Effects of Lead Exposure on the Expression of Phospholipid Hydroperoxidase Glutathione Peroxidase mRNA in the Rat Brain

Joong Koo Kang*, Donggeun Sul{dagger}, Jong Koo Kang{ddagger}, Sang-Yoon Nam{ddagger}, Hae-Joon Kim{dagger} and Eunil Lee{dagger},1

* Department of Neurology, Asan Medical Center, University of Ulsan, Seoul, 138–736 Republic of Korea; {dagger} Department of Preventive Medicine, College of Medicine and Institute for Environmental Health, Medical Science Research Center, Korea University, Seoul, 136–701 Republic of Korea; {ddagger} Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, 361–763, Republic of Korea

Received August 18, 2003; accepted February 4, 2004


    ABSTRACT
 TOP
 ABSTRACT
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative damage associated with lead in the brain has been proposed as a possible mechanism of lead toxicity. Of the many antioxidant enzymes, phospholipid hydroperoxidase glutathione peroxidase (PHGPx) is known to protect cells from lipid peroxide–mediated damage by catalyzing lipid peroxide reduction. In this study, the effects of lead on the activity and expression of PHGPx mRNA were investigated in the brains of rats exposed to lead for 8 weeks. Male Sprague-Dawley rats (3 week old, n = 40) were randomly divided into four groups of 10 and treated with four different concentrations of lead in drinking water: a low dose (0.1% lead acetate), a medium dose (0.3% lead acetate), and a high dose (1.0% lead acetate), and a control group (0% lead acetate). We compared the four groups in terms of body and brain weight, lead concentrations in the brain and blood, and the activities of superoxide dismutase (SOD), gluthatione peroxidase (GPx), and PHGPx mRNA in the brain. Phospholipid hydroperoxidase glutathione peroxidase was found to have a dominant role in lead exposure. We also performed in situ hybridization of PHGPx mRNA in the brain to identity PHGPx mRNA active sites. We found that the level of PHGPx mRNA in brain increased in the medium- and low-dose groups, but decreased in the high-dose group versus the non-lead-treated control group. These results suggest that lead exposure increases the expression of PHGPx mRNA in the low- and medium-dose groups without inducing structural changes, and that the reduced expression of PHGPx mRNA in the high-dose group was associated with structural damage. An In situ hybridization study showed that PHGPx mRNA in the brain is expressed mainly in the white matter of the cerebral hemisphere and in the Purkinje cells of the cerebellar hemispheres; these sites are known to be the vulnerable to lead toxicity.

Key Words: brain; gluthatione peroxidase; lead; oxidative damage; phospholipid hydroperoxidase; glutathione peroxidase; superoxide dismutase.


    Introduction
 TOP
 ABSTRACT
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lead is one of the most clinically important heavy metals, because it induces a broad range of physiological, biochemical, and behavioral dysfunctions. The central nervous system has been identified as a major target site for lead effects in developing organisms (Feldman and White, 1992Go; Koller, 1990Go; Lilis et al., 1978Go; Yokoyama et al., 1997Go). Moreover, recent studies have proposed that oxidative damage due to an impaired oxidant/antioxidant balance is a mechanism of lead toxicity (Adonaylo and Oteiza, 1999Go; Gurer and Ercal, 2000Go; Wang et al., 2001Go). Resistance to oxidative stress depends on the status of operative antioxidant systems in cells and tissues. It prevents the uncontrolled formation of free radicals and the activation of oxygen species or inhibits their reactions with the biological structure (Chaudière and Ferrari-Iliou, 1999Go). Several studies have shown that free radical–mediated damage to cell components is an aspect of lead toxicity (Bechara et al., 1993Go; Hermes-Lima et al., 1991Go; Jiun and Hsien, 1994Go; Somashekaraiah et al., 1992Go; West et al., 1994Go)

It has been also reported that lead exposure has a dose–response relationship with changes in antioxidant enzyme levels (Adonaylo and Oteiza, 1999Go; Bechara et al., 1993Go) and in their activities. These enzymes include SOD, catalase, and GPx, the expressions of which were found to be changed in the presence of some antioxidant molecules in animals (Hsu, 1985Go; McGowan and Donaldson 1986Go) and in workers exposed to lead (Bechara et al., 1993Go; Ito et al., 1985Go; Monteiro et al., 1985Go; Sugawara et al., 1991Go). In addition, a recent study showed that the activity of brain GPx in workers exposed to lead was significantly higher than in control subjects, but that brain SOD activity was similar in the two groups (Adonaylo and Oteiza, 1999Go).

