic leukocytes (12-15). The inflammatory reaction is particularly striking in bronchial tissue that had been exposed to particulates and irritants in tobacco smoke and to mineral fibers (13). The mechanisms by which inflammatory leukocytes exert their carcinogenic effects have not been elucidated but some likely pathways can be proposed. Phagocytic leukocytes produce a highly complex mixture of growth and differentiation factors as well as biologically active arachidonic acid metabolites (16). In addition, they possess the capacity to release large amounts of active oxygen (AO) in an oxidative burst (17). Current evidence suggests that AO and arachidonic acid metabolites are important in tumorigenesis. Low molecular weight antioxidants, antioxidant enzymes, and antiinflammatory agents that inhibit arachidonic acid metabolism are anticarcinogenic in several experimental systems (18-20). The notion that AO from phagocytes may be an important carcinogen is supported by the finding that an extracellular burst of AO produced by xanthine/xanthine oxidase (X/XO) is a potent promoter for initiated mouse embryo 10T1/2 fibroblasts and mouse epidermal JB6 cells (21,22). Furthermore, the carcinogenic effect of cocultured neutrophils on 10T1/2 cells has been directly demonstrated (23). While hydrogen peroxide (H2O2) alone is a weak promoter for initiated mouse skin, several xenobiotic organic endo- and hydroperoxides possess considerable potential as promoters and progressors (18,24,25).
Polycyclic aromatic hydrocarbons (PAHs) represent important etiologic agents in lung cancer induced by tobacco smoke. They are metabolically activated to epoxy intermediates that form covalent adducts to DNA. In addition, PAH metabolites and other aromatic compounds that have the potential to form quinoid intermediates can induce oxidative DNA damage. This was shown for benzo[a]pyrene, which caused the formation of 5-hydroxymethyluracil at a high rate in the DNA of human epithelioid lung cells A549 (26,27).
As mentioned above, the cellular antioxidant defenses are bound to play a role in oxidant carcinogenesis. Epidemiologic studies on serum antioxidants and diet suggest that an elevated level of vitamins E and ß-carotene reduce mortality due to cancer in the lung and colon (28,29). On the other hand, levels of antioxidant enzymes in tumors have yielded inconclusive results, although it had been observed that the activity of Mn-superoxide dismutase was often below that of normal tissues (30). In view of the multiple stages and targets where oxidants can act in carcinogenesis, it may not be astonishing that the effect of the antioxidant defense depends on the cell type and tissue. For example, high antioxidant capacity is expected to protect the DNA from oxidative damage and mutagenesis but at the same time it may protect "initiated" cells from excessive oxidant toxicity and favor their clonal expansion in tumor promotion (11,31,32). Furthermore, the cellular antioxidant system consists of multiple interacting and interdependent components (10). Therefore, the biological consequences of the variation of a single component "out of the cellular context" is difficult to predict.
In this article we review progress made in our laboratory in the understanding of the role of the antioxidant enzymes Cu, Zn-superoxide dismutase (SOD), catalase (CAT), and Se-glutathione peroxidase (GPx) in oxidant carcinogenesis. To evaluate their individual contributions inside the cell, we prepared single and double transfectants with moderately increased levels of one or two antioxidant enzymes. With the help of these transfectants, we defined their role in the protection from oxidant-induced chromosomal aberrations, DNA strand breakage, growth inhibition, and the transcriptional inducibility of the growth- and differentiation-related protooncogene c-fos.
