Effects of hydroxyurea on deoxynucleotide pools of mouse fibroblasts.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
V Bianchi
Publications and source records attributed to V Bianchi.
Increased mutagenicity of chromium compounds by nitrilotriacetic acid.
Nitrilotriacetic acid trisodium salt (NTA), which is a substitute for polyphosphates in household laundry detergents, and N-nitrosoiminodiacetic acid (NIDA), a derivative of NTA produced by metabolism of soil microorganisms, were tested for in vitro mutagenicity in bacteria and yeasts. No gene reversions in five strains of Salmonella typhimurium (TA 1535, TA1537, TA1538, TA98, and TA100), no forward gene mutations in Schizosaccharomyces pombe P1, and no mitotic gene conversions at two loci in Saccharomyces cerevisiae D4 were induced by NTA (up to 870 micrograms/plate or 40 micrograms/ml) and NIDA (up to 2,000 micrograms/plate or 1,000 micrograms/ml), independently of the presence of rat liver metabolic activation. The influence of NTA on the mutagenic and clastogenic activity of several chromium compounds was examined in the Salmonella/microsome assay and in the sister chromatid exchange (SCE) assay in mammalian cell cultures (Chinese hamster ovary [CHO] line). NTA does not affect the genetic inactivity of water-soluble Cr(III) (Cr2[SO4]3) and the direct mutagenicity of soluble Cr(VI) (Na2CrO4,K2Cr2O7) compounds. The very insoluble Cr(VI) compounds PbCrO4 and PbCrO4 X PbO are instead clearly mutagenic in the Salmonella/microsome assay (TA100 strain) only in the presence of NTA or NaOH. The mutagenicity of lead chromates is correlated with the amounts of Cr(VI) solubilized by NTA or alkali, as detected by the colorimetric reaction with diphenylcarbazide and atomic absorption spectrophotometry. In the SCE assay, the insoluble lead chromates are directly clastogenic owing to prolonged treatment conditions and cellular endocytosis. The chromosome-damaging activity of PbCrO4 is significantly increased by NTA but not by NaOH.
Acetates↗
Interaction of nitrilotriacetic acid with heavy metals in the induction of sister chromatid exchanges in cultured mammalian cells.
The ability of nitrilotriacetic acid trisodium salt (NTA) to induce sister chromatid exchanges (SCE) and to influence the induction of SCE by heavy metal compounds was evaluated with mammalian cell cultures. In accordance with the generally negative results obtained by other investigators on the mutagenic effects of NTA, no increase of SCE frequency was observed in Chinese hamster cells (CHO line) or in primary cultures of mouse (BALB/c and BALB/Mo strains) lymphocytes, after treatment with NTA at subtoxic concentrations (2 X 10(-3) M and 10(-3) M, respectively). The induction of SCE by salts of heavy metals (Cd, Hg, Ni, Pb) was evaluated with CHO cells. Soluble (CdCl2, HgCl2, NiCl2, Pb [CH3COO]2) and insoluble (CdCO3, HgCl, NiCO3, PbSO4) compounds were tested, and, with the exception of Pb(CH3COO)2, all were found to increase the frequency of SCE. NTA apparently did not affect the ability of the soluble metal compounds to induce SCE, but it significantly increased the frequency of SCE induced by treatment with all the insoluble salts.
Acetates↗
Effects of nitrilotriacetic acid on the induction of gene mutations and sister-chromatid exchanges by insoluble chromium compounds.
The influence of nitrilotriacetic acid trisodium salt (NTA) on the mutagenic and clastogenic activity of several water-insoluble or poorly soluble chromium compounds was determined by means of the Salmonella/microsome assay (plate test on TA100 strain) and the sister-chromatid exchange (SCE) test in mammalian cell cultures (CHO line). NTA in itself did not induce gene mutations nor did it increase the frequency of SCE. Cr(VI) compounds (Pb, Ba, Zn, Sr and Ca chromates) and an industrial Cr(VI) pigment, chromium orange (containing PbCrO4 PbO), were inactive or scarcely active mutagens in the Salmonella/microsome test when dissolved in water, but they were increasingly mutagenic when solubilized by 0.5 N NaOH or NTA (10 or 100 mg/ml). Also, the mutagenic activity of Cr(VI), contaminating an industrial Cr(III) pigment (chromite), was slightly enhanced by NTA. Mutagenicity of chromates was correlated with the amounts of Cr(VI) solubilized by NTA or alkali, as determined by the colorimetric reaction with diphenylcarbazide and atomic absorption spectrophotometry, and was decreased by incubation with microsomes, due to reduction of Cr(VI) to the genetically inactive Cr(III) form. In the SCE assay, the insoluble or poorly soluble Ba, Zn, Sr and Ca chromates and the insoluble Cr(VI) pigments zinc yellow (containing ZnCrO4 Zn(OH2], chromium yellow and molybdenum orange (both containing PbCrO4) were directly clastogenic due to cellular endocytosis taking place in prolonged treatments, and NTA significantly increased their chromosome-damaging activity.
