Cancer in Japan: Prevalence, prevention and the role of heterocyclic amines in human carcinogenesis.

In this article, three topics are being studied in order to understand the present state of cancer in Japan. First, the statistics on cancer mortality indicates that the mortality from cancer in young individuals has been decreasing during the last 50 years, although the total mortality from cancer has been steadily and steeply increasing. Second, epidemiological analyses of cancer causes in Japan indicated that 50 % of cancer cases are preventable, and that prevention of infection and refraining from tobacco smoking will reduce cancer mortality by about 40 %. Third, mutagenic/carcinogenic heterocyclic amines present in cooked meat/fish have been suggested to be carcinogenic in humans in many epidemiological studies carried out in Japan and other countries.

Modification of the ³²P-postlabeling method to detect a single adduct species as a single spot.

The original (32)P-postlabeling method developed by Randerath and his colleagues has been modified to detect a single type of adduct as a single spot in thin-layer chromatography (TLC), because some types of adducts gave multiple adduct spots by the original method. In the remodified methods, DNA is first digested with micrococcal nuclease and phosphodiesterase II and then labeled with [γ-(32)P]ATP under standard or adduct intensification conditions. Since the labeled digest includes adducted mono-, di-, and/or oligo-deoxynucleotides, it is further treated with phosphatase and phosphodiesterase prior to TLC. The labeled digest is treated with nuclease P1 (NP1) in Method I, with T4 polynucleotide kinase and NP1 in Method II, and then with phosphodiesterase I in both cases and subjected to TLC. The advantage of these methods is that the number of adduct species formed can be estimated by TLC.

Genotoxicity evaluation of sesamin and episesamin.

Sesamin is a major lignan that is present in sesame seeds and oil. Sesamin is partially converted to its stereoisomer, episesamin, during the refining process of non-roasted sesame seed oil. We evaluated the genotoxicity of these substances through the following tests: a bacterial reverse mutation assay (Ames test), a chromosomal aberration test in cultured Chinese hamster lung cells (CHL/IU), a bone marrow micronucleus (MN) test in Crlj:CD1 (ICR) mice, and a comet assay using the liver of Sprague-Dawley (SD) rats. Episesamin showed negative results in the Ames test with and without S9 mix, in the in vitro chromosomal aberration test with and without S9 mix, and in the in vivo comet assay. Sesamin showed negative results in the Ames test with and without S9 mix. In the in vitro chromosomal aberration test, sesamin did not induce chromosomal aberrations in the absence of S9 mix, but induced structural abnormalities at cytotoxic concentrations in the presence of S9 mix. Oral administration of sesamin at doses up to 2.0g/kg did not cause a significant increase in either the percentage of micronucleated polychromatic erythrocytes in the in vivo bone marrow MN test or in the % DNA in the comet tails in the in vivo comet assay of liver cells. These findings indicate that sesamin does not damage DNA in vivo and that sesamin and episesamin have no genotoxic activity.

Induction of abnormal nuclear shapes in two distinct modes by overexpression of serine/threonine protein phosphatase 5 in Hela cells.

Okadaic acid-sensitve serine/threonine protein phosphatase 5 (PP5) is expressed ubiquitously in various tissues and is considered to participate in many cellular processes. PP5 has a catalytic domain in the C-terminal region and three tetratricopeptide repeat (TPR) motifs in the N-terminal region, which are suspected to function as a protein-protein interaction domain. Physiological roles of PP5 are still largely unknown, although several PP5-binding proteins were reported and a few in vivo functions of PP5 were suggested. In the present study, the effects of expression of the full-length wild-type PP5 fused with EGFP (EGFP-PP5(WT)) and its phosphatase-dead mutant EGFP-PP5(H304A) were investigated. Transient expression of either EGFP-PP5(WT) or EGFP-PP5(H304A) in HeLa cells induced deformed nuclei with a 10-fold frequency compared to that of EGFP. Abnormal-shaped nuclei were also substantially increased by induced moderate expression of PP5 in tet-on HeLa cells. Many HeLa cells expressing EGFP-PP5(WT) possessed multi-nuclei separated from each other by nuclear membrane, while expression of EGFP-PP5(H304A) induced deformed nuclei which were multiple-like in shape, but not separated completely and were surrounded by one nuclear membrane. These results suggest that PP5 plays important roles at the M-phase of the cell cycle, especially in separation of chromosomes and formation of nuclear membrane.

