ACTA FAC. MED. NAISS. 2003; 20 (3): 183-188

Original article

EFFECTS OF CAPTOPRIL ON MEMBRANE-ASSOCIATED ENZYMES IN LEAD-INDUCED HEPATOTOXICITY IN RATS
Tatjana Jevtović-Stoimenov, Gordana Kocić, Dušica Pavlović, Ivana Stojanović, Tatjana Cvetković, Dučan Sokolović, Jelena Bašić Institute of Biochemistry, School of Medicine, University of Niš
Ass. Dr Tatjana Jevtović Stoimenov, tjevtovic@yahoo.com

INTRODUCTION

Lead (Pb), a well-known environmental toxin, is one of the major hazards for human health. Although lead as an etiological factor was identified as early as 200 BC, it remains a common entity even today. In the past centuries lead acetate was added to wine to sweeten it. Retained bullets can result in lead poisoning because synovial fluid appears to be a good solvent for lead (1). The environmental sources of lead are urban air due to the use of leaded gasoline, soil contaminated with exterior lead paint, the water supply due to lead plumbing, and house dust in homes with interior lead paint (2). More than 1 million workers in over 100 different occupations have been exposed to lead (mining, spray painting, recycling, and radiator repair). Swallowing lead household objects, such as lead curtain weights have poisoned children. Daily lead intake is approximately 300 mg. When daily intake rises to 1000 mg the symptoms of lead intoxication appear (3). Inhalation is most important route of lead intoxication compared to ingestion. The absorbed lead is mainly (80% to 85%) taken up by bone and developing teeth in children; the blood accumulates 5% to 10%, and the remainder is distributed throughout the soft tissues.
The critical interest in lead poisoning arises from the fact that industrialization led to general population lead poisoning by increasing the whole body lead content from 2 to 200 mg.
Lead induces a wide range of physiological, biochemical and behavioral dysfunctions. The major effects of lead are related to its multiple biochemical effects:
• high affinity for sulfhydryl groups (inhibition of d-aminolevulinic acid dehydratase and Ferro-oxidase)(4);
• competition with calcium ions (especially in bones, nerve transmission and brain development);
• inhibition of membrane-associated enzymes (5'-nucleotidase, sodium-potassium ion pumps)(5);
• impaired metabolism of calcitriol.
Although clinical symptoms of lead poisoning are well known, the molecular mechanisms underlying the toxicity still need to be investigated. Recent studies suggest that oxidative stress is a potential contributor to lead toxicity and that lead can change the pro-oxidant/ antioxidant balance in the biological tissues (6). Inorganic Pb acetate is a pro-oxidant, and peroxidation damage of cellular membrane lipids leading to membrane fragility and permeability, is a likely consequence of Pb poisoning. The assumption of oxidative stress as a mechanism in lead toxicity suggests that antioxidants might play an important role in therapy.
Captopril, an angiotensin-converting enzyme inhibitor (ACE-i) widely used in anti-hypertensive therapy, has been postulated as a free radical scavenger (7). Furthermore, recent studies have indicated that SH-containing ACE-inhibitor also modulates antioxidative defense system.
In the present study, we investigated in vivo effects of captopril on lead-induced liver injury and membrane-associated enzyme activity. The selected membrane-associated enzymes and parameters of liver function such as alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), 5'-nucleotidase (5'-NT) activity and plasma urea, albumins and total proteins level were measured in blood and liver of lead exposed rats. In addition, the ability of captopril to raise the d-aminolevulinic acid dehydratase (ALA-D) activity of Pb-exposed animals was examined.
 

