Carcinogenesis vol.13 no.10 pp.1725-1729, 1992

The effect of sodium selenite on cell proliferation and transformation of primary rat tracheal epithelial cells

Songyun Zhu, Thomas E.Gray and Paul Nettesheim' Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA 'To whom correspondence should be addressed

Introduction Selenium is an essential trace element for mammalian species (1). It has been known for many years that it is important for growui of various cell types in culture (2). Since Clayton and Baumann first reported inhibition of azo dye-induced liver cancer by selenium in 1949 (3), selenium compounds have attracted considerable attention as possible chemopreventive agents in carcinogenesis. Epidemiological investigations have strongly suggested an inverse relationship between the geographical distribution of selenium in forage crops and the death rates from large intestine, rectum, prostate, breast and ovary cancers and from leukemias (4-7). Animal studies in various species have shown that selenium compounds effectively prevent chemically as well as virally induced carcinogenesis in mammary gland, liver, stomach, intestine, colon, pancreas, skin and oral cavity (8-13). As an integral component of redox-type enzymes such as glutathione peroxidase, selenium is important in protecting cells from reactive oxygen species (for a recent review see ref. 14). Although epidemiological evidence has suggested a weak, but statistically significant negative correlation between dietary •Abbreviations: RTE, rat tracheal epithelial; EGV, enhanced growth variants; NNK, 4-(mcthylnitrosamino)-l-(3-pyridyr)-l-butanone; CSFM, complete serumfree medium; DMSO, dimethylsulfoxide; CFE, colony-forming efficiency; RCFE, relative CFE; TF, transformation frequency; RTF, relative TF; CFU, colonyforming units.

Materials and methods Establishment of RTE cell cultures Primary cultures of RTE cells in complete serum-free medium (CSFM) were established as previously described (29). Briefly, RTE cells were obtained from tracheas of 10-15 week old male, pathogen-free Fischer 344 rats. The lumenal lining was dissociated by 4°C overnight digestion with 1 % protease (Type XIV, Sigma, St Louis, MO). The epithelial cells were flushed from the tracheas, washed and suspended in culture medium, counted visually and plated into 60 mm dishes with CSFM which was composed of Ham's F12 medium (GIBCO, Grand Island, NY) supplemented with epidermal growth factor (5 ng/ml, Collaborative Research, Bedford, MA), insulin (10/ig/ml), hydrocortisone (0.1 jig/ml), transferrin (5 /tg/ml), cholera toxin (0.1 jig/ml), phosphoethanolamine and ethanolamine (80 (iM each), bovine serum albumin (3 mg/ml), HEPES (15 mM, pH 7.2) and CaCl2 (°-8 m M) d\ purchased from Sigma. The medium was also supplemented with 1 % (v/v) bovine pituitary extract prepared as described (30) from whole bovine pituitaries (Pel Freeze, Rogers, AR). Penicillin/streptomycin (1%, v/v, GIBCO) were added to all media. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Chemicals and treatment Sodium selenite (Na2SeO3, Sigma) was dissolved in distilled water before use and diluted with CSFM. NNK (Chemsyn Labs., Lenexa, KS) was dissolved in dimethylsulfoxide (DMSO) and further dilutions were made with CSFM (the final concentration of DMSO in the medium was 0.25%). All chemical treatments (NNK alone, Na2SeO3 alone or NNK plus Na2SeO3 simultaneously in the cotreatment experiments) were started 24 h after plating and were continued for 7 days without medium change. In the post-treatment study, cells were exposed to NNK 24 h after plating and continued for 7 days without medium change, and then maintained in a selection medium (see below) for an additional 4 weeks with weekly medium change.


