Interaction of nickel with mutagens in the induction of sister chromatid exchanges in human lymphocytes.

In this study, individual treatments of human lymphocytes with Ni(II) [0.5-25 microM], Cr(VI) [0.65-1.30 microM], UV-light or X-rays induced SCEs in a dose-dependent fashion, and combined treatments of Ni(II) with Cr(VI), UV-light or X-rays interacted antagonistically. Nickel, at environmentally relevant exposure levels, can have the effect in complex mixtures of reducing an otherwise positive SCE response and could lead to underestimating human exposures to certain classes of chemicals or radiation. Furthermore, our data indicate that antagonism may occur when human lymphocytes are exposed simultaneously to Ni(II) and Cr(VI), suggesting an explanation for epidemiological studies reporting conflicting results for cytogenetic effects in lymphocytes of workers exposed to chromium and nickel.

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms.

Ni(II) induced changes in cell cycle duration and sister-chromatid exchanges in cultured human lymphocytes.

Investigations from our laboratory and others have shown that Ni(II) treatments of cultured human lymphocytes produced a relatively small but significant increase in SCE frequency. Based on the known effects of Ni(II) on DNA replication, we evaluated whether Ni(II) produced a cell cycle delay in lymphocytes. Human lymphocytes of three normal subjects were exposed to 5, 10, and 25 microM of NiSO4 in culture medium and scored for the percent of metaphases in the first (M1), second (M2), and third (M3) cell cycle for harvest times spaced every 4 h from 36 to 72 h after culture initiation. Cell cycle duration was studied using Tice's BISACK method with certain modifications. All three doses of NiSO4 caused a delay of nearly 1.5 h in the initiation of cell division, but only 25 microM NiSO4 caused a lengthening in the cell cycle time of nearly 4 h for completion of the first cycle. Only at the highest dose of Ni(II) was there a significant increase in the SCE frequency compared to the control. When the proliferation rate index (PRI) was examined, the effect of 5 or 10 microM Ni(II) was negligible while the 25 microM concentration caused a suppression in the proliferation rate. The effect of Ni(II) on the cell cycle was much more pronounced than on the PRI. A significant increase in SCE frequency was observed only for the concentration of Ni(II) that caused a pronounced cell cycle delay, a result that is consistent with prior studies showing higher SCE responses for chemical treatments that lengthen the cell cycle.

Somatic cell mutation in workers occupationally exposed to mercury vapors.

Somatic cell mutation in human peripheral lymphocytes is one of the tools used recently in the biological monitoring of the work environment. The scope of this review was to test whether biomonitoring methods are sensitive to the presence of mercury (Hg) in the body. We used the following techniques: micronucleus frequency (MN), sister chromatid exchanges (SCE), and hypoxanthine guanine phosphoribosyl transferase (HGPRT) assay in human lymphocytes. A total number of 30 male workers exposed to Hg vapors in chloralkali industry had been selected and compared with 30 control subjects. The concentration of mercury in urine (HgU) was used as a biological index of exposure. The exposed group showed higher levels of MN (32.0 +/- 1.7), SCE (7.3 +/- 0.2),and HGPRT mutations (0.94 +/- 0.01) then the nonexposed controls. We recommend the introduction of somatic cell mutation analysis in the periodic medical examination of workers exposed to Hg vapors.

A single stranded DNA binding protein isolated from HeLa cells facilitates Ni2+ activation of DNA polymerases in vitro.

The divalent nickel ion (Ni2+) is one of several metal ions that can substitute for Mg2+ in the activation of DNA polymerases in vitro, but usually with very low efficiency. We have purified and partially characterized a Ni(2+)-binding protein (p40) from HeLa cell extracts that can specifically enhance the polymerase activity of DNA polymerase alpha (pol alpha) and other DNA polymerases in response to Ni2+. This protein, with a molecular mass of 40 kDa, is a single stranded DNA binding protein that binds to a M13 DNA template-primer with an optimum stoichiometry of approximately 90 equiv of protein per equiv of DNA template and enhances the affinity of pol alpha for the primer-template. In the presence of Ni2+, p40 exhibits an increased affinity for DNA. The p40 increased by 3- to 6-fold the rates at which pol alpha and the Klenow fragment of Escherichia coli DNA polymerase I (KF) replicate different DNA templates in response to Ni2+. The low processivity of Ni(2+)-activated pol on primed M13 ssDNA was also enhanced by the presence of p40. The rates of Ni(2+)-dependent replication by inherently more processive enzymes, DNA polymerase delta and T4 DNA polymerase, were not significantly increased by p40 when M13 ssDNA was used as a template; however, p40 did increase the activity of T4 polymerase on an activated calf thymus DNA template. The protein did not stimulate Mg(2+)-activated DNA replication.

