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Carcinogenesis, Vol. 23, No. 9, 1419-1425, September 2002
© 2002 Oxford University Press

CANCER BIOLOGY

Induction of DNA polymerase ß-dependent base excision repair in response to oxidative stress in vivo

Diane C. Cabelof1, Julian J. Raffoul1, Sunitha Yanamadala1, ZhongMao Guo2 and Ahmad R. Heydari1,3

1 Department of Nutrition and Food Science, 3009 Science Hall, Wayne State University, Detroit, MI 48202, USA and
2 Department of Physiology, University of Texas Health Science Center at San Antonio, TX 78284, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Base excision repair (BER) is the DNA repair pathway primarily responsible for repairing small base modifications and abasic sites caused by normal cellular metabolism or environmental insult. Strong evidence supports the requirement of DNA polymerase ß (ß-pol) in the BER pathway involving single nucleotide gap filling DNA synthesis in mammalian systems. In this study, we examine the relationship between oxidative stress, cellular levels of ß-pol and BER to determine whether oxidizing agents can upregulate BER capacity in vivo. Intraperitoneal injection of 2-nitropropane (2-NP, 100 mg/kg), an oxidative stress-inducing agent, in C57BL/6 mice was found to generate 8-hydroxydeoxyguanosine (8-OHdG) in liver tissue (4-fold increase, P < 0.001). We also observed a 4–5-fold increase in levels of DNA single strand breaks in 2-NP treated animals. The protein level of the tumor suppressor gene, p53 was also induced in liver by 2-NP (2.1-fold, P < 0.01), indicating an induction of DNA damage. In addition, we observed a 2–3-fold increase in mutant frequency in the lacI gene after exposure to 2-NP. Interestingly, an increase in DNA damage upregulated the level of ß-pol as well as BER capacity (42%, P < 0.05). These results suggest that ß-pol and BER can be upregulated in response to oxidative stress in vivo. Furthermore, data show that heterozygous ß-pol knockout (ß-pol+/–) mice express higher levels of p53 in response to 2-NP as compared with wild-type littermates. While the knockout and wild-type mice display similar levels of 8-OHdG after 2-NP exposure, the ß-pol+/– mice exhibit a significant increase in DNA single strand breaks. These findings suggest that in mice, a reduction in ß-pol expression results in a higher accumulation of DNA damage by 2-NP, thus establishing the importance of the ß-pol-dependent BER pathway in repairing oxidative damage.

Abbreviations: BER, base excision repair; 8-OHdG, 8-hydroxydeoxyguanosine; 2-NP, 2-nitropropane


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Maintenance of genomic integrity is essential to survival, as 80–90% of all cancers may be caused by damage to DNA (1). Both exogenous agents and endogenous metabolic processes generate electrophiles, which attack nucleophilic centers in the cell, causing damage to macromolecules such as nucleic acids, proteins and lipids. Certain types of DNA damage, including oxidized, lost or alkylated bases occur at alarming rates. Left unchecked, this damage would probably be incompatible with life. On a percentage basis, remarkably few of these lesions become fixed into the genome. The explanation is that the cell has at its disposal an array of elaborate DNA repair mechanisms. The base excision repair (BER) pathway is believed to be primarily responsible for repairing small, non helix-distorting lesions in the DNA that arise spontaneously or that are induced. It has been estimated that the BER pathway is responsible for the repair of as many as 1 million nucleotides per cell per day (2) emphasizing its importance in maintaining stability in the genome.

