Oxidative stress and DNA damage induced by a drinking-water chlorination disinfection byproduct 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in mice

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a water chlorine disinfection byproduct, can induce DNA damage (e.g., modification of nucleotides and DNA strand breaks) and subsequent DNA repair in vitro. However, the underlying mechanism(s) how DNA damage is induced by MX is unknown. We hypothesized that MX may cause oxidative stress that leads to DNA damage in vivo. In the present study, we exposed groups of mice to MX at concentrations of 0 (solvent control), 11 (low), 33 (medium) and 99 (high) mg/kg b.w. by single intraperitoneal injection. After treating the mice for 3h, we detected cellular levels of malondialdehyde (MDA) and glutathione (GSH) to assess oxidative stress in the target cells. In addition, we also evaluated DNA damage using single cell gel electrophoresis (SCGE or Comet assay). We found that the levels of DNA damage in all cell types were correlated positively with levels of MDA but negatively with levels of GSH (P<0.05 for all). Also, there were negative correlations between levels of MDA and GSH (r=-0.995 for liver cells, -0.916 for kidney cells, -0.975 for intestine cells, respectively; P<0.05 for all but kidney cells). Our findings suggest that MX may induce DNA damage by the mechanism of causing cellular oxidative stress as measured by increased MDA and decreased GSH, at least in mice.     

Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.     

Poly(ADP-ribose) Polymerase (PARP-1) in Homologous Recombination and as a Target for Cancer Therapy

Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumours. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumours.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.     

Changes in nuclear chromatin related to apoptosis or necrosis induced by the DNA topoisomerase II inhibitor fostriecin in MOLT-4 and HL-60 cells are revealed by altered DNA sensitivity to denaturation.

The antitumor drug fostriecin (phosphotrienin, FST) has been reported to exert its cytostatic and cytotoxic effects via inhibition of DNA topoisomerase II. The sensitivity of human lymphocytic leukemic MOLT-4 and promyelocytic HL-60 leukemic cells to a wide range of FST concentrations was studied by analyzing the cell cycle-specific effects and changes in nuclear chromatin induced by this inhibitor. The latter was evaluated by assaying the sensitivity of DNA in situ to acid-induced denaturation cytofluorimetrically, with the use of the metachromatic fluorochrome acridine orange (AO), which differentially stains double-stranded and denatured DNA. The cytostatic effects were observed soon after addition of FST (at concentrations of 1-30 microM for MOLT-4 cultures and 1-5 microM for HL-60 cultures) as a perturbation of cell progression through S and G2 phases of the cell cycle. Cell progression through the cycle was halted at greater than 30 microM FST in MOLT-4 cultures and at greater than 5 microM in HL-60 cultures; the effect was instantaneous and affected all phases of the cycle, so that no changes in the cell cycle distribution were apparent with increasing length of exposure to the drug. Instead, at these high FST concentrations, immediate cytotoxic effects became evident, manifesting either as cell apoptosis or necrosis. Apoptosis was observed only in the case of HL-60 cells, at FST concentrations of 5-100 microM, and was characterized by markedly increased sensitivity of DNA to denaturation combined with a decrease in overall DNA stainability, either with the DNA-specific dye DAPI or with AO, indicative of the activation of endogenous nucleases. Necrotic cell death was observed at FST concentrations of 1 mM and at greater than 30 microM for HL-60 and MOLT-4 cells, respectively: in both cases the overall DNA stainability, with either DAPI or AO, was unchanged compared to the control, but their DNA was very sensitive to denaturation. Interestingly, DNA in G2 and late S phase MOLT-4 cells, which were undergoing necrotic death, was much more sensitive to denaturation than was DNA in G1 cells of this lineage. The data indicate that chromatin changes induced by DNA topoisomerase II inhibitors in cells that undergo apoptotic or necrotic death can be conveniently monitored by the assay of DNA in situ sensitivity to denaturation.     

