FABAD J. Pharm. Sci., 28, 175-182, 2003 RESEARCH ARTICLE
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Gülhan CENG‹Z*, fiebnem fi. ÇEÇEN*, Tülin SÖYLEMEZO⁄LU*°,
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SSuummmmaarryy :: The objectives of this study were a) to enlighten the toxicity mechanism of cocaine related to free radical formation (50 mg/kg, i.p.), b) to investigate the effect of ethanol (3 g/kg, i.p.) on its toxicity and c) to compare pre- and post-nifedipine administration by the assessments of malondialdehyde (MDA), nonprotein sulfydryl groups (NP-SH) and total sulfydryl (T-SH) levels. All tissue MDA levels in the group dosed with cocaine increased significantly (p<0.001) except for brain MDA level (p<0.05), whereas a decrease was observed in all tissue NP-SH levels (p<0.001) and T-SH levels (p<0.001 in all examined tissues except brain (p<0.05). After "ethanol+cocaine" treat- ment, MDA levels in liver, heart (p<0.001) and brain (p<0.01) increased. The same group had lower NP-SH (liver, heart p<0.001, brain p<0.01 and kidney p<0.05) and T-SH levels (brain p<0.001, liver and kidney p<0.01 and heart p<0.05) compared with both cocaine-administered group and control group. Pre- and post-treatment with nifedipine prevented cocaine-induced increases in MDA levels in all tissues (p<0.001 except heart) significantly. Pre-treatment with nifedipine caused a significant elevation in NP-SH levels only in liver (p<0.001) and heart (p<0.01) and T-SH levels in kidney, heart, brain (p<0.001) and liver (p<0.01). Post-niphedipine administration showed a significant increase in NP-SH levels in liver (p<0.001) and in T-SH levels in liver, kidney, brain (p<0.001) and heart (p<0.05).
K
Keeyywwoorrddss:: Cocaine, cocaethylene, oxidative stress, gluthat- hione, nifedipine.
Received : 25.12.2003 Revised : 28.4.2004 Accepted : 11.5.2004
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Özzeett:: : Bu çal›flman›n amac› malondialdehit (MDA), proteine ba¤l› olmayan sülfidril gruplar› (NP-SH) ve total sülfidril grup- lar›n›n(T-SH) düzeylerini de¤erlendirerek a) kokainin (50 mg/kg, i.p.) serbest radikal oluflumuna ba¤l› toksisite mekaniz- mas›n› ayd›nlatmak b) etanolün (3g/kg, i.p.) kokainin toksisite- si üzerine etkilerini araflt›rmak ve c) pre ve post-nifedipin uygu- lamalar›n› karfl›laflt›rmakt›r. Kokain uygulanan tüm dokularda MDA düzeyinde anlaml› bir art›fl gözlenirken (tüm dokularda p<0.001 yaln›z beyinde p<0.05), NP-SH (p<0.001) ve T-SH (p<0.001, yaln›z beyinde p<0.05) düzeyleri azalm›flt›r. Eta- nol kokain uygulanan grupta ise karaci¤er, kalp (p<0,001) ve beyin (p<0,01) MDA düzeyleri artm›flt›r. Ayn› grupta kokain uygulanan grup ve kontrol grubuna göre NP-SH (karaci¤er, kalp p<0.001, beyin p<0.01 ve böbrek p<0.05) ve T-SH (be- yin p<0.001, karaci¤er ve böbrek p<0.01, kalp p<0.05) dü- zeylerinde düflüfller gözlenmifltir. Pre ve post nifedipin uygula- nan gruplarda kokaine ba¤l› MDA düzeylerindeki art›fl (kalp hariç tüm dokularda p<0.001) anlaml› derecede azalm›flt›r.
Pre-nifedipin uygulanan grupta sadece karaci¤er (p<0.001) ve kalp NP-SH ve böbrek, kalp, beyin (p<0.001) ve karaci¤er (p<0.01) T-SH düzeylerinde iyileflmeler görülmüfltür. Post-ni- fedipin uygulamas› sonucu karaci¤er NP-SH (p<0.001) ve ka- raci¤er, böbrek, beyin (p<0.001) ve kalp (p<0.05) T-SH dü- zeylerinde anlaml› yükselmeler görülmüfltür.
A
Annaahhttaarr kkeelliimmeelleerr:: Kokain, kokaetilen, oksidatif stres, glutat- yon, nifedipin.
IINNTTRROODDUUCCTTIIOONN
Cocaine is a natural alkaloid and a stimulant of the central nervous system, and is toxic to most of the systems including heart, liver, neuromuscular sys- tem and kidney1,2,3. Its widespread abuse causes medical and social problems1.
