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.

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.

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.

Queuine-containing isoacceptor of tyrosine tRNA in Drosophila melanogaster. Alteration of levels by divalent cations.

Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(-), ones of tRNATyr, tRNAHis and tRNAAsp of Drosophila melanogaster. Of the other divalent cations examined, Sr2+, Ni2+, Cu2+, Zn2+ and Hg2+, only Hg2+ failed to cause an increase in Q(+)tRNATyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responded to divalent ions; Hg2+ as well as Cd2+, Sr2+ and Zn2+ caused an increase in Q(+)tRNATyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd2+-containing diet, they formed significantly more Q(+)tRNATyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the 'hard-soft' classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(-) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation.

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.

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.

Quantitative aspects of metal ion content and toxicity in Drosophila.

As a basis for both a genetic and a biochemical approach to a study of metal ion effects, a method for quantitating the toxic response of Drosophila to metal ions was developed. The response to 13 metal ions has been examined, including several chemical groups from the periodic table: the IIb ions Zn2+, Cd2+, and Hg2+; the IIa ions, Be2+, Mg2+, Sr2+, and Ba2+; the transition elements, Ni2+, Cu2+, Co2+, and Mn2+: and trivalent ions, Y3+ and Cr3+. The standard test procedure provides estimates of the median lethal concentration (LC50) and the range of the tolerance distribution both of which are obtained by the method of probit transformation. Range is defined as the change in concentration between the LC2.5 and the LC97.5. Estimation of range as the measure of the limits of the tolerance distribution was utilized to measure variation in the response to metals with time. Genetic characterization of strains will require strains of flies without overlapping tolerance ranges. Although there was a general trend of increasing values of range with increasing LC50 values, the exceptions (e.g., Cr3+ and Be2+) indicated that several factors may be involved in determining the range of lethal responses. Elemental analyses have been performed on flies before and after metal ion treatments to establish the amount of metal ion taken up and baselines for comparison. This study provides additional evidence that Drosophila is an appropriate organism for the study of specific biochemical alterations induced by metals, e.g., the cadmium-induced increase in Q(+)tRNAs.

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.

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.

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.

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.

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.

Toxic and biochemical effects of divalent metal ions in Drosophila: correlation to effects in mice and to chemical softness parameters.

The mechanism of toxicity of 11 divalent cations was evaluated by determining the effects of dietary administration to Drosophila melanogaster and measurement of the frequency of lethality at 4 days, alterations in the developmental patterns of proteins, and changes in specific transfer RNAs. The relative effectiveness of divalent cations to kill Drosophila is significantly correlated to the relative values of the coordinate bond energy of the metal ions. The resistance of Drosophila to cadmium toxicity appears to be genetically determined since different inbred strains vary markedly. Also, the resistance is maximal in the young adult. Two different genetic strains seem to respond to different cations (Cd2+, Hg2+, Cu2+, Co2+, Ba2+, and Sr2+) in a similar manner. Basic mechanisms of toxicity may be studied in Drosophila as well as mice since the chemical properties of the metals reflect their toxic effects on the former as closely as the latter.

Investigation of correlations between chemical parameters of metal ions and acute toxicity in mice and Drosophila.

We are studying correlations between physicochemical properties associated with metal ions and observed toxicity. In order to test correlations, we obtained, under uniform conditions, LD50 values for acute toxicity in mice for 24 metal ions. The new data show a better correlation between LD50 and Pearson and Mawby's softness parameter sigma p, defined using chemical concepts of hard and soft acids and bases, than had been obtained by others. From a wide range of physicochemical parameters, the electrode potential can give almost as good a correlation as sigma p. Better correlations might exist for parameters more relevant to biological systems.

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.

In vitro assessment of the toxicity of metal compounds : III. Effects of metals on DNA structure and function in intact cells.

