
==== Front
BMC Anesthesiol
BMC Anesthesiol
BMC Anesthesiology
1471-2253
BioMed Central London

2704
10.1186/s12871-024-02704-5
Research
Different ethanol exposure durations affect cytochrome P450 2E1-mediated sevoflurane metabolism in rat liver
Jiang Wei 1
Zhang Min 1
Cao Rui 1
Wang Xinghao 1
Zuo Youbo zuoyb2519@163.com

12
1 https://ror.org/05k3sdc46 grid.449525.b 0000 0004 1798 4472 Department of Anesthesiology, School of Clinical Medicine of North Sichuan Medical College, Nanchong, 637000 Sichuan China
2 https://ror.org/01673gn35 grid.413387.a 0000 0004 1758 177X Department of Anesthesiology, Affiliated Hospital of North Sichuan Medical College, Nanchong, 637000 Sichuan China
10 9 2024
10 9 2024
2024
24 3213 3 2024
27 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Chronic alcohol users often exhibit an increased minimum alveolar concentration (MAC) of sevoflurane, yet the specific mechanism remains unclear. It has been reported that ethanol exposure can upregulate the protein expression and enzyme activity of cytochrome P450 2E1 (CYP2E1). CYP2E1 is a key enzyme that converts 2–5% of sevoflurane into equimolar amounts of hexafluoroisopropanol (HFIP) and F−. This study aims to explore whether ethanol exposure could alter sevoflurane metabolism through CYP2E1 modulation, potentially explaining the increased MAC observed in alcohol users.

Methods

Eighty adult male Sprague-Dawley (SD) rats were randomly divided into two groups and received either 50% ethanol (dose: 3 g/kg) or 0.9% saline twice daily by gavage. After 1, 2, 3, and 4 weeks of gavage, ten rats were randomly selected from each group to undergo 1-hour anesthesia with 2.3% sevoflurane. Blood samples were collected after anesthesia to measure the concentration of free HFIP using gas chromatography. Additionally, the left lobe tissue of the liver was collected for the analysis of CYP2E1 protein expression by Western blot and CYP2E1 enzyme activity by colorimetric assay. Correlations between these parameters were analyzed using Pearson’s correlation.

Results

In the ethanol group, CYP2E1 expression, activity, and the concentration of free HFIP were significantly higher at all time points compared to the control group (P < 0.05), except for protein expression in the first week (P > 0.05). Within-group comparisons indicated no significant changes in any of the parameters for the control group (P > 0.05). In the ethanol group, there was no difference in free HFIP concentration between the first and second weeks (P > 0.05), but a significant increase was observed in the third and fourth weeks (P < 0.01); protein expression and enzyme activity significantly varied over time, especially showing a notable increase from the first to the third and fourth weeks (P < 0.05). Correlation analysis revealed strong positive correlations between free HFIP concentration and CYP2E1 activity (r = 0.7898), free HFIP concentration and CYP2E1 expression (r = 0.8418), and CYP2E1 activity and expression (r = 0.8740), all with P < 0.001.

Conclusions

Ethanol exposure increased both the expression and enzymatic activity of CYP2E1, consequently enhancing the metabolism of sevoflurane.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12871-024-02704-5.

Keywords

Ethanol
CYP2E1
Sevoflurane
Hexafluoroisopropanol (HFIP)
Inhalation anesthesia
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pmcBackground

Alcohol abuse is a global public health problem, placing a significant burden on both society and individual health. In 2016, around 2.3 billion individuals were identified as alcohol consumers, with alcohol-related diseases resulting in an estimated 3 million deaths [1, 2]. In medical environments, particularly during emergency surgeries, anesthesiologists frequently encounter difficulties in managing patients with underreported alcohol abuse [3, 4]. Long-term drinkers exhibit increased tolerance to various anesthetic drugs, including sevoflurane, fentanyl, and propofol, necessitating higher dosages to achieve effective anesthesia [5, 6]. For example, they have a higher minimum alveolar concentration (MAC) of sevoflurane, a phenomenon whose underlying mechanism remains to be fully elucidated [7, 8].

