2 cells

CANCER LETTERS

ELSEVIER

Cancer Letters 103 (1996) 65-69

Arsenic and chromium enhance transformation of bovine papillomavirus DNA-transfected C3WlOT1/2 cells L.A. KowalskW, S.-S. Tsanp*, A.J. Davisona,b2c aB.C. Cancer Research Centre, Epidemiology and Cancer Prevention, 601 West 10th Ave., Vancouver; BC, Canada bBioenergetics Research Laboratory, Simon Fraser University, Bumaby, BC VSA 1S6, Cunada ‘University of Northern B.C., Department of Chemistry, 3333 University Way, Prince George, BC V2N 429, Canada

Received7 September1995;revisionreceived13February1996;accepted14February1996

Abstract

Tumor promoters such as phorbol esters,teleocidin and okadaic acid increasethe numbers of multilayered, transformed foci produced by BPV DNA-transfected C3WlOTU2 cells. We questioned whether arsenic and chromium, which are known human carcinogens also enhance transformation of BPV DNA-transfected C3WlOT1/2 cells. Cr(II1) potassium sulfate at 100pM enhanced transformation by l.Cfold, but Cr(VI) as potassium chromate did not enhance transformation, although toxicity of potassium chromate may have prevented enhancementof transformation. Sodium arsenite (As(II1)) at 5 PM and sodium arsenate(As(V)) at 25 PM both enhancedneoplastic transformation by 6-fold. By comparison, in previous studies, sodium orthovanadate(V(V)) or vanadyl sulfate (V(IV)) at 4pM enhancednumbersof transformedfoci by 25-50-fold. The comparatively strong enhancementof transformation by vanadium and phorbol esterssuggeststhat neoplastic transformation may occur by mechanismsthat are common to thesecompoundsincluding alteration of tyrosine phosphorylation. Keywords:

Arsenic; Chromium; Transformation; Bovine papillomavirus DNA-transfectedC3H/lOT1/2 cells

1. Introduction Carcinogenic metals such as arsenic, chromium and vanadium contaminate the environment and workplace. The interaction between these metals and tumor viruses is a significant health concern. We have previously shown that neoplastic transformation of C3WlOT1/2 cells transfected with BPV DNA is greatly enhanced by tumor promoters such as mezerein [I], teleocidin and okadaic acid [2].

* Corresponding author.

Elsevier Science Ireland Ltd. PII: SO304-3835(96)04189-4

Both sodium orthovanadate (V(V)) and vanadyl sulfate (V(IV)) enhance numbers of transformed foci in BPV DNA-transfected C3WlOT1/2 cells by 2550-fold [3]. This suggests that numbers of foci developed by BPV DNA transfected C3H/lOT1/2 cells may be increased by carcinogenic metals. Both chromium [4] and arsenic [5] are known human carcinogens. Like vanadium, they form oxyanions. Both chromium and arsenic are synergistic with other carcinogens and with transforming viruses. Cr(V1) and As(II1) enhance transformation of Syrian hamster embryo (SHE) cells infected with SA7 virus [6]. Cr(V1) enhances transformation of SHE cells induced

L.A. Kowalski et al. /Cancer Letters 103 (1996) 65-69

66

by benzo[a]pyrene [7]. As(II1) increasesclastogenicity and mutagenicity of DNA cross-linking agents[8] and mutagenicity of ultraviolet light [9,10]. Arsenic also activates Herpes simplex and Herpes zoster [ 111. Taken together, this suggeststhat arsenic and chromium may enhance transformation of BPV DNA transfectedC3WlOT1/2 cells. 2. Methods 2.1. Cell culture and transfection

C3WlOT1/2 mouse embryo fibroblasts from the American Type Culture Collection were grown at 37°C 97% sir/3% C02, in Dulbecco’s modified essential medium (Flow) supplemented with 10% fetal calf serum (Gibco). Medium also contained 20 mM HEPES (N-2-hydroxyethylpiperazine-N’-2ethane-sulfonic acid) (Sigma), 9.96 U/ml of penicillin G (Sigma) and 72Spg/ml. of streptomycin sulfate (Sigma). C3WlOT1/2 cells of passages16-23 were used for our experiments. For each experiment, cells were seeded on to plates of 100 mm diameter. After 48 h, cells were transfected with pdBPV-1 (142-6) containing the complete BPV-1 genome cloned into the plasmid pML2d at the BamHI site, using the method described previously [l]. Controls were transfected with only sheared calf thymus DNA (Sigma) [12]. After transfection, medium was removed, cultures were exposed to 15% glycerol in HBS buffer for 2 min, washed twice with medium, then left overnight in fresh medium as described previously [l]. On the following day, cells were subcultured at a dilution of 1:17 and mezerein, chromium or arsenic compounds were added. Cultures were incubated for 21 days continuously exposed to arsenic, chromium or mezerein (as a positive control) with twice-weekly changesof medium. After each experiment, cultures were stained for l-2 h with 0.025% methylene blue in 50% methanol for visualization of foci. Plates were air-dried and foci scored.

