Surfactant proteins (SPs) are essential for the proper structure and respiratory function of the lungs. There are four subtypes of SPs: SP-A, SP-B, SP-C, and SP-D. The expectorant drug ambroxol hydrochloride is clinically used to stimulate pulmonary surfactant and airway serous secretion. In addition, previous studies showed that ambroxol regulated SP production and attenuated pulmonary inflammation, with ambroxol hydrochloride being found to suppress quartz-induced lung inflammation via stimulation of pulmonary surfactant and airway serous secretion. In this study, we investigated the expression of SP-A, SP-B, SP-C, and SP-D in neoplastic and inflammatory lung lesions in rodents, as well as their possible application as potential markers for diagnostic purposes. SP-B and SP-C showed strong expression in lung hyperplasia and adenoma, whereas SP-A and SP-D were expressed in the mucus or exudates of inflammatory alveoli. Rodent tumorigenic hyperplasic tissues induced by various carcinogens were positive for napsin A, an aspartic proteinase involved in the maturation of SP-B; this indicated a focal increase in type II pneumocytes in the lungs. Therefore, high expression of napsin A in the alveolar walls may serve as a useful marker for prediction of the tumorigenic potential of lung hyperplasia in rodents.

In research to develop healthy foods or preventive medicines from edible and medicinal herbs in Okinawa, we focused on the antioxidant activities of those bioresources. We first confirmed that the herbal antioxidant activities of such herbs increased upon ultraviolet irradiation treatment. This observation explains the high antioxidant activity of Okinawan vegetables, which grow under exposure to stronger ultraviolet light compared with those in other prefectures in Japan. Antidiabetic, hepatoprotective, cancer preventive, and cardioprotective actions were clarified using herbal extracts, and quercetin, chlorogenic acid, and gallic acid derivatives were isolated as antioxidant components from the herbs. Dimerumic acid was also isolated from the mold Monascus anka. All these antioxidants showed strong radical scavenging activities in vitro and beneficial effects in animal models. However, the concentrations of these compounds used in vivo seemed to be too low to have a physiologically important antioxidant effect based on their radical scavenging activities in vitro. Therefore, I performed a literature survey of antioxidant activities in vivo. Accumulating evidence has emerged that antioxidant phytochemicals show not only radical scavenging activities in vitro but also pleiotropic actions in vivo. The multitargeted, beneficial effects of antioxidant phytochemicals can be rationally explained using the xenohormesis concept, in which phytochemicals are the products of plant evolutionary adaptation to stress in plants, and their ability to induce a stress-adaptive response has been evolutionarily conserved in animals.

Ambroxol hydrochloride (AH) is an expectorant drug used to stimulate pulmonary surfactant and serous airway secretion. Surfactant proteins (SPs) are essential for maintaining respiratory structure and function, although SP expression has also been reported in lung inflammatory and proliferative lesions. To determine whether AH exerts modulatory effects on these lung lesions, we examined its effects on pleural thickening induced by intrathoracic administration of dipotassium titanate (TISMO) in A/JJmsSlc (A/J) mice. We also analyzed the modulatory effects of AH on neoplastic lung lesions induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice and by N-nitrosobis (2-hydroxypropyl) amine (DHPN) in F344/DuCrlCrj (F344) rats. A/J mice treated with TISMO showed decreased body weight, increased white blood cell (WBC) counts, and pleural thickening caused by pleuritis and poor general condition. However, A/J mice treated with TISMO + 120 ppm showed significant recovery of body weight and WBC counts to the same levels as those of A/J mice not treated with TISMO, although no significant differences were observed in histopathological changes including the immunohistopathological expression of IL-1β in the lung and maximum pleural thickness regardless of AH treatment. In the NNK and DHPN experiments, no significant differences in body weight, hematology, plasma biochemistry, and histopathological changes were associated with AH concentration. These results suggest that AH potentially exerts anti-inflammatory effects but does not have a direct suppressive effect on lung tumorigenesis in rodents.

