Stent assisted coil embolization for a dissecting cerebral aneurysm of middle cerebral artery: A case report and the literature review.

Background: Dissecting aneurysms of the middle cerebral artery (MCA) are very rare. We herein report a case of an unruptured dissecting aneurysm of the MCA treated by stent-assisted coil embolization. Case Description: A 65-year-old man with no history of trauma presented with a headache. Time-of-flight imaging revealed a dissecting cerebral aneurysm in the right M1 segment of the MCA, and the aneurysm had increased in size within a short time. We treated the aneurysm by endovascular stenting with coils, and the patient developed no neurological deficits. Conclusion: Because of the potential involvement of the lenticulostriate artery (LSA) in the area of dissection, choosing the best treatment (such as direct surgery or endovascular treatment) may be challenging. Treatment efficacy depends on whether the LSA is affected and on the length of the dissection. In our case, the dissection did not involve the LSA and could therefore be treated by stent-assisted coil embolization.

An aggressive dural arteriovenous fistula manifested by unilateral subcortical calcification and cerebral edema: A case report.

Unilateral subcortical calcifications are unique radiographic findings indicating specific focal pathologies. When the lesion is accompanied by edema, cerebral neoplasm usually leads to a differential diagnosis. This report presents a case of unilateral subcortical calcification and edema that resulted in cerebral hemorrhage and a subsequent diagnosis of an aggressive dural arteriovenous fistula. A man in his 60s presented with left hemianopsia and a progressive headache for over 6 months. Initial computed tomography revealed unilateral subcortical calcification and cerebral edema in the right occipital lobe, raising the suspicion of oligodendroglioma. However, 10 days later, a cerebral hemorrhage occurred in the lesion. Magnetic resonance imaging revealed flow void clusters and dilatation of the bilateral external carotid arteries and cortical veins, indicating a dural arteriovenous fistula. Cerebral angiography confirmed the presence of a parasagittal dural arteriovenous fistula (Borden type III). The patient was successfully treated with trans-arterial embolization using Onyx. Thus, calcifications with edema are more commonly associated with cerebral neoplasms; however, in this case, they indicated the presence of a dural arteriovenous fistula with severe corticovenous reflux. The presented case highlights the importance of recognizing these imaging features in dural arteriovenous fistulas and raises awareness of the potential danger of early hemorrhage after diagnosis. Therefore, timely evaluation of cranial vessels is essential in cases of unilateral subcortical calcification and edema to facilitate the early detection and management of aggressive dural arteriovenous fistulas.

The impact of hormonal dynamics and serum sodium fluctuations on symptomatic vasospasm after subarachnoid hemorrhage.

BACKGROUND: Symptomatic vasospasm (SVS) is a major cause of morbidity and mortality in aneurysmal subarachnoid hemorrhage (SAH), and serum sodium frequently decreases before SVS. Serum sodium changes might be regulated by sodium metabolism-related hormones. This multi-institutional prospective cohort study therefore investigated the measurement of sodium metabolism-related hormones to elucidate the pathophysiology of serum sodium changes in SAH. METHODS: SAH patients were treated with clipping or coiling from September 2017 to August 2020 at five hospitals. The laboratory data of 133 SAH patients were collected over 14 days and correlations between changes in serum sodium, sodium metabolism-related hormones (plasma adrenocorticotropic hormone (ACTH), serum cortisol, plasma arginine vasopressin (AVP)), and SVS were determined. Serum sodium concentrations were measured every day and serum sodium levels >135 mEq/L were maintained until day 14. RESULTS: Of the 133 patients, 18 developed SVS within 14 days of subarachnoid hemorrhage onset (SVS group) and 115 did not suffer from SVS (non-SVS group). Circulating AVP, ACTH, and cortisol concentrations were significantly higher on day 1 in the SVS group compared with the non-SVS group. Fluctuations in serum sodium in the SVS group were significantly higher than those in the non-SVS group. There were antiparallel fluctuations in serum sodium and potassium from days 2 to 14. CONCLUSIONS: Elevated levels of ACTH/cortisol and AVP on day 1 may be predictive markers for the occurrence of SVS. Multiple logistic regression analysis showed that serum sodium fluctuations were associated with SVS occurrence. Serum sodium fluctuations were associated with stress-related hormonal dynamics. (249 words).

Endovascular Internal Trapping by Low-Concentration N-butyl-2-Cyanoacrylate for a Ruptured Giant Common Carotid Artery Pseudoaneurysm.

