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Investigation of propofol, fentanyl, and midazolam-related toxicity and the protective effect of midazolam on THLE-2 cell lines

  • Asu Özgültekin ORCID logo EMAIL logo , Asuman İnan ORCID logo , Kubra Bozali ORCID logo , Beyza Nur Özkan ORCID logo and Eray Metin Güler ORCID logo
Published/Copyright: March 20, 2025
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 50 Issue 2

Abstract

Objectives

The widespread use of propofol, fentanyl, and midazolam in intensive care units necessitates a thorough understanding of their potential toxic effects. These sedative-hypnotic agents are frequently administered in combination to manage critically ill patients, raising concerns about their cumulative toxicity. To address this, we investigated the cytotoxic and genotoxic effects of these drugs, both individually and in combination, on a human liver epithelial cell line (THLE-2). The liver, as a major organ involved in drug metabolism, is particularly vulnerable to drug-induced toxicity. By evaluating the impact of these agents on liver cells, we aim to gain insights into their potential adverse effects and inform clinical practice.

Methods

Cells were treated with increasing concentrations of each drug, as well as with their combination, over a 72 h incubation period. Cell viability, oxidative stress, antioxidant defense mechanisms and apoptotic activity and potential genotoxicity were explored using various assays.

Results

A dose dependent increase in cytotoxicity, intracellular reactive oxygen species production, apoptotic activity, and DNA damage were detected in all treatment groups (p<0.05). Higher concentrations of the study drugs were associated with marked increases in oxidative stress and apoptotic markers. There was a concomitant reduction in intracellular glutathione levels, suggesting a depletion of the cells’ antioxidant defenses. When these drugs were administered in combination, the cytotoxic and genotoxic effects appeared to be mitigated, indicating a potential protective interaction between these agents, particularly involving midazolam.

Conclusions

The study drugs caused dose-dependent hepatotoxicity, induced DNA damage and apoptosis and consequently decreased cell viability.

Keywords: cytotoxiciy; hepatotoxicity; fentanyl; propofol; midazolam

Introduction

In the clinical practice of Intensive Care Units (ICUs), continuous infusion of sedative, analgesic, and hypnotic agents, such as propofol, fentanyl, and midazolam, is a standard protocol for patient management, particularly those undergoing mechanical ventilation or invasive procedures. While these drugs are typically initiated at therapeutic doses, there are instances when the clinical necessity arises to increase the dosage in order to achieve and maintain the desired effects. In certain cases, combination therapies involving these agents are preferred to mitigate the potential side effects arising from dose escalation [1], 2].

Despite their therapeutic benefits, concerns persist regarding the potential cytotoxic effects of these drugs, especially when used at high doses and/or for extended periods. Limited information exists about their combined effects and potential interactions at the cellular level. ICU patients, already vulnerable to complications like hypoxia and exposure to hepatotoxic drugs, may be at increased risk of adverse drug reactions. Therefore, it is crucial to investigate the potential harmful effects of prolonged drug infusion on vital organs [3], 4].

As one of the most commonly used hypnotic agents in ICU settings, propofol is primarily metabolized in the liver. Numerous studies have demonstrated the efficacy of propofol in mitigating hepatic ischemia-reperfusion injury and enhancing liver function. This is achieved by reducing oxidative damage and boosting the liver’s antioxidant capacity [5], 6]. At clinically relevant doses, propofol has been shown to possess antioxidant and anti-inflammatory properties, contributing to its cytoprotective effects [7], [8], [9], [10]. However, at higher concentrations or with cumulative dosing, propofol’s cytoprotective effects may reverse, leading to cytotoxicity, as observed in various cell types and under different experimental conditions [11].

Midazolam, a short-acting benzodiazepine, similarly exhibits both in vitro and in vivo cytoprotective effects by enhancing the levels of several key antioxidant proteins. However, midazolam also demonstrates pro-oxidant properties at higher doses, which can lead to apoptosis and oxidative damage [12], [13], [14], [15].

It has been demonstrated that fentanyl, an opioid analgesic, is associated with mitochondrial dysfunction and cytotoxic effects, which contribute, in certain cases, to hepatic and renal dysfunction. Moreover, the neurotoxic effects of fentanyl, which are caused by its ability to induce inflammation and oxidative stress, have been extensively documented in the scientific literature [16], [17], [18].

