Role of P53 functionality in the genotoxicity of metronidazole and its hydroxy metabolite

P53 mediates several biological processes for preservation of genetic stability such as the induction of cell cycle arrest, DNA repair or apoptosis in response to DNA damage. The antiparasitic drug, 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole, MTZ) is able to increase lymphocyte proliferation inducing at the same time chromosomal aberrations. Trying to understand this unexpected event we used cell lines with different P53 functionality, determining the proliferation capacity and the induction of micronuclei (MN) after the treatment with MTZ or its hydroxy metabolite. Our results show that MTZ increased proliferation in a dose response manner in all P53 functional cell lines without inducing changes on the levels of P53 nor MN. However, MTZ hydroxy metabolite induced a dose response increase of P53 and MN, while cell proliferation was not increased. Several studies have shown that the hydroxy metabolite is more potent than MTZ itself. Only in cell lines that do not have a functional P53, MTZ and its metabolite increased both cell proliferation and MN. MTZ use is increasing and its carcinogenicity has not been discarded. Our data indicate that MTZ hydroxy metabolite is potentially a carcinogen and needs to be further studied.     

The kinetics of DNA damage by bleomycin in mammalian cells.

The kinetics of DNA damage by bleomycin (BLM) was assessed by measuring the amount of DNA breakage induced by BLM at different doses, treatment lengths, and treatment temperatures. DNA degradation was measured with the alkaline unwinding method. Comparison of the curves of DNA cleavage by BLM leads to the conclusion that low doses (1-5 micrograms/ml) and short treatments (5-15 min) produce marked damage in the DNA. High increases in BLM concentration produce relatively small increases in DNA damage above the levels obtained with low doses. Extension of treatment times does not increase the DNA degradation above the rate observed with 15-min treatments. The repair of DNA damage starts at about 15 min after the initiation of treatment. The mending of DNA breaks is very fast and extensive when BLM is no longer present. Repair not only implies the closing of DNA nicks, but very likely the degradation of the BLM molecules intercalated in the DNA interrupting the reactions responsible for the generation of free radicals. Persistence of BLM in the cell environment facilitates the replacement of degraded BLM molecules by new ones. This produces the persistent production of free radicals and the establishment of a balance between the processes of DNA damage and repair.     

Changes in minimal alveolar concentration of isoflurane following treatment with medetomidine and tiletamine/zolazepam, epidural morphine or systemic buprenorphine in pigs

The aim of this study was to determine the changes in minimal alveolar concentration (MAC) of isoflurane after treatment with medetomidine and tiletamine/zolazepam (MTZ), epidural morphine or systemic buprenorphine in 11 healthy crossbred pigs. The first part of this study was to measure the baseline values in pigs induced with isoflurane (5%) by face mask and maintained with isoflurane in air and oxygen for 2 h (ISO). Baseline isoflurane MAC was determined using mechanical stimulation. Thereafter, each pig was randomly chosen for a crossover test in which the same animal received three different treatments with at least one week in between treatments. The three treatments were as follows: induction of anaesthesia with medetomidine (0.05 mg kg(-1)) and tiletamine/zolazepam (2.5 mg kg(-1) each) given intramuscularly (MTZ); MTZ followed by epidural morphine (0.1 mg kg(-1); MTZ/M); and MTZ followed by intramuscular buprenorphine (0.1 mg kg(-1); MTZ/B). All pigs were maintained with isoflurane in oxygen and air for 2 h and their lungs were mechanically ventilated. The end-tidal isoflurane concentration, respiratory rate, inspiratory and expiratory O2 and CO2 concentrations, heart rate (HR) and arterial blood pressure were recorded every 10 min. Arterial blood gases were analysed every 20 min. Among the treatment groups, differences in isoflurane MAC were tested using GLM and Tukey's method for further comparison; P < 0.05 was adopted as significant. Isoflurane MAC was 1.9 +/- 0.3%. MTZ reduced isoflurane MAC to 0.6 +/- 0.1%. Additional morphine or buprenorphine reduced the MTZ isoflurane MAC further to 0.4 +/- 0.2 and 0.3 +/- 0.1%, respectively. During MTZ, MTZ/M and MTZ/B mean arterial blood pressure was higher and the alveolar-arterial oxygen tension difference was lower compared with ISO. In conclusion, induction of anaesthesia with MTZ reduced the isoflurane MAC in pigs by 68%. Additional epidural morphine or systemic buprenorphine decreased MTZ isoflurane MAC by 33 and 50%, respectively.     

