Antioxidant status of liver, erythrocytes, and blood serum of rats in acute methanol intoxication

Alcohol, Vol. 14, No. 5, pp. 431-437, 1997 © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/97 $17.00 + .00

PII S0741-8329(96)00149-8

E LS EV I ER

Antioxidant Status of Liver, Erythrocytes, and Blood Serum of Rats in Acute Methanol Intoxication E. S K R Z Y D L E W S K A

AND

R. F A R B I S Z E W S K I

D e p a r t m e n t o f Analytical Chemistry, Medical A c a d e m y , 15-230 Biatystok 8, P.O. B o x 14, Poland R e c e i v e d 16 F e b r u a r y 1996; A c c e p t e d 5 July 1996 SKRZYDLEWSKA, E. AND R. FARBISZEWSKI. Antioxidant status of liver, erythrocytes, and blood serum of rats in acute methanol intoxication. ALCOHOL 14(5) 431-437, 1997.--SOD, CAT, GSH-Px, GSSG-R, ascorbic acid, o~-tocopherol, nonprotein- and protein-bound sulfhydryl compounds, and TBA-rs content in the liver, erythrocytes, and blood serum of rats treated with methanol after 6, 12, and 24 h and 2, 5, and 7 days were investigated. Furthemore, hematological parameters of erythrocytes were analysed. GSH-Px, GSSG-R, sulfhydryl compounds, and ascorbic acid in the liver, erythrocytes, and in blood serum were significantly decreased. In addition, Cu,Zn-SOD and tocopherol in erythrocytes were diminished, whereas TBA-rs in the three biological materials was enhanced. Simultaneously, erythrocytes amount, hemoglobin level, hematocrit, and MCV were reduced. These results indicate that methanol in rats leads to the impairment of antioxidant mechanisms in the liver, erythrocytes, and blood serum. © 1997 Elsevier Science Inc. Methanol

Antioxidant enzymes

SH groups

Ascorbic acid

M E T H A N O L is oxidized by alcohol dehydrogenase, microsomat ethanol oxidizing system (MEOS), and catalase, mainly in the liver. In rats, a catalase-peroxidase system is considered to be responsible for oxidizing methanol to formaldehyde. The latter is then oxidized by formaldehyde dehydrogenase, an enzyme that requires reduced glutathione as a cofactor. Other dehydrogenases play a role in oxidation of aldehydes in liver cytosol and mitochondria. This process involves augmentation of reduced N A D H and leads to manifestation of oxidase activity, including xanthine oxidase, which is a principal source of superoxide anion formation that may be involved in lipid peroxidation (23). Erythrocytes are constantly being subjected to various types of oxidative stress, such as inhaled oxidants, ingested chemicals, and accidentally methanol. Although rat erythrocytes contain an abundance of catalase they are incapable of oxidizing methanol (28). In addition, released leukotrienes from infiltrated PMN leukocytes of the liver in the presence of excess of aldehydes formed during acute methanol intoxication are of relevant importance in lipid peroxidation of membranes. It is known that products of this process are toxic to the liver (8).

c~-Tocopherol

Rat

Methanol toxicity, either acute or chronic, is characterized by a severe derangement of subcellular metabolism and structural alteration of different cells. This fact can be associated with mitochondrial injury, especially with regard to the cell respiratory chain; partial inhibition of this chain may cause increased auto-oxidation of a redox carrier, resulting in increased production of oxygen radicals (14,29). Lipid peroxidation is considered to be an efficient triggering mechanism of the disassembly of microsomal membranes and cytochrome P-450. A n inverse correlation exists between the steady-state concentration of lipid peroxidation product and cytochrome P-450 content in the liver endoplasmic reticulum membranes (12). This lipid peroxidation can induce a chain reaction ineluding activation of endogenous phospholipases and proteases, resulting in disassembly of cytochrome P-450 (6). In addition, cytochrome P-450 also catalyzes the reductive transformation of foreign compounds. This type of pathway results mostly in toxification due to reactive free radicals. Data on the antioxidant defense potential in the liver, erythrocytes, and blood serum after methanol intoxication in rats are lacking. In this report, particular attention has been

Requests for reprints should be addressed to R. Farbiszewski, Department of Analytical Chemistry, Medical Academy, P.O. Box 14, 15-230 Bialystok 8, Poland. 431

