©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of All Forms of HeLa Cell Mitochondrial DNA and Assessment of Damage to It Caused by Hydrogen Peroxide Treatment of Mitochondria or Cells (*)

(Received for publication, November 2, 1994; and in revised form, February 4, 1995)

Yoshihiro Higuchi (§) Stuart Linn (¶)

From the Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, California 94720-3202

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 H(2)O(2). 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(2)O(2) which were just lethal, neither increased DNA damage nor inactivation of cytochrome c oxidase was observed.


INTRODUCTION

Mitochondrial DNA (mtDNA) (^1)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 H(2)O(2). H(2)O(2), 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 bulletOH 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).


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's medium and fetal calf serum were obtained from Life Technologies, Inc. DNase I, ABTS, horseradish peroxidase, phenylmethylsulfonyl fluoride, and NADH were obtained from Sigma, snake venom phosphodiesterase and Escherichia coli alkaline phosphatase were from Worthington, proteinase K, DNase-free RNase A, and P1-nuclease were from Boehringer Mannheim. Reagents for PCR were from Perkin Elmer and Pharmacia LKB Biotechnol.

Purification of mtDNA

HeLa cells (10^9 cells) were grown in spinner culture in 4 liters of Joklik's minimum essential medium modified for suspension culture (JRH BioSciences) containing 4.5 g of glucose/liter supplemented with 5% newborn bovine serum. The cells were harvested by centrifugation at 4,000 revolutions/min in a Sorvall H-6000 rotor for 10 min, washed twice with PBS by centrifugation at 2600 times g for 10 min, resuspended in 20 ml of MgRSB buffer (10 mM NaCl, 1.5 mM MgCl(2), 10 mM Tris-HCl, pH 7.5), allowed to stand at 4 °C for 10 min, and then disrupted with a Dounce glass homogenizer, type 1A, with 15-20 strokes. Disruption was verified by microscopic examination. Twenty-six ml of 2.5 times MSB buffer (0.525 MD-mannitol, 175 mM sucrose, 12.5 mM EDTA, 12.5 mM Tris-HCl, pH 7.5) was added, and diluted homogenate was centrifuged at 1,000 times g for 10 min to remove cell debris. The supernatant fluid was further centrifuged at 18,000 times g for 20 min in a GSA rotor, and the pellet was suspended in 5 ml of EDTA-free MSB containing 10 mM MgCl(2) and 0.5 mM phenylmethylsulfonyl fluoride. The suspension was digested with 6,000 Kunitz units of DNase I (Sigma) at 37 °C for 30 min. After digestion, the suspension was washed three times in 10 volumes of MSB by centrifugation at 13,000 times g for 20 min. The washed pellet was resuspended in 4 ml of MSB and applied on top of sucrose layers consisting of 15 ml of 1.5 M sucrose (lower layer) and 15 ml of 1 M sucrose (upper layer), both containing 10 mM Tris-HCl, pH 7.4, 5 mM EDTA. After centrifugation at 25,000 revolutions/min for 30 min in a Beckman SW28 rotor at 4 °C, the interface fraction was collected and washed twice with 4 volumes of MSB by centrifugation at 18,000 times g for 20 min.

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 times 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 times 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.

Treatment of HeLa Cells or Mitochondria with H(2)O(2)

HeLa cells (10^9 cells) were suspended in 167 ml of PBS and exposed to 3 mM H(2)O(2) once at 0 min or three times at 0, 6, 15 min. After a total incubation of 30 min, the cells were washed twice with PBS by centrifugation and mtDNA isolated. To treat mitochondria, the organelles prepared from 10^9 HeLa cells were suspended in 167 ml of PBS and treated with H(2)O(2) under the same conditions as whole cells. mtDNA was isolated immediately after treatment.

