Solubility of Low-solubility Chromates and Their Clastogenic Activity in Cultured Cells

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1 Industrial Health, 1983, 21, Solubility of Low-solubility Chromates and Their Clastogenic Activity in Cultured Cells Kimiko KOSHI and Kenji IWASAKI National Institute of Industrial Health, 21-1, Nagao 6-chome, Tama-ku, Kawasaki-shi, Kanagawa 214, Japan (Received March 1, 1983) Abstract : Low-solubility chromates, such as lead chromate, zinc chromate and calcium chromate, which are potent carcinogens, have been examined for their solubility and clastogenic effects in a cultured Chinese hamster cell line (Don) as compared with those of potassium chromate, which is a high-solubility chromate and a weak carcinogen. Lead chromate was slightly soluble in water, Tyrode's solution, protein or amino acid solution, fetal calf serum and culture medium. Zinc chromate and calcium chromate were less soluble in serum and Tyrode's solution than in water. There were good correlations between the frequencies of chromosomal aberrations in the cells treated with the above chromates and chromium concentrations dissolved from these chromates in culture medium. When the frequency of chromosomal aberration in the cells exposed to the above chromates was compared on equal amounts of dissolved chromium concentrations in culture medium, the frequency of chromosomal aberration was the most remarkable with lead chromate, followed by zinc chromate and potassium chromate and then by calcium chromate. On the column chromatographs by gel filtration on Sephadex G-200, the chromium dissolved from all the above chromates in culture medium was present with low molecular fraction. From the above results, it was assumed that the carcinogenicity of low-solubility chromates might be related to their low solubility in body fluid. Key words : Solubility-Chromosomal aberration-gel filtration-chromate Cultured cell INTRODUCTION It has already been demonstrated that potassium chromate (K2CrO4) and chromium trioxide (CrO3), highly soluble hexavalent chromates, have potently clastogenic activity1-3). However, this finding has not yet been confirmed definitely in experiments on carcinogenicity. On the other hand, it has been shown that lowsolubility chromates, such as calcium chromate (CaCrO4), lead chromate (PbCrO4) and zinc chromate (ZnCrO4), are carcinogenic4-6). As for the mutagenicity of these chromates, PbCrO4 did not show mutagenic activity when its resistance to 8-azaguanine was tested with V79 cells1). However, when PbCrO4 dissolved in

2 58 K. KOSHI AND K. IWASAKI sodium hydroxide solution was added to the cell culture, it was clastogenic7). Furthermore, there has been a report stating that ZnCrO4 is slightly mutagenic1). No report on the mutagenicity or clastrogenicity of CaCrO4 has been published. The above findings were obtained based on qualitative experiments, and the relationship between the clastogenic activity of the chromates and the concentration of dissolved chromium (Cr) in culture medium was not clear. Therefore, in this paper, low-solubility chromates, such as PbCrO4, ZnCrO4 and CaCrO4, were examined for their solubility in the solution of biological substances, clastogenic activity in a cultured Chinese hamster cell line (Don) and the existing state of soluble Cr in culture medium as compared with potassium chromate. MATERIALS AND METHODS 1. Chemicals and biological materials The chromate samples used were PbCrO4 (Merck, Germany), ZnCrO4 (Shimakyu Pure Chemical Co., Japan), CaCrO4 (Kanto Chemical Co. Inc., Japan) and K2CrO4 (Kokusan Chemical Work Ltd., Japan). It was confirmed by X-ray diffraction analysis and infrared analysis that CaCrO, did not contain any water of crystallization. Crystalline bovine albumin was obtained from Sigma Chemical Co. (U.S.A.) and fetal calf serum (FCS) and Eagle's minimal essential medium (MEM) from Gibco Co. (U.S.A). Glycine and disodium ethylenediaminetetraacetate (NaEDTA) were obtained from Wako Chemical Industries Ltd. (Japan). 2. Solubility experiments Distilled water, Tyrode's solution, aqueous solutions containing 2% albumin, 2% glycine and 0.1 % NaEDTA, Tyrode's solution containing 2% albumin, FCS and MEM containing 10% FCS were used as solvents. Two mg of each chromate were suspended in 10 ml of each solution, and the suspensions were dispersed for two minutes by ultrasonic vibration (29 KHz, 45W). The resultant dispersed suspensions were centrifuged at 60,000 r.p.m. for 30 minutes to separate the supernatant. The metal content of each supernatant was determined with an atomic spectrophotometer using an acetylene flame. 3. Culture, chromosome preparation, and Cr determination in culture medium A pseudo-deploid Chinese hamster cell line (Don) was used. The cells were grown in MEM supplemented with 10% FCS. The cells (5 ~ 105 cells) per Falcon plastic flask were seeded, and, following day chromate suspensions at various concentrations were added to the cultures. The cultures were incubated for 48 hours at 37 Ž in an atomosphere of 5% CO2 in air, and 0.07 ƒêg/ml of colcemide were added for the final two hours. After incubation, the cells were collected by scraping with a rubber policeman, and the cultures were centrifuged at 1,500 r.p.m. for

