In vivo effects of mercury (II) on deoxyuridine triphosphate nucleotidohydrolase, DNA polymerase (alpha, beta), and uracil-DNA glycosylase activities in cultured human cells: relationship to DNA damage, DNA repair, and cytotoxicity

Abstract

The effect of mercuric acetate on the activities of deoxyuridine triphosphate nucleotidohydrolase (dUTPase), DNA polymerase (alpha, beta), and uracil-DNA glycosylase has been studied in cultured human KB cells. There was a dose- and time-dependent inactivation of both dUTPase and DNA polymerase alpha activities by mercuric acetate. In cells exposed to low concentrations (10 microM) of mercuric acetate, dUTPase was most sensitive to inhibition with 30% of the activity being inhibited after a 1-hr exposure. At higher concentrations or for longer exposure times, DNA polymerase alpha was most sensitive to inhibition with greater than 60% of the activity being inhibited by 25 microM mercuric acetate after a 15-min exposure. There was no inhibition of DNA polymerase beta or uracil-DNA glycosylase activities in cells exposed to 50 microM mercuric acetate for 90 min. In fact, there was a time- and dose-dependent activation of uracil-DNA glycosylase activity with maximum activation occurring in cells exposed to 50 microM mercuric acetate. The inhibition of dUTPase and DNA polymerase alpha activities and the activation of uracil-DNA glycosylase activity correlated with the induction of single-strand breaks in DNA by mercuric acetate and with the decrease in cell viability.

Fig. 1

Effect of mercuric acetate on dUTPase, DNA polymerase (α,β), and uracil-DNA glycosylase activities in KB cells. Intact cells were exposed to various concentrations of mercuric acetate for the times indicated. Cell extracts were prepared and enzymatic assays were performed as described in Materials and Methods. Extracts were assayed simultaneously for the various enzymes. The values represent the average ± the standard deviation for three experiments. A, dUTPase; B, DNA polymerase α; C, uracil-DNA glycosylase; D, DNA polymerase β. △ untreated; ○, 5 μM; ●, 10 μM; ■, 25 μM; □, 50 μM mercuric acetate.

Fig. 2

Effect of mercuric acetate on protein synthesis. Cells were preexposed to various concentrations of mercuric acetate for the times indicated and protein synthesis was then determined as described in Materials and Methods. The values represent the average of at least two experiments ± the standard deviation. [³H]Leucine incorporation in untreated controls was 55,563 ± 2,351 cpm. Symbols are as in Fig. 1.

Fig. 3

Effect of rnercuric acetate on DNA synthesis. Cells were exposed to various concentrations of mercuric acetate for 30 min and simultaneously pulsed with [³H]thymidine. DNA synthesis was determined as described in Materials and Methods. The values represent the average ± the standard deviation of at least two experiments.

Fig. 4

Induction of SSBs in DNA by mercuric acetate. Alkaline elution analysis of DNA was performed as described in Materials and Methods. Values represent the average of at least two experiments. A, 5 μM mercuric acetate; B, 10 μM mercuric acetate; C, 25 μM mercuric acetate; D, 50 μM mercuric acetate. For simplicity, the untreated controls ○ are shown only in A. Mercury (II) exposure was: ▲, 15 min; △, 30 min; □, 60 min; and ■, 90 min.

Fig. 5

Concentration- and time-dependent formation of SSBs in DNA exposed to mercuric acetate. The SSF was determined from the alkaline elution profiles as described in Materials and Methods. A. Concentration-dependent formation of SSB. ●, 15 min; ▲, 30 min; ○, 60 min; △, 90 min. B. Time-dependent formation of SSB. ○, 5 μM; △, 10 μM;, ●, 25 μM; ▲, 50 μM.