Scanning electron microscopy of wet tissue, wet SEM (WETSEM^TM), is a new technology that permits ultrastructural examination of fully hydrated biological samples. This review provides a description of this innovative achievement in relation to its reported and potential applications in pathology and toxicology. Possessing LM, SEM and TEM functionality, wet SEM provides a resolution that enables visualizing common ultrastructural targets of disease and toxicity, such as mitochondria and the cytoskeleton. Additionally, this confluence of multiple microscopic modalities allows for a continuous spectrum of inspection options for all structural aspects of cells and tissues. Broad overviews of the sample surface smoothly interdigitate with a detailed scrutiny of the cell interior. Elemental microanalysis and immuno-gold labeling are also possible with the wet SEM, which facilitates diagnostic applications and structural-functional correlations. Importantly, all these options are possible on the same sample during a single scoping session using one instrument. Since hydrated material is being directly examined, there is no need for embedment and microtome sectioning, so that preparation time is minimal. With such a rapid preparation phase and multifaceted inspection options, wet SEM appears particularly advantageous for the multi-arm design of drug development and safety studies. Naturally, as in any new form of microscopy, limitations exist that should be appreciated, and the image repertoire will need to be defined so that the most suitable and practical applications can be identified. Within its niche, wet SEM is likely to serve as a uniquely useful tool in pathological investigation.

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For over 35 years, many synthetic and natural chemicals have been tested by government agencies, private companies and research institutes for carcinogenic activity in rats and mice in classical 2 year studies as part of a toxicity profile ultimately used for human toxicity and carcinogenicity risk assessment. With an increasing number of pharmaceutical and agricultural chemicals shown to be carcinogenic in these bioassays, research into the mechanisms of toxicity and carcinogenesis has intensified. The relevance of the induced tumors in rodents has been questioned after much research. Research has provided evidence to some scientists that doses used in the bioassays may represent situations where toxicity pathways do not develop in humans exposed to levels of these chemicals, toxicity itself may create situations where tumors develop only under those situations, species specific responses may exist, and tumors induced may not be relevant to human risk. Regulatory agencies have considered these and other factors when preparing regulatory decisions on regulation of these chemicals. Thus, the USA FDA often has approved drugs despite their carcinogenicity in rodents and the USA EPA has explored many situations where considerations of the mechanisms of carcinogenesis in rodents and humans play a role in their regulatory decisions. Unfortunately, much of the decisions are based on unproven and hypothetical mechanisms of carcinogenesis in rodents and humans. Despite this situation, the impact of these decisions on future considerations and decisions for regulation of chemicals suggests that the US regulatory agencies consider that the occurrence of increased incidences of tumors in standard 2 year rodent carcinogenesis bioassay is often not relevant to human carcinogenesis risk assessment.

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The purpose of this study was to investigate the potential effects of the anti-bone resorptive agent alendronate (ALN) and the steroidal anti-inflammatory agent prednisolone (PL) on rheumatoid arthritis (RA) in mice with an anti-type II collagen (CII) antibody-induced model of arthritis. Arthritis was induced in female BALB/c mice with anti-CII antibody and LPS. ALN (1 mg/kg) was subcutaneously and PL (0.3, 3 mg/kg) orally administered once a day for 12 days. Severity of arthritis was evaluated on Days 6, 10 and 12, and paws and femoral bone were collected on Day 12 for histopathological examination and microcomputed tomography (micro-CT) analysis. ALN significantly inhibited bone destruction in the paws, but did not inhibit paw edema, synovial proliferation, or inflammatory cell infiltration. On the other hand, PL inhibited paw edema, synovial proliferation and inflammatory cell infiltration, but had no effect on bone destruction in the paws or bone loss in the femoral bone. ALN inhibited bone destruction in this RA model, with prominent amelioration of bone destruction and osteoclast reduction. PL inhibited inflammation, but not bone destruction in the paws. In addition, PL induced bone loss in the femoral bone as determined by micro-CT analysis. Our findings suggest that not only anti-inflammation effects but also direct inhibition of bone destruction is important for the treatment of arthritis.

