Sensitivities of peripheral lymphocytes from healthy humans and cancer patients to induction of sister chromatid exchanges by genotoxicants.
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Adult↗
Biomedical subjects
A Oikawa
Publications and source records attributed to A Oikawa.
Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens.
3-Aminoharman (3AH, 3-amino-1-methyl-9H-pyrido[3,4-b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.
Biotransformation↗
Stimulation of tyrosinase activity of cultured melanoma cells by lysosomotropic agents.
The tyrosinase (EC 1.14.18.1) activity of cell-free extracts (TyH) of B16 melanoma cells cultured in the presence of 5 to 10 mM ammonium chloride was considerably higher than that of cells from control cultures. This increase in TyH in the presence of ammonium chloride seemed to be due to de novo synthesis of the enzyme, because it was inhibited by 1 microgram/ml of cycloheximide. In the presence of the latter, however, ammonium chloride did increase the tyrosinase activity of living cells in culture (TyC) resulting in about threefold increase in the TyC/TyH ratio, a measure of the extent of tyrosinase reaction exerted by the enzyme present in living cells. This higher TyC/TyH ratio induced by ammonium chloride was also observed in the absence of cycloheximide. Similar increases in TyH, TyC, and TyC/TyH occurred in the presence of methylamine or ethylamine instead of ammonium chloride, but not in the presence of tetraethylammonium chloride, and also in culture medium of higher pH. The apparently similar effects of lysosomotropic bases and medium of higher pH on the TyC/TyH ratio suggest that there are some mechanisms that control the intramelanosomal pH lower than the cytoplasmic pH.
Ammonium Chloride↗
Differential features of sister-chromatid exchange responses to ultraviolet radiation and caffeine in xeroderma pigmentosum lymphoblastoid cell lines.
Sister-chromatid exchange (SCE) induced by ultraviolet (UV) irradiation and viability after UV irradiation were studied in lymphoblastoid cell lines derived from 7 patients with xeroderma pigmentosum (XP) and 6 normal donors. UV irradiation caused significant increases of SCEs in both XP and normal cells. In 3 XP cell lines, which were deficient in unscheduled DNA synthesis (UDS) and sensitive to the killing effect of UV, very high SCE frequencies were observed after UV irradiation. Cells from a patient with the De Sanctis-Cacchione syndrome were the most sensitive to UV in terms of both SCE induction and cell killing. In 2 of 4 UDS-proficient XP cell lines tested, the incidences of UV-induced SCEs were similar to those in normal cell lines, but in 2 other UDS-proficient lines from 2 XP patients with skin cancer, the frequencies of UV-induced SCEs were significantly higher than in normal cells. Continuous post-UV treatment with 1 mM caffeine markedly enhanced UV-induced SCEs in 3 of 4 UDS-proficient XP cell lines but had only slight effects on cells from the 4th UDS-proficient XP patient and from normal individuals.
Caffeine↗
Actions of amino-beta-carbolines on induction of sister-chromatid exchanges.
Synthetic 3-aminoharman and 3-aminonorharman (amino-beta-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-beta-carbolines are ranked between 2-amino-alpha-carboline and 2-amino-6-methyl-9a-aza-delta-carboline (Glu-P-2) and much lower than 3-amino-gamma-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-beta-carboline, harman and norharman had very weak, if any, SCE-inducer activity. Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[a]pyrene.
Animals↗
Presence of 3-amino-beta-carbolines (3-amino-9H-pyrido[3,4-b]indoles), effectors in the induction of sister-chromatid exchanges, in protein pyrolysates.
3-Amino-9H-pyrido[3,4-b]indole (3-aminonorharman) and 3-amino-1-methyl-9H-pyrido[3,4-b]indole (3-aminoharman), effectors for induction of sister-chromatid exchanges in human lymphoblastoid cells, were identified in pyrolysates of casein by high-performance liquid chromatography and UV spectrophotometry, together with some other mutagenic/carcinogenic carbolines. In pyrolysates from a preparation of zein, which contained little tryptophan, only norharman and harman were found in small amounts.
Animals↗
Sensitivities of peripheral lymphocytes from healthy humans to induction of sister chromatid exchanges by chemicals.
The spontaneous sister chromatid exchanges (SCEs) and SCEs induced by 50 microM methyl methanesulfonate, 1 microM 4-nitroquinoline 1-oxide, and 5 microM 3-amino-1-methyl-5H-pyrido[4,3-b]indole were examined in phytohemagglutinin-activated peripheral lymphocytes from 53 healthy adult donors. The means of the mean spontaneous SCEs and SCEs induced by methyl methanesulfonate, 4-nitroquinoline 1-oxide, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole in cells of these donors had coefficients of variability of 12, 14, 24, and 28%, respectively. Thus, it was possible to estimate the SCE-inducing activities of chemicals in peripheral lymphocytes from a population of healthy humans under the defined experimental conditions. These facts provide one of the useful parameters for evaluating the genotoxicities of chemicals to human beings, a genetically heterogeneous species. Cells from two of the donors were considerably more sensitive to particular chemicals than were those from other donors, consistently giving higher numbers of induced SCEs in two repeat examinations.
