Affiliations of authors: Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, and the Ottawa Regional Cancer Centre, Ontario, Canada.
Correspondence to: H. Chaim Birnboim, M.D., M.Sc., Ottawa Regional Cancer Centre, 501 Smyth Rd., Ottawa, ON, K1H 8L6, Canada (e-mail: birnboim{at}uottawa.ca).
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ABSTRACT |
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INTRODUCTION |
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Many types of chronic inflammatory conditions in humans predispose to malignancy (1315). Neutrophils, monocytes, and macrophages, present at sites of inflammation, generate a variety of oxidants and nitrosants, which, as a class, are known to be genotoxic and mutagenic (16,17). Similar cell types are often found in solid tumors. Using a mouse tumor model, we have recently shown (18) that the number of infiltrating neutrophils and the level of nitric oxide synthase correlate positively with an increase in hypoxanthine phosphoribosyltransferase (hprt) mutation frequency in the tumor cells, suggesting that neutrophil-derived reactive oxygen and nitrogen species were involved. The Mutatect model is a series of related cell lines that can be grown as subcutaneous tumors in syngeneic C57BL/6 mice; the system permits ready detection of mutations arising in vivo (19). This model was the first to show a quantitative increase in mutation frequency as a result of factors in the tumor microenvironment when compared with the same cells in culture (20). We now report on the antimutagenic effect of dietary vitamin E in this experimental model.
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MATERIALS AND METHODS |
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Construction of an interleukin 8 (IL-8)-expressing Mutatect TM-28 cell line. Human IL-8 complementary DNA (cDNA) #328322 from I.M.A.G.E. Consortium (Lawrence Livermore National Laboratories, Livermore, CA) was used as the starting material (21). Errors in the cDNA were corrected, and the cDNA was cloned into vector pTRE (Clontech Laboratories, Inc., Palo Alto, CA) to create pTRE-IL8. Tetracycline-responsive transcriptional activator pTET-off (Clontech Laboratories, Inc.) was stably transfected into Mutatect MC-TGS17-51 cells to create the MT-6 cell line. pTRE-IL8 was introduced into the MT-6 cells, and clone TM-28 was selected. The TM-28 clone produces biologically active IL-8, as determined by an in vitro chemotaxis assay for human neutrophils (Haqqani AS, Sandhu JK, Birnboim HC: unpublished data).
Mutatect tumor formation, detection of mutants, and myeloperoxidase measurement. Tumors from Mutatect MN-11, MT-6, and TM-28 cells were established by subcutaneous injection of cells into the flanks of C57BL/6 female mice, 810 weeks of age (Charles River Laboratories, Quebec, Canada). Tumors were excised when they reached 1 cm in size (23 weeks, depending on the cell line); single-cell suspensions were prepared by gentle mechanical dispersion of the tumor fragments with a plastic syringe. Tumor cells were established in culture for 24 days, and the frequency of mutations arising in vivo was then estimated from the number of colonies capable of growth in 6-TG medium, as described earlier (20). Mutation frequency is expressed as the number of 6-thioguanine-resistant colonies per 1 x 105 clonable tumor cells. The same tumor cell suspension was also used for measurement of myeloperoxidase activity, a marker of neutrophil infiltration (22).
Vitamin E supplements to the diet.
Dietary vitamin E (d--tocopherol acetate; Novartis, Mississauga, ON, Canada) was administered by diluting it in soy oil and adding the liquid to dry standard rodent chow (Charles River Laboratories). Animals were isolated, one per cage, to ensure that the pellet was consumed. Control animals were treated similarly. Vitamin E supplements were started 7 days before tumor cell injection and were continued until the animals were killed (i.e., a total of 34 weeks, depending on the cell line). Control animals received 0.45 IU of vitamin E per day from their standard chow (90 IU of vitamin E/kg chow). Vitamin E-supplemented animals received an additional 2 IU of vitamin E (
-tocopherol acetate) per mouse per day (400 IU vitamin E/kg chow); they were estimated to have also received 0.50 mg of
-tocopherol and 0.25 mg
-tocopherol from the 20 µL of soybean oil used as a vehicle. Control animals received neither vehicle nor supplementary tocopherol. Experiments were carried out at the Animal Care and Veterinary Service of the University of Ottawa in accordance with guidelines of the Canadian Council on Animal Care. Vitamin E determinations were carried out with the use of a high-performance liquid chromatography method (23).
