DNA Synthesis In Human Lymphocytes Research Articles

Unscheduled DNA synthesis in human lymphocytes treated with cigarette smoke condensate and its semivolatile fraction.

A cigarette smoke condensate (CSC) and the semivolatile fraction (SVF) from the CSC were tested for their ability to induce unscheduled DNA synthesis (UDS) in human lymphocytes. Exposed cells were incubated with tritiated thymidine and the UDS was determined by liquid scintillation counting of precipitated DNA. The tests were performed with and without metabolic activation by liver fraction (S9) from rats induced with Aroclor 1254. No UDS activity was detected, neither for the CSC nor for the SVF. Two control substances, benzo(a)pyrene and methyl methanesulfonate, increased the UDS activity in tests with and without metabolic activation, respectively. The scintillometric method used for detection of UDS was not sufficiently sensitive to be used as a tool for genetic risk assessment of CSC fractions.

Final Report of the Safety Assessment of Niacinamide and Niacin1

Niacinamide (aka nicotinamide) and Niacin (aka nicotinic acid) are heterocyclic aromatic compounds which function in cosmetics primarily as hair and skin conditioning agents. Niacinamide is used in around 30 cosmetic formulations including shampoos, hair tonics, skin moisturizers, and cleansing formulations. Niacin is used in a few similar product types. The concentration of use of Niacinamide varies from a low of 0.0001% in night preparations to a high of 3% in body and hand creams, lotions, powders and sprays. Niacin concentrations of use range from 0.01% in body and hand creams, lotions, powders and sprays to 0.1% in paste masks (mud packs). Both ingredients are accepted for use in cosmetics in Japan and the European Union. Both are GRAS direct food additives and nutrient and/or dietary supplements. Niacinamide may be used in clinical treatment of hypercholesteremia and Niacin in prevention of pellegra and treatment of certain psychological disorders. Both ingredients are readily absorbed from skin, blood, and the intestines and widely distribute throughout the body. Metabolites include N1-methylnicotinamide and N1-methyl-4-pyridone-3-carboxamide. Excretion is primarily through the urinary tract. While Niacinamide ismore toxic than Niacinin acute toxicity studies, both are relatively non-toxic. Short-term oral, parenteral, or dermal toxicity studies did not identify significant irreversible effects. Niacinamide, evaluated in an in vitro test to predict ocular irritation, was not an acute ocular hazard. Animal testing of Niacinamide in rabbits in actual formulations produced mostly non-irritant reactions, with only some marginally irritating responses. Skin irritation tests of up to 2.5% Niacinamide in rabbits produced only marginal irritation. Skin sensitization tests of Niacinamide at 5% during induction and 20% during challenge were negative in guinea pigs. Neither cosmetic ingredient was mutagenic in Ames tests, with or without metabolic activation. Niacinamide and Niacin at 2 mg/ml were negative in a chromosome aberration test in Chinese hamster ovary cells, but did produce large structural chromosome aberrations at 3 mg/ml. Niacinamide induced sister chromatid exchanges in Chinese hamster ovary cells, but Niacin did not. Under certain circumstances, Niacinamide can cause an increase in unscheduled DNA synthesis in human lymphocytes treated with UV or a nitrosoguanidine compound. Niacinamide itself was not carcinogenic when administered (1%) in the drinking water of mice. No data on the carcinogenic effect of Niacin were available. Niacinamide can moderate the induction of tumors by established carcinogens. Niacinamide in combination with streptozotocin (a nitrosourea compound) or with heliotrine (a pyrrolizidine alkaloid), produced pancreatic islet tumors. On the other hand, Niacinamide reduced the renal adenomas produced by streptozotocin; and intestinal and bladder tumors induced by a preparation of bracken fern. Niacinamide evaluated in in vitro test systems did affect development, but Niacinamide reduced the reproductive/developmental toxicity of 2-aminonicotinamide-amino-1,3,4-thiadiazole hydrochloride and urethane. Clinical testing of Niacinamide produced no stinging sensation at concentrations up to 10%, use tests produced no irritation at concentrations up to 5%, and a 21-day cumulative irritation test at concentrations up to 5% resulted in no irritancy. Niacinamide was not a sensitizer, nor was it a photosensitizer. The CIR Expert Panel considered that Niacinamide and Niacin are sufficiently similar from a toxicologic standpoint to combine the available data and reach a conclusion on the safety of both as cosmetic ingredients. Overall, these ingredients are non-toxic at levels considerably higher than would be experienced in cosmetic products. Clinical testing confirms that these ingredients are not significant skin irritants, sensitizers or photosensitizers. While certain formulations were marginal to slight ocular irritants, other formulations were not. Niacinamide, while not carcinogenic alone, can modulate the induction of tumors by certain established carcinogens. The Panel noted that the doses in these studies are high relative to the low concentrations at which Niacinamide is used in cosmetic formulations. In neither case (tumor protection or tumor promotion) are these findings considered relevant to the use of Niacinamide at its current low concentrations of use in cosmetics. Both ingredients were considered safe as used in cosmetics.

Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes

Deoxycytidine kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-β- d-arabinofuranosylcytosine, difluorodeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl–Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

Activation of deoxycytidine kinase during inhibition of DNA synthesis in human lymphocytes.

Deoxycytidine kinase was shown to be activated during 2-chlorodeoxyadenosine (CdA) treatment of human lymphocytes, under the conditions when the DNA synthesis is inhibited. As the increase of dCK activity was shown in crude protein extracts, without an increase in the amount of dCK protein, shown by immunostaining after SDS-PAGE, a secondary modification of the protein structure was considered. NaF treatment of cells in the concentration range of 5-20 mM gave a similar activation of dCK, suggesting a possible role of phosphatases and/or a possibility of a G-protein related phenomenon. Using the same conditions, no effect of CdA or NaF was found on the thymidine kinase activity of cell extracts. Alternatively, activation of catabolic pathways could be considered, however, the increase in dCK activity was not influenced either by the removal of 5'-nucleotidases, or by the inhibition of deaminases.

Geranylgeranylpyrophosphate, a Metabolite of Mevalonate, Regulates the Cell Cycle Progression and DNA Synthesis in Human Lymphocytes

We investigated the role of the intrinsic mevalonate cascade in DNA synthesis and cell cycle progression in human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). PHA stimulated the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA prior to the DNA synthesis (S phase). Pravastatin, an HMG-CoA reductase inhibitor, inhibited DNA synthesis and blocked the entry to S phase in PHA-stimulated PBMC. Mevalonate restored these inhibitory effects. Thus, we examined two major metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), using a novel liposome system for uptake into the cells. GGPP, not FPP, restored the pravastatin-induced inhibitions. These data indicated that 1) the intrinsic mevalonate cascade plays critical roles for the entry to S phase and DNA synthesis, and that 2) GGPP is an essential metabolite of mevalonate cascade for cell cycle progression in PBMC stimulated by PHA.

Activation of deoxycytidine kinase during inhibition of DNA synthesis in human lymphocytes

Deoxycytidine kinase was shown to be activated during 2-chlorodeoxyadenosine (CdA) treatment of human lymphocytes, under the conditions when the DNA synthesis is inhibited. As the increase of dCK activity was shown in crude protein extracts, without an increase in the amount of dCK protein, shown by immunostaining after SDS-PAGE, a secondary modification of the protein structure was considered. NaF treatment of cells in the concentration range of 5–20 mM gave a similar activation of dCK, suggesting a possible role of phosphatases and/or a possibility of a G-protein related phenomenon. Using the same conditions, no effect of CdA or NaF was found on the thymidine kinase activity of cell extracts. Alternatively, activation of catabolic pathways could be considered, however, the increase in dCK activity was not influenced either by the removal of 5′-nucleotidases, or by the inhibition of deaminases.

