Mutagenesis Advance Access originally published online on February 14, 2008
Mutagenesis 2008 23(4):249-260; doi:10.1093/mutage/gen004
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Occupational exposure to mineral fibres. Biomarkers of oxidative damage and antioxidant defence and associations with DNA damage and repair
1Research Base of Slovak Medical University, Limbová 12, 833 03 Bratislava, Slovakia 2Department of Nutrition, University of Oslo, PO Box 1046 Blindern, 0316 Oslo, Norway 3Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia 4Norwegian Institute of Air Research, Instituttveien 18, NO-2027 Kjeller, Norway
In order to study the effect of mineral wool exposure on oxidative DNA damage and lipid peroxidation, an epidemiological study was conducted in a mineral wool factory in Slovakia. Altogether 141 subjects were investigated (21–58 years old), 43 controls (20 men and 23 women: 27 non-smokers, 16 smokers) and 98 exposed (75 men and 23 women: 61 non-smokers, 37 smokers). We found higher malondialdehyde (MDA) levels in the group of all exposed workers (P = 0.025) and in exposed non-smokers (P = 0.003) and a significantly suppressed activity of ceruloplasmin oxidase (P = 0.02, P < 0.02, respectively) and catalase (CAT) (P = 0.04, P = 0.01, respectively) in these groups. The activity of glutathione S-transferase (GST) was affected by exposure to mineral wool; levels were significantly lower in all exposed subjects (P = 0.04), in the exposed non-smokers (P = 0.03) and in exposed men (P < 0.01). Concentrations of vitamin C in plasma and the ferric-reducing activity of plasma (FRAP) were not affected by the mineral wool exposure. There was a significant negative correlation between the activity of glutathione peroxidase (GPX) and MDA in the whole group (P < 0.01) and in the exposed group and between CAT activity and MDA in all subjects (P < 0.01). GST activity correlated inversely with oxidized pyrimidines in lymphocyte DNA, in almost all subgroups. We found significant negative correlations between DNA repair and GPX in all subjects (P = 0.03) as well as in control men (P < 0.03) and between DNA repair and CAT in all control subjects (P < 0.02) and in control men (P < 0.01). Interestingly, we found a positive correlation between DNA repair and MDA in all subjects (P < 0.01) and in all exposed subjects (P < 0.03). The presented results indicate that mineral wool exposure induces an increase in oxidative damage to biomolecules especially in the group of male non-smokers. However, optimal levels of antioxidants could have a protective effect. Biomarkers such as MDA, antioxidant enzymes and antioxidant vitamins measured in blood may be useful biomarkers of oxidative stress and antioxidant protection. We do not recommend FRAP as a marker of antioxidant status as interference from other constituents can provide false or confusing results. Our study supports the idea that there might also be other mechanisms by which antioxidant enzymes (especially GST) protect cells against oxidative DNA damage.
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Introduction |
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Free radicals and peroxides are clearly involved in physiological phenomena such as synthesis of prostaglandins and thromboxanes (1
) and in pathogenesis of various diseases, including atherosclerosis, inflammatory diseases and cancer. They are also thought to participate in aging processes. The biological effects of these highly reactive compounds are controlled in vivo by a wide spectrum of antioxidative defence mechanisms: vitamins E and C, carotenoids, metabolites such as glutathione and uric acid and antioxidant enzymes (2,3). Among these enzymes, superoxide dismutase (SOD; EC 1.15.1.1) catalyzes dismutation of the superoxide anion (O–.) into hydrogen peroxide (H2O2), catalase (CAT; EC 1.11.1.6) breaks down H2O2 and glutathione peroxidase (GPX; EC 1.11.1.9) both detoxifies H2O2 and converts lipid hydroperoxides to non-toxic alcohols. In several clinical studies, one or several of these antioxidant enzymes were measured in blood as possible biological indicators, especially concerning hyperlipidaemia and atherosclerosis (4), alcoholism (5–8), diabetes (9,10), Down syndrome (11,12), cancer (13,14) and Alzheimer disease (15). However, data concerning the biological variability of these enzymes in blood of apparently healthy subjects are scarce and have often been obtained from restricted populations (16–18).
Oxidative stress occurs when reactive oxygen species (ROS) are formed in amounts that exceed the capacity of the antioxidant defence system to remove them. The detrimental effects of ROS typically are assessed by the presence of oxidatively altered biomolecules. Oxidative DNA damage formed during oxidative stress probably plays a critical role in carcinogenesis (19). During oxidative stress in vivo or when ROS react with DNA in vitro, several types of DNA damage are formed, including strand breaks and small base lesions (20). 8-Oxoguanine constitutes one of the most easily formed oxidative DNA lesions and can be detected by high-performance liquid chromatography (HPLC) in both urine and tissues after oxidative stress (19). Alternatively, oxidative DNA lesions can be detected by the enzyme-modified single-cell gel electrophoresis (comet) assay (21).
The adverse effects that arise from exposure to asbestos have stimulated the development of substitute materials, man-made mineral fibres (MMMFs). However, little is known about the health effects of these fibres. The potentially harmful effects of all types of respirable fibres are at present one of the most important fields of interest in industrial hygiene (22).