BER is a tightly coordinated sequence of events that results in the repair of a single damaged base. This process is initiated by glycosylase-mediated recognition of a damaged base, or by the presence of an abasic site. In the predominant BER pathway, which involves the removal and insertion of a single base, removal of the damaged base is completed by a monofunctional glycosylase. An endonuclease incises the DNA, creating a transient single strand break in the DNA. When a bifunctional glycosylase initiates BER the damaged base is removed and the DNA backbone is incised in the same step, leaving a 3' residue that must be processed prior to completion of repair. Both strand incision in monofunctional glycosylase-initiated BER and 3' end-trimming in bifunctional glycosylase-initiated BER appear to be accomplished by Ape1. At this point DNA polymerase ß (ß-pol) completes repair synthesis (3), and in monofunctional glycosylase-initiated BER is followed by ß-pol-mediated excision of the deoxyribose phosphate (dRp) flap created during repair, which is the rate-limiting step in this predominant subpathway (4). In bifunctional glycosylase-initiated BER, Ape1 appears to perform the rate-limiting step (5). Additionally, these two BER rate-limiting enzymes have been shown to physically interact, and Ape1 stimulates the rate-determining activity of ß-pol (6). In a minor BER subpathway involving the insertion of >1 nucleotide, the precise role of ß-pol is less clear. Certain modifications of abasic sites render the DNA resistant to ß-pol-mediated dRp excision, but may still rely on ß-pol for repair synthesis. It is suggested that in the presence of a 3' blocking lesion—a lesion not processed by either ß-pol or Ape1—ß-pol becomes processive, synthesizing a DNA strand that displaces the old strand, thus creating a flap (7). This flap may be excised by FEN1 followed by ligation with DNA Ligase I or DNA Ligase III/XRCCI to complete the repair process. FEN1 has been determined to be essential for long-patch repair (7–9) and has also been shown to stimulate ß-pol activity (10).

This research addresses the role of BER and ß-pol in the removal of oxidized bases and is the first to address the induction of both ß-pol and BER in vivo. It has been demonstrated previously that BER enzymes are inducible in cells by both alkylating agents (11) and by oxidizing agents (12). In an animal model of ischemia/reperfusion, Lin et al. (13) have demonstrated induction of ß-pol, suggesting that the BER pathway may be induced in response to oxidative stress. To further investigate the role of ß-pol and BER in repair of oxidative damage, we have used an oxidizing agent, 2-nitropropane (2-NP), to determine whether oxidative damage in vivo induces BER and upregulates ß-pol. While 2-NP has been shown to induce DNA synthesis (14) we are interested in specifically and directly measuring whether 2-NP induces repair synthesis by the BER pathway.

We find that in response to in vivo exposure to 2-NP, both BER activity and ß-pol levels are increased. Induction of both BER and ß-pol suggests that a ß-pol-dependent BER pathway is critical in the repair of oxidative damage. Furthermore, in order to more directly test the importance of ß-pol and BER in the repair of oxidative damage, we have measured the effects of 2-NP in an animal with reduced levels of ß-pol. The ß-pol heterozygous knockout mouse (ß-pol+/–) developed by Rajewsky's lab (15) exhibits reductions in both ß-pol level and BER activity (16). Importantly, we find that the ß-pol+/– mice accumulate more damage in response to 2-NP than do the wild-type mice, clearly demonstrating that a ß-pol-dependent pathway plays a role in the repair of oxidative damage in a whole animal model.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
Experiments were performed in male C57BL/6-specific pathogen-free mice in accordance with the NIH guidelines for the use and care of laboratory animals with approval from the Wayne State University Animal Investigation Committee. Mice were maintained on a 12 h light/dark cycle and were fed a semi-purified diet. The DNA polymerase ß heterozygous knockout mice were constructed by deleting the promoter and the first exon of the DNA polymerase ß gene and were produced using embryonic stem cells from the 129 strain of mice, which were injected into blastocytes of the C57BL/6 strain of mice (15). The ß-pol+/– mouse produced has been back-crossed to C57BL/6 mice so that >98% of the genetic background is C57BL/6. Animals were subjected to an i.p. injection of 100 mg/kg body weight 2-NP (Aldrich Chemical Company, CAS registry number 79-46-9, Milwaukee, WI) or olive oil vehicle. After 24 h, animals were killed by cervical dislocation. Tissues were flash frozen in liquid nitrogen.

