Neal Adam, Rountree Austin M, Philips Craig W, Kavanagh Terrance J, Williams Dominic P, Newham Peter, Khalil Gamal, Cook Daniel L, Sweet Ian R
*Department of Medicine, University of Washington, Seattle, Washington, 98195;
Center for Commercialization, University of Washington, Seattle, Washington 98195;
Toxicol Sci. 2015 Dec;148(2):594-602. doi: 10.1093/toxsci/kfv208. Epub 2015 Sep 22.
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.
在临床前筛选过程中，普遍需要检测药物的毒性作用。我们提出，提高异生物素毒性的敏感性并改善体外生理模拟，将能更准确地评估接近体内药理学暴露水平的细胞生物化学潜在毒性扰动。重要的是，此类细胞病理学测量避免了激活在与体内效应无关的超药理学水平介导毒性的机制。我们提出一种灵敏的方法，使用流动培养系统和先进的氧传感系统，来测量氧消耗率（OCR）的变化，OCR是一个反映潜在危害的成熟参数，用于响应药理学水平药物的暴露。我们用大鼠肝切片测试了二甲双胍和对乙酰氨基酚来说明该方法。该方法的特点包括在连续流动室中对肝切片在48小时内连续且非常稳定地测量OCR，能够分辨低至0.3%/小时的变化。在广泛浓度范围内对二甲双胍抑制OCR的动力学建模揭示了一种慢速和快速机制，其中快速机制仅在浓度高于0.6 mM时激活。对于这两种药物，少量抑制是可逆的，但更高程度的下降是不可逆的。总体而言，该研究突出了测量低水平毒性的优势，从而避免基于在超药理学水平测试药物的效应而对药物毒性进行的常见推断。