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of mouse spermatogenic cells to O6-alkylating agents

The O(6)-alkylguanine-DNA alkyltransferase inactivator O(6)-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea to study the role of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O(6)-alkylating agents. Exposure of the mice to 1, 3-bis(2-chloroethyl)-1-nitrosourea or O(6)-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O(6)-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O(6)-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O(6)-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1, 3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O(6)-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.     

Repopulation in murine skin after X-ray treatments with multiple fractions per day

The kinetics of repopulation of clonogens in skin after fractionated X-ray exposures was studied in a series of experiments using a top-up design. The feet of mice were exposed to small X-ray doses (1.5 or 2 Gy), given two or three times a day on consecutive days with a minimum interfraction interval of 8 h. A single top-up dose of d(4)-Be neutrons was then given at various intervals after the last X-ray fraction, typically on Days 1,4,8, 15, and 19. The acute skin reaction produced was scored an analyzed by both a standard 23-day averaging and a 7-day averaging procedure. Either method gave similar results and led to the same conclusions. The amount of top-up dose needed to produce a fixed skin reaction was used as a measure of the net effect of the X-ray treatments. This net effect is a result of the initial reduction in skin clonogens due to X rays, and their repopulation before the top-up dose was given. Repopulation was not detected during any of these courses of fractionated treatment, up to an overall time of at least 12 and possibly 16 days. On completion of X-ray schedules lasting 6-16 days, repopulation started 4 days later. In contrast, this delay lengthened to approximately 8 days for shorter overall treatment times of 3-4 days. Once repopulation started, it proceeded rapidly over 11 days so that by 15 days after the cessation of X rays, the skin was restored almost to its normal state with respect to radiosensitivity. The residual damage from Day 15 to Day 19 postirradiation was 3-13% of a full-effect level. The rate of repopulation can be expressed as a clonogen doubling time (Tclon), assuming that an average skin reaction of 1.5 is equivalent to a clonogen surviving fraction of 1.7 x 10(-5). Tclon varied inversely with the amount of initial damage inflicted by the X rays, with the shortest values (1-1.3 days) seen following X-ray doses that gave an initial damage level of 60-80% of full effect. These data are consistent with a hypothesis that damage is "sensed" only 10-12 days after the first X-ray fraction, which provides the stimulus for repopulation of the target cells in the basal layer, the keratinoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo.

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.     

Repair of radiation-induced DNA damage in rat epidermis as a function of age

The rate of repair of radiation-induced DNA damage in proliferating rat epidermal cells diminished progressively with increasing age of the animal. The dorsal skin was irradiated with 1200 rad of 0.8 MeV electrons at various ages, and the amount of DNA damage was determined as a function of time after irradiation by the method of alkaline unwinding followed by S1 nuclease digestion. The amount of DNA damage immediately after irradiation was not age dependent, while the rate of damage removal from the DNA decreased with increasing age. By fitting an exponential function to the relative amount of undamaged DNA as a function of time after irradiation, DNA repair halftimes of 20, 27, 69 and 107 min were obtained for 28, 100-, 200-, and 400-day-old animals, respectively.     

The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation 1-10 Gy

Mitotic compaction of chromatin was generated by treatment of cells with nocodazole. Alternatively, chromatin structure was altered by incubating cells in 500 mM NaCl. The irradiation response in the dose range of 1-10 Gy was measured by colony assay and by a modified fluorometric analysis of DNA unwinding (FADU) assay which measures the amount of undamaged DNA by EtBr fluorescence. Cell survival curves of irradiated CHO-K1 cells showed that treatment with nocodazole increases radiosensitivity as indicated by a decrease of the mean inactivation dose (D) from 4.446 to 4.376. Nocodazole treatment increased the initial radiation-induced DNA damage detected by the FADU assay from 7% to 13%. In repair-defective xrs1 cells, the same conditions increased the radiosensitivity from 1.209 to 0.7836 and the initial DNA damage from 43% to 57%. Alterations to chromatin structure by hypertonic medium increased radiosensitivity in CHO-K1 cells from of 4.446 to 3.092 and the initial DNA damage from 7% to 15%. In xrs1 cells these conditions caused radiosensitivity to decrease from 1.209 to 1.609 and the initial DNA damage to decrease from 43% to 36%. Disruption of chromatin structure by hypertonic treatment was found to be time-dependent. A threefold increase of exposure time to hypertonic medium from 40 to 120 min increased the initial DNA damage in CHO-K1 cells from 7% to 18% but decreased initial DNA damage in xrs1 cells from 43% to 21%. Perturbation of chromatin structure with hypertonic treatment has been shown to increase the radiosensitivity and the initial DNA damage in repair-competent CHO-K1 cells and decrease the radiosensitivity and DNA damage in repair-defective xrs1 cells. Hypertonic treatment thus abolishes differences in chromatin structure between cell lines and differences in initial DNA damage. Radiosensitivity and initial DNA damage are correlated ( r(2)=0.92; p=0.0026) and this correlation also holds when chromatin compaction is altered. The experiments demonstrate that initial DNA damage and chromatin structure are major determinants of radiosensitivity.     