Isolation of crude nuclear extract
All tissues were handled on ice or at 4°C during isolation of nuclear proteins. Tissues were homogenized in a homogenization buffer (10 mM HEPES pH 8.0, 1.5 mM MgCl2, 10 mM NaCl, 10 mM NaS2O5, 0.5 mM DTT, 0.5 mM PMSF, 1 µg/ml pepstatin A) and then centrifuged for 10 min at 10 000 g at 4°C. The pellet was mixed with 1.5 vol homogenization buffer plus 1 M NaCl, homogenized again, then centrifuged at 100 000 g at 4°C. The nuclear proteins were then precipitated by addition of 40% (NH)4SO4, and stirring for 30 min. Precipitated materials were collected by centrifugation at 15 000 g for 20 min at 4°C. The resultant pellet was dissolved in a minimal volume of dialysis buffer (20 mM Tris–100 mM KCl pH 8.0, 10 mM NaS2O5, 0.1 mM DTT, 0.1 mM PMSF, 1 µg/ml pepstatin A) and dialyzed against the buffer for 1 h at 4°C using Slide-A-Lyzer® Dialysis Cassettes (Pierce, Rockford, IL). Insoluble materials were removed by centrifugation at 12 000 g for 10 min at 4°C. The supernatant was stored at –20°C for use in repair assay and western blot analysis. Protein concentration of nuclear extracts was determined as described previously (17).

Western blot analysis
Nuclear extracts from livers of control and 2-NP animals were subjected to SDS–PAGE and transferred to nitrocellulose using a Bio-Rad semi-dry transfer apparatus according to the manufacturer's protocol (Bio-Rad, Hercules, CA). SDS–PAGE was conducted in duplicate; one gel was stained with Coomassie Blue to ensure equal loading of protein, the other gel was used to quantify p53 or ß-pol protein levels. Western blot analysis was accomplished using affinity purified monoclonal anti-sera developed against mouse p53 (pAB 240, Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal anti-sera developed against rat ß-pol (NeoMarkers, Fremont, CA). The bands were detected using an Alpha Innotech MultiImageTM system after incubation in SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Band intensity was quantified using a Molecular Dynamics densitometer (Molecular Dynamics, Sunnyvale, CA) and the data were expressed as the integrated intensity of the band per microgram of protein loaded.

In vitro analysis of BER (G:U mismatch repair assay)
Radio end-labeled 30 bp oligonucleotides (upper strand: 5'-ATATACCGC GGUCGGCCGATCAAGCTTATTdd-3'; lower strand: 3'-ddTATATGGCGCCGGCCGGCTAGTTCGAATAA-5') containing a G:U mismatch were incubated in a reaction mixture (100 mM Tris pH 7.5, 5 mM MgCl2, 1 mM DTT, 0.1 mM ATP, 0.5 mM NAD, 20 µM dNTPs, 5 mM diTris-phosphocreatine, 10 U creatine phosphokinase, 50 µg protein) with nuclear extract from hepatocytes of either control animals or animals injected with 2-NP. The reaction mixtures were incubated for 10 min at 37°C, and the duplex oligonucleotides were extracted with phenol–chloroform and precipitated. The purified oligonucleotides were treated with 50 U of HpaII for 30 min, and the duplex oligonucleotides were separated by electrophoresis on a denaturing 12% polyacrylamide sequencing gel, as described previously (17). Reaction products were visualized and quantified using a Bio-Rad Molecular Imager®. The data are expressed as machine counts per microgram of protein.

Quantification of 8-OHdG
We used a protocol that incorporates D-mannitol as an oxygen radical scavenger during the isolation of DNA to avoid introducing oxidative damage during extraction. The isolated DNA was digested to nucleosides using nuclease P1 and alkaline phosphatase (18). The concentration of 8-OHdG in the DNA hydrolysate was determined by high performance liquid chromatography with electrochemical detection (HPLC-ECD) as described by Shigenaga et al. (19) and modified by Adachi et al. (20).

