Effect of Myxin on Deoxyribonucleic Acid Synthesis in Escherichia coli Infected with T4 Bacteriophage

Exposure of Escherichia coli cells to myxin results in the almost complete inhibition of new deoxyribonucleic acid (DNA) synthesis, extensive degradation of pre-existing intracellular DNA, and a rapid loss of viability in these cells (9). After exposure to myxin for 30 min (<1% survivors and >25% degradation of DNA), infection of these cells by T4 bacteriophage results in the renewal of DNA synthesis at a rate essentially equal to that found in T4-infected cells in the absence of myxin. This DNA was characterized as T4 DNA by hybridization and by hydroxyapatite chromatography. These results suggest that the primary site of action of myxin does not involve the biochemical pathways involved in either the energy metabolism or the biosynthesis of DNA precursors in the uninfected host cell. The yield of infectious T4 particles was reduced when myxin was present during multiplication. This effect may be partly accounted for by the finding that a significant fraction of the T4 DNA synthesized in the presence of myxin is apparently not properly enclosed by the bacteriophage protein coat since it is shown to be degraded by exogenous nuclease.     

Mode of Action of Myxin on Escherichia coli

The effect of the new antibiotic, myxin, on the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in Escherichia coli (strains B and 15T(-)) was examined. Within 7 min of the addition of myxin at 5 mug/ml, the synthesis of new bacterial DNA was almost completely inhibited. This was followed by an extensive degradation of the pre-existing DNA to an acid-soluble form. All of the evidence indicated that the primary effect of the antibiotic was on cellular DNA. The synthesis of RNA was completely inhibited after 15 min of exposure to myxin (5 mug/ml), and the synthesis of protein was markedly reduced after 30 min. There was no measurable breakdown of either RNA or protein in the myxin-treated cells. A marked stimulation of (14)C-uracil incorporation was found in the presence of myxin in 15T(-) cells only. This did not result from an increased rate of RNA synthesis but was due to an increase in the proportion of exogenous uracil, relative to endogenous uracil, incorporated into cellular RNA. This probably reflected a partial inhibition of the biosynthesis of uridine monophosphate from orotate. At 4.5 mug of myxin per ml and with 0.8 x 10(8) cells per ml, 50% of the antibiotic was reduced in 15 min from the biologically active oxidized form to the biologically inactive state. Under these conditions, a maximum of 0.6% (27 mumug/ml) of the myxin was retained in the cells.     

Smoke treatment triggers the release of a novel DNA damaging factor by lymphocytes

Abstact Organic fuel smoke is a hazardous agent, which pushes the cells towards“prooxidant state', leading to 4,46,400 strand breaks/cell/day as against 47,000 strand breaks/cell/day produced by constitutive oxygen radicals. This prooxidants scenario switches on a plethora of intercellular events. Here we report a novel DNA damaging factor released by lymphocytes, upon treatment with smoke condensate. Human lymphocytes, when exposed to cow dung cake smoke condensate, were found to release a low molecular weight factor into the media at 20 min of exposure. The conditioned media, displayed a propensity of inducing DNA damage in fresh, normal lymphocytes, which were not exposed to any damaging agent. The above DNA damaging effect of the conditioned media was not due to any residual presence of Polycyclic Aromatic Hydrocarbons, which were present in the smoke. The release of this factor was in correlation with the DNA damaging event, taking place in the cells. This secondary DNA damaging factor had a molecular weight less than 5 kd. The factor had the cell death inducing propensity when allowed to act on lymphocytes     

Effects of rifampicin, streptolydigin and actinomycin D on the replication of Col E1 plasmid DNA in Escherichia coli

We recently reported (Clewell et al., 1972) on an inhibitory effect of rifampicin on Col E1 plasmid replication. The present study represents a further characterization of this phenomenon as well as a study of the effects of two other known inhibitors of RNA synthesis, Streptolydigin and actinomycin D.During treatment of cells with chloramphenicol the colicinogenic factor E₁ (Col E1) continues to replicate for more than ton hours. During this time 4 to 5 S RNA is also synthesized. When varying concentrations of rifampicin were included during chloramphenicol treatment, inhibition of plasmid DNA synthesis correlated very closely with inhibition of cellular RNA synthesis. Similar experiments testing the effects of Streptolydigin and actinomycin D (during chloramphenicol treatment) showed that cellular RNA synthesis was at least 100 times more sensitive to these drugs than was plasmid DNA synthesis.When actively growing cells (i.e. cells not treated with chloramphenicol) were treated with a high concentration of rifampicin (250 μg/ml), chromosomal DNA synthesis continued to an extent representing about a 50% increase in DNA, while plasmid DNA synthesis appeared to stop abruptly.     