Ethanol is primarily metabolized in the body by alcohol dehydrogenase (ADH) and the Microsomal Ethanol Oxidizing System (MEOS) enzyme cytochrome P450 2E1 (CYP2E1) [9, 10]. CYP2E1 is a substrate-inducible enzyme, and long-term or excessive ethanol exposure can increase its expression and activity [11, 12]. It is noteworthy that CYP2E1 not only participates in the metabolism of ethanol but also serves as a critical enzyme for the metabolism of sevoflurane [13].

Sevoflurane, a widely used inhalation anesthetic, undergoes metabolism primarily through CYP2E1, with 2–5% being converted into equimolar production of hexafluoroisopropanol (HFIP) and inorganic fluoride (F−) [14]. The majority of HFIP is conjugated with glucuronic acid and subsequently excreted in the urine. However, about 15% or less of HFIP remains in the bloodstream in its free form, noted for its high solubility. This characteristic is demonstrated by a saline/gas partition coefficient of 263 and a blood/gas partition coefficient of 452 [15, 16]. Additionally, free HFIP has a half-life of 19 h, indicating its slow elimination from the body [17, 18]. These characteristics establish free HFIP as a reliable biological marker for evaluating the metabolic degradation of sevoflurane.

In this study, our primary objective is to investigate the impact of ethanol exposure on the expression and enzymatic activity of the CYP2E1 protein, as well as the concentration of free HFIP in the bloodstream. By exploring these elements, we intend to clarify the relationship between the duration of ethanol exposure and sevoflurane metabolism.

Methods

Materials

Sevoflurane (Hengrui, China), HFIP (Sigma Aldrich, US), pentobarbital sodium (xiya, China), Anti-CYP2E1 Antibody (Abcam, ab28146,), Anti-β-actin Antibody (Abclonal, AC026). Goat Anti-Rabbit IgG (H + L) HRP (Affinity, S0001), HRP Goat Anti-Mouse IgG (H + L) (Abclonal, AS003), BCA protein Assay Kit (Beyotime, P0009).

Animal

All experimental procedures involving animals were approved by the North Sichuan Medical College (NSMC) Animal Ethics Committee under license number [2023]018. All animal experiments adhered to the ARRIVE guidelines and were conducted in accordance with the guidelines of the National Institutes of Health. Measures were taken to minimize the number of animals used and to reduce their suffering. A total of 80 healthy adult male SD rats, weighing 180–210 g, were obtained from the Animal Experimental Center of North Sichuan Medical College. The rats were housed in an Animal Facility and acclimatized to a 12-hour light-dark cycle, with temperatures maintained between 20 and 24℃ and humidity at 50 ± 10%. Food and water were provided ad libitum. The rats underwent a one-week acclimatization period in the laboratory environment before the commencement of the experiments.

Fig. 1 Experimental protocol

Experimental groups and treatments

After a one-week acclimatization period, 80 rats were randomly divided into two groups by an independent party using a random number generator: an ethanol group (n = 40) and a control group (n = 40). Rats in the ethanol group received 50% ethanol via oral gavage at a dosage of 3 g/kg twice daily, with an 8-hour interval, for a continuous period of 4 weeks [19]. In contrast, the control group was given 0.9% saline solution via oral gavage, adjusted for their body weight (Fig. 1). The gavage procedure was performed by researchers not involved in the study’s daily observations.

