Print version ISSN 0034-7094
Rev. Bras. Anestesiol. vol.54 no.5 Campinas Sept./Oct. 2004
Thiobarbituric acid reactive substances as an index of lipid peroxidation in sevoflurane-treated rats*
Determinación de las substancias reativas al ácido tiobarbitúrico como indicador de la peroxidación lipídica en ratones tratados con sevoflurano
Francisco José Lucena Bezerra, M.D.I; Adriana Augusto Rezende, M.D.II; Sara Jane Rodrigues, M.D.III; Maria das Graças Almeida, M.D.II
IAnestesiologista do Hospital Universitário
Onofre Lopes da UFRN
IIProfessor Doutor do Departamento de Análises Clínicas e Toxicológicas do Centro de Ciências da Saúde da UFRN
IIIProfessor Mestre do Departamento de Anatomia Patológica do Centro de Ciências da Saúde da UFRN
BACKGROUND AND OBJECTIVES: Sevoflurane
is a fluorinated ether with low blood solubility and biotransformed by an oxidative
enzymatic liver system involving cytochrome P450 2E1. Lipid peroxidation occurs
during ethers biotransformation process under action of cytochrome P450, a possible
mechanism for liver and kidney toxicity promoted by such compounds. This study
aimed at determining the levels of substances reactive to thiobarbituric acid
(TBARS), as an index for lipid peroxidation in sevoflurane-treated rats, previously
treated or not with isoniazid, enzymatic inducer of cytochrome P450 2E1.
METHODS: Forty two male Wistar rats were randomly distributed in 4 groups receiving respectively: G1 - 1 L.min-1/60 minutes of 100% oxygen for 5 consecutive days; G2 - 4% sevoflurane in 1 L.min-1/60 minutes of 100% oxygen for 5 consecutive days; G3 - intraperitoneal isoniazid (50 mg.kg-1/day) for 4 consecutive days and then treated as G1; G4 - intraperitoneal isoniazid (50 mg.kg-1/day) for 4 consecutive days and then treated as G2. Animals were sacrificed 12 hours after the last treatment, plasma was collected for TBARS analysis and the liver left lobe and both kidneys were removed for histological evaluation.
RESULTS: Results have shown increased TBARS levels in G3 and G4, with mild increase in G2. Histological evaluation has revealed focal liver necrosis in rats pretreated with isoniazid (G3).
CONCLUSION: Sevoflurane has promoted lipid peroxidation only when associated to isoniazid.
Key Words: ANESTHETICS, Volatile: sevoflurane; ANIMAL: rat; DRUGS: isoniazid; METABOLISM: lipid peroxidation
JUSTIFICATIVA Y OBJETIVOS: El sevoflurano
es un éter fluorado de baja solubilidad sanguínea y su biotransformación
ocurre por medio del sistema enzimático hepático oxidativo que envuelve
el citocromo P450 2E1. La peroxidación lipídica ocurre durante el
proceso de biotransformación dos éteres sobre la acción del citocromo
P450, uno de los posibles mecanismos de toxicidad hepática y renal promovida
por eses compuestos. El objetivo de este estudio fue determinar los niveles
de substancias reactivas al ácido tiobarbitúrico (SRAT), como indicador
de la peroxidación lipídica, en ratones que recibieron sevoflurano,
previamente tratados o no con isoniazida, inductora enzimática del citocromo
MÉTODO: Los animales fueron distribuidos aleatoriamente en 4 grupos que recibieron respectivamente: G1 - oxígeno a 100% 1 l.min-1/60 minutos por 5 días consecutivos; G2 - sevoflurano a 4% en oxígeno a 100%, 1 l.min-1/60 minutos por 5 días consecutivos; G3 - isoniazida (50 mg.kg-1.dia) por vía intraperitoneal durante 4 días consecutivos, en seguida fue tratado como el G1, en G4 - isoniazida 50 mg.kg-1.dia por vía intraperitoneal durante 4 días consecutivos, siendo tratado, posteriormente, como el G2. Después de 12 horas del último tratamiento, se sacrificaran los animales y fue colectado el plasma para la análisis de las SRAT, siendo removido el lóbulo izquierdo del hígado y de los riñones para examen histológico.
RESULTADOS: Los resultados mostraron aumento en las tasas de SRAT en el G3 y G4, con elevación discreta en G2. El estudio histológico reveló necrosis focal en el hígado de ratones pre-tratados con isoniazida (G3).
