Modulation of hepatic ABC transporters by Eruca vesicaria intake: Potential diet-drug interactions.

The aim of this work was to evaluate whether oral administration of Eruca vesicaria, a species of rocket cultivated in Argentina, could modify cyclophosphamide (CP)-induced genotoxicity through modulation of hepatic ABC transporters. Daily oral administration of E. vesicaria fresh leaves juice (1.0, 1.4 and 2.0  g/kg) for 14 days did not alter genotoxicity biomarkers -alkaline comet assay and micronucleus test -in neither male nor female mice. Instead, repeated intake of this cruciferous decreased CP-induced DNA damage dose-dependently and it caused hepatic overexpression of P-glycoprotein (P-gp; 1.4 and 2.0  g/kg) and multidrug resistance protein 2 (MRP2; 2.0  g/kg), but not breast cancer resistance protein (Bcrp). The antigenotoxic effect of E. vesicaria was prevented by 50 mg/kg verapamil (P-gp inhibitor) or 10 mg/kg indomethacin (MRP2 inhibitor). In turn, CP-induced cytotoxicity (10 mM, 24 h) on human hepatoma cells (HepG2/C3A) was significantly reduced by preincubation with E. vesicaria (1.4 mg/ml; 48 h); this effect was absent when CP was coincubated with 35 µM verapamil, 80 µM indomethacin or 10 µM KO-143 (BCRP inhibitor). Altogether, these results allow us to demonstrate that repeated intake of E. vesicaria exhibited antigenotoxicity, at least in part, by induction of hepatic ABC transporters in vivo in mice as well as in vitro in human liver cells. This could account for other diet-drug interactions.

In vivo antigenotoxic activity of Diplotaxis tenuifolia against cyclophosphamide-induced DNA damage: Relevance of modulation of hepatic ABC efflux transporters.

Several epidemiological studies have demonstrated that a diet with high contents of cruciferous vegetables (which belong to the Brassicaceae family) may reduce the incidence of cancer and neurodegenerative diseases. However, some authors have postulated that they might bring about toxic effects. Therefore, the aim of this study was to assess the effects of chronic administration of Diplotaxis tenuifolia (wild rocket), a species found in Argentina, concerning its putative genotoxicity or antigenotoxicity against the DNA damage inducer cyclophosphamide, and its ability to modulate the hepatic expression of ABC efflux transporters on mice. The alkaline comet assay and the micronucleus test were used as genotoxicity biomarkers, and the ABC transporter expression was analyzed by Western-blotting. D. tenuifolia juice exhibited no genotoxicity in any of the three tested doses (p > 0.05), showing instead a protective effect against genotoxic damage induced by cyclophosphamide (p < 0.001) in a dose-dependent behavior. Furthermore, hepatic expression of ABCB1 remained unchanged in both sexes at every dose, whereas ABCG2 expression increased in females (p < 0.05) and males (p < 0.01) at the highest dose. Regarding ABCC2, sex-related differences were observed (p < 0.05), its expression decreasing in females (p < 0.05) and increasing in males (p < 0.05). The modulation of these transporters may contribute to the antigenotoxic effects of D. tenuifolia since they act as universal detoxifiers, excreting xenobiotics to the cellular exterior. Phytochemicals present in the juice such as glucosinolates, quercetin and kaempherol may be responsible for these beneficial effects.

Development and Validation of a Computational Model Ensemble for the Early Detection of BCRP/ABCG2 Substrates during the Drug Design Stage.

Breast Cancer Resistance Protein (BCRP) is an ATP-dependent efflux transporter linked to the multidrug resistance phenomenon in many diseases such as epilepsy and cancer and a potential source of drug interactions. For these reasons, the early identification of substrates and nonsubstrates of this transporter during the drug discovery stage is of great interest. We have developed a computational nonlinear model ensemble based on conformational independent molecular descriptors using a combined strategy of genetic algorithms, J48 decision tree classifiers, and data fusion. The best model ensemble consists in averaging the ranking of the 12 decision trees that showed the best performance on the training set, which also demonstrated a good performance for the test set. It was experimentally validated using the ex vivo everted rat intestinal sac model. Five anticonvulsant drugs classified as nonsubstrates for BRCP by the model ensemble were experimentally evaluated, and none of them proved to be a BCRP substrate under the experimental conditions used, thus confirming the predictive ability of the model ensemble. The model ensemble reported here is a potentially valuable tool to be used as an in silico ADME filter in computer-aided drug discovery campaigns intended to overcome BCRP-mediated multidrug resistance issues and to prevent drug-drug interactions.

