Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX.

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

Immunohistochemistry of γ-H2AX as a method of early detection of urinary bladder carcinogenicity in mice.

Phosphorylated histone H2AX (γ-H2AX) has been demonstrated as a DNA damage marker both in vitro and in vivo. We previously reported the effects of genotoxic carcinogens in the urinary bladder of rats by immunohistochemical analysis of γ-H2AX using samples from 28-day repeated-dose tests. To evaluate the application of γ-H2AX as a biomarker of carcinogenicity in the bladder, we examined species differences in γ-H2AX formation in the urinary bladder of mice. Six-week-old male B6C3F1 mice were treated orally with 12 chemicals for 4 weeks. Immunohistochemical analysis demonstrated that N-butyl-N-(4-hydroxybutyl)nitrosamine, p-cresidine and 2-acetylaminofluorene (2-AAF), classified as genotoxic bladder carcinogens, induced significant increases in γ-H2AX levels in the bladder urothelium. In contrast, genotoxic (2-nitroanisole, glycidol, N-nitrosodiethylamine and acrylamide) and non-genotoxic (dimethylarsinic acid and melamine) non-bladder carcinogens did not upregulate γ-H2AX. Importantly, 2-nitroanisole, a potent genotoxic bladder carcinogen in rats, significantly increased the proportion of γ-H2AX-positive cells in rats only, reflecting differences in carcinogenicity in the urinary bladder between rats and mice. Significant upregulation of γ-H2AX was also induced by uracil, a non-genotoxic bladder carcinogen that may be associated with cell proliferation, as demonstrated by increased Ki67 expression. 2-AAF caused γ-H2AX formation mainly in the superficial layer, together with reduced and disorganized expression of uroplakin III, unlike in rats, suggesting the mouse-specific cytotoxicity of 2-AAF in umbrella cells. These results suggest γ-H2AX is a useful biomarker reflecting species differences in carcinogenicity in the urinary bladder.

Dietary intake of iodine-enriched eggs decreases the incidence of mouse mammary tumors caused by the activated ErbB2 oncogene.

Human epigenetic studies suggest that consumption of seaweed prevents mammary cancer, which possibly is explained by iodine daily intake. In this study, we evaluated the efficacy of dietary intake of iodine-enriched eggs on mammary tumor incidence caused by the expression of activated type ErbB2. Female transgenic mice were divided into three groups, and fed a basic diet, a diet supplemented with ordinary eggs, or with iodine-enriched eggs. The number of mammary tumors greater than 5 mm in diameter was recorded in mice at 6 months of age. We report that the average number of mammary tumors per mouse was significantly lower in the iodine-enriched egg-added diet group than in either the basic diet or ordinary egg diet groups. These results indicate that iodine intake through livestock-derived products can reduce the incidence of mammary cancers caused by the expression of activated type ErbB2.

Size-dependent acute toxicity of silver nanoparticles in mice.

In this study, we aimed to evaluate changes in the acute toxicity of intraperitoneally administered silver nanoparticles (AgNPs) of varying sizes in BALB/c mice. Seven-week-old female BALB/c mice were intraperitoneally administered AgNPs measuring 10, 60, or 100 nm in diameter (0.2 mg/mouse) and then sacrificed 1, 3, or 6 h after treatment. In mice administered 10 nm AgNPs, reduced activity and piloerection were observed at 5 h post administration, and lowered body temperature was observed at 6 h post administration, with histopathological changes of congestion, vacuolation, single cell necrosis, and focal necrosis in the liver; congestion in the spleen; and apoptosis in the thymus cortex. These histopathological changes were not evident following administration of either 60 or 100 nm AgNPs. These results suggested that smaller AgNPs, e.g., those measuring 10 nm in diameter, had higher acute toxicity in mice.

Spontaneous infarcted adenoma of the mammary gland in a Wistar Hannover GALAS rat.

