Changes in Selenium and Antioxidant Status during ...

Minerals and Trace Elements

Changes in Selenium and Antioxidant Status during DMBA-Induced Mammary Carcinogenesis in Rats1'2 MARYR.

L'ABBÉ,* f PETER W. F. FISCHER,*f AND EDOARDO R. CHAVEZf

'Nutrition Research Division, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario K1A 012 and ^Department of Animal Science, Macdonald College ofMcGHI university, Ste. Anne de Bellevue, Quebec H9X ICO, Canada

INDEXING KEY WORDS: •selenium •cancer •glutathione peroxidase •Superoxide dismutase •lipid peroxidation •rat •7,12-dimethylbenz(a)anthracene

Two types of epidemiological studies have investi gated the relationship between selenium and cancer. A number of studies have measured selenium levels in the blood of cancer patients and compared them with matched, healthy controls (1-3). Pancreatic, liver, stomach, breast and rectal cancer patients had signif icantly lower blood selenium levels than controls. In the second type of investigation, prospective studies in the United States (4), Finland (5, 6) and the Netherlands (7) have found serum selenium levels to be significantly lower, prior to the development of cancer, in individ 0022-3166/89 $3.00 ©1989 American Institute of Nutrition.

'Publication

No. 276 of the Bureau of Nutritional

Sciences.

^Presented in part at the 29th Annual Meeting of the Canadian Federation of Biological Societies, Guelph, Ontario, June 16-20, 1986: Can. Fed. Biol. Soc. Pioc. 29: 103 |abs. PA-99).

Received 14 June 1988. Accepted 9 February 1989.

766

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uals who subsequently developed cancer, compared to matched controls. Comparisons of blood selenium in cancer patients and controls, however, cannot distin guish an effect of cancer on blood selenium levels from an effect of selenium on cancer (8). Determining the activity of an enzyme offers several advantages over measures of an element in blood. The activity of a trace element-requiring metalloenzyme measures the amount of metabolically active metal for which a specific function is known, and thus more accurately assesses status (9). The amount of enzyme activity can then be compared with a physiological con sequence which, in the case of the present study, is the development of tumors. Thus, the objective of the present experiment was to determine if there is a relationship between glutathione peroxidase (GSHPx) and Superoxide dismutase (SOD) activity and carcinogenesis. Rats were fed graded amounts of selenium and the blood activity of these enzymes was measured every 4 wk during 7,12-dimethylbenzfo(anthracene (DMBA)-induced mammary carcinogenesis. These measures were used to deter mine if there were changes in these enzymes due to carcinogen administration. The activity of these en zymes was also examined at the end of the experiment to determine if there were differences between rats with tumors and those without tumors. Finally, the analysis of blood samples obtained before the appearance of palpable tumors allowed us to determine if there were differences between control rats, DMBA-treated rats that developed tumors, and DMBA-treated rats that re mained tumor free. Lipid peroxidation was also as sessed by measuring urinary and erythrocyte malondialdehyde.

ABSTRACT Female weanling Sprague-Dawley rats were used to examine the changes that occurred in selenium and antioxidant status during the development of 7,12dimethylbenz(a)anthracene (DMBA)-induced mammary tumors. Animals were fed an AIN-76 diet, modified to con tain 20% fat (3:1 wt/wt, lard:corn oil) and 0.1, 0.035, 0.1, 1.0, 2.0 or 4.0 mg Se/kg diet. At wk 5, rats in groups 26 were administered by intragastric tube 4.32 mg of DMBA dissolved in corn oil. Control rats received corn oil only. A blood sample was removed from the tail vein and analyzed for selenium-dependent glutathione peroxidase (SeGSHPx) and Superoxide dismutase (SOD) activity at wk 5, and every 4 wk until wk 25. At the end of the experiment, rats were classified by tumor status, and each diet group was subdivided into two groups: those rats remaining free of tumors for 25 wk and those with tumors. DMBA treat ment caused an initial decrease in erythrocyte SeGSHPx and SOD activity compared to untreated control rats. SeGSHPx activity in rats with tumors remained lower than controls, while SeGSHPX activity increased in rats with no tumors. These changes, however, were not associated with any changes in lipid peroxidation. J. riuti: 119: 766— 771, 1989.