Acetates↗
Accuracy of UV-induced DNA repair in V79 cells with imbalance of deoxynucleotide pools.
Chemical mutagens generally cause nucleotide pool imbalance. We postulated that this effect might enhance the mutagenic effect by reducing the accuracy of DNA-repair synthesis. We used an inducer of DNA repair which causes minor pool modifications, namely UV light, and imbalanced the nucleotide pools by incubating UV-irradiated V79 cells with thymidine or deoxycytidine (10(-5)-10(-2) M) during the early phases of repair. The effects on pool sizes of the incubation with deoxynucleosides were determined by directly measuring the 4 deoxynucleoside triphosphates in cell extracts. The impairment of repair accuracy was evaluated by comparing the frequency of mutations at the HGPRT locus (induction of resistance to 6TG) in irradiated cells incubated with deoxynucleosides or allowed to carry on repair synthesis in nucleoside-free medium. Despite the marked imbalance of pyrimidine nucleotide pools, an increase of mutations was observed only with the highest concentrations of thymidine and deoxycytidine. Such an increase was much lower than that reported in the case of facilitation by excess nucleosides of chemically induced mutagenesis. The results indicate that UV-induced repair is scarcely affected by precursor biases.
Animals↗
Genetic effects of chromium tannins.
Seventeen tannins used in the hide and leather industry, most of which contain mainly Cr(III) sulphates, were tested for the ability to directly induce gene mutations in Salmonella typhimurium (TA 100 strain) and chromosomal effects (sister chromatid exchanges, SCE) in cultured hamster cells (CHO line). Total chromium [Cr(III) + Cr(VI)] content and contaminating Cr(VI) were determined spectrophotometrically by reaction with diphenylcarbazide. None of the tested compounds induced gene mutations, whereas eight tannins were able to increase significantly the frequency of SCE. A contamination with Cr(VI) was detected in four compounds (from 30 up to 100 parts of Cr(VI) per 10(6) parts of compound), insufficient to be revealed by the Salmonella assay but sufficient to account for the observed SCE increase. On the other hand, the increase of SCE induced by the other four tannins could not be explained by the level of Cr(VI) contamination, and can be ascribed to other impurities present in those industrial compounds. These four tannins did not induce gene mutations in the S. typhimurium assay even when strain TA 98 was used in addition to TA 100, independently of microsomal activation. By prolonging the time of the SCE assay from 30 to 48 h in order to facilitate Cr(III) endocytosis, a significant increase of the SCE frequency was induced by an analytical-grade Cr(III) reagent (chromium chloride), absolutely uncontaminated by Cr(VI), as well as by three Cr(III) tannins, otherwise inactive in the SCE assay.
Animals↗
Genetic effects of procarbazine in the yeast Saccharomyces cerevisiae, strain D4.
Procarbazine ( PCZ ) was tested for its ability to induce mitotic gene conversions at the ade and trp loci of Saccharomyces cerevisiae, strain D4. The influence of the following factors was examined: growth phase of the yeast cells (log vs stationary phase), pH of the treatment solution, and addition of mouse S9 fractions prepared from different organs. The drug was found more toxic and mutagenic at low doses (up to 25 mg/ml) for log phase cells, and scarcely toxic but highly mutagenic, even at high doses, for stationary phase cells. PCZ activity was reduced by acidic pH, and suppressed by S9 mix. Gene conversions were also analyzed in the intrasanguineous host-mediated assay performed in mice orally administered with PCZ . In such conditions PCZ was ineffective in stimulating mitotic gene conversions, probably owing to its inactivation in the acidic environment of the gastroenteric tract.