Unfolding of higher DNA structures formed by the d(CGG) triplet repeat by UP1 protein.

Fragile X syndrome is caused by expansion of a d(CGG) triplet repeat in the 5'-untranslated region of the first exon of the FMR1 gene resulting in silencing of the gene. The d(CGG) repeat has been reported to form hairpin and quadruplex structures in vitro, and formation of these higher structures could be responsible for its unstable expansion in the syndrome, although molecular mechanisms underlying the repeat expansion still remain elusive. We have previously proved that UP1, a proteolytic product of hnRNP A1, unfolds the intramolecular quadruplex structures of d(GGCAG)5 and d(TTAGGG)4 and abrogates the arrest of DNA synthesis at d(GGG)n sites. Here, we demonstrate that the d(CGG) repeat forms a peculiar DNA structure, which deviates from the canonical B-form structure. In addition, UP1 was demonstrated by CD spectrum analysis to unfold this characteristic higher structure of the d(CGG) repeat and to abrogate the arrest of DNA synthesis at the site. This ability of UP1 suggests that unfolding of unusual DNA structures of a triplet repeat is required for DNA synthesis processes.

Effect of chlorophyllin-chitosan on excretion of dioxins in a healthy man.

We investigated the usefulness of chitosan and chlorophyllin-chitosan (chl-chitosan) administration for reduction of the body burden of environmental dioxins, including polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/ Fs) and coplanar polychlorinated biphenyls (Co-PCBs), by examining the excretion levels in the feces and sebum of a healthy man. The volunteer ate the same three meals every day during the 40-d experiment, which was composed of five phases (I-V) of 8 d each. In phase I (days 1-8), the volunteer was given only the basal diet. In phases II-V, 0.2 g of chitosan, 0.6 g of chitosan, 0.2 g of chl-chitosan, and 0.6 g of chl-chitosan, respectively, were administered immediately after each meal. We measured daily the amount of dioxins occurring in the feces and sebum during the last 5 d of each phase. The total toxicity equivalency (TEQ) of the dioxin in phases I-V were 27, 26, 38, 36, and 67 pg/d in the feces and 20, 19, 16, 16, and 14 pg/d in the sebum, compared with 74 pg/d in the food. The excretion of dioxins in the feces was significantly increased in phases III, IV, and V, being 140% (p < 0.05), 135% (p < 0.05), and 249% (p < 0.01) of the control level (phase I). Although the dioxin in the sebum was slightly decreased in phase V as compared with the control level, the total amount of excreted dioxin in feces and sebum was increased significantly in phase V, being 174% of the control level, which is almost the same level as that in the food. This indicates that chl-chitosan can prevent accumulation of dioxin, at least at the intake level of normal foods.

Modification of the 32P-postlabeling method to detect a single adduct species as a single spot.

The original 32P-postlabeling method developed by Randerath and his colleagues has been modified to detect a single type of adduct as a single spot in thin-layer chromatography (TLC), because some types of adducts gave multiple adduct spots by the original method. In the remodified methods, DNA is first digested with micrococcal nuclease and phophodiesterase II and then labeled with [gamma-32P]ATP under standard or adduct-intensification conditions. Since the labeled digest includes adducted mono-, di-, and/or oligo-deoxynucleotides, it is further treated with phosphatase and phosphodiesterase prior to TLC. The labeled digest is treated with nuclease P1 (NP1) in method I, and with T4 polynucleotide kinase and NP1 in method II, and then with phosphodiesterase I in both cases, and subjected to TLC. The advantage of these methods is that the number of adduct species formed can be estimated by TLC.

LRP130, a single-stranded DNA/RNA-binding protein, localizes at the outer nuclear and endoplasmic reticulum membrane, and interacts with mRNA in vivo.

