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Ca2+ cytochemical study on hepatotoxicity of halothane and sevoflurane in enzyme-induced hopoxic rats
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INTRODUCTION It is now recognized that two types of halothane-induced hepatic dysfunction exist[1,2]. A mild sub clinic at form manifested by abnormal biochemical indices of hepatic function could be caused by toxic products of halothane metabolism, possibly determined by genetic factors, or by hepatic hypoxia resulting from an imbalance between hepatic oxygen supply and demand. A much rarer fulminant form may occur with severe necrosis which may prove fatal[3]. It is probable that this form results from an immune reaction: an oxidative metabolite binds covalently to liver proteins, producing a hepten which in turn provokes and immune reaction and the formation of a circulating antibody. A major question being addressed in hepato-cellular injury is whether a unifying mechanism exists which involves a loss of regulation of cellular Ca2+ levels. In this regard, alteration of Ca2+ homeostasis have been proposed to play a major role in cell injury induced by a diversity of situations such as chemical intoxication and abnormal physiological states such as ischemia.[4,5] Animal studies have provided evidence supporting a role for altered calcium fluxes in the mechanism of halothane-induced liver injury. In guinea pigs, hepatic calcium content was increased significantly 24 h after exposure to halothane. Subsequent changes in liver calcium were proportionat to the severity of liver necrosis, as determined morphologically[6]. More recently it was shown that halothane, enflurane and isoflurane each stimulated a significant, dose–dependent release of radiolabelled calcium from internal calcium stores in isolated rat hepatocytes[7]. Further evidence in support of the calciogenic hypothesis of cell injury is offered by studies in which the administration of a calcium channel blocker reduced the extent of hepatic necrosis animal exposed to hepatotoxic agents, including halothane[8]. For better understanding of the mechanism of liver injury attributed to halothane and sevoflurane hepatotoxicity, we used cytochemical methods to evaluate the changes of intracellular calcium, and corresponding hepatic histopathological changes in enzyme-induced hypoxic rats. MATERIALS AND METHODS Animal model The protocol was approved by the institutional Animal Care and Use committee. Adult mate Sprague-Dawley rats weighting approximately 150-160 g were obtained from the Animal Center of the Second Military Medical University and maintained on a 12 hour dark-light cycle. The animals had free access to water and diet of Wayne rodent food. To induce the hepatic microsomal drug-metabolizing enzymes, 48 animals were given phenobarbital (1mg/mL) in their drinking water for 10 d prior to any experimentation[9]. For exposure to halothane and sevoflurane, animals were placed into 35L plexiglasss cages in groups of eight per cage . These animals were randomly divided into six groups of eight and anesthetized for 1 h with O2/ N2/1.2MAC anesthetic agents according to the following schedule: Group NC(normal controled),21% O2/79% N2;Group HC(hypoxic controled),14% O2/86% N2; Group NNS(normal sevoflurane),21% O2/79% N2/1.2MAC sevoflurane; Group HS(hypoxic sevoflurane) ,14% O2/86% N2/1.2MAC sevoflurane; Group NH(noamal holathane), 21% O2/79% N2/1.2MAC halothane; Group HH(hypoxic halothane), 14% O2/86% N2/1.2MAC halothane,. The anesthetic , nitrogen and oxygen were delivered to the chamber by Dräger anesthetic machine at a flow rate of 4L/min. The chamber concentrations of O2/CO2,halothane and sevoflurane were monitored with a calibrated Capnormac Ultima. After anesthesia or the appropriate exposure, the animals were returned to their metal cages. They were sacrificed by decapitation 24 h after anesthesia. Blood from the trunks was collected into dried beakers, and livers were rapidly removed and placed in chilled petridishes. Serum was separated from clotted blood and assayed for ALT by automated methods in the Department of Clinical Chemistry. For histological examination , liver samples were collected into 10% phosphate-buffered formation, fixed and mounted in paraffin blocks. Tissue sections were stained with hematoxylin and erosin, Gomori trichome and