Analysis of DNA single strand breaks (Comet assay)
DNA damage in hepatocytes was measured by single-cell gel electrophoresis as described by Singh et al. (21) with modifications by Poli et al. (22). Degreased slides were dipped in 1% normal melting agarose. The cells (~2x105) were washed twice with PBSG (phosphate-buffered saline glycerol) and mixed with 100 µl of 0.7% low melting agarose and placed on the first layer. Low melting agarose (100 µl) was added to the top layer. The cells were lysed at 4°C in the dark, for at least 1 h, in an ice-cold freshly prepared solution of 2.5 M NaCl, 10 mM Na2EDTA, 10 mM Tris–HCl, 1% Triton X-100 and 10% DMSO, pH 10. The slides were placed on a horizontal gel electrophoresis unit. The DNA was allowed to unwind for 20 min in an electrophoresis alkaline buffer (1 mM Na2EDTA, 300 mM NaOH, pH 13) and subjected to electrophoresis for 20 min at 0.78 V/cm and 300 mA. The slides were then washed in a neutralization buffer (0.4 M Tris–HCl, pH 7.5), dried and then fixed by immersion in absolute methanol for at least 60 s. Immediately before examination, the DNA was stained by 100 µl ethidium bromide (2 µl/ml) and DNA migration was measured for 40–50 cells (selected at random) on two to three microscope slides, under a fluorescence microscope (Laborlux 11; Leitz, Wetzlar, Germany) and evaluated by computer analysis. For each cell, the total length of the head and tail and the nuclear diameter was determined as described previously (23).

Mutation analysis
The mutation analysis was performed as described by Cabelof et al. (17). In brief, genomic DNA was isolated using the RecoverEase DNA isolation kit per manufacturer's protocol. The LacI transgene was recovered from genomic liver DNA by the Transpack in vitro packaging kit from Stratagene, according to manufacturer's protocol. Packaged phage was mixed with Escherichia coli SCS-8 cells from Stratagene and plated on NZY agar assay trays containing X-gal. To determine the mutant frequency, the total number of mutant plaques was divided by the total number of plaque-forming units.

Statistical analysis
Statistical significance between means was determined using ANOVA followed by the Fisher's least significant difference test where appropriate (24). Data for the Comet assay are reported as mean ± standard error of the mean. The differences between control and treated and/or between wild-type and knockout mice were analyzed by two factor analysis of variance followed by Sidak's multiple comparison test. A P-value <0.01 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Analysis of DNA damage in response to 2-NP
Because we are interested in determining the significance of BER in the repair of oxidative damage, we have selected 2-NP to induce oxidative DNA damage. 2-NP has been shown to induce high levels of 8-OHdG, a commonly occurring pre-mutagenic lesion. Additionally, 2-NP induces aminated guanines, which are prone to subsequent oxidation, and hydrazinohypoxanthine (25–27). Because these damages represent small base lesions, they are probable substrates for the BER pathway. In addition to the induction of DNA base damage, 2-NP is mutagenic as determined by the Ames test (28), and hepatocarcinogenic in rats (29). Sakano et al. (30) have demonstrated that metal-mediated oxidative stress is instrumental in determining the carcinogenicity of 2-NP. That is, the oxidative damage induced by 2-NP appears to be responsible for its carcinogenicity.

In agreement with data from others, we find that 100 mg/kg i.p. administration of 2-NP results in a 4–5-fold induction in levels of 8-OHdG (Figure 1Go) 24 h after injection. Deng et al. (31) have demonstrated an ~4-fold increase in 8-OHdG levels 24 h after administration of 100 mg/kg 2-NP. It is interesting to note that at the 6 h time point, the increase in 8-OHdG levels was nearly 9-fold (31). This rapid decrease in 8-OHdG levels represents removal, i.e. DNA repair, of this damaged base. During BER-mediated repair of the 8-OHdG lesion, the bifunctional glycosylase, OGG1, removes the damaged base and subsequently incises the DNA backbone, creating a strand break that persists until DNA repair is complete. In line with the concept of DNA repair-induced strand breaks, i.e. repair intermediates, we have used the Comet assay (single-cell gel electrophoresis) to measure the level of DNA single strand breaks. We observe a 4–5-fold increase in levels of DNA single strand breaks 24 h after 2-NP administration (Figure 1Go).