Selenoglutathione peroxidase is one of the most abundant antioxidant enzymes in the brain. This family includes the classic enzyme selenoglutathione peroxidase-I and a more recently characterized PHGPx. Of the four groups of known glutathione peroxidase (Holben and Smith 1999Go), PHGPx is the only known intracellular antioxidant enzyme that can directly reduce peroxidized phospholipids and cholesterol in membranes (Arthur et al., 1993Go; Thomas et al., 1990Go; Yagi et al., 1996Go). The primary role of PHGPx predominates in somatic tissues, where it plays a key role in preventing membrane oxidative damage (Adonaylo and Oteiza, 1999Go; Chambers et al., 1986Go; Chu et al., 1992Go; Holben and Smith, 1999Go; Knopp et al., 1997Go; Roveri et al., 1992Go).

Recently, PHGPx activity has been observed to be high in testis, which showed 45-fold higher PHGPx mRNA levels than the liver (Cockell et al., 1996Go; Lei et al., 1995Go). Additionally, PHGPx may have an important role in the brain as an antioxidative enzyme for protecting against oxidative damage. However, its role and relationship with toxic materials such as lead has not been clearly established. To our knowledge, no reports are available concerning the relationship between PHGPx and lead toxicity and the effects of lead on the expression and alteratons of PHGPx mRNA in the brain.

Therefore, we undertook this study to investigate changing PHGPx mRNA expression in the lead-exposed rat brain. We also evaluated the correlation between changes in the PHGPx mRNA levels and histological changes in the brain occurring after exposure to different lead doses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Sprague-Dawley rats (3 weeks old, n = 40) purchased from Charles River Japan (Yokohama, Japan) and were allowed to acclimate in the animal facility for a week before use. They were housed in a rat microisolator housing system for 8 weeks (temperature: 24 ± 2°C, humidity: 50 ± 10%, and 12 h day and night cycles).

Lead treatment. The rats, weighing 80–85 g, were randomly divided into four groups: low-dose (0.1% lead acetate), medium-dose (0.3% lead acetate), high-dose (1.0% lead acetate), and control (0% lead acetate) groups. The low-, medium-, and high-dose groups received 0.1%, 0.3%, and 1.0% lead acetate in distilled drinking water, respectively, but the control group received distilled drinking water only. All the rats freely received the same standard laboratory feed (MF, Oriental Yeast. Co., Tokyo). Body weight was measured every 3 days. All animal experiments were conducted in compliance with Guide For the Care and Use of Laboratory Animals of the U.S. National Research Council (National Research Council, 1996Go).

Blood collection and organ preparations. At the end of the 8-week exposure period, the rats were sacrificed and bloods were collected. Several factors including the level of hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were measured in whole blood by a hematological autoanalyzer (Celltac2; Nihon Koden, Japan). The brains were removed and weighed.

Lead concentration on blood and brain tissues. Lead concentration was measured in blood and brain tissues of rats. Brain tissues were digested with concentrated nitric acid and hydrogen peroxide mixture (5:1, v/v). Lead concentration in the digested tissue mixture was measured by inductive coupled plasma mass spectrometer analysis (PQ2 Turbo; VG ELEMENTAL, UK). Results were expressed as ng/g brain tissue. Blood lead levels were determined by using atomic absorption spectrophotometer (AAS 4100ZL; PerkinElmer, U.S.A.).

Total RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was extracted with the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Reverse transcription of mRNA and amplification of cDNA were performed using a Pelter thermal cycler (MJ Research Inc.). 1 µg of total RNA was reverse transcribed by using the 1st Strand cDNA Synthesis Kit (Boehringer Mannheim, Germany), and the cDNA samples were subjected to PCR under the following conditions: 10x PCR buffer, 0.2 mM dNTP mixture, 10 pmol of sense (5'-ATGCACGAATTCTCAGCCAAG-3') and antisense primer (5'-GGCAGGTCCTTCTCTAT-3') (Nam et al., 1997Go) for PHGPx, sense (5'-TACATTGTTTGAGAAGTGCG-3') and antisense (5'-GACAGCAGGGTTTCTATGTC-3') for GPx (Chambers et al., 1986Go), and sense (5'-CAATACACAAGGCTGTACCA-3') antisense (5'-TGCTCTCCTGAGAGTGAGAT-3') for SOD (Benedetto et al., 1991Go), and 0.5 U of Taq DNA polymerase. All PCR mixtures were heated at 94°C for 1 min, and cycled 33 times at 94°C for 1 min, at 57°C for 1 min, and at 72°C for 1 min 30 s, followed by an additional elongation step at 72°C for 7 min. GAPDH primers, sense primer (5'-AACGGATTTGGCCGTATTGG-3'), and antisense primer (5'-AGCCTTCTCCATGGTGGTGAAGAC-3') were used as a standard. The amplification product was separated electrophoretically on 1.0% agarose gels with ethidium bromide and analyzed by photography.