Animals↗
Induction of sister-chromatid exchanges in human lymphocytes exposed in vitro and in vivo to therapeutic ultrasound.
The induction of sister-chromatid exchanges (SCEs), chromosomal aberrations and cell-cycle delay was determined in human lymphocytes after treatment in vitro and in vivo with therapeutic ultrasound (u.s.). In vitro treatments (1 W/cm2; 0.860 MHz; for 40-160 sec) were performed on unstimulated lymphocytes from 9 donors: a statistically significant, dose-dependent increase in SCE frequency was produced, whereas no induction of chromosomal aberrations nor alteration of the distribution of 1st, 2nd and 3rd division metaphases were observed. The same increase in the frequency of SCEs was detected by treating in vitro stimulated lymphocytes with u.s. The effects of in vivo exposure to u.s. were detected on lymphocytes from 10 patients before, during and after u.s. therapy (0.6-1.0 W/cm2; 0.860 MHz; from 8 to 20 applications lasting 5-6 min each). SCE frequency was statistically significantly increased in all patients at mid-therapy, without a further increase during the second half of therapeutic cycle, and was restored to pretreatment level 3 months after the end of u.s. therapy. No increase in chromosomal aberrations was noticed during and after u.s. therapy, whereas erratic delays of the cell cycle were observed, not clearly related to u.s. application or SCE levels. A linear relationship was found between SCE frequency and age in 21 healthy donors.
Cell Cycle↗
Trivalent chromium is neither cytotoxic nor mutagenic in permeabilized hamster fibroblasts.
BHK cells became reversibly permeable by a 30-min incubation in hypertonic medium. During permeabilization they were exposed to water-soluble Cr(VI) (K2Cr2O7) and Cr(III) (CrCl3). Thymidine uptake in the intracellular nucleotide pool, DNA replication, DNA damage and repair and sister-chromatid exchanges (SCE) were examined to detect the cytotoxic and genetic effects of Cr compounds. Cr(III) remained inactive also in permeabilized cells. An apparent induction of DNA damage by Cr(III), suggested by the Painter's test, was considered unreliable. Cr(VI) cytotoxic and genetic activity was enhanced in permeabilized cells, as demonstrated by increased inhibition of DNA replication and higher frequency of SCE.
Animals↗
A cytogenetic study on workers exposed to low concentrations of benzene.
A cytogenetic study was performed on 22 healthy workers engaged in benzene production and exposed to low concentrations of benzene, ranging from 0.2 to 12.4 p.p.m. (threshold limit value 10 p.p.m.). Workers were divided into two groups according to the different levels of exposure, inferrable also from the concentration of benzene in the alveolar air and the levels of urinary phenols. Each exposed subject was paired with a suitable control, living in the same area and of similar smoking habits and age. No statistically significant increase of sister chromatid exchange (SCE) frequency was observed in the exposed groups. In controls, SCE frequency was positively correlated with age and smoking habits. Among structural chromosomal aberrations, only the chromosome-type ones were significantly higher in exposed than in control subjects, and their increase was still significant when gaps were discarded.
Adult↗
Workers exposed to ethylene oxide have increased incidence of sister chromatid exchange.
We have determined the frequencies of sister chromatid exchange (SCE) in 41 workers engaged in the sterilization of medical equipment. Each exposed subject was paired with a control matched for age, smoking habits and sex. The frequencies of SCE were markedly higher in the exposed than the control workers. The exposed subjects were divided into two groups according to time-weighted average concentrations of ethylene oxide (EtO) in the air, that were determined at the work place of each worker during the 5 min following the sterilizer opening, during the entire sterilization cycle or during the 8-h working day. One group (19 subjects) was exposed to a range of 3.7-35.5 ppm (mean, 15.8 +/- 9.8 for a sterilization cycle) and showed a mean of 13.0 +/- 1.8 SCE compared to 10.2 +/- 1.2 in controls (variance, F = 31.2; p less than 0.001). The second group (22 subjects) was exposed to 0.3 - 2.6 ppm (mean, 1.1 +/- 1.0) and had a mean of 11.0 +/- 1.6 SCE compared to 9.8 +/- 1.4 in controls (F = 4.7; p less than 0.05). Statistical evaluation of the SCE frequencies correlates SCE induction with the level of EtO exposure and also with smoking and age. Our data show that EtO can induce human chromosomal damage at very low environmental exposure levels, lower than the present Italian official limits and lower than those found even in the most technologically advanced hospitals.