Mineral wool, made from natural basic material, is used mainly for thermal and acoustic insulation and fire protection of roofs, walls and floors. Mineral wool, formerly regarded by the International Agency for Research on Cancer as a possibly carcinogenic material (in the classification group 2B), was reassessed in 2001 and is now assigned to group 3, i.e. not classifiable as to human carcinogenicity. There is only weak epidemiological evidence linking mineral wool exposure with human cancer (23). There appear to have been few studies of the possible genotoxicity of MMMF. In principle, fibres might induce carcinogenesis directly, or via inflammation, with its associated release of reactive oxygen, damage to DNA and cell proliferation; on the other hand, mineral wool fibres are cleared rapidly from the rodent lung (24). In recent in vitro experiments in rat alveolar macrophages, mineral wool was not cytotoxic (25).
As part of an EC-funded investigation into the possible consequences to health of exposure to mineral fibres, we have monitored various biomarkers related to genotoxicity in 98 workers exposed to mineral wool during its manufacture in a factory in Nova Bana (Slovakia) and 43 control employees of the same factory working in administrative or other jobs with minimal exposure to mineral wool (26). Strand breaks in lymphocyte DNA were higher in exposed compared to control non-smokers, but there was no effect of exposure on specific damage to bases in DNA (oxidation and alkylation), nor on chromosome aberrations. The frequency of micronuclei was higher in women in the control group than in mineral wool-exposed women. DNA repair 8-oxoguanine DNA glycosylase (OGG) activity was unaffected by exposure, but was negatively correlated with micronucleus frequency, implying that unrepaired 8-oxoguanine contributes to micronucleus formation. The conclusion from this study was that, overall, mineral wool exposure has no clearly deleterious effect on genetic stability in humans. Here we present data on additional biomarkers of oxidative damage and antioxidant defence in relation to exposure to mineral wool and compare them with previously measured genetic stability markers, namely DNA damage and repair.
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Materials and methods |
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Subject selection and samplings
Workers with at least 5 years exposure to mineral wool at an industrial plant in Nova Bana, Slovakia, were recruited for this study. The group of 141 healthy adult volunteers (average age 40.8, ranging from 21 to 58) consisted of 43 control clerical workers from the factory (20 men and 23 women: 27 non-smokers, 16 smokers) and 98 exposed (75 men and 23 women: 61 non-smokers, 37 smokers) (Table I). The sampling was carried out in September 2000. The participants were interviewed by trained personnel. Each participant provided detailed information on working histories, physical activity, active and passive smoking and alcohol consumption, exposure to environmental materials, health condition, occupation, education and other socioeconomic variables. All workers underwent clinical examination, including a functional spirometry test, radiological and immunological examination. All subjects contributed a single blood sample in autumn. A urine sample was used for measurement of cotinine to determine smoking status. Blood was collected by venipuncture from fasted subjects, with ethylenediaminetetraacetic acid (EDTA) as anticoagulant, and used for isolation of plasma, lymphocytes and erythrocytes for both markers of genetic stability and markers of oxidative damage and antioxidant protection. Both exposed and referent samples were collected on the same day to minimize the influence of experimental variation. The study was blinded and all subjects, questionnaires and samples were coded prior to coming to the clinic for examination or to the laboratory for further analyses. The study was conducted in a good laboratory practice laboratory following standard operating procedures.
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All study participants signed an informed consent form. This study was approved by the Ethical Committee of the Research base of the Slovak Medical University (the Institute of Preventive and Clinical Medicine) in Bratislava.
Exposure in workplace and personal dosimetry
Exposure to mineral fibres and polycyclic aromatic hydrocarbon (PAH) in the workplace was detected using high-volume stationary samplers (GPS-1; Graseby-Andersen, Atlanta, GA, USA) as well as by personal dosimeters. Concentrations of mineral fibres as well as PAH exposure were measured four times a year (including sampling time) in the mineral wool factory. Two samples of indoor air (one in a production hall and one in an administrative building) were collected in each season.
Personal dosimetry was carried out at the time of sampling in the mineral wool factory. Personal air samples for fibres as well as PAHs were collected by personal samplers both at the workplace (8 h) and at home (8 h).
PAH measurements. Thirteen PAH congeners (fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[a]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene) collected by personal and high-volume samplers were determined by high-resolution gas chromatography/low-resolution mass spectrometry in the selected ion monitoring mode isotope dilution method according to United States Environmental Protection Agency TO-13 method.
Fibre determination. Air particulate samples were taken on membrane filters. Samples were evaluated using a microscope with phase contrast (Nikon, Tokyo, Japan) according to the Reference Method for the Determination of Airborne Asbestos Fibre Concentration at Workplaces by Light Microscopy (Membrane Filter Method), AIA 1979 (London, UK). Fibre identification, size distribution and quantification were confirmed by an electron scanning microscope combined with X-ray microanalysis. All fibres were counted for artificial mineral fibres (basalt glass) and then sorted to respirable and non-respirable.
