Development of novel rectal/uterine clamping device.

We developed a novel clamping device for laparoscopic surgery, free from conventional pinch structure, capable of uniformly occluding any ductal organ. This study aimed to evaluate performance of the new clamper compared to the pinch-type clamper. The new clamper consists of two metal bars with ties at each end, which enables parallel clamping. A resected porcine stomach was used, with an infusion tube at the anal end to increase intra-luminal pressure. The oral side of the stomach was clamped with either the new clamper or the pinch-type clamper, and their performances were evaluated in qualitative and semi-quantitative manner. Qualitative evaluation involved imaging the clamping site at intra-gastric pressures from 0 to 15 mmHg using microfocus computed tomography. The new clamper showed no gap even under increased intra-luminal pressure, while the pinch-type clamper showed a gap on the distal side. Quantitative evaluation measured bursting pressure under continuous air insufflation. Air leakages were observed in the new clamper at higher intra-luminal pressures than in the pinch-type clamper (46.1 mmHg vs. 13.6 mmHg, P < 0.01). Our new clamping device showed superior performance in preclinical setting compared to the conventional pinch-type clamper. We are currently working on its design freezing and aiming for early commercialization.

Fast Competitive Swimmers Demonstrate a Diminished Diving Reflex.

Competitive swimmers complete 50-m front crawl swimming without breathing or with a limited number of breaths. Breath holding during exercise can trigger diving reflex including bradycardia and diminished active muscle blood flow, whereas oxygen supply to vital organ such as brain is maintained. We hypothesized that swimmers achieving faster time in 50-m front crawl with limited number of breaths demonstrate a blunted diving reflex of cardiac and active muscle blood flow responses with elevated cerebral perfusion to counteract peripheral and central fatigues. Twenty-eight competitive swimmers (12 females) underwent a 50-m front crawl swimming time trial with minimum respiratory interruptions, following which they were categorized into two groups: Fast (n = 13) and Slow (n = 15). Additionally, they performed knee extension exercises with maximal voluntary breath- holding, wherein leg blood flow (Doppler ultrasound), cardiac output (Modelflow), heart rate (electrocardiogram), and middle cerebral artery mean blood velocity (transcranial Doppler ultrasound) were evaluated. The pattern of leg blood flow response differed between the two groups (p = 0.031) with the Fast group experiencing a delayed onset of reductions in leg blood flow (p = 0.035). The onset of bradycardia was also delayed in the Fast group (p = 0.014), with this group demonstrating a higher value of the lowest heart rate (between-trial difference in average: 15.9 [3.73, 28.2] beats/min) and cardiac output (between-trial difference in median: 2.84 L/min) (both, p ≤ 0.013). Middle cerebral artery mean blood velocity was similar between the groups (all p ≥ 0.112). We show that swimmers with superior performance in 50-m front crawl swim with limited breaths display a diminished diving reflex.

Chromosome aberrations cause tumorigenesis through chromosomal rearrangements in a hepatocarcinogenesis rat model.

Chromosome aberrations (CAs), a genotoxic potential of carcinogens, are believed to contribute to tumorigenesis by chromosomal rearrangements through micronucleus formation. However, there is no direct evidence that proves the involvement of CAs in tumorigenesis in vivo. In the current study, we sought to clarify the involvement of CAs in chemical carcinogenesis using a rat model with a pure CA-inducer hepatocarcinogen, acetamide. Whole-genome analysis indicated that hepatic tumors induced by acetamide treatment for 26-30 weeks showed a broad range of copy number alterations in various chromosomes. In contrast, hepatic tumors induced by a typical mutagen (diethylnitrosamine) followed by a nonmutagen (phenobarbital) did not show such mutational patterns. Additionally, structural alterations such as translocations were observed more frequently in the acetamide-induced tumors. Moreover, most of the acetamide-induced tumors expressed c-Myc and/or MDM2 protein due to the copy number gain of each oncogene. These results suggest the occurrence of chromosomal rearrangements and subsequent oncogene amplification in the acetamide-induced tumors. Taken together, the results indicate that CAs are directly involved in tumorigenesis through chromosomal rearrangements in an acetamide-induced hepatocarcinogenesis rat model.

