Polarization change of femtosecond laser pulses in air measured by electric-field-induced second-harmonic generation.

We experimentally demonstrated the polarization change of femtosecond laser pulses in air by using electric-field-induced second-harmonic generation (E-FISHG) for the first time to our knowledge. The polarization change from linear to elliptical was observed at the laser intensity over the filamentation threshold. These results suggest that the polarization change can occur by the birefringence caused by filamentation. This phenomenon can be used for new applications such as an ultra-fast and precise three-dimensional electric field measurement by E-FISHG. In addition, E-FISHG can be an excellent tool to investigate the characteristics of femtosecond laser propagation such as filamentation.

A carcinogenicity study of diphenylarsinic acid in C57BL/6J mice in drinking water for 78 weeks.

Diphenylarsinic acid (DPAA), a neurotoxic organic arsenical, is present in groundwater and soil in some regions of Japan owing to illegal dumping. The present study evaluated the potential carcinogenicity of DPAA, including investigating whether bile duct hyperplasia in the liver that was observed in a chronic study on 52 week mouse, develops into a tumor when administered to mice in their drinking water for 78 weeks. DPAA was administered to 4 groups of male and female C57BL/6J mice at concentrations of 0, 6.25, 12.5, and 25 ppm in drinking water for 78 weeks. A significant decrease in the survival rate was found for females in the 25 ppm DPAA group. Body weights of males in the 25 ppm and females in the 12.5 and 25 ppm DPAA groups were significantly lower than those of the controls. Histopathological evaluation of neoplasms in all tissues showed no significant increase in tumor incidence in any organ or tissue of 6.25, 12.5, or 25 ppm DPAA-treated male or female mice. In conclusion, the present study demonstrated that DPAA is not carcinogenic to male or female C57BL/6J mice. Taken together with the fact that the toxic effect of DPAA is predominantly restricted to the central nervous system in humans, and the finding that DPAA was not carcinogenic in a previous 104-week rat carcinogenicity study, our results suggest that DPAA is unlikely to be carcinogenic in humans.

Response biomarkers of inhalation exposure to cigarette smoke in the mouse lung.

Cigarette smoking is known to increase the risk of cancer and chronic obstructive pulmonary disease (COPD). In this study, we evaluated the effects of short-term nose-only inhalation exposure to cigarette smoke in mice. Male 10-week-old C57BL mice were exposed to clean air (control) or mainstream cigarette smoke for 1 h/day, 5 days/week, for 2 or 4 weeks. Exposure to cigarette smoke increased the number of inflammatory cells, especially neutrophils, in the bronchoalveolar lavage fluid, increased inflammatory cell infiltration foci, and caused an increase in the thickness of the peripheral bronchial epithelium. Microarray gene expression analysis indicated that smoke exposure induced inflammatory responses, including leukocyte migration and activation of phagocytes and myeloid cells, as early as two weeks after the initiation of exposure. Importantly, chemokine (C-C motif) ligand 17, resistin-like alpha, and lipocalin 2 were upregulated and may serve as useful markers of the toxic effects of exposure to cigarette smoke before pulmonary histological changes become evident.

Electric-field-induced second-harmonic generation using high-intensity femtosecond laser pulses over the observable optical breakdown threshold.

We investigated the performance of electric-field-induced second-harmonic generation (E-FISHG) by spectroscopic measurement using high-intensity femtosecond laser pulses. The second-harmonic intensity increased quadratically versus the applied electric field, as expected from the theory, up to 15 kV/cm with the laser energy up to 2.5 mJ, which is ∼5 times higher than the observable optical breakdown threshold. In addition, when the laser energy was 2.8 mJ, ∼80 times signal intensity at 0.23 mJ was obtained. These results suggest that the electric-field measurement by E-FISHG with high-intensity second harmonics is expected by using high-intensity laser pulses above the observable optical breakdown threshold. Spectroscopic measurement shows no E-FISHG of white light generated by self-phase modulation in laser-induced filament.