Two different pathways are responsible for cocaine metabolism. Cocaine is mainly metabolized to its pharmacologically inactive and non-toxic metabo- lites benzoylecgonine, ecgonine and ecgonine methyl esther by non-specific plasma and tissue estherases4,5,6. Another minor pathway is an oxida- tive process by which cocaine (about 10%) is bioacti-
* Ankara University, Institute of Forensic Medicine, 06260, Dikimevi, Ankara - TURKEY
° Corresponding author e-mail: [email protected]
vated in the liver by various oxidative steps to nor- cocaine which is further metabolized to N-hydrox- ynorcocaine and to a free radical norcocaine nitrox- ide. The first step in the production of the toxic metabolite is believed to be the demethylation of cocaine by P-450 monooxygenase system to norco- caine. This metabolite may oxidize to N-hydrox- ynorcocaine and then to the free radical norcocaine nitroxide by a flavin-containing monooxygenase or by P-450 monooxygenase at the expense of NADPH5,6,7(Figure 1). In its presence the cell may enter a redox cycling with the final result of lipid peroxidation, gluthathione depletion, cytotoxicity and finally cell death.
FFiigguurree 11:: Hydrolytic and oxidative metabolism of cocaine6.
Cocaine may exert some of its toxic effect by lipid peroxidation8. Lipid peroxidation (LPO) is consi- dered as the deterioration of membrane phospho- lipids as a result of free radical attack.
Malondialdehyde (MDA), which is a decomposition product of LPO, is still used as an indicator of free radical damage9.
There is increasing evidence that the liver is the tar- get organ for cocaine toxicity10. It is put forward that simultaneous oxidation reactions are responsible for cocaine toxicity6,11.
Gluthathione (GSH) is one of the most important endogenous antioxidant defense system of the organism against cytotoxicity induced by chemicals.
It is reported that it plays an important role in the
cell function and in protection of the organism against hepatic damage induced by cocaine. Lipid peroxidation caused by cocaine depletes GSH to critical levels12.
Another potent metabolite of cocaine is cocaethyl- ene. In the presence of ethyl alcohol, cocaine is metabolized to its ethyl homolog, cocaethylene. The transesterification of cocaine and ethanol by car- boxyesterases in the liver results in cocaethylene for- mation (Figure 2). It is also reported to be toxic to laboratory animals such as mice and rats13,14.
FFiigguurree 22:: Metabolic pathway of cocaethylene in mam- mals15.
Numerous experimental data strongly suggest that calcium antagonists may also be useful in oxidative hepatocellular injury16 and renal injury17, in addi- tion to the treatment of cardiovascular disease18, neurological and psychiatric disorders19, and opioid and ethanol dependence20.
In the present study, the toxicity mechanism of cocaine related to free radical formation, effect of ethanol on its toxicity and pre- and post-nifedipine therapies were assessed by measuring MDA, non- protein sulfydryl groups (NP-SH) and total sul- fydryl (TSH) levels.
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Maatteerriiaallss aanndd MMeetthhooddss
Animals
Mice used in this study were housed and cared for in accordance with Refik Saydam H›fz›s›hha Institute, Animal Care Unit, which applies the guidelines of the National Institutes of Health on Laboratory Animal Welfare. Procedures involving
animals and their care were conducted in conformi- ty with international laws and policies and the stud- ies on animals accepted by the Ethic Council of Ankara University, Veterinary Faculty (30.04.2001, No: 2001/16). Local breed albino male mice, weigh- ing 20-25 g, were housed in group cages under nor- mal laboratory conditions (temperature 20-22°C, natural day-night cycle), and given free access to commercial chow and water. The food was with- drawn 12-16 h before the experiment.
Treatment
Animals were divided into five groups consisting of nine mice in each and exposed as follows: (1) received saline only (controls); (2) received 50 mg/kg cocaine; (3) received 3 g/kg ethanol half hour prior to 50 mg/kg cocaine administration; (4) received 50 mg/kg nifedipine one hour prior to 50 mg/kg cocaine administration and (5) received 50 mg/kg nifedipine one hour after 50 mg/kg cocaine administration. All administrations were done intraperitoneally. Mice were sacrificed by an over- dose of diethyl ether six hours after the last injec- tions. Liver, kidney, heart and brain were removed and washed in an ice cold 0.9% NaCl.
Homogenization
Tissues of animals were immediately excised and chilled in ice-cold 0.9% NaCl. After washing with 0.9% NaCl, 0.5 g of wet tissue was weighed exactly and homogenized in 4.5 ml of 0.25 M sucrose to obtain a 10% w/v suspension in order to measure LPO in tissues. 0.2 g tissue was homogenized with 8 ml 0.02 M Na2EDTA to measure GSH level.
