Cytotoxic effect of three arsenic compounds in HeLa human tumor and bacterial cells

Numerous epidemiological studies suggest that arsenic (As) compounds are carcinogens, however, recent data have renewed the interest in their anticarcinogenic properties. The cytotoxic effects of three arsenic compounds were assessed: sodium arsenite, sodium arsenate and sodium cacodylate, representing the trivalent and pentavalent species of arsenic, along with a dimethylated pentavalent arsenic species. HeLa cells and Salmonella typhimurium (strains TA98 and TA100) were exposed to As compounds and the cytotoxic effects were evaluated. Alterations on RNA and DNA synthesis in HeLa cells were also examined. All arsenic compounds produced a dose-dependent inhibition on colony formation and DNA synthesis in HeLa cells, yet any of them significantly influenced RNA synthesis in these cells. No evidence of arsenic-induced mutagenicity or antimutagenicity was observed using the Ames assay. In bacterial cells, only sodium arsenite caused a dose-dependent inhibition of colony formation.Collectively, these results indicate that in both, HeLa and S. typhimurium cell systems, only trivalent sodium arsenite can act as an effective inhibitor of cell growth. The possible mechanism(s) of the cytotoxic effect of arsenite in these two different cell systems might be due to its reactivity with intracellular sulfhydryl groups.     

Role of the Alkali Labile Sites,Reactive Oxygen Species and Antioxidants in DNA Damage Induced by Methylated Trivalent Metabolites of Inorganic Arsenic

In the last decade arsenic metabolism has become an important matter of discussion. Methylation of inorganic arsenic (iAs) to monomethylarsonic acid (MMA^V) and dimethylarsinic acid (DMA^V) is considered to decrease arsenic toxicity. However, in addition to these pentavalent metabolites, the trivalent metabolites monomethylarsonous (MMA^III) and dimethylarsinous acid (DMA^III) have been identified recently as intermediates in the metabolic pathway of arsenic in cultured human cells. To examine the role of oxidative damage in the generation of DNA strand breaks by methylated trivalent arsenic metabolites, we treated human lymphocytes with both metabolites at non-cytotoxic concentrations. We further tested whether these effects are sensitive to modulation by the antioxidants ascorbate (Vitamin C) and selenomethionine (Se-Met). Both trivalent metabolites produced oxidative stress related DNA damage, consisting of single strand breaks and alkali-labile sites, with MMA^III being more potent at low concentrations than DMA^III. Neither MMA^III nor DMA^III induced DNA-double strand breaks. The oxidative stress response profiles of the metabolites were parallel as determined by lipid peroxidation induction. MMA^III induced peroxidation from the lowest concentration tested, while effects of DMA^III were apparent only at concentrations above 10 μM. The antioxidant Se-Met exhibited a more pronounced inhibition of trivalent arsenic metabolite-induced oxidative-DNA damage than did vitamin C. The present findings suggest that DNA damage by methylated trivalent metabolites at non-cytotoxic concentrations may be mediated by a mix of reactive oxygen and nitrogen oxidized species.     

Arsenite and its biomethylated metabolites interfere with the formation and repair of stable BPDE-induced DNA adducts in human cells and impair XPAzf and Fpg

The underlying mechanisms of arsenic carcinogenicity are only poorly understood and especially the role of biomethylation is still a matter of debate. Besides the induction of oxidative DNA damage the interference with DNA repair processes have been proposed to contribute to arsenic-induced carcinogenicity. Within the present study the effects of arsenite and its mono- and dimethylated trivalent and pentavalent metabolites on BPDE-induced DNA adduct formation and repair has been investigated and compared in cultured human lung cells. Whereas only arsenite and MMA(III) increased BPDE-DNA adduct formation, arsenite (>/=5 microM), the trivalent (>/=2.5 microM) and the pentavalent (>/=250 microM) metabolites diminished their repair at non-cytotoxic concentrations. As potential molecular targets, interactions with the zinc finger domain of the human XPA protein (XPAzf) and the Escherichia coli zinc finger protein Fpg, involved in NER and BER, respectively, have been investigated. All trivalent arsenicals were able to release zinc from XPAzf; furthermore, MMA(III) and DMA(III) inhibited the activity of isolated Fpg. Altogether the results suggest that besides arsenite, especially the trivalent methylated metabolites may contribute to diminished NER at low concentrations.     

