Unscheduled DNA synthesis in human leucocytes

Polymorphonuclear leucocytes have been induced to synthesize new DNA by exposure to UV light. Preliminary observations (not included) also indicate that 6-MeV electrons and incubation with the radiomimetic agent methyl methanesulfonate (MMS) are effective agents for inducing unscheduled DNA synthesis (UDS). A study of the kinetics of UV-induced DNA synthesis suggests that there are at least two processes operating, one fast and essentially complete within the first 1–2 h and the second lasting at least 8 h.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

Age-related changes in unscheduled DNA synthesis by rat hepatocytes

Ultraviolet-induced unscheduled DNA synthesis was studied as a function of age in hepatocytes isolated from 6- to 32-months-old rats. Unscheduled DNA synthesis was measured by both DNA specific activity and autoradiography. Using both procedures, a significant decline in unscheduled DNA synthesis was observed after 14 months of age.     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

Mitochondrial DNA mutations in individuals occupationally exposed to ionizing radiation

Mutations in a 443-bp amplicon of the hypervariable region HVR1 of the D-loop of mitochondrial DNA (mtDNA) were quantified in DNA extracted from peripheral blood samples of 10 retired radiation workers who had accumulated external radiation doses of >0.9 Sv over the course of their working life and were compared to the levels of mutations in 10 control individuals matched for age and smoking status. The mutation rate in the 10 exposed individuals was 9.92 x 10(-5) mutations/ nucleotide, and for the controls it was 8.65 x 10(-5) mutations/ nucleotide, with a procedural error rate of 2.65 x 10(-5) mutations/nucleotide. No increase in mtDNA mutations due to radiation exposure was detectable (P = 0.640). In contrast, chromosomal translocation frequencies, a validated radiobiological technique for retrospective dosimetric purposes, were significantly elevated in the exposed individuals. This suggests that mutations identified through sequencing of mtDNA in peripheral blood lymphocytes do not represent a promising genetic marker of DNA damage after low-dose or low-dose-rate exposures to ionizing radiation. There was an increase in singleton mutations above that attributable to procedural error in both exposed and control groups that is likely to reflect age-related somatic mutation.     

Kinetics of nitric oxide binding to R-state hemoglobin

Despite earlier work indicating otherwise, some recent reports have suggested that nitric oxide (NO) binds to hemoglobin cooperatively. In particular, it has been suggested that, under physiological conditions, NO binds to the high-affinity R-state hemoglobin as much as 100 times faster than to the low-affinity T-state hemoglobin. This rapid NO binding could provide a means of preserving NO bioactivity. However, using a flash-flow photolysis technique, we have determined that the rate of NO binding to normal adult R-state hemoglobin is (2.1 +/- 0.1) x 10(7) (s(-1) M(-1)), which is essentially the same as that reported for T-state NO binding. (c)2002 Elsevier Science (USA).     

DNA repair synthesis and chromosomal aberrations induced in vivo by triethylenemelamine

The alkylating agent, triethylenemelamine (TEM), was studied for its ability to induce unscheduled DNA (repair) synthesis (UDS) in vivo in rat lymphocytes. Somatic cytogenetic alterations were analyzed (in bone marrow) and compared with UDS as a function of TEM dosage. UDS was evaluated through the use of autoradiography; cytogenetic alterations were studied in metaphase bone marrow chromosome preparations.Data indicated that the degree of UDS is a direct function of TEM dosage up to a rate-limiting concentration, at which point it ceases to be dose dependent. Except for a deviation at the highest dose level tested, the extent of cytogenetic damage was directly and linearly related to TEM dose. Between the control and intermediate (0.2 mg/kg) dose levels, UDS response increased II-fold while cytogenetic damage showed only a 4-fold increase; this disparity diminished with increasing TEM dose. In the lower dose levels, therefore, the greater relative sensitivity of UDS evaluation in the detection of genetic activity may be indicated. Patterns of UDS response observed through the in vivo assay developed in this study were found to be analogous to those established in in vitro studies.     

Genotoxic effects of inhaled ethylene oxide, propylene oxide and butylene oxide on germ cells: sensitivity of genetic endpoints in relation to dose and repair status

We report here results on forward mutation induction (recessive lethal mutations, RL) in Drosophila spermatozoa and spermatids by the three 1,2-alkyl-epoxides ethylene oxide (EO), propylene oxide (PO) and butylene oxide (BO), at doses ranging from 47 to 24,000 ppm h for EO, 375 to 48,000 ppm h for PO, and 24,000 to 91,200 ppm h for BO. The results indicate for EO mutation induction at doses 500-fold below the LD₅₀. In crosses of mutagenized NER⁺ males with NER⁺ females, the 500-fold increase in EO dose from 47 ppm h to 24,000 ppm h resulted in no more than a 17-fold enhanced mutant frequency in spermatozoa. This flat dose–response relationship is primarily the result of efficient repair of EO-induced DNA adducts in the fertilized egg, as was evident from the up to 40-fold or 240-fold increased mutant frequencies above NER⁻ or NER⁺ background levels, respectively, in crosses with NER⁻ females. With decreasing dose, / ratios decreased from 9 to 14 at high doses down to ≈1 at the two lowest doses, indicating that a small fraction of premutagenic lesions induced by EO cannot be repaired by the NER system of Drosophila. Linear extrapolation from high to low EO exposure led to an underestimation of the mutation frequency actually observed at low doses. The pattern of EO-induced ring chromosome loss (CL) differed in two respects from that observed for forward mutations: (a) an increase in CL frequencies was observed only at the two highest EO exposure levels, and (b) inactivation of the NER pathway by the mus201 mutant had no measurable effect on the occurrence of CL. The absence of a potentiating effect of mus201 on EO-induced clastogenicity suggests the formation of clastogenic DNA lesions not causing point mutations, and which are not repaired by NER. Consistent with an inversed correlation of reactivities towards N7-guanine and chain length of 1,2-alkyl-epoxides, the relative mutagenic efficiencies of EO:PO:BO are 100:7.2:1.8 for the NER⁺ groups, and 100:20:0.7 in the absence of NER. Although in Drosophila germ cells EO is also more effective as a clastogen than PO, the difference (EO:PO=100:58) is much smaller than for recessive mutations. These results provide another argument that DNA lesions generating base substitutions as opposed to those causing clastogenic damage may not be the same for these agents.     

In vivo and in vitro ethylene oxide exposure of human lymphocytes assessed by chemical stimulation of unscheduled DNA synthesis

Factory workers exposed to ethylene oxide (EO), 0.5–1.0 ppm in factory air, together with matched controls from the same factory, were examined for evidence of toxic exposure by measurement of unscheduled DNA synthesis (UDS) induced by N-acetoxy-2-acetylaminofluorene (NA-AAF) and of chromosome aberrations in peripheral lymphocytes.The total chromatid gaps plus breaks were significantly elevated and NA-AAF-induced UDS was significantly reduced in the EO-exposed group as compared with the unexposed control group. The NA-AAF-induced UDS values negatively correlated to the duration (yr) of EO exposure (r = ?0.45, p < 0.02) and the number of chromosome breaks (r = ?0.61, p < 0.05), indicating an inhibition in vivo of DNA-repair capacity by EO. These data were verified in vitro by biochemical and autoradiographic studies of EO-induced UDS in human blood cells. Above 2 mM EO, UDS was inhibited in lymphocytes whether they were cultured for 24 or 122 h after alkylation with EO. Even at the subtoxic EO dose of 0.1 mM, lymphocytes were sensitized to additional exposures of NA-AAF, so that cytotoxicity was increased to 40% compared with 5% for the controls even though UDS was unaffected.It is concluded that EO was toxic to lymphocytes, even when they were sensitized at non-toxic EO doses to the cytotoxic action of other mutagens (e.g. NA-AAF), and the cells that did survive above 2 mM EO were inhibited in their DNA-repair capacity as judged by reduced UDS.     

