Selenium in carcinogenesis

Biochimica et BiophysicaActa, 738 (1984) 2 0 3 - 2 1 7

203

Elsevier

BBA87124

S E L E N I U M IN C A R C I N O G E N E S I S L.N. VERNIE

Division of Chemical Carcinogenesis, The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, 121 Plesmanlaan, 1066 CX Amsterdam (The Netherlands) ( R e c e i v e d O c t o b e r 2nd, 1984)

Contents I.

Introduction

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II.

S e l e n i u m as a n a n t i o x i d a n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203 205

III. S e l e n i u m as a c a r c i n o g e n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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IV. S e l e n i u m as a n a n t i c a r c i n o g e n in h u m a n s : e p i d e m i o l o g i c a l studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. T o t a l p o p u l a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. C a n c e r p a t i e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. S e l e n i u m t r e a t m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. S e l e n i u m s t a t u s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

206 206 207 207 207

V.

208 208 208 209 210 210

S e l e n i u m as a n a n t i c a r c i n o g e n in a n i m a l studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. C a n c e r of the b r e a s t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Virally i n d u c e d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. C h e m i c a l l y i n d u c e d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. T r a n s p l a n t e d t u m o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. O t h e r t u m o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VI. S e l e n i u m as a c y t o t o x i c a g e n t

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VII. C o n c l u d i n g r e m a r k s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213

Acknowledgements ............................................................................

214

References ..................................................................................

214

I. Introduction

In recent years selenium has attained increasing interest as a chemical and naturally occurring Abbreviations: MeDAB, 3'-methyl-4-dimethylaminoazobenzene; D M B A , 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e ; G S H , g l u t a thione. 0 3 0 4 - 4 1 9 X / 8 4 / $ 0 3 . 0 0 © 1984 Elsevier Science P u b l i s h e r s B.V.

compound with potential capacity to reduce carcinogenesis. The first description of selenium was published by Berzelius [1], who obtained a reddish precipitate in 1817 while burning pyrite in a sulfuric acid plant. Upon analysis this precipitate turned out to be a new element. He called the element after Selene, the Greek goddess of the moon, as tellurium, discovered earlier, was named

204

after the goddess of earth, Tellus. Selenium is an element of subgroup VIA of the periodic chart. This subgroup comprises the nonmetals oxygen (O) and sulfur (S), the metalloid selenium (Se) and the metals tellurium (Te) and polonium (Po). Among these elements the close relationship in chemical properties of selenium and sulfur is most striking. Due to this chemical similarity no discrimination between sulfur and selenium is made in certain biological processes and incorporation of selenium and sulfur into proteins is parallel [2]. There is a wide distribution of selenium throughout the earth's crust, similar to that of sulfur. However, the ratio of S : Se has been calculated as 6000:1 [3]. The concentration of selenium on earth averages 0.09 ppm [4] but shows a large range [5,6]. Concentrations of selenium in soil range from less than 0.1 ppm in the seleniumdeficient areas of New Zealand to as high as 1200 ppm in the organic soil in Ireland [4]. Various plants, growing on selenium-rich soil, absorb and also accumulate selenium. In plants it is found as organic compounds and as selenium-containing amino acids [2]. The selenium content of plants differs widely among species. Accumulator plants can incorporate several thousands (1000-10000) ppm of selenium per dry weight [7]. Cereals, vegetables and grasses growing in seleniferous areas also may contain high levels of selenium [7]. In 1957 Schwarz and Foltz [8] discovered that selenium was an essential trace element for animals. Selenium-deficiency diseases, commonly referred to as selenium-responsive disease, such as muscular dystrophy and liver necrosis, were recognized (reviewed in Ref. 9). Selenium deficiency in animals occurs in many countries of the world and it has been estimated that selenium deficiency has caused more economic losses than selenium toxicity [10]. Recently selenium deficiency has also been observed in man. This selenium-responsive disease, known as Keshan disease, is a juvenile cardiomyopathy occurring in the Heilongjiang province of the People's Republic of China. It caused the death of many young children [11-13], Since the administration of 0.5-1 mg selenium per week fully prevented this disorder, selenium appears to be an essential trace element in man. To prevent deficiency diseases, a human requirement of about 60 120 /xg per day is suggested [14]. Selenium

uptake from food largely depends on the chemical form of selenium present. The bioavailability to humans of selenium from different dietary sources has been discussed by Levander [15] and Young et al. [16]. Selenium is also highly toxic to animals and man. The LDs0 of selenium salts in several species ranges from 0.4 to 6.4 mg Se/kg [17] and depends on the selenium compound used [18]. A statement from the U.S. Food and Nutrition Board [14] estimates that overt chronic selenium intoxication would result in human beings after long-term consumption of 2.4-3.0 mg daily. An endemic disease due to a selenium uptake of about 5 mg per day, affecting 50% of the inhabitants of several villages in the Hubei province of the People's Republic of China, has been described [19]. After the discovery of the high selenium content of many plants, selenium has been blamed for the illness and death of livestock. This led to investigations on the toxicity of selenium in the 1930s. As this coincided also with an increasing interest in chemically induced tumors, the carcinogenicity of selenium was studied, resulting in the identification of selenium as a possible carcinogenic agent (see below). Paradoxically, however, further studies revealed that selenium could act as an anticarcinogenic agent as well. In 1949, Clayton and Baumann [20] were the first to report a reduction in the incidence of chemically induced liver tumors by selenium. This investigation was performed to study the possible additive effect between selenium and the azo dye MeDAB. In two separate experiments 50% reduction in liver tumor incidence was noticed in the rats given selenium, compared to those that did not receive selenium. The result obviously surprised the authors, as they stated that 'selenium retarded the formation of tumors induced by MeDAB in spite of the reported carcinogenicity of the element itself'. No follow-up experiments were conducted and for a long time this study did not draw the attention it would have merited. As far as I know, Shamberger and Rudolph [21] were the first, in 1966, who deliberately used selenium to counteract chemically induced tumor development. In skin-tumor-promoting studies, it was found that sodium selenide, topically applied, markedly reduced the number of tumors, induced