DNA damage produced by cadmium in a human fetal hepatic cell line

Cadmium (Cd) is one of the most important heavy metal environmental toxicants. It alters a wide variety of cellular and biochemical processes. The objective of this work was to study DNA damage and recovery after acute and chronic CdCl2 treatment in a human fetal hepatic cell line (WRL-68 cells). Using the alkaline microgel electrophoresis assay that detects DNA single-strand breaks and/or alkali-labile sites in individual cells, we evaluated for levels of DNA damage. The mean migration length in control cells was 35.37+/-1. 43 microm (8% damaged cells), whereas the mean migration in cells treated with 0.005 microM CdCl2 for 3 h (acute low dose) was 65. 87+/-2.07 microm (88% damaged cells). Treatment with 0.01 microM CdCl2 for the same time (acute high dose) increased the mean migration length to 125.79+/-2.91 microm (92% damaged cells). However, a 0.005 microM CdCl2 treatment for 7 days (chronic treatment) only increased 65% DNA migration to 58.38+/-2.59 microm (88% damaged nucleus). Lipoperoxidative damage expressed as malondialdehyde (MDA) production per milligram of protein was 15. 7+/-2.6 for control cells, whereas in Cd-treated cells the values were 20.2+/-2.4 (acute low dose), 22.9+/-2.2 (acute high dose), and 22.6+/-2.1 (chronic treatment). To study the repair of DNA damage, cells were washed with 0.01 microM meso-2,3-dimercaptosuccinic acid (DMSA), and fresh Dulbecco's modified essential medium (DMEM) added. The percentage of damaged cells diminished after 90 min, with DNA migration returning to control values by 120 min. Cd treatment produced DNA single-strand breaks and the damage was greater in acute high dose treated cells. Lipid peroxidation values did not correlate with DNA single-strand breaks.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.     

Early liver alterations induced by the sex-dependent hepatocarcinogen beta-blocker ZAMI 1305

Liver alterations occurring after 1, 6 or 10 days of treatment with the hepatocarcinogen beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butyl-amino-propan-2-ol (ZAMI 1305) were studied in male and female Wistar rats. In agreement with its sex-dependent oncogenicity, ZAMI 1305 administration causes DNA damage in the liver of the female but not of the male rat, with the only exception of 2 out of 4 males treated for 6 days. In female rat, the amount of DNA damage increases from 1 to 6 days of treatment, being unchanged at 10 days; a small portion of DNA is however damaged. ZAMI 1305 administration to female rat induces also: (i) an increase of the relative liver weight, of the DNA and RNA synthesizing activity; (ii) a decrease of the number of hepatocytes in mitosis; (iii) a minimal oval cell hyperplasia. When the same parameters were studied in ZAMI 1305-treated male rats, they were unaffected or changed to a less extent in respect to female rats.     

Testing drug effects against hypoxic damage of cultured neurons during long-term recovery

Cultured neurons of chick cerebral embryo hemispheres were used to study drug effects against neuronal damage caused by hypoxia during long-term recovery. Sodium cyanide (NaCN, 1 mmol/l) induces hypoxia-like conditions by inhibiting oxydative phosphorylation. The sensitivity of the cultured neurons against this type of hypoxia was determined after 3, 4, 5 and 6 days of cultivation followed by 4, 3, 2 days and 1 day of recovery, respectively. The ATP level and the viability of cells as well as the total cell number and the protein content of the cultures were used to characterize the extent of posthypoxic neuronal damage. A hypoxic period of 30 min after 4 days of cultivation followed by 3 days of recovery seemed to be appropriate for determining protective drug effects. The drug effects obtained were comparable to those from in vivo models of cerebral ischemia or hypoxia. The results suggest that cultured neurons exposed to hypoxia and to long-term recovery could be suitable for studying post-hypoxic neuronal damage as well as neuroprotective drug effects.     

Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase.

X-ray-induced DNA base damage can be detected using endonuclease III and formamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sensitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method that detects DNA damage in individual cells. In using this approach to measure the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiation-induced strand breaks, base damage was detected more easily after higher doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen peroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently when few direct strand breaks were present. Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as direct strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximately 1.0, and (2) the presence of direct DNA strand breaks (>2000-4000 per cell) prevents accurate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

Effect of caffeine on in vivo processing of alkylated bases in proliferating plant cells

DNA damage was induced by either 2 mM ethylmethanesulfonate or 1 Gy of gamma-irradiation in Allium cepa L. root meristems. The percentage of DNA that migrated towards the anode during microelectrophoresis after alkali denaturation (pH approximately 13.5) of the isolated nuclei (comet assay) reflects the amount of single strand breaks present in them. There was some DNA migration (12.8+/-2.4%) in untreated roots. This percentage doubled at the end of 1.5 h treatment with the mono-functional alkylating agent 2 mM ethylmethanesulfonate, and trebled after a single exposure to 1 Gy of gamma-rays. A proportion of the DNA migration caused by these two treatments was reversed (repaired) by a 2 h long period of in vivo recovery. However, when 5 mM caffeine was applied after removal of the alkylating agent, the amount of DNA migrating to the comet tail over the same 2 h period was almost double that at the onset of recovery. In both control and irradiated nuclei, caffeine also increased the initial level of DNA migration in the comet assay, but to a lesser extent. These results indicate that caffeine increases the DNA damage that accumulates during the processing of alkylated bases and, to a lesser extent, of the DNA bases damaged by gamma-irradiation. Thus, the potentiation effect of caffeine on induced chromosomal damage may not just be due to caffeine-induced cancellation of the G2 checkpoint, but also to a direct effect this methylxantine has on the processing of DNA damage.     

Application of conductance measurements to drug mode of action studies

The cytotoxic properties of 1-(2-nitro-1-imidazolyl)-3-(1-aziridinyl)-2-propanol (RSU-1069) on wild-type Escherichia coli and mutants defective in specific DNA repair have been studied. Comparison of the conductance curves obtained for each mutant strain with various drug concentrations allows determination of the processes involved in the repair of drug-induced damage and from this the type of damage induced may be determined. Automated conductance measurements can be used as a model system for studying modes of drug action.     

Investigation of effects of Lacidipine, Ramipril and Valsartan on DNA damage and oxidative stress occurred in acute and chronic periods following isoproterenol-induced myocardial infarct in rats

In this study, effects of Lacidipine (LAC), Ramipril (RAM) and Valsartan (VAL) on DNA damage and oxidative stress occurred in acute and chronic periods after isoproterenol (ISO)-induced myocardial infarct (MI) were investigated in rats. LAC, RAM and VAL had been administered by oral gavage at 3, 3 and 30 mg/kg doses, respectively, in acute and chronic periods following MI. In acute MI model, LAC, RAM and VAL had been administered once per day to rat groups during 30 days. On days 29 and 30, the rats of the acute MI control and drug treatment groups were administered 180 mg/kg ISO, subcutaneously at an interval of 24 h. In chronic MI model, LAC, RAM and VAL had been administered to rat groups during 30 days, and on the 1st and 2nd days, the rats of the chronic MI control and drug treatment groups were administered ISO, by the same way. After this period, routine biochemistry indicators of MI, alanin aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase-isoenzymes (CK-MB), troponin I (TnI) and nitric oxide (NO), oxidative stress indicator, has been measured in the serums obtained from rat’s blood. Also, 7,8-Dihydro-8-oxo-guanine (8-OHGua), which is an indicator of DNA damage level, has been determined in whole blood. After MI diagnosis, the relationships among the 8-OHGua, NO and clinic MI indicators have been determined. Results have been evaluated by comparing with that of control group. In control groups, the clinic MI indicators have been found to be statistically higher than the drug groups. In parallel to this increase in MI indicators, there have been determined a significant decrease in NO levels and an increase in 8-OHGua level. There was no significant difference in the rat groups which received drugs without MI induction. We have observed that the level of 8-OHGua which increased after MI in both acute and chronic periods decreased by LAC, RAM and VAL when compared to acute and chronic MI control groups. In conclusion, it has been determined that oxidative stress has been increased after ISO induced MI model and this stress reduces NO and even damages DNA. LAC, RAM and VAL may decrease the severity of MI and prevent DNA damage by reducing oxidative stress.     

