BIOLOGICAL TRACE ELEMENT RESEARCH 3, 185-196 (1981)

Effects of Dietary Selenium and of Lead on the Genesis of Spontaneous Mammary Tumors in Mice G. N. SCHRAUZER,* K. KUEHN, AND D. HAMM Department of Chemistry, University of California at San Diego, Revelle College, La Jolla, California 92093 Accepted May 4, 1981

Abstract Selenium added to the diet significantly lowers the incidence of spontaneous mammary adenocarcinoma in female inbred C3H/St mice infected with the Bittner Milk Factor. Lead, 5 ppm, added to the drinking water in the form of the acetate, diminishes the uptake of selenium and reduces its anticarcinogenic effects, causing mammary tumors to appear with the same high incidence as in Se-unsupplemented controis. At higher lead concentrations in the drinking water (25 ppm), the overall tumor incidence is lowered, but tumor growth is significantly accelerated and the survival of tumor-bearing mice is shortened. Under the conditions of administration chosen, lead acts as a selenium antagonist and lowers the concentrations of selenium in liver, kidney, and spleen. The deposition of selenium, copper, and arsenic in bone is increased as compared to lead-unexposed controls. Index Entries: Selenium, effect on mammary tumorigenesis; lead, effect on mammary tumorigenesis; mammary tumors, effects of Se and Pb on; tumorigenesis, effects of Se and Pb on mammary; mice, C3H/St, effects of Se and Pb on mammary tumorigenesis in; liver, Se in; kidney, Se in; spleen, Se in; bone; Se, Cu, and As in; Cu, in bone; As, in bone.

Introduction Selenium in the form o f selenite in the drinking water has been shown previously to prevent or retard the genesis o f spontaneous m a m m a r y adenocarcinoma in female inbred C3H/St mice infected with the Bittner Milk Factor (1-3). Similar 9 1981 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4992/81/0900--0185502.40

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SCHRAUZER, KUEHN, AND HAMM

anticarcinogenic effects of selenium were also observed on addition to the diet in organically bound forms normally present in foods (4), and since selenium exerts its anticarcinogenic effects at levels that are still in the normal nutritional range, it may be considered a "nutritional cancer-protecting agent" (5). The anticarcinogenic effects of selenium are counteracted by a number of elements that have been shown in other studies to possess selenium-antagonistic properties, typically by arsenic (6), but also by high levels of zinc (7). In principle, all elements or compounds that possess a high chemical affinity for selenium may be expected to act as selenium antagonists. Since some of the heavy metals that fall into this category are common "environmental pollutants" or are observed in certain locations at higher than average concentrations, we are presently investigating the effects of such elements together with selenium in the C3H/St mammary tumor model system. In the present paper we describe the results of life-term studies with mice exposed to two concentrations of lead in the drinking water. Lead is of special interest because of the significant variations of human exposure to lead in various forms arising from its widespread industrial uses and its presence in certain foods, and because lead-cancer relationships have been the subject of much interest.

Experimental Animals and Diets Weanling female inbred C3H/St mice infected with the Bittner Milk Particle were obtained from, and housed in, the animal care facilities of UCSD Cancer Center. The animals were kept in specially cleaned plastic cages to minimize exposure to extraneous heavy metals, with only three animals at most per cage to minimize stress through overcrowding. The animals received selenium-supplemented Torula yeast diets whose composition is given in Table 1. Selenium was added in form of "Nutrition 21 High Selenium Yeast", purchased from CELL-LIFE Corp., San Diego, Calif., in amounts calculated to increase the selenium content of the diets to 0.15 and 1.0 ppm, respectively. The selenium content of each batch of diet was checked by analysis of wet-ashed samples, using the fluorimetric assay of Olson (8). The basal lead content of the diets, determined by atomic absorption spectroscopy, varied only slightly from batch to batch, and was on the average 0.245 ppm. All animals received the same diet and water over their entire postweaning life-span ad libitum. The experimental groups III and IV received 5 and 25 ppm of lead in form of lead acetate in the water. The water also contained 3 mL 5% acetic acid/L to keep the lead in solution. The controls in groups I and II received deionized water with 3 mL 5% acetic acid/L.