205 with DMBA and croton oil. Shamberger extended his study in a subsequent paper [22] and a start was made for further epidemiological and animal studies. The purpose of this paper is to review the action of selenium as an anticarcinogenic agent. For those who are interested in other aspects of selenium, many excellent review articles on metabolism in animals and man [23] and biochemical and biological [24-26], clinical [27], environmental [6,28] and toxic [3,29] aspects are available. II. Selenium as an antioxidant

Prior to the discovery of selenium as an integral part of the enzyme glutathione peroxidase, the antioxidant properties of selenium compounds, in relation to vitamin E, were extensively studied and discussed by Tappel [30] and Tappel and Caldwell [31]. Damage to the unsaturated fatty acids of subcellular membranes by lipid peroxidation induced by free radicals could be prevented by vitamin E and selenium. Although the original hypothesis of the antioxidant action of selenium has receded into the background, the essence of it should not be forgotten. It has been shown that selenium compounds, including the selenoamino acids, which can be incorporated into proteins, act as free-radical scavengers [30,31]. An increase in the amount of selenium given to experimental animals leads to an increase in the biosynthesis of such biologically active selenium compounds which afford protection against free radical-induced peroxidation. As many carcinogens produce free radicals in vivo, selenium compounds can act as free-radical traps by scavenging free radicals and converting them to stable compounds. In 1973 Rotruck et al. [32] and Floh6 et al. [33] almost simultaneously reported that selenium is present in the enzyme glutathione peroxidase. This enzyme reduces a wide variety of organic hydroperoxides in addition to H202 [34]. Glutathione peroxidase was the first selenium-containing enzyme discovered in mammalian systems, and as selenium acts as an antioxidant it was thought that this was the main metabolic role of selenium. Since more enzymes, e.g., catalase and superoxide dismutase, can protect the cell against oxidative

damage, glutathione peroxidase may not be as important in this defence system as originally proposed [35]. Glutathione peroxidase from sheep and bovine erythrocytes contains 4 gatom of selenium per mol [33,36]. The way selenium is incorporated into this enzyme is still controversial. Forstrom et al. [37] reported that selenium in reduced glutathione peroxidase is present as selenocysteine. Hawkes et al. [38] and Hawkes and Tappel [39] obtained evidence that selenocysteine is incorporated into the enzyme by the normal translational pathway using a specific selenocysteyl-tRNA. But Sunde and Hoekstra [40,41] suggested that selenium is incorporated by a post-translational process in which selenium is attached enzymatically to an amino acid residue already present in the molecule. Dietary supplementation and depletion experiments with experimental animals have shown an increase and decrease, respectively, in glutathione peroxidase activity. However, beyond a certain selenium concentration in food or drinking water, a plateau value in the glutathione peroxidase activity is reached [42]. Human studies have given similar results [43-45]. Thomson reported a good correlation between glutathione peroxidase activity and the blood selenium levels of 222 residents of New Zealand with blood selenium levels ranging from 2 to 10 #g/100 ml [46]. But at levels over 10 ~tg/100 ml there was no longer a correlation between the two parameters. This indicates the existence, both in animals and in man, of a selenium blood level above which no further increase in glutathione peroxidase activity occurs. III. Selenium as a carcinogen Undoubtedly the knowledge of the high toxicity of selenium has led to serious concern in early reports about the possible carcinogenicity of this element. In 1943, Nelson and co-workers [47] reported the development of hepatic cell adenoma and low-grade carcinoma in rat livers when feeding a seleniferous diet. In discussing this paper, Shapiro [48] suggested that the observed lesions might have represented hepatic regeneration rather than neoplasia. Again, in 1946, alarming news was published by Seifter and colleagues [49], who described the appearance of thyroid adenomas in

206 rats receiving 0.05 %-0.1% bis(4-acetaminophenyl)selenium dihydroxide, corresponding to 103-207 p p m selenium in their diet for 10 days. However, no control group was studied and it is quite possible that this selenium compound has carcinogenic properties which are not related to selenium (cf. Ref. 50). It took until 1960 before the next reports concerning tumor induction in experimental animals after selenium administration were published [51-53]. As discussed by Shapiro [48], none of these papers provides conclusive evidence for tumor induction by selenium. In a study reporting the incidence of cancer death among industrial workers exposed to selenium over a period of 10 years, Cooper and Glover [54] found that six workers died of malignant neoplasms of various types. Since the expected number of cancer deaths was 5.1, these results do not support an increased tumor incidence due to selenium. In 1975 the International Agency for Research against Cancer, after evaluating the biological data relevant to the carcinogenic risk of selenium in man, concluded that ' t h e available data provided no suggestion that selenium is carcinogenic in man' [55].