Protective effect of carboxymethyl-glucan (CM-G) against DNA damage in patients with advanced prostate cancer

Carboxymethyl-glucan (CM-G) is a soluble derivative from Saccharomyces cerevisiae (1 → 3)(1 → 6)-β-D-glucan. The protective efficiency of CM-G against DNA damage in cells from patients with advanced prostate cancer (PCa), and undergoing Androgen Deprivation Therapy (ADT), was evaluated. DNA damage scores were obtained by the comet assay, both before and after treatment with CM-G. The reduction in DNA damage, ranging from 18% to 87%, with an average of 59%, was not related to the increased number of leukocytes in peripheral blood. The results demonstrate for the first time the protective effect of CM-G against DNA damage in patients with advanced PCa. Among smokers, three presented the highest reduction in DNA damage after treatment with CM-G. There was no observable relationship between DNA damage scores before and after treatment, and age, alcoholism and radiotherapy.     

Cisplatin-evoked DNA fragmentation in normal and cancer cells and its modulation by free radical scavengers and the tyrosine kinase inhibitor STI571

Cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP) is well studied anticancer drug, whose activity can be attributed to its ability to form adducts with DNA, but this drug can also form DNA-damaging free radicals, however this mechanism of cisplatin action is far less explored. Using the comet assay we studied cisplatin-induced DNA damage in the presence of spin traps: DMPO and PBN, Vitamins A, C and E as well as the tyrosine kinases inhibitor STI571 in normal human lymphocytes and leukemic K562 cells. The latter cells express the BCR/ABL fusion protein, which can be a target of the tyrosine kinase inhibitor STI571. A 20 h incubation with cisplatin at 1-10 microM induced DNA cross-links and DNA fragmentation in normal and cancer cells. Cisplatin could induce intra- and interstrand DNA-DNA cross-links as well as DNA-protein cross-links. DNA damage in K562 cells was more pronounced than in normal lymphocytes. In the presence of spin traps and vitamins we noticed a decrease in the DNA fragmentation in both cell types. Co-treatment of the lymphocytes with cisplatin at 10 microM and STI571 at 0.25 microg/ml caused an increase of DNA fragmentation in comparison with DNA fragmentation induced by cisplatin alone. In the case of K562 cells, an increase of DNA fragmentation was observed after treatment with cisplatin at 1 microM. Our results indicate that the free radicals scavengers could decrease DNA fragmentation induced by cisplatin in the normal and cancer cells, but probably they have no effect on DNA cross-linking induced by the drug. The results obtained with the BCR/ABL inhibitor suggest that K562 cells could be more sensitive towards co-treatment of cisplatin and STI571. Our results suggest also that aside from the BCR/ABL other factors such as p53 level, signal transduction pathways and DNA repair processes can be responsible for the increased sensitivity of K562 cells to cisplatin compared with normal lymphocytes.     

Effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus DNA replication.

The effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus (EBV) DNA replication in the lymphoblastoid cell lines P3HR-1 and Raji is reported. Acyclovir at a concentration of 100 microM completely inhibited EBV DNA synthesis in superinfected Raji cells, but did not inhibit DNA synthesis in mock-infected cells. The number of EBV genome equivalents per cell in the virus-producing cell line P3HR-1 was significantly reduced by acyclovir, whereas the number of latent EBV genomes in Raji cells was not affected by the drug. In situ cytohybridization performed on untreated P3HR-1 cultures revealed the presence of relatively large amounts of EBV DNA in 15 to 20% of the cells. After a 100 microM drug treatment, no P3HR-1 cells contained levels of EBV DNA detectable by in situ cytohybridization. Indirect immunofluorescence studies demonstrated that during treatment with 100 microM acyclovir for 7 days, the percentage of P3HR-1 cells expressing viral capsid antigen was reduced. The EBV DNA remaining in P3HR-1 cells after treatment with 100 microM acyclovir (approximately 14 genomes per cell) had the properties of covalently closed circular DNA with an average molecular weight of 108 X 10(6), as determined by contour length measurements.