a reticulin stain. Coded liver sections were examined by one of us without knowledge of the experimental details. The necrosis and denaturation of the slides of each section were quantitatively estimated by Weibel’ stereological method[10]. Calcium cytochemistry A portion of the right anterior lobe was cut into 0.5 mm blocks. The specimens were treated in cold fixative consisting of 25% glutaraloehyde in 0.9 mol/L potassium oxalate adjusted to pH 7.4 with 1 mol/L potassium hydroxide. Sucrose was added to 1.4% final concentration. Fixation was done for 4 h at 4℃. The specimens were subsequently kept in a cold mixture of 1% osmium tetroxide and 2% potassium pyroantimonate for 2 h, and osmium tetroxide and 1%potassium ferrocyanide for 1 h. Thereafter, the specimens were rinsed for 15 min with distilled water adusted to PH 10 with 1 mol/L potassium hydroxide, dehydrated in cold ethanol series, and routinely embedded in Epon-812 or Spur. EDX microanalysis The thin section for calcium cytochemistry, approximately 100 nm in thickness, were left unstained and coated with carbon, films in a vacuum evaporator. EDX microanalysis was performed under an analytical electron microscope (Hitachi-800) equipped with an energy-dispersive X-ray detecting system ( EDAX, type 9100/60). The acceleration voltage was 100 KV and the probe current was for 100s and evaluation of the energy-dispersive X-ray spectra performed by a computer program[11]. Statiatical analysis Data are expressed as the mean + SE. The data were analyzed with analyses of variance. Means were compared with Fisher’s least significant difference test . P values of less than 0.05 were considered significant. RESULTS Hepatotoxicity Under conditions of hypoxia and induction of the microsomal enzymes, halothane anesthesia produced extensive hepatic injury . Within 24 h after exposure to halothane at 14% O2, all the rats had many areas of hepatic necrosis that radiated from the central veins . The stereological measurement of necrosis and denaturation in Group HH increased significantly as compared with the clusters of lymphocytes, histocytes and neutrophils, and often encircled by a layer of swollen hepatocytes containing single large vcuoles, strands of degenerating: cytoplasm, and eccentric intact of pyknotic nuclei. Accompanying the morphologic damage was an increase in serum glutamic pyruvic transminase (P<0.01 Table 1). No statistically significant histologic change was found for the following variables: normal control,hypoxic control, halothane anesthesia at 21% O2, sevoflurane exposure at 21% O2 (Figure1). Calcium cytochemistry In Group NC and NS , calcium precipitation was located in nucleus with mitochondria and cytoplasm as fine particles . In Group HC and HS , intracellular calcium increased slightly . But after exposure to 21% O2/ N2/1.2MAC halothane or 14% O2/ N2/1.2MAC halothane, more and more calcium precipitated in the seriously calcified cytoplasm and mitochondria, especially, in Group HH, a large amount of calcium deposition was shown in cytoplasm and mitochondria (Figure 2) . EDX microanalysis Qualitative analyses were performed in nucleus, mitochondria and cytoplasm. The characteristic emission of calcium (Kal) was observed. Neither sodium nor portassium was present. Semi-quantitative analyses were performed in mitochondria and cytoplasm. The calcium emission analyses are shown in Table 2. The amount of cellular calcium increased in Group HH (P<0.01) and there was positive linear correlation between the calcium in mitochondria and the calcium in cytoplasm . DISCUSSION The principle of calcium cytochemical technique is to use potassium pyroantimenate to deposite intracellular cations and can contribute to the understanding of cellular cation redistribution resulting from physiologic and pathologic stimuli. Because the precipitation by potassium pyroantimonate of cations in nonspecific , and careful choice of reaction conditions for calcium cytochemistry is very important, also, it must be done in conjunction with analytical techniques such as X-ray analyses to ascertain whether other cations are deposited[12] . When tissue is first fixed with glutaraldehyde and potassium pyroantimonate at low temperature (4℃) , better results can be acquired. Thus, the precipitation of calcium with cytochemical methods combined with EDX microanalysis is valuable in