Diethyl ether has been a widely used anesthetic in animal research. It provides a simple way to anesthetize animals and allows for more effective blood collection by cardiac puncture because the heart is still beating during the collection. The American Veterinary Medical Association Panel on Euthanasia currently promotes the use of isoflurane as a replacement for diethyl ether because of explosion hazards and increased toxicity ether presents (1).
Ether affects several enzymes of the P450 complex when animals are treated for a prolonged period (2,3), and the subfamily 2E1 seems to be the most affected. It has been shown that exposure of rats to ether anesthesia for 6 min results in increase of CYP2E1 activity by 60% (4).
Isoflurane is mainly metabolized by CYP2E1 in human liver microsomes (5), and there is evidence to suggest it is metabolized in a similar way by rat liver microsomes (6). In fact, exposure of rats to isoflurane 3 times daily for 3 days resulted in a 1.5-fold increase in N-nitrosodimethylamine activity (2).
Although prolonged exposure of rats to both ether and isoflurane seems to significantly affect the activity of certain P450 enzymes, there are no reports regarding brief exposure (e.g., the length of time needed to collect blood and harvest tissues) of animals to these drugs and the effects on these enzymes. Therefore, the objective of this study was to determine whether a brief exposure to one of these anesthetics would result in alteration of the activities of CYP2E1, NADH-ferricyanide reductase, and NADPH-cytochrome c reductase.
This study was approved by the Animal Care and Use Committee of the University of Minnesota. Male Wistar rats (n = 30), aged 3-4 wk, were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Animals were housed individually in wire cages in rooms maintained at 20°C ± 2°C with a relative humidity of 50% ± 10% and a 12-h light/dark cycle. Food and water were available ad libitum throughout the study. Rats were fed a purified diet (AIN-93G) for 6 wk. At the end of this period they were divided into 3 groups (n = 10). Each group was killed using one of three anesthetics: carbon dioxide gas (CO2), diethyl ether, or isoflurane. For treatment with CO2, the rats were put inside a closed chamber to which CO2 gas was vented. They were kept in the chamber until loss of the righting reflex (approximately 60 s). This treatment group was used as the control group. For treatment with diethyl ether, rats were put in a closed 9”× 4” jar, containing 3 paper towels saturated with 40 mL of diethyl ether. The same procedure was used for treatment with isoflurane, and the animals were kept inside the closed jars until loss of righting reflex (average exposure time was 75 s for both treatments).
Microsomal isolation was performed using the method described by Prasad et al. (7). Livers were perfused in situ with ice-cold 50 mM Tris/150 mM KCl buffer (pH 7.5), and homogenized in Tris/KCl buffer and phenylmethylsulphonyl fluoride was added to inhibit protease activity. The microsomal fraction was obtained by ultracentrifugation of the 10,000g supernatant at 105,000g for 70 min. The microsomal pellet was resuspended in Tris/glycerol buffer containing 0.01% butylated hydroxytoluene, and stored at –18°C. Before analyses, microsomes were washed by ultracentrifugation at 105,000g with 0.1 mM sodium pyrophosphate/1 mM EDTA buffer.
To assay the activity of CYP2E1 in the microsomal fractions, a kinetic assay for p-nitrophenol hydroxylase was performed. The method was described by Allis and Robinson (8) and consists of a simplified version of the method of Reinke and Moyer (9). Briefly, 0.1 mM p-nitrophenol was mixed with 0.1 M phosphate buffer (pH 6.8) and the microsomal fraction (0.2 mg protein/mL) in a 1-cm spectrophotometer cuvette and incubated for 5 min at 37°C. The reaction was initiated by adding NADPH to a final concentration of 0.1 mM. The reaction mixture was incubated for another 5 min at 37°C and then absorbance of p-nitrocatechol was measured at 480 nm for 10 min on a Cary 50 spectrophotometer. Enzyme activity was calculated using the extinction coefficient of 3.57 mM−1 · cm−1 for p-nitrocatechol. The assays for NADH-ferricyanide reductase, and NADPH-cytochrome c reductase were performed as described by Crankshaw et al. (10). Enzyme activities were calculated using extinction coefficients of 21.1 mM−1 · cm−1 for cytochrome c, and 1.02 mM−1 · cm−1 for ferricyanide.
Comparisons among groups were made by one-way analysis of variance using SigmaStat software, version 3.0 (Systat Software Inc., Point Richmond, CA).
Results and Discussion
Table 1 shows the effects of CO2, diethyl ether, and isoflurane anesthesia on the P450 enzymes. Although the enzyme activities were slightly numerically greater after isoflurane treatment, no statistically significant differences were found among groups for any of the enzyme activities. These data suggest that a brief exposure of rats to either diethyl ether or isoflurane (approximately 75 s) does not significantly affect the activity of CYP2E1 or P450 reductases compared with CO2 exposure. Although previous studies in rats exposed to ether or isoflurane have reported increased P450 enzyme activity, the exposure times in these studies were much longer than in ours (2,4). Our study indicates that either diethyl ether or isoflurane may be used to briefly anesthetize rats (up to 75 seconds of exposure time) in studies involving measurement of cytochrome P450 enzymes, without concern for induction of enzyme activity by the anesthetic. In this case, time of exposure and amount of anesthetic used should be limited to those described in this report.
1. AVMA Panel on Euthanasia. 2000 report of the AVMA Panel on Euthanasia. Am Vet Med Assoc 2001;218:669–96.
2. Brady JF, Lee MJ, Li M, et al. Diethyl ether as a substrate for acetone/ethanol-inducible cytochrome P-450 and as an inducer for cytochrome(s) P-450. Mol Pharmacol 1987;33:148–54.
3. Loch JM, Potter J, Bachmann KA. The influence of anesthetic agents of rat hepatic cytochromes P450 in vivo. Pharmacology 1995;50:146–53.
4. Liu PT, Ioannides C, Shavila, J. et al. Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney. Biochem Pharmacol 1993;45:871–7.
5. Kharasch ED, Thummel KE. Identification of cytochrome P450 2E1 as the predominant enzyme catalyzing human liver microsomal defluorination of sevoflurane, isoflurane, and methoxyflurane. Anesthesiology 1993;79:795–807.
6. Bradshaw JJ, Ivanetich KM. Isoflurane: a comparison of its metabolism by human and rat hepatic cytochrome P-450. Anesth Analg 1984;63:805–13.
7. Prasad JS, Crankshaw DL, Erickson RR, et al. Studies on the effect of chronic consumption of moderate amounts of ethanol on male rat hepatic microsomal drug-metabolizing activity. Biochem Pharmacol 1985;34:3427–31.
8. Allis JW, Robinson BL. A kinetic assay for p-nitrophenol hydroxylase in rat liver microsomes. Anal Biochem 1994;219:49–52.
9. Reinke LA, Moyer MJ. p-Nitrophenol hydroxylation: a microsomal oxidation which is highly inducible by ethanol. Drug Metabo Dispos 1985;13:548–52.
10. Crankshaw DL, Hetnarski HK, Wilkinson CF. Microsomal NADPH-cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania). Insect Biochem 1979;9:43–8.