Lipid peroxidation in tissues
The method of Ohkawa et al.21 as modified by Jamall and Smith22was used to determine MDA in tissue samples. MDA, formed from the breakdown of polyunsaturated fatty acids, serves as a conve-
nient index for determining the extent of the peroxi- dation reaction. MDA has been identified as the product of LPO that reacts with thiobarbituric acid (TBA) to give red species absorbing at 532 nm. The tissue extract supernatant was obtained by a two step-centrifugation first at 1000 x g for 10 mins and then at 2000 x g for 30 mins at 4°C. 0.20 ml of super- natant was transferred to a vial and was mixed with 0.20 ml sodium dodecyl sulfate solution (SDS; 8.1%), 1.50 ml of acetic acid solution (CH3COOH; 20%
v/v, adjusted to pH 3.5 with NaOH), and 1.50 ml of 0.8% (w/v) solution of TBA, and the final volume was adjusted to 4.0 ml with distilled water. Each vial was tightly capped and heated in a boiling water bath for 60 mins. The vials were then cooled under running water. Equal volumes of tissue blank or test sample and 10% trichloroacetic acid (TCA) were centrifuged at 1000 x g for 10 mins. The absorbance of the supernatant was measured at 532 nm against tissue blank. Tissue blank was processed using the same experimental procedure except that the TBA solution was replaced with distilled water. 1,1,3,3- tetraethoxypropan was used as the standard for the calibration curve. The tissue TBA reactive products were expressed as nmoles of MDA/g wet weight.
Non-protein -SH groups in tissues
NP-SH was measured using the method of Sedlak and Lindsay23. The purpose of this method is to measure nitromercaptobenzoic acid anion, which gives intense yellow color at 412 nm resulting from the reaction of sulfydryl groups with Ellman’s reagent. Aliquots of 5.0 ml of the homogenates were mixed in 15.0 ml test tubes with 4.0 ml distilled H2O and 1.0 ml of 50% TCA. The tubes were centrifuged for 15 mins at approximately 3000 x g. Two ml of supernatant was mixed with 4.0 ml of 0.4 M Tris buffer (pH: 8.9), and 0.1 ml Ellman’s reagent, (5,5’- dithiobis-2-nitro-benzoic acid) - DTNB was added, and the sample was shaken. The absorbance was read within 5 mins after the addition of DTNB at 412 nm against a reagent blank with no
homogenate. The tissue NP-SH levels were expressed as µmol/g wet tissue.
Total -SH groups in tissues
T-SH was also measured by the method of Sedlak and Lindsay23. Aliquots of 0.5 ml of the tissue homogenates were mixed in 15.0 ml test tubes with 1.5 ml of 0.2 M Tris buffer, pH 8.2, and 0.1 ml of 0.01 M DTNB. The mixture was brought to 10.0 ml with 7.9 ml of absolute methanol. A reagent blank (with- out sample) was prepared in a similar manner. The test tubes were stoppered with rubber caps and allowed to stand 15 mins for color development.
Reaction mixtures were centrifuged at 3000 g at room temperature for 15 mins. The absorbance of the clear filtrates or supernatants was read at 412 nm against a reagent blank. The tissue T-SH levels were expressed as µmol/g wet tissue.
Statistical analysis
The data obtained were analyzed by one-way analy- sis of variance (ANOVA) and Student-Newman- Keul’s-Multiple comparison test for the possible sig- nificant interrelation between the various groups.
The data were analyzed with the help of Instat com- puter software.
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Reessuullttss aanndd DDiissccuussssiioonn
Previous studies have already demonstrated that oxidative damage plays an important role in the cytotoxicity of cocaine2. Lipid peroxidation is stimu- lated in vitro and in vivo during the metabolism of cocaine6. A futile redox reaction is set up between the oxidation and reduction of N-hydroxynorcocaine and norcocaine nitroxide radical to each other conti- nously. As a result: a) cellular NADPH is lost b) hydrogen peroxide (H2O2) is produced c) GSH is depleted and gluthathione disulfide (GSSG) increas- es in the reduction process of H2O2 to H2O d) GSH:GSSG normal ratio is ruined e) gluthathione
peroxidase and reductase activity decrease f) LPO is stimulated by H2O2 and superoxide radical (O2-.) formation g) cellular membranes are damaged and hepatic enzyme activities decrease and h) cellular death occurs10,24.
