Abstract
Oxidative damage to DNA has been associated with neurodegenerative diseases. Developmental exposure to lead (Pb) has been shown to elevate the Alzheimer's disease (AD) related β-amyloid peptide (Aβ), which is known to generate reactive oxygen species in the aging brain. This study measures the lifetime cerebral 8-hydroxy-2′-deoxyguanosine (oxo 8 dG) levels and the activity of the DNA repair enzyme 8-oxoguanine DNA glycosylase (Ogg1) in rats developmentally exposed to Pb. Oxo 8 dG was transiently modulated early in life (Postnatal day 5), but was later elevated 20 months after exposure to Pb had ceased, while Ogg1 activity was not altered. Furthermore, an age-dependent loss in the inverse correlation between Ogg1 activity and oxo 8 dG accumulation was observed. The effect of Pb on oxo 8 dG levels did not occur if animals were exposed to Pb in old age. These increases in DNA damage occurred in the absence of any Pb-induced changes in copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), and reduced-form glutathion (GSH). These data suggest that oxidative damage and neurodegeneration in the aging brain could be impacted by the developmental disturbances.
Key words: Alzheimer • epigenetic • free radicals • methylation • Ogg1 • oxidative stress lzheimer's disease (AD) is a progressive neurodegenerative disorder whose clinical manifestations appear in old age. However, the initial events that trigger this disease may begin very early in life. The long latency of neurodegenerative diseases such as AD suggests that neurodegeneration is similar to cancer, in which a significant asymptomatic period (perhaps decades) elapses before a progressive accumulation of damage becomes clinically detectable (1). The sporadic nature of most AD cases strongly argues for an environmental link that may exaggerate normal age-related processes in the brain. Furthermore, the progressive and latent nature of the disease suggests that the triggering event occurs much earlier in time than the appearance of visible symptoms. Therefore, it is important to identify these environmental trigger(s) and to pinpoint the period during which such factors pose the greatest risk and the mechanism(s) involved therein.
Aging is the primary known risk factor for AD, and the concept that oxidative damage is an important mechanism involved with many aging diseases has been amply demonstrated. It is, however, presumed that the accumulation of oxidative damage is simply part of the overall aging process, which subsequently diminishes functional capacities. Recently, we have reported that developmental exposure of rats to the xenobiotic metal lead (Pb) resulted in a delayed overexpression (20 months later) in β-amyloid precursor protein (APP) mRNA, which was accompanied by an elevation in the levels of APP and its amyloidogenic β-amyloid (Aβ) product (2). Notably, we have also found that aged monkeys exposed to Pb as infants also express higher levels of APP mRNA, APP, and Aβ (unpublished data). The toxic effects of Aβ are partially mediated through the generation of free radicals (3). Therefore, enhancement of Aβ levels late in life due to developmental events may play an important role to influence the redox balance in the aging brain. Alternatively, epigenetic changes on the DNA molecule early in life may make it more susceptible to damage in old age. In addition, gene regulation and DNA damage in the aging human brain has also been a subject of a recent study (4).
Pb is an environmental neurotoxicant known to produce detrimental effects in the nervous system (5-9). Animal models of Pb exposure during development, from birth through weaning, have further shown a sequel of neurological deficits that remain well into adulthood when blood Pb levels are undetectable (10). The aim of this study was to evaluate some of the major indicators of antioxidant defense along with the degree of oxidative damage to DNA, as determined by the levels of oxo 8 dG, and the associated activity of the DNA repair enzyme Ogg1 in the brain cerebral cortical tissue of rats exposed to Pb during postnatal brain development or during adulthood. These experiments were designed to test the hypothesis that early exposure to a neurotoxicant determines the outcome of oxidative damage late in life.
MATERIALS AND METHODS
Timed-pregnant Long-Evans hooded rats were obtained from Charles River Laboratories (Wilmington, MA). Twenty-four hours following birth was designated as postnatal day 1 (PND 1). All pups were pooled, and new litters consisting of 10 males were randomly selected and placed with each dam. The animals were divided into three groups. Dams of the control group (Con) received unsupplemented tap water. The Con group was given ordinary tap water after weaning. The second group (Pb-E) was exposed to 0.2% lead acetate (Sigma, St. Louis, MO) from PND 1 through PND 20 through the drinking water of the dam. The third group (Pb-L) was exposed to Pb from 18 to 20 months of age. Food and water were freely available throughout the study (2, 11, 12) . The animals were individually housed at constant temperature (21±2°C) and relative humidity (50±10%) with a 12-hour light/dark cycle (0700-1900 h). On PND 5, 20, 60, 350, and 600, randomly selected pups/adults/senescent animals were decapitated following exposure to carbon dioxide and hypothermia and brain regions were dissected, frozen on dry-ice, and stored at -80°C. The animal exposure protocol and the maintenance of the animal facilities were regularly monitored by the Institutional Animal Care and Use Committee of the University of Rhode Island.