It is well known that fasting substantially affects the metabolism of drugs and chemicals. Food restriction also affects drug kinetics, such as absorption, metabolism, and excretion, and therefore, it can potentially modulate the onset of chemical toxicity or drug-induced adverse reactions. In the present study, the expression of drug-metabolizing enzyme genes and total glutathione content in the liver, which are related to toxicity induced by overdose of the hepatotoxic drug acetaminophen (N-acetyl-p-aminophenol; APAP), were examined in rats reared under different feeding conditions: ad libitum feeding, 16-h fasting, and food restriction (fed 70% of the average intake of ad libitum feeding for 10 days) conditions. The rats under food restriction conditions as well as fasted rats showed significantly higher expression of Cyp2e1, the gene encoding the enzyme that metabolizes APAP to its toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). They also had lower levels of liver total glutathione, which detoxifies NAPQI. In contrast, the gene expression of UDP-glucuronosyltransferase 1A6 (Ugt1a6), sulfotransferase 1A1 (Sult1a1), and glutathione S-transferase M1 (Gstm1) was not affected by food restriction or fasting. When APAP was administered (800 mg/kg), histopathological changes were not observed in rats fed ad libitum, while hepatocellular necrosis was observed in most of the rats treated with APAP after fasting or food restriction. Taken together, these results suggest that not only fasting but also food restriction exacerbate APAP-induced acute liver injury, probably by the induction of CYP2E1 and the reduction of liver glutathione contents, in rodents.

Propolis is a resin-like material produced by honey bees from bud exudates and sap of plants and their own secretions. An ethanol extract of Brazilian green propolis (EEBGP) contains prenylated phenylpropanoids and flavonoids and has antioxidative and anti-inflammatory effects. Acetaminophen (N-acetyl-p-aminophenol; APAP) is a typical hepatotoxic drug, and APAP-treated rats are widely used as a model of drug-induced liver injury. Oxidative stress and inflammatory reactions cause APAP-induced hepatocellular necrosis and are also related to expansion of the lesion. In the present study, we investigated the preventive effects of EEBGP on APAP-induced hepatocellular necrosis in rats and the protective mechanism including the expression of antioxidative enzyme genes and inflammation-related genes. A histological analysis revealed that administration 0.3% EEBGP in the diet for seven days reduced centrilobular hepatocellular necrosis with inflammatory cell infiltration induced by oral administration of APAP (800 mg/kg) and significantly reduced the area of necrosis. EEBGP administration did not significantly change the mRNA expression levels of antioxidant enzyme genes in the liver of APAP-treated rats but decreased the mRNA expression of cytokines including Il10 and Il1b, with a significant difference in Il10 expression. In addition, the decrease in the mRNA levels of the Il1b and Il10 genes significantly correlated with the decrease in the percentage of hepatocellular necrosis. These findings suggest that EEBGP could suppress APAP-induced hepatocellular necrosis by modulating cytokine expression.

Metabolic diseases including nonalcoholic steatohepatitis develop due to various environmental factors. In particular, the westernization of food is closely related to the development of these diseases. In this study, we investigated pathophysiological changes in the livers of Zucker fatty (ZF) rats induced by feeding Western diets. Male ZF rats were fed a sucrose/fat/cholesterol-enriched diet (Western diet, WD) or standard diet (SD) for 18 weeks, from 7 to 25 weeks of age. Body weight, food intake, and biochemical parameters were periodically measured, histopathological analyses were performed at 25 weeks, and mRNA expression in the liver was determined. ZF rats fed the WD (ZF-WD rats) developed obesity, hyperinsulinemia, hyperglycemia, and hyperlipidemia, and their alanine aminotransferase and aspartate aminotransferase levels increased compared with those of ZF rats fed the SD (ZF-SD rats). Hepatic lesions including fibrosis and necrosis were observed in the ZF-WD rats at 25 weeks; however, fibrosis and necrosis were not observed in the ZF-SD rats. Oxidative stress markers also increased in the livers of ZF-WD rats. Hepatic mRNA expression related to inflammation and fibrosis increased in the ZF-WD rats; however, mRNA expression related to lipid synthesis decreased. Microsomal triglyceride transfer protein mRNA levels in the ZF-WD rats also decreased. In Zucker lean rats fed the WD, similar changes were observed in the liver; however, the hepatic changes were not serious compared with ZF-WD rats. In conclusion, hepatic lesions, such as inflammation, fibrosis, and necrosis, were observed in the ZF-WD rats. The sucrose/fat/cholesterol-enriched diet induced significant lipotoxicity in the livers of animals in this insulin-resistant model.