LRP130 (also known as a LRPPRC) is an RNA and single-stranded DNA-binding protein, and recently identified as a candidate gene responsible for the Leigh syndrome, a French-Canadian type cytochrome c oxidase deficiency. However, the biological function of LRP130 still remains largely unresolved. In the present study, we found that the C-terminal half of the mouse LRP130 located within a 120 amino acid sequence (a.a. 845-964) binds to synthetic RNA homopolymers, poly(G), poly(U), and poly(C), as well as r(CUGCC)(6). Assessment of the subcellular localization indicated both nuclear/endoplasmic reticulum (ER) and mitochondrial fractions to be positive. To further analyze the subcellular localization of LRP130, a nuclear/ER fraction was fractionated into the nucleoplasm (NP) and nuclear envelope (NE)/ER, and the latter was further separated into outer nuclear membrane (ONM)/ER and inner nuclear membrane (INM) by treatment with Triton X-100. LRP130 was detectable in all three fractions, and the distribution pattern was in good accordance with that known for ONM/ER proteins. Interestingly, immunostaining of HeLa cells demonstrated nuclear rim staining of LRP130, specifically at the outside of the NE and also at ER, and association of LRP130 with poly(A)(+) RNA was restricted only to the ONM/ER fraction. Overexpression of full-length mouse LRP130 fused with EGFP resulted in nuclear accumulation of poly(A)(+) RNA in HeLa cells. Taking all these results together, it is suggested that LRP130, a novel type of RNA-binding protein, associates with mRNA/mRNP complexes at the outside of NE and ER, and plays a role in control of mRNA metabolisms.

Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish.

Research leading to the discovery of a series of mutagenic and carcinogenic heterocyclic amines (HCAs) was inspired by the idea that smoke produced during cooking of food, especially meat or fish, might be carcinogenic. More than ten kinds of HCAs, actually produced by cooking or heating of meat or fish, have now been isolated and their structures determined, most being previously unregistered compounds. They are highly mutagenic towards Salmonella typhimurium in the presence of S9 mix and are also mutagenic in vitro and in vivo toward mammalian cells. HCAs have now been chemically synthesized in quantity and subjected to long-term animal testing. When HCAs were fed in the diet, rodents developed cancers in many organs, including the colon, breast and prostate, and one HCA produced hepatomas in monkeys. The lesions exhibited alteration in genes including Apc, beta-catenin and Ha-ras, and these changes provide clues to the induction mechanisms. The HCAs are oxidized to hydroxyamino derivatives by cytochrome P450s, and further converted to ester forms by acetyltransferase and sulfotransferase. Eventually, they produce DNA adducts through the formation of N-C bonds at guanine bases. There are HCA-sensitive and resistant strains of rodents and a search for the responsible genes is now under way. While the content of HCAs in dishes consumed in ordinary life is low and not sufficient in itself to explain human cancer, the coexistence of many other mutagens/carcinogens of either autobiotic or xenobiotic type and the possibility that HCAs induce genomic instability and heightened sensitivity to tumor promoters suggest that avoidance of exposure to HCAs or reduction of HCAs' biological effects as far as possible are to be highly recommended. Usage of microwave ovens for cooking and supplementation of the diet, for example with soy-isoflavones, which have been found to suppress the occurrence of HCA-induced breast cancers, should be encouraged. Advice to the general public about how to reduce the carcinogenic load imposed by HCAs would be an important contribution to cancer prevention.

Global gene expression analysis of rat colon cancers induced by a food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Colon cancers develop after accumulation of multiple genetic and epigenetic alterations in colon epithelial cells. To shed light on global changes in gene expression of colon cancers and to gain further insight into the molecular mechanisms underlying colon carcinogenesis, we have conducted a comprehensive microarray analysis of mRNA using a rat colon cancer model with the food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Of 8749 genes or ESTs on a high density oligonucleotide microarray, 27 and 46 were over- and underexpressed, respectively, by > or =3-fold in colon cancers in common in two rat strains with distinct susceptibility to PhIP carcinogenesis. For example, genes involved in inflammation and matrix proteases and a cell cycle regulator gene, cyclin D2, were highly expressed in colon cancers. In contrast, genes encoding structural proteins, muscle-related proteins, matrix-composing and mucin-like proteins were underexpressed. Interestingly, a subset of genes whose expression is characteristic of Paneth cells, i.e. the defensins and matrilysin, were highly overexpressed in colon cancers. The presence of defensin 3 and defensin 5 transcripts in cancer cells could also be confirmed by in situ mRNA hybridization. Furthermore, Alcian blue/periodic acid Schiff base (AB-PAS) staining and immunohistochemical analysis with an anti-lysozyme antibody demonstrated Paneth cells in the cancer tissues. AB-PAS-positive cells were also observed in high grade dysplastic aberrant crypt foci, which are considered to be preneoplastic lesions of the colon. Our results suggest that Paneth cell differentiation in colon epithelial cells could be an early morphological change in cryptic cells during colon carcinogenesis.