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The effects of sodium selenite (N^SeOj) on cell proliferation and the development of preneoplastic transformed variants were studied in primary cultures of rat tracheal epithelial cells. Results revealed a Diphasic effect of Na2SeO3 on cell proliferation: at concentrations between 6 x 10~8 and 6 x 10~ 6 M, it stimulated and at concentrations of — 2 x 10~ 5 and above it inhibited cell proliferation (presumably due to toxicity). Nontoxic concentrations of Na2SeO3 (6 x 10~ 8 -6 x 10~7 M) significantly reduced the spontaneous transformation frequency. Transformation induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyI)-l-butanone (NNK) was effectively inhibited by nontoxic as well as toxic concentrations of Na2SeOj. Treatment of cultures with N^SeC^ after cessation of NNK exposure, i.e. during the selection period, also significantly reduced the transformation frequency. These experiments show that the inhibition of transformation by Na2SeO3 is not the result of an antiproliferative effect. They further indicate that the inhibitory effect occurs even when the chemical treatment occurs during the 'postinitiation' phase. Thus the inhibition of transformation by Na2SeO3 cannot solely be explained by its effects on drug metabolism.

selenium intake and lung cancer incidence (5,15), the preventive effects of selenium on experimentally induced respiratory tract cancers have not been thoroughly studied. Birt et al. reported that the bis(2-oxopropyl)-nitrosamine-induced lung adenocarcinoma incidence in rats decreased from 4/29 in rats given low selenium diet to 0/30 in rats given a diet high in selenium (P < 0.05; 16). In contrast Thompson and Becci (17) and Beems (18) failed to observe any chemopreventive effect of selenium on lung cancer development in hamsters. Our laboratory has developed an in vitro transformation model of rat tracheal epithelial cells (RTE*) (19), which has proven to be very useful in the study of mechanisms of multistage carcinogenesis and has also been used as a screening system to identify carcinogenic chemicals (20) and chemopreventive agents (21). In this cell culture system the first identifiable transformed colonies, so-called enhanced growth variants (EGV), are detected 5 weeks after exposure of primary tracheal cells to transforming agents. The main purpose of the studies described here was to determine whether sodium selenite is capable of suppressing spontaneous as well as 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone (NNK)-induced transformation of RTE cells. This chemical was recently shown to transform RTE cells following in vivo as well as in vitro exposure (22). At the same time we also wanted to determine whether sodium selenite had an effect on proliferation of tracheal epithelium similar to that described for other epithelial cells (23-28).

S.Zhu.T.E.Gray and P.Netteshdm

Statistical analysis The differences of CFE as well as the number of transformants per culture among different treatments were evaluated by the two-tailed Student's (-test for statistical significance. The correlation between the background CFEs in untreated controls and the CFEs in selenium-treated cultures was examined by linear regression analysis.

Results Effect of sodium selenite on cell proliferation Figure 1 summarizes all data on the effects of Na2SeO3 on CFE of RTE cells obtained throughout the course of our studies. At concentrations ranging from 6.0 x 10~9 M to 1.2 x 10~5 M Na2SeO3 was nontoxic and in many instances increased CFE. At concentrations of 1.8 X 10~5 M and above the chemical caused significant reductions in CFE. The enhancement of CFE by Na2SeC«3, which was most clearly observed at concentrations of 6 x 10~8M to 6 x 10~6 M, is analyzed in detail in Figure 2. It can be seen that the enhancement effect was most pronounced when the CFE of untreated control cells, which can vary

considerably from experiment to experiment, was low, i.e. 4-fold increase in TF (RTF 4.71). In experiments summarized in Figure 4, cultures were exposed simultaneously to NNK and Na2SeO3 (started 24 h after plating and continued for 7 days) to investigate the effect of Na2SeO3 on NNK-induced transformation. Untreated cultures and cultures treated with either NNK or Na2SeO3 alone were used as controls in each experiment. Data from these three separate experiments were



NajSeO3(M) Fig. 1. Effect of Na2SeO3 on colony-forming efficiency (CFE). Results are the means obtained from at least two and in several cases > 10 experiments. Vertical bars represented ± SE. Statistically significant at P < 0.05 (a), P < 0.001 (b) compared with controls.





Control CFE (%) Fig. 2. Relationship between CFE in control cultures and the stimulatory effects of Na2SeOj on colony formation. Data are from the same experiments given in Figure 1. Na2SeO3 concentrations: 6 x 10~8 M (O), 6 x 10~7 M (A), 6 x 1Q~6 M (D). Curve is fitted by regression analysis.