Impact of arsenite and its methylated metabolites on PARP-1 activity, PARP-1 gene expression and poly(ADP-ribosyl)ation in cultured human cells

The underlying mechanisms of arsenic carcinogenicity are still not fully understood. Mechanisms currently discussed include the induction of oxidative DNA damage and the interference with DNA repair pathways. Still unclear is the role of biomethylation, which has long been considered to be one major detoxification process. Methylated arsenicals have recently been shown to interfere with DNA repair in cellular and subcellular systems, but up to now no DNA repair protein has been identified being particular sensitive towards methylated arsenicals in cultured cells. Here we report that the trivalent methylated metabolites MMA(III) and DMA(III) inhibit poly(ADP-ribosyl)ation in cultured human HeLa S3 cells at concentrations as low as 1nM, thereby showing for the first time an inactivation of an enzymatic reaction related to DNA repair by the trivalent methylated arsenicals at very low environmentally relevant concentrations. In contrast the pentavalent metabolites MMA(V) and DMA(V) showed no such effects up to high micromolar concentrations. All investigated arsenicals did not alter gene expression of PARP-1. However, all trivalent arsenicals were able to inhibit the activity of isolated PARP-1, indicating that the observed decrease in poly(ADP-ribosyl)ation in cultures human cells, predominantly mediated by PARP-1, is likely due to changes in the activity of PARP-1. Since poly(ADP-ribosyl)ation plays a major role in DNA repair, cell cycle control and thus in the maintenance of genomic stability, these findings could in part explain DNA repair inhibition and the genotoxic and carcinogenic effects of arsenic.     

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 1×10^?5 M, 1×10^?6 or 2×10^?6 M, and 0.8×10^?6 or 1×10^?6 M for sodium arsenite exposure of 1 h, 3 d, and 6 d, respectively; for sodium arsenate, 1× 10^?5 M, 1×10^?5 M, and 2×10^?6 or 5×10^?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).     

Arsenicals affect base excision repair by several mechanisms

Inorganic arsenic is a strong, widespread human carcinogen. How exactly inorganic arsenic exerts carcinogenicity in humans is as yet unclear, but it is thought to be closely related to its metabolism. At exposure-relevant concentrations arsenic is neither directly DNA reactive nor mutagenic. Thus, more likely epigenetic and indirect genotoxic effects, among others a modulation of the cellular DNA damage response and DNA repair, are important molecular mechanisms contributing to its carcinogenicity. In the present study, we investigated the impact of arsenic on several base excision repair (BER) key players in cultured human lung cells. For the first time gene expression, protein level and in case of human 8-oxoguanine DNA glycosylase 1 (hOGG1) protein function was examined in one study, comparing inorganic arsenite and its trivalent and pentavalent mono- and dimethylated metabolites, also taking into account their cellular bioavailability. Our data clearly show that arsenite and its metabolites can affect several cellular endpoints related to DNA repair. Thus, cellular OGG activity was most sensitively affected by dimethylarsinic acid (DMA(V)), DNA ligase IIIα (LIGIIIα) protein level by arsenite and X-ray cross complementing protein 1 (XRCC1 protein) content by monomethylarsonic acid (MMA(V)), with significant effects starting at ≥3.2μM cellular arsenic. With respect to MMA(V), to our knowledge these effects are the most sensitive endpoints, related to DNA damage response, that have been identified so far. In contrast to earlier nucleotide excision repair related studies, the trivalent methylated metabolites exerted strong effects on the investigated BER key players only at cytotoxic concentrations. In summary, our data point out that after mixed arsenic species exposure, a realistic scenario after oral inorganic arsenic intake in humans, DNA repair might be affected by different mechanisms and therefore very effectively, which might facilitate the carcinogenic process of inorganic arsenic.     

Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells

We investigated the lethal, UV killing-potentiating and repair-inhibiting effects of trivalent arsenic trioxide (As2O3) and pentavalent sodium arsenate (Na2HAsO4) in normal human and xeroderma pigmentosum (XP) fibroblasts. The presence of As2O3 for 24 h after UV irradiation inhibited the thymine dimer excision from the DNA of normal and XP variant cells and thus the subsequent unscheduled DNA synthesis (UDS): excision inhibitions were partial, 30-40%, at a physiological dose of 1 microgram/ml and 100% at a supralethal dose of 5 micrograms/ml. Correspondingly, As2O3 also potentiated the lethal effect of UV on excision-proficient normal and XP variant cells in a concentration-dependent manner, but not on excision-defective XP group A cells. Na2HAsO4 (As5+) was approximately an order of magnitude less effective in preventing all the above repair events than As2O3 (As3+) which is highly affinic to SH-containing proteins. The above results provide the first evidence that arsenic inhibits the excision of pyrimidine dimers. Partially repair-suppressing small doses of As2O3 (0.5 microgram/ml) and Na2HAsO4 (5 micrograms/ml) enhanced co-mutagenically the UV induction of 6-thioguanine-resistant mutations of V79 Chinese hamster cells. Thus, such a repair inhibition may be one of the basic mechanisms for the co-mutagenicity and presumably co-carcinogenicity of arsenic. XP group A and variant strains showed a unique higher sensitivity to As2O3 and Na2HAsO4 killing by a yet unidentified mechanism.     