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes in vitro

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes were biphasic: The chemicals at very low concentrations enhanced DNA synthesis, whereas higher concentrations inhibited DNA synthesis. The concentrations of arsenicals at which the maximum stimulating effect was found were 1 x 10(-5) M, 1 x 10(-6) or 2 x 10(-6) M, and 0.8 x 10(-6) or 1 x 10(-6) M for sodium arsenite exposure of 1 h, 3 d, and 6 d, respectively; for sodium arsenate, 1 x 10(-5) M, 1 x 10(-5) M, and 2 x 10(-6) or 5 x 10(-6) M, respectively. Arsenicals must be present for the entire 6-d culture period to produce maximum stimulation of DNA synthesis in human lymphocytes. The longer exposure of the lymphocytes to arsenicals, the lower the concentrations of arsenicals at which the maximum stimulating effect on DNA synthesis was found. Stimulating effect of trivalent arsenic (sodium arsenite) on DNA synthesis was stronger than pentavalent arsenic (sodium arsenate), and the stronger the effect of trivalent arsenic than pentavalent, the longer exposure of the cells to the chemicals. Both sodium arsenite and sodium arsenate stimulated DNA synthesis in human lymphocytes to a lower degree than phytohemagglutinin (PHA).

Effects of arsenic on DNA synthesis in human lymphocytes.

Effects of arsenic on DNA synthesis in human lymphocytes were biphasic: Either trivalent (arsenic trioxide and sodium arsenite) or pentavalent (sodium arsenate) arsenic compounds at very low concentrations enhanced DNA synthesis in human lymphocytes stimulated by phytohemagglutinin (PHA), whereas higher concentrations inhibited DNA synthesis. There were differences among individual susceptibilities to arsenic-induced DNA synthesis. Either stimulating or inhibiting effects of trivalent arsenic on DNA synthesis in PHA-stimulated lymphocytes were always stronger than those of pentavalent arsenic. Both trivalent and pentavalent arsenic could be rapidly taken up into the human lymphocytes and immediately stimulate or inhibit DNA synthesis. A possible dual effect of arsenic at very low concentrations as both comutagen and inhibitor of mutagenesis is discussed.

Effects of arsenic on DNA synthesis in human lymphocytes stimulated by phytohemagglutinin.

Effects of arsenic on DNA synthesis in human lymphocytes were biphasic: either trivalent (arsenic trioxide and sodium arsenite) or pentavalent (sodium arsenate) arsenic compounds at very low concentrations enhanced DNA synthesis in human lymphocytes stimulated by phytohemagglutinin (PHA), whereas higher concentrations inhibited DNA synthesis. There were differences among individual susceptibilities to arsenic-induced DNA synthesis. Either stimulating or inhibiting effects of trivalent arsenic on DNA synthesis in PHA-stimulated lymphocytes were always stronger than those of pentavalent arsenic. It was also shown that both trivalent and pentavalent arsenic could be rapidly taken up into the human lymphocytes, and immediately stimulated or inhibited DNA synthesis. A possible dual effect of arsenic at very low concentrations as both comutagen and inhibitor of mutagenesis is discussed.

Reduced mitogen stimulation of DNA synthesis in human lymphocytes by a human recombinant interleukin-1 receptor antagonist

