Chemosensitivity of three patient-derived primary cultures of canine pericardial mesothelioma by single-agent and combination treatment.

Introduction: Canine mesothelioma is a rare malignant tumor that mostly affects body cavities, such as the pericardial and pleural cavities. Chemotherapy plays a crucial role in the treatment of canine mesotheliomas. We aimed to compare the antitumor effects of single-agent and combination chemotherapeutic agents on patient-derived primary cultures of canine pericardial mesothelioma established in this study. We planned to generate xenograft models for future studies. Material and methods: Effusion samples were collected from three dogs with histologically diagnosed pericardial mesothelioma and used for primary culture. Cultured cells were characterized by immunostaining for pan-cytokeratin AE1/AE3, vimentin, Wilms' tumor suppressor gene 1 (WT1), and cytokeratin 5 (CK5). To assess the tumorigenic properties of cells in the effusion and generate a xenograft model, the cell suspension was injected into a severe combined immunodeficient (SCID) mouse either subcutaneously (SC) or intraperitoneally (IP). Lastly, chemosensitivity of established primary cultures against four drugs, doxorubicin, vinorelbine, carboplatin, and gemcitabine, by single-agent treatment as well as combination treatment of carboplatin at a fixed concentration, either 10 or 100 µM, and gemcitabine at different concentrations ranging from 0-1000 µM was assessed by cell viability assay. Results: Primary cultures were successfully generated and characterized by dual positivity for AE1/AE3 and vimentin and positive staining for WT-1 and CK5, confirming the mesothelial origin of the cells. In the xenograft models, SC mouse developed a subcutaneous mass, whereas IP mouse developed multiple intraperitoneal nodules. The masses were histopathologically consistent with mesotheliomas. The chemosensitivity assay revealed that carboplatin had the highest anti-tumor effects among the four tested single-agent treatments. Furthermore, carboplatin at 100 µM combined with gemcitabine at clinically relevant doses demonstrated the augmented anti-tumor effects compared to single-agent treatment. Discussion and conclusion: Primary cultures and xenograft models generated in this study could be useful tools for in vitro and in vivo studies of canine mesothelioma. Carboplatin is a highly effective chemotherapeutic agent against canine mesothelioma when used as a sole agent and in combination with gemcitabine.

Cyanoacrylate closure for arteriovenous fistula in the lower extremity with saphenous vein insufficiency.

A lower extremity arteriovenous fistula (AVF) is sometimes associated with venous disease following venous hypertension, especially when the saphenous vein is the main return route. This can cause venous dilation, leading to valve insufficiency. A complete cure can be difficult in cases with multiple vascular branches. We report three surgical cases of lower extremity AVF with saphenous vein insufficiency. All patients had saphenous vein insufficiency with long duration leg symptoms and underwent full-length occlusion of saphenous vein using cyanoacrylate closure. Substantial improvements in leg symptoms and appearance were observed immediately after surgery in all three patients. Cyanoacrylate closure could be a treatment option for lower extremity AVF.

Derivation of a new model of lung adenocarcinoma using canine lung cancer organoids for translational research in pulmonary medicine.

Canine primary lung cancer (cPLC) is a rare malignant tumor in dogs, and exhibits poor prognosis. Effective therapeutic drugs against cPLC have not been established yet. Also, cPLC resembles human lung cancer in histopathological characteristics and gene expression profiles and thus could be an important research model for this disease. Three-dimensional organoid culture is known to recapitulate the tissue dynamics in vivo. We, therefore, tried to generate cPLC organoids (cPLCO) for analyzing the profiles of cPLC. After samples from cPLC and the corresponding normal lung tissue were collected, cPLCO were successfully generated, which recapitulated the tissue architecture of cPLC, expressed lung adenocarcinoma marker (TTF1), and exhibited tumorigenesis in vivo. The sensitivity of cPLCO to anti-cancer drugs was different among strains. RNA-sequencing analysis showed significantly upregulated 11 genes in cPLCO compared with canine normal lung organoids (cNLO). Moreover, cPLCO were enriched with the MEK-signaling pathway compared with cNLO. The MEK inhibitor, trametinib decreased the viability of several strains of cPLCO and inhibited the growth of cPLC xenografts. Collectively, our established cPLCO model might be a useful tool for identifying novel biomarkers for cPLC and a new research model for dog and human lung cancer.

