Stepanka Vlckova

Markers of oxidative damage of nucleic acids and proteins among workers exposed to TiO2 (nano) particles

Objective The use of nanotechnology is growing enormously and occupational physicians have an increasing interest in evaluating potential hazards and finding biomarkers of effect in workers exposed to nanoparticles. Methods A study was carried out with 36 workers exposed to (nano)TiO2 pigment and 45 controls. Condensate (EBC) titanium and markers of oxidation of nucleic acids (including 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), 5-hydroxymethyl uracil (5-OHMeU)) and proteins (such as o-tyrosine (o-Tyr), 3-chlorotyrosine (3-ClTyr) and 3-nitrotyrosine (3-NOTyr)) were analysed from samples of their exhaled breath. Results In the production workshops, the median total mass 2012 and 2013 TiO2 concentrations were 0.65 and 0.40 mg/m3, respectively. The median numbers of concentrations measured by the scanning mobility particle sizer (SMPS) and aerodynamic particle sizer (APS) were 1.98×104 and 2.32×104 particles/cm3, respectively; and about 80% of those particles were smaller than 100 nm in diameter. In the research workspace, lower aerosol concentrations (0.16 mg/m3 and 1.32×104 particles/cm3) were found. Titanium in the EBC was significantly higher in production workers (p<0.001) than in research workers and unexposed controls. Accordingly, most EBC oxidative stress markers, including in the preshift samples, were higher in production workers than in the two other groups. Multiple regression analysis confirmed an association between the production of TiO2 and the levels of studied biomarkers. Conclusions The concentration of titanium in EBC may serve as a direct exposure marker in workers producing TiO2 pigment; the markers of oxidative stress reflect the local biological effect of (nano)TiO2 in the respiratory tract of the exposed workers.

Human urinary biomarkers of dioxin exposure: Analysis by metabolomics and biologically driven data dimensionality reduction

Untargeted metabolomic approaches offer new opportunities for a deeper understanding of the molecular events related to toxic exposure. This study proposes a metabolomic investigation of biochemical alterations occurring in urine as a result of dioxin toxicity. Urine samples were collected from Czech chemical workers submitted to severe dioxin occupational exposure in a herbicide production plant in the late 1960s. Experiments were carried out with ultra-high pressure liquid chromatography (UHPLC) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry. A chemistry-driven feature selection was applied to focus on steroid-related metabolites. Supervised multivariate data analysis allowed biomarkers, mainly related to bile acids, to be highlighted. These results supported the hypothesis of liver damage and oxidative stress for long-term dioxin toxicity. As a second step of data analysis, the information gained from the urine analysis of Victor Yushchenko after his poisoning was examined. A subset of relevant urinary markers of acute dioxin toxicity from this extreme phenotype, including glucuro- and sulfo-conjugated endogenous steroid metabolites and bile acids, was assessed for its ability to detect long-term effects of exposure. The metabolomic strategy presented in this work allowed the determination of metabolic patterns related to dioxin effects in human and the discovery of highly predictive subsets of biologically meaningful and clinically relevant compounds. These results are expected to provide valuable information for a deeper understanding of the molecular events related to dioxin toxicity. Furthermore, it presents an original methodology of data dimensionality reduction by using extreme phenotype as a guide to select relevant features prior to data modeling (biologically driven data reduction).

Oxidative stress markers are elevated in exhaled breath condensate of workers exposed to nanoparticles during iron oxide pigment production

