A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocyte/DNA repair assay

Methapyrilene and four related antihistamines were evaluated for their ability to cause DNA repair measured autoradiographically as unscheduled DNA synthesis (UDS) in primary cultures of Fischer-344 rat hepatocytes. Methapyrilene failed to induce UDS at all doses tested while pyrilamine and tripelennamine induced a concentration-dependent increase in DNA repair. Doxylamine and thenyldiamine, previously untested in this system, induced a weak response at the highest non-toxic doses tested. Methapyrilene was clearly cytotoxic at doses of 100 microM and higher, as judged by morphology, and precursor incorporation into RNA and protein. Precursor incorporation into RNA was irreversibly inhibited 90% and 55% at 1000 microM and 100 microM methapyrilene, respectively, while precursor incorporation into protein was inhibited 80% and 60%. These data verify the genotoxicity of pyrilamine and tripelennamine and the failure of methapyrilene to elicit DNA repair, and suggest that doxylamine and thenyldiamine may be weak DNA-damaging agents.     

A study of the potential genotoxicity of methapyrilene and related antihistamines using the hepatocytes/DNA repair assay

Methapyrilene and four related antihistamines were evaluated for their ability to cause DNA repair measured autoradiographically as unschedules DNA synthesis (UDS) in primary cultures of Fischer-344 rat hepatocytes. Methapyrilene failed to induce UDS at all doses tested while pyrilamine and tripelennamine induced a concentration-dependent increase in DNA repair. Doxylamine and thenyldiamine, previously untested in this system, induced a weak response at the highest non-toxic doses tested. Methapyrilene was clearly cytotoxic at doses of 100 μM and higher, as judged by morphology, and precursor incorporation into RNA and protein. Precursor incorporation into RNA was irreversibly inhibited 90% and 55% at 1000 μM and 100 μM methapyrilene, respectively, while precursor incorporation into protein was inhibited 80% and 60%. These data verify the genotoxicity of pyrilamine and tripelennamine and the failure of methapyrilene to elicit DNA repair, and suggest that doxylamine and thenyldiamine may be weak DNA-damaging agents.     

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by chemicals carcinogenic to the rat thyroid

Five chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the same Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the five test compounds: methimazole from 2.5 to 10mM; nitrobenzene, potassium bromate, N,N'-diethylthiourea and ethylenethiourea from 1.25 to 5mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in potassium bromate-exposed thyroid cells from all the three donors and in those from two of three donors with either nitrobenzene or ethylenethiourea, but did not match the criteria for a positive response in thyroid cells from any of the donors with methimazole and N,N'-diethylthiourea. Consistently with their ability to induce thyroid tumors, all the five test compounds, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid. These findings suggest that the five test compounds might be carcinogenic to thyroid in humans.     

Human hepatocyte primary cultures in toxicity assessment

Forty-two compounds of various chemical families were tested for their cytotoxicity and for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes obtained from 74 human donors, and a comparison was carried out with data provided in the same experimental conditions by rat hepatocytes. The results indicate that for the majority of chemicals the intraspecies variability was greater than the average interspecies difference. Some chemicals, however, produced quite different effects in the hepatocytes of the two species, and this suggests that rat hepatocytes might be sometimes inappropriate predictor of the human response.     

Species,sex and inter-individual differences in DNA repair induced by nine sex steroids in primary cultures of rat and human hepatocytes

Sex steroids, due to the generally negative responses observed in routinely employed standard genotoxicity assays, are considered epigenetic carcinogens. Some doubts on this conviction are raised by the results of recent studies providing evidence that cyproterone acetate and two structural analogues, chlormadinone acetate and megestrol acetate, are genotoxic in female rats but only for the liver, and in primary human hepatocytes from donors of both genders. The experimental evidence suggests that the metabolic activation of these molecules to reactive species and the consequent formation of DNA adducts occur only in the intact hepatocyte. Since the possibility that other sex steroids cause a liver-specific genotoxic effect cannot be ruled out a priori, we investigated nine drugs of this family for their ability to induce DNA repair synthesis in primary cultures of rat and human hepatocytes. Each steroid was tested in cultures from at least two male and two female donors of each species. Hepatocytes were exposed for 20h to sub-toxic concentrations ranging from 1 to 50 micro M, and DNA repair induction was measured by quantitative autoradiography. In primary rat hepatocytes, induction of DNA repair indicative of a frankly positive response was detected in cultures from: 2/2 males and 3/3 females with drospirenone, 2/2 males and 1/2 females with ethinylestradiol, 1/2 males and 1/2 females with oxymetholone, 1/2 males with norethisterone, 1/4 females with progesterone, and 1/4 males with methyltestosterone. Consistent negative responses were obtained with testosterone and stanozolol. A few inconclusive responses were observed in rat hepatocytes exposed to progesterone, medroxyprogesterone, norethisterone, methyltestosterone and oxymetholone. In contrast, under the same experimental conditions the nine sex steroids provided frankly negative responses in the large majority of cultures of primary hepatocytes from both male and female human donors; the only exceptions being the inconclusive responses obtained in cultures from two of the donors exposed to norethisterone and to ethinylestradiol, and from one of the donors exposed to testosterone, methyltestosterone, and stanozolol. These results and previous findings concerning cyproterone and its structural analogues suggest that sex steroids differ for their ability to induce DNA repair, and that their genotoxicity may be: (i) different in rat and human hepatocytes, (ii) dependent on the sex of the donor, and (iii) affected by inter-individual variability.     