MATERIALS AND METHODS

The experiment was performed on 12 weeks old female Srague Dawley rats weighing 150 g. The animals were housed in steel cages, in a temperature-controlled room (22°C) with a 12-hour light: dark cycle. The animals were divided into four groups: Group I (n=5) served as the control one, rats were treated with physiological saline (0.85% NaCl) intra-peritoneally (i.p.); group II (n=5) was treated with Pb-acetate (25mg/kg b.w. i.p. daily during 5 days); group III received captopril (100mg/kg b.w.i.p.) whereas the IV group of animals were treated with both agents i.p. simultaneously. After five days the animals were anesthetized, blood samples were collected via abdominal aorta and liver was removed for analyses. Hepatic tissue was homogenized on ice with a Teflon homogenizer.
Analysis
The activity of ALA-D, 5'-NT, g-GT and ALP was measured in 10% liver homogenate and g-GT, ALP, urea, albumins and total proteins were measured in plasma applying the adequate spectrophotometrical methods. Hepatic and blood ALA-D activity was analyzed using the method described by Mitchell et al. (8). The activity of 5'-NT was measured spectrophotometrically based on phosphorous liberation with the use of 10 mmol AMP as a substrate in barbiturate buffer pH 7.73 modified for tissue homogenate according to the method of Wood and Williams (10). ALP was estimated by the method of Bodansky (1933) based on measuring of inorganic P liberated from beta-disodium glycerol phosphate (11).
The tissue protein concentration was determined applying the method of Lowry et al. (12) using bovine serum albumin as a standard.
The results were presented as mean ± SD. The statistical significance was evaluated by the Student's t-test for paired samples.
 

RESULTS

Table 1 displays ALA-D activity assay. As it was shown, Pb-acetate alone caused significant decrease of d - aminolevulinic acid dehydratase in liver tissue (p<0.001), blood (p<0.01) and bone marrow (p<0.05). In rats, treated with captopril and Pb-acetate the activity of ALA-D was completely recovered in liver and bone marrow, but not in blood.

Table 1. The effect of captopril on ALA-D activity in liver, blood and bone marrow in lead exposed rats

*** p<0.001; **p<0.01;*p<0.05 Pb-acetate vs. control

Table 2 displays the results of alkaline phosphatase activity in liver and plasma. The activity of liver alkaline phosphatase in lead-exposed rats is significantly increased (p<0.05) in comparison with the control group of animals, although plasma activity of this enzyme didn't change in all investigated groups of rats. Furthermore, captopril treated lead-exposed animals showed reduced ALP activity compared to the rat treated only with Pb (p<0.05).
 

Table 2. The effect of lead exposure on liver and plasma ALP activity in the presence or absence of captopril treatment

*p<0.05; Pb-acetate vs. control ^o p<0.05; Pb+Captopril vs. control
^o p<0.05 Pb+Captopril vs. Pb-acetate

The activity of 5'-NT in rat liver treated with toxic doses of Pb-acetate and protective doses of captopril is showed in Table 3. As it can clearly be seen, the activity of 5'-NT in the liver of lead induced injury was significantly reduced (p<0.05). During the simultaneous treatment with both agents (captopril and lead-acetate) the activity of 5'-NT is almost completely recovered in relation to the activity in liver of lead exposed animals.
 

Table 3. The effect of captopril on liver 5’-NT activity in lead treated rats

***p<0.001; vs. control
^o p<0.05; Pb+Captopril vs. Pb-acetate

Table 4. The effect of captopril on plasma urea, total proteins and albumins level in lead exposed rats

*** p<0.001; **p<0.01;*p<0.05 Pb-acetate vs. control

In group treated with Pb-acetate the activity of plasma and liver g-GT was significantly increased (p<0.05) compared to the control values (Table 4). However, after captopril treatment of lead-exposed animals the activity was similar to the control group.
The levels of plasma urea did not show any statistical differences among the investigated groups. However, the level of plasma proteins and albumins in lead exposed rats were significantly lower than in the control group (p<0.01). In animals treated with both agents, the levels of the mentioned parameters were more reduced (p<0.01) in relation to the control group.
 