Treating carotid blowout syndrome following rupture of giant pseudoaneurysms is difficult because the destroyed parent artery precludes conventional treatment. We present a patient with a ruptured giant pseudoaneurysm that we occluded using a modified internal trapping technique with low-concentration N-butyl-2-cyanoacrylate (NBCA) and a minimum number of coils. An 80-year-old man with a history of chemoradiation therapy for oropharyngeal cancer presented with several episodes of active bleeding from the subsequent tracheostomy site. Radiological examination revealed a giant right common carotid artery (CCA) pseudoaneurysm. Endovascular internal trapping was performed using both NBCA and coils under proximal flow control. We slowly injected 9 ml of low-concentration NBCA, which subsequently filled the entire pseudoaneurysm. We then injected an additional 2 ml of NBCA into the proximal CCA to achieve complete obliteration. No re-bleeding was observed during the 6-month follow-up. Endovascular internal trapping using low-concentration NBCA was feasible to treat a giant CCA pseudoaneurysm. The injected low-concentration NBCA filled the entire pseudoaneurysm without the risk of catheter entrapment.

[Two Cases of Bilateral Orbital Metastases of Breast Cancer].

Two cases of breast cancer with bilateral orbital metastases associated with intracranial metastases are presented. Case 1:A 61-year-old woman who was diagnosed with breast cancer 14 years earlier presented with rapid deterioration of visual acuity, eye pain, and limitation of left-sided extraocular motility. Magnetic resonance(MR)images showed an enhanced lesion in the left orbital apex, ethmoid sinus, and right middle fossa. The first gamma knife radiotherapy(35 Gy, 5 Fr)was performed successfully, but was followed by recurrence 18 months later in the right intraorbital, where newly formed iso-intensity masses in the extraconal compartment were found. The second gamma knife radiosurgery was performed for three masses(20 Gy). Case 2:A 35-year-old woman with breast cancer who was diagnosed 22 months earlier was treated for meningeal carcinomatosis by whole-brain radiation(30 Gy, 10 Fr)and intrathecal chemotherapy. Eight months later, swelling in both eyelids and limitation of extraocular motility developed rapidly. MR imaging revealed an infiltrating lesion in the cone with heterogenous signal that was encasing, but not infiltrating the optic nerves. The extraconal lesion extended into the soft tissue of the lower eye lid. She expired one week after diagnosis. With the increasing number of long-term survivors with breast cancer, intraorbital metastases may be found during the course of treatment for intracranial lesions. Understanding the unique clinical presentation and characteristic MR findings of this rare entity are emphasized.

Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFß signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.

Supratentorial extraventricular WHO grade III (anaplastic) ependymoma 17 years after total removal of WHO grade II ependymoma of the fourth ventricle.

We report a WHO grade III ependymoma of the supratentorial interhemispheric fissure and grew to form a large mass with anaplastic transformation without local recurrence 17 years after the total removal of a fourth ventricular WHO grade II ependymoma. We emphasize the necessity of long-term follow-up, even in benign ependymomas.

Diagnostic yield and morbidity by neuronavigation-guided frameless stereotactic biopsy using magnetic resonance imaging and by frame-based computed tomography-guided stereotactic biopsy.

BACKGROUND: We compared the diagnostic yield and morbidity by frame-based computed tomography-guided stereotactic biopsy (CTSTB) with Brown-Roberts-Wells (BRW) unit and by neuronavigation-guided frameless stereotactic biopsy (NSTB) using magnetic resonance imaging (MRI). METHODS: The subjects' age range was 15-83 years. CTSTB with BRW unit was performed for 59 tumors (58 cases, 1988-2007). NSTB was performed for 38 tumors (35 cases, 2007-2013) with the needle sheath attached to the head holder. By NSTB, target locations of sampling points and trajectories were confirmed by using MRI. Diffusion tensor imaging-based fiber tractography was used to achieve safe trajectories. STB by using BRW did not visualize the trajectory virtually; however, the planning images for NSTB were able to show the trajectory virtually before the procedure. RESULTS: Histological diagnoses were established for 93 tumors at the first biopsy. The diagnostic yield was 94.9% by CTSTB and 97.4% by NSTB (P = 0.944). The morbidity rate was 5.1% by CTSTB and 0% by NSTB (P = 0.417). The absolute risk reduction was 23.1% by NSTB when the targets were basal ganglia (putamen, globus pallidus) or thalamus. In the cases of glioma for which the targets were basal ganglia (putamen, globus pallidus) or thalamus, the absolute risk reduction by NSTB was 30%. CONCLUSIONS: There was no significant difference between CTSTB and NSTB concerning the diagnostic yield and morbidity. However, when the target is the basal ganglia (putamen, globus pallidus) or thalamus and glioma is suspected, NSTB by using MRI with virtual trajectory is preferable to CTSTB concerning morbidity.