At the end of each week (1, 2, 3, and 4 weeks after gavage), ten rats from each group were randomly selected by an independent individual. Following a 12-hour fasting period, rats were transferred to the animal operating room. There, they were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). Upon cessation of movement, a tracheotomy was performed, and a 14# PE tube was inserted, then connected through a T-tube to a small animal ventilator, initiating mechanical ventilation. Before starting mechanical ventilation, a simulated lung was used with an oxygen flow of 0.5 L/min. The vaporizer was adjusted to maintain the sevoflurane concentration at 2.3% in the circuit, as measured by gas chromatography (GC) [20]. The respiratory parameters were established as follows: a tidal volume (VT) of 10 ml/kg, a respiratory rate (RR) of 65 breaths per minute, an inspiratory-to-expiratory ratio of 1:2, and a positive end-expiratory pressure maintained at 0 [21, 22]. Pulse rate, oxygen saturation, rectal temperature, and ventilatory pressures, including mean and plateau pressures, were continuously monitored. Rectal temperature was measured via a rectal thermometer and regulated within the range of 36.5–37.5℃ using a circulating heating pad. Sevoflurane concentration in the circuit was measured at 5, 15, 30, and 45 min after the start of anesthesia using GC, with necessary vaporizer adjustments made to maintain the sevoflurane concentration at approximately 2.3%. After one hour of anesthesia, 5 ml of blood was collected via ventral aortic puncture using a sterile, sealed syringe for the analysis of free HFIP and sevoflurane concentrations. The left lateral lobe of the liver was subsequently excised, immediately frozen at -80℃, and preserved for later analysis of CYP2E1 protein expression by Western blot and CYP2E1 enzyme activity using a colorimetric assay. Following the collection of tissues, euthanasia was conducted using sodium pentobarbital (150 mg/kg, iv) while the rats were under sevoflurane anesthesia.

The concentrations of free HFIP and sevoflurane

Gas chromatograph (Agient 7890 A, Beijing, China), six-port valve, and capillary column (HP-5) were used in the study. The operating parameters were set as follows: injector temperature: 70℃, column oven temperature: 30℃, hydrogen flame detector temperature: 200℃, hydrogen flow rate: 30 mL/min, air flow rate: 300 mL/min, and nitrogen flow rate: 20 mL/min. Using these parameters, high concentrations of sevoflurane and low concentrations of HFIP can be distinctly separated in the chromatogram.

The laboratory temperature was maintained at 25℃. A small quantity of HFIP liquid sample was extracted and weighed, then injected into a sealed conical bottle under negative pressure (0.08Mpa) and left to stand until completely evaporated. After a stabilization period, the pressure inside and outside the conical bottle was equalized without any leakage. The concentration of the prepared standard gas was calculated using the formula C = W × R ×T / (MW × Vx). C represents the concentration of the HFIP standard gas. W is the weight of the HFIP liquid sample. R is the ideal gas constant, valued at 0.08206 L·atm/(mol·K). T denotes the temperature in Kelvin, which is 298 K in this study. MW stands for the molar mass of HFIP, which is 168.05 g/mol. Vx is the volume of the sealed conical bottle. The standard gas was repeatedly diluted by half. Subsequently, a gas chromatograph was used to construct the calibration curve equation, which relates the concentration to the corresponding peak area. This calibration curve equation can be utilized to detect the concentration of free HFIP in blood. It is crucial to ensure that the concentration range encompasses the expected analyte concentration, and R2 > 0.999 [16].

After blood collection, air was drawn into the syringe already containing 5 ml of blood to increase its volume to 17.5 ml. The syringe was then placed in a constant-temperature air bath oscillator set at 37℃ and oscillated to promote the evaporation of free HFIP. After 90 min, additional air was drawn to adjust the volume to 20 ml, and the oscillation continued for another 60 min. This process ensures the gradual evaporation of free HFIP from the blood into the gas phase, reaching equilibrium between the two phases. The gas within the syringe was then analyzed using a gas chromatograph, and the concentration of HFIP in the gas phase (Cg) was determined using the previously established calibration curve equation. The concentration of free HFIP in the blood (C) was calculated with the formula C = (Vg / V + λ) × Cg, where Vg is the volume of gas in the syringe after equilibration (15 ml), V represents the total volume of gas and blood within the syringe (20 ml), and λ is the blood/gas partition coefficient of free HFIP in rats (452) [16]. The standard curve establishment and GC detection method for sevoflurane is the same as that for HFIP.