3 SOLUBILITY AND CLASTOGENIC ACTIVITY OF CI-IROMATES 59 five minutes to separate the cells. These cells were treated with M calcium chloride for 15 minutes and then fixed with a mixture of ethanol and acetic acid (3:1) at 0 Ž. Following this, chromosome spreads were prepared and stained with Giemsa's solution by conventional methods. Chromosome aberrations were examined on 50 metaphases for each dose. When a metaphase had more than one chromatid or chromosome aberration, it was scored as an abnormal metaphase. In the classification of chromatid or chromosome aberration, however, each different chromatid or chromosome aberration in one cell was scored separately. The supernatants of the cultures used for the chromosome study were recentrifuged at 60,000 r.p.m. for 30 minutes, and the Cr contents in the supernatants were determined by an atomic absorption spectrophotometer. 4. Column chromatography The ultra-centrifuged supernatants of the cultures used for the chromosome study were further analyzed by gel filtration. Gel filtration was carried out on a 50 cm ~ 2.5 cm column packed with Sephadex G-200. Elution was performed with 0.1 M Tris-HCl buffer (ph 8.0) containing 0.02% sodium azide. The flow rate was maintained at 20 ml/hr. The fractions were collected with a fraction-collector (LKB Co.), and each fraction volume was kept at 5.1 ml (85 drops). The protein concentrations in the collected fractions were determined as the absorbency at 280 nm with a spectrophotometer. Gel filtration calibration was performed using y-globulin [molecular weight (M.W.), 160,000], albumin (M.W. 60,000) and myoglobulin (M.W., 16,000) as standards. The relationship between the elution volume and the molecular weight was linear. The metal contents of the fractions were determined with an atomic absorption spectrophotometer. RESULTS 1. Dissolution of chromate samples The results obtained in the solubility experiments with the chromate samples are shown in Table 1. PbCrO4 was dissolved in a very small amount in each solvent. The amount of dissolved PbCrO4 was almost the same in all the solvents except NaEDTA. In the case of NaEDTA, the ratio of Pb to Cr in the solution was almost the same as that when PbCrO4 was added to the solvent. In the other solvents, however, the ratios of Pb to Cr in the solutions were lower than that in PbCrO4. CaCrO4 was more easily dissolved in water than in Tyrode's solution (TS). The amount of dissolved CaCrO4 in water was approximately 10 times as much as that in TS. Furthermore, the amounts of dissolved CaCrO4 in 2% albumin-ts, FCS and 10% FCS-MEM were smaller than in TS. On the contrary, CaCrO4 was dissolved in larger quantities in glycine solution and NaEDTA

