Carcinogenesis vol.14 no.9 pp.
1733-1735, 1993
ACCELERATED PAPER
Direct enzymic detection of endogenous oxidative base damage in
human lymphocyte DNA
Andrew R.Collins, Susan J.Duthie and
Victoria L.Dobson
Rowett Research Institute, Greenbum Road, Bucksburn, Aberdeen
AB2 9SB, UK
The endogenous production of oxidative damage in DNA by
free radicals released as a by-product of respiration is a likely
cause of mutations which, if they occur in appropriate genes,
may lead to cancer. Using an endonuclease specific for
oxidized pyrimidines, in conjunction with the highly sensitive
method of single cell gel electrophoresis, we have detected
significant oxidative damage in untreated, freshly isolated
lymphocytes from normal, healthy individuals.
Oxidative damage to DNA results from free radical attack
following exposure to ionizing radiation or to agents such as
H2O2 that release active oxygen species. The hydroxyl radical
is thought to be responsible for most of the damage, which takes
the form of strand breaks and oxidized bases. Ames has suggested
that significant oxidative damage occurs in vivo as a result of
endogenous free radical attack (1) and contributes to the aetiology
of cancer. Antioxidants, largely of dietary origin, may, by
scavenging free radicals, limit the damage incurred by the DNA
and hence protect against mutagenesis and cancer.
The rate of DNA damage can be gauged from measurement
of the products of its repair in urine, making allowance for the
presence of derivatives of oxidized nucleic acids from the diet
(2). However, this does not shed light on the amount of damage
present in cellular DNA at any time—arguably a determinant of
mutation risk—since this depends on the turnover time (i.e. the
time between occurrence and repair of damage). Direct
measurement of the steady-state level of damage in cellular DNA
is a more meaningful indicator of oxidative stress.
We have devised an assay to monitor peripheral human
lymphocytes for the presence of oxidative DNA damage received
in vivo, by converting oxidized bases to strand breaks using
endonuclease HI, which specifically nicks DNA at sites of
oxidized pyrimidines (3). The enzyme was purified using FPLC
after the method of Asahara et al. (4) and used at a final protein
concentration of 1 /tg/ml. Breaks are detected by the 'comet
assay', single cell gel electrophoresis (5). After embedding cells
in a layer of agarose on a microscope slide, they are lysed widi
detergent and treated with high salt, leaving 'nucleoids' containing
intact supercoiled loops of DNA (6). If strand breaks are present,
the supercoiling is relaxed and, on electrophoresis at high pH,
loops of DNA extend towards the anode, giving the appearance
of the tail of a comet when visualized byfluorescencemicroscopy.
The more breaks that are present, the greater is the intensity of
DNA in the tail. The procedure was as described (7) except mat,
after lysis in 2.5 M NaCl, 0.1 M Na2EDTA, 10 mM
Tris-HCl, 1% Triton X-100, pH 10.0, for 1 h at 4°C, slides
were washed 3 X in endonuclease buffer (40 mM HEPES-KOH,
0.1 M KC1, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin,
© Oxford University Press
pH 8.0), drained and the agarose covered with 25 ii\ of either
buffer or endonuclease HI in buffer, sealed with a cover glass
and incubated for 30 min at 37°C. Further steps (alkaline
electrophoresis, neutralization and staining with DAPI) were as
described (7). Cells were examined with a Zeiss Axioskop
Downloaded from https://academic.oup.com/carcin/article/14/9/1733/334111 by OUP-USA Mirror user on 20 May 2024
fluorescence microscope.
The method was developed with HeLa cells (grown in GMEM
with 5% fetal calf serum, 5% newborn calf serum). Five minutes
treatment on ice with 100 jtM H2O2 in PBS produces comets
with highly extended and intense tails (Figure 1A, filled bars),
reflecting free radical-induced strand breaks. Treatment on ice
minimizes the possibility of cellular processing of damage. One
hour of subsequent incubation at 37°C, however, allows rejoining
of strand breaks and the resulting comets show little damage
(Figure 1A, open bars). Figure 1(B) gives the results of treating
cells with different doses of H2O2 and then incubating them,
after lysis in agarose, with endonuclease HI or with buffer alone.
There is a dramatic H2O2-dependent increase in the damage seen
with the enzyme (filled bars) compared with buffer alone (hatched
bars). The extra sites revealed as breaks in this way represent
oxidized bases, which persist in DNA after the initial strand
breaks are rejoined. [The damage seen with buffer after 100 /tM
H2O2 (Figure IB, bottom left panel) is somewhat greater than
is seen without in-gel incubation (Figure 1A, right panel), perhaps
reflecting a residual endogenous endonuclease activity.] In the
case of cells not treated with H2O2, no significant increase in
DNA breakage occurs when the cells are incubated with
endonuclease El, indicating that HeLa cells do not carry
detectable unrepaired oxidative damage to pyrimidines and
confirming the specificity of the enzyme.
To examine DNA damage in lymphocytes, 30 /d of blood from
a finger-prick sample was mixed with 1 ml of culture medium
(RPMI1640 with 10% fetal calf serum) in a microcentrifuge tube
and kept on ice for 30 min. Lymphocytes were separated by
centrifugation (200 g, 3 min, 4°C) over a layer of 100 id of
Histopaque 1077. Cells retrieved from above this layer were
mixed with PBS and centrifuged again. The pelleted cells,
suspended in low melting point agarose, were then treated in the
same way as the HeLa cells.
In marked contrast to HeLa cells, untreated lymphocytes
isolated from normal human subjects contain many sites sensitive
to endonuclease HI. Figure 2 shows these sites revealed as DNA
breaks with endonuclease HI. Most comets are scored in the more
severely damaged classes. In an antioxidant supplementation trial
currently in progress, 100 male volunteers have been assayed
in this way. In every case extra sites are revealed by digestion
with endonuclease HI compared with buffer alone. In both HeLa
cell and lymphocyte experiments, increasing the concentration
of enzyme had no effect on the appearance of comets (results
not shown) and we therefore assume that all accessible lesions
are detected. Proteins, which could block access of the
endonuclease to the lesions, are absent from the nucleoids and
while nucleoid DNA is supercoiled, it is known that supercoiling
is not a bar to the enzyme's action on plasmid DNA (4). To make
1733
A.R.Collins et al.

