In vitro induction of chromosome damage by sulphasalazine in human lymphocytes

Two different endpoints, sister-chromatid exchange and micronucleus induction, were measured in human peripheral blood lymphocytes stimulated to divide in short-term in vitro cultures. The cultures were exposed to sulphasalazine and 6 of its metabolites for 72 h in the absence of any exogenous metabolic activation system. Analysis of the sister-chromatid exchange and micronuclei frequencies clearly indicates that sulphasalazine itself is capable of inducing both sister-chromatid exchange and micronuclei while sulphapyridine and its acetylated metabolites only induce sister-chromatid exchange. 5-Aminosalicylic acid, the therapeutic moiety of sulphasalazine, and its acetylated metabolite did not induce either sister-chromatid exchange or micronuclei at the concentrations tested. The data from these in vitro experiments are discussed in relation to the previously reported elevations in sister-chromatid exchange and micronucleus frequencies in inflammatory bowel disease patients receiving sulphasalazine therapy.     

The cytogenetic effect of bleomycin on human peripheral lymphocytes in vitro and in vivo.

The cytogenetic effect of bleomycin (BLM) in human lymphocytes was studied after exposure to different doses during the G0 and G2 phases. BLM produced a marked specific effect on the cell cycle. The main aberration types after exposure in tg0 were dicentrics and deletions; and after exposure in G2, open chromatid breaks. A linear dose--response was calculated for all these aberration types as well as for the number of aberrant cells. In the G2 experiments, partially and totally pulverized cells also increased linearly with dose. The intercellular distributions of the most frequent aberration types after exposure in G0 and G2--the dicentrics and chromatid breaks, respectively--showed over-dispersion. These results show that the cytogenetic effect of BLM may be compared with that of densely ionizing irradiation. Preliminary results of chromosome analysis of three cancer patients in the course of BLM therapy showed effects similar to those in the G0 experiments.     

Induction of complete and incomplete chromosome aberrations by bleomycin in human lymphocytes

Bleomycin (BLM) is a clastogenic compound, which due to the overdispersion in the cell distribution of induced dicentrics has been compared to the effect of high-LET radiation. Recently, it has been described that in fibroblast derived cell lines BLM induces incomplete chromosome elements more efficiently than any type of ionizing radiation. The objective of the present study was to evaluate in human lymphocytes the induction of dicentrics and incomplete chromosome elements by BLM. Peripheral blood samples have been treated with different concentrations of BLM. Two cytogenetic techniques were applied, fluorescence plus Giemsa (FPG) and FISH using pan-centromeric and pan-telomeric probes. The observed frequency of dicentric equivalents increases linearly with the BLM concentration, and for all BLM concentrations the distribution of dicentric equivalents was overdispersed. In the FISH study the ratio between total incomplete elements and multicentrics was 0.27. The overdispersion in the dicentric cell distribution, and the linear BLM-concentration dependence of dicentrics can be compared to the effect of high-LET radiation, on the contrary the ratio of incomplete elements and multicentrics is similar to the one induced by low-LET radiation (~0.40). The elevated proportion of interstitial deletions in relation to total acentric fragments, higher than any type of ionizing radiation could be a characteristic signature of the clastogenic effect of BLM.     

Hyperthermic enhancement of chromosome damage and lack of effect on sister-chromatid exchanges induced by bleomycin in Chinese hamster cells in vitro.

Chinese hamster cells, M-3, were treated with BLM (1--4 micrograms/ml) for 30 min to 1 h at 37 degrees or 43 degrees C. After treatment, the cells were reincubated at 37 degrees until recovery. The material treated at 43 degrees showed increased damage expressed as chromosome and chromatid-type breaks and exchanges. Since the amount of BLM entering the cell at 37 degrees is supposedly similar to that which enters the cell at 43 degrees, the enhanced damage is the result of true synergism, and not the facilitation of the drug's entry into the cell.     

Cytogenetic effect of bleomycin on human leukocytes in vitro.