The liver plays a crucial role in maintaining homeostasis and metabolism in ICU patients. As the primary organ responsible for processing exogenous and endogenous substances, it is particularly vulnerable to oxidative stress, which can contribute to liver injury [13]. Prolonged exposure to sedative-analgesic agents, such as propofol, fentanyl, and midazolam, can further exacerbate this risk. The objective of this study was to examine the potential cytotoxic effects of these drugs on liver cells. To this end, the impact of the drugs on THLE-2 cells, a model of healthy human liver epithelial cells, was investigated.

Previous studies have primarily investigated the cytotoxic and genotoxic effects of propofol, fentanyl, and midazolam on cancer cells [12], [19], [20], [21], neurons [22], endothelial cells [23], and cardiomyocytes [24]. However, limited research has focused on the impact of these drugs on healthy liver cells, the primary site of their metabolism. Moreover, information on the combined effects of these drugs is scarce despite their frequent co-administration in ICU settings. This study aims to address these knowledge gaps by evaluating the cytotoxic and genotoxic effects of propofol, fentanyl, and midazolam on human liver epithelial cells (THLE-2) that we believe will contribute valuable knowledge to the literature.

Materials and methods

THLE-2 cell line maintenence and treatments

The hepatocyte-derived THLE-2 cell line (Transformed Human Liver Epitelial-2) (CRL-2706™, American Type Culture Collection) was used to evaluate the cytotoxic effects of propofol, fentanyl, and midazolam. These immortalized human liver epithelial cells are mainly utilized for in vitro pharmacotoxicological studies to assess the potential adverse effects of drugs on liver function [25].

Propofol (United States Pharmacopeia (USP) Reference Standard, Sigma 1,572,503), fentanyl (Fentanyl citrate salt, Sigma F3886), and midazolam (Pharmacopeia (USP) Reference Standard, Sigma 1443602) were administered to THLE-2 cells at increasing concentrations (1–20 μg/mL for propofol, 1–40 µM for fentanyl, and 10–50 µM for midazolam). Combination treatments were also performed, using the lowest cytotoxic dose of midazolam in combination with propofol and fentanyl. Cells were incubated for 72 h under standard cell culture conditions.

Propofol was initially prepared and administered in μg/mL because this unit is commonly reported in clinical and pharmacological research and facilitates direct comparison with the available literature [26], 27]. On the other hand, the expression of fentanyl and midazolam in molarity (µM) facilitates the standardization and interpretation of their effects at the molecular level, particularly when comparing drugs with different molecular weights.

In the combination therapy group, the lowest cytotoxic dose of midazolam was combined with propofol and fentanyl to assess their interactive effects on the cell line. All treated THLE-2 cells were incubated for 72 h under standard cell culture conditions. All experiments were conducted in quadruplicate to ensure the reproducibility of results.

Cell line maintenance

The THLE-2 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10 % Fetal Bovine Serum (FBS) and 1 % penicillin-streptomycin (P/S). Cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO2 to maintain optimal growth conditions. Cell confluency was monitored daily, and passages were conducted upon reaching 70–80 % confluence.

Cytotoxicity assay

The ATP assay (Cell-Titer-Glo® 2.0 Cell Viability Assay, Promega) was employed to assess the cytotoxicity of propofol, fentanyl, and midazolam on the THLE-2 cell line. Cells were seeded at 7,000 cells per well in 96-well opaque white plates and treated with propofol (1–20 μg/mL), fentanyl (1–40 µM), and midazolam (10–50 µM), as well as their combination treatment doses. Following the 72-h incubation at 37 °C in 5 % CO2, 100 μL of ATP solution was added to each well, and the plates were incubated for 10 min. Cytotoxicity was measured by determining the luminescence output using a flash multimode reader (BioTek Synergy™, HTX Multimode Reader, U.S.A.), with results expressed as relative luminescence units (RLU) [28].

Intracellular reactive oxygen species (iROS) levels

To evaluate intracellular reactive oxygen species (iROS) production, cells were treated with incremental doses of propofol (1–20 μg/mL), fentanyl (1–40 µM), midazolam (10–50 µM), and combination treatment doses. After 72 h of incubation, the media was aspirated, and the wells were washed three times with 1 × PBS. 2, 7-Dichlorodihydrofluorescein diacetate (H2DCF-DA) (Sigma Aldrich, Germany) was used as the fluorescent probe for ROS detection. A 10 μM concentration of H2DCF-DA was added to each well, and the plates were incubated at 37 °C. The DCF fluorescence intensity was measured using a fluorescence plate reader set at Ex: 488 nm and Em: 525 nm (BioTek Synergy™ HTX Multimode Reader, USA). ROS levels were expressed as relative fluorescence units (RFU) [28].