Western blotting assay

After weighing, liver tissues were placed into homogenization tubes. Then, 3 mm steel beads and Radio-Immunoprecipitation Assay (RIPA) buffer with protease inhibitors were added at a weight-to-buffer ratio of 1:10. The tubes were then homogenized using a high-speed, low-temperature tissue homogenizer at -20 °C for 60 s per cycle, repeated four times, and then lysed on ice for 30 min. Centrifugation at 4 °C and 12,000 rpm was subsequently performed for 10 min. The supernatant was collected and stored at -80 °C. Protein concentration was assessed using the BCA Protein Assay Kit. SDS-polyacrylamide gel electrophoresis (PAGE) was conducted with a 5% stacking gel and a 10% resolving gel, loading 30 µg of protein per well. After electrophoresis, proteins were transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk in Tris-Buffered Saline with Tween 20 (TBST) on a shaker for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies against CYP2E1 (1:5000, Abcam, UK) and β-actin (1:50000, Abclonal, China). Following this, it was incubated with secondary antibodies for 2 h at room temperature: Goat Anti-Rabbit IgG (H + L) HRP (1:5000, Affinity, USA) and HRP Goat Anti-Mouse IgG (H + L) (1:5000, Abclonal, China). The ECL reagent was evenly applied to the membrane, and signal detection was performed using the Fusion FX system (Vilber Lourmat). Band densities were quantified using ImageJ software. With β-actin serving as the internal control, the grayscale ratio of CYP2E1 to β-actin was computed to determine relative protein expression levels [23].

The enzyme activity of CYP2E1

The activity of p-nitrophenol hydroxylase (p-NPH), as the diagnostic marker for CYP2E1, involves the hydroxylation of p-nitrophenol to p-nitrocatechol. This reaction product can be detected at a wavelength of 510 nm, allowing for the quantitative measurement of CYP2E1 activity using spectrophotometry [24, 25]. This experimental procedure was performed using the GMS18021.1 kit from GeneMe (Shanghai). 480 µl of GENMED buffer, reaction liquid, and base color liquid were added into a 1.5 ml centrifuge tube. 20 µl of the supernatant with a protein concentration of 100 µg was added to the sample tube, while 20 µl of negative control containing DDC (diethyldithiocarbamate) was added to the background tube. DDC is a selective mechanism-based inhibitor of CYP2E1 and significantly inhibits the CYP2E1-catalyzed p-nitrophenol hydroxylase (p-NPH) activity. After incubating at 37℃ for 30 min, 100 µl of stop solution was added, followed by centrifugation at 10,000 g for 5 min. The supernatant was then transferred and mixed with 300 µl of neutralizing liquid. The resulting mixture was immediately measured using a spectrophotometer (Shimadzu UV-26001, Suzhou, China). The enzyme activity of the sample was calculated based on the absorbance values (sample tube reading - background tube reading) and the extinction coefficient of 9.53 L/(mmol·cm) for p-nitrocatechol, expressed as nmol p-nitrocatechol/min/mg protein.

Statistical analysis

We used G-Power software to calculate the sample size, choosing a one-tailed t-test with an anticipated power of 0.8 and a significance level of 0.05. Based on the mean concentration of free HFIP observed in our preliminary study, the total sample size was determined to be 80.

Statistical analysis was performed using SPSS 26.0 software. The Shapiro-Wilk test was used to assess the normality of the data. Results are presented as mean ± standard deviation (x ± s). Two-way repeated measures analysis of variance (2-way RM-ANOVA) was conducted to examine the interaction between groups and time. When the F statistic was significant, Bonferroni correction was used for multiple comparisons. Correlations between the measured variables were analyzed using Pearson’s correlation, with P < 0.05 indicating statistical significance.

Results

During the experimental period, no accidental deaths occurred among the rats. Rats in the ethanol group exhibited signs of drowsiness, reduced activity levels, decreased appetite, and irritability, with a significantly slower rate of weight gain compared to the control group (P < 0.001, Fig. 2). During sevoflurane inhalation anesthesia, the rats’ toe end skin appeared ruddy. The rats’ pulse rates remained above 254 bpm, and their rectal temperatures were maintained within the range of 36.5–37.5℃. All rats consistently maintained an oxygen saturation level above 98%, while the airway pressure stayed within a 0–2 mmHg range. Statistical analysis included data from all 80 rats.