Interindividual differences in initial DNA repair capacity when evaluating H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H₂O₂)-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H₂O₂, and there was a significant decrease in the H₂O₂-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H₂O₂-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H₂O₂-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.     

Genotoxicity testing of chloramphenicol in rodent and human cells

The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphenicol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of DNA fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced DNA lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of 1/2 LD50 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.     

Role of DNA-dependent protein kinase in neuronal survival

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.     

Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5-10mM), hydrazine (HZ; 0.5-4mM), cadmium sulfate (CD; 31.2 and 62.5muM), 4,4'-methylene bis (2-chloroaniline) (MOCA; 31.2-125muM), isobutyl nitrite (IBN; 7.8-31.2muM) and tetranitromethane (TNM; 1.9-15.6muM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.     

Sister-chromatid exchanges in a special form of xeroderma pigmentosum (form II)

The frequency of sister-chromatid exchanges (SCE) was studied in peripheral blood lymphocytes from a xeroderma pigmentosum (form II, XPII) patient. The cells were irradiated with UV or X-rays. In some experiments novobiocin (NB), inhibitor of topoisomerase II, or caffeine (CA), inhibitor of DNA repair were added to the cultures. The level of spontaneous SCE in the patient's lymphocytes was found to be significantly increased in comparison to that in the cells from normal donors. The inhibitors and UV-light caused a rise in the frequency of SCE in the cells taken from normal donors and except for NB, in the lymphocytes from the patient XPII. X-Rays did not increase SCE frequency in normal lymphocytes and lowered it in the patient's cells. SCE frequency rose when inhibitors of DNA replication and repair were used in combination with mutagens.     

DNA strained breakage repair in ataxia telangiectasia fibroblast-like cells.

Human diploid fibroblast-like cells derived from four patients with the genetic disease ataxia telangiectasia and from two non-mutant donors were examined for the repair of X-ray induced strand breaks in DNA. The ataxia telangiectasia cultures showed no significant differences from the non-mutant cultures in the kinetics and extent of strand repair. This suggests that the increased spontaneous and X-ray induced chromatid aberrations observed in ataxia telangiectasia cells are not caused by a defect in the repair of single strand breaks as might be suspected from a general model of aberration production.     

Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity

It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a relatively long lag period between exposure and cell death. This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size. Experiments reported here demonstrate that cell death can be prevented if 50 micro M CdSO(4) is removed from the growth medium during the lag phase, suggesting that an irreversible apoptotic trigger is delivered within 24 h, between the third and fourth days of cadmium treatment. The post-cadmium recovery phase was characterized by DNA repair at the level of 50-200 kb and increased telomerase activity. Analysis of high-molecular-weight DNA by pulsed-field-gel electrophoresis revealed that the majority of DNA strand breaks was repaired within 48 h after cadmium withdrawal. Telomerase activity increased 2.5-fold in the recovery phase, but elevated levels were also found in cell extracts from apoptotic cells suggesting that telomerase might be associated with DNA repair, but it is not capable of inhibiting ongoing apoptosis. Limited exposure of TBY-2 cells to cadmium elicits non-random DNA damage of relatively high magnitude that can be repaired. It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient genotoxic stress.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

Energy transfer in monomeric phycoerythrocyanin

We describe the involvement of poly(ADP-ribose)polymerase 1 and 2 (PARP-1 and -2) and poly(ADP-ribose)glycohydrolase (PARG) in the response of rat germinal cells to the action of the NO donors, 3-morpholino-sydnonimine (SIN-1) and spermine nonoate (SNO). Primary spermatocytes and round spermatids showed a differential sensitivity to DNA damage induced by acute exposure to SIN-1 and SNO. Spermatocytes were able to repair DNA damage caused by the release of NO from SNO but neither spermatocytes nor spermatids could recover from the release of NO and O2*- from SIN-1. Addition of the PARPs inhibitor, 3-aminobenzamide, and the PARG inhibitor, gallotannin (GT), to germ cell cultures impaired DNA repair significantly. Consistent with the DNA repair seen in primary spermatocytes, both SIN-1 and SNO induced PARPs activation in these cells. In the case of SIN-1, there was an immediate but transient response while SNO induced a delayed but more sustained increase in PARPs activity. Chronic exposure of spermatocytes to SIN-1 and SNO, however, committed the cells to apoptosis, which coincided with proteolysis of PARP-1. The data indicate a dual role for PARPs and PARG in germinal cells as key proteins in processes that sense and repair DNA damage as well as in the commitment to apoptosis following prolonged oxidative stress.     