Establishment of an experimental model of canine malignant mesothelioma organoid culture using a three-dimensional culture method.

Canine malignant mesothelioma (cMM) is a rare and drug-resistant malignant tumor. Due to few patients and experimental models, there have not been enough studies to demonstrate the pathogenesis of the disease and novel effective treatment for cMM. Since cMM resembles human MM (hMM) in histopathological characteristics, it is also considered a promising research model of hMM. Compared with conventional 2-dimensional (2D) culture methods, 3-dimensional (3D) organoid culture can recapitulate the properties of original tumor tissues. However, cMM organoids have never been developed. In the present study, we for the first time generated cMM organoids using the pleural effusion samples. Organoids from individual MM dogs were successfully generated. They exhibited the characteristics of MM and expressed mesothelial cell markers, such as WT-1 and mesothelin. The sensitivity to anti-cancer drugs was different in each strain of cMM organoids. RNA sequencing analysis showed cell adhesion molecule pathways were specifically upregulated in cMM organoids compared with their corresponding 2D cultured cells. Among these genes, the expression level of E-cadherin was drastically higher in the organoids than that in the 2D cells. In conclusion, our established cMM organoids might become a new experimental tool to provide new insights into canine and human MM therapy.

Effect of edaravone against cisplatin-induced chronic renal injury.

Cisplatin has been widely used as an anticancer agent for a wide range of tumors, but it had nephrotoxicity that was mainly caused by oxidative stress. Edaravone, a free radical scavenger, has reportedly been validated to have a protective effect against renal injury induced by reactive oxygen species. However, most of these reports are against AKI, and few studies have examined the effect of chronic renal injury. In this study, we investigate the effect of edaravone on cisplatin nephropathy in the chronic phase. Twenty-five male Wistar rats were divided into five groups: control, cisplatin, cisplatin + edaravone 1 mg kg-1, cisplatin + edaravone 10 mg kg-1, and cisplatin + edaravone 100 mg kg-1. Edaravone was administrated intraperitoneally every other day for 5 weeks, starting 1 week before cisplatin administration (6 mg kg-1, i.p.). As a result, proximal tubule injury, interstitial fibrosis, and mononuclear cell infiltration were ameliorated histologically in the group of rats treated with high edaravone dose. In the cisplatin group, the number of α-SMA-, CD68-, and CD3-positive cells increased markedly compared with the Control group, but these numbers were significantly decreased by higher doses of co-administered edaravone. While there was no clear mRNA expression variation in antioxidant enzymes, the apoptosis-promoting factors, caspase8, were markedly reduced in the high-dose edaravone co-administration group compared with the cisplatin group. In conclusion, our results suggested that cisplatin-induced renal injury in the chronic phase was ameliorated by edaravone.

Pericardial Mesothelioma in a Dog: The Feasibility of Ultrasonography in Monitoring Tumor Progression.

A 6-year-old neutered male Yorkshire Terrier presented with recurrent pericardial effusion. Although clinical examinations including computed tomography were inconclusive, an exploratory thoracotomy revealed multiple small nodules and plaques on the inner surface of the pericardial sac (Day 1). A subtotal pericardiectomy was performed to prevent cardiac tamponade due to the increasing pericardial effusion, and the resected section of the pericardium was histopathologically diagnosed with mesothelioma. After surgery, chemotherapy with intrathoracic carboplatin was commenced. During the course of the treatment, a detailed follow-up ultrasonographic scan was performed to detect early lesions disseminated on the pleura, originating from the primary pericardial mesothelioma. On Day 101, the minute pleural nodules, which were disseminated lesions as predicted, were successfully imaged by ultrasonography. As the clinical stage advanced, the nodules were observed to gradually increase in size and number, implying tumor progression. These observations highlight the feasibility of ultrasonography in detecting minute disseminated lesions at an early stage, monitoring tumor progression, and thereby, predicting the prognosis of canine pericardial mesothelioma.