Spontaneous adenolipoma of the mammary gland in the male F344 rat.

A 110-week-old male F344 rat from the high-dose group of a 104-week carcinogenicity study, exhibited a spontaneously occurring subcutaneous mass in the left axilla extending to the chest. Histologically, the mass was well-demarcated from the adjacent mammary tissue and slightly encapsulated without evidence of infiltration into the surrounding tissues. The mass contained both epithelial and adipose components. The epithelial component consisted of ductal structures of various sizes lined by a single layer of flattened to cuboidal epithelial cells with relatively clear or vacuolated cytoplasm. These ductal structures were well-intermingled with an adipose component that consisted of a uniform monomorphic cell population of mature adipocytes. Both cell types were well-differentiated and did not exhibit cellular atypia. Within the mass, fibrous connective tissue was found in the stroma with infiltration of numerous mast cells. Based on these findings, the mass was diagnosed as an adenolipoma of the mammary gland.

Dimethylarsinic acid (DMA) enhanced lung carcinogenesis via histone H3K9 modification in a transplacental mouse model.

Pregnant CD-1 mice received 200 ppm dimethylarsinic acid (DMA) in the drinking water from gestation day 8-18, and tumor formation was assessed in offspring at the age of 84 weeks. DMA elevated the incidence of lung adenocarcinoma (10.0%) and total tumors (33.3%) in male offspring compared to male control offspring (1.9 and 15.1%, respectively). DMA also elevated the incidence of hepatocellular carcinoma (10.0%) in male offspring compared to male control offspring (0.0%). DMA and its metabolites were detected in the lungs of transplacental DMA-treated neonatal mice. Transplacental DMA exposure increased cell proliferation in the epithelium in the lungs of both neonatal and 6-week-old male mice. Microarray and real-time PCR analyses detected high expression of keratin 8 (Krt8) in the lungs of both neonatal and 6-week-old DMA-treated mice. Western blot analysis indicated that DMA elevated methylation of histone H3K9, but not H3K27, in the lungs of male mice. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) analysis using an H3K9me3 antibody found differences in heterochromatin formation between mice exposed to DMA and the controls. Notably, ChIP-seq analysis also found regions of lower heterochromatin formation in DMA-treated mice, and one of these regions contained the Krt8 gene, agreeing with the results obtained by microarray analysis. High expression of Krt8 was also detected in adenoma and adenocarcinoma of the lung in male offspring. Overall, these data indicate that transplacental DMA treatment enhanced lung and liver carcinogenesis in male mice. In the lung, DMA caused aberrant methylation of histone H3K9, increased Krt8 expression, and enhanced cell proliferation.

Chronic dietary toxicity and carcinogenicity studies of dammar resin in F344 rats.

Dammar resin is a natural food additive and flavoring substance present in many foods and drinks. The present study evaluates the chronic toxicity and carcinogenicity of dietary dammar resin in F344 rats. Dietary concentrations in the 52-week chronic toxicity study were 0, 0.03, 0.125, 0.5, or 2%. The major treatment-related deleterious effects were body weight suppression, increased relative liver weight, and low hemoglobin levels in males and females. Foci of cellular alteration in the liver were observed in the male 2% group, but not in any other group. The no-observed-adverse-effect level for chronic toxicity was 0.125% for males (200.4 mg/kg b.w./day) and females (241.9 mg/kg b.w./day). Dietary concentrations in the 104-week carcinogenicity study were 0, 0.03, 0.5, or 2%. Dammar resin induced hemorrhagic diathesis in males and females, possibly via the inhibition of extrinsic and intrinsic coagulation pathways. Incidences of hepatocellular adenomas and carcinomas were significantly increased in the male 2% group, but not in any other group. In the 4-week subacute toxicity study, the livers of male rat-fed diet-containing 2% dammar resin had increased levels of protein oxidation and increased the expression of two anti-apoptotic and seven cytochrome P450 (CYP) genes. There was also an increased tendency of oxidative DNA damage. These findings demonstrate that dammar resin is hepatocarcinogenic in male F344 rats and underlines the roles of inhibition of apoptosis, induction of CYP enzymes, and oxidative stress in dammar resin-induced hepatocarcinogenesis.