The monokine interleukin-1 is produced by monocytes/macrophages after antigen/LPS stimulation and is an important early signal for the activation of resting T cells to become antigen specific T cells. However, little is known about the regulation and inhibition of IL-1. Recently, a new monokine has been described, generated by human macrophages, called interleukin-1 receptor antagonist (IL-1ra). This new monokine adheres to IL-1 in solution and blocks IL-1 receptor binding. IL-1ra is a glycoprotein structurally similar to IL-1 beta but having no interleukin-1-like activity. Using as a model mitogen (PHA 20 micrograms/ml)-stimulated lymphocyte DNA synthesis, we found that hrIL-1ra (30 min lymphocyte pretreatment) inhibits [3H]thymidine incorporation in a dose-dependent manner. This effect is most probably due to the inhibition of endogenous IL-1, which is a very important signal for T cell activation. The inhibition was maximum at the highest hrIL-1ra concentration used (250 ng/ml). However, when hrIL-1ra was added 2 h after PHA (20 micrograms/ml), a little, if any, inhibition of lymphocyte proliferation was found. The addition of hrIL-1ra simultaneously to the cell cultures with [3H]thymidine [( 3H]TdR) 6 h before the end of culture incubation did not significantly modify the results compared to the cells treated with PHA alone, indicating no interference of hrIL-1ra on [3H]TdR lymphocyte incorporation. We also found that the antibody anti-IL-1 beta inhibits mitogen stimulated lymphocyte DNA synthesis in dose-dependent concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Ciprofloxacin-induced photosensitivity: in vitro and in vivo studies

Ciprofloxacin is one of the new series of broad-spectrum antibiotic quinolones, chemically related to nalidixic acid and which may, therefore, induce photosensitization of human skin. Three in vitro tests for phototoxicity: the destruction of histidine, killing of mouse peritoneal macrophages and inhibition of PHA-stimulated DNA synthesis in human lymphocytes have demonstrated this photosensitizing potential with UVA irradiation at an order of magnitude lower than that for nalidixic acid. The Candida albicans test and photohaemolysis were negative. Controlled irradiation monochromator phototesting of 12 subjects, before, during and after taking ciprofloxacin showed subclinical photosensitivity with significantly lowered minimal 24 h erythema doses at 335 +/- 30 nm, 365 +/- 30 nm and 400 +/- 30 nm but not at 305 +/- 5 nm or above 400 +/- 30 nm.

The effects of phorbol ester and Ca ionophore on c-fos and c-myc expression and on DNA synthesis in human lymphocytes are not directly related

Phorbol ester activates human lymphocyte proliferation as measured by [ 3H]thymidine incorporation and blast transformation. Phorbol ester and calcium ionophore A 23187 induce c-fos and c-myc expression in these cells as shown by the measure of the specific mRNA levels and of the c-fos nuclear protein amount. The combination of these two agents has a synergistic effect on c-fos but not on c-myc expression. Lymphocyte stimulation by phorbol ester for a short time induces c-fos and c-myc expression, but has no effect on [ 3H]thymidine incorporation. This result indicates that the induction of c-fos and c-myc expression is not sufficient to commit these cells to DNA synthesis.

Induction of immunoglobulin secretion and DNA synthesis in human lymphocytes in vitro by cytomegalovirus preparations.

Immunoglobulin secretion as evidenced by plaque-forming cells (PFC) in an indirect haemolysis-in-gel assay, and DNA synthesis were induced in human blood lymphocytes by the following preparations of cytomegalovirus (CMV): Nucleocapside Nc-CMV antigen, membrane M-CMV antigen, crude C-CMV preparations and CMV-incubated adherent cells. Peak stimulations occurred around day 6 in culture. Nc-CMV and M-CMV only stimulated PFC and DNA synthesis in lymphocytes from CMV seropositive individuals. C-CMV also stimulated lymphocytes from CMV seronegative individuals but gave better responses in lymphocytes from CMV seropositive individuals. CMV-incubated adherent cells occasionally stimulated lymphocytes from CMV seronegative individuals but always in CMV seropositive blood donors. Nc-, M-, and C-CMV gave high numbers of PFC in B cells enriched by sheep erythrocyte sedimentation and in co-cultures of enriched T and B cells. Almost no PFC were induced in enriched T cells. DNA synthesis induced by all the three antigenic CMV preparations increased after removal of adherent cells. Strong DNA synthesis was induced in enriched T cells compared to almost none in enriched B cells. It is concluded that some pure CMV preparations act as antigens and more crude preparations induce polyclonal activation. Different CMV preparations may be used to diagnose CMV, to study immune reactivity against CMV and as a model for CMV infections in vitro.