Fig. 2 The graph illustrates the mean (SD) weekly weight (g) rats subjected to different gavage regimens. At corresponding time points, the ethanol gavage group showed significantly lower weights compared to the control group, denoting a statistically significant difference (P < 0.001)

Fig. 3 GC chromatogram of a blood sample of a sevoflurane-anesthetized rat

Free HFIP concentration

The GC chromatogram of free HFIP in the blood sample of a rat anesthetized with sevoflurane is displayed as shown in Fig. 3.

In the examination of free HFIP concentration, 2-way RM-ANOVA was utilized to analyze any interaction between the ‘Groups’ (Ethanol vs. Control) and ‘Time’ (across four weeks). Mauchly’s Test was employed to ascertain the assumption of sphericity, which the data successfully fulfilled (P = 0.411). The analysis revealed significant effects due to group differences (F = 500.22, P < 0.001, Table 1), time variations (F = 9.20, P < 0.001, Table 1), and crucially, the interaction between these elements (F = 9.29, P < 0.001, Table 1), indicating distinct variations in free HFIP concentration between the ethanol and control groups across the observed periods.

Table 1 The free HFIP concentration in the blood of the two groups of rats (mean, SD; n = 10)

	Control(%)	Ethanol(%)	F	P	
1week	0.019 ± 0.003	0.026 ± 0.002C	33.74	<0.001	
2weeks	0.017 ± 0.004	0.030 ± 0.002BC	99.83	<0.001	
3weeks	0.018 ± 0.002	0.035 ± 0.003A	221.52	<0.001	
4weeks	0.018 ± 0.003	0.035 ± 0.003AB	118.94	<0.001	
F	0.69	28.14			
P	>0.05	<0.001			
overall test	
group (F,P)	500.22	<0.001			
time (F,P)	9.20	<0.001			
interact (F,P)	9.29	<0.001			
The results of the two-way RM ANOVA demonstrated a significant interaction between different gavage treatments and time on the generation of free HFIP in the blood (F = 9.29, P < 0.001). Bonferroni correction was applied for multiple comparisons. For within-group comparisons, differences were denoted using alphabetical letters (A, B, C). Groups sharing the same letter indicate non-significant differences, while different letters indicate significant differences (P < 0.01). Although the ethanol group values at weeks 3 and 4 appear identical, they are rounded values. The comparison between weeks 2 and 4 yielded a P value of 0.051, which is not statistically significant

After identifying significant F-values, Bonferroni correction was applied for comprehensive comparisons. This demonstrated that the ethanol group consistently had higher free HFIP concentrations than the control at all measured points (F values ranging from 33.74 to 221.52, P < 0.001, Table 1). Within-group analysis revealed constant HFIP levels in the control group throughout the study (F = 0.69, P > 0.05, Table 1), in marked contrast to the ethanol group, which experienced significant fluctuations (F = 28.14, P < 0.001, Table 1). Specifically, HFIP concentrations in the ethanol group progressively increased, peaking in the third week. Although no significant differences were noted between weeks 1 and 2 (P > 0.05, Table 1), significant differences emerged between week 1 and both weeks 3 and 4, as well as between weeks 2 and 3 (P < 0.01, Table 1).

Collectively, these findings suggest that prolonged ethanol consumption increased free B-HFIP concentration.

