JOURNAL OF MEDICINAL FOOD J Med Food 11 (4) 2008, 761–766 © Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2008.0051
Effects of Mikania glomerata Spreng. and Mikania laevigata Schultz Bip. ex Baker (Asteraceae) Extracts on Pulmonary Inflammation and Oxidative Stress Caused by Acute Coal Dust Exposure Tiago P. Freitas,1 Paulo C. Silveira,2 Luís G. Rocha,2 Gislaine T. Rezin,1 João Rocha,1 Vanilde Citadini-Zanette,3 Pedro T. Romão,4 Felipe Dal-Pizzol,1 Ricardo A. Pinho,2 Vanessa M. Andrade,4 and Emilio L. Streck1 Laboratórios de 1Fisiopatologia Experimental, 2Fisiologia e Bioquímica do Exercício, and 4Imunologia e Mutagênese and 3Herbário Pe. Dr. Raulino Reitz, Universidade do Extremo Sul Catarinense, Criciúma, Santa Catarina, Brazil ABSTRACT Several studies have reported biological effects of Mikania glomerata and Mikania laevigata, used in Brazilian folk medicine for respiratory diseases. Pneumoconiosis is characterized by pulmonary inflammation caused by coal dust exposure. In this work, we evaluated the effect of pretreatment with M. glomerata and M. laevigata extracts (MGE and MLE, respectively) (100 mg/kg, s.c.) on inflammatory and oxidative stress parameters in lung of rats subjected to a single coal dust intratracheal instillation. Rats were pretreated for 2 weeks with saline solution, MGE, or MLE. On day 15, the animals were anesthetized, and gross mineral coal dust or saline solutions were administered directly in the lung by intratracheal instillation. Fifteen days after coal dust instillation, the animals were killed. Bronchoalveolar lavage (BAL) was obtained; total cell count and lactate dehydrogenase (LDH) activity were determined. In the lung, myeloperoxidase activity, thiobarbituric acidreactive substances (TBARS) level, and protein carbonyl and sulfhydryl contents were evaluated. In BAL of treated animals, we verified an increased total cell count and LDH activity. MGE and MLE prevented the increase in cell count, but only MLE prevented the increase in LDH. Myeloperoxidase and TBARS levels were not affected, protein carbonylation was increased, and the protein thiol levels were decreased by acute coal dust intratracheal administration. The findings also suggest that both extracts present an important protective effect on the oxidation of thiol groups. Moreover, pretreatment with MGE and MLE also diminished lung inflammatory infiltration induced by coal dust, as assessed by histopathologic analyses. The present study indicates that M. glomerata and M. laevigata might become good candidates for the prevention of lung oxidative injury caused by coal dust exposure. KEY WORDS:
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coal dust
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Mikania glomerata
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Mikania laevigata
INTRODUCTION
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oxidative stress
fected by the production of oxidants during the combustion of oxygen.5 As stated above, this response is mediated by the activation of macrophages and the recruitment of polymorphonuclear cells, which increase inflammatory mediators and the formation of reactive oxygen species.6 In this context, it was demonstrated that antioxidants may play an important protective effect on oxidative damage induced by acute exposure to coal dust.3,4 Mikania glomerata and Mikania laevigata are popularly known as “guaco.” These plants are medicinal plants used in Brazilian folk medicine for several inflammatory and allergic conditions, particularly of the respiratory system. Phytochemically, the presence of coumarins, terpenes, and organic acids were reported, especially in the leaves.7,8 These plants have been widely used, and their folk indications in asthma and bronchitis are probably due to its bronchodilating properties.9 Anti-inflammatory10 and anti-ulcerogenic11 activities have also been reported. An interesting work from
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OAL MINERS are commonly exposed to coal dust that originates within the mine. The coal dust exposure is related to coal workers’ pneumoconiosis, a disease of coal miners characterized by the aggregation of macrophages near the respiratory bronchioles. Inflammation is a consistent feature of the pulmonary response to respirable coal dust.1,2 Some studies performed in our laboratory have previously shown that the parameters of oxidative damage to pulmonary function are altered following inhalation of industrial particles, such as coal.3,4 Following the inhalation of atmospheric pollutants, the lungs are the first targets af-