Progression process and safety assessment adaptation of endometrial lesions in ENU-induced 2-stage uterine carcinogenicity in a Tg-rasH2 mouse model.

Although acotiamide hydrochloride hydrate (acotiamide-HH) has not been reported to have genotoxic findings in any of the genotoxicity studies or treatment-related toxicological findings in reproductive and developmental studies, suspicious uterine tumorigenesis was observed in the results of a long-term rat carcinogenicity study. To clarify the uterine tumorigenesis of acotiamide-HH, we performed a 2-stage uterine carcinogenicity model in the transgenic rasH2 mouse initiated by N-Ethyl-N-nitrosourea (ENU). This model facilitated the short-term detection of uterine carcinogenic potential, and it appears to be a very useful testing method for assessing the safety of chemicals that may affect uterine tumorigenesis. However, there have not been many reports on this model, and accumulation of case studies using this model is recommended to support its usability. In this study, we performed this carcinogenesis model to not only confirm uterine tumorigenesis of acotiamide-HH but also to confirm the reliability of the model. The results of this study revealed that the endometrial adenocarcinoma found in the long-term rat carcinogenicity study possibly arose spontaneously. Also, we confirmed early induction of a uterine tumor as in previous reports and confirmed that 26 weeks is the appropriate treatment period for this rasH2 mouse model according to time-course observations of uterine tumor development.

Accumulation of brown pigment-laden macrophages associated with vascular lesions in the lungs of cynomolgus monkeys(Macaca fascicularis).

Accumulation of macrophages containing brown pigments in the lungs is a well-known spontaneous lesion found in cynomolgus monkey. However, its pathogenesis has not been clearly described. In our survey, brown pigment-laden macrophages were found in the lungs of 4 out of 43 cases. Brown pigments were mostly found in the macrophages of the perivascular interstitium, which proved to be hemosiderin. Some small- to medium-sized vessels that exhibited prominent accumulation of brown pigment-laden macrophages showed degeneration and necrosis of the smooth muscle cells of tunica media. Furthermore, ruptures of the internal and external elastic laminae were seen in some of the vessels. These findings suggested that partial fragmentation of the vascular elastic lamina followed by degeneration and necrosis of the tunica media caused blood leakage leading to the accumulation of hemosiderin-laden macrophages in the perivascular interstitium of the lungs.

Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation.

Nonalcoholic steatohepatitis (NASH) is characterized by combined pathology of steatosis, lobular inflammation, fibrosis, and hepatocellular degeneration, with systemic symptoms of diabetes or hyperlipidemia, all in the absence of alcohol abuse. Given the therapeutic importance and conflicting findings regarding the potential for healing the histopathologic features of NASH in humans, particularly fibrosis, we investigated the reversibility of NASH-related findings in Wistar rats fed a choline-deficient and iron-supplemented l-amino acid-defined (CDAA) diet for 12weeks, with a recovery period of 7weeks, during which the diets were switched to a choline-sufficient and iron-supplemented l-amino acid-defined (CSAA) one. Analysis showed that steatosis and inflammation were significantly resolved by the end of the recovery period, along with decreases in AST and ALT activities within 4weeks. In contrast, fibrosis remained even after the recovery period, to an extent similar to that in continuously CDAA-fed animals. Real-time reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical investigations revealed that expression of some factors indicating oxidative stress (CYP2E1, 4-HNE, and iNOS) were elevated, whereas catalase and SOD1 were decreased, and a hypoxic state and CD34-positive neovascularization were evident even after the recovery period, although the fibrogenesis pathway by activated α-SMA-positive hepatic stellate cells via TGF-ß and TIMPs decreased to the CSAA group level. In conclusion, persistent fibrosis was noted after the recovery period of 7weeks, possibly due to sustained hypoxia and oxidative stress supposedly caused by capillarization. Otherwise, histopathological features of steatosis and inflammation, as well as serum AST and ALT activities, were recovered.