Cloning of carrier cells infected with oncolytic adenovirus driven by midkine promoter and biosafety studies.

BACKGROUND: A549 carrier cells infected with oncolytic adenovirus can induce complete tumor reduction of subcutaneous ovarian tumors but not intraperitoneal disseminated ovarian tumors. This appears to be a result of the insufficient antitumor effect of A549 carrier cells. Therefore, in the present study, we cloned a novel carrier cell with the aim of improving the antitumor effects. METHODS: Carrier cells infected with oncolytic adenovirus AdE3-midkine with a midkine promoter were cloned by limiting dilution. We examined the antitumor effects of these cells on subcutaneous and intraperitoneal OVHM ovarian tumors in a syngeneic mouse model. Biosafety tests were conducted in beagle dogs and rabbits. RESULTS: We cloned EHMK-51-35 carrier cells with 10-fold higher antitumor effects compared to A549 carrier cells in vitro. EHMK-51-35 carrier cells co-infected with AdE3-midkine and Ad-mGM-CSF induced a 100% complete tumor reduction in subcutaneous tumors and a 60% reduction of intraperitoneal disseminated tumors. Single-dose acute toxicity test on beagle dogs with EHMK-51-35 carrier cells co-infected with AdE3-midkine and Ad-cGM-CSF showed no serious side effects. Biologically active adenoviruses were not detected in the blood, saliva, feces, urine or whole organs. In a chronic toxicity test, VX2 tumors in rabbits were injected five times with EHMK-51-35 carrier cells infected with AdE3-midkine and these rabbits showed no serious side effects. CONCLUSIONS: Significant antitumor effects and safety of cloned EHMK-51-35 carrier cells were confirmed in intraperitoneal ovarian tumors and toxicity tests, respectively. These findings will be extended to preclinical efficacy studies using dogs and cats, with the aim of conducting human clinical trials on refractory solid tumors.

Inhibitory effect of propolis on the development of AA amyloidosis.

In the several types of amyloidoses, participation of oxidative stresses in the pathogenesis and the effect of antioxidants on amyloidosis have been reported. Meanwhile, the relationship between oxidative stresses and pathogenesis of amyloid A (AA) amyloidosis is still unclear. In this study, we used an antioxidant, Brazilian propolis, to investigate the inhibitory effects on AA amyloidosis. The results showed that AA deposition was inhibited by administration of propolis. Increased expression of antioxidant markers was detected in molecular biological examinations of mice treated with propolis. Although serum amyloid A (SAA) levels were strongly correlated with the immunoreactive area of AA deposits in the control group, the correlation was weaker in the propolis-treated groups. In addition, there were no changes in SAA levels between the control group and the propolis-treated groups. The results indicate that propolis, an antioxidant, may induce inhibitory effects against AA amyloidosis.

Spontaneous, Experimentally Induced, and Transmissible AA Amyloidosis in Japanese Quail ( Coturnix japonica).

The authors describe a spontaneous case of amyloid A (AA) amyloidosis in an adult female Japanese quail ( Coturnix japonica). The bird developed AA amyloidosis secondary to chronic peritonitis caused by a Gram-negative bacillus infection. Mild amyloid deposition was also identified in the intestinal tract of apparently healthy adult individuals, suggesting that quail may develop intestinal amyloidosis with age. Based on these observations, it was hypothesized that quail can develop AA amyloidosis following inflammatory stimulation with lipopolysaccharide (LPS). Therefore, adult quail were repeatedly injected with LPS and the development of AA amyloidosis was confirmed. The amyloid deposition in this model increased when quail amyloid was intravenously injected as an amyloid-enhancing factor. The experiments were repeated with young quail, but amyloid deposits were not observed following LPS injections. However, AA amyloidosis did develop when quail amyloid was injected in addition to LPS. These results indicated that adult quail develop AA amyloidosis after inflammatory stimulation with LPS. Furthermore, quail AA amyloidosis was shown to have transmissibility regardless of age. Interestingly, the authors found that administration of chicken amyloid fibrils also induced AA amyloidosis in young quail. This is the first report of cross-species transmission of avian AA amyloidosis.