Assay of cotinine in urine
Concentration of cotinine was measured by a radioimmunoassay method using a commercial kit from Brandeis University (Waltham, MA, USA). The method is based on competition between labelled and unlabelled cotinine for limited antibody-binding sites. To the standard diluted with buffer, 3H-cotinine and anti-cotinine-CDI-thyroglobulin were added. After incubation, normal rabbit serum and goat ‘Minnie’ anti-rabbit-
-globulins were added. The reaction mixture was incubated overnight at 4°C. The sediment was diluted in 0.1 M NaOH. After adding scintillation reagent, the activity of the cotinine–antibody complex was measured. For every set of samples, a new calibration curve was made. The concentration of cotinine is expressed as milligrams of cotinine per millilitre creatinine in urine. Creatinine (two-point rate test) was measured with a VITROS 250 biochemical analyser.
Biochemical screening
Serum biochemical markers were measured including aspartate aminotransaminase, alanine aminotransferase, -glutamyltransferase, alkaline phosphatase, amylase, urea, creatinine, albumin, total protein, total bilirubin, cholesterol, triacetylglycerols and glucose. Urine osmolarity and uric acid were measured. Biochemical analyses were performed in a Vitros 250 analyser (Johnson & Johnson, Rochester, NY, USA). The body mass index (BMI) was calculated as body weight (kg)/height (m2).
Isolation of lymphocytes
After centrifugation of blood for collection of plasma (frozen as aliquots at –80°C), the buffy coat was recovered and mixed with RPMI 1640 medium with 10% foetal calf serum (FCS) before layering over an equal volume of Lymphoprep (Nycomed, Oslo, Norway) and centrifuging at 700 x g for 20 min at 20°C. The layer above the Lymphoprep, containing lymphocytes, was removed, diluted with medium and centrifuged at 700 x g for 15 min at 20°C. Lymphocytes were used immediately for estimation of DNA damage, while surplus cells were suspended in 90% FCS and 10% dimethyl sulphoxide, divided into aliquots and frozen slowly to –80°C. For the DNA repair assay, lymphocytes (5 x 106 in 50-µl aliquots) were snap frozen in liquid nitrogen in 45 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.4 M KCl, 1 mM EDTA, 0.1 mM dithiothreitol and 10% glycerol, pH 7.8. The remaining blood cells were stored frozen at –80°C for additional analyses.
Measurement of markers of oxidative stress and antioxidant protection
Measurement of antioxidant enzyme activity.
After plasma separation, erythrocytes were washed three times with isotonic saline (0.9% sodium chloride). Centrifugation for each washing was at 3000 r.p.m. for 10 min at 4°C. Erythrocytes were then lysed with hypotonic solution (distilled water) and the haemolysate was used (at suitable dilutions) for measurement of activities of antioxidant enzymes. The activity of GPX was determined by the kinetic method according to Paglia and Valentine (27), CAT was measured spectrophotometrically by a modified method of Cavarocchi et al. (28) and glutathione S-transferase (GST) by a kinetic method according to Habig et al. (29). The activity of SOD was estimated by a commercial test kit (Randox Lab. Ltd, Grumlin, UK).
Measurement of total antioxidant capacity, FRAP.
The index of the combined non-enzymatic antioxidant capacity of plasma [ferric-reducing activity of plasma (FRAP)] was measured spectrophotometrically according to Benzie and Strain (30). Ferric to ferrous ion reduction at low pH causes formation of a coloured ferrous–tripyridyltriazine complex. FRAP values were obtained by comparing the absorbance at 593 nm in test samples with solutions containing ferrous ions in known concentration.
Measurement of ceruloplasmin oxidase.
Ceruloplasmin (CPL) oxidase activity in plasma was assayed with the use of o-dianisidine dihydrochloride according to the method of Schosinsky et al. (31).
Measurement of antioxidant micronutrients.
Plasma vitamin C (32), 
-tocopherol, -tocopherol, β-carotene, retinol, xanthophyll and lycopene were detected by HPLC (33).
Measurement of malondialdehyde.
Lipid peroxidation was determined as levels of malondialdehyde (MDA) by a modified HPLC method in plasma (34).
Measurement of DNA damage and repair.
DNA damage was measured using the comet assay (single-cell alkaline gel electrophoresis) as previously described (35,36), and these data were presented previously (26). In addition to frank DNA strand breaks, oxidized bases were measured by conversion to breaks using endonuclease III (Endo III, which recognizes oxidized pyrimidines) or formamidopyrimidine DNA glycosylase (FPG, specific for altered purines including formamidopyrimidines and 8-oxoguanine). The enzyme 3-methyladenine DNA glycosylase II (AlkA) was used in a similar way to analyse DNA alkylation. Net enzyme-sensitive sites were calculated by subtracting the comet score after incubation with buffer alone from the score with enzyme. Reference standard from frozen human lymphocytes was used and slides run together with coded samples.
OGG activity was measured using an in vitro assay based on the ability of a cell-free lymphocyte extract to incise substrate DNA containing 8-oxoguanine. The increase in DNA breaks during 10-min incubation, measured with the comet assay, was taken as the measure of repair incision for statistical analysis (36,37).
