* Department of Neurology, Asan Medical Center, University of Ulsan, Seoul, 138736 Republic of Korea; Department of Preventive Medicine, College of Medicine and Institute for Environmental Health, Medical Science Research Center, Korea University, Seoul, 136701 Republic of Korea;
Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, 361763, Republic of Korea
Received August 18, 2003; accepted February 4, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: brain; gluthatione peroxidase; lead; oxidative damage; phospholipid hydroperoxidase; glutathione peroxidase; superoxide dismutase.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been also reported that lead exposure has a doseresponse relationship with changes in antioxidant enzyme levels (Adonaylo and Oteiza, 1999; Bechara et al., 1993
) 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, 1985
; McGowan and Donaldson 1986
) and in workers exposed to lead (Bechara et al., 1993
; Ito et al., 1985
; Monteiro et al., 1985
; Sugawara et al., 1991
). 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, 1999
).
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 1999), PHGPx is the only known intracellular antioxidant enzyme that can directly reduce peroxidized phospholipids and cholesterol in membranes (Arthur et al., 1993
; Thomas et al., 1990
; Yagi et al., 1996
). The primary role of PHGPx predominates in somatic tissues, where it plays a key role in preventing membrane oxidative damage (Adonaylo and Oteiza, 1999
; Chambers et al., 1986
; Chu et al., 1992
; Holben and Smith, 1999
; Knopp et al., 1997
; Roveri et al., 1992
).
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., 1996; Lei et al., 1995
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lead treatment. The rats, weighing 8085 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, 1996).
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., 1997) for PHGPx, sense (5'-TACATTGTTTGAGAAGTGCG-3') and antisense (5'-GACAGCAGGGTTTCTATGTC-3') for GPx (Chambers et al., 1986
), and sense (5'-CAATACACAAGGCTGTACCA-3') antisense (5'-TGCTCTCCTGAGAGTGAGAT-3') for SOD (Benedetto et al., 1991
), 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., 1998) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 100125 µg/dl, which is a clinically toxic level (Goyer, 1995). 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, 1995
). 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., 1989
; Goyer, 1995
). In addition, the brain lead level is 0.5 µg/g, which is similar to that observed previously (Yun et al., 2000
).
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, 1995; Goetz and Washburn, 1999
; Popoff et al., 1963
). In addition, PHGPx mRNA remains stable despite malnutrition, as was found in various studies of selenium deficiency (Baigelius-Flohe, 1999
; Bermano et al., 1999
; Mitchell et al., 1997
). 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, 1995; Goetz and Washburn, 1999
; Popoff et al., 1963
), 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., 1998).
Phospholipid hydroperoxidase glutathione peroxidase is thought to contribute to the enzymatic defenses against oxidative damage to mitochondria (Chu, 1994), and cellular PHGPx activity was found to be correlated positively with cell survival after singlet oxygen exposure (Wang et al., 2001
). 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, 1987), 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., 1983
), and it often causes focal and diffuse changes in the white matter, including myelin sheath fragmentation and reactive astrocytosis (Popoff et al., 1963
). 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, 1995
; Goetz and Washburn, 1999
; Popoff et al., 1963
). 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, 1995
; Goetz and Washburn, 1999
; Popoff et al., 1963
).
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 |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed at E-mail: eunil{at}korea.ac.kr.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arthur, J. R., Nicol, F., and Beckett, C. J. (1993). Selenium deficiency, thyroid hormone metabolism, and thyroid hormone deiodinases. Am. J. Clin. Nutr. 57, 236239.
Baigelius-Flohe, R. (1999). Tissue-specific functions of individual glutathione peroxidases. Free Radical Biol. Med. 27, 951965.[CrossRef][ISI][Medline]
Bechara, E. J., Medeiros, M. H., Monteiro, H. P., Hermes-Lima, M., Pereira, B., and Demasi, M. (1993). A free radical hypothesis of lead poisoning and inborn porphyrias associated with 5-aminolevulinic acid overload. Quim Nova 16, 385392.
Benedetto, M. T., Anzai, Y., and Gordon, J. W. (1991). Isolation and analysis of the mouse genomic sequence encoding Cu(2 +)-Zn2 + superoxide dismutase. Gene 99, 191195.[CrossRef][ISI][Medline]
Bermano, G., Nicol, F., Dyer, J. A., Sunde, R. A., Beckett, G. J., Arthur, J. R., and Hesketh, J. E. (1999). Tissue-specific regulation of selenoenzyme gene expression during selenium deficiency in rats. Biochem. J. 311(Pt 2), 425430.