Splenic hemangiosarcoma in a young sprague-dawley rat.

The present report describes a rare case of spontaneous hemangiosarcoma in a nine-week-old male Sprague-Dawley rat. At necropsy, multiple white nodules of various sizes were observed on and within the enlarged spleen and liver and were histopathologically determined to be composed of spindle- to oval-shaped cells that showed invasive growth without encapsulation and were arranged solidly but partially in whorls or faint alveolar patterns with vascular-like spaces containing small clefts or erythrocytes in the tumor mass. Immunohistochemical analysis revealed that most of the tumor cells were strongly positive for vimentin, von Willebrand factor (vWF) and CD34 but negative for podoplanin. In addition, electron microscopic examination revealed the presence of Weibel-Palade bodies in the cytoplasm of the tumor cells. Based on these findings, this case was diagnosed as a hemangiosarcoma. The splenic masses were larger than the hepatic ones, with tumor cells mainly observed at periportal regions with tumor embolism in the liver, suggesting that primary hemangiosarcoma initially developed in the spleen before metastasizing.

Transitional gene expression profiling in ovarian follicle during ovulation in normal-cycle rats.

Evaluation of ovarian toxicity requires an understanding of the physiological changes related to the estrous cycle in the ovary. The authors investigated the transitional gene expression profile of ovulatory follicles in rats that show normal estrous cyclicity. Ovaries were collected at 10:00 and 22:00 on the proestrus day and at 10:00 on the estrus day. Ovarian follicles or early corpora lutea were isolated using laser microdissection, and extracted total RNA was analyzed using microarray technology. Clustering analysis revealed four different expression patterns: transient up- or down-regulation only at 22:00 on the proestrus day (pattern 1), up- or down-regulation only at 10:00 on the estrus day (pattern 2), continuous increase at 22:00 on the proestrus day and at 10:00 on the estrus day (pattern 3), and up- or down-regulation at 22:00 on the proestrus day and level maintenance at 10:00 on the estrus day (pattern 4). In addition, these probe sets were functionally categorized in each pattern using the Ingenuity Pathways Analysis database. These data will aid in understanding the physiology of ovulation and may be useful in assessing ovarian toxicity and its mechanism, such as in investigations of chemical-induced ovulatory impairment.

Abdominal cysticercosis in a cynomolgus monkey.

Cysticercus tenuicollis, the larval form of Taenia hydatigena, was observed in a 5-year-old male cynomolgus monkey used in a toxicity study for a safety assessment of a pharmaceutical. The animal was born and raised in a primate colony in China. A pale yellow cyst filled with more than 100ml of pale yellow fluid was found in the abdominal cavity in the autopsy. The cyst was found attached to the greater omentum, and it was double layered. Histopathologically, the outer layer was a part of the greater omentum, and the inner layer was the bladder wall of a cysticercus with a well developed scolex. A partial sequence of mitochondrial NADH dehydrogenase subunit 1 showed a high homology to the same region of Taenia hydatigena (1.5-3.3%).

A Case of Spontaneous Malignant Hibernoma in a Crl:CD(SD)IGS Rat.

A firm, tan, well-circumscribed mass that measured 25 × 30 × 35 mm was observed in the thoracic cavity of a 53-week-old male Crl:CD(SD) IGS rat. Histologically, the mass was encapsulated by fibrous tissue and contained fibrovascular septae. Tumor cells were compactly arranged, and most were oval to polygonal in shape with multivacuolated cytoplasm and a centrally located nucleus. In some parts of the tumor, marked cellular atypia and frequent mitoses were evident. Vacuoles in cytoplasm were positive for oil red O. The tumor cells were characterized ultrastructurally by abundant, round to oval mitochondria with transverse closely-packed cristae. Tumor cells were immunohistochemically positive for uncoupling protein 1 (UCP-1). Several thrombi and hemorrhagic or necrotic foci were also observed within the tumor mass. Vascular invasion of the tumor capsule was observed; however, invasion of surrounding tissues or metastases were not observed. Based on the pathology findings, this case was diagnosed as a malignant hibernoma.

