(Received for publication, November 2, 1994; and in revised form, February 4, 1995)
From the
A purification scheme for mitochondrial DNA (mtDNA) was designed
which maximized the yield of all forms of the DNA while minimizing
damage to the DNA during its isolation. Treatment of intact
mitochondria with DNase I removed nuclear DNA and the avoidance of
phenol and the isolation by CsCl density gradients in the absence of
ethidium bromide and subsequent detection by Southern Hydridization
dot-blots minimized DNA damage. Four different mtDNA forms free of
apparent nuclear DNA were obtained: closed circular (I), open circular
(II), linear (III), and a large multimer complex (C) which were
characterized by agarose gel electrophoresis and electron microscopy.
Using this procedure, mtDNA was obtained from both whole cells or
intact mitochondria treated with HO
.
Significant fragmentation was observed after treatment at 37 °C,
but not at 0 °C, and more damage was observed when treating whole
cells than isolated mitochondria. Very low levels of
8-hydroxydeoxyguanosine were observed in all cases. However, at doses
of H
O
which were just lethal, neither increased
DNA damage nor inactivation of cytochrome c oxidase was
observed.
Mitochondrial DNA (mtDNA) ()has been discussed as a
target of oxygen radicals, and recently a number of age-related
diseases have been associated with mutations in the mtDNA (Miquel,
1992), suggesting that somatic mtDNA mutations and deletions could be
an important component of aging and degenerative diseases (Cortopassi et al., 1992; Miquel, 1992; Wallace, 1992).
Mitochondria consume more than 90% of the cell's oxygen, and the
respiratory chain in mitochondria is a major source of oxygen radicals
(Chance et al., 1979).
The mitochondrial genome in
animals has a high rate of mutation possibly because mtDNA is not
complexed to histones and hence may be more susceptible to attack by
active oxygen than chromosomal DNA (Richer, 1992; Brown et
al., 1979; Clayton, 1984). Therefore, to study
mitochondrial DNA damage, a method for the purification of mtDNA from
cells or tissues without the introduction of such damage during the
isolation is needed. Most procedures for the isolation of mtDNA take
advantage of the unique physical properties of the closed circular form
of mtDNA. However, such procedures result in the isolation of only a
subset (less than 50%) of the mtDNA; most of the DNA is actually in a
nicked or more complex state. Indeed these latter molecules may contain
the majority of the damages. Moreover, the use of ethidium bromide or
phenol in commonly used procedures for mtDNA isolation may introduce
oxidative damages. We thus undertook to devise a procedure which
recovers all forms of mtDNA in good yield and which avoids the use of
potential free radical-generating chemicals. We then used the procedure
to test the effect on mtDNA of treatment of HeLa cells or purified
mitochondria from these cells with HO
.
H
O
, thought to be a source of oxidative damage
to cellular membranes, proteins, and DNA, is by itself relatively
inactive with these cellular constituents. However, in the presence of
reduced transition metals such as Fe
and
Cu
it generates
OH and related species that are
highly reactive with organic compounds (Fenton, 1894; Halliwell and
Gutteridge, 1990). Such damaging species can also be generated with
Fe
and NADH in lieu of Fe
(Imlay
and Linn, 1988).
The pellet (mitochondrial
fraction) was suspended in 3 ml of STE buffer (100 mM NaCl, 10
mM EDTA, 50 mM Tris-HCl, pH 7.4) and solubilized at
37 °C for 10 min by the addition of 330 µl of 10% SDS. CsCl
(0.75 g) was added to the solubilized suspension, and the mixture was
chilled to 0 °C for 10 min after dissolving the CsCl completely.
After centrifugation at 12,000 g for 10 min, an
additional 2.8 g of CsCl was added, and the volume was adjusted to 4.5
ml with STE to give a final CsCl concentration of 4.5 M. After
centrifugation at 45,000 revolutions/min in a Beckman SW 50.1 rotor for
24 h at 20 °C, the material in the tube was divided into
18-20 equal fractions by drop collection, and the fractions were
monitored for mtDNA by dot-blot Southern hybridization. mtDNA fractions
were dialyzed extensively against STE.
For cases where ethidium bromide was used, 500 µg of ethidium bromide was added to the CsCl sample solution for ultracentrifugation. After centrifugation, the bands which were visualized under UV light (366 nm) were collected by aspirating with a syringe. After removing ethidium bromide by three extractions with n-butanol, the DNA fraction was dialyzed against STE.
