This student paper was written as an assignment in the graduate course

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1 77:222 Spring 2003 Free Radicals in Biology and Medicine Page 0 This student paper was written as an assignment in the graduate course Free Radicals in Biology and Medicine (77:222, Spring 2003) offered by the Free Radical and Radiation Biology Program B-180 Med Labs The University of Iowa Iowa City, IA Spring 2003 Term Instructors: GARRY R. BUETTNER, Ph.D. LARRY W. OBERLEY, Ph.D. with guest lectures from: Drs. Freya Q. Schafer, Douglas R. Spitz, and Frederick E. Domann The Fine Print: Because this is a paper written by a beginning student as an assignment, there are no guarantees that everything is absolutely correct and accurate. In view of the possibility of human error or changes in our knowledge due to continued research, neither the author nor The University of Iowa nor any other party who has been involved in the preparation or publication of this work warrants that the information contained herein is in every respect accurate or complete, and they are not responsible for any errors or omissions or for the results obtained from the use of such information. Readers are encouraged to confirm the information contained herein with other sources. All material contained in this paper is copyright of the author, or the owner of the source that the material was taken from. This work is not intended as a threat to the ownership of said copyrights.

2 G. Chauhan Selenium 1 Selenium: The essential poison by GAURAV CHAUHAN 4133, SC Department of Chemical and Biochemical Engineering The University of Iowa Iowa City, IA For: 077:222, Spring 2003 April 7, 2003 Abbreviations: SDS-PAGE: sulfate-polyacrylamide gel electrophoresis DAN: 2,4-diaminonaphthalene HPLC: high performance liquid chromatography FLD: fluorimetric detection Se: Selenium

3 G. Chauhan Selenium 2 Table of Contents: Page no. 1. Abbreviations 1 2. Abstract 2 3. Introduction 3 4. Selenium Chemistry 3 5. Selenium toxicity 4 6. Selenium carcinostatic activity 5 7. Detection of selenium compounds 7 8. Summary 8 9. References 10 Abstract Selenium is an essential dietary nutrient for most animals and humans, which is incorporated into twelve or more known proteins or enzymes as an amino acid, selenocysteine. Selenium toxicity was first confirmed in 1933 to occur in livestock that consumed plants of the genus Astragalus, Xylorrhiza, Oonopsis and Stanleya. Later, selenium was identified as an essential nutrient for laboratory rats and soon thereafter for chickens and sheep, whereas essentiality for mammalian species was not established until 1973, with the discovery that glutathione peroxidase contained selenium and epidemiological evidence suggested that selenium possessed anticarcinogenic effects. In 1988, the observation was made that oxidation of glutathione by selenite produced superoxide, opening a new area for selenium research. This paper primarily focuses on the toxicity and carcinostatic activity of selenium.

4 G. Chauhan Selenium 3 Introduction Selenium is an essential dietary nutrient for most animals and humans, which is incorporated into twelve or more known proteins or enzymes as an amino acid, selenocysteine. Selenium toxicity was first confirmed in 1933 to occur in livestock that consumed plants of the genus Astragalus, Xylorrhiza, Oonopsis and Stanleya [1]. However, in 1957, it was discovered that selenium was an essential nutrient for laboratory rats to prevent dietary necrosis and over a period of time it was found to be an essential nutrient for many mammalian species. In 1973, selenium was found to be a component of glutathione peroxidase in the form of selenoamino acid, selenocysteine. Dietary selenium from the inorganic salts and organic selenium compounds are metabolized into selenocysteine. In 1988, the observation was made that oxidation of glutathione by selenite produced superoxide, opening a new area for selenium research [1]. This paper primarily focuses on the selenium toxicity and carcinostatic activity. Selenium Chemistry [2] Elemental selenium has both metallic and non-metallic properties and is considered a metalloid. It is located between the metals telluriam and polonium and the non-metals oxygen and sulfur by group, and between arsenic and bromine by period. Selenium has proved to be particularly suitable for biological experimentation because of its relatively long half-life (120 days). Selenium shows allotropy, existing in amorphous as well as three crystalline states (alphamonoclinic, beta-monoclinic, and hexagonal). Elemental selenium can be oxidized to +4 or +6 oxidation states. In +4 states it exists as dioxide (SeO 2 ), selenious acid (H 2 SeO 3 ), or selenite (SeO 2-3 ) salts, while in +6 oxidation states it exists as selenate (SeO 2-4 ) salts. In its most reduced state (-2) selenium exists as selenide (Se 2- ).

