Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

In vitro studies of biological effects of cigarette smoke condensate: II. Induction of sister-chromatid exchanges in human lymphocytes by weakly acidic,semivolatile constituents

Cigarette smoke condensate is known to enhance the frequency of sister-chromatid exchanges (SCE) in human lymphocytes in vitro and some of the activity has been found in the most volatile part of the particulate phase, the semivolatile fraction. In this study we have investigated the chemical composition and the SCE-inducing activity of the weakly acidic, semivolatile fraction of a cigarette smoke condensate. A number of individual weakly acidic compounds were also tested for their SCE-inducing effects.The weakly acidic fraction was separated by preparative gel chromatography into 11 subfractions (F1–F11). The chemical composition was determined by gas chromatography and gas chromatography-mass spectrometry. Measurements of the effects on SCE in human lymphocytes were used to evaluate the genotoxic effects. All fractions except F11 induced SCE in a dose-dependent way. The most active fraction was F4 which contained mainly alkyl-2-hydroxy-2-cyclopenten-1-ones.The individual compounds to be tested for induction of SCE were selected on the basis of their abundance in the weakly acidic subfractions and on the basis of their occurrence in the environment. Of 23 tested compounds, most of which were alkylphenols, 7 induced SCE, i.e., catechol, 2-(1-propenyl)phenol, cyclotene, maltol, isoeugenol, 2-methoxyphenol (guaiacol) and vanillin. Many of these are important flavor components that occur not only in tobacco and tobacco smoke but also in food, candies, beverages and perfumes.     

DNA repair replication,DNA breaks and sister-chromatid exchange in human cells treated with adriamycin in vitro

The effects of adriamycin (AM) on DNA repair replication, the frequency of sister-chromatid exchange (SCE), the rate of cell proliferation and the frequency of DNA strand breaks were studied in human cells in vitro. No repair replication was observed in lymphocytes exposed to AM in concentrations up to 10^?3 moles/1. DNA repair replication induced by UV and alkylating agents was not affected by a concentration of AM that completely inhibited cell proliferation (10^?6 moles/1).Fibroblasts exposed to AM at 10^?4 moles/1 in the presence of hydroxyurea showed an increase of strand breaks and cross-links in DNA. When AM was added to UV-irradiated fibroblasts, there was an increase of DNA strand breaks in addition to the breaks caused by UV alone. Similar effects were observed in lymphocytes.A dose-dependent increase of SCE was observed in lymphocytes exposed to low concentrations of AM (<10^?7 moles/1). At higher concentrations the increase of SCE levelled off, and cell proliferation became severely inhibited. There was no evidence of removal of SCE-inducing damage in cells exposed to AM during G₀ or G₁. The level of SCE induced in the third cell cycle after treatment with AM was not different from that induced during the first two cell cycles.These results suggest that the various genotoxic and cytotoxic effects of AM are caused by different types of cellular damage. Moreover, AM-induced DNA damage persists for several cell cycles in human cells in vitro and seems to be resistant to repair activity.     

In vitro studies of biological effects of cigarette smoke condensate. II. Induction of sister-chromatid exchanges in human lymphocytes by weakly acidic, semivolatile constituents

Cigarette smoke condensate is known to enhance the frequency of sister-chromatid exchanges (SCE) in human lymphocytes in vitro and some of the activity has been found in the most volatile part of the particulate phase, the semivolatile fraction. In this study we have investigated the chemical composition and the SCE-inducing activity of the weakly acidic, semivolatile fraction of a cigarette smoke condensate. A number of individual weakly acidic compounds were also tested for their SCE-inducing effects. The weakly acidic fraction was separated by preparative gel chromatography into 11 subfractions (F1-F11). The chemical composition was determined by gas chromatography and gas chromatography-mass spectrometry. Measurements of the effects on SCE in human lymphocytes were used to evaluate the genotoxic effects. All fractions except F11 induced SCE in a dose-dependent way. The most active fraction was F4 which contained mainly alkyl-2-hydroxy-2-cyclopenten-1-ones. The individual compounds to be tested for induction of SCE were selected on the basis of their abundance in the weakly acidic subfractions and on the basis of their occurrence in the environment. Of 23 tested compounds, most of which were alkylphenols, 7 induced SCE, i.e., catechol, 2-(1-propenyl)phenol, cyclotene, maltol, isoeugenol, 2-methoxyphenol (guaiacol) and vanillin. Many of these are important flavor components that occur not only in tobacco and tobacco smoke but also in food, candies, beverages and perfumes.     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

Different SCE-inducing effects of HN2 and MMS in early and late G1 in human lymphocytes

The induction of SCE was studied in PHA-stimulated human lymphocytes exposed to nitrogen mustard (HN2) or methyl methanesulfonate (MMS) for various time periods in the G1 phase. HN2 was found to induce about 10 times more SCE when cells were exposed in late G1 (24 h after PHA) as compared to early G1 (immediately after PHA). In contrast, only a small difference was observed between cells exposed to MMS in late or early G1. The results suggest that different types of SCE-inducing alkylating damage agents are removed at widely different rates in human G1-lymphocytes.     