IV. Selenium as an anticarcinogen in humans: epidemiological studies

IVA. Total population After the observation that selenium was a tumor-reducing agent in skin-tumor-promoting studies, Shamberger and Frost [56] suggested that 'if selenium had an effect on public health, areas adequate or deficient could be expected to show different disease incidences or death rates'. Indeed they showed that the selenium content of forage crops in different parts of the United States is negatively correlated with the cancer death rate in the same area. Furthermore, an inverse relationship between selenium levels in human blood and cancer death rates was noticed. This study was followed by a more extensive investigation [57]. The ratio of the age-adjusted expected cancer deaths to the observed cancer deaths was determined in large and small cities in high-selenium areas with matched cities in low-selenium regions.

Variables such as degree of urbanization, air pollution and racial make-up were eliminated. This study showed for both sexes a sharp reduction in deaths due to cancer of the small intestine, stomach, large intestine, rectum, bladder, urinary tract and kidney in high-selenium areas. In males also a reduction in cancer death from pharynx and esophagus and in females of the cervix, uterus, breast and ovary was observed. In general the same results were obtained in a comparison of the age-adjusted cancer death rates by site of origin of high- and low-selenium provinces in Canada [58,59]. It was noted, however, that the highselenium areas also had less air pollution [59]. In 1971, Schrauzer and Rhead [60] found that an alleged plasma cancer test which measured the time required for the reduction of methylene blue by human plasma was in fact a crude assay for the plasma selenium concentration. This led Schrauzer et al. [61] to suggest that cancer patients have lower circulating selenium levels than normal individuals and that selenium is a cancer-protecting agent. Schrauzer et al. [62] correlated next the human dietary selenium intake in 27 countries with the cancer incidence at 17 major sites. Although there are uncertainties in the estimation of the selenium intakes - the per-capita food consumption data were taken from the Food and Agriculture Organization of the United Nations and the same average selenium concentration was assumed to be present in food from the different countries - a significant negative correlation was observed between selenium intakes and cancer of the large intestine, rectum, prostate, breast, ovary and lung, as well as leukemia. If the blood selenium levels from humans in different areas of the United States and in different countries were correlated with death due to several malignancies, a similar inverse interdependence was found. Selenium is not the only element linked with cancer incidence. Selenium is also known as an antagonist of many metals. This induced Schrauzer et al. [63] to extend the dietary intake studies to zinc, cadmium, copper, chromium, manganese and arsenic. They concluded that trace elements have both direct and inverse correlations with cancer incidence and can counteract selenium in its cancer-protecting effect. Jansson et al. [64] compared the geographical distribution of gastrointestinal cancer and breast

207 cancer with the distribution of selenium deficiency. They noted that regions with high colorectal and breast cancer rates are also deficient in selenium and concluded that there is 'a good deal of epidemiological evidence indicating that selenium helps to prevent human cancer, especially of the intestines and the breast'. In 1975 the International agency for Research against Cancer judged that 'the evidence for negative correlation between regional cancer death rates and selenium is not convincing' [55]. In fact, there are two questions. First, does an increase in dietary selenium intake reduce the cancer incidence, and secondly, does a lack of selenium intake cause cancer? The epidemiological studies are accompanied by numerous animal studies (see below) and there is strong evidence that the first question has to be answered positively. The second question has been discussed by Schrauzer [65] in connection with the low dietary selenium intake in New Zealand. He pointed out that a low selenium intake does not cause cancer but merely increases the susceptibility to cancer induction by endogenous and exogenous factors.

IVB. Cancer patients IVB1. Selenium treatment After a communication from Wassermann et al. [66], who in 1911 had already successfully treated tumor-bearing mice with selenium, Delbet [67] reported in 1912 the treatment of cancer patients with this compound. However, a local application of sodium selenate to a cancer patient led to fatal complications. Treatment of patients with colloidal selenium and sodium tellurate was also unsuccessful. In 1956 Weisberger and Suhrland [68] treated acute and chronic leukemia patients with selenocystine, the selenium analog of cystine [68], using an average daily dose of 100 mg for 10-57 days. In all patients a rapid and striking decrease in the number of leukocytes was observed. They even saw an effect in patients refractory to other chemotherapeutic agents. However, the acute toxic effects of selenocystine, such as nausea, vomiting and in one patient acute myocardial infarction, were so severe that the administration had to be discontinued before complete remission was obtained.

In the following years several selenium compounds and selenium analogues of known chemotherapeutic agents, for example 6-selenopurine versus 6-mercaptopurine or selenoguanine versus thioguanine, have been prepared and tested in animal systems [69]. In general these selenium compounds did not show a higher antitumor activity than the parent compound. More recently the structure of the antitumor agent cis-dichlorodiammineplatinum(II) (cisplatin) was modified by replacing the chlorides with selenium [70]. In mice this new compound did not show good antitumor activity. Naganuma and coworkers [71,72] proposed the use of selenium to protect against the renal toxicity of cis-platin, which is the dose-limiting side-effect of this drug. In general, heavy metals can produce severe organ disturbance and it has been established that metal toxicity can be alleviated by selenium [73,74]. One daily injection of selenite indeed protected mice against a lethal dose of cis-platin, while its antitumor activity was hardly affected. Berry et al. [75] found that i.p. injection of selenium 4 h prior to the injection of cis-platin allowed the administration of high doses of cis-platin, which improved the antitumor effect. It might be possible that in this way selenium can be used clinically, not as an anticancer agent but to protect against side-effects of chemotherapeutic agents containing heavy metals.