Rat brain cerebral cortical tissue samples were homogenized in 10 mM EDTA (ethylenediaminetetraacetic acid disodium salt) buffer (pH 7.0), using a VirTis ® VirSonic Ultrasonic Cell Disruptor 100. Following centrifugation at 20,817 g, at 4°C for 15 min, supernatants were aliquoted for glutathione, reduced form (GSH) and superoxide dismutase (SOD) activity analysis. Pellets were stored at -80°C until subsequent extractions were performed. Protein concentration in each sample was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA).
Protein was precipitated from supernatant samples collected as described above in 6.5% sulfosalicylic acid (SSA), and non-protein thiol content quantified following centrifugation at 10,621 g, at 4°C for 10 min. Total levels of reduced-form glutathion (GSH) in each sample were measured as described previously (13). Briefly, supernatants were added to a reaction mixture containing 2-nitro-5-thiobenzoic acid as a chromogenic substance. Absorbance was determined at 412 nm using a SpectraMax ® 340 96-well plate reader (Molecular Devices Corp., Sunnyvale, CA). The content of GSH in the samples was quantified by comparing absorption with a standard curve constructed with known amounts of GSH. Data were expressed as nanomoles per milligram of protein.
Total SOD activity was measured as described previously with slight modifications (14). Briefly, aliquots of the supernatant obtained from the brain cerebral cortical tissue homogenates prepared as described above, were incubated for 20 min in a 25°C water bath with 150 µl phosphate buffer (POB) (65 mM), 15 µl xanthine (1.5 mM in POB), and 15 µl hydroxylamine chloride (1.0 mM in POB). The reaction was initiated by the addition of 75 µl of xanthine oxidase (0.2 mg protein/ml). An aliquot of (100 µl) of the incubation mixture was added to a mixture of 100 µl sulfanilic acid (19 mM) and 100 µl of α-napthylamine (7 mM) in a 96-well plate, and the absorbance was determined, following a 20-min incubation at room temperature, at 539 nm, using a SpectraMax ® 340 96-well plate reader (Molecular Devices). The activity of total SOD was determined from a standard curve of percent inhibition of nitrite formation vs. SOD activity constructed using known amounts of purified SOD. SOD2 activity was differentiated from SOD1 activity with the addition of potassium cyanide (KCN, 5 mM) in the incubation mixture. The difference between the total activity and the KCN-inhibited enzyme activity was defined as SOD1. Data were expressed as units of SOD activity per milligram of protein.
Determination of oxidative damage to DNA (oxo 8 dG levels) and Ogg1 activity Oxo 8 dG levels and Ogg1 activity were quantified in an aliquot of the pellet from the samples prepared as outlined above using the procedure described previously (15). Briefly, DNA glycosylase was extracted from pellets with homogenization at 4°C in extraction buffer containing 20 mM Trizma-base; pH 8.0 (2-Amino-2-[hydroxymethyl]-1,3-propanediol), 1 mM . Following the addition of 2.5 M potassium chloride (KCl), the homogenate was rocked for 30 min at 4°C, and aliquots of supernatant were collected after centrifugation at 20,817 g for 30 min and stored along with the pellet at -80°C for use with the oxo 8 dG repair assay. Ogg1 activity was determined using a synthetic probe containing oxo 8 dG (Trevigen, Gaithersburg, MD) labeled with γ-32 P at the 5′-end, using polynucleotide T4 polynucleotide kinase (Roche). The probe used has the nucleotide sequence 5′-GAACTAGTGOATCCCCCGGGCTGC-3′ (O=oxo 8 dG) and was annealed to its corresponding complementary oligonucleotide before the nicking reaction was performed. The nicking reaction was initiated by incubating 30 µg of protein extract and the double-stranded labeled probe for 2 h at 37°C and stopped by placing the samples in ice. Aliquots of loading buffer containing 90% formamide, 10 mM NaOH, and blue-orange dye (Promega, Madison, WI) were then added to each sample. After 5 min heating at 95°C, samples were chilled and loaded into a polyacrylamide gel (20%) with 7 M urea and 1× TBE and run at 400 mV for 2 h. Gels were quantified using FLA-3000 Series Fuji Film Fluorescent Image Analyzer and analysis software. The capacity of the extract to remove oxo 8 dG was expressed as percentage of the cleaved synthetic probe (containing oxo 8 dG) to the total probe used (densitometric units).