Synthesis of rare earth doped yttrium-vanadate nanoparticles encapsulated within apoferritin.

Luminescent europium (Eu) and dysprosium (Dy) doped yttrium-vanadate (Y-V) nanoparticles (NPs) were synthesized in the cavity of the protein, apoferritin. Y-V NPs were synthesized by incubating a solution of apoferritin with Y(3+) and VO3(-) ions in the presence of ethylene diamine-N-N'-diacetic acid (EDDA). EDDA plays an important role in preventing Y-vanadate precipitation in bulk solution by chelating the Y(3+) ions. Using high resolution electron microscopy, the obtained NPs in the apoferritin cavities were confirmed to be amorphous, and to consist of Y and V. Eu-doped Y-V (Y-V:Eu) NPs were synthesized by the same procedure as Y-V NPs, except that Eu(NO3)3 was added. Y-V:Eu NPs exhibited a strong absorption peak due to the O-V charge transfer transition and remarkable luminescence at 618 nm due to the (5)D0 → (7)F2 transition. The luminescence lifetime of Y:Eu and Y-V:Eu NPs measured in H2O and D2O solution showed reduction of non-radiative transition to the O-H vibration in Y-V:Eu NPs. Accordingly, Y-V NPs showed strong luminescence compared to Y:Eu NPs. Dy-doped Y-V NPs were also synthesized in apoferritin cavities and showed luminescence peaks at 482 nm and 572 nm, corresponding to (4)F9/2 → (6)H15/2 and (4)F9/2 → (6)H13/2 transitions. These NPs stably dispersed in water solution since their aggregation was prevented by the protein shell. NPs encapsulated in the protein are likely to be biocompatible and would have significant potential for biological imaging applications.

Evaluation of axillary lymph nodes by diffusion-weighted MRI using ultrasmall superparamagnetic iron oxide in patients with breast cancer: initial clinical experience.

PURPOSE: To investigate the diagnostic performance and clinical feasibility of diffusion-weighted magnetic resonance imaging (MRI) using ultrasmall superparamagnetic iron oxide (USPIO) in the evaluation of axillary lymph nodes (ALNs) in patients with breast cancer. MATERIALS AND METHODS: Sixteen patients with known breast cancer underwent 1.5 T MRI. Axial diffusion-weighted images (DWIs) and conventional T1- and T2*-weighted images (CIs) were acquired before and 24-36 hours after intravenous administration of USPIO. Detection of ALNs was evaluated on DWIs in comparison with CIs. The apparent diffusion coefficient values (ADCvs) of the nonmetastatic and metastatic nodes in precontrast DWIs were determined. The diagnostic performance of DWI using USPIO was compared with that of CIs using USPIO with pathological correlation. RESULTS: Out of a total of 286 ALNs, 216/286 (76%) nodes were detected on DWIs and 238/286 (83%) on CIs. The differences in the ADCvs between metastatic and nonmetastatic nodes were not significant (P = 0.06). Sensitivity of CIs and DWIs using USPIO were respectively 70% and 83%, specificity 98% and 98%, and overall accuracy 93% and 95%. CONCLUSION: Although the detection on DWIs of ALNs in patients with breast cancer was inferior compared to CIs, the sensitivity and accuracy of DWIs using USPIO were superior in the diagnosis of ALNs metastasis.

Rapid deposition of glomerular IgA in BALB/c mice by nivalenol and its modifying effect on high IgA strain (HIGA) mice.