Tetronic® 904-containing polymeric micelles overcome the overexpression of ABCG2 in the blood-brain barrier of rats and boost the penetration of the antiretroviral efavirenz into the CNS.

AIM: To assess the involvement of ABCG2 in the pharmacokinetics of efavirenz in the blood-brain barrier (BBB) and investigate a nanotechnology strategy to overcome its overexpression under a model of chronic oral administration. Materials & methods A model of chronic efavirenz (EFV) administration was established in male Sprague-Dawley rats treated with a daily oral dose over 5 days. Then, different treatments were conducted and drug concentrations in plasma and brain measured. RESULTS: Chronic treatment with oral EFV led to the overexpression of ABCG2 in the BBB that was reverted after a brief washout period. Moreover, gefitinib and the polymeric amphiphile Tetronic(®) 904 significantly inhibited the activity of the pump and potentiated the accumulation of EFV in CNS. The same effect was observed when the drug was administered within mixed micelles containing TetronicT904 as the main component. CONCLUSION: Tetronic 904-containing polymeric micelles overcame the overexpression of ABCG2 in the BBB caused by chronic administration of EFV then boosting its penetration into the CNS.

Phytoestrogens enhance the vascular actions of the endocannabinoid anandamide in mesenteric beds of female rats.

In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular adrenergic hyperactivity that usually precedes the onset of primary hypertension, the oral administration (3 daily doses) of either 10 mg/kg genistein or 20 mg/kg daidzein potentiated the anandamide-induced reduction of contractility to NA in female but not in male rats. Oral treatment with phytoestrogens also restored the vascular effects of anandamide as well as the mesenteric content of calcitonin gene-related peptide (CGRP) that were reduced after ovariectomy. The enhancement of anandamide effects caused by phytoestrogens was prevented by the concomitant administration of the estrogen receptor antagonist fulvestrant (2.5 mg/kg, s.c., 3 daily doses). It is concluded that, in the vasculature of female rats, phytoestrogens produced an estrogen-receptor-dependent enhancement of the anandamide-vascular actions that involves the modulation of CGRP levels and appears to be relevant whenever an adrenergic hyperactivity occurs.

Efavirenz is a substrate and in turn modulates the expression of the efflux transporter ABCG2/BCRP in the gastrointestinal tract of the rat.

The oral bioavailability of the antiretroviral efavirenz (EFV) undergoes high inter and intra-individual variability, this fact supporting its therapeutic drug monitoring. Previously, it was demonstrated that the encapsulation of EFV within polymeric micelles increases the oral bioavailability of the drug. The breast cancer resistant protein (BCRP, ABCG2) is known to be inhibited by EFV in vitro. Since ABCG2 is profusely expressed in the gastrointestinal tract, the aim of the present work was to thoroughly investigate whether the intestinal permeability of EFV is modulated by ABCG2. The functional role of ABCG2 in mediating the transport of EFV at the intestinal level was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C (5-10µM), significantly potentiated the mucosal-to-serosal permeation of the drug in everted gut sacs; (b) a five-day oral treatment with 20mg/kg EFV promotes the over-expression of ABCG2 in about 100%, this phenomenon being accompanied by a clear decline in the intestinal permeability of the antiretroviral and (c) the normalization of the ABCG2 expression within 24h after the last administration of EFV was coincident with the recovery of the ability of the drug to permeate through the small intestine wall. Interestingly, no interactions between EFV and P-glycoprotein (ABCB1) were apparent. Since the intestinal permeability of a drug could be associated with its in vivo absorbability, we suggest that the oral absorption of EFV is affected by modifications in the ABCG2 intestinal expression contributing to the intra-individual bioavailability variations.

Increases in vanilloid TRPV1 receptor protein and CGRP content during endotoxemia in rats.