Appearance of sex-determining region Y-box 9 (SOX9)- and glutathione S-transferase placental form (GST-P)-positive hepatocytes as possible carcinogenic events in the early stage of furan-induced hepatocarcinogenesis.

Furan, the basic skeleton of various flavoring agents, induces cholangiocellular tumors with higher incidences in the caudate lobe and hepatocellular tumors without the lobe specificity in rats, but the mechanism is unclear. We investigated the lobe distribution of possible carcinogenic events. Furan caused proliferation/infiltration of oval and inflammatory cells prominently in the caudate lobe as early as 4 weeks and cholangiofibrosis in this lobe at 8 weeks. In vivo mutagenicity assays using DNA extracted from the caudate or left lateral lobe of male gpt delta rats, the reporter gene-transgenic rats, treated with 8 mg/kg furan for 4 or 8 weeks showed negative outcomes. The distribution of glutathione S-transferase placental form (GST-P)-positive or sex-determining region Y-box 9 (SOX9)-positive hepatocytes was examined. Significant increases in the number of GST-P-positive hepatocytes were observed in all lobes of furan-treated rats at 8 weeks. By contrast, SOX9-positive hepatocytes, liver injury-inducible progenitor cells, were also found in all lobes of treated rats, the incidences of which were by far the highest in the caudate lobe. In addition, some of these hepatocytes also co-expressed delta like 1 homolog (DLK1), a hepatoblast marker, particularly in areas with a predominant presence of inflammatory cells. Overall, furan induced liver injury, leading to the appearance of SOX9-positive hepatocytes, some of which were subjected to dedifferentiation in the inflammatory microenvironment of a cholangiocarcinoma-prone lobe. Thus, the appearance of SOX9-positive hepatocytes together with GST-P-positive hepatocytes could be initial events in furan-induced hepatocarcinogenesis via non-genotoxic mechanisms.

Menthol alleviates post-race elevations in muscle soreness and metabolic and respiratory stress during running.

PURPOSE: We evaluated (1) whether participating in middle- and long-distance running races augments muscle soreness, oxygen cost, respiration, and exercise exertion during subsequent running, and (2) if post-race menthol application alleviates these responses in long-distance runners. METHODS: Eleven long-distance runners completed a 1500-m race on day 1 and a 3000-m race on day 2. On day 3 (post-race day), either a 4% menthol solution (Post-race menthol) or a placebo solution (Post-race placebo) serving as a vehicle control, was applied to their lower leg skin, and their perceptual and physiological responses were evaluated. The identical assessment with the placebo solution was also conducted without race participation (No-race placebo). RESULTS: The integrated muscle soreness index increased in the Post-race placebo compared to the No-race placebo (P < 0.001), but this response was absent in the Post-race menthol (P = 0.058). Oxygen uptake during treadmill running tended to be higher (4.3%) in the Post-race placebo vs. No-race placebo (P = 0.074). Oxygen uptake was 5.4% lower in the Post-race menthol compared to the Post-race placebo (P = 0.018). Minute ventilation during treadmill running was 6.7-7.6% higher in the Post-race placebo compared to No-race placebo, whereas it was 6.6-9.0% lower in the Post-race menthol vs. Post-race placebo (all P ≤ 0.001). The rate of perceived exertion was 7.0% lower in the Post-race menthol vs. Post-race placebo (P = 0.007). CONCLUSIONS: Middle- and long-distance races can subsequently elevate muscle soreness and induce respiratory and metabolic stress, but post-race menthol application to the lower legs can mitigate these responses and reduce exercise exertion in long-distance runners.

Possible contribution of 8-hydroxydeoxyguanosine to gene mutations in the kidney DNA of gpt delta rats following potassium bromate treatment.