Manuscript received 1 February 2008. Revision accepted 31 March 2008. Address reprint requests to: Prof. Emilio L. Streck, Laboratório de Fisiopatologia Experimental, Universidade do Extremo Sul Catarinense, 88806-000, Criciúma, SC, Brazil, E-mail:
[email protected]
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Fierro et al.12 reported that a fraction obtained from the ethanolic extract of M. glomerata is effective in inhibiting immunologic inflammation but did not affect the acute inflammatory response in lung of rats. In this context, Santos et al.13 have also recently shown that Mikania extracts present important effects in a mouse allergic pneumonitis model. Finally, another significant biological effect is the antimicrobial activity against bacteria and yeasts14–16 and protozoans, such as Leishmania amazonensis and Trypanosoma cruzi.17 Considering that coal dust exposure induces an inflammatory response in lungs and that M. glomerata and M. laevigata are plants used in Brazilian folk medicine for several inflammatory conditions of the respiratory system, this study aimed to investigate whether extracts from these plants present any effect on inflammatory and oxidative damage indicators in the lungs of rats acutely exposed to coal dust.
MATERIALS AND METHODS Identification and extraction of plant material Aerial parts (leaves) of M. glomerata and M. laevigata were collected in Grão Pará, SC, Brazil. Voucher specimens of M. glomerata and M. laevigata were deposited at the Herbarium Pe. Dr. Raulino Reitz, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil under numbers CRI 7380 and CRI 7379, respectively. The leaves were allowed to dry under air circulation (40°C) for 3 days and powdered. The resulting powder (400 g) was subjected to dynamic maceration with 2 L of ethanol/water (70:30, vol/vol) solution during a 3-hour period. The extracts were filtered, and this procedure was repeated twice. The solvent was evaporated in a rotary evaporator, and the residue was dissolved in distilled water.15,18
Coal dust preparation One kilogram of gross coal was collected from Carboniferous Cooperminas, located in the municipal district of Criciúma. Samples of 300 g were triturated in a mill of spheres for 3 hours, at a frequency of 25 Hz. The coal was analyzed by the Analyses of Soil and Fertilizers Laboratory at the Universidade do Extremo Sul Catarinense and presented the following mineralogical characteristics: copper (0.003%), iron (2.480%), zinc (0.003%), and silica (27.3%). The coal dust used in the experiments was of diameter less than 15 m.
treatment with subcutaneous administration of M. glomerata extract (MGE) and intratracheal instillation of saline; (4) pretreatment with subcutaneous administration of MGE and intratracheal instillation of coal dust; (5) pretreatment with subcutaneous administration of M. laevigata extract (MLE) and intratracheal instillation of saline; and (6) pretreatment with subcutaneous administration of MLE and intratracheal instillation of coal dust. All studies were performed in accordance with National Institutes of Health guidelines and with the approval of the Ethics Committee from the Universidade do Extremo Sul Catarinense.
Pretreatment with M. glomerata and M. laevigata and coal dust instillation Rats were pretreated for 2 weeks with saline solution (groups 1 and 2), MGE (100 mg/kg of body weight, s.c.) (groups 3 and 4), and MLE (100 mg/kg of body weight, s.c.) (groups 5 and 6). The dose was chosen based on the work performed by Fierro et al.,12 which showed that MGE was effective in inhibiting lung immunologic inflammation caused by ovalbumin. On day 15, the animals were anesthetized with ketamine (80 mg/kg of body weight, i.p.) and xylazine (20 mg/kg body weight, i.p.). Gross mineral coal dust (3 mg/0.3 mL of saline) (groups 2, 4, and 6) or saline solution (0.3 mL) (groups 1, 3, and 5) was administered directly in the lung by intratracheal instillation, according to our previous protocol.3
Bronchoalveolar lavage (BAL) Fifteen days after coal dust instillation, the animals were anesthetized with a ketamine/xylazine overdose. BAL was conducted as published previously.19 Briefly, after cannulation of the trachea, the thorax was opened, and the lungs of rats were lavaged with phosphate-buffered saline, pH 7.4. Pooled samples were centrifuged at 300 g for 10 minutes, and the cell pellets were washed twice and resuspended.
