Abstract
Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay (®) (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and interesting research lines in biology and human physiology with the potential for significant benefits for both space and terrestrial medicine.
Highlights
Critical medical situations can occur in space missions[1]
In each gravitational condition four different assays were performed: fully activated transporter or “Multi-drug resistance-associated protein 2 (MRP2)+”, simple diffusion/control or “MRPdef”, ATP-independent transport or “noATP”, BZM inhibition assay samples or “BZM”. To our knowledge, this is the first validation study of a biotechnological approach aimed at evaluating human ABC transporters-mediated active transmembrane transport capabilities in microgravity
Despite the high signal variability expected, the precision attained by our electromechanical system and the enhanced sensibility of the detection protocol used, enabled us to detect net MRP2 transport activity in the short microgravity periods provided by parabolic flights
Summary
Critical medical situations can occur in space missions[1]. In such situations, adequate treatment may require the use of drugs with potential severe side effects. Pharmacokinetics and pharmacodynamics can be modified under weightlessness conditions by many factors[2,3], which may lead to undesired pharmacological effects These parameters should be assessed carefully in order to ensure safe use of medications in space. ABC (ATP-Binding Cassette) transporters are a large family of trans-membrane proteins widely present in many life forms, from bacteria to mammals. In humans, they play important roles in many physiological processes and in drug pharmacokinetics, pharmacodynamics, and drug-to-drug interactions of many currently used medications in oncology, emergency medicine, critical care and general practice[6,7]. Multi-drug resistance-associated protein 2 (MRP2) is a well-studied representative of the ABC transporters family, by which transport of glucuronide, sulphate and gluthationeconjugated drugs is made against a concentration gradient requiring adenosine-tri-phosphate (ATP) hydrolysis[8]
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