Adult↗
Sister chromatid exchanges and chromosomal aberrations induced by mitomycin C in mouse lymphocytes carrying a leukemogenic virus.
The induction of chromosomal damage (sister chromatid exchanges (SCEs), chromosomal aberrations, and micronuclei) in T lymphocytes from mouse spleen was analyzed after treatment in vivo with different concentrations of mitomycin C (MMC). Lymphocytes were derived from BALB/Mo mice, which carry an endogenous type C retrovirus (Moloney murine leukemia virus, M-MuLV), and from BALB/c mice (controls, M-MuLV-free). Chromosomal damage was determined in vitro on lymphocytes stimulated with concanavalin A (Con A) and incubated for two generation cycles with bromodeoxyuridine (BUdR). The baseline frequency of SCEs was significantly higher in untreated BALB/Mo than in BALB/c lymphocytes. The frequencies of SCEs were significantly increased by increasing doses of MMC in both BALB/c and BALB/Mo T lymphocytes. Treatment with a low dose of MMC (0.3 mg/kg) produced an additive effect on SCE frequency in BALB/Mo lymphocytes, which was gradually suppressed by increasing the MMC concentration (3-5 mg/kg). Indeed, the levels of SCEs became significantly lower in BALB/Mo than in BALB/c lymphocytes at the highest MMC concentration tested (10 mg/kg), indicating that a negative synergistic effect was eventually produced. Chromosomal aberrations (breaks and total aberrations) were significantly increased by the highest MMC doses (5-10 mg/kg) and were more frequent in BALB/Mo than in BALB/c lymphocytes at 10 mg/kg MMC. The frequencies of micronuclei were increased by all MMC doses and were significantly higher in BALB/Mo than in BALB/c lymphocytes at 10 mg/kg MMC. These results are referred to interferences of M-MuLV and MMC with the function of enzymes, such as DNA topoisomerases, involved in the mechanism of SCE production.
Animals↗
The scintillometric evaluation of DNA repair synthesis can be distorted by changes of thymidine pool radioactivity.
The induction of DNA repair synthesis by UV radiation and methylmethane sulphonate (MMS) in mammalian cell lines of human (EUE, HeLa, FT, KB) and hamster (CHO, BHK) origin has been evaluated by means of autoradiography and the scintillometric procedure which implied the use of hydroxyurea (HU) to suppress DNA replication. While with UV radiation both methods produce concordant positive results, in the case of MMS the evidence of DNA repair synthesis obtained from the autoradiograms is occasionally accompanied by a lack of increase of DNA radioactivity in the treated cultures, as detected by scintillation counting. In such instances MMS is shown to reverse the enhancement of pool radioactivity in the cultures incubated with HU and even to reduce the radioactivity of thymidine pool below control values. By normalizing DNA radioactivities on the basis of pool variations, the discrepancy between autoradiography and scintillation counting is solved. The chromatographic analysis of thymidine pool components justifies the normalization procedure as it demonstrates that also in cultures treated with MMS or MMS + HU pool variations closely parallel the variations of thymidine triphosphate (dTTP) level. The normalization of DNA radioactivities based on the overall pool radioactivities gives an improved evaluation of the actual rate of DNA synthesis. It can be recommended for screening studies of DNA repair inducers because it allows one to correct false negative results without producing false positive data. Compared with the dTTP levels, overall pool radioactivities used as normalizing factors still produce an underestimate of DNA repair when high doses of MMS are applied to hamster cell cultures.
Animals↗
Induction of DNA repair synthesis by ultraviolet radiation and methylmethanesulphonate in cultured mouse lymphocytes.
The induction of DNA repair synthesis by UV-radiation and methyl-methanesulphonate (MMS) was studied in mouse lymphocytes and leukemic cells by means of autoradiography and scintillation counting, after labelling in vitro with tritiated thymidine ([3H]dThd). Repair stimulation was detected by both procedures in LSTRA and YC8 leukemic cell lines as well as in primary fibroblasts of BALB/c and BALB/Mo mice. No stimulation was observed in primary cultures of lymphocytes from the spleen, thymus and lymph-nodes of the same mice. In primary lymphocytes neither stimulation with concanavalin A (Con A) nor pre-incubation with 5-bromodeoxyuridine (BUdR) were effective in making evident DNA repair. The data put into question the reliability of the repair test for the prediction of carcinogenic potential of chemicals.