8-Hydroxydeoxyguanosine (8-OHdG) is well known not only as an effective biomarker of oxidative stress but also as a mutagenic DNA modification. Incorporation of dAMP at the opposite site of 8-OHdG induces G>T or A>C transversions. However, in vivo analyses of gene mutations caused by potassium bromate (KBrO3), which can induce 8-OHdG at carcinogenic target sites, showed that G>T was prominent in the small intestines of mice, but not in the kidneys of rats. Because KBrO3 was a much clearer carcinogen in the kidneys of rats, detailed analyses of gene mutations in the kidney DNA of rats treated with KBrO3 could improve our understanding of oxidative stress-mediated carcinogenesis. In the current study, site-specific reporter gene mutation assays were performed in the kidneys of gpt delta rats treated with KBrO3. Groups of 5 gpt delta rats were treated with KBrO3 at concentrations of 0, 125, 250, or 500 ppm in the drinking water for 9 weeks. At necropsy, the kidneys were macroscopically divided into the cortex and medulla. 8-OHdG levels in DNA extracted from the cortex were dramatically elevated at concentrations of 250 ppm and higher compared with those from the medulla. Cortex-specific increases in mutant frequencies in gpt and red/gam genes were found at 500 ppm. Mutation spectrum and sequence analyses of their mutants demonstrated significant elevations in A>T transversions in the gpt gene and single base deletions at guanine or adenine in the gpt or red/gam genes. While A>T transversions and single base deletions of adenine may result from the oxidized modification of adenine, the contribution of 8-OHdG to gene mutations was limited despite possible participation of the 8-OHdG repair process in guanine deletion.

Timeframe Analysis of Novel Synthetic Cannabinoids Effects: A Study on Behavioral Response and Endogenous Cannabinoids Disruption.

This study investigates the impact of SCs consumption by assessing the effects of three novel synthetic cannabinoids (SCs); MDMB-CHMINACA, 5F-ADB-PINACA, and APICA post-drug treatment. SCs are known for their rapid onset (<1 min) and prolonged duration (≥5 h). Therefore, this research aimed to assess behavioral responses and their correlation with endocannabinoids (ECs) accumulation in the hippocampus, and EC's metabolic enzymes alteration at different timeframes (1-3-5-h) following drug administration. Different extents of locomotive disruption and sustained anxiety-like symptoms were observed throughout all-encompassing timeframes of drug administration. Notably, MDMB-CHMINACA induced significant memory impairment at 1 and 3 h. Elevated levels of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were detected 1 h post-MDMB-CHMINACA and 5F-ADB-PINACA administration. Reduced mRNA expression levels of fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL) (AEA and 2-AG degrading enzymes, respectively), and brain-derived neurotrophic factor (BDNF) occurred at 1 h, with FAAH levels remaining reduced at 3 h. These findings suggest a connection between increased EC content and decreased BDNF expression following SC exposure. Cognitive disruption, particularly motor coordination decline and progressive loss manifested in a time-dependent manner across all the analyzed SCs. Our study highlights the importance of adopting a temporal framework when assessing the effects of SCs.

Formation of hepatocyte cytoplasmic inclusions and their contribution to methylcarbamate-induced hepatocarcinogenesis in F344 rats.

