Sevoflurane is biotransformed by guinea pig liver slices but causes minimal cytotoxicity.

Guinea pig liver slices were used to evaluate the biotransformation and hepatotoxic potential of sevoflurane. Precision-cut liver slices (250-300 microns thick) were incubated in sealed roller vials in buffer at 37 degrees C under 95% O2. Sevoflurane was added to produce 0.9 or 2.1 mM medium concentrations. After incubation (6-24 h), the intracellular K+ content and protein synthesis were determined, along with the defluorination of sevoflurane. Isoflurane was included for comparative purposes. Sevoflurane (2.1 mM) and isoflurane (2.3 mM) had no effect on slice K+ content, but both anesthetics depressed protein synthesis. The biotransformation of sevoflurane was maximal at 95% O2, with threefold more F- produced from sevoflurane than isoflurane. Sevoflurane appears to have a minimal effect on the guinea pig liver slices, which is consistent with in vivo studies in which minimal or no hepatotoxicity has been observed.

Plasma inorganic fluoride with sevoflurane anesthesia: correlation with indices of hepatic and renal function.

The biotransformation and plasma inorganic fluoride ion production of sevoflurane (the new volatile anesthetic) during and after surgical anesthesia was studied in 50 ASA I or II surgical patients. Twenty-five additional patients served as controls by receiving isoflurane. Sevoflurane or isoflurane was administered with a semiclosed (total gas flow, 2 L/min O2) circle absorption system for durations of 1.0 to greater than 7.0 minimal alveolar concentration (MAC) hours for surgical anesthesia (sevoflurane MAC, 2.05%; isoflurane MAC, 1.15%). Preoperative and postoperative blood urea nitrogen and creatinine concentrations were determined. Blood samples were obtained during and after anesthesia in both groups for determining anesthetic blood concentration analysis and plasma fluoride level. Plasma fluoride concentrations did not significantly increase during isoflurane anesthesia. Sevoflurane biotransformation produced a mean peak plasma inorganic fluoride concentration of 29.3 +/- 1.8 mumol/L, 2 h after anesthesia, which decreased to 18 mumol/L concentration by 8 h after anesthesia. The peak plasma inorganic fluoride ion concentration correlated with duration of sevoflurane anesthetic exposure. Five patients given sevoflurane had peak levels transiently exceeding 50 mumol/L, and one of these had a history of ingesting drugs potentially producing hepatic enzyme induction. No increases in postoperative levels of creatinine, blood urea nitrogen, direct bilirubin, or hepatic transaminase and no changes in serum electrolyte level occurred in either anesthetic group. Indirect bilirubin concentration increased significantly after sevoflurane anesthesia, but the increase was not of clinical significance (from 0.30 +/- 0.03 to 0.38 +/- 0.06 mg/dL). Indirect bilirubin concentrations did not increase after isoflurane anesthesia; the concentrations reached 0.31 +/- 0.04 mg/dL and did not differ significantly from those found with sevoflurane.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of halothane and other volatile anaesthetics on protein synthesis and secretion in guinea pig liver slices.

We have investigated the effect of volatile anaesthetics on protein synthesis and secretion in Hartley male guinea pig liver slices. The slices (250-300 microns thick) were incubated in sealed roller vials containing Krebs-Henseleit buffer at 37 degrees C under 95% oxygen. Volatile anaesthetics were vaporized in the vials to produce constant concentrations in the medium. Halothane 1-2.1 mmol litre-1 produced a concentration-related decrease in protein synthesis (3H-leucine incorporation) and secretion. Deuterated halothane (d-halothane), which is less biotransformed, was less inhibiting than halothane: uptake of the 3H-leucine was not affected but its incorporation into the nascent peptide was inhibited. Enflurane 2.2 mmol litre-1, isoflurane 2.2 mmol litre-1 and sevoflurane 2.1 mmol litre-1 also inhibited protein synthesis, but to a lesser extent than halothane and d-halothane. We conclude that alterations in protein synthesis and secretion are an early and sensitive indicator of cellular injury by volatile anaesthetics in liver slices.

Minimal biotransformation and toxicity of desflurane in guinea pig liver slices.

