Lu Y Y, Yang J L
Institute of Biomedical Sciences, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
J Cell Biochem. 1995 Apr;57(4):655-65. doi: 10.1002/jcb.240570410.
Chromium(VI) resistant Chinese hamster ovary (CHO) cell lines were established in this study by exposing parental CHO-K1 cells to sequential increases in CrO3 concentration. The final concentration of CrO3 used for selection was 7 microM for Cr7 and 16 microM for Cr16 cells. Cr16-1 was a subclone derived from Cr16 cells. Next, these resistant cells were cultured in media without CrO3 for more than 6 months. The resistance of these cells to CrO3 was determined by colony-forming ability following a 24-h treatment. The LD50 of CrO3 for chromium(VI) resistant cells was at least 25-fold higher than that of the parental cells. The cellular growth rate, chromosome number, and the hprt mutation frequency of these chromium(VI) resistant cells were quite similar to their parental cells. The glutathione level, glutathione S-transferase, catalase activity, and metallothionine mRNA level in Cr7 and Cr16-1 cells were not significantly different from their parental cells. Furthermore, Cr16-1 cells were as sensitive as CHO-K1 cells to free-radical generating agents, including hydrogen peroxide, nickel chloride, and methanesulfonate methyl ester, and emetine, i.e., a protein synthesis inhibitor. The uptake of chromium(VI) and the remaining amount of this metal in these resistant and the parental cell lines were assayed by atomic absorption spectrophotometry. Experimental results indicated that a vastly smaller amount of CrO3 entered the resistant cell lines than their parental cells did. A comparison was made of the sulfate uptake abilities of CHO-K1 and chromium(VI) resistant cell lines. These results revealed that the uptake of sulfate anion was substantially reduced in Cr7 and Cr16-1 cells. Extracellular chloride reduced sulfate uptake in CHO-K1 but not in Cr16-1 cells. Therefore, the major causative for chromium(VI) resistance in these resistant cells could possibly be due to the defects in SO4(2-)/C1- transport system for uptake chromium(VI).
在本研究中，通过将亲本中国仓鼠卵巢（CHO）-K1细胞暴露于逐步增加的三氧化铬（CrO₃）浓度下，建立了抗六价铬的CHO细胞系。用于筛选的CrO₃最终浓度，Cr7细胞为7微摩尔，Cr16细胞为16微摩尔。Cr16 - 1是从Cr16细胞衍生而来的亚克隆。接下来，将这些抗性细胞在不含CrO₃的培养基中培养6个月以上。通过24小时处理后的集落形成能力来测定这些细胞对CrO₃的抗性。抗六价铬细胞对CrO₃的半数致死剂量（LD50）比亲本细胞至少高25倍。这些抗六价铬细胞的细胞生长速率、染色体数目和次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（hprt）突变频率与其亲本细胞非常相似。Cr7和Cr16 - 1细胞中的谷胱甘肽水平、谷胱甘肽S - 转移酶、过氧化氢酶活性和金属硫蛋白mRNA水平与其亲本细胞无显著差异。此外，Cr16 - 1细胞与CHO - K1细胞对包括过氧化氢、氯化镍、甲磺酸甲酯和依米丁（一种蛋白质合成抑制剂）在内的自由基生成剂同样敏感。通过原子吸收分光光度法测定这些抗性细胞系和亲本细胞系中六价铬的摄取量以及该金属的剩余量。实验结果表明，进入抗性细胞系的CrO₃量远少于亲本细胞。对CHO - K1细胞系和抗六价铬细胞系的硫酸盐摄取能力进行了比较。这些结果表明，Cr7和Cr16 - 1细胞中硫酸根阴离子的摄取量大幅降低。细胞外氯离子降低了CHO - K1细胞中的硫酸盐摄取，但对Cr16 - 1细胞无此作用。因此，这些抗性细胞中抗六价铬的主要原因可能是摄取六价铬的SO4(2 - )/Cl - 转运系统存在缺陷。
