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Mechanisms and Biomarkers (WG 4) page 36
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permeabilisation/damage resulting in cell death. Ethidium bromide (EtBr) and acridine orange
can be used in conjunction to demonstrate apoptotic as well as cytotoxic events via cellular
uptake as a result of membrane damage. EtBr has been used to demonstrate a link between
membrane and DNA damage by ROS which was modulated by ß-carotene and lycopene
(Lowe et al., 1999).
DNA Damage
Intervention studies where DNA damage is an intermediate biomarker can employ gross
changes in DNA as an indicator of disease progression.
Comet Assay - The comet assay measures single and double strand breaks, by a single cell gel
electrophoresis technique and is a useful indicator of oxidative DNA damage (Gedik et al.,
1998). The traditional comet assay has been used in my laboratory to detect DNA strand
breaks in HT29 cells challenged with xanthine-xanthine oxidase. Subsequent incubations with
ß-carotene and lycopene reduced DNA damage at low to physiological levels, but showed a
progressive loss of protection with increasing carotenoid concentration (Lowe et al., 1999).
The method can be further refined to measure oxidised bases by incubating the ROS stressed
cells with endonuclease III and formamidopyrimidine glycosylase for detection of oxidised
pyrimidines and purines respectively (Kruszewski et al., 1998). Supplementation with vitamin
C, E and ß-carotene has been shown to reduce lymphocyte DNA damage in a smoking
intervention trial using the modified comet assay (Duthie et al., 1996). Lymphocytes from the
supplemented subjects were also resistant to in vitro strand breakage induced by H2O2.
A challenging future development of this sensitive method is the application of fluorescent
in-situ hybridisation (FISH) to investigate the integrity of specific genes within the damaged
DNA (McKelvey-Martin et al., 1998).
Micronucleus Assay (MN) - Similar to the comet assay this cytogenetic technique can be used
as an intermediate end point in in vivo studies. Several laboratories have demonstrated
protective effects of ß-carotene against X and γ-ray induced MN formation in mice and
isolated human lymphocytes (Umegaki et al., 1997. Konopacka et al., 1998) β-carotene was
shown to have no effect on spontaneous chromosomal damage in peripheral blood
lymphocytes from 30 healthy non smoking human donors, using the MN assay (Odagiri and
Uchida, 1998). Xue et al. (1998) revealed interesting differences in MN frequency depending
on the carotenoid isomers used, the all trans isomer appeared to increase MN formation