Enhancement of UV and chromate mutagenesis by nickel ions in the Chinese hamster HGPRT Assay †

1. The HGPRT (Hypoxanthine‐Guanine‐Phospho‐Ribosyl‐Transferase) assay with Chinese Hamster V79 cells was used to measure the mutagenic effects of UV irradiation, potassium dichromate and nickel chloride. The agents were tested separately and in the combinations of UV plus nickel and dichromate plus nickel.

2. UV, Cr(VI) and Ni(II) were confirmed to be mutagenic in the V79 cell assay. The combination of UV(5J/m²) and Ni(II) (0.5 mM) caused a mutation rate 11.2 times above that corresponding to the sum of the individual mutation rates of these agents. The combined action of Cr(VI) (0.1 mM) and Ni(II) (0.5 mM) produced a mutation rate 2.8 fold above that corresponding to the sum of the individual rates of the separate agents.

3. The enhancing effect of nickel chloride on the mutagenicity of UV or Cr(VI) is interpreted by an interference of Ni(II) with the repair of DNA lesions.     

Model reactions of chromium compounds with mammalian and bacterial cells†

Chromate uptake, reduction, cytotoxicity and mutagenicity were studied with human red blood cells, Chinese hamster ovary (CHO) cells and/or Salmonella typhimurium mutant cells. All cell types rapidly took up chromates whereas chromium(III) salts were excluded under the experimental conditions. Red blood cells reduced and accumulated chromium from chromate. At concentrations above 0.1 mM, chromate inactivated the red cell chromate carrier. Chromate above 0.01 mM inhibited CHO cell proliferation irrespective of the cations present. Chromate and two chromium(III) complexes were mutagenic with Salmonella mutants in the Ames’ assay. A model for chromate metabolism and genotoxicity is proposed.     

Uptake and genotoxicity of micromolar concentrations of cobalt chloride in mammalian cells

Literature data concerning the genotoxicity of cobalt salts have been conflicting. To establish appropriate incubation conditions, we conducted a series of uptake studies, before genotoxicity was determined by DNA strand break induction in HeLa cells and mutagenicity in V79 Chinese hamster cells. Co(II) is taken up by HeLa cells in a concentration‐dependent manner and is accumulated inside the cell. The uptake is preceded by a fast association step to the outer membrane, with no saturation up to 24 h. DNA strand breaks as determined by nucleoid sedimentation are induced at concentrations as low as 50μMCoCl₂. The induction is time‐dependent, showing the highest number of breaks after 4h incubation with no further increase up to 24h. CoCl₂ is mutagenic at the HPRT‐locus, enhancing the spontaneous mutation frequency 4.2‐fold at 100μ?. Besides direct interactions with DNA, the mutagenicity of CoCl₂ could also be due to a decrease in the Fidelity of DNA polymerisation.     

Biochemical speciation in chromium genotoxicity

The biochemical speciation of chromium compounds in mammalian cells is discussed with respect to uptake, metabolism, DNA binding and damaging. Whereas soluble hexavalent chromium is taken up rapidly and accumulated intracellularly after its reduction, compounds of trivalent chromium penetrate biomembranes about three orders of magnitude slower. Cr(VI) after its uptake is metabolised by electron donating compounds via Cr(V) to Cr(III) compounds. Chromium from various Cr(III) compounds, but not chromate, binds to chromatin in isolated cell nuclei. The DNA‐protein crosslinks and DNA strand breaks observed in rat liver and kidney after chromate administration are also found in vitro, when Cr(III) compounds (but not chromate) interacts with isolated nuclei. In the Chinese Hamster cell HGPRT mutation assay, three out of four tested Cr(III) complexes were found to be mutagenic. In a direct DNA strand break assay with supercoiled bacteriophage PM 2 DNA, neither chromate nor the four Cr(III) compounds tested caused nicks. However, the combined action of chromate plus glutathione as well as the isolated complex of pentavalent chromium, Na₄Cr(glutathione)₄, did cause DNA breaks. Reactive oxygen species are inferred to be the ultimate DNA nicking agents in this assay. In conclusion there appear to be two mechanisms of chromate genotoxicity; one with direct DNA damage caused by Cr(V) species and one via DNA‐protein crosslinks formed with Cr(III), the final reduction state of chromate.     