Markers of oxidative stress and inflammation were analysed in the exhaled breath condensate (EBC) and urine samples of 14 workers (mean age 43  ±  7 years) exposed to iron oxide aerosol for an average of 10  ±  4 years and 14 controls (mean age 39  ±  4 years) by liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (LC-ESI-MS/MS) after solid-phase extraction. Aerosol exposure in the workplace was measured by particle size spectrometers, a scanning mobility particle sizer (SMPS) and an aerodynamic particle sizer (APS), and by aerosol concentration monitors, P-TRAK and DustTRAK DRX. Total aerosol concentrations in workplace locations varied greatly in both time and space. The median mass concentration was 0.083 mg m(-3) (IQR 0.063-0.133 mg m(-3)) and the median particle concentration was 66 800 particles cm(-3) (IQR 16,900-86,900 particles cm(-3)). In addition, more than 80% of particles were smaller than 100 nm in diameter. Markers of oxidative stress, malondialdehyde (MDA), 4-hydroxy-trans-hexenale (HHE), 4-hydroxy-trans-nonenale (HNE), 8-isoProstaglandin F2α (8-isoprostane) and aldehydes C6-C12, in addition to markers of nucleic acid oxidation, including 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), 5-hydroxymethyl uracil (5-OHMeU), and of proteins, such as o-tyrosine (o-Tyr), 3-chlorotyrosine (3-ClTyr), and 3-nitrotyrosine (3-NOTyr) were analysed in EBC and urine by LC-ESI-MS/MS. Almost all markers of lipid, nucleic acid and protein oxidation were elevated in the EBC of workers comparing with control subjects. Elevated markers were MDA, HNE, HHE, C6-C10, 8-isoprostane, 8-OHdG, 8-OHG, 5-OHMeU, 3-ClTyr, 3-NOTyr, o-Tyr (all p  <  0.001), and C11 (p  <  0.05). Only aldehyde C12 and the pH of samples did not differ between groups. Markers in urine were not elevated. These findings suggest the adverse effects of nano iron oxide aerosol exposure and support the utility of oxidative stress biomarkers in EBC. The analysis of urine oxidative stress biomarkers does not support the presence of systemic oxidative stress in iron oxide pigment production workers.

Markers of lipid oxidative damage in the exhaled breath condensate of nano TiO2 production workers

Abstract Nanoscale titanium dioxide (nanoTiO2) is a commercially important nanomaterial. Animal studies have documented lung injury and inflammation, oxidative stress, cytotoxicity and genotoxicity. Yet, human health data are scarce and quantitative risk assessments and biomonitoring of exposure are lacking. NanoTiO2 is classified by IARC as a group 2B, possible human carcinogen. In our earlier studies we documented an increase in markers of inflammation, as well as DNA and protein oxidative damage, in exhaled breath condensate (EBC) of workers exposed nanoTiO2. This study focuses on biomarkers of lipid oxidation. Several established lipid oxidative markers (malondialdehyde, 4-hydroxy-trans-hexenal, 4-hydroxy-trans-nonenal, 8-isoProstaglandin F2α and aldehydes C6–C12) were studied in EBC and urine of 34 workers and 45 comparable controls. The median particle number concentration in the production line ranged from 1.98 × 104 to 2.32 × 104 particles/cm3 with ∼80% of the particles <100 nm in diameter. Mass concentration varied between 0.40 and 0.65 mg/m3. All 11 markers of lipid oxidation were elevated in production workers relative to the controls (p < 0.001). A significant dose-dependent association was found between exposure to TiO2 and markers of lipid oxidation in the EBC. These markers were not elevated in the urine samples. Lipid oxidation in the EBC of workers exposed to (nano)TiO2 complements our earlier findings on DNA and protein damage. These results are consistent with the oxidative stress hypothesis and suggest lung injury at the molecular level. Further studies should focus on clinical markers of potential disease progression. EBC has reemerged as a sensitive technique for noninvasive monitoring of workers exposed to engineered nanoparticles.

Markers of lipid oxidative damage among office workers exposed intermittently to air pollutants including nanoTiO2 particles

Abstract Nanoscale titanium dioxide (nanoTiO2) is a commercially important nanomaterial used in numerous applications. Experimental studies with nanotitania have documented lung injury and inflammation, oxidative stress, and genotoxicity. Production workers in TiO2 manufacturing with a high proportion of nanoparticles and a mixture of other air pollutants, such as gases and organic aerosols, had increased markers of oxidative stress, including DNA and protein damage, as well as lipid peroxidation in their exhaled breath condensate (EBC) compared to unexposed controls. Office workers were observed to get intermittent exposures to nanoTiO2 during their process monitoring. The aim of this study was to investigate the impact of such short-term exposures on the markers of health effects in office workers relative to production workers from the same factory. Twenty-two office employees were examined. They were occupationally exposed to (nano)TiO2 aerosol during their daily visits of the production area for an average of 14±9 min/day. Median particle number concentration in office workers while in the production area was 2.32×104/cm3. About 80% of the particles were <100 nm in diameter. A panel of biomarkers of lipid oxidation, specifically malondialdehyde (MDA), 4-hydroxy-trans-hexenal (HHE), 4-hydroxy-trans-nonenal (HNE), 8-isoprostaglandin F2α (8-isoprostane), and aldehydes C6−C12, were studied in the EBC and urine of office workers and 14 unexposed controls. Nine markers of lipid oxidation were elevated in the EBC of office employees relative to controls (p<0.05); only 8-isoprostane and C11 were not increased. Significant association was found in the multivariate analysis between their employment in the TiO2 production plant and EBC markers of lipid oxidation. No association was seen with age, lifestyle factors, or environmental air contamination. The EBC markers in office employees reached about 50% of the levels measured in production workers, and the difference between production workers and office employees was highly significant (p<0.001). None of these biomarkers were elevated in urine. The approach presented here seems to be very sensitive and useful for non-invasive monitoring of employees exposed to air pollutants, including gases, organic aerosols, and nanoTiO2, and may prove useful for routine biomonitoring purposes. Among them, aldehydes C6, C8, C9, and C10 appear to be the most sensitive markers of lipid oxidation in similar occupational cohorts. One major challenge with sensitive biomonitoring techniques, however, is their non-specificity and difficulty in interpreting the meaning of their physiological values in the context of chronic disease development and damage-repair kinetics.