Experimental Design Groups I and II served as the controls (see Table 2). Groups III and IV show the effects of lead at two concentrations chosen to produce only marginal chronic toxic-

SE AND P8 IN MAMMARYTUMORIGENESIS

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Table 1 Composition of Basal Test Diet (Teklad # 170698) a Component

g/kg

Torula yeast Sucrose Lard, tocopherol-stripped Mineral mix, Hubbell-Mendel-Wakeman(Cat # 170790) Thiamine.HC1 Riboflavin Pyridoxine.HC1 Calcium panthothenate Niacin Biotin Folic acid Vitamin Blz (0.1% trituration in mannitol) Choline chloride Dry vitamin A palmitate (500,000 U/g) Dry vitamin De (500,000 U/g) Dry vitamin E acetate (500 U/g) Menadione

300 598.29 50.0 50.0 0.0004 0.0025 0.002 0.02 0.1 0.001 0.002 0.1 1.0 0.028 0.0064 0.44 0.001

aSelenium was added in form of selenium-containing Brewers yeast to bring the basal Se-content to 0.15 and 1.0 ppm, respectively. To make the diets strictly comparable, selenium-free Brewers yeast was added to 0.15 ppm Se yeast. The commercial Se-yeast contained 240 ppm Se, as determined by analysis. Table 2 Experimental Design of Study of Selenium and Lead Interactions in Female Inbred C3H/St Mice Se content of diet, ppm Group No.

0.15

I (control) II (control) III IV

x

1.0 x x x

Pb content of water, ppm 0

5

25

x x

ity effects on life-term administration. At the start o f the exposure, the mice were 6 weeks o f age. T h e y were monitored with respect to weight gain, and food and water consumption. At 6 months o f age, blood samples were collected from the eyes for whole-blood A L A D (delta-aminolevulinic acid dehydratase) determinations, using a modification (9) o f the technique originated by Granick (10). The an-

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imals were checked for mammary tumors by palpation once per week. Mammary tumor growth rates were measured by recording the tumor diameters with a caliper. Survival times of tumor-bearing animals as well as of tumor-free animals were also recorded. One randomly selected surviving old animal from each group was sacrificed and autopsied. Tissue slides from liver and kidney were prepared for histological examination. Selected organs (liver, kidney, and spleen) were collected and analyzed for Pb, Cu, and Zn by atomic absorption analysis. One femur from each of the animals autopsied was sent to a commercial microanalytical laboratory (Health Evaluations Inc., Hayward, Calif.) for trace element analysis by X-ray fluorescence.

Results In Table 3, the weights of the mice are shown at different ages. No statistically significant differences between the average weights were observed for any of the groups studied. The food and water consumptions in all groups were also identical. The cumulative survival rates in groups I-IV are shown in Fig. 1. It may be seen that the cumulative survival in group III was shorter than in the other groups. The Table 3 Weight Averages of Mice in Grams a Experimental groups Age, months 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

I

II "

III

IV

17 21 21 24 30 32 34 36 37 38 38 38 36 36 34 31

18 21 24 27 30 32 34 37 38 38 38 37 36 35 33 32

19 22.5 25 27.5 29.5 30.5 32 33 35 35 34.5 33 32.5 31 30 28

18 22.5 26 28 30 32 32.5 35 35.5 35 34.5 33 32 31 29 29

~The standard deviation for any group of mice any month was less than or equal to 3 g.

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cumulative nontumor mortality was also higher in group III than in the remaining groups (Fig. 2). In Table 4, the incidence of spontaneous mammary tumors is listed for each group together with other relevant information. Figure 3 shows the cumulative tumor incidence. The mean life-span after tumor onset was shorter in the animals of group IV than in those of group II (P < 0.01). The tumors appeared earlier in group IV than in all other groups, but the cumulative tumor incidence was lower. Average tumor growth rates are shown in Fig. 4 and were found to be significantly faster in group IV than in the remaining groups (P < 0.01). The selenium concentrations of liver, spleen, and kidney of animals from groups I-IV are given in Table 5 together with the Cu, Zn, and Pb concentrations and whole-blood ALAD activities. Selenium and lead had no effect on the concentrations of copper and zinc in liver and kidneys. Selenium concentrations in the livers, kidneys, and spleens of lead-exposed mice were lower than in the 1 ppm Se controls. The lead concentrations in the livers of lead-exposed animals were higher than in the controls, the whole-blood ALAD activities were lower in the lead-exposed animals, as expected (see Table 5). Histological analyses of slices from randomly selected animals receiving lead in the drinking water did not exhibit discernible differences from those of animals in the control groups. The kidneys of animals fed 25 ppm Pb showed somewhat enlarged tubules, but no gross signs of renal damage. Table 6 shows the results of the analysis of the femurs of randomly selected animals from groups I-IV. It may be seen that the iron and zinc concentrations are not affected by lead exposure. However, copper, selenium, and arsenic levels are higher in the bones of the lead animals.