4 60 K. KOSHI AND K. IWASAKI Table 1. Solubility of chromates in various solutions solution than in water. In the case of NaEDTA, the substance added to the solvent was almost dissolved. In the case of ZnCrO4, the quantities of dissolved Cr and Zn were remarkably smaller in TS than in water. In this case, the amounts were slightly larger when protein or glycine was added to either water or TS than when water alone or TS alone was used. In the case of CaCrO4 and ZnCrO4, the ratio of Ca:Cr or Zn:Cr in the solutions was almost the same as that of the added materials. K2CrO4 was almost completely dissolved in each solvent. 2. Effects of concentration of dissolved Cr on chromosomal aberrations in cultured cells The relationships between the concentration of dissolved Cr in the culture medium and the frequency of chromosomal aberrations (aberrant metaphases) are shown in Fig. 1. There were good correlations between the frequency of aberrant metaphases in cultured cells and the Cr concentrations dissolved from all chromate samples. At the same concentration of dissolved Cr in the culture medium, chromosomal aberrations were most frequent with PbCrO4, followed by ZnCrO4 and K2CrO4. The frequency was the lowest with CaCrO4. Chromosomal aberrations are illustrated by dividing them into the sum of gap and break and the exchange. With regard to the relationship between the concentration of dissolved Cr in the culture medium and the frequency of gap and break (Fig. 2), the clastogenic activity of each chromate is in accordance with that shown in Fig. 1. With regard to the relationship between the concentration of dissolved Cr in the culture medium and the frequency of exchange (Fig. 3), this chromosomal aberration occurred when the dissolved Cr concentration was 0.08 Đg/ml in the case of PbCrO4. On the other hand, it occurred when the concentration of Cr was 0.29 Đg/ml in the case of other chromate samples.

5 SOLUBILITY AND CLASTOGENIC ACTIVITY OF CHROMATES 61 Fig. 1. The relationships between the frequency of aberrant metaphases and dissolved Cr concentration in the culture medium. Fig. 2. The relationship between the frequency of gap and break and dissolved Cr concentration in the culture medium.

6 62 K. KOSHI AND K. IWASAKI Fig. 3. The relationship between the frequency of exchange and dissolved Cr concentration in the culture medium. 3. Column chromatography of chromates dissolved in culture medium Supernatant from culture treated with CaCrO4 or ZnCrO4 was analyzed by gel filtration. The results are shown in Figs. 4 and 5. As can be seen in these figures, in these media, Cr was not bound to coexistent serum protein and existed in the fraction of low molecular weight. In the case of ZnCrO4, most of the Zn coexisted with serum albumin, and a small part of Zn coexisted with Cr. In the case of CaCrO4, Ca and Cr existed in the same fraction. In the case of PbCrO4, atomic absorption analysis did not reveal detectable Pb in the fractions of culture medium obtained by gel filtration. Therefore, after PbCrO4 dissolved in NaOH solution was added to the culture medium, PbCrO4 was separated by gel filtration. The Chromatogram in the case of PbCrO4 is given in Fig. 6. Cr existed in the fraction of low molecular weight. Most of the Pb coexisted with serum albumin, and an extremely small part of Pb existed only in the fraction of low molecular weight. In the case of the supernatant from the culture with K4CrO4 as well, Cr was not bound to coexistent serum protein and existed in the fraction of low molecular weight. DISCUSSION It was thought that PbCrO4 was insoluble in aqueous solutions. However, our experiment has revealed that Pb,CrO4 is soluble in an extremely small quantity in water, TS, FCS, culture medium and TS containing albumin. In the case of CaCrO4 and ZnCrO4, approximately half of the total amounts added to water was dissolved. On the other hand, the amounts of dissolved CaCrO4 and. ZnCrO4 in TS were approximately 1/10 or 1/8 times as much as that in water, respectively. Further-

7 SOLUBILITY AND CLASTOGENIC ACTIVITY OF CHROMATES 63 Fig. 4. Chromatogram of the supernatant of the culture with ZnCrO4. Fig. 5. Chromatogram of the supernatant of the culture with CaCrO4. Fig. 6. Chromatogram of the supernatant of the culture with PbCrO4.

