Molecular Pathological Differences in Global Gene Expression between Two Sustained Proliferative Lesions, Nodular Regenerative Hepatocellular Hyperplasia and Hepatocellular Adenoma, in Mice.

Long-term exposure to piperonyl butoxide (PBO) induces multiple nodular masses along with hepatocellular tumors in the liver of mice. The histopathological features of the nodules led to our diagnosis of nodular regenerative hepatocellular hyperplasia (NRH). However, because of the lack of data on the biological characteristics of NRH, whether this lesion is truly nonneoplastic remains unknown. In this study, the molecular characteristics of NRH were compared with those of hepatocellular adenoma (HCA) by global gene expression analysis. Six-week-old male ICR mice were fed a diet containing 6,000 ppm PBO for 43 weeks to induce NRH and HCA development. Complementary DNA microarray analysis was performed using messenger RNA extracted from NRH and HCA frozen sections collected by laser microdissection. Hierarchical cluster analysis showed that all NRH samples clustered together but were separate from the HCA cluster. Pathway analysis revealed activation of the cell cycle and Delta-Notch signaling in both lesions, but the latter was more upregulated in HCA. Downregulation of cytochrome p450 enzymes was observed in NRH, but not in HCA. These results imply that NRH differs from HCA in terms of not only morphological but also molecular characteristics.

Role of oxidative stress in the chemical structure-related genotoxicity of nitrofurantoin in Nrf2-deficient gpt delta mice.

Despite its antimicrobial activity, nitrofurantoin (NFT) is a renal carcinogen in rats. Oxidative stress induced by reduction of the nitro group of NFT may contribute to its genotoxicity. This is supported by our recent results indicating that the structure of the nitrofuran plays a key role in NFT-induced genotoxicity, and oxidative DNA damage is involved in renal carcinogenesis. Nuclear factor erythroid 2-related factor 2 (NRF2) regulates cellular responses to oxidative stress. To clarify the role of oxidative stress in the chemical structure-related genotoxic mechanism of NFT, we performed reporter gene mutation assays for NFT and 5-nitro-2-furaldehyde (NFA) using Nrf2-proficient and Nrf2-deficient gpt delta mice. NFT administration for 13 weeks resulted in a significant increase in 8-hydroxydeoxyguanosine (8-OHdG; a marker of oxidative stress) and gpt mutant frequency only in the kidneys of Nrf2-/- mice. The mutation spectrum, characterized by increased substitutions at guanine bases, suggested that oxidative stress is involved in NFT-induced genotoxicity. However, NFA did not increase the mutation frequency in the kidneys, despite the increased 8-OHdG in NFA-treated Nrf2-/- mice. Thus, it is unlikely that oxidative stress is involved in the genotoxic mechanism of NFA. These results imply that nitro reduction plays a key role in the genotoxicity of NFT, but the lack of a role of oxidative stress in the genotoxicity of NFA indicates a potential role of side chain interactions in oxidative stress caused by nitro reduction. These findings provide a basis for the development of safe nitrofurans.

Mechanisms of oxidative stress-induced in vivo mutagenicity by potassium bromate and nitrofurantoin.

Oxidative stress is well known as a key factor of chemical carcinogenesis. However, the actual role of oxidative stress in carcinogenesis, such as oxidative stress-related in vivo mutagenicity, remains unclear. It has been reported that 8-hydroxydeoxyguanosine (8-OHdG), an oxidized DNA lesion, might contribute to chemical carcinogenesis. Potassium bromate (KBrO3) and nitrofurantoin (NFT) are known as renal carcinogens in rats. Our previous studies showed an increase in mutant frequencies accompanied by an increased level of 8-OHdG in the kidneys of rodents following KBrO3 or NFT exposure. Furthermore, KBrO3 and NFT induced different types of gene mutations. Thus, in the present study, we performed reporter gene mutation assays and 8-OHdG measurements following KBrO3 or NFT exposure using Nrf2-proficient and Nrf2-deficient mice to clarify the relationship between KBrO3- or NFT-induced oxidative stress and subsequent genotoxicity. Administration of 1,500 ppm of KBrO3 in drinking water resulted in an increase in deletion mutations accompanied by an increase in 8-OHdG level, and administration of 2,500 ppm of NFT in diet induced an increase in guanine base substitution mutations without elevation of the 8-OHdG level in Nrf2-deficient mice. These results demonstrated that the formation of 8-OHdG, which resulted from the oxidizing potential of KBrO3, was directly involved in the increase in deletion mutations, although factors related to oxidative stress other than 8-OHdG might be crucial for NFT-induced guanine base substitution mutations. The present study provides new insight into oxidative stress-related in vivo mutagenicity.