The effect of divalent nickel (Ni2+) on in vitro DNA replication by DNA polymerase alpha.

The effects of the carcinogenic metal nickel on DNA polymerase alpha (pol alpha) activity and fidelity have been analyzed. In the absence of Mg2+, the presence of Ni2+ ions at concentrations below 0.25 mM gave rise to a dose-dependent activation of pol alpha as monitored by [3H]dTMP incorporation into an activated DNA template. The apparent Km for Ni(2+)-dependent pol alpha incorporation of dTTP was estimated to be 25 microM, which was about 10 times higher than the Km for Mg2+ (2.3 microM). Above 0.25 mM, Ni2+ caused a dose-dependent inhibition of pol alpha activity and the Ki was calculated to be 1.5 mM. Scatchard analyses showed that Ni2+ binds to affinity-purified pol alpha and associated proteins at two tight binding sites with a Kd of approximately 50 microM and at eight weak binding sites with a Kd of approximately 4 mM. In the presence of 2 mM Mg2+, the addition of Ni2+ to the reactions caused an inhibition of polymerase activity. The inhibition patterns tended to switch from competitive to mixed-type to noncompetitive as a function of Ni2+ concentration. Lastly, Ni2+ increased the incorporation of the modified nucleotide dideoxy-CMP in reactions using varying ratios of dideoxy-CTP/dCTP.

The stimulatory effect of nickel chloride on DNA replication in human HeLa cells and Escherichia coli.

An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2). Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E. coli in a time- and concentration-dependent manner. The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types. In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent [3H]dTMP incorporation into activated calf thymus DNA. However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment. In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract.

Mutagenicity of soluble and insoluble nickel compounds at the gpt locus in G12 Chinese hamster cells.

Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful. In this report we describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E. coli gpt gene. This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants. When G12 cells were treated with insoluble particles of crystalline nickel sulfide < 5 microns in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background. Of 48 mutant gpt(-) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR). The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred. When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background. This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms of nickel.

The Malmö biomarker programme.

Evaluation of pro-oxidant/antioxidant dietary factors in relation to individual susceptibility to cancer and cardiovascular disease is justified based on a review of the literature. This working hypothesis is amendable to further scientific validation from biomarkers with end-point sensitivity to oxygen radicals. So far, the biomarker programme developed around this theme may be divided into three distinct classes: (1) Markers of genotoxic exposure estimate DNA damage either directly as a biologically effective dose, or indirectly by estimating aberrant cellular functions that lead to accumulation of DNA damage. The examples included are ADP-ribosylation in mononuclear leucocytes (R. Pero, A. Olsson), oxidative DNA damage (K. Frenkel), gene expression in lymphocytes (S. Garte, G. Cosma), serum alpha macroglobulin (W. Troll) and oxidized DNA damage and repair (N. Christie). (2) Markers of genetic predisposition have been shown to have genetic inheritance patterns that relate to individual susceptibility to cancer or cardiovascular disease. The examples included are glutathione transferase mu phenotyping (R. Pero, J. Seidegård) and poly (ADP-ribose) polymerase pseudogene polymorphism (M. Smulson). (3) Markers of dietary status have been validated to estimate the amount of a particular nutrient or xenobiotic in the diet that has been taken up and metabolized or distributed to body fluids or tissues. The example included here is niacin nutriture (E. Jacobson, M. Jacobson). This biomarker is presented in Section 5 (pp. 59-62) of the Malmö Diet and Cancer Programme Minisymposium reported in this issue of the journal.

Repair of X-ray induced DNA strand damage by isolated rat splenic lymphocytes.