The aim of the present study was to determine whether the transient receptor potential vanilloid (TRPV1) receptor protein as well as the calcitonin gene-related peptide (CGRP) content could be enhanced after the i.p. administration of 5 mg/kg lipopolysaccharide (LPS) to Sprague-Dawley rats. In tongue tissue, used as a representative model of TRPV1 receptors expression, there was a significant increase in the abundance of TRPV1 receptor protein 6 h after LPS administration. In mesenteric arteries, the density of the CGRP-positive nerves as well as the release of CGRP induced by 10 microM anandamide was also significantly increased 6 h after LPS administration. The relaxant responses induced by anandamide in mesenteric beds isolated from either untreated or LPS-treated rats were abolished after a 2 h exposure to 10 microM capsaicin. Moreover, anandamide-induced relaxations of untreated mesenteries were potentiated by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 0.1 microM), but not by its inactive analogue 4alpha-phorbol (0.1 microM). The potentiation of anandamide effects caused by the PKC activator was accompanied by a significant increase in the overflow of CGRP induced by anandamide in the untreated rats. It is proposed that the overexpression of the TRPV1 receptors and the increased content of CGRP could contribute to the enhancement of anandamide effects during the endotoxemic shock. An eventual phosphorylation event linked to the overflow of CGRP could also participate in the enhanced relaxation caused by anandamide in endotoxemia.

Participation of CGRP and prostanoids in the sex-linked differences of vascular anandamide effects in mesenteric beds of Sprague-Dawley rats.

The in vitro exposure to anandamide elicits greater relaxations in mesenteric beds isolated from female compared to male rats. The present work shows that in mesenteric beds precontracted with noradrenaline the removal of endothelium increased the relaxation caused by anandamide in male and ovariectomized female but not in sham-operated female rats. The nitric oxide synthase inhibition with 100 microM N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) and the sensory in vivo denervation through neonatal administration of capsaicin also reduced anandamide-induced relaxations but these effects had the same extent in male and in female mesenteries. The content of calcitonin gene related peptide (CGRP) in mesenteric beds, that was higher in intact female than in male rats, was reduced by ovariectomy and restored to control values 21 days after a 3 weekly i.m. administration of 450 microg/kg 17beta-oestradiol. This latter treatment also increased CGRP content in mesenteries from males up to the same levels observed in females. The basal release of CGRP in mesenteric beds was equivalent in either sex, but the exposure to anandamide increased CGRP release solely in female mesenteries. The ratio prostacyclin/thromboxane A(2) was selectively reduced in mesenteries from male rats after exposure to anandamide, due to the decrease of the tissue levels of prostacyclin. Moreover, the cyclooxygenase-2 inhibitor 0.1 microM N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulphonamide (NS-398) diminished the relaxations caused by anandamide solely in female rats. It is proposed that relaxing factors such as CGRP and prostacyclin contribute to the higher relaxations caused by anandamide in the vasculature of female rats.

Sex-linked differences in the vasorelaxant effects of anandamide in vascular mesenteric beds: role of oestrogens.

Anandamide (0.01 to 10 microM) caused greater concentration-dependent reductions of the contractile-induced responses to noradrenaline in female than in male mesenteric vascular beds isolated from adult Sprague-Dawley rats. Greater relaxant responses in females were also induced by the vanilloid TRPV1 receptor agonist capsaicin (0.01 to 10 microM), whereas no sex differences were observed for the relaxations caused by either acetylcholine or sodium nitroprusside. The effect of anandamide in either sex was reduced by the vanilloid TRPV1 receptor antagonist capsazepine but not by the cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A). In males, the anandamide-induced relaxations were potentiated by in vitro exposure during 5 min to 0.5 microM 17beta-oestradiol and unmodified by the protein synthesis inhibitor cycloheximide. The vasorelaxant effects of anandamide in female rats were decreased by ovariectomy. This decrease was prevented by in vivo treatment with 17beta-oestradiol-3-benzoate (450 microg/kg i.m., once a week during 3 weeks) and counteracted by in vitro exposure to oestrogen. In vivo treatment with 17beta-oestradiol also potentiated anandamide-induced responses in males. In conclusion, this study shows an oestrogen-dependent sensitivity to the vanilloid TRPV1 receptor-mediated vasorelaxant effects of anandamide in the mesenteric vasculature of Sprague-Dawley rats, that could be mediated by both genomic and non-genomic mechanisms.