Statistical analysis
To test for significant differences between groups, we used the independent samples t-test for normally distributed data and the Mann–Whitney U-test for non-normally distributed data. Differences between three groups were tested by one-way analysis of variance and by Bonferroni's test if equal variances were assumed or Tamhane's test if equal variances were not assumed. For variables with normal distribution Pearson's and for non-normally distributed variables Spearman's correlation coefficients were calculated. SPSS 13.0 software was used for statistical analysis. The data are expressed as means ± SEM. Differences with P < 0.05 were considered to be statistically significant.
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Results |
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Measurement of exposure to PAHs and fibres in workplace and personal dosimetry
Exposure to mineral wool in the factory was monitored for several decades by National Public Health Institutions using optic microscope, but no published results are available.
The levels of mineral fibres in the workplace were much higher in the past than when measured during this study. In the present study mineral fibre as well as PAH exposures were measured four times a year (including the time of the blood sampling). The presence of basalt fibres was confirmed in the wool factory, although all measured levels of basalt fibres were very low (10–1000 times below the Slovak occupational limits).
Total PAH levels in production and office workplaces determined in different seasons were also very low (Table II). Summed PAH concentrations in autumn air samples collected by stationary samplers were 146 ng/m3 in a production hall and 53 ng/m3 in an office room. Results of personal dosimetry of subjects working in the mineral wool factory are documented in Figure 1. Personal monitoring was carried out altogether in 29 workers of an industrial plant. Mean values of 161 ng/m3 (the sum of the 13 PAH congeners) and 148 ng/m3 were found in air samples collected by personal samplers worn by the smoking and non-smoking male workers, respectively (8-h sampling at work), whereas mean values were 89 and 26 ng/m3, respectively, for 8-h samples collected inside their flats/houses. Mean values of 106 and 82 ng/m3 were found in smoking and non-smoking male clerks, respectively (8-h sampling at work), whereas the concentrations were 75 and 53 ng/m3, respectively, in the 8-h samples collected inside their flats/houses. Summed PAH concentrations in air samples collected by personal samplers carried by factory workers were 216 ng/m3 in the production area and 89 ng/m3 in the office area.
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Clinical examination: effect of exposure and smoking on clinical markers
No pathological changes were found on X-rays of workers exposed to mineral wool. Spirometrical examination also did not show any changes. Forced expiratory vital capacity and forced expiratory volume 1 second markers in exposed workers in the mineral wool factory did not differ from controls. Systolic blood pressure showed interesting variations. Moderately elevated (P = 0.067) systolic blood pressure was found among the exposed group in comparison with the control group. Moreover, systolic blood pressure in smokers among the factory controls and in all exposed (smokers and non-smokers) was above the normal reference range. Mean values of systolic blood pressure in exposed and control smokers reached 140 mm Hg. Smoking exposed workers had significantly higher pulse frequency than non-smoking exposed colleagues. There was no significant difference in BMI between exposed and control groups, nor between any subgroups. However, the mean BMI in all groups and subgroups exceeded 25. The lowest BMI were seen in control women (25.76 ± 0.96) and the highest in exposed women (27.93 ± 1.16).
People who worked with mineral wool had significantly elevated serum levels of urea in all exposed groups except women (Table III). Men, both exposed and controls, had higher levels of urea compared to the corresponding group of women. Other biochemical parameters did not show any changes related to exposure to mineral fibres (Table III).
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Measurement of lipid peroxidation and antioxidant defence markers
Lipid peroxidation measured as level of MDA in plasma shows elevation in the group of all exposed workers (P = 0.03) and in exposed non-smokers (P = 0.003) (Figure 3).
We measured plasma concentrations of vitamin C and tocopherols, carotenoids and other micronutrients; FRAP; CPL oxidase activity; and the activities of antioxidant enzymes, GST, GPX, SOD and CAT in erythrocytes. The results are presented in Tables IV and V. The activity of GST was affected by exposure to mineral wool; GST levels were significantly lower in all exposed subjects, in the exposed men and exposed non-smokers. Control men had higher levels of GST than control women. Activities of CPL oxidase and CAT were significantly suppressed in all exposed subjects. This effect was also seen specifically in non-smokers. Control women had higher activities of CPL than men. Activities of SOD, GPX and FRAP were not affected by the mineral wool exposure. There were significant differences only between men and women.
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Concentrations of vitamins in plasma are shown in Table V. There were no significant differences between exposed and control subjects, except for exposed non-smokers who had higher levels of
-tocopherol compared to control non-smokers and control smokers who had higher levels of β-carotene compared to exposed smokers. Among exposed subjects, women had higher levels of vitamin C but lower levels of retinol compared to men. Control smokers had higher levels of -tocopherol compared to control non-smokers. Women had higher levels of β-carotene, both in exposed as well as control groups compared to men, as did exposed non-smokers compared to smokers (Table V).
Association between different markers in exposed and control groups and subgroups
In order to assess which markers of oxidative stress, antioxidant defence and genetic stability are the most relevant in relation to exposure to mineral fibres, we sought associations of different biochemical markers, lipid peroxidation (MDA), markers of antioxidant protection and of genetic stability, in exposed and control groups and different subgroups (Tables VI–XIII). All subjects, groups and subgroups were analysed in the same way. We only report those results where significant differences are seen.