Although a number of chemicals can alter DNA repair function, little is known about the effect of chronic, low dose exposure to environmental agents on DNA repair capacity. Lymphocytes provide a potential target population to study the effects of chronic exposures to low doses of toxic chemicals since they are an easily obtainable cell population. Prior to investigating the repair capacity of chemically exposed lymphocytes, the repair by chemically naive lymphocytes has been characterized. In the present study, the DNA repair capacity of isolated rat lymphocytes was characterized. The capacity of these cells to repair single-strand DNA breaks (SSB) was determined after in vitro treatments with X-rays. The effect of in vitro exposure to 3-aminobenzamide (3-AB) on DNA repair capacity was also assessed. The levels of induced SSB and their repair were determined using the alkaline elution technique. Splenic lymphocytes were isolated and placed in culture medium 18 h prior to assessment of repair capacity, but were not stimulated with mitogens. A dose-dependent increase in SSB was observed following exposure of lymphocytes to 300 or 600 rad. The rate of SSB repair was analyzed after a dose of 400 rad. Approximately 80% of the DNA strand break repair was completed within 60 min. The half-time for repair of these lesions by lymphocytes was determined to be 21.3 min. Exposure to 3-AB resulted in a decrease in the rate of repair of the X-ray-induced strand breakage. Although no SSB were detected at the end of a 1-h 3-AB treatment of non-irradiated cells, significant accumulation of SSB was observed after a 2-h treatment. The characterization of DNA repair in rat lymphocytes following in vitro exposure to X-rays will allow us to investigate the effects of chronic, in vivo toxicant exposure on the capacity of isolated lymphocytes to repair DNA damage produced by X-rays.

The effect of Ni(II) on DNA replication.

The cellular regulation of DNA replication is governed in part by the availability of essential metal ions. A continuous supply of Mg(II) ions is necessary for the efficient and faithful replication of parental strands during S-phase as well as during the repair of DNA damage. A metal ion such as Ni(II) may interfere with the replication process by binding to sites on proteins at which essential ions normally bind. Binding at these sites by a toxic metal ion may produce inappropriate responses from the replication proteins and thus alter the normal balance in one or more of the microsteps comprising DNA synthesis. We have studied the effect of Ni(II) on DNA replication in a reconstituted in vitro system using a HeLa cell extract as a source of polymerase activity on a template of activated calf thymus DNA. Ni(II) has an initial stimulatory effect that is followed by an overall inhibition of the incorporation of DNA precursors. These results suggest that Ni(II), similar to Mg(II) may have more than one binding site, but that the binding of Ni(II) to replication proteins may significantly alter the timing of events in DNA synthesis.

Effects of nickel sulfate, lead sulfate, and sodium arsenite alone and with UV light on sister chromatid exchanges in cultured human lymphocytes.

Sister chromatid exchanges (SCE) have been examined in human lymphocytes following in vitro treatments with metal salts, nickel sulfate, lead sulfate and sodium arsenite. All of the metal salts produced significant increases in the SCE frequencies over the levels for untreated lymphocytes. The SCE frequencies were also examined for metal treatments combined with ultraviolet light (200 ergs/mm2). For the lead treatments combined with the UV dose selected, an additive SCE response was observed compared to the SCE responses for UV or metal alone. The nickel and arsenite treatments combined with UV produced a less than additive SCE response for most concentrations tested. These results suggest that nickel or arsenite present in complex mixtures may reduce the SCE response to other compounds in the mixture normally capable of producing a much stronger SCE response and therefore lead to an underestimate of the effects of chemical exposure.

Chromosomal changes in cell lines from mouse tumors induced by nickel sulfide and methylcholanthrene.

Rhabdomyosarcomas were induced in mice by intramuscular injections of crystalline nickel sulfide and 3-methylcholanthrene. At early passage, karyotypes were performed by G-banding for four nickel sulfide cell lines and for three 3-methylcholanthrene cell lines. Six cell lines were near-diploid and one nickel sulfide line was near-tetraploid. Three of the nickel sulfide cell lines were characterized by a rearranged marker chromosome which was present in a majority of the cells of each line. The rearrangements leading to the formation of marker chromosomes were different in each nickel sulfide cell line but involved chromosome 4 in two of the nickel sulfide cell lines. Extra copies of chromosome 15 were present in two nickel sulfide cell lines. Possible rearrangement and/or gene activation was examined for the c-mos oncogene on chromosome 4 and the c-myc oncogene on chromosome 15, but no alteration or activation was observed. None of the 3-methylcholanthrene cell lines contained rearranged marker chromosomes; however, one MCA cell line did contain large numbers of double minutes. In all cell lines, minichromosomes (small atypical acrocentric chromosomes) were observed that contained distinct centromeric regions but no other G-positive bands.

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide.

Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the effects of Hg(II) on DNA repair induced in Chinese hamster ovary cells by ultraviolet or X-rays.