432

SKRZYDLEWSKA AND FARBISZEWSKI

drawn to the antioxidant status expressed in enzyme activities and in low molecular substances in liver, erythrocytes, and blood serum of rats after one dose of methanol, which were observed for 7 consecutive days. Methanol concentration, hematologic parameters, and acid base balance in the blood during this intoxication have been also analysed. METHOD

Male Wistar rats (approximately 200 g b.wt.) fed on a standard diet (containing no antioxidants but 0.55% of cysteine and methionine) were used. All procedures were in strict accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. Sixty animals were given (by the intragastric route) 50% methanol (6 g/kg b.wt.) in isotonic NaC1 solution with syringe through a plastic tube. This quantity of methanol is used in experimental acute methanol intoxication (17). Equivalent volume of saline was given to 10 control rats. The intoxicated rats were divided into six groups (10 rats in each group). A t 6, 12, and 24 h, and 2, 5, and 7 days after methanol administration, the animals were sacrificed under ether anaesthesia; their livers were removed quickly and placed in iced 0.15 M NaC1, perfused with the same solution to remove the blood cells, then blotted on filter paper, weighed, and homogenized in 9 ml of ice-cold 0.25 M sucrose and 0.15 M NaC1 with addition of 6 Ixl of 250 mM BHT in ethanol (to prevent formation of new peroxides during the assay). Homogenization procedure was performed under completely standardized conditions. Ten percent homogenates were centrifugated at 10,000 × g for 15 min at 4°C and the supernatant was kept on ice until it was assayed. Blood for biochemical and hematological measurements was taken by cardiac puncture into containers with E D T A . Erythrocytes were separated by centrifugation and washed with 5 ml of 0.15 M NaC1. Erythrocytes were lysed by the addition of 2.0 ml of ice-cold distilled water. Cell membranes and hemoglobin were removed from the resulting mixtures by extraction with 1.0 ml of cold ethanol and 0.6 ml of cold chloroform followed by centrifugation at 31,000 × g for 60 min at 4°C, as described by McCord and Fridovich (16). The supernatant was used to analyse superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R). Cu,Zn-SOD (EC.1.15.1.1) activity in the liver tissue, erythrocytes, and in blood serum was determined by the method of Misra and Fridovich (20) as modified by Sykes et al. (27), which measures the activity of cell cytosolic SOD. Mn-SOD of the liver mitochondria is known to be destroyed during this procedure. A standard curve for SOD activity was made using SOD from bovine erythrocytes (Sigma Biochemicals, St. Louis, MO). One unit of SOD was defined as the amount of the enzyme that inhibits by 50% epinephrine oxidation to adrenochrome. The enzyme activity was expressed in units per milligram of protein for liver and serum or per milligram of hemoglobin for erythrocytes. Catalase (EC.1.11.1.9) activity was measured after 30-min preincubation of the postmitochondrial fraction of the liver homogenate with 1% Triton X-100 by measuring the decrease in absorbance of hydrogen peroxide at 240 nm (1). The rates were determined at 25°C using 10 mM hydrogen peroxide and the activity was expressed as micromoles of H202 decomposed/minute per milligram of protein for the liver and serum and per milligram of hemoglobin for erythrocytes. Glutathione peroxidase (EC. 1.11.1.6) activity was measured in the liver, erythrocytes, and serum specrophotometrically

