Comparison of DNA Lesions and Cytotoxicity Induced by Calcium Chromate in Human, Mouse, and Hamster Cell Lines1

[CANCER RESEARCH 46, 4547-4551, September 1986) Comparison of DNA Lesions and Cytotoxicity Induced by Calcium Chromate in Human, Mouse, and Hamster Cell Lines1

Masayasu Sugiyama,2 Xin-Wei Wang,3 and Max Costa3'4

Department of Pharmacology; University of Texas Medical School at Houston, Houston, Texas 77025

ABSTRACT are significant differences in the toxic action of metal ions such as chromate among cells originating from different species. DNA lesions, cytotoxicity, and cellular uptake of CaCrO4 were com In the present study we have compared the cytotoxicity, pared in Chinese hamster ovary, in mouse embryo fibroblast C3H10T'/2, cellular uptake, and DNA damage induced by CaCrO4 in hu and in human osteosarcoma cells. The concentration of CaCrO4 that reduced colony formation by 50% was 2- or 3-fold less in human osteo man, mouse, and hamster cell lines, utilizing the alkaline elution sarcoma cells than in C3H 10T'/2 or Chinese hamster ovary cells. Alkaline technique. Our findings illustrate that the human cells were the elution studies showed that CaCrO4 induced DNA single strand breaks most sensitive of the cell lines examined in terms of induction in a concentration dependent manner in all three cell lines. However, the of DNA damage and cytotoxicity. These results are explained, human cells exhibited four times more breaks than the hamster cells and in part, by the greater uptake of chromate in the human cell two times more than ( 311 III I ' cells when the CaCrO4 exposure condi lines, but these uptake studies cannot entirely explain the tions were equivalent. Alkaline elution studies also demonstrated the differences observed. formation of DNA-protein cross-links by CaCrO4 in all three cell lines. In hamster and mouse cells the induction of these DNA-protein cross links was dependent on concentrations that ranged from 5 to 50 >tM for MATERIALS AND METHODS 6 h; however, the cross-links were saturated at 25 n\i in human cells and at 50 MMin mouse and hamster cells. The level of cross-links was four Chemicals. The radioisotope [14C]deoxythymidine (58 mCi/mmol) times greater in the human cells compared to the mouse cells and was a and Na2Cr5'O4 were obtained from New England Nuclear (Boston, factor of 2 greater in the hamster cells compared to the mouse cells. The MA) and ICN Chemical Co. (Irvine, CA), respectively. Free acid EDTA, uptake of CaCrO4 was linear with respect to time and concentration. and sodium dodecyl sulfate were acquired from Sigma Chemical Co. Uptake of CaCrO4 was equivalent in the human and mouse cells, but was (St. Louis, MO). Tetrapropyl ammonium hydroxide (10% aqueous a factor of 4 less in the hamster cells. The single strand breaks were solution) was purchased from RSA Chemical Co. (Ardsley, NY). Poly almost entirely repaired after an 18-h incubation in metal free medium carbonate filters were from Nucleopore (Pleasanton, CA), and polyvinyl in all three cell lines, whereas DNA-protein cross-links persisted in these chloride filters were from Millipore Corp. (Bedford, MA). Proteinase cell lines in proportion to their initial levels. These results demonstrate K was obtained from EM Laboratories, Inc. (Elmsford, NY). Bovine differences in the sensitivity of human, hamster, and mouse cells to serum and a-minimal essential medium were from Hazleton, Inc. CaCrO4 and suggest that the repair-resistant DNA-protein cross-link (Denver, PA), and trypsin was obtained from Gibco, Inc. (Grand Island, may be important in mediating the long term toxic and carcinogenic NY). Liquiscint was acquired from National Diagnostics, Inc. (Som- effects of CaCrO4. merville, NJ). Cell Culture. The AA8 CHO5,1 OT'/2,and HOS cells were