The effect of relatively nontoxic levels of HgCl2 on semiconservative DNA synthesis and on DNA repair induced following treatment of intact cells with X-ray or ultraviolet (UV) light has been studied in cultured Chinese hamster ovary cells. In the presence of 1 microM HgCl2 the repair of DNA strand breaks induced by 450 rads of X-rays was reduced by 37%. If a treatment of 2.5 microM HgCl2 was given to cells for only 15 min prior to a 450-rad irradiation, the rate of repair was reduced even further with only 25% of the breaks being repaired in the first hour following irradiation. When comparable treatments of HgCl2 were given to Chinese hamster ovary cells in conjunction with UV irradiation there was no significant effect on either the number of initial strand scission events or the return to high molecular weight DNA following completion of repair. Only after exposure of cells to toxic levels of Hg(II) (higher concentrations or longer treatments) was there measurable inhibition of UV-induced repair as evidenced by a reduced rate of ligation of DNA to a high molecular weight form. Inhibition of the endonuclease step of UV repair was not observed since Hg(II)-treated cells exhibited the same level of strand scission immediately following UV as cells not treated with Hg(II). The observed differences in the effects of Hg(II) on two pathways for DNA repair indicate that the potential for synergistic action between Hg(II) and other DNA damaging agents will be determined in part by the repair pathways induced by each agent. Additionally, it was found that inhibition of semiconservative synthesis also occurs at low concentrations of HgCl2 similar to those affecting X-ray-induced repair. The presence of Hg-DNA adducts in the DNA at these concentrations may cause a reduction in normal replication to facilitate DNA repair.

Genetic and physiological parameters associated with cadmium toxicity in Drosophila melanogaster.

Two strains of Drosophila melanogaster represent the extremes in resistance and sensitivity to the lethal effects of CdCl2. The strain containing the mutations vermilion and brown (v; bw) and the strain Austin had LC50's of 3.3 and 1.3 mM CdCl2, respectively. The three major chromosomes from these two strains were assorted genetically into the six possible combinations. The measured LC50's for CdCl2 for these six genotypes fell into two groups according to the X chromosome; those containing the X chromosome from v; bw had LC50's 0.5-1.0 mM greater than those in which the X chromosome was from Austin. Since the parent strains differed by 2 mM, we suggest that the X chromosome is a major, but not the sole, site of genes to produce resistance to CdCl2. When 109Cd was in the diet the uptake by v; bw and Austin over 2 days was the same. After 4 days of uptake, the Austin strain excreted the 109Cd five times faster than v; bw but the six genotypes did not differ appreciably in excretion rate from one another and resembled the sensitive parent Austin more than the resistant one. Thus a second process is indicated that distinguishes resistance to CdCl2 that apparently is not associated with the X chromosome.

Mechanism of HgCl2 cytotoxicity in cultured mammalian cells.

Treatment of intact Chinese hamster ovary cells with HgCl2 produced a rapid, concentration-dependent induction of DNA single-strand breaks (SSB) as revealed by alkaline elution analysis. Direct addition of HgCl2 to cell lysates did not result in DNA strand breaks. HgCl2 treatment of cells also caused a rapid leakage of superoxide radicals that were detected in their media by measurement of the reduction of exogenously added cytochrome c. There was a linear relationship between the production of radicals and the induction of DNA strand breaks, and there were also excellent temporal correlations in these parameters. Addition of oxygen radical scavengers, such as the enzymes superoxide dismutase and catalase, to the extracellular media significantly reduced the extent of DNA damage caused by HgCl2 without a similar attenuation of its uptake into cells, as did the autoclaved enzymes. Similarly, addition of radical scavengers such as glycerol or ascorbate inhibited the DNA damage but also reduced the uptake of the metal by almost the same degree. Thus, because of secondary effects on uptake of the metal, the radical scavenger experiments could not address the importance of oxygen radicals in the DNA damage caused by HgCl2. SSB were enhanced when cells were treated with HgCl2 and diethylmaleate or diethyldithiocarbamate, agents that deplete cellular reduced glutathione or inhibit the intracellular activity of superoxide dismutase, respectively. Thus, DNA damage in cells rendered sensitive to radicals was greater when these cultures were subsequently treated with HgCl2. The binding of 203HgCl2 to the DNA of intact Chinese hamster ovary cells was also studied. These studies were made possible by the relatively high stability of Hg(II) interaction with DNA and by utilizing a gentle method of DNA isolation that minimized redistribution of intracellular Hg(II) complexes after cells were lysed. The amount of Hg(II) bound to DNA varied from approximately 7 to 35 Hg atoms per 10(4) base pairs (bp) at concentrations of HgCl2 that have been previously shown to produce between 1 SSB/10(7) bp and 1 SSB/10(6) bp. The Hg(II)-DNA adducts were relatively stable complexes, since they resisted treatment with 0.1 M EDTA and 1 M NaCl and were stable to precipitation of the DNA with ethanol and trichloroacetic acid. However, the Hg(II) was released from the DNA when it was degraded enzymatically to mononucleosides, suggesting that the Hg(II)-DNA bonds formed in the cell were not truly covalent and that the strength of Hg(II) binding to DNA depended upon polynucleotide structure.(ABSTRACT TRUNCATED AT 400 WORDS)

6-Thioguanine-induced DNA damage as a determinant of cytotoxicity in cultured Chinese hamster ovary cells.