Studies on the potential risk of amyloidosis from exposure to silk fibroin.

Amyloid A (AA) amyloidosis can be induced by the administration of amyloid fibrils to animals under inflammatory conditions. Silk fibroin (SF) is a main component protein of bombic silk and has amyloid-like features. The amyloidogenesis of SF solution in mice has been previously reported. Recently, the biochemical properties of silk have attracted increasing attention, and research and development have been undertaken regarding applications other than as a clothing material. However, the risk of AA amyloidosis from exposure to SF-related products is unknown. In this study, we examined the amyloidogenesis of several SF-related products that vary in preparation method or route of injection in a mouse model of amyloidosis. The results revealed that amyloid deposits were rarely observed in mice exposed to SF solution or feed supplemented with SF powder. On the other hand, heavy amyloid deposits were observed in some mice implanted with SF non-woven fabric by abdominal operation. Congo red staining of SF solutions under polarized light and electron microscopy indicated that SF solution in this study had no amyloid-like structures. We found that SF-related products occasionally promote amyloidogenesis, but have a low potential for amyloidosis.

Amyloidosis enhancing activity of bovine amyloid A fibrils in C3H/HeN mice and cynomolgus monkeys (Macaca fascicularis).

BACKGROUND: In experimentally induced cases of AA amyloidosis, the development of disease is enhanced by the administration of homogenous or heterogeneous amyloid fibrils. In recent years, cross-species transmission of animal amyloidosis into human has become of particular concern. METHODS: Cynomolgus monkeys (Macaca fascicularis) and C3H/HeN mice were inoculated with bovine amyloid fibrils under acute inflammation. RESULTS: Amyloid A deposits were not detected in any of the monkeys, but mild-to-severe AA deposits were found in all mice. CONCLUSIONS: These results suggest that unlike in rodents, cross-species transmission of AA amyloidosis is less likely to develop, at least during acute inflammation, in primates.

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model.

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-ß/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of Ki-67(+) proliferating cells within GST-P(+) foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P(+) foci (area) to 48.9-49.4% and Ki-67(+) cells to 55.5-61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN(-) foci, phospho-Akt substrate(+) foci, phospho-Smad3(-) foci and Smad4(-) foci in GST-P(+) foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN(-) foci in GST-P(+) foci to 59.8-72.2% and Smad4(-) foci to 62.4-71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate(+) foci in GST-P(+) foci to 75.2-75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-ß/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.

Immunohistochemical characterization of multicentric hepatocholangiocellular adenoma in a pig.

Three spherical opaque-white tumor nodules were found in close proximity to each other in the liver of a breeding sow, postslaughter, at a veterinary food inspection. The tumor nodules were circumscribed and histologically consisted of discrete hepatocellular and cholangiocellular nests, in association with polygonal-to-oval-shaped cells with slight cellular atypia. Immunohistochemically, all cellular components were negative for carcinoembryonic antigen, but positive for p53. Both cholangiocytes and oval-shaped cells were immunoreactive to anti-cytokeratin antibodies AE1/AE3 and MNF116. In addition, cholangiocytes were exclusively immunoreactive to anti-cytokeratin antibody CAM5.2, and hepatocytes were positive for MNF116 and hepatocyte paraffin 1. All neoplastic cells were positive for the hepatic progenitor cell markers, α-1-fetoprotein, sal-like protein 4, and epithelial cell adhesion molecule. From these results, the present case was diagnosed as hepatocholangiocellular adenoma, arising from epithelial cells of the canals of Hering.