Methylcarbamate (MC), a reaction product between dimethyl dicarbonate and ammonia or ammonium ion, is a potent hepatocarcinogen in F344 rats. Various genotoxicity tests have shown negative results for MC. Although previous studies have described the effects of MC on the liver, including the formation of characteristic basophilic cytoplasmic inclusions (CIs) in hepatocytes, the toxicological significance of CIs and their involvement in hepatocarcinogenesis remain unclear. In the current study, to elucidate the mechanisms of MC hepatocarcinogenesis, we examined hepatotoxicity and genotoxicity after 4 weeks of administration of MC using gpt delta rats with an F344 genetic background as a reporter gene transgenic animal model. Histopathologically, single-cell necrosis, karyomegaly, and the formation of CIs positive for Feulgen staining were observed in hepatocytes at the carcinogenic dose, demonstrating the hepatotoxicity of MC. CIs were also detected as large micronuclei in liver micronucleus tests but not in the bone marrow, suggesting that MC could cause chromosomal instability specifically in the livers of rats. Reporter gene mutation assays demonstrated that MC did not induce mutagenicity even in the liver. Immunofluorescence analyses revealed that CIs exhibited loss of nuclear envelope integrity, increased heterochromatinization, and accumulation of DNA damage. An increase in liver STING protein levels suggested an effect on the cyclic GMP-AMP synthase/stimulator of interferon genes innate immune pathway. Overall, these data demonstrated the possible occurrence of chromothripsis-like chromosomal rearrangements via CIs. Thus, the formation of CIs could be a crucial event in the early stage of MC-induced hepatocarcinogenesis in F344 rats.

Site-specific genotoxicity of rubiadin: localization and histopathological changes in the kidneys of rats.

Rubiadin (Rub) is a genotoxic component of madder color (MC) that is extracted from the root of Rubia tinctorum L. MC induces renal tumors and preneoplastic lesions that are found in the proximal tubule of the outer stripe of the outer medulla (OSOM), suggesting that the renal carcinogenicity of MC is site specific. To clarify the involvement of Rub in renal carcinogenesis of MC, we examined the distribution of Rub in the kidney of male gpt delta rats that were treated with Rub for 28 days. We used desorption electrospray ionization quadrupole time-of-flight mass spectrometry imaging (DESI-Q-TOF-MSI), along with the histopathological analysis, immunohistochemical staining, and reporter gene mutation assays of the kidney. DESI-Q-TOF-MSI revealed that Rub and its metabolites, lucidin and Rub-sulfation, were specifically distributed in the OSOM. Histopathologically, karyomegaly characterized by enlarged nuclear and microvesicular vacuolar degeneration occurred in proximal tubule epithelial cells in the OSOM. The ɤ-H2AX- and p21-positive cells were also found in the OSOM rather than the cortex. Although dose-dependent increases in gpt and Spi- mutant frequencies were observed in both the medulla and cortex, the mutant frequencies in the medulla were significantly higher. The mutation spectra of gpt mutants showed that A:T-T:A transversion was predominant in Rub-induced gene mutations, consistent with those of MC. Overall, the data showed that the distribution of Rub and its metabolites resulted in site-specific histopathological changes, DNA damage, and gene mutations, suggesting that the distribution of genotoxic components and metabolites is responsible for the site-specific renal carcinogenesis of MC.

A 13-week comprehensive toxicity study with adductome analysis demonstrates the toxicity, genotoxicity, and carcinogenicity of the natural flavoring agent elemicin.

Elemicin, an alkenylbenzene flavoring, exists naturally in foods, herbs, and spices. Some alkenylbenzenes are hepatotoxic and hepatocarcinogenic in rodents. However, few studies have examined the toxicology of elemicin. In the current study, we comprehensively evaluated the general toxicity, genotoxicity, and carcinogenicity of elemicin using gpt delta rats and DNA adductome analysis. Groups of 10 male F344 gpt delta rats were treated with elemicin by gavage at a dose of 0, 25, 100, or 400 mg/kg bw/day for 13 weeks. Liver weights were significantly increased with histopathological changes in groups receiving 100 mg/kg bw/day or more. Significant increases in serum hepatotoxic parameters were observed in the 400 mg/kg bw/day group. Based on the observed changes in liver weights, 18.6 mg/kg bw was identified as the low benchmark dose. Significant increases in the number and area of glutathione S-transferase placental form-positive foci and gpt mutant frequencies were apparent only in the 400 mg/kg/day group, although elemicin-specific DNA adducts were detected from the lowest dose, suggesting that elemicin exhibited hepatocarcinogenicity in rats only at higher doses. Because elemicin showed no mutagenicity at lower doses, there was an adequate safety margin between the acceptable daily intake and the estimated daily intake of elemicin.