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 1,4-dihydropyridine derivative reduces DNA damage and stimulates DNA repair in human cells in vitro

Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiation-induced mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at 14.9+/-1.2 and 10.3+/-0.8mM, respectively, but did not show a cytotoxic effect at concentrations <100 microM. Alkaline single-cell gel electrophoresis (comet) assays showed that AV-153 reduced the number of DNA strand breaks in untreated cells and also in cells exposed to 2 Gy of gamma-radiation, 100 microM ethylmethane sulfonate (EMS), or 100 microM H2O2. DNA damage was reduced by up to 87% at AV-153 concentrations between 1 and 10nM, and a positive dose-effect relationship was seen between 0.01 and 1 nM. Comparison of the kinetics of DNA strand-break rejoining in the presence and absence of AV-153 revealed a considerable influence on the rate of repair. In view of the resemblance of this compound's structure to that of dihydronicotinamide, a substrate for poly(ADP-rybose)polymerase, the modulation of DNA repair by AV-153 could involve an influence on poly(ADP)ribosylation.     

Induction of TK mutations in human lymphoblastoid TK6 cells by the rat carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX)

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water, is carcinogenic in rats and genotoxic in mammalian cells in vitro. In the current study, the mechanism of genotoxicity of MX in human lymphoblastoid TK6 cells was investigated by use of the Comet assay, the micronucleus test, and the thymidine kinase (TK) gene-mutation assay. MX induced a concentration-dependent increase in micronuclei and TK mutations. The lowest effective concentrations in the MN test and the TK gene-mutation assay were 37.5μM and 25μM, respectively. In the Comet assay, a slight although not statistically significant increase was observed in the level of DNA damage induced by MX in the concentration range of 25-62.5μM. Molecular analysis of the TK mutants revealed that MX induced primarily point mutations or other small intragenic mutations (61%), while most of the remaining TK mutants (32%) were large deletions at the TK locus, leading to the hemizygous-type loss-of-heterozygosity (LOH) mutations. These findings show that aside from inducing point mutations, MX also generates LOH at the TK locus in human cells and may thus cause the inactivation of tumour-suppressor genes by LOH.     

Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.     

Translocation of XRCC1 and DNA ligase IIIalpha from centrosomes to chromosomes in response to DNA damage in mitotic human cells

DNA single-strand breaks (SSBs) are the most frequent lesions caused by oxidative DNA damage. They disrupt DNA replication, give rise to double-strand breaks and lead to cell death and genomic instability. It has been shown that the XRCC1 protein plays a key role in SSBs repair. We have recently shown in living human cells that XRCC1 accumulates at SSBs in a fully poly(ADP-ribose) (PAR) synthesis-dependent manner and that the accumulation of XRCC1 at SSBs is essential for further repair processes. Here, we show that XRCC1 and its partner protein, DNA ligase IIIα, localize at the centrosomes and their vicinity in metaphase cells and disappear during anaphase. Although the function of these proteins in centrosomes during metaphase is unknown, this centrosomal localization is PAR-dependent, because neither of the proteins is observed in the centrosomes in the presence of PAR polymerase inhibitors. On treatment of metaphase cells with H₂O₂, XRCC1 and DNA ligase IIIα translocate immediately from the centrosomes to mitotic chromosomes. These results show for the first time that the repair of SSBs is present in the early mitotic chromosomes and that there is a dynamic response of XRCC1 and DNA ligase IIIα to SSBs, in which these proteins are recruited from the centrosomes, where metaphase-dependent activation of PAR polymerase occurs, to mitotic chromosomes, by SSBs-dependent activation of PAR polymerase.     

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.     

