California Sea Grant College Program

The classic calcein-AM accumulation assay of multidrug resistance studies has been used in several vertebrate systems but not yet in zebrafish {Danio rerio) or their embryos. Here we describe the adaptation of this assay for use in zebrafish and include details on the approach used to develop an epifluorescence microscopy protocol for viewing and measuring fluorescence of treated embryos — the development of which facilitated further investigations into the early zebrafish embryo's capacity for multidrug resistance as a defense mechanism. Multidrug resistance (MDR) transporters of the multidrug resistanceassociated protein (Mrp) and P-glycoprotein (P-gp) families were examined during early development in zebrafish. We investigated transport activity in the presence and absence of MDR inhibitors MK571 (MRP) and PSC833 (P-gp): Mrp-mediated transport activity was most apparent in the pre-midblastula transition (pre-MBT) embryo, although limited activity was also evident post-midblastula transition (post- MBT), and P-gp-mediated transport was not detectable at either developmental stage. These findings indicate that MDR is active in the early embryo and provides defensive capabilities against accumulation of select toxicants. While investigating the existence and behavior of transcript expression of select MDR-affiliated genes in the early developing zebrafish (Danio rerio) embryo, results of normalizing transcript expression according to an established conservative method seemed to suggest that transcript expression was occurring during early development, although patterns in expression were not observable and an exact behavior was unknown. We therefore alternately explored a course of data treatment involving a non-conservative approach to normalization calculations that accounted only for inter-experimental variation. Though both experimental approaches were valid, they did not show the same interpretation of data. Therefore a power analysis based investigation of suitable calculation methods for comparatively analyzing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) data was used to assess the most appropriate analysis method for the experimental design: transcript expression of the MDR-affiliated genes abcbl, abccl, abcc2, and abcg2a in early developing zebrafish. We concluded that transcript expression of select MDR genes is measurable in the early zebrafish embryo and that experimental design should take into consideration that the selection of a course of data treatment, in the form of normalization calculations, affects calculated expression values.