For cases where phenol/chloroform extraction was used, the mitochondrial fraction was suspended in 3 ml of STE and incubated with 330 µl of 10% SDS and 400 µl of proteinase K (10 mg/ml) at 50 °C for 3 h. After incubation, the digest was extracted with 3 ml of phenol saturated with TE (1 mM EDTA, 10 mM Tris-HCl, pH 7.4) by shaking gently for 30 min. The extraction was repeated twice and then once with 3 ml of phenol/chloroform (1:1). The DNA in the aqueous phase was precipitated by the addition of 370 µl of 3 M sodium acetate and 8 ml of 99.5% ethanol followed by incubation for 30 min at -80 °C. The precipitate was collected by centrifugation, washed with 3 ml of 70% ethanol, dried, and resuspended in STE.
mtDNA fractions were treated with 3 µg
of RNase A (DNase free) at 37 °C for 2 h, and then the digest was
loaded onto a Sephadex G-150 column (0.7 18 cm) previously
equilibrated with STE to remove the digested RNA. Elution was carried
out at a flow rate of 60 µl/min, and mtDNA was monitored by
dot-blot Southern hybridization. Purified mtDNA was stored at -20
°C.
For dot-blot
Southern hybridization of samples from CsCl density gradient
centrifugations and Sephadex G-150 gel filtration, 2 µl of the
fractionated sample solution was heated at 95 °C for 5 min, chilled
in ice-water, and spotted onto a Hybond N membrane
which was prewetted with 5
SSPE (50 mM sodium
phosphate, 5 mM EDTA, and 0.9 M NaCl, pH 7.7)
according to the manual accompanying the Hybond N
membranes. The membrane was then dried, placed onto two
thicknesses of Whatman 3MM filters soaked in denaturing solution (1.5 M NaCl, 0.5 M NaOH), left for 5 min, and then
transferred to filter papers soaked in neutralizing solution (1.5 M NaCl, 1 mM EDTA, 0.5 M Tris-HCl, pH 7.2), and
dried under air. Next, the membrane was placed on a pad of filter paper
(two to three pieces thick) soaked in 0.4 M NaOH for 30 min to
be fixed, and then rinsed briefly by immersion in 5
SSPE for
less than 1 min and dried under air.
After transfers were complete,
the membranes were placed in plastic bags filled with hybridization
buffer (5 SSPE, 5
Denhardt's solution containing
0.1% Ficoll 400, 0.1% polyvinylpyrolidone, and 0.1% bovine serum
albumin, 0.5% SDS) supplemented 20 µg/ml of denatured salmon sperm
DNA and prehybridized in a shaking water bath at 65 °C for at least
60 min. After prehybridization, the membranes were hybridized in
hybridization buffer at 65 °C for 15 h with a
P-labeled mtDNA probe which had been denatured by boiling
for 5 min. After hybridization, the membrane was washed twice with 2
SSPE and 0.1% SDS at room temperature, and finally with SSPE
and 0.1% SDS at 65 °C for 15 min. The membrane was subjected to
autoradiography with Kodak x-ray film and an intensifying screen at
-80 °C.
Figure 1: Summary of procedures for purification of mtDNA from HeLa cells. Experimental details are under ``Experimental Procedures.''
When the CsCl-ethidium bromide gradients containing mitochondria lysed with SDS were visualized under UV illumination (366 nm), two bands were observed, a heavy one, band a, and a lighter one, band b. As described below, band a contained covalently closed, circular mtDNA which had little contamination with nuclear DNA. Band b contained nuclear DNA and relaxed circular, linear, and complex forms of mtDNA. Band a had approximately 5-7% the amount of DNA of band b in the case of DNA prepared without DNase I treatment of the mitochondria. By treating the mitochondrial fraction with DNase I before the solubilization with SDS, however, most contaminating nuclear DNA in band b was removed and band b had only nicked or complex forms of mtDNA (see Fig. 2, lane 6 below).