Fig. 4 Influence of different gavage methods and time on CYP2E1 protein expression, analyzed by two-way RM-ANOVA with Bonferroni correction. Relative CYP2E1 expression in livers normalized to β-actin as an internal control. Data presented as mean (SD); n = 3. For within-group comparisons, differences were denoted using alphabetical letters (a, b, c). Groups sharing the same letter indicate non-significant differences, while different letters indicate significant differences (P < 0.05). No significant differences across four time points in control group. * indicates between-group differences at the same time points (P < 0.05). The blots came from separate membranes for optimal sensitivity. We maintained consistent loading and uniform processing for reliability. Full-length blots are presented in Supplementary Fig. 1

CYP2E1 protein expression

To investigate the potential influence of ethanol on CYP2E1, we employed the Western Blot method to assess the expression of CYP2E1 protein in the rat liver. Our findings demonstrated that as the duration of gastric administration extended, the CYP2E1 protein expression in the ethanol group gradually amplified, reaching the highest levels by week 4 (Fig. 4). Within the ethanol group, a significant divergence was discerned between weeks 1–2 and weeks 3–4 (P < 0.05, Fig. 4). In contrast, no significant differences were noted within the control group (P > 0.05, Fig. 4). In comparisons between the groups, the ethanol group displayed substantially higher CYP2E1 expression compared to the control group at weeks 2, 3, and 4 (P < 0.05, Fig. 4). These results indicate that continuous gastric administration of ethanol augments the expression levels of CYP2E1.

Fig. 5 The changes in rat liver CYP2E1 activity at different gavage durations. Data are presented as mean (SD); n = 10. For within-group comparisons, differences were denoted using alphabetical letters (A, B, C). Groups sharing the same letter indicate non-significant differences, while different letters indicate significant differences (P < 0.01). *** represents significant between-group differences (P < 0.001)

CYP2E1 enzyme activity

Our study utilized two-way RM-ANOVA to assess CYP2E1 enzyme activity, examining interactions between groups (Ethanol vs. Control) and time over four weeks. Mauchly’s Test confirmed the sphericity assumption (P = 0.063), indicating equal variance across group combinations. Significant findings included effects due to group differences (F = 110.55, P < 0.001), time (F = 4.90, P < 0.01), and their interaction (F = 3.75, P < 0.05), showing distinct changes in CYP2E1 enzyme activity over time between the groups, markedly altered by ethanol exposure.

Further analysis with Bonferroni correction revealed that the ethanol group consistently exhibited higher CYP2E1 activity compared to the control across all time points (F values ranging from 26.39 to 66.15, P < 0.001). Within-group contrasts showed no significant changes in enzyme activity for the control (F = 0.087, P > 0.05), unlike the ethanol group, which demonstrated significant variations (F = 17.48, P < 0.001). Statistically significant differences were observed between week 1 and weeks 3–4 (P < 0.01, Fig. 5). The results highlight that continuous gastric administration of ethanol enhances the enzymatic activity of CYP2E1.

Sevoflurane concentration

Table 2 The sevoflurane concentration in the blood of the two groups of rats (mean, SD; n = 10)

	Control (%)	Ethanol (%)	
1week	1.729 ± 0.050	1.734 ± 0.047	
2weeks	1.728 ± 0.048	1.725 ± 0.038	
3weeks	1.737 ± 0.047	1.724 ± 0.044	
4weeks	1.750 ± 0.035	1.743 ± 0.043	
overall test	
group(F,P)	0.38	>0.05	
time(F,P)	0.66	>0.05	
interact(F,P)	0.10	>0.05	

The GC chromatogram (Fig. 3) shows the second peak at 2.141, which corresponds to sevoflurane. The data are detailed in Table 2. The results of the two-way repeated measures ANOVA (RM ANOVA) indicate that, after implementing strict control measures and a standardized inhalation process, there was no significant interaction between different gavage treatments and time on the sevoflurane concentration in the blood (F = 0.10, P > 0.05). Neither the main effect of time (F = 0.66, P > 0.05) nor the main effect of group (F = 0.38, P > 0.05) was statistically significant (Figs. 6 and 7).

