Max Costa

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.

In vitro assessment of the toxicity of metal compounds

A review of the activity of metal compounds in mammalian cell transformation assays has been completed. Results from these assays appear to correlate well with the known carcinogenic activity displayed by specific metal compounds in vivo. Studies of cell transformation in vitro may provide information pertaining to the mechanism of the induction of carcinogenesis by certain metals.

Factors influencing the phagocytosis, neoplastic transformation, and cytotoxicity of particulate nickel compounds in tissue culture systems

Seven particulate nickel compounds were studied for their cell transformation activity using cultured Syrian hamster embryo cells and for their phagocytotic activity in cultured Chinese hamster ovary cells. The crystalline nickel compounds (αNi3S2, αNiS, and Ni3Se2) had significantly more cell transforming activity and were more actively phagocytized than the other nickel compounds examined (amorphous NiS, metallic Ni, Ni3O2, and NiO). Therefore, the crystalline structure of nickel compounds is one factor influencing their toxic activity upon biological systems. A second influencing factor was the particle size of the water-insoluble nickel compounds. Particles of crystalline αNiS ranging from 2 to 4 μm were phagocytized six times more than αNiS particles having mean diameters of 5–6 μm. Differences in amorphous NiS particle size had little effect on its already low susceptibility to be phagocytized by cells and ability to cause a reduction of cell plating efficiency. The presence of Mn dust inhibited the neoplastic transformation of crystalline nickel sulfide and also reduced the phagocytosis of crystalline αNiS and αNi3S2 particles by cultured cells. The phagocytosis of crystalline NiS particles was inhibited by the presence of amorphous NiS, Mn or MnCl2. Therefore, the presence of noncarcinogenic metals which are not themselves actively phagocytized diminishes the transforming effects of crystalline metal compounds probably by reducing their internalization. Various metabolic inhibitors such as dansylcadaverine, cycloheximide, and actinomycin D reduced the phagocytosis of crystalline αNiS.

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.

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.

The induction of DNA strand breakage by nickel compounds in cultured Chinese hamster ovary cells.

Both NiCl2 and crystalline alphaNiS induced DNA strand breaks in cultured Chinese hamster ovary (CHO) cells. Alkaline sucrose gradient analysis of [3H]thymidine radiolabelled DNA isolated from cells exposed to NiCl2 at 1 microgram/ml for only 2 h indicated a high degree of DNA strand breakage. Similarly crystalline alphaNiS caused substantial strand breakage at 1 microgram/ml following a 24-h treatment interval. These nickel compounds caused DNA strand breaks at concentrations which did not significantly impair normal cellular division. A concentration-dependent effect upon the number and average size of DNA fragments was obtained with both NiCl2 and crystalline alphaNiS. Since DNA strand breakage occurred at such low concentrations, these results suggest that nickel compounds which cause cellular transformation have highly selective and specific effects upon DNA structure.

Cytoplasmic dissolution of phagocytized crystalline nickel sulfide particles: A prerequisite for nuclear uptake of nickel

The intracellular fate of particulate crystalline alpha NiS, an inducer of neoplastic transformation which is readily phagocytized by cultured cells, was compared with that of particulate amorphous NiS, which does not have these properties. Amorphous and crystalline NiS both dissolve slowly in complete medium; phagocytized alpha NiS particles remain in the cytoplasm, where they dissolve more rapidly than extracellular particles. Thus the selective phagocytosis of alpha NiS accounts for both high intracellular particle accumulation and high levels of soluble Ni relative to the surrounding medium. Since phagocytized alpha NiS particles do not enter the nucleus, dissolution in the cytoplasm may represent an activation step in carcinogenesis, forming soluble Ni which diffuses into the nucleus. Dissolution products from phagocytized alpha NiS were detected in subcellular fractions isolated from treated cells; the highest levels were found in the nuclei, mitochondria, and lysosomes. That the Ni in the subcellular fractions was dissolved is suggested by the fact that dissolution products from phagocytized alpha NiS were detected in nuclei after centrifugation on sucrose pads, which substantially reduced contamination from cytoplasmic alpha NiS particles. Cytoplasmic dissolution of alpha NiS was enhanced by prior exposure of cells to the same compound. Loss of visible particles from cells was compared with loss of total Ni by use of alpha 63 NiS particles; the particles disappeared from almost half the cells during the first 2 d of treatment, while the total radioactivity associated with the cells and the total number of cells in the monolayer remained the same. The accelerated dissolution of alpha NiS after exposure to the same particles may be due to enhancement of lysosomal enzyme activity by particle phagocytosis. A 20-30% increase in intracellular acid phosphatase activity was observed after treatment with crystalline, but not amorphous, NiS, suggesting enhanced lysosomal activity.