To quantify oxo 8 dG levels in each sample, the pellet from the glycosylase extraction was first treated with DNAase-free RNAase followed by digestion with proteinase K. The protein fraction was then separated from DNA by three consecutive organic extractions. Following this step, the DNA was precipitated by adding two volumes of ethanol (with respect to the aqueous volume) and incubated overnight at -20°C. The purified DNA was prepared for HPLC analysis by resolving it into deoxynucleoside components. The amount of oxo 8 dG and 2'-deoxyguanosine (2-dG) was calculated by comparing the peak area of oxo 8 dG and 2-dG obtained from the enzymatic hydrolysate of the DNA sample to a calibration curve for both compounds. Levels of oxo 8 dG in the samples were expressed relative to the content of 2-dG, e.g., the molar ratio of oxo 8 dG/2-dG (fmol oxo 8 dG/nmol of 2-dG). The HPLC method used for analysis of the samples was as follows. The mobile phase consisted of 100 mM sodium acetate, pH 5.2, with 5% methanol. Flow rate was kept at 1 mL/min using a Model 582 Solvent Delivery Module (ESA, Chelmsford, MA). DNA was analyzed using a reverse phase YMCbasic HPLC column (4.6×150 mm) with a 3 μm particle size (YMC Inc. Wilmington, NC). Oxo 8 dG and 2-dG were detected by a Model 5600A CoulArray Detector (ESA) with three model 6210 four-channel electrochemical cells, potentials were set at 175, 200, and 250 V for oxo 8 dG and at 785, 850, and 890 V for 2-dG. Data were recorded, stored, and analyzed with CoulArray for Windows 32 Software (ESA). Data are expressed as femtomoles of oxo 8 dG per nanomoles of 2-dG.

that may exaggerate normal age-related processes in the brain. Furthermore, the progressive and latent nature of the disease suggests that the triggering event occurs much earlier in time than the appearance of visible symptoms. Therefore, it is important to identify these environmental trigger(s) and to pinpoint the period during which such factors pose the greatest risk and the mechanism(s) involved therein.
Aging is the primary known risk factor for AD, and the concept that oxidative damage is an important mechanism involved with many aging diseases has been amply demonstrated. It is, however, presumed that the accumulation of oxidative damage is simply part of the overall aging process, which subsequently diminishes functional capacities. Recently, we have reported that developmental exposure of rats to the xenobiotic metal lead (Pb) resulted in a delayed overexpression (20 months later) in β-amyloid precursor protein (APP) mRNA, which was accompanied by an elevation in the levels of APP and its amyloidogenic β-amyloid (Aβ) product (2) . Notably, we have also found that aged monkeys exposed to Pb as infants also express higher levels of APP mRNA, APP, and Aβ (unpublished data). The toxic effects of Aβ are partially mediated through the generation of free radicals (3) . Therefore, enhancement of Aβ levels late in life due to developmental events may play an important role to influence the redox balance in the aging brain. Alternatively, epigenetic changes on the DNA molecule early in life may make it more susceptible to damage in old age. In addition, gene regulation and DNA damage in the aging human brain has also been a subject of a recent study (4) .
Pb is an environmental neurotoxicant known to produce detrimental effects in the nervous system (5) (6) (7) (8) (9) . Animal models of Pb exposure during development, from birth through weaning, have further shown a sequel of neurological deficits that remain well into adulthood when blood Pb levels are undetectable (10) . The aim of this study was to evaluate some of the major indicators of antioxidant defense along with the degree of oxidative damage to DNA, as determined by the levels of oxo 8 dG, and the associated activity of the DNA repair enzyme Ogg1 in the brain cerebral cortical tissue of rats exposed to Pb during postnatal brain development or during adulthood. These experiments were designed to test the hypothesis that early exposure to a neurotoxicant determines the outcome of oxidative damage late in life.
MATERIALS AND METHODS

Lead exposure
Timed-pregnant Long-Evans hooded rats were obtained from Charles River Laboratories (Wilmington, MA). Twenty-four hours following birth was designated as postnatal day 1 (PND 1). All pups were pooled, and new litters consisting of 10 males were randomly selected and placed with each dam. The animals were divided into three groups. Dams of the control group (Con) received unsupplemented tap water. The Con group was given ordinary tap water after weaning. The second group (Pb-E) was exposed to 0.2% lead acetate (Sigma, St. Louis, MO) from PND 1 through PND 20 through the drinking water of the dam. The third group (Pb-L) was exposed to Pb from 18 to 20 months of age. Food and water were freely available throughout the study (2, 11, 12) . The animals were individually housed at constant temperature (21±2°C) and relative humidity (50±10%) with a 12-hour light/dark cycle (0700-1900 h). On PND 5, 20, 60, 350, and 600, randomly selected pups/adults/senescent animals were decapitated following exposure to carbon dioxide and hypothermia and brain regions were dissected, frozen on dry-ice, and stored at -80°C. The animal exposure protocol and the maintenance of the animal facilities were regularly monitored by the Institutional Animal Care and Use Committee of the University of Rhode Island.
