Affiliations of authors: Ottawa Regional Cancer Centre and the Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada (CCYS, HCB); Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada (ASH); Nutrition Research Division, Health Canada, Ontario, Canada (NH); Division of Nutritional Sciences, Cornell University, Ithaca, NY (JES, RSP).
Correspondence to: H. Chaim Birnboim, MD, MSc, Ottawa Regional Cancer Centre, 503 Smyth Rd., Ottawa, ON, Canada K1H 1C4 (e-mail: birnboim{at}uottawa.ca)
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ABSTRACT |
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Inflammatory cells in tissues release reactive nitrogen oxide species in response to the presence of foreign microorganisms or as a result of autoimmune reactions. It has long been recognized that chronic or recurrent inflammation associated with low-grade infection or autoimmune disease is an important risk factor for cancer in humans (1013). Because inflammatory cells also frequently infiltrate established solid tumors, we hypothesized that reactive nitrogen oxide species generated by such cells contribute to tumor progression by inducing genetic damage in tumor cells.
The mouse Mutatect tumor model is a transplantable fibrosarcoma developed as an experimental paradigm to study the contributions of tumor-infiltrating leukocytes and the reactive nitrogen oxide species they produce on genetic instability (14). These subcutaneous tumors become infiltrated with leukocytes, predominantly neutrophils, which express inducible nitric oxide synthase, the principal source of reactive nitrogen oxide species. The number of neutrophils is strongly associated with the mutation frequency at the hypoxanthine phosphoribosyltransferase (Hprt) locus in the tumor cells (15). When injected into mice, Mutatect cell lines engineered to express human interleukin 8 (IL-8) produce tumors with high levels of neutrophil infiltration and correspondingly high Hprt gene mutation frequencies (16). The high-frequency loss of IL-8 transgene-containing cells that occurs in these tumors may be due to a combination of generalized genotoxicity and selective cytotoxicity against IL-8secreting tumor cells by neutrophil-derived reactive nitrogen oxide species (16). Additional evidence of an increase in reactive nitrogen oxide species in these tumors is the presence of protein nitrotyrosine (1517). We have previously shown that Mutatect tumor cells from mice fed dietary supplements of D--tocopherol acetate display a decrease in Hprt gene mutation frequency and in IL-8 transgene loss compared with tumor cells from mice fed no such supplements (18,19). Here we examine in greater detail the effects of
-tocopherol acetate on Hprt gene mutation frequency and IL-8 transgene loss in the Mutatect tumor model and compare them with the effects of
-tocopherol.
Derivation of the mouse Mutatect tumor model and Mutatect cell lines have been described previously (16,20). Mutatect TM-28 cells, a clone that expresses human IL-8 (16), were injected subcutaneously into 6- to 8-week-old C57BL/6 mice (typically 9 per group; Charles River Laboratories, Quebec, Canada). Tumors were harvested when they reached 1 cm in size (typically at 2.5 to 3 weeks after injection). Dietary vitamin E supplements (0, 25, 50, or 100 mg/kg body weight per day) were added to the tocopherol-stripped rodent pellets that were fed to the mice from 7 days before injection of tumor cells until the mice were killed and their tumors harvested (i.e., a total of 3.5 to 4 weeks) (18). Dietary supplementation with tocopherol had no observable effects on tumor volumes or on mouse behavior or survival (data not shown). The cellular fractions of the tumors were analyzed for Hprt gene mutation frequencies (20) and myeloperoxidase (MPO) activity (21) as described elsewhere (19). Two-tailed nonparametric tests were used for all statistical analyses. Nonparametric KruskalWallis tests were used to compare three or more unpaired groups. Where P values were less than .05, we used Dunns multiple comparison post hoc test to compare values between two groups. We used Analyse-it (version 1.65; www.analyse-it.com) to calculate 95% confidence intervals (CIs) and Graphpad Prism (version 3; Graphpad Software, San Diego, CA; www.graphpad.com) to perform all other statistical calculations. Experiments were carried out at the Animal Care and Veterinary Services of the National Research Council (Ottawa, ON, Canada) in accordance with guidelines of the Canadian Council on Animal Care.