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  		Fig. 1. Measurement of DNA damage in wild-type mice in response to 2-NP. The experiment was conducted using liver tissue or hepatocytes isolated from mice injected with 2-NP and their non-injected counterparts as described in Materials and methods. (A) The concentration of 8-OHdG in the genomic DNA obtained from liver tissue was determined by HPLC using an electrochemical detector and the data are expressed as values relative to the amount of 2dG detected by UV absorbance at 290 nm. (B) The induction of DNA strand breaks by 2-NP was determined by quantifying the relative amount and spatial distribution of DNA that migrated out of the nuclei of hepatocytes obtained from control and 2-NP-treated mice. Values represent an average (± SEM) for data obtained from at least three animals in each group. {square}, i.p. injection of 2-NP; {blacksquare}, control, i.e. no injection; 2-NP, 2-nitropropane; ß-pol, DNA polymerase ß; 8-OHdG, 7,8-dihydro-8-oxoguanine; *, value significantly different from control at P < 0.01.

 
We have measured p53 protein levels as an additional measure of damage induction as this protein is stabilized via phosphorylation in response to DNA damage (32). We find that in response to 100 mg/kg 2-NP there is a significant increase in p53 protein levels (Figure 2Go). We have observed an even greater-fold increase in p53 levels in rats in response to 2-NP (Figure 2Go), which is in line with evidence that rats are more responsive to the effects of 2-NP than are mice (33,34). This increased stabilization of p53 in response to a DNA damaging agent that induces the BER pathway is particularly interesting in light of accumulating evidence that p53 plays a direct role in BER (35,36).



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  		Fig. 2. Effect of 2-NP in wild type mice on p53 protein levels. Nuclear extracts were isolated from liver tissue of mice injected with 2-NP (lanes 2, 4 and 6) and their untreated counterparts (lanes 1, 3 and 5) as described in Materials and methods. (A) The level of p53 protein in 20 µg of nuclear extract was determined by western blot analysis using an antibody against p53 protein and a SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Samples were pooled from extracts obtained from three to four animals in each group. Experiments were completed in F344 rats as well. Nuclear extracts were isolated from liver tissue of untreated rats (lanes 7, 9 and 11) and from rats injected with 2-NP (lanes 8, 10 and 12) and western blot analysis was completed as above. (B) The relative level of p53 protein in liver tissue was quantified using an Alpha Innotech MultiImageTM system, and the data were normalized based on the amount of protein loaded on each gel. Values represent an average (± SEM) for data obtained from at least three animals in each group. +, {square}, i.p. injection of 2-NP; –, {blacksquare}, control, i.e. no injection; 2-NP, 2-nitropropane; *, value significantly different from control at P < 0.01.

 
Analysis of mutant frequency in response to 2-NP
Having determined that DNA damage is induced in response to 2-NP in our animals, we were interested in determining whether induction in damage would correspond to an increase in mutant frequency. We have measured mutant frequency in the LacI gene 2 weeks after exposure to 100 mg/kg body weight 2-NP. We observe that 2-NP results in a 3-fold induction in mutant frequency in the liver of animals exposed to this carcinogen, demonstrating that damage induced by 2-NP becomes fixed in the genome (Table IGo).


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  		Table I. Mutant frequencies in liver tissues obtained from wild-type mice exposed to 2-NP
 
Analysis of DNA repair activity in response to oxidative damage
A central question to be addressed is whether the presence of oxidative DNA damage induces the BER pathway. To measure BER activity we use an in vitro G:U mismatch assay as described in Materials and methods. If the nuclear extract induces repair (G:U->G:C) a HpaII restriction site is created. Thus, upon digestion with HpaII, repair capacity can be quantified. The 12mer band indicates repair and the 30mer band (uncut by HpaII) represents no repair. We find that nuclear extracts from 2-NP treated animals induce a statistically significant (P < 0.01) increase in BER activity as compared with the level of BER activity observed in untreated animals (Figure 3Go). This shows that BER is induced by DNA damage sustained in vivo, and in particular by 2-NP which induces oxidative damage.