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12-O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, CF from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic aldehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.     

Fecapentaene causes sister-chromatid exchanges in human lymphocytes

Fecapentaenes are potent mutagenic compounds found in human feces that are considered as potential colon carcinogens. It is demonstrated that a synthetic racemic all-trans fecapentaene-12 (fec-12) causes a strong dose-dependent increase in the frequency of sister-chromatid exchanges (SCE) in human lymphocytes exposed at different stages of the cell cycle. The SCE-inducing capacity is consistent with published results on the DNA-damaging activity of fec-12 such as formation of DNA single-strand breaks and interstrand cross-links.     

Caffeine induces sister-chromatid exchanges during the whole S-phase of the cell cycle

The capacity of caffeine to induce sister chromatid exchanges (SCEs) in different cell cycle stages and the proliferation kinetics were studied. Continuous treatment with this xanthine during the whole second cycle significantly increased the baseline SCE frequency. Pulse-treatment experiments showed that the induction of SCEs by caffeine, which was dose-dependent, was restricted to the S-phase of the cell cycle without effect on G1 or G2 cells. Moreover, unlike other SCE-inducing agents, such as DNA-synthesis inhibitors and DNA-damaging agents, caffeine produced similar SCE increases in cells treated at different times throughout the S-phase. In the light of Painter's model for SCE formation and the known effects of caffeine on the DNA replication pattern, the most likely mechanism of SCE induction by caffeine is an increase in the number of DNA-replication sites.     

Genotoxicity assessment of new synthesized acridine derivative--3,6-diamino-10-methyl-9,10-dihydroacridine.

A new synthesized acridine derivative, 3,6-diamino-10-methyl-9, 10-dihydroacridine (AcrH), was tested for in vitro reverse mutations with Salmonella TA strains, chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes, and for in vivo chromosome aberrations in bone marrow of mice. Using the classic plate incorporation method, mutagenicity of AcrH in bacterial cells (TA97a, TA98, TA100 and TA102) was observed in the experiments performed with, and without, rat liver S9 metabolic activation. The reverse mutation assay showed no difference in mutagenic activity between AcrH and acriflavine (Acr(+)) in the test with TA97. The results of in vitro chromosome aberrations assay revealed potential clastogenicity. The test using macroculture of human lymphocytes induced mainly chromatid gaps. The experiments with human lymphocytes revealed SCE-inducing effect of AcrH and Acr(+). In an in vivo study, AcrH given intraperitoneally to Balb/c mice did not cause any significant increase in the percentage of cells with aberrations compared to the negative control.     

Cell line dependent involvement of ceramide in ultraviolet light-induced apoptosis

Ultraviolet light (UV) activates an acid sphingomyelinase (ASMase) pathway, which hydrolyzes sphingomyeline to ceramide. Ceramide has been found to be a second messenger, which activates the c-jun N-terminal kinase (JNK) that is required for apoptotic cell death. However, the role of ceramide in UV-induced JNK activation and apoptosis remains controversial. In this study, we examined the correlation among ceramide production, JNK activation and cell apoptosis after UV-irradiation in three cell lines: 293 (kidney), Jurkat (lymphocytes) and MCF-7 (breast) were used in this study. The ceramide production was analyzed using the diacylglycerol kinase assay method. The JNK activation was measured by Western blot analysis using an antibody specifically recognizing phosphorylated JNK. Cell apoptosis was determined by morphological change or flow cytometry. Our data show that UV-irradiation induces ceramide production in both 293 and Jurkat cells. Inhibition of ceramide production by desipramine (25–50 M) reduced UV-induced JNK activation in both 293 and Jurkat cells; and protects 293 cells from UV-induced apoptosis. However, inhibition of ceramide production does not prevent Jurkat cells from UV-induced apoptosis. In addition, our data demonstrates that UV-irradiation induces JNK activation and apoptosis of MCF-7 cells without production of detectable amounts of ceramide after UV-irradiation. These results suggest that UV-induced JNK activation and apoptosis can be mediated through a ceramide dependent or an independent pathway.     

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.     

Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet light-induced sister-chromatid exchanges in Chinese hamster cells

Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet light (UV)-induced sister-chromatid exchanges (SCE) were examined in a Chinese hamster cell line, V79 B-1. The inhibitors used were hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (ara-C), aphidicolin (APC), 2',3'-dideoxythymidine triphosphate (ddTTP), neocarzinostatin (NCS), novobiocin (NB) and cycloheximide (CHX). HU, ara-C, and APC increased spontaneous SCE frequency, and had a synergistic effect on UV-induced SCE frequency. DdTTP, NCS and NB failed to show any statistically significant effect on either spontaneous or UV-induced SCE frequencies, though NCS and NB did slightly increase both spontaneous and UV-induced SCE frequencies. On the contrary, CHX decreased spontaneous SCE frequency, and more drastically, also UV-induced SCE frequency. These results are interpreted with respect to the replicating fork of DNA, a structure postulated to be involved in the formation of spontaneous and UV-induced SCE. A new model for SCE formation is proposed.     