Destruction of quadruplex by proteins, and its biological implications in replication and telomere maintenance.

The minisatellite DNA Pc-1 consists of tandem repeats of d(GGCAG). We previously reported that a d(GGCAG)n strand folds into an intramolecular quadruplex under physiological conditions and that during replication the progression of DNA polymerase is blocked by the quadruplex in vitro. Therefore, the formation of the quadruplex was supposed to be responsible for the hypermutable features of Pc-1. Then, we have identified proteins that bind to Pc-1, one of which is hnRNP A1. Here, we have demonstrated that hnRNP A1 destroys the quadruplex of Pc-1 on binding and abrogates the arrest of DNA polymerase at the repeat. Thus, hnRNP A1 functions as if it is a chaperon to assist Pc-1 DNA to form the proper folding suitable for replication. We have also found that hnRNP A1 and a related protein, hnRNP D, destroy the quadruplex of telomere DNA, which suggests the involvement of these proteins in telomere maintenance as DNA chaperons.

Characterization of dysplastic aberrant crypt foci in the rat colon induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

The multistage model of colon carcinogenesis is well established in both humans and experimental animals, and aberrant crypt foci (ACF) are generally assumed to be putative preneoplastic lesions of the colon. However, morphological analyses of ACF have suggested that they are highly heterogeneous in nature and their role in tumorigenesis is still controversial. To better understand the biological significance of ACF in carcinogenesis, morphological and genetic analyses were performed using a rat colon cancer model induced by a food-borne colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). ACF of different sizes were collected at weeks 6, 18, 25, and 32 after three cycles of 2-week PhIP feeding (400 ppm in diet) with 4-week intervals on a high-fat diet, and a total of 110 ACF, representing approximately three-quarters of the total ACF, were subjected to histological evaluation. Thirty (27%) were diagnosed as dysplastic ACF, based on cytological and structural abnormalities of crypts. Dysplastic ACF were detected even at week 6 (0.4 per rat), and the numbers increased slightly at later time points, being 0.8, 1.4, and 0.8 per rat at weeks 18, 25, and 32, respectively. The sizes of these dysplastic ACF varied widely from 1 to 16 crypts and 50% (15 of 30) were composed of less than 4 crypts. Immunohistochemical analysis revealed that 83% (25 of 30) of dysplastic ACF demonstrated beta-catenin accumulation; 22 only in the cytoplasm and 3 in both the cytoplasm and nucleus, the latter manifesting a higher grade of dysplasia as compared with the former. Seven dysplastic ACF harbored beta-catenin mutations at codon 32, 34, or 36 in exon 2, and one had an Apc mutation at the boundary of intron 10 and exon 11. Mutations at these sites were also commonly found in colon tumors induced by PhIP. The results of our present study indicate that dysplastic ACF, which accounted for approximately one-fourth of the total ACF, are preneoplastic lesions of colon cancers induced by PhIP in rats.

Effective pretreatment of human serum samples for dioxin analysis by solid phase extraction and blue-chitin column cleanup.

For the analysis of dioxins, polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/Fs) and coplanar polychlorinated biphenyls (Co-PCBs), devising simple and economical methods is important, especially for mass screening of human exposure. Pretreatment of samples, namely the extraction and cleanup methods that are widely used at present, needs to be improved for savings in time, manpower, and solvent consumption. In the present study, we applied solid phase extraction (SPE) using octadecyl (C18) and a blue-chitin column in place of liquid-liquid extraction (LE) and an active-carbon column with serum samples, frequently used for assessment of human exposure. Efficacy of the new pretreatment methods was demonstrated by successful high resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS) of the major 17 PCDDs/Fs and 12 Co-PCBs that are on the list of WHO/IPCS (1997) hazardous dioxins with toxic equivalent factor (TEF) values. SPE is timesaving and requires less manpower and organic solvent as compared with the LE that is presently widely used. Concerning cleanup with blue-chitin, the amount of toluene applied as eluent could be reduced to 1/3, as compared with the active-carbon case. The combination of SPE and blue-chitin for pretreatment of serum saves time and manpower, is accurate and uses less organic solvent than LE with active carbon cleanup.

Differential gene expression profiles in colon epithelium of two rat strains with distinct susceptibility to colon carcinogenesis after exposure to PhIP in combination with dietary high fat.

Colon cancers develop through accumulation of multiple genetic and epigenetic alterations in colon epithelial cells, and the environment of the genetically altered epithelial cells may also have a substantial impact on their further development to cancer. In the present study, groups of 6-week-old F344 and ACI male rats, the former strain being susceptible to colon carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and the latter being relatively resistant, were subjected to a long-term carcinogenesis experiment using our intermittent feeding protocol of PhIP in combination with a high-fat diet, which serves as a relevant risk factor that promotes the development of colon cancers. Animals were sacrificed at 60 weeks, and global gene expression analyses of normal parts of colon epithelial tissues were conducted using a high-density oligonucleotide microarray to elucidate the differential gene expression profile (environment) in normal colonic regions between F344 and ACI strains. Of 8799 entries on the RatU34A array, 74 genes exhibited 3-fold or greater variation. A subset of genes encoding ribosomal RNAs and proteins were highly preferentially expressed in the F344 strain. In addition, genes encoding fatty acid binding proteins and the peroxisome membrane protein 70 appeared up-regulated in the susceptible F344 strain. In the ACI strain, a mismatch repair gene, Msh2, was preferentially expressed, at approximately 20-fold the F344 level, along with a gene encoding a detoxification enzyme, catechol-O-methyltransferase. The combined effects of the repertoire of these differentially expressed genes in normal colon epithelial tissues may account for the distinct susceptibilities of F344 and ACI strains to colon carcinogenesis.

Beta-catenin mutations in liver tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in CDF1 mice.

Heterocyclic amines are potent mutagens and carcinogens formed in cooked protein rich foods. In this study, we screened liver tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in CDF1 mice for beta-catenin and APC mutations and other genetic alterations shown to occur in human hepatocellular carcinomas (HCC), including mutations in the p53 and H-ras genes, c-myc amplification and E-cadherin promoter methylation. SSCP followed by direct DNA sequencing revealed mutations in exon 2 of the beta-catenin gene in 2 of 16 liver tumors (12.5%). Promoter methylation of the E-cadherin gene was detected in one liver tumor induced by MeIQ. There were no mutations in the mutation cluster region of the APC gene, in exons 5-8 of the p53 gene, or in codons 12, 13 and 61 of the H-ras gene, nor c-myc amplification in any of liver tumors induced by MeIQ. These data indicate that except for the occasional disruption of the Wnt pathway through beta-catenin mutations, the genetic pathways involved in the development of HCC differ significantly between human liver cancer and tumors induced in mice by MeIQ, but do not rule out the possibility that heterocyclic amines constitute a carcinogenic risk factor in humans.

Soy protein isolate enhances hepatic copper accumulation and cell damage in LEC rats.

In a series of experiments, the effects of soy protein isolate (SPI), defatted soy (DFS) or SPI supplemented with L-methionine (SPIM) were examined in the Long-Evans rat with a cinnamon coat color (LEC rat), a model animal of Wilson's disease with a hereditary defect in the Atp7b gene resulting in defective copper metabolism and copper accumulation in hepatocytes. Milk casein in the control AIN-93G diet (20 g/100 g) was totally or 60% replaced by the soy products, SPI, DFS or SPIM (L-Met added to be equal to that in the control diet) beginning when rats were 6 wk old. Copper and iron concentrations in SPI and DFS were measured and the concentrations of these metals in the salt mix were adjusted so that test and the control diets had the same final concentrations. Food intake did not differ among groups. Rats were euthanized when they became moribund with jaundice. Survival time in the SPI diet group was shorter (14.0 +/- 0.8 wk) than in the control group (19.1 +/- 1.7 wk) (P < 0.001), and that in the DFS diet group was intermediate (16.0 +/- 1.7 wk). Survival time in the SPIM diet group did not differ from that of the SPI diet group. Copper concentrations in the livers of rats in the SPI and SPIM diet groups were approximately 80% higher than in rats fed the control diet. Liver iron concentrations did not differ among the groups. The results, including histological analyses, indicate that SPI enhances copper uptake into the liver cells and promotes liver cell damage in LEC rats. However, this did not occur in the livers of F344 rats with wild-type Atp7b. Recommendations to individuals suffering from Wilson's disease to avoid consuming soy protein may be warranted.