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Biochemical parameters
We found associations between urea and antioxidant vitamins. There was a significant negative correlation of urea with vitamin C in all subjects (n = 135, r = –0.192, P = 0.03), all controls (n = 42, r = –0.305, P = 0.05) and control non-smokers (n = 26, r = –0.424, P = 0.03), with β-carotene in control non-smokers (n = 26, r = –0.395, P = 0.046). A positive correlation between urea and
-tocopherol was found only in the group of control women (n = 23, r = 0.569, P = 0.005). The level of glucose positively correlates with cholesterol and with MDA in many groups and in almost all groups and subgroups with triglycerides (Table VI). The level of glucose is also associated with BMI as well as correlating with age (Table VI).
Antioxidant capacity
The total antioxidant capacity of plasma (FRAP) surprisingly showed a positive correlation with lipid parameters cholesterol and triglycerides in nearly all investigated subgroups and groups (Table VII); it was also associated in many groups with a marker of lipid peroxidation, MDA (Table IX). There were positive correlations of FRAP with glucose and BMI in all subjects, all exposed and controls, all men, exposed men, control women, all smokers and all non-smokers as well as exposed smokers. FRAP correlated with age in all subjects, all men and in non-smoker controls (Table VII).
We also found, as expected, positive correlations of FRAP with -tocopherol in all main groups and many subgroups. Correlations of FRAP with urea were found in all subjects, all exposed, non-smokers and exposed non-smokers. β-Carotene correlated negatively with FRAP in all subjects, all exposed subjects and in non-smokers. No correlations with ascorbic acid or bilirubin were found (Table VIII).
Lipid peroxidation
There was a significant negative correlation between levels of MDA and the activity of GPX in all subjects and in exposed group, in exposed smokers, exposed men and in male non-smokers. MDA correlated inversely also with CAT activity in all subjects, in non-smokers, in exposed non-smokers and in non-smoking women (Table IX).
As already mentioned, we found a positive correlation between lipid peroxidation measured as level of MDA and FRAP in all investigated subjects, all exposed, smokers, exposed smokers, all women, exposed women and non-smoking women (Table IX).
DNA damage and biochemical markers and markers of antioxidant defence
It should be mentioned here that the results of assays for DNA damage are taken from a previous publication (26) where we reported no difference of FPG or Endo III sites between exposed and controls. However, levels of strand breaks of exposed subjects were higher than controls (Figure 2).
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FPG-sensitive sites correlated positively with BMI in controls (n = 42, r = 0.333, P = 0.03), men (n = 93, r = 0.374, P = 0.001), exposed men (n = 73, r = 0.332, P = 0.004), control men (n = 20, r = 0.506, P = 0.03) and smokers (n = 51, r = 0.289, P = 0.04). The same positive correlation was found with Endo III-sensitive sites in the exposed group (n = 96, r = 0.223, P = 0.03) and in exposed smokers (n = 37, r = 0.344, P = 0.04).
We found an association of DNA damage with age. The level of FPG-sensitive sites correlated with age in all control subjects (n = 42, r = 0.389, P = 0.01), in all men (n = 95, r = 0.240, P = 0.02), in control men (n = 20, r = 0.481, P = 0.03) and in non-smoker controls (n = 27, r = 0.539, P = 0.004). The positive association between age and Endo III-sensitive sites was found only in the exposed smokers group (n = 37, r = 0.362, P = 0.03).
We found a positive correlation between FPG sites and cholesterol in exposed men (n = 70, r = 0.414, P = 0.001) but a negative correlation in exposed women (n = 19, r = –0.527, P = 0.02). A positive association of FPG-sensitive sites with triglycerides was found in the control group (n = 38, r = 0.443, P = 0.005), in control women (n = 21, r = 0.479, P = 0.03) and in non-smoking controls (n = 24, r = 0.557, P = 0.005). Triglycerides correlated positively also with Endo III sites in the group of non-smoking controls (n = 24, r = 0.557, P = 0.005).
Bilirubin correlated positively with FPG sites in the entire exposed group (n = 89, r = 0.210, P = 0.01), in control smokers (n = 14, r = 0.542, P = 0.05) and in exposed non-smokers (n = 54, r = 0.368, P = 0.006).
The level of total proteins in plasma correlated inversely with both FPG as well as Endo III-sensitive sites in all women (n = 40, r = –0.318, P = 0.05, respectively, r = –0.370, P = 0.02) and in exposed women (n = 19, r = –0.495, P = 0.03, respectively, r = –0.483, P = 0.036). Urea concentrations correlated negatively with Endo III-sensitive sites only in the group of control non-smokers (n = 24, r = –0.418, P = 0.042).
There were interesting associations between oxidative DNA damage markers and antioxidant enzymes. GST activity correlated inversely with oxidized purines measured as FPG-sensitive sites in all smokers and in male smokers. The association between DNA damage and GST activity was more pronounced when measuring Endo III sites (oxidized pyrimidines). We found an inverse correlation in almost all subgroups: all subjects, exposed, all women, all men, all non-smokers, exposed men, control non-smokers and women non-smokers. On the other hand, the level of FPG-sensitive sites correlated positively with antioxidant enzyme GPX in the control group, women, control women, control non-smokers and women non-smokers (Table X).