Influx of 5'-deoxy-5'-methylthioadenosine into HL-60 human leukemia cells and erythrocytes

The influx of 5'-deoxy-5'-methylthioadenosine (MeSAdo) into human HL-60 leukemia cells and erythrocytes was characterized in order to determine whether it is facilitated by the nonspecific nucleoside carrier system or by a separate transporter, as suggested by other reports. Initial velocities were measured at room temperature by means of inhibitor-stop and oil-stop assays. MeSAdo influx was strongly inhibited by Ado, dAdo, and nucleoside transport inhibitors including nitrobenzylthioinosine and dipyridamole. Ade was inhibitory only at concentrations in excess of 1 mM. Loss of nucleoside transport capacity during differentiation of HL-60 cells was accompanied by a corresponding decrease in MeSAdo influx rates. These results indicate that MeSAdo influx was mediated by the nonspecific nucleoside transport system. The kinetic data were consistent with a single saturable carrier and yielded Km values of 74 and 184 microM and Vmax values of 424 and 48 pmols/10(6) cells/min with HL-60 cells and erythrocytes, respectively, after correction for a substantial passive diffusion component, which accounted for over 50% of the influx of 1 mM MeSAdo. The passive diffusion of MeSAdo in the presence of a transport inhibitor was not rate-limiting for the salvage of 50 microM MeSAdo to methionine when HL-60 cells were cultured in methionine-deficient medium. The large contribution of passive diffusion to the influx of MeSAdo is consistent with its unusually high octanol/water partition ratio (5.7-fold greater than that of Ado).     

Poly(ADP-ribose) Polymerase inhibitors preserve nicotinamide adenine dinucleotide and adenosine 5'-triphosphate pools in DNA-damaged cells: mechanism of stimulation of unscheduled DNA synthesis

Inhibitors of poly(ADP-ribose) polymerase stimulated the level of DNA, RNA, and protein synthesis in DNA-damaged L1210 cells but had negligible effects in undamaged L1210 cells. The poly(ADP-ribose) polymerase inhibitors stimulated DNA repair synthesis after cells were exposed to high concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (68 and 136 microM) but not after exposure to low concentrations (13.6 and 34 microM). When the L1210 cells were exposed to 136 microM N-methyl-N'-nitro-N-nitrosoguanidine, the activation of poly(ADP-ribose) polymerase resulted in the rapid depletion of oxidized nicotinamide adenine dinucleotide (NAD+) levels and subsequent depletion of adenosine 5'-triphosphate (ATP) pools. After low doses of N-methyl-N'-nitro-N-nitrosoguanidine (13.6 microM), there were only small decreases in NAD+ and ATP. Poly(ADP-ribose) polymerase inhibitors prevented the rapid fall in NAD+ and ATP pools. This preservation of the ATP pool has a permissive effect on energy-dependent functions and accounts for the apparent stimulation of DNA, RNA, and protein synthesis. Thus, the mechanism by which poly(ADP-ribose) polymerase inhibitors stimulate DNA, RNA, and protein synthesis in DNA-damaged cells appears to be mediated by their ability to prevent the drastic depletion of NAD+ pools that occurs in heavily damaged cells, thereby preserving the cells' ability to generate ATP and maintain energy-dependent processes.     

Fe(II)-induced DNA damage in alpha-synuclein-transfected human dopaminergic BE(2)-M17 neuroblastoma cells: detection by the Comet assay

Lewy bodies in the brains of patients with Parkinson's disease (PD) contain aggregates of alpha-synuclein (alpha-syn). Missense mutations (A53T or A30P) in the gene encoding alpha-syn are responsible for rare, inherited forms of PD. In this study, we explored the susceptibility of untransfected human dopaminergic BE(2)-M17 neuroblastoma cells, cells transfected with vector only, or cells transfected with wild-type alpha-syn, A30P alpha-syn or A53T alpha-syn to Fe(II)-induced DNA damage in the form of single-strand breaks (SSBs). DNA SSBs were detected following 2-h treatments with various concentrations of Fe(II) (0.01-100.0 microm), using the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) microm). Fe(II) treatment induced significant increases in CTL in cells transfected with A30P alpha-syn or A53T alpha-syn, even at the lowest concentrations of Fe(II) tested. In comparison, untransfected cells, vector control cells or cells transfected with wild-type alpha-syn exhibited increases in SSBs only when exposed to concentrations of 1.0 microm Fe(II) and above. Even when exposed to higher concentrations (10.0-100.0 microm) of Fe(II), untransfected cells, vector control cells or cells transfected with wild-type alpha-syn were less susceptible to DNA-damage induction than cells transfected with A30P alpha-syn or A53T alpha-syn. Incorporation of DNA-repair inhibitors, hydroxyurea and cytosine arabinoside, enhanced the sensitivity of DNA damage detection. Susceptibility to Fe(II)-induced DNA damage appeared to be dependent on alpha-syn status because cells transfected with wild-type alpha-syn or A53T alpha-syn were equally susceptible to the damaging effects of the mitochondrial respiratory chain inhibitor rotenone. Overall, our data are suggestive of an enhanced susceptibility to the toxic effects of Fe(II) in neuroblastoma cells transfected with mutant alpha-syn associated with inherited forms of PD.     

Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli

Ethidium bromide (EtBr) and SYBR Green I are nucleic acid gel stains and used commonly in combination with UV-illumination. EtBr preferentially induces frameshift mutations but only in the presence of an exogenous metabolic activation system, while SYBR Green I is a very weak mutagen that induces frameshift mutations. We found that EtBr and SYBR Green I, without an added metabolic activation system, strongly potentiated the base-substitution mutations induced by UV-irradiation in E. coli B/r WP2 cells. Each DNA stain alone showed no mutagenicity to the strain. Base-substitutions induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and 4-nitroquinoline-1-oxide were similarly potentiated by EtBr and SYBR Green I. SYBR Green I had a much greater effect. No enhancing effects were observed on mutations induced by mitomycin C, cisplatin, transplatin, cumene hydroperoxide, base analogs, and alkylating agents. Another DNA stain, acridine orange, showed similar enhancing effects on UV- and MX-mutagenicity, but no effect was observed for 4',6-diamidino-2-phenylindole (DAPI). UV- and MX-induced mutations in E. coli WP2s (uvrA), which is defective in nucleotide excision repair activity, were not potentiated by the addition of EtBr, SYBR Green I, or acridine orange. Those nucleic acid stains might inhibit the nucleotide excision repair of DNA damaged by UV and MX treatment.     

Timely termination of repair DNA synthesis by ATAD5 is important in oxidative DNA damage-induced single-strand break repair

Reactive oxygen species (ROS) generate oxidized bases and single-strand breaks (SSBs), which are fixed by base excision repair (BER) and SSB repair (SSBR), respectively. Although excision and repair of damaged bases have been extensively studied, the function of the sliding clamp, proliferating cell nuclear antigen (PCNA), including loading/unloading, remains unclear. We report that, in addition to PCNA loading by replication factor complex C (RFC), timely PCNA unloading by the ATPase family AAA domain-containing protein 5 (ATAD5)-RFC–like complex is important for the repair of ROS-induced SSBs. We found that PCNA was loaded at hydrogen peroxide (H₂O₂)-generated direct SSBs after the 3′-terminus was converted to the hydroxyl moiety by end-processing enzymes. However, PCNA loading rarely occurred during BER of oxidized or alkylated bases. ATAD5-depleted cells were sensitive to acute H₂O₂ treatment but not methyl methanesulfonate treatment. Unexpectedly, when PCNA remained on DNA as a result of ATAD5 depletion, H₂O₂-induced repair DNA synthesis increased in cancerous and normal cells. Based on higher H₂O₂-induced DNA breakage and SSBR protein enrichment by ATAD5 depletion, we propose that extended repair DNA synthesis increases the likelihood of DNA polymerase stalling, shown by increased PCNA monoubiquitination, and consequently, harmful nick structures are more frequent.     