Sample preparation
Rat brain cerebral cortical tissue samples were homogenized in 10 mM EDTA (ethylenediaminetetraacetic acid disodium salt) buffer (pH 7.0), using a VirTis ® VirSonic Ultrasonic Cell Disruptor 100. Following centrifugation at 20,817 g, at 4°C for 15 min, supernatants were aliquoted for glutathione, reduced form (GSH) and superoxide dismutase (SOD) activity analysis. Pellets were stored at -80°C until subsequent extractions were performed. Protein concentration in each sample was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA).
Quantification of GSH levels and SOD activity
Protein was precipitated from supernatant samples collected as described above in 6.5% sulfosalicylic acid (SSA), and non-protein thiol content quantified following centrifugation at 10,621 g, at 4°C for 10 min. Total levels of reduced-form glutathion (GSH) in each sample were measured as described previously (13) . Briefly, supernatants were added to a reaction mixture containing 2-nitro-5-thiobenzoic acid as a chromogenic substance. Absorbance was determined at 412 nm using a SpectraMax ® 340 96-well plate reader (Molecular Devices Corp., Sunnyvale, CA). The content of GSH in the samples was quantified by comparing absorption with a standard curve constructed with known amounts of GSH. Data were expressed as nanomoles per milligram of protein.
Total SOD activity was measured as described previously with slight modifications (14) . Briefly, aliquots of the supernatant obtained from the brain cerebral cortical tissue homogenates prepared as described above, were incubated for 20 min in a 25°C water bath with 150 µl phosphate buffer (POB) (65 mM), 15 µl xanthine (1.5 mM in POB), and 15 µl hydroxylamine chloride (1.0 mM in POB). The reaction was initiated by the addition of 75 µl of xanthine oxidase (0.2 mg protein/ml). An aliquot of (100 µl) of the incubation mixture was added to a mixture of 100 µl sulfanilic acid (19 mM) and 100 µl of α-napthylamine (7 mM) in a 96-well plate, and the absorbance was determined, following a 20-min incubation at room temperature, at 539 nm, using a SpectraMax ® 340 96-well plate reader (Molecular Devices). The activity of total SOD was determined from a standard curve of percent inhibition of nitrite formation vs. SOD activity constructed using known amounts of purified SOD. SOD2 activity was differentiated from SOD1 activity with the addition of potassium cyanide (KCN, 5 mM) in the incubation mixture. The difference between the total activity and the KCN-inhibited enzyme activity was defined as SOD1. Data were expressed as units of SOD activity per milligram of protein.
Determination of oxidative damage to DNA (oxo 8 dG levels) and Ogg1 activity Oxo 8 dG levels and Ogg1 activity were quantified in an aliquot of the pellet from the samples prepared as outlined above using the procedure described previously (15) . Briefly, DNA glycosylase was extracted from pellets with homogenization at 4°C in extraction buffer containing 20 mM Trizma-base; pH 8.0 (2-Amino-2-[hydroxymethyl]-1,3-propanediol), 1 mM
, 50% glycerol, and 1.3% protease inhibitor cocktail (Roche Life Science, Indianapolis, IN). Following the addition of 2.5 M potassium chloride (KCl), the homogenate was rocked for 30 min at 4°C, and aliquots of supernatant were collected after centrifugation at 20,817 g for 30 min and stored along with the pellet at -80°C for use with the oxo 8 dG repair assay. Ogg1 activity was determined using a synthetic probe containing oxo 8 dG (Trevigen, Gaithersburg, MD) labeled with γ-32 P at the 5′-end, using polynucleotide T4 polynucleotide kinase (Roche). The probe used has the nucleotide sequence 5′-GAACTAGTGOATCCCCCGGGCTGC-3′ (O=oxo 8 dG) and was annealed to its corresponding complementary oligonucleotide before the nicking reaction was performed. The nicking reaction was initiated by incubating 30 µg of protein extract and the double-stranded labeled probe for 2 h at 37°C and stopped by placing the samples in ice. Aliquots of loading buffer containing 90% formamide, 10 mM NaOH, and blue-orange dye (Promega, Madison, WI) were then added to each sample. After 5 min heating at 95°C, samples were chilled and loaded into a polyacrylamide gel (20%) with 7 M urea and 1× TBE and run at 400 mV for 2 h. Gels were quantified using FLA-3000 Series Fuji Film Fluorescent Image Analyzer and analysis software. The capacity of the extract to remove oxo 8 dG was expressed as percentage of the cleaved synthetic probe (containing oxo 8 dG) to the total probe used (densitometric units).