Human leukocytes treated with bleomycin (BLM) for clinical use, at concentrations of 0.1, 0.5, 1.0, 10 and 50 mug/ml were studied. Both chromosome- and chromatid-type aberrations were observed. The groups of larger chromosomes were more affected at every concentration. At dosages from 0.1 to 10 mug/ml no significant difference of effects on chromosomes was observed. However, a dose-difference of about 500 times showed significant differences in effect both on the degree of chromosomal aberration and on mitotic indices.     

The effect of paracetamol on oxidative damage in human peripheral lymphocytes

Unscheduled DNA synthesis (UDSox) and lipid peroxidation (LPO) induced by non-enzymatic activation of molecular oxygen (Fe2+ +H2O2) were measured in human peripheral lymphocytes from healthy volunteers. The effect of paracetamol (PC) in a final concentration range of 0.05-10 mmole/l on these oxidative processes and on DNA repair induced by MNNG (UDSmut) was investigated. The level of induced LPO was measured by the thiobarbituric acid assay, UDSox and UDSmut were determined by scintillometric measurement of incorporated [methyl-3H]thymidine into damaged DNA. PC at concentrations lower than 1 mmole/l significantly potentiates the non-enzymatically induced LPO and UDSox with the maximum of the activation being around 0.1 mmole/l. In contrast, PC at concentrations higher than 1 mmole/l exhibits an inhibitory effect on both LPO and UDSox. On the other hand, concentrations higher than 1 mmole/l significantly suppressed DNA-repair synthesis induced by MNNG.     

The effect of increased intakes of polyunsaturated fatty acids and vitamin E on DNA damage in human lymphocytes.

The effect of increasing dietary intakes of polyunsaturated fatty acids (PUFAs) and vitamin E on indices of oxidative DNA damage was investigated. Twenty-one healthy male, nonsmokers aged 28.9 +/- 1.3 years participated in a free-living, split plot/change over trial in which half the volunteers consumed diets containing 5% PUFA as food energy for 4 wk and, after a 10 wk washout period, consumed a 15% PUFA diet for another 4 wk. The other volunteers followed an identical protocol, except that they consumed the 15% PUFA diet first. The diets were provided to volunteers either with or without an additional 80 mg dalpha-tocopherol acetate/day; otherwise total fat, carbohydrates, protein, and basal vitamin E contents remained unchanged. DNA damage induced by 200 microM H(2)O(2) in lymphocytes from volunteers as well as endogenous DNA damage in the form of oxidized pyrimidines, measured by alkaline single-cell gel electrophoresis (the comet assay), significantly decreased after consumption of the 5% PUFA diet (P<0.001 and P=0.01, respectively), but significantly increased after consumption of the 15% PUFA diet when alpha-tocopherol levels were in the range of 5-7 mg/day (P=0. 008 and P=0.03, respectively). These changes were abolished by an additional 80 mg dalpha-tocopherol/day. This study indicates that increasing dietary levels of PUFA to 15% may adversely affect some indices of DNA stability. However, increasing the dietary intake of vitamin E by 80 mg/day ameliorates the damaging effects of PUFA. -Jenkinson, A. McE., Collins, A. R., Duthie, S. J., Wahle, K. W. J., Duthie, G. G. The effect of increased intakes of polyunsaturated fatty acids and vitamin E on DNA damage in human lymphocytes.     

Comparative studies on the specificity of anticlastogenic action in human lymphocytes in culture

Summary Comparative studies on human lymphocyte cultures yielded a certain specificity of the anticlastogenic action of the SH compounds 1-cysteine, cysteamine, and -aminoethylisothiouronium (AET) as well as of the amide 1-asparagine and the amino acid 1-methionine. This specific anticlastogenic activity manifested itself in specific changes of the spectrum of aberration types induced by the clastogens and of the pattern of intercellular distribution of the induced aberrations. It was clearly dependent on the concentration of the anticlastogens but was also influenced by the used clastogen. The use of different culture media yielded some quantitative influences on the anticlastogenic activity, but fundamental changes in the spectrum of anticlastogenic action have not been observed except with cysteamine. The patterns of activity ascertained for the different anticlastogens specifically differed from those changes in the spectrum and pattern of distribution of aberrations induced by a mere reduction of the concentration for instance of Trenimon. Therefore a direct reaction between the protectors and the clastogen Trenimon as the cause of the observed anticlastogenic action was again excluded. The presented data are also discussed under the aspects of the hypotheses of aberration induction as well as of their importance for further antimutagen research.Some parts of this paper have been supported by grants of Deutsche Forschungsgemeinschaft.     

