DNA damage in leukocytes of workers occupationally exposed to 1-bromopropane

1-bromopropane (1-BP; n-propyl bromide) (CAS No. 106-94-5) is an alternative to ozone-depleting chlorofluorocarbons that has a variety of potential applications as a degreasing agent for metals and electronics, and as a solvent vehicle for spray adhesives. Its isomer, 2-brompropane (2-BP; isopropyl bromide) (CAS No. 75-26-3) impairs antioxidant cellular defenses, enhances lipid peroxidation, and causes DNA damage in vitro. The present study had two aims. The first was to assess DNA damage in human leukocytes exposed in vitro to 1- or 2-BP. DNA damage was also assessed in peripheral leukocytes from workers with occupational exposure to 1-BP. In the latter assessment, start-of- and end-of-work week blood and urine samples were collected from 41 and 22 workers at two facilities where 1-BP was used as a solvent for spray adhesives in foam cushion fabrication. Exposure to 1-BP was assessed from personal-breathing zone samples collected for 1-3 days up to 8h per day for calculation of 8h time weighted average (TWA) 1-BP concentrations. Bromide (Br) was measured in blood and urine as a biomarker of exposure. Overall, 1-BP TWA concentrations ranged from 0.2 to 271 parts per million (ppm) at facility A, and from 4 to 27 ppm at facility B. The highest exposures were to workers classified as sprayers. 1-BP TWA concentrations were statistically significantly correlated with blood and urine Br concentrations. The comet assay was used to estimate DNA damage. In vitro, 1- or 2-BP induced a statistically significant increase in DNA damage at 1mM. In 1-BP exposed workers, start-of- and end-of-workweek comet endpoints were stratified based on job classification. There were no significant differences in DNA damage in leukocytes between workers classified as sprayers (high 1-BP exposure) and those classified as non-sprayers (low 1-BP exposure). At the facility with the high exposures, comparison of end-of-week values with start-of-week values using paired analysis revealed non-sprayers had significantly increased comet tail moments, and sprayers had significantly increased comet tail moment dispersion coefficients. A multivariate analysis included combining the data sets from both facilities, log transformation of 1-BP exposure indices, and the use of multiple linear regression models for each combination of DNA damage and exposure indices including exposure quartiles. The covariates were gender, age, smoking status, facility, and glutathione S-transferase M1 and T1 (GSTM1, GSTT1) polymorphisms. In the regression models, start-of-week comet tail moment in leukocytes was significantly associated with serum Br quartiles. End-of-week comet tail moment was significantly associated with 1-BP TWA quartiles, and serum Br quartiles. Gender, facility, and GSTM1 had a significant effect in one or more models. Additional associations were not identified from assessment of dispersion coefficients. In vitro and in vivo results provide limited evidence that 1-BP exposure may pose a small risk for increasing DNA damage.     

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.     

Oxygen effect in heat-mediated damage to DNA

The effects of gassing conditions in DNA solution on the major types of heat-mediated DNA damage (depurination of DNA, generation of 8-oxoguanine, cytosine deamination with the formation of uracil) have been studied by ELISA, column liquid chromatography, and spectrophotometry. It was found that the number of DNA lesions depends on oxygen concentration in solution; i.e., the oxygen effect takes place. The heat-induced generation of hydrogen peroxide in water increased after the addition of D20 and decreased by the action of various 1O2 quenchers, suggesting that singlet oxygen is involved in the heat-induced production of reactive oxygen species (ROS) in water. The data obtained favor the hypothesis that all the types of heat-induced damage to DNA are due to a common mechanism associated with the heat-mediated generation of reactive oxygen species in solution.     