Fig. 6 Correlation between CYP2E1 activity and free HFIP concentration (r = 0.7898, P < 0.001). The linear regression equation is Y = 122.4X + 0.1075 (r² = 0.6239, P < 0.001). Correlation between CYP2E1 expression and free HFIP concentration (r = 0.8418, P < 0.001). The linear regression equation is Y = 193.9X − 2.609 (r² = 0.7087, P < 0.001)

Fig. 7 Correlation between CYP2E1 activity and CYP2E1 expression (r = 0.8740, P < 0.001). The linear regression equation is Y = 0.7710X + 1.220 (r² = 0.7639, P < 0.001)

To examine the relationships between free B-HFIP levels and liver CYP2E1 activity and expression, we conducted Pearson correlation analyses, which indicated significant positive correlations between free B-HFIP levels and CYP2E1 activity (r = 0.7898), free B-HFIP levels and CYP2E1 expression (r = 0.8418), and CYP2E1 activity and expression (r = 0.8740), all with P < 0.001, underscoring the potential role of CYP2E1 in the metabolism of sevoflurane.

Discussion

To explore the impact of prolonged ethanol exposure on the metabolism of sevoflurane, we conducted a four-week ethanol gavage study in rats, periodically assessing changes in the expression and activity of CYP2E1, as well as variations in the concentration of free HFIP. The findings reveal that with the extension of ethanol gavage duration, enzyme activity and the concentration of free HFIP significantly increased during the initial three weeks, while protein expression continued to rise throughout the four weeks. These results suggest that ethanol exposure augments the metabolism of sevoflurane through the upregulation of CYP2E1.

Alcohol metabolism primarily involves converting alcohol to acetaldehyde by ADH. However, when ADH is saturated, MEOS, notably CYP2E1, plays a crucial role in converting alcohol to acetaldehyde as a secondary pathway [10]. Our experimental findings, along with those from other studies, underscore the substrate-inducible nature of CYP2E1, demonstrating that ethanol exposure significantly upregulates CYP2E1 expression and activity. Lu et al. observed a marked increase in CYP2E1 protein expression in rats continuously fed with ethanol at 9 g/kg BW per rat over an 8-week period, showing higher levels than in the control group [26]. Furthermore, the in vitro experiments conducted by Jin et al. reveal that a short exposure to 50 mM ethanol for 6 h can induce a 150% increase in CYP2E1 protein expression in SVGA astrocytes [27]. Similarly, Ge et al.’s research, involving continuous gastric administration of 50% (v/v) ethanol at 8–12 ml/kg/day to rats for six weeks, reported a 96% increase in CYP2E1 activity [25]. Additionally, I. Dupont and colleagues, utilizing the 6-hydroxychlorzoxazone test, measured CYP2E1 activity and found it increased by four-fold in alcoholics compared to non-alcoholic patients [28]. These outcomes collectively highlight the significant role of CYP2E1 in alcohol metabolism, illustrating how ethanol exposure increases the enzyme’s expression and activity.

Our experimental results showed an increase in the concentration of free HFIP, suggesting an acceleration of sevoflurane metabolism. This observation corresponds with the findings of Malan TP et al., where continuous administration of 16% ethanol to rats for a 14-day period, followed by sevoflurane anesthesia, resulted in a 68% increase in serum fluoride ions [29]. Previous studies assessing sevoflurane metabolism often measured fluoride ion concentrations. However, this approach lacks specificity as fluoride ions are not unique markers and are prevalent in various environmental sources, including water, tea, beverages, fluoride supplements, and dental products. Furthermore, once absorbed, approximately 50% of fluoride ions deposit in bones and teeth within 24 h [30]. In contrast, free HFIP originates solely from the metabolism of sevoflurane and exhibits a slow metabolic rate, making it a reliable indicator of blood HFIP concentration and the extent of sevoflurane metabolism.

The increase in free HFIP concentration indicates an accelerated metabolism of sevoflurane, necessitating higher concentrations of sevoflurane to maintain a stable blood/brain partial pressure difference. This may partially explain the observed increase in the MAC of sevoflurane in chronic alcohol users. The significance of enzymatic interactions and alterations in sevoflurane metabolism has received less attention. Previous studies on how alcohol changes the effectiveness of anesthetics have mainly focused on the impact of ethanol on GABAA receptors, the primary ionotropic receptors for fast inhibitory neurotransmission in the central nervous system. GABAA receptors, which are targets for both alcohol and sevoflurane, have specific ethanol binding sites. Alcohol enhances Cl− influx, mimicking GABAergic substances. Chronic alcohol consumption reduces the sensitivity of GABAA receptors to GABAergic substances, leading to an increased tolerance to sevoflurane [31–33].