100 mM stock solutions in glass distilled water. Mezerein (Sigma) was dissolved in dimethylsulfoxide at a concentration of 0.2 mg/ml and diluted with medium. Controls received 0.5 rig/ml of mezerein for 21 days. 2.3. Determinations of toxicity

To determine toxicity of chromium or arsenic compoundsto transfectedcells, C3WlOT1/2 cells of passage 16 were transfected with BPV DNA. One day later, 200 cells were seededinto 60 mm dishes in normal medium without arsenic or chromium. Twenty-four hours after seeding 200 cells, various concentrations of test compounds were added to the medium. Cultures were incubated for a further 8 days, with two changes of medium, continuously exposed to test compounds. Cultures were then stained, dried, and colonies containing more than 25 cells counted. 3. Results

Sodium arsenite (As(II1)) stimulated a dosedependent increase in transformed foci to 6-fold at 5pM (Figs. 1 and 2). In an assessmentof toxicity, percent colony forming efficiency decreasedsharply above 7pM (Table 1). Sodium arsenate(As(V)) increased numbers of transformed foci to 6-fold at 25,uM (Figs. 2 and 3). Percent colony forming efficiency decreasedsharply above 25 PM (Table 1). Chromium potassium sulfate (Cr(II1)) at 202OOpM enhanced numbers of transformed foci by 1.6fold. (Table 2). Percent colony forming efficiency was not altered by concentrations of Cr(II1) compounds up to 250pM (Table 3) (P > 0.01). Potassium chromate (Cr(V1)) up to 2,uM did not enhance focus formation (Table 2) (P c 0.05). However, percent colony forming efficiency was significantly decreasedat 0.1 PM and fell abruptly to 0% at 2pM (Table 3) (P < 0.05). Potassium chromate is clearly very toxic to cells. Therefore, toxicity may have masked enhancementof transformation by potassiumchromate.

2.2. Addition of compounds 4. Discussion

K2Cr04, NaAsO*, and Na2HAs04*7H20were prepared as 10 or 100 mM stock solutions in glass distilled

water. CrK(S04)12H20

was prepared as a

Chromium compounds enhance transformation of BPV DNA transfected C!3H/lOT1/2 cells but not to

67

LA. Kowalski et al. / Cancer L-ettersIO3 (I 9%) 65-69 18 16 ‘G P 14 6 & 12 2 10 2 i% 8 E F 6 a 4

0 0123456 Arsenite concentration

-

BPV DNA

t

(Micromolar)

CT DNA only

Fig. 1. Average number of foci as a function of arsenitc concentration. Cells at passage 17 were seededat a density of 2.2 X lo5 cells/100 mm plate and transfectcd with 1pg of BPV DNA/plate. They were subculturcd 21 h later at 1:17, then exposed continuously to arsenite for 21 days. Cells transfectedwith BPV DNA but not exposed to arsenitc developed an average of 2.7 foci/60 mm plate. Cells exposed continuously to 0.5 ngM of mezerein developed an average of 91 foci/60 mm plate. Values were determined in triplicate. Error bars arc standarderrors of the mean. P < 0.001 by analysis of variance.

the same extent as vanadium or mezerein. Cr(II1) at 1OOpM enhancedtransformation by 1.4-fold. Cr(VI) up to 1PM did not enhancetransformation, although this may have been due to toxicity. In other studies, the effects of chromium compounds on cell transformation varied depending upon types of cells. Cr(V1) at 2.6pM increased transformation frequency of SHE cells initiated by benzo[a]pyrene, but 3pM Cr(II1) was ineffective [7]. Cr(V1) at 10pM increased transformation of SHE cells infected with SA7 [6]. Conversely, Cr(V1) at 0.5 PM did not transform (untransfected) C3H/lOT1/2 cells [13]. Our observations are generally consistent with these previous studies. Nonetheless, the biological significance of such a modest enhancementof transformation is open to question. As(V) at 25 PM increasednumbersof transformed foci by 6-fold. In previous studies, As(V) at 75,~M produced a 10% increase in transformation of SHE cells [ 141. As(V) at 1OOpM transformed SHE cells infected with Simian adenovirus 7 [6]. In our studies,

As(II1) at 1PM enhanced transformation by 6-fold. As(II1) at 4,uM enhanced transformation of SHE cells by 10% [31]. As(II1) at 3pM enhanced transformation of SHE cells infected with Simian adenovirus 7 [6]. Therefore, enhancement of numbers of transformed foci in BPV DNA-transfected C3WlOT1/2 cells by As(V) and As(II1) is consistent with enhancement of transformation in previous studies. Enhancementof transformation was significantly different for vanadium (25-50-fold), arsenic (6-fold) and chromium (1.4-fold). There are at least four possible hypothesesfor the difference in strength of enhancement:differential pcrmeabilities of the cells to the metals, differences in metal binding within the cells, differential toxicity to cells, or different mechanismsof interaction with BPV DNA or other cellular processes.Although all three metals form oxyanions, their biochemical effects are very differa

0

1 PM

0.1 pM

1PM

5PM

b

5uM

Fig. 2. Enhancementof numbersof foci in BPV DNA transfected C3B/lOT1/2 cells exposed continuously to various concentrations of (a) arseniteand (b) arsenatefor 21 days.

L.A. Kowalski et al. /Cancer Letters 103 (1996) 65-69

68

Table 2 Numbers of foci formed by BPV DNA-transfected C3WlOT112 cells exposed for 21 days to various concentrations of chromium compounds CrK(SO&

-0 5 10 15 20 25 30 Arsenate concentration (Micromolar)

/--t-BPV

DNA

t

CT DNA only

Fig. 3. Average number of foci as a function of arsenate concentration. Cells at passage 17 were seeded at a density of 2.2 x lo5 cells/100 mm plate and transfected with 1 pg of BPV DNA /plate. They were subcultured 21 h later at 1:17, then exposed continuously to arsenate for 21 days. Cells transfected with BPV DNA but not exposed to arsenate developed an average of 2.7 foci/60 mm plate. Cells exposed continuously to 0.5 r&ml of mezerein developed an average of 91 foci/60 mm plate. Values were determined in triplicate. Error bars are standard errors of the mean. Pi 0.001 by analysis of variance.

K2Cr04

yM cont.

Foci

0 20 100 200

13? 16 f 19 + 19*

I 0.3 0.3 1

pM cont.

Foci

0 0.5 I 2

135 1 llt2 9&l 321

Positive controls of transfected cells exposed to 0.5 rig/ml of mezerein developed an average of 52 foci per 60 mm dish. Controls transfected only with carrier DNA (calf thymus DNA) and exposed to various concentrations of CrK(SO& and K$rO4 developed a maximum of one focus. The experiment was done three times, each time in triplicate with similar results. Values reported are averages of 3 plates with standard deviations. P < 0.05, determined by t-tests for all values as compared with untreated controls except for 20yM Cr(lI1) and 0.5 PM Cr (VI) for which P > 0.05.

ent. Vanadium has wide-ranging effects on tyrosine phosphorylation [ 15-171 and generates active oxygen species [ 18,191. Arsenic inhibits DNA repair and

causes endoreduplication [8,20,21]. Among its many other effects, chromium binds to DNA [22] and also generates active oxygen species [23]. We are investigating the mechanisms of interaction between these metals and BPV DNA in the enhancement of BPV DNA mediated transformation. The comparatively strong enhancement of transformation by vanadium points to a mechanism involving alteration of tyrosine phosphorylation.

Table 1

Table 3

Number of colonies formed by 200 BPV DNA-transfected C3WlOT1/2 cells exposed to arsenic for 8 days

Number of colonies formed by 200 BPV DNA-transfected C3WlOT112 cells exposed to chromium for 8 days

NaAsO,

CrK(S04)

PM cont.

Colonies

0 0.1 1 5 IO

55lt.2 52 -c 10 55 f 2 42 f 4 S+l

0 5 10 25 50

K2Cr04

Colonies

yM cont.

Colonies

PM

Colonies

55 + 2 58 f 10 55 f 3 45 f 3 5+2

0 50 100 200 250

55 + 2 56 + 2 54 f. 5 52 + 10 53 +7

0 0.1 0.5 I 2

55 + 2 42 f 5 40* 10 16+2 0

Toxicity experiments were done twice, each time in triplicate. Values reported are averages of 4-6 plates with standard errors of the mean. Toxicity for arsenite is significant above 5 PM, P < 0.05 and for arsenate above 25pM, P < 0.01, determined by Itests.

Toxicity experiments were done twice, each time in triplicate. Values reported are averages of 4-6 plates with standard errors of the mean. Toxicity of Cr(II1) was not significant (P > 0.05) for 250pM and toxicity of Cr(V1) was significant at 20.1 PM (P < O.OS),both determined by f-tests.