Spontaneous massive infarction of mammary gland tumors has been reported to occur infrequently in humans. A subcutaneous mass (18 × 17 × 10 mm) was observed in the right axilla extending to the chest region of a 110-week-old female Wistar Hannover GALAS rat. Histopathologically, a well-circumscribed mass with lobular structures was present in the subcutis. Most of the mass was occupied by extensive coagulative necrosis of neoplastic cells with relatively uniform acinar and ductal structures. Although each necrotic acinar structure was separated by reticular fibers, periacinar stromal collagen fibers were not abundant. Considering the site of occurrence and histological features, the necrotic tissue was diagnosed as adenoma of the mammary gland. The necrotic region lacked hemorrhage and obvious inflammatory cell infiltration, indicating the necrosis was caused by infarction. Although multiple necrosis and focal infarction are occasionally observed in large-sized tumors in rodents, especially in adenocarcinomas, the present case was characteristic, with the massive infarction involving most parts of the tumor despite the relatively small size and low atypia of neoplastic cells. This is a rare case of spontaneous infarcted adenoma of the mammary gland in rats histologically resembling human cases.

Lgr4 is required for endometrial receptivity acquired through ovarian hormone signaling.

Previously, using the Keratin5-Cre transgenic mouse model we reported that female Lgr4-conditional KO mice (Lgr4(K5 KO)) showed subfertility with defective stromal decidualization due to abnormal development of the uterine gland. However, the impact of the LGR4 defect on luminal epithelial cells was not investigated in the previous report. Here, we focused on the receptive state of the luminal epithelium in Lgr4(K5 KO) mice that received ovarian hormone treatment. In Lgr4(K5 KO) mice, progesterone failed to inhibit the luminal epithelial cell proliferation. Immunohistochemical and qRT-PCR analyses revealed down-regulated progesterone signaling in the uterus of Lgr4(K5 KO) mice. These results demonstrated that LGR4 is essential for the acquisition of endometrial receptivity through ovarian hormone signaling.

LGR4 expressed in uterine epithelium is necessary for uterine gland development and contributes to decidualization in mice.

In previous work we generated mice with a tissue specific ablation of a leucine-rich repeat containing G-protein-coupled receptor 4 (Lgr4) using the Keratin-5 (K5) Cre transgenic mouse strain (Lgr4(K5 KO)). Interestingly, the Lgr4(K5 KO) female mice were subfertile, and their embryos had impaired development. Notably, the contributions of uterine development to the subfertility phenotype were not elucidated in the previous report. In a readdress, the following study explores uterine aberration in Lgr4(K5 KO) female mice. Histological analysis revealed that the uteri of Lgr4(K5 KO) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands. Furthermore, Lgr4 deletion led to the reduced expression of morphoregulatory genes related to the Wnt signaling pathway. Additionally, the uteri of the Lgr4(K5 KO) mice lost the ability to undergo induced decidualization. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis and administration of recombinant leukemia inhibitory factor (LIF) demonstrated that the impaired decidualization in Lgr4(K5 KO) mice resulted from the decreased secretion of LIF concurrent with a reduction in uterine gland count. Thus, we propose that LGR4 contributes to uterine gland development, which supports decidualization during pregnancy.

LGR4 is required for the cell survival of the peripheral mesenchyme at the embryonic stages of nephrogenesis.

In mice, homozygous Lgr4 inactivation results in hypoplastic kidneys. To understand better the role of LGR4 in kidney development, we performed an analysis of kidneys in Lgr4-/- embryos. We stained Lgr4-/- kidneys with anti-WT1 and anti-Cleaved Caspase3 antibodies at E16.5, and observed that the structures of the cap mesenchyme were disrupted and that apoptosis increased. In addition, the expression of PAX2, an anti-apoptotic factor in kidney development, was also significantly decreased at E16.5. We found that the LGR4 defect caused an increase in apoptosis in the peripheral mesenchyme during kidney development.