Neurological and Neurophysiological Findings in Workers with Chronic 2,3,7,8-TCDD Intoxication 50 Years after Exposure

The last eight survivors of 80 workers accidentally exposed to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) during production of herbicides based on trichlorophenoxyacetic acid in 1965–1967 in a chemical factory were followed. All were men, mean age 72.4 ± 1.3 years. Their current median TCDD blood level was 112 (46–390) pg/g lipids. Neurological examination revealed central nervous system impairment in all individuals and signs of polyneuropathy in 87.5%, which was confirmed by a nerve conduction study (NCS) in 75%. A Lanthony test demonstrated acquired dyschromatopsia in 87.5% of the patients, with deterioration of mean colour confusion index (CCI) from 1.52 ± 0.39 in 2010 to 1.73 ± 0.41 in 2016. Single‐photon emission computer tomography (SPECT) of the brain showed focal reduction of perfusion in various brain locations in all patients and worsening in six patients. Visual‐evoked potentials (VEP) was abnormal in 62.6% of individuals. Most patients complained of psychological problems. The neuropsychological test battery showed most positive impairments in the Trail Making Test evaluating processing speed (average level in the range of mild neurocognitive impairment), which correlated with mean CCI (p < 0.05). Conclusion: Fifty years after exposure, blood levels of TCDD are still 10 times higher than the general population. NCS, VEP, Lanthony test and SPECT findings deteriorated from examination of these patients in 2004 and in 2010. The total of abnormal tests per patient in 2016 is very high. Minor differences among patients and their reduced count may explain why the number of impairments in 2016 does not correlate with TCDD blood level.

Markers of Oxidative Stress in the Exhaled Breath Condensate of Workers Handling Nanocomposites

Researchers in nanocomposite processing may inhale a variety of chemical agents, including nanoparticles. This study investigated airway oxidative stress status in the exhaled breath condensate (EBC). Nineteen employees (42.4 ± 11.4 y/o), working in nanocomposites research for 18.0 ± 10.3 years were examined pre-shift and post-shift on a random workday, together with nineteen controls (45.5 ± 11.7 y/o). Panels of oxidative stress biomarkers derived from lipids, nucleic acids, and proteins were analyzed in the EBC. Aerosol exposures were monitored during three major nanoparticle generation operations: smelting and welding (workshop 1) and nanocomposite machining (workshop 2) using a suite of real-time and integrated instruments. Mass concentrations during these operations were 0.120, 1.840, and 0.804 mg/m3, respectively. Median particle number concentrations were 4.8 × 104, 1.3 × 105, and 5.4 × 105 particles/cm3, respectively. Nanoparticles accounted for 95, 40, and 61%, respectively, with prevailing Fe and Mn. All markers of nucleic acid and protein oxidation, malondialdehyde, and aldehydes C6–C13 were elevated, already in the pre-shift samples relative to controls in both workshops. Significant post-shift elevations were documented in lipid oxidation markers. Significant associations were found between working in nanocomposite synthesis and EBC biomarkers. More research is needed to understand the contribution of nanoparticles from nanocomposite processing in inducing oxidative stress, relative to other co-exposures generated during welding, smelting, and secondary oxidation processes, in these workshops.

