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

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

DNA methylation profiles in a group of workers occupationally exposed to nanoparticles

The risk of exposure to nanoparticles (NPs) has rapidly increased during the last decade due to the vast use of nanomaterials (NMs) in many areas of human life. Despite this fact, human biomonitoring studies focused on the effect of NP exposure on DNA alterations are still rare. Furthermore, there are virtually no epigenetic data available. In this study, we investigated global and gene-specific DNA methylation profiles in a group of 20 long-term (mean 14.5 years) exposed, nanocomposite, research workers and in 20 controls. Both groups were sampled twice/day (pre-shift and post-shift) in September 2018. We applied Infinium Methylation Assay, using the Infinium MethylationEPIC BeadChips with more than 850,000 CpG loci, for identification of the DNA methylation pattern in the studied groups. Aerosol exposure monitoring, including two nanosized fractions, was also performed as proof of acute NP exposure. The obtained array data showed significant differences in methylation between the exposed and control groups related to long-term exposure, specifically 341 CpG loci were hypomethylated and 364 hypermethylated. The most significant CpG differences were mainly detected in genes involved in lipid metabolism, the immune system, lung functions, signaling pathways, cancer development and xenobiotic detoxification. In contrast, short-term acute NP exposure was not accompanied by DNA methylation changes. In summary, long-term (years) exposure to NP is associated with DNA epigenetic alterations

Mutations in p53, p53 protein overexpression and breast cancer survival

p53 is an important tumor-suppressor gene that encodes p53 protein, a molecule involved in cell cycle regulation, and has been inconsistently linked to breast cancer survival. Using archived tumor tissue from a population-based sample of 859 women diagnosed with breast cancer between 1996–1997, we determined p53 mutations in exons 5–8 and p53 protein overexpression. We examined the association of p53 mutations with overexpression and selected tumor clinical parameters. We assessed whether either p53 marker was associated with survival through 2002, adjusting for other tumor markers and prognostic factors. The prevalence of protein overexpression in the tumor was 36% (307/859) and any p53 mutation was 15% (128/859). p53 overexpression was positively associated with the presence of any p53 mutation (odds ratio (OR)=2.2, 95% confidence interval (CI)=1.5–3.2), particularly missense mutations (OR=7.0, 95%CI=3.6–13.7). Negative estrogen and progesterone receptor status (ER/PR) was positively associated with both p53 protein overexpression (OR = 2.6, 95% CI = 1.7–4.0), and p53 mutation (OR = 3.9, 95% CI = 2.4–6.5). Any p53 mutation and missense mutations, but not p53 protein overexpression, were associated with breast cancer-specific mortality (Hazard ratio HR=1.7, 95%CI=1.0–2.8; HR=2.0, 95%CI=1.1–3.6, respectively) and all-cause mortality (HR=1.5, 95%CI=1.0–2.4; HR=2.0, 95%CI=1.2–3.4, respectively); nonsense mutations were associated only with breast cancer-specific mortality (HR=3.0, 95%CI=1.1–8.1). These associations however did not remain after adjusting for ER/PR status. Thus, in this population-based cohort of women with breast cancer, although p53 protein overexpression and p53 mutations were associated with each other, neither independently impacted breast-cancer specific or all-causing mortality after considering ER/PR status

Associations between Polycyclic Aromatic Hydrocarbon-Related Exposures and \u3cem\u3ep53\u3c/em\u3e Mutations in Breast Tumors

BACKGROUND: Previous studies have suggested that polycyclic aromatic hydrocarbons (PAHs) may be associated with breast cancer. However, the carcinogenicity of PAHs on the human breast remains unclear. Certain carcinogens may be associated with specific mutation patterns in the p53 tumor suppressor gene, thereby contributing information about disease etiology. OBJECTIVES: We hypothesized that associations of PAH-related exposures with breast cancer would differ according to tumor p53 mutation status, effect, type, and number. METHODS: We examined this possibility in a population-based case–control study using polytomous logistic regression. As previously reported, 151 p53 mutations among 859 tumors were identified using Surveyor nuclease and confirmed by sequencing. RESULTS: We found that participants with p53 mutations were less likely to be exposed to PAHs (assessed by smoking status in 859 cases and 1,556 controls, grilled/smoked meat intake in 822 cases and 1,475 controls, and PAH–DNA adducts in peripheral mononuclear cells in 487 cases and 941 controls) than participants without p53 mutations. For example, active and passive smoking was associated with p53 mutation–negative [odds ratio (OR) = 1.55; 95% confidence interval (CI), 1.11–2.15] but not p53 mutation–positive (OR = 0.77; 95% CI, 0.43–1.38) cancer (ratio of the ORs = 0.50, p \u3c 0.05). However, frameshift mutations, mutation number, G:C→A:T transitions at CpG sites, and insertions/deletions were consistently elevated among exposed subjects. CONCLUSIONS: These findings suggest that PAHs may be associated with specific breast tumor p53 mutation subgroups rather than with overall p53 mutations and may also be related to breast cancer through mechanisms other than p53 mutation

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)