L.A. Kowalski et al. /Cancer Letters 103 (1996) 65-69

References [1] Tsang, S.-S. and Stich, H.F. (1988) Enhancement of bovine papillomavirus-induced cell transformation by tumor promoters. Cancer Lett., 43.93-98. [2] Tsang, S.-S., Stich, H.F. and Fujiki, H. (1991) Cell transformation induced by bovine papillomavirus DNA as an assay for tumor promoters and chemopreventive agents. Cancer Det. Prev., 15.423-428. [3] Kowalski, L.A., Tsang, S.-S. and Davison, A.J. (1994) Ascorbate and retinol suppress BPV DNA-mediated transformation of C3WlOTl12 cells. Carcinogenesis, submitted. [4] World Health Organization, International Agency for Research on Cancer (1980) Some metal and metallic compounds. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, 23,205-323. [5] World Health Organization, International Agency for Research on Cancer (1980) Some metal and metallic compounds. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, 23.39-141. [6] Caste, B.C., Meyer, J. and Dipaolo, J.A. (1979) Enhancement of viral transformation for evaluation of the carcinogenic or mutagenic potential of inorganic metal salts. Cancer Res., 39, 193-198. [7] Rivedal, E. and Sanner, T. (1981) Metal salts as promoters of in vitro morphological transformation of hamster embryo cells initiated by benzo(a)pytene. Cancer Res., 41, 295t& 2953. [8] Landolph, J.R. (1989) Molecular and cellular mechanisms of transformation of C3H/lOTl/2 Cl 8 and diploid human tibroblasts by unique carcinogenic, nonmutagenic metal compounds. A review. Biol. Trace Elem. Res., 21,459467. [9] Rossman, T.G. (1981) Enhancement of UV mutagenesis by low concentrations of arsenite in E. coli. Mutat. Res., 91, 207. [IO] Lee, T.C., Huang, R.Y. and Jan, K.Y. (1985) Sodium arsenite enhances the cytotoxicity, clastogenicity and 6thioguanidine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells. Mutat. Res., 148, 83. [l 11 Stohrer, G. (1991) Arsenic: opportunity for risk assessment. Arch. Toxicol., 65, 525-531. [12] Zhou, R.-P., Kan, N. Papas, T. and Duesberg, P. (1985) Mutagenesis of avian carcinoma virus MH2: only one of two potential transforming genes @gag-myc) transforms fibroblasts. Proc. Natl. Acad. Sci. USA, 82, 6389-6393.

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[13] Patierno, S.R., Banh, D. and Landolph, J. (1988) Transformation of C3WlOTl12 mouse embryo cells to focus formation and anchorage independence by insoluble lead chromate but not soluble calcium chromate: relationship to mutagenesis and internalization of lead chromate particles. Cancer Res., 48.5280-5288. [14] Lee, T.C., Oshimura, M. and Barrett, J.C. (1985) Comparison of arsenic-induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis (London), 6, 1421-1426. [15] Yang, DC., Brown, A.B. and Chan, T.M. (1989) Stimulation of tyrosine-specific protein phosphorylation and phosphatidylinositol phosphorylation by orthovanadate in rat liver plasma membrane. Arch. Biochem. Biophys., 274, 659-662. [16] Tracey, A.S. and Gresser, M.J. (1986) Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on systems regulated by tyrosine phosphorylation. Proc. Nat]. Acad. Sci. USA, 83.609613. [17] Swamp, G., Cohen, S. and Garbers, D.L. (1982) Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem. Biophys. Res. Commun., 107, 1104 1109. [I81 Ramasarma, T., MacKellar, W.C. and Crane, F.L. (1981) Vanadate-stimulated NADH oxidation in plasma membrane. Biochim. Biophys. Acta, 646,88-98. [19] Liochev, S.I. and Ivancheva, E.A. (1988) A study on the mechanism of the vanadate-dependent NADH oxidation. Free Rad. Biol. Med., 5, 349-354. [20] Okui, T. and Fujiwara, Y. (1986) Inhibition of human excision DNA repair by inorganic arsenic and comutagenic effect in V79 Chinese hamster cells. Mutat. Res., 172,69. [21] Snyder, R.D., Davis, G.F. and Lachmann, P.L. (1989) Inhibition by metals of x-ray and ultraviolet-induced DNA repair in human cells. Biol. Trace Elem. Res., 21,389. [22] Bridgewater, L.C., Manning, F.C.R., Woo, E.S. and Patiemo, S.R. (1994) DNA polymerase arrest by adducted trivalent chromium. Mol. Carcinogen,, 9, 122-133. [23] Shi, L., Dalal, N.S. and Vallyathan, V. (1991) One electron reduction of carcinogen chromate by microsomes, mitochondria, and Escherichia coli: identification of Cr(V) and OH radical. Arch. Bioch. Biophys., 287, 381-386.