investigating the mechanisms of hepatocellular injuries. Stereological methods provide the means of efficiently producting quantitative data on the internal structure of organs, tissues ,and cells. These methods can easily be applied to cytological work at the light or electron microscope level of resolution[13]. Although particular caution is indicated in avoiding systematic errors which may results from inadequate preparation, section thickness, and so on, the results are generally very reliable. Cytosolic Ca2+ concentration of hepatocytes may increase under hypoxic condition, which might have been due to the changes of membrane functions, such as Ca2+- ATPase activity, Na+-Ca2+exchange system[14]. but we didn’t observe that cellular and mitochondrial calcium significantly increased in the Group HC. Three main causes are follows (1) The degree of hypoxia (14% O2) is not severe; (2) Hypoxia differs ischemia .The substance which synthetize ATP do not exhaust; (3) Ca2+-ATPase activity may partly recover 24 h after hypoxic exposure for 1 h. It has been proposed that hepatic damage induced by volatile anesthetic agents occurs secondary to disruption of mechanism which maintain cellular calcium homeostasis. the a retrospectivestudies provide clear evidence that halothane can elevate cytosolic free Ca2+ by release of calcium from internal calcium stores and uptake of calcium from extracellular medium[15]. Recent work has demonstrated has loss of sarcoplasmic reticilums Ca2+-ATPase activity work has demonstrated that loss of sarcoplasmic reticulums Ca2+-ATPase activity by oxidizing agents resulted direct oxidation of thiol groups on the ATPase, and not from lipid peroxidation. Halothane is also an oxidizing agent, but a single unified theory or mechanism for this increase of cytosolic Ca2+ can not be postulated; sevoflurane is a new , nonflammable ,fluorinated inhalation anesthetic agent. Sevoflurane undergoes considerable less metabolism and less disturbed Ca2+ homestasis than halothane could be relevant to the mechanism of its less hepatotoxicity[16]. Cell injury mechanisms of loss of Ca2+ homeostasis are based on the following (1) significant evidence now exists that correlates blebbing of plasma membranes with hepatocellular injury. Although not well understood on a molecular basis , plasma membrane blebbing is thought by Nicotera[17] and coworkers to involve cytoskeletal proteins , Ca2+ione and Ca2+dependent proteases (2) The cytoskeletal is therefore expected to be a target for cytoplasmic calcium ions that promote changes in cell shape. Most of the kown cytoskeletal receptors for calcium are associated with the actin filament system and these may be important in regulating many types of cell mortility, including locomotion, phagocytosis and secretion (3) Cytosolic free Ca2+ is now seen to play an increasingly important and fundamental role in the integrated control of membrane permeability and the cellular response to stimulation. Channel of discrete unitary conductance and selectivity activated by an increase in cytosolic free Ca2+ are responsible for K+ efflux transfer, and Na+ influx ,respectively (4) The elevation of mitochondrial Ca2+ can influence mitochondrial respiration by changing activities of three matrix enzymes ,pyruvate dehydrogenase, 2-oxoglularate dehydrogenase and isocitrate dehydrogenase (5) Ca2+ can activate proteases and endonucleases (6 ) Ca2+ can enhance formation of active oxygen species , ect. Kawahara[18] and co-workers found that calcium channel blocking agent inhibit the production of radical intermediates during the reductive metabolism of halothane . These results suggest that calcium can activate the thereductive metabolism of halothane .In addition , it can cause elevated by radical intermediates .Thus we consider that the vicous circle of peroxidation activated by radical intermediates and elevation of cytosolic calcium may be the basis of halothane –induced hepatotoxicity. Table 1 Serum ALT levels, the stereological measurement of hepatic morphology in enzyme-induced hypoxic rats