Modifying effects of liver tumor promotion in rats subjected to co-administration of indole-3-carbinol and phenobarbital.

Indole-3-carbinol (I3C) and phenobarbital (PB) are cytochrome P450 (CYP) 1A and CYP2B inducers, respectively, and have liver tumor-promoting effects in rats. In this study, we investigated the modifying effects on tumor promotion by I3C and PB co-administration. Six-week-old male F344 rats received a single intraperitoneal injection of N-diethylnitrosamine for initiation treatment. Two weeks after the initiation, rats were given no tumor-promoting agents (DEN alone), I3C (2,500 or 5,000 ppm in diet), PB (60 or 120 ppm in drinking water), or 2,500 ppm I3C + 60 ppm PB for 6 weeks. One week after the I3C/PB treatments, all animals underwent a two-thirds partial hepatectomy. The number and area of liver cell foci positive for glutathione S-transferase placental form (GST-P(+) foci) were not significantly fluctuated in the PB+I3C group in the isoadditive statistical model. On the contrary, the mRNA levels of Cyp2b1/2 and Nqo1 were suppressed and enhanced, respectively, in the PB+I3C group in the isoadditive model, but there was no enhancement in the microsomal reactive oxygen species (ROS) production, thiobarbituric acid-reactive substance levels, and Ki-67(+) cell ratio in this group. The results suggest that the co-administration of I3C and PB causes no modifying effects in liver tumor promotion in rats.

Maternal single injection of N-methyl-N-nitrosourea to cause microcephaly in offspring induces transient aberration of hippocampal neurogenesis in mice.

N-Methyl-N-nitrosourea (MNU) is an alkylating agent having antiproliferative cytotoxity targeting the neural stem/progenitor cells to cause microcephaly by maternal exposure. This study investigated the effect of transient exposure to MNU on the process of hippocampal neurogenesis in later life using mice. Pregnant mice received a single injection of MNU at 0, 5 and 10 mg/kg body weight, intraperitoneally on gestational day 14, and their offspring were examined on postnatal day (PND) 21 and PND 77. On PND 21, offspring displayed microcephaly and hippocampal formation hypoplasia at 10 mg/kg, decrease of doublecortin (Dcx)(+) cells in the dentate subgranular zone from 5mg/kg, and decrease of TUNEL(+) apoptotic cells and increase of transcript expression of anti-apoptotic Bcl-2 at 10 mg/kg in the dentate gyrus. In the dentate hilus, numbers of reelin(+) or parvalbumin (Pvalb)(+) interneurons or neuron-specific nuclear protein(+) neurons increased at 10 mg/kg. Microcephaly and hippocampal formation hypoplasia continued through PND 77 at 10 mg/kg. Thus, apart from the massive cell killing at the migratory stream causing microcephaly, MNU may decrease Dcx(+) cells reflecting disruption of the differentiation process of late-stage neuronal progenitors and immature granule cells through defective molecular functions by gene mutations. Increase of reelin(+) and Pvalb(+) cells may reflect the disruption of neurogenesis and following neuronal migration. All of the granule cell lineage and interneuron changes disappeared at the adult stage on PND 77 suggesting that MNU mainly targets transient populations of highly proliferative progenitor cells but hardly affects their stem cells having self-renewal ability.

Suppressive effect of liver tumor-promoting activities in rats subjected to combined administration of phenobarbital and piperonyl butoxide.

