Trj. Ollikainen et al., DNA SINGLE-STRAND BREAKS AND ADENINE-NUCLEOTIDE DEPLETION AS INDEXES OF OXIDANT EFFECTS ON HUMAN LUNG-CELLS, Free radical biology & medicine, 24(7-8), 1998, pp. 1088-1096
The comet assay (single cell gel electrophoresis) is a novel method to
assess DNA strand breaks in single cells. We studied the oxidant sens
itivity of cultured primary and transformed (MeT-5A) human pleural mes
othelial cells, as well as primary and transformed (BEAS 2B) human bro
nchial epithelial cells, and compared the results obtained with the Co
met assay to other markers of oxidant effects on cells, such as deplet
ion of intracellular high-energy nucleotides (ATP, ADP, AMP), accumula
tion of products of nucleotide catabolism (xanthine, hypoxanthine, uri
c acid), and release of lactate dehydrogenase (LDH). The cells were ex
posed for 5 min to 4 h to 50-500 mu M H2O2 or to 5-50 mu M menadione.
Significant tail moment increase, which is a marker of DNA strand brea
ks in the Comet assay, and intracellular nucleotide depletion occurred
simultaneously in MeT-5A and BEAS 2B cells during the first 30-60 min
of exposure to H2O2 and menadione, In the Comet assay variation betwe
en the individual cells could be detected. LDH release, a marker of ce
ll injury, showed that mesothelial cells were far more sensitive than
epithelial cells to oxidant-induced lytic cell injury. MeT-5A and BEAS
2B cells contained similar intracellular antioxidant enzyme activitie
s, which may explain their similar oxidant sensitivity in the Comet as
say. A significant increase (164%) in the tail moment was delectable i
n MeT-5A cells exposed to 50 mu M H2O2 for 30 min. This returned to co
ntrol level during the 4 h of continuing exposure. A 30 min exposure t
o 25 mu M menadione caused a 61% increase in the mean toil moment but,
unlike with H2O2, the change was irreversible during the following 4
h incubation. We conclude that human pleural mesothelial cells and bro
nchial epithelial cells show similar oxidant sensitivity when assessed
by the Comet assay, but various oxidants differ in their potency in c
ausing DNA breaks in these cells. (C) 1998 Elsevier Science Inc.