IVB2. Selenium status The inverse relationship between cancer incidence and regional selenium content in forage crops, soil or blood might suggest a lower selenium blood level in cancer patients than in healthy individuals. Indeed, Shamberger et al. [76] found significantly lower blood selenium levels in patients with gastrointestinal cancer than in healthy controls. The lower selenium blood levels could be due to a diminished resorption of selenium but the authors showed that patients with various other gastrointestinal disorders had normal blood selenium values. No difference from the control group was found in breast cancer patients, however, nor in patients with rectal carcinoma, sarcomas, or several other diseases. On the other hand, patients with liver metastases had significantly lower blood selenium levels and it seems

208 likely that the occurrence of metastases is accompanied by low blood selenium values. Selenium levels were also determined in sera of patients with various diseases by McConnell et al. [77]. Significantly lower selenium values were found in sera of patients with carcinoma of several sites of origin or gastrointestinal cancer relative to the control group. These results confirm Shamberger's observations of a decreased blood selenium content in patients with gastrointestinal cancer. In a subsequent study by Broghamer et al. [78] the authors concluded that lower selenium levels were more frequently associated with advanced disease. In a later study by McConnell et al. [79] significantly lower serum selenium concentrations in breast cancer patients than in healthy controls were found but a correlation between the selenium level and the extent of metastasis was absent. A lower selenium blood or serum level has not been found in all studies. In patients with reticuloendothelial neoplasms even higher serum selenium values were observed than in the control group [80]. Sullivan et al. [81] noted normal serum selenium levels in cancer patients, while Capel and Williams [82] reported a decrease in serum but an increase in erythrocyte selenium levels in stage III breast cancer patients receiving radiotherapy compared to healthy controls and in patients with benign lesions of the breast. Willet and coworkers [83] used a different approach to study a possible correlation between serum selenium levels and the occurrence of cancer. From 10 940 people taking part in a screening and subsequent follow-up program for hypertension, serum samples were collected in 1973 and 1974. The subjects were followed for 5 years and during this period 111 cases of cancer were detected. Matched controls, 210 persons, generally two controls for each patient, were taken from the original group. The selenium value in the control group (mean: 0.136 ~ g / m l ) was slightly but significantly ( P = 0.02) higher than in the patient group (0.129 ~ g / m l ) . After arranging the cancer cases according to localization of the primary tumor the strongest correlation appeared to exist between low selenium and gastrointestinal cancer ( P = 0.01). No correlation was observed between breast cancer and selenium serum levels ( P = 0.90). This is in contrast with the results of McConnell et al.

[79] and Shamberger et al. [76]. It is important to note that the serum samples were collected months to years prior to cancer diagnosis, making it unlikely that the low selenium levels are a result of overt disease or its medical treatment. The latter possibility was investigated by Vernie et al. [84]. In breast cancer patients, who were in a good clinical condition and received maintenance therapy, selenium levels were measured in whole blood and plasma, and the glutathione peroxidase activity was measured prior to therapy and during 10 consecutive courses (259 days). No significant changes were found during this period. In conclusion, the main correlation between selenium status and cancer incidence that has emerged from these epidemiological studies is a significant correlation between low serum selenium and the risk of developing gastrointestinal cancer. In general, however, caution has to be taken when interpreting low selenium levels in cancer patients. These might be dependent on the clinical condition of the patient, and healthy persons do not always seem to be the proper control group. V. S e l e n i u m as an anticarcinogen in animal studies

VA. Cancer of the breast VA1. Virally induced Following early reports in the 1970s, an increasing number of papers have appeared during recent years concerning the anticarcinogen properties of selenium in virally as well as chemically induced m a m m a r y tumors. The effect of selenium supplementation on mouse m a m m a r y tumorigenesis was first examined by Schrauzer and Ishmael [85]. They investigated the influence of 2 p p m selenium in drinking water on C 3 H / S t mice, which show a high incidence (82%) of m a m m a r y adenocarcinomas. The addition of selenium reduced the tumor incidence to 10% (3 out of 30) but did not inhibit the growth of transplanted m a m m a r y tumors. Tumors appeared in selenium-treated animals at the age of 9 months, while in the control group tumors were first seen between 12 and 16 months. In a parallel study with 10 ppm arsenic, a reduction of the tumor incidence was also noticed but, in contrast, the development of the spontaneous tumors was enhanced. The corn-