To quantify oxo 8 dG levels in each sample, the pellet from the glycosylase extraction was first treated with DNAase-free RNAase followed by digestion with proteinase K. The protein fraction was then separated from DNA by three consecutive organic extractions. Following this step, the DNA was precipitated by adding two volumes of ethanol (with respect to the aqueous volume) and incubated overnight at -20°C. The purified DNA was prepared for HPLC analysis by resolving it into deoxynucleoside components. The amount of oxo 8 dG and 2'-deoxyguanosine (2-dG) was calculated by comparing the peak area of oxo 8 dG and 2-dG obtained from the enzymatic hydrolysate of the DNA sample to a calibration curve for both compounds. Levels of oxo 8 dG in the samples were expressed relative to the content of 2-dG, e.g., the molar ratio of oxo 8 dG/2-dG (fmol oxo 8 dG/nmol of 2-dG). The HPLC method used for analysis of the samples was as follows. The mobile phase consisted of 100 mM sodium acetate, pH 5.2, with 5% methanol. Flow rate was kept at 1 mL/min using a Model 582 Solvent Delivery Module (ESA, Chelmsford, MA). DNA was analyzed using a reverse phase YMCbasic HPLC column (4.6×150 mm) with a 3 μm particle size (YMC Inc. Wilmington, NC). Oxo 8 dG and 2-dG were detected by a Model 5600A CoulArray Detector (ESA) with three model 6210 four-channel electrochemical cells, potentials were set at 175, 200, and 250 V for oxo 8 dG and at 785, 850, and 890 V for 2-dG. Data were recorded, stored, and analyzed with CoulArray for Windows 32 Software (ESA). Data are expressed as femtomoles of oxo upstream of the +1 TSS were obtained for genes whose product levels had been shown previously (12, (18) (19) (20) (21) to respond to early Pb treatment, specifically Amyloid β precursor protein (APP) (22) , methyl CpG binding protein 2 (MECP2), ornithine decarboxylase (ODC), proteolipid protein 1 (PLP), stimulating protein 1/specificity protein (SP1), and glutamate receptor, ionotropic, N-methyl D-aspartate 1 (NMDAR1). All sequences were obtained from the human DNA reference assembly (GenBank, assembly 35.1). Total cumulative CpG sites were determined for each sequence at each bp before the TSS, and totals averaged within each of the two groups ("non-responding" vs. "responding"). Cumulative CpG content was further determined at −100, −200, −400, −800, −1200, −1600, and −2000 bp before the TSS and subject to t test at each of these points.
The selected region (2 kb upstream of +1 TSS) was not chosen solely based on genomic database search, as we have functionally characterized 5′-flanking regions of both the BACE1 and APP genes and determined cell-type differentiated activity for the 2 kb upstream region for each of these two promoters (16, 23).
Statistical analysis
All data were analyzed using Prism GraphPad 4.0 software (Graph pad Software Inc., San Diego, CA). Oxo 8 dG data was analyzed by two-way ANOVA followed by a Turkey's post hoc test to compare the effects among various treatments; values denoted with an asterisk are significantly different from their corresponding controls (P<0.05). Pearson's correlation analysis was also used to evaluate the relationship between oxo 8 dG and Ogg1 for individual samples; asterisk denotes significant correlation amongst samples (P<0.05).
RESULTS

Postnatal Pb exposure and oxo 8 dG accumulation
To identify whether oxidation of DNA was a potential result of reactive oxidizing species produced by Pb exposure, the oxidized guanine adduct oxo 8 dG was evaluated at discrete ages following postnatal exposure to Pb in the rat cerebral cortex. Figure 1A and B represents chromatograms of injected samples of DNA from 20-month-old rat brain cerebral cortical tissue of control and animals exposed to Pb during development. A comparative analysis of the overlay of 2 dG chromatogram vs. oxo 8 dG chromatogram suggests elevated oxo 8 dG levels in developmentally Pb-exposed animals vs. control animals. Lifelong activity profiles of oxo 8 dG and OGG1 were determined, as well as oxo 8 dG levels were determined in control rats and those exposed to Pb from birth until weaning (Fig. 2) . On PND 5, a significant reduction in oxo 8 dG levels was found in developmentally Pb-exposed animals. However, oxo 8 dG levels followed an age-dependent increasing trend and were significantly higher at 20 months of age as compared with controls ( Fig. 2A) . The concentration of Pb in the blood (46 µg/dl) and tissue (0.41 µg/g wet weight) were increased in the developing animals during exposure but were at background levels at 20 months of age (blood=<2.0 µg/dl and tissue =<0.2 µg/g wet weight).