Phosphorylation of protein phosphatase 2A facilitated an early stage of chemical carcinogenesis.

Protein phosphatase 2A (PP2A) is a serine-threonine phosphatase that regulates cell signaling pathways. Its inactivation is correlated with tumor malignancy, possibly due to the effects on cell differentiation and malignant cell transformation. Therefore, it has been noted that PP2A could be a promising target for cancer therapy. In our previous study of the hepatocarcinogen estragole (ES), cell proliferation may be required to convert ES-specific DNA adducts to mutations. To explore the trigger for cell proliferation, gpt delta rats were administered ES by gavage at doses of 3, 30 and 300mg/kg/day for 4weeks. ES-induced cell proliferation and gene mutations were observed at only the high dose whereas ES-specific DNA adducts were detected in a dose-dependent manner. Western blot analyses revealed activation of the Akt and ERK pathways without activation of upstream regulators, such as c-Raf, PKC and, PI3K. Phosphorylation of the PP2A C subunit at Tyr307 was found along with phosphorylation of Src. The overall data might imply that PP2A inactivation is responsible for cell cycle progression through activation of the Akt and ERK pathways at high doses of ES. Based on γ-H2AX immunohistochemistry and Western blot analysis for Rad51 protein, the resultant mutation spectra showed large deletion mutations that might result from double strand breaks of DNA. Thus, it is likely that inactivation of PP2A resulted in acceleration and exacerbation of gene mutations. We conclude that PP2A might contribute to an early stage of chemical carcinogenesis, suggesting that PP2A could be a molecular target of primary cancer prevention.

Acrylamide induces specific DNA adduct formation and gene mutations in a carcinogenic target site, the mouse lung.

Acrylamide (AA) is a contaminant in heated foods and is carcinogenic in multiple organs of rodents. There have been many reports regarding AA-induced DNA modification and genotoxicity. However, the data are insufficient to understand fully the relationship between the two events. A recent report demonstrated carcinogenicity in the mouse lung. The lung is advantageous for investigation of AA-induced genotoxicity because DNA adduct levels are relatively high in this organ. In the present study, reporter gene mutation assays and quantitative analyses of specific DNA adducts were performed in the lungs of mature gpt delta mice treated with AA at doses of 100, 200 and 400 p.p.m. in drinking water for 4 weeks. N7-GA-Gua was detected in all AA-treated mice in a dose-dependent manner. gpt mutant frequencies (MFs) were significantly increased in the middle- and high-dose groups. In the analysis of mutation spectra, significant increases in GC-TA transversions and single base deletion mutations were observed in the high-dose group. Spi(-) MFs were significantly increased in the high-dose group. Analysis of Spi(-) mutants revealed significant increases in the frequencies of single base deletion mutation in runs of G/C and A/T. Analyses of immature mice under the same experimental conditions showed that there were no differences of susceptibility to AA-induced genotoxicity in the two age classes. The overall data clearly show the causal relationship between AA-induced DNA adducts and the gene mutations at carcinogenic target sites.

Immunohistochemical study on the secretory host defense system of bactericidal peptides in rat digestive organs.

To clarify the fundamental regulation mechanism against indigenous bacterial proliferation in the alimentary tract, we immunohistochemically examined the localization of 4 bactericidal peptides (BP) in the rat digestive exocrine glands. In the upper alimentary tract, lysozyme was detected in the gustatory, extraorbital lacrimal and parotid glands. Secretory phospholipase A2 (sPLA2) was detected in the extraorbital lacrimal glands. ß-defensin1 was detected in the gustatory and extraorbital lacrimal glands. ß-defensin2 was detected in the Harderian glands. In the stomach, ß-defensins were detected in the gastric superficial epithelial cells. In the small and large intestines, only lysozyme and sPLA2 were detected in the Paneth cells. In the cecum, all 4 BP were detected in the middle to apical portions of the crypts, and only sPLA2 was detected in the basal portion. No BP were localized in other exocrine glands associated with the alimentary tract. In addition, all 4 BP were also detected in the columnar epithelial cells of the apical portions of intestinal villi. In the intestinal superficial epithelial cells, lysozyme and ß-defensins were detected in the ascending colon, whereas only ß-defensin1 was detected in the descending colon and rectum. These results suggest that BP are mainly secreted from exocrine tissues in the initial portion of the digestive tract and play a role in host defense against indigenous bacteria throughout the digestive tract. Part of the BP in the chyme might be absorbed by the epithelium at the most inner sites of mucosae in the small and large intestines.

