Kinetics of excision repair of UV-induced DNA damage, measured using the comet assay

The kinetics of UV- (254 nm) irradiation-induced DNA single-strand breaks (SSBs), generated during the excision repair of UV-induced DNA damage, in leukemic lymphocytes and in normal blood mononuclear cells (MNCs) were studied using the alkaline comet assay. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. The cytotoxicity of UV irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from eight healthy donors using the incorporation of radioactive leucine in 4-day cultures. A remarkable difference in excision repair capability was observed between normal and leukemic lymphocytes. In contrast to normal lymphocytes, there was always a subpopulation of CLL cells that did not complete the repair of UV-induced DNA damage during the 24-h repair period. Furthermore, differences were also recorded between UV-sensitive and UV-resistant CLL cases. The differences in DNA migration between the maximum increase (59-77 microm) and that at 24 h after irradiation (21-66 microm) was statistically significant in two of three patients exhibiting UV-resistance. Correspondingly, only in one of three patients exhibiting UV-sensitivity was the difference in DNA migration statistically significant (maximum increase: 44-107 microm, vs. 24 h after: 42-100 microm). Our results confirm an abnormal pattern of the CLL cell response to UV irradiation. Furthermore, we identified defective processing of UV-induced DNA damage in CLL versus normal lymphocytes, particularly in UV-sensitive cases.     

Characterization of biotinylated repair regions in reversibly permeabilized human fibroblasts.

We have examined the incorporation of biotinyl-11-deoxyuridine triphosphate (BiodUTP) into excision repair patches of UV-irradiated confluent human fibroblasts. Cells were reversibly permeabilized to BiodUTP with lysolecithin, and biotin was detected in DNA on nylon filters using a streptavidin/alkaline phosphatase colorimetric assay. Following a UV dose of 12 J/m2, maximum incorporation of BioUTP occurred at a lysolecithin concentration (80-100 micrograms/mL) similar to that for incorporation of dTTP. Incorporation of BiodUTP into repair patches increased with UV dose up to 4 and 8 J/m2 in two normal human fibroblast strains, while no incorporation of BiodUTP was observed in xeroderma pigmentosum (group A) human fibroblasts. The repair-incorporated biotin was not removed from the DNA over a 48-h period, and only slowly disappeared after longer times (approximately 30% in 72 h), while little of the biotin remained in cells induced to divide. Furthermore, the stability of the biotin in repaired DNA was unaffected by a second dose of UV radiation several hours after the biotin-labeling period to induce a "second round" of excision repair. Exonuclease III digestion and gap-filling with DNA polymerase I indicate that the majority of biotin-labeled repair patches (approximately 80%) are rapidly ligated in confluent human cells. However, the remaining patches were not ligated after a 24-h chase period, in contrast to dTTP-labeled repair patches. The BiodUMP repair label in both chromatin and DNA is preferentially digested by staphylococcal nuclease, preventing the use of this enzyme for nucleosome mapping in these regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in nucleotide and base DNA excision repair observed during mitogenic stimulation of bovine lymphocytes.

The bromodeoxyuridine density-shift technique was used to examine nucleotide and base DNA excision repair in quiescent and lectin stimulated bovine lymphocytes damaged with either ultraviolet light or dimethyl sulfate (DMS). Compared to a number of human cell lines, quiescent lymphocytes were less proficient in the repair of both types of damage. Repair replication was enhanced upon mitogenic stimulation, but both the amount and time course of the increase in repair depended upon the damaging agent used. A 2-3-fold increase in UV light induced repair replication occurred early during stimulation and subsided only gradually as stimulation proceeded. However, the profile of DMS induced repair increased 7-fold and then decreased, in parallel with measurements of lectin-stimulated DNA replication. Estimates of average repair patch sizes showed that quiescent lymphocytes produced smaller patches of 7 nucleotides in response to DMS damage while UV light irradiation resulted in repair patches of 20 nucleotides. During stimulation, patch sizes appeared to increase to maximum values of 45 and 33 nucleotides in response to UV light and DMS, respectively, one day prior to the peak of DNA replication. These increases in patch size were followed by a gradual decrease towards unstimulated levels. However, the appearance of a DNA species of intermediate density in the gradient profiles made the interpretation of repair patch sizes in stimulated cells difficult. These results are discussed as evidence not only for differences in the mechanisms of nucleotide and base excision repair but also for changes in repair as the cell progresses through the cell cycle.     