Preparation of in situ hybridization probe. To prepare RNA probes for in situ hybridization analysis, a pCRII vector (Invitrogen, Tokyo) containing a mouse PHGPx cDNA clone (S1-As2 fragment (Nam et al., 1998Go) was linearized with BamHI or EcoRV restriction enzyme. Digoxigenin (DIG)-labeled sense (5'-ATGCACGAATTCTCAGCCAAG-3') and antisense (5'-GGCAGGTCCTTCTCTAT-3') riboprobes for PHGPx were generated by in vitro transcription in the presence of T7 or Sp6 RNA polymerase (Boehringer Mannheim, Germany) at 37°C for 60 min.

In Situ Hybridization. In situ hybridization was performed on 5 µm sections of rat brain. Deparaffinized sections were incubated in 10 µg/ml of proteinase K in buffer (10 mM Tris-HCl, pH 8.0; and 1 mM EDTA) at 37°C for 5 to 30 min and treated with 4% paraformaldehyde/PBS for 10 min; in 0.2 M HCl for 10 min; and in 0.25% acetic anhydride/0.1 M triethanolamine (pH 8.0) for 15 min. Tissue sections were then prehybridized in 50% formamide/2X SSC at 42°C for 30 min and hybridized at 42°C for 16 h by adding either the DIG-labeled sense or antisense riboprobes for PHGPx in the following solution: 50% formamide, 10 mM Tris-HCl (pH 7.6), 200 µm/ml of yeast tRNA, 1x Denhart's reagent, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA (PH 8.0) in DEPC-treated water. After hybridization, sections were rinsed in 50% formamide/2x SSC at 42°C for 20 min three times and treated with 10 µm/ml of RNase A in buffer (10 mM Tris-HCl; pH 7.6, 500 mM NaCl, and 1 mM EDTA) at 37°C for 30 min. The sections were washed twice in 2x SSC at 42°C for 20 min. Thereafter, each section was incubated with 7.5 U/ml of FITC conjugated anti-DIG (Boehringer Mannheim, Germany), counterstained with 20µl/ml propidium iodide (Sigma, St. Louis, MO), and examined by a confocal scanning microscope. The experiments were repeated at least three times.

Histological examination. The brains were fixed in Bouin's fixative solution and washed with saturated lithium carbonate in 70% ethyl alcohol for 24 h to remove the excess fixatives. The tissues were dehydrated through increasing concentrations of ethyl alcohol (70%, 80%, 90%, and 100%), cleared in xylene, infiltrated in paraffin and Paraplast by an automatic tissue processor (Shandon Co., U.S.A.), and embedded in the paraffin wax with an embedding machine (Leica Co., Germany). The tissue blocks were cut at 5 µm in thickness on a rotary microtome (Leica Co.), stained with hematoxylin and eosin (H&E), and observed under a light microscope (Leica Co.).

Statistical analysis. Statistical analyses were carried out with SAS version 8.2. To test for significant differences between groups, we used the t-test, the analysis of variance (ANOVA) and Duncan's multiple comparison.


    RESULTS
 TOP
 ABSTRACT
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of Lead on Body Weight and the Relative Brain Weight
Rats were examined after 8 weeks of exposure. Most of the animals in each group appeared to be in good health, despite a marked reduction in the body weights of the animals in the high-dose group. After lead acetate treatment for 8 weeks, the average body weights in the low-, medium-, and high-dose groups and in the control group were; 352.6 ± 21.5 g, and 344.4 ± 22.9 g, 256.2 ± 38.2 g, 344.1 ± 31.9 g, respectively (Fig. 1). Body weights in the high-dose group were significant lower than those of the other groups (p < 0.0001). No statistically significant differences were found between the body weights of the low-dose, medium-dose. and control groups.