Phenobarbital (PB) is a cytochrome P450 (CYP) 2B inducer, and piperonyl butoxide (PBO) is a CYP1A/2B inducer. These inducers have liver tumor-promoting effects in rats. In this study, we performed a rat two-stage liver carcinogenesis bioassay to examine the tumor-promoting effect of PB and PBO co-administration. Male rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) for initiation. Two weeks after DEN administration, rats were given PB (60 or 120 ppm in drinking water), PBO (1,250 or 2,500 ppm in diet) or 60 ppm PB+1,250 ppm PBO for 6 weeks. One week after the PB/PBO treatment, all rats were subjected to a two-thirds partial hepatectomy. To evaluate the effect of the combined administration, we used two statistical additive models. In the isoadditive model, the average values of the area of GST-P positive foci in the PB+PBO group were significantly lower than those in the High PB or High PBO groups. In the heteroadditive model, the net values of Cyp1a1 mRNA level and microsomal reactive oxygen species (ROS) production in the PB+PBO group were significantly lower than the sum of those in the Low PB or Low PBO groups. On the contrary, there was no interactive effect in the PCNA-positive hepatocyte ratio, mRNA levels of Cyp2b1/2, Gstm3, Gpx2 and Nqo1, and the level of thiobarbituric acid-reactive substances in the PB+PBO group. These results suggest that PB and PBO co-administration causes suppressive effects in liver tumor-promoting activity in rats resulting from inhibited microsomal ROS production because of suppression of CYP1A induction.

Potentials and limitations of nonclinical safety assessment for predicting clinical adverse drug reactions: correlation analysis of 142 approved drugs in Japan.

The objective of this study was to elucidate the range of abilities of nonclinical safety assessment for predicting adverse drug reactions (ADRs) in humans. The dataset included 1256 ADRs with an incidence rate of 5% or more collected from 142 drugs approved in Japan from 2001 to 2010 (excluding anticancer agents and vaccines). Gastrointestinal, neurological and hepatobiliary ADRs were relatively common, followed by hematological, cutaneous, systemic and cardiovascular ADRs in the dataset. The analysis revealed that 48% of ADRs were predictable based on a comprehensive nonclinical safety assessment considering animal toxicity. Hematological and ocular ADRs, infection, and application site reactions showed a correlation of more than 70%, while musculoskeletal, respiratory and neurological ADRs showed a correlation of less than 30%. In addition to subjective patient perceptions, several laboratory parameters routinely monitored both in animals and humans showed a lower correlation, e.g., abnormalities in hepatobiliary and metabolic parameters, and blood pressure increase. Large molecule drugs showed lower correlation than small molecule drugs; ADRs were observed in various organs and consideration of pharmacological action did not significantly contribute to the prediction. It was also confirmed that the current standard of toxicology testing regarding dosing duration and dose level is adequate to detect concordant animal toxicity. This study collectively demonstrated a significant value of nonclinical safety assessment in predicting ADRs in humans. It also identified the subset of ADRs with poor predictability, highlighting the need for advanced testing that enables successful translation of animal toxicity to clinical settings with better accuracy and sensitivity.

Liver tumor promoting effect of orphenadrine in rats and its possible mechanism of action including CAR activation and oxidative stress.

Orphenadrine (ORPH), an anticholinergic agent, is a cytochrome P450 (CYP) 2B inducer. CYP2B inducers are known to have liver tumor-promoting effects in rats. In this study, we performed a rat two-stage liver carcinogenesis bioassay to examine the tumor-promoting effect of ORPH and to clarify its possible mechanism of action. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) as an initiation treatment. Two weeks after DEN administration, rats were fed a diet containing ORPH (0, 750, or 1,500 ppm) for 6 weeks. One week after the ORPH-administration rats were subjected to two-thirds partial hepatectomy for the acceleration of hepatocellular proliferation. The number and area of glutathione S-transferase placental form-positive foci significantly increased in the DEN-ORPH groups. Real-time RT-PCR revealed increased mRNA expression levels of Cyp2b1/2, Mrp2 and Cyclin D1 in the DEN-ORPH groups and of Gpx2 and Gstm3 in the DEN-High ORPH group. Microsomal reactive oxygen species (ROS) production and oxidative stress markers such as thiobarbituric acid-reactive substances and 8-hydroxydeoxyguanosine were increased in the DEN-High ORPH group. Immunohistochemically, constitutively active/androstane receptor (CAR) were clearly localized in the nuclei of hepatocytes in the DEN-ORPH groups. These results suggest that ORPH causes nuclear translocation of CAR resulting in the induction of the liver tumor-promoting activity. Furthermore, oxidative stress resulting from ROS production is also involved in the liver tumor-promoting activity of ORPH.