CONCLUSIONES: El sevoflurano promovió peroxidación lipídica apenas cuando asociado a la isoniazida.
Unsaturated fatty acids peroxidation in lipid membranes is a process known as lipid peroxidation. This process promotes severe cell membrane changes resulting in loss of fluidity, changes in secreting function and transmembrane ionic gradients. In addition, the loss of ion selectivity has been observed with organelles content release, leading to cytotoxic product formation and even cell death 1.
Recent studies have related liver and renal toxicity induced by anesthetic drugs such as halothane 2 and ethers such as isoflurane 3 to the attack of polyunsaturated fatty acids, present in plasma membranes, to oxygen reactive species produced during biotransformation of such agents, indicating that this could be one mechanism involved in tissue injuries.
Sevoflurane (CH2F-O-CH(CF3)2; fluoromethyll 2,2,2-trifluoro-1-[trifluoromethyl] ethyl ether) is a fluorinated inhalational anesthetic agent, with low blood solubility, which justifies its widespread use in pediatric and outpatient anesthesia (fast induction and emergence) 4,5. Its biotransformation, especially catalyzed by cytochrome P450 2E1, is low (2% to 5%) and predominantly hepatic, with production of inorganic fluorine and organic fluorine: hexafluoroisopropanol (HFIP) 6. However, inorganic fluorine is able to produce lipid peroxidation in different organs of rats, such as liver, brain and intestines 7.
In the liver, monoxygenases, such as isoform P450 2E1, have high NADPH-oxidase activity through which oxygen reactive species are produced, such as superoxide anion (O2-) and hydrogen peroxide (H2O2) and, as a consequence, may induce potentially noxious lipid peroxidation 8.
Considering that there are no reports on lipoperoxidation after multiple exposures to sevoflurane, this study aimed at evaluating lipid peroxidation in the plasma of Wistar rats treated with sevoflurane and submitted to cytochrome P450 2E1 enzymatic induction with isoniazid.
After the Animal Research Ethics Committee approval, 42 male Wistar rats weighing in mean 280 g and aged 90 days were involved in this study.
Animals received water and food ad libitum and were submitted to light/dark cycle of 12 hours in the experimental animals facility, Sciences Center, Federal University of Rio Grande do Norte (UFRN). Rats were anesthetized in a 3200 cm3 glass chamber with 1 L.min-1 of 100% oxygen and a gauged HB44 (Abbott, Madison, WI-USA) vaporizer.
After 1-hour exposure, variable according to the group, rats received 1 L.min-1 of 100% oxygen until postural reflex recovery, when they were placed in their cages.
Animals were distributed in 4 groups: G1, control group, received 1 L.min-1 of 100% oxygen for 5 consecutive days; G2 received 4% sevoflurane (1.8 MAC/h) in 1 L.min-1 of 100% oxygen for 5 consecutive days; G3 received intraperitoneal isoniazid (50 mg.kg-1/day) for 4 consecutive days and was then treated with 1 L.min-1 of 100% oxygen for 5 consecutive days; G4 received intraperitoneal isoniazid (50 mg.kg-1/day) for 4 consecutive days and was then treated with 4% sevoflurane in 1 L.min-1 of 100% oxygen for 5 consecutive days. Rats were sacrificed 12 hours after the last exposure by cervical displacement and were submitted to laparotomy for portal vein blood collection and kidneys and liver left lobe removal.
Heparinized blood was centrifuged 1100 x g at 4 ºC during 10 minutes and isolated plasma was used to determine thiobarbituric acid reactive substances (TBARS) through colorimetric determination of the product of the reaction between thiobarbituric acid and malonyldialdehyde produced during peroxidased lipid breakdown. After blood collection, plasma was separated and submitted to 0.86% thiobarbituric acid reaction. Absorbance was read at 535 nm and TBARS concentration was calculated 9, considering a molar extinction coefficient e = 0,156 µM-1.cm-1. Values were expressed in nmol.L-1.
Liver and kidney specimens were fixed in 10% formalin and taken to the Pathologic Anatomy Lab, UFRN, where the same pathologist would analyze the specimens without knowing the group they belonged to. Tissue samples were soaked in paraffin, cut into 4 µm slices and died with hematoxylin-eosin (HE) by Massons trychromatic. Sections were then coded and pathologic changes were evaluated by optic microscopy.
Analysis of Variance was used to compare among groups and Mann-Whitney U test was used to compare between two groups, being significant p < 0.05.
Plasma TBARS concentration in control group rats and rats treated with sevoflurane and submitted or not to pretreatment with isoniazid (INH) is shown in table I.
