DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.

Chronic respiratory diseases and quality of life in elderly nursing home residents.

Few studies have assessed the quality of life (QOL) related to chronic respiratory diseases in the elderly. In the framework of the geriatric study on the health effects of air quality in elderly care centers (GERIA) study, a questionnaire was completed by elderly subjects from 53 selected nursing homes. It included various sections in order to assess respiratory complaints, QOL (World Health Organization QOL (WHOQOL)-BREF), and the cognitive and depression status. The outcome variables were the presence of a score lower than 50 (<50) in each of the WHOQOL-BREF domains (physical health, psychological health, social relationships, and environmental health). Chronic bronchitis, frequent cough, current wheezing, asthma, and allergic rhinitis were considered as potential risk factors. The surveyed sample was (n = 887) 79% female, with a mean age of 84 years (SD: 7 years). In the multivariable analysis, a score of <50 in the physical domain was associated with wheezing in the previous 12 months (odds ratio (OR): 2.03, confidence interval (CI): 1.25-3.31) and asthma (OR: 1.95, CI: 1.12-3.38). The psychological domain was related with a frequent cough (OR: 1.43, CI: 0.95-2.91). A score of <50 in the environmental domain was associated with chronic bronchitis (OR: 2.89, CI: 1.34-6.23) and emphysema (OR: 3.89, CI: 1.27-11.88). In view of these findings, the presence of respiratory diseases seems to be an important risk factor for a low QOL among elderly nursing home residents.

Efectos genotóxicos e inmunotóxicos de la exposición laboral al plomo / Genotoxic and immunotoxic of occupational exposure to lead

Objetivo. Evaluar los efectos genotóxicos e inmunotóxicos asociados a la exposición laboral al plomo.Métodos. Se ha realizado el ensayo de mutación en el receptor de las células T (TCR) y se han determinado las variacionesen los porcentajes de diferentes subpoblaciones linfocitarias y en las concentraciones de ciertas citoquinas circulantesen plasma sanguíneo mediante citometría de fl ujo en 30 trabajadores expuestos procedentes de 2 empresas y en 30 trabajadoresno expuestos como grupo control.Resultados: Los individuos expuestos mostraron frecuencias de mutación signifi cativamente mayores que los controles(media±error estándar: 20,88±3,58 vs. 12,98±2,88), independientemente de su empresa de procedencia, así como menorporcentaje de linfocitos CD8+ (%medio±error estándar: 31,97±1,70 vs. 36,70±1,30), descenso que sólo mantuvo signifi caciónen una de las empresas. Las concentraciones de las citoquinas analizadas fueron en general mayores en los expuestosque en los controles, siendo signifi cativo el incremento para IL-2 (media±error estándar: 1,09±0,26 vs. 0,25±0,17 pg/ml)e IL-10 (media±error estándar: 2,88±1,14 vs. 0,58±0,23 pg/ml), con diferencias entre empresas. Los efectos de la exposiciónen cuanto a frecuencia de mutación y concentraciones de IL-2 e IL-10 fueron mayores en los individuos no fumadores,apuntando a una menor susceptibilidad de los fumadores posiblemente como consecuencia de la potenciación de los mecanismosde reparación por el contacto crónico con el humo del tabaco.Conclusiones. Los resultados indican que la exposición laboral a plomo induce mutagenicidad y alteraciones en parámetrosinmunológicos, sugiriendo la necesidad de aplicación de medidas para la eliminación o disminución de los nivelesde plomo en los lugares de trabajo

Objective. To evaluate genotoxic and immunotoxic effects associated with occupational exposure to lead.Methods. A T-cell receptor (TCR) mutation assay was performed, and variations in the percentages of different lymphocytesubpopulations and in the concentrations of certain plasma circulating cytokines determined by fl ow cytometry in30 exposed individuals from 2 factories and 30 controls.Results. Exposed individuals showed signifi cantly higher levels of mutation frequency than controls (mean±standard error:20.88±3.58 vs. 12.98±2.88), regardless of their factory; the percentage of CD8+ lymphocytes was also lower (mean%±standard error: 31.97±1.70 vs. 36.70±1.30), although statistical signifi cance was observed in only one factory. Concentrations ofanalysed cytokines were generally higher among exposed individuals than controls. The increase was signifi cant for IL-2(mean±standard error: 1.09±0.26 vs. 0.25±0.17) and IL-10 (mean±standard error: 2.88±1.14 vs. 0.58±0.23), with differencesbetween factories. Exposure effects regarding mutation frequency and IL-2 and IL-10 concentrations were greater amongnonsmokers, suggesting a lower susceptibility of smokers as a consequence of strengthening of repair mechanisms throughchronic contact with tobacco smoke.Conclusions. Occupational exposure to lead induces mutagenicity and alterations in immunological parameters, suggestingthe need to apply measures for the elimination or decrease of lead levels in workplaces