Histological assessment After BAL obtaining, the animals were killed by decapitation, and lungs were quickly removed and stored at –70°C for biochemical analysis. For histological examinations, the lungs were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. An experienced pathologist performed blinded histopathologic analyses.
Animals and study design
Total cell count
Male Wistar rats obtained from the Central Animal House of the Universidade do Extremo Sul Catarinense were caged in groups of five, provided with commercial rat chow and water ad libitum, and maintained on a 12-hour light:12-hour dark cycle at a temperature of 22 1°C. The animals were divided into six groups: (1) pretreatment with subcutaneous administration of saline and intratracheal instillation of saline; (2) pretreatment with subcutaneous administration of saline and intratracheal instillation of coal dust; (3) pre-
The total number of cells present in BAL was obtained with a hemacytometer chamber immediately after the sample was obtained. BAL cells were adjusted to a concentration of 5 105 cells/mL in phosphate-buffered saline.
Lactate dehydrogenase (LDH) activity LDH was determined in BAL with the aid of a specific kit obtained from Labtest Diagnóstica SA (Lagoa Santa,
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MIKANIA SPECIES AND COAL DUST EXPOSURE TABLE 1. Group Saline saline Saline coal dust MGE saline MGE coal dust MLE saline MLE coal dust
CELLULAR
AND
BIOCHEMICAL PARAMETERS
IN
Total protein (g/mL) 598.6 549.5 617.5 593.4 577.2 560.6
BAL
OF
RATS AFTER COAL DUST EXPOSURE
LDH activity (U/mL)
81.3 47.5 36.2 28.8 26.9 71.7
14.8 56.6 15.2 43.6 23.0 23.4
Total cell count (cells/L)
3.8 9.2* 4.9# 9.9** 6.1# 9.1#
11.5 27.6 17.3 14.7 11.6 9.5
3.7 6.1* 2.5# 4.7# 2.0# 4.0#
Data are mean SD values (n 5). Different from control (saline saline): *P .01, **P .05. Different from coal dust intratracheal instillation control (saline coal dust): #P .01.
Myeloperoxidase activity The activity of myeloperoxidase was assessed by the method of Trush et al.,20 by using 3,3,5,5-tetramethylbenzidine as a substrate and measured spectrophotometrically at 650 nm. Tissue myeloperoxidase activity is used as an indicator of neutrophil infiltration. Data are presented as U/mg of tissue. All samples were assayed in duplicate.
Thiobarbituric acid-reactive substances (TBARS) levels The formation of TBARS during an acid-heating reaction is widely adopted as a sensitive method for measurement of lipid peroxidation, as previously described by Draper and Hadley.21 In brief, the samples were mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid. Subsequently, they were heated in a boiling water bath for 30 minutes. Malondialdehyde equivalents were determined by the absorbance at 532 nm using 1,1,3,3-tetramethoxypropane as an external standard. Results were expressed as malondialdehyde equivalents (nmol/mg of protein). All samples were assayed in duplicate.
Protein carbonyl content The oxidative damage to proteins was assessed by the determination of content of carbonyl groups, based on the reaction with dinitrophenylhydrazine, as previously described by Levine et al.22 In brief, proteins were precipitated by addition of 20% trichloroacetic acid and redissolved in dinitrophenylhydrazine, and the absorbance was monitored at 370 nm. All samples were assayed in duplicate.
saline (pH 7.4) containing 1 mM EDTA. Then, 30 L of 10 mM 5,5-dithio-bis(2-nitrobenzoic acid), prepared in a 0.2 M potassium phosphate solution (pH 8.0), was added. Subsequently, a 30-minute incubation at room temperature in a dark room was performed. Absorption was measured at 412 nm. The sulfhydryl content is inversely correlated to oxidative damage to proteins. Results were reported as nmol of thionitrobenzoic acid/mg of protein. All samples were assayed in duplicate.
