This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.

• MeSH
• buněčné linie MeSH
• kometový test metody   MeSH
• lidé MeSH
• oprava DNA * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The phenolic compounds of methanolic extracts of Salvia pomifera and Salvia fruticosa were identified by liquid chromatography tandem mass spectrometry. Carnosic acid and its metabolite carnosol were the most abundant terpene phenolic compounds of S. fruticosa, while they were completely absent in S. pomifera. The main terpene phenolic constituent of S. pomifera was 12-O-methylcarnosic acid and its mass/mass fragmentation pathway was explained. The detailed mechanism of carnosic acid oxidation to carnosol was suggested. The effects of Salvia extracts and/or carnosic acid, the main diterpene phenolic component of S. fruticosa, on the proliferation and cell cycle of two melanoma cell lines (A375, Mel JuSo) and human fibroblast cell line (HFF) were investigated by MTT assay, PI-exclusion assay and flow cytometry cell cycle analysis. Extract of S. fruticosa more efficiently than S. pomifera extract reduced the proliferation of the human melanoma cells. Carnosic acid showed the most significant effect. The first evidence that carnosic acid affects microtubule dynamics and arrests the cell cycle in the G2/M phase was provided. Collectively, our results demonstrate that these two Salvia species are plants of medicinal interest with perspective for further investigation. Carnosic acid could be the compound responsible for the biological activities of S. fruticosa extracts.

• Klíčová slova
• 12-O-methylcarnosic acid, LC-MS, Salvia fruticosa, Salvia pomifera, cancer, carnosic acid, cell cycle, cytotoxicity, melanoma, microtubules,
• MeSH
• antitumorózní látky chemie   izolace a purifikace   farmakologie   MeSH
• buněčné linie MeSH
• diterpeny abietanové chemie   izolace a purifikace   farmakologie   MeSH
• epitelové buňky účinky léků   patologie   MeSH
• fenoly chemie   izolace a purifikace   farmakologie   MeSH
• fibroblasty cytologie   účinky léků   MeSH
• inhibiční koncentrace 50 MeSH
• kontrolní body fáze G2 buněčného cyklu účinky léků   MeSH
• lidé MeSH
• methanol chemie   MeSH
• nádorové buněčné linie MeSH
• nadzemní části rostlin chemie   MeSH
• oxidace-redukce MeSH
• proliferace buněk účinky léků   MeSH
• rostlinné extrakty chemie   MeSH
• rozpouštědla chemie   MeSH
• šalvěj chemie   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky MeSH
• carnosol MeSH Prohlížeč
• diterpeny abietanové MeSH
• fenoly MeSH
• methanol MeSH
• rostlinné extrakty MeSH
• rozpouštědla MeSH
• salvin MeSH Prohlížeč

Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations.

• Klíčová slova
• DNA repair, animal studies, base excision repair (BER), clinical studies, comet assay, human biomonitoring, nucleotide excision repair (NER), occupational studies,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH