Time-Dependent Effects of Anesthetic Isoflurane on Reactive Oxygen Species Levels in HEK-293 Cells

Abstract

The inhalation anesthetic isoflurane has been reported to induce caspase activation and apoptosis, which may lead to learning and memory impairment. However, the underlying mechanisms of these effects are largely unknown. Isoflurane has been shown to induce elevation of cytosol calcium levels, accumulation of reactive oxygen species (ROS), opening of the mitochondrial permeability transition pore, reduction in mitochondria membrane potential, and release of cytochrome c. The time course of these effects, however, remains to be determined. Therefore, we performed a pilot study to determine the effects of treatment with isoflurane for various times on ROS levels in HEK-293 cells. The cells were treated with 2% isoflurane plus 21% O2 and 5% CO2 for 15, 30, 60, or 90 min. We then used fluorescence imaging and microplate fluorometer to detect ROS levels. We show that 2% isoflurane for 60 or 90 min, but not 15 or 30 min, induced ROS accumulation in the cells. These data illustrated that isoflurane could cause time-dependent effects on ROS levels. These findings have established a system to further determine the time course effects of isoflurane on cellular and mitochondria function. Ultimately, the studies would elucidate, at least partially, the underlying mechanisms of isoflurane-induced cellular toxicity.

Figure 1

Fluorescence imaging studies of isoflurane’s effects on ROS levels in HEK 293 Cells. The green fluorescence image shows the of ROS levels in cells. (1) shows the ROS images in the cells treated with control condition; (2) shows the ROS images in the cells treated with 2% isoflurane for 15 min; (3) shows the ROS images in the cells treated with 2% isoflurane for 30 min; (4) shows the ROS image in the cells treated with 2% isoflurane for 60 min; (5) shows the ROS images in the cells treated with 2% isoflurane for 90 min. The treatments with 2% isoflurane for 15 and 30 min (2,3) do not induce visible increases in ROS levels inside the cells as compared to the control condition (1). The treatments with 2% isoflurane for 60 and 90 min (4,5) lead to accumulation of ROS inside the cells as compared to the control condition (1).

Figure 2

Fluorescence reading studies of isoflurane’s effects on ROS levels in HEK 293 Cells. (A) A fluorescence assay shows that the treatment with 2% isoflurane for 15 min (black bar) does not increase ROS levels compared with the control condition (white bar); (B) A fluorescence assay shows that treatment with 2% isoflurane for 30 min (black bar) does not increase ROS levels compared with the control condition (white bar); (C) Treatment with 2% isoflurane for 60 min (black bar) increases ROS levels compared with the control condition (white bar) (** p = 0.001); (D) Treatment with 2% isoflurane for 90 min (black bar) increases ROS levels compared with the control condition (white bar) (** p = 0.0001).