Urinary 8-oxo-7,8-dihydro-2\u27-deoxyguanosine analysis by an improved ELISA: does assay standardization reduce inter-laboratory variability?

ELISA is commonly used for the detection of urinary 8-oxo-7,8-dihydro-2\u27-deoxyguanosine (8-oxodG), a marker of whole body oxidative stress. However, the method has been criticized for high inter-laboratory variability and poor agreement with chromatographic techniques. We performed an inter-laboratory comparison of 8-oxodG assessed in 30 urine samples and a urine spiked with four different concentrations of 8-oxodG by ELISA using standardized experimental conditions, including: sample pre-treatment with solid-phase extraction (SPE), performing analysis using a commercial kit from a single manufacturer and strict temperature control during the assay. We further compared the ELISA results with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed tentative identification of compounds that may contribute to the discrepancy between both methods. For all but one participating laboratory (Data 1) we observed consistent ELISA results lying mostly within 1SD of the mean 8-oxodG concentration. Mean 8-oxodG levels assessed by ELISA correlated with the data obtained by HPLC-MS/MS (R=0.679, p\u3c0.001). The correlation improved when Data 1 were excluded from the analysis (R=0.749, p\u3c0.001). We identified three outlying urine samples; one with an ELISA 8-oxodG concentration lower, and two with 8-oxodG levels higher, than those measured by HPLC-MS/MS. Omitting these samples further improved inter-methodology agreement (R=0.869, p\u3c0.001). In the outliers with high 8-oxodG estimates various aromatic and heterocyclic compounds were tentatively identified using gas chromatography-mass spectrometry (GC-MS). Application of authentic standards revealed the presence of saccharides, including d-glucose and d-galactose as putative interfering substances. In summary, assay standardization improved ELISA inter-laboratory agreement, although some variability is still observed. There are still compounds contributing to overestimation of 8-oxodG by ELISA, but only in some urine samples. Thus, despite significant improvement, ELISA still should not be considered a robust alternative to chromatographic techniques

Oxidative Stress and Antioxidant Response in Populations of the Czech Republic Exposed to Various Levels of Environmental Pollutants

We aimed to identify the variables that modify levels of oxidatively damaged DNA and lipid peroxidation in subjects living in diverse localities of the Czech Republic (a rural area, a metropolitan locality, and an industrial region). The sampling of a total of 126 policemen was conducted twice in two sampling seasons. Personal characteristics, concentrations of particulate matter of aerodynamic diameter <2.5 µm and benzo[a]pyrene in the ambient air, activities of antioxidant mechanisms (superoxide dismutase, catalase, glutathione peroxidase, and antioxidant capacity), levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), concentrations of persistent organic pollutants in blood plasma, and urinary levels of polycyclic aromatic hydrocarbon metabolites were investigated as parameters potentially affecting the markers of DNA oxidation (8-oxo-7,8-dihydro-2′-deoxyguanosine) and lipid peroxidation (15-F2t-isoprostane). The levels of oxidative stress markers mostly differed between the localities in the individual sampling seasons. Multivariate linear regression analysis revealed IL-6, a pro-inflammatory cytokine, as a factor with the most pronounced effects on oxidative stress parameters. The role of other variables, including environmental pollutants, was minor. In conclusion, our study showed that oxidative damage to macromolecules was affected by processes related to inflammation; however, we did not identify a specific environmental factor responsible for the pro-inflammatory response in the organism

The Differential Effect of Carbon Dots on Gene Expression and DNA Methylation of Human Embryonic Lung Fibroblasts as a Function of Surface Charge and Dose

This study presents a toxicological evaluation of two types of carbon dots (CD), similar in size (<10 nm) but differing in surface charge. Whole-genome mRNA and miRNA expression (RNAseq), as well as gene-specific DNA methylation changes, were analyzed in human embryonic lung fibroblasts (HEL 12469) after 4 h and 24 h exposure to concentrations of 10 and 50 µg/mL (for positive charged CD; pCD) or 10 and 100 µg/mL (for negative charged CD, nCD). The results showed a distinct response for the tested nanomaterials (NMs). The exposure to pCD induced the expression of a substantially lower number of mRNAs than those to nCD, with few commonly differentially expressed genes between the two CDs. For both CDs, the number of deregulated mRNAs increased with the dose and exposure time. The pathway analysis revealed a deregulation of processes associated with immune response, tumorigenesis and cell cycle regulation, after exposure to pCD. For nCD treatment, pathways relating to cell proliferation, apoptosis, oxidative stress, gene expression, and cycle regulation were detected. The expression of miRNAs followed a similar pattern: more pronounced changes after nCD exposure and few commonly differentially expressed miRNAs between the two CDs. For both CDs the pathway analysis based on miRNA-mRNA interactions, showed a deregulation of cancer-related pathways, immune processes and processes involved in extracellular matrix interactions. DNA methylation was not affected by exposure to any of the two CDs. In summary, although the tested CDs induced distinct responses on the level of mRNA and miRNA expression, pathway analyses revealed a potential common biological impact of both NMs independent of their surface charge

Maternal Diet Quality and the Health Status of Newborns

The maternal diet during pregnancy affects neonatal health status. The objective of this study was to assess the nutritional quality of the maternal diet, and its contamination by persistent organic pollutants (POPs), in pregnant women living in two areas of the Czech Republic with different levels of air pollution, and subsequently to assess the relationship of these two factors with birth weight and neonatal oxidative stress. To determine the level of oxidative stress, 8-isoprostane concentrations in umbilical cord plasma were measured. The overall nutritional quality of the maternal diet was not optimal. Of the nutritional factors, protein intake proved to be the most significant showing a positive relationship with birth weight, and a negative relationship with the oxidative stress of newborns. Dietary contamination by persistent organic pollutants was low and showed no statistically significant relationship with birth weight. Only one of the 67 analyzed POPs, namely the insecticide dichlorodiphenyltrichloroethane (DDT), showed a statistically significant positive relationship with the level of neonatal oxidative stress

Measurement of 8-oxo-7,8-dihydro-2 '-deoxyguanosine in urine by an improved ELISA

53rd Congress of the European-Societies-of-Toxicology (EUROTOX) -- SEP 10-13, 2017 -- Bratislava, SLOVAKIAWOS: 000425486700105European Soc ToxicolGA CRGrant Agency of the Czech Republic [16-14631S]Supported by GA CR (16-14631S)

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes