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Annals of Occupational Hygiene Advance Access originally published online on January 30, 2006
Annals of Occupational Hygiene 2006 50(4):405-409; doi:10.1093/annhyg/mei079
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© 2006 British Occupational Hygiene Society Published by Oxford University Press

Original Article

Oxidative Stress in Mouse Brain Exposed to Lead

JUNQING WANG1,*, JUNHUI WU2 and ZHAOMING ZHANG1

1 College of Life Science and Technology, Shanxi University, Taiyuan 030006, P.R.China; 2 Hospital of Chinese People's Liberation Army 61768, Sanya 572011, P.R.China

* Author to whom correspondence should be addressed. Tel: +86 351 4169226; fax: +86 351 4169226; e-mail: junqingw{at}tom.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study was carried out to investigate effects of developmental Pb-exposure on antioxidant enzyme activities of mice brain. BALB dams were exposed to 600 p.p.m. of Pb-acetate in drinking water during pregnancy and lactation. Pb-exposure presented significant increase of plasma and brain Pb and 5-aminolevulinic acid (ALA) concentrations of weaned pups. In Pb-exposed 21-day-old pups, activities of superoxide dismutase, glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Re) decreased significantly in hypothalamus, corpora quadrigemina and corpus striatum compared with Na-exposed pups. Regarding 70-day-old pups, Pb-exposure had different effects on antioxidant enzymes of the three brain regions. The activities of GSH-Px and GSH-Re in corpora quadrigemina and GSH-Re in hypothalamus of Pb-exposure group did not decrease significantly. That meant that the lead employed might make occurrence of long-term effect on the antioxidant enzymes possible. The result also implied a correlation between ALA and oxidative stress in mice brain. Based on these results, it seemed that oxidative stress because of decreased antioxidant function, induced by significant accumulation of ALA, might be the main mechanism involved in mice brain neurotoxicity induced by developmental Pb-exposure.

Keywords: 5-aminolevulinic acid • antioxidant enzymes • lead exposure • oxidative stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Lead (Pb) is a toxic heavy metal widely distributed in the environment. Exposure to lead has been known to adversely affect human health in urbanized communities. Lead poisoning is a potential factor in brain damage, mental impairment and severe behavioral problems, as well as anemia, kidney insufficiency, neuromuscular weakness, and coma (Liuji et al., 2002Go). Many authors tentatively attribute the neurological symptoms of lead poisoning to the ability of 5-aminolevulinic acid (ALA) to inhibit either the K+-stimulated release of {gamma}-aminobutyric acid (GABA) from preloaded rat brain synaptosomes or the binding of GABA to synaptic membranes (Brennan et al., 1990Go). Moreover, the developing organism presents a 5-fold greater absorption of Pb and lacks a functional blood brain barrier (Lockitch, 1993Go). Perinatal exposure to low levels of lead has been involved in behavioral and neurochemical alterations detected in both suckling and adult rats (Moreira et al., 2001Go). One possible molecular mechanism involved in the lead neurotoxicity is the disruption of the prooxidant/antioxidant balance (Adonaylo and Oteiza, 1999Go), which can lead to brain injury via oxidative damage to critical biomolecules, such as lipids, proteins and DNA. To date, the studies that have evaluated oxidative stress in the brain following Pb-exposure were carried out in adult animals (Gurer and Ercal, 2000Go). Few studies investigated oxidative stress in weaned and adult pups whose dams were exposed to lead during pregnancy and lactation. This study was carried out to investigate (i) the effect of developmental Pb-exposure on the brain antioxidant system (ii) and correlation between ALA concentration and oxidative stress in brain of weaned and adult mice.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals and tissues preparation
BALB mice were obtained from the Hainan Medicine University and used as the parent generation. The animals were mated at the age of 70 days (two females and one male per cage). On pregnancy Day 0 (determined by the presence of sperm in vaginal smears) the dams were divided in Na- or Pb-exposed group and were housed singly. The drinking water of dams was adulterated with 600 p.p.m. Pb acetate, or 750 p.p.m. Na acetate to equalize acetate exposure for the two groups throughout pregnancy (19–21 days) and lactation (20 days).The decision to give all dams the same lead dose during pregnancy and lactation was based on prior studies in our laboratory showing that pups' blood lead levels remained relatively low and constant across a wide range of maternal employed (150–550 p.p.m.) (Lasley et al., 2001Go) and did not increase until very large doses are used. 0.5 ml of glacial acetic acid was added while stirring to prepare 1000 ml of both solutions (Na and Pb) to prevent formation of lead precipitate. At birth six pups were culled from each litter. Both Na- and Pb-exposure groups had at least three litters, 18 pups. All pups were weaned at 20 days old. The pups' weight gain, fluid and food consumption from fourth to 10th week after birth were recorded weekly. At 21 and 70 days old, blood collected from orbital venous plexiform were treated with anticoagulant (4% sodium citrate, 1:16), and centrifuged (10 min, 1200x g, 0°C). The supernatant was used for plasma Pb and ALA determination. Additionally nine pups selected randomly from each group were then anesthetized with pentobarbital sodium (60 mg kg–1, intraperitoneally). After perfusion through the ascending aorta with 140 mmol l–1 phosphate-buffered saline (PBS, pH 7.4), the brain was removed and dissected by method of Glowinski and Iversen (1966)Go into three regions of interest: hypothalamus, corpora quadrigemina and corpus striatum, which might be associated to animal behavior and respond differently to oxidative stress (Sandhir et al., 1994Go; Slawomir et al., 2004Go). Tissues from three littermates were pooled, weighed and homogenized (1 mg tissue/4 ml buffer) in ice-cold 140 mmol l–1 PBS in a homogenizer (Cole Parmer Instrument Co., Chicago). The tissue, maintained at –4°C throughout dissection and homogenization, was centrifuged for 15 min (100x g, 0°C), then supernatant collected was centrifuged again at 1800x g for 15 min at 0°C. The final supernatant was kept at –4°C for enzyme assays, brain Pb and ALA analysis.