Influence of GSTM1 genotype on comet assay and chromosome aberrations after induction by bleomycin in cultured human lymphocytes

Investigators have demonstrated that the mutagen sensitivity assay, based on the quantification of bleomycin (BLM)-induced chromatid breaks in short-term cultured peripheral lymphocytes, can be a marker of cancer susceptibility. Although many factors can contribute to variability in human biomonitoring studies, genetic susceptibility (the influence of polymorphic metabolising genes on response to environmental mutagens) should be considered whenever appropriate. Glutathione-S-transferases (GSTs) encode a family of detoxifying phase II enzymes catalysing the conjugation of glutathione to electrophilic compounds. Studies on Caucasians indicate that about 45% of individuals lack the glutathione-S-transferase M1 (GSTM1, null) enzyme, and are therefore, theoretically at a higher risk to the toxic effects of chemicals. The aim of the present study was to investigate this hypothesis further by evaluating whether the GSTM1 genotype influences the backround level of DNA damage and the induction of chromosomal aberrations by BLM in peripheral-blood lymphocytes. The alkaline comet assay was used to evaluate background levels of DNA damage in unstimulated lymphocytes while standard cytogenetic techniques were used in mitogen-stimulated lymphocytes treated with BLM. Without BLM treatment, individuals with the GSTM1 null genotype had no significant difference in frequencies of damaged cells by comparison to individuals with the GSTM1 genotype. Also, no significant differences between the two groups of individuals (GSTM1 positive and GSTM1 null) were observed for BLM-induced chromosomal aberrations.     

Regulation of various proteolytic pathways by insulin and amino acids in human fibroblasts

Intracellular protein degradation is a regulated process with several proteolytic pathways. Although regulation of macroautophagy has been investigated in some detail in hepatocytes and in few other cells, less is known on this regulation in other cells and proteolytic pathways. We show that in human fibroblasts insulin and amino acids reduce protein degradation by different signalling pathways and that this inhibition proceeds in part via the mammalian target of rapamycin, especially with amino acids, which probably increase lysosomal pH. Moreover, the regulatory amino acids (Phe, Arg, Met, Tyr, Trp and Cys) are partially different from other cells. Finally, and in addition to macroautophagy, insulin and amino acids modify, to different extents and sometimes in opposite directions, the activities of other proteolytic pathways.     

Inhibition of chromosome repair by caffeine or isonicotinic acid hydrazide on chromosome damage induced by mitomycin C in human lymphocytes

The frequency of interchromatic exchanges induced by mitomycin C in cultured human lymphocytes was markedly lowered in the presence of caffeine or isonicotinic acid hydrazide (INH) during a post-treatment period. The autoradiographic experiment showed that the decrease in the exchange frequency did not result from delaying or cell-killing effects by the post-treatment with caffeine or INH. Therefore, it may deduced that the exchange formation closely related to a process sensitive to caffeine or INH.     

The effect of selected amino acids on in vitro motility of mouse sperm

The effect of glutamic acid, alanine, glycine, arginine, leucine, and cystine on the motility of mouse sperm was investigated. In comparison with the control, sperm motility was increased only following incubation in glutamic acid at a concentration of 0.05 mg/ml, concentrations of 0.25 mg/ml and 1.00 mg/ml decreasing the motility of spermatozoa. Alanine, glycine, arginine, and leucine at all concentration used decreased the percentage of motile sperms, while cystine did not cause any visible changes in their motility.