Fungal volatilization of trivalent and pentavalent arsenic under laboratory conditions

Production of volatile derivatives of arsenic was studied using pure cultures of different fungal strains under laboratory conditions. Arsenic was used in its trivalent and pentavalent forms to evaluate the effect of arsenic valency on its biovolatilization. The average amount of volatilized arsenic for all fungal strains ranged from 0.026 mg to 0.257 mg and 0.024 mg to 0.191 mg of trivalent and pentavalent arsenic, respectively. These results show that approximately 23% of arsenic was volatilized from all culture media originally enriched with approximately 4 and 17 mg L(-1) of arsenic in trivalent form. The average amount of biovolatilized arsenic from culture media originally enriched with 4 and 17 mg L(-1) of arsenic in pentavalent form was 24% and 16%, respectively. The order of ability of arsenic biovolatilization is Neosartorya fischeri > Aspergillus clavatus > Aspergillus niger. Toxicity and fungal resistance to trivalent and pentavalent arsenic were also evaluated based on radial growth and biomass weight.     

Cytokine production and DNA synthesis by Human peripheral Lymphocytes in response to Palmitic,Stearic, Oleic,and Linoleic acid

Effects of palmitic, stearic, oleic, and linoleic acid on mitogen-induced DNA synthesis, on production of IL-1β, IL-2, IFN-gamma, and TNF-α, and on IL-2R expression were determined in human peripheral lymphocytes. Free fatty acids (FFA) were added over a wide range of concentrations to cells cultured under serum free conditions with fatty acid free albumin. DNA synthesis was stimulated by low and inhibited by high FFA concentrations. Physiologica concentrations were stimulatory, except for linoleic acid. Cytokine production became affected by all FFA tested. Palmitic acid enhanced the release of IFN-gamma at concentrations that diminished TNF-α production. Saturated fatty acids were significantly more potent than unsaturated fatty acids in affecting cytokine production. IFN-gamma secretion was significantly more stimulated or inhibited by the various FFA compared with the other cytokines. IL-2R expression correlated with the production of IL-2. When tested in combination, stimulatory as well as inhibitory effects of the individual FFA became attenuated. It is suggested that palmitic, stearic, oleic, and linoleic acid are physiological regulators of DNA synthesis and cytokine release in human peripheral lymphocytes. Modulation of FFA ratios may be an effective means for the fine tuning of the immune system. As secretory mechanisms of cytokines appear to exhibit substrate specificity for FFA, the release of individual cytokines may be selectively influenced by FFA. © 1994 Wiley-Liss, Inc.     

Pentavalent methylated arsenicals are substrates of human AQP9

Liver aquaglyceroporin AQP9 facilitates movement of trivalent inorganic arsenite (As^III) and organic monomethylarsonous acid (MAs^III). However, the transport pathway for the two major pentavalent arsenic cellular metabolites, MAs^V and DMAs^V, remains unknown in mammals. These products of arsenic metabolism, in particular DMAs^V, are the major arsenicals excreted in the urine of mammals. In this study, we examined the uptake of the two pentavalent organic arsenicals by human AQP9 in Xenopus laevis oocytes. Xenopus laevis oocytes microinjected with AQP9 cRNA exhibited uptake of both MAs^V and DMAs^V in a pH-dependent manner. The rate of transport was much higher at acidic pH (pH5.5) than at neutral pH. Hg(II), an aquaporin inhibitor, inhibited transport of As^III, MAs^III, MAs^V and DMAs^V via AQP9. However, phloretin, which inhibits water and glycerol permeation via AQP9, can only inhibit transport of pentavalent MAs^V and DMAs^V but not trivalent As^III and MAs^III, indicating the translocation mechanisms of these arsenic species are not exactly the same. Reagents such as FCCP, valinomycin and nigericin that dissipate transmembrane proton potential or change the transmemebrane pH gradient did not significantly inhibit all arsenic transport via AQP9, suggesting the transport of pentavalent arsenic is not proton coupled. The results suggest that in addition to the initial uptake of trivalent inorganic As^III inside cells, AQP9 plays a dual role in the detoxification of arsenic metabolites by facilitating efflux from cells.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.     

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.     