8 64 K. KOSHI AND K. IWASAKI more, CaCrO4 was dissolved in smaller quantities in 2% albumin-ts, FCS and 10% FCS-MEM than in TS. These results suggest that not only PbCr04, but also CaCrO4 and ZnCrO4 are dissolved slowly in body fluids. In this experiment, atomic absorption analysis of the amount of Cr was influenced by the concentration of the coexisting ion. Therefore, the concentration of the ion of the standard solution was adjusted to that of the sample measured. Of the tested chromate samples, PbCrO4 has the most potent clastogenic activity. This finding agrees with the experimental results of Douglas7), in which sister chromatid exchange in cells occurred less frequently with K2CrO4 than with PbCrO4 dissolved in NaOH solution, which was added to CHO cells. Newbold et al.1) could not induce 8-azaguanine-resistance in V79 cells by PbCrO4. However, this experiment confirmed that PbCrO4 has adequate clastogenic activity even at low concentrations at which PbCrO4 can be dissolved in culture medium. The fact that PbCrO4 has more potent clastogenic activity than other chromates suggests the possibility of the coaction of Pb and Cr. As for the toxicity of cells, our previous experiment8) confirmed that the toxicity of lead dioxide (PbO2) in cells does not decrease when Pb is bound to serum protein. Therefore, even if Pb in the culture with PbCrO4 is bound to albumin, as shown chromatographically, there is no denying that Pb is toxic to cells. On the other hand, the cell toxicity of Zn decreased when Zn was bound to serum protein, as also shown in the previous paper. Since Zn in the supernatant from culture treated with ZnCrO4 was bound to protein, there is a possibility that the toxicity of Zn decreased. Newbold1) reported that ZnCrO4 is less mutagenic than K2CrO4. However, this experiment revealed that the clastogenic activity of ZnCrO4 was almost the same as that of K2CrO4. In the existing state of Cr in culture media, and in the clastogenic activity, there was no difference between K2CrO4 and other carcinogenic chromates, such as CaCrO4, ZnCrO4 and PbCrO4. It might be probable that the carcinogenicity of PbCrO4, ZnCrO4 and CaCrO4 is due to the low solubility of these chromates in body fluids. Furthermore, when the toxicity and the clastogenic activity of these chromates in cultures are evaluated,the amount of Cr dissolved in culture medium must be determined quantitatively. As shown in the present solubility experiments, the amount of Cr dissolved differs depending upon the salts, amino acids and proteins in the culture medium. This should be considered when the cellular effects of these chromates are compared. ACKNOWLEDGEMENT The authors are very grateful to Mrs. K. Suzuki for her skilled technical assistance.

9 SOLUBILITY AND CLASTOGENIC ACTIVITY OF CHROMATES 65 REFERENCES 1) Newbold, R. F., Amos, J. and Connell, J. R. (1979). The cytogenetic, mutagenic and clastogenic effects of chromium-containing compounds on mammalian cells in culture, Mutation Res., 67, 55. 2) Tsuda, H. and Kato, K. (1977). Chromosomal aberrations and morphological transformation in hamster embryonic cells treated with transformation in hamster embryonic cells treated with potassium dichromate in vitro, Mutation Res., 46, 87. 3) Koshi, K. (1979). Effects of fume particles from stainless steel welding on sister chromatid exchanges and chromosome aberrations in cultured Chinese hamster cells, Ind. Health, 17, 39. 4) Hueper, W. C. (1961). Environmental carcinogenesis and cancer, Cancer Res., 21, ) Furst, A., Schauder, M. and Sasmore, D. P. (1976). Tumorigenic activity of lead chromate, Cancer Res., 36, ) Dalager, A. N., Mason, T. J., Fraumeni, J. F. and Hoover, R. (1980). Cancer mortality among workers exposed to zinc chromate paints, J. Occup. Med., 22, 25. 7) Douglas, G. R., Bell, R. D. L., Grant, C. E., Wytsma, J. M. and Bora, K. C. (1980). Effect of lead chromate on chromosome aberration, sister chromatid exchange and DNA damage in mammalian cells in vitro, Mutation Res., 77, )Takahashi,H. andkoshi,k.(1981).solubilityandcelltoxicity ofcobalt, zinc and lead, Ind. Health, 19, 47.