Patterns of unscheduled DNA synthesis in mouse embryo cells associated with in vitro aging and with spontaneous transformation to a continuous cell line

Monolayer cell cultures derived from B/C mouse embryos were examined for the ability to repair ultraviolet light-induced DNA damage (50–250 erg/mm²) during in vitro aging and subsequent alteration to a continuous cell line. Excision repair was measured by incubating the cultures with [³H]TdR and measuring DNA specific activity, and by performing quantitative autoradiography. DNA repair capacity declined during in vitro aging, and the level of repair correlated with the fraction of cells which retained the capacity to undergo scheduled DNA synthesis. This result indicates that mouse cells aged in vitro undergo a decline in their ability to repair UV-induced DNA damage comparable to that seen in cultured human fibroblasts. In cultures which spontaneously altered into continuously proliferating cell lines, DNA repair capacity increased to high levels, as did the fraction of cells capable of initiating scheduled DNA synthesis.     

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.     

125IdUrd-induced chromosome fragments, assayed by premature chromosome condensation, and DNA double-strand breaks have similar repair kinetics in G1-phase CHO-cells

The effect of 125I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G1-phase CHO-cells. For this purpose a population of mitotic cells was allowed to divide and progress through S-phase in the presence of 125IdUrd. Cells were subsequently transferred to conditioned medium (C-med) obtained from plateau-phase cultures that allowed cells to divide and accumulate in G1-phase in a non-cycling state. To accumulate 125I-induced damage, cells were kept frozen at -80 degrees C. Freezing was carried out using a new method that optimally preserves cell integrity. After various times of cold storage, cells were thawed and assayed for survival, DNA and chromosome damage, either immediately or after various times in C-med. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragment was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation.     

Incorporation of (3H)thymidine stimulated by ultraviolet radiation into human fibroblast cultures.

We studied DNA repair synthesis after ultraviolet irradiation in human fibroblasts cultured in vitro by measuring the ultraviolet-stimulated incorporation of [3H]thymidine into cells in which the semi-conservative DNA replication was inhibited by hydroxyurea. Experiments performed with five fibroblasts lines derived from healthy donors showed a relatively fast initial process ( that is completed within 1 h for 100 erg/mm2 and within 2 h for 500 erg/mm2) and a subsequent slower process, evident between 2 and 6 h after irradiation. The repair capacity of normal cells is expressed by the difference between the values of incorporation (in presence of hydroxyurea) of irradiated and control cells. The pattern of repair was similar in all five cell lines: repair capacity was positive and the amount of repair synthesis increased with incubation time after UV irratiation. Similar experiments were performed with fibroblasts derived from five patients with the classical xeroderma pigmentosum (XP) and from one patient with the De Sanctis-Cacchione syndrome. Normal and XP cells could be distinguished according to whether they displayed a positive or negative value of repair synthesis and/or according to the degree of the slope of the repair synthesis curve as a function of the incubation time after irradiation. We conclude that the technique used in our experiments can demonstrate in a rapid and simple way a defect in the repair capacity in fibroblast cultures; the data are in good agreement with those obtained in the same XP cell lines by other authors [9], who have measured unscheduled DNA synthesis in autoradiographs and repair replication after addition of BUdR.     

Differential repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in DNA of human fibroblasts exposed to ultraviolet light and 4-nitroquinoline 1-oxide

The extent of DNA excision repair was determined in dermal fibroblast strains from clinically normal and xeroderma pigmentosum (XP; complementation group A) human donors after single or combined exposures to 254-nm ultraviolet light and 4-nitroquinoline 1-oxide (4NQO). The repair was monitored by incubation of the treated cultures in the presence of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of long-patch excision repair, followed by quantitation of araC-accumulated DNA single-strand breaks (representing repair events) by velocity sedimentation analysis in alkaline sucrose gradients. The amount of repair in normal fibroblast strains increased as a function of UV fluence and reached a plateau at 15 J/m2; strand breaks were not detected when these same cultures were irradiated with as much as 60 J/m2 UV and incubated in the absence of araC, implying that an initial (incision) step is rate-limiting in the repair of UV damage. In normal fibroblasts (i) the incidence of araC-detectable lesions removed during fixed intervals following exposure to 4NQO (4 microM; 30 min) was approximately 2.5 times greater than that seen following irradiation with repair-saturating fluences (greater than or equal to 15 J/m2) of UV-rays; and (ii) the amount of repair in cultures treated simultaneously with 4NQO (0.5-6 microM; 30 min) and a repair-saturating fluence of UV (20 J/m2) was found to approach the sum of that arising from exposure to each separately. The XP cells (XP12BE) exhibited a deficiency in the removal of araC-detectable DNA lesions following exposure to either of the carcinogens. Since araC is known to inhibit the repair of alkali-stable 4NQO-DNA adducts (i.e., lesions assumed to be removed by the UV-like excision pathway) but not that of alkali-labile sites (i.e., DNA lesions operated on by the X-ray-like repair pathway), our results strongly imply that the multistep excision-repair pathway operative on UV photoproducts in human fibroblasts differs from that responsible for removing alkali-stable (araC-detectable) 4NQO adducts by at least one step, presumably the rate-limiting incision reaction mediated by a lesion-recognizing endonuclease.     

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.