Induction of mutations by glyceryl trinitrate and by molsidomine in MN-11 tumor-bearing animals. On day 12 after injection of MN-11 cells, mice received an intraperitoneal injection of either injectable glyceryl trinitrate (Sabex, Boucherville, Quebec) or molsidomine (Hoechst Marion Roussel Canada, Laval, Quebec). Control mice were given an injection of phosphate-buffered saline. Two days after treatment, tumors were recovered and established in culture as described above. Cells were cultured for 8 days to allow expression of the mutant phenotype before challenge with 6-thioguanine.
Statistical analyses. Nonparametric tests were used for the statistical analyses. Two groups were compared by use of the MannWhitney U test. Correlation between two variables was determined with the use of the Spearman rank coefficient. All P values shown are two-tailed. A P value of <.05 was considered to be statistically significant, and a P value of <.01 was considered to be highly statistically significant. Confidence intervals (CIs) of median differences were calculated with the use of Analyse-it Version 1.5 (www.analyse-it.com). Other statistical calculations were done with the use of GraphPad Prism Version 3 (GraphPad Software, San Diego, CA; www.graphpad.com).
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RESULTS |
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Induction of mutations by nitric oxide-donating drugs and effect of vitamin E.
Nitric oxide and related reactive nitrogen oxide species are potentially mutagenic (16,17). We have shown (24) that nitrovasodilator drugs, glyceryl trinitrate and sodium nitroprusside, were very effective at inducing mutations in cultured MN-11 cells. To assess the in vivo mutagenicity of such drugs, we gave MN-11 tumor-bearing mice an intraperitoneal injection of either glyceryl trinitrate or molsidomine (a chemically unrelated nitrovasodilator drug) 2 days before tumor excision. Both glyceryl trinitrate and molsidomine caused a dose-dependent increase in mutation frequency. The maximum effect was observed at the highest dose tested: Glyceryl trinitrate at a dose of 5 mg/kg induced about 130 mutations, and molsidomine at a dose of 100 mg/kg induced about 50 mutations per 1 x 105 cells over spontaneous levels. Vitamin E was tested for its ability to protect against mutagenicity induced by glyceryl trinitrate (Fig. 2). Tumor cells from animals whose diets were supplemented with vitamin E had a 61.4% reduction in median mutation frequency (P = .058) (72% if the average control frequency is subtracted). (The difference between medians = 192.7 [95% CI = 10.3 to 442.5].) These results indicate that an antioxidant, vitamin E, can reduce the mutation frequency induced by glyceryl trinitrate, a drug that releases nitric oxide. No change in tumor neutrophil content following glyceryl trinitrate treatment compared with controls was seen, as determined histologically (18) (data not shown).
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DISCUSSION |
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Vitamin E is an important group of lipophilic antioxidants found at high concentration in polyunsaturated vegetable oils (1). Dietary supplements usually consist of natural or synthetic -tocopherol ester. In clinical trials, a small benefit of low-dose vitamin E supplements (50 IU per day) in fatal cardiovascular diseases has been reported (25), but evidence for its utility in reducing cancer risk has been inconclusive (9,26,27). Our report is the first, to our knowledge, to show an unequivocal protective effect of dietary vitamin E supplements (2 IU per mouse per day for 34 weeks) on mutation frequency in an experimental tumora relatively short period of dietary supplementation with vitamin E (d-
-tocopherol acetate) was able to reduce markedly the level of mutant cells arising in vivo. In a transgenic mouse model designed to study spontaneously arising mutants in different normal tissues, 3 months of dietary supplementation with vitamin E (4.1 IU per mouse per day) failed to detectably reduce the mutation frequency at a lacI transgenic marker locus (28). This striking difference strongly implies that the mechanism of induction of spontaneously arising mutations in normal tissues is different from spontaneously arising mutations in a tumor, presumably attributable to factors in the tumor microenvironment.