Figure 2: Agarose gel electrophoresis of mtDNA obtained from HeLa cells by various purification methods. DNase I, Proteinase K/phenol, EtBr, and Dot Hyb. refer to the procedures for purification and detection depicted in Fig. 1. Bands a and b indicate the fractions obtained after CsCl gradient centrifugation with EtBr present as described in the text. The left panel shows the DNA displayed by EtBr and UV light. The right panel shows the DNA of the same gel displayed by Southern hybridization with a mtDNA probe. DNA amounts present in lanes 1, 4, and 7 were approximately 5 µg. The DNA present in the other tracks varied from roughly 0.2 to 0.5 µg. However, due to uncertainties in these concentrations (since they were estimated by UV absorption), the gels should be taken to assess the relative amounts of each form and contaminating nuclear DNA, not yields of mtDNA.
Figure 3: Characterization of the forms of mtDNA by restriction enzyme digestion. A, the restriction enzyme map of human mtDNA. (-) indicates the mtDNA probe utilized for Southern blots. Restriction site locations are BamHI 14 and 259; PstI, 6,915 and 9,025; HindIII, 6,023, 11,680, and 12,570; XbaI, 1,193, 2,953, 7,440, 8,286, and 10,256. B, mtDNA restriction fragments as seen after AGE and EtBr staining. mtDNA (0.2 µg) was digested with 10 units of each restriction enzymes in 10 µl of appropriate buffer at 37 °C for 60 min. C, the DNA of the same gel visualized by Southern hybridization to the probe shown in A.
Without DNase I treatment of intact mitochondria, large amounts of contaminating nuclear DNA are present (lanes 1, 4, and 7). This contamination could be removed by equilibrium centrifugation in CsCl-EtBr (lane 3), but then Forms I and III DNA are lost (lane 3) and some mitochondrial DNA appears to band with the nuclear DNA in band b (lane 4). The loss of Forms I and III may have been due to the presence of EtBr during visualization, since Forms I and III were recovered if bands a and b were located by Southern hybridization (lanes 5 and 6). Since bands a and b both appeared to have mitochondrial DNA and since there appeared to be a risk of damage to DNA by ethidium bromide plus light, the safest procedure appeared to be to eliminate the EtBr from the CsCl gradients and to monitor the presence of the mtDNA with Southern blots (lane 8).
Note that treatment of solubilized, DNase I-treated mitochondria with proteinase K and then phenol gave reasonable preparations of mitochondrial DNA (lane 2). This procedure might be practical when maximum yields of total mtDNA in all forms is desired and the problem of the potential for some oxidative damage due to contaminants in the phenol is not important.
The mtDNA was also examined by electronmicroscopy. Fig. 4shows a sample micrograph. The Form C molecule would be exactly 2 unit lengths if the length of the linear portion is doubled. Many such molecules have been observed with varying ratios of size of the circular compared to the linear portions, although the two circular portions on a given molecule are equal in size. These could be recombination intermediates or unresolved replication products which had collapsed into a four-stranded structure. The distributions of the four forms of mtDNA by observation in the electron microscope correspond well with the relative amounts of each mtDNA band on AGE of the same preparation (data not shown).
Figure 4: Electron microscope observation of purified mtDNA. mtDNA purified as used for the sample of lane 8 of Fig. 2was observed in the electron microscope. The bar indicates 500 nm.
Figure 5:
Depletion of exogenous
HO
from the culture medium of HeLa cells. Three
mM H
O
was added to a suspension of
HeLa cells (6
10
cells/ml) in PBS, three times, as
indicated, at 37 °C (
-
) or at 0 °C (
-
);
or once at 0 min at 37 °C (
) or
at 0 °C (
).
H
O
concentrations were determined as described
under ``Experimental Procedures.'' (
) indicates
H
O
added to PBS without cells and held at 37
°C.
If the medium
were made 3 mM HO
at 0 °C,
however, the H
O
reduction was slower than that
at 37 °C, becoming roughly 0.9 mM at 6 min and 20
µM 30 min after the addition (Fig. 5, closed
circles). If H
O
were replenished at 6 and
15 min (at 0 °C), the total consumption was 8.6 mM. Hence
there was no loss of efficiency in H
O
degradation during the 30 min at 0 °C.
In parallel studies
the effect of the exposure to HO
on viability
was examined by monitoring colony-forming ability (Table 1).
Cells treated with 3 mM H
O
at 37
°C lost colony-forming ability 15 min after treatment. However, if
treated three times with 3 mM H
O
at 0
°C (at 0, 6, and 15 min) cells had about 40% survival (as judged by
colony-forming ability) 30 min after the first addition of
H
O
. Hence, loss of colony-forming ability is
quite temperature-dependent: even though H
O
is
more rapidly removed at 37 °C, toxicity is more apparent at that
temperature.