Agarose Gel Electrophoresis

mtDNA (0.2-0.5 µg) was dissolved in a mixture of 9 µl of TE and 1 µl of gel loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, 30% (v/v) glycerol), and then loaded into a well of a 0.5% agarose gel in TBE buffer (89 mM Tris boric acid, 2 mM EDTA, pH 8.0). Electrophoresis was performed in TBE for 50 min at 80 V. After electrophoresis, the gel was stained with ethidium bromide (0.5 µg/ml in TBE) and photographed. A HindIII digest of a DNA or a 1-kb DNA ladder (Life Technologies, Inc.) were used as size markers.

Southern Hybridization

After electrophoresis, agarose gels were placed in an appropriate volume of 0.25 M HCl until the dye changed color and then left for an additional 10 min. After washing with distilled water, the gel was placed on a wick made from two sheets of Whatman 3MM chromatographic paper and solvated with alkali transfer buffer (0.4 M NaOH). Hybond N nylon membrane (Amersham Corp.) was cut to the exact size of the gel and placed on top of it. Two sheets of 3MM paper were also cut to size, wetted with alkaline transfer buffer, and placed on top of the membrane together with three sheets of dried 3MM paper.

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 times 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 times 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 times SSPE, 5 times 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 times 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.

PCR Product

The nucleotide sequences of the oligonucleotides for PCR primers were Primer A, L11778m AACTACGAACGTATTCACAG(C+T)CG; Primer B, H12625m GAATTCTATGATGGATCA(G+T)GT, where L and H, correspond to the light and heavy strands, respectively, of mitochondrial DNA (m), and the accompanying number reflects the position of the 3`-end according to the numbering system for human mtDNA (Anderson et al., 1981). The 847-bp probe (nucleotides 12625-11778) was amplified from 0.1 ng of human mtDNA purified from HeLa cells with a GeneAmp amplification reagent (Perkin-Elmer) together with the four nucleotides (0.2 mM each) in a DNA thermal cycler (Perkin-Elmer) using the following cycle profile: denature at 93 °C for 1 min, anneal at 55 °C for 1 min, extension at 72 °C for 1 min, for a total of 30 cycles. The mtDNA probe was labeled with 30 µCi of [alpha-P]dCTP (3000 Ci/mmol) during the PCR reaction. The labeled DNA fragment was purified with a Quick Spin column (Boehringer Mannheim) packed with Sephadex G-50.

Electron Microscopic Observation of mtDNA

The DNA concentration was adjusted to 2 µg/ml with 5 mM HEPES KOH and 10 mM MgCl(2), pH. 7.5, and cytochrome c was added to the final concentration of 10 µg/ml, and samples were spread by the aqueous drop method of Thresher and Griffith (1992). A 20-µl sample was loaded onto a 400-mesh copper-tabbed grid and allowed to sit for 1 min. Two µl of 10 times spermidine buffer (25 mM spermidine, 750 mM NaCl, 500 mM KCl, 100 mM Tris-HCl, pH 7.9, 10 mM MgCl(2), and 2.5 mM CaCl(2)) was added to the sample and then after 30 s, 20 µl of 1% uranyl acetate was added and the grid was put immediately into distilled water for 3 min. The sample was dehydrated stepwise with 25, 50, 75, and 100% ethanol for 5 min each and placed onto filter paper to dry. The DNA was then observed with a JEOL 1200 EXII transmission type electron microscope.

Enzyme Digestion of mtDNA for Detection of Base Damages

Five to 10 µg of mtDNA dissolved in 150 µl of 10 mM Tris-HCl buffer, pH 7.5, containing 10 mM MgCl(2) and 0.1 mM CaCl(2) was digested with 3 units of DNase I at 37 °C for 30 min, then with 0.9 unit of P1-nuclease for 60 min after adding 4.8 µl of 1 M sodium acetate to the digestion solution. Finally, digestion was completed with a mixture of 0.5 unit of snake venom phosphodiesterase and 1.45 units of bacterial alkaline phosphatase for 30 min after adjusting the digestion solution to pH 8.0 with 1 M Trizma (Tris base). The digested mtDNA solution was stored at -20 °C until use.

