Cadmium is a catalytic inhibitor of DNA topoisomerase II

Abstract

Cadmium (Cd(2+)) is a highly toxic and carcinogenic metal that is an environmental and occupational hazard. DNA topoisomerase II is an essential nuclear enzyme and its inhibition can result in the formation of genotoxic and recombinogenic DNA double strand breaks. In this study we showed that cadmium chloride strongly inhibited the DNA decatenation activity of human topoisomerase IIα in the low micromolar concentration range and that its inhibitory effects were reduced by glutathione. Because the activity of topoisomerase II is strongly inhibited by thiol-reactive compounds this result suggested that cadmium may be binding to critical topoisomerase II cysteine thiols. Cadmium, however, did not stabilize DNA-topoisomerase II covalent complexes, as measured by the lack of formation of DNA double strand breaks. Hence, it is not likely to be a topoisomerase II poison. Consistent with the idea that cadmium cytotoxicity may be modulated by glutathione levels, buthionine sulfoximine pretreatment to decrease glutathione levels resulted in a greatly increased cadmium-induced cytotoxicity in K562 cells. The results of this study suggest that cadmium may exert some of its cell growth inhibitory, and possibly its toxicity and carcinogenicity, by inhibiting topoisomerase IIα through reaction with critical cysteine thiols.

Fig. 1

(A) Inhibition of the decatenation activity of topoisomerase IIα by cadmium chloride and the affect of GSH on the inhibition as measured in a fluorescence assay. The fluorescence measures the amount of decatenated kDNA minicircles in the supernatant of the centrifuged assay mixture. The reaction mixture contained no added GSH (○) or 500 μM GSH (●). The smooth lines are from non-linear least squares fits to a 4-parameter logistic equation. In the absence and presence of GSH the curve fitting yielded IC₅₀ values of 26 ± 4 μM and 149 ± 69 μM, respectively. A limit of the number of samples that can be handled in a single experiment do not allow for multiple replicates for each cadmium chloride treatment. Thus, while the result shown is from a single experiment, results from 3 replicate determinations on different days gave an average 4.6 ± 0.6-fold increase in the IC₅₀ value in the presence of added GSH. (B) Fluorescent image of a gel showing inhibition of the decatenation activity of topoisomerase IIα by cadmium chloride and the effect of GSH on this inhibition. Curve fitting yielded an IC₅₀ of 50 μM and 122 μM in the absence and presence of GSH, respectively. In this ethidium bromide gel assay topoisomerase IIα was present in the reaction mixture for all lanes but lanes 1 and 10. In the absence of topoisomerase IIα the extremely high molecular weight catenated kDNA did not move from the origin. When the decatenation was highly inhibited, as in lanes 8, 9 and 18, most of the kDNA remained at the origin. In the other lanes, where there is only partial or no inhibition of topoisomerase II, there are higher order catenanes (containing 2, 3, 4 or more catenated minicircles) that are present that move slower on the gel than the fully decatenated kDNA. ORI is the gel origin, NC is nicked circular DNA and OC is open circular DNA. In the absence of topoisomerase IIα in the assay mixture the highly catenated kDNA does not move from the origin.

Fig. 2

Fluorescent image of an ethidium bromide-stained gel showing the effect of cadmium chloride and etoposide on the topoisomerase IIα-mediated cleavage of supercoiled (SC) pBR322 plasmid DNA. Topoisomerase IIα (Topo IIα) was present in the reaction mixture for all lanes but lane 2. As shown in lane 1, etoposide treatment produced significant amounts of linear DNA (LIN). A small amount of nicked circular (NC) is normally present in the pBR322 DNA. A densitometric analysis of the linear DNA bands showed that treatment with either 0.1 or 1 mM cadmium chloride produced much less linear DNA than the etoposide positive control (lane 1) and amounts of linear DNA about equal to that of topoisomerase IIα alone (lane 3). At the high concentration of topoisomerase II used in this assay topoisomerase II induced catenation of the pBR322 DNA to form catenated DNA which can now be seen at the gel origin (ORI). In this ethidium bromide gel relaxed DNA (RLX) ran slightly ahead of supercoiled DNA.

Fig. 3

Comparison of the effects of cadmium chloride on the growth inhibitory effects on a K562 and K/VP.5 cells with reduced levels of topoisomerase II. K562 (○) and K/VP.5 (●) cells were treated with cadmium chloride for 72 h prior to the assessment of growth inhibition by an MTS assay. The curved lines are non-linear least squares fits to logistic equations and yield IC₅₀ values of 19.0 ± 0.9 and 12.2 ± 0.4 μM, respectively for K562 and K/VP.5 cells.

Fig. 4

Effect of reducing intracellular GSH levels in K562 cells increases the growth inhibitory effects of cadmium chloride. K562 cells were preincubated either without (○), or with 5 mM OTC (□), or with 100 μM BSO (●) for 24 h and then treated for 72 h with cadmium chloride. The data shown are an average of 3 replicates. One other experiment performed on a different day with a BSO treatment yielded similar results. The slightly lower absorbance for both OTC and BSO treatments indicates that these compounds slightly reduced the growth of the K562 cells. The curved lines are non-linear least squares fits to 4-parameter logistic equations and yield IC₅₀ values of 18 ± 4, 0.77 ± 0.12, and 23 ± 4 μM for K562 cells not treated with BSO, or treated with BSO, or treated with OTC, respectively. Error bars are SEs.