Hypomethylation of repair patches in HeLa cells

In HeLa cells, under conditions where normal semiconservative synthesis is suppressed by hydroxyurea, the excision repair process after irradiation by UV results in a small amount of incorporation of nucleotides into nonreplicated DNA. By labelling the cytosine moieties of these repair patches, and measuring the ratio between cytosine and 5-methylcytosine, we have found that the level of methylation of cytosine in repair patches five hours after UV-irradiation of the cells is about half of that observed in normal semiconservatively synthesized DNA.     

Accessory cells reduce lipoprotein suppression of lymphocyte activation

Plasma lipoproteins of d less than or equal to 1.063 g/ml suppress lymphocyte activation triggered in vitro by polyclonal T cell mitogens. The extent of suppression decreases as the number of accessory cells per culture increases. Accessory cells isolated by glass adherence and by counter-flow centrifugation reduce lipoprotein suppression to the same extent. Modulation of lipoprotein suppression by accessory cells is independent of the amount and type of polyclonal activator. Reduction of lipoprotein suppression requires viable accessory cells and that they be present with lymphocytes, mitogen and lipoproteins during the initial 24-h culture period. It is within this same time period that lipoproteins exert their suppressive effect. Accessory cells isolated from a patient with the homozygous form (receptor-defective) of familial hypercholesterolemia also reduce the extent of lipoprotein suppression, suggesting that modulation is not mediated by the classic low density lipoprotein receptor. There appear to be at least two mechanisms by which accessory cells may alter lipoprotein suppression of T lymphocyte activation: by secretion of a soluble factor, probably not interleukin 1, that decreases the extent of suppression and by direct modification of the population of suppressive lipoproteins. Neither mechanism accounts for the lipoprotein-enhanced activation that occurs when cultures contain approximately equal numbers of T lymphocytes and accessory cells.     

KINETICS OF THE INCORPORATION OF SOME TRITIATED NUCLEIC ACID PRECURSORS AND [3H]LYSINE INTO MOUSE BRAIN CELLS FOLLOWING INTRAPERITONEAL INJECTION

Abstract— Intraperitoneal injection into white mice of the same amount of radioactivity (0.5 mCi) of [³H]uridine and [³H]lysine demonstrated by autoradiography that there was a much greater labelling of nerve cells from lysine than from uridine. For uridine, the choroid plexus cell nuclei gave maximal labelling within 1 h, with a decrease after 6 h. The plexus nuclei of lysine-injected animals gave almost the same amount of labelling during the experimental period of 48 h. In nerve cells, labelling from uridine increased in the nuclei up to 18 h after injection and there was an almost parallel increase in the labelling in the cytoplasm and neuropil. These results are compared with earlier reports on the results from intravenous injection of uridine. In lysine-injected animals the nerve cell nuclei and cytoplasm showed a fairly constant amount of label over 48 h, but the neuropil counts increased steeply. The activity of the blood was determined by scintillation counting during the 48-h period, and, as with uridine injection, was found to be almost constant over this period. A small series of animals was injected with 0.5 mCi of [³H]uracil, [³H]guanine, [³H]guanosine or [³H]cytidine for comparison. The autoradiograms from animals injected with these bases showed very slight labelling; that from guanosine was heavy in plexus nuclei, slight in nerve cells, and from cytidine it was heavy in plexus cells and moderate in nerve cells.     

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts

The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.     