Protein determination Protein content was determined by the method described by Lowry et al.24 using bovine serum albumin as the standard.
Statistical analysis Data were analyzed by one-way analysis of variance followed by Tukey’s test when F values were significant. All
2.00 Myeloperoxidase activity [U/mg tissue]
Brazil). The reading was made starting from an enzymatic system with a kinetic method, according to the technical orientations observed in the bulletin of the commercial kit. LDH activity was used as an index of cell death in BAL.
1.50
1.00
0.50
0.00
Protein sulfhydryl content The assay is based on the reduction of 5,5-dithio-bis(2nitrobenzoic acid) by thiols, which in turn become oxidized (disulfide), generating a yellow derivative whose absorption is measured spectrophotometrically at 412 nm.23 In brief, 50 L of homogenate was added to 1 mL of phosphate-buffered
Saline Saline
Saline Coal dust
MGE Saline
MGE Coal dust
MLE Saline
MLE Coal dust
FIG. 1. Myeloperoxidase activity in lung after coal dust instillation. Rats were subjected to intratracheal administration of coal dust or saline. Animals were assigned to receive MGE or MLE, and the lung was removed to determine myeloperoxidase activity as described in Materials and Methods. Data are mean SD values for six animals.
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Several studies have reported important biological effects of M. glomerata and M. laevigata, popularly known as “guaco.” The indications for M. glomerata and M. laevigata are asthma and bronchitis, especially because these medicinal plants present bronchodilating properties.9 Anti-inflammatory10 and antimicrobial14–17 activities for these plants have also been reported. Pneumoconiosis, a disease of coal miners, is characterized by pulmonary inflammation caused by coal dust exposure.1,2 The lung is a primary target of inhaled oxidants such as coal dust. The role of reactive oxygen species in pneumoconiosis has also been reported. The main source of these species is lung inflammation, a process that occurs after exposure to many agents.6 In this work we evaluated total protein levels, LDH activity, and total cell count in BAL of rats pretreated with MGE and MLE and subjected to acute coal dust exposure. As seen in Table 1, total protein levels were not altered by coal dust intratracheal instillation. LDH activity was increased by coal dust intratracheal instillation, suggesting that coal dust exposure induces cellular death. Moreover, MLE pretreatment prevented this effect, but MGE did not. Finally, we verified that total cell count was increased in coal dust-exposed rats. Both MGE and MLE prevented the increase in cell count. These results are evidence that coal dust leads to inflammation and cellular death in lungs of rats and that M. laevigata presents a protective effect in these parameters.
TBARS [nmol MDA/mg protein]
0.40
80 Protein carbonyl content [units/mg protein]
RESULTS AND DISCUSSION
A
70 60
*
50
*
*
40 30 #
20
#
10 0 Saline Saline
Saline Coal dust
MGE Saline
MGE Coal dust
MLE Saline
MLE Coal dust
B Protein sulfhydryl content [nmol TNB/mg protein]
analyses were performed using the Statistical Package for the Social Science (SPSS) software (SPSS, Chicago, IL).
800 700 600
# #
500
# #
400
*
300 200 100 0 Saline Saline
Saline Coal dust
MGE Saline
MGE Coal dust
MLE Saline
MLE Coal dust
0.30
FIG. 3. (A) Protein carbonyl and (B) sulfhydryl content in lung after coal dust instillation. Rats were subjected to intratracheal administration of coal dust or saline. Animals were assigned to receive MGE or MLE and the lung was removed to determine protein carbonyl and sulfhydryl contents as described in Materials and Methods. Data are mean SD values for six animals. TNB, thionitrobenzoic acid. *Different from control (saline saline), P .01 (Tukey’s test). #Different from coal dust intratracheal instillation control (saline coal dust), P .01 (Tukey’s test).