Intracellular glutathione (iGSH) levels

The intracellular glutathione (iGSH) levels were measured using the GSH/GSSG-Glo™ Assay kit (Promega, USA) following the manufacturer’s instructions [29]. Cells were seeded in 96-well white plates at a density of 6,000 cells per well. After treatment with propofol (1–20 μg/mL), fentanyl (1–40 µM), midazolam (10–50 µM), and their combination doses for 72 h, the GSH solution was added to each well. iGSH levels were quantified using a luminometric method with a multiplate reader (BioTek Synergy™ HTX Multimode Reader, USA).

Apoptosis assay

Apoptosis was evaluated using the acridine orange/ethidium bromide double staining method, as described by McGahon et al. [30]. To determine the apoptotic activity of the substances, 5 × 104 THLE-2 cells were seeded per well in 6-well plates. The cells were incubated with propofol (1–20 μg/mL), fentanyl (1–40 µM), midazolam (10–50 µM) and combination treatments for 72 h. Following treatment, the culture medium was aspirated, and the cells were washed with 1 × PBS. Cells were subsequently detached using 0.25 % trypsin-EDTA and washed again with 1 × PBS. For apoptotic analysis, the cells were stained with acridine orange/ethidium bromide (AO/EB) at a 1:1 ratio and visualized using a fluorescence microscope (Nikon Eclipse Ts2, Japan). Green fluorescence indicated viable cells, red fluorescence identified necrotic cells, and yellow fluorescence marked apoptotic cells. Apoptotic activity was determined by calculating the percentage of apoptotic cells relative to the total cell population based on the nuclear morphology of at least 150 cells per treatment condition.

DNA damage assay (Comet assay)

DNA damage was assessed using the Comet assay as described by Singh et al. [31]. Cells treated with propofol (1–20 μg/mL), fentanyl (1–40 µM), midazolam (10–50 µM), and their combination doses were suspended in 0.65 % low melting point agarose and spread on microscope slides pre-coated with 1 % normal-melting point agarose. The slides were subjected to cell lysis followed by electrophoresis (26 V, 300 mA at +4 °C) to induce DNA migration. After neutralization, the slides were stained with ethidium bromide (Sigma Aldrich, Germany), and images of the cells were captured using a fluorescence microscope (Nikon Eclipse Ts2, Japan). DNA damage was quantified using the Comet Assay IV analysis software. Comet Assay IV analysis software measures DNA damage by calculating the area of ​​fluorescence intensity in the tail regions of the comet, excluding the head. The data represents the percentage of total DNA fluorescence in the tail.

Statistical analysis

All experiments were conducted in quadruplicate, and the resulting data are presented as the mean ± standard deviation (mean ± SD). Statistical analyses were conducted using one-way analysis of variance (One-Way ANOVA) to determine the significance of differences between treatment groups. Prior to conducting ANOVA, the normality of the data was assessed using the Shapiro-Wilk test. Post-hoc analyses were performed using Tukey’s HSD test to identify specific group differences. Significance thresholds were set as p<0.05, p<0.01, and p<0.001, with p-values <0.05 considered statistically significant. These thresholds were used to indicate varying levels of statistical significance in the text. Correlations between the measured parameters were assessed using the Pearson correlation coefficient. Data analysis was carried out using SPSS software (Version 25.0, Chicago, USA).

Results

Propofol, fentanyl, and midazolam exhibited dose-dependent cytotoxicity, significantly reducing cell viability at higher concentrations (Figure 1). Propofol was the most potent, reducing cell viability by 50 % at a concentration of 5 μg/mL. Further increases in concentration led to more pronounced cytotoxicity, with significant reductions observed at 10 μg/mL (p<0.01) and 20 μg/mL (p<0.001).