Pathway of nickel uptake influences its interaction with heterochromatic DNA

Exposure of intact Chinese hamster ovary cells to water-soluble NiCl2 and to particulate crystalline NiS induced a concentration-dependent incidence of chromosomal aberrations which included gaps, breaks, and exchanges. Exposure of cells to crystalline NiS particles caused a high incidence of chromatid exchanges and dicentrics and produced what appears to be an effect on the condensation state of the heterochromatic long arm of the X chromosome. Treatment of cells with NiCl2 did not cause any significant effect on the long arm of the X chromosome, and there was a much lower incidence of the dicentric type of chromosomal aberrations compared to NiS. To examine whether the fragmentation/decondensation of the long arm of the X chromosome produced by crystalline NiS particles was due to a phagocytic pathway of uptake of NiS particles, cells were treated with NiCl2-albumin complexes that had been encapsulated in liposomes. Although treatment of cells with NiCl2-albumin complexes yielded higher intracellular nickel levels than were obtained by treatment of cells with NiCl2, at comparable intracellular levels fragmentation/decondensation of the heterochromatic long arm of the X chromosome was observed when nickel (II) was delivered by way of a liposome but not when cells were treated with unencapsulated NiCl2. Ionic nickel alone irrespective of its delivery mechanism exhibited some preference for heterochromatin, since there was a higher incidence of aberrations observed in the heterochromatic centromeric region of chromosomes. These observations suggest that the pathway of delivery of Ni2+ from NiS particles may be responsible for a preferential interaction of this metal with heterochromatin leading to an effect on the condensation state/fragmentation of the heterochromatic long arm of the X chromosome.

Analysis of metal-induced DNA lesions and DNA-repair replication in mammalian cells.

The potency of several metal compounds in causing lesions in DNA either directly or by exposure of intact cultured cells has been examined using the neutral conditions of nucleoid gradient sedimentation. HgCl2 was clearly the most potent inducer of single-strand breakage when added to isolated nucleoids or when nucleoids were prepared from cells treated with this compound. CaCrO4 , however, caused DNA-strand breaks in nucleoids isolated from cells treated with this agent but did not induce DNA strand breaks when added directly to nucleoids. Although less potent than HgCl2, NiCl2 also caused significant single strand breakage in isolated nucleoids or in nucleoids prepared from cells treated with this metal. Since strand breakage of DNA in intact cells may occur secondary to activation of DNA-dependent nucleases during repair replication, CsCl gradient density sedimentation was utilized to examine whether repair processes were induced by exposure of cells to NiCl2, HgCl2 and CaCrO4 . CaCrO4 and NiCl2 induced substantial DNA-repair activity at concentrations and exposure times where DNA lesions could not be detected whereas HgCl2 induced a 10-fold lower level of DNA-repair activity compared to CaCrO4 at optimal concentrations which again were below the concentrations of this metal that produced measurable DNA lesions. Both the induction of DNA-repair activity and DNA-strand breakage by these metals was concentration- and time-dependent. These results demonstrate some unique aspects of the interaction of HgCl2, NiCl2 and CaCrO4 with the DNA of intact cells and point to the possible important correlation of induction of DNA repair to carcinogenesis since nickel and chromate have clearly been implicated as carcinogens and induce considerable repair whereas HgCl2 is not considered a carcinogen and induces the least DNA repair despite its potency in producing DNA 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 McCoys 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 McCoys 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.