0.20
0.10
0.00 Saline Saline
Saline Coal dust
MGE Saline
MGE Coal dust
MLE Saline
MLE Coal dust
FIG. 2. TBARS in lung after coal dust instillation. Rats were subjected to intratracheal administration of coal dust or saline. Animals were assigned to receive MGE or MLE, and the lung was removed to determine TBARS as described in Materials and Methods. Data are mean SD values for six animals. MDA, malondialdehyde.
We also measured myeloperoxidase activity, another parameter of inflammation, in lung of rats. Figure 1 shows that this enzyme was not affected by coal dust intratracheal instillation. Moreover, pretreatment with MGE or MLE did not affect myeloperoxidase. We also measured oxidative stress parameters in lung of rats, namely, TBARS and protein carbonyl and sulfhydryl contents. As seen in Figure 2,
MIKANIA SPECIES AND COAL DUST EXPOSURE
TBARS levels were not altered by coal dust exposure, indicating that lipid peroxidation is not present in lungs of rats 15 days after intratracheal instillation of coal dust. Pinho et al.4 reported increased TBARS levels in lung of rats exposed to coal dust. However, in this work the rats were killed 30 days after coal dust exposure, and the regimen of coal dust administration was not the same (3 days/week for 3 weeks). Our results also showed that protein carbonyl and sulfhydryl contents were affected by coal dust intratracheal instillation. The findings indicate that rats subjected to coal dust acute exposure presented higher levels of protein carbonyl content, and MGE and MLE pretreatment did not prevent such effect (Fig. 3A). In agreement with our results, Pinho et al.3 also showed increased protein carbonyl groups after different times in rats subjected to coal dust exposure. Finally, Figure 3B shows that coal dust induced oxidation of sulfhydryl groups, since protein thiol content was significantly decreased in the lungs of these animals. Moreover, MGE and MLE prevented this effect, leading to speculation that these extracts may present a protective role in oxidation of thiol groups. These findings show that MGE and MLE may be more effective in the prevention of oxidation of sulfhydryl groups than in the carbonylation of amino acids residues in proteins caused by coal dust acute exposure. Regarding to TBARS and protein carbonyl content results, it is important to state that our findings are in accordance to previous reports from our laboratory.3 Our findings are also in accordance with another study in which Suyenaga et al.10 showed that M. laevigata presented an anti-inflammatory effect. Moreover, there is a lack of information about M. laevigata properties. The present study indicates that M. glomerata and M. laevigata might become good candidates for the prevention of lung oxidative injury caused by coal dust exposure.
ACKNOWLEDGMENTS The present work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico and the Universidade do Extremo Sul Catarinense.
AUTHOR DISCLOSURE STATEMENT No competing financial interests exist.
REFERENCES 1. Ghanem MM, Battelli LA, Mercer RR, Scabilloni JF, Kashon ML, Ma JYC, Nath J, Hubbs AF: Apoptosis and Bax expression are increased by coal dust in the polycyclic aromatic hydrocarbon–exposed lung. Environ Health Perspect 2006;114:1367– 1373. 2. Rom WN, Bitterman PB, Rennard SI, Cantin A, Crystal RG: Characterization of the lower respiratory tract inflammation of nonsmoking individuals with interstitial lung disease associated with chronic inhalation of inorganic dusts. Am Rev Respir Dis 1987;136:1429–1434.