using a technique based on Paglia and Valentine (22) whereas G S H formation was assayed by measurement of conversion of N A D P H to NADP. The enzyme activity was expressed as micromoles of NADPH/minute per milligram of protein for the liver and per milligram of hemoglobin for erythrocytes. Glutathione reductase (EC.1.6.4.2) activity was measured by the method of Mize and Langdon by monitoring the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm (21). Sulfhydryl compounds were estimated according to Ellman using 5,5'-dithiobis(nitrobenzoic acid) (DTNB) in whole and in previously deproteinized (with perchloric acid) liver homogenates and blood serum (7). Liver and serum ascorbic acid was measured according to Kyaw (13). Serum tocopherol was analysed by HPLC (24). Lipid peroxidation in the fiver, erythrocytes, and blood serum was assayed by the method described by Buege and Aust (3). The following hematological parameters were estimated: total erythrocytes count by chamber method, hemoglobin level (Hb) by the cyanomethaemoglobin method, and hematocrit by micromethod. The mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were also calculated. Blood methanol was measured in a Pye Unicam gas chromatograph equipped with a flame ionization detector (column: glass capillary, length 1.5 m, liquid phase carbowax 20M on anacrom 90/100). The column temperature was 90°C with injector and detector temperatures of 200°C and 180°C, respectively. Isopropanol was added to each blood sample as the internal standard. Blood p H was estimated in an acid-balance analyser (CibaCorning type 288). Diagnostic Cormway test was used for assessment of blood serum alanine (ALT) and aspartate aminotransferase (AST) activities. The results were expressed as mean _+ SD. Statistical analysis was performed using Student's t-test for unpaired data, and values of p < 0.001 to p < 0.05 were considered significant. RESULTS The liver activity of Cu,Zn-SOD and C A T after 6, 12, and 24 h of methanol ingestion was significantly increased compared with the control group (Table 1), but the activity of GSH-Px and GSSG-R in the same time was decreased and did not reach control values 7 days after methanol intoxication. Nonprotein- and protein-bound sulfhydryl compounds and ascorbic acid were significantly decreased 5 days after the intoxication and returned to normal by 7 days. Lipid peroxidation in the liver was enhanced by methanol treatment as evidenced by the significant increase in TBA-rs formation. The effect of methanol ingestion on the antioxidant status of erythrocytes is shown in Table 2. The activity of Cu,Zn-SOD, GSH-Px, and GSSG-R was significantly decreased after methanol intoxication. The GSSG-R activity and the sulfhydryl compounds, which were mostly reduced 24 h after methanol intake, dropped to 0.44 nmol NADPH/min × mg of Hb, and 298 nmol/ ml, respectively. Sulfhydryl compounds were significantly decreased during 7 days of erythrocyte observation. TBA-rs content was elevated in the highest degree 2 days after methanol ingestion. These values were enhanced even after 7 days. Table 3 shows the serum antioxidant potential from control rats and animals treated with methanol. The activity of

METHANOL

EFFECTS ON ANTIOXIDANT

433

MECHANISMS

TABLE 1 ANTIOXIDANT PARAMETERS IN THE LIVER FROM CONTROL RATS AND ANIMALS TREATED WITH METHANOL Intoxication Time Parameters Analysed Cu, Zn-SOD (U/mg of protein) CAT (U/mg of protein) GSH-Px (ixmol NADPH/ min × mg of protein) GSSG-R (nmol NADPH/min × mg protein) Nonprotein sulfhydryl compound (txmol/g of tissue) Protein sulfhydryl compounds (p~mol/g of tissue) Ascorbic acid (p~g/g of tissue) TBA-rs (nmol/g of tissue)

6h

12 h

24 h

2 Days

12.0 _+ 0.5

14.4 ± 0.6§

13.8 ± 0.6§

13.2 ± 0.5§

12.0 ± 0.5

12.0 -+ 0.5

12.0 ± 0.5

232 _+ 16

324 ± 14§

320 ± 16§

311 ± 13§

283 ± 13§

260 ± 14§

235 ± 14

132.8 ± 10.7

119.2 ± 10.8'

85.0 ± 12.7§

74.1 ± 10§

87.8 ± 8.1§

108.0 ± 8.6§

116.6 ± 6.7§

38.3 ± 3.1

28.0 ± 3.3§

24.2 ± 3.2

17.1 ± 2.3§

22.0 ± 2.6§

33.1 ± 2.9t

34.8 ± 2.7*

4.47 ± 0.40

3.92 ± 0.46*

3.48 ± 0.48§

3.07 ± 0.46§

3.50 ± 0.45§

4.08 ± 0.34*

4.20 ± 0.39

15.5 + 1.1 81.0 ± 4.7 84.1 ± 8.2

13.9 ± 1.2t 80.6 ± 4.6 90.4 ± 8.7

13.4 ± 1.0§ 73.5 ± 4.35 92.8 ± 7.7*

14.6 ± 0.9 78.9 ± 4.7 86.3 ± 6.7

11.5 ± 1.0§ 78.2 ± 5.0 109.0 ± 8.8§

10.1 ± 0.9§ 71.1 ± 4.8§ 117.9 _+ 11.0§

5 Days

7 Days

Control

12.0 ± 0.9§ 70.5 ± 4.6§ 101.2 ± 7.7§

Significantly different from control value: *p < 0.05, tP < 0.01; 5P < 0.005; §p < 0.001.

