Large scale enzyme based xenobiotic identification for exposomics.

Advances in genomics have revealed many of the genetic underpinnings of human disease, but exposomics methods are currently inadequate to obtain a similar level of understanding of environmental contributions to human disease. Exposomics methods are limited by low abundance of xenobiotic metabolites and lack of authentic standards, which precludes identification using solely mass spectrometry-based criteria. Here, we develop and validate a method for enzymatic generation of xenobiotic metabolites for use with high-resolution mass spectrometry (HRMS) for chemical identification. Generated xenobiotic metabolites were used to confirm identities of respective metabolites in mice and human samples based upon accurate mass, retention time and co-occurrence with related xenobiotic metabolites. The results establish a generally applicable enzyme-based identification (EBI) for mass spectrometry identification of xenobiotic metabolites and could complement existing criteria for chemical identification.

EphB2 receptor tyrosine kinase promotes hepatic fibrogenesis in mice via activation of hepatic stellate cells.

Hepatic fibrosis is the result of an excessive wound-healing response subsequent to chronic liver injury. A feature of liver fibrogenesis is the secretion and deposition of extracellular matrix proteins by activated hepatic stellate cells (HSCs). Here we report that upregulation of EphB2 is a prominent feature of two mouse models of hepatic fibrosis and also observed in humans with liver cirrhosis. EphB2 is upregulated and activated in mouse HSCs following chronic carbon tetrachloride (CCl4) exposure. Moreover, we show that EphB2 deficiency attenuates liver fibrosis and inflammation and this is correlated with an overall reduction in pro-fibrotic markers, inflammatory chemokines and cytokines. In an in vitro system of HSCs activation we observed an impaired proliferation and sub-optimal differentiation into fibrogenic myofibroblasts of HSCs isolated from EphB2-/- mice compared to HSCs isolated from wild type mice. This supports the hypothesis that EphB2 promotes liver fibrosis partly via activation of HSCs. Cellular apoptosis which is generally observed during the regression of liver fibrogenesis was increased in liver specimens of CCl4-treated EphB2-/- mice compared to littermate controls. This data is suggestive of an active repair/regeneration system in the absence of EphB2. Altogether, our data validate this novel pro-fibrotic function of EphB2 receptor tyrosine kinase.

The receptor tyrosine kinase EphB2 promotes hepatic fibrosis in mice.

UNLABELLED: Beyond the well-defined role of the Eph (erythropoietin-producing hepatocellular) receptor tyrosine kinases in developmental processes, cell motility, cell trafficking/adhesion, and cancer, nothing is known about their involvement in liver pathologies. During blood-stage rodent malaria infection we have found that EphB2 transcripts and proteins were up-regulated in the liver, a result likely driven by elevated surface expression on immune cells including macrophages. This was significant for malaria pathogenesis because EphB2(-/-) mice were protected from malaria-induced liver fibrosis despite having a similar liver parasite burden compared with littermate control mice. This protection was correlated with a defect in the inflammatory potential of hepatocytes from EphB2(-/-) mice resulting in a reduction in adhesion molecules, chemokine/chemokine receptor RNA levels, and infiltration of leukocytes including macrophages/Kupffer cells, which mediate liver fibrosis during rodent malaria infections. These observations are recapitulated in the well-established carbon tetrachloride model of liver fibrosis in which EphB2(-/-) carbon tetrachloride-treated mice showed a significant reduction of liver fibrosis compared to carbon tetrachloride-treated littermate mice. Depletion of macrophages by clodronate-liposomes abrogates liver EphB2 messenger RNA and protein up-regulation and fibrosis in malaria-infected mice. CONCLUSION: During rodent malaria, EphB2 expression promotes malaria-associated liver fibrosis; to our knowledge, our data are the first to implicate the EphB family of receptor tyrosine kinases in liver fibrosis or in the pathogenesis of malaria infection.