DISCUSSION

In the present study we investigated the protective effects of captopril on liver membrane-associated enzymes (ALP, 5'-NT, GGT) in liver and plasma as well as the levels of plasma urea, total proteins and albumens in rats exposed to the toxic doses of lead acetate.
Liver is the major lead deposition organ besides teeth, bones and kidneys. Inorganic lead could provoke very serious functional and anatomic disturbances in liver and the whole body as well, since liver plays many important roles (metabolic, synthetic, defense and detoxification). Several lines of evidence suggest that cellular damage mediated by oxidants may be involved in some of the pathology associated with Pb intoxication (13). Oxidative stress in lead poisoning may occur due to a direct participation of Pb in free radical-mediated reactions or as a consequence of d-aminolevulinic acid accumulation, a metabolite that can release Fe2+ from ferritin (14), and induce oxidative damage to different organic molecules like proteins, lipids and nucleic acids (15). The inhibition of regulatory enzymes in heme biosynthesis pathway serves as potentially important biological markers of lead exposure and cell injury. d-aminolevulinic acid dehydratase (EC 4.2.1.24) represents a key regulatory enzyme of heme synthesis pathway. In the present study we have found a significant decrease of ALA-D activity in all investigated tissue. Completely recovered activity was found in liver and bone marrow, but not in blood of lead exposed rats treated with captopril, most probably because of long erythrocyte half-life (about 120 days).
In lead poisoning there were some serious disturbances in synthetic liver function as reduced protein synthesis, inadequate cholecalciferol hydroxylation, the decrease of Hb and Fe2+ levels as well as the decrease of alkaline and acid phosphatase activity in plasma (16). Furthermore, during the lead intoxication, liver phosphatases change both their localization and activity as a result of adaptation to metabolic, structural and functional changes in hepatocytes (17).
A number of data showed that captopril reacts rapidly with hydroxyl radical, slowly with superoxide anion and it is a power scavenger of hypochlorous acid, hydrogen peroxide and singlet oxygen (18). It is well known that after Pb absorption, the thiol group of hepatic glutathione (GSH), particularly, reacts with lead and helps in the elimination of this toxic metal from the body.
In the experimental model of the acute lead poisoning our results revealed significant changes in membrane-associated enzymes, the increase of ALP and GGT (p<0.05) and the decrease of 5'-NT (p<0.05). The increase of ALP activity, metalloenzyme that needs Zn2+ and Mg2+ for optimal activity, could be a consequence of direct effect of Pb. The investigation of Wielgus-Serafinska & Sterelec (19) showed that Pb could interact with Zn and increase ALP activity. The activity of liver ALP in rats treated with both agents was significantly lower compared to the lead exposed animals, probably as a consequence of the protective effects of captopril on lead action (SH group of captopril binds Pb). Although in rats treated with captopril the activity of ALP is slightly elevated, witch is in accordance with literature data dealing with hepatotoxic and cholestatic effects of this drug (20).
The activity of specific phosphatase (5'-NT) in rat liver treated with Pb-acetate intraperitoneally was significantly decreased (p<0.05) in relation to the control group. Since the optimal activity of 5'-NT requires histidine, serine and cysteine in an active site, and having in mind that one of the mechanism of lead-toxicity is binding SH group of cysteine, the decreased activity of 5'-NT could be a consequence of Pb competitive inhibition. Whereas captopril (SH-containing agent) increases, the reduced glutathione (GSH) in liver and erythrocytes, completely recover 5'-NT activity in rat liver, treated with captopril and lead, which in turn could be a consequence of GSH accumulation in cell.
Pb also caused the increase of liver and plasma activity of g-GT (p<0.05) in relation to the control group. The elevation of this enzyme could be a result of lead-induction of g-GT in liver (21).
Our results didn't confirm any disturbances of synthetic function (urea, total proteins and albumins). However, even if statistically lower level of total proteins and albumens (p<0.01) could be a result of a disturbance in kidney function, those facts still remain to be established.
 

Conclusion
Stability and integrity of hepatocyte membrane is both a priority and an essential prerequisite for accomplishing all liver vital functions. Disturbance of liver membrane enzymes activity (ALP, 5'-NT and g-GT) leads to disarrangement in the transport of physiologic substrates, necessary for normal liver function. Lead hepatotoxicity could be a consequence of Pb direct action on liver membrane enzymes (ALP, 5'-NT and g-GT).
The assumption of oxidative damage as a mechanism in lead toxicity suggests that SH-containing antioxidative agent captopril may play an important role in the therapy of lead poisoning.
 