G S H - P x a n d G S S G - R b e c a m e gradually d e c r e a s e d after m e t h a n o l intoxication, w h e r e a s t h e activity of S O D was gradually i n c r e a s e d after 6, 12, a n d 24 h. T h e c o n c e n t r a t i o n s of ascorbic acid, et-tocopherol, n o n p r o t e i n - a n d p r o t e i n - b o u n d sulfhydryl c o m p o u n d s w e r e decreased. A s c o r b i c acid a n d a t o c o p h e r o l c o n c e n t r a t i o n s w e r e still r e d u c e d after 7 days comp a r e d with the c o n t r o l group, w h e r e a s n o n p r o t e i n sulfhydryl c o m p o u n d s level was r e s t o r e d to n o r m a l values in this time. T B A - r s c o n t e n t s were i n c r e a s e d d u r i n g o b s e r v a t i o n . Following m e t h a n o l i n g e s t i o n a d e c r e a s e of e r y t h r o c y t e a m o u n t in the blood, h e m o g l o b i n level, h e m a t o c r i t , a n d M C V was f o u n d ( T a b l e 4). A f t e r a single dose of m e t h a n o l we o b s e r v e d in rats t h e red u c t i o n of b l o o d p H f r o m 7.37 to 7.24 after 6 h, 7.19 after 12 h, 7.22 after 24 a n d 48 h, a n d 7.33 after 7 days of m e t h a n o l intoxication (Fig. 1 ). T h e b l o o d m e t h a n o l level was high d u r i n g the

first day of intoxication ( a b o u t 450 mg/100 ml). A f t e r 2 days it was d e c r e a s e d to 150 mg/100 ml a n d after 5 days it d r o p p e d to zero. M o r e o v e r , t h e b l o o d acid base i m b a l a n c e was evident. Results of s e r u m e n z y m e tests suggest d a m a g e to p a r e n chymal liver cells in rats receiving m e t h a n o l (Fig. 2). T h e activity of all e n z y m e s e x a m i n e d was significantly i n c r e a s e d d u r i n g 5 days after m e t h a n o l intoxication. DISCUSSION Toxic effects of m e t h a n o l o n h u m a n a n d a n i m a l s o r g a n i s m h a v e b e e n well d o c u m e n t e d a n d c o n f i r m e d by basic b i o c h e m ical researches, m o r p h o l o g i c a l studies, a n d in m o s t clinical exa m i n a t i o n s d u r i n g accidental i n t o x i c a t i o n in h u m a n . T h e influence of m e t h a n o l o n a n a n t i o x i d a n t status e x p r e s s e d in e n z y m e s activities ( S O D , G S H - R , G S S G - R , a n d C A T ) as

TABLE 2 ANTIOXIDANT PARAMETERS OF ERYTHROCYTES FROM CONTROL RATS AND ANIMALS TREATED WITH METHANOL intoxication Time Parameters Analysed Cu, Zn-SOD (U/mg of Hb) CAT (U/mg of Hb) GSH-Px 0zmol NADPH m i n x mg of Hb) GSSG-R (nmol NADPH min × mg of Hb) Nonprotein sulfhydryl compounds (nmol/ml) TBA-rs (nmol/ml)

Control

6h

12 h

24 h

2 Days

5 Days

7 Days

2.58 _+ 0.21

2.21 ± 0.23t

1.8 ± 0.215

1.95 ± 0.205

2.12 _+ 0.205

2.48 _+ 0.21

2.63 _+ 0.19

5.99 ± 0.32

8.76 ± 0.495

9.92 ± 0.485

10.23 ± 0.535

9.17 ± 0.495

7.21 ± 0A25

6.13 ± 0.31

3.27 _+ 2.0

27.9 ± 3.15

20.4 _+ 2.85

13.5 +_ 2.65

22.1 _+ 2.55

28.3 _+ 2.55

30.7 _+ 2.4

2.19 ± 0.17

1.55 ± 0.21:~

0.93 ± 0.165

0.44 ± 0.095

1.07 + 0.115

1.51 _+ 0.115

1.96 ± 0.13t

558 +_ 31 162 _+ 9

451 _+ 435 178 ± 15"

367 ± 355 203 ± 155

298 _+ 345 234 _+ 145

339 _+ 325 259 _+ 155

427 ± 325 216 ± 135

503 ± 315 182 ± 115

Significantly different from control value: *p < 0.01; tP < 0.005; 5P < 0.001.