Table 4 Effects of Selenium and Lead at Different Concentrations Experimental groups: Se content and Pb exposure, ppm Number of animals Average weight at 14 mo, g • 3 Deaths prior to tumor age No. of tumors at end of study Tumor onset age, months • 0.5 Lifespan after tumor onset, days Age at death of last survivor, months Percent tumor incidence, %c

I 0.15/0

II 1.0/0

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III 1.0/5

IV 1.0/25

30 30 32.5 32 10 0 13 6 8 6.5 42 • 27 19 • 18.7 23 b 67~ 20

~Significantlydifferent (P < 0.01) relative to group II (X 2 contingencytest). bLast surviving animal sacrificed at 23 months of age. CCorrected for survival.

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II

III

IV

Selenium concentrations in ppm, wet weight Liver Kidney Spleen

1.20 • 0.15 1.46 - 0.03 1.35 +- 0.08

Liver Kidney

6.2/23.7 15.0/15.0

1.92 • 0.11 2.01 • 0.05 1.46 • 0.07

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1.25 • .01 1.46 • .05 1.05 • .06

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~Moles ~-ALAD transformed/min-LRBc.

1.87 25

194

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Table 6 Trace Element Content'~ of Femurs of Animals in Groups I-IV b Group Element Fe

Cu Zn Se Pb As

I 5 0.5 16 0.05 0.8 0.04

• • 0.2 • 2 --- 0.02 • • 0.02

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• • 0.2 • 2 • 0.03 ---0.1 -+ 0.02

III 5 ---2 1.0--- 0.4 17 • 2 0.1 --- 0.02 2.0---+0.1 0.1 - 0.05

IV 6 2 15 0.2 5 0.2

• • 0.4 • 3 • 0.1 ---0.5 --• 0.1

aln mg/100 g, with error limits. OPerformed by Health Evaluations, Inc., Hayward, Calif., by X-ray fluorescence.

Discussion The present study demonstrates that the anticarcinogenic effects of selenium against mammary tumorigenesis by the Bittner Milk Factor are counteracted by 5 ppm of lead in form of the acetate in the drinking water. In the group of animals thus exposed, tumors appear at the age of 8.5 months, i.e., up to 5 months earlier than in the 1 ppm Se controls (Fig. 3). Most tumors appeared between the 8th and 11 th month. In the remaining 7 months of the experiment, tumors were produced at a significantly slower rate. However, during this time, a high tumor-unrelated mortality occurred, as may be seen in Figs. 1 and 2. On exposure to 25 ppm of lead, the first tumor was observed with an even shorter latency period than in the 5 ppm Pb group, i.e., at the age of only 6.5 months, but in the subsequent 12 months of observation only 5 more tumors appeared, causing the survival-corrected tumor incidence to be significantly lower than in the 5 ppm Pb group. However, the tumor growth rates in the 25 ppm Pb group were significantly greater, and the survival of tumor-beating animals was shorter than in the other groups. Under our conditions of administration, lead exhibits selenium-antagonistic effects, as evidenced by the lower selenium concentrations in the livers, kidneys, and spleens of lead exposed mice (see Table 5). Lead concentrations in the liver increase with increasing lead exposure. This causes a depression of the liver selenium concentrations, but has no effect on the concentrations of copper and zinc. In addition, lead also depresses ALAD activity. In bone, lead also influences selenium, copper, and arsenic levels, causing increased deposition, especially at the 25 ppm level (see Table 6). We attribute the higher cancer incidence of the animals in the 5 ppm Pb group to the selenium-antagonistic effects of lead. Similar observations have been made previously on simultaneous administration of 2 ppm selenium (as selenite) and 2 ppm of arsenic (as arsenite) (6). However, whereas 5 ppm lead produced a high