In these experiments, colony-forming ability, the most
quantitative method for determining killing, was utilized. However, 7
days are required to monitor colony formation. Microscopic examination
of the cells shortly after the exposure to HO
and trypan blue exclusion observations suggested that many cells,
though committed to die during the 7-day period and unable to divide,
were not immediately killed. This behavior is similar to that observed
with E. coli exposed to H
O
at these
doses. In that case, limited filamentous growth occurred following
treatment, suggesting that the cells suffered high levels of DNA damage
which ultimately resulted in their death (Imlay and Linn, 1987).
Figure 6:
Agarose gel electrophoresis of mtDNA from
HeLa cells treated with HO
. Cells were exposed
to 3 mM H
O
as in Fig. 5at 0
min only (+) or at 0, 6, and 15 min (+++). The
mtDNA was isolated, subjected to AGE, and visualized by staining with
EtBr (left) and Southern hybridization (right).
To investigate a base modification of mtDNA after
treatment of HeLa cells with HO
, 8-OH-dG was
determined in the mtDNA, by enzymatic hydrolysis followed by HPLC and
electrochemical detection. The 8-OH-dG/dG ratios in mtDNA from HeLa
cells treated with H
O
under various conditions
are given in Table 2. The level of 8-OH-dG was quite low and was
not increased significantly by exposure to H
O
,
even when treated three times with 3 mM H
O
at 37 °C. Hence, even under conditions for which no viable
cells remain (Table 1) and the DNA is extremely fragmented (Fig. 6), no significant increase in the dG analog was observed.
As an independent assessment of the disruption of mitochondria after
treatment of whole cells with HO
, mitochondrial
cytochrome oxidase activity was examined (Table 3). In all cases
the cytochrome c oxidase activity was not significantly
changed. Hence, the mitochondria seem to have been essentially
biologically functional.
Figure 7:
Agarose gel electrophoresis of mtDNA from
isolated mitochondria treated with HO
. Isolated
intact mitochondria were exposed to 3 mM H
O
at 0 min only (+) or at 0, 6, and 15 min
(+++) at a concentration of 44 µg of mitochondrial
protein/ml in 100 ml of PBS. The mtDNA was then isolated and displayed
as in Fig. 6. Lane 5 had less sample applied, but this
fact does reflect a lower yield of DNA from the
mitochondria.
Finally, the effect on
mtDNA integrity of exposing isolated mitochondria to
HO
plus iron and/or NADH was examined (Fig. 8). DNA from mitochondria treated three times with 3
mM H
O
in the presence of NADH and
ferrous iron showed severe degradation at 37 and 0 °C, with
somewhat more at 37 °C. Fe
/NADH was less
effective, while H
O
alone did not induce damage
as expected from the experiment in Fig. 8. Evidently,
H
O
treatment of mitochondria effectively
degrades mtDNA damages only if exogenous iron and a reductant is
provided.
Figure 8:
Agarose gel electrophoresis of mtDNA from
isolated mitochondria treated with HO
and
reduced iron. Isolated mitochondria were exposed to 3 mM H
O
at 0 min only (+) or at 0, 6, and
15 min (+++) with 1.5 mM FeSO
, or
1.5 mM FeCl
and 3 mM NADH as indicated,
as described in Fig. 7. After treatment, the mtDNA was isolated
and displayed as in Fig. 6with 0.5 µg of mtDNA in each lane
except for lane 2 which contained 0.2
µg.
Several methods for the isolation and purification of mtDNA
are in general use (Palva and Palva, 1985; Bogenhagen and Clayton,
1974; Myers et al., 1988). In order to maximize the
yield of all forms of mtDNA and to minimize damage to the DNA during
the process, we altered these procedures. Hypotonic buffer together
with the D-mannitol as a scavenger of oxygen radicals were
used with Dounce homogenization to open the cells. DNase I treatment of
the intact mitochondria prior to their isolation by sucrose gradient
centrifugation was used to remove all detectable contaminating nuclear
DNA. Whereas phenol-chloroform extraction of crude solubilized
mitochondria produced mtDNA which appeared similar by AGE to that
obtained by CsCl equilibrium centrifugation without EtBr, it has been
reported that phenol-chloroform extraction of DNA and UV irradiation
increases the frequency of 8-OH-dG in DNA 20- and 60-fold, respectively
(Claycamp, 1992; Fischer-Nielson et al., 1992).