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Cytotoxicity assays in primary RTE cell cultures The colony-forming efficiency (CFE) assay was used to determine the toxicity of the chemicals. Primary RTE cells were plated (0.5-2.0 x 10* cells/dish) in CSFM and exposed to NNK and/or Na2SeO3 as described above. Seven days after the start of the exposure, cultures (three to five replicates) were fixed and stained and the number of colonies ( > 2 0 cells) surviving treatment was counted. The data were expressed either as CFE (the number of colonies per dish/the number of cells plated per dish x 100%) or as relative CFE (RCFE = CFE in treated cultures/CFE in control cultures). To determine the cell number per colony, cultures were exposed to Na2SeO3 for 7 days as above, and then dissociated by trypsin-EDTA treatment. The cell number per culture was determined by visual counting. The average cell number per colony (as an indicator of colony size) was determined by dividing the number of cells per culture by the number of colonies, per culture. Transformation assays in primary RTE cell cultures RTE cells (0.5-2.0 x 104 cells/60 mm dish, 20-32 replicates per group) were plated and treated for 7 days with the test chemicals singly or in combination as described above. At the end of exposure, the culture medium was changed to a selection medium composed of Ham's F12 medium supplemented with insulin (1 /ig/rnl), hydrocortisone (0.1 fig/ml), 5% fetal bovine serum (Hazleton Biologies, Inc., Lenexa, KS) and penicillin/streptomycin/fungizone (1%, v/v, GIBCO). Cultures were maintained in this medium, with weekly medium change, for an additional 4 weeks before they were fixed, stained and scored. The transformed colonies, termed EGV, appeared as large colonies, containing tightly packed, small, deeply basophilic cells having high nuctearcytoplasm ratios (31,32). Data were expressed as the number of transformants per culture, transformation frequency (TF = the total number of transformants per group divided by the total number of colony-forming units surviving treatment per group X 100%) and relative transformation frequency (RTF) which is the TF of the treated group divided by the TF of the untreated control group.

Effects of sodium setenite on RTE cells

expressed as RTFs. At the two concentrations tested (experiment A and B, 2.5 x 10~4 M; experiment C, 5.0 X 1(T4 M), NNK increased the transformation frequency 3- to 8-fold over controls. In nine out of nine groups Na2SeO3 significantly inhibited the transforming effect of NNK as indicated by a 4 0 - 6 0 % reduction of RTF in the NNK-Na 2 SeO 3 treated cultures as compared with that in cultures exposed to NNK only. The inhibitory effect was seen with nontoxic as well as toxic concentrations of Na2SeO3, and was independent of the CFE in the untreated cultures (results not shown). To determine the effects of sequential exposure, NNK exposure followed by N^SeC^ treatment, cultures were treated for 1 week with NNK and then, after changing the media, to Na2SeO3 for 4 weeks (with medium change weekly), i.e. during the selection period. Results summarized in Table II demonstrate that the post-treatment of Na2SeO3 effectively inhibited NNK-induced cell transformation by ~60%.

Table I. Effect of Na2SeO3 on spontaneous cell transformation




2.75 1.85

1.65* 0.15**

0.48 0.07

6.0 x 10~7

1.24 0.84 0.75 1.00 1.36 0.84 0.56 2.75 0.33 1.95 0.33 1.14 1.85

0.05* 0.05* 0.70 0.57* 0.55** 0.05** 1.05 1.45** 0.26 1.45 0.28 0.54** 0.30**

0.07 0.06 1.33 0.36 0.57 0.04 1.50 0.43 0.64 0.52 0.75 0.51 0.12

6.0 x 10" 6

0.41 1.85 0.70 2.76 0.06

0.09* 0.65** 1.05 2.56 0.12

0.15 0.30 1.21 0.75 2.00

6.0 x 10"




(M) Fig. 3. Effect of Na2SeO3 on colony size (cell number per colony). • , mean ± SE of three experiments in which the CFEs in untreated cultures were high (4.8 ± 0.4%); cells per colony in control: 165 ± 3 (mean ± SE). • , mean ± SE of three experiments in which the CFEs in untreated cultures were low (2.2 ± 0.1%); cells per colony in control: 121 ± 14 (mean ± SE). Compared with untreated cultures, *P < 0.05, **P < 0.001.



Na2SeO3 (M)

"Based on 2 0 - 2 8 dishes per group. *P < 0.05, **P < 0.001 compared with controls.


