Implications
(i) Genotoxicity, carcinogenesis
Focus
(i) Oxidative modification of DNA
Models
(i) Direct (in vitro) effect ® cell culture models ® animal models ® human volunteers
Why DNA damage?
Significant levels of DNA damage leading to mutations and carcinogenesis are caused by endogenous oxidants. It is estimated that half of all human cancers are due to oxidative DNA damage. The level of oxidative DNA damage can be estimated by measuring the amount of 8-hydroxy-2´-deoxyguanosine (8-HdG). Several independent studies have shown that the level of 8-HdG is elevated in a variety of human tumors and premalignancies and, in accordance, e.g. due to tobacco smoking, Helicobacter pylori infection, and exposure to chemical carcinogens. Therefore, the 8-HdG –assay seems to offer one suitable biomarker to be used in experimental studies as an indicator of the risk of cancer.
Effects on DNA oxidation in vitro
Test:
(i) Determination of DNA damage by electrophoresis
Aim: To find out if the test compound causes oxidation of DNA or, alternatively, if the test compound can prevent experimentally induced DNA oxidation.
Procedure: The relative amount of 8-HdG is used as an index of DNA oxidation. Oxidation of chemically pure DNA and DNA in tissue homogenates will be monitored with time. Oxidation of DNA is induced experimentally by chemicals or irradiation. The results will be presented as comparison of the potency of the test compound with that of a simultaneously tested known antioxidant.
Effects on DNA oxidation in cell culture models
Tests:
(i) Single-stranded DNA breaks by Comet assay
(ii) DNA damage by electrophoresis
(iii) Determination of 8-HdG
Aim: To find out if the test compound causes DNA oxidation or, alternatively, if the test compound can prevent experimentally induced DNA oxidation in living cells.
Procedure: Studies will be done using a cell line suitable for the purposes of testing. DNA oxidation is induced experimentally by chemicals. The results will be presented as comparison of the potency of the test compound with that of a simultaneously tested known antioxidant.
Effects on DNA oxidation in experimental animals
Test:
(i) 8-HdG in leucocytes and tissues
Aim: To find out if the test compound causes DNA oxidation or, alternatively, if the test compound can prevent experimentally induced DNA oxidation in tissues of experimental animals.
Procedure: Studies will be done in animal models (usually rats or mice) that suit best for the purposes of testing. The test compound will be administered with feed or drinking water. DNA oxidation is affected experimentally by vitamin E deficiency and/or chemical prooxidant. At termination, DNA will be isolated from animal tissues and analyzed for the relative amount of oxidized DNA.