Inhibition of DNA alkylation damage with inorganic salts

Human exposure to alkylating agents metabolized from tobacco- and food-borne carcinogens occurs regularly. Dietary inorganic compounds such as selenium and vanadium have been shown previously to provide chemoprotective benefits in rat and human trials. Here, we present biochemical data on the ability of inorganic compounds to protect DNA from alkylation damage. An enzyme cleavage assay is used to observe alkylated DNA. Simple salts (e.g., NaCl or NiCl₂) did not prevent DNA alkylation, whereas anionic oxo species (e.g., Na₂SeO₄ or Na₃VO₄) did inhibit alkylation. We propose that these oxo species behave as nucleophilic targets for the electrophilic alkylating agents, thereby preventing DNA damage.     

The effects of nickel oxide nanoparticles on structural changes,heme degradation,aggregation of hemoglobin and expression of apoptotic genes in lymphocytes

Abstract

Nickel oxide nanoparticles (NiO NPs) have received great interests in medical and biotechnological applications. However, their adverse impacts against biological systems have not been well-explored. Herein, the influence of NiO NPs on structural changes, heme degradation and aggregation of hemoglobin (Hb) was evaluated by UV-visible (Vis) spectroscopy, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), and molecular modeling investigations. Also, the morphological changes and expression of Bax/Bcl-2 mRNA in human lymphocyte cell exposed to NiO NPs were assayed by DAPI staining and quantitative real-time PCR (qPCR), respectively. The UV-Vis study depicted that NiO NPs resulted in the displacement of aromatic residues and heme groups and production of the pro-aggregatory species. Intrinsic and Thioflavin T (ThT) fluorescence studies revealed that NiO NPs resulted in heme degradation and amorphous aggregation of Hb, respectively, which the latter result was also confirmed by TEM study. Moreover, far UV-CD study depicted that NiO NPs lead to substantial secondary structural changes of Hb. Furthermore, near UV-CD displayed that NiO NPs cause quaternary conformational changes of Hb as well as heme displacement. Molecular modelling study also approved that NiO NPs resulted in structural alterations of Hb and heme deformation. Moreover, morphological and genotoxicity assays revealed that the DNA fragmentation and expression ratio of Bax/Bcl-2 mRNA increased in lymphocyte cells treated with NiO NPs for 24?hr. In conclusion, this study indicates that NiO NPs may affect the biological media and their applications should be limited.

Communicated by Ramaswamy H. Sarma     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.     

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - AFB₁ aflatoxin B₁ - ARG autoradiography - DMN dimethylnitrosamine - LSC liquid scintillation counting - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 4-NQO 4-nitroquinoline-1-oxide - PCA perchloric acid - TCA trichloroacetic acid - UDS unscheduled DNA synthesis     

Chromosome aberrations in individuals occupationally exposed to ethylene oxide, and in a large control population

Chromosome aberration frequencies in 61 employees potentially exposed to ethylene oxide (ETO) were compared with those in unexposed control groups. We studied 3 worksites with differing historical ambient levels of ETO. Within worksites, groups were classified as high potential exposed, low potential exposed, or controls. Further control groups including an off-site community control group were added to give a total of 304 control individuals. Blood samples were drawn several times over a 24-month period. Aberrations were analyzed in 100 cells per sample after culture for 48-51 h. Worksites I, II and III respectively represented increasing levels of potential ETO exposure. At worksites I and II, no consistent differences in aberration frequencies were found among groups. At worksite III aberration frequencies in potentially exposed individuals were significantly increased compared with controls. The frequencies of cells with aberrations were 5.6% for the 2 individuals in the high potential exposure category and 2.6% for 23 persons in the low potential exposure group. The overall frequency of cells with aberrations in the matched control individuals was 1.4%. In the total control group of 304 individuals we found significant increases in aberrations associated with smoking and with increasing age. We have also reported previously an association between sister-chromatid exchange (SCE) frequency and ETO exposure (Stolley et al., 1984). When aberration frequencies were compared with levels of SCEs there was only a weak overall association. The correlation was found in potentially exposed but not in control groups, and for any individual, one observation could not be used to predict the other.     