Rats treated with oxygen and sevoflurane presented high TBARS rates, although not significant as compared to G1 treated with oxygen alone. Conversely, animals treated with isoniazid alone - G3 - had increase in TBARS concentration as compared to animals treated with oxygen alone - G1. However, animals pretreated with isoniazid followed by sevoflurane - G4 - had a significant increase as compared to G1 (p < 0.055) and approximately 30% increase as compared to G2. These results suggest that, although sevoflurane increases TBARS rates, a significant percentage of this increase was observed in the presence of isoniazid, thus indicating the active presence of lipid peroxidation.
Cross-sections (Figure 1, Figure 2, Figure 3 and Figure 4) have shown for animals treated with sevoflurane - G2 - congestion in sinusoidal and hepatic vein, and for animals pretreated with isoniazid - G3 - focal liver necrosis and lymphocytic infiltrate. In liver cross-sections of animals treated with oxygen - G1 - and animals pretreated with isoniazid and treated with sevoflurane - G4 - no histological abnormalities have been found.
In the center of the figure, focal necrosis of one hepatocyte with lymphocytic infiltrate may be observed.
Kidney histological evaluation (Figure 5, Figure 6, Figure 7 and Figure 8) of groups G2, G3 and G4 has shown glomerular congestion. There have been no histological abnormalities in animals treated with oxygen - G1.
It has been observed during the experiments that animals treated with 4% sevoflurane have shown high TBARS levels, although without statistical significance. This increase, however, has been significant in the presence of isoniazid, an enzymatic inducer of cytochrome P450 2E1, allowing to suggest that pretreatment with isoniazid has been responsible for increased lipid peroxidation observed among different groups; sevoflurane may also induce significant lipid peroxidation, but only when in association with isoniazid (Table I).
Sevoflurane may trigger lipoperoxidation 10, possibly through its metabolites, such as inorganic fluorine, since it was able to induce lipid peroxidation in different tissues, such as liver, brain and intestine of rats 7, probably by acting on iron compartmentation 11.
It has also been reported that isoform P450 2E1, sevoflurane biotransformation catalyst, is recognized by the production of oxygen reactive species, such as superoxide anion (O2-) and hydrogen peroxide (H2O2) 12, in addition to the hydroxyl radical (OH) and the ability to trigger lipid peroxidation, especially in the presence of transition metals 8,13. So, in addition to fluorines direct action, lipid peroxidation observed in G4 rats, may have been caused by increased cytochrome P450 2E1 activity induced by isoniazid.
Conversely, Wang et al. 14 using isolated hearts of rats treated with 1.5 MAC sevoflurane, submitted to ischemia and reperfusion, have found decreased lipid peroxidation levels in rats treated with sevoflurane as compared to control group. According to Allaouchiche et al. 15 sevoflurane induces less local and systemic oxidative stress in swine as compared to desflurane, suggesting the presence of some antioxidant action.
It is worth highlighting that there are studies trying to relate halothanes liver toxicity to lipid peroxidation 16. In addition to the above-mentioned agents, enflurane has also been reported as lipid peroxidation inducer 17. More recent studies have tried to show the potential antioxidant activity of other anesthetic agents such as propofol, midazolam, ketamine and vecuronium 18,19.
The fact that no G4 animals had liver necrosis may be related to enzymatic inhibition in isoniazid biotransformation path, promoted by sevoflurane or some of its biotransformation products, such as fluorine or HFIP, as well as to the fact that this halogenate is able to maintain oxygen supply to tissues even in situations when liver blood flow may be decreased 20.
Acetylisoniazid, first metabolite formed by isoniazid, suffers enzymatic hydrolysis to form isonicotinic acid and acetylhydrazin 21. Acetylisoniazid and acetylhydrazin are able to produce dose-dependent liver necrosis in rats 22.
Could sevoflurane or its biotransformation products be inhibiting isoniazid conversion into acetylisoniazid? Or even, would they be contributing for increased isonicotinic acid formation, which is not hepatotoxic, as from acetylisoniazid, as opposed to acetylhydrazin?
The evaluation of these compounds concentration in this group of animals could help explaining these hypotheses in further studies. Isoniazid is an enzymatic inducer specific of cytochrome P450 2E1 able to temporarily increase tissue toxicity by increasing the formation of products derived of the biotransformation of the drug used. In theory, increasing its biotransformation increases the formation of its bioproducts, worsening adverse effects. For sevoflurane, isoniazid was used as enzymatic inducer because it increases sevofluranes defluorination rate in 0.5 to 4 times, in a dose and time dependent way 23.
