Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies

Abstract

Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

Salivary Leucocytes as In Vitro Model to Evaluate Nanoparticle-Induced DNA Damage

“…The possible interference of the NPs with the comet assay procedure and hOGG1 enzyme activity was checked prior the experiments. Frozen cells were used since a recent study demonstrated that cryopreserved salivary leucocytes show similar sensitivity to DNA damage induction by genotoxic agents than fresh cells and, consequently, both can be equally employed as appropriate biomatrices to detect DNA damage by the comet assay [ 14 ].…”

“…Since the inhalation pathway represents the main route involved in xenobiotic systemic uptake when any type of NPs are dispersed in air, salivary leucocytes seem to be a suited biomatrix to in vitro simulate and evaluate genotoxic effects of environmental or occupational exposure to NPs. They are also easy to obtain, cost-effective, and can be collected non-invasively without requiring highly trained personnel [ 14 ]. Still, to date, they have not been used before in assessing in vitro nanotoxicity.…”

“…Negative controls (culture medium) were included in each experiment. Positive controls used were 50 µg/mL MMS for 3 h in the case of primary DNA damage, and 335 µg/mL KBrO 3 for 1 h in the case of oxidative DNA damage [ 14 ]. Trypan blue exclusion technique was used to assess cell viability after treatments.…”

“…Recently, human salivary leucocytes have been proposed as a non-invasive adequate alternative to peripheral blood leucocytes to evaluate the genotoxic effects of recent exposure to environmental contaminants, particularly those involving inhalatory or oral exposure routes [ 13 ]. Moreover, this cell type might be also useful for in vitro testing, and indeed our group has recently proved that leucocytes isolated from saliva samples, both fresh and cryopreserved, can be employed as appropriate biomatrices to detect primary and oxidative DNA damage caused by different mechanisms by the comet assay [ 14 ]. However, their suitability and the specific protocol to be employed for in vitro nanogenotoxicity screening have not been described yet.…”

Measuring DNA modifications with the comet assay: a compendium of protocols

“…Centrifuge the oral rinses (15 min, 1,100g, at 4 °C), wash the cell pellet with cold PBS and resuspend in RPMI 1640 cell culture medium. Leukocytes are isolated from the cell suspension by standard density gradient centrifugation 328,329 • Buccal cells: before sampling, the subject should perform two consecutive rinses with water (room temperature (RT), ~22 °C). The sample is collected with a cytobrush or toothbrush c CRITICAL The initial collection/scraping of both cheeks (using separate brushes) is discarded.…”