We also found significant positive correlations of DNA damage, both FPG- and Endo III-sensitive sites, with -tocopherol and retinol in several groups. Interestingly, an inverse correlation of FPG as well as Endo III sites with β-carotene was found in several groups. A negative correlation of Endo III-sensitive sites was found with xanthophyll in smokers and control smokers (Table XI).
DNA repair and biochemical markers and markers of antioxidant defence
An interesting association between DNA repair capacity (incisions per 10 min at 8-oxoguanine in DNA, measured with the comet assay as the activity of a lymphocyte extract on a DNA substrate containing 8-oxo-dG) and the level of lipid peroxidation in plasma was found in all investigated subjects, in all exposed subjects and in all male subjects. There were also positive correlations between DNA repair capacity and serum cholesterol in all subjects and in exposed, as well as with triglycerides in exposed and exposed smoker groups (Table XII).
On the other hand, we found a significant negative correlation between DNA repair capacity and GPX in all subjects as well as in control men and CAT in all control subjects, control men and control smokers. The relationships between DNA repair rate and antioxidant micronutrients ascorbic acid, -tocopherol, β-carotene and retinol are also shown in Table XIII. There are negative correlations of DNA repair rate with ascorbic acid and β-carotene in several groups and subgroups and positive correlations with -tocopherol and retinol in smokers and non-smokers (Table XIII).
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Discussion |
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Reactive oxygen intermediates together with other proinflammatory mediators are proposed to be involved in the mechanism of action of industrial fibrous dusts. The effect of occupational exposure to mineral wool on DNA damage and repair (measured in lymphocytes) was already published by Dusinska et al. (26
). Though lymphocytes are surrogate cells and not target cells, we believe that they reflect overall body exposure. In this case, blood circulates to all organs including lung where mineral fibres deposit. Mineral wool exposure did not increase levels of oxidized bases in lymphocyte DNA as measured with the comet assay, nor induce cytogenetic damage. Nevertheless, results showed that exposure to mineral wool led to an increase in DNA strand breaks in the lymphocytes of investigated subjects (Figure 2). When analysed according to sex and smoking habit, this effect was apparent in the group of non-smokers. DNA repair of oxidative damage did not differ between exposed and controls. However, DNA repair capacity in lymphocytes of exposed subjects was higher in men compared to women. DNA repair capacity was higher in control compared to exposed men (26). In order to investigate the possible mechanism of mineral wool exposure, we focused in the present paper on markers of oxidative stress and antioxidant defence and compared them with oxidative DNA damage and repair. We were also interested in which markers of oxidative stress, antioxidant defence and genetic stability are the most relevant and related to exposure to mineral fibres, and therefore, we examined associations between different biochemical markers, lipid peroxidation (MDA), markers of antioxidant protection and of genetic stability in exposed and control groups and in different subgroups.
We analysed typical biochemical and clinical markers which could be related to exposure to mineral wool. Sampling of the same group and all analyses were done at the same time as already published results of markers of genetic stability (26). We measured plasma concentrations of vitamin C, -tocopherol, β-carotene, retinol, xanthophyll and lycopene; FRAP; CPL assayed for its oxidase activity; MDA levels in plasma; and the activities of antioxidant enzymes, GST, GPX, SOD and CAT in erythrocytes.
Effect of exposure and smoking on clinical markers
All 141 volunteers in our study were relatively healthy. However, though exposure to mineral wool was very low, we found a moderate elevation of systolic blood pressure above the normal reference range among the exposed group, as well as in smokers and a significantly higher mean pulse frequency in smokers. An interesting association of blood glucose levels with cholesterol, triglycerides and MDA in all subjects, in all exposed and also in many other subgroups (Table VI) shows the relation of biochemical and clinical markers. In our previous study with diabetes II patients, we found an association of markers of oxidative DNA damage with blood glucose levels (38).
The mean BMI for all investigated subjects, both women and men, was slightly above the recommended range. Several clinical and biochemical parameters correlated with BMI such as blood glucose, MDA, FRAP, FPG and Endo III-sensitive sites. Moreover, blood glucose levels and also FRAP as well as DNA damage were associated with age.
MDA as a marker of lipid peroxidation
MDA is a physiologic ketoaldehyde produced by peroxidative decomposition of unsaturated lipids as a byproduct of arachidonate metabolism. The excess MDA produced as a result of tissue injury can combine with free amino groups of proteins, producing MDA-modified protein adducts (39). The clinical relevance of the reaction between MDA and proteins is highlighted in atherosclerosis, which is a major cause of coronary heart disease and strokes. Plasma MDA concentrations are increased in diabetes mellitus, and MDA can be found in the atherosclerotic lesions (39,40). One consequence of oxidative stress and lipid peroxidation is the formation of DNA adducts. Since DNA is believed to be the target molecule for carcinogens, endogenous DNA adducts derived from oxidative stress, lipid peroxidation and other sources have been proposed to contribute to the aetiology of human cancers (41).