maintained in a humidified atmosphere of 95% air-5% CO2 at 37Â°Cin Â«-minimal INTRODUCTION essential medium supplemented with 10% fetal bovine serum and a 1% solution of penicillin-streptomycin- Fungizone (Gibco). lOTVi or HOS Chromium compounds have been shown to cause tumors and cells used in all experiments were at passages 12-19 or 49-59, respec to produce a number of toxic effects in both humans and tively. HOS cells were a generous gift from Dr. D. Blair (National animals (1-3). Chromate potently induces transformation and Cancer Institute, Frederick, MD). mutation in cultured mammalian cells of hamster (4-6) and Alkaline Elution. The alkaline elution technique for analysis of DNA human (7) origin. Chromium compounds have been shown to lesions was performed as described by Kohn et al. (22) with minor induce sister chromatid exchanges and chromosomal aberration modifications. Cells were seeded into 100-mm diameter tissue culture in cell lines of human (7-10), mouse (11), and hamster (5, 6, 9, dishes and incubated for 48 h with [14C]thymidine (0.02 ^Ci/ml). 10, 12-15) origin. Logarithmically growing cells were washed and incubated in complete In cultures of human (9, 16-18), as well as mouse (17), and growth medium in the absence of radioactivity for 2 h prior to treatment hamster cells (19-21), the carcinogen chromate induced DNA with CaCrO4. Cells were treated with CaCrO4 in complete growth single strand breaks and DNA-protein cross-links. However, medium or SGM consisting of 50 mM 4-(2-hydroxyethyl)-I-piperazineethanesulfonic acid buffer at pH 7.2 with (mM) NaCl, 100; KCI, 5; there have been only a few studies that compared the toxic CaCI2, 2; and glucose, 5. Following treatment, cells were washed twice effects of chromium in cultured cells originating from different and removed from the monolayer by scraping with a rubber policeman species (9, 22). In fact, these studies have illustrated that chro into ice cold Puck's saline A (5 mM NaHCO3-6 mM glucose-5 mM KC1- mate was more toxic to the human cells than to the hamster 140 mM NaCl, pH 7.2). At this stage the cytotoxicity was assessed by cells. Therefore, it is important to further assess whether there trypan blue exclusion. To study DNA damage by alkaline elution a volume of 4-5 ml containing 0.5-1.0 x IO6cells was deposited onto 25- Received 3/4/86; revised 5/21/86; accepted 5/30/86. mm polycarbonate filters (when DNA was assayed for strand breaks) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in or polyvinyl chloride filters (when DNA was assayed for cross-links). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Filters were rinsed with ice cold saline A and the cells were then lysed 1This work was supported by Grant CA 29581 from the National Cancer directly onto the filters by passage of 5 ml of 2% sodium dodecyl sulfate Institute and by Grant R812127 from the United States Environmental Protection solution containing 0.025 M EDTA. DNA was eluted at a flow rate of Agency. 2 Present address: Department of Medical Biochemistry, Kurume University 0.028 ml/min with 25 ml of a solution that contained both 0.025 M School of Medicine, 67 Asahi-machi. Kurume 830. Japan. EDTA and 2% tetrapropyl ammonium hydroxide (pH 12.15). Fractions 3 Present address: Department of Environmental Medicine, New York Uni versity Medical Center, 550 First Avenue, New York, NY 10016. 'The abbreviations used are: CHO. Chinese hamster ovary; lOT'/z, mouse 4 To whom requests for reprints should be addressed, at Department of embryo fibroblast C3H lOT'/z; HOS, human osteosarcoma; SGM, salts/glucose Environmental Medicine, NYU Medical Center, 550 First Avenue, New York, medium; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SSF, strand NY 10016. scission factor; CLF, cross-link factor. 4547