Increased Constitutive Antioxidant Defense in Promotable Mouse Epidermal Cells
A first indication that the cellular antioxidant defense affects the capacity of oxidants to stimulate the growth of epithelial cells was obtained in a study comparing promotable and nonpromotable mouse epidermal cells JB6. When we measured the specific activities of Cu,Zn-SOD, CAT, and GPx in monolayer cultures of JB6 cells, we discovered that the promotable clone 41 contained approximately twice the activity of SOD and CAT relative to the nonpromotable clone 30, whereas the activities of GPx were comparable. The activity data were confirmed by Western blots that indicated elevated protein levels of SOD and CAT in clone 41 cells. Northern blots indicated that the higher amounts of CAT and SOD in clone 41 were due to increased stationary concentrations of mRNAs for these genes. We concluded that the antioxidant defense of JB6 clone 41 is superior to that of clone 30. The difference between the two clones is particularly remarkable, because the two antioxidant enzymes SOD and CAT are increased coordinately in clone 41. Because the product of the action of SOD is H2O2, an increase in its activity is only beneficial to the cell if it is counterbalanced by a sufficient capacity for the destruction of H2O2. This is apparently accomplished in clone 41 by an increase in CAT (32). It should be mentioned that SOD and CAT may mutually protect each other from inactivation by active oxygen (33). The difference in the constitutive antioxidant defense of the two epidermal cell clones may play a role in their promotability by oxidants (31,32). In support of this concept, we recently found that oxidants produced by X/XO caused higher cytotoxicity and cell death in nonpromotable clone 30 cells (22). A rise in cytosolic ionized calcium (Ca2+)i preceding bleb formation was sustained for 8 to 10 min longer in nonpromotable relative to promotable cells according to digital imaging fluorescence microscopy. We concluded that the superior antioxidant defense of promotable clone 41 protects it from more severe deregulation of (Ca2+)i and, as a consequence, from excessive cytotoxicity following exposure to oxidant promoters (P Jain, S Chang, I Berezesky, P Cerutti, B Trump, unpublished data).
Effect of the Antioxidant Defense on Oxidant-induced Chromosome and DNA Breakage
The genome represents one of the most vulnerable targets to oxidants which cause permanent structural damage to DNA as well as transient changes in gene expression. The sensitivity of the genome to oxidants is modulated by the cellular antioxidant defense. To study the effect of the major antioxidant enzymes on genome vulnerability we constructed genetic variants of promotable mouse epidermal cells JB6 clone 41 by transfection with cDNAs coding for human Cu,Zn-SOD, CAT, and bovine Se-GPx. Different resistance cassettes were used to construct each of the three expression vectors, allowing the preparation of single- and double transfectants with increased complements of one or two enzymes. This represents a crucial feature of our experimental design since it allows dissection of the individual contributions of these interacting enzymes to the overall antioxidant defense. To stay in the physiologic range, we chose stable transfectants with moderate 2- to 3-fold increases in antioxidant enzyme activities for in-depth characterization. These clones were completely analyzed on the molecular and biochemical level [(11,34); P Amstad, R Moret, P Cerutti, unpublished data].
Northern blots of total RNA from these clones revealed increased expression of Cu,Zn-SOD, CAT, or GPx, respectively, and Southern blots indicated the presence of the transfected DNA. Western blots with antibodies against the human proteins showed the presence of bands corresponding to the human enzymes in addition to the cross-reacting endogenous mouse proteins. For the antioxidant enzyme activities of the transfectants, we refer to the original report (34) from our laboratory. Below we describe the effects of the modulation of the antioxidant defense on oxidant-induced chromosome and DNA damage.
Cu,Zn-SOD Transfectants Are Sensitized to Oxidant-induced Chromosomal Damage while CAT and GPx Transfectants Are Protected. Cytogenetic analysis revealed that the Cu,Zn-SOD transfectants SOD3 and SOD15 were sensitized to chromosomal damage induced by oxidant. Two- to threefold higher SOD activities resulted in a 3- to 5-fold increase in total chromosomal aberrations after exposure to X/XO relative to the parent strain (scored: breaks, iso-breaks, gaps,
iso-gaps, exchanges; Y Oya, P Cerutti, unpublished data). Similarly, the SOD transfectants were sensitized to X/XO- induced DNA strand breaks as measured by the alkaline elution method (34).
In contrast, stable transfectants with increased levels of CAT were protected from X/XO-induced chromosome and DNA breakage. For example, in transfectant CAT4 with approximately 3-fold increased CAT activity, the oxidant-induced total chromosomal aberration frequency was reduced 4-fold relative to the parent strain (Y Oya, P Cerutti, unpublished data). As expected, CAT transfectants also were more resistant to oxidant-induced DNA strand breakage. Interestingly, a SODCAT double transfectant derived from CAT4 was more resistant than the parent strain to DNA breakage. These results indicate that the balance between SOD and CAT plays a crucial role for the overall vulnerability of the genome to a mixture of O2
- and H2O2 produced extracellularly by X/XO (34). As a follow-up to our observation, Mao et al. (35) studied the efficacy of conjugates of Cu,Zn-SOD and CAT as protectors from ischemia-reperfusion damage. They found that the enzyme conjugate supplied better protection than Cu,Zn-SOD alone.