The observed increase in free HFIP concentration raises concerns due to its notable solubility and extended half-life [15–17]. Administering high doses of sevoflurane over lengthy periods in individuals with chronic alcohol use could lead to significant free HFIP accumulation. Importantly, Eger EI et al. have reported that the MAC of free HFIP is only 0.0044%, highlight the risk of excessive anesthesia or delayed recovery [15], These concerns need further clinical studies for investigation.

CYP2E1 is not only involved in the metabolism of ethanol and sevoflurane, but also participates in the biotransformation processes of many other drugs [10]. The upregulation of CYP2E1 expression and enzyme activity could affect their metabolic rates. This study establishes a foundation for future investigations into the effects of ethanol exposure on the processing of drugs metabolized by CYP2E1.

Administering ethanol at a dosage of 3 g/kg twice daily has been shown to maintain stable blood ethanol levels, reduce the risk of mortality from ethanol overdose in rats, and significantly influence CYP2E1 expression levels [19, 34]. This study utilized tracheal intubation for sevoflurane delivery, instead of the anesthesia chamber frequently used in previous sevoflurane metabolism experiments. The anesthesia chamber might cause respiratory depression and reduce minute ventilation, potentially lowering the alveolar concentration of sevoflurane. Tracheal intubation with mechanical ventilation allows for precise control of the concentration of sevoflurane in the circuit, reducing bias caused by weight differences due to alcohol gavage.

This study has some limitations. First, there are species differences between humans and rats. Although the literature indicates that the CYP2E1 gene is highly conserved across multiple species, and rats are considered a reliable model for studying CYP2E1 expression, catalytic function, and substrate screening [24, 35], the direct applicability of animal model research results to humans remains uncertain.

Secondly, in calculating the concentration of free HFIP, this study utilized the blood/gas partition coefficient of 452, as measured in another experiment [16]. However, this coefficient may vary among individual rats, which could potentially impact the study results.

Lastly, the study measured the concentration of free HFIP in rat blood only 1 h after anesthesia. Although free HFIP can be detected within 5 min post-inhalation anesthesia, its peak concentration typically occurs 5.5 h afterwards [14]. The choice of this time point may have limited a comprehensive understanding of the dynamic changes in free HFIP concentration.

Conclusions

This study observed that after ethanol gavage, there was a significant increase in the expression and enzymatic activity of rat CYP2E1 protein, along with a significant rise in the concentration of free HFIP, suggesting an acceleration in the metabolism of sevoflurane. This metabolic change may be a potential reason for the observed increase in the MAC of sevoflurane in chronic drinkers.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Acknowledgements

The authors would like to express their gratitude to North Sichuan Medical College for providing resources for this research. The authors would also like to acknowledge the support and assistance provided by the Animal Center during this study.

Author contributions

WJ conducted the literature review, designed the study, participated in the molecular biology studies, and drafted the manuscript. MZ processed data, analyzed and interpreted the findings. RC performed animal experiments and collected data. XHW performed animal experiments. YBZ designed the experiment, managed the project administration, and revised the manuscript. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

The datasets used and/or analyzed during this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments adhered to the ARRIVE guidelines and were conducted in accordance with the guidelines of the National Institutes of Health. All experimental procedures involving animals were approved by the NSMC Animal Ethics Committee under license number [2023]018.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Abbreviations

ADH Alcohol dehydrogenase

MEOS Microsomal Ethanol Oxidizing System

MAC Minimum alveolar concentration

CYP450 2E1 Cytochrome P450 2E1

HFIP Hexafluoroisopropanol

F− Fluoride ions

GABA γ-aminobutyric acid

NSMC North Sichuan Medical College

RIPA buffer Radio-Immunoprecipitation Assay Buffer

TBST Tris-Buffered Saline with Tween 20

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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