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. DNA DAMAGE INDUCED BY CaCrO4 of approximately 2.5 ml were collected and radioactivity of these tration increased there was more strand breakage. In agreement fractions was determined by scintillation counting after the addition of with the results shown in Fig. 1, HOS cells exhibited the most 7 ml of Liquiscint and 0.7 ml of glacial acetic acid. The filters were breaks at all concentrations, while 10T'/2 cells were intermedi digested for l h at 60"C in 0.4 ml of l N HC1 and neutralized with 2.5 ml of 0.4 N NaOH at 25Â°Cfor 1 h. Radioactivity in the filters was then ate and CHO cells were the least sensitive. Assessment of DNA-Protein Cross-Links in Various Cell determined by scintillation counting in 10 ml of Liquiscint containing 1.0 ml of glacial acetic acid. The degree and nature of DNA cross-links Lines. Fig. 3 shows the concentration-dependent effects of were assessed by utilizing a test dose of X-rays (300 rails) and proteinase CaCrO4 on the induction of DNA-protein cross-links in CHO K as previously described (15). To quantitate the extent of DNA single cells which is measured by the increased retention of DNA strand breaks and DNA-protein cross-links, the strand scission factor from cultures treated with CaCrO4 and irradiated with a test (SSF) and cross-link factor (CLF) were calculated from the alkaline dose of 300 rads. Since the treatment of all cell lysates with elution patterns according to the following relationship: SSF = log . I proteinase K totally reversed the observed cross-links, it was B, CLF = [log A - log AA"]/[logA - log BX], where A is the amount of presumed that the increased retention of DNA was due to DNA retained in the sixth fraction of untreated sample and B is the DNA-protein cross-links and not to DNA-DNA cross-links DNA retained in the sixth fraction of the CaCrO4-treated samples; AX (not shown). CHO and lOT'/z cells had increased DNA-protein is the amount of DNA retained in the sixth fraction of untreated X cross-links when the concentration of CaCrO4 was raised to 50 irradiated cells, and li.\ is the amount of DNA retained in the sixth fraction of CaCrO4-treated X-irradiated cells (23). UM; however, these cross-links reached a maximum in HOS Colony-forming Assay. Following treatment with CaCrO4, cells were cells at 25 /Â¿M,whileCHO and lOT'/z cells required 50 /J.Mto rinsed twice with fresh medium and then trypsinized. Cell number was attain a maximal level of cross-links. At equivalent exposure determined with a Coulter particle counter or a hemocytometer. An conditions, CaCrO4 induced more DNA-protein cross-links in appropriate number of cells (200-1200) were then plated into culture CHO cells than in 10T'/2 cells (Fig. 3). The HOS cells exhibited dishes and allowed to form colonies. Cells were fixed with 95% ethanol the most sensitivity in the CaCrO4 induction of DNA-protein and stained with a 0.2% crystal violet solution. The number of surviving cross-links (Fig. 3). colonies (>50 cells) in each plate was expressed as a function of the Uptake of ('hnmiatc in Various Cell Lines. In an attempt to original number of cells plated. This survival was then compared to understand the differences in the effects of CaCrO4 among the that of untreated cells. Plating efficiencies for untreated CHO cells ranged from 95 to 105%, while normal 10T'/2 cells had plating efficien cell lines, the uptake of this metal was examined. Fig. 4 shows cies ranging from 11 to 19%, and HOS cells plated with an efficiency that the uptake of chromate increased linearly with treatment of 28-32%. time (1-24 h) as well as concentration (25 and 50 pM). With Cellular Uptake of "Cr. Cells were incubated with 2 nCi of 51Cr at respect to time and concentration the uptake of chromate was various concentrations, then washed twice with SGM, and dislodged the least in CHO cells but was of an equivalent level in 10T'/2 from the dishes by scraping. The cell number was determined, and the and HOS cells. Table 1 shows the cellular uptake of chromate cellular uptake of s'Cr was estimated by the radioactivity detected in a was two to three times greater in SGM than in complete tissue Beckman gamma counter. culture growth medium. Cytotoxicity of CaCrO4 in Various Cell Lines. Fig. 5 examines the survival of the three cell lines treated with various concen RESULTS trations of CaCrO4 for 6 h in complete tissue culture medium. Measurement of DNA Single Strand Breaks in Various Cell HOS cells were the most sensitive to CaCrO4, while 10T'/2 cells Lines. Logarithmically growing CHO, lOT'/z, and HOS cells were intermediate, and CHO cells were the least sensitive. A were treated with CaCrO4, and DNA single strand breaks were 50% survival level was found for HOS, lOT'/z, and CHO cells measured by the alkaline elution method. Fig. 1 shows that at CaCrO4 concentrations of 15, 30, and 45 fiM, respectively. CaCrO4 induced different amounts of DNA single strand breaks There were no colonies formed in HOS cells 10 to 12 days in each of the three cell lines after they had been exposed to following treatment with 50 n\t CaCrO4. The viability of CaCrO4 in either a SGM or complete culture medium. At CaCrO4 treated or untreated cells was equivalent based upon equivalent CaCrO4 exposure concentration HOS cells exhibited trypan blue exclusion studies. There was also initially little a greater degree of strand breakage based upon the elution effect of CaCrO4 on cell numbers even at levels that strikingly kinetics illustrated in Fig. 1. 10T'/2 cells were intermediate reduced plating efficiency (i.e., 50 (iM CaCrO4 in HOS cells for while CHO cells exhibited the least sensitivity (Fig. 1). Fig. 2 6h). shows the effect of concentration on the induction of strand Repair of DNA Lesions in Various Cell Lines. Fig. 6 illustrates breaks by CaCrO4 in the CHO cells. As the exposure concen the repair of single strand breaks and DNA-protein cross-links

