Effect of Adriamycin treatment on the lifetime of pyrene butyric acid in single living cells

Abstract

We investigated the fluorescence lifetime of pyrene butyric acid (PBA) using various O2 concentrations in cells. Both in living and freshly fixed cells, PBA lifetime decreased with oxygen concentration. We recorded decay curves in single cells and measured PBA lifetime and NAD(P)H intensity values. Under nitrogen atmosphere, the probe lifetime differences (199 and 209 ns in living and freshly fixed cells, respectively) suggest a supplemental pathway for the deactivation of the probe when the cell functions are not stopped. We propose reactive oxygen species (ROS) to be the additional quenchers that cause this decrease. We further studied the effect of drugs generating ROS the anthracycline doxorubicin (adriamycin). For living cells, PBA lifetime decreased after adriamycin (ADR) treatment (200 and 1000 ng/ml). This supports our hypothesis that under nitrogen atmosphere and for freshly fixed cells, PBA lifetimes increase to an unchanging value due to absence of quenchers.

New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: A comparison between hypoxic and normoxic cells

“…Figure 1B). We had previously observed similar reduction of PBA fluorescence lifetimes already at 4 h, in the same conditions of cell treatment [13]. Then, the means and SD remained stables during the 144 h of treatment (mean: Â148 ns; SD: Â4 ns), corresponding to ROS quantities two-fold the control value.…”

“…In a complex cellular environment, oxygen but also several ROS can deactivate PBA fluorescence [13,14]. If we make the simple assumption of two independent quenchers, we obtain the intracellular total ROS variation by comparing two treatment conditions [14]:…”

“…Thus, the method is less prone to errors and shows good sensitivity compared with other methods that use fluorescence intensity. PBA is a fluorescent probe with a long fluorescence lifetime depending on the concentration of molecular oxygen, but also on the intracellular level of ROS [13]. Fluorescence lifetime measurements of PBA in cells had permitted an evaluation of the ROS variations under various conditions [14].…”

Fluorescence Lifetime Measurements and Biological Imaging

“…Oxygen partial pressure in cells is low, ranging from 1.7-2.3 kPa for intracellular oxygen to 0.7 - 1.3 kPa in mitochondria 354. Such low partial pressure is close to the sensitivity level of pyrene to oxygen (1 kPa)355 and therefore pyrenes cannot be used for quantitative measurements in cells, although such attempts have been undertaken 198,356…”

“…354 Such low partial pressure is close to the sensitivity level of pyrene to oxygen (1 kPa), 355 and therefore, pyrenes cannot be used for quantitative measurements in cells, although such attempts have been undertaken. 198,356…”