Management of abdominal gas leakage from surgical trocars in laparoscopic surgery: a preclinical study.

Introduction: There is an ongoing concern about the potential infectious risk due to pneumoperitoneal gas leakage from surgical trocars in laparoscopic surgery. We aimed to visually confirm the presence of leakage from trocars and investigate the changes in the leakage scale according to intra-abdominal pressures and trocar types. Material and methods: We established a porcine pneumoperitoneum model and performed experimental forceps manipulation using 5-mm grasping forceps with 12-mm trocars. The gas leakage, if any, was imaged using a Schlieren optical system, which can visualize minute gas flow invisible to the naked eye. For measuring the scale, we calculated the gas leakage velocity and area using image analysis software. Four types of unused and exhausted disposable trocars were compared. Results: Gas leakage was observed from trocars during forceps insertion and removal. Both the gas leakage velocity and area increased as the intra-abdominal pressure increased. Every type of trocar we handled was associated with gas leakage, and exhausted disposable trocars had the largest scale gas leakage. Conclusions: We confirmed gas leakage from trocars during device traffic. The scale of leakage increased with high intra-abdominal pressure and with the use of exhausted trocars. Current protection against gas leakage may not be sufficient and new surgical safety measures and device development may be needed in the future.

Dimethylglycine, a Methionine Metabolite, Participates in the Suppressive Effect of Methionine on 1-Fluoro-2,4-dinitrobenzene-Induced Dermatitis.

Allergic contact dermatitis (ACD) is a common skin disorder caused by contact with allergens. The optimal treatment for ACD is to avoid contact with allergens. However, in some cases, avoiding exposure is not possible when the allergens are unknown. Therefore, establishing treatment methods other than allergen avoidance is important. We previously reported that the continuous administration of methionine, an essential amino acid, in a mouse model of atopic dermatitis alleviated its symptoms. In the present study, we investigated the effect of methionine on a mouse model of ACD caused by 1-fluoro-2,4-dinitrobenzene (DNFB). Differences in the effect of methionine were observed in DNFB-induced ACD model mice based on the mouse strain used. This difference was attributed to the suppression of hepatic dimethylglycine (DMG) production, which is associated with the suppression of hepatic betaine-homocysteine methyltransferase (Bhmt) expression by ACD. Although we did not reveal the mechanism underlying DMG suppression, our study suggests the presence of interactions between the liver and skin in dermatitis, such as the regulation of hepatic metabolic enzyme expression in dermatitis and the alleviation of dermatitis symptoms by the hepatic metabolism status of DMG.

α-Lipoic acid eliminates dioxin-induced offspring sexual immaturity by improving abnormalities in folic acid metabolism.

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes developmental and reproductive disorders in pups due to the attenuated luteinizing hormone (LH) production during the perinatal stage; however, the administration of α-lipoic acid (LA) to TCDD-exposed pregnant rats reversed the attenuated LH production. Therefore, reproductive disorders in pups are expected to be ameliorated with LA supplementation. To address this issue, pregnant rats orally received low dose TCDD at gestational day 15 (GD15) and proceeded to parturition. The control received a corn oil vehicle. To examine the preventive effects of LA, supplementation with LA was provided until postnatal day 21. In this study, we demonstrated that maternal administration of LA restored the sexually dimorphic behavior of male and female offspring. TCDD-induced LA insufficiency is likely a direct cause of TCDD reproductive toxicity. In the analysis to clarify the mechanism of the decrease in LA, we found evidence suggesting that TCDD inhibits the synthesis and increases the utilization of S-adenosylmethionine (SAM), a cofactor for LA synthesis, resulting in a decrease in the SAM level. Furthermore, folate metabolism, which is involved in SAM synthesis, is disrupted by TCDD, which may adversely affect infant growth. Maternal supplementation of LA restored SAM to its original level in the fetal hypothalamus; in turn, SAM ameliorated abnormal folate consumption and suppressed aryl hydrocarbon receptor activation induced by TCDD. The study demonstrates that the application of LA could prevent and recover next-generation dioxin reproductive toxicity, which provides the potential to establish effective protective measures against dioxin toxicity.