Mechanisms of arsenic-induced cell transformation

Arsenic is a well-established carcinogen in humans, but there is little evidence for its carcinogenicity in animals and it is inactive as an initiator or tumor promoter in two-stage models of carcinogenicity in mice. Studies with cells in culture have provided some possible mechanisms by which arsenic and arsenical compounds may exert a carcinogenic activity. Sodium arsenite and sodium arsenate were observed to induce morphological transformation of Syrian hamster embryo cells in a dose-dependent manner. The trivalent sodium arsenite was greater than tenfold more potent than the pentavalent sodium arsenate. The compounds also exhibited toxicity; however, transformation was observed at nontoxic as well as toxic doses. At low doses, enhanced colony forming efficiency of the cells was observed. To understand the mechanism of arsenic-induced transformation, the genetic effects of the two arsenicals were examined over the same doses that induced transformation. No arsenic-induced gene mutations were detected at two genetic loci. However, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges, were induced by the arsenicals with similar dose responses. These results support a possible role for chromosomal changes in arsenic-induced transformation. The two arsenic salts also induced another form of mutation-gene amplification. Both sodium arsenite and sodium arsenate induced a high frequency of methotrexate-resistant 3T6 cells, which were shown to have amplified copies of the dihydrofolate reductase gene. The ability of arsenic to induce gene amplification may relate to its carcinogenic effects in humans since amplification of oncogenes is observed in many human tumors. Epidemiological studies suggest that arsenic acts late in the carcinogenic process in humans and oncogene amplification correlates with the progression of tumors. These observations lead us to propose the hypothesis that arsenic acts as a tumor progressor, rather than a tumor initiator or tumor promoter. Arsenic-induced chromosome aberrations or gene amplifications may play a role in tumor progression.     

Cytochalasin sensitive structures and lymphocyte activation.

In purified human peripheral blood lymphocytes, low (0.01–10 μM) concentrations of cytochalasins A B, E and D (CA, CB, CD, CE) produced marked augmentation of transport and metabolic responses to phytohemagglutinin (PHA) and concanavalin A (ConA) including effects on DNA synthesis, cAMP accumulation, phosphatidylinositol turnover and sodium-dependent amino acid transport. At high concentrations (10–100 μM) these same responses were inhibited. Cytochalasin effects were minimal or absent if lectin was not present indicating that these agents are acting by modulating the action of the lectin rather than through a direct effect on cell metabolism. Using [¹²⁵I]ConA, the number of lectin molecules bound per cell was shown not to be altered by the cytochalasins. Taken together with the previously reported effects of the cytochalasins on calcium uptake in lectin stimulated lymphocytes, these observations suggest that microfilaments (or related cytochalasin sensitive structures) play an important role in the modulation of lymphocyte activation.     

In vitro cultured cocklebur (Xanthium strumarium L.) responses to dimercaptopropanesulfonic acid and monosodium methanearsonate

Monosodium methanearsonate (MSMA) is an effective herbicide used for the control of Xanthium strumarium (cocklebur), a serious weed problem in cotton production. Recently, MSMA-resistant cocklebur was reported, which reduced MSMA effectiveness in the cocklebur control strategy. The mode of action of MSMA may involve the reduction of the pentavalent arsenic in MSMA to a trivalent form, which may then form a complex with sulfhydryl-containing enzymes. Dimercaptopro-panesulfonic acid (DMPS) chelates trivalent arsenic, resulting in a nontoxic complex. The objective of this study was to determine the effect of DMPS and MSMA on the growth of in vitro grown cocklebur shoot and callus tissues. Lateral and terminal shoots were grown on basal Murashige and Skoog medium containing 6.25 mg L^–1 (0.0428 mm) MSMA alone and with various concentrations of DMPS. Callus was cultured on basal callus medium also containing 6.25 mg L^–1 MSMA alone and an equimolar concentration of DMPS. Shoot injury symptoms, dry root weight, and fresh weight of callus were recorded. Susceptible cocklebur shoots showed severe discoloration and death when treated with MSMA. Resistant shoots and cultured shoot tips were not adversely affected. DMPS alone did not inhibit growth of these tissues. Callus induced from susceptible tissue was inhibited by MSMA and DMPS alone. In all combinations of DMPS and MSMA tested, DMPS did not counteract the MSMA response. Thus, the pentavalent form of arsenic in MSMA apparently is not reduced to the trivalent form by the plant and hence is not involved in the mechanism of actionAbbreviations MSMA monosodium methanearsonate - DMPS dimercaptopropanesulfonic acid - SM shoot medium - CM callus medium - ANOVA analysis of variance     

The phosphatidylinositol response and proliferation of oxidative enzyme-activated human T lymphocytes: suppression by plasma lipoproteins

