Quantification of Low-Level Drug Effects Using Real-Time, in vitro Measurement of Oxygen Consumption Rate

Abstract

There is a general need to detect toxic effects of drugs during preclinical screening. We propose that increased sensitivity of xenobiotics toxicity combined with improved in vitro physiological recapitulation will more accurately assess potentially toxic perturbations of cellular biochemistry that are near in vivo pharmacological exposure levels. Importantly, measurement of such cytopathologies avoids activating mechanisms mediating toxicity at suprapharmacologic levels not relevant to in vivo effects. We present a sensitive method to measure changes in oxygen consumption rate (OCR), a well-established parameter reflecting a potential hazard, in response to exposure to pharmacologic levels of drugs using a flow culture system and state of the art oxygen sensing system. We tested metformin and acetaminophen on rat liver slices to illustrate the method. The features of the method include continuous and very stable measurement of OCR over the course of 48 h in liver slices in a continuous flow chamber with the ability to resolve changes as small as 0.3%/h. Kinetic modeling of metformin inhibition of OCR over a wide range of concentrations revealed both a slow and fast mechanism, where the fast mechanism activated only at concentrations above 0.6 mM. For both drugs, small amounts of inhibition were reversible, but higher decrements were irreversible. Overall the study highlights the advantages of measuring low-level toxicity so as to avoid the common extrapolations made about drug toxicity based on effects of drugs tested at suprapharmacologic levels.

Keywords: acetaminophen; drug toxicity; liver slices; mathematical modeling; metformin; oxygen consumption rate.

FIG. 1.

Schematic of flow culture/assessment system. The components of the environmental housing of the flow system are contained in a plexiglass container (consisting of a left and right compartments that contain the perifusate and is temperature controlled at 42°C by a convective fan and a heating strip (left) and the lung and the perifusion chambers which are submersed in bath water that is temperature controlled at 37°C by an immersion heater (right). The parts of the flow conduit from the pump inflow, through the bubble trap, lung and perifusion chamber to the fraction collector are shown as follows. 1, PEEK Tubing (1/16 × 0.04 × 16 in, 1538XL, IDEX, Lake Forest, Illinois). 2, Conical adaptor, 1/16” to 0.5–0.8 mm union (P-794, IDEX). 3, Tygon pump tubing (Masterflex Tygon E-LFL Tubing, 0.25 mm I.D. × 15 in, 06447-12, Cole-Parmer, Vernon Hills, Illinois). 4, PEEK Tubing (1/16 × 0.04 × 24 in, 1538XL, IDEX). 5, PharMed BPT Tubing (0.8 mm I.D. × 0.75 in, L/S #13, 6508-13, Cole-Parmer). 6, Stainless steel tubing (0.062 (O.D)/0.020 (I.D.) × 1.5 in, (WAT025592, Waters, Milford, Massachusetts). 7, Barbed connector (1/16 to 1/16 in, P-801X, IDEX). 8, Silastic Laboratory Tubing, 0.062 I.D./0.095 O.D. × 10 in, 508-007, Dow Corning, Midland, Michigan). 9, HPFA+ Tubing, 0.062 (O.D.) × 0.02 (I.D.) × 12 in (PM-1451-F, IDEX). 10, Upchurch Scientific Reducer Barbed, 1/16 × 1/8 (P-807X). 11, PharMed BPT Tubing (3.1 mm I.D × 1 in. L/S #16, 6508-16, Cole-Parmer). 12, Frits for bubble trap: porous polyethylene (1/4 in thick, SPEH-4894, Small Parts, Inc, Logansport, Indiana). 13, HPFA+ Tubing, 0.062 (O.D.) × 0.02 (I.D.) × 60 in (PM-1451-F, IDEX). Only 1 channel is shown, but in practice, 8 channels were run at a time.

FIG. 2.

Kinetic profile of OCR of control liver slices placed in to the flow system immediately after harvesting. Liver slices (n = 3 from separate animals done on separate days) were perifused in culture media for 40 h. OCR stabilized after 18–20 h and from then on maintained a very constant value (steady-state values of OCR were 0.22 ± 0.04 nmol/min/mg liver.

FIG. 3.

Determination of inflow concentration of oxygen using periodic flow rate changes. Rather than track the inflow for each individual flow channel and use up available optical detection channels, inflow concentration was determined by periodically doubling the flow rate and calculating the inflow concentration as described in the Methods. Data show the comparison of the calculated value to baseline prior to loading the liver, and subsequent to inhibiting respiration with cyanide.

FIG. 4.

High resolution of effects of metformin on liver OCR. To maximize the sensitivity to detect low levels of changes in OCR due to the exposure to drugs, the difference between data obtained simultaneously in the presence and absence of 0.3 mM metformin was calculated. This data processing removed the effects of inflow concentration drift and other factors that affected all chambers equally which contributed to short- and long-term noise that ranged around 3%, and increased the resolution of the specific affects of the drug.

Fig. 5.

OCR by rat liver during exposure to various concentrations of metformin. Top, After equilibrating the liver in the flow system for 20 h, metformin was added to the inflow at various concentrations as indicated, and the responses of OCR were continuously assessed during 10 h of drug exposure. The n shown for each concentration indicates the number of separate perifusions run on different days using liver slices from different animals. Sufficient replicates were carried out at 0.3 mM metformin (n = 9) to run statistical analysis on the OCR decrement average over the final 15 min of the experiment (** denotes P < .001). Data were fitted to the multi-exponential decay model (equation 1) for each concentration of metformin. Bottom, Kinetic and dose–response analysis of OCR profiles: distinguishing multiple mechanisms. Values of rate constants for the slow and fast inhibition are plotted as a function of metformin concentration.

FIG. 6.

Reversibility after exposure to 0.3 or 2.5 mM metformin. After exposure to metformin for 10 h, the liver was perifused in the absence of the drug for an additional 12 h. Data are average of 3 separate experiments done on different days and the error bars are plus or minus the SE.

FIG. 7.

OCR by rat liver during exposure to various concentrations of acetaminophen. After equilibrating the liver in the flow system for 20 h, the continuous responses to acetaminophen of OCR during 7 h of continuous assessment. Data was fitted to the multiexponential decay model (equation 1) for each concentration of acetaminophen.