Focal necrosis and inflammatory infiltrate have only been observed in G3 animals, indicating higher isoniazid hepatotoxic potential, as opposed to G4 where sevoflurane seems to have had a protective effect over isoniazids ability to produce liver necrosis (Figure 3). According to the literature, isoniazid is able to produce dose/time-dependent liver necrosis 24,25.
In terms of renal architecture, there has been vascular congestion in all studied groups. There have been no classic pathologic signs of fluorine-induced nephropathy, such as pyknosis, karyorrhexis, renal tubules regeneration and dilatation or even hypereosinophilia in all studied groups after acute exposure with a time period of 5 hours. This result is in line with the literature, which describes rats exposed to 1.5% sevoflurane without soda lime during 15 hours with no histological evidence of kidney injury 26.
Our results contribute with evidences that the use of sevoflurane in patients in treatment with isoniazid or in chronic use of alcohol, both cytochrome P450 2E1 inducers, may lead to oxidative stress, since the antioxidant defense would be unable to fight pro-oxidant substances generated in a higher proportion. In addition, this exposure, if prolonged or repeated, could promote erythrocytic injuries with consequent vascular complications. According to Yesilkaya et al. 27 changes in erythrocytic deformation ability could result in tissue perfusion problems and lead to vascular complications in the postanesthetic period. Lipid peroxidation in erythrocytes has been also associated to decreased red blood cells useful life and hemolytic states 28.
Sevoflurane associated to isoniazid should also be considered for individuals with glucose-6-phosphate dehydrogenase enzyme (G6PD) deficiency, because they havent satisfactory NADPH production and may be more susceptible to peroxidating injuries than normal individuals, especially in red blood cells 29. NADPH is produced by G6PD during pentoses cycle and is used as hydrogen donor to biologic constituents, such as plasma membranes, preventing their structural damage.
The metabolic pathway of sevoflurane is more safe in men as compared to rats, due to the higher b-lyase content of those rodents kidneys 30. Possibly, its single use or during balanced anesthesia is still safe, because sevoflurane has not induced significant lipid peroxidation in the animals treated with it as unique agent.
01. Ferreira ALA, Matsubara SL - Radicais livres: conceitos, doenças relacionadas, sistema de defesa e estresse oxidativo. Rev Ass Med Brasil, 1997;43:61-68. [ Links ]
02. Durak I, Guven T, Birey M et al - Halothane hepatotoxicity and hepatic free radical metabolism in guinea pigs: the effects of vitamin E. Can J Anaesth, 1996;43:741-748. [ Links ]
03. Durak I, Ozturk HS, Dikmen B et al - Isoflurane impairs antioxidant defence system in guinea pig kidney. Can J Anaesth, 1999;46:797-802. [ Links ]
04. Delfino J, Vale NB, Magalhães E et al - Estudo comparativo entre sevoflurano e halotano para cirurgia pediátrica de curta duração. Rev Bras de Anestesiol, 1997;47:10-15. [ Links ]
05. Goa KL, Noble S, Spencer CM - Sevoflurane in paediatric anaesthesia: a review. Paediatr Drugs, 1999;1:127-153. [ Links ]
06. Kenna JG, Jones RM - The organ toxicity of inhaled anesthetics. Anesth Analg, 1995;81:(Suppl)S51-S66. [ Links ]
07. Shayiq RM, Raza H, Kidwai AM - Fluoride and lipid peroxidation: a comparative study in different rat tissues. Bull Environ Contam Toxicol, 1986;37:70-76. [ Links ]
08. Dai Y, Rashba-Step J, Cederbaum AI - Stable expression of human cytochrome P4502E1 in HepG2 cells: characterization of catalytic activities and production of reactive oxygen intermediates. Biochemistry, 1993;32:6928-6937. [ Links ]
09. Bernheim F, Bernheim MLC, Wilbur KM - The reaction between thiobarbituric acid and the oxidation products of certain lipids. J Biol Chem, 1948;174:257-264. [ Links ]