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It has been speculated that oxidative stress is involved in the liver tumor-promoting effect of β-naphthoflavone (BNF; 5,6-benzoflavone) in rats, because of its strong induction of cytochrome P450 (CYP) 1A enzymes. In order to clarify the mechanism of liver tumor promoting effects of BNF, male F344 rats were initiated with a single intraperitoneal injection of 200 mg/kg diethylnitrosamine (DEN), and fed diet containing 2% of BNF for 6 weeks staring 2 weeks after injection. Two/third partial hepatectomy was perfomed at Week 3 after the treatment of BNF. Low-density Rat Toxicology & Drug Resistance Microarray and quantitative analyses of mRNA expressions of the selected genes using real-time reverse transcription (RT) -PCR were carried out on total RNAs extracted from the livers of F344 rats. Collected liver tissues were subjected to light microscopic examinations (hematoxylin and eosin staining), immunohistochemistries of proliferating cell nuclear antigen (PCNA), glutathione S-transferase placental form (GST-P) and CYP1A1, and Schmorl staining to identify lipofuscin. Gene expression analysis showed that 7 genes (CYP1A1, CYP1A2, CYP1B1, Gstm2 (GST mu2), Gstm3, glutathione peroxidase (Gpx)2 and NAD(P)H dehydrogenase, quinone 1(Nqo1)) were up-regulated (> 1.5 fold), and 4 genes (8-oxoguanine DNA glycosylase (Ogg1), Gpx1, peroxiredoxin (Prdx) 1 and P450 oxidoreductase (Por)) were down-regulated (< 0.67 fold) in the DEN + BNF group rats as compared with the DEN alone group. In immunohistochemical analyses, the numbers of foci positive for GST-P, the rate of PCNA-positive cells, and the rate of CYP1A1-positive cells were significantly increased in the DEN + BNF group, as compared to the corresponding control. Furthermore, deposition of lipofuscin, positive for Schmorl staining, was moderately increased in the DEN + BNF group. These results confirm the possibility that oxidative stress is involved in the liver tumor-promoting effect of BNF in rats.

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In order to validate early biomarkers for chemical carcinogenesis, alterations of DNA damage and subsequent cell replication induced by 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) were sequentially investigated in the rat colon and liver. Male 6-week-old Sprague Dawley rats were singly administered by gavage 300 mg/kg bw PhIP and control rats received vehicle alone. All rats were euthanized after 4-72 hr, and the organs were removed for histopathological examination, immunohistochemistry and electron microscopic observation. Immunohistochemically, in the colon, PhIP-DNA adduct already appeared at 4 hr and the positive ratios peaked at 24 hr after the PhIP exposure. Nucleolar alteration, demonstrable by electron microscopy as segregation of nucleolar components into granular and fibrillar compartments, was evident in cells of the target organ colon. Sequential observation clarified that such alteration was highest in frequency after 48 hr in colon cells, suggesting that nucleolar segregation occurs subsequent to generation of DNA adduction. Following these events, Ki-67-labeling in the colon was significantly increased at 72 hr. No significant PhIP-DNA adduct formation, nucleolar alteration or cell proliferation were noted in colons of the control rats nor in livers regardless of the PhIP treatment. Our results thus indicate an identity between the target cells for PhIP-DNA adduct formation, nucleolar segregation and enhanced cell replication, which correlated with DNA damage. These biomarkers could be useful for predicting the target organs of chemical carcinogenesis.

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This study was designed to rapidly screen for chemopreventive effects of three natural products, cyanidine-3-glycoside, acetoside and rosemaric acid, against 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP)-induced colonic, pancreatic and prostatic carcinogenesis in rats, using DNA adduct and cell proliferation as end-points. Ten-week-old F344 male rats were maintained for 2 weeks on a powdered basal diet containing 0.03% PhIP alone, PhIP together with 1% or 5% cyanidine-3-glycoside, acetoside or rosemaric acid, 1% or 5% cyanidine-3-glycoside, acetoside or rosemaric acid alone or basal diet. Immunohistochemically, PhIP DNA adduct-positive cells as well as BrdU-positive cells induced by PhIP treatment were significantly (P<0.05) reduced by the combined treatment with 5% cyanidine-3-glycoside in the proximal colon and pancreatic acinar cells as compared to the PhIP alone group values. In addition, combined treatment with 5% cyanidine-3-glycoside significantly (P<0.05) decreased numbers of BrdU-positive cells in the ventral prostate. Combined treatment with 5% rosemaric acid also significantly (P<0.05) reduced PhIP DNA adduct-positive cells in the proximal and distal colon, and ventral prostate as well as BrdU-positive cells in the lateral prostate and exocrine pancreas at 5%, and in the distal colon with 1% or 5%. However, co-treatment with acetoside did not significantly affect these parameters under the present experimental conditions. These results suggest that cyanidine-3-glycoside and rosemaric acid may have the potential to prevent PhIP-induced carcinogenesis in the colon, prostate and/or pancreas.