209 bination of 2 ppm arsenic and 2 ppm selenium in the drinking water also increased the incidence of mammary tumors [86]. In a subsequent study the effect of higher selenium concentrations on C3H mice was investigated [87]. Even at 15 ppm, a toxic level, a reduction of the tumor incidence to 33% was obtained. Simultaneous addition of 200 ppm zinc to mice which received 5 ppm selenium abolished its tumor-protective effect. This result shows the antagonist effect of zinc on the prevention of tumorigenesis in mice and correlates with epidemiological studies concerning human breast cancer mortalities in relation to selenium and zinc [63]. By diminishing the uptake of selenium, lead also reduced the anticarcinogenic effect of selenium in C 3H / S t mice [88]. A change of the laboratory diet with a concomitant increase in the selenium content of 0.15 ppm to 0.45 ppm resulted in a drop in the spontaneous tumor incidence in C 3 H / S t mice from 80-100% to 42% [89]. Addition of selenium to the drinking water gave a dose-proportional decrease in the tumor incidence. Medina and Shepherd [90] and Medina et al. [91] confirmed the findings of Schrauzer and coworkers in Balb/cf C 3H mice given 2 and 6 ppm selenium in the drinking water. The tumor incidence in the control mice, 82%, declined to 48% and 12%, respectively. Whanger et al. [92] tested the effect of selenium supplementation combined with six different types of diet on the mammary tumor incidence in C3H mice. Administration of selenium resulted in a decreased percentage of tumor-bearing mice from 64% to 25% in one diet only, indicating that the type of diet influences the tumor-inhibiting effect of selenium. It is of interest that with the fat-supplemented diets selenium significantly increased the glutathione peroxidase activity in liver, kidney and red blood cells and the selenium content of liver and kidney, but it did not decrease the tumor incidence. In continuation of prior experiments Schrauzer et al. [93] changed the selenium content of the food from 0.15 ppm selenium to 1.00 ppm and vice versa at 13.8 weeks, halfway through the life-time of the mice. The mammary tumor incidence in both groups was compared to that in mice which received 0.15 ppm or 1.00 ppm selenium over the entire experimental period. Mice

maintained on a diet of 0.15 ppm selenium developed a 77% mammary tumor incidence, while a diet of 1.0 ppm selenium gave a significantly lower tumor incidence of 27%. If the amount of selenium in the diet, halfway through the experiment, was switched from 1.00 to 0.15 ppm selenium, tumors began to appear 3 weeks after the diet switch and a tumor incidence of 69% was observed. It turned out that this group developed mammary tumors at the same rate as the 0.15 ppm selenium group. The switch from 0.15 to 1.00 ppm selenium still resulted in a significantly lower, 46%, tumor incidence than in the control group. From these experiments the important conclusion was drawn that 'dietary selenium prevents and retards tumor development only as long as it is supplied in adequate amounts consistent with its role as a non-accumulative trace nutrient'.

VA2. Chemically induced Mammary tumors have been induced in rats and mice by various carcinogens: acetylaminofluorene [94,95], D M B A [91,96-106] and N-methyl-N-nitrosourea [103,107,108]. The effect of selenium administration was studied and reductions in the chemically induced tumor incidences of over 50% were no exception. Moreover, a synergistic effect of selenium and vitamin A [108,109] or vitamin E [110] in the chemoprevention of mammary carcinogenesis in rats has been reported. These effects were not caused by a diminished food intake due to the selenium administration [100,106-109]. To elucidate the mechanism by which selenium exerts its anticarcinogenic effect, Ip and Sinha [98] studied the inhibition by selenium of DMBA-induced mammary carcinogenesis in rats fed either a 5% or 25% corn oil diet. In both groups selenium reduced the number of tumors, but had no effect on the malondialdehyde content (which is a final product of lipid peroxidation) nor on the glutathione peroxidase activity in the mammary gland. The authors concluded that 'the inhibitory action of selenium is not mediated by its antioxidant action in lipid metabolism'. Lane and Medina [111] showed that the glutathione peroxidase level in mammary and hepatic tissues of mice increased when the dietary selenium increased from 0.02 ppm to 0.1 ppm. Beyond 0.1 ppm selenium no

210 further increase was observed, but addition of selenium (0.5-2.0 ppm) to a diet containing 0.15 ppm selenium inhibited DMBA-induced mammary carcinogenesis [112]. There was no correlation between administration of selenium to mice and the reduction in mammary tumors on the one hand and the glutathione peroxidase activity on the other. Thus the chemopreventive effect of selenium could not be attributed to glutathione peroxidase activity [105]. In several reports the effect of selenium on the initiation and promotion phases of DMBA-induced mammary carcinogenesis has been investigated in order to define the stage of tumorigenesis that is sensitive to selenium-mediated inhibition [100,103,104,112]. Ip [100] administered DMBA to rats at zero time and tested the effect of selenium supplementation for six different periods of time during the carcinogenic process. The greatest inhibition was obtained when selenium was supplemented during weeks - 2 to +24 (66%), - 2 to + 12 (47%) and + 2 to + 24 (41%). Ip concluded that selenium can inhibit both initiation and promotion phases of carcinogenesis but a continuous intake of selenium is necessary to achieve maximal inhibition. In addition he studied the effect of selenium supplementation on the reappearance of mammary tumors which had regressed after ovariectomy [100]. Selenium addition to the diet immediately after endocrine ablation inhibited the reappearance of mammary tumors. Welsch et al. [104] treated rats at 60 days of age with DMBA and added selenium to the drinking water from day 30 to day 90 and from day 90 to day 150. In all groups a similar, but significantly reduced, number of mammary carcinomas per rat was found and the authors concluded that selenium inhibits the early promotion phases of mammary carcinogenesis. These results were confirmed by Medina and Lane [112] in DMBA-treated B a l b / c mice. Selenium was given in drinking water during, after, and during + after DMBA treatment. The tumor incidences for these three groups were reduced from 42% to 25%, 8% and 13%, respectively. The authors concluded that selenium is an effective chemopreventive agent in the post-initiation phases of chemical carcinogenesis. The same result was obtained by Thompson and Becci [107] and Thompson et al. [103] using N-methyl-N-nitrosourea as a carcinogen.

