Phosphodiesterase (PDE) 4 inhibitors are a class of drugs that can provide novel therapies for asthma and chronic obstructive pulmonary disease. Their development is frequently hampered by the induction of vascular toxicity in rat mesenteric tissue during preclinical studies. Whereas these vascular lesions in rats have been well characterized histologically, little is known about their pathogenesis and in turn, sensitive and specific biomarkers for preclinical and clinical monitoring do not exist. In order to investigate the early molecular mechanisms underlying vascular injury, time-course studies were performed by treating rats for 2-24 h with high doses of the PDE4 inhibitor CI-1044. Transcriptomics analyses in mesenteric tissue were performed using oligonucleotide microarray and real-time RT-PCR technologies and compared to histopathological observations. In addition, protein measurements were performed in serum samples to identify soluble biomarkers of vascular injury. Our results indicate that molecular alterations preceded the histological observations of inflammatory and necrotic lesions in mesenteric arteries. Some gene expression changes suggest that the development of the lesions could follow a primary modulation of the vascular tone in response to the pharmacological effect of the compound. Activation of genes coding for pro- and antioxidant enzymes, cytokines, adhesion molecules, and tissue inhibitor of metalloproteinase 1 (TIMP-1) indicates that biomechanical stimuli may contribute to vascular oxidant stress, inflammation, and tissue remodeling. TIMP-1 appeared to be an early and sensitive predictive biomarker of the inflammatory and the tissue remodeling components of PDE4 inhibitor-induced vascular injury.

The cytotoxicity profile of various chemical entities was evaluated using two in vitro hepatocyte models. Liverbeads is a cryopreserved model consisting of primary hepatocytes entrapped in alginate beads. WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma (Fao) and human fibroblasts (WI38). Various reference hepatotoxicants were tested and ranked according to their equivalent concentration 50 (EC50) for various biochemical endpoints (lactate dehydrogenase (LDH) release, 3-(4,5 dimethylthiazol 2yl)-2,5-diphenyl-2H tetrazolium bromure (MTT) activity, adenosine triphosphate (ATP) and glutathione (GSH) levels). The ranking obtained was comparable in both models and consistent with previously published results on hepatocyte monolayers. Ketoconazole, erythromycin estolate, retinoic acid, telithromycin and alpha-naphthyl-isothiocyanate were among the most toxic chemicals in both models, with an EC50 < 200 microM. Troleandomycin, spiramycin, erythromycin, diclofenac, taurodeoxycholate, warfarin, galactosamine, valproic acid and isoniazid were found to be less toxic. Few marked differences, potentially linked to metabolism pathways, were observed between EC50s in the two models for compounds such as cyclosporine A (10 and > 831 microM) and warfarin (5904 and 1489 microM) in WIF-B9 and Liverbeads, respectively. The results obtained indicate that Liverbeads and WIF-B9 cells are reliable in vitro models to evaluate the hepatotoxic potential of a wide range of chemicals, irrespective of structure and pharmaceutical class.

Background information. WIF‐B9 is a hybrid cell line obtained by fusion of rat hepatoma cells (Fao) and human fibroblasts (WI38). It exhibits the structural and functional characteristics of differentiated hepatocytes, including active bile canaliculi. The aim of the present study was to characterize the WIF‐B9 cell line as a model for analysing drug‐induced hepatic effects. The drug metabolism potential of WIF‐B9 cells was identified by studying the rat and human CYP (cytochrome P450) mRNA constitutive expression profile and induction potential after exposure to reference inducers. The morphological alterations provoked by chemical entities were also characterized.