In xenograft models, orthotopic (ORT) engraftment is thought to provide a different tumor microenvironment compared with subcutaneous (SC) engraftment. We attempted to characterize the biological difference between OE19 (adenocarcinoma of the gastroesophageal junction) SC and ORT models by pathological analysis and CASTIN (CAncer-STromal INteractome) analysis, which is a novel method developed to analyze the tumor-stroma interactome framework. In SC models, SCID mice were inoculated subcutaneously with OE19 cells, and tumor tissues were sampled at 3 weeks. In ORT models, SCID mice were inoculated under the serosal membrane of the stomach wall, and tumor tissues were sampled at 3 and 6 weeks after engraftment. Results from the two models were then compared. Histopathologically, the SC tumors were well circumscribed from the adjacent tissue, with scant stroma and the formation of large ductal structures. In contrast, the ORT tumors were less circumscribed, with small ductal structures invading into abundant stroma. Then we compared the transcriptome profiles of human tumor cells with the mouse stromal cells of each model by species-specific RNA sequencing. With CASTIN analysis, we successfully identified several interactions that are known to affect the tumor microenvironment as being selectively enhanced in the ORT model. In conclusion, pathological analysis and CASTIN analysis revealed that ORT models of OE19 cells have a more invasive character and enhanced interaction with stromal cells compared with SC models.

In neonatal rats, glutamate could induce retinal thinning depending on the development stage, and the severity peaked at treatment on postnatal day (PND) 8. To elucidate the molecular mechanism of retinal thinning induced by L-glutamate in neonatal rats, we investigated the time-course gene expression profile in the developing retina in addition to initial histopathological changes. Histopathologically, apoptotic cells in the inner retina were observed at 6 hours after treatment on PNDs 4, 6 and 8, and inflammatory cell infiltration was noted at 24 hours. Comprehensive gene expression analysis conducted on PNDs 4 and 8 indicated that cell death/proliferation- and inflammation-related genes were upregulated and that neuron development- and neurotransmitter-related genes were downregulated. Furthermore, quantitative RT-PCR analysis of apoptosis- and inflammation-related genes performed on PNDs 4, 6, 8, 10 and 12 showed that the time-course changes of the gene expression ratios of Gadd45b and Ccl3 seemed to be related to histopathological changes of the retina induced by L-glutamate. These results revealed that the association of initial histopathological changes with the gene expression profile in the retina induced by L-glutamate and that Gadd45b and Ccl3 are considered to participate in retinal thinning induced by L-glutamate in neonatal rats.

Cardiotoxicity is a concern in the development of microtubule-disassembling agents (MDAs) as vascular-disrupting agents of tumors. This study investigated cardiotoxicity in rats induced by a single-dose of combretastatin A4 disodium phosphate (CA4DP), an MDA and discussed the use of this rat model in nonclinical studies of MDAs. First, CA4DP (120 mg/kg) was administered to rats intravenously, and cardiac histopathology and blood biomarkers were examined after 0.5, 24, and 72 h. Next, CA4DP (120 mg/kg) was administered to rats intravenously, and the electrocardiography and echocardiography results were analyzed. The results showed that at 0.5 h after dosing, plasma creatine kinase (CK), CK-muscle/brain (CK-MB), and fatty acid binding protein 3 levels increased. At 24 h, lactate dehydrogenase (LDH)-1, CK, and CK-MB levels increased, and multifocal vacuolar degeneration of myocardial cells was observed in the apical inner layer. At 72 h, LDH-1 levels were increased, and multifocal myocardial necrosis was observed in the interventricular septum and inner layer of the apex of left ventricular wall. Furthermore, at 0.5 h, heart rate (HR), ejection fraction (EF), and cardiac output (CO) decreased. At 24 h, CO decreased. Finally, at 72 h, HR, EF, and CO decreased, and depression of the T-wave amplitude was observed. In conclusion, myocardial injury, bradycardia, and depressed cardiac function were induced in rats by a single-dose of CA4DP. The lesion distribution and electrocardiographic features suggested that myocardial injury was induced by ischemia. These findings are similar to MDA-induced cardiotoxicity in humans, and this rat model will prove useful in studies of the cardiotoxicity in humans.

The demand for board certified toxicologic pathologists and expansion of IT capabilities has resulted in an increase of “virtual” and “distance” pathology (telepathology), and a need for greater flexibility in real time consultations. Newly developed microscope-integrated telepathology systems enable geographically remote stakeholders to view the live histopathology slide as seen by the microscope user. Simultaneous online viewing, annotation, and dialog between pathologists and study experts facilitates good science and economic benefits by enabling more timely and informed clinical decisions.