Figure 1: Cytotoxicity levels in THLE-2 cells treated with propofol (P), fentanyl (F), midazolam and the combination doses. Data are represented as mean ± SD of four independent experiments. *p<0.05, <0.001, ***p<0.001 compared to the control group (RLU: relative luminescent unit).
Figure 1:

Cytotoxicity levels in THLE-2 cells treated with propofol (P), fentanyl (F), midazolam and the combination doses. Data are represented as mean ± SD of four independent experiments. *p<0.05, <0.001, ***p<0.001 compared to the control group (RLU: relative luminescent unit).

Fentanyl also exhibited dose-dependent cytotoxicity, with significant reductions in cell viability at higher concentrations. At 20 µM and 40 µM, fentanyl significantly reduced cell viability (p<0.001). These findings suggest that while fentanyl may be less potent than propofol at lower concentrations, its cytotoxic effects become more pronounced at higher doses, which are commonly used in clinical settings.

Midazolam, a commonly used sedative, also exhibited dose-dependent cytotoxicity. However, it was less potent than propofol and fentanyl, with significant effects observed only at higher concentrations (30 µM and above). At 40 µM and 50 µM, midazolam significantly reduced cell viability (p<0.01 and p<0.001, respectively). This suggests that while midazolam may be generally safe at therapeutic doses, its prolonged use at higher doses could potentially lead to adverse effects.

When administered in combination, propofol, fentanyl, and midazolam exhibited a synergistic cytotoxic effect, particularly at higher doses. The combination treatments resulted in a significant decrease in cell viability (p<0.001), suggesting that the combined use of these drugs may increase the risk of cell damage. This finding has important clinical implications, as these drugs are often used together in ICU settings. Careful monitoring of drug dosages is crucial to minimize potential adverse effects.

Propofol, fentanyl, and midazolam significantly increased iROS levels in a dose-dependent manner, as shown in Figure 2. These findings showed that each drug significantly elevated iROS levels in a concentration-dependent manner, with notable variations in their potency and thresholds for inducing oxidative stress.

Figure 2: The iROS levels in THLE-2 cells treated with propofol (P), fentanyl (F), midazolam and the combination doses. Data are represented as mean ± SD of four independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to the control group (RFU: Relative fluorescence unit).
Figure 2:

The iROS levels in THLE-2 cells treated with propofol (P), fentanyl (F), midazolam and the combination doses. Data are represented as mean ± SD of four independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to the control group (RFU: Relative fluorescence unit).

Propofol significantly increased ROS levels at concentrations as low as 2.5 μg/mL, with the most pronounced effects observed at 10 μg/mL and 20 μg/mL (p<0.001). This suggests that propofol can induce oxidative stress even at relatively low doses, potentially contributing to cellular damage, especially during prolonged or high-dose clinical use. The dose-dependent increase in ROS production aligns with propofol’s known effects on mitochondrial function and redox balance, emphasizing the importance of careful dose management.

Fentanyl also significantly increased ROS levels in a dose-dependent manner, with a notable effect at concentrations as low as 1 µM. At higher concentrations (20 µM and 40 µM), ROS production was significantly elevated (p<0.001). This suggests that fentanyl, in addition to its analgesic effects, can induce oxidative stress, potentially leading to mitochondrial dysfunction and cellular damage. This is particularly concerning in ICU settings, where fentanyl is often used at escalating doses for prolonged periods.

The sedative midazolam was observed to increase ROS levels, though only at high concentrations (20 µM and 50 µM, p<0.001). While midazolam has been shown to have cytoprotective effects at therapeutic doses, these findings suggest that higher doses may induce oxidative stress, potentially leading to cellular damage.

When administered in combination, propofol, fentanyl, and midazolam synergistically increased ROS production at all tested doses. This suggests that the combined use of these drugs may significantly exacerbate oxidative stress, potentially leading to increased cellular damage and tissue injury. Therefore, careful dosing strategies and monitoring of oxidative stress markers are crucial for patients receiving multi-drug sedation protocols.

In Figure 3, propofol, fentanyl, and midazolam doses statistically decreased iGSH levels in the healthy THLE-2 cell line (p<0.05; p<0.01). The results of the assays are presented as µM iGSH Eq.

Figure 3: The iGSH levels in THLE-2 cells treated with propofol (P), fentanyl (F), midazolam and the combination doses. Data are represented as mean ± SD of four independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to the control group.
Figure 3:

The iGSH levels in THLE-2 cells treated with propofol (P), fentanyl (F), midazolam and the combination doses. Data are represented as mean ± SD of four independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to the control group.