SE AND ANTIOXID ANT STATUS DURING CARCINOGENESIS

METHODS

767

Erythrocyte 80

RESULTS The effects of DMBA administration and the devel opment of tumors on erythrocyte SeGSHPx and SOD activities were determined by comparing the activity observed in vehicle-treated control animals (group 1) with the activity in DMBA-treated rats fed the same amount of selenium. The data for erythrocyte SeGSHPx activity at the various time intervals while tumors were developing are shown in Figure 1. DMBA administra tion caused an initial decline in erythrocyte activity in

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FIGURE 1 Effects of DMBA treatment and tumor status on erythrocyte and plasma SeGSHPx activity at various time intervals after DMBA administration. Values are means ± SEM; means at each week with different letters are signifi cantly different (P < 0.05). All rats were fed the recommended amount of selenium (0.1 mg/kg diet) for 25 wk. Vehicle-treated control rats (solid bars, n = 20) were dosed with the same volume of corn oil as DMBA-treated rats. DMBA-treated rats were administered an intragastric dose of 4.32 mg of DMBA dissolved in corn oil at 56 d of age (wk 5) and were classified by tumor status: NT rats (hatched bars, n = 2) were DMBAtreated rats that remained free of tumors for 25 wk; WT rats (open bars, n = 17) had one or more malignant tumors at wk 25.

both the WT and NT groups compared to the untreated control rats at wk 9 (P < 0.05). By wk 13, however, SeGSHPx activity was higher in the NT animals than either the control or WT group. Erythrocyte SeGSHPx activity remained higher in the NT group than in the WT or control rats until the end of the experiment. SeGSHPx activity in plasma, however, did not vary significantly with tumor status or with DMBA admin istration (Fig. 1). The effects of DMBA administration and tumor sta tus on erythrocyte Cu-Zn SOD activity at various time intervals while tumors were developing are shown in

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Animals and diets. Weanling female Sprague-Dawley rats (Crl:CD(SD)BR, Charles River Canada, St. Con stant, Quebec) were fed a casein-based diet containing 20% fat (3:1 wt/wt, lardxorn oil) and varying amounts of selenium. Selenium was added as sodium selenite to all diets except the basal diet, which contained 0.035 mg Se/kg diet. The diets contained 0.1, 0.035, 0.1, 1.0, 2.0, and 4.0 mg Se/kg diet (10), as determined fluorimetrically (11). Rats in groups 2-6 were given 4.32 mg of DMBA, dissolved in corn oil, by intragastric tube at wk 5. Control rats (group 1) were fed 0.1 mg Se/kg diet, and were treated with the same volume of corn oil without DMBA. Fasting (approximately 16 h) blood samples were taken every 4 wk from wk 5-25, and the activity of plasma selenium-dependent (SeGSHPx) and total glutathione peroxidase, erythrocyte SeGSHPx and SOD determined. Details of the diets, tumor induction, blood sampling and biochemical methods were given previously (10). Only rats with histologically confirmed malignancies were considered to be tumor bearing (10). Statistical analysis. Data, were analyzed by analysis of variance for unbalanced data using the ANOVA II or PCANOVA programs (Human Systems Dynamics, Northridge, CA). Significant differences between means were determined by the Studentized Range Test or by the method of Least Significant Differences at the 0.05 critical difference level (12). Factors examined were the effects of dietary selenium and tumor status. All DMBAtreated rats that had no expressible tumors for the 25wk duration of the experiment were considered tumor free (NT group), while all rats that had one or more malignant tumors were considered to be tumor bearing (WT group). In order to separate those changes which were due to the administration of carcinogen from changes associated with tumor development, compar isons were made between vehicle-treated control rats, DMBA-treated NT and DMBA-treated WT rats fed the recommended level of selenium (0.1 mg Se/kg diet). Where there were no significant differences due to di etary selenium, data were pooled and comparisons were made between the untreated control, NT and WT rats from all dietary selenium groups.

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TABLE 1 Effect of dietary selenium and tumor status on glutathione peroxidase activity in liver and spleen of rats treated with DMBA and fed graded amounts of selenium for 25 u A' P-values

Selenium, mg/kg diet status2NT Control3734 DMBA4386 EnzymeSeGSHPxNon-SeGSHPxTotal

