Study on the Use of Cellular Respiration as a Surrogate Biomarker in Drug Development

Research Article

Austin J Clin Pathol. 2014;1(3): 1012.

Study on the Use of Cellular Respiration as a Surrogate Biomarker in Drug Development

Al-Hammadi S1, Alfazari AS2, Shaban S3 and Souid A-K1*

1Department of Pediatrics, UAE University, UAE

2Department of Medicine, UAE University, UAE

3Department of Medical Education, UAE University, UAE

*Corresponding author: Souid A-K, Department of Pediatrics, UAE University, Tawam campus, Al-Ain, Abu Dhabi, UAE

Received: May 25, 2014; Accepted: June 30, 2014; Published: July 02, 2014;


Cellular respiration (mitochondrial O2 consumption or oxidative phosphorylation) is the process of delivering nutrients and O2 to the mitochondria, oxidation of reduced metabolic fuels, passage of electrons to O2, and synthesis of ATP. These vital processes can serve as surrogate biomarkers for identifying cellular responses to drugs. This study investigated divergent compounds to learn whether their modes-of-action or adverse events could be linked to altered cellular bioenergetics. Small tissue fragments were collected from various organs of C57BL/6 mice in RPMI with and without designated drugs. Cellular respiration was then measured with the aid of a phosphorescence O2 analyzer, using Pd (II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin as an O2 probe. The phosphoinositide 3-kinase/mammalian target of rapamycin (Pi3K/mTOR) inhibitor GSK2126458 (50nM) repressed heart cellular respiration (p<0.001). This finding is consistent with the role of Pi3K/PTEN/Akt/mTOR pathway in cellular metabolism including insulin-dependent glucose uptake. The β2-adrenergic receptor agonist salbutamol (29 μM) and the bronchodilator magnesium sulphate (20mM) both inhibited lung cellular respiration (p≤0.006). The N-methyl-D-aspartate receptor antagonist ketamine (210 to 1,050 μM) had no significant effect on forebrain cellular respiration (p≤0.132).Although valproate induced histologic liver abnormalities, a therapeutic concentration of the drug (600 μM) had no effect on hepatocyte respiration (p=0.916). Similarly, the hepatotoxic drug azidothymidine (a nucleoside reverse transcriptase inhibitor; 100 μM) had no effect on hepatocyte respiration (p=0.297). Thus, cellular respiration can sense the activity and toxicity of some drugs. This analytical tool needs to be incorporated in drug development as a pharmacodynamic measure in targeted and off-targeted tissues.

Keywords: PI3K/mTOR Inhibitors; Salbutamol; MgSO4; Ketamine; Valproate; Azidothymidine


Pi3K: Phosphoinositide 3-kinase; mTOR: Mammalian Target of Rapamycin; NMDA: N-methyl-D-aspartate; ATP: Adenosine 5'-triphoshate; NRTI: Nucleoside Reverse Transcriptase Inhibito


Studies addressing the effects of drugs on cellular bioenergetics [the metabolic reactions involved in energy conversion or transformation including cellular respiration and accompanying adenosine 5'-triphoshate (ATP) synthesis] are limited [1-2]. These vital processes are involved in many drug mechanisms and adverse events. Therefore, it is reasonable to incorporate measurements of cellular energy into drug development programs. This study investigated the effects of several drugs on cellular respiration. Its main purpose was to use mitochondrial O2 consumption as a surrogate biomarker for assessing drug activity and toxicity.

Cellular respiration has been previously measured in specimens from the heart, liver, and kidney[3-4]. Therefore, drugs that target these organs are potential candidates for testing. In contrast, the hematopoietic system and pancreas are not readily permissible organs a brief account of these compounds is given below.

GSK2126458 potently inhibits phosphoinositide 3-kinase (Pi3K) and mammalian target of rapamycin (mTOR) [5]. This novel class of drugs is in clinical trials for treatment of various human diseases, as monotherapy or in combination with other cytotoxic agents. This therapeutic approach targets critical processes, such as survival pathways (e.g., Pi3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK) and cellular bioenergetics. For example, engagement of insulin with its receptor activates Pi3K, allowing cellular glucose uptake and other insulin signaling. Consistently, the activities of Pi3K inhibitors are expected to include impaired cellular bioenergetics [6-8]. GSK2126458 was studied here using heart cellular respiration since this organ consumes high metabolic energy and this type of drugs is known to have cardiac toxicity [9-10].

Salbutamol sulphate (a selective β2-adrenergic receptor agonist) and magnesium sulphate (Epsom salt) are routinely used medications for treatment of bronchospasm [11]. Plasma salbutamol concentration following repetitive dosing is about 0.2 μM [12]; the drug however, is commonly given by repetitive inhalations, which result in higher lung tissue concentrations. Therapeutic serum magnesium sulphate concentrations range from 2.0 to 3.5mM; higher dosing is associated with loss of deep tendon reflexes and cardiorespiratory arrest [13]. To our knowledge, the effects of these drugs on lung tissue cellular respiration have not been adequately previously studied.

Adverse events of the antiepileptic drug valproate include hepatotoxicity, mitochondrial dysfunction and complex metabolic derangements [14-15]. Its direct toxicity has been demonstrated in isolated liver mitochondria [16]. In cultured fibroblasts, the drug increases mitochondrial biogenesis [17]. Its effect on cellular respiration in isolated liver tissue is unknown. In one study, peak plasma valproate levels ranged from 465 μM to 1,118 μM [18].