Our study shows that the plasma level of MDA is slightly elevated in all exposed groups and significantly in all exposed workers, as well as in exposed non-smokers (Figure 3). Higher oxidative damage in these groups is possibly the consequence of significantly suppressed activity of CPL oxidase and CAT in these groups (Table IV). MDA indeed correlated inversely with CAT activity in all subjects, in non-smokers, in exposed non-smokers and in non-smoking women (Table IX). The activity of GST was also apparently affected by exposure to mineral wool, being relatively suppressed in all exposed subjects, in exposed men and in exposed non-smokers. In contrast, GPX and SOD activities measured in erythrocytes did not differ between exposed and control workers. There was a negative correlation between the activity of GPX and MDA which suggests that the higher enzyme activity can protect against lipid peroxidation. Similar results have been obtained in workers exposed to nickel (42), cadmium (43), chromium (44) and manganese (45). The results of the present study suggest that increased plasma lipid peroxidation and decreased erythrocyte antioxidant levels could be used as biomarkers of oxidative stress in exposed workers. In contrast, antioxidant enzymic activities were not considered a suitable marker for chromium exposure (43–45).
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The higher MDA level and suppressed GST, CPL and CAT activities found in our study seem together to indicate elevated oxidative stress in blood cells of exposed subjects and could be suggested as useful markers of oxidative stress and antioxidant protection.
FRAP as marker of total antioxidant capacity
The FRAP assay was established by Benzie and Strain (30) as an index of total antioxidant capacity of plasma. It is quick and easy to perform, and the reaction is reproducible and linearly related to the molar concentration of the antioxidants present. There is also no activity-changing interaction between antioxidants in this system. Therefore, FRAP was suggested as a useful marker to measure antioxidant capacity in cells. This marker was used in several population studies including ours (46,47). Benzie and Strain (30) estimated the per cent contribution of plasma antioxidants to total FRAP (Table XIV) where uric acid represents 60% of total FRAP, ascorbic acid 15% and proteins 10%.
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In our study, we did not find any difference in FRAP values between exposed and controls but men had significantly higher levels of FRAP compared to women in both exposed and control groups (Table IV). FRAP correlated strongly with
-tocopherol, urea and total proteins (Table VIII) in many groups and subgroups. The correlation with proteins was expected in view of its 10% contribution to FRAP. The strong correlation of FRAP with -tocopherol is also not surprising, though it was reported as contributing to FRAP only 5% (30). Our results suggest that perhaps the contribution of -tocopherol to total FRAP might be higher than 5%. An expected correlation between FRAP and vitamin C was not found in any group or subgroup which might be perhaps a result of the negative association we found between urea and vitamin C. FRAP correlates positively with urea (Table VIII). We also found that FRAP inversely correlates with β-carotene. This also might indirectly result from a negative correlation between urea and β-carotene, though we found this only in the group of non-smoking controls. The total antioxidant capacity of plasma (FRAP) surprisingly correlated positively with the lipid parameters cholesterol and triglycerides in nearly all investigated subgroups and groups, and it is therefore not surprising that it positively correlates with BMI in almost all groups and subgroups, as well as with age (Table VII). We would expect total antioxidant capacity to correlate negatively with MDA, but the opposite was true in our study (Table IX). This is likely to be the consequence of the strong positive correlation between FRAP and lipid parameters. Our results clearly show that FRAP as a marker of total antioxidant capacity in human population studies cannot be recommended due to the interference with other biochemical, mainly lipid parameters.
Plasma antioxidants
Concentrations of antioxidant micronutrients (except for -tocopherol in one group) were not affected by the mineral wool exposure (Table V). To assess the contribution of micronutrients to antioxidant status, it should be taken into consideration that together they can form synergistic complexes in cells and organisms, e.g. vitamin C can regenerate oxidized vitamin E (48). Therefore, assessment of antioxidant status depends on measurement of not just one particular micronutrient but of a complex battery of antioxidant compounds.
DNA damage and repair: an index of antioxidant protection—role of GST
DNA damage in lymphocytes is a biomarker of exposure to DNA-damaging agents. The link between DNA damage and the risk of cancer, and possibly other diseases, has yet to be established definitively through epidemiological studies, though there is clearly a mechanism for such a link. We believe that oxidative damage measured in lymphocytes can accurately reflect the overall level of exposure to reactive oxygen in the body. Many events downstream from the initial event of DNA damage help to determine whether a particular damage results in carcinogenic change. One of the events is DNA repair. We measured DNA strand breaks and apurinic/apyrimidinic sites using the alkaline comet assay and sites sensitive to the enzymes FPG- and Endo III, i.e. oxidized purines and pyrimidines. We also measured the capacity of a lymphocyte extract for repair of oxidized guanine.
We found an interesting positive correlation of DNA damage with cholesterol and triglycerides which suggests that lipid parameters can directly contribute to the induction of oxidative DNA damage. Similar results were found in rats with high cholesterol diet (
tetina et al., in preparation). On the other hand, DNA damage (strand breaks, FPG- or Endo III-sensitive sites) did not correlate with plasma levels of MDA. It is not surprising that both FPG- and Endo III-sensitive sites correlated with BMI and age since cholesterol and triglycerides are associated with BMI (49–51) as we also found in this study.