The phosphatidylinositol (PI) response and DNA synthesis of neuraminidase and galactose oxidase (NAGO)-stimulated human T lymphocytes are suppressed by low density lipoproteins (LDL). To understand the mechanism of lymphocyte activation more fully, the PI response and DNA synthesis and suppression of these events by LDL in NAGO-stimulated T lymphocytes were characterized. Between 30 min and 6 hr after NAGO stimulation, there was an increase of 32Pi incorporation into PI without increased incorporation into the phosphorylated forms of PI or into other phospholipids. DNA synthesis as determined by [3H]thymidine incorporation depended on the lymphocyte-accessory monocyte ratio and total cell density. Optimal stimulation of the PI response and DNA synthesis occurred at the same concentration of neuraminidase and galactose oxidase. While the PI response was only partially suppressed by LDL with optimal suppression at 10 to 20 micrograms of protein/ml, DNA synthesis was completely suppressed although at much higher LDL concentrations, greater than 100 micrograms protein/ml. As monocyte numbers are increased, LDL suppression of DNA synthesis is decreased. The ability of NAGO to stimulate the PI response and DNA synthesis in a similar way, and the suppression of both events by LDL, suggests the PI response is important for lymphocyte activation and proliferation. Stimulation of human T lymphocytes by oxidative mitogens, neuraminidase, and galactose oxidase caused increased phosphatidylinositol metabolism and increased DNA synthesis. Both responses were suppressed by low density lipoproteins.     

Inhibition of lymphocyte activation by cyclosporin A: interference with the early activation of the membrane phospholipid metabolism in rabbit lymphocytes stimulated with concanavalin A, anti-rabbit immunoglobulin or the Ca2+ ionophore A 23187

Rabbit lymphocytes from the mesenteric lymph nodes were stimulated with concanavalin A, goat anti-rabbit immunoglobulin, or the Ca2+ ionophore A 23187. The stimulated incorporation of labeled uridine into RNA as well as of labeled thymidine into DNA was suppressed within a dose range of 40-1000 ng/ml cyclosporin A in both Con A-stimulated T lymphocytes and in anti-immunoglobulin-stimulated B lymphocytes, without affecting the resting cells. A 23187-stimulated rabbit lymphocytes proved to be more sensitive to cyclosporin A. At 40 ng/ml the immunosuppressive drug was effective in inhibiting elevated incorporation of labeled nucleosides into macromolecules in ionophore-stimulated cells. Cyclosporin A, at the same concentrations that were effective in inhibiting stimulated RNA and DNA synthesis, suppressed one of the earliest events occurring in stimulated lymphocytes, i.e., enhanced incorporation of unsaturated fatty acids into membrane phospholipids. Whereas cyclosporin A significantly inhibited the incorporation of arachidonic acid into phosphatidylcholine and phosphatidylethanolamine in concanavalin A-, anti-immunoglobulin-, and A 23187-stimulated cells, it proved to be ineffective in inhibiting the incorporation of arachidonate into phosphatidylinositol. The data indicate that cyclosporin A inhibits both T- and B-cell stimulation by interfering with a common target, e.g., the early activation of membrane phospholipid metabolism of rabbit lymphocytes.     

Rearrangements of chromatin structure in newly repaired regions of deoxyribonucleic acid in human cells treated with sodium butyrate or hydroxyurea

The rate and extent of redistribution of repair-incorporated nucleotides within chromatin during very early times (10-45 min) after ultraviolet irradiation were examined in normal human fibroblasts treated with 20 mM sodium butyrate, or 2-10 mM hydroxyurea, and compared to results for untreated cells. Under these conditions, DNA replicative synthesis is reduced to very low levels in each case. However, DNA repair synthesis is stimulated by sodium butyrate and partially inhibited by hydroxyurea. Furthermore, in the sodium butyrate treated cells, the core histones are maximally hyperacetylated. Using methods previously described by us, it was found that treatment with sodium butyrate had little or no effect on either the rate or the extent of redistribution of repair-incorporated nucleotides during this early time interval. On the other hand, there was a 1.7-2.5-fold decrease in the rate of redistribution of these nucleotides in cells treated with hydroxyurea; the extent of redistribution was unchanged in these cells. Since hydroxyurea has been shown to decrease the rate of completion of "repair patches" in mammalian cells, these results indicate that nucleosome rearrangement in newly repaired regions of DNA does not occur until after the final stages of the excision repair process are completed. Furthermore, hyperacetylation of the core histones in a large fraction of the total chromatin prior to DNA damage and repair synthesis does not appear to alter the rate or extent of nucleosome core formation in newly repaired regions of DNA.     

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.