Cadmium resistance correlated with cadmium uptake and thionein binding in CHO cell variants Cdr20F4 and Cdr30F9

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Abstract
Cadmium‐resistant populations were derived by exposing monolayer cultures of Chinese hamster ovary (CHO) cells to stepwise increments of Cd2+ concentration, [Cd2+]. Clones Cdr20F4 and Cdr30F9 were selected from populations showing normal growth rates in 20 and 30 μM Cd2+, respectively. These variants were compared with CHO and with Cdr2C10, a previously derived clone, in terms of cell survival and Cd2+ uptake and partitioning as functions of [Cd2+]. The maximum [Cd2+] to which the various cells may be exposed in suspension culture without effects on growth or survival was 0.2, 2, 26, and 40 μM for CHO, Cdr2C10, Cdr20F4, and Cdr30F9, respectively. Patterns of survival as functions of time exposure to toxic levels of Cd2+ differed—survival of CHO decreased exponentially with time, that of Cdr2C10 displayed a shoulder followed by biphasic exponentially decreasing components, and those of Cdr30F9 and Cdr20F4 also had shoulders followed by exponentially decreasing components and, at longer times, by exponentially increasing components indicative of repopulation. The surival patterns for the resistant variants indicated that (1) there was an initial refractory period (0–4 h) during which no cells were killed, and (2) cell killing occurred primarily during the first 6–10 h of exposure. Thereafter, few were killed and survivors outgrew the populations. Studies of Cd2+ uptake and partitioning among intracellular forms provided an explanation for such survival patterns. Investigation of the role of metallothionein (MT) in conferring protection against Cd2+‐mediated cytotoxicity showed that none of the cells is protected significantly by preexisting thlonein. During the first hour of uptake, no protection is ascribable to significant thionein sequestration of Intracellular Cd2+ in any of the populations. In the resistant populations, however, Cd2+ appears in MT earlier and/or more rapidly than in CHO. As a consequence, the non‐MT Cd2+ forms do not continue to increase in these species. Within 4 h of exposure, nuclear and cytoplasmic non‐MT Cd3+ concentration level off or decrease in the resistant cells, but continue to increase in CHO. However, none of the cells have identical nuclear or cytoplasmic non‐MT Cd2+ contents when exposed to their maximum subtoxic [Cd2+] for 8 h. These levels are significantly higher in the resistant cells, Indicating that there are proportionately more ligands (other than MT) that complex Cd2+ in a nontoxic form in these cells. Such ligands have high molecular weights (presumably proteins), either preexist or are very rapidly induced in cells exposed to Cd2+, and do not detectably Increase once MT appears. Possible roles of such ligands in Cd2+ detoxification and/or thlonein induction are discussed.