To confirm that the increase in oxo 8 dG levels was a latent effect of developmental Pb exposure, oxo 8 dG levels were also determined in DNA isolated from the cerebral cortices of animals exposed to Pb from 18 to 20 months of age (Pb-L group). No significant oxo 8 
dG accumulation

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was found in the Pb-L group as compared with control animals (Fig. 2C) , although Pb concentrations in the blood and the brain cerebral cortical tissue of these animals were significantly elevated (blood Pb=60 µg/dl and tissue Pb=0.32 µg/g wet weight of tissue).
Neonatal Pb exposure and DNA repair
In an effort to better characterize potentially compromised mechanisms that might account for the accumulation of oxo 8 dG in the neonatally exposed old animals, the activity of the oxo 8 dGspecific DNA repair enzyme Ogg1 was evaluated. Lifetime Ogg1 activity, assayed concomitantly with oxo 8 dG analysis, was not significantly different between controls and developmentally Pb-exposed animals (Fig. 2B) . The highest Ogg1 activity was observed in the PND 5 age group, and a hyperbolic trend of decreasing Ogg1 activity up to 12 months of age and increased activity between 12 and 20 months of age was observed. Pearson's correlation analysis of oxo 8 dG levels as compared with Ogg1 activity in each sample shows significant correlation in control animals that is lost with neonatal Pb exposure in all age groups excluding PND 5 ( Fig. 3A  and B) . The PND 5 group was excluded from correlation analysis due to the high levels of both oxo 8 dG and Ogg1 activity in control animals presumably due to high cellular turnover at this early age in development ( Fig. 2A and B insets) . The coefficient of determination (r 2 ) was reduced from 0.7284 to 0.2258 with Pb exposure indicative of an uncoupling of DNA damage and repair.
Cumulative gene promoter CpG content vs. response to developmental Pb exposure
Based on established studies, we have divided the genes into two categories, specifically a "responding group" and a "non-responding group". In the responding group, we included those genes, such as APP, that showed latent delayed overexpression of the gene with early Pb exposure. In the non-responding group, we included those genes, such as β-amyloid cleaving enzyme (BACE1) and SOD1/SOD2, which failed to show this property of delayed overexpression. We compared respective CpG methylation between these groups.
When mean cumulative CpG content of respective 2 kb 5′-flanking regions was compared between "non-responding" and "responding" genes, "non-responding" genes tended to have greater cumulative CpG than did "responding" genes (Fig. 4) . This difference was significant (P<0.05) by t test at 100 bp upstream of TSS. It was not significant at 200 or more bp upstream of the TSS. This finding suggests that the mechanism for the effects of developmental Pb exposure may depend upon hypomethylation of the proximal promoter region (less than 300 bp upstream of the TSS). A more refined association among response to developmental Pb exposure, CpG methylation, and the presence or absence of specific SP-family, zinc finger, and methylation-selective transcription factor binding sites may likewise exist.
Antioxidants and neonatal Pb exposure
Additional analysis of key antioxidants was also evaluated in an attempt to further characterize the factors participating in the oxidative status of the brain cerebral cortical tissues of developmentally Pb-exposed rats. Table 1 lists the levels of reduced GSH for each age category of rats in both the Pb-exposed and control groups. Postnatal Pb exposure does not appear to significantly alter the levels of GSH in the brain cerebral cortical tissue of these rats at any of the
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age groups evaluated. Levels of GSH remain relatively constant through the ages evaluated with the exception of the PND 60 samples, which demonstrate ~30 percent of the total GSH found in the same tissue of other age groups. Activities of both SOD1 and SOD2 were also unchanged in Pb-exposed rat brain cerebral cortical tissue samples at each age group assayed (see Table 2 ).
DISCUSSION
The concept that events early in life could have lasting effects that would determine adult disease is a logical one to make. In 1989, Barker showed that lower birth weights and obesity in humans were associated with an increased risk of death from congestive heart failure and stroke (24) . More epidemiological studies have also established the link between fetal under-nutrition and other diseases such as diabetes (25, 26) . Therefore, oxidative damage can be viewed as a pathological outcome that is usually evident during the process of aging while the preceding initiating event may have occurred during early stages of brain development.