In summary, the conclusion must be drawn that the effect of selenium is not mediated through the antioxidant action of glutathione peroxidase, and secondly that events after the initiation steps of carcinogenesis are involved. VA 3. Transplanted tumors

Ip et al. [113] fed female W / F U rats with a diet containing selenium (0.02-2.0 ppm). After 6 weeks a standardized piece of MT-W9B mammary tumor was implanted subcutaneously into the right axillary region. The increase in tumor volume was delayed in the rats supplemented with 2.0 ppm but with less than 0.02 ppm or 0.1 ppm selenium no difference in tumor growth was observed. The growth of the rats was not influenced by the selenium supplementation and no histological change in the tumors was found. The reduction in tumor growth was not accompanied by an increase in glutathione peroxidase activity in liver tissue. Medina and Shepherd [96] tested the effect of selenium supplementation on the growth of mammary adenocarcinomas in Balb/c, BD2F 1 and Balb/cf C3H mice. Out of 36 mammary tumors examined the growth rate of three tumors was decreased, while that of one tumor was enhanced. Medina et al. [91] and Medina and Lane [112] found that the growth of some but not all preneoplastic nodules was reduced by selenium supplementation. Watrach et al. [114] observed a reduction in the average tumor volume by about 75% in selenium-treated athymic mice implanted with canine mammary carcinoma. These experiments show that selenium not only reduced the induction of mammary tumorigenesis but also inhibited the growth of transplants. At the same time it must be noted that many transplanted tumors or preneoplastic nodules were not sensitive to selenium supplementation. The reason for this is unknown, but Medina and Lane [112] ruled out a difference in glutathione peroxidase activity. VB. Other tumors

About 30 years after the observation by Clayton and Baumann [20] that selenium reduced the induction of liver tumors by MeDAB, the results were confirmed by Griffin and Jacobs [115]. The

211 tumor incidence was reduced from 92% to 46% and 67%, respectively, by including selenium in the diet as sodium selenite or as a high-selenium-containing yeast. The effect of selenium supplementation, 4 ppm, in drinking water, was investigated when given throughout or during the early or later stages of azo dye administration [116]. A reduction in the tumor incidence from 90% to 14%, 54% and 67%, respectively, was observed, showing that the best protection was obtained when selenium was given during the whole period of azo dye administration. Hgrr et al. [94,95] reported that selenium addition to food reduced the number of hepatomas in rats given acetylaminofluorene and that tumors developed more slowly at the highest concentration used. While confirmed by Marshall et al. [117], others failed to produce similar results (cf. Ref. 118). The addition of selenium to the drinking water, to prevent a suspected selenium deficiency in New Zealand, reduced the number of spontaneous intestinal adenocarcinomas observed at autopsy [119] almost to zero. Subsequent studies showed that selenium also inhibited tumors of the intestines induced with various carcinogens such as methylazoxymethanolacetate [120-123], 1,2-dimethylhydrazine [120-122,124,125], azoxymethane [126,127] and bis(2-oxopropyl)nitrosamine [128]. Jacobs et al. [120] compared the effects of selenium supplementation on colon tumor induction by 1,2-dimethylhydrazine and methylazoxymethanolacetate. Both chemicals are highly specific colon carcinogens but differ in their metabolic conversion. Methylazoxymethanolacetate is the proximate alkylating carcinogen formed after in vivo oxidative activation of 1,2-dimethylhydrazine. Addition of selenium to the drinking water significantly reduced the tumor incidence in the 1,2-dimethylhydrazine-treated rats, but not so after treatment with methylazoxymethanolacetate. However, in the latter case the average number of tumors per rat decreased from 5.2 to 2.8. These results imply that selenium must act beyond the activation of 1,2-dimethylhydrazine to methylazoxymethanolacetate. This conclusion was supported by Harbach and Swenberg [129], although these authors found that selenium shifted 1,2-dimethylhydrazine metabolism to an increased

amount of azomethane and a decreased amount of CO 2 exhaled. Studies on the effect of selenium on the levels of enzymes involved in the metabolism of acetylaminofluorene and N-hydroxyacetylaminofluorene are not decisive. Daoud and Griffin [130] reported that selenium enhanced the glucuronyltransferase activity by 100%, inhibited the p-nitrophenylsulfotransferase by 50%, but did not change the acetylaminofluorene deacylase activity. In contrast, Besbris et al. [131] found that selenium-deficient rats exhibited a higher glucuronyltransferase but the same sulfotransferase activity as selenium-supplemented rats. The finding that selenium administration shifted the urinary metabolites from N-hydroxyacetylaminofluorene to a greater amount of non-carcinogenic acetylaminofluorene metabolites [132] is in accordance with the finding of an enhanced glucuronyltransferase activity. However, whether there is a changed metabolism or not, several reports showed that the carcinogen-DNA binding of acetylaminofluorene [130,132,133] or benz[a]-pyrene [134] is not changed by selenium supplementation. Moreover, selenium did not protect against acetylaminofluorene- or methylazoxymethanolacetate-induced inhibition of RNA and DNA synthesis in liver and colon [123]. Protection has been found against the occurrence of single-strand breaks in liver DNA induced by acetylaminofluorene [133] and in colon DNA induced by bis(2-oxopropyl)nitrosamine [128]. Selenium compounds are radical scavengers [30,31] and it is not unlikely that selenium affords protection against indirect damage, such as single-strand breaks, caused by radicals which are produced during carcinogen metabolism. Taken together, there is still no sufficient evidence that the selenium-mediated inhibition of carcinogenesis is due to an altered activation of carcinogens or interaction with DNA. VI. Selenium as a cytotoxic agent

