Effects of inhalation anesthetics halothane, sevoflurane, and isoflurane on human cell lines

Abstract

Cytotoxic and antiproliferative effects of halothane, isoflurane, and sevoflurane in anesthetic doses on human colon carcinoma (Caco-2), larynx carcinoma (HEp-2), pancreatic carcinoma cells (MIA PaCa-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), and normal fibroblasts were investigated. Cells were exposed to anesthetic gas mixture consisting of O₂: N2O (35:60 vol.%), halothane (1.5 vol.%) or isoflurane (2.0 vol.%) or sevoflurane (3.0 vol.%), and CO₂ (5 vol.%), for 2, 4, and 6 h. Cytotoxicity of anesthetics was analyzed by validated tetrazolium dye assay MTT test. All anesthetics expressed cytotoxic effects on treated tumor cells in time and cell line dependent manner. Growth suppression in cells exposed to halothane was enhanced in HEp-2 (to 67.7%), Caco-2 (to 76.3%), and SW620 cells (to 80.9%), and was minimal in normal fibroblasts (to 89.4%). Antiproliferative activity of halothane was measured via radioactive precursors incorporation assay. In Caco-2 cells treated by halothane, decrease in DNA synthesis (52.4%, p = 0.001), RNA synthesis (39.2%, p < 0.001), and protein synthesis (19.2%, p = 0.004) was observed. In HEp-2 cells, DNA and RNA syntheses were decreased to 72.5% and 79.9%, whereas protein synthesis was 14.0% of control (p < 0.001). In SW620 cells, protein synthesis after 4 h was 24.4% (p = 0.007). A DNA fragmentation was observed in Caco-2 and MIA PaCa-2 cells. Exposition of phosphatidylserine on outer lipid bilayer plasma membrane of tumor cell treated by halothane proved apoptosis as mode of cell death.

Introduction

Inhaled anesthetics are probably the most used anesthetics, alone or as a part of a balanced anesthesia. Adverse reactions of inhaled anesthetics, especially mutagenesis and carcinogenesis, were usually in the focus of anesthetic community (Doll and Peto, 1977). The major reason was that inhaled anesthetics were suspected to induce tumors, spontaneous abortions, and congenital anomalies in chronically exposed anesthetic stuff. The first animal and epidemiological studies on effects of chronic exposure to subanesthetic concentration of inhaled anesthetics failed to prove such hypothesis (ASA ad hoc committee, 1974, Mazze et al., 1986, Eger et al., 1978).

The use of inhaled anesthetics in anesthesia for cancer diseases was commonly investigated (Van de Louw et al., 1998, Kvolik et al., 2003). Most observations referring to the use of inhaled anesthetics in cancer surgery and diagnostics were directed to side effects observing, although those could be clinically irrelevant (Fisher et al., 1985). In the clinical setting, the choice of the anesthetic agent may be important in the view of mutagenic potential, impaired metastatic capability, and growth of pre-existing tumor cells. Tumor growth is particularly important in extensive cancer surgery resulting in immune and organ dysfunctions (Melamed et al., 2003).

The major problem concerning in vitro investigations on effects of inhaled anesthetics on tumor growth was a study design. Exposure protocols and the vapor concentration of test chemicals were poorly monitored and different from clinical setting in several points. Some authors have bound anesthetics into drug-micelle complexes with toxic substance DMSO, which allowed concentration adjustment (O'Leary et al., 2000). Others used very high concentrations of anesthetics or prolonged exposure e.g. 3% halothane for 24 h to pronounce toxic effects (Muckter et al., 1998). Hack et al. (1981) used halothane 3 vol.%, enflurane and isoflurane 5 vol.%, and methoxyflurane 2 vol.% in O2: N2O 20: 78% and CO₂ 2 vol.%.

Investigations in vivo are usually more complex. Numerous factors were different from operative setting: e.g. inhaled anesthetics were delivered in concentrations lower than used in clinical practice over prolonged time, so that spontaneous breathing was allowed (Eger et al., 1978, Hack et al., 1981). In the study of Moudgil and Singal (1997), researching metastatic alterations after exposure to inhaled anesthetics nonexposed tumor cells was inoculated in the anesthetized organism. As experimental conditions were distinctive from those in the anesthesia during surgery, given results were contradictory.

Therefore in this study, clinically relevant doses of halothane, isoflurane, and sevoflurane were used. It was conducted to compare growth effects of single exposure of human cell lines to inhaled anesthetics. Antiproliferative effects on human tumor and normal cells in vitro were measured after 2, 4, and 6 h of exposure to anesthetics. A cell line specific effect and mechanism of death of treated tumor cells were investigated. Our observations pointed that three inhaled anesthetics expressed quantitatively different inhibitory effect against cell lines, and induced apoptosis as a mode of cell death.