Influence of iron-overload on DNA damage and its repair in human leukocytes in vitro

Iron is an important element that modulates the production of reactive oxygen species, which are thought to play a causative role in biological processes such as mutagenesis and carcinogenesis. The potential genotoxicity of dietary iron has been seldom studied in human leukocyte and only few reports have investigated in human colon tumor cells. Therefore, DNA damage and repair capacity of human leukocytes were examined using comet assay for screening the potential toxicity of various iron-overloads such as ferric-nitrilotriacetate (Fe-NTA), FeSO(4), hemoglobin and myoglobin, and compared with 200μM of H(2)O(2) and HNE. The iron-overloads tested were not cytotoxic in the range of 10-1000 microM by trypan blue exclusion assay. The exposure of leukocytes to Fe-NTA (500 and 1000 microM), FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) for 30 min induced significantly higher DNA damage than NC. Treatment with 500 and 1000 microM of Fe-NTA showed a similar genotoxic effect to H(2)O(2), and a significant higher genotoxic effect than HNE. The genotoxicity of FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) was not significantly different from that of H(2)O(2) and HNE. Iron-overloads generated DNA strand break were rejoined from the first 1h. Their genotoxic effect was not observed at 24h. These data from this study provide additional information on the genotoxicity of iron-overloads and self-repair capacity in human leukocytes.     

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes

The colorimetric assay previously described by Mosmann for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lytic mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.     

DNA damage in human leukocytes after ischemia/reperfusion injury

Leukocytes have been shown to play an important role in the development of tissue injury after ischemia and reperfusion (I/R). In the present study, the effects of tourniquet-ischemia on induction of DNA damage in peripheral leukocytes and on respiratory burst of neutrophils in humans were examined. The DNA damage was measured as increased migration of DNA using the single-cell gel-electrophoresis technique (comet assay). Intracellular production of reactive oxygen species by neutrophils was measured flow-cytometrically using dihydrorhodamine 123 as indicator. Postischemic, significantly increased migration of DNA was found in leukocytes of 20 patients (tourniquet-ischemia of the lower limb for 65-130 min, anterior-cruciate-ligament-reconstruction) and in 10 experiments (1 volunteer, repeated tourniquet-ischemia of the upper limb for 60 min, no operation). DNA effects were most pronounced 5-30 min after tourniquet release, and then declined over a 2 h period, but did not return to preischemic baseline values. A similar time course showed the oxidative status of unstimulated granulocytes during reperfusion. Simultaneously, opposing changes were measured in formyl peptide (f-MLP)- or phorbol ester (PMA)-stimulated granulocytes, which showed a significantly declined respiratory burst reaction after tourniquet-release indicating preactivation of neutrophils by IR. Our data suggest that IR induces genotoxic effects in human leukocytes presumably in response to oxidative stress during reperfusion.     

Numerical study to evaluate the effect of a surface-based sensor on arterial tonometry

Abstract

Arterial tonometry is a widely used non-invasive blood pressure measurement method. In contrast to the cuff-based method, it is possible to obtain a continuous pressure profile with respect to systolic and diastolic pressures using this method. However, due to a requirement of arterial tonometry—that a sensor needs to be placed directly above a blood vessel—placement error is inevitable if the measurement device is only capable of measuring local regions. This study assumed that the plate sensor is flexible, thus reducing the placement error. We investigated the pressure distribution along the wrist surface rather than the local region through the contact simulation between the flexible plate sensor and the wrist. As a result, we concluded that there is a unique pressure distribution for any specific wrist, regardless of the length and position of the plate, and that it is possible to measure the blood pressure using the response at the wrist surface to the pressure inside the radial artery.     

Evidence that DNA damage is associated to phenylalanine blood levels in leukocytes from phenylketonuric patients

Phenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism, biochemically characterized by the accumulation of Phe and its metabolites in blood and tissues of affected patients. Treatment for PKU consists of a protein restricted diet supplemented with a mixture containing essential amino acids (other than Phe) and micronutrients. In recent years several authors have studied the pathomechanisms of the disease and demonstrated the existence of lipid and protein oxidative damage in PKU patients. In this work we investigated the in vivo and in vitro effects of Phe on DNA damage determined by the alkaline comet assay using silver staining and visual scoring. We found a dose-dependent effect of Phe on DNA damage in leukocytes from normal individuals incubated with different concentrations of Phe. Additionally, by analyzing blood leukocytes from two groups of treated PKU patients based on their blood Phe levels, we verified that the DNA damage index was significantly higher in PKU patients with high Phe blood levels (DI = 68.2 ± 12.3), compared to well-treated patients and the control group (healthy individuals). Furthermore, well-treated PKU patients had greater DNA damage (DI = 44.9 ± 7.6) relatively to controls (DI = 12.7 ± 4.1). Our present in vitro and in vivo findings indicate that DNA damage occurs in peripheral blood from PKU patients and is associated to Phe blood levels.     