434

SKRZYDLEWSKA

AND FARBISZEWSKI

TABLE 3 ANTIOXIDANT PARAMETERS IN BLOOD SERUM FROM CONTROL RATS AND ANIMALS TREATED WITH METHANOL Intoxication Time Parameters Analysed

Control

SOD (U/mg of protein) GSH-Px (ixmol NADPH/ min × mg of protein) GSSG-R (nmol NADPH/ min × mg of protein) Nonprotein sulfhydryl compounds (ixmol/ml) Protein sulfhydryl compounds (p~mol/ml) Ascorbic acid (mg/dl) a-Tocopherol (mg/dl) TBA-rs (nmol/ml)

6h

12 h

24 h

0.82 _+ 0.06

1.13 ± 0.10§

1.07 ± 0.10§§

14.6 ± 0.7

12.8 _+ 1.0§

0.85 ± 0.05

2 Days

5 Days

7 Days

0.97 ± 0.09

0.97 ± 0.08

0.83 _+ 0.06

0.75 _ 0.06

10.9 _+ 0.9§

9.4 _+ 0.8§

9.7 _+ 0.8§

10.9 ± 0.8§

12.7 +_ 0.7§

0.68 ± 0.07§

0.51 ± 0.05§

0.49 ± 0.05§

0.55 ± 0.05§

0.69 ± 0.05§

0.79 _+ 0.06*

0.29 ± 0.02

0.20 ± 0.03§

0.14 ± 0.02§

0.12 ± 0.01§

0.15 ± 0.01§

0.23 +_ 0.01§

0.27 ± 0.02

2.19 2.74 1.66 2.15

2.00 2.35 1.62 2.45

1.67 2.07 1.47 2.71

1.37 1.86 1.17 2.89

1.42 1.93 1.08 2.97

1.53 2.18 1.22 2.67

1.89 2.37 1.45 2.41

± 0.14 ± 0.20 ± 0.11 _+ 0.15

± ± ± ±

0.17" 0.24§ 0.15 0.235

± 0.15§ ± 0.22§ ± 0.13t _+ 0.22§

± ± ± ±

0.13§ 0.20 0.12§ 0.23§

÷ 0.13§ ± 0.18 ± 0.11§ _+ 0.23§

± ± ± ±

0.11§ 0.18§ 0.11§ 0.22§

_+ 0.12§ ± 0.18§ ± 0.10§ ± 0.185

Significantly different from control value: *p < 0.05; tp < 0.01; Sp < 0.005; §p < 0.001.

well as m o l e c u l a r substances in the liver, erythrocytes, a n d ser u m of rats has n o t b e e n e v a l u a t e d to date. A direct c o n n e c tion b e t w e e n m e t h a n o l a n d t h e f o r m a t i o n of s u p e r o x i d e a n i o n radicals has n o t b e e n clearly e s t a b l i s h e d either. It is k n o w n t h a t m a m m a l i a n r e d cells are n o t able to oxidize m e t h a n o l , d u e to the a b s e n c e of a specific m e t h a n o l oxidase. B u t enz y m e - l o a d e d e r y t h r o c y t e s a t t a i n t h e ability to detoxify m e t h a nol (15) a n d so they b e c a m e m o r e effective t h a n any p r o p h y laxis against m e t h a n o l p o i s o n i n g k n o w n at p r e s e n t . O u r studies reveal t h a t o n e dose of 6 g/kg b.wt. m e t h a n o l in rats leads to changes in a n t i o x i d a n t m e c h a n i s m s in the liver, erythrocytes, a n d serum. It seems t h a t t h e increase in C A T a n d S O D activities in the liver and especially the increase in erythrocytes C A T activities could result f r o m g r e a t e r availability of active site cofactors (i.e., t r a n s i t i o n m e t a l ions t h a t are decisive factors controlling the expression of the e n z y m e activity). O x i d a t i v e stress in rats i n t o x i c a t e d with m e t h a n o l t e n d s to e n h a n c e the n e e d for copp e r to sustain C u , Z n - S O D activities t h a t in the p r e s e n c e of a b u n d a n c e of cofactors could increase the activity. C o p p e r a n d iron ions in oxidative stress can b e t r a n s l o c a t e d from ceru l o p l a s m i n a n d iron-consisting h e m p r o t e i n s a n d thus b e c o m e