Lipid peroxidation in cocaine-induced hepatic injury was provided by the findings such as the for- mation of conjugated dienes and the generation of TBARS (thiobarbituric acid reactive substances).
Boelsterli and Göldlin reported that cocaine induces NADPH-dependent production of reactive oxygen species (ROS, especially H2O2and O2-,) in the hepat- ic microsomal systems and in homogenates of cul- tured hepatocytes derived from phenobarbital- pre- treated rats24.
Devi and Chan demonstrated that acute administra- tion of cocaine (40 mg/kg) increased TBARS level.
They also reported that reactive metabolism may be responsible for the cocaine-induced oxidative stress and liver necrosis1.
Present results correlate well with these previous findings. There was a significant increase in liver, kidney, heart (p<0.001) and brain (p<0.05) MDA le- vels in the group administered cocaine compared to the control group (Figure 3).
FFiigguurree 33:: MDA levels in tissues exposed to cocaine and cocaine+ethanol and effects of pre- and post- nifedipine administration on MDA levels in mice exposed to cocaine. Results are obtained as the mean (±SEM) of nine mice per group.
Gluthathione is the most important and abundant nonprotein thiol that serves as a hepatocellular defense against reactive electrophilic species derived from a variety of hepatotoxic drugs and chemicals24. It is reported that cocaine depresses hepatic GSH concentration. This is proposed to result from the depletion of GSH by a futile redox cycle including cocaine metabolites and reactive oxygen species25.
There may be several reasons which induce and/or cause depletion in GSH levels in tissues:
• Oxidative metabolites of cocaine (e.g. norcocaine nitroxide) may have depleted GSH by entering redox cycling reactions.
• Formed H2O2 rapidly reduces to H2O by glu- tathione peroxidase (GSH-Px); however, during this reaction GSH would also turn into GSSG.
Back formation of GSH from GSSG by glu- tathione reductase enzyme would not be proba- ble because of the depletion in NADPH stores.
Therefore, the equilibrium between GSH:GSSG will be ruined.
• Excessive depletion of NADPH in cocaine metabolism may result in the depletion of GSH to critical levels.
In a study of Devi and Chan, oxidative stress was shown to develop in hepatic mitochondria of rats exposed to cocaine (25 mg/kg, 5 injections, 3 days)7.
In the current study, cocaine treatment induced a remarkable decrease in liver, heart (p<0.001) brain and kidney (p<0.01) NP-SH levels and in liver, heart, kidney (p<0.001) and brain T-SH levels com- pared to the control group (Figures 4 and 5).
FFiigguurree 44:: NP-SH levels in tissues exposed to cocaine and
"cocaine+ethanol" and effects of pre- and post- nifedipine administration on NP-SH levels in mice exposed to cocaine. Results are obtained as the mean (±SEM) of nine mice per group.
According to clinical and experimental studies, it is well documented that an increase in oxidative stress results in myocardial contractile functions. There is increasing evidence that ROS plays an important role in ischemia-reperfusion injury26. Histopathological investigations reported by Devi and Chan showed that neutrophils formed in myocardial cavities in rats exposed to cocaine which indicates that i.p. administration of cocaine causes reperfusion damage26. In addition they also demon- strated that it causes an increase in apoptic cell num- ber in the rat heart. It is now well known that oxida- tive stress is closely related with the apoptic process.
In a study performed by Barroso-Moguel et al., it was shown that in rats exposed to cocaine chroni- cally, as a result of capillary lysis which is followed by leakage of erythrocytes, brain lesions were induced3.
In the group exposed to cocaine, brain MDA levels increased (p<0.001), whereas a significant decrease was observed in NP-SH and T-SH levels. There may be several reasons for these findings, such as:
a) The brain is a highly oxygenated organ and takes most of its energy from oxidative metabolism of the mitochondrial respiratory chain
b) It cannot readily neutralize H2O2 and O2-, because of its poor enzyme activities such as catalase and superoxide dismutase.
c) Brain membrane lipids are rich in polyunsatura- ted fatty acids27.
Cocaine, as a result of rhabdomyolysis, induces var- ious renal types of damages including acute tubular necrosis28. As a result of blockage of the reuptake mechanism of noradrenaline at nerve endings caused by cocaine, the catecholamine level increas- es, which leads to vasoconstriction, edema and glomerular and tubular lesions3.
Experimental models show that ethanol increases the hepatotoxic effect induced by cocaine. Mice fed on liquid diet (including ethanol) gained more sen- sitivity to hepatotoxicity induced by cocaine. This sensitivity was shown by an increase in serum ALT levels and centrilobular necrosis24.