We first examined the effect of increasing doses of dietary -tocopherol acetate on Hprt gene mutation frequency in cells isolated from Mutatect TM-28 tumors in two separate experiments (Table 1). Hprt gene mutation frequencies were statistically significantly lower in tumor cells from mice fed 50 mg/kg
-tocopherol (P<.001 [experiment 1]; P = .009 [experiment 2]) or 100 mg/kg
-tocopherol (P<.001 [experiment 1]; P = .002 [experiment 2]) than in tumor cells from mice fed 0 mg/kg
-tocopherol (Table 1, experiments 1 and 2). Nonparametric two-factor (experiment [exp, random], dose [D, fixed]) Model III analysis of variance revealed a statistically significant difference in the overall Hprt gene mutation frequency between the two experiments (ScheirerRayHare Hexp = 12.2, df = 1, P<.001). However, in both experiments, the Hprt gene mutation frequency decreased with increasing
-tocopherol dose (Table 1, experiments 1 and 2).
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To examine the effects of dietary tocopherol on the number of neutrophils in tumors, we measured the activity of MPO, a neutrophil-specific marker (21), in single-cell and stromal fractions of Mutatect TM-28 tumors from mice fed - or
-tocopherol. There was a statistically significant decrease in MPO activity in the single-cell fraction with increasing dose of
-tocopherol (KruskalWallis pooled over experiment = 35.1, df = 3, P<.001). A statistically significant decrease in MPO was observed in the single-cell fraction of tumors from mice fed 50 or 100 mg/kg
-tocopherol, as compared with mice fed 0 mg/kg
-tocopherol in two independent experiments (Table 1, experiments 1 and 2). However, MPO activity in stromal fractions was unaffected by
-tocopherol at doses up to 100 mg/kg (data not shown). By contrast, we observed no detectable effect of mixed
-tocopherols on MPO activity in either single-cell (Table 1, experiment 3) or stromal fractions (data not shown). In addition, compared with 0 mg/kg/day of either form of tocopherol, pure
-tocopherol at 100 mg/kg/day statistically significantly decreased tumor MPO activity, whereas pure
-tocopherol at 100 mg/kg/day did not (Table 1, experiment 4).
We also measured the levels of tocopherols in the plasma and tumors of the tumor-bearing mice fed different forms and doses of vitamin E (Table 2). Overall, plasma -tocopherol levels as a function of dose did not vary statistically significantly between experiments 1 and 2 (Hexp = 0.17, df = 1, P = .68) (Table 2). In both experiments, we observed a saturating doseresponse relationship between dietary dose of
-tocopherol acetate and the plasma
-tocopherol level (KruskalWallis [pooled] = 48.4, df = 3, P<.001). Similarly, tumor
-tocopherol levels did not differ between experiments 1 and 2 (Hexp = 0.24, df = 1, P = .62); we observed a general increase in tumor levels of
-tocopherol with increasing dose of dietary
-tocopherol (KruskalWallis [pooled] = 31.3, df = 3, P< .001). We observed a saturating doseresponse relationship between the dietary dose of a mixture of
- and
-tocopherols and the plasma levels of each tocopherol (KruskalWallis [
-tocopherol] = 25.6, df = 3, P<.001); (KruskalWallis [
-tocopherol] = 22.6, df = 3, P< .001) (Table 2, experiment 3). However, the tumor levels of
-tocopherol or
-tocopherol levels did not vary statistically significantly as a function of dose (KruskalWallis [
-tocopherol] = 2.6, df = 3, P = .46; KruskalWallis [
-tocopherol] = 3.2, df = 3, P = .36). Compared with mice fed a diet not supplemented with either tocopherol, mice fed a diet supplemented with pure
-tocopherol at 100 mg/kg body weight per day only had a statistically significant increase in median plasma and tumor
-tocopherol levels (median plasma
-tocopherol levels = 1.8 nmoles/mL [100 mg/kg group] and 0 nmoles/mL [0 mg/kg group]; difference = 1.8 nmoles/mL [95% CI = 1.0 to 2.0 nmoles/mL], MannWhitney U [plasma] = 2.0, P<.001; median tumor
-tocopherol levels = 7.4 nmoles/mL [100 mg/kg group] and 0.4 nmoles/mL [0 mg/kg group]; difference = 7.0 nmoles/mL [95% CI = 5.3 to 10.1 nmoles/mL], MannWhitney U [tumor] = 0.001, P = .003 [Table 2, experiment 4]).