HPLC Analysis

8-OH-dG analysis by HPLC was carried out according to Shigenaga et al.(1990) with a Water Associates model 625 HPLC solvent delivery system. Separations were performed with a linear gradient of 2.5-6.25% methanol in 50 mM potassium phosphate, pH 5.5, over 20 min and a Supelco (Bellefonte, PA) LC-18DB Supelcosil 3 µm column (4.6 mm times 15 cm) equipped with a LC-18DB precolumn cartridge assembly at a flow rate of 1.0 ml/min with a back pressure of approximately 2400 pounds/square inch. Electrochemical detection of 8-OH-dG utilized an ESA (Bedford, MA) model 5100 Coulochem detector equipped with a 5011 analytical cell with potentials of electrodes 1 and 2 adjusted to 0.1 and 0.4, respectively. UV detection at 260 nm utilized a Kratos (Westwood, NJ) UV detector.

Determination of H(2)O(2)

Quantitation of H(2)O(2) was by the ABTS-horseradish peroxidase method described by Putter and Becker(1983). One-hundred µl of sample solution was added to 900 µl of ABTS-horseradish peroxidase reaction mixture (2.5 mM ABTS, 0.2 unit of horseradish peroxidase, 0.1 M sodium acetate, pH 4.7). The mixture was allowed to stand at 37 °C for 20 min, and then the absorbance of the mixture was measured at 420 nm.

Cytotoxic Assay

Colony forming ability was used as a cytotoxic assay. HeLa cells (3.6 times 10^6 cells) were suspended in 600 µl of PBS and then treated with H(2)O(2) as described. After treatment, 500 cells from each sample were plated onto a dish (55 cm^2) in 10 ml of Dulbecco's modified Eagle's medium supplemented with 4.5 g glucose/liter and 10% fetal calf serum and cultured at 37 °C in 5% CO(2) in humidified air for 7 days. Colonies of more than 50 cells were fixed with 50% methanol, stained with 0.2% crystal violet, and counted.

Assay for Cytochrome c Oxidase

Mitochondria suspended in 0.1 M Tris-HCl, pH 7.4, were disrupted by freezing and thawing three times as described above. Reduced cytochrome c solution prepared as described by Darly-Usmar et al.(1987) was placed in a cuvette at a concentration of 0.5-10 µM in 0.2 M Tris-HCl, pH 7.4, containing 0.3% Tween 80, and the spectrum was recorded from 500 to 600 nm to ensure that cytochrome c was fully reduced. An appropriate amount of mitochondrial homogenate prepared as above was added to the cuvette, and the rate of decrease in absorbance at 550 nm was monitored.


RESULTS

Preparation and Characterization of Mitochondrial DNA

Purification of mtDNA

In order to study damage accumulated in mtDNA, it was necessary to devise a purification method which would account for all forms of the DNA (not just the covalently closed circular molecules), yet not introduce damage into that DNA. The procedure which was developed and several alternatives for comparison are summarized in Fig. 1. HeLa cells were disrupted in hypotonic buffer by Dounce homogenization and, after removing nuclear and membrane fractions by centrifugation, nuclear DNA was digested away with DNase I. The mitochondrial fraction was then isolated by stepwise sucrose density gradient centrifugation and solubilized with 1% SDS. Three subsequent methods were then compared, particularly for monitoring the presence of mtDNA damage. The first procedure used proteinase K digestion and phenol-chloroform extraction. The second used CsCl density gradient centrifugation with ethidium bromide. The third used CsCl equilibrium centrifugation without ethidium bromide and Southern hybridization dot-blotting to detect the mtDNA fraction after the centrifugation. All methods concluded with RNase A digestion and Sephadex G-150 filtration.


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.



Comparison of mtDNAs Obtained by Various Purification Methods

mtDNAs obtained by the three purification procedures schematized in Fig. 1were subjected to electrophoresis on a polyacrylamide gel and visualized simultaneously with ethidium bromide (Fig. 2, left) and by Southern hybridization with a 847-bp mtDNA probe (bp 11,778-12,625) (Fig. 2, right). Four bands of mitochondrial DNA are resolved: bands I, III, and II migrate to positions expected for closed circular, linear, and open circular unit length mitochondrial genomes (see also discussion of Fig. 3, below). In addition, a large amount complex mitochondrial DNA remains at the top of the gel which appears to be made up of multimers and possible replication and/or recombination intermediates (see below).