Biotransformation and hepatotoxicity of desflurane were evaluated in the guinea pig liver slice culture system. Liver slices (250-300 microns) were prepared from 600-650-g male Hartley guinea pigs. The slices were incubated in sealed vials in a Krebs-Henseleit buffer at 37 degrees C under 95% O2. Desflurane was vaporized to produce media concentrations of 0.7-2.3 mM. After incubation (3-24 h) viability of the slices was determined (K+ content; protein synthesis secretion) along with the biotransformation of desflurane (F-). Isoflurane (2.3 mM) was included in the studies for comparative purposes. Although desflurane caused a mild concentration-related reduction in slice K+ content (1.1-2.2 mM; 20%-40% of control), the effects were less than those produced by 2.3 mM isoflurane (50% of control). High concentrations of desflurane decreased protein synthesis at the first 9 h of incubation, and isoflurane decreased protein synthesis throughout the incubation period. Neither anesthetic affected protein secretion. The biotransformation of desflurane was minimal with threefold less F- produced from desflurane than isoflurane.

Inhibition of protein synthesis and secretion by volatile anesthetics in guinea pig liver slices.

The decrease in protein synthesis and secretion caused by volatile anesthetics was investigated using Hartley male guinea pig liver slices. Precision-cut liver slices (250-300 mM thick) were incubated in sealed roller vials (3 slices/vial) containing Krebs-Hensleit buffer at 37 degrees C under 95% O2 atmosphere. Volatile anesthetics were injected through a teflon septa cap on a filter paper wick and vaporized to produce constant concentration in the medium. A concentration (1-2.1 mM) and time related (0-24) decrease in protein synthesis (3H-leucine incorporation) and secretion by halothane and d-halothane was observed. d-Halothane was less inhibiting than halothane. Inhibition was not on the uptake of the 3H-leucine but with its incorporation in the nascent peptide. The effects of enflurane (2.2 mM), isoflurane (2.2 mM), and sevoflurane (1.3 mM) on protein synthesis and secretion were also studied. The rank order of decrease in protein synthesis caused by the volatile anesthetics studied was halothane greater than isoflurane greater than enflurane greater than sevofluane greater than d-halothane. Enflurane, isoflurane, and sevoflurane increased the protein secretion while halothane and d-halothane caused a pronounced decrease. Alterations in protein synthesis and secretion appears to be an early and sensitive indicator of cytotoxin injury.

Biotransformation of halothane in guinea pig liver slices.

The biotransformation of halothane was studied using liver slices. Precision-cut Hartley male guinea pig liver slices (1 cm diameter; 250-300 microns thick) were incubated in sealed roller vials containing supplemented Krebs-Henseleit buffer at 37 degrees C under different O2 tensions (2.5, 21, and 95%). After a 1-hr preincubation, halothane was vaporized in the vial producing a 1.9 mM medium concentration. Halothane metabolites (Br-, trifluoroacetic acid, F-) were measured at 2, 4, and 6 hr. Viability of the incubated slices was verified by determining intracellular K+ content and levels of cytochrome P-450, which were maintained under 95% O2 atmosphere but decreased with lower O2 tensions (2.5%). The highest fluoride production was 300 +/- 22 pmol/mg slice weight/6 hr at low O2 tension (2.5%). Defluorination decreased with increasing O2 tension to undetectable levels under 95% O2. Production of the oxidative metabolite, trifluoroacetic acid, was highest at 95% O2 (2.35 +/- 0.17 nmol/mg slice weight/6 hr). Trifluoroacetic acid production decreased with decreasing O2 tension. Br- production was the highest at 21% O2 (1.8 +/- 0.13 nmol/mg slice weight/6 hr). Production of Br- was not dependent on the O2 tension. The guinea pig slices are capable of biotransforming halothane (oxidative/reductive); therefore, this in vitro system appears suitable for studying the biotransformation of halothane.

Toxicity of halothane in guinea pig liver slices.

Guinea pigs have proven to be a reliable model of halothane associated hepatotoxicity. An in vitro system with Hartley male guinea pig liver tissue was designed to assess the toxicity of halothane and other volatile anesthetics in the target organ. Precision-cut guinea pig liver slices (250-300 microns) were incubated in sealed roller vials containing Krebs-Henseleit buffer (plus vitamins, amino acids, glutamine, gentamycin) at 37 degrees C, under 95%, 21% and 5% O2/CO2 atmospheres. Halothane (10-15 microliters) was injected through a Teflon septa cap on a filter paper wick and vaporized. Viability of the slices was monitored by measuring intracellular K+ content which was maintained under 95% O2 up to 24 h. A dose- and time-related decrease in intracellular slice K+ by 1.9, 2.1, 2.7 mM halothane in the media was observed. At 2.7 mM halothane a direct physio-chemical effect may be occurring since incubating liver slices from allylisopropyl-acetamide-treated animals did not protect against the drop in intracellular K+. Concentration/time responses of halothane, d-halothane, enflurane, isoflurane and sevoflurane were compared. Sevoflurane had no effect on the liver slice K+ content up to 24 h while the other anesthetics caused the following rank-order decrease in intracellular K+ content: halothane greater than isoflurane and enflurane greater than d-halothane. Precision-cut cultured guinea pig liver slices offer a system where the target tissue for intoxication by anesthetics can be examined for its susceptibility and mechanism of intoxication.