**P<0.01 vs Normal Control(NC); Value are mean ±SEM Table 2 EDX semi-quantitative analyses of calcium content in mitochondria and cytoplasm
**P<0.01 vs Normal Control(NC)Group; Value are mean ± SEM Figure 1 Cytostructure morphological change of rats liver(*2,000). The liver of rats anesthetized with 14% O2/86% N2/1.2MAC Halothane had extensive centribular necrosis and denaturation(HH Group,seen in picture A), and there was a increase in serum glutamic pyruvic transminase accompanying the morphologic damage, but no marked change was found in liver morphological inhaled with 21% O2/79% N2 (NC Group, seen in picture B).  Fig 2 Hepatic Ca2+ cytochemical changes in rats of rats (*20,000). The liver of rats anesthetized with 14% O2/86% N2/1.2MAC Halothane had a large amount of Calcium deposition was show in cytoplasm and mitochondria(HH Group, picture A) while in liver of rats inhaled with 21% O2/79% N2 (NC Group, picture B), calcium precipitation was mainly located in nucleus with mitochondria and cytoplasm as fine particles.   REFERENCE 1 Bond GR. Hepatitis, rash and eosinophilia following trichloroethylene exposure: a case report and speculation on mechanistic similarity to halothane induced hepatitis. J Toxicol Clin Toxicol. 1996;34:461-466 [PMID: 8699563] 2 Kenna JG. The molecular basis of halothane-induced hepatitis. Biochem Soc Trans. 1991 ;19:191-195[ PMID: 2037145] 3 El-Bassiouni EA, Abo-Ollo MM, Helmy MH, Ismail S, Ramadan MI. Changes in the defense against free radicals in the liver and plasma of the dog during hypoxia and/or halothane anaesthesia. Toxicology. 1998;128: 25-34[ PMID: 9704903] 4 Iaizzo PA, Seewald MJ, Olsen R, Wedel DJ, Chapman DE, Berggren M, Eichinger HM, Powis G. Enhanced mobilization of intracellular Ca2+ induced by halothane in hepatocytes isolated from swine susceptible to malignant hyperthermia. Anesthesiology. 1991;74:531-538[ PMID: 2001032] 5 Frost L, Prendergast D, Farrell G. Halothane hepatitis: damage to peripheral blood mononuclear cells produced by electrophilic drug metabolites is Ca(2+)-dependent. J Gastroenterol Hepatol. 1989;4:1-9.[ PMID: 2490933] 6 Frost L, Mahoney J, Field J, Farrell GC. Impaired bile flow and disordered hepatic calcium homeostasis are early features of halothane-induced liver injury in guinea pigs. Hepatology. 1996;23:80-86[ PMID: 8550053] 7 Araki M, Inaba H, Kon S, Imai M, Mizuguchi T. Effects of volatile anesthetics on the calcium ionophore A23187-mediated alterations in hepatic flow and metabolism in the perfused liver in fasted rats. Acta Anaesthesiol Scand. 1997;41:55-61[ PMID: 9061115] 8 Satorres J, Perez-Mateo M, Mayol MJ, Esteban A, Graells ML. Protective effect of diltiazem against acetaminophen hepatotoxicity in mice. Liver. 1995;15:16-19[ PMID: 7776852] 9 Yamazoe Y, Shimada M, Murayama N, Yamauchi K, Kato R. Alteration of hepatic drug metabolizing activities and contents of cytochrome P-450 isozymes by neonatal monosodium glutamate treatment. Biochem Pharmacol. 1988;37:1687-1691[ PMID: 3377831] 10 Baranska W, Baran W, Skopinski P, Ziemba H. Morphometric analysis of satellite cells in rat skeletal muscles: soleus and extensor digitorum longus. Acta Anat (Basel). 1997;160: 88-94[ PMID: 9673706] 11 Jonas L, Fulda G, Kroning G, Merkord J, Nizze H. Electron microscopic detection of tin accumulation in biliopancreatic concrements after induction of chronic pancreatitis in rats by di-n-butyltin dichloride. Ultrastruct Pathol. 2002;26:89-98.[ PMID: 12036097] 12 Musetti R, Favali MA. Cytochemical localization of calcium and X-ray microanalysis of Catharanthus roseus L. infected with phytoplasmas. Micron. 2003; 34:387-393[ PMID: 14680925] 13 Makita T, Itagaki S, Ohokawa T. X-ray microanalysis and ultrastructural localization of cisplatin in liver and kidney of the rat. Jpn J Cancer Res. 1985;76:895-901[ PMID: 3932289] 14 Chang YJ, Chang KJ. Calcium concentration in rat liver mitochondria during anoxic incubation. J Formos Med Assoc. 2002;101:136-143[ PMID: 12099205] 15 Byrne AM, Lemasters JJ, Nieminen AL.Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes. Hepatology. 1999;29:1523-31[ PMID: 10216138] 16 Johansson JS, Zou H. Partitioning of four modern volatile general anesthetics into solvents that model buried amino acid side-chains. Biophys Chem. 1999;79:107-116[ PMID: 10389237] 17 Volbracht C, Leist M, Nicotera P. ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation. Mol Med. 1999;5:477-489[ PMID: 10449809] 18 Sugimura M, Kitayama S, Morita K, Irifune M, Takarada T, Kawahara M, Dohi T. Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes. Toxicol Lett. 2001;123:69-76.[ PMID: 11514107]
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