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  		Fig. 3. Effect of 2-NP in wild-type animals on BER activity. The experiment was conducted using crude nuclear extracts isolated from liver tissue of mice as described in Materials and methods. (A) Schematic diagram of G:U mismatch repair assay. (B) An autoradiograph of a sequence gel indicating repair activity as visualized by the appearance of a 12b fragment. Samples were pooled from extracts obtained from three to four animals in each group. (C) The relative level of BER in liver tissue of mice was quantified using a Bio-Rad Molecular Imager® and the data were normalized based on the amount of protein used in each reaction. Values represent an average (± SEM) for data obtained from at least three animals in each group. +, {square}, i.p. injection of 2-NP; –, {blacksquare}, control, i.e. no injection; negative control, G:U, G:U mismatch oligonucleotide incubated in the absence of nuclear extract and treated with HpaII restriction endonuclease; 2-NP, 2-nitropropane; ß-pol, DNA polymerase ß; *, value significantly different from control at P < 0.01.

 
Analysis of ß-pol protein levels in response to oxidative damage
Because DNA polymerase ß (ß-pol) has been determined to be the polymerase responsible for repair synthesis in short-patch BER (3), and probable in pre-replicative, long-patch BER as well (7,37,38) we have measured the effects of 2-NP on ß-pol protein levels. We find that in response to 100 mg/kg 2-NP, ß-pol protein levels are significantly increased (P < 0.01; Figure 4Go) 24 h after injection. We see a similar effect in rats as well (Figure 4Go). This upregulation of ß-pol by 2-NP suggests that ß-pol is necessary for the repair of 2-NP-induced oxidative damage.



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  		Fig. 4. Effect of 2-NP in wild-type mice on ß-pol protein levels. Nuclear extracts were isolated from liver tissue of mice injected with 2-NP and their non-injected counterparts as described in Materials and methods. (A) The level of the 39 kDa ß-pol protein in 20 µg of nuclear extract was determined by western blot analysis using an antibody against ß-pol protein and a SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Samples were pooled from extracts obtained from three to four animals in each group. Experiments were completed in F344 rats as well. Nuclear extracts were isolated from liver tissue of untreated mice and rats (lanes 1, 3, 5, 7, 9 and 11) and from mice and rats injected with 2-NP (lanes 2, 4, 6, 8, 10 and 12) and western blot analysis was completed as above. (B) The relative level of ß-pol protein in liver tissue from mice and rats was quantified using an Alpha Innotech MultiImageTM system, and the data were normalized based on the amount of protein loaded on each gel. Values represent an average (± SEM) for data obtained from at least three animals in each group. +, {square}, i.p. injection of 2-NP; –, {blacksquare}, control, i.e. no injection; 2-NP, 2-nitropropane; ß-pol, DNA polymerase ß; *, value significantly different from control at P < 0.01.

 
Analysis of the effects of oxidative damage in ß-pol+/– mice
To further demonstrate the role of ß-pol in processing oxidative damage, we have used a ß-pol+/– mouse model developed by Rajewsky's laboratory (15). This mouse has been characterized by our laboratory as having a 40–50% reduction in both mRNA and protein levels of ß-pol, and exhibits a 40–50% reduction in repair activity (16). Repeating the 2-NP experiments in the ß-pol+/– mice provides interesting information. After exposure to 2-NP, the knockout and wild-type mice display similar 8-OHdG levels (Figure 5Go), demonstrating that the initial step of BER is properly regulated in our model. Interestingly, Lin et al. (13) have shown that the BER glycosylase responsible for removal of 8-OHdG, OGG1, is upregulated in response to oxidative stress, and this regulation appears to be unaffected in our model. What we do observe in response to oxidative stress in the ß-pol+/– mice is a significant increase in the level of DNA single strand breaks (Figure 6Go). These single strand breaks may represent BER repair intermediates, where the glycosylase/lyase activity of OGG1 creates the strand incision, but subsequent DNA repair synthesis is delayed as a result of limiting amounts of ß-pol in the ß-pol+/– mice. The increased persistence of DNA damage in the mice lacking ß-pol demonstrates an important role for ß-pol in the processing of 2-NP-induced damage, i.e. oxidative damage.