Lectin histochemical detection of special sugars on the mucosal surfaces of the rat alimentary tract.

Surfaces of the most luminal positions of mucosae are fundamental settlement sites of indigenous bacteria throughout the rat alimentary tract. In these positions, also epithelial cell-shedding sites, the special sugar expression in the glycocalyx is very important as it provides possible ligands of bacterial lectins for attachment to epithelial cells. Therefore, the sugar expression in glycocalyx of epithelial cells was lectin-histochemically surveyed using 21 lectins throughout the rat alimentary tract. From the tongue to the nonglandular part of the stomach, α-D-Man, α-D-Glc and α-D-GalNAc were detected on the surface of the keratinized stratified squamous epithelium. In the glandular part of the stomach, α-D-Man, ß-D-Gal-4GlcNAc, D-Gal, D-GalNAc, D-GlcNAc, α-L-Fuc- α-D-Gal-ß(1-4)GlcNAc and bisected triantennary N-glycans were detected on the surface of gastric superficial epithelial cells. From the duodenum to the ileum, (GlcNAc)(2-4) was expressed exclusively on the epithelial cells in the apical portions of the intestinal villi. From the cecum to the rectum, α-D-Man, ß-D-Gal-4GlcNAc, D-Gal, D-GalNAc, α-D-Gal(1-3)D-GalNAc, (GalNAc)(n) and NeuNAc were expressed on the intestinal superficial epithelial cells. These results suggest that special sugars are expressed on the most luminal portions of mucosae as exclusive epithelial cell-shedding sites, and that sugar expression differs among the various segments of the alimentary tract. These site differences might reflect differences in resident bacterial species in the rat alimentary tract.

Histoplanimetrical study on the relationship between the cell kinetics of villous columnar epithelial cells and the proliferation of indigenous bacteria in rat small intestine.

The relationship between the kinetics of villous columnar epithelial cells and the expansion of colonies of indigenous bacteria from the narrow apical portions of intestinal villi was immunohistochemically and histoplanimetrically investigated in the small intestine of bromodeoxyuridine administred Wistar rats. As a result, the lifespan of villous columnar epithelial cells was slightly shorter in the distal ileum than in other portions of small intestine, accompanying the minimum height of the intestinal villi of the distal ileum in the small intestine. The migration speed of villous columnar epithelial cells was significantly decreased toward the distal small intestine. The migration speed in the distal ileum was about one-fourth of that in the duodenum. The migration speed of the villous columnar epithelial cells was greater and their lifespans were shorter in the sites with wide expansion of the indigenous bacterial colony from the narrow apical portions of the intestinal villi than that in sites with no or less expansion. Additionally, the expansion of the indigenous bacterial colony from narrow villous apices also immediately shortened the heights of the intestinal villi. These findings suggest that the migration speed of villous columnar epithelial cells might contribute to the regulation of the settlement of bacteria at the villous apices and the inevitable proliferation of indigenous bacteria at the intervillous spaces in the rat small intestine.

Histoplanimetrical study on the spatial relationship of distribution of indigenous bacteria with mucosal lymphatic follicles in alimentary tract of rat.

The spatial relationship between the distribution of indigenous bacteria (IB) and the situation of mucosal lymphatic follicles (LF) is histoplanimetrically studied in the rat alimentary tract. From the oral cavity to the nonglandular part of the stomach, IB adhered to the corneal layer of the most luminal mucosa. In the glandular part of the stomach, IB adhered only to the most luminal mucosa but not in the gastric pits. In the small intestine, IB consistently adhered around the apices of both intestinal villi and the domes, and their amounts decreased toward their basal portions. No IB entered the intestinal crypts. In the large intestine, IB consistently adhered to the most luminal mucosa. Numerous IB were suspended in the intestinal crypts of both the cecum and the proximal colon, whereas there were no IB in the crypts of the distal colon and the rectum. When IB spread over the basal portions of the intestinal villi, IB with the same morphology were detected on the neighboring LF, whereas no bacteria were detected on the neighboring LF, when IB were located in the apical to middle portions of the intestinal villi. This close relationship between the distribution of IB and mucosal LF was also observed in the large intestine. These results suggest that the most luminal mucosae are a fundamental settlement site of IB throughout the alimentary tract and that the hyperproliferation of IB's colonies might be detected by neighboring LF in the rat intestine.