Enzyme assays
The final supernatant was used for the activity evaluation of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Re) by Lasley's method (2001). SOD result is expressed as 1 U mg–1 of protein and GSH-Px and GSH-Re results as 1000 mU mg–1 of protein. All chemicals were purchased from Sigma (St Louis, MO).

Plasma and brain Pb and ALA analysis
Plasma and brain Pb concentrations were determined by atomic absorption spectrophotometry (Shimadzu AA6300, Japan). Analytic conditions are shown in Table 1. Analytic conditions for plasma and brain ALA measurement by HPLC (Shimadzu LC 10AT vp, Tokyo) are shown in Table 2.


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  		Table 1. Analytic conditions for plasma and brain Pb by graphite furnace atomic absorption spectrophotometry

 

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  		Table 2. Analytic conditions for plasma and brain ALA by HPLC

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Plasma and brain Pb and ALA levels
The plasma and brain Pb and ALA concentrations of 21 and 70 days old pups are shown in Table 3. This shows that plasma and brain Pb and ALA concentrations for the 21-day-old Pb-exposure group were significantly higher than the Na-exposure group (P < 0.05). Regarding 70-day-old Pb-exposure pups, these concentrations were lower than 21-old-day Pb-exposure group; plasma and brain Pb concentrations were still significantly higher than in the Na-exposed group (P < 0.05), but the differences for ALA concentrations were not. Similarly Cristine et al. (1997)Go has reported that the plasma ALA concentration from lead exposed workers was 7.1-fold higher than the control. The results also implied that blood lead level could be used as surrogate of brain lead level (Diane et al., 2004Go).


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  		Table 3. The plasma Pb and ALA concentrations of mice at 21 and 70 days old exposed to Na and Pb acetate

 
At the same time the body weight gain, fluid and food consumption levels of weaned pups were also analyzed (Fig. 1). The results showed that fluid consumption of Pb-exposure group decreased significantly (P < 0.05) compared with Na-exposure group, but body weight gain and food intake were insignificantly different between two groups(P > 0.05).