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FIG. 1. Body weight of rats treated with four different concentrations of lead acetate (0%, 0.1%, 0.3%, and 1.0% of lead acetate; n = 10 in each group). *p < 0.0001 by ANOVA between the control group and the high-dose group, which was treated with 1.0% lead acetate.

 
The ratio of brain weight to body weight in the low-dose, medium-dose, high-dose, and control groups were 0.59 ± 0.04%, 0.60 ± 0.04%, 0.74 ± 0.09%, and 0.60 ± 0.11%, respectively (Fig. 2). This ratio was significantly higher in the high-dose group than in other groups (p < 0.03).



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FIG. 2. The ratio of brain weight to body weight of rats treated with four different concentration of lead acetate (0%, 0.1%, 0.3%, and 1.0% lead acetate; n = 10 in each group). *p < 0.03 by ANOVA between the control group and the high-dose group, which was treated with 1.0% lead acetate.

 
Concentration of Lead in the Blood and Brain Tissues
Figures 3 and 4 show the concentrations of lead in the blood and brain tissues of treated rats. Mean blood lead levels in the low-, medium-, and high-dose groups and in the control groups were 84.5 ± 35.6 µg/dl, 117.9 ± 38.6 µg/dl, 366.2 ± 100.8 µg/dl, and 2.3 ± 0.9 µg/dl, respectively (Fig. 1), and corresponding lead concentrations in brain tissues were 187.0 ± 73.1 ng/g, 443.6 ± 169.0 ng/g, 1238.6 ± 465.85 ng/g, and 45.6 ± 12.6 ng/g. Lead levels in the blood and brain correlated significantly with the administered dose.



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FIG. 3. Lead concentrations in the blood of rats treated with four different concentrations of lead acetate (0%, 0.1%, 0.3%, 1.0% lead acetate; n = 10 in each group).

 


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FIG. 4. Lead concentration in the brains of rats treated with four different concentrations of lead acetate (0%, 0.1%, 0.3%, 1.0% lead acetate; n = 10 in each group).

 
Effects of Lead on Hematological Components
Table 1 shows the profile of hematological parameters in rats treated with four different concentrations of lead for 8 weeks. The values of hemoglobin, hematocrit, MCV, and MCH in rats treated with medium and high doses of lead acetate were significantly lower than the values in the control group except for the hematocrit value in rats treated with a medium dose of lead acetate for 8 weeks. Rats treated with a low dose of lead acetate showed a statistically significant decrease in MCV and MCH versus the control (p < 0.05). This result suggests that MCV and MCH are more sensitive indicators than hemoglobin or hematocrit in rats exposed to lead acetate (Table 1).


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TABLE 1 The Profile of Hematological Parameters in Rats Treated with Four Different Concentrations of Lead Acetate for 8 Weeks

 
Quantitative Changes PHGPx, SOD, and GPx mRNA in the Brain
Reverse transcriptase PCR analysis using specific primers for the PHGPx, SOD, and GPx cDNA sequences were performed on the total RNAs isolated from the brains of animals in each group. The expressions of PHGPx mRNA and GADPH mRNA are shown in Figure 5. Phospholipid hydroperoxidase glutathione peroxidase mRNA expression was detected in all brains in all groups. The expression level of brain PHGPx mRNA was elevated in the low- and medium-dose groups (p < 0.05) but was depressed in the high-dose group (p < 0.05) (Fig. 5).



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FIG. 5. PHGPx mRNA expression patterns in the brains of rats treated with four different concentrations of lead acetate (0%, 0.1%, 0.3%, 1.0% lead acetate; n = 10 in each group). *The p value by ANOVA was 0.007, and Duncan's multiple comparison showed that high-dose group(1.0 %) and the medium-dose group (0.3%) had statistically significant differences from the control and low-dose group (p < 0.05).

 
The expressions of SOD and GPx mRNA and GADPH mRNA are shown in Figure 6 and 7. Their expression levels changed in ways unrelated to that of PHGPx mRNA in brain. In addition, they showed similar expression in the low- and medium-dose groups. However, the expression levels of SOD and GPx were reduced in the high-dose group, as was that of PHGPx mRNA.



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FIG. 6. Superoxide dismutase mRNA expression patterns in the brains of rats treated with four different concentrations of lead acetate (0%, 0.1%, 0.3%, 1.0% lead acetate; n = 10 in each group). *The p value by ANOVA was 0.008, and Duncan's multiple comparison showed that only the high-dose group(1.0%) had a statistically significant difference with the control, low-dose and medium-dose groups (p < 0.05).