It is surmised that chronic alcohol consumption induces cocaine metabolism. Therefore, acute liver damage induced by cocaine will also be increased by chronic alcohol consumption. It has been put for- ward that acute administration of ethanol increases O2-. radical formation24. Intraperitoneal administra- tion of cocaine and ethanol significantly increased MDA levels in liver, heart (p<0.001) and brain (p<0.01) compared to the cocaine treated group. No significant change was observed in kidneys. There were also significant decreases in NP-SH (liver, kid- ney, heart p<0.001; brain p<0.01) and T-SH levels (liver and kidney p<0.01; heart p<0.05 and brain p<0.001) in mice treated with both ethanol and cocaine (Figures 3, 4, 5).
FFiigguurree 55:: T-SH levels in tissues exposed to cocaine and
"cocaine+ethanol" and effects of pre- and post- nifedipine therapies on T-SH levels in mice exposed to cocaine. Results are obtained as the mean (±SEM) of nine mice per group.
Present results suggest that LPO may be an alterna- tive mechanism in cocaine toxicity, and tissue MDA, NP-SH and T-SH levels may be assessed as the bio- markers of cellular damage in tissues.
Most toxic agents can disrupt intracellular calcium homeostasis by causing enhanced influx of extracel- lular calcium or by inhibiting the transport sys- tems29,30.
As previously stated, development of LPO would produce loss of essential thiols, which in turn would result in impairment of the calcium sequestration activity. Homeostasis of cellular calcium distur- bance generally leads to cell death. A number of recent studies have demonstrated that calcium channel blockers protect the tissues against the cytotoxic effects31. Lipid peroxides diminish mem- brane fluidity and increase nonspecific permeability to ions, especially Ca2+. Furthermore, they may oxi- dize various thiol groups that are required for activ- ities of enzymes in the membrane. There are studies showing that calcium channel blockers having lipophilic structure may destroy the lipid peroxide chain by attaching the lipid parts of the mem- brane18. Therefore, if the cell damage is caused by free radicals and/or by an increase in the calcium amount, it is probable that calcium channel blockers
may prevent the tissue toxicity caused by the free radicals. In a previous study, it was shown that a piperazine derivative calcium channel blocker, cin- narizine, prevents LPO in rat liver homogenate32. Post administration of nimodipine reversed the car- diovascular effects of cocaine. When nimodipine was used as an antidote, it normalized plasma cate- cholamine concentration, which had been elevated by cocaine. Calcium channel antagonists show their protective effect by inhibiting the release of cate- cholamines from the tissues and the vasoconstric- tion caused by them33. John et al. reported that cin- narizine, prevented LPO in the rat liver32. It was also reported that dihydropyridine types of calcium channel blockers decrease the production of MDA in tissues34. Antiperoxidant effect of the same group of drugs has also been shown experimentally35.
It is reported that functional and morphological car- diac toxicity induced by intravenous administration of cocaine can be prevented by nitrendipine treat- ment. Nitrendipine has a stabilizing effect on the central nervous system besides its cardioprotective effects in cocaine intoxication. For these reasons;
these dihydropyridine types of calcium antagonists might be considered for clinical trials in the treat- ment of acute cocaine toxicity in man36.
In the current study nifedipine a calcium channel antagonist, was used to observe if it has any amelio- rating effect on cocaine-induced toxicity. Towards this aim, pre- and post-nifedipine administrations were applied to mice which were exposed to cocaine.
In the group which was pretreated with nifedipine, statistical changes were observed in MDA levels in all tissues (p<0.001) except heart. In the group which was treated with nifedipine after cocaine adminis- tration, significant alterations were observed in liver, kidney (p<0.001), and brain (p<0.01) (Figure 3). Pre-nifedipine therapy increased NP-SH in liver (p<0.001) and in heart (p<0.01) and T-SH levels in
heart, brain, kidney (p<0.001) and liver (p<0.01). No significant change was observed in brain and kid- neys. Post-nifedipine therapy, while increasing NP- SH level in liver (p<0.001), it did not show any effect in other tissues. However, it did increase T-SH lev- els in all other tissues (liver, kidney, brain p<0.001;
heart p<0.05).
Trouve and Nahas reported that nitrendipine was an effective antidote when given five minutes after a lethal dose of cocaine37.
According to the results of our study, both pre- and post- treatments of nifedipine were observed to have an effective antidotal activity in cocaine- induced toxicity. However, pre-treatment of nifedipine was evaluated to have a more effective protection if MDA (liver p<0.01, brain p<0.05), NP- SH (liver p<0.001, heart p<0.05) and T-SH (liver p<0.05, kidney and heart p<0.01) data are compared to post-nifedipine treatment.
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