The assessment of apoptosis revealed a significant increase in the percentage of apoptotic cells in THLE-2 cells treated with propofol, fentanyl, and midazolam, as compared to the untreated control group (Figure 4). The data indicated a statistically significant increase in apoptosis across all treatment groups, with a clear dose-dependent relationship (p<0.001). As the doses of each agent were escalated, the number of apoptotic cells respectively increased, demonstrating that these drugs induce apoptosis in a concentration-dependent manner.

Figure 4: Effect of propofol (P), fentanyl (F), midazolam and the combination doses on apoptosis, and representative immunofluorescence image of apoptosis of combination concentration propofol (P), fentanyl (F), 20 μM midazolam in THLE-2 cell line. For differences in THLE-2 cells *p<0.05, **p<0.01, ***p<0.001 compared to the control group. Data are represented as mean ± SD of four independent experiments.
Figure 4:

Effect of propofol (P), fentanyl (F), midazolam and the combination doses on apoptosis, and representative immunofluorescence image of apoptosis of combination concentration propofol (P), fentanyl (F), 20 μM midazolam in THLE-2 cell line. For differences in THLE-2 cells *p<0.05, **p<0.01, ***p<0.001 compared to the control group. Data are represented as mean ± SD of four independent experiments.

A detailed screening of apoptotic cells was performed by examining a minimum of 150 cells per dose, ensuring reliable quantification of apoptotic events. The increasing percentage of apoptotic cells with higher drug concentrations highlights the cytotoxic potential of propofol, fentanyl, and midazolam when used individually and in combination. These findings are consistent with the known pro-apoptotic mechanisms of these agents, particularly at supratherapeutic doses, where they may induce cellular stress, disrupt mitochondrial function, and activate caspase pathways leading to programmed cell death.

The observed dose-dependent increase in apoptosis suggests that prolonged or high-dose use of these agents in clinical settings, such as in ICU sedation or analgesia protocols, could exacerbate cellular damage, potentially contributing to organ dysfunction over time. This is particularly pertinent when these agents are administered concurrently, as the cumulative cytotoxicty effects may further intensify apoptotic pathways, thereby raising concerns about their long-term safety in critically ill patients.

These results underscore the importance of careful dose titration and the potential need for alternative therapeutic strategies to minimize drug-induced apoptosis, especially in situations where these agents are used for extended periods. Further research is required to elucidate the underlying molecular mechanisms of apoptosis induced by these drugs and to identify potential protective strategies. This will facilitate the optimization of patient outcomes.

In addition to the observed cytotoxic and apoptotic effects, the Comet Assay results revealed a dose-dependent increase in comet tail moment, further suggesting that propofol, fentanyl, midazolam, and their combination treatments exerted significant genotoxic effects on THLE-2 cells (Figure 5). The comet tail moment is a well-established marker of DNA strand breaks and serves as an indicator of genomic instability. The present study demonstrated that all three drugs, when administered individually and in combination, caused significant DNA damage in a dose-dependent manner.

Figure 5: THLE-2 cells were treated with different propofol (P), fentanyl (F), midazolam and the combination doses (1–50 μg/mL) for 72 h. Results are presented as comet tail moment in percentage. The representative immunofluorescence image of apoptosis of combination concentration propofol (P), fentanyl (F), 20 μM midazolam in THLE-2 cell line are shown. Data are represented as mean ± SD of four independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to the control group.
Figure 5:

THLE-2 cells were treated with different propofol (P), fentanyl (F), midazolam and the combination doses (1–50 μg/mL) for 72 h. Results are presented as comet tail moment in percentage. The representative immunofluorescence image of apoptosis of combination concentration propofol (P), fentanyl (F), 20 μM midazolam in THLE-2 cell line are shown. Data are represented as mean ± SD of four independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to the control group.

The dose-dependent increase in comet tail moment underscores the potential for these drugs to cause genotoxicity, which could lead to mutagenesis or carcinogenesis under prolonged exposure. The results clearly demonstrate that as the doses of propofol, fentanyl, and midazolam were escalated, the magnitude of DNA damage also increased, indicating a cumulative genotoxic effect. This finding is particularly concerning in the context of long-term ICU treatment, where these agents are often administered continuously over extended periods.