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A body of evidence suggests that dietary supplementation with vitamin E reduces the incidence of prostate cancer, but there is controversy regarding which form is more effective in cancer prevention (6,22,23). -Tocopherol is usually considered the most biologically active form of vitamin E, and it is the form administered as dietary supplements. In humans,
-tocopherol is metabolically favored over
-tocopherol because
-tocopherol is preferentially absorbed and secreted into the plasma from the liver by hepatic
-tocopherol-transfer protein (24). However,
-tocopherol possesses electrophile-trapping and nitrogen dioxide-radicaltrapping properties that are different from those of
-tocopherol (25,26). In addition, ingestion of
-tocopherol has been reported to result in a reduction in serum levels of
-tocopherol (22,2729). We found no statistically significant difference in plasma levels of
-tocopherol between mice that received 100 mg/kg
-tocopherol:34 mg/kg
-tocopherol (median plasma concentration = 1.6 nmol/mL; Table 2, experiment 3) and mice that received pure 100 mg/kg
-tocopherol (median plasma concentration = 1.8 nmol/mL; Table 2, experiment 4) (difference in median plasma concentrations = 0.2 nmol/mL [95% CI = 0.85 to 1.03 nmol/mL], P = .97 [MannWhitney U test]). However, we found that there was a statistically significant difference in the tumor levels of
-tocopherol between mice that were fed 100 mg/kg
-tocopherol:34 mg/kg
-tocopherol (median tumor concentration = 0.7 nmol/mg [95% CI = 0.2 to 1.5 nmol/mg]; Table 2, experiment 3) and mice that were fed pure 100 mg/kg
-tocopherol (median tumor concentration = 7.4 nmol/mg [95% CI = 4.8 to 12.7 nmol/mg]; Table 2, experiment 4) (difference in median tumor concentrations = 6.7 nmol/mg [95% CI = 5.0 to 10.0 nmol/mg], P = .0002 [MannWhitney U test]). This finding is consistent with the notion that high doses of
-tocopherol may displace
-tocopherol (23). In addition, results of a recent nested case-control study provided evidence that
-tocopherol may be more protective against prostate cancer than
-tocopherol (6). Our findings, that dietary
-tocopherol had no effect on Hprt gene mutation frequency, MPO activity, or IL-8 transgene loss in our animal model, may simply reflect our inability to achieve a sufficiently high level of
-tocopherol in either plasma or tumors. Thus, our results are not inconsistent with suggestions that
-tocopherol might have important health benefits (22).
In the Mutatect tumor model, -tocopherol (both 50 mg and 100 mg) statistically significantly decreased mutation frequency and MPO activity but 100 mg was required to reduce transgene loss. Earlier, we suggested that neutrophils are responsible both for genotoxicity in tumor cells and selective elimination of IL-8 secreting tumor cells (16). In this Communication, we demonstrate that a higher dose of
-tocopherol is required to reduce transgene loss compared with the dose required to lower mutation frequency. This could be explained by our earlier suggestion that multiple events, such as a combination of genotoxicity and selective cytotoxicity, are involved in transgene loss (16). Our findings about the dose-dependent effects of
-tocopherol and its possible ability to displace
-tocopherol may have implications for the design of future human chemoprevention studies with vitamin E and the interpretation of results from the ongoing SELECT prevention trial for prostate cancer (9,30,31).
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NOTES |
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We thank Dr. Scott Findlay for his important contribution as a statistical consultant in the writing of the manuscript.
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Manuscript received April 11, 2003; revised March 16, 2004; accepted March 22, 2004.
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