Accumulation and turnover of metabolites of toluene and xylene in nasal mucosa and olfactory bulb in the mouse.

Autoradiography of male mice following inhalation of the radioactively labelled solvents, toluene, xylene, and styrene, revealed an accumulation of non-volatile metabolites in the nasal mucosa and olfactory bulb of the brain. Since no accumulation occurred after benzene inhalation, it was assumed that the activity represented aromatic acids, which are known metabolites of these solvents. This was supported by the finding that also radioactive benzoic acid (main metabolite of toluene) and salicylic acid accumulated in the olfactory bulb. High-performance liquid chromatography revealed that after toluene inhalation (for 1 hr), nasal mucosa and olfactory bulb contained mainly benzoic acid, with a strong accumulation in relation to blood plasma, and considerably less of its glycine conjugate, hippuric acid. After xylene inhalation, on the other hand, methyl hippuric acid dominated over the non-conjugated metabolite, toluic acid. The results indicate a specific, possibly axonal flow-mediated transport of aromatic acids from the nasal mucosa to the olfactory lobe of the brain. The toxicological significance of these results remains to be studied.

Covalent binding of four DDD isomers in the mouse lung: lack of structure specificity.

Previous studies have shown that o,p'-DDD is activated and covalently bound in the mouse lung. In order to examine the structure dependency of the selective lung binding, the 14C-labelled DDD isomers p,p'-DDD, m,p'-DDD and o,m'-DDD were injected intravenously into female C57B1 mice and covalent binding was measured. Autoradiography of solvent-extracted tape-sections showed that all isomers were selectively and covalently bound in the lung alveolar region. As determined by exhaustive extraction of homogenized tissue, maximal binding was observed 4 hr after injection, although the lung/liver concentration ratio increased for 12 days. Covalent protein binding was also observed in vitro, implying that the activation of DDD to a reactive metabolite takes place in the target organ. Since the aryl-chlorine substitution pattern did not change the selective lung binding, bioactivation of DDD may take place at the ethane side-chain.

Epithelial binding of 1,2-dichloroethane in mice.

The metabolism and binding of 14C-labelled 1,2-dichloroethane (DCE) in female C57BL-mice were studied. As shown by whole-body autoradiography with heated and organic solvent-extracted tissue sections of i.v. injected mice, a selective localization of non-volatile and bound DCE-metabolites occurred in the nasal olfactory mucosa and the tracheo-bronchial epithelium. Low levels of metabolites were also present in the epithelia of the upper alimentary tract, vagina and eyelid, and in the liver and kidney. A decreased mucosal and epithelial binding was observed after pretreatment with metyrapone, indicating that the binding might be due to an oxidative metabolism of DCE. The quantitated levels of in vivo binding were considerably lower in mice injected i.p. with DCE, as compared to mice given equimolar doses of 14C-labelled 1,2-dibromoethane. In vitro experiments with 1000 g supernatants from various tissues showed that the nasal mucosa has a marked ability to activate DCE into products that become irreversibly bound to the tissue. It is proposed that the nasal olfactory mucosa is a target tissue for toxicity of DCE.

Extrahepatic sites of metabolism of halothane in the rat.

Rats were given 14C-halothane intravenously and whole-body autoradiography with freeze-dried sections, or with sections extracted in trichloroacetic acid, water, and organic solvents, was carried out to trace tissues accumulating halothane metabolites. In vitro incubations of tissue homogenates were performed to examine the capacity by the various organs to form tissue-bound 14C from the 14C-halothane. Autoradiography of isolated organs after incubation with 14C-halothane was performed to study the tissue localization of halothane metabolites formed under in vitro conditions. A localization of halothane metabolites was observed in several extrahepatic tissues in vivo, and the in vitro experiments showed a capacity by the same tissues to transform 14C-halothane to metabolites that bind strongly to tissue components. In addition to the liver, the other tissues shown to have a marked halothane-metabolizing capacity were the nasal mucosa, lateral nasal gland, mucosa of the tongue, cheek, soft palate (but not the hard palate), pharynx, larynx, oesophagus, and the tracheo-bronchial mucosa. The in vivo data obtained indicated a diffusion of the halothane over the walls of the large intestine and the caecum, followed by the formation of apparently reductive metabolites by intestinal microbes and a binding of the metabolites to the intestinal contents. The localization of halothane metabolites in the upper alimentary and respiratory pathways is correlated to the presence of cytochrome P-450 at these sites.