Moreover, the combination treatments produced a synergistic increase in DNA damage, as indicated by the heightened comet tail moments observed in these groups. This suggests that the combined use of these drugs may amplify their genotoxic potential, further complicating their safety profile when used in combination therapy. Given that DNA damage is a precursor to cellular dysfunction and disease progression, this study highlights the critical need for careful monitoring and dose management when these agents are used, particularly in vulnerable patient populations.

Our findings emphasize the importance of further research into the molecular mechanisms driving the genotoxic effects of propofol, fentanyl, and midazolam. Investigating potential protective strategies against genotoxicity, such as the co-administration of antioxidants or genoprotective agents, could be vital in mitigating the adverse effects associated with these drugs.

Discussion

Patients in the ICU are often particularly vulnerable to hepatotoxic drugs due to pre-existing liver damage associated with underlying diseases. These patients frequently require multiple medications, increasing the risk of drug interactions and potential organ toxicities. The continuous administration of drugs used for sedation and analgesia over extended periods of time further exacerbates concerns about cytotoxicity. Although generally considered safe in clinical practice, propofol, midazolam, and fentanyl have been associated with a range of adverse effects.

When administered at high doses or for prolonged durations, these drugs can lead to more severe adverse effects: Propofol has been associated with propofol infusion syndrome, QT prolongation on electrocardiograms, and hypotension; midazolam can result in delayed awakening and respiratory depression; and fentanyl has been linked to hemodynamic instability and respiratory depression. Furthermore, overuse of these drugs has been correlated with increased mortality rates and prolonged ICU and hospital stays [3], 4], 32]. Despite their clinical utility, the precise mechanisms of cell damage induced by these agents remain poorly understood.

In this study, we utilized a cytotoxicity screening model based on the THLE-2 cell line to explore the effects of propofol, midazolam, and fentanyl on several key parameters, including iROS and iGSH levels, cell viability, DNA damage, and apoptosis.

Propofol has been identified as a potential cause of idiosyncratic acute liver injury in rare cases. Additionally, when administered in high doses over prolonged periods, it can contribute to the development of propofol infusion syndrome, which is thought to result from mitochondrial injury affecting the liver. Although fentanyl has not been linked to liver toxicity in large-scale clinical trials, its metabolism through CYP 3A4 poses a risk of increased toxicity, especially when CYP 3A4 inhibitors are co-administered. Midazolam is generally considered safe, with clinically significant liver injury being extremely rare, likely due to its relatively short duration of use and the low doses typically administered [33].

The liver plays a critical role in drug detoxification, generating reactive oxygen species as intermediate byproducts. Under normal physiological conditions, ROS are neutralized by the mitochondrial antioxidant defense system. However, certain drugs, including those used for sedation and analgesia, can produce ROS at levels that overwhelm the cellular antioxidant capacity, leading to oxidative stress, cell death, and, ultimately, organ damage. In our study, all three drugs demonstrated dose-dependent cytotoxicity. The viability of cells was found to decline significantly at doses of propofol and midazolam that were medium to high, as well as at doses of fentanyl that were low to high. These cytotoxic effects remained consistent when the three agents were used in combination. Recent in vitro studies on the hepatotoxicity of opioids and sedatives have shown that midazolam has a relatively mild impact on hepatocyte viability compared to fentanyl and propofol. While the decrease in hepatocyte viability was only slightly significant, this effect was more pronounced with other opioids such as remifentanil and sufentanil. Notably, no significant decrease in cell count was observed with propofol, fentanyl, or midazolam in this study, which aligns with the low hepatotoxicity typically associated with these drugs [34].

Midazolam has been shown to reduce pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in both in vitro and in vivo models. For instance, continuous infusion of midazolam over 48 h significantly lowered these cytokines’ levels [35]. In lipopolysaccharide (LPS) and galactosamine-induced acute liver injury models, midazolam reduced serum ALT and liver TNF-α levels, thereby protecting against liver injury [5]. Additionally, midazolam has been shown to inhibit oxidative stress, reduce ROS levels, and protect neurons from apoptosis in various neuroprotective models [36], 37]. It has also been found to alleviate CCl4-induced acute liver injury by enhancing the Nrf2 signaling pathway, suggesting its potential as a hepatoprotective agent [13].