4-Nitroquinoline-1-oxide↗
Chromatin structure interferes with excision of abnormal bases from DNA.
Cell-free extracts of human lymphoblastoid cells NL3 excised almost all uracil residues from free DNA with misincorporated dUMP, but only about half the uracil residues from nuclei, chromatin and reconstituted chromatin with dUMP-misincorporated DNA. This difference in susceptibility to uracil-DNA glycosylase of free and complexed DNAs was similar to the difference in susceptibility of free and complexed methylated DNAs to 3-methyladenine-DNA glycosylase. Methylated poly(dA-dT) was also protected by formation of complexes with calf thymus chromosomal proteins. It seems that the nucleosome structure prevents the action of DNA glycosylases. The very high sensitivity of PBS1 phage DNA, which contains uracil as a natural component, in complexes with calf thymus chromosomal proteins as well as in the free form [1] was confirmed. This high sensitivity seems ascribable to the high uracil content of PBS1 DNA. Methylated nucleosome monomers and dimers, and reconstituted nucleosome monomers containing methylated DNA of about 150 bp length, were considerably more resistant to 3-methyladenine-DNA glycosylase than chromatin reconstituted from methylated DNA of longer chain length. This may be due to the lower proportion of linker regions of free form stretches of the DNA chain in nucleosome oligomers.
Cell Line↗
Induction of supermelanin synthesis and morphological changes in interspecific reconstituted cells and its reversal by tumor promoter.
Chloramphenicol-resistant (CAPr) reconstituted cells and cybrids were isolated by fusion of karyoplasts (or intact cells) of mouse amelanotic melanoma B16 cells with cytoplasts of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) -deficient, CAPr rat myoblastic cells, L6TG.CAPr, and double selection in HAT medium containing CAP. Reconstituted cells or cybrids exhibited unique cellular arrangement, and about one third of the isolated clones expressed high tyrosinase activity and marked melanin synthesis, although the parental mouse cells expressed low tyrosinase activity and the parental rat cells did not express tyrosinase activity. These phenotypic changes have been stable for more than a year. The phenotypic reversions of these clonal cells were induced by treatment with a tumor promoter. There were changes in the morphology of the treated cells to that of the mouse B16 cells and extinction of tyrosinase activity and melanin synthesis in pigmented clonal cells. These phenotypic changes and reversions induced by a promoter were repeatedly reversible.
Animals↗
In vitro induction of HLA-restricted cytotoxic T lymphocytes against autologous Epstein-Barr Virus transformed B lymphoblastoid cell line.
Cytotoxic T lymphocytes (CTL) against autologous EBV-transformed B lymphoblastoid cell line (LCL) were induced in vitro by culturing peripheral blood lymphocytes (PBL) of healthy donors together with mitomycin C-treated autologous LCL for 6 days. The cytotoxic cells developed only from the E-rosette-positive fraction but not from the negative fraction of PBL. These CTL killed autologous LCL but not PWM-stimulated autologous PBL. In addition, the CTL killed allogeneic LCL when at least 1 of the HLA-A antigens was identical with that of the LCL of CTL donor. However, identity of HLA-B and HLA-C antigens was not enough for a significant killing of allogeneic LCL. The specificity of the CTL was also confirmed by a cold target inhibition test. These results indicated that the CTL induced specifically recognized EBV-transformed cells with HLA restriction.
Animals↗
Action of DNA glycosylases on chromatin structure.
Nuclei of human lymphoblastoid cells NL3 incorporated dUMP into DNA in vitro. The rate and extent of this incorporation were less than those of dTMP, presumably due to the presence of dUTPase and uracil-DNA glycosylase in nuclear preparations. NL3 cell extracts released only about a half of the misincorporated uracil as free base from nuclei, their chromatin and chromatin reconstituted from dUMP-containing DNA and calf thymus chromosomal protein, though almost all uracil from reconstituted chromatin containing PBS1 phage DNA. The cell extracts also release 10-40% of 3-methyladenine from methylated nucleosome monomers and dimers, and chromatins reconstituted from methylated DNA and poly (dA . dT). The shorter molecule lost fewer 3-methyladenine.
Cell Line↗
Inhibitors of poly(adenosine diphosphate ribose) polymerase induce sister chromatid exchanges.
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Animals↗
Synthesis and degradation of tyrosinase in cultured melanoma cells.