Saturable accumulation of retinoic acid in neural and neural crest derived cells in early embryonic development.

Retinoic acid (RA) binds to a cytosolic protein distinguishable from the cellular retinol (R) binding protein. Recent studies, showing an influence by R and RA on genomic expression, suggest an interaction with the cell nucleus mediated by the specific binding proteins in a manner resembling that of steroid hormones. RA can irreversibly stimulate in vitro differentiation of teratocarcinoma cells and support early embryonic development in vitamin A depleted animals. This study demonstrates a saturable, highly specific and regional accumulation of RA in the neuroepithelium and developing CNS that occurs in early but not in late fetal development in the mouse. The results suggest that a binding protein, or some other cellular mechanism for accumulation of RA is expressed in the neural cells only during restricted periods of development. High levels are recorded also in regions where cranial neural crest cells are known to migrate, and later in the visceral arches and maxillary areas, the mesenchyme of which is known to be partly derived from migrating cranial neural crest cells. The specific accumulation of RA in embryonic neural and cranial neural crest cells is in line with animal experiments and human clinical data, showing that retinoids specifically impair CNS, eye, ear, and facial development.

Distribution of halothane and the metabolites trifluoroacetic acid and bromide in the conceptus after halothane inhalation by pregnant mice.

Mice in late stage of gestation were exposed to halothane at various concentrations for 1 hr, and were killed at different time intervals after discontinuance of inhalation. The concentration of halothane in maternal plasma decreased rapidly, and in the amniotic fluid the halothane never reached more than 20% of maternal plasma levels. Trifluoroacetic acid (TFA) and bromide, formed mainly by maternal metabolism of halothane, accumulated in foetus and amniotic fluid with time, and reached plateau levels in amniotic fluid between 4 and 24 hrs. TFA infused intravenously to the mother reached higher levels in amniotic fluid after long survival times, than in maternal plasma. Equilibrium dialysis experiments showed that TFA and trichloroacetic acid (TCA) (previously shown to accumulate in amniotic fluid) were bound to amniotic fluid macromolecules only to approximately 20-30 percent. This was at the same magnitude (or lower) as compared to binding in maternal plasma, suggesting that such binding did not contribute to the observed retention in the amniotic fluid. Other possible explanations for the slow accumulation and long-term retention in amniotic fluid are transport by bulk flow via foetus, excretion via foetal urine, or paraplacentally through endometrium and foetal membranes, followed by trapping in the amniotic fluid. The significance of this accumulation of metabolites of halogenated organic solvents and halothane for their foetotoxicity is not clear.

Trichloroacetic acid accumulates in murine amniotic fluid after tri- and tetrachloroethylene inhalation.

The distribution of trichloroethylene (Tri) and tetrachloroethylene (Tetra) and their metabolites have been studied in pregnant mice by means of whole-body autoradiography (14C-labelled Tri and Tetra) and gas chromatography, with special emphasis on possible uptake and retention in the foetoplacental unit. Volatile (non-metabolized) activity appeared at short intervals after a 10 min. or 1 hr inhalation period in foetus and amniotic fluid. Most notable, however, was a strong accumulation and retention (peak at 4 hrs) in amniotic fluid of the metabolite trichloroacetic acid (TCA) after inhalation of either of the solvents. The main metabolite of Tri, trichloroethanol (TCE) (or conjugates), did not accumulate specifically as compared to maternal plasma. TCA infused intravenously in the maternal plasma was accumulated in amniotic fluid, but less pronounced than after Tri and Tetra inhalation, indicating that some metabolism of Tri and Tetra to TCA may occur in the foetoplacental unit. The results suggest that TCA may be transported to the foetus partly paraplacentally through foetal membranes and amniotic fluid, with the possibility of foetal swallowing or absorption through the skin. Foetal urinary activity also suggests that circulation between foetus and amniotic fluid may contribute to the long-term retention in the foetoplacental unit. In the mother, after inhalation exposures, and in intraperitoneally injected newborn mice, non-extractable radioactivity was found in the respiratory tract, liver, and kidney, indicating binding to these organs through metabolism.