Diabetes, Cardiovascular Disorders and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Body Burden in Czech Patients 50 Years After the Intoxication

The correlation between 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) intoxication and the parameters of metabolic impairment was examined in the last eight male survivors of 80 workers exposed to TCDD during the production of herbicides in a chemical factory in 1965–1967. Their median TCDD blood level was 112 (46–390) pg/g lipids, and the median TCDD body deposit was 3.9 (0.8–11.7) μg. This puts these patients into the most severely intoxicated group of subjects, according to back‐calculated levels of TCDD. The median TCDD blood level in eight controls was 12 pg/g (<0.10 to 22.2 pg/g). Markers of metabolic impairment – diabetes, dyslipidaemia, arterial hypertension, carotid artery plaque, skin microvascular reactivity, eye fundus hypertensive angiopathy and history of coronary heart disease – were assessed and compared to a general male population of comparable age. Measured parameters compared with a population of comparable age were as follows: prevalence of diabetes (62.5% versus 17.6%), arterial hypertension (87.5% versus 71.8%), dyslipidaemia (87.5% versus 88.8%), history of coronary heart disease (62.5% versus 26.0%) and eye fundus hypertension angiopathy (50% versus 14%). All eight patients (100% versus 43%) developed plaques in carotid arteries, six had stenosis >50% and two had a carotid intervention (stenting or endarterectomy). Total cholesterol levels decreased compared to the earlier study this patient group in 2008, most likely due to a more intensive use of lipid‐lowering drugs. Several metabolic parameters were higher (diabetes as much as 3.5‐fold) in the group of severely TCDD‐intoxicated subjects than in a general population of comparable age. This suggests that TCDD plays a role in the development of metabolic impairment and vascular changes.

Deep Airway Inflammation and Respiratory Disorders in Nanocomposite Workers

Thousands of researchers and workers worldwide are employed in nanocomposites manufacturing, yet little is known about their respiratory health. Aerosol exposures were characterized using real time and integrated instruments. Aerosol mass concentration ranged from 0.120 mg/m3 to 1.840 mg/m3 during nanocomposite machining processes; median particle number concentration ranged from 4.8 × 104 to 5.4 × 105 particles/cm3. The proportion of nanoparticles varied by process from 40 to 95%. Twenty employees, working in nanocomposite materials research were examined pre-shift and post-shift using spirometry and fractional exhaled nitric oxide (FeNO) in parallel with 21 controls. Pro-inflammatory leukotrienes (LT) type B4, C4, D4, and E4; tumor necrosis factor (TNF); interleukins; and anti-inflammatory lipoxins (LXA4 and LXB4) were analyzed in their exhaled breath condensate (EBC). Chronic bronchitis was present in 20% of researchers, but not in controls. A significant decrease in forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) was found in researchers post-shift (p ˂ 0.05). Post-shift EBC samples were higher for TNF (p ˂ 0.001), LTB4 (p ˂ 0.001), and LTE4 (p ˂ 0.01) compared with controls. Nanocomposites production was associated with LTB4 (p ˂ 0.001), LTE4 (p ˂ 0.05), and TNF (p ˂ 0.001), in addition to pre-shift LTD4 and LXB4 (both p ˂ 0.05). Spirometry documented minor, but significant, post-shift lung impairment. TNF and LTB4 were the most robust markers of biological effects. Proper ventilation and respiratory protection are required during nanocomposites processing.

Is Chelation Therapy Efficient for the Treatment of Intravenous Metallic Mercury Intoxication

The efficacy of treatment for intravenous elemental mercury intoxication has not been fully studied with regard to clinical outcome, and treatment recommendations vary. We treated a 41‐year‐old man with a history of drug abuse and depression who attempted suicide using 1 mL (13.53 g) metallic Hg i.v. He was admitted to the hospital 2 months later for dyspnoea and thoracic pain and was diagnosed with pneumonia. Hg deposits were seen in the lungs and extra‐pulmonary organs. His blood level (372 μg/L) exceeded the population level of 5 μg/L by more than 70 times. Dimercaptopropane sulphonate sodium (DMPS; 600 mg/day orally) was administered for 14 days. One year later, the patient presented with dyspnoea on exertion, fatigue, depression and impaired sleep. His chest X‐ray showed multiple opacities (size up to 2.8 cm), and psychological testing revealed a selective cognitive deficit in the area of visual attentiveness, flexibility, source memory and impairment of the motor speed of the dominant upper extremity. Mercury blood level was 158 μg/L and mercury urine output was 1380 μg/24 hr. DMPS (800 mg/day orally) was administered for 40 days; the patient eliminated up to 18 mg Hg/day. His Hg blood level and Hg urine output belong to the highest among reported cases. In spite of the therapy, the patients blood Hg, complaints and psychological tests showed no improvement. This case report confirms that DMPS does not effectively remove intravenous deposits of metallic Hg.