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

Automation and validation of micronucleus detection in the 3D EpiDerm™ human reconstructed skin assay and correl

Recent restrictions on the testing of cosmetic ingredients in animals have resulted in the need to test the genotoxic potential of chemicals exclusively in vitro prior to licensing. However, as current in vitro tests produce some misleading positive results, sole reliance on such tests could prevent some chemicals with safe or beneficial exposure levels from being marketed. The 3D human reconstructed skin micronucleus (RSMN) assay is a promising new in vitro approach designed to assess genotoxicity of dermally applied compounds. The assay utilises a highly differentiated in vitro model of the human epidermis. For the first time, we have applied automated micronucleus detection to this assay using MetaSystems Metafer Slide Scanning Platform (Metafer), demonstrating concordance with manual scoring. The RSMN assay's fixation protocol was found to be compatible with the Metafer, providing a considerably shorter alternative to the recommended Metafer protocol. Lowest observed genotoxic effect levels (LOGELs) were observed for mitomycin-C at 4.8 μg/ ml and methyl methanesulfonate (MMS) at 1750 μg/ml when applied topically to the skin surface. In-medium dosing with MMS produced a LOGEL of 20 μg/ml, which was very similar to the topical LOGEL when considering the total mass of MMS added. Comparisons between 3D medium and 2D LOGELs resulted in a 7-fold difference in total mass of MMS applied to each system, suggesting a protective function of the 3D microarchitecture. Interestingly, hydrogen peroxide (H2O 2), a positive clastogen in 2D systems, tested negative in this assay. A non-genotoxic carcinogen, methyl carbamate, produced negative results, as expected. We also demonstrated expression of the DNA repair protein N-methylpurine-DNA glycosylase in EpiDerm™. Our preliminary validation here demonstrates that the RSMN assay may be a valuable followup to the current in vitro test battery, and together with its automation, could contribute to minimising unnecessary in vivo tests by reducing in vitro misleading positives. © The Author 2014

Biomarkers of exposure and effect—interpretation in human risk assessment

The effect of exposure to carcinogenic polycyclic aromatic hydrocarbons adsorbed onto respirable air particles (PM2.5, diameter < 2.5 μm) on DNA adducts and chromosomal aberrations was repeatedly studied in Prague, Czech Republic, in groups of policemen working in the downtown area and in bus drivers. Personal exposure was evaluated using personal samplers during working shifts. DNA adducts were analyzed in lymphocytes by the 32P-postlabeling assay and chromosomal aberrations were analyzed by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH). The impact of environmental pollution on DNA adducts and chromosomal aberrations was studied in a total of 950 subjects. Our results suggest that the environmental exposure of nonsmokers to concentrations higher than 1 ng benzo[a]pyrene/m3 represents a risk of DNA damage, as indicated by an increase in DNA adducts and the genomic frequency of translocations determined by FISH

Chapter Molecular Epidemiology and Air Pollution

Biotechnolog

The Molecular Mechanisms of Adaptive Response Related to Environmental Stress

none6siThe exposure of living organisms to environmental stress triggers defensive responses resulting in the activation of protective processes. Whenever the exposure occurs at low doses, defensive effects overwhelm the adverse effects of the exposure; this adaptive situation is referred to as "hormesis". Environmental, physical, and nutritional hormetins lead to the stimulation and strengthening of the maintenance and repair systems in cells and tissues. Exercise, heat, and irradiation are examples of physical hormetins, which activate heat shock-, DNA repair-, and anti-oxidative-stress responses. The health promoting effect of many bio-actives in fruits and vegetables can be seen as the effect of mildly toxic compounds triggering this adaptive stimulus. Numerous studies indicate that living organisms possess the ability to adapt to adverse environmental conditions, as exemplified by the fact that DNA damage and gene expression profiling in populations living in the environment with high levels of air pollution do not correspond to the concentrations of pollutants. The molecular mechanisms of the hormetic response include modulation of (a) transcription factor Nrf2 activating the synthesis of glutathione and the subsequent protection of the cell; (b) DNA methylation; and (c) microRNA. These findings provide evidence that hormesis is a toxicological event, occurring at low exposure doses to environmental stressors, having the benefit for the maintenance of a healthy status.mixedRossnerova, Andrea; Izzotti, Alberto; Pulliero, Alessandra; Bast, Aalt; Rattan, S I S; Rossner, PavelRossnerova, Andrea; Izzotti, Alberto; Pulliero, Alessandra; Bast, Aalt; Rattan, S I S; Rossner, Pave

Chapter Molecular Epidemiology and Air Pollution

Biotechnolog