Quantitative study of deoxycytidine incorporation in large and small lymphocytes of the mouse

Using radioautographic smear preparations of thymocytes and mesenteric lymph node (MLN) cells labelled with three different tritiated pyrimidine deoxyribonucleosides, the incorporation of DNA precursors was studied separately on large lymphocytes and small lymphocytes. Radioautographic reaction due to generally tritiated deoxycytidine ( [G-3H]CdR) labelling in vivo in large lymphocytes was more intense than that in small lymphocytes. When mice were sacrificed 6 hr after the administration of tritiated thymidine ( [3H]TdR), small lymphocytes were labelled more heavily than large lymphocytes. However, labelling intensity with [3H]TdR in large lymphocytes was greatly enhanced by the administration of 5-fluoro-deoxyuridine, whereas in small lymphocytes labelling intensity was only fairly enhanced by the same treatment. When cells were incubated in vitro with 5-tritium labelled deoxycytidine [( 5-3H]CdR) for 10 min, there was no significant difference in labelling intensities between large and small lymphocytes. In the case of [G-3H]CdR incorporation, the labelling intensity in large lymphocytes was found to be significantly stronger than that in small lymphocytes. Large as well as small lymphocytes incorporated [3H]TdR very well in vitro. However, addition of 5 X 0 X 10(-5) M of non-radioactive CdR to the medium greatly decreased the incorporation of [3H]TdR by large lymphocytes, whereas the effect of non-radioactive CdR in small lymphocytes was not so marked as that in large lymphocytes. Furthermore, the [3H]TdR-labelling percentages were decreased at the same rate by the addition of non-radioactive CdR in both large and small lymphocytes. These results indicate that large lymphocytes and a proportion of small lymphocytes have a strong tendency to convert CdR to thymidine mono-phosphate, which is utilized for DNA synthesis, whereas this ability is relatively weak in the rest of small lymphocytes. Thus, it is probably that this metabolic ability changes during the transition of the large lymphocyte to the small lymphocyte.     

Repair of (6-4)photoproducts correlates with split-dose recovery in UV-irradiated normal and hypersensitive rodent cells

Chinese hamster ovary cells and two UV-hypersensitive derivatives were used to determine the importance of DNA excision repair for split-dose recovery. In the wild-type cells 75% of the maximum theoretical recovery was observed when the fractions were delivered at 2-h intervals. Very little recovery was evident in the two hypersensitive cell lines. Using radioimmunoassays specific for (6-4)photoproducts and cyclobutane dimers, the ability of UV-irradiated repair-deficient cells representing 5 complementation groups to repair these 2 photoproducts was determined. Removal of antibody-binding sites specific for (6-4)photoproducts was 80% complete in 6 h and was defective in the UV-sensitive cells. In contrast, only 20-60% of antibody-binding sites specific for cyclobutane dimers were removed 18 h post-irradiation, and the extent of removal was the same in normal and defective cell lines. We conclude that repair of (6-4)photoproducts accounts for split-dose recovery. In addition, we conclude that a consequence of DNA repair in CHO cells is modification rather than removal of cyclobutane dimers.     

DNA repair in cultured mouse cells of increasing population doubling level

Cultures of mouse cells of various population doubling levels (PDL) were examined for DNA-repair capabilities as estimated by (i) the excision of pyrimidine dimers; (ii) unscheduled DNA synthesis (UDS) in response to UV-irradiation or N-methyl-N'-nitrosoguanidine (MNNG) treatment; (iii) the levels of two DNA-repair enzyme activities, uracil DNA glycosylase and AP endonuclease. The responses to ultraviolet light and MNNG decreased rapidly within the first two PDL and more slowly thereafter until essentially no repair was detected by PDL 12. A continuous cell line which emerged from the cultured cells after a crises period had some restoration of repair capability. The amount of uracil DNA glycosylase activity decreased by approximately 40% before the crises period then decreased by 90% in the continuous cell line. In contrast, the amount of AP endonuclease activity present in the precrises cells showed no significant change until PDL 12, then increased 6-7-fold in the continuous cell line.     

Macromolecular synthesis in Streptomyces antibioticus: in vitro systems for aminoacylation and translation from young and old cells.