â€”â€¢â€”-.____untreated

CHO

Fig. 1. Analysis by alkaline elution of DNA single strand breaks induced in various cell lines treated with CaCrO4. HOS, lOT'/j, and CHO cells were treated for 2 h with 50 *JM CaCKX. in SGM (A), or in complete culture medium for 6 h with 50 Â»iMCaCrO4(B), or for 24 h with 25 JIMCaCKX, (Q. Following these exposures, cellular DNA was analyzed by al kaline elution. Prior to elution, cell lysates were incubated with proteinase K to eliminate the retention of DNA caused by the induction of DNA-protein cross-links. 10 15 20 5 10 15 20 15 20

ELUTION VOLUME Imi

4548

HOS

oc O H U 2

g C/1 u O)

TREATMENT TIME (HR) Fig. 4. Cellular uptake of CaCrO4 in various cell lines. HOS, lOT'/a, and CHO cells were treated in complete tissue culture medium for various time intervals 100 with 25 or 50 ^M CaCrO4. Following treatment, cellular uptake of "Cr was 25 50 75 determined from the radioactivity present in 10" cells. This experiment was CaCr04 luMI repeated one additional time. Fig. 2. Comparison of the effect of CaCrO4 concentration on the extent of DNA single strand breaks in various cell lines. HOS, lOT'/z, and CHO cells were Table 1 Comparison of the uptake of"Cr in various cell lines treated for 6 h with ( 'Â¡i<'r()4 in complete growth medium. Following treatment, Cells were treated with 50 //\i CaCrO4 for 2 h in either complete medium or the cellular DNA was analyzed by alkaline elution as illustrated in Fig. 1. The SGM. Following treatment, the cellular uptake of "Cr was determined. Each DNA single strand breaks induced by CaCrO4 were expressed as strand scission factor. Ban, SD. a, P < 0.01 compared to lOTVz or CHO cells, b, P < 0.05 value shown in the table is the mean of three determinations. compared to CHO cells (Student's / test). Uptake (nmol/10'cells)

HOS IOTV4 CHO Complete growth medium 6.14 5.79 1.83 SGM 11.5 15.43 5.56

100

10 25 50 75

CaCrO4 l>iMI Fig. 3. Comparison of the induction of DNA-protein cross-links by CaCrO4 3 in three cell lines. HOS, lOT'/z, and CHO cells were treated for 6 h in complete M tissue culture growth media with the concentrations of CaCrO4 shown in the figures. Following treatment, DNA-protein cross-links were examined by alkaline 20 elution as described in "Materials and Methods." DNA-protein cross-links were expressed as cross-link factor. Bars, SD. a, P < 0.05 compared to lOT'/z or CHO cells. b,P< 0.05 compared to lOT'A cells, c, P < 0.05 compared to lOT'/i cells.

at 18 h following removal of CaCrO4. Note that essentially all the single strand breaks were repaired during this time interval, 10 whereas the DNA-protein cross-links persisted in proportion 10 20 30 40 50

to their initial levels. In contrast to single strand breaks there CaCr04 luMI was apparently no repair of DNA-protein cross-links. Fig. 5. Effect of CaCKX, on cell survival. HOS, lOT'/i, and CHO cells were treated for 6 h with CaCrO4 in complete growth medium. Following treatment, cells were plated to form colonies for 8-12 days as described in "Materials and DISCUSSION Methods." Bars, SD. Previous studies demonstrated that treatment of cultured mammalian cells with chromium compounds resulted in DNA three different cell lines of human, mouse, and hamster origin. single strand breaks and DNA-protein cross-links but not in CaCrO4 induced a different level of both DNA single strand DNA interstrand cross-links (17, 21). In the present study, the breaks and DNA-protein cross-links in each of these three cell DNA lesions and cytotoxicity of CaCrO4 were compared in lines. 4549