The increased interest in selenium as a toxic and concomitantly potential anticarcinogenic agent has also led to several studies on the effect of selenium on cells in various tissue cultures and on ascites tumor cells. Gruenwedel and Cruikshank

212 [135] showed that the DNA, RNA and protein synthesis of intact HeLa cells strongly decreased at selenite concentrations above 5 - 1 0 - 6 M and at incubation times of more than 6 h. A similar effect of selenium was reported by Medina and Oborn [136]: selenite at 10 -5 M inhibited the growth of three established mammary cell lines and the growth of preneoplastic and neoplastic cells in monolayer cultures. There appeared to be a difference in response in various cell lines and a correlation was found between the sensitivity of the cells in culture and the corresponding tumors in mice (cf. Ref. 90). At low selenium concentrations, 5 • 10 -8 M, a growth stimulation in some of the cell lines in tissue culture was noted. This correlated with a previous observation that selenium was essential for the growth of diploid human fibroblasts [137]. Poirier and Milner [138] incubated cultured Ehrlich ascites tumor cells with various concentrations of sodium selenite, selenium dioxide, selenocystine, selenomethionine and sodium selenate. Except for sodium selenate, increasing selenium concentrations diminished cell viabilities. Most effective were sodium selenite and selenium dioxide, which reduced the cell viability by about 90% after 4 h of incubation. Further studies showed that these compounds also suppressed Ehrlich ascites tumor cells growth in mice when injected intraperitoneally [139]. The efficacy of the selenium compounds in inhibiting the growth of L1210 cells in vitro and in vivo was investigated [140]. In vitro sodium selenite and selenium dioxide were more effective than selenocysteine and sodium selenate. Also in vivo sodium selenite was more inhibitory than sodium selenate, selenocysteine or selenomethionine. Selenite has been shown to react with thiols, and the corresponding selenotrisulfides are formed [141,142]. Since biological systems contain thiols, such as GSH and cysteine, Vernie et al. [143] suggested that the selenium-containing reaction products are the inhibitors. GSH reacts with sodium selenite and two products are formed which have been identified as oxidized glutathione and selenodiglutathione [144]. Selenodiglutathione inhibits amino acid incorporation in a cell-free system derived from rat liver [145]. Inhibition appeared to be due to a specific inactivation of elongation factor 2 [146]. In tissue culture a strong

inhibition of protein synthesis of intact 3T3-f cells was found [147]. Inhibition was obtained at 5.8. 10 -6 M selenodiglutathione after 15 min of incubation and could not be reversed by removing selenodiglutathione. Subsequent studies by Vernie et al. [148] with intact L1210 and P815 cells demonstrated that various reaction products of thiols with selenite inhibited protein synthesis and led to cell death. A pronounced difference in inhibitory effect was observed with the selenium compounds, and a variety of effects of the same product tested on several cell lines was noted. Maximum inhibition of protein synthesis of intact L1210 cells was found at a selenium concentration of 0.25-10 -6 M with the reaction product of selenite and 203-dimercapto-l-propanol. Selenite itself was less effective than the reaction products derived from it. In view of the reported inhibition of amino acid incorporation in a cell-free system the inhibition of protein synthesis in intact cells was taken as a parameter to study the effect of the various reaction products. However, the possibility cannot be excluded that other metabolic activities were also inhibited. HiSgberg and Kristoferson [149], studying the toxic or nearly toxic concentrations of selenite on isolated hepatocytes, reported that selenite inhibited amino acid uptake of the cells and that this effect can be potentiated by low molecular weight thiols. Vernie et al. [143] tested the reaction product of selenite and GSH, selenodiglutathione, or cysteine, selenodicysteine, on in vivo ascites malignant mouse lymphoid cells. Intraperitoneal injections of these two compounds in mice, 1 h after the inoculation of the tumor cells, appeared to inhibit tumor growth and to increase the life-span of treated compared to untreated control mice up to 6 days. As the doubling time of these tumor cells in mice is about 18 h, such a survival suggests a considerable tumor cell death. This assumption could be confirmed by calculation of cell counts in the ascites fluid of the mice. Poirier and Milner [150] compared the antitumorigenic properties of sodium selenite, selenodiglutathione, sodium selenide, dimethylselenide and selenocysteine by measuring their inhibition of Ehrlich ascites tumor ceils growth in mice. Sodium selenite and selenodiglutathione showed the highest activity. Sodium selenite injected intraperitoneally also reduced solid Ehrlich ascites tumor cells