m o r e available to t h e i r a n t i o x i d a n t enzymes. T h e e v i d e n c e indicates t h a t cofactors, especially copper, exert m a j o r regulation o v e r C u , Z n - S O D expression (11) a n d t h e r e f o r e favourably c h a n g e its reductive potential. D u r i n g the first day of intoxication a very high m e t h a n o l level was o b s e r v e d in t h e blood. T h e c h a n g e s in e r y t h r o c y t e s s t r u c t u r e a n d d e c r e a s e in different e n z y m e activity were n o t e d in the s a m e time. It seems this is caused by direct a n d inactivating m e t h a n o l effect o n the erythrocytes. Particularly e r y t h r o c y t e s e x h i b i t e d significantly l o w e r e d activity of key antioxidant d e f e n s e enzymes, C u , Z n - S O D , G S H - P x , G S S G - R , a n d of ascorbate, a - t o c o p h e r o l , a n d n o n p r o t e i n - a n d p r o t e i n b o u n d sulfhydryl c o m p o u n d s after m e t h a n o l intoxication. T h e r e d u c t i o n of C u , Z n - S O D , G S H - P x , a n d G S S G - R activities in e r y t h r o c y t e s of rats after m e t h a n o l is p r o b a b l y conn e c t e d with d a m a g e to t h e structure of these e n z y m e s and, hence, with a d e c r e a s e of t h e i r activity in vivo. R e d u c e d activity of e r y t h r o c y t e s C u , Z n - S O D m a y also b e due to a n inhibited biosynthesis of e n z y m e molecules by m e t h a n o l or by its m e t a b o l i t e s a n d / o r to t h e effect of s u p e r o x i d e radicals, which m a y directly alter t h e i r catalytic potential. T h e d a m a g e to the C u , Z n - S O D m o l e c u l e m i g h t h a v e b e e n related to the hy-

TABLE 4 HEMATAOLOGICAL PARAMETERS IN CONTROL RATS AND ANIMALS TREATED WITH METHANOL Intoxication Time Parameters Analysed Erythrocytes 106/mm 3 Hb (g/dl) HCT (%) MCV (1~3) MCH (ixg) MCHC (g/ml)

Control

8.07 15.8 43.1 53.3 19.9 37.4

± ± ± ± ± ±

0.94 1.7 3.7 5.8 1.8 3.1

6h

6.56 13.1 33.7 50.8 20.1 39.6

± 0.975 _+ 1.85 ± 3.9§ +_ 5.9 ± 2.0 ± 3.7

12 h

6.03 12.8 31.4 49.1 24.2 46.6

± 1.10§ _+ 1.85 + 4.1§ _+ 6.2 _+ 2.7§ ± 4.5§

24 h

4.90 12.2 25.1 45.7 27.8 54.5

_+ 0.91§ ± 1.6§ _+ 3.6§ ± 6.4* ± 2.5§ ± 5.3§

Significantly different from control value: *p < 0.05; t p < 0.01; 5p < 0.005; §p < 0.001.

2 Days

5.91 13.9 28.2 47.8 23.8 49.2

± 0.95§ _+ 1.5 ± 3.7§ ± 6.3 ± 2.1§ ± 5.0§

5 Days

6.86 14.1 34.8 50.8 20.6 40.2

± ± ± + ± ±

0.87t 1.5" 3.6 6.0 2.0 4.1

7 Days

7.84 15.2 39.9 52.1 19.8 38.1

± 0.85 ± 1.6 ± 3.9 ± 6.0 _+ 2.0 ± 3.6

M E T H A N O L EFFECTS ON A N T I O X I D A N T M E C H A N I S M S

435 7,5

500 7,4 400

7,3

'~ 300

="

E 200

7,2

100

7,1

0

I control

6h

7 12h

2days

24h

5days

7days

intoxication time --e--methanol --II--- pH

FIG. 1. Methanol concentration and pH in the blood of control rats and animals treated with methanol. *Significantly different from control value (p < 0.001).

droxyl radical, the product of reaction of superoxide and hydrogen peroxide. Cu,Zn-SOD selectively scavenges superoxide anion radicals and the resulting hydrogen peroxide is removed from the erythrocytes mainly by the GSH-Px system (9), and this suggests the importance of the N A D P H - G S H - P x system in scavenging toxic species of oxygen. The superoxide radical is the primary free radical species involved in various tissue injuries. It undergoes spontaneous dismutation to produce hydrogen peroxide and, in the presence of iron ions, hydrogen radicals

(26), which are the most widely proposed initiators of lipid peroxidation that is evaluated by measurement of the level of thiobarbituric acid-reactive substances. The key enzyme in the maintenance of the liver environment in the reduced state is GSSG-R, which catalyses the conversion of oxidized glutathione form (GSSG) to reduced glutathione form (GSH) using N A D P H (18). This form, being a powerful reducing agent, plays a major role in the cellular defense against exogenous oxidant (18). The activity of GSSG-R in our studies correlates with the nonprotein- and protein-bond

300

250

20O

150

100

50 0 control

6h

12h

24h

2days

5days

7days

intoxication time [] A L T fm A S T

FIG. 2. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in control rats and animals treated with methanol. *Significantly different from control value (p < 0.005); **significantly different from control value (p < 0.001).

