430s Blochemical Society Transactions ( 1 995) 23
Phenol isolation of DNA yields higher levels of
50-I
&oxodeoxyguanosine compared to p r o m E isolation.
MONICA T. FINNEGAN, KARL E. HERBERT, MARK D.
EVANS and JOSEPH LUNEC.
Division of Chemical Pathology. Centre for Mechanisms of
Human Toxicity, University of Leicester, Hodgkin Building,
PO Box 138, Lancaster Road, Leicester, LEI 9HN.
Pronase E
Oxidative damage is thought to play a role in the aetiology of
aging and a number of diseases including cancer, chronic
inflammation, ischemia, degenerative arterial and autoimmune
diseases [I]. 8-oxodeoxyguanosine (8-oxodG), an oxidative
DNA adduct. has gained much popularity as a biomarker of
damage to DNA. HPLC combined with electrochemical
detection provides a selective and sensitive method of
measuring 8-oxodG [2]. It has been reported that phenol
extraction of DNA may cause sensitization of DNA to
subsquent oxidative damage and result in higher 8-oxodG
levels [3]. In this study we compare phenol isolation with
pronase. E isolation of DNA [4].
Human peripheral blood mononuclear cells (PBMC) were
isolated from whole blood using Histopaque 1077 and treated
using two different exposure protocols to induce oxidative
stress. First, the mononuclear cells were treated on ice, to
reduce DNA repair, with H202 (400vM) for 15min. Second,
PBMC were exposed on ice to a Vickrad %
' o source, and
treated with gamma irradiation at a rate of 0.48Gylsec (OGy,
2OGy, 20OGy). Mononuclear cell DNA was extracted using
the pronase E method of Kendall et al, [4] or the phenol
method based on a procedure described by Winyard et al [S].
DNA was enzymatically digested to the deoxynucleoside level
[6]; micrococcal nuclease (0.14units/4pg DNA) was used in
place of N.crussa endonuclease. The method for reversedphase HPLC of the DNA deoxynucleosides was modified from
the procedure of Floyd et a1 [7].
The levels of DNA extracted using either method were not
significantly different (data not shown). The results in Figure 1
show that lower levels of 8-oxodG were observed in H202
treated and untreated PBMC and naked calf thymus DNA
when DNA was isolated using the pronase E method
compared to the phenol method. In the experiments where the
cells were treated with hydrogen peroxide in the presence of
foetal calf serum (FCS) a response to treatment with H202
was observed (p< 0.05) in that 8-oxodG levels were increased
but only for phenol extraction of DNA (Figure 1). Similarly
for y-irradiated cells the levels of 8-oxodG observed using
pronase E extraction were lower than the levels observed
using the phenol method (p< 0.05) (Figure 2). A dose
response was suggested using phenol isolation from yirradiated PBMC 0-2OOGy (Figure 2). Higher levels of 8oxodG were observed on extraction of naked calf thymus
DNA using the phenol method (p< 0.05) but no increase in 8oxodG proportional to increased irradiation was seen for
phenol extracts of DNA.
The importance of establishing a reliable method to measure
Abbreviations used: 8-oxodG, 8-oxodeoxyguanosine; HPLC,
high performance liquid chromatography; PBMC, peripheral
blood mononuclear cells; FCS, foetal calf serum; H202,
hydrogen peroxide; Gy, Gray; N.crassa,neurospora crassa.
Phenol
Figurc 1: Tlus graph show the lewls of
8-0xodG in PBMC or naked DNA treated
wthJOOph4 hvlrogcn pcroude tor ISmin
on ice tsEh d u e represents the mean
+SEM for three scp.ratc experiments
20,
(DNA) (PHMC)
(DNA) (PHMC)
Pronase E
Phenol
Figure 2: y-trrdatton of PHMC or DNA.
dtrect comparisonhetwen pronase I: and
phenol tsolatlon EBch value represents
the mean +SEM for three seperate
exprnments
8-oxodG at ultra-low levels is clear. We are currently using
selective and sensitive antibodies to specific DNA damage
products and immunocytochemistry to detect oxidative
products of DNA damage in cells. This would appear to be a
promising approach, because these methods limit the potential
for artefactual production of DNA damage caused by
manipulations inherent in other methods. In conclusion,
phenol isolation of DNA appeared to cause artificially high
levels of the oxidative DNA adduct 8-oxodG. Lower levels of
8-oxodG were detected using pronase E extraction of DNA
from PBMC or naked calf thymus DNA solutions, but such
levels were very often at or near the limit of detection of the
assay.
The authors gratefully acknowledge the financial support of
the Arthritis and Rheumatism Council and the Medical
Research Council.
1. Richter, C., Park, J-W. & Ames, B.N. (1088) Proc. Nail.
Acad. Sci. USA. 85,6465-6467.
2. Kasai, H., Crain, P.F., Kuchino, Y., Nishimura, S.,
Ootsuyama, A. & Tanaoka, H. (1986) Carcinogenesis, 7,
1849- 1851.
3. Claycamp, H.G. (1992) Carcinogenesis, 13. 1289- 1292.
4. Kendall, T.L., Byerley, D.J. & Dean, R. (1991) Anal.
Biochem.l95,74-76.
5. Winyard, P.G.. Perrett, D., Blake, D.R., Harris, G . &
Chipman, J.K. (1990) Anal. Proc. 27.224-227.
6. Faux, S.P.. Francis, J.E., Smith. A.G. & Chipman, J.K.
(1992) Carcinogenesis. 13.2.247-250,
7. Floyd, R.A., Watson, J.J.. Wong, P.K., Atmiller, D.H. &
Rickard R.C. (1986) Free Rad. Res. Comms. 1. 163-172.