The present study demonstrates that the brain cerebral cortical tissue of aging rats exposed to Pb during development accumulates a greater amount of oxo 8 dG as compared with unexposed animals. This increased accumulation of DNA damage is not reproduced, however, in animals exposed to Pb during old age, which provides evidence of a window of susceptibility during development that renders this population of neurons compromised later in life. The phenomenon of early exposure to Pb induced changes later in life is consistent with a recent report (2) in which developmental exposure of rats to Pb resulted in a delayed over-expression (20 months later) in APP and its amyloidogenic Aβ product. Similar to oxidative damage to DNA, both APP and Aβ were unresponsive to Pb exposure during old age (2) . Because Aβ toxicity is mediated through the generation of free radicals, these data suggests a potential link between the developmentally based elevation of Aβ in old age and the enhancement of oxidative DNA damage.
Another potential explanation for the elevated oxo 8 dG levels detected in the mature rat brain cerebral cortical tissue exposed to Pb during development is the uncoupling of the dynamic inverse relationship between the activity of Ogg1 and oxo 8 dG accumulation. Previous studies correlating oxo 8 dG and Ogg1 activity in discrete brain regions of mice have found similar evidence of this fundamental co-regulation (13) . In vitro evidence corroborating a loss in Ogg1 activity, upon exposure to the pesticide dieldrin, with an increase in the accumulation of oxo 8 dG in differentiated, neuronal like PC12 cells has also been shown (27) . The overall pattern of oxo 8 dG and Ogg1 activity across the lifespan of mature animals presented in this study, with the exception of early development, is indicative of this inverse relationship (Fig. 2) .
A recent study has suggested that DNA damage may reduce the expression of selectively vulnerable genes involved in learning, memory, and neuronal survival, initiating a program of brain aging that starts early in adult life (4) . Age-dependent accumulation of oxo 8 dG in various organs, including the brain, has been extensively studied in rat and mouse models (28) (29) (30) ). An age-dependent decline in the activity of base excision repair (BER) has also been demonstrated in rat brains which is accompanied by a down-regulation in the expression of Ogg1 resulting in oxo 8 dG accumulation (31) . Our results demonstrate an overall decrease in Ogg1 activity in the 12 month adult rat brain cerebral cortical tissue as compared with the activity in developing animals which is followed by an increase in Ogg1 activity between the 12 and 20 month old age groups. However the increase in Ogg1 activity tends to be greater in the Pb-exposed group as compared with the control. This trend toward an increased activity of Ogg1 in the oldest age group of Pb-exposed animals is a possible compensatory mechanism in response to elevated oxo 8 dG levels and a plausible explanation for the loss of the relationship between oxo 8 dG and Ogg1 following postnatal Pb exposure.
While this relationship between oxo 8 dG and Ogg1 activity appears disturbed by developmental Pb exposure, it is not clear how this reprogramming occurs without any direct effects on Ogg1 activity. Preliminary experiments in our lab, which used CpG dinucleotides in a DNA sequences in which methylcytosine and oxo 8 dG replaced an unmethylated or oxidized CpG, demonstrated that Ogg1 was unable to remove the oxo 8 dG if it was preceded by a methylcytosine (unpublished data). This finding suggested that if some CpGs are methylated early in life due to Pb exposure, their adjacent Gs would be un-repaired if subjected to oxidation later in life. This would explain the accumulation of oxidative damage to DNA in the aging brain following developmental exposure to Pb, in the absence of alterations in glutathione levels or the activities of antioxidant enzymes. These findings are consistent with developmental Pb exposure models evaluating antioxidants such as SOD, glutathione reductase (Gred), and glutathione oxidase (Gpx) in the rat hippocampus and hypothalamus, which have reported that these free radical scavengers are not affected in adult animals (10) . Within this experimental paradigm, the observed increase in oxidative damage to DNA in adult animals does not appear to be a factor of decreased antioxidant capacity in the cerebral cortex but rather a product of epigenetic modulations, which mask the damaged DNA.
An increase in oxidized DNA, marked by oxo 8 dG, in brain regions of patients with ageassociated neurodegenerative disorders such as AD and Parkinson's disease (PD) has been previously reported (32) (33) (34) . Studies in cell lines from Caucasian subjects have shown an increase in mitochondrial DNA damage following oxidative challenge. There was also another study on the effect of oxidative stress on DNA damage in lymphoblastoid cell lines from a Nigerian AD population by determining levels of 8-hydroxy-2′-deoxyguanosine and deoxyguanosine (dG) (35) . It is proposed that African populations are less vulnerable to chemical-induced oxidative DNA damage. The prevalence of AD is lower in Nigerians than in African-Americans (36).
It has been proposed that an accumulation of oxo 8 dG in the AD brain might be a result of a decrease in the activity of Ogg1, which suggests an involvement of the relationship between oxo 8 dG and Ogg1 activity as a mechanism within the pathogenesis of this neurodegenerative disease. This study suggests that oxidative stress exhibited in aging rats due to developmental Pb exposure may be explained by a loss of the vital relationship between oxo 8 dG accumulation and Ogg1 activity as well as latent build-up in Aβ levels. These disturbances originate early in life and may influence the potential onset of neurodegenerative diseases and behavioral alterations associated with aging.