Our data are best explained by postulating a dual action of vitamin E. Vitamin E acts as a scavenger of nitric oxide or other reactive nitrogen oxide species, since it strongly inhibited glyceryl trinitrate-induced mutagenesis in the absence of any change in neutrophil content (Fig. 2). A second mechanism is suggested by the results shown in Fig. 1
, where tumor cell suspensions from vitamin E-supplemented mice had a markedly lower level of myeloperoxidase (a neutrophil-specific marker) than those from control mice. The latter observations suggest that vitamin E may affect the number or distribution of neutrophils in tumors, possibly by altering the expression of cell adhesion molecules. A clinical study supports the latter possibility. Vitamin E (600 IU) given for 8 days to patients prior to surgery for aortic abdominal aneurysm attenuated the influx of neutrophils associated with ischemia-reperfusion injury of the lower limbs (29). The level of E-selectin (a cell adhesion molecule) and ICAM-1 (i.e., intercellular adhesion molecule-1), molecules responsible for adhesion of circulating neutrophils to endothelium, was reported to be lower in patients receiving vitamin E. Changes in neutrophil distribution and adhesion after vitamin E supplementation are currently being explored in the Mutatect model. Although we have also shown an in vitro protective effect of vitamin E on glyceryl trinitrate-induced mutagenesis (unpublished data), the possibility of a second mode of action of vitamin E could not have been predicted without the use of this animal model.
A strong positive correlation between the number of tumor-infiltrating neutrophils (measured by direct counting in histologic sections or by the level of myeloperoxidase in tumor homogenates) and the hprt mutation frequency was consistently seen in this report (Fig. 1, D) and in another published study from our laboratory (18). However, in both cases, the slope of the correlation line between mutation frequency and the absolute neutrophil count and/or level of myeloperoxidase varied considerably between experiments. It has been reported that neutrophil emigration can be profoundly affected by systemic factors. For example, intraperitoneal administration of lipopolysaccharide completely abrogated a local neutrophil response at the site of injection of IL-8 (30). We, therefore, postulate that inapparent differences between batches of C57BL/6 mice from the same supplier may have appreciably affected neutrophil recruitment and/or the state of activation of the neutrophils.
Chronic inflammatory conditions, such as ulcerative colitis, Crohn's disease, Schistosomiasis, and Helicobacter pylori infection (1315), are important risk factors for cancer, possibly because of mutagenic reactive oxygen and nitrogen species generated by inflammatory cells. Rheumatoid arthritis is a chronic inflammatory disease associated with malignancy (31), in which a high frequency of mutations has been found (32). Vitamin E may protect against the development of cancers associated with inflammatory conditions by scavenging these reactive species. However, an additional mechanism by which vitamin E may be protective is suggested by the results presented in Fig. 1: Once tumors become established, vitamin E may reduce mutations by affecting the infiltration of neutrophils or other inflammatory cells.
In summary, our results suggest that vitamin E may exert antimutagenic/anticancer properties by two distinct mechanisms. Both of these mechanisms should be taken into account in the planning of future clinical trials.
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NOTES |
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We thank Donna Grant and Denise Proulx for their skillfull technical assistance. We also thank Dr. Nick Hidiroglou and Rene Madere of the Nutrition Section, Health Canada, for performing plasma vitamin E analyses.
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Manuscript received January 28, 2000; revised June 7, 2000; accepted June 20, 2000.
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