Formation of reactive oxygen species in L929 cells after exposure to 900 MHz RF radiation with and without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone

The aim of this study was to investigate the induction of reactive oxygen species in murine L929 fibrosarcoma cells exposed to radiofrequency (RF) radiation at 900 MHz, with or without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent environmental carcinogen produced during chlorination of drinking water. Both continuous-wave and GSM mobile phone signals were applied for 10 or 30 min at specific absorption rates of 0.3 and 1 W/kg. Simultaneous sham exposures were performed for each exposure condition. MX treatment was performed at a subtoxic level of 500 microM, and the RF-field exposure was carried out during the first 10 or 30 min of the chemical treatment. The formation of reactive oxygen species was followed soon after the exposure and at different harvesting times until 1 h after RF-field treatment. The studied provided no indication that 900 MHz RF-field exposure, either alone or in combination with MX, induced formation of reactive oxygen species under any of the experimental conditions investigated. In contrast, exposure to MX resulted in a statistically significant increase in the formation of reactive oxygen species for all the treatment durations investigated, confirming that MX is an inductor of oxidative stress in L929 cells.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

Double-edged sword of chemosensitizer: increase of multidrug resistance protein (MRP) in leukemic cells by an MRP inhibitor probenecid

The multidrug resistance protein (MRP) is a drug efflux membrane pump conferring multidrug resistance to tumor cells. Clinical trials have been undertaken to improve the effectiveness of chemotherapy by adding an MRP inhibitor to the treatment regimen. This study attempted not only to determine novel resistance mechanisms in MRP-overexpressing AML cells (AML-2/DX100) by chronic exposure to doxorubicin in the presence of an MRP inhibitor probenecid but also to find out whether probenecid could increase MRP levels. AML-2/DXPBA cultured in the presence of probenecid (600 microM) and doxorubicin (100 ng/ml) showed a higher level of the multidrug resistance (MDR) phenotype when compared to AML-2/DX100. AML-2/DXPBA showed increased levels of MRP compared to those of AML-2/DX100. Probenecid increased the MRP levels without an increase in MRP mRNA in AML-2/WT in both a time- and dose-dependent manner. Of the MRP inhibitors including probenecid, ofloxacin, erythromycin, and rifampicin used in this study, only probenecid showed a marked chemosensitizing effect in AML-2/DX100 but not in HL-60/Adr, suggesting that the chemosensitizing effects of the MRP inhibitors vary according to the type of resistant cells. The maximum noncytotoxic concentrations of these MRP inhibitors increased the MRP levels to various degrees in both AML-2/WT and HL-60/WT. However, the chemosensitizing effects of the MRP inhibitors were not correlated with their MRP-increasing effects. Altogether, MRP inhibitors such as probenecid have been shown to function as a double-edged sword, indicating that they are not only an effective chemosensitizer of MRP-associated MDR tumor cells but also an MRP activator. Therefore caution should be taken whenever using MRP inhibitors to reverse MRP-mediated multidrug resistance in clinical cancer chemotherapy as well as when used to inhibit MRP expression in vitro.     

Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays

Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta (polbeta) are thought to be critical elements in the repair of these SSBs and base damages. XRCC1-deficient cells display a radiosensitive phenotype, while proliferating polbeta-deficient cells are not more radiosensitive. We have recently shown that cells deficient in polbeta display increased radiosensitivity when confluent. In addition, cells expressing a dominant negative to polbeta have been found to be radiosensitized. Here we show that repair of radiation-induced lesions is inhibited in extracts with altered polbeta or XRCC1 status, as measured by an in vitro repair assay employing irradiated plasmid DNA. Extracts from XRCC1-deficient cells showed a dramatically reduced capacity to repair ionizing radiation-induced DNA damage. Extracts deficient in polbeta or containing a dominant negative to polbeta also showed reduced repair of radiation-induced SSBs. Irradiated repaired plasmid DNA showed increased incorporation of radioactive nucleotides, indicating use of an alternative long-patch repair pathway. These data show a deficiency in repair of ionizing radiation damage in extracts from cells deficient or altered in polbeta activity, implying that increased radiosensitivity resulted from radiation damage repair deficiencies.