Oxidative DNA damage in polymorphonuclear leukocytes of patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disorder characterized by recurrent, inflammatory self-limited episodes of fever and other symptoms. This disease is caused by more than 25 mutations in the gene MEFV. During fever attacks, there is a substantial influx of polymorphonuclear leukocytes into the affected tissues. Attack-free periods are accompanied by the up-regulation of neutrophil and monocyte phagocytic activity and oxidative burst. These facts led us to hypothesize that oxidative damage by free radicals to DNA may accumulate in FMF patients. To test this hypothesis, we investigated oxidative DNA damage in polymorphonuclear leukocytes of FMF patients during the attack-free period in comparison with FMF-free control individuals. DNA was isolated from polymorphonuclear leukocytes of 17 FMF patients and 10 control individuals. DNA samples were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of various typical oxidatively induced products of DNA. We show, for the first time, that FMF patients accumulate statistically significant levels of these lesions in their DNA when compared to FMF-free control individuals. This work suggests that the persistent oxidative stress with excess production of free radicals in FMF patients may lead to accumulation of oxidative DNA damage. Defective DNA repair may also contribute to this phenomenon, perhaps due to mutations in the MEFV gene. The accumulation of mutagenic and cytotoxic DNA lesions may contribute to increased mutations and apoptosis in FMF patients, thus to worsening of the disease and well-being of the patients. Future research should deal with prevention of oxidative DNA damage and apoptosis in FMF patients, and also the elucidation of a possible role of DNA repair in this disease.     

DNA damage and aberrant crypt foci as putative biomarkers to evaluate the chemopreventive effect of annatto (Bixa orellana L.) in rat colon carcinogenesis

Chemoprevention opens new perspectives in the prevention of cancer and other degenerative diseases. Use of target-organ biological models at the histological and genetic levels can markedly facilitate the identification of such potential chemopreventive agents. Colon cancer is one of the highest incidence rates throughout the world and some evidences have indicated carotenoids as possible agents that decrease the risk of colorectal cancer. In the present study, we evaluate the activity of annatto (Bixa orellana L.), a natural food colorant rich in carotenoid, on the formation of aberrant crypt foci (ACF) induced by dimethylhydrazine (DMH) in rat colon. Further, we investigate, the effect of annatto on DMH-induced DNA damage, by the comet assay. Male Wistar rats were given s.c. injections of DMH (40 mg/kg body wt.) twice a week for 2 weeks to induce ACF. They also received experimental diets containing annatto at 20, 200 or 1000 ppm for five 5 weeks before (pre-treatment), or 10 weeks after (post-treatment) DMH treatment. In both protocols the rats were sacrificed on week 15th. For the comet assay, the animals were fed with the same experimental diets for 2 weeks. Four hours before the sacrifice, the animals received an s.c. injection of DMH (40 mg/kg body wt.). Under such conditions, dietary administration of 1000 ppm annatto neither induce DNA damage in blood and colon cells nor aberrant crypt foci in rat distal colon. Conversely, annatto was successful in inhibiting the number of crypts/colon (animal), but not in the incidence of DMH-induced ACF, mainly when administered after DMH. However, no antigenotoxic effect was observed in colon cells. These findings suggest possible chemopreventive effects of annatto through their modulation of the cryptal cell proliferation but not at the initiation stage of colon carcinogenesis.     