In vitro systems for the aminoacylation of transfer ribonucleic acid (tRNA) and for polypeptide synthesis have been constructed from young (12-h cultures, not producing actinomycin) and old (48-h cultures, producing actinomycin) cells of Streptomyces antibioticus. When Escherichia coli aminoacyl-tRNA synthetases were used to acylate S. antibioticus tRNA's, it was observed that, per absorbance unit of tRNA, the tRNA's from 48-h cells had a lower ability to accept the amino acids, leucine, serine, pheynlalanine, methionine, and valine than did the tRNA's from 12-h cells. Individual differences were observed between aminoacyl-tRNA synthetases from 12-h cells and those from 48-h cells with respect to the rate and extent of aminoacylation of E. coli tRNA with the five amino acids listed above. In vitro systems for the synthesis of polyphenylalanine have been constructed from 12- and 48-h cells. Ribsomes and soluble enzymes from 12-h cells are more efficient than those from 48-h cells in supporting polyphenylalanine synthesis, and, although the activity of both systems can be stimulated by the addition of E. coli tRNA, the higher level of incorporation observed in the unstimulated 12-h system (ribosomes and soluble enzymes) is maintained. Indeed, the difference in capacity for polyphenylalanine synthesis between in vitro systems from 12- and 48-h cells is greater when the systems are maximally stimulated by E. coli tRNA. Cross-mixing experiments reveal that enzymes from 48-h cells support a slightly higher level of polyphenylalanine synthesis than enzymes from 12-h cells with ribosomes from either cell type, and that the ribosomes are the primary agents responsible for the decreased efficiency of the in vito system from 48-h cells are compared with that from 12-h cells. To determine whether ribosome-associated factors were responsible for the relative inefficiency of the ribosomes from 48-h cells in translation, salt-washed ribosomes from 12- and 48-h cells were examined for their abilities to catalyze polyphenylalanine synthesis. Even after salt washing, ribosomes from 12-h cells were about five times higher in specific activity (counts per minute of polyphenylalanine synthesized per absorbance at 260 nm of ribosomes) than equivalent amounts of ribosomes from 48-h cells. Analysis of the proteins of salt-washed ribosomes of the two cell types by acrylamide gel electrophoresis suggests that the relative amounts of individual proteins present on ribosomes from 12-h cells are different from the amounts present on ribosomes from 48-h cells. These results are discussed in terms of the regulation of translation in S. antibioticus.     

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham's F12 medium with 10(-8) M retinoic acid (RA+), or without retinoic acid (RA-). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA- cultures, but the large cells became flat in RA- medium on day 2. On days 3-5, thymidine incorporation and total labelling indices were less in RA- than RA+ cultures, and on days 4-6, cell numbers were decreased in RA- cultures. On day 3, the large cells of the RA- colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA- colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA- colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA- colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA+ cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA- cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA- cultures on days 5 and 6, respectively. The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a "basal cell hyperplasia".(ABSTRACT TRUNCATED AT 400 WORDS)     

A study of the effect of caffeine upon excision repair of damaged DNA.