 


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FIG. 7. GPx mRNA expression patterns in the brains of rats treated with four different concentrations of lead acetate (0%, 0.1%, 0.3%, 1.0% lead acetate; n = 10 in each group). *The p value by ANOVA was 0.032, and Duncan's multiple comparison showed that only the 1.0% high-dose group had statistically significant difference from the control, low-, and medium-dose groups (p < 0.05).

 
In Situ Hybridization in the Brain Tissues
To determine the expression pattern of PHGPx mRNA, in situ hybridization using DIG-labeled riboprobes for PHGPx was performed in the brain of 11-week-old rats treated with lead. The expression of brain PHGPx mRNA was mainly observed in the white matter of the cerebral hemisphere (Fig. 8A, 8B, 8C, and 8D) and the Purkinje cells of the cerebellar hemisphere (Fig. 9A, 9B, 9C, and 9D). Phospholipid hydroperoxidase glutathione peroxidase mRNA signals in the low- and medium-dose groups were higher than in the high-dose or control groups, and PHGPx mRNA expression in the high-dose group was lower than in the control group.



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FIG. 8. In situ hybridization analysis by using DIG-labeled riboprobe in the white matter of the cerebral hemisphere (x100). (A) 1.0% lead acetate; (B) 0.3% lead acetate; (C) 0.1% lead acetate; and (D) 0% lead acetate (control).

 


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FIG. 9. In situ hybridization analysis by using DIG-labeled riboprobe in the cerebellar hemisphere (x100). The PHGPx mRNA signal was mainly observed in Purkinje cells. (A) 1.0% lead acetate; (B) 0.3% lead acetate; (C) 0.1% lead acetate; (D) 0% lead acetate (control).

 
Histopathological Findings in the Brain
Microscopically, the brains of the low- and medium-dose groups showed no definite histopathological abnormalities. However, the brains of animals in the high-dose group showed diffuse vacuolar degeneration and increased neovascularization of the cerebral white matter (Figs. 10A and 10B), and hippocampus (Figs. 11A and 11B) and cerebellar Purkinje cell degeneration (Figs. 12A and 12B). In addition, neuronal degeneration and mild spongy changes in brain stem nuclei, such as in spinal trigeminal and facial nuclei, were observed in the high-dose group (data not shown).



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FIG. 10. Histopathological findings in the cerebral white matter of rats treated with lead acetate. (A) 0% lead acetate; (B) 1.0% lead acetate. Diffuse vacuolar degeneration was evident (H&E x100).

 


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FIG. 11. Histopathological findings in the hippocampus of rats treated with lead acetate. (A) 0% lead acetate; (B) 1.0% lead acetate (H&E x100).

 


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FIG. 12. Histopathological findings in the cerebellar Purkinje cells in rats treated with lead acetate. (A) 0% lead acetate; (B) 1.0% lead acetate. Degenerated cells were observed (H&E x100).

 

    DISCUSSION
 TOP
 ABSTRACT
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, 3-week-old rats were treated with various concentrations of lead acetate for 8 weeks, and the relationship between the activity of PHGPx and lead exposure was investigated.

A previous study revealed that the blood levels of adult mice administered 0.5% lead acetate solution as the sole drinking fluid for 30 days was 100–125 µg/dl, which is a clinically toxic level (Goyer, 1995Go). We provided the medium-dose group in our study with 0.3% lead acetate based on the presumption that over 8 weeks this might result in a blood level of slightly higher than 100 µg/dl (Goyer, 1995Go). Our findings show that the blood lead level of rats administered lead acetate at this level is about 117.85 µg/dl, which is similar to that observed in previous studies (Draski et al., 1989Go; Goyer, 1995Go). In addition, the brain lead level is 0.5 µg/g, which is similar to that observed previously (Yun et al., 2000Go).

The mean body weight of the high-dose group was significantly lower than that of the other groups. It is possible this was caused by a reduction in the PHGPx mRNA level due to malnutrition. However, the distribution and patterns of microscopic structural changes observed in the high-dose group were consistent with the previous finding of structural damage associated with lead toxicity (Goyer, 1995Go; Goetz and Washburn, 1999Go; Popoff et al., 1963Go). In addition, PHGPx mRNA remains stable despite malnutrition, as was found in various studies of selenium deficiency (Baigelius-Flohe, 1999Go; Bermano et al., 1999Go; Mitchell et al., 1997Go). Therefore, PHGPx mRNA is probably a good stable toxic indicator, despite the weight change and nutritional deficiency.