Animals↗
Effects of potassium dichromate on ATP content of mammalian cells cultured in vitro.
In order to elucidate the mechanism of the cytotoxic activity of hexavalent chromium (Cr(VI)), the alterations of intracellular ATP levels induced by potassium dichromate in cultured hamster fibroblasts (BHK line) have been studied. Two kinds of treatment procedures were adopted: (1) BHK cell suspensions were exposed to 0.05--1.00 mM K2Cr2O7 in Hanks' balanced salt solution (BSS) for up to 180 min and ATP concentrations were determined immediately after the exposure to Cr(VI). A decrease of ATP content was observed with 0.25--12.00 mM K2Cr2O7 but only in the case of the highest dose was it related in a linear fashion to the duration of the treatment. (2) Cells were preincubated in BSS for 30 min with 0.05--1.00 mM dichromate. They were then reincubated in Eagle's minimal essential medium (MEM) for up to 180 min and ATP was measured at different time points. Immediately after the exposure to chromium all the treated cultures showed a depletion of ATP content. However while the cells treated with 0.25--0.25 mM dichromate rapidly resumed ATP levels very similar to that of the control, no recovery was detected in cells treated with 0.50 and 1.0 mM K2Cr2O7, even after 180 min. The observed effects have been attributed to the oxidizing activity of Cr(VI), which subtracts electrons from electron donors involved in metabolic pathways producing ATP, and to the ability of Cr(III), deriving from Cr(VI) reduction, to form stable coordination complexes with ATP precursors and enzymes involved in ATP synthesis.
Adenosine Triphosphate↗
Hypocupremia-related hypochromic anemia during D-penicillamine treatment.
Explore the source record for details and available documents.
Anemia, Hypochromic↗
Nucleotide pool unbalance induced in cultured cells by treatments with different chemicals.
Intracellular nucleotide pools directly influence a great number of cellular activities, and their size and composition are easily altered by chemical agents. However, they are seldom examined when chemical toxicity is investigated. The importance of evaluating the effects produced at the nucleotide pool level is also discussed. Some examples are reported in which the activity of a tested chemical on nucleic acid metabolism could be detected only by taking into account the modifications induced in the precursor pool. One example is the case of hexavalent chromium, which inhibits DNA and RNA syntheses, but also affects the uptake of exogenous nucleosides, thus modifying the labelling pattern of nucleic acids in treated cells. A similar situation is observed with alkylating agents (e.g. MMS and EMS), whose induction of DNA repair synthesis is sometimes masked by the concomitant depletion of labelled nucleotides in the intracellular pool.
Animals↗
Effects of hexavalent chromium on the adenylate pool of hamster fibroblasts.
The mechanisms through which Cr(VI) reduces the intracellular levels of ATP in hamster fibroblasts (BHK 21 line) were investigated. Leakage of pool components during treatment of cells prelabelled with [3H]adenosine was examined. Adenylate pool composition was analyzed by thin-layer chromatography (TLC) in cells incubated with [3H]adenosine immediately after treatment with Cr(VI), and the endogenous concentrations of different nucleotides were determined by high-performance liquid chromatography (HPLC). Interference of Cr(VI) with different steps of ATP biosynthesis could be assessed. Treated cells showed increased loss of radioactive hypoxanthine and inosine in the treatment medium, but no nucleotides were detected outside the cells. A marked unbalance of the endogenous adenylate pool was produced by chromium, consisting in a strong decrease of ATP, accompanied by a very pronounced increase of ADP and AMP. Treatment with Cr(VI) caused an altered distribution of label among the different adenylate pool components, especially an accumulation of radioactivity in the ADP and AMP fractions. Also other effects on the soluble pool were detected by HPLC analysis, namely the reduction of NAD content and variations in the guanylate pool which closely paralleled the ones observed in the adenylate pool. Energy charge values, calculates on the basis of HPLC and TLC data, allowed to detect a specific inhibition of ADP phosphorylation to ATP. An interference with the early steps of adenylate synthesis could be inferred from the enhanced amounts of hypoxanthine and inosine in the treated cultures.
Adenine Nucleotides↗