Verification of a false positive in a two-year rat carcinogenicity study using dual control groups.

There is sometimes controversy over whether or not statistically significant responses produced in carcinogenicity studies have biologically significance. Ambiguous results from our previous two-year oral carcinogenicity study on acotiamide hydrochloride hydrate (acotiamide-HH), a prokinetic drug for functional dyspepsia, in rats made it unclear whether the drug may exhibit uterine carcinogenicity. To check this finding, we performed a second long-term carcinogenicity study using two identical control groups to more accurately evaluate uterine carcinogenesis by considering the incidence of spontaneous neoplasms. Female Fischer 344 rats were divided into three groups: the two control groups (control 1 and 2) were administered vehicle (0.5% w/v methylcellulose) and the acotiamide-HH-treated group was administered 2,000 mg/kg/day of acotiamide-HH by oral gavage for two years. Among all groups, the incidence of endometrial adenocarcinoma (EmA) was highest in the control 2 group, followed by the acotiamide-HH-treated group and the control 1 group. Moreover, acotiamide-HH did not affect the incidence of precursor lesions of EmA. In cases where an ambiguous difference is observed, the use of two control groups allows for a more informed interpretation of the findings in the drug-treated groups. The outcomes in this study strongly support the hypothesis that the increase in EmA in rats treated with acotiamide-HH in our previous study is unrelated to administration of the drug.

The possible mechanism of enhanced carcinogenesis induced by genotoxic carcinogens in rasH2 mice.

Microarray and RT-PCR analyses were performed for the transgene and Ras-related genes in forestomach squamous cell carcinomas (SCCs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rasH2 mice; these results were compared with our previous molecular data of N-ethyl-N-nitrosourea-induced forestomach SCCs and urethane-induced lung adenomas in rasH2 mice. Overexpression of the transgene was detected in the DMBA-induced SCCs, suggesting that the transgene plays an important role in enhanced carcinogenesis in rasH2 mice. In addition, the mouse endogenous ras genes were up-regulated in the DMBA-induced SCCs, and are probably involved in the tumorigenesis of forestomach SCCs. Genes such as osteopontin, Cks1b, Tpm1, Reck, gelsolin, and amphiregulin that were commonly altered in these three different carcinogen-induced tumors may contribute to the development of tumors in rasH2 mice.

Possible involvement of oxidative stress in piperonyl butoxide induced hepatocarcinogenesis in rats.

To clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by piperonyl butoxide (PBO), male F344 rats were administered an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Two weeks later, the rats were administered a PBO-containing (0, 1, or 2%) diet for 6 weeks and subjected to a two-third partial hepatectomy 1 week later. After sacrificing them on week 8, their livers were histopathologically examined and analyzed for gene expression using a microarray and real-time RT-PCR. Reactive oxygen species (ROS) products were also measured using liver microsomes. Hepatocytes exhibited centrilobular hypertrophy and increased glutathione S-transferase placental form (GST-P) positive foci formation. ROS products increased significantly in liver microsomes. In the microarray analysis, the expressions of genes related to metabolism and oxidative stress - NAD(P)H dehydrogenase, quinone 1 (Nqo1), UDP-glucuronosyltransferase (UDPGTR-2), glutathione peroxidase 2 (Gpx2), glutathione reductase (GRx) - multidrug resistance associated protein 3 (Abcc3), and solute carrier family 7 (cationic amino acid transporter, y+ system) member 5 (Slc7a5) were up-regulated in the PBO group in comparison to the 0% PBO group; this was confirmed by real-time RT-PCR. Additionally, a significant up-regulation of stress response related genes such as CYP1A1 was observed in PBO-treated groups in real-time RT-PCR. HPLC analysis revealed that the level of 8-OHdG in the 2% PBO group was significantly higher than that in the 0% PBO group. This suggests that PBO has the potential to generate ROS via metabolic pathways and induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in rats.

