Optimized noninvasive procedures to measure DNA damage in comet assay.

The comet assay is a well-established, simple and sensitive method to measure DNA damage in single cell and is commonly used in human trials to investigate the effects of pollution, occupational hazards and potential genoprotective agents. Peripheral blood lymphocytes are most commonly used in human biomonitoring studies, but lymphocytes collected from the mouth offer a potentially attractive, noninvasive alternative. The aim of the current study was to develop a buccal cell lymphocyte comet assay procedure. Cells were collected from mouthwash of three healthy volunteers and tested individually. The comet assay was performed under different pH and times of alkaline treatment, electrophoresis run times and hydrogen peroxide concentrations. Optimal conditions for buccal lymphocytes in comet assay were found to be pH >13 for unwinding and electrophoresis buffers, 10-min alkaline unwinding treatment and 20-min electrophoresis run time. We successfully utilized our optimized assay conditions to demonstrate the genoprotective activity of quercetin. This newly established procedure offers an alternative noninvasive sampling method for the investigation of DNA protection and/or damaging effect.

Bioscreening of Australian olive mill waste extracts: biophenol content, antioxidant, antimicrobial and molluscicidal activities.

The biophenolic fraction was characterized in olive mill waste (OMW) obtained as a by-product from processing of Mission and Frantoio olive fruit. OMW produced from the Mission fruit contained higher total phenol content. Individual biophenols with the exception of verbascoside and a hydroxytyrosol-secoiridoid were also present at higher concentrations in the OMW produced from Mission cultivar. Antioxidant activities were measured in aqueous (DPPH) and emulsion (BCBT) systems. The Frantoio extract was more active than the Mission extract in the DPPH assay - EC(50) values were 28.3+/-1.7 ppm and 34.7+/-1.7 ppm, respectively. Activities were reversed in the BCBT, with the Mission extract (EC(50) 60.6+/-2.3 ppm) more potent than the Frantoio extract (EC(50) 79.9+/-2.0 ppm), and this may be related to the more lipophilic nature of the Mission extract. Both extracts showed broad spectrum antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa; whereas individual biophenols (hydroxytyrosol, luteolin, oleuropein) showed more limited activity. Molluscicidal activity was measured against Isidorella newcombi and LD(50) values were 424 ppm and 541 ppm for Mission and Frantoio extracts, respectively. The results suggest that OMW may be utilised as a source of bioactive compounds.