5 G. Chauhan Selenium 4 Numerous organoselenium compounds can be prepared from elemental selenium by addition, displacement or substitution reactions. Also, selenium halides can be used to prepare organoselenium compounds by addition reactions to C=C double bonds, or by electrophilic substitutions of hydrogen in aliphatic or aromatic species. Selenium toxicity Various mechanisms have been proposed to explain why selenium is toxic. In 1941 Painter EP proposed that selenium toxicity was due to its interaction with thiols as shown in equation set 1 [1]. These reactions occur spontaneously and do not require oxygen. 4GSH + SeO 2 GSSeSG + GSSG + 2H 2 O 4GSH + SeO 3 GSSeSG + 2OH - + H 2 O (1a) (1b) In 1968, Ganther HE proposed that the toxicity was due to the interaction of selenium with disulfides of proteins in forming selenotrisulfides (RSSeSR), reaction similar to that shown for selenoglutathione in equation 1b [3]. Selenotrisulfides can be reduced to excess thiols or by cellular glutathione reductase to form highly reactive selenopersulfides (GSSeH). In 1989, Seko et al. proposed that selenite reacts with glutathione and then H 2 Se to produce superoxide as shown in equation 2 [4]. 4GSH GSH GSH O 2 (2) SeO 3 2- GSSeSG GSSeH H 2 Se Se 0 GSSG GSSG GSSG Later on, Spallholz et al. confirmed the reaction by using lucigenin amplified luminescence for the detection of superoxide [1]. Selenocysteine has subsequently been shown to react with GSH under different conditions of ph and thiol concentrations to produce superoxide. They also experimentally surveyed a number of other compounds other then selenite to see if they react O 2 -

6 G. Chauhan Selenium 5 with GSH to produce superoxide [5] and are tabulated in Table 1. With the help of these results they developed a hypothesis that all the selenium compounds which react with thiols ultimately produce superoxide and/or hydrogen peroxide and are thus toxic, whereas selenium compounds which do not react with thiols do not produce any superoxide and/or hydrogen peroxide and are thus non-toxic [1]. The literature available supports this generalized hypothesis for selenium toxicity. Table 1: Glutathione oxidase activity of selenium compunds [5] Superoxide is produced (toxic) Superoxide is not produced (non-toxic) Selenite Elemental selenium Selenium dioxide Selenate Selenocystine Selenoethionine Selenocystamine Selenomethionine Diselenodiproprionic acid Selenobetaine Selenium carcinostatic activity Evidence for carcinostatic activity of Selenium compounds: Research over last 20 years have shown that dietary selenium can prevent or reduce the incidence of naturally occurring and both chemically and virally induced cancer [8]. Most but not all selenium compounds have carcinostatic activity, which arrests cancer cell growth and prevent or reduce induced carcinogenesis. In humans, supplemental levels of 200-µg selenium/day have been reported to exhibit carcinostatic activity, which is 2-3 times of the amount of normal dietary levels. Milner et al. showed with the help of an experiment that most carcinostatic forms of selenium are inorganic salts: selenite and selenium dioxide. Selenate, selenocystine and selenomethionine are less effective as carcinostatic agents in preventing tumor growth. The experiment is shown in Table 2 and it can be seen that even a small dose of selenite and selenium dioxide prevent tumor growth [7].

7 G. Chauhan Selenium 6 Table 2: Tumor incidence in mice injected with selenium compounds [7] Treatment Experiment 1 Experiment 2 Experiment 3 Control 10/10 (infected/total-mice) 5/5 5/5 Inorganic forms Dose, µg/g weight Selenium dioxide 0/10 0/5 1/ 4 Sodium selenite 0/10 0/5 0/3 Sodium selenate 0/10 0/5 2/5 Organic forms Selenomethionine 0/10 5/5 5/5 selenocystine 0/10 0/5 2/5 Later on, they found out that even more potent against cancer cells than selenite is selenodiglutathione. Selenodiglutathione readily arrests cancer cell growth than selenite or any other selenium compound. All inorganic selenium compounds that express carcinostatic activity against cancer cells in vivo do so by interaction with thiol compounds and generation of free radical species [9]. Mechanism of carcinostatic activity of selenium compounds: There are a number of hypotheses that have been postulated to account for the experimental data that selenium prevents cancer. Five hypotheses seem to be possible to account for selenium s chemopreventative [9]. The five hypotheses postulated (as shown in Figure 1) are: (1) selenium s antioxidant role as a component of the glutathione peroxidase enzymes, (2) selenium s enhancement of immunity, (3) selenium s effect on the metabolism of carcinogens, (4) selenium s interactions that affect protein synthesis and the cycle of cell division, and (5) the formation of anti-cancer selenium metabolites. Chemoprevention takes place in the cell cycle apoptosis area of the supranutritional dose range. This dose range provides a metabolic level of catalytically active selenols and their redox cycle produce oxidative stress and induce apoptosis in more sensitive cancer cells. Chemoprevention likely occurs because of a differential