Despite its antioxidant properties, propofol has been suggested for uses beyond sedation, including its potential application in treating glioblastoma, where it induces apoptosis through mechanisms involving Bcl-2, Bax, and caspase activation [21], 38]. In this study, propofol and fentanyl both induced apoptosis, with the number of apoptotic cells increasing in a dose-dependent manner. The pro-apoptotic effects of midazolam were observed only at medium to high doses. However, when administered in combination with propofol and fentanyl, midazolam appeared to attenuate the apoptotic effects of these agents.

The GSH system, essential for antioxidant defense, plays a pivotal role in ROS detoxification. Previous studies have demonstrated that propofol enhances GSH activity [39], [40], [41], [42]. In our study, iGSH levels decreased significantly with medium to high doses of propofol, high doses of fentanyl, and the highest dose of midazolam. Interestingly, despite propofol’s known genoprotective effects in previous studies [43], our results indicate that propofol, fentanyl, midazolam, and their combination doses exhibited significant genotoxic effects on liver epithelial cells. Fentanyl and propofol-induced DNA damage at lower doses, while midazolam demonstrated genotoxicity at medium to high concentrations. Notably, the genotoxic effects did not differ when these agents were used in combination.

Conclusions

The administration of propofol, fentanyl, and midazolam at varying doses resulted in the induction of hepatotoxicity, DNA damage, and apoptosis, ultimately leading to a reduction in cell viability. These cytotoxic and genotoxic effects were more pronounced for propofol and fentanyl, especially at medium and high doses. In contrast, the lowest cytotoxic dose of 20 mcg/mL midazolam showed a protective effect when administered with the other two agents. This protective effect was especially significant in terms of apoptosis.

Corresponding author: Ass. Prof. Dr. Asu Özgültekin, Department of Anesthesiology and Intensive Care, Haydarpasa Numune Health Application and Research Center, University of Health Sciences Turkey, Istanbul, Türkiye, E-mail:

  1. Research ethics: The local Institutional Review Board deemed the study exempt from review.

  2. Informed consent: Not applicable.

  3. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: None declared.

  7. Data availability: Not applicable.

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Received: 2024-09-19
Accepted: 2024-12-06
Published Online: 2025-03-20

© 2025 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/tjb-2024-0234
Keywords for this article
cytotoxiciy; hepatotoxicity; fentanyl; propofol; midazolam
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Review
  3. Interaction of fetuin-A with obesity related insulin resistance and diabetes mellitus
  4. Research Articles
  5. Preanalytical errors in pediatric blood sampling: a systematic review of common challenges and risks
  6. The promotive role of reticulocalbin 3 (RCN3) in the pathogenesis of keloid via TGFβ1/Smad2/Smad7 signaling pathway in vitro
  7. Investigation into drug resistance to cisplatin in cancer stem cell-enriched population in non-small cell lung cancer
  8. The cytotoxic and antiproliferative effect of Polygala saponin XLIV on the human colorectal carcinoma cell line
  9. Development of HEK293T cell reference materials for β-thalassemia genetic testing using prime editing
  10. Investigation of propofol, fentanyl, and midazolam-related toxicity and the protective effect of midazolam on THLE-2 cell lines
  11. Leucine-rich α-2-glycoprotein 1 can be a novel angiogenic mediator in autosomal dominant polycystic kidney disease
  12. Asiaticoside reverses the inhibition effect of miR-184 on proliferation, migration and AKT phosphorylation of HTR-8/Svneo cells
  13. Perioperative D-dimer levels and head and neck cancer surgery: a prospective observational study
  14. Comprehensive analysis of KRT15 in pan-cancer and verification in lung adenocarcinoma
  15. Age and sex-dependent effects of nitrosative stress on the osmotic fragility of human red blood cells
  16. Evaluation of oxidative stress parameters in older patients with urinary incontinence
  17. Determination of reference change values for thyroid-related biomarkers: TSH, fT3, fT4, Tg, Anti-Tg, and Anti-TPO
  18. An evaluation of serum boron level in pregnancies with severe pre-eclampsia
  19. Involvement of SIRT3/FOXO1 and TLR4/NF-κB/NLRP3 pathways in protective effects of Δ(9)-tetrahydrocannabinol on diabetic cardiomyopathy in rats
  20. Evaluating the potential therapeutic effect of Rosa damascena Mill. essential oil on acetic acid-induced ulcerative colitis in rats
  21. Assessment of blood collection competency in phlebotomists: a survey study
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