Several negative associations of both FPG- and Endo III-sensitive sites with β-carotene and xanthophyll suggest the protective effect of antioxidant vitamins. On the other hand, a positive correlation with -tocopherol and retinol in many groups of investigated subjects, both with FPG and Endo III sites (Table XI), suggests that more detailed studies are needed, which include measurement of activities of several antioxidant enzymes together with overall antioxidant status.
A negative correlation of FPG- and Endo III-sensitive sites with GST levels implies that antioxidant enzymes may play an important role in protection against oxidative DNA damage. Indeed, we found an inverse correlation between activity of antioxidant enzyme GST and oxidized purines and pyrimidines (Table X). GST has an important role in detoxification of xenobiotics, drugs and carcinogens and thus protects the cells against redox cycling and oxidative stress (52,53). Xenobiotic-metabolizing enzymes play a major role in regulating the toxic, oxidative damaging, mutagenic and neoplastic effects of chemical carcinogens. Mounting evidence has indicated that the induction of phase 2 detoxification enzymes such as GSTs results in protection against toxicity and chemical carcinogenesis, especially, during the initiation phase. The GSTs are a family of enzymes that catalyze the nucleophilic addition of the thiol of GSH to a variety of electrophiles (54). It seems that GST may have also some other role in protection of DNA against oxidative DNA damage. Recently, it was found that -class GSTs bind with the dinitrosyl–diglutathionyl–iron complex in rat hepatocytes and that a significant part of the bound complex is also associated with the nuclear fraction (55,56). Stella et al. (56) using confocal and electron microscopy reported nuclear localization of GSTs in these cells. Surprisingly, they found that a considerable amount of GST corresponding to 10% of the cytosolic pool is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly -class GSTs are involved in this double modality of interaction. A quantitative analysis of the membrane-bound GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent a considerable novelty in cell physiology. The authors conclude that the interception of potentially noxious compounds to prevent DNA damage could be the possible physiological role of the perinuclear and intra-nuclear localization of GSTs.
The interesting positive correlation between DNA repair capacity of lymphocyte extract (on 8-oxoguanine-containing substrate DNA) and MDA, cholesterol and triglycerides reported here suggests that elevated oxidative damage can stimulate DNA repair (Table XII).
On the contrary, negative correlations between the capacity for repair of oxidized DNA and GPX and CAT (Table XIII) may suggest that these enzymes protect DNA against oxidative DNA damage and thus indirectly correlate with DNA repair. This could be true with CAT but not with GPX as we found a positive correlation of the enzyme activity with FPG-sensitive sites (Table X) and a negative correlation with repair rate (Table XIII).
The possible relationship between DNA repair rate and antioxidant micronutrients such as vitamin C, -tocopherol, β-carotene and retinol (Table XIII) is still confusing. The negative correlations of DNA repair rate with ascorbic acid and β-carotene might suggest that the higher the level of these micronutrients, the lower the level of DNA damage (as we found with β-carotene) and, consequently, DNA repair activity. In the case of -tocopherol and retinol, we found a positive association both with DNA damage (FPG- as well as Endo III-sensitive sites) and also with DNA repair capacity for oxidative DNA damage. More data are needed to provide a sufficient explanation.
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TopIntroductionMaterials and methodsResultsDiscussionConclusionFundingReferences |
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The results presented indicate that mineral wool exposure induces an increase in oxidative damage of biomolecules especially in the group of male non-smokers. However, the optimal levels of antioxidants could have a protective effect. We assume that indicators such as MDA, antioxidant enzymes and antioxidant vitamins are useful markers to detect the level of oxidative stress and antioxidant protection in blood cells. On the other hand, FRAP does not seem a good marker of total antioxidant capacity in population studies as the possible interference with other parameters can provide false or confusing results. Our study also shows that additionally to an antioxidant role, there might also be other mechanisms by which antioxidant enzymes (especially GST) protect cells against oxidative DNA damage.
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TopIntroductionMaterials and methodsResultsDiscussionConclusionFundingReferences |
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European Union (project FIBRETOX no. QLK4-1999-01629).
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Acknowledgments |
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We thank all participants in the mineral wool factory (Nova Bana, Slovakia), as well as the management, for their enthusiastic participation. We thank all staff at the Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava for help with conducting the study, and with processing of samples. Personal sampling, fibre analysis and medical investigation were carried out with help of the National Institutes of Health (Banská Bystrica, Nitra and Ziar nad Hronom). We thank Mrs Anna Morávková and Anna Ga
iová, Renáta Mátéová, Kristína Gaval'ová, Zuzana Ro
táová, L'ubica Mikloková, Jarmila Jantoková and Viera Machálková for their excellent technical help. We also thank Dr Blaí
ek for MDA analyses. Conflict of interest statement: None declared. |
Notes |
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* To whom correspondence should be addressed. Tel/Fax: +421 2 59369270; Email: maria.dusinska{at}szu.sk
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Received on October 20, 2007; revised on December 21, 2007; accepted on December 21, 2007.
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