DNA repair in the UV irradiation of human peripheral blood lymphocytes (healthy donors and xeroderma pigmentosum patients) in relation to the dedifferentiation process in phytohemagglutinin exposure

UV-induced unscheduled DNA synthesis has been studied in lymphocytes of healthy donors and of Xeroderma pigmentosum patients (the classic form, and a form with an increased sensitivity to gamma-ray). In order to study the influence of PHA-induced differentiation on repair capacity of cells, lymphocytes were cultured in the presence or in the absence of PHA. The data obtained show that PHA-induced differentiation of human lymphocytes leads to an increase in the intensity of repair after UV-irradiation of these cells, and, accordingly, the repair is completed in a shorter time. In the case of the classic form of Xeroderma pigmentosum, the effect of differentiation on the repair level is more distinct, but no effect is observed in cells of Xeroderma pigmentosum sensitive both to UV and gamma-ray.     

Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.     

Increased frequency of acetaldehyde-induced sister-chromatid exchanges in human lymphocytes treated with an aldehyde dehydrogenase inhibitor

Acetaldehyde, the first metabolite of ethanol oxidation, in concentrations ranging from 100 microM to 400 microM caused a dose-dependent linear increase in the frequency of sister-chromatid exchanges (SCE) in cultured human peripheral lymphocytes. The SCE frequency was on an average 2-fold higher when the cells were exposed to the acetaldehyde after 24 h incubation instead of at the time of mitogen stimulation (0 h). When acetaldehyde was added together with the potent aldehyde dehydrogenase inhibitor 1-aminocyclopropanol (0.1 mM), the SCE response was significantly (p less than 0.05) increased. The present results indicate that acetaldehyde is metabolized within human lymphocytes, and, moreover, that alcohol consumption during treatment with drugs that inactivate aldehyde dehydrogenase may cause a further increased incidence of acetaldehyde-induced SCE and concomitant lesions.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

Induction of sister-chromatid exchange (SCE) and cell-cycle inhibition in mouse peripheral blood B lymphocytes exposed to mutagenic carcinogens in vivo

To determine the sensitivity of the mouse peripheral blood lymphocyte (PBL) culture system, male B6C3f1 mice were injected i.p. with either 2-acetylaminofluorene (AAF) (20, 40, 80, 160 mg/kg), benzo[a]pyrene (BP) 25, 75, 150, 300 mg/kg), dichlorvos (DCV) (5, 15, 25, 35 mg/kg), ethyl methanesulfonate (EMS) (10, 30, 90, 180, 270 mg/kg), or N-nitrosomorpholine (NM) (37.5, 75, 150, 300 mg/kg) dissolved in either RPMI 1640 (DCV, EMS, NM) or sunflower oil (AAF, BP). 24 h later blood was removed by cardiac puncture, and the lymphocytes were cultured in the presence of lipopolysaccharide for analysis of SCE in B lymphocytes. All 4 mutagenic carcinogens (AAF, BP, EMS, NM) induced significant dose-related increases in SCE frequency. DCV, a potent neurotoxicant, caused no change in the baseline SCE frequency. At the highest concentration of each chemical examined, AAF caused a 1.6-fold increase, EMS a 1.8-fold increase, NM a 3.0-fold increase, and BP a 3.1-fold increase in SCE frequency compared to concurrent controls. A comparison of these results for PBLs with those reported in the literature for bone marrow cells indicates that PBLs offer a good quantitative and qualitative estimate of the SCE-inducing potential for these 5 compounds in bone marrow cells.     

Increased sister chromatid exchange in human lymphocyte cultures infected with mycoplasma

The effects of fermenting, poorly arginine-utilizing Mycoplasma fermentans and arginine-utilizing Mycoplasma salivarium on the frequency of sister chromatid exchange (SCE) in cultured human lymphocytes were examined. M. fermentans caused no apparent mitosis inhibition of lymphocytes and the increase in SCE frequency was dependent on the inoculum size of the mycoplasma. An evident increase in SCE frequency was observed in lymphocytes infected with smaller inoculum sizes of M. salivarium whereas there was mitosis inhibition of lymphocytes infected with larger inoculum sizes of the mycoplasma. In lymphocyte cultures infected with M. salivarium, the addition of arginine to the culture medium reduced mitosis inhibition but did not diminish the increase in SCE frequency, indicating that arginine depletion was not involved in causing the induction of SCEs in mycoplasma-infected lymphocytes. With regard to the genetic effectiveness of SCE, these results suggested that mycoplasmas are capable of inducing cytogenetic changes in infected host cells.