(A

60min
(0 75
• + buffer + enzyme
Omin "5
incubation • incubation
no repair 1 h repair
1 50

m 25
s
B 3 4 0 1

Comet class

Downloaded from https://academic.oup.com/carcin/article/14/9/1733/334111 by OUP-USA Mirror user on 20 May 2024

Fig. 2. Lymphocytes. Accumulated oxidative damage revealed with
endonuclease III. Cells were incubated (in gel) with buffer or endonuclease
in.

Table I. DNA break frequencies

CO H 2 O 2 concn Repair Enzyme Breaks/1012 Da

.2 incubation
o
£ HeLa cells 100 , >1300
O 100, 120
(0 0 50
0)
0 50
10 /ti 70
10^; 160
"55 30 PL. 210
o 30^: 710
100, 370
100, 780
Lymphocytes 0 110
0 670

Comet analysis was calibrated against HeLa cells containing determined

amounts of DNA breakage (7). Class 0 (no detectable tail) corresponds to
40 breaks or less; class 1, to 170 breaks; class 2, to 650; class 3, to 870;
class 4, to 1300 or more per 1012 Da of DNA. The visual scoring was
confirmed by computer analysis of representative images in terms of % total
DNA in the tail [a parameter shown (10) to be linearly related to the
frequency of breaks], using a system developed by Confocal Technologies
Ltd.

