Chromosomal passports provide new insights into diffusion of emmer wheat.

Emmer wheat, Triticum dicoccon schrank (syn. T. dicoccum (schrank) schÜbl.), is one of the earliest domesticated crops, harboring a wide range of genetic diversity and agronomically valuable traits. The crop, however, is currently largely neglected. We provide a wealth of karyotypic information from a comprehensive collection of emmer wheat and related taxa. In addition to C-banding polymorphisms, we identified 43 variants of chromosomal rearrangements in T. dicoccon; among them 26 (60.4%) were novel. The T7A:5B translocation was most abundant in Western Europe and the Mediterranean. The plant genetic resources investigated here might become important in the future for wheat improvement. Based on cluster analysis four major karyotypic groups were discriminated within the T. dicoccon genepool, each harboring characteristic C-banding patterns and translocation spectra: the balkan, asian, european and ethiopian groups. We postulate four major diffusion routes of the crop and discuss their migration out of the Fertile Crescent considering latest archaeobotanical findings.

A novel MDCKII in vitro model for assessing ABCG2-drug interactions and regulation of ABCG2 transport activity in the caprine mammary gland by environmental pollutants and pesticides.

The ABC efflux transporter ABCG2 represents the main route for active secretion of xenobiotics into milk. Thus, ABCG2 regulation by aryl hydrocarbon receptor (AhR) ligands including ubiquitously environmental pollutants is of great toxicological relevance. However, no adequate in vitro model is as yet available to study AhR-dependent ABCG2 regulation in dairy animals. In this study, we therefore systematically investigated the effect of various environmental contaminants and pesticides on ABCG2 efflux activity in MDCKII cells stably expressing mammary ABCG2 from dairy goats. The AhR-agonists TCDD, Aroclor 1254, prochloraz, and iprodione caused a dose- and time-dependent increase in EROD activity. Moreover, TCDD and prochloraz significantly stimulated ABCG2 transport activity through a dose- and time-dependent induction of transporter gene expression. AhR inhibitors like CH223191 significantly reversed TCDD- and prochloraz-induced stimulation of ABCG2 efflux activity. In contrast, non-AhR activators such as PCB 101 had no significant effect on EROD activity, ABCG2 gene expression or transporter activity. As we identified various anthelmintics including monepantel as potential ABCG2 substrates this regulatory mechanism may result in increased milk residues of potentially harmful xenobiotics. Thus, MDCKII-cABCG2 cells may represent a suitable in vitro model to study mammary ABCG2 secretory activity and its potential regulation by AhR-activating contaminants.

Assessment of ABCG2-mediated transport of xenobiotics across the blood-milk barrier of dairy animals using a new MDCKII in vitro model.

The ATP-binding cassette (ABC) efflux transporter ABCG2 represents the main route for active secretion of drugs and toxins across the blood-milk barrier, thereby producing a potential health risk for dairy consumers through formation of relevant residues in milk. However, no suitable in vitro model is as yet available to systematically investigate ABCG2-mediated transport of xenobiotics into milk of dairy animals. We recently cloned ABCG2 from the lactating mammary gland of dairy cows (bABCG2) and goats (cABCG2). Thus, the objective of this study was to generate a suitable blood-milk barrier in vitro model using polarized MDCKII monolayers stably expressing mammary bABCG2 or cABCG2. ABCG2 protein was localized by confocal microscopy to the apical and lateral plasma membrane of polarized MDCKII cells. Intact barrier function of MDCKII-bABCG2 and MDCKII-cABCG2 monolayers was confirmed by determination of cell permeability of transcellular marker propranolol and paracellular marker atenolol which was ≤1 %. In flux assays, ABCG2 substrate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) showed preferential basolateral to apical (B > A) transport in ABCG2-MDCKII cells. This apically directed PhIP transport was significantly inhibited by ABCG2 inhibitor fumitremorgin C (FTC) or the flavonoid equol. PhIP B > A transport in MDCKII-bABCG2 monolayers was additionally decreased by ABCG2 inhibitor Ko143. The fluoroquinolone antibiotic enrofloxacin was identified as a substrate of ruminant mammary ABCG2. The analgesic drug sodium salicylate was shown to be substrate of bABCG2 but not of cABCG2. Thus, the generated mammary ABCG2-expressing MDCKII cells represent a valuable tool to study active secretion of drugs and toxins into milk.

Fungicide prochloraz and environmental pollutant dioxin induce the ABCG2 transporter in bovine mammary epithelial cells by the arylhydrocarbon receptor signaling pathway.

The molecular mechanisms by which environmental pollutants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or widely used imidazole fungicide prochloraz display their toxic effects in vertebrates are still not well understood. Using computer analysis, we recently identified nuclear aryl hydrocarbon receptor (AhR) binding sites termed "dioxin response elements" (DREs) in the 5'-untranslated region (5'-UTR) of efflux transporter ABCG2 (Accession No. EU570105) from the bovine mammary gland. As these regulatory motifs mediate regulation of target genes by AhR agonists including TCDD and prochloraz, we have systematically investigated the effect of both contaminants on functional ABCG2 transport activity in primary bovine mammary epithelial cells. TCDD or prochloraz doubled ABCG2-mediated Hoechst H33342 secretion. This effect was almost completely reversed by specific ABCG2 inhibitor Ko143. In further mechanistic studies, we showed that this induction was due to binding of activated AhR to DRE sequences in the ABCG2 5'-UTR. Receptor binding was significantly reduced by specific AhR antagonist salicyl amide. Induction of AhR by TCDD and prochloraz resulted in a time- and dose-dependent increase of ABCG2 gene expression and transporter protein levels. As ABCG2 represents the main mammary transporter for xenobiotics including drugs and toxins, exposure to prevalent AhR agonists may enhance transporter-mediated secretion of potential harmful compounds into milk. Through identification of mammary ABCG2 as a novel target gene of pesticide prochloraz and dioxin, our results may therefore help to improve the protection of breast-feeding infants and the consumer of dairy products.

Determination of functional ABCG2 activity and assessment of drug-ABCG2 interactions in dairy animals using a novel MDCKII in vitro model.

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is a member of the ATP-binding cassette (ABC) family of efflux carriers that mediates cellular extrusion of various drugs and toxins. In the mammary gland, ABCG2 is expressed at the apical membrane of alveolar epithelial cells and is induced during lactation. It is well established that ABCG2 plays the main role in active secretion of xenobiotics into milk of humans and mice. In contrast, no detailed information is as yet available about functional activity and substrate spectrum of ABCG2 in dairy animals. Therefore, we cloned full-length ABCG2 from bovine, ovine and caprine lactating mammary gland tissues using rapid amplification of complementary DNA (cDNA) ends polymerase chain reaction. The generated full-length ABCG2 cDNA constructs were stably transduced in MDCKII cells. Functional ABCG2 efflux activity was demonstrated with the Hoechst H33342 accumulation assay using the specific ABCG2 inhibitor Ko143. The established ruminant MDCKII-ABCG2 cell culture models in conjunction with the H33342 transport assay showed interaction of various drugs such as cefalexin and albendazole with bABCG2, oABCG2 or cABCG2. Moreover, the flavonoids equol and quercetin exhibited interaction with all ruminant ABCG2 clones. Altogether, our generated cell culture models allowed rapid and high-throughput screening of potential ruminant ABCG2 substrates and thus increase the understanding of carrier-associated secretion of xenobiotics into milk.