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Discussion It is well established and has been demonstrated in a number of tumor induction models that selenium compounds can inhibit the initiation as well as the promotion phases of carcinogenesis (9,33). Inhibition of initiation of carcinogenesis by selenium compounds may at least in part, be mediated through an increase in antioxidant activity of glutathione peroxidase, a selenoenzyme. Inhibition of the promotion phase of carcinogenesis by selenium compounds remains entirely unexplained. Cell culture studies with mammary and also prostate epithelial cells have shown that low concentrations of selenium in the range of 10~8 M can stimulate growth of normal, preneoplastic and neoplastic cells. In contrast, high concentrations of 10 —10~4 M selenium were found to be growth inhibitory for normal and in some cases also for tumor cells (23—28). Our interest in examining the effects of Na2SeO3 on transformation of RTE cells was spurred by a review of the literature which indicated, that the tracheo-bronchial epithelium may possibly be one of the few tissues which does not benefit from the antineoplastic effects of selenium (17,18).

We therefore decided to examine the effect of Na2SeOj on cell proliferation and transformation of rat tracheal epithelium using the RTE cell culture system. While quite a number of studies are reported in the literature describing the effect of selenium compounds on proliferation of normal, preneoplastic and neoplastic cells of various origins, surprisingly few experiments are reported on inhibition of the transformation process itself. One of the notable exceptions is the study by Chatterjee and Banerjee (34). These investigators described that selenium inhibits the induction of hyperplastic alveolar nodules in cultured mouse mammary glands (which are thought to be preneoplastic lesions) by dimethylbenz[a]anthracene at selenium concentrations of 10~ 6 -10" 5 M, but increases the incidence of such nodules at concentrations of 10~8 —10~7 M. We found, as has been reported by other investigators working with different cell culture systems (23-28), that sodium selenite has a biphasic effect on cell proliferation. Concentrations of 6 x 10~8—10~6 M increased colony formation of primary RTE cells; concentrations of 1.8 x 10~5 M and above decreased colony formation (which is a measure of proliferation activity). This biphasic effect of Na2SeO3 on cell proliferation was also indicated by the measurement of the colony size (cell number per colony). Interestingly, the enhancement effect on colony formation as well as on colony size by low concentrations of Na2SeO3 was most pronounced when the CFE of untreated cultures was low, i.e. 2.0-2.5% or less. In contrast, the growth inhibitory effect of high concentrations of Na2SeOj was independent of the CFE in control cultures. Since we do not know what causes the fluctuation in CFE in the RTE cell system we hesitate to speculate why the growth stimulatory effect of selenium is so much more pronounced when growth of the cultures is poor. Rat tracheal cell cultures can spontaneously undergo transformation. The frequency of spontaneous cell transformation varies considerably from experiment to experiment. Na2SeO3 inhibited

S.Zhu.T.E.Gray and P.Nettesbdni

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Fig. 4. Relative transformation frequencies in cultures co-treated with NNK and Na2SeO3. NNK concentrations used: 2.5 x 10~4 M (experiments A and B); 5 x 10~ 4 M (experiment C) Na2SeO3 concentrations used: (1), 6 x 10" 7 M, (2), 1.2 x 1(T 5 M, (3), 3 x 1CT5 M. The spontaneous transformation frequencies in experiments A, B and C were 0.18, 0.85 and 0.28% respectively.

Table n. Effect of Na2SeO3 post-treatment on NNK-induced cell transformation NNK (M)

Na2SeO3 (M)

Transformants per culture


0 0 5.0 x 10" 4 5.0 x 10" 4

0 6.0 x 10~7 0 6.0 x 10~ 7

0.15 0.14 0.59 0.21*

1.00 1.00 5.00 1.75

•Significantly lower than cultures exposed to NNK alone (P < 0.01).

spontaneous transformation reproducibly, but only at low concentrations, i.e. < 6 X 10" 6 M. At these levels, Na 2 Se0 3 stimulated growth of RTE cells (see Figure 1). Na2SeO3 concentrations which inhibited growth (3 X 10~5 M) did not inhibit spontaneous transformation. In contrast, NNK-induced transformation was inhibited by both low and high concentrations of Na2SeC>3. In the studies reported here, Na2SeO3 had two effects on RTE cell cultures. First, at low concentrations it stimulated cell growth, particularly in cultures with low CFE; and second, it inhibited transformation of RTE cells. This occurred regardless of whether the cultures had a high or low CFE. Taken together this suggests that the inhibition of transfor1728