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  		Fig. 5. Measurement of 8-OHdG in genomic DNA in response to 2-NP in ß-pol+/– and wild-type mice. The experiment was conducted using genomic DNA obtained from liver tissues of ß-pol+/– mice and their wild-type counterparts with an i.p. injection of 100 mg/kg of 2-NP or of vehicle, i.e. control as described in Materials and methods. The concentration of 8-OHdG in the DNA hydrosylate was determined by HPLC using an electrochemical detector, and the data are expressed as values relative to the amount of 2dG detected by UV absorbance at 290 nm. Values represent an average (± SEM) for data obtained from at least three animals in each group. ß-pol+/–, {square}, DNA polymerase ß knockout mice; WT, {blacksquare}, wild-type mice; 2-NP, 2-nitropropane; ß-pol, DNA polymerase ß; 8-OHdG, 7,8-dihydro-8-oxoguanine.

 


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  		Fig. 6. Time-dependent measurement of DNA single-strand breaks in response to 2-NP in ß-pol+/– and wild-type mice. The experiment was conducted using hepatocytes isolated from ß-pol+/– knockout mice and their wild-type counterparts with an i.p. injection of 100 mg/kg of 2-NP or of vehicle, i.e. control as described in Materials and methods. The time-dependent induction of DNA strand breaks by 2-NP was determined by quantifying the relative amount and spatial distribution of DNA that migrated out of the nuclei of cells. Values represent an average (± SEM) for data obtained from at least three animals in each group. ß-pol+/–, {square}, ß-pol+/– mice; WT, {blacksquare}, wild-type mice; 2-NP, 2-nitropropane; ß-pol, DNA polymerase ß; *, value significantly different from control at P < 0.01.

 
As an additional measure of sustained damage after oxidant exposure in the ß-pol+/– mice we have measured p53 protein levels in response to 2-NP in ß-pol+/– and wild-type mice. While p53 protein levels are increased in response to 2-NP in wild-type animals (above, Figure 2Go), this increase is even greater in the ß-pol+/– animals (Figure 7Go). This indicates that more damage is sustained in the ß-pol+/– mice in response to oxidative stress. There also may be another explanation for increased p53 levels in response to DNA damage in the ß-pol+/– mice related to the interrelationship between p53 and BER. We show that as BER activity is induced in response to 2-NP, p53 protein levels also increase. This provides the first evidence that the apparent coordination of BER activity and p53 activity observed in cell systems also occurs in a whole animal model.



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  		Fig. 7. Effect of 2-NP on the level of p53 induction in the ß-pol+/– and wild-type mice. Nuclear extracts were isolated from hepatocytes of ß-pol+/– mice and their wild-type counterparts as described in Materials and methods. (A) The level of p53 protein in 20 µg of nuclear extract was determined by western blot analysis using an antibody against p53 protein and a SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Samples were pooled from extracts obtained from three to four animals in each group. (B) The relative level of p53 protein in liver tissue was quantified using an Alpha Innotech MultiImageTM system, and the data were normalized based on the amount of protein loaded on each gel. Values represent an average (± SEM) for data obtained from at least three animals in each group. ß-pol+/–, {square}, ß-pol+/– mice; WT, {blacksquare}, wild-type mice; 2-NP, 2-nitropropane; ß-pol, DNA polymerase ß; *, value significantly different from control at P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Understanding the mechanisms for processing oxidative damage is likely to be important in elucidating mechanisms of both cancer and aging. We have used the DNA damaging agent 2-NP—which induces a high level of oxidative damage—in order to advance our knowledge of these processes. In this study we show that 2-NP causes induction in BER activity and an upregulation of ß-pol protein levels in a whole animal model. Previously, Lin et al. (13) demonstrated an upregulation of ß-pol protein in response to ischemia/reperfusion, which suggested that BER activity would likewise be upregulated in response to oxidative stress, and data presented herein confirms this. Additionally, we show that in an animal model deficient in BER activity, more damage accumulates in response to oxidative stress, clearly establishing a role for a ß-pol-dependent pathway in the repair of oxidative damage. In support, Chen et al. (12) have shown in fibroblasts that lipopolysaccharide-induced oxidative damage induces ß-pol levels and that more damage accumulates in the ß-pol-null fibroblasts.

