Uptake of irradiated human low density lipoprotein by cultured Chinese hamster V79 cells

Specific binding and degradation of native and gamma-rays irradiated (100-2000 rad; 100 rad/min; 137Cs) human low density lipoprotein by Chinese hamster V79 cells and mouse peritoneal macrophage line, J774G were studied. Low density lipoproteins were labeled with 125I for studying the specific binding and subsequent degradation. The specific binding and degradation of irradiated 125I-low density lipoproteins (mixed with irradiated native lipoprotein) by Chinese hamster V79 cells are considerably reduced. The uptake depends on the concentration of thiobarbutaric acid-reactive products generated in the irradiated lipoproteins which in turn depends on the concentration of carotenoids. In contrast the rate of uptake of oxidized low density lipoproteins is enhanced by Chinese hamster macrophages. The alteration in the surface amino groups of apo-B of low density lipoprotein either due to direct damage of peptide bonds by gamma-rays or via interaction with lipid peroxides (generated in the core upon irradiation) are invoked as possible mechanisms for the reduction in specific binding and subsequent degradation by V79 cells.     

Recovery of thioguanine-resistant cells from Chinese hamster V79 spheroids

Multicell spheroids may prove useful in evaluting the interactions of mutagens with cells exposed in a tissue-like environment. However, direct comparisons among populations of Chinese hamster V79 spheroids of different sizes or with monolayers are complicated by the observation that as spheroids enlarge, the fraction of mutant cells resistant to 6-thioguanine (TG^r) gradually decreases from about 5 in 10⁵ to less than 1 in 10⁵. There appear to be at least 2 explanations for these observations. First, TG^r cells grow less well as spheroids than do 6-thioguanine-sensitive (TG^s) cells. Second, the clonal nature of spheroid growth means that small samples fo spheroids are likely to contain fewer pre-existing TG^r cells.     

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO(4)) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100-400 microM NiSO(4) for 24h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay.Aneuploid cells were induced in a significant fraction of the cell population 24-48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO(4). The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose.These results indicate that NiSO(4) has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.     

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells

1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a non-carcinogen showing some evidence of in vitro genotoxicity. We tested this compound in Chinese hamster V79 cells at 20-80 micrograms/ml with two endpoints: sister-chromatid exchange (SCE) and thioguanine resistance (TGR), with 5 repeat experiments. The SCE values in the presence of rat or hamster hepatocytes were consistently above the 95% and usually the 99% upper confidence limits for the corresponding control. Without hepatocyte activation, the control upper confidence limits were not exceeded except in one experiment in which the control SCE value was unusually low. TGR was scored both as proportion of plates with mutant colonies and as number of mutant colonies per plate. In 2 of 5 experiments, these values exceeded control 95% or 99% upper confidence limits; on the other hand, these values were substantially lower than those of the positive controls, dimethylbenz[a]anthracene (2.6 micrograms/ml) with activation and ethyl methanesulfonate (155 microgram/ml), which is direct-acting. For TGR, activation of 1-NN by either rat or hamster hepatocytes produced inconsistent results. Overall we would consider this compound to be a weak genotoxin, to which a cancer bioassay would be expected to be relatively insensitive.     

Mutagenicity of hydrogen peroxide in V79 Chinese hamster cells

Hydrogen peroxide (H2O2) was investigated for its potential to induce gene mutations in V79 Chinese hamster cells. Exposure of 2-3 X 10(6) cells/100-mm dish to 0.5-4.0 mM H2O2 for 1 h resulted in a concentration-dependent increase in the frequency of 6-thioguanine-resistant clones. At 4 mM H2O2 the mutation frequency was increased about 6-fold above that in controls and survival of the cells was reduced by 50%. Cytotoxicity was markedly increased at lower cell densities. When only 100-200 cells/100-mm dish were exposed to H2O2 for 1 h, 50% were killed at an H2O2 concentration as low as 60 microM. The results show that mutagenicity of H2O2 in mammalian cells in vitro has escaped attention previously because the concentrations tested were too low, presumably because the likely toxicity of H2O2 to V79 cells treated at high cell densities was overestimated.     