SE AND PB IN MAMMARY TUMORIGENESIS

195

tumor-unrelated mortality, no such effect was observed in the mice exposed to arsenic nor in the animals exposed to 25 ppm lead. The fact that the exposure to 25 ppm lowers the overall tumor incidence suggests that lead has cytotoxic properties against certain malignant cell populations. However, surviving malignant cell lines give rise to more rapidly growing tumors. A lower incidence of mammary tumors with greatly enhanced tumor growth rates was also observed in mice receiving 10 ppm of arsenic (as arsenite) in the drinking water (6). Thus, there are parallels in the behavior of arsenic and of lead at intermediate dosage, but not at low exposure levels. It is of interest to note that previous workers initially failed to observe clear effects of lead on selenium metabolism. Thus, selenium did not restore the reduced ALAD activity caused by lead poisoning in Japanese Quail (9), nor was an effect seen of injected lead acetate on metabolism of injected selenite in the rat (11). However, Cerklewsky and Forbes showed that selenium was mildly protective against the toxic effects of lead at low levels, but exaggerated lead toxicity at excessive levels (12). The interactions between lead and selenium depend on the method of administration and the chemical forms of the elements chosen. Our study shows that Pb 2+ in the drinking water antagonizes selenium in the organic forms in which it occurs normally in foods. The diminution of Se uptake by Pb 2+ under our experimental conditions is significant, but does not generate acute selenium deficiency. However, interactions between lead and selenium in vivo must also be considered since these may have effects on the physiological functions of selenium. At the 5 ppm Pb 2+ exposure level, this appears to generate a special condition characterized by increased susceptibility to tumor virus and higher mortality from tumor-unrelated causes. At the 25 ppm Pb 2+ exposure level, compensatory changes appear to take place that render the animals more resistant to tumor development as well as tumor-unrelated mortality. The underlying mechanism(s) for these changes are not yet understood. Historically, lead-containing preparations have been used in cancer therapy by W. Blair Bell and his associates more than 50 years ago (13). In a number of cases, tumor regressions and objective remissions have been documented. The treatment was abandoned, following the deaths of treated patients from lead toxicity. However, our work suggests that lead has anticarcinogenic properties at levels producing only marginal chronic toxicity. In this context, a report of Matarazzo et al. (14) is also of interest. These authors found that Pb 2+, Cd 2+, and C r 3+ at concentrations of 0.01-5 ppm in the drinking water of mice caused increased lymphocyte transformation as measured by DNA, RNA, or protein turnover. When challenged with a plastocytoma tumor cell line, the mortality rate of.the metal-exposed mice was significantly reduced. It was suggested that B-cell, antibody dependent, and T-cell antibody independent cytotoxicity as a result of increased lymphocyte transformation is responsible for the observed effects. The fact that three different metal ions elicit similar effects suggests that their action is indirect. Nevertheless, further studies with lead are necessary, especially at low dosage levels, where lead may no longer have seleniumantagonistic effects and functions as an essential nutrient (15,16).

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SCHRAUZER, KUEHN, AND HAMM

Acknowledgments This work was supported by Grant No. 8000420 from the U.S. Department of Agriculture. We thank Messrs. J. G. Palmer, J. E. McGinness, and G. Nakonechny for skillful experimental assistance.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

G. N. Schrauzer and D. Ishmael, Ann. Clin. Lab. Sci. 4, 443 (1974). G. N. Schrauzer, D. A. White, and C. J. Schneider, Bioinorg. Chem. 8, 387 (1978). D. Medina and F. Shepherd, Cancer Lett. 8, 241 (1980). G. N. Schrauzer, J. E. McGinness, and K. Kuehn, Carcinogenesis 1, 199 (1980). G. N. Schrauzer, Advan. Nutr. Res. 2, 219 (1979). G. N. Schrauzer, D. A. White, J. E. McGinness, C. J. Schneider, and L. J. Bell, Bioinorg. Chem. 9, 245 (1978). G. N. Schrauzer, D. A. White, and C. J. Schneider, Bioinorg. Chem. 6, 265 (1976). O. E. Olson, J. Assoc. Off. Anal. Chem. 52, 627 (1969). C. L. Stone and J. H. Soares, Jr., Poultry Sci. 55, 341 (1976). S. Granick, S. Sassa, J. L. Granick, R. D. Levere, and A. Kappas, Proc. Natl. Acad. Sci. USA 69, 2381 (1972). O. A. Levander, Environmental Health Perspectives 29, 115 (1979). F. L. Cerklewski and R. M. Forbes, J. Nutr. 106, 778 (1976). W. Blair Bell, Some Aspects of the Cancer Problem, Bailliere, Tindall and Cox, London, 1930. W. J. Matarazzo, T. M. Carbone, and I. Gray, Trace Subst. Envir. Health XIII, 382 (1979). K. Schwarz, in Clinical Chemistry and Chemical Toxicology of Metals (S. S. Brown, ed.), Elsevier-North Holland, New York, 1977, pp. 3-22. A. Reichhnayr-Lais and M. Kirchgessner, Arch. Tierernaehrg. in press, 1981.