We recently evaluated the effect of increasing the cellular complement in GPX on the oxidant sensitivity of the parent strain JB6 clone 41 and its SOD transfectants SOD3 and SOD15. Sensitivity to DNA strand breakage and killing by X/XO was reversely related to the ratio of activities GPx over SOD. A GPx transfectant with a GPx/SOD ratio of 3.8 was very strongly protected. The hypersensitivity of the SOD clones with a GPx/SOD ratio of 0.4 was corrected or overcorrected by secondary transfection with bovine seleno-GPx resulting in increased activity ratios GPx/SOD of 1 to 2.4. Our results indicate that small deviations from the physiologic activity ratios of GPx/SOD have a dramatic effect on the resistance of cells to oxidant-induced damage to the genome and cell killing (P Amstad, R Moret, P Cerutti, unpublished data). X/XO produces a large burst of active oxygen close to the cell surface and it is conceivable that lipid peroxidation in the membrane and the formation of long-lived clastogenic products are on the pathway to DNA breakage (36-38). Indeed, the protective action of GPx might be due in part to its capacity to destroy clastogenic lipid hydroperoxides. Our results are in agreement with the recent finding that the transfection of human GPx into human breast carcinoma cells rendered them more resistant to menadione-induced DNA breakage (39).
A satisfactory interpretation of these results requires an understanding of the reasons for the toxicity of high levels of Cu,Zn-SOD. The compensatory effect of CAT and GPx suggests that overproduction of H2O2 by O2- dismutation might be responsible for SOD toxicity (34,35). Alternatively, overscavenging of hydroperoxy radical HO2 (the conjugate acid of O2-) by excess SOD may reduce radical chain termination and result in increased lipid peroxidation (40). Finally, the inherent peroxidatic activity of Cu,Zn-SOD could play a role (41). However, the fact that both excess Cu,Zn- and Mn-SOD have been shown to be toxic argues against this possibility since the latter enzyme lacks peroxidatic activity (40). While we favor the interpretation that the genotoxicity of elevated levels of Cu,Zn-SOD in cells exposed to O2- plus H2O2 is due to overproduction of H2O2, we do not imply that DNA is the immediate target for attack by H2O2 (42) or its radical derivatives. The fact that the chelation of intracellular Ca2+ strongly inhibits DNA breakage by H2O2 suggests that the activation of Ca2+-dependent endonucleases plays a role. It should be noted that evidence in the literature supports the notion that overexpression of SOD can sensitize rather than protect cells from oxidative stress. For example, a 5-fold increase in Mn-SOD or a 10-fold increase in Fe-SOD sensitized E. coli to paraquat toxicity (43,44). While transfection of Cu,Zn-SOD into HeLa cells resulted in overall resistance to paraquat, the degree of protection was not proportional to the increase in enzyme activity (45).
Effect of Antioxidant Defense on Oxidant-induced Growth Inhibition and Cell Killing. In general, oxidants are cytostatic and cytotoxic, although under exceptional circumstances they can promote growth and facilitate the clonal expansion of initiated cells in carcinogenesis (22,31, 46,47). Membrane lipid peroxidation, which causes disruption of membrane integrity, increased permeability to ions, and surface bleb formation as well as chromosomal breakage are major mechanisms leading ultimately to growth inhibition and cell death (3). Of course, the antioxidant defenses are expected to modulate the sensitivity of cells to these cytopathologic effects of oxidants. As mentioned above, the oxidant promotability of clone 41 of JB6 mouse epidermal cells may in part be due to its elevated content in SOD and CAT. The enhanced antioxidant defense may protect these cells from excessive toxicity but still allow the activation of the signal transduction pathways that are required for growth stimulation (32).
We have further explored the role of the major antioxidant enzymes in the protection from inhibition of cell growth and killing with the help of our collection of stable antioxidant enzyme transfectants. We measured the effect of an extracellular burst of oxidant produced by X/XO on growth in monolayer cultures and on the survival of colony forming ability. As a whole, our results are in qualitative agreement with those described above for chromosomal and DNA breakage: a) overexpression of Cu,Zn-SOD sensitized to growth inhibition and killing (34); b) elevated levels of CAT or GPx protected from growth inhibition and killing; c) transfection of SOD clones with either CAT or GPx corrected or even overcorrected their hypersensitivity
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