8 G. Chauhan Selenium 7 sensitivity between cancer and normal cells to metabolically generate selenols that induce apoptosis [9]. Figure 1: Two-stage model for the roles of Se in cancer prevention [8,9] Detection of Selenium compounds Techniques used in separation and detection of selenoproteins in mammalian tissues include gel filtration chromatography, affinity chromatography, electrophoresis and immunoassay. However, affinity chromatography and gel filtration can be used to separate only a group of proteins from a mixture, and they suffer from low resolution for protein separation [6]. In addition, a large volume of effluents used during separation dilutes the sample, making the subsequent steps more difficult. Electrophoresis, such as SDS-PAGE, is a high-resolution technique for separating numerous proteins from biological sample simultaneously, but the amount of protein in each band is so small that a very sensitive method for Se determination is required. Gao et al.

9 G. Chauhan Selenium 8 suggested an approach for Se determination. The approach includes protein separation by SDS- PAGE and sensitive determination of selenium in the protein bands. The method for the determination of selenium consisted of a pre-column derivatization with 2,4-diaminonaphthalene (DAN), followed by separation using high performance liquid chromatography (HPLC), with fluorimetric detection (FLD). A calibration curve was obtained in a range of ng Se and a linear relationship between Se-selenite dosage and fluorescent intensity was observed (Figure 2). Figure 2: Calibration of Se determination at ng levels [6] Summary Selenium is an essential dietary nutrient for most animals and humans, which is incorporated into twelve or more known proteins or enzymes as an amino acid, selenocysteine. Most, but not all selenium from selenium compounds can be efficiently incorporated into selenocysteine. The major dietary forms of selenium are L-selenomethionine from cereal and animal protein and L- selenocysteine from animal protein. Fruits and vegetables generally contain very low levels of selenium. At supranutritional levels of dietary selenium, most but not all selenium compounds can reduce the incidence of naturally occurring, viral or chemically induced cancer in both

10 G. Chauhan Selenium 9 animals and humans. A common characteristic of all selenium compounds that express significant experimental carcinostatic activity in vitro or in vivo is their interaction with thiols and the generation of free radical species. Selenodiglutathione is the most potent selenium compound against cancer cells and readily arrests their growth as compared to selenite and any other selenium compound. At higher levels of dietary intake many selenium compounds can become toxic. In dealing with selenium we must remember the words of Paracelsus ( ), The dosage makes either a poison or a remedy [9].

11 G. Chauhan Selenium 10 References 1. Spallholz JE. (1994) On the nature of selenium toxicity and carcinostatic activity. Free Radic Biol Med. 17: Subcomittee on selenium (1983). Selenium in nutrition. Revised ed. Washington DC; National academy press. 3. Ganther HE. (1968) Selenotrusulfides. Formation by reaction of thiols with selenious acid. Biochem. 7: Seko Y, Saito YKJ, Imura N. (1989) Active oxygen generation by the reaction of selenite with reduced glutathione in vitro. In: Wendel A, ed. Selenium in Biology and Medicine. Berlin: Springer-Verlag. pp Spallholz JE, Zhang X, Boylan LM (1993) Generation of oxyradicals by selenium compounds. FASEB J. A290: Gao Y, Wang Z. (2000) Separation and detection of selenium-containing proteins in human serum. J Anal Chem. 367: Milner JA, Greeder GA, Poirier KA. (1981) Selenium and transplantable tumors. In: Spallholz JE, Martin JL, Ganther HE, ed. Selenium in biology and medicine, Eds. CT: AVI Publishig Co.; pp: Spallholz JE (2001). Selenium and the prevention of cancer. Part I: Evidence for the carcinostatic activity of Se compouonds. The Bulletin of Selenium-Tellurium Development Association. ISSN May Spallholz JE (2001). Selenium and the prevention of Cancer. Part II: Mechanisms for the carcinostatic activity of Se compouonds. The bulletin of Selenium-Tellurium development association. ISSN October 2001.