In the present study, sodium selenite exhibited its suppressing effect on NNK-induced cell transformation not only when it was added to the cultures simultaneously with NNK (co-treatment), but also when it was added to the medium after cessation of NNK exposure (post-treatment). Thus selenium was able to inhibit chemically induced cell transformation both, during the initiation as well as the expression stages. In in vivo studies it has been shown that selenium inhibits the initiation as well as the promotion of mammary carcinogenesis (9,33). The transformed colonies in the RTE cell system, also referred to by us as EGV, were previously shown to be preneoplastic (for review see ref. 19). These EGV colonies are not as yet neoplastic, however upon subculturing they become highly tumorigenic (37). Thus the data presented in this report indicate that selenium inhibits an early stage of neoplastic transformation of RTE cells. In summary, our studies showed that sodium selenite has biphasic effects on cell proliferation and inhibits both spontaneous and chemically induced in vitro transformation of RTE cells. The mechanisms by which it does so are entirely unclear. Resolving this enigma presents a challenging task for future investigations.

References l.Ganther.H.E., Hafeman.D.G., Lawrence, R. A., Serfass.R.E., and Hoekstra.W.G. (1976) Selenium and glutathione peroxidase in health and disease—a review. In Prasa^VS. (ed.), Truce Elements in Human Health and Disease, New York, Academic Press, Inc, pp. 165-234. 2. McKeehan.W.L., HamUton.W.G. and Ham.R.G. (1976) Selenium is an essential trace nutrient for growth of Wl-38 diploid human fibroblasts. Proc. Natl. Acad. Sd. USA, 73, 2023-2027. 3. Clayton.C.C. and Baumann.C.A. (1949) Diet and azo dye tumors: Effect of diet during a period when the dye is not fed. Cancer Res., 9, 575-582.

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mation of RTE cells by sodium selenite is independent of its effects on cell proliferation. These findings pose several intriguing questions none of which we are currently able to resolve. First of all, why does a selenium concentration, which increases cell growth, inhibit cell transformation, rather than enhancing it? A great deal of evidence has accumulated over the years indicating that increased cell proliferation during the initiation phase increases the probability of neoplastic transformation, and the promotion phase is virtually defined as a phase of proliferative expansion of clones of initiated cells. The awareness of the importance of cell proliferation in carcinogenesis has been heightened by a recent, vigorous debate (35,36). Secondly, why does a high selenium concentration inhibit NNK-induced transformation, but not spontaneous transformation, while low selenium concentrations inhibit both? One possible explanation is that the effect of high selenium concentrations is exclusively to inhibit the metabolic conversion of the nitrosamine to DNA-damaging metabolites, while low concentrations of selenium interfere not so much with drug metabolism but instead inhibit some cellular process essential for the development of transformed colonies, regardless of whether they are spontaneous or chemically induced. In the in vitro cell transformation study with mouse mammary glands (34) in which selenium was used to inhibit DMBA-induced transformation, it did so at relatively high concentrations of 10~ 6 -10~ 5 M which were growth inhibitory. At 10~8 and 10~7 M, concentrations which are growth stimulatory (see 20-25 and also this study) selenium stimulated the development of presumed preneoplastic mammary nodules. This is in marked contrast to the effects of selenium on RTE cell transformation because in the RTE cell system inhibition of transformation occurred at nontoxic concentrations which are growth stimulatory to normal cells.

Effects of sodium sdenhe on RTE cells 33. Ip.C. and White.G. (1987) Mammary cancer chemoprevention by inorganic and organic selenium: Single agent treatment or in combination with vitamin E and their effects on in vitro immune functions. Carcinogenesis, 8, 1763-1766. 34. Chatterjee.M. and Banerjee.M.R. (1982) Selenium mediated dose-inhibition of 7,12slimethylbenz(a)anthracene-induced transformation of mammary cells in organ culture. Cancer Lett., 17, 187-195. 35. Ames.B.N. and Gold.L.S. (1990) Too many rodent carcinogens. Mhogenesis increases mutagenesis. Science, 249, 970-971. 36. Cohen.S.M. and Ellwein.L.B. (1990) Cell proliferation in carcinogenesis. Science, 249, 1007-1011. 37. Walkcr.C. and Nettesheim.P. (1989) In vitro neoplastic progression of transformants generated by diverse carcinogens. Cancer Res., 49, 4427—4430. Received on April 24, 1992; revised on June 26, 1992; accepted on June 29, 1992

