PBTK model-based analysis of CYP3A4 induction and the toxicokinetics of the pyrrolizidine alkaloid retrorsine in man

Cytochrome P450 (CYP)3A4 induction by drugs and pesticides plays a critical role in the enhancement of pyrrolizidine alkaloid (PA) toxicity as it leads to increased formation of hepatotoxic dehydro-PA metabolites. Addressing the need for a quantitative analysis of this interaction, we developed a physiologically-based toxicokinetic (PBTK) model. Specifically, the model describes the impact of the well-characterized CYP3A4 inducer rifampicin on the kinetics of retrorsine, which is a prototypic PA and contaminant in herbal teas. Based on consumption data, the kinetics after daily intake of retrorsine were simulated with concomitant rifampicin treatment. Strongest impact on retrorsine kinetics (plasma AUC24 and Cmax reduced to 67% and 74% compared to the rifampicin-free reference) was predicted directly after withdrawal of rifampicin. At this time point, the competitive inhibitory effect of rifampicin stopped, while CYP3A4 induction was still near its maximum. Due to the impacted metabolism kinetics, the cumulative formation of intestinal retrorsine CYP3A4 metabolites increased to 254% (from 10 to 25 nmol), while the cumulative formation of hepatic CYP3A4 metabolites was not affected (57 nmol). Return to baseline PA toxicokinetics was predicted 14 days after stop of a 14-day rifampicin treatment. In conclusion, the PBTK model showed to be a promising tool to assess the dynamic interplay of enzyme induction and toxification pathways

The pyrrolizidine alkaloid senecionine induces CYP-dependent destruction of sinusoidal endothelial cells and cholestasis in mice

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.</p

Evolution of the eukaryotic ARP2/3 activators of the WASP family: WASP, WAVE, WASH, and WHAMM, and the proposed new family members WAWH and WAML

<p>Abstract</p> <p>Background</p> <p>WASP family proteins stimulate the actin-nucleating activity of the ARP2/3 complex. They include members of the well-known WASP and WAVE/Scar proteins, and the recently identified WASH and WHAMM proteins. WASP family proteins contain family specific N-terminal domains followed by proline-rich regions and C-terminal VCA domains that harbour the ARP2/3-activating regions.</p> <p>Results</p> <p>To reveal the evolution of ARP2/3 activation by WASP family proteins we performed a "holistic" analysis by manually assembling and annotating all homologs in most of the eukaryotic genomes available. We have identified two new families: the WAML proteins (WASP and MIM like), which combine the membrane-deforming and actin bundling functions of the IMD domains with the ARP2/3-activating VCA regions, and the WAWH protein (WASP without WH1 domain) that have been identified in amoebae, Apusozoa, and the anole lizard. Surprisingly, with one exception we did not identify any alternative splice forms for WASP family proteins, which is in strong contrast to other actin-binding proteins like Ena/VASP, MIM, or NHS proteins that share domains with WASP proteins.</p> <p>Conclusions</p> <p>Our analysis showed that the last common ancestor of the eukaryotes must have contained a homolog of WASP, WAVE, and WASH. Specific families have subsequently been lost in many taxa like the WASPs in plants, algae, Stramenopiles, and Euglenozoa, and the WASH proteins in fungi. The WHAMM proteins are metazoa specific and have most probably been invented by the Eumetazoa. The diversity of WASP family proteins has strongly been increased by many species- and taxon-specific gene duplications and multimerisations. All data is freely accessible via <url>http://www.cymobase.org</url>.</p

Identification of microRNAs Implicated in Modulating Senecionine-Induced Liver Toxicity in HepaRG Cells

1,2-unsaturated Pyrrolizidine Alkaloids (PAs) are secondary plant metabolites that occur as food contaminants. Upon consumption, they can cause severe liver damage. PAs have been shown to induce apoptosis, to have cytotoxic and genotoxic effects, and to impair bile acid homeostasis in the human hepatoma cell line HepaRG. The major mode of action of PAs is DNA- and protein-adduct formation. Beyond that, nuclear receptor activation has only been observed for one receptor and two PAs, yielding the possibility that other cellular mediators are involved in PA-mediated toxicity. Here, the mode of action of Senecionine (Sc), a prominent and ubiquitous representative of hepatotoxic PAs, was investigated by analyzing 7 hepatic microRNAs (miRNAs) in HepaRG cells. Ultimately, 11 target genes that were predicted with Ingenuity Pathway Analysis software (IPA) were found to be significantly downregulated, while their assigned miRNAs showed significant upregulation of gene expression. According to IPA, these targets are positively correlated with apoptosis and cellular death and are involved in diseases such as hepatocellular carcinoma. Subsequent antagomiR-inhibition analysis revealed a significant correlation between PA-induced miRNA-4434 induction and P21-Activated Kinase-1 (PAK1) downregulation. PAK1 downregulation is usually associated with cell cycle arrest, suggesting a new function of Sc-mediated toxicity in human liver cells