Chambers, I., Frampton, J., Goldfarb, P., Affara, N., McBain, W., and Harrison, P. R. (1986). The structure of the mouse glutathione peroxidase gene: The selenocysteine in the active site is encoded by the termination code TGA. EMBO J. 5, 12211227.[Abstract]
Chaudière, J., and Ferrari-Iliou, R. (1999). Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem. Toxicol. 37, 949962.[CrossRef][ISI][Medline]
Chu, F. F., Esworthy, R. S., Doroshow, J. H., Doan, K., and Liu, X. F. (1992). Expression of placenta glutathione peroxidase in human liver in addition to kidney, heart, lung, and breast in human and rodents. Blood 79, 32333238.[Abstract]
Chu, F. F. (1994). The human glutathione peroxidase genes GPX2, GPX3, GPX4 map to chromosomes 14, 5, 19, respectively. Cytogenet. Cell Genet. 66, 9698.[ISI][Medline]
Cockell, K. A., Brash, A. R., Burk, R. F. (1996). Influence of selenium status on activity of phospholipids hydroperoxide glutathione peroxidase in rat liver and testis in comparison with other selenoproteins. Nutr. Biochem. 7, 333338.[CrossRef]
Draski, L. J., Burright, R. G., and Donovick, P. J. (1989). The influence of prenatal and/or postnatal exposure to lead on behavior of preweaning mice. Physiol. Behav. 45, 711715.[CrossRef][ISI][Medline]
Feldman, R. G., and White, R. F. (1992). Lead neurotoxicity and disorders of learning, J Child Neurol. 7, 354359.[ISI][Medline]
Goetz, C. C., and Washburn, K. R. (1999). Metal and neurotoxicology. In Medical Neurotoxicology (P. G. Blain, and J. B. Harris, Eds), Arnold, New York, pp. 181186.
Goyer, R. A. (1995). Toxic effects of metals. In Casarett and Doull's Toxicology (M. O. Amdur, J. Doull, and C. D. Klaassen, Eds), McGraw-Hill, International Edition, pp. 623680.
Gurer, H., and Ercal, N. (2000). Can antioxidants be beneficial in the treatment of lead poisoning? Free Radical Biol. Med. 29, 927945.[CrossRef][ISI][Medline]
Hermes-Lima, M., Pereira, B., and Bechara, E. J. (1991). Are free radicals involved in lead poisoning? Xenobiotica 21, 10851090.[ISI][Medline]
Holben, D. H., and Smith, A. M. (1999). The diverse role of selenium within selenoproteins: A review. J. Am. Diet. Assoc. 99, 836843.[CrossRef][ISI][Medline]
Hsu, J. M. (1985). Lead toxicity related to glutathione metabolism. J. Nutr. 111, 2633.
Ito, Y., Niiya, Y., Kurita, H., Shima, S., and Sarai, S. (1985). Serum lipid peroxide level and blood superoxide dismutase activity in workers with occupational exposure to lead. Int. Arch. Occup. Environ. Health 56, 119127.[ISI][Medline]
Jiun, Y. S., and Hsien, L. T. (1994). Lipid peroxidation in workers exposed to lead. Arch. Environ. Health 49, 256259.[ISI][Medline]
Knopp, E. A., Arndt, T. L., Eng, K. L., Caldwell, M., LeBoeuf, R. C., Deeb, S. S., O'Brien, K. D. (1997). Murine phospolipid hydroperoxide gluthathione peroxidase: cDNA sequence, tissue expression, and mapping. Mammalian Genome 10, 601605.[CrossRef]
Koller, L. D. (1990). The immunotoxic effects of lead in lead-exposed laboratory animals. Ann. N. Y. Acad. Sci. 587, 160167.[ISI][Medline]
Lilis, R., Einsinger, J., Blumberg, W., Fischbein, A., and Selikoff, I.J. (1978). Hemoglobin, serum iron, and zinc protoporphyrin in lead-exposed workers. Environ. Health Perspect. 25, 97102.[ISI][Medline]
Lei, X. G., Evenson, J. K., Thomson, K. M., Sunde, R. A. (1995). Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differently regulated in rats by dietary selenium. J. Nutr. 125, 14381446.[ISI][Medline]
McGowan, C., and Donaldson, W. E. (1986). Changes in organ nonprotein sulfhydryl and glutathione concentrations during acute and chronic administration of inorganic lead to chicks. Biol. Trace Elem. Res. 10, 3746.[ISI]
Mitchell, J. H., Nicol, F., Beckett, G. J., and Arthur, J. R. (1997). Selenium and iodine deficiencies: Effects on brain and browm adipose tissue selenoenzyme activity and expression. J. Endocrinol. 155, 255263.