Evidence that ultraviolet light-induced DNA replication death of recA bacteria is prevented by protein synthesis in repair-proficient bacteria

The ultraviolet light (UV) survival curve of Escherichia coli WP10 recA trp is almost biphasic, with a greatly reduced shoulder but demonstrating a transition to a decreased slope with increasing fluences, indicating the presence in the culture of a low frequency of resistant cells. Treatment of the culture with chloramphenicol before UV exposure brought almost all of the cells to a high degree of UV resistance, by bringing them to the end of their DNA replication cycle. The survival curves of the repair-proficient E. coli WP2 trp showed a similar pattern with chloramphenicol treatment or tryptophan starvation before UV exposure, but only if protein synthesis were blocked by chloramphenicol for 60 min after UV exposure. The results suggest that when recA/lexA-regulon induction is prevented, either by the recA mutation or by inhibition of protein synthesis after UV exposure, death occurs unless the cells are in the resistant state characteristic of bacteria at the end of their DNA replication cycle. With repair-proficient bacteria treated before UV exposure with chloramphenicol, when protein synthesis is not blocked after UV exposure, a marked expansion of the shoulder occurs because of the function of another resistance-conferring mechanism. This mechanism also depends on the recA+ gene since expansion of the shoulder does not occur in recA bacteria when protein synthesis is inhibited before UV exposure.     

DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2)

Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2). As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis.     

Chloramphenicol effects on DNA replication in UV-damaged bacteria

Increasing UV-doses to cultures of Escherichia coli strain B/r decreased progressively the amount of DNA which was formed in the presence of chloramphenicol (160 μg/ml) from the amount formed in unirradiated control cultures in chloramphenicol-containing medium. This is attributed to the progressive inactivation of active sites of DNA replication by UV. In order to form DNA the bacteria must then replicate from the chromosomal fixed origin, an activity which requires protein synthesis and thus cannot occur in the presence of chloramphenicol. Such damage was shown to be subject to photoreactivation after lower UV-doses and thus is the pyrimidine dimer. After higher doses non-photoreversible lesions began to accumulate so that all such damage became non-photoreversible after 96 erg/mm². The rate of synthesis of DNA in the presence of chloramphenicol was shown to be very close to the rate shown by bacteria incubated in the absence of chloramphenicol, indicating that all active sites of replication remaining after UV-damage remain active in the presence of chloramphenicol, as expected if the limiting effect of chloramphenicol is on initiation at the chromosomal origin and not due to reduction in rate of DNA replication.A much lower concentration of chloramphenicol (2 μg/ml) blocking only the chloramphenicol-sensitive event in control of DNA replication described by Ward and Glaser¹⁵, imposed a limitation in DNA accumulation in the culture of somewhat less than a doubling, as would be expected if the antibiotic at this concentration does not block the chloramphenicol-resistant control event. DNA degradation occured with incubation of bacteria given a UV-dose sufficient to inactivate all active DNA replication sites on their chromosomes, when in medium containing chloramphenicol concentrations (above 20 μg/ml) sufficient to block the chloramphenicol-resistant control event. Such breakdown resulted in death. The damage responsible for such death and DNA breakdown was not photoreversible after this dose, supporting the hypothesis that breakdown results from non-photoreversible inactivation of active DNA replication sites. This was in contrast to increased death in UV-damaged bacteria promoted by nalidixic acid, a specific inhibitor of DNA replication, which could be prevented in part by light exposure after the same UV-dose.     

Rescue of mitomycin C- or psoralen-inactivated Micrococcus radiodurans by additional exposure to radiation or alkylating agents

The processing of damaged DNA was altered in a mitomycin C-sensitive mutant (mtcA) of Micrococcus radiodurans. Even though the mutant retained resistance to 254-nm UV radiation, it did not, in contrast to the wild-type strain, show any excessive DNA degradation or cell death when incubated with chloramphenicol after sublethal doses of either UV light or mitomycin C. The results suggest the constitutive synthesis of an enzyme system responsible for wild-type proficiency in the repair of mitomycin C-induced damage. An alternative system able to repair damage caused by mitomycin C was demonstrated in the mtcA background. In this strain, additional damage inflicted upon the cellular DNA effected a massive rescue of cells previously inactivated by mitomycin C. Rescue was provoked by ionizing radiation, by UV light, or by simple alkylating agents. Cells treated with psoralen plus near-UV radiation could be rescued only when inactivation was due primarily to psoralen-DNA interstrand cross-links rather than to monoadducts. The rescue of inactivated cells was prevented in the presence of chloramphenicol. These results can be interpreted most readily in terms of an alternative repair system able to overcome DNA interstrand cross-links produced by mitomycin C or psoralen plus near-UV light, but induced only by the more abundant number of damages produced by radiation or simple alkylating agents.     

Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay

The alkaline single cell gel electrophoresis (comet) assay was applied to study genotoxic properties of two inhalation anesthetics-halothane and isoflurane-in human peripheral blood lymphocytes (PBL). The cells were exposed in vitro to either halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) at concentrations 0.1-10 mM in DMSO. The anesthetics-induced DNA strand breaks as well as alkali-labile sites were measured as total comet length (i.e., increase of a DNA migration). Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner. In experiments conducted at two different electrophoretic conditions (0. 56 and 0.78 V/cm), halothane was able to increase DNA migration to a higher extent than isoflurane. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA degradation due to cell death. For this reason a contribution of toxicity in the observed effects was examined. We tested whether the exposed PBL were able to repair halothane- and isoflurane-induced DNA damage. The treated cells were incubated in a drug-free medium at 37 degrees C for 120 min to allow processing of the induced DNA damage. PBL exposed to isoflurane at 1 mM were able to complete repair within 60 min whereas for halothane a similar result was obtained at a concentration lower by one order of magnitude: the cells exposed to halothane at 1 mM removed the damage within 120 min only partly. We conclude that the increase of DNA migration induced in PBL by isoflurane at 1 mM and by halothane at 0.1 mM was not a result of cell death-associated DNA degradation but was caused by genotoxic action of the drugs. The DNA damage detected after the exposure to halothane at 1 mM was in part a result of DNA fragmentation due to cell death.     

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro.

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.     

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.     

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.     

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following³H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA. Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells. The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis. These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.     

Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress

Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase α from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.     

Response of human keratinocytes to extremely low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine

Since alkylating agents are widely present in the environment and constitute a continuous challenge to genome integrity, cells and organisms have developed defense mechanisms to remove such lesions. We monitored the response of human keratinocytes to a very low concentration of a methylating agent, namely 2.5 nM N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The effect of a 60-min exposure of quiescent cells to 2.5 nM MNNG was studied in terms of DNA integrity, poly(ADP-ribose) metabolism, clonogenic survival and DNA synthesis. We observed two waves of DNA strand break formation and resealing. Interestingly, the amount of DNA strand breaks in exposed cells was lower than in unexposed control cells. This phenomenon was also observed when cells were exposed to MNNG in the presence of a protein synthesis inhibitor, or when they were maintained on ice during the treatment. A dose of 2.5 nM MNNG stimulated poly(ADP-ribose) turnover, reduced the intracellular NAD⁺ content, stimulated DNA synthesis and caused a remarkable increase in clonogenic survival. Thus, the cellular responses to extremely low concentrations of MNNG differ sharply from those observed at higher doses of this carcinogen. We conclude that the very low dose response cannot be extrapolated from usual dose-response analyses.     

Inhibition and recovery of DNA synthesis in human tumor cell lines following radiation exposure

Eight human tumor cell lines with radiosensitivities (D0) ranging from 1 to 3 Gy were analyzed for their response to radiation-induced inhibition of DNA synthesis. These cell lines differ in their sensitivity to induction of DNA double-strand breaks and in the rate at which they rejoin DNA double-strand breaks. Fifty-gray doses of gamma rays induced between 35 and 75% inhibition in rates of DNA synthesis. The magnitude of the inhibition was not related to cellular radiosensitivity, frequency of initial DNA double-strand breaks, or the rate of rejoining of DNA double-strand breaks. All the cell lines studied had similar kinetics of recovery from inhibition of DNA synthesis following radiation exposure. These results suggest that factors other than or in addition to frequency of DNA double-strand breaks are important in the control of DNA synthesis following exposure to ionizing radiation in human tumor cell lines.     

Influence of cellular differentiation on repair of ultraviolet-induced DNA damage in murine proadipocytes

The effects of cellular differentiation on the repair of DNA damage induced by uv radiation were investigated in the murine 3T3-T proadipocyte cell culture system. Upon exposure to human plasma, actively cycling 3T3-T cells (stem cells) undergo growth arrest, which is followed by terminal differentiation into lipid-laden adipocytes. In response to uv irradiation, the level of unscheduled DNA synthesis is significantly lower in adipocytes as compared to stem cells. The alkaline elution assay was used to monitor the appearance of repair-induced strand breaks in 3T3-T cells after uv irradiation. DNA strand breaks were detected in stem cells by 4 min post-uv with essentially no further increase after 8 min. When terminally differentiated adipocytes were irradiated and allowed to repair, however, more strand breaks were present at 4 min and, in marked contrast to stem cells, continued to accumulate in adipocytes for at least 16 min post-uv. Inhibition of repair-replication with hydroxyurea and cytosine arabinoside significantly increased accumulation of repair-induced strand breaks in stem cells, yet had little effect on this accumulation in adipocytes. For stem cells and adipocytes, incision activity was linear out to at least 10 Jm-2 without saturation. These data suggested that 3T3-T cell differentiation is accompanied by a defect in some postincision process of the excision-repair pathway.     

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.