To clarify the underlying mechanisms of IgA nephropathy (IgAN) induced by nivalenol (NIV), a trichothecene mycotoxin, we examined the time and dose relationships of glomerular deposition of IgA by NIV in BALB/c mice (Experiment 1), and also evaluated the modification of NIV on spontaneous IgAN in an inbred murine model, a high IgA strain (HIGA), during its early stage of pathogenesis (Experiment 2). In Experiment 1, female BALB/c mice were given a diet containing 0, 12, or 24 ppm concentration of NIV for 4 or 8 weeks. An increase in serum IgA levels was found at 24 ppm from 4 weeks. At week 8 of treatment, dose-dependent increases in serum IgA levels and glomerular deposition of IgA and IgG were observed without accompanying histopathological glomerular changes. On the other hand, in Experiment 2, control HIGA mice exhibited rather high levels of serum IgA as compared with BALB/c mice from 4 weeks of experiment as well as glomerular deposition of IgA and IgG and mesangial proliferation as revealed at week 8. NIV at 24ppm further increased serum IgA in this strain; however, it did not enhance glomerular immunoglobulin deposition or histopathological lesion. These results suggest that NIV-induced increase of serum IgA levels may be primarily responsible for glomerular immunoglobulin deposition; however, NIV does not enhance glomerular IgA deposition that may lead to exacerbation of predisposed IgAN in the short term, irrespective of the further elevation of serum IgA from the high basal levels.

Immunohistochemical analyses at the late stage of tumor promotion by oxfendazole in a rat hepatocarcinogenesis model.

The present study was performed to characterize immunohistochemically the expression levels of molecules related to not only xenobiotic and antioxidant functions but also cell proliferation and apoptosis in neoplastic lesions induced by the benzimidazole anthelmintic, oxfendazole (OX), at the late stage of its tumor promotion in a rat hepatocarcinogenesis model. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and 2 weeks later they were fed a diet containing 0% (basal diet) or 0.05% OX for 26 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and killed at week 28. Histopathologically, OX increased the incidence and multiplicity of altered foci (4.0- and 3.6-fold, respectively) and hepatocellular adenomas (HCAs) (3.0- and 5.5-fold, respectively). OX treatment induced 5.2- and 5.6-fold increases in the number of proliferating cell nuclear antigen (PCNA)-positive cells and single-stranded DNA (ssDNA)-positive cells in HCAs compared with the surrounding tissue, respectively. Staining for the cell cycle regulators P21 and C/EBPα and the AhR-regulated CYP1A1 molecules decreased but increased reactivity of the Nrf2-regulated, detoxifing/antioxidant molecules aldo-keto reductase 7 (AKR7) and glutathione peroxidase 2 (GPX2) were also seen in HCAs compared with the surrounding hepatocytes. These results suggest that dysregulation of cell proliferation and apoptosis and escape from oxidative stress elicited by OX treatment play an important role in OX-induced hepatocarcinogenesis in rats.

Chemical category approach of genotoxicity studies for branched alkylphenols.

A chemical category is a group of chemicals whose toxicological properties are expected to be similar or follow a regular pattern as a result of structural similarity. The category approach is beneficial for decreasing in the resource of risk assessment for huge amount of unevaluated existing chemicals, and also in the use of all kinds of animal tests including even in vivo genotoxicity tests from a point of view of the animal welfare. The present paper reports the results of in vivo micronucleus tests of o-sec-butylphenol (CAS:89-72-5) and 2-isopropyl-5-methylphenol (CAS:89-83-8) and discusses genotoxic potential of seven alkylphenols, o-sec-butylphenol, 2-isopropyl-5-methylphenol, p-sec-butylphenol (CAS:99-71-8), 2-tert-butylphenol (CAS:88-18-6), 2, 4-di-tert-butylphenol (CAS:96-76-4), 4-tert-butylphenol (CAS:98-54-4) and 6-tert-butyl-m-cresole (CAS:88-60-8) by the category approach. Based on the negative results of in vivo micronucleus tests, it can be concluded that these category chemicals are not likely clastogenic in vivo. Further in vivo micronucleus assays on untested substances may not be required by using the category approach, but further supporting information such as physicochemical profiles and (Q) SAR predictions may be necessary to strengthen the rationale for the category approach.

The threshold dose for liver tumor promoting effects of dicyclanil in ICR mice.

