In addition to the respiratory tract tissues, the following organs were fixed, processed, and histopathologically examined to clarify whether long-term MS inhalation induced any extra-pulmonary carcinogenesis by in this mTOR inhibitor model: both adrenal glands, aorta abdominalis, bone (os femoris with joint), bone marrow (cervical, thoracic,

sternum, lumbar, os femoris), brain (cerebrum, cerebellum, brain stem, hippocampus, paraventricular parts), caecum, diaphragm, ductus thoracicus, both epididymes, eye with optic nerve, gall bladder, gross lesions observed, Harderian glands, heart (left and right ventricle, septum), small intestine (duodenum, jejunum, ileum), large intestine (colon, rectum), both kidneys and ureters, both lacrimal glands (infraorbitalis, extraorbitalis), liver, lymph nodes (axillary, bronchial, cervical, inguinal, mandibular, mediastinal, mesenteric, paraaortic, poplietus), mammary gland, mucosa (mouth), muscle (skeletal), nerve (sciatic), esophagus, olfactory bulb, both ovaries and the mesovarium, Idelalisib concentration pancreas, pituitary, both preputial glands, prostate, salivary glands (submandibular, parotid), both seminal vesicles, skin, spleen, sternum, stomach and forestomach, both testes, thymus, both thyroids (including parathyroids), tissue masses or tumors, tongue (inclusive base), urethra, urinary bladder, uterus (including cervix and

both uterine horns and oviducts), and vagina. Histopathological Montelukast Sodium preparations of respiratory tract organs were performed at Philip Morris Research Laboratories GmbH, Cologne, Germany. The histopathological evaluation was performed by Histovia GmbH, Overath, Germany. All pulmonary proliferative lesions were classified according to international classifications and criteria (Brambilla et al., 2001 and Dungworth

et al., 2001). Histopathological preparations of non-respiratory tract organs were performed by the Laboratory of Pharmacology and Toxicology GmbH & Co KG, Hamburg, Germany. The histopathological examination was performed by Toxicologic Pathology Consultancy (Kiel, Germany). Classification of neoplastic lesions in the various organs except the lungs was performed (according to the criteria defined by Mohr, 2001). All histopathological examinations were performed without knowledge of the treatment groups. Gene expression analysis was performed on lung tumor samples from the MS-300 and sham control groups. Frozen sections (20 μm) from whole lung tissue were placed on sterilized glass slides and stained with cresyl violet. Total tissues from single lung tumors were collected from these slides using laser capture micro-dissection (Zeiss, Oberkochen, Germany) except of one slide, which was preserved for the histopathological characterization of the tumor. The tumor tissues were immediately lysed with Qiazol lysis buffer (Qiagen, Hilden, Germany).