In this context, it is paradoxically noteworthy that when comparing cumulative CpG content of respective 2kb 5′-flanking regions between "non-responding" and "responding" genes indicated that the latter group of genes tended to have lower cumulative CpG than did "responding" genes. This difference was significant at −100, but not at −200, −400, −800, −1600, and −2000 bp upstream of TSS, which suggests that mechanisms for the effects of developmental Pb exposure may also depend upon hypomethylation of the immediately proximal upstream promoter region. One potential alternative explanation is analogous to a "flock defense", wherein greater density of potential methylation targets would reduce the likelihood of a specific critically positioned CpG dinucleotide being subject to a single random methylation event. Genes with lower 5′-flanking CpG density would thus be more susceptible to a single methylation event that significantly alters binding capacity of potentially important nuclear factors.
The presence of the CpG density distinction in the proximal upstream region is not to be taken to discount any potential contribution of the 5′-untranslated region (UTR) of these genes to Pb response, as evidence has been shown for transcriptional regulatory activity for the APP gene 5′-UTR (37, 38) . In addition, the APP 5′-UTR has been shown to have metallomediated posttranscriptional activity in response to iron, zinc, and copper (39) (40) (41) , suggesting a potential role for Pb interaction with the 5′-UTR of the "responding" genes.
In the context of overall AD etiology, up-regulation of "responding" genes by developmental Pb exposure, whatever the mechanism, would result in greater expression of genes such as SP1 and APP. Higher levels of APP would result in greater Aβ concentrations, which would facilitate Aβ aggregation. Aβ alone or in aggregates is neurotoxic and leads to oxidative damage. In addition, in susceptible species, these higher levels of Aβ my lead to greater formation of amyloid plaque and resultant disease (Fig. 5) . Animals were exposed to 0 (control) or 200 ppm lead acetate (Pb-E) from birth through weaning, and the cerebral cortical tissue was were collected at specific time points (PND 5, 20, 60, 350, and 600). Samples were analyzed for levels of GSH (nmol/mg of total protein) as described in Materials and Methods, and the data shown were the mean ± SEM of 3-4 independent determinations. 3 . Correlation analysis of DNA damage and DNA repair in rat brain cerebral cortex. Animals were exposed to 0 (control) or 200 ppm lead acetate from birth through weaning (Pb-E) and the brain cerebral cortical tissues were collected at specific time points (postnatal day 20, 50, 350, and 600). Correlation analysis of oxo 8 dG (fmol/nmol) levels represented on y-axis and Ogg1 activity (% synthetic oligo cleaved) represented on x-axis for rat brain cerebral cortical samples (all age groups excluding postnatal day 5). Data from (A) control and (B) Pb-E samples were subjected to linear regression and correlation analysis. Equations from linear regression analysis and coefficients of determination (r 2 ) are presented for both control and Pb-E group samples. Pearson's correlation analysis of both groups showed significant correlation of oxo 8 dG levels and Ogg1 activity for (A) control samples only as shown by an "*" (P<0.05). flanking sequences of 2 kb length for 12 genes were obtained from the reference human genomic sequence database (GenBank, build 35.1). Mean CpG content for "non-responsive" and "responsive" genes to Pb was calculated and graphed as described in the text. In addition, content at −100, −200, −400, −800, −1200, −1600, and −2000 bp before the transcription start site (TSS) was subject to statistical analysis, as described in the text. Error bars represent standard errors of the mean. Specific probability scores generated by t tests are indicated at each of the six points analyzed. Pb exposure. Early life exposure to Pb may lead to developmental reprogramming in the genes that are responsive to Pb, thereby resulting in overexpression of the APP gene (a gene responsive to Pb) in the old age potentially through hypomethylation, which is established during development. This overexpression of APP gene results in an increase in APP production and its amyloidogenic cleavage product, Aβ peptide during senescence. Aβ is a cytotoxic agent, and its toxicity is mediated through the generation of free radicals. The elevation of Aβ may result in an accumulation of oxo 8 dG, whereas the levels of Ogg1 remain unaltered, thereby uncoupling the dynamic relationship between the activity of Ogg1 and oxo 8 dG accumulation. This imbalance between the activity of Ogg1 and the accumulation of oxo 8 dG could lead to the oxidative DNA damage, which may further lead to neuronal cell loss and neurodegeneration. Furthermore, in species susceptible to plaque formation, the increase in the levels of Aβ enhances aggregation leading to the formation of plaques in the brain.
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