Mutagenicity of 4-hydroxynonenal in V79 Chinese hamster cells

4-Hydroxynonenal (HNE), a major product of the peroxidation of liver microsomal lipids, was examined for mutagenic activity at the hypoxanthine-guanine phosphoribosyltransferase locus in V79 Chinese hamster lung cells. At concentrations ranging from 10 to 45 microM, HNE induced a dose-dependent increase in the number of mutations to 6-thioguanine resistance, which reached the level of 4.7X baseline at the highest concentration tested.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Chromosome aberrations induced by pyrolysates of carbohydrates in Chinese hamster V79 cells

The clastogenic activity of some pyrolysates of carbohydrates was examined in cultured Chinese hamster V79 cells. These pyrolysates include levoglucosan (LG-I), levoglucosenone (LG-II), furfural (FF), 5-(hydroxymethyl)-2-furfural (HMF), glyoxal (GL), methylglyoxal (MGL), 3-deoxy-D-glucosone (DG) and thiazolidine (TZ). LG-I did not induce a significant number of chromosome aberrations at doses up to 8000 micrograms/ml. In contrast, the related compound LG-II induced aberrations and reduced mitosis in a dose-dependent fashion at around 1/2000 of the LG-I doses. Both furan derivatives, FF and HMF, and both glyoxal derivatives, GL and MGL, induced a significant number of chromosome aberrations and a significant lowering of mitotic activity. Among these compounds, FF and MGL showed stronger clastogenic activity than HMF and GL, respectively. DG slightly but positively induced chromosome aberrations. TZ was one of the most potent clastogens among the compounds examined in this study, showing the highest incidence of aberrant cells with many exchanges at doses inducing a significant lowering of mitotic activity. The results of this study indicate the need for a re-evaluation of the thermal decomposition of carbohydrates as a source of genotoxic contaminants.     

Inhibition of recovery from potentially lethal damage by chemicals in Chinese hamster V79 A cells

Summary The inhibition of recovery from potentially lethal damage and the influence of mutation induction by lactate, pyruvate, novobiocin, and nalidixic acid was studied in the stationary phase of Chinese hamster V79 A cells. Lactate and pyruvate were selected to elucidate their possible involvement in the inhibition of recovery from PLD since high levels of lactate and pyruvate accumulate due to increased aerobic and anaerobic glycolysis in tumours. Effects of novobiocin and nalidixic acid were also studied to elucidate the possible role of an enzyme similar to DNA gyrase in the potentially lethal damage recovery of V79 cells. The inhibition of recovery depends on drug concentration and is complete with 20 mM of lactate and pyruvate and 20 µM of nalidixic acid and novobiocin. The chemicals seem to interfere with an early step in the recovery process. Incubation with novobiocin in a post-irradiation period does not change the mutation frequency significantly whereas lactate and pyruvate demonstrate a slight increase. Cells incubated with nalidixic acid showed a significant increase in mutation frequency at 24 h post-irradiation recovery time.Alexander von Humboldt Fellow: Present address: Department of Bio-Science, H. P. University, Summer-Hill, Simla, India     

Elimination of MNU-induced mutational lesions in V79 Chinese hamster cells

Studies were performed on the non-linear dose response for gene mutations induced by low doses of monofunctional methylating agents in V79 Chinese hamster cells. When treatment with methylnitrosourea was applied at the beginning of the S phase in synchronized cells, a linear dose-response curve was obtained, whereas application of the dose after gene replication resulted in a strong reduction of the number of induced mutations. Additional time for repair resulted in reduced dose response of MNU, indicating that an error-free repair process operates on methylated DNA in V79 Chinese hamster cells.     

Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells

The tannins, delphinidin and procyanidin were isolated from flowers of white clover (Trifolium repens) and the leaves of Arnot Bristly Locust (Robina fertilis) respectively, and tested for mutagenic properties in a range of systems. There was no evidence for either compound causing significant levels of frameshift or base-pair mutagenesis in bacterial mutagenicity assays, although both were weakly positive in a bacterial DNA-repair test. Both compounds very slightly increased the frequency of petite mutagenesis in Saccharomyces cerevisiae strain D5. In V79 Chinese hamster cells, both were efficient inducers of micronuclei. In each of these test systems, increasing the potential of the compound for metabolic activation by addition of 'S9' mix had little effect on toxicity or mutagenicity of either tannin. It would seem that potential chromosome-breaking activity of condensed tannins could represent a carcinogenic hazard for animals grazing on pastures of white clover in flower. It may also have wider implications for human carcinogenesis by some, if not all, condensed tannins.     

Further studies on tetracycline-induced mutation in V79 Chinese hamster cells

The interaction between ultraviolet light and tetracycline in producing cell killing and mutation has been studied in V79 Chinese hamster cells. It has been established that these agents act independently of each other. Cycloheximide altered the response to tetracycline in the fractionation experiment: when cycloheximide was not present, fractionation of TC treatment resulted in a higher mutation yield but no change in survival level; in the presence of cycloheximide, however, mutation was greatly reduced but survival increased. The results were taken to indicate that for tetracycline action to take place, de novo protein synthesis during tetracycline treatment was necessary. Caffeine had no influence on tetracycline-induced lethality or mutagenicity. This observation was considered to suggest that tetracycline did not affect cellular repair processes.     