Figure 1
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  		Fig. 1. Weekly body weight gain (A), fluid (B) and food consumption (C) of pups from fourth to 10th week after birth exposed to Na (filled bars) and Pb (open bars). Columns represent mean values and bars represent standard errors.

 
Antioxidant enzymes activity
The effect of Pb-exposure on antioxidant enzyme activities of the hypothalamus of 21- and 70-day-old pups were shown in Fig. 2A. It indicated activities of SOD, GSH-Px and GSH-Re decreased (P < 0.05) in 21-day-old pups exposed to lead compared with Na-exposure group. The activities of SOD and GSH-Px also decreased (P < 0.05) in 70-day-old pups exposed to lead compared with Na-exposure group. Moreover, 70-day-old pups presented higher SOD, GSH-Px and GSH-Re activities than 21-day-old pups exposed to lead (P < 0.05). But 70-day-old Pb-exposure pups presented no significant difference in activity of GSH-Re compared with Na-exposed pups (P > 0.05).


Figure 2
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  		Fig. 2. Activities of SOD, GSH-Px and GSH-Re in the hypothalamus (A), corpora quadrigemina (B) and corpus striatum (C). SOD was expressed as U mg–1 and GSH-Px and GSH-Re as mU mg–1. Closed bars, 21-day-old Na-exposure; open bars, 21-day-old Pb-exposure; dotted bars, 70-day-old Na-exposure; striped bars, 70-day-old Pb-exposure.

 
Figure 2B showed a significant effect of Pb-exposure on activities of antioxidant enzymes in corpora quedrigemina. The activities of SOD, GSH-Px and GSH-Re decreased in 21-day-old pups exposed to lead compared with Na-exposure group (P < 0.05). But there were no statistical differences in activities of GSH-Px and GSH-Re between two groups at 70 days old (P > 0.05), for the activities of the antioxidant enzymes in adult pups increased than weaned pups in Pb-exposure group (P < 0.05).

The Pb-exposure effects on corpus striatum were shown in Fig. 2C. The results indicated activities of SOD, GSH-Px and GSH-Re all decreased significantly in both 21- and 70-day-old pups exposed to lead (P < 0.05), whatever activities of three enzymes increased significantly in Pb-exposure pups 70 days old (P < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study examined the plasma and brain Pb and ALA concentrations and antioxidant system status in three brain regions of weaned and adult mice whose dams were exposed to lead during pregnancy and lactation. The results indicated that the regimen of Pb-exposure induced a decreased fluid intake of pups, but it caused no effect on body weight gain and food intake of pups. Meanwhile plasma and brain ALA concentrations increased significantly in 21-day-old Pb-exposure pups, but insignificantly in 70-day-old pups. The decrease of plasma and brain Pb were possibly due to excretion through kidney, skin or dejecta. Some authors have associated increase of lead concentration with the manifestation observed in neurological disturbances (Hammond and Dietrich, 1990Go). It seemed that lead presented no effect on nutrition status and growth of developmental Pb-exposure pups. The effects of Pb-exposure on antioxidant function of hypothalamus, corpora quedrigemina and corpus striatum were different, which was in agreement with Sandhir's report (Sandhir et al., 1994Go). The statistically significant effect of Pb-exposure induced decrease in activities of SOD, GSH-Px and GSH-Rein three brain regions of 21-day-old pups. But for 70-day-old Pb-exposure pups, whose plasma lead concentration was significantly higher, the activities of GSH-Re in hypothalamus and GSH-Px and GSH-Re in corpora quedrigemina were not significantly decreased compared with Na-exposure pups. That meant lead employed in the tissue might make occurrence of long-term effect on the antioxidant enzymes possible.

This was in agreement with measurements described by Roberts et al. (2001)Go who stated that concentration of blood lead was a reliable indicator for the so-called internal lead dose. A correlation between ALA concentration and lead level also observed in this study would be useful especially for future clinical studies to demonstrate the relationship between ALA and symptoms of plumbism in brain or nervous system. Some authors have associated increase of plasma or brain ALA concentration with some manifestations observed in plumbism, e.g. hematopoietic damage and neurological disturbances (Rothenberg et al., 2000Go; Theodore & Jay, 2003Go).