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Flumequine (FL), a quinolone antibiotic used for veterinary treatment of bacterial infections, has been reported to exert hepatocarcinogenicity in mice, and oxidative stress has been considered as a possible underlying mechanism. To elucidate the tumor-promoting mechanism of FL in mice, we employed a two-stage liver carcinogenesis model. Livers from mice were subjected to histopathological examinations, histochemistry for γ-glutamyltranspeptidase (GGT), and immunohistochemistry for PCNA. In addition, gene expression analyses were performed using low-density cDNA microarrays for mouse stress and toxicity and drug metabolism, and quantitative real-time RT-PCR analyses. On histopathological examinations, centrilobular hypertrophy, vacuolation, and mitotic figures of liver cells were observed in animals receiving FL, with or without DEN-initiation. The number of GGT- and PCNA-positive hepatocytes in the DEN+FL group was significantly higher than that in the DEN alone group. In the gene expression analysis, animals of the DEN+FL group showed significant up-regulations of oxidative and metabolic stress related genes, such as glutathione S-transferase (Gst) α2, Gstμ2, microsomal epoxide hydrolase (Ephx1), NAD(P)H:quinone oxidoreductase 1 (Nqo1), γ-glutamylcysteine synthetase heavy subunit (γGCSh), and NF-E2 related factor 2 (Nrf2), as well as extracellular signal regulated kinase 5 (ERK5) and c-Jun, as compared to the DEN alone group. Additionally, the in vitro measurement of reactive oxygen species (ROS) generated in the mouse liver microsomes showed a significant increase of ROS production induced by FL. These results support our previous hypothesis that oxidative stress plays an important role in FL-induced hepatocarcinogenesis in mice.

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Epidermal growth factor receptor (EGFR), a receptor protein tyrosine kinase, is a transmembrane protein. Recent studies indicate that mutations in the gene encoding EGFR are present in several human cancers. To assess the involvement of these mutations in the development of rat tumors, we looked for the presence of mutations in exons 18-21, a region which encodes the tyrosine kinase domain of Egfr, in lung and liver tumors in rats. Lung adenocarcinomas were induced in rats by exposure to N-nitrosobis(2-hydroxypropyl)amine (BHP). We also induced hepatocellular carcinomas (HCCs) in rats with multiple hepatocarcinogens and a choline-deficient L-amino acid-defined (CDAA) diet. Genomic DNA was extracted from 12 lung adenocarcinomas, 8 HCCs induced by multiple hepatocarcinogens and 8 HCCs induced by the CDAA diet. To identify mutations in Egfr, polymerase chain reaction (PCR)- single-strand conformation polymorphism (SSCP) analysis was performed. No mutations were detected throughout exons 18-21, in either lung or liver tumors in rats. These results suggest that alterations to Egfr might not be involved in the development of lung and liver tumors in rats.

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Effects of octreotide acetate, a somatostatin analog, on the development of spontaneous pancreatitis were investigated in WBN/Kob rats. Delivery of the agent continuously for 28 days via osmotic pumps implanted subcutaneously at 6 μg/day (group 1), 3 μg/day (group 2) or 0 μg/day (saline) (group 3) resulted in comparable body weight gain in all three groups. Relative weights of the liver, kidney, testis, spleen and pancreas also did not significantly differ between the treatments. Blood glucose levels were lowered by the high, but not the low dose treatment, while plasma somatostatin levels were remarkably increased in both the octreotide treatment groups. Remarkable hemorrhage, inflammatory cell infiltration, fibrosis, vacuolation of acinar cells and ductular proliferation were observed in the pancreas of control rats in group 3. However, these findings were consistently less intense in the octreotide treatment groups, in line with morphometric data showing fibrotic areas to be significantly (P<0.01) reduced. Immunohistochemically, collagen fibers in the intralobular space were mainly of type-III and mixed with α-smooth muscle actin, reflecting fibrosis in all groups. The present experiment demonstrated that octreotide inhibits spontaneous pancreatitis in WBN/Kob rats.

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