In contrast, Sobol et al. (39) observed a lack of sensitivity to hydrogen peroxide in ß-pol-null fibroblasts, which seemed to contradict a role for a ß-pol-dependent pathway in the repair of oxidative damage. However, in revisiting this question Horton et al. (40) have found that while early passage ß-pol-null cells are not sensitive to H2O2, late passage cells are. This is supported by Fortini et al. (41) who have demonstrated moderate sensitivity to hydrogen peroxide in ß-pol-null fibroblasts. Perhaps these variations in sensitivity are a function of the spectrum of damage induced by H2O2. Some of the damage induced by H2O2, in particular the single strand breaks, may be repaired by direct ligation (and not by BER) as suggested by the observation that EM-CII cells which are deficient in DNA Ligase III are sensitive to single strand breaks (42). It is also possible that H2O2-induced damage may be repaired by an alternative BER pathway termed `long-patch' repair. Yet, the role of FEN1 (DNase IV) appears to be crucial in long-patch repair, and deletion of the yeast homolog of FEN1 does not increase sensitivity to hydrogen peroxide (43), suggesting that long-patch repair does not predominate in the repair of hydrogen peroxide-induced damage. Finally, one must consider whether the cytotoxic effects of H2O2 may be independent of the DNA damage/repair effects of oxidant exposure. This possibility is supported by the observation that early passage ß-pol-null cells show no increased cytotoxicity in response to H2O2, but do show very different repair capacity for 8-OHdG than do the wild-type cells (40).

Oxidative damage can also be repaired by a transcription–coupled mechanism. It has been demonstrated that 8-OHdG lesions occurring on the transcribed strand of actively transcribed genes are repaired by transcription–coupled repair (44). It is certainly reasonable to consider that a variety of repair mechanisms would exist to remove damage believed to be as detrimental as oxidative damage. That is, perhaps in actively transcribed genes, a CSB-dependent mechanism is essential for repair; in actively replicating cells, translesion synthesis, mismatch repair and/or PCNA/pol delta-dependent repair may be essential for repair; and in post-mitotic, resting or slowly dividing cells a ß-pol-dependent mechanism may be essential for repair. In agreement with the role for ß-pol-dependent repair in slowly dividing or resting cells, we have studied the effect of an oxidizing agent in liver tissue and we have found that at the level of the whole animal a ß-pol deficiency results in sensitivity to oxidative damage. This clearly demonstrates an essential role for ß-pol-dependent BER in processing oxidative damage in vivo.

Another area of intense investigation is the role of p53 in BER. Offer et al. (35) and Zhou et al. (36) have demonstrated that BER activity within cells correlates tightly with p53 levels. We have shown here that increases in p53 levels correspond to increases in BER activity, demonstrating for the first time that the effect seen by others in cell culture is also occurring in an animal model. One function of an increase in p53 protein levels is to halt the cell cycle in G1, allowing time for the cell to repair its damage prior to DNA replication. It seems that this may be a very tightly coordinated effort as it is now known that p53 is capable of stimulating BER activity in vitro, interacting directly with ß-pol, and stabilizing the interaction between ß-pol and abasic DNA in an Ape1-dependent manner (36). The data suggest a critical role for p53 in the proper functioning of the BER pathway and in BER-dependent maintenance of genomic integrity. In light of the data, we find it very interesting that the ß-pol knockout mice have an increased `p53 response' after induction of DNA damage. Because of the interrelationship between p53 and BER, this increase in p53 in the ß-pol+/– mice may describe a potentially important mechanism, perhaps suggesting a p53-mediated effort to increase BER activity in an animal with reduced BER capacity.


   Notes
 
3 To whom correspondence should be addressed Email: ahmad.heydari{at}wayne.edu Back


   Acknowledgments
 
We thank Drs Samuel H.Wilson and Robert W.Sobol of the National Institute of Environmental Health Sciences, National Institutes of Health, USA, for their generous gifts of the DNA polymerase ß antibody and of the DNA polymerase ß-pol+/– knockout mice. This work was supported in part by grants from the American Institute for Cancer Research (AICR 97A113), the National Institute on Aging (AG14242), and by a Pilot Project Program grant from the Wayne State University Environmental Health Sciences Center for Molecular and Cellular Toxicology with Human Applications (ES 06639).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 19, 2002; revised May 9, 2002; accepted May 15, 2002.


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