Giant electromagnetic vortex and MeV monoenergetic electrons generated by short laser pulses in underdense plasma near quarter critical density region.

Very efficient generation of monoenergetic, about 1MeV , electrons from underdense plasma with its electron density close to the critical, when irradiated by an intense femtosecond laser pulse, is found via two dimensional particle-in-cell simulation. The stimulated Raman scattering of a laser pulse with frequency omega< or =2omega(pl max) gives rise to a giant electromagnetic vortex. In contrast to electron acceleration by the well-known laser pulse wake, injected plasma electrons are accelerated up to vortex ponderomotive potential forming a quite monoenergetic distribution. A relatively high charge of such an electron source makes very efficient generation of soft gamma rays with homega>300 keV .

Focusing quality of a split short laser pulse.

For multiple laser pulse experiments, it is necessary to split a laser pulse. In order to split a short laser pulse without stretching the pulse width, the laser pulse should not pass through thick materials. For this reason, a pellicle beam splitter (BS) and/or a mirror with a hole are required as a BS for the short laser pulse. The focusing qualities of the laser pulse after passing through the pellicle BS and the mirror with a hole are the same as without the BS's. The laser pulse quality reflected by the BSs should be considered for the laser pulse. A pellicle BS is a thin foil, so, it is weak against vibrations. One should be careful about airflows and isolation from vibration sources. The spot size of the reflected laser pulse is consistent with the size reflected by a normal mirror. The energy loss is about 10% compared with a normal mirror. A mirror with a hole is strong against external vibrations. The reflected laser pulse has a doughnut shape. The reflected laser pulse is interfered due to the shape. In order to cleanly focus the laser pulse, the inside size of the doughnut should be smaller than a half size of the outside portion of the doughnut.

Parous mammary glands exhibit distinct alterations in gene expression and proliferation responsiveness to carcinogenic stimuli in Lewis rats.

Early full-term pregnancy affords lifetime protection against the development of breast cancer. Parity-induced protection can be reproduced in a carcinogen-induced rat mammary carcinoma model, but the molecular mechanisms of this protection against carcinogenic stimuli in rat mammary glands have not been fully characterized. To gain a better understanding of these molecular mechanisms, we used an oligonucleotide microarray to examine gene expression in parous and age-matched virgin (AMV) mammary glands of Lewis rats before and after carcinogen (N-methyl-N-nitrosourea; MNU) treatment. Parous mammary glands before MNU treatment showed up-regulation of multiple differentiation-related genes, such as whey acidic protein (Wap), casein beta (Csn2), casein gamma (Csng), lipopolysaccharide binding protein (Lbp), secreted phosphoprotein 1 (Spp1) and glycosylation-dependent cell adhesion molecule 1 (Glycam1). Also, parous mammary glands before MNU treatment exhibited down-regulation of growth-related genes such as regenerating islet-derived 3 alpha (Reg3a), mesothelin (Msln), insulin-like growth factor 2 (Igf2) and insulin-like growth factor binding protein 4 (Igfbp4). After MNU treatment, AMV mammary glands exhibited up-regulation of growth-related genes, such as Msln, cell division cycle 2 homolog A (Cdc2a), Igf2, Igfbp4, stathmin 1 (Stmn1) and homeobox, msh-like 1 (Msx1), whereas expression of these genes remained low in parous mammary glands. AMV mammary glands also exhibited marked up-regulation of Cdc2a and Stmn1 in response to MNU. After MNU treatment, the PCNA labeling index increased significantly in AMV mammary epithelial cells (13.7+/-1.1%), but remained low in parous mammary glands (3.6+/-0.4%). The response of AMV mammary glands to carcinogenic stimuli includes up-regulation of growth-related genes and increased cell proliferation. The lack of a similar response in parous mammary glands may explain parity-induced protection against mammary tumor development.