To determine the threshold dose of dicyclanil (DC) that induces hepatocellular tumor-promoting effects associated with reactive oxygen species (ROS) generation via their metabolic pathways, partial hepatectomized ICR male mice were fed diets containing 0, 187.5, 375 or 750 ppm DC after an intraperitoneal injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Immunohistochemically, the proliferating cell nuclear antigen (PCNA)-positive cell ratio was significantly increased in the DEN + 750 ppm DC group compared with the DEN alone group. However, significant increases in the number of gamma-glutamyltranspeptidase (GGT)-positive cells and formation of microsomal ROS were not observed in the DEN + DC groups compared with the DEN alone group. Real-time polymerase chain reaction (RT-PCR) showed that the expression of Cyp1a1, Cyp1a2, and OGG1genes was significantly up-regulated in mice given diets containing 375 ppm DC or more, 187.5 ppm DC or more, and 750 ppm DC, respectively. These results suggest that the threshold dose of DC that induces ROS-mediated liver tumor promotion in mice is more than 750 ppm, although expression of the Cyp1a2 gene, which is related to ROS generation, was up-regulated in the liver of mice, even at a DC dose of 187.5 ppm.

Elevation of cell proliferation via generation of reactive oxygen species by piperonyl butoxide contributes to its liver tumor-promoting effects in mice.

Piperonyl butoxide (PBO) is a pesticide synergist used with pyrethroids as a domestic insecticide, and it acts as a non-genotoxic hepatocarcinogen in rats and mice. To clarify whether oxidative stress is involved in the liver tumor-promoting effect of PBO in mice, male mice were subjected to two-thirds partial hepatectomy, followed by N-diethylnitrosamine (DEN) treatment, and given a diet containing 0.6% PBO for 25 weeks. The incidences of cytokeratin (CK) 8/18-positive foci, adenomas, and carcinomas significantly increased in the DEN + PBO group compared with the DEN-alone group. The PCNA-positive ratio significantly increased in non-tumor hepatocytes, CK8/18-positive foci and adenomas in the DEN + PBO group compared with the DEN-alone group. PBO increased reactive oxygen species (ROS) production in microsomes but did not change oxidative DNA damage as assessed by 8-hydroxydeoxyguanosine (8-OHdG). In real-time RT-PCR, PBO upregulated the expression of genes related to metabolism, such as Cytochrome P450 1a1, 2a5, and 2b10, and metabolic stress, such as Por and Nqo1, but downregulated Egfr and Ogg1. PBO also increased early response genes downstream of mitogen-activated protein kinase (MAPK), such as c-Myc that is induced by excessive ROS production, and G1/S transition-related genes, such as E2f1 and Ccnd1. Thus, PBO can generate ROS via the metabolic pathway without any induction of oxidative DNA damage, activate cell growth, increase c-Myc- and E2F1-related pathways, and act as a liver tumor promoter of DEN-induced hepatocarcinogenesis in mice.

Antioxidant enzymatically modified isoquercitrin or melatonin supplementation reduces oxidative stress-mediated hepatocellular tumor promotion of oxfendazole in rats.

To clarify whether enzymatically modified isoquercitrin (EMIQ) or melatonin (MLT) supplementation reduces oxidative stress-mediated hepatocellular tumor-promoting effect of oxfendazole (OX), a benzimidazole anthelmintic, male rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing OX (500 ppm) for 10 weeks with or without EMIQ (2,000 ppm) or MLT (100 ppm) in the drinking water after DEN initiation. One week after the commencement of the administration of OX, rats were subjected to two-thirds of partial hepatectomy. The number of GST-P-positive foci promoted by OX was significantly inhibited by the combined antioxidant EMIQ or MLT administration, and the area of GST-P-positive foci was inhibited by the administration of MLT. Real-time RT-PCR analysis revealed decreases in mRNA expression levels of cytochrome P450, family 2, subfamily b, polypeptide 2 (Cyp2b2) and malic enzyme 1 (Me1) in the DEN-OX-EMIQ and DEN-OX-MLT groups and decreases in mRNA expression levels of Cyp1a1 and aldo-keto reductase family 7, member A3 (Akr7a3) in the DEN-OX-MLT group compared to those in the DEN-OX group. In in vitro ROS production assay, inhibited production of NADPH-dependent ROS was observed by the treatment with EMIQ or MLT. These results suggest that coadministration of EMIQ or MLT suppresses the hepatocellular tumor-promoting activity of OX in rats through the decrease in ROS production by the activation of CYPs.

Cytokeratin 8/18 is a useful immunohistochemical marker for hepatocellular proliferative lesions in mice.