Competition effect in DNA damage response

We have built an ion-microbeam for studies of the nuclear topography and kinetics of double-strand break repair at the single cell level. Here, we show that a first and a second, delayed single ion exposure at different nuclear sites led to comparable accumulations of phospho-ATM, γ-H2AX and Mdc1 at both earlier (e) and later (l) microirradiated sites. In contrast, accumulations of 53BP1 and the recombination protein Rad51 were strongly reduced at l-sites. This apparent competition effect is accompanied by a reduced amount of 53BP1 in undamaged areas of the irradiated nuclei. We suggest that a critically limited pool size combined with strong binding at irradiated sites leads to the exhaustion of unbound factors freely roaming the nuclear space. The undersupply of these factors at l-sites requires in addition a long-lasting binding at e-sites or a weaker binding at l-sites. The observed effects suggest that DNA damage response at individual nuclear sites depends on the time course of damage load. This may have implications for therapeutic radiation treatments. Christoph Greubel, Volker Hable and Guido A. Drexler contributed equally to this work.     

Oxygen effect in heat-induced DNA damage

The major kinds of heat-induced damage to DNA (depurination, guanine oxidation to 8-oxoguanine, cytosine deamination to uracil) were shown to depend in their extent on the oxygen content in solution. Formation of hydrogen peroxide in water upon heating was enhanced in the presence of D₂O and decreased by various scavengers of singlet oxygen, corroborating the involvement of ¹O₂ in the thermal generation of reactive oxygen species. The aggregate data indicate that all kinds of heat-induced DNA damage in solution arise through this common mechanism.     

An in vitro approach to study chromosomal DNA damage

In this study a simple electrophoresis approach has been proposed for assessing DNA damage per chromosome in vitro. Novel procedures of gel casting, sample loading, electrophoresis and quantification of damage have been suggested. Sets of Saccharomyces cerevisiae chromosomes subjected to DNA damage by Bleomycin, Co⁶⁰--radiation alone and in combination with Hoechst were studied in detail. Statistical analyses showed that damage induced by Bleomycin bore linear positive correlation with %GA (r=0.97) and %GT (r=0.61) contents of chromosomes. Samples pre-treated with Hoechst showed much less damage by Co⁶⁰--irradiation as compared to samples not treated with Hoechst but exposed to Co⁶⁰--irradiation. The `protective effect of Hoechst' bore linear positive correlation (r=0.8) with %TAT content of chromosomes.     

Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke

The objective of the present study was to use the comet assay to evaluate the steady-state level of DNA damage in peripheral blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke. A total of 20 rats were distributed into four experimental groups (n=5 rats/group): non-diabetic (control) and diabetic exposed to filtered air; non-diabetic and diabetic exposed to cigarette smoke. A pancreatic beta (beta)-cytotoxic agent, streptozotocin (40 mg/kg b.w.) was used to induce experimental diabetes in rats. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. At the end of the 2-month exposure period, each rat was anesthetized and humanely killed to obtain blood samples for genotoxicity analysis using the alkaline comet assay. Blood leukocytes sampled from diabetic rats presented higher DNA damage values (tail moment=0.57+/-0.05; tail length=19.92+/-0.41, p<0.05) compared to control rats (tail moment=0.34+/-0.02; tail length=17.42+/-0.33). Non-diabetic (tail moment=0.43+/-0.04, p>0.05) and diabetic rats (tail moment=0.41+/-0.03, p>0.05) exposed to cigarette smoke presented non-significant increases in DNA damage levels compared to control group. In conclusion, our data show that the exposure of diabetic rats to cigarette smoke produced no additional genotoxicity in peripheral blood cells of female Wistar rats.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.     

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.     

The effect of the adhesion pili of Yersinia pestis on the physiological activity of the leukocytes and macrophages in experimental animals

The data on the influence of the preparation of Y. pestis adhesion pili on peritoneal macrophages in white mice and guinea pigs are presented. Y. pestis adhesion pili have been found to induce the dose-dependent increase of cell chemiluminescence. They have also been found to induce a number of biochemical changes in target cells: the secretion of myeloperoxidase, an increase in the activity of cAMP-dependent protein kinases.