Although several reports have suggested that oxidative stress may be involved in lead toxicity (Goyer, 1995Go; Goetz and Washburn, 1999Go; Popoff et al., 1963Go), no reports are available on the relationship between lead exposure and PHGPx changes. In the present study, the expression of PHGPx mRNA in the brain was found to be higher in the low- and medium-dose groups, but lower in the high-dose group than in the control group. These findings suggested that lead exposure increased the expression of PHGPx mRNA in the low- and medium-dose groups set against a background of no structural damage, but that it reduced the expression of PHGPx mRNA in the high-dose group because of structural brain tissue damage caused by lead toxicity and accumulation. In addition, the microscopic morphology of brain in the low- and medium-dose groups showed no significant differences from the control, yet the activity of PHGPx mRNA increased in the low- and medium-dose groups versus the control. The microscopic results of the brain examination performed in the high-dose group clearly demonstrate structural damage to brain tissues and reduced PHGPx mRNA activity versus the control. Also, the amount of lead accumulation in brain tissues was highest in the high-dose exposure group.

Although PHGPx activity might increase the resistance of cells to oxidative damage, we did not identify the origin of the PHGPx, i.e., whether it originated from the cytosol or the mitochondria, although it was reported recently that the activity of PHGPx mRNA is similar in mitchodrial and non-mitochondrial fractions in brain (Nam et al., 1998Go).

Phospholipid hydroperoxidase glutathione peroxidase is thought to contribute to the enzymatic defenses against oxidative damage to mitochondria (Chu, 1994Go), and cellular PHGPx activity was found to be correlated positively with cell survival after singlet oxygen exposure (Wang et al., 2001Go). Further study to identify the type of PHGPx mRNA involved is required.

The brain is a susceptible target, in part because it contains relatively low levels of enzymes that are capable of protecting it against oxidative stress (Savolainen, 1987Go), and in part because of its high myelin-associated content, which makes it vulnerable to the propagation of peroxidative events. Lead exposure results in a reduction in the accumulation of brain myelin in the developing brain (Toews et al., 1983Go), and it often causes focal and diffuse changes in the white matter, including myelin sheath fragmentation and reactive astrocytosis (Popoff et al., 1963Go). Gray matter, including the neurons of Ammon's horn of the hippocampus, basal ganglia, pons, medulla, and Purkinje cells of cerebellum also undergoes degeneration and cell loss after exposure to high levels of lead (Goyer, 1995Go; Goetz and Washburn, 1999Go; Popoff et al., 1963Go). We found that the brains of the high-dose group in our study showed diffuse vacuolar degeneration of the cerebral white matter, degeneration of the hippocampus and cerebellar Purkinje cells, and neuronal degeneration, and that mild spongy changes were produced in brain stem nuclei, such as in the spinal trigeminal and facial nuclei. Moreover, the pattern of such microscopic changes in the high-dose group was similar to changes reported previously (Goyer, 1995Go; Goetz and Washburn, 1999Go; Popoff et al., 1963Go).

Cytoplasmic selenoglutathione peroxidase I in mouse is localized in the adult brain, but no reports are available describing the localization of PHGPx mRNA in the rat brain. In situ hybridization performed during the present study showed that cerebellar Purkinje cells and the white matter of the cerebral hemispheres were the main PHGPx mRNA expression sites, and these are also the regions most vulnerable to lead toxicity. Thus, the observed location of PHGPx mRNA activity correlated well with that of areas vulnerable to lead toxicity.

In summary, we found that PHGPx mRNA was generally more expressed in the lead-exposed groups than in the control group, but not in animals exposed to the highest lead levels. These findings were obtained by in situ hybridization and confirmed by PCR. We also found a similar relationship between lead toxicity and PHGPx enzyme expression. However, we do not provide evidence of PHGPx enzyme activity at the protein level because of the non-availability of antibodies. Moreover, a comparison of other antioxidant enzymes, i.e., SOD and GPx, demonstrated PHGPx has a dominant role in lead exposure.


    ACKNOWLEDGMENTS
 
This work was supported by the Medical Research Center for Environmental Toxico-Genomics & Proteomics of Korea University.


    NOTES
 

1 To whom correspondence should be addressed at E-mail: eunil{at}korea.ac.kr.


    REFERENCES
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 ABSTRACT
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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