Therefore phenol-chloroform extraction and/or exposure to UV light
would be unsuitable for the investigation of DNA base modification
caused by oxidative stress. Likewise, dot-blot Southern hybridization
was used to detect mtDNA after CsCl density gradient centrifugation in
order to avoid mtDNA damage induced by the UV radiation necessary to
detect the ethidium bromideDNA complexes. Audic and Giacomoni
(1993) have reported that UV irradiation introduces DNA nicking
especially in the presence of iron and oxygen, and we find that EtBr
also enhances this nicking.
mtDNA prepared from HeLa cells by the method described in this paper contains four different forms. Proteinase K treatment of the mtDNA preparation did not change the relative distribution of among mtDNA of these forms, but restriction enzyme digestion converted each of them totally to unit length, linear molecules. Therefore, Forms I-III and C each appear to consist of such unit length molecules or integral multiples of such. In particular, there is no evidence that Form C is either bound to protein or aggregated due to an association with proteins.
Radloff et al.(1967) observed complex mtDNA in HeLa cells during the first serious characterization of mtDNA structure. However, later characterization in Mouse L cells suggested that these forms contained intermediates in all stages of replication (Robberson et al., 1972), i.e. of different sizes. However, a preponderance of dimer molecules was noted in leukocytes from patients with chronic granulocytic leukemia, but not normal leukocytes (Radloff et al., 1967). It would be worthwhile, with the current availability of the Southern hybridization technique and restriction enzymes, to reinvestigate quantitatively and to compare the structures of these complex forms in normal blood, leukemic blood, and cultured cell lines in order to resolve these apparently conflicting observations.
Hydrogen peroxide added to the medium of HeLa cells to
3 mM disappeared within 5 min at 37 °C. At 0 °C
virtually all of the HO
was gone in 30 min.
Temperature-dependent enzymes such as catalase in the peroxisome and
GSH peroxidase in the cytoplasm and mitochondria are presumably
responsible for this depletion. What was unexpected, however, was that
even at doses of H
O
which were lethal, DNA
damage as measured by strand breaks or 8-OH-dG induction were minimal.
Indeed, the levels of 8-OH-dG were considerably lower than those
observed by Richter et al.(1988) to be present in rat liver
mtDNA without exogenous oxidative stress. Possibly the damage was
introduced to the rat liver mtDNA during its isolation, or possibly
there is a good deal more 8-OH-dG in rat liver mtDNA, than in HeLa
mtDNA. However, we would not expect that if a repair system for 8-OH-dG
were operative (Tchou and Grollman, 1993), so many as several such
residues/mitochondrial genome (Richter et al., 1988)
would normally be present.
Cytochrome c oxidase is present
in the inner membrane of mitochondria. It has reported that cytochrome c oxidase activity dropped by more than 40% when exposed to
600 nmol of OH/mg of inner mitochondrial membrane protein during
-radiation (Zhang et al., 1990). In the case of
exposure of HeLa cells to H
O
, the activity of
cytochrome c oxidase was not changed significantly. This
result suggests that oxidative stress by the exogenous
H
O
was not strong enough to damage cytochrome c oxidase even at lethal doses.
mtDNA was not fragmented by
the treatment of isolated mitochondria three times with 3 mM HO
at 37 °C, indicating that some
intracellular, extramitochondrial factor(s) are needed to induce mtDNA
damage. While these factors are unknown, Fe
or
Cu
ions are required for Fenton-type reactions with
H
O
. Indeed, H
O
induced
mtDNA fragmentation even at 0 °C when Fe
or
Fe
and NADH were also included. Apparently, a
cellular environment for the mitochondria must be present for reduced
transition metals to be available in the mitochondria. The increased
damage at 37 °C of mtDNA in whole cells could reflect a requirement
for metabolism to generate this reduction, or it could mean that after
H
O
treatment, mitochondrial DNases (Beaufay et al., 1959; Linn 1994; Curtis and Smellie, 1966;
Low et al., 1987) or topoisomerase (Lin and Castora,
1991) are disrupted from their controlled state and act to damage
mtDNA.
In conclusion, it would appear that damage to mtDNA is not a
primary factor in cell toxicity by HO
. However,
these studies were done in cell culture with an immortal polyploid cell
line, HeLa. They now should be repeated with whole animal tissues
before extrapolations from cultured cell- to tissue mitochondria can be
drawn.