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. DNA DAMAGE INDUCED BY CaCrO4 DNA-protein cross-link levels may be due to such a reductive metabolism whose affinity or capacity might vary with each cell

HOS type. Since there was no survival of HOS cells at 50 J/M CaCrO4, it was possible that this lack of dependency of DNA-protein tr O cross-links on the concentration of CaCrO4 may be due to cell death, as well as to the loss of cells containing DNA cross-links CHO or the limited cellular uptake of chromium at toxic levels. O Z However, since HOS cells treated with CaCrO4 exhibited linear -i M 10TJ uptake of chromate at 25 and 50 UMand there was no difference in viability of cells by trypan exclusion test or in cell growth 3) immediately following CaCrO4 exposure, the former possibili ties remain unlikely. It is possible that at the highest concentra tion of chromate used, the DNA or protein may have become saturated with chromium ions, resulting in an absence of reac

10 25 50 tive sites for the formation of DNA-protein cross-links. How ever, there was still a dose dependency in the induction of single CaCrO4 l strand breaks at concentrations where cross-links were satu Fig. 6. Repair of DNA lesions induced in various cell lines by CaCrO4. HOS, rated. Previous studies with CHO cells have also shown that 10T'/2. and CHO cells were treated for 6 h with CaCrO4 in complete growth the formation of DNA-protein cross-links required time, medium. Cellular DNA was analyzed by alkaline elution after an additional 18-h incubation in CaCrO4-free medium. DNA single strand breaks and DNA-protein whereas strand breaks were at a maximal level immediately crosslinks were expressed as strand scission factor and cross-link factor, respec following treatment and could always be directly related to the tively. Bars, SD. a, P < 0.05 compared to 10T'/2 or CHO cells. concentration of CaCrO4 (21). In the present study, the order of sensitivity to DNA-protein cross-links in the cell line exam These findings are consistent with those in the literature ined was not consistent with the sensitivity to the formation of showing that human cells were more sensitive to sister chro- CaCrO4 induced single strand breaks. Other studies have shown in:it id exchange, to DNA single strand breaks, as well as to the that DNA single strand breaks induced by chromium com cytotoxicity when compared with hamster cells (9,22). We have pounds were associated with the cellular levels of glutathione extended these preliminary findings by conducting a more and cytochrome P-450, whereas protein cross-links were not thorough examination of DNA lesions with the alkaline elution dependent upon the levels of these components (26). These technique and by assessing metal uptake as well as cytotoxicity results suggest that DNA-protein cross-links may be formed by in three different cell lines. While single strand breaks and chromium by a different mechanism from that of the single cytotoxicity studies indicated that the following could be ranked strand breaks. Since DNA-protein cross-links were saturated at in decreasing order of sensitivity: (a) HOS, (b) 10T'/2, and (c) different levels in each cell line, it was important to test a CHO cells; the uptake of chromate was equivalent in HOS and number of concentrations when DNA-protein cross-linking ac 10T'/2 cells but was lower in CHO cells. The volume and surface tivity of CaCrO4 was compared in each cell type. area of each of these cells may, partially, explain the observed Recently, we have reported that CaCrO4 induced DNA dam differences in the metal uptake, since HOS cells were smaller age as well as cytotoxicity in CHO cells predominantly during than 10T'/2 cells, but both of these cells were much larger than the early S phase of the cell cycle (21). Transcriptionally active CHO cells. For example a confluent monolayer of CHO cells euchromatic DNA is known to be replicated during early S in a 100-mm tissue culture plate may contain about 20 x IO6 phase. Previous clastogenic studies have also showed that chro cells, while a similarly confluent monolayer plate of HOS cells mium compounds induced the highest frequency of sister chro- could contain 10-15 x IO6, and the maximal concentration of matid exchanges during the early S phase of the cell cycle of 10T'/2 cells that a 100-mm plate would hold is 4-6 x IO6cells. human cells (8). Sen and Costa (15) have shown that a majority Differences in the size of cells is also obvious at the level of of CaCrO4 induced sister chromatid exchanges were not local light microscopy. A major factor for these differences is prob ized in heterochromatic regions of CHO chromosomes. There ably related to spreading of the cells onto the monolayer and fore, it was possible that the sites of chromium interaction with not so much to large differences in cell volume. The larger more euchromatin may differ in each of the cell types examined. The spread out cells will have a greater surface area for chromate differences in sensitivity to CaCrO4 of the three cell lines exposure and this may have contributed to the amount of examined may also be due to other factors such as their tissue chromate uptake. CaCrO4 was not as readily taken up by the of origin. cells in normal tissue culture growth medium as it was in a The frequency of chromate induced single strand breaks SGM, but this may be due to the inhibitory effects of ligands correlated with its cytotoxicity in each of the three cell lines. such as amino acids that bind chromate and prevent it from However, DNA single strand breaks in all three cell types were entering cells. repaired after removal of CaCrO4. In contrast, DNA-protein It is well known that chromate which contains hexavalent cross-links were not repaired and, in fact, increased in propor chromium can enter the cells by the sulfate transport system, tion to the concentration of CaCrO4. Therefore, cytotoxicity of and it is then reduced to the trivalent form by intracellular CaCrO4 may be associated with onset of repair-resistant DNA- reductive metabolism perhaps involving the microsomal en protein cross-links. zyme system (24, 25). Since in vitro experiments show that only Since the frequency of mutation and transformation is related the trivalent form of chromium can form a stable ternary to DNA damage present during the period of DNA replication, complex with DNA and protein (17, 25), the formation of persistent DNA lesions which are not easily repaired would, DNA-protein cross-links may strongly depend upon this reduc therefore, have the greatest impact in permanently altering tive metabolism. Consequently the differences observed in the DNA structure and function. DNA-protein cross-links in all 4550