Enhanced liver tumor promotion activity in rats subjected to combined administration of phenobarbital and orphenadrine.

Phenobarbital (PB) and orphenadrine (ORPH) are cytochrome P450 (CYP) 2B inducers and have liver tumor-promoting effects in rats. In this study, we performed a rat two-stage liver carcinogenesis bioassay to examine the tumor-promoting effect of PB and ORPH co-administration. Twelve male rats per group were given an intraperitoneal injection of N-diethylnitrosamine (DEN) for initiation. Two-week after DEN administration, rats were given PB (60 or 120 ppm in drinking water), ORPH (750 or 1,500 ppm in diet) or 60 ppm PB+750 ppm ORPH for 6-week. One-week after the PB/ORPH treatment, all rats were subjected to two-thirds partial hepatectomy. To evaluate the effect of the combined administration, we used two statistical models: a heteroadditive model and an isoadditive model. In the heteroadditive model, the net values of the number and area of glutathione S-transferase placental form (GST-P) positive foci, Cyp2b1/2, Gstm3 and Gpx2 mRNA levels, microsomal reactive oxygen species (ROS) production and thiobarbituric acid-reactive substances level in the PB+ORPH group were significantly higher than the sum of the net values of those in the Low PB and Low ORPH groups. In the isoadditive model, the average values of the area of GST-P positive foci and PCNA positive hepatocyte ratio and Gstm3 mRNA level in the PB+ORPH group were significantly higher than the average values of those in the High PB and High ORPH groups. These results suggest that PB and ORPH co-administration causes synergistic effects in liver tumor-promoting activity in rats resulting from oxidative stress due to enhanced microsomal ROS production.

Involvement of multiple cell cycle aberrations in early preneoplastic liver cell lesions by tumor promotion with thioacetamide in a two-stage rat hepatocarcinogenesis model.

Thioacetamide (TAA) induces oxidative stress and hepatocarcinogenicity in rats. We previously reported that TAA promotion caused various disruptions in cell cycle protein expression in rats, including downregulation of p16(Ink4a), which is associated with intraexonic hypermethylation in hepatocellular proliferative lesions. This study further investigated the contribution of cell cycle aberrations associated with early hepatocarcinogenic processes induced by TAA using antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), in a two-stage rat hepatocarcinogenesis model. TAA-promotion after initiation with N-diethylnitrosamine increased the number and area of hepatocellular foci immunoreactive for glutathione S-transferase placental form (GST-P) and the numbers of proliferating and apoptotic cells. Co-treatment with EMIQ and ALA suppressed these increases. TAA-induced formation of p16(Ink4a-) foci in concordance with GST-P(+) foci was not suppressed by co-treatment with EMIQ or ALA. TAA-promotion increased cellular distributions of cell proliferation marker Ki-67, G2/M and spindle checkpoint proteins (phosphorylated checkpoint kinase 1 and Mad2), the DNA damage-related protein phosphorylated histone H2AX, and G2-M phase-related proteins (topoisomerase IIα, phosphorylated histone H3 and Cdc2) within GST-P(+) foci, and co-treatment with EMIQ or ALA suppressed these increases. These results suggest that downregulation of p16(Ink4a) may allow selective proliferation of preneoplastic cells by TAA promotion. However, antioxidants did not counteract this gene control. Moreover, effective suppression of TAA-induced cellular population changes within preneoplastic lesions by antioxidants may reflect facilitation of cell cycling and accumulation of DNA damage causing the activation of cell cycle checkpoints, leading to G2 and M phase arrest at the early stages of hepatocarcinogenesis promoted by TAA.