The horizontal arrow bars indicate the periods of sunitinib administration

first time conclusive experimental demonstration that an ABC transporter acts as a heme transporter in C. elegans, yeast, zebrafish and mammalian cells. Drug detoxification in eukaryotic cells is generally described as a process that involves three steps: the chemical modification of xenobiotics, followed by conjugation to anionic groups such as glutathione, glucuronate or sulfate, and finally, excretion by ABC transporters. Resistance to insecticides has been shown to involve enzymes from the first two classes, including esterases, P-450 cytochromes and glutathione-S-transferases, but the participation of ABC transporters in the development of resistance only recently has been acknowledged. Here, we show that heme transport from the digestive vesicle to the hemosome is mediated by RmABCB10, an ABC transporter of the subfamily B10 that is upregulated in a tick cell line resistant to ivermectin and is also up-regulated in an acaricide-resistant tick strain obtained from the field. We also present evidence that a common acaricide, amitraz, is accumulated into the hemosomes through a pathway that uses the same ABC transporter, in this way outlining a novel type of resistance to pesticides. Materials and Methods Ethics Statement All animal care and experimental protocols were conducted following the guidelines of the institutional care and use committee and were approved under the registry # 14403/protocolo 07. Technicians dedicated to the animal facility at Faculdade de Veterinria from UFRGS carried out all aspects related to cattle husbandry under strict guidelines to insure careful and consistent handling of the animals. Animals R.microplus of the Porto Alegre strain,, free of Babesia spp. and Anaplasma spp., were reared on calves obtained from a tick-free area and maintained at the Faculdade de Veterinria of Universidade Federal do Rio Grande do Sul, Brazil. Fully engorged adult females were kept in Petri dishes at 28C and 80% relative humidity until use. The animals were handled in compliance with the UFRGS and EMBRAPA review committee for animal care. The amitraz-resistant strain of R. microplus was collected from a farm in the Ibirapu district, Bahia state, Brazil, and maintained on calves reared in a tick-free area at Empresa Brasileira de Agropecuria from Juiz de Fora, Brazil. Amitraz resistance was evaluated using the adult immersion test, a bioassay applied to fully engorged female ticks, ML-128 web performed as described by Drummond et al.. Briefly, groups of 10 females were immersed for 5 min in the acaricide solutions, usually using the acaricide concentrations recommended by the manufacturer. The control group was immersed in distilled water. The viability was estimated using the Porto Alegre strain as a susceptible standard, which showed 0% survival to all types of acaricides tested. The effect of pesticides on the Ibirapu strain, in contrast, ranged from a lack of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19756382 resistance to some organophosphates, moderate resistance to pyrethroids and 100% survival to 85 M of amitraz. Digest cell culture The digest cell culture was performed as described by Lara et al. Briefly, fully engorged females on the second day after a blood meal were rinsed in 70% ethanol for 1 min and dissected in sterile phosphate buffered saline containing 200 U mL1 streptomycin and penicillin. Midguts were isolated in sterile PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19754643 Petri dishes, and digest cells were detached from the gut wall with sterile tweezers. The cells were carefully collected using a Pasteur pipette