It was supposed that oxidative stress was one possible mechanism for lead neurotoxicity. Pb-induced oxidative stress might result from accumulation of ALA, a potential endogenous source of free radicals, induced by inhibition of lead to ALA dehydratase. Overload of ALA seemed to be involved in the neurological disturbances, which leads to inhibition GABA release from synaptosomes and blocking GABA receptor. Nihei et al. (2001)Go have reported ALA can cause oxidative stress to rat brain. Additionally, direct interaction of lead to biological membranes was to induce lipid peroxidation. Pb-exposure might also induce decrease in activities of free radical scavenging enzymes. This mainly attributed to high affinity of lead to sulfhydryl-groups in these enzymes. Moreira et al. (2001)Go also demonstrated that brain antioxidant defenses (SOD and GSH-Px) from individuals exposed to lead decreased significantly and a nonlinear relationship between blood lead and SOD. Meanwhile, Slawomir et al. (2004)Go observed an increased activity of SOD in people protractedly exposed to lead (mean 15 ± 10 years), which seemed to be an adoptive mechanism against the raised amount of production of reactive oxygen species (ROS) caused by lead.

Although the biochemical basis for explaining correlation between lead level and SOD was not evident, this study supposed the antioxidant defenses damage in Pb-exposure might be a deleterious effect of oxygen radicals generated by ALA oxidation. The involvement of ROS in lead poisoning has been addressed by Schwartz et al. (2000)Go who found a decrease in GSH-Re and an increase in oxidized glutathione (GSSG) concentrations in lead acetate-treated rats. In addition, they also found the effects were reduced by treatment with N-acetylcysteine, a precursor of GSH. This provided a possibility of antioxidant therapy for individuals exposed to lead, for GSH/GSSG, an important component of antioxidant defense system in mammalian cells, was considered a sensitive indicator of oxidative stress (Wilson et al., 2000Go). Overproduction and accumulation of ALA in acute intermittent porphyria could be an origin of endogenous source of ROS, which can then exert their oxidative damage to cell structures. Princ et al. (1998) who investigated the induction of lipid peroxidation by exposure of cerebellum minimal tissue units to 1.0 mmol l–1 ALA suggested that protein damage was promoted by ALA auto-oxidation. Our findings provided the experimental evidence of involvement of ALA-promoted ROS in the damage of proteins related to porphyrin biosynthesis, mainly ALA dehydratase. Oxidation of this enzyme would lead to further accumulation of ALA, which might be the origin of the well-known neuropsychiatric manifestations.

Therefore, lead induced oxidative stress might result from accumulation of ALA induced by Pb-exposure. Then the activities of antioxidant enzymes scavenging free radicals, mainly generated by ALA oxidation, decreased significantly. Together with previous results showing oxidative stress associated with ALA accumulation, this study provided a strong evidence for the contribution of ALA to the prooxidant effects and toxicity of lead in mice brain.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Based on the presented results, it seemed that oxidative stress due to decreased antioxidant function might occur in weaned and adult mice whose dams were exposed to lead during pregnancy and lactation. Behavioral alterations as well as neurochemical alterations in different brain regions in weaned and adult rats exposed to a similar Pb-exposure regimen employed in this study have been detected in previous work (data not published), which also suggested oxidative stress to be an important mechanism involved in the neurotoxicity in mice brain induced by Pb-exposure during maternal pregnancy and lactation.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
REFERENCES
 
The authors wished to thank the Hainan Hospital for their donation of experimental animals. This work was supported by National Research Foundation of Ministry of Science and Technology of China (2001BA540C).

Received August 8, 2005; in final form December 22, 2005


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

Adonaylo VN, Oteiza PI. (1999) Pb2+ promotes lipid oxidation and alterations in membrane physical properties. Toxicology; 132: 19–32.[CrossRef][ISI][Medline]

Brennan MJ, Cantrill RC, Kramer S. (1990) Effect of 5-aminolevulinic acid on GABA receptor binding in synaptic plasma membranes. Int J Biochem; 22: 833–835.