Histoplanimetrical study on the relationship between cellular kinetics of epithelial cells and proliferation of indigenous bacteria in the rat colon.

The aim of this study was to clarify the regulatory effects of epithelial kinetics on indigenous bacterial proliferation in the large intestine. The lifespan, migration speed and proliferation rate of crypt epithelial cells in the initial 20% of the colon (proximal colon) and the 50% of the colon (middle colon) in bromodeoxyuridine-administrated rats were histoplanimetrically and chronologically compared. The proximal colon possessed well-developed mucosal folds and a large amount of indigenous bacteria which filled the crypt lumen, whereas no folds or bacteria were found to occupy the crypt lumen in the middle colon. The cell lifespans were 32.2, 42.5 and 33.6 hr in the apical and the basal parts of the mucosal folds of the proximal colon, and in the middle colon, respectively. The migration speeds were 4.2, 2.1 and 3.3 microm/hr, respectively, while the appearance frequencies of proliferating cell nuclear antigen (PCNA)-positive crypt epithelial cells were 35.0, 24.6 and 33.8%. These findings suggest that the lifespan was shortened and the migration speed increased in the most luminal mucosa of colon, contributing to the elimination of the adhered bacteria from the most luminal mucosa. By contrast, the elongation of the lifespan and deceleration of the migration of epithelial cells in the basal parts of the mucosal folds might contribute to reliable settlement of indigenous bacteria, resulting in the maintenance of a large amount of indigenous bacteria in the lumen of the proximal colon.

DNA modifications that do not cause gene mutations confer the potential for mutagenicity by combined treatment with food chemicals.

Cell proliferation plays a key role in fixing mutations induced by DNA damage. We clarified whether this phenomenon occurred after combined treatment with chemicals in food. The effects of antibiotic flumequine (FL), a residue of veterinary medicinal products in foodstuffs, on mutagenicity in the liver were examined in mice treated with estragole (ES), a natural food flavouring compound. Gpt delta mice were orally administered 10 or 100 mg/kg/day ES and simultaneously fed a diet containing 0.4% FL for 4 weeks. Proliferating cell nuclear antigen-positive cells and cell cycle-related genes were additively increased in the livers of combined treatment groups as compared with high-dose ES or FL groups. Mutant frequencies (MFs) in gpt after cotreatment with low-dose ES and FL were significantly increased, although treatment with ES alone increased MFs only in the high-dose group. Sult1a1 mRNA levels were unchanged after FL treatment. Liquid chromatography with tandem-mass spectrometry analysis showed that FL did not affect the amount of ES-specific DNA adducts in the livers, indicating that FL treatment did not influence metabolic pathways of ES. Thus, enhancement of the mutagenic potential of a chemical by chemical-induced cell proliferation may occur as a result of the combined effects of chemicals in food.

An immunohistochemical detection of actin and myosin in the indigenous bacteria-adhering sites of microvillous columnar epithelial cells in Peyer's patches and intestinal villi in the rat jejunoileum.

The mechanism of physical elimination of indigenous bacteria was ultrastructurally and immunohistochemically investigated in microvillous columnar epithelial cells of Peyer's patches and intestinal villi of the rat jejunoileum. From ultrastructural observation, the microfilaments accumulated to form several electron-dense layers beneath the bacteria adhering to the cell membrane, which was slightly invaginated in the epithelial cells of Peyer's patches and intestinal villi. As the microfilamentous layers were forming, the end portions of invaginations were deformed into a cone-shape and were finally collapsed. At the same time, the end portions of the adhered bacteria were also deformed into cone-shapes. The bacterial cells were moved back toward the invagination orifices with no morphological change in their inner structure. From immunohistochemical observation, beta-actin and nonmuscle-type myosin were detected at the thin layer just beneath the invaginated cell membrane. These findings suggest that indigenous bacteria which adhere to epithelial cells are removed by only a physical action of actin and myosin filaments, but are not killed. This bacterial cell removal system might lead to the establishment of a settlement of indigenous bacteria on host cells.

In vivo reporter gene mutation and micronucleus assays in gpt delta mice treated with a flame retardant decabromodiphenyl ether.

Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, have been widely used as additive flame retardants. Recently, the use of brominated flame retardants has been restricted or prohibited under various legislative acts because of the persistence, bioaccumulation potential, and toxicity of these compounds. However, there are also additional concerns regarding environmental contamination and human exposure to PBDEs resulting from informal recycling technology. Decabromodiphenyl ether (decaBDE), one type of PBDE, has carcinogenic potential in the livers of rodents. Although one study has shown that decaBDE exerts genotoxic effects, the other in vitro and in vivo studies were negative for such effects. Thus, it remains unknown whether genotoxic mechanisms are involved in decaBDE-induced hepatocarcinogenesis in rodents. In this study, to explore the genotoxicity of decaBDE in mice, particularly in the context of carcinogenesis, we performed micronucleus assays in the bone marrow and reporter gene mutation assays in the liver using gpt delta mice treated with decaBDE at carcinogenic doses for 28days. Our results demonstrated negative results in micronucleus tests and reporter gene mutation assays. Thus, decaBDE did not exert genotoxic effects at carcinogenic target sites and did not show positive results in conventional in vivo genotoxicity tests in mice for 4-week treatment. Overall, comprehensive evaluation using in vivo genotoxicity data in rats and our data indicated that nongenotoxic mechanisms may be responsible for decaBDE-induced hepatocarcinogenesis.

Effects of Nrf2 silencing on oxidative stress-associated intestinal carcinogenesis in mice.

To assess the risk of colorectal cancer in humans with inactivation of NRF2, Nrf2-proficient (Nrf2(+/+) ) and -deficient (Nrf2(-/-) ) mice were exposed to potassium bromate (KBrO3 ) at concentrations of 750 or 1500 ppm for 52 weeks. Neoplastic proliferative lesions were observed in the small intestine and exhibited accumulations of ß-catenin and cyclin D1. The lesions had characteristics similar to those in experimental models of human hereditary colorectal cancer. An additional 13-week study was performed to examine the role of Nrf2 in the effects of oxidative stress. Significant increase in combined incidences of preneoplastic and neoplastic lesions in Nrf2(-/-) mice administered high-dose KBrO3 . In the short-term study, although 8-hydroxydeoxyguanosine (8-OHdG) levels in the epithelial DNA of Nrf2(-/-) mice at the high dose were significantly lower than those of the corresponding Nrf2(+/+) mice, the difference was very small. mRNA levels of Nrf2-regulated genes were increased in Nrf2(+/+) mice. Overexpression of cyclooxygenase 2 (COX2) and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells in the jejunal crypts were observed in Nrf2(-/-) mice administered high-dose KBrO3 . Overall, these data suggested that individuals having single-nucleotide polymorphisms in NRF2 may have a risk of colorectal cancer to some extent.

Role of p53 in the progression from ochratoxin A-induced DNA damage to gene mutations in the kidneys of mice.

Carcinogenic doses of ochratoxin A (OTA) cause increases of mutant frequencies (MFs) of the red/gam gene (Spi(-)) in the kidneys of p53-deficient gpt delta mice, but not in p53-proficient mice. Here, we investigated the role of p53 in the progression from OTA-induced DNA damage to gene mutations. To this end, p53-proficient and -deficient mice were administered 5 mg/kg OTA for 3 days or 4 weeks by gavage. After 3 days of administration, comet assays were performed and there were no differences in the degrees of OTA-induced DNA damage between p53-proficient and -deficient mice. However, the frequencies of γ-H2AX-positive tubular epithelial cells in p53-deficient mice were significantly higher than those in p53-proficient mice, implying that p53 inhibited the progression from DNA damage to DNA double-strand breaks (DSBs). Evaluation of global gene expression and relevant mRNA/protein expression levels demonstrated that OTA increased the expression of Cdkn1a, which encodes the p21 protein, in p53-proficient mice, but not in p53-deficient mice. Moreover, in p53-deficient mice, mRNA levels of cell cycle progression and DSB repair (homologous recombination repair [HR])-related genes were significantly increased. Thus, G1/S arrest due to activation of the p53/p21 pathway may contribute to the prevention of DSBs in p53-proficient mice. In addition, single base deletions/insertions/substitutions were predominant, possibly due to HR. Overall, these results suggested that OTA induced DSBs at the carcinogenic target site in mice and that p53/p21-mediated cell cycle control prevented an increase in the formation of DSBs, leading to gene mutations.

Alteration in the apoptosis process of rat esophageal epithelium with hyperproliferation of indigenous bacteria under a physiological condition.