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4. Shamberger.RJ., Tytko.S.A. and Willis.C.E. (1976) Annoxidants and cancer. Arch. Environ. Health, 31, 231-235. 5.Schrauzer,G.N., White,D.A. and Schneider.C.J. (1977) Cancer mortality correlation studies—m. Statistical associations with dietary selenium intakes. Bioinorg. Qxem., 7, 23-34. 6. Stampfer.M.J., Colditz.G.A. and Willett.W.C. (1987) The epidemiology of selenium cancer. Cancer Surv., 6, 623—633. 7. Clark.L.C. (1985) The epidemiology of selenium and cancer. Fed. Proc., 44, 2584-2589. 8. Ip,C (1981) Factors influencing the anticarcinogenic efficacy of selenium on dime«hyibenz(a)anihracene-induced mammary tumorigenesis in rats. Cancer Res., 41, 2683-2686. 9. Ip,C. (1985) Selenium inhibition of chemical carcinogenesis. Fed. Proc., 44, 2573-2578. 10. Medina,D. and Lane.H.W. (1983) Stage specificity of selenium-mediated inhibition of mouse mammary tumorigenesis. Biol. Trace Element Res., 5, 297-306. 11. Griffin.A.C. and Jacobs,M.M. (1977) Effect of selenium on azo hepatocarcinogenesis. Cancer Lett., 3, 177-181. 12. Baldwin.S. and Parker.R.S. (1987) Influence of dietary fat and selenium in initiation and promotion of aflatoxin Bl-induced preneoplastic foci in rat liver. Carcinogenesis, 8, 101 -107. 13.NayinU., El-Bayoumy,K., Sugie.S., Cohen.L.A. and Reddy.B.S. (1989) Chemoprevention of experimental mammary carcinogenesis by the synthetic organoselenium compound, benzylselenicyanate, in rats. Carcinogenesis, 10, 509-512. 14. Stadtman.T.C. (1990) Selenium biochemistry. Annu. Rev. Biochem., 59, 111-127. 15. Schrauzer.G.N. (1977) Trace elements, nutrition and cancer: Perspectives of prevention. Adv. Exp. Med Biol., 91, 323-344. 16. Birt.D.F., Lawson.T.A., Julius.A.D., Runice.C.E. and Salmasi.S. (1982) Inhibition by dietary selenium of colon cancer induced in the rat by bis(2-oxopropyl)nitrosamine. Cancer Res., 42, 4455-4459. 17. Thompson.H.J. and Becci.P.J. (1979) Effect of graded dietary levels of selenium on tracheal carcinomas induced by 1-methyl-l-nitrosourea. Cancer Lett., 7, 215-219. 18. Beems.R.B. (1986) Dietary selenium and benz(a)pyrene-induced respiratory tract tumors in hamsters. Carcinogenesis, 7, 485—489. 19. Ncttesheim.P. and BarretU.C. (1984) Tracheal epithelial cell transformation: A model system for studies on neoplastic progression. CRC Crit. Rev. Toxicol., 12, 215-219. 20. Steele.V.E., AmokU.T. and Mass.M.J. (1988) In vivo and in vitro characteristics of early carcinogen-induced piemalignant phenotypes in cultured rat tracheal epithelial cells. Carcinogenesis, 9, 1121-1127. 21. Steele,V.E., KeUoff.GJ., Willdnson.B.P. and AmokU.T. (1990) Inhibition of transformation in cultured rat tracheal epithelial cells by potential chemopreventive agents. Cancer Res., 50, 2068—2074. 22. Zhu.S.Y., Cunningham.M.L., Gray.T.E. and Nettesheim.P. (1991) Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)l-

The effect of sodium selenite on cell proliferation and transformation of primary rat tracheal epithelial cells.

The effects of sodium selenite (Na2SeO3) on cell proliferation and the development of preneoplastic transformed variants were studied in primary cultu...
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