Cristine A, Gilmar C, Adriana MP. (1997) Correlation between plasma 5-aminolevulinic acid concentration and indicator of oxidative stress in lead-exposed workers. Clin Chem; 13:1196–1202.

Diane ES, Myla SS, Donald S. (2004) Reductions in blood lead estimate reductions in brain lead after repeated succimer regimens in a rodent model of childhood lead exposure. Environ Health Perspect; 112: 302–8.[Medline]

Glowinski J, Iversen LL. (1966) Regional studies of catecholamines in the rat brain. J Neurochem; 13: 655–9.[ISI][Medline]

Gurer H, Ercal N. (2000) Can antioxidants be beneficial in the treatment of lead poisoning? Free Radic Biol Med; 29: 927–45.[CrossRef][ISI][Medline]

Hammond PB, Dietrich KN. (1990) Lead exposure in early life: health consequences. Rev Environ Contam Toxicol; 115: 91–124.[ISI][Medline]

Lasley SM, Green MC, Gilbert ME. (2001) Rat hippocampal NMDA receptor binding as a function of chronic lead exposure level. Neurotoxicol Teratol; 23: 185–9.[CrossRef][ISI][Medline]

Liuji C, Xianqiang Y, Hongli J. (2002) Tea catechins protect against lead-induced cytotoxicity, lipid peroxidation, and membrane fluidity in HepG2 cells. Toxicological Sci; 69: 149–56.[Abstract/Free Full Text]

Lockitch G. (1993) Perspectives on lead toxicity. Clin Biochem; 26: 371–81.[CrossRef][ISI][Medline]

Moreira EG, Vassilieff I, Vassilieff VS. (2001) Developmental lead exposure: behavioral alterations in theshort- and long-term. Neurotoxicol. Teratol; 23: 489–95.[CrossRef][ISI][Medline]

Nihei MK, McGlothan JL, Toscano CD et al. (2001) Low level Pb2+ exposure affects hippocampal protein kinase Caf gene and protein expression in rats. Neurosci Lett; 298: 212–6.[CrossRef][ISI][Medline]

Princ FG, Juknat AA, Amitrano AA et al. (1998) Effect of reactive oxygen species promoted by delta-aminolevulinic acid on porphyrin biosynthesis and glucose uptake in rat cerebellum. Gen Pharmacol; 31: 143–8.[Medline]

Roberts JR, Reigart JR, Ebeling M et al. (2001) Time required for blood lead levels to decline in nonchelated children. J Toxicol Clin Toxicol; 39: 153–60.[CrossRef][ISI][Medline]

Rothenberg SJ, Poblano A, Schnaas L. (2000) Brainstem auditory evoked response at five years and prenatal and postnatal blood lead. Neurotoxicol Teratol; 22: 503–10.[CrossRef][ISI][Medline]

Sandhir R, Julka D, Gill KD. (1994) Lipoperoxidative damage on lead exposure in rat brain and its implications on membrane bound enzymes. Pharmacol Toxicol; 74: 66–71.[ISI][Medline]

Schwartz BS, Lee BK, Lee GS. (2000) Associations of blood lead, dimercaptosuccinic acidchelatable lead and tibia lead with polymorphisms in the vitamin D receptor and 5-aminolevulinic acid dehydratase genes. Environ Health Perspect; 108: 949–54.

Slawomir K, Ewa B, Aleksandra K et al. (2004) Activity of SOD and catalase in prople protractedly exposed to lead compounds. Ann Agric Environ Med; 11: 291–6.[ISI][Medline]

Theodore IL, Jay SS. (2003) Lead neurotoxicity in children: basic mechanisms and clinical correlates. Brain; 126: 5–19.[Abstract/Free Full Text]

Wilson MA, Johnston MV, Goldstein GW. (2000) Neonatal lead exposure impairs development of rodent barrel field cortex. Proc Natl Acad Sci USA; 97: 5540–5.[Abstract/Free Full Text]


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