A review has been compiled illustrating the directions taken in examining the genotoxic effects of metals and their compounds centering only on those studies pertaining to effects of metals and their compounds on DNA structure and function, such as the induction of DNA strand breaks, production of DNA-protein crosslinks, induction of chromosomal aberrations, and sister chromatid exchanges. Although it is premature to declare a cause and effect relationship between the carcinogenic activity of metals and their ability to induce one or more lesions in DNA, strong evidence is emerging to suggest such a relationship. Low concentrations of metals induce the appearance of DNA lesions, such as strand breaks and crosslinks, or induce sister chromatid exchanges or DNA repair synthesis. Assays based upon these events constitute extremely sensitive probes for genotoxic effects of metals and their compounds. These effects of metals on DNA are consistent with the currently accepted mechanism of chemical carcinogenesis, allowing the acquisition and propagation of altered DNA function. The lack of complete information on the activity of metals in producing DNA lesions allow only preliminary conclusions to be drawn. Certain compounds containing potentially or actually carcinogenic elements, such as Ni, Be, As, Cr, Cd, and to a minor extent Pb, have yielded positive responses in one or more DNA lesion assays. At relatively nontoxic levels of Ni and Cr, considerable evidence suggests that multiple types of DNA lesions are induced.

In vitro assessment of the toxicity of metal compounds : IV. Disposition of metals in cells: Interactions with membranes, glutathione, metallothionein, and DNA.

This review has focused on several parameters related to the delivery of carcinogenic metal compounds to the cell nucleus as a basis for understanding the intermediates formed between metals and cellular components and the effect of these intermediates on DNA structure and function. Emphasis has been placed on metal interactions at the cellular membrane, including lipid peroxidation, metal interactions with glutathione and their relation to membrane injury, and metal effects on the membrane bound enzyme, Na(+)/K(+) ATPase. Metal binding to metallothionein is also considered, particularly as related to transport and utilization of metal ions and to genetic defects in these processes exemplified in Menkes disease. The ability of cadmium to induce the synthesis of metallothionein more strongly than zinc is also discussed in relation to other toxic and carcinogenic metals. The effects of metal ions on purified DNA and RNA polymerase systems are presented with some of the recent studies using biological ligand-metal complexes. This review points out the importance of considering how metals affect in vitro systems when presented as ionic forms or complexed to relevant biological ligands.

Evidence for nonrandom alterations in a fraction of the highly repetitive DNA of a eukaryote.

Although the DNA of the red crab Geryon quinquedens has no patent satellites, a large fraction (approximately 40%) is highly repetitive. Treatment of total DNA by Hind III produces fragments comprising 5-6% of the genome. While the sizes of some of these fragments form an arithmetic series based on an 81 bp repeating unit, the amounts of the multimers differ significantly from distributions observed for multimeric series in the DNAs of other eukaryotes. In red crab DNA, the amounts of some of the multimers suggest that they may have undergone as much as four times the divergence as the others. Other data, however, are more compatible with the conclusion that there has been selective amplification of segments of highly repeated DNA which results in the enhanced amount of specific multimers. These results indicate the presence of a nonrandom process in the evolution of the highly repetitive DNA. Selective mutation alone seems insufficient to explain these results.

Selective amplification of variants of a complex repeating unit in DNA of a crustacean.

The nucleotide sequence of the repeating unit of a fraction of the highly repetitive DNA of the red crab, Geryon quinquedens, is reported. Treatment of total DNA with HindIII nuclease produced an 81-base-pair monomer and multimers to the size of an octamer. Several of the multimers contained large amounts of fragments of variant sequences, which cannot easily be explained by random mutation alone. That the alterations were not random was corroborated by divergence measurements made on the distribution of Hha I nuclease sites within several multimers. The analyses showed that a fraction of each of them is characterized by 4% divergence, while the amounts of dimer, tetramer, and octamer suggest that they have undergone 2-4 times more divergence than that. These results, coupled with the data on sequence variants that are more prevalent in the dimer, indicate that amplification of divergent repeating units could easily explain enhanced amounts of selected multimers.

Interspersion of highly repetitive DNA with single copy DNA in the genome of the red crab, Geryon quinquedens.

Kinetic analysis of the reassociation of 420 nucleotide (NT) long fragments has shown that essentially all of the repetitive sequences of the DNA of the red crab Geryon quinquedens are highly repetitive. There are negligible amounts of low and intermediate repetitive DNAs. Though atypical of most eukaryotes, this pattern has been observed in all other brachyurans (true crabs) studied (1,2). The major repetitive component is subdivided into short runs of 300 NT and longer runs of greater than 1200 NT while the minor component has an average sequence length of 400 NT. Both components reassociate at rates commonly observed for satellite DNAs. Unique among eukaryotes the organization of the genome includes single copy DNA contiguous to short runs (approximately 300 NT) of both repetitive components. Although patent satellites are not present, subsets of the repetitive DNA have been isolated by either restriction endonuclease digestion or by centrifugation in Ag+ or Hg2+/Cs2SO4 density gradients.