Monteiro, H. P., Abdalla, D. S. P., Arcuri, A. S., and Bechara, E. J. H. (1985). Oxygen toxicity related to exposure to lead. Clin. Chem. 31, 16731676.
Nam, S. Y., Nakamuta, N., Kurohmaru, M., and Hayashi, Y. (1997). Cloning and sequencing of mouse cDNA encoding a phospholipid hydroperoxide glutathione peroxidase. Gene 198, 245249.[CrossRef][ISI][Medline]
Nam, S. Y., Fujisawa, M., Kim, J. S., Kurohmaru, M., and Hayashi, Y. (1998). Expression pattern of phospholipid hydroperoxide glutathione peroxidase messenger ribonucleic acid in mouse testis. Biol. Reprod. 58, 12721276.[Abstract]
Nam, S. Y., Kurohmaru, M., and Hayashi, Y. (1998). Testicular selenoproteins: Expression and distribution. In Reproductive Biology Update (H. Miyamoto, and N. Manabe, Eds.), Shoukadoh Booksellers Company, Japan, pp. 239245.
National Research Council. (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC.
Popoff, N., Weinberg, S., and Feigin, I. (1963). Pathologic observations in lead encephalopathy: With specific reference to the vascular changes. Neurology 13, 101112.[ISI][Medline]
Roveri, A., Casasco, A., Maiorino, M., Dalan, P., Calligaro, A., and Ursini, F. (1992). Phospholipid hydroperoxide glutathione peroxidase of rat testis. J. Biol. Chem. 267, 61426146.
Savolainen, H. (1987). Superoxide dismutase and glutathione peroxidase activities in rat brain. Res. Commun. Chem. Pathol. Pharmacol. 21, 173176.
Somashekaraiah, B. V., Padmaja, K., and Prasad, R. K. (1992). Lead induced lipid peroxidation and antioxidant defense components of developing chicken embryos. Free Radical Biol. Med. 13, 107114.[CrossRef][ISI][Medline]
Sugawara, E., Nakamura, K., Miyake, T., Fukumura, A., and Seki, Y. (1991). Lipid peroxidation and concentration of glutathione in erythrocytes from workers exposed to lead. Br. J. Ind. Med. 48, 239242.[ISI][Medline]
Thomas, J. P., Maiorino, M., Ursini, F., and Girotti, A. W. (1990). Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides. J. Biol. Chem. 265, 454461.
Toews. A. D., Blaker. W. D., Thomas. D. J., Gaynor. J. J., Krigman, M. R., Mushak, P., and Morell, P. (1983). Myelin deficits produced by early postnatal exposure to inorganic lead or triethyltin are persistent. J. Neurochem. 41, 816822.[ISI][Medline]
Wang, H. P., Qian, S. Y., Schafer, F. Q., Domann, F. E., Oberley, L. W., and Buettner. G. R. (2001). Phospholipid hydroperoxide glutathione peroxidase protects against singlet oxygen-induced cell damage of photodynamic therapy. Free Radical Biol. Med. 30, 825835.[CrossRef][ISI][Medline]
West, W. L., Knight, E. M., Edwards, C. H., Manning, M., Spurlock, B., James, H., Johnson, A. A., Oyemade, U. J., and Cole, O. J. (1994). Maternal low level lead and pregnancy outcomes. J. Nutr. 124, 981S986S.[Medline]
Yagi, K., Komura, S., Kojima, H., Sun, Q., Nagata, N., Ohishi, N., and Nishikimi, M. (1996). Expression of human phospholipid hydroperoxide glutathione peroxidase gene for protection of host cells from lipid hydroperoxidemediated injury. Biochem. Biophys. Res. Commun. 219, 486491.[CrossRef][ISI][Medline]
Yokoyama, K., Araki, S., Muraka, M., Morita, Y., Katsuno, N., Tanigawa, T., Mori, N., Yokota, J., Ito, A., and Sakata, E. (1997). Subclinical vestibulo-cerebellar, anterior cerebellar lobe and spinocerebellar effects in lead workers in relation to current and past exposure. Neurotoxicology 18, 371380.[ISI][Medline]
Yun, S. W., Gartner, U., Arendt, T., and Hoyer, S. (2000). Increases in vulnerability of middle-aged rat brain to lead by cerebral energy depletion. Brain Res. Bull. 52, 371378.[CrossRef][ISI][Medline]
|