10. Sato N, Fujii K, Yuge O - In vivo and in vitro sevoflurane-induced lipid peroxidation in guinea-pig liver microsomes. Pharmacol Toxicol, 1994;75:366-370. [ Links ]
11. Kundu D, Hallinan T - Fluoride or GTP-gamma-S markedly stimulate lipid peroxidation catalysed by endogenous iron in rat liver microsomes. Biochem Soc Trans, 1995;23:541S. [ Links ]
12. Ekstrom G, Ingelman-Sundberg M - Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450llE1). Biochem Pharmacol, 1989;38:1313-1319. [ Links ]
13. Arimoto T, Yoshikawa T, Takano H et al - Generation of reactive oxygen species and 8-hydroxy-2´-deoxyguanosine formation from diesel exhaust particle components in L1210 cells. Jpn J Pharmacol, 1999;80:49-54. [ Links ]
14. Wang J, Zeng YM, Li H - Effects of sevoflurane and halothane on contracture function, SOD activity, MDA content on isolated ischemic rat hearts. Anesthesiology, 1997;87:3A. [ Links ]
15. Allaouchiche B, Debon R, Goudable J et al - Oxidative stress status during exposure to propofol, sevoflurane and desflurane. Anesth Analg, 2001;93:981-985. [ Links ]
16. Wood CL, Gandolfi AJ, Van Dyke RA - Lipid biding of a halothane metabolite. Relationship to lipid peroxidation in vitro. Drug Metab Dispos, 1976;4:305-313. [ Links ]
17. Naziroglu M, Gunay C - The levels of some antioxidant vitamins, glutathione peroxidase and lipoperoxidase during anaesthesia of dogs. Cell Biochem Funct, 1999;17:207-212. [ Links ]
18. Tsuchiya M, Asada A, Kasahara E et al - Antioxidant protection of propofol and its recycling in erythrocyte membranes. Am J Respir Crit Care Med, 2002;165:54-60. [ Links ]
19. Kang MY, Tsuchiya M, Packer L et al - In vitro study on antioxidant potential of various drugs used in the perioperative period. Acta Anaesthesiol Scand, 1998;42:4-12. [ Links ]
20. Kharasch ED, Frink EJ, Artru A et al - Long-duration low-flow sevoflurane and isoflurane effects on postoperative renal and hepatic function. Anesth Analg, 2001;93:1511-1520. [ Links ]
21. Timbrell JA, Mitchell JR, Snodgrass WR et al - Isoniazid hepatotoxicity: the relationship between covalent binding and metabolism in vivo. J Pharmacol Exp Ther, 1980;213:364-369. [ Links ]
22. Mitchell JR, Zimmerman HJ, Ishak KG et al - Isoniazid liver injury: clinical spectrum, pathology, and probable pathogenesis. Ann Intern Med, 1976;84:181-192. [ Links ]
23. Rice SA, Sbordone L, Mazze RI - Metabolism by rat hepatic microsomes of fluorinated ether anesthetics following isoniazida administration. Anesthesiology, 1980;53:489-493. [ Links ]
24. Walubo A, Smith P, Folb PI - The role of oxygen free radicals in isoniazida-induced hepatotoxicity. Methods Find Exp Clin Pharmacol, 1998;20:649-655. [ Links ]
25. Sarich TC, Zhou T, Adams SP et al - A model of isoniazid-induced hepatotoxicity in rabbits. J Pharmacol Toxicol Methods, 1995;34:109-116. [ Links ]
26. Bermejo-Alvarez MA, Rodrigues-Dintén MJ, Brime-Casanueva JI et al - Efectos renales de la anestesia prolongada com sevoflurane en ratas Wistar. Rev Esp Anestesiol Reanim, 1999;46:241-246. [ Links ]
27. Yesilkaya A, Ertug Z, Yegin A et al - Deformability and oxidant stress in the red blood cells under the influence of halothane and isoflurane anesthesia. Gen Pharmacol, 1998;31:33-36. [ Links ]
28. Saralakumari D, Rao PR - Erythrocyte glutathione metabolism in human chronic fluoride toxicity. Biochem Int, 1991;23:349-357. [ Links ]
29. Luzzatto L, Mehta A - The Metabolic Basis of Inherited Disease, 2nd Ed, New York McGraw-Hill, 1995; 225-229. [ Links ]
30. Gentz BA, Malan TP - Renal toxicity with sevoflurane: a storm in a teacup? Drugs, 2001;61:2155-2162. [ Links ]
Dr. Francisco José Lucena Bezerra
Rua Almirante Tamandaré, 146/202 Lagoa Nova
59054-560 Natal, Brazil
Submitted for publication September 9, 2003
Accepted for publication March 5, 2004
* Received from Departamento de Análises Clínicas e Toxicológicas do Centro de Ciências da Saúde da Universidade Federal do Rio Grande do Norte, Natal, RN