Phenytoin potentiates accessory cell dependent DNA-synthesis in human lymphocytes in vitro.

The effect of phenytoin and its major metabolite p-HPPH on concanavalin A (ConA) induced DNA-synthesis of human lymphocytes was studied in vitro. In lymphocyte cultures depleted of phagocytizing cells by iron treatment PHT and p-HPPH, in pharmacologic concentrations, caused a depression of the mitogen response measured as uptake of 3H-thymidine. In contrast, the presence of phagocytizing mononuclear cells during ConA-stimulation in the presence of PHT and p-HPPH caused a potentiation of ConA induced DNA-synthesis. These effects of phenytoin on immunocompetent cells may be of significance in the pathogenesis of the PHT-induced gingival overgrowth, since earlier we have reported the presence of large infiltrates of lymphocytes, mainly T-lymphocytes in gingival biopsies from clinically non-inflamed PHT-induced gingival overgrowth.

The influence of selenium and caffeine on chemical carcinogenesis in rats, mutagenesis in bacteria, and unscheduled DNA synthesis in human lymphocytes

The influence of sodium selenite (Na2SeO3) and caffeine on chemical carcinogenesis induced in rats by diethylnitrosamine (DEN), N-nitrosomorpholine (NM), andN-methyl-N-nitro-N-nitrosoguanidine (MNNG) was investigated. A dose-dependent inhibitory effect of Na2SeO3 (l-10 ppm) on hepatocarcinogenesis induced by DEN was demonstrated. Na2SeO3 also increased the latency period for stomach tumor formation in rats treated with MNNG. Combined treatment of rats with Na2SeO3 plus vitamin C added to the diet resulted in a slight inhibition of NM-induced liver carcinogenesis. Supplementation of diet with Na2SeO3 plus butylated hydroxytoluene, vitamin C, and vitamin E did not reveal any additive inhibitory effect compared to the inhibitory effect of Na2SeO3 given alone. Caffeine (600 rag/L) reduced the number of liver tumors induced in rats by DEN. Preliminary experiments have indicated that combined treatment of rats with selenium and caffeine could result in more effective inhibition of DEN-induced liver carcinogenesis.Further experiments are being conducted to study the influence of selenium and caffeine on mutagenic activity of 1-methyl-l-nitrosourea (MNU) inSalmonella typhimurium TA 1535. The pretreatment of bacteria cells with Na2SeO3 (3-10 p.g/mL) increased the mutagenic response of bacteria to MNU. A synergistic stimulation of mutagenic activity of MNU was observed in bacteria pretreated simultaneously with Na2SeO3 and caffeine.In addition the influence of Na2SeO3 on UDS induced by DEN in human lymphocytes was investigated. The trace element inhibited the UDS up to 82%.The possible role of potentiation by NazSeO3 of the cell killing effect of DEN in inhibition of liver carcinogenesis was discussed.

Inhibition of unscheduled dna synthesis in human lymphocytes by chemical carcinogens

Freshly isolated human peripheral lymphocytes were treated with an alkylating agent immediately after collection and subsequently treated with UV radiation. This system was used because it represents a method for assaying damage in cells immediately after their removal from the host. The amount of UV-induced repair was measured as unscheduled DNA synthesis (UDS) by incorporation of [3H]deoxythymidine into the cellular DNA. The alkylating agents beta-propiolactone (BPL) and methyl methane-sulfonate (MMS) inhibited UDS at concentrations of 0.08 mM and 0.6 mM, respectively. Lower concentrations had no effect. Lymphocytes allowed to remain in culture medium after treatment with the alkylating agents did not recover the ability to perform UV-induced UDS even when cells were irradiated 48 h after carcinogen treatment. The decrease in UV-induced UDS resulting from alkylating agent treatment could not be attributed to cell death.