436

S K R Z Y D L E W S K A AND F A R B I S Z E W S K I

sulfhydryl compounds. They are essential for the optimal functioning of numerous SH-dependent enzymes. The results of the present study show that one dose of methanol elicits an increase of malondialdehyde content (MDA) (measured as TBA-rs) in the liver, RBCs, and serum, but the greater increase was in RBCs. On the following days the M D A level decreased gradually. RBCs are susceptible to methanol toxicity as the result of the high polyunsaturated fatty acid contents of membrane and the high concentration of oxygen and Hb; the later is a potentially powerful promoter of oxidative processes (5). Furthermore, membrane proteins are sensitive to covalent modification by lipid peroxidation products. M D A is metabolised in a living organisms in many nonenzymatic and enzymatic pathways. It can be oxidatively metabolized only in the liver to CO2 and H20. In this process aldehyde dehydrogenases first convert M D A to malonic acid semialdehyde, which spontaneously decarboxylates to acetaldehyde. Then this is further oxidised to acetate and to CO2 and H20 (25). The diminution of sulfhydryl compounds in erythrocytes, liver, and serum results in oxidative damage of cell and defective transport of some amino acids into cell. Expression of amino acid transport deficiency was shown to precede the decrease of intracellular SH groups (10). The sulfhydryl compounds participate in the degradation of hydrogen peroxide, which is continuously formed in small amounts in the course of metabolic pathway and also under the influence of some drugs. Sulfhydryl compounds also protect hemoglobin against oxidative denaturation of its protein. It should be emphasised that the diminution of sulfhydryl compounds is greater in erythrocytes than in the liver and in serum. It is suggested that the liver cells possess greater range of protecting system against the toxic methanol. Changes in some hematological parameters are connected with this phenomenon. Erythrocytes subjected to various

chemical factors can be rapidly transformed into a broad spectrum of transitional shape, which may alter the hemodynamic properties of blood and decrease the cell survival in the circulation (2). The lipid bilayer of erythrocyte membrane and/or its underlying cytoskeletal protein network may be directly responsible for the control of cell shape and specific rheologic properties. Methanol, like ethanol, has deleterious effects on the red blood cells (unpublished data). It is well known that ascorbic acid is beneficial in reducing oxidative stress but, on the other hand, it can be harmful depending on the sensitive balance of its concentration. It can react with aqueous peroxyl radical and restores the antioxidant properties of fat-soluble c~-tocopherol. Therefore, it interrupts the radical chain reaction of lipid peroxidation. The effect of ascorbate is complex and the relative contribution of antioxidant and prooxidant effects may depend on a number of cofactors, including transition metal status (19,30). The decrease of ascorbic acid concentration can diminish the cellular resistance of liver cells to oxidative damage. This phenomenon is associated with parallel increase in TBA-rs. These data are in agreement with results of other authors that showed the interrelationship between ascorbate acid and TBA-rs content (4). Methanol administered to rats in a dose 1.5 g/kg g.wt. elicited almost the same changes in the antioxidant status of the liver and erythrocytes as in the dose applied in the present study. In conclusion, we hypothesize that the oxidative stress induced by metabolism of methanol creates changes in the liver, erythrocytes, and blood serum of rats, which in turn lead to changes in some enzymatic and nonenzymatic antioxidant defense system. ACKNOWLEDGEMENT This work was supported by a grant from Polish Committee of Scientific Research No. 4P05B04011.