Similarly, hepatic failure associated with the nucleoside reverse transcriptase inhibitors (NRTI), such as azidothymidine has been attributed to mitochondrial toxicity, which includes inhibition of mitochondrial DNA polymerases [19-20]. In one study, the plasma azidothymidine level was about 8 μM [21]. Valproate and azidothymidine are studied here using liver cellular respiration; (they are tested alone and in combination since both are potentially hepatotoxic). The N-methyl-D-aspartate receptor antagonist (NMDA) ketamine is studied using forebrain cellular respiration [22], as previously described [23].


Reagents and solutions

The Pi3K/mTOR inhibitor GSK2126458 (m.w. 505.5; cat. #HY- 10297) was purchased from MedChem Express, LLC (Princeton, NJ); the compound was dissolved in DMSO at 5 mg/mL (9.9mM) and stored at -20°C. Salbutamol BP (as sulphate, m.w. 576.7) 0.5% w/v (8.7mM) was purchased from Glaxo Operations UK Limited (Barnard, UK); the drug was stored at 25oC. Magnesium Sulphate Injection (as pentahydrate, m.w. 246.5) 50% w/v (2.0 M) was purchased from Martindale Pharmaceuticals (Romford, UK); the drug was stored at 25oC. Ketamine HCl Injection (m.w. 274.2) containing 50 mg ketamine base (m.w. 237.7) per mL (210mM) was purchased from JHP Pharmaceuticals (Rochester, MI, US). Sodium valproate (400 mg powder and solvent for solution for injection vials; m.w. 166.2) was purchased from Sanofi (Surrey, UK); the drug was dissolved in Water for Injection immediately before use and the powder was stored at 25oC in air-sealed vial. The antiretroviral drug azidothymidine (AZT; m.w. 267.2; 10 mg/mL of Water for Injection; 37.4mM) was purchased from Glaxo Operations UK Limited (Barnard, UK).

Pd (II) complex of meso-tetra- (4-sulfonatophenyl) -tetrabenzoporphyrin (Pd phosphor) was purchased from Porphyrin Products (Logan, UT). Pd phosphor (2.5 mg/mL = 2mM), NaCN (1.0 M) and glucose oxidase (10 mg/mL) solutions were prepared as previously described and stored at -20°C [3-4]. RPMI 1640 medium and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).


C57BL/6 (9-10 weeks old) mice were housed at the animal facility in rooms maintained at 22°C, 60% humidity and 12-h light-dark cycles. The mice had ad libitum access to standard rodent chow and filtered water. The study received approval from the Animal Ethics Committee - United Arab Emirates University - College of Medicine and Health Sciences.

Tissue collection and processing

Urethane (25% w/v, 100 μL per 10 g) was used to anesthetize the mice. Tissue fragments (about 10 to 25 mg each) were then cut manually with sterile scalpels (Swann-Morton, Sheffield, England) and immediately processed for measuring cellular respiration in the presence and absence of designated concentrations of the studied drugs. Alternately, liver specimens were incubated at 37°C in 50mL RPMI (continuously gassed with 95% O2: 5% CO2) with and without the drugs for up to 6 h. At designated times; samples were removed from the incubation solution and processed for measuring cellular respiration. Some samples were also processed for histology (hematoxylin & eosin staining), cellular ATP and cellular GSH as previously described [3-4].

Cellular respiration

Phosphorescence O2 analyzer was used to measure cellular mitochondrial O2 consumption as previously described [3-4]. Briefly, samples were exposed to 600/min flashes. O2 detection was with the aid of Pd phosphor (absorption 625 nm; emission maximum 800 nm). The phosphorescence was detected by Hamamatsu photomultiplier tube. The phosphorescence decay rate (1/τ) was single exponential; 1/τ was linear with dissolved O2: 1/τ = 1/τ° + kq[O2], 1/τ = phosphorescence decay rate in presence of O2, 1/τ° = phosphorescence decay rate in absence of O2, and kq = second-order O2 quenching rate constant (s-1 μM-1) [24]. A Microsoft Visual Basic 6 program was developed with the aid of Microsoft Access Database 2007 and Universal Library components (Universal Library for Measurements Computing Devices). These tools allowed direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board) [25]. O2 measurements were performed at 37°C in glass vials sealed from air. Respiratory substrates were endogenous metabolic fuels and the glucose present in RPMI. [O2] decreased linearly with time; this zero-order process was inhibited by cyanide (CN), confirming O2 consumption occurred in the mitochondrial respiratory chain. The rate of respiration (k; μM O2 min-1) was the negative of the slope d [O2]/dt. The value of k was divided by specimen weights, giving kc (μM O2 min-1 mg-1). The data were analyzed on SPSS statistical package (version 19), using the nonparametric (two independent samples) Mann-Whitney test.


Figure 1A shows representative runs of heart muscle cellular mitochondrial O2 consumption with and without the Pi3K/mTOR inhibitor GSK2126458. Each run represented a specimen that was collected from a C57BL/6 mouse and immediately processed for measuring cellular respiration in the presence and absence of 50nM GSK2126458. A summary of the results is shown in Figure 1B and Table 1. The rate of respiration (kc; μM O2 min-1 mg-1; mean ± SD) without addition was 0.19 ± 0.04 (n = 14 mice) and with the addition of GSK2126458 was 0.08 ± 0.03 (n = 17 mice; p<0.001). Thus, GSK2126458 decreased heart cellular respiration by about 58%.