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.

Characterization of the Forms of mtDNA by Restriction Enzyme Digestion and Electron Microscopy

mtDNA obtained using DNase I treatment and dot-blot Southern hybridization after CsCl density gradients was analyzed by restriction enzyme digestion. The human mtDNA genome has one BamHI site, two PstI sites, three HindIII sites, and five XbaI sites as shown in Fig. 3A. As shown in Fig. 3, B and C, when the mixture of all of mtDNA forms (I-III and complex, lane 1) is treated with BamHI, only a single, 16.6-kbp band is formed (lane 2), i.e. Forms I, II, and C have each been converted to unit length, linear Form III molecules. In a similar manner, PstI produced two fragments 14.5 and 2.0 kbp), HindIII produced three fragments (10.2, 5.5, and 0.89 kbp), and XbaI produced five fragments (7.5, 4.5, 2.0, 1.8, and 0.8 kbp) (Fig. 3B), and, as shown in Fig. 3C, the corresponding autoradiogram indicated only the presence of the single band which would be expected to hybridize to the probe made up of nucleotides 11,778-12,625 (see Fig. 3A). The lack of other apparent material in any of these digests (neither bands nor random sized pieces) shows that Form C consists largely of multimers, virtually complete, unresolved replication intermediates, or, conceivably, recombination intermediates.

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.



Studies of Oxidative Damage to Mitochondrial DNA

Cytotoxic Effect of H(2)O(2)

The preparation procedure outlined above was applied to the study of damage to mtDNA after treatment of HeLa cells with hydrogen peroxide under various conditions. First, the fate of hydrogen peroxide added to the incubation medium (PBS) of HeLa cells was investigated (Fig. 5). After the addition of hydrogen peroxide to 3 mM to 6 times 10^6 HeLa cells/ml, the H(2)O(2) concentration became undetectable within 5 min at 37 °C. When the 3 mM H(2)O(2) was replenished at 6 and at 15 min after the first addition, the concentration reduced similarly in each case (Fig. 5, open circles).


Figure 5: Depletion of exogenous H(2)O(2) from the culture medium of HeLa cells. Three mM H(2)O(2) was added to a suspension of HeLa cells (6 times 10^6 cells/ml) in PBS, three times, as indicated, at 37 °C (circle-circle) or at 0 °C (bullet-bullet); or once at 0 min at 37 °C (circlebulletbulletbulletbulletcircle) or at 0 °C (bulletbulletbulletbulletbulletbullet). H(2)O(2) concentrations were determined as described under ``Experimental Procedures.'' (box) indicates H(2)O(2) added to PBS without cells and held at 37 °C.



If the medium were made 3 mM H(2)O(2) at 0 °C, however, the H(2)O(2) 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(2)O(2) 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(2)O(2) degradation during the 30 min at 0 °C.

In parallel studies the effect of the exposure to H(2)O(2) on viability was examined by monitoring colony-forming ability (Table 1). Cells treated with 3 mM H(2)O(2) at 37 °C lost colony-forming ability 15 min after treatment. However, if treated three times with 3 mM H(2)O(2) 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(2)O(2). Hence, loss of colony-forming ability is quite temperature-dependent: even though H(2)O(2) 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 H(2)O(2) 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(2)O(2) 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).

mtDNA Damage Induced by Treatment of Whole Cells with Hydrogen Peroxide

mtDNAs were prepared by the procedure used for the sample of Fig. 3, lane 8, after treatment of HeLa cells with H(2)O(2), and then analyzed by AGE (Fig. 6). Cells treated at 0 °C exhibited no apparent mtDNA degradation by this technique. Likewise, cells treated once at 37 °C showed no apparent mtDNA degradation, in spite of a severe loss of viability (Table 1). However, cells treated three times at 37 °C showed severe degradation of the mtDNA. From these results we conclude that within the sensitivity of this assay, strand breaks do not occur with exposures to H(2)O(2) that kill the cells (37 °C, one exposure). Evidently, strand breaks occur only with multiple exposures, perhaps arising after cells have become unable to replicate.