From measurement of oxidative damage to deoxycytidine in

0 1 2 3 4 0 1 2 3 4
Fig. 1. HeLa cells. DNA strand breaks visualized by single cell gel
electrophoresis. (A) Cells treated with 100 /*M H 2 O 2 , with or without 1 h
repair incubation. (B) Cells incubated for 1 h after treatment with doses of
H 2 O 2 as shown and incubated (in gel) with buffer or enzyme. One hundred
comets on each slide were scored according to tail intensity into classes
0—4, representing increasing amounts of damage. Error bars represent
standard errors of the mean value for each class from three experiments.
this assay quantitative we have compared the comets with those
produced following known amounts of DNA breakage (7). Table
I gives DNA break frequencies for the comet distributions shown
in Figures 1 and 2. It should be noted that these figures are
estimates; a more definitive calibration (based on lymphocytes
rather than HeLa cells) is in progress.
DNA isolated from human leucocytes (8) it is estimated that tens
of thousands of oxidized pyrimidines are present in the
approximately 1012 Da of DNA in each cell. However, Lindahl
has recently pointed out (9) that such estimates are prone to
serious artefactual errors owing to the likely oxidation of DNA
during isolation with reagents such as phenol/chloroform. Our
procedure minimizes the exposure of DNA to oxidative
conditions, as is confirmed by the absence of detectable damage
in HeLa cells and in cultured lymphoblastoid GM1899 cells put
through the same procedure as in the isolation of lymphocytes
(results not shown). We therefore regard our estimate of several
hundred oxidized pyrimidines per cell as a more realistic one.
The lack of a significant burden of free radical damage in HeLa
cells might indicate that they possess a higher level of antioxidant
defences than lymphocytes. However, HeLa cells are markedly
more susceptible to H2O2-induced DNA breakage than
lymphocytes (unpublished observations). Another possibility is
that HeLa cells are able to repair damage more efficiently. Little
is yet known of the kinetics of base excision repair in either cell
type.

1734
 Oxidative base damage detection

Applications of this modified comet assay include screening

for oxidative base damage in molecular epidemiological studies
or in monitoring effects of irradiation in cancer therapy. The assay
might also be employed in genotoxicity testing, with die inclusion
of enzymes specific for certain kinds of lesion, to characterize
the damage caused by suspect carcinogens. Finally, it will be
possible to investigate excision repair pathways by following
removal of the appropriate lesions from the DNA with this assay.

Acknowledgements
This work was supported by the Scottish Office, Agriculture and Fisheries
Department, and the Ministry of Agriculture, Fisheries and Food. We thank Dr
R.Cunningham for the bacterial strain overproducing endonuclease HI.

Downloaded from https://academic.oup.com/carcin/article/14/9/1733/334111 by OUP-USA Mirror user on 20 May 2024

References
l.Ames.B.N. (1983) Dietary carcinogens and anticarcinogens. Science, 221,
1256-1264.
2.Park,E.-M., Shigenaga.M.K., Degan.P., Korn.T.S., KitzlerJ.W.,
Wehr.C.M., Kolachana.P. and Ames.B.N. (1992) The assay of excised
oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives
from biological fluids with a monoclonal antibody column. Proc. NatlAcad.
Sci. USA, 89, 3375-3379.
3.Doetsch,P.W., Henner.W.D., Cunningham,R.P., Toney.J.H. and
Helland,D.E. (1987) A highly conserved endonuclease activity present in
Escherichia coli, bovine, and human cells recognizes oxidative DNA damage
at sites of pyrimidines. Mol. Cell. Biol., 7, 26-32.
4.Asahara,H., Wistort,P.M., Bank,J.F., Bakerian,R.H. and Cunningham.R.P.
(1989) Purification and characterization of Escherichia coli endonuclease III
from the cloned nth gene. Biochemistry, 28, 4444-4449.
5.Singh,N.P., McCoy,M.T., Tice.R.R. and Schneider,E.L. (1988) A simple
technique for quantitation of low levels of DNA damage in individual cells.
Exp. Cell Res., 175, 184-191.
6. Cook.P.R. and Brazell,I.A. (1976) Conformational constraints in nuclear DNA.
J. Cell Sci., 22, 287-302.
7.Gedik,C.M., Ewen,S.W.B. and CoUins.A.R. (1992) Single-cell gel
electrophoresis applied to the analysis of UV-C damage and its repair in human
cells. Int. J. Radiat. Biol, 62, 313-320.
8. Wagner,J.R., Hu,C.-C. and Ames,B.N. (1992) Endogenous oxidative damage
of deoxycytidine in DNA. Proc. NatlAcad. Sci. USA, 89, 3380-3384.
9. Lindahl,T. (1993) Instability and decay of the primary structure of DNA.
Nature, 362, 709-715.
10.Olive,P.L., BanathJ.P. and Durand,R.E. (1990) Heterogeneity in radiation-
induced DNA damage and repair in tumor and normal cells measured using
the 'comet' assay. Radiat. Res., 122, 86-94.

Received on May 27, 1993; revised on July 19, 1993; accepted on July 20, 1993

1735