The apoptosis process in rat esophageal epithelium was investigated using enzyme-immunohistochemistry and transmission electron microscopy. As a result, Fas and Fas-L were expressed in the epithelial cell membrane and cytoplasm from the stratum spinosum (SS) to the stratum granulosum (SG). No TNF-R1 show immunopositivity in the cell membranes. TNF-α and caspase-8 were not observed in any layer. Caspase-10, cleaved caspase-3, XIAP and DNase-1 were found in the epithelial cytoplasm from the SS to the SG, whereas Bid, Apaf-1 and cleaved caspase-9 were detected only in the SG. Cytochrome c was observed as cytoplasmic granular positivity from the stratum basale (SB) and altered into homogeneous immunopositivity in the SG. Bcl-2 and Bcl-X immunopositivity was detected in cytoplasm from the SB to the SG. Immunoreactions of Bak in the cytoplasm and Bax beneath the cell membrane were observed from the upper portion of the SS with increasing intensity toward the SG. In the sites with the hyperproliferation of indigenous bacteria, TNF-R1, TNF-α and caspase-8 were detected in the SG and the immunopositive intensities of Bid, Apaf-1 and cleaved caspase-9 were altered to be strong. Prominently swollen cells and decreased mitochondria were ultrastructurally confirmed in the uppermost layers of stratum corneum. These findings suggest that the Fas-Fas-L-interaction initially induces apoptosis through a mitochondria-independent pathway and secondarily through a mitochondria-dependent pathway, leading to eventual epithelial cell death in the rat esophageal epithelium. The bacterial stimuli probably enhance the mitochondria-dependent pathway through the TNF-R1-TNF-α interaction.

Immunohistochemical detection of toll-like receptor-2, -4 and -9 in exocrine glands associated with rat alimentary tract.

Localization of Toll-like receptors (TLRs) in the exocrine glands associated with the rat alimentary tract was immunohistochemically studied using anti-TLR antibodies. TLR-2, -4 and -9 were detected in the secretory granules of acinar cells or the luminal substances of the gustatory gland, extraorbital lacrimal gland, Harderian gland, proper gastric gland and pancreas. TLR-2 and -9 were also detected in the mucous acinar cells of the sublingual gland. Positivity for all TLRs was found in the striated borders of columnar epithelial cells and the luminal substances of the intestinal crypts throughout the small intestine, and also in the goblet cells throughout the large intestine. Only TLR-4 was detected in the secretory granules of Paneth cells. A reduction of TLR-4-positive secretory granules and the formation of TLR-4-positive vacuoles were found in the ileal Paneth cells under the hyper-proliferation of indigenous bacteria. In the apical to middle intervillous portions of the ileum, Gram-positive bacterial colonies were significantly more abundant than Gram-negative bacterial colonies, whereas this difference disappeared in the basal intervillous portions. These findings suggest that there are distribution differences in the secretory sources of soluble TLRs that possibly neutralize their luminal ligands, in the rat alimentary tract. Therefore, the bacterial ligand-recognition system composed of the membranous TLRs of villous columnar epithelial cells and soluble TLRs from crypt epithelial cells might contribute to host defense mechanisms for the selective elimination of Gram-positive bacteria rather than Gram-negative bacteria in the rat small intestine.

Immunohistochemical and histoplanimetrical study on the spatial relationship between the settlement of indigenous bacteria and the secretion of bactericidal peptides in rat alimentary tract.

To clarify the regulatory mechanism by bactericidal peptides secretion, the secretion of bactericidal peptides was immunohistochemically and histoplanimetrically compared with the degree of Gram-positive/negative bacterial colonization throughout the rat alimentary tract. In the associated exocrine glands from the oral cavity to the stomach, no comparable differences were observed under the changes of development of indigenous bacterial colonies. In the small intestine, immunopositive granules for lysozyme and secretory phospholipase A2 (sPLA2) were markedly decreased, whereas immunopositive vacuoles in the Paneth cells were more increased at sites with hyper-development of indigenous bacterial colonies in the intervillous spaces than at sites with no or less development. No changes in exocrine glands were observed in the large intestine because of the constant existence of large quantities of bacteria. Gram-positive bacterial colonies on the mucosal surfaces were dominant from the oral cavity to the stomach. Gram-negative bacteria were dominant in the large intestine, and the distributions of both Gram-positive and negative bacteria were intermediate in the small intestine. These findings suggest that lysozyme and sPLA2 secreted from the Paneth cells contribute to the regulation of the proliferation of indigenous bacteria in the intervillous spaces of the small intestine, and that the inversion of distributions of Gram-positive and -negative bacteria in the alimentary tract might be caused by the secretion of lysozyme and sPLA2 in the small intestine.