1. REFERENCES
   
   1. Robertson WO. Chronic poisoning: trace metals and others. In Goldman: Cecil Textbook of Medicine, ed. W. B. Saunders Company, 2000.
   2. Tong S, von Schirnding YE, Prapamontol T. Environmental lead exposure: a public health problem of global dimensions. 2000; 78: 1068-1077.
   3. Varagic V, Miloševic M: Trovanje olovom. U. Varagic M. (ed), Farmakologija, Beograd, 2000: 636-637.
   4. Chmielnicka J, Zareba G, Nasiadek M. Combined effect of tin and lead on heme biosynthesis in rats. Ecotoxicol Environ Saf 1994; 29(2):165-173.
   5. Fischbein A. Occupational and environmental lead exposure. In Rom WN (ed): Environmental and Occupational Medicine, 2nd ed. Boston, Little, Brown, 1992; 735.
   6. Aykin-Burns N, Laegeler A, Kellogg G, Ercal N. Oxidative Effects of Lead in Young and Adult Fisher 344 Rats. Arch Environ Contam Toxicol 2003; 44: 417-420.
   7. Bagchi D, Prasad R, Das DK. Direct scavenging of free radicals by captopril, an angiotensin converting enzyme inhibitor. Biochem Biophys Res Com 1989; 158: 52-57.
   8. Mitchell RA, Drake JE, Wittlin LA. et al. Erythrocyte porphobilinogen synthase (delta-aminolevulinate dehydratase) activity: a reliable and quantitative indicator of lead exposure in humans. Clin Chem. 1977 Jan; 23(1):105-111.
   9. Wood D, Williams Y. Colorimetric determination of serum 5'-nucleotidase without deproteinisation. Clin Chem 1981; 24: 701-703.
   10. Bowers GN, McCombe RD. Measurement of total alkaline phosphatase activity in human serum. Clin Chem 1975; 21: 1988-1995.
   11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
   12. Hermes-Lima M, Pereira B, Bechara JH. Are free radicals involved in lead poisoning? Xenobiotica 1991; 21: 1085-1090.
   13. Oteiza PI, Kleinman CG, Demasi M, Bechara EJH. 5-Aminolevulinica acid induces iron release from ferritin. Arch Biochem Biophys 1995; 316: 607-611.
   14. Vercesi AE, Castillo RF, Meinicke AR, Valle VGR, Hermes-Lima M, Bechara EJH. Oxidative damage of mitochondria induced by 5-aminolevulinic acid: role of Ca2+ and membrane protein thiols. Biochem Biophys Acta 1994; 1188: 86-92.
   15. Corpas I, Benito MJ, Marquina D. et al. Gestational and Lactational Lead Intoxication Produces Alterations in the Hepatic System of Rat Pups. Ecotoxicol Environ Saf 2002; 51:35-43.
   16. Jarrar BM, Mahmoud YN. Histochemical demonstration of changes in the activity of hepatic phosphatases induced by experimental lead poisoning in male white rats (Rattus norvegicus). Toxicol Ind Health, 2000; 16; 7-15.
   17. Mira MS, Silva MM, Queiroz MJ, Manso CF. Angiotensin converting enzyme-inhibitors as oxygen free radical scavengers. Free Radic Res Commun 1993; 19:173-181.
   18. Wielgus-Serafinska E, Strzelec M. Zinc and lead interactions on alkaline phosphatase activity (E.C. 3.1.3.1.) in rats. Acta Physiol Pol, 1982; 33:425-439.
   19. Gonzalez de la Puente M. Fatal hepatotoxicity associated with enalapril (Vasotec). Ann Pharmacother, 2001; 35; 1492-1492.
   20. Columbano A, Ledda-Columbano GM, Ennas MG. et al. Modulation of the activity of hepatic gamma-glutamyl transpeptidase, adenosine triphosphatase, placental glutathione S-transferase and adenylate cyclase by acute administration of lead nitrate. Basic Appl HistoChem 1988; 32 (4): 501-510.