The tyrosinase (EC 1.14.18.1) activity of cultured B-16 mouse melanoma cells (C2M) in the stationary phase depends greatly on whether the culture medium contains glucose or galactose. The activity in medium containing galactose was about ten times that in medium containing glucose at pH 7.2. This difference in tyrosinase activity was concluded to be due to a shift of balance between synthesis and degradation of the enzyme. Experiments were conducted with stationary phase cultures in the presence of cycloheximide. The melanoma cells did not synthesize tyrosinase in medium containing glucose in the stationary phase. But when they were cultured under identical conditions, except that glucose was replaced by galactose, they continued to synthesize tyrosinase. The rate of synthesis in medium containing galactose at pH 6.3 was one third of that in the same medium at about pH 7, in which the increase in specific activity of tyrosinase per day was about 30 nmoles/mg cell protein per hr. The rate of degradation of the enzyme was practically the same in medium containing glucose as in medium containing galactose, and largely depended on the pH of the culture medium. At pH 6.3, the half-life was about one third of that at pH 7.2, where it was about 1.8 days. The degradation at acidic pH values was much reduced by ammonium salt and was strongly inhibited by the protease inhibitor, leupeptin.
Ammonium Chloride↗
Induction of sister-chromatid exchanges by mutagens from amino acid and protein pyrolysates.
Sister-chromatid exchanges (SCEs) in a permanent cell line of human lymphoblastoid cells were induced by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-9H-pyrido[2,3-b]indole (2-amino-alpha-carboline). The first two compounds were found in tryptophan pyrolysates, the third in a glutamic acid pyrolysate and the last in a globulin pyrolysate. All these compounds required the metabolic activation system (S9 mix) for induction of SCE. Trp-P-2 had the highest SCE-inducing activity of these chemicals (approximately equivalent to that of aflatoxin B1), followed by Trp-P-1, Glu-P-1 and then 2-amino-alpha-carboline.
Carbolines↗
Establishment of autoantibody-producing cell lines from peripheral blood lymphocytes of patients with systemic lupus erythematosus.
Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.
Adolescent↗
Epstein-Barr virus-transformed human lymphoblastoid cells for study of sister chromatid exchange and their evaluation as a test system.
Epstein-Barr virus-transformed human lymphoblastoid cell lines are suitable for detection of sister chromatid exchange (SCE) induced by mutagens-carcinogens because they have shown a stable chromosome number and stable frequency of spontaneous SCE for more than two years in culture. Their spontaneous and induced SCE frequencies were practically the same as those of phytohemagglutinin-stimulated lymphocytes from the same blood donors. The SCE responses of one established cell line, NL3, to 13 typical mutagens and five nonmutagens were examined. This cell line responded to all the mutagens tested but not to the nonmutagens. The SCE-inducing activities of these chemicals were well correlated with their mutagenic activities assayed with the Salmonella system by Ames' and Sugimura's groups, although there were a few but significant deviations.
Carcinogens↗
Actions of human DNA glycosylases on uracil-containing DNA, methylated DNA and their reconstituted chromatins.
Extracts of human lymphoblastoid cells catalyzed complete release of uracil (Ura) from PBS1 DNA, which contains Ura instead of thymine as a normal component (Ura-DNA), and 3-methyladenine (3-MeAde) from DNA methylated with methyl methanesulfonate (Me-DNA). These two activities, Ura-DNA glycosylase and 3-MeAde-DNA glycosylase, differed in heat stability. Cell extracts released Ura more rapidly and 3-MeAde more slowly from alkali-denatured preparations of Ura- and Me-DNA, respectively, than from native DNA's. On incubation with reconstituted chromatins, prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein by salt gradient dialysis, cell extracts released all the Ura but only about half of the 3-MeAde residues, although both these chromatins were degraded by micrococcal nuclease until about half of the nucleotides became acid soluble. The activities of Ura-DNA and 3-MeAde-DNA glycosylase of xeroderma pigmentosum cells were similar to those of normal cells.
Animals↗
DNA glycosylases of human lymphoblastoid cells.
Lymphoblastoid cell lines were established after transformation by Epstein-Barr virus of peripheral lymphocytes from xeroderma pigmentosum (XP) patients and normal donors. These lines expressed B-lymphocyte characteristics. Typical characteristics related to XP of these cell lines were not altered by transformation. Extracts of these cells catalyzed release of uracil (Ura) and 3-methyladenine (3MeAde) from Ura-containing DNA (Ura-DNA) and methylated DNA (Me-DNA), respectively. These two activities, Ura-DNA glycosylase and 3MeAde-DNA glycosylase, differed in heat stability. Extracts released Ura more rapidly and 3MeAde more slowly from a single-stranded DNA than from a double-stranded DNA. On incubation with reconstituted chromatins prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein, cell extracts released all the Ura but about half the 3MeAde residues. The activity levels of these two enzymes of XP cells were similar to those of normal cells.
B-Lymphocytes↗