Study of reparative DNA synthesis in lymphocytes of persons occupationally exposed to radiation

The UDS efficiency in lymphocytes of professionals chronically exposed to gamma/neutron radiation, as well as for a control cohort was estimated. A credible reduction of UV-induced UDS (KUV) index as compared to the control was demonstrated. This shows an invalid repair state of blood cells in professionals. As for the control cohort, the decreasing tendency of UDS index with age was found. The correlative analysis of UDS index dependence upon an absorbed dose (based on physical dosimetry data) revealed the trend towards repair index reduction along with a higher total absorbed doze, that is followed by UDS index coming onto a plateau. It was also demonstrated that after a sharp and/or accidental irradiation UDS index reduces as compared to permanent portioned irradiation. It was not observed the influence of smoking upon UDS efficiency in blood lymphocytes neither for control, nor for experimental group. The analysis of vitamin therapy of the both cohorts showed that such therapy raises UDS index of the control (i.e., non-professional) group. Besides, it was found out an additional positive effect of vitamin therapy, i.e. doze leveling in long-term perspective after irradiation. The most expressed KUV/doze correlation was characteristic for professionals, who do not take vitamins and do not smoke. This proves that in blood cells of professionals it is preserved a dependence of repair system invalidity upon the absorbed doze without similar external factors.     

Repair of alkylation damage in Saccharomyces cerevisiae

Summary Repair of methylated bases in Saccharomyces cerevisiae was measured by two methods: in vitro in cell extracts, and in vivo, by determining the loss of methylated bases from yeast DNA after treatment of stationary cultures with [³H]-N-methyl-N-nitro-N-nitrosoguanidine. Whereas no repair activity could be detected by the in vitro method, the methylated bases were removed in vivo very efficiently. These contradictory results of in vitro and in vivo repair measurements suggest that either the repair enzymes of yeast are sufficiently different from those of bacteria and mammalian cells that they are not active in the in vitro assay, or that methylated bases are repaired in yeast by a different pathway.     

Micronuclei frequency in lymphocytes of individuals occupationally exposed to pesticides.

Pesticides have been widely used in developing countries over the years. A large amount of these remains in the environment and organisms. Pesticide pollution is detrimental to human health. The effects can be seen on a short or a long-term basis and the symptoms can vary from headache to cancer. Only a minority of studies focuses on their genotoxic effect. This study assesses the genotoxic effect of the pesticides used at banana-packaging plants with binucleate micronuclei assay using cultured lymphocytes. The studied population included 32 exposed and 37 unexposed women from Costa Rica. There is no significant difference between the two groups. However, women who worked at the packaging plant and had stillbirths or spontaneous abortions were 1.45 times more (alpha = 0.06) likely to have an increased micronuclei frequency than their coworkers who lacked those disorders; this may indicate genetic susceptibility. In vitro pesticides studies and susceptibility biomarkers are needed to identify subgroups with higher risks.     

Comet assay for assessment of DNA damage in greenhouse workers exposed to pesticides

Purpose: The main goal of the present study was to determine DNA damage in pesticide-exposed greenhouse workers and pesticides non-exposed controls.

Materials and methods: The DNA damage was measured by alkaline comet assay method (pH?>?13) in 41 greenhouse workers and 45 non-exposed individuals as the control. Pesticide exposure was assessed by duration of working in the greenhouse and pesticide application in the greenhouse time. DNA damage was estimated by arbitrary unit and damage frequency.

Results: Arbitrary unit and damage frequency were consistently significantly higher in greenhouse workers than those of the controls (p?=?0.001). In terms of gender in greenhouse, DNA damage of female workers was significantly higher than those in male workers (p?<?0.05). We found significant correlation between DNA damage and working hours spent. Multiple linear regression analysis showed that working hours in the greenhouse as an indication of pesticide exposure were significantly associated with the DNA damage, which can be attributed to the genotoxic potential of the pesticide mixture.

Conclusions: The comet assay is sensitive to detect the damage exposed to chronic effect of pesticides in greenhouse workers. Significant DNA damage was obtained for the exposed group, which was associated with the pesticide exposure.     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P < 0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P < 0.05 or P < 0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90–8.50), 3.43 (2.31–8.29) and 2.04 (0.09–3.83) μm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14–11.81), 3.41 (1.25–11.07) and 0.53 (0.13–3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P < 0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P < 0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.