Figure 6: Agarose gel electrophoresis of mtDNA from HeLa cells treated with H(2)O(2). Cells were exposed to 3 mM H(2)O(2) 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 H(2)O(2), 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(2)O(2) 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(2)O(2), even when treated three times with 3 mM H(2)O(2) 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 H(2)O(2), 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.



mtDNA Damage Induced by Hydrogen Peroxide Treatment of Isolated Mitochondria

The effect on mtDNA of H(2)O(2) treatment of isolated mitochondria at 37 and at 0 °C was examined (Fig. 7). No observable fragmentation of the mtDNA was seen even under conditions for which treatment of whole cells gave DNA fragmentation. It appears that mtDNA is considerably more resistant to H(2)O(2) treatment of isolated organelles than of whole cells.


Figure 7: Agarose gel electrophoresis of mtDNA from isolated mitochondria treated with H(2)O(2). Isolated intact mitochondria were exposed to 3 mM H(2)O(2) 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 H(2)O(2) plus iron and/or NADH was examined (Fig. 8). DNA from mitochondria treated three times with 3 mM H(2)O(2) 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(2)O(2) alone did not induce damage as expected from the experiment in Fig. 8. Evidently, H(2)O(2) 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 H(2)O(2) and reduced iron. Isolated mitochondria were exposed to 3 mM H(2)O(2) at 0 min only (+) or at 0, 6, and 15 min (+++) with 1.5 mM FeSO(4), or 1.5 mM FeCl(3) 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.




DISCUSSION

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 bromidebulletDNA 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 H(2)O(2) 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(2)O(2) 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 bulletOH/mg of inner mitochondrial membrane protein during -radiation (Zhang et al., 1990). In the case of exposure of HeLa cells to H(2)O(2), the activity of cytochrome c oxidase was not changed significantly. This result suggests that oxidative stress by the exogenous H(2)O(2) 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 H(2)O(2) 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(2)O(2). Indeed, H(2)O(2) 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(2)O(2) 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 H(2)O(2). 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.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants R29GM19020 and P30ES011896, United States Department of Energy Grant 92ER61458, and the Hokkoku Foundation for Cancer Research, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pharmacology, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920, Japan.

To whom correspondence should be addressed. Tel.: 510-642-7583; Fax: 510-643-5035; LinnS{at}mendel.berkeley.edu.

(^1)
The abbreviations used are: mtDNA, mitochondria DNA; ABTS, 2-2`-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid); AGE, agarose gel electrophoresis; EtBr, ethidium bromide; 8-OH-dG, 8-hydroxy-2`-deoxyguanosine; PBS, phosphate buffered saline; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s); HPLC, high performance liquid chromatography; kbp, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Drs. Mark Shigenaga and Yunghuei Chen for their help with HPLC analyses and EM observations, respectively. We also thank Dr. Yoshinori Kumazawa for providing primer oligonucleotides for PCR analysis and Ann Fisher for her expertise with tissue culture.