The mechanism of action of 6-thioguanine (TG) has been examined in cultured Chinese hamster ovary cells by direct measurement of the incorporation of the compound into DNA and by analysis of the resulting DNA damage. The predominant lesions as monitored by alkaline elution were DNA strand breaks. Very few, if any, interstrand or DNA-protein cross-links could be definitively observed. The cytotoxicity of TG as measured by colony-forming ability appeared closely related with its incorporation into DNA and the DNA strand scission events. As TG concentrations were increased, cytotoxicity, DNA incorporation, and strand scission reached a plateau; this result is consistent with earlier reports that TG produces a reversible block of DNA synthesis. Strand breaks appeared to be related to the incorporation of TG into DNA, since the addition of 1 microM cycloheximide during a 24-hr treatment with 3 microM TG prevented the cytotoxicity, prevented incorporation of TG into DNA, and eliminated the strand breaks. Alkali-labile sites were detected in the DNA of TG-treated cells by alkaline elution at pH 12.8, suggesting that depurination of TG residues by a glycosylase mechanism may occur. It is also postulated that TG residues are recognizable by the long-patch repair system, since UV-sensitive cells deficient for long-patch repair were more sensitive to TG than were wildtype cells. Furthermore, caffeine (1 mM) was shown to enhance the lethality of TG (3 microM), as monitored by colony formation, without altering levels of TG incorporation into DNA or the strand scission as measured immediately after treatment. This result, coupled with the known delayed cytotoxic response of TG, suggests that gaps may occur in newly synthesized DNA opposite TG residues and that the repair of these gaps by a postreplication repair mechanism is inhibited by caffeine.

Use of mammalian DNA repair-deficient mutants to assess the effects of toxic metal compounds on DNA.

Wild-type and repair-deficient cell lines ( EM9 ) of Chinese Hamster Ovary cells were utilized to assess cytotoxic responses towards metals that produce lesions in DNA. Alkaline elution studies indicated that both CaCrO4 and HgCl2 induced single-strand breaks in the DNA. CaCrO4 and HgCl2 treatments of intact Chinese hamster ovary cells also caused the induction of DNA cross links. The mutant cells, which are thought to have a defect in the repair polymerase enzyme and therefore exhibit greater sensitivity towards a variety of agents that produce lesions in the DNA such as X-rays and ultraviolet-light, also displayed a greater sensitivity, compared to wild-type cells, towards the cytotoxic response of HgCl2 and CaCrO4 . For example, the IC50 (concentration producing a 50% growth inhibition) following exposure for 6-hr to CaCrO4 or 1 hr to HgCl2 was 3.4-fold or 1.8- to 3.9-fold greater in wild-type cells compared to repair-deficient cells respectively. Mutant cells compared to wild-type cells were not more sensitive to growth inhibition by agents whose primary site of action was not at the DNA level (i.e. amphotericin B, trifluoroperazine and cycloheximide). The DNA crosslinks induced by exposure to 10 microM CaCrO4 for 6 hr were almost completely repaired in wild-type cells within 24 hr, whereas in similarly exposed mutant cells this lesion was initially more pronounced and was only partially repaired following a 24-hr recovery period in the absence of CaCrO4 . The repair of single-strand breaks induced by CaCrO4 was more rapid and similar in both wild-type and mutant cells. Since Hg(II) inhibits repair of single-strand breaks, we could not study repair of this lesion induced by this agent; however, at very low concentrations (1 microM) binding of 203Hg(II) to DNA was greater in the mutant cells compared to the wild-type cells. Following removal of 203Hg(II) from the media, mutant cells generally retained more 203Hg bound to DNA relative to the total 203Hg(II) present in the cell. These results demonstrate that an important toxic action of CaCrO4 and HgCl2 involves injury to DNA since the concentrations of these metals causing measurable DNA damage were consistent with their respective cytotoxic concentrations and DNA repair-deficient mutants displayed both enhanced cytotoxicity and decreased repair of metal-induced lesions.

Characterization of DNA lesions produced by HgCl2 in cell culture systems.

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.