Enhancement of mitogen-induced lymphocyte proliferation after preincubation is due to altered sensitivity to prostaglandins

Twenty-four hour preincubation at 37°C enhances the mitogen-induced DNA synthesis of human lymphocytes. PGE 1, given simultaneously with Con A at the start of lymphocyte culture, inhibits the DNA synthesis. After 24 h preincubation of cells, PGE 1 fails to decrease the DNA synthesis. Similarly, preincubation abolishes the effect of indomethacin increasing DNA synthesis of freshly separated lymphocytes. The intracellular cAMP level of human mononuclear leukocytes rapidly decreases during in vitro incubation at 37°C. PGE 1 elevates the intracellular cAMP of freshly separated lymphocytes to 45 times of its starting level. After 24 h preincubation of cells at 37°C, PGE 1 elevates cAMP to a lesser extent. This change of PGE 1 action may explain the fact that the effect of exogenous PGE 1 and endogenous prostaglandins (the production of which can be inhibited by indomethacin) diminishes after incubation of lymphocytes. It is very likely that the change of the effect of prostaglandins produced in lymphocyte cultures and the spontaneous decrease of intracellular cAMP level explains the enhancement of mitogen-induced lymphocyte proliferation after 24 h preincubation at 37°C.

Mitogenic leukoagglutinin from Phaseolus vulgaris binds to a pentasaccharide unit in N-acetyllactosamine-type glycoprotein glycans.

The carbohydrate binding specificity of leukoagglutinin (La; Phaseolus vulgaris isolectin L4) was studied by using quantitative precipitation and precipitation-inhibition. A series of purified glycopeptides and synthetic oligosaccharides were used as inhibitors. The minimal structural unit required for La binding was the disaccharide GlcNac(1 leads to beta 2)Man. Additions for this basic unit of different sugar residues gave a positive or negative contribution to binding. The most complementary structure was the pentasaccharide (formula: see text). This pentasaccharide units occurs in tetraantennary N-acetyllactosamine-type glycoprotein glycans. Glycoproteins containing such structures were accordingly precipitated by La. Selected glycopeptides and oligosaccharides were also tested as inhibitors of La-induced DNA synthesis in human lymphocytes. The pattern of inhibition was essentially the same as that obtained by precipitation-inhibition, indicating that binding to lymphocytes via the carbohydrate binding site of the lectin is an essential step in the activation process.

Dehydroepiandrosterone and 16 alpha-bromo-epiandrosterone: inhibitors of Epstein-Barr virus-induced transformation of human lymphocytes.

Dehydroepiandrosterone (DHEA), a major adrenal secretory product in men and women, is a potent inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH). Long-term treatment with this steroid has previously been found to suppress spontaneous breast cancer development in C3H mice. DHEA is now shown to inhibit Epstein-Barr virus (EBV)-induced morphologic transformation and stimulation of DNA synthesis in human lymphocytes. 16 alpha-Br-epiandrosterone, a DHEA analog that is about 60 times as potent as DHEA as an inhibitor of G6PDH, is much more effective as an inhibitor of EBV-induced transformation.

Stimulation of DNA Synthesis in Human Lymphocytes by an Insect Virus Antigen 12

Synthesis of DNA in lymphocytes isolated from adult human peripheral blood was detected by measuring incorporation of tritiated thymidine after cell cultures were exposed to polyhedra of a nuclear polyhedrosis virus of Autographa californica . Similar results obtained with lymphocytes isolated from human umbilical cord blood suggests that this mitogenic activity is nonspecific. No DNA synthesis was evident when lymphocyte cultures were treated with enveloped or unenveloped nucleocapsids. Observation of lymphocytes cultured over a 3D-day period, in the presence of baculovirus, provided no evidence of permanently transformed cells.