Genotoxicity of 2,4,6-trichlorophenol in V79 Chinese hamster cells.

The genotoxicity of the rodent carcinogen 2,4,6-trichlorophenol (TCP) was studied without exogenous metabolic activation in V79 Chinese hamster cells. TCP did not induce mutation at the hprt locus to 6-thioguanine resistance or structural chromosome aberrations. However, it produced statistically significant, dose-related increases in hyperdiploidy and micronuclei. From these results it appears that TCP causes chromosome malsegregation as its major mode of genotoxic action.     

Mutagenicity of some intercalating agents in Chinese hamster V79 cells

ICR 170 and ICR 191, but not 9-aminoacridine or chloroquine, induced both 6-thioguanine- and, to a smaller extent, ouabain-resistance in Chinese hamster V79 cells. These results indicate that covalent binding to DNA is necessary for intercalating agents to induce mutation in this cell line, and that this assay can distinguish potential carcinogens from non-carcinogenic analogues of this chemical type. The induction of ouabain-resistance by both ICR 170 and ICR 191 indicates that these frameshift mutagens induce base-pair substitution to some extent in V79 cells.     

Mutagenic characterization of cholesterol epoxides in Chinese hamster V79 cells

The uptake, metabolism and alkylating properties of the diastereomeric cholesterol epoxides were studied using Chinese hamster lung fibroblasts (V79 cells). Specific emphasis is given to the comparative cyto- and geno-toxic effects of cholesterol 5 beta,6 beta-epoxide (beta CE) and cholesterol 5 alpha,6 alpha-epoxide (alpha CE) and data are provided for the first time indicating that beta CE can induce more 6-thioguanine-resistant cells than alpha CE. Cholesterol 5 beta,6 beta-epoxide induced colonies of cells resistant to 6-thioguanine at 2-3-fold the frequencies observed with the alpha-isomer, but neither compound produced ouabain-resistant colonies. The cytotoxicity (LD50) of alpha CE was estimated to be 45-50 microM whereas beta CE displayed an LD50 of 25-29 microM. Inhibition of DNA synthesis (IC50) was observed over the same dose ranges as the LD50 for each epoxide isomer. The epoxides were assimilated by cells to an equal extent, however, beta CE was metabolized to cholestane 3 beta,5 alpha-6 beta-triol twice as rapidly as the alpha-isomer. Both epoxides reacted with 4-(4'-nitrobenzyl)-pyridine to a similar extent, and with identical nucleophilic selectivity at pH 7.4, but their alkylating activity was estimated on this basis to be two orders of magnitude less than methyl methanesulfonate. Binding experiments with the DNA or cultured V79 cells or with calf-thymus DNA indicated that interactions were noncovalent and DNA binding did not correlate with the potency of the epoxides to induce the 6-thioguanine-resistant phenotype. Our results could be interpreted as indicating that both cholesterol epoxide isomers are weak mutagens or that they might induce some epigenetic event repressing the hypoxanthine guanine-phosphoribosyltransferase gene. The similarity of the epoxides' alkylating activity and their DNA-binding properties are inconsistent with their different potencies in inducing the 6-thioguanine-resistant phenotype, suggesting that the mechanism leading to this phenotype is not necessarily the result of DNA alkylation.     

Structural and functional properties of hemoglobins from unicellular organisms as revealed by resonance Raman spectroscopy

Hemoglobins have been discovered in organisms from virtually all kingdoms. Their presence in unicellular organisms suggests that the gene for hemoglobin is very ancient and that the hemoglobins must have functions other than oxygen transport, in view of the fact that O2 delivery is a diffusion-controlled process in these organisms. Based on sequence alignment, three groups of hemoglobins have been characterized in unicellular organisms. The group-one hemoglobins, termed truncated hemoglobins, consist of proteins with 110-140 amino acid residues and a novel two-over-two alpha-helical sandwich motif. The group-two hemoglobins, termed flavohemoglobins, consist of a hemoglobin domain, with a classical three-over-three alpha-helical sandwich motif, and a flavin-containing reductase domain that is covalently attached to it. The group-three hemoglobins consist of myoglobin-like proteins that have high sequence homology and structural similarity to the hemoglobin domain of flavohemoglobins. In this review, recent resonance Raman studies of each group of these proteins are presented. Their implications are discussed in the context of the structural and functional properties of these novel hemoglobins.