Effects of soy protein isolate on LEC rats, a model of Wilson disease: mechanisms underlying enhancement of liver cell damage.

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.

Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1.

The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)(n) folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible for the hypermutable feature of Pc-1 and other MNs with similar repetitive units. On the other hand, we have isolated six MN Pc-1 binding proteins (MNBPs) from nuclear extracts of NIH 3T3 cells. Here, we describe one of those MNBPs, MNBP-B, that binds to the single-stranded d(GGCAG)(n). Amino acid sequences of seven proteolytic peptide fragments of MNBP-B were determined, and the cDNA clones were isolated. MNBP-B was proven identical to the single-stranded DNA-binding protein, UP1. Recombinant UP1 bound to single-stranded d(GGCAG)(n) and other G-rich repetitive sequences, such as d(GTCAGG)(n) and d(GTTAGG)(n). In addition, UP1 was demonstrated by CD spectrum analysis to unfold the intramolecular quadruplex structure of d(GGCAG)(5) and d(TTAGGG)(4) and to abrogate the arrest of DNA synthesis at the d(GGG)(n) site. This ability of UP1 suggests that unfolding of quadruplex DNA is required for DNA synthesis processes.

A rat colon cancer model induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines contained in cooked meat and fish, and induces aberrant crypt foci (ACF), putative preneoplastic lesions of the colon, and colon cancers in male rats when administered orally. As has been reported previously, F344 rats are susceptible to induction of ACF by PhIP, while ACI rats being relatively resistant. Approximately one-fourth of ACF induced by PhIP in F344 rats are dysplastic; exhibiting lesions with structural distortion of the crypt, decrease of goblet cells, nuclear stratification and enlargement of nuclei. Dysplastic ACF demonstrate beta-catenin accumulation, mainly in the cytoplasm, and increased cell proliferation in crypts. These dysplastic ACF are, therefore, strongly considered to be putative preneoplastic lesions of the colon.A genetic trait affecting the susceptibility to colon carcinogenesis in F344 rats was mapped to chromosome 16, between D16Rat17 and D16Wox3, using the number of ACF as a surrogate biomarker for colon carcinogenesis. Since the number of dysplastic lesions is well correlated with the total number of ACF, being approximately one-fourth of the total ACF as described above in F344 rats and will be described elsewhere in ACI rats, the gene involved in the susceptibility to ACF induction may possibly be partly responsible for the susceptibility to colon carcinogenesis by PhIP. We, thus, tentatively referred the name of the candidate susceptibility gene on rat chromosome 16 as susceptibility to colon tumor (Sct). In the present study, the colonic lesions induced by PhIP were well refined histologically and genetically, and the multi-step profiles of colon cancer development by PhIP were well characterized and revealed to be similar to the multi-step model of colon carcinogenesis in humans. The PhIP-induced colon cancer model in rats, thus contributes as a relevant tool to elucidate genetic factors responsible for susceptibility to colon carcinogenesis in human. Other unknown genetic or epigenetic alterations, which are essential for the development of early lesions of colon carcinogenesis, could also be clarified using this system.

Transient forebrain ischemia induces expression of serine/threonine protein phosphatase 1 mRNA in the vulnerable regions of gerbil brain.

Apoptosis is thought to be implicated in delayed neuronal cell death following transient forebrain ischemia. Recently, apoptosis in neurons induced by an inhibitor of serine/threonine (ser/thr) protein phosphatases (PPs) has been reported. In this study, we investigated the effect of transient forebrain ischemia on the expression of ser/thr PPs in the brain of Mongolian gerbils. At 24 h after 5-min bilateral carotid artery occlusion, Northern blotting analysis revealed the increase of PP1 mRNA expression in the vulnerable CA1 region of the hippocampus and striatum, but not in the cortex and CA3 region. In contrast, the protein level of PP1 detected by Western blotting analysis decreased in all regions. We conclude that the inhibition in PPs expression in the vulnerable regions may affect cell death after transient forebrain ischemia.