Tumor DMBA1.0DMBALiver,

mu/mg protein 826 ±57 814 ±45 644 46172±27 768 ± WTNT 12876± ±14 25 9998 119 ± 6± ± WTNT 133981 46213± ±71 54 GSHPxGSHTSeGSHPxNon-SeGSHPxTotal 34250 763 ± 51± ± WTNT 901230 ±1 12 7138± 202 8Spleen, ±9167 243± ± WTNT mu/mg protein 153 ±1 157 ±3 550 ± ±10 123 746 ± 140 438 ±3 154 ± WTNT 4189 ± ±5 ±6 5± 3191 49 ± 4191 53 ± 492054± WTNT ±60.035 ±15 6193± 7 GSHPxTumor 172 ±50.1 ±548 203± ±62.0 WT0.1 35142±

±45 431 3973± ±5 10459 82 ± ±50 513 48162± ±3 209 14145±

DMBA897 23 787185 40± ± 20 11± ± 1591081 33 946302 50± ± 21 ± 15± 271165

status653 DMBA ±690 ±140 4222 ± 119 ±793 0.271 ± ±338 809 4024 ± 328 ±186 2018

Diet < 0.01

< 0.09

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< 0.01

< 0.01

< 0.05

< 0.01

ns

2 < 0.01 ± 2± 15751± 170 ±69 1015 3 ± ns 3± ± 51216207± ±260 52 938 < 0.01 4 ± ±54.0 222 ±23 1D

Tx5nsnsnsnsnsnsns

< 0.05 ns ns

'Values are means ±SEMof 2-12 rats per group; ns, not significantly different. 2Rats were classified according to the presence or absence of tumors. NT denotes those rats treated with DMBA, but remained free of tumors for 25 wk; WT refers to all rats that had one or more malignant tumors at wk 25. 3Vehicle-treated control rats were dosed with the same volume of corn oil as DMBA-treated rats. 4DMBA-treated rats were administered an intragastric dose of 4.32 mg of 7,12-dimethylbenz(u)anthracene dissolved in corn oil at 56 d of age (wk 5). 5Diet x Tumor interaction.

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FIGURE 2 Effects of DMBA treatment and tumor status on erythrocyte Cu-Zn SOD activity at various time intervals after DMBA administration. Values are means ±SEM;means at each week with different letters are significantly different (P < 0.05). Data from rats fed all dietary levels of selenium were pooled as there was no significant difference due to dietary selenium. Control rats (solid bars, n = 7-12) were dosed with the same volume of corn oil as DMBA-treated rats. DMBA-treated rats were administered an intragastric dose of 4.32 mg of DMBA dissolved in corn oil at 56 d of age (wk 5) and were classified by tumor status: NT rats (hatched bars, n = 8-15) were DMBA-treated rats that remained free of tumors for 25 wk; WT rats (open bars, n = 21-34) had one or more malignant tumors present at wk 25.

Figure 2. No significant differences were seen 4 wk after DMBA administration (wk 9) but, by wk 13, the activity was significantly lower in the DMBA-treated rats than in the untreated controls. This reduction in erythrocyte SOD activity in DMBA-treated rats continued until the end of the experiment, regardless of whether or not tumors eventually developed. No significant differ ences that could be attributed to the tumor status of the DMBA-treated rats were seen. The effects of dietary selenium and the presence or absence of tumors at wk 25 on tissue GSHPx and glutathione transferase (GSHT) activity are shown in Table 1. In most diet groups, with the exception of the se lenium-deficient and 4.0 mg Se/kg diet groups, liver SeGSHPx activity, as well as the nonselenium-dependent form of the enzyme, was lower in the tumor-bearing rats than in the tumor-free rats. These reductions were reflected in significantly reduced total GSHPx activity (P < 0.05). GSHT activity in the liver, however, did not vary significantly with tumor status. No significant differences in GSHPx activity due to the presence or absence of tumors were evident in kidneys or heart at the end of the experiment. There was a significant re duction in spleen SeGSHPx activity in the tumor-bear ing rats compared to the tumor-free rats (P < 0.05). Liver and spleen SeGSHPx activity also showed similar dif ferences with tumor status for control, DMBA-treated NT and DMBA-treated WT rats fed the recommended

SE AND ANTIOXIDANT STATUS DURING CARCINOGENESIS

769

TABLE 2 Effects of tumor status on tissue Superoxide dismutase activity 25 wk after DMBA administration1 Tumor status2 114.16 =

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protein4.222.876.9515.07.70.20.3*b0.3>b0.60.922.7 SODMnSODTotal SODCu-Zn SODMnSODTotal SODControln

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0.13.69 ± 0.4»7.72 ± 0.4b13.7 ±

1.16.8 ± 0.722.9 ± ±1.3NT

0.96.0 ± 0.619.6 ± ±1.2P-valuens