11. Umeda, M., and Nishimura, M. Inducibility of chromosomal aberrations by three cell lines resisted repair even at low noncytotoxic concen metal compounds in cultured mammalian cells. MutÃ¢t.Res., 67: 221-229, trations, whereas single strand breaks were repaired at all 1979. concentrations. These findings suggest that the DNA-protein 12. Levis, A. G., and Majone, F. Cytotoxic and clastogenic effects of soluble cross-link is a very important lesion that may permanently alter chromium compounds on mammalian cell cultures. Br. J. Cancer, 40: 523- 533, 1979. the process of genetic transfer. 13. Tsuda, H., and Kalo, K. Potassium dichromate induced chromosome aber rations and its control with sodium in hamster embryonic cells in vitro. Gann, 67:469, 1976. ACKNOWLEDGMENTS 14. Majone, F., and Levis, A. G. Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds. MutÃ¢t.Res., 67: 231-238, 1979. The authors would like to thank Kitty Goldstein for excellent secre 15. Sen, P., and Costa, M. Incidence and localization of sister chromatid ex tarial assistance and Dr. P. Sen for her criticism of this manuscript. changes induced by nickel and chromium compounds. Carcinogenesis, in press, 1986. 16. Whiting, R. F., Stich, H. F., and Koropatnick, D. J. DNA damage and DNA repair in cultured human cells exposed to chromate. Chem.-Biol. Interact., REFERENCES 26:267-280, 1979. 17. Fornace, A. J., Seres, D. S., Lechner, J. F., and Harris, C. C. DNA-protein 1. Leonard, A., and Lauwerys, R. R. Carcinogenicity and mutagenicity of crosslinking by chromium salts. Chem.-Biol. Interact., 36: 345-354. 1981. chromium. Mutai. Res., 76:227-239, 1980. 18. Fornace, A. J., Jr. Detection of DNA single strand breaks produced during 2. Doll, R. Problems of epidemiolÃ³gica! evidence. Environ. Health Perspect., the repair of damage by DNA protein cross-linking agents. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. Comparison of DNA Lesions and Cytotoxicity Induced by Calcium Chromate in Human, Mouse, and Hamster Cell Lines

Masayasu Sugiyama, Xin-Wei Wang and Max Costa

Cancer Res 1986;46:4547-4551.

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