Placental transfer and distribution of toluene, xylene and benzene, and their metabolites during gestation in mice.

The distribution of radioactivity in pregnant mice was registered at different time intervals (0-24 h) after a 10 min period of inhalation of 14C-toluene, -xylene, and -benzene. Autoradiographic and liquid scintillation methods were used to make possible the distinction between volatile, water-soluble and firmly tissue-bound radioactivity. Toluene, xylene, as well as benzene reached high concentrations immediately after inhalation in lipid-rich tissues (brain and fat) and well perfused organs (e.g., liver and kidney) but were rapidly eliminated resulting in low concentrations at 1 h in all maternal tissues, except fat. Metabolites reached peak levels around 30 min to 1 h after inhalation, but were also relatively rapidly eliminated. One exception from this general trend was a retention of firmly tissue-bound metabolites in maternal liver and kidney after benzene inhalation. Another exception was the very strong accumulation of water-soluble metabolites at 4 and 24 h in the nasal mucosa and olfactory bulb after inhalation of toluene and xylene. Volatile radioactivity was observed in the placenta and fetuses immediately and up to 1 h after inhalation of all the three studied solvents at all stages of gestation. The fetal levels were, however, much lower than in maternal tissues. In early gestation an even distribution pattern was observed, while the fetal liver reached higher concentration than other fetal tissues in late gestation. In similarity with maternal tissues, fetal tissues reached the highest levels of metabolites 30 min to 1 h after inhalation. A retention in uterine fluid was seen at 4 h. Otherwise no retention of metabolites was observed in the feto-placental unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of chloroform and methyl chloroform and their metabolites in pregnant mice.

The distribution of radioactivity in pregnant mice was registered at different time intervals (0-24 h) after a 10-min period of inhalation of 14C-labelled chloroform and methyl chloroform. Autoradiographic and liquid scintillation methods were used to make possible the distinction between volatile (non-metabolized), water-soluble and firmly tissue-bound radioactivity. Methyl chloroform was retained longer in fat as compared to chloroform. Metabolites of chloroform were present in a much greater abundance than those of methyl chloroform and they were found preferentially in the respiratory tract (nasal mucosa, trachea and bronchi), liver and excretory organs. Tissue-bound activity after chloroform inhalation or i.p. injection to newborn mice was found in the respiratory tract and centrilobular areas of the liver. Volatile radioactivity was observed in the placenta and fetuses at short time intervals after inhalation of both chloroform and methyl chloroform at all stages of gestation. While a low level of radioactive metabolites of methyl chloroform was observed in the fetoplacental unit, metabolites of chloroform accumulated with time. This fact was especially marked in the amniotic fluid, where the peak level of radioactivity was observed at 4 h. In early gestation, metabolites accumulated in the embryonic neural tissues. Tissue-bound metabolites of chloroform were observed in the fetal respiratory epithelium in late gestation, indicating a capacity for drug metabolism in these cells in the late fetal period.

Accumulation in murine amniotic fluid of halothane and its metabolites.

The distribution of radioactivity in pregnant mice was registered at 0, 4, and 24 hrs after a 10 min. period of inhalation of 14C-halothane. Autoradiographic methods were used to allow to distinguish between the distribution of volatile (non-metabolize) halothane, water-soluble metabolites, and firmly tissue-bound metabolites. While volatile radioactivity was seen predominantly at short survival intervals, e.g. in body fat, blood, brain and liver, metabolites accumulated with time. Peak values occurred at 4 hrs in most organs (measured with liquid scintillation as well). The most remarkable findings were the high concentrations of radioactivity in amniotic fluid (and the ocular fluids of adults) with peak values at 4 hrs and rather high concentrations still prevailing at 24 hrs after inhalation. It is assumed that this activity represents only partly volaile halothane and mostly non-volatile metabolites. High activity of metabolites was seen in the neuroepithelium of the embryo in early gestation. Firmly tissue-bound metabolites, still remaining after washing the tissues with trichloroacetic acid and organic solvents, were found in the nasal mucosa, trachea and bronchial tree and in (presumably centrilobular) zones of the liver of adults after inhalation and 5-day old mice after intraperitoneal injection, indicating the formation of reactive metabolites in these organs. Firmly tissue-bound activity was not observed in the corresponding foetal organs.