In order to clarify whether cytokeratin (CK) 8/18 is a useful immunohistochemical marker for hepatocellular proliferative lesions in mice, partially hepatectomized male ICR mice were given 0.6% piperonyl butoxide (PBO) for 8 (Experiment I) or 25 weeks (Experiment II) after N-diethylnitrosamine (DEN) initiation treatment, and the livers were subjected to histological examinations on hematoxylin and eosin (HE) stained sections, CK8/18 immunohistochemistry and gamma-glutamyl transpeptidase (GGT) histochemistry. In Experiment I, the multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive for CK8/18 was 10.17 and 18.50, respectively, while that of hepatocellular foci in frozen sections which were observed in HE-stained sections and positive/negative for GGT was 6.17 and 8.17, respectively. In Experiment II, the total multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive/negative for CK8/18 was 4.47 and 23.17, respectively, while that of hepatocellular foci in frozen sections which were observed in HE-stained sections and positive/negative for GGT was 2.50 and 3.50, respectively. Most of the hepatocellular adenomas and carcinomas observed in HE-stained sections were positive for CK8/18, but some of the adenomas were negative for CK8/18. These findings indicate that more hepatocellular proliferative lesions can be detected in CK8/18 immunohistochemistry in addition to those observed in HE-stained sections, and suggest that CK8/18 may become a useful immunohistochemical marker for detecting hepatocellular proliferative lesions in mice.

Involvement of glycogen synthase kinase-3beta signaling and aberrant nucleocytoplasmic localization of retinoblastoma protein in tumor promotion in a rat two-stage thyroid carcinogenesis model.

To investigate the cell cycle kinetics during the tumor promotion process induced by hypothyroidism in a rat model of thyroid follicular cell carcinogenesis, immunohistochemical analysis of cell cycle molecules and related signaling molecules was performed in conjunction with analysis of cell proliferation activity in an initiation-promotion model. Male F344 rats were injected with N-bis(2-hydroxypropyl)nitrosamine, and one week later treated with 6-propyl-2-thiouracil (PTU) at 12ppm in the drinking water for 4, 10 or 15 weeks. At each time point, proliferative lesions increased the expression of cyclin A, cyclin D, cyclin E and cyclin-dependent kinase (Cdk)-2, in association with the development of lesion stages from the early focal hyperplasia to the late carcinoma, while a subpopulation of proliferative lesions showed decreased numbers of both cell division cycle-2- and Ki-67-positive cells at week 15 compared with that at week 10, suggesting a reduced promoting effect of serum thyroid-stimulating hormone in the sensitive cellular population after long-term exposure to PTU. On the other hand, increased immunolocalization of phosphorylated and inactive glycogen synthase kinase (GSK)-3beta was observed in a subpopulation of proliferative lesions, in parallel with the cyclins and Cdk2. Nuclear immunoreactivity of phosphorylated and inactive retinoblastoma (Rb) protein was also increased in association with lesion development, with carcinomas showing increased cytoplasmic localization. The results suggest that proliferative lesions activate the cell cycle machinery following tumor promotion via a regulatory mechanism involving inactivation of GSK3beta and Rb protein, the latter signaling mechanism involving its aberrant nucleocytoplasmic transport for the acquisition of a malignant phenotype.

Mechanistic study on hepatocarcinogenesis of piperonyl butoxide in mice.

To clarify the mechanism of piperonyl butoxide (PBO)-induced hepatocarcinogenesis in mice, male mice were subjected to a two-thirds partial hepatectomy, N-diethylnitrosamine (DEN) initiation, and a diet containing 0.6% PBO for eight weeks. The incidence of gamma-glutamyl transpeptidase (GGT)-positive foci and PCNA-positive cells was significantly increased in the DEN + PBO group compared with the DEN-alone group. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis showed up-regulation of genes related to metabolism, such as cytochrome P450 1A1 and 2B10, and metabolic stress, such as Por, Nqo1, Nrf2, abcc3, and abcc4. Early responsive genes downstream of mitogen-activated protein kinase (MAPK), such as c-fos, c-jun, c-myc, and activating transcription factor 3 (ATF3), were also up-regulated in this group. Positive immunohistochemical staining for ATF3 was diffusely observed in nonproliferating hepatocytes of the DEN + PBO group, but altered foci were negative or weakly positive for ATF3. The nuclei of hepatocytes within ATF3-negative foci were positive for cyclin D. Thus PBO can induce oxidative stress, activate the MAPK pathway, and increase ATF3 transcript levels in hepatocytes outside the altered foci during the early stage of PBO-induced hepatocarcinogenesis in mice.