213

growth if the cells were injected subcutaneously on the left dorsal side. This experiment ruled out a direct toxic effect of sodium selenite on the tumor cells but, in view of the high reactivity of selenite with cellular components such as thiols, also suggested that selenite is not the ultimate inhibitor. The longevity of mice inoculated intraperitoneally with Ehrlich ascites tumor cells preincubated together with either sodium selenite, selenodiglutathione or dimethylselenide was only significantly increased in the case of selenodiglutathione-pretreated cells. Furthermore, selenodiglutathione delayed the first appearance of tumors most effectively [150]. VII. Concluding remarks During half a century the public image of selenium gradually evolved from that of a highly toxic and carcinogenic element to an essential trace element with antioxidant and anticarcinogenic properties (Fig. 1). Growth inhibition of virally as well as chemically induced neoplasia has been observed in rat and mice. In general, selenium manifests its effect in the form of a lower tumor incidence, a reduction in the tumor yield and a longer latency period. These results have been observed with tumor induction at several sites (breast, bowel, liver, lung and skin) and have supported the earlier epidemiological findings. Originally the antioxidant action of selenium via

C f ciN0 E^

Fig. 1. The public image of selenium.

the enzyme glutathione peroxidase, in analogy with the anticarcinogenic properties of other antioxidants (cf. Refs. 21,151), was an attractive hypothesis. However, in several studies it has been shown that a relationship between glutathione peroxidase activity and the antitumorigenic effect was absent [98,102,105,106,110,111]. Various in vivo experiments have been performed to study the effect of selenium on the binding of carcinogens, or their metabolites, to D N A [123,129,130,132-134], on DNA-strand breaks [132,133] and repair mechanism [128, 133,152] and on the reduction of DNA and RNA synthesis [123] caused by carcinogens. In addition, experiments in vitro have been performed to determine whether selenium affects the metabolism of carcinogens to less mutagenic products as assessed after incubation with microsomal preparations of liver [118,153,154]. Although alterations have been observed, these experiments do not explain the reduction of virally induced carcinogenesis, the tumor growth inhibition of transplanted tumors, the reduced reappearance of mammary tumors which have regressed after ovariectomy nor the reversed situation that upon withdrawal of selenium addition the reduction of tumorigenesis is halted. Looking for a common mechanism - which does not necessarily exist in the anticarcinogenic action of selenium - an effect on the initiating action of carcinogens is not likely and it is most

"S"ELEN !1"1PI'

214 p r o b a b l e that selenium acts on cell proliferation. This has been specified b y M e d i n a a n d L a n e [112], who suggest an i n h i b i t i o n of cell growth b y an i n h i b i t i o n of D N A synthesis. These authors also suggested that at low, physiological doses, selenium is necessary to m a i n t a i n the a n t i o x i d a n t function of glutathione p e r o x i d a s e b u t at higher doses the antiproliferative effect of selenium becomes manifest. I n h i b i t i o n of t u m o r cell g r o w t h in vitro a n d in vivo has been o b s e r v e d a n d a difference in effect on various cell lines has been n o t e d [148]. Vernie et al. [147,148] d e m o n s t r a t e d in vitro a n d Poirier a n d Milner [150] in vitro and in vivo that the first p r o d u c t of selenite m e t a b o l i s m , selenodiglutathione, is a better cell-growth-inhibiting c o m p o u n d t h a n selenite itself. A l s o in vivo it is likely that s e l e n o d i g l u t a t h i o n e is f o r m e d after ingestion of selenite [144,155]. A further reaction of selenod i g l u t a t h i o n e with other cellular c o m p o n e n t s leading to the synthesis of o r g a n o s e l e n o c o m p o u n d s with i n h i b i t o r y capacities has to be considered. This is not easily studied, as only p a r t of the selenium a d m i n i s t e r e d to animals might be converted to the u l t i m a t e i n h i b i t o r y c o m p o n e n t . A final a n d i m p o r t a n t question is whether selenium can be used to reduce the h u m a n t u m o r incidence a n d whether selenium a d m i n i s t r a t i o n should be advocated. Selenium a n d cancer prevention have been the subject of several p u b l i c a t i o n s [156-160]. A l t h o u g h it has been p o i n t e d out that selenium is a highly toxic agent a n d caution has to be taken, this c a n n o t be an overriding o b j e c t i o n against s u p p l e m e n t a t i o n of the diet with low doses of selenium. T o define the p o t e n t i a l c o n t r i b u t i o n of selenium to cancer incidence in man, p r o s p e c t i v e r a n d o mized studies are required, p r e f e r a b l y in a lowselenium a r e a with a high incidence of breast, liver or bowel cancer, cancer types on which a selenium effect m a y be expected. T h e cost, logistic p r o b l e m s a n d legal c o m p l i c a t i o n s of such a study will be f o r m i d a b l e b u t the e n c o u r a g i n g e p i d e m i o l o g i c a l a n d a n i m a l studies s u m m a r i z e d in this review p r o vide a m p l e j u s t i f i c a t i o n for such an undertaking.

Acknowledgements I wish to t h a n k Drs. P. Borst, E. K r i e k a n d U A . Smets for their critical c o m m e n t s . I a m grateful to

Miss. G. M e i j e r i n k for t y p i n g this m a n u s c r i p t and R. Slagter for d r a w i n g the cartoon.

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