REFERENCES

  1. Anderson, S., Bankier, A. T., Barrell, B. G., Bruikn, M. H. L., Couson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R., and Young, I. G. (1981) Nature 290, 457-465 [Medline] [Order article via Infotrieve]
  2. Audic, A., and Giacomoni, P. U. (1993) Photochem. Photobiol. 57, 508-512 [Medline] [Order article via Infotrieve]
  3. Beaufay, H., Bendall, D. S., Baudhuin, P., and Duve, C. (1959) Biochem. J. 73, 623-628 [Medline] [Order article via Infotrieve]
  4. Bogenhagen, D., and Clayton, D. A. (1974) J. Biol. Chem. 249, 7991-7995 [Abstract/Free Full Text]
  5. Brown, W. M., George, M., Jr., and Wilson, A. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1967-1971 [Abstract]
  6. Chance, B., Sies, H., and Boveris, A. (1979) Phys. Rev. 59, 527-605 [Free Full Text]
  7. Claycamp, H. G. (1992) Carcinogenesis 13, 1289-1292 [Abstract]
  8. Clayton, P. A. (1984) Annu. Rev. Biochem. 53, 573-594 [CrossRef][Medline] [Order article via Infotrieve]
  9. Cortopassi, G. A., Shibata, D., Soong, N.-W., and Arnheim, N. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7370-7374 [Abstract]
  10. Curtis, P. J., and Smellie, R. M. S. (1966) Biochem. J. 98, 818-825 [Medline] [Order article via Infotrieve]
  11. Darley-Usmar, V. M., Capaldi, R. A., Takamiya, S., Millett, F., Wilson, M. T., Malatesta, F., and Sarti, P. (1987) Mitochondria, A Practical Approach (Darley-Usmar, V. M., Rickwood, D., and Wilson, M. T., eds) pp. 113-152, IRL Press, Oxford
  12. Fenton, H. J. H. (1894) J. Chem. Soc. 65, 899-910
  13. Fischer-Nielsen, A., Poulsen, H. E., and Loft, S. (1992) Free Rad. Biol. & Med. 13, 121-126
  14. Halliwell, B., and Gutteridge, J. M. C. (1990) Methods Enzymol. 86, 1-85
  15. Imlay, J. A., and Linn, S. (1987) J. Bacteriol. 169, 2967-2976 [Medline] [Order article via Infotrieve]
  16. Imlay, J. A., and Linn, S. (1988) Science 240, 1302-1309 [Medline] [Order article via Infotrieve]
  17. Lin, J. H., and Castora, F. J. (1991) Biochem. Biophys. Res. Commun. 176, 690-697 [Medline] [Order article via Infotrieve]
  18. Linn, S. (1994) in Molecular Aspects of Aging, (Esser, K., and Martin, G. M., eds), pp. 191-194, John Wiley and Sons, Ltd., Chichester
  19. Low, R. L., Cummings, O.-W., and King, T. C. (1987) J. Biol. Chem. 262, 16164-16170 [Abstract/Free Full Text]
  20. Miquel, J. (1992) Mutat. Res. 275, 209-216
  21. Myers, K. A., Saffhill, R., and O'Connor, P. J. (1988) Carcinogenesis 9, 285-292 [Abstract]
  22. Palva, T. K., and Palva, E. T. (1985) FEBS Lett. 192, 267-270 [CrossRef][Medline] [Order article via Infotrieve]
  23. Putter, J., and Becker, R. (1983) in Methods of Enzymatic Analysis, Vol. 3, (Bergmeyer, H. U., ed) pp. 286-293, Verlag Chemie GmbH, Weinheim
  24. Radloff, R., Bauer, W., and Vinograd, J. (1967) Proc. Natl. Acad. Sci. U. S. A. 57, 1514-1521 [Medline] [Order article via Infotrieve]
  25. Richer, C. (1992) Mutat. Res. 275, 249-255
  26. Richter, C., Park, J. W., and Ames, B. N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6465-6467 [Abstract]
  27. Robberson, D. L., Kasamatsu, H., and Vinograd, J. (1972) Proc. Natl. Acad. Aci. U. S. A. 69, 737-741 [Abstract]
  28. Shigenaga, M., Park, J. W., Cundy, K. C., Gimeno, C. J., and Ames, B. N. (1990) Methods Enzymol. 186, 521-530 [Medline] [Order article via Infotrieve]
  29. Tchou, J., and Grollman, A. P. (1993) Mutat. Res. 299, 277-287 [Medline] [Order article via Infotrieve]
  30. Thresher, R., and Griffith, J. (1992) Methods Enzymol. 211, 481-491 [Medline] [Order article via Infotrieve]
  31. Wallace, D. C. (1992) Science 265, 628-632
  32. Zhang, Y., Marcillat, O., Giulivi, C., Ernster, L., and Davies, K. J. A. (1990) J. Biol. Chem. 265, 16330-16336 [Abstract/Free Full Text]

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