Cultured mammalian cells incur damage to their DNA when exposed to ultraviolet light or adduct-producing mutagens such as 4-nitroquinoline-1-oxide (4NQO). At least two processes are important in repair of such damage: post-replication repair and excision repair. Many researchers have reported that caffeine inhibits the former process, which occurs in connection with semiconservative DNA replication, especially in rodent cell lines such as mouse lymphoma or Chinese hamster. Excision repair is not generally considered caffeine-sensitive, although the data are somewhat conflicting because some studies had used rodent cells, which show little or no excision repair, or human cells in which alternate repair processes may have been operating.Human peripherhal blood lymphocytes from healthy donors were treated with UV light or 4NQO in order to produce pyrimidine dimers or adducts. Caffeine at concentrations of 0.75–3.0 mM was included in some cultures. The cells treated with caffeine were incubated for 90 min prior to mutagen treatment and for the entire period thereafter until cell harvests. [³H]Thymidine was added and the uptake quantitated as a measure of DNA repair. DNA replication was inhibited by hydroxyurea, so that only excision repair was measured by this method. Separate plates of cells not exposed to mutagens exhibited negligible or low thymidine uptakes.Following harvest, the cells were lysed and the DNA extracted. The DNA released was measured spectrophotometrically and then placed into liquid-scintillation counter (LSC) vials for measurement of incorporated radioactivity. Resulting cpm/μ DNA were compared for cells with and without caffeine. Lymphocytes from patients with systemic lupus erythematosus (SLE), who previously had demonstrated reduced levels of excision repair under these conditions, were also tested with caffeine. Caffeine did not inhibit repair by normal lymphocytes and the reduced repair seen in the SLE patients was not further reduced in its presence.In a series of pulse-chase experiments, some cells were treated with 4NQO and allowed to incubate with [³H]thymidine for 3 h and were harvested at the end of this period, while others were given a 13-h chase i n cold thymidine before harvest. The cpm/μg DNA for both groups were virtually identical, both in the presence and absence of 2.0 mM caffeine.     

A comparison of the response of unstimulated and stimulated T-lymphocytes and fibroblasts from normal, xeroderma pigmentosum and trichothiodystrophy donors to the lethal action of UV-C.

Unstimulated T-lymphocytes from normal donors are significantly more sensitive to the lethal effects of UV-C than either stimulated T-lymphocytes or fibroblasts as judged by colony-forming ability. Data from other studies suggest that excision repair is more effective in stimulated than unstimulated T-lymphocytes leading to the prediction that these differences in survival should be minimal in cells established from excision defective donors. The prediction was met with XP6BR, a donor of unknown complementation group. For 3 XP's from complementation group D, however, enhanced survival in stimulated T-cells was observed. With cells from an excision-defective TTD who was included in complementation group D of XP both fibroblasts and unstimulated T-lymphocytes were hypersensitive. For a second excision defective TTD patient who was excluded from complementation group D, the unstimulated T-lymphocytes were more resistant than those of normal donors although the fibroblasts were hypersensitive. These results suggest that the in vitro response of stimulated T-lymphocytes or fibroblasts may not reflect the in vivo response of cells, as measured by the response of unstimulated T-lymphocytes.     

Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase α and thymidine kinase activities, as well as a high rate of incorporation of [³H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [³H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G₁ peak of DNA distribution had a significant amount of incorporated [³H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [³H]thymidine.     

The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml.When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.     

Early events in thymocyte activation. II. Changes in nonhistone chromatin proteins induced by a thymus-dependent human serum factor.

Previously, it has been shown that a human thymus-dependent serum factor (SF), isolated from peripheral blood and acting on precursors of mature T lymphocytes, induces an increase in the synthesis of cyclic AMP and proteins in thymocytes. We have now investigated the action of SF on the incorporation of 3H-leucine and 32P-orthophosphate into nuclear proteins of thymocytes after 15 to 240 min of culture. SF induced a rapid increase in the synthesis and phosphorylation of nuclear proteins, especially in the phosphorylated nonhistone chromatin proteins (P-NHCP). Electrophoretic patterns in polyacrylamide gels of the P-NHCP fractions, extracted from the chromatin of the stimulated cells, showed that proteins with m.w. higher than 50 x 10(3) were synthesized to a larger extent as compared with unstimulated cells. These data suggest that SF acts specifically on the synthesis of P-NHCP and may in this way control DNA-template activity.     

Unscheduled DNA synthesis in growing roots and stored embryos of Vicia faba after the action of maleic hydrazide and methyl methanesulphonate

Growing roots of Vicia faba were treated with MH for 5 h, washed for 2 h and exposed to 3H-thymidine (3H-TdR) for additional 2-h periods at 7 h, 24 h and 32 h after the onset of MH treatment, to label DNA. As the replicative DNA synthesis was suppressed by HU, an enhancement of 3H-TdR incorporation into nuclear DNA above the control, as determined by microautoradiography, was considered to be due to unscheduled DNA synthesis induced by the mutagen. A significantly higher incorporation of 3H-TdR into DNA of MH-treated roots occurred, when labelling was applied 7 h after the MH action, whereas at 24 h only slight and at 32 h no enhancement of DNA labelling above control was registered. A 3-14-day storage with 50% water content of V. faba seeds exposed to MH or MMS resulted in a recovery from mutagen-induced chromosomal damage and a significantly higher incorporation of 3H-TdR into nuclear DNA. This supports the hypothesis that recovery from MH- and MMS-induced chromosomal damage is mediated by excision repair during seed storage.     

