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Quantification of Low-Level Drug Effects Using Real-Time, in vitro Measurement of Oxygen Consumption Rate
Adam Neal1, Austin M Rountree1, Craig W Philips2
1*Department of Medicine, University of Washington, Seattle, Washington, 98195;
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.
Insights
This study introduces a sensitive method to detect low-level drug toxicity by measuring oxygen consumption rate (OCR) in liver slices. This approach provides more accurate preclinical toxicity assessments relevant to in vivo drug exposure levels.
Area of Science:
- Biochemistry
- Toxicology
- Pharmacology
Background:
- Preclinical drug screening requires sensitive methods to detect toxic effects.
- Current in vitro models often use suprapharmacologic drug levels, limiting relevance to in vivo conditions.
- Accurate assessment of cellular biochemistry perturbations near pharmacologic levels is crucial.
Purpose of the Study:
- To develop and validate a sensitive method for measuring drug-induced toxicity at pharmacologic levels.
- To assess cellular toxicity by monitoring oxygen consumption rate (OCR) in rat liver slices.
- To compare drug toxicity mechanisms at low vs. high concentrations.
Main Methods:
- Utilized a continuous flow culture system with advanced oxygen sensing for stable OCR measurement over 48 hours.
- Applied the method to rat liver slices exposed to metformin and acetaminophen at pharmacologic concentrations.
- Employed kinetic modeling to analyze OCR changes and determine drug-specific inhibition mechanisms.
Main Results:
- The method achieved high sensitivity, resolving OCR changes as small as 0.3%/h.
- Metformin exhibited both slow and fast inhibition mechanisms, with the fast mechanism appearing above 0.6 mM.
- Drug-induced OCR inhibition was dose-dependent, with higher decrements being irreversible.
Conclusions:
- Measuring low-level drug toxicity using OCR in a flow culture system offers a more physiologically relevant preclinical assessment.
- This approach avoids artifacts from suprapharmacologic drug concentrations, improving the accuracy of toxicity predictions.
- The findings highlight the importance of evaluating drug effects at concentrations relevant to in vivo exposure.
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