Cytochrome P-450-mediated ethoxy-resorufin Odeethylase activity measured in non-fixed, non-frozen sections using a fluorescence microscope
A highly sensitive method, based on the use of a microscope fluorimeter and fluorogenic substrates, is being developed to measure rates of drug-metabolising enzyme reactions in small areas of cells in thin liver sections. The results described here illustrate a method in which reaction rates were measured in small groups of approximately 25 hepatocytes. The development of the method has been based initially on the measurement of the cytochrome. P-450-mediated ethoxyresorufin O-deethylase reaction in mouse hepatocytes. Fluorogenic substrates are known, however, for several other drug-metabolising enzymes and alternative fluorogenic substrates, namely pentoxyresorufin and benzyloxyresorufin, for different forms of P-450 have been used in this study. There has been significant improvement over an earlier method (Burke et al., 1983) of sectioning non-fixed, non-frozen pieces of liver. The left lobe of a mouse liver was stuck with cyanoacrylate glue to the Teflon stage of the Vibraslice cutting machine. The Teflon stage and attached liver lobe were immersed in a bath of Krebs-Henseleit physiological buffer. The specimen bath was moved towards the vibrating blade which cut through the liver so producing a section which was transferred with a fine brush to the surface of a slide of encapsulating resin on to which it was pinned. The section of liver was then superfused with the Krebs-Henseleit solution at 37 C on the heated stage of a microscope. The tip (10-12 um diameter) of a micropipette containing a solution of ethoxyresoruf in DMSO was manoeuvred onto the surface of the section using a micromanipulator. Selection of the cells to be measured and placement of the micropipette were carried out in transmitted light. The microscope was then switched to epi-illumination fluorescence mode. Ethoxyresoruf in was expelled from the micropipette on to the section by the application of a 2-5 second pulse of pressurised Nitrogen gas (approximately 10 lbf/in2). The volume of ethoxyresoruf in expelled was estimated as 0.405 0.027 ml using a combination of [3Hj-harmine and 1 mM ethoxyresorufin in the micropipette. The spread of substrate and the ensuing O-dealkylation reaction were confined to a circular area of the tissue approximately 100 um in diameter, comprising about 25 hepatocytes. During, the reaction, the fluorescence of the forming metabolite, resorufin, was measured continuously by means of a computerised photomultiplier system attached to the microscope. The measurement of fluorescence was from the surface layer of cells only. Since the method is at a stage in development, various, pieces of apparatus were subject to change. The excitation and emission filters were chosen so that the selected wavelengths of light were more appropriate for the metabolite resorufin than the substrate ethoxyresorufin. For excitation, a combination of a Vickers OG515 long wave pass filter (50% transmission between 370 nm and 560 nm), a Vickers TRITC exciter (50% transmission between 380 nm and 560 nm) and a Balzer TRITC dichroic mirror (10% transmission between 490 nm and 570 nm) were used. For emission, a Balzer TRITC dichroic mirror and a Barr and Stroud DL7S narrow band pass filter (maximum transmission of 49.2% at 590.1 nm) were used. The filters provided epi-illumination (incident) excitation light of 515-560 nm and selected emitted fluorescence of 580-600 run. A photomultiplier converted the photons of light into an electrical signal which was converted to a digital signal. Ideally the photomultiplier tube in use at each of the improvements was maximally sensitive to the emission wavelengths of resorufin. The digital signal was displayed either on a pen recorder or on a microcomputer, both of which resulted in a graph of relative fluorescence against time. From this graph three measurements were taken. These were: i) the initial reaction velocity (rate of linear rise of fluorescence intensity). ii) the highest fluorescence intensity attained, and iii) the time to reach the highest fluorescence intensity after the cessation of ethoxyresorufin application. The initial velocity had a closer correlation with the maximum reading (r = 0.75) than with the time at which the maximum occurred (r = 0.43). The results encompassed a wide range of values but did follow a normal distribution which meant that the Student's t-test could be used to test for significant differences between means. Means s.e. mean initial velocities measured in centrilobular and periportal regions of liver sections from control mice, were 2.7 0.32 and 1.9 0.28 fluor. unitsmin-1 respectively, suggesting no' difference between centrilobular and periportal regions. Pretreatment with MC increased these values to 55.0 + 3.5 and 49.9 3.3 respectively but again suggested no centrilobular/periportal difference, -Naphthoflavone produced less of an effect causing an increase from 11.2 1.7 to 52.9 10.6 in centrilobular regions and from 7.3 1.0 to 51.7 10.0 in periportal regions.