Persistent effects of ouabain treatment on human lymphocytes: synthesis of DNA, RNA and protein in stimulated and unstimulated cells.

Pretreatment of human lymphocytes for 2 days in 2 X 10(-6)M ouabain caused irreversible loss of their subsequent capacity to stimulate in the mixed lymphocyte reaction (MLR). Pretreatment for the same period with 10(-7)M ouabain resulted in an enhanced incorporation of thymidine into DNA of the responding cells in the MLR; this effect was also on the stimulating cells, as previously reported by Christen et al. (Cell, Immunol. 19, 137-142 (1975)). Pretreatment of stimulating lymphocytes with 10(-7)M ouabain caused a persistent but reversible inhibition of the synthesis of RNA and protein in the MLR; peak incorporation of labelled uridine or alanine reached the same level as that of the control cultures, but 24 h later. Exactly the same persistent but reversible inhibition was found in the case of DNA syntheis of cells pretreated with 10(-7)M ouabain and then stimulated by antigens (streptolysin-O and varidase) or by mitogens (phytohemagglutinin and concanavalin A); the same level of incorporation of labelled thymidine occurred but 24-48 h later than in the case of the controls. Pretreatment with the cardiotonic steroid under these conditions also resulted in a pronounced inhibition of the basal, unstimulated levels of RNA and protein synthesis in the case of both control lymphocytes and those which had been treated with mitomycin C. The effects of ouabain pretreatment on basal RNA and protein synthesis were identical for both 2 X 10(-6)M and for 10(-7)M; the effect of pretreatment of stimulating cells with these two concentrations was completely opposite: irreversible inhibition of the proliferative response of allogeneic responding cells at the former concentration and delayed activation at the latter.     

Repair of X-ray induced DNA strand damage by isolated rat splenic lymphocytes.

Although a number of chemicals can alter DNA repair function, little is known about the effect of chronic, low dose exposure to environmental agents on DNA repair capacity. Lymphocytes provide a potential target population to study the effects of chronic exposures to low doses of toxic chemicals since they are an easily obtainable cell population. Prior to investigating the repair capacity of chemically exposed lymphocytes, the repair by chemically naive lymphocytes has been characterized. In the present study, the DNA repair capacity of isolated rat lymphocytes was characterized. The capacity of these cells to repair single-strand DNA breaks (SSB) was determined after in vitro treatments with X-rays. The effect of in vitro exposure to 3-aminobenzamide (3-AB) on DNA repair capacity was also assessed. The levels of induced SSB and their repair were determined using the alkaline elution technique. Splenic lymphocytes were isolated and placed in culture medium 18 h prior to assessment of repair capacity, but were not stimulated with mitogens. A dose-dependent increase in SSB was observed following exposure of lymphocytes to 300 or 600 rad. The rate of SSB repair was analyzed after a dose of 400 rad. Approximately 80% of the DNA strand break repair was completed within 60 min. The half-time for repair of these lesions by lymphocytes was determined to be 21.3 min. Exposure to 3-AB resulted in a decrease in the rate of repair of the X-ray-induced strand breakage. Although no SSB were detected at the end of a 1-h 3-AB treatment of non-irradiated cells, significant accumulation of SSB was observed after a 2-h treatment. The characterization of DNA repair in rat lymphocytes following in vitro exposure to X-rays will allow us to investigate the effects of chronic, in vivo toxicant exposure on the capacity of isolated lymphocytes to repair DNA damage produced by X-rays.