REFERENCES

1. Aebi, H.: Catalase in vitro. Methods Enzymol. 105:141-146;1984. 2. Branemark, P. I.; Bagge, V.: Intravascular rheology of erythrocytes in man. Blood Cells 3:97-130; 1977. 3. Buege, J. A.; Aust, S. D.: Microsomal lipid peroxidation. Methods Enzymol. 52:302-310; 1978. 4. Cadenas, S.; Rojas, C.; Perez-Campo, R.; Lopez-Torres, M.; Barja, G.: Effect of dietary vitamin C and catalase inhibition on antioxidants and molecular markers of oxidative damage in guinea pigs. Free Radic. Res. 21:109-118; 1994. 5. Clemens, M. R.; Waller, H. D.: Lipid peroxidation in erythrocytes. Chem. Phys. Lipids 45:251-268; 1987. 6. Davies, K. J. A.: Protein damage and degradation by oxygen radicals. I. General aspects. J. Biol. Chem. 262:9895-9901; 1987. 7. Ellman, G. L.: Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70-77; 1959. 8. Esterbauer, H.; Gebicki, J.; Puhl, H.; Jurgens, G.: The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13:341-390;1992. 9. Fridovich, I.; Freeman, B.: Antioxidant defenses in the lung. Annu. Rev. Physiol. 48:693-702; 1986. 10. Haest, C. W.; Kamp, D.; Deuticke, B.: Formation of disulfide bonds between glutathione and membrane SH groups in human erythrocytes. Biochim. Biophys. Acta 557:363-371; 1979. 11. Harris, E. D.: Regulation of antioxidant enzymes. FASEB J. 6:2675-2683; 1992. 12. Kagan, V. E.; Kotelevtsev, S. V.; Kozlov, Iu. P.: Rol' fermentativnogo perekisnogo okisleniia fosfolipidovv mekhanizme razborki

membran endoplazmaticheskogoretikuluma in vivo. Dokl. Acad. Nauk SSSR 217:213-216;1974. 13. Kyaw, A.: A simple colorimetric method for ascorbic acid determination in blood plasma. Clin. Chim. Acta 86:153-157; 1978. 14. Liesivuori, J.; Savolainen, H.: Methanol and formic acid toxicity: biochemical mechanisms. Pharmocol. Toxicol. 69:157-163; 1991. 15. Magnani, M.; Fazi, A,; Magnani, F.; Rossi, L.; Mancini, U.: Methanol detoxification by enzyme-loaded erythrocytes. Biotechnol. Appl. Biochem. 18:217-226;1993. 16. McCord, J. M.; Fridovich, I.: The generation of superoxide radicals during the autooxidation of hemoglobin. J. Biol. Chem. 247:69606962; 1972. 17. McMartin, K. E.; Makar, A. B.; Martin-Amat, G.; Palese, M.; Tephly, T. R.: Methanol poisoning. I. The role of formic acid in the development of metabolic acidosis in the monkey and the reversal by 4-methylpyrazole. Biochem. Med. 13:319-333;1975. 18. Meister, A.: Selective modification of glutathione metabolism. Science 220:471-477; 1983. 19. Minotti, G.; Aust, S. I.: The requirement for iron (III) in the initiation of lipid peroxidation by iron (II) and hydrogen peroxide. J. Biol. Chem. 262:1098-1104; t987. 20. Misra, H. P.; Fridovich, I.: The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutation. J. Biol. Chem. 247:3170-3174;1972. 21. Mize, C. E.; Langdon, R. G.: Hepatic glutathione reductase. I. Purification and general kinetic properties. J. Biol. Chem. 237:1589-1595; 1%2.

METHANOL

EFFECTS ON ANTIOXIDANT

MECHANISMS

22. Paglia, D. E.; Valentine, W. N.: Studies on the quantitative and qualitative charakterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70:158-169; 1967. 23. Poli, G.: Liver damage due to free radicals. Br. Med. Bull. 49:604609; 1993. 24. Rudy, I. L.; Ibarra, F.; Zeigler, M.; Howard, J.; Argyle, C.: Simultaneous determination of retinol, retinyl palmitate and alphatocopherol in serum or plasma by reversed phase high performance liquid chromatography. LC-CG Intern. 3:36-38; 1990. 25. Siu, G. M.; Draper, H.: Metabolism of malondehyde in vivo and in vitro. Lipids 17:349-355; 1982. 26. Southorn, P. A.; Powis, G.: Free radicals in medicine. I. Chemical nature and biological reactions. Mayo Clin. Proc. 63:381-389; 1988.

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27. Sykes, J. A.; McCormac, F. X.; O'Brien, T. J.: Preliminary study of the superoxide dismutase content of some human tumors. Cancer Res. 38:2759-2762; 1978. 28. Tephly, T. R.; Atkins, M.; Mannering, G. J.; Parks, R. E.: Activation of a catalase peoxidative pathway for the oxidation of alcohols in mammalian erythrocytes. Biochem. Pharmacol. 14:435444; 1965. 29. Tephly, T. R.: The toxicity of methanol. Life Sci. 48:1031-1041; 1991. 30. Young, I. S.; Torney, J. J.; Trimble, E. R.: The effect of ascorbate supplementation on oxidative stress in the streptozotocin diabetic rat. Free Radic. Biol. Med. 13:41-46: 1992.