Implications
(i) Biosafety
(ii) In vitro toxicity
Focus
(i) Cytotoxicity (prooxidant activity)
(ii) Cytoprotectivity (elimination of oxidative stress)
Models
(i) Primary cell culture (rat, pig and/or human hepatocyte; rat cardiomyocyte)
(ii) Cell line (fibroblast (3T3 and Balb); keratinocyte (HaCaT); endothelial cell (HUVEC); HL-60; HepG2; HeLa)
Why testing of cytotoxicity/cytoprotectivity?
In vitro screening of cytotoxic properties of the test substance/extract/phytopreparation can partially substitute in vivo test. Using primary cell cultures or cell lines derived from various sources opens a large variability of detection of cytotoxic properties of tested samples. On these cell models where pathological alterations (e.g. oxidative stress) had been induced, the cytoprotectivity of a compound/extract can be tested.
Assessment of cell toxicity/protectivity
Tests:
(i) Viability – damage of cell membrane – leakage of LDH in medium; mitochondrial activity measured by MTT assay (reduction of tetrazolium salt); Neutral red dye retention assay; damage of mitochondria – leakage of AST
(ii) Redox status of cell – content of glutathione
(iii) Energy status of cell – intracellular level of ADP and ATP
(iv) Oxidative damage of cell membranes – lipoperoxidation – assessed as TBARS
(v) Effect on cell proliferation (DNA synthesis) – incorporation of bromo-deoxyuridine into DNA by immunoassay
(vi) DNA damage – single – stranded DNA breaks – Comet assay
Aim: To assess if the test substance/extract/phytopreparation will display cytotoxic/ cytoprotective effect against the oxidative stress induced in cells.
Procedure: Cell cultures will be used for the assessment of cytotoxicity and/or cytoprotectivity of substance/extract/phytopreparation. Their cytoprotective effect will be monitored on cell cultures intoxicated by model toxins, e.g. tert-butyl hydroperoxide or UV exposure. The cell viability, intracellular GSH, ADP and ATP level, and lipid peroxidation products will be used for the evaluation. Antiproliferative effect will be evaluated on proliferative cells by incorporation of bromo-deoxyuridine into DNA. DNA damage will be detected by Comet assay.