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3. Pinho RA, Bonatto F, Andrades M, Frota MLC Jr, Ritter C, Klamt F, Dal-Pizzol F, Uldrich-Kulczynski JM, Moreira JCF: Lung oxidative response after acute coal dust exposure. Environ Res 2004;96:290–297. 4. Pinho RA, Silveira PCL, Silva LA, Streck EL, Dal-Pizzol F, Moreira JCF: N-Acetylcysteine and deferoxamine reduce pulmonary oxidative stress and inflammation in rats after coal dust exposure. Environ Res 2005;99:355–360. 5. Mossman BT: Introduction to serial reviews on the role of reactive oxygen and nitrogen species (ROS/RNS) in lung injury and diseases. Free Radic Biol Med 2003;34:1115–1116. 6. Tao F, Gonzalez-Flecha B, Kobzik L: Reactive oxygen species in pulmonary inflammation by ambient particulates. Free Radic Biol Med 2003;35:327–340. 7. Celeghini RMS, Vilegas JHY, Lanças FM: Extraction and quantitative HPLC analysis of coumarin in hydroalcoholic extracts of Mikania glomerata Spreng. (“guaco”) leaves. J Braz Chem Soc 2001;12:706–709. 8. Osório AC, Martins JLS: Determinação de cumarina em extrato fluido e tintura de guaco por espectrofotometria derivada de primeira ordem. Rev Bras Ciênc Farm 2004;40:481–486. 9. Soares de Moura R, Costa SS, Jansen JM, Silva CA, Lopes CS, Bernardo-Filho M, Nascimento da Silva V, Criddle DN, Portela BN, Rubenich LM, Araujo RG, Carvalho LC: Bronchodilator activity of Mikania glomerata Sprengel on human bronchi and guinea-pig trachea. J Pharm Pharmacol 2002;54:249–256. 10. Suyenaga ES, Reche E, Farias FM, Schapoval EE, Chaves CG, Henriques AT: Antiinflammatory investigation of some species of Mikania. Phytother Res 2002;16:519–523. 11. Bighetti AE, Antonio MA, Kohn LK, Rehder VL, Foglio MA, Possenti A, Vilela L, Carvalho JE: Antiulcerogenic activity of a crude hydroalcoholic extract and coumarin isolated from Mikania laevigata Schultz Bip. Phytomedicine 2005;12:72–77. 12. Fierro IM, Silva ACB, Lopes CS, Soares de Moura R, Barja-Fidalgo C: Studies on the anti-allergic activity of Mikania glomerata. J Ethnopharmacol 1999;66:19–24. 13. Santos SC, Krueger CL, Steil AA, Kreuger MR, Biavatti MW, Wisniewski A Jr: LC characterisation of guaco medicinal extracts, Mikania laevigata and M. glomerata, and their effects on allergic pneumonitis. Planta Med 2006;72:679–684. 14. Holetz FB, Pessini GL, Sanches NR, Cortez DAG, Nakamura CV, Dias-Filho BP: Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz 2002;97:1027–1031. 15. Yatsuda R, Rosalen PL, Cury JA, Murata RM, Rehder VLG, Melo LV, Kooc H: Effects of Mikania genus plants on growth and cell adherence of mutans streptococci. J Ethnopharmacol 2005;97: 183–189. 16. Betoni JEC, Mantovani RP, Barbosa LN, Di Stasi LC, Fernandes A Jr: Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases. Mem Inst Oswaldo Cruz 2006;101:387–390. 17. Luize PS, Tiuman TS, Morello LG, Maza PK, Ueda-Nakamura T, Dias-Filho BP, Cortez DAG, Mello JCP, Nakamura CV: Effects of medicinal plant extracts on growth of Leishmania (L.) amazonensis and Trypanosoma cruzi. Rev Bras Ciênc Farm 2005;41:85–94. 18. Duarte MCT, Figueira GM, Sartoratto A, Rehder VLG, Delarmelina C: Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol 2005;97:305–311.
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19. Dörger M, Allmeling AM, Kiefmann R, Münzing S, Mesmer K, Krombach F: Early inflammatory response to asbestos exposure in rat and hamster lung: role of inducible nitric oxide synthase. Toxicol Appl Pharmacol 2002;181:93–105. 20. Trush MA, Egner PA, Kensler TW: Myeloperoxidase as a biomarker of skin irritation and inflammation. Food Chem Toxicol 1994;32:143–147. 21. Draper HH, Hadley M: Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990;186:421– 431.
22. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, Ahn BW, Stadtman ER: Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464–478. 23. Aksenov MY, Markesbery WR: Change in thiol content and expression of glutathione redox system gene in the hippocampus and cerebellum in Alzheimer’s disease. Neurosci Lett 2001; 302:141–145. 24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–275.