Rapid derivatization of phosphorus-containing amino acid herbicides in plasma and urine using microwave heating.

We developed a derivatization technique that involves microwave heating to reduce the overall forensic analysis time of phosphorus-containing amino acid herbicides (PAAHs). Combined with an extraction method that uses titanium (IV) oxide (TiO2), we were able to obtain a practical analytical method for PAAHs and their metabolites in samples intended for poisoning cases. The optimized derivatization conditions were 700 W power and 5-min irradiation time, which is a significant time-saving. The plasma samples extracted using TiO2-packed Tip columns and derivatized under the optimized conditions had an intra-day accuracy and precision within 9.3% and 9.0%, respectively. The intermediate accuracy and precision were within 8.8% and 8.5%, respectively, and the recoveries were more than 91.2%. Similarly, for urine samples, the intra-day accuracy and precision were within 13.3% and 9.1%, respectively. The intermediate accuracy and precision were within 13.6% and 10.3%, respectively, and finally, the recoveries were more than 88.2%. In addition to reducing the pretreatment time, this method was suitable for reducing the overall labor burden on laboratories responsible for routine analysis because of its stable validation data.

Comprehensive analysis of the general toxicity, genotoxicity, and carcinogenicity of 3-acetyl-2,5-dimethylfuran in male gpt delta rats.

The safety of flavoring agents has been evaluated according to classification by chemical structure and using a decision tree approach. The genotoxic potential found in some flavoring agents has highlighted the importance of efficient toxicity studies. We performed a comprehensive toxicity analysis using reporter gene transgenic rats to assess the safety of 3-acetyl-2,5-dimethylfuran (ADF), a flavoring agent exhibiting genotoxic potential in silico and in vitro assays. Male F344 gpt delta rats were given 0, 30, or 300 mg/kg body weight/day ADF by gavage for 13 weeks. In serum biochemistry analyses, triglyceride, total cholesterol, phospholipid, and total protein levels and albumin/globulin ratios were significantly altered in the 30 and 300 mg/kg groups. Histopathologically, nasal cavity toxicity and hepatocellular hypertrophy were observed in the 300 mg/kg group. In the livers of 300 mg/kg group, a significant increase in gpt mutant frequencies were observed along with ADF-specific DNA adduct formation. The number and area of glutathione S-transferase placental form-positive foci were significantly increased in the same group. Thus, ADF affected nasal cavity, liver, and lipid metabolism and showed genotoxicity and possible carcinogenicity in the liver. Overall, our comprehensive toxicity study using gpt delta rats provided insights into the safety evaluation of ADF.

Toxicity, genotoxicity, and carcinogenicity of 2-methylfuran in a 90-day comprehensive toxicity study in gpt delta rats.

2-Methylfuran (2-MF) exists naturally in foods and is used as a flavoring agent. Furan, the core structure of 2-MF, possesses hepatocarcinogenicity in rodents. Accumulation of toxicological information on furan derivatives is needed to elucidate their carcinogenic mode of action. In the current study, we examined the comprehensive toxicological studies of 2-MF using gpt delta rats. 2-MF was intragastrically administered to groups of 10 male and 10 female Sprague-Dawley gpt delta rats at a dose of 0, 1.2, 6, or 30 mg/kg/day for 13 weeks. Effects of 2-MF on the hepatobiliary system including an increase in serum alkaline phosphatase were observed in the 6 and 30 mg/kg groups, and cholangiofibrosis was found in the 30 mg/kg group. The no observed adverse effect level was set at 1.2 mg/kg/day for both sexes and 1.14 mg/kg/day was determined as the benchmark dose low. The acceptable daily intake was calculated to be 11.4 µg/kg/day. Increases in the number and areas of glutathione S-transferase placental form-positive foci in the 30 mg/kg group were apparent, suggesting the hepatocarcinogenicity of 2-MF in rats. By contrast, the lack of increase in in vivo mutagenicity in the liver implied that 2-MF hepatocarcinogenesis may not involve genotoxic mechanisms.

Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4.

Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics and pharmacogenomics research.

[Role and potential of histopathological specimens in the toxicological evaluation of pharmaceuticals and chemicals].

With the development of molecular-targeted drugs, the demand for utilization of histopathological specimens and pathological diagnosis is increasing in the field of clinical pathology. In particular, in case of companion diagnostics, the results of immunohistochemical staining have become beyond diagnostic assistance, definitive diagnosis, and now indispensable for selection of therapeutic agents. Histopathological examination also serves an important role in non-clinical toxicological evaluation. Particularly, it is pivotal for obtaining data on organ-specific toxicity and carcinogenicity. On the other hand, a weight of evidence approach is currently being considered in International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) S1 as an option to replace the 2-year rat carcinogenicity study, in which case the evaluation of the 26-week-repeated dose toxicity study will be more important. In this article, We would like to introduce the usefulness of γ-H2AX-based rat bladder carcinogenicity evaluation using histopathological specimens obtain from 28-day repeated-dose study and the application of mass spectrometric imaging currently under consideration in our laboratory, thus discuss the role of histopathological examination in non-clinical toxicological and safety evaluation and its future perspective.

Visualization of the distribution of anthraquinone components from madder roots in rat kidneys by desorption electrospray ionization-time-of-flight mass spectrometry imaging.

Madder color (MC), a natural dye isolated from Rubia tinctorum, is a potent carcinogen that targets the outer stripe of outer medulla (OSOM) in the kidneys of rats. To clarify the role of MC components in renal carcinogenesis, we examined distributions of MC components and metabolites in the kidneys of rats treated with MC using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). Alizarin, lucidin, munjistin, nordamnacanthal, purpurin, pseudopurpurin, rubiadin, and some other metabolites detected and identified by liquid chromatography time-of-flight MS analysis of rat serum 1 h after MC administration were subjected to DESI-MSI. This analysis enabled visualization of the distribution of anthraquinones in the kidney, and the ion images showed a characteristic distribution according to their chemical structure. Among the components, lucidin and rubiadin specifically localized in the OSOM, suggesting that their genotoxicity was a direct cause of MC carcinogenesis. Alizarin showed greater distribution in the OSOM than the cortex and may therefore participate in renal carcinogenicity owing to its tumor-promoting activity. Overall, our data suggested that the distribution of carcinogenic components to the OSOM was responsible for the site-specific renal carcinogenicity of MC and that DESI-MSI analysis may be a powerful tool for exploring the mechanisms of chemical carcinogenesis.

Usefulness of microfocus computed tomography in life science research: preliminary study using murine micro-hepatic tumor models.

PURPOSE: Microfocus computed tomography (micro-CT) has not been widely used at high radiation intensity (industrial micro-CT) in life science fields. In this preliminary study, we investigated its potential value in the detection of micro-hepatic tumors in a mouse model. METHODS: The liver with micro-hepatic tumors was surgically resected en-bloc from mice, and examined with industrial micro-CT and lower intensity micro-CT (small animal micro-CT). The number of hepatic tumors was manually counted on serial images. Then, the accuracy of each technique was determined by preparing matching liver sections and comparing the number of tumors identified in a conventional pathological examination. RESULTS: The number of hepatic tumors evaluated with industrial micro-CT showed high concordance with the results of the pathological examinations (intraclass correlation coefficient [ICC]: 0.984; 95% confidence interval [CI] 0.959-0.994). On the other hand, the number of hepatic tumors evaluated with the small animal micro-CT showed low concordance with the number identified in the pathological examinations (ICC: 0.533; 95% CI 0.181-0.815). CONCLUSION: Industrial micro-CT improved the detection of small structures in resected specimens, and might be a promising solution for life science research.