Sorption,cellular distribution,and cytotoxicity of imidazolium ionic liquids in mammalian cells – influence of lipophilicity

In order to assist an integrated development of ionic liquids (ILs), a study on the sorption, distribution, and cytotoxicity of a series of 1-alkyl-3-methyl imidazolium tetrafluoroborates with C6 rat glioma cells has been performed. Cellular sorption and distribution among three cellular fractions (cytosol, nuclei, and membranes) were analysed by reversed-phase HPLC (RP-HPLC). Compounds with longer 1-alkyl substituents were sorbed with higher enrichment factors and sorption coefficients per protein than those with shorter 1-alkyl chains. The 1-octyl-3-methyl imidazolium cation (C₈MIM) was enriched 17-folds whereas C₆MIM and C₄MIM were enriched by factors of 3.5 and 2.3, respectively. After fractionation of cells by centrifugation, about 8% of C₈MIM was found in the nuclear fractions. The cytotoxicity as estimated by the tetrazolium reductase assay was increasing with the lengths of the 1-alkyl chains from C₄MIM to C₁₀MIM. Consistently, cell proliferation rates were decreasing with increasing lengths of the 1-alkyl chains. The results reveal the correlations between lipophilicity, cellular sorption, and cytotoxicity.     

Chromium binding by calf thymus nuclei and effects on chromatin

1. No binding of chromium was detected after incubation of calf thymus nuclei with hexavalent chromium up to 0.5 mM.

2. Chromium was readily taken up and tightly bound after incubation with trivalent chromium.

3. In a DNA‐filter binding assay, increasing amounts of chromium and DNA were bound with increasing chromium trichloride concentrations incubated with the nuclei.

4. Treatment with proteinase K abolished the increase in DNA retention induced by trivalent chromium.

5. It is concluded that trivalent chromium is the ultimate genetoxic agent after chromate uptake by living cells.

     

Fluorescence studies of dye displacement from DNA by chromium(III) complexes: Evidence for cation induced DNA condensations

The interactions of 10 different chromium(III) complexes with isolated calf thymus DNA have been analysed by studying the electronic and fluoresence spectra of intercalated ethidiumbromide. Triply charged cationic complexes including: [Cr(urea)₆]Cl₃.3H₂O, [Cr(1,10‐phenanthroline)₃](ClO₄)₃.2H₂O, [Cr(2,2'‐bipyridyl)₃] (ClO₄)₃.2H₂O, [Cr(ethylendiamine)₃]Cl₃.3.5H₂O and [Cr(NH₃)₆](NO₃)₃ displaced the dye from DNA. Similar effects were observed in experiments using the non‐intercalating dye bisbenzimidazole ("Hoechst 33258"). However, singly charged cationic, anionic and uncharged chromium(III) complexes such as: cis‐[Cr(1,10‐phenanthroline)₂Cl₂]Cl.2H₂O, cis‐[Cr(2,2'‐bipyridyl)₂Cl₂]Cl.2H₂O, [Cr(glutathione)₂]Na₂, [Cr(cysteine)₂]Na.2H₂O and [Cr(glycine)₃] were unable to displace both ethidiumbromide and bisbenzimidazole from DNA. There was no evidence for the formation of co‐ordinate bonds between chromium(III) and DNA for any of the above complexes. The charge and type of ligand are important in controlling the interaction of chromium(III) with isolated DNA in vitro. Our findings indicate that the outer sphere interaction of a chromium(III) complex with DNA is weak and unlikely to be the mechanism by which chromate causes DNA impairments in vivo and in vitro.     

Electrophoretic and liquid chromatographic characterization of red cell membrane proteins modified by chromate

Besides its effects of the genetic material and function, chromate also immediately affects the structure and function of cell membranes. With human erythrocytes, chromate produces alterations in cell size and shape, it impairs the anion transport function, and it causes modifications in membrane constituents. The proteins have been analysed by one‐ and two‐dimensional gel electrophoresis and by fast protein liquid chromatography (FPLC). The main chromate effects are the crosslinking of proteins including the membrane protein bands 1 and 2 (spectrin) and haemoglobin. Furthermore, a 40.000 D membrane protein fraction is modified. Chromate may react directly with membrane constituents but evidence also points to the formation of reactive oxygen compounds which in turn may react with proteins and lipids of the cell membrane.