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The gestagenic and antiandrogenic drug cyproterone acetate (CPA is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue™ transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue™ rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 ± 4) x 10-6 spontaneous mutations was increased to a maximum of (80 ± 8) x 10-6 mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 ± 5) and (27 ± 5) x 10-6, respectively. The mutation frequency at doses of 50 and 25 CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of 12 000 ± 3000 adducts per 109 nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations. [ABSTRACT FROM PUBLISHER]

The effect of di(2-ethylhexyl)phthalate (DEHP) on diethyl nitrosainlne (DEN)-initiated preneoplastic liver lesions with expression of gamma-glutamyltranspeptidase (GGTase) and loss of adenosine trlphosphatase (ATPase) as well as alterations of hepatic carbohydrate metabolism in male and female Sprague-Dawley rats have been investigated. Two treatment schedules have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic islands and by the biochemical determination of alterations in enzyme activities of liver homogenates and of serum, the last indicating hepatotoxicity. For initiation, a single dose of DEN was given, followed by treatment with various doses of DEHP given three times weekly by gavage for 7 or 11 consecutive weeks. As histochemical enzyme markers, the expression of positive GGTase as well as the deficiency in ATPase were used for identification of liver foci. The weanling female rats (protocol A) were found to be more sensitive to the carcinogenic effect of DEN in view of foci incidence than the mature male rats which underwent partial hepatectomy prior to DEN application. The administration of 200 mg DEHP/kg body increased the incidence of ATPase-deficient foci in both male and female rats; however, concentrations of 1000 and 2000 mg DEHP/kg decreased the incidence of liver foci. The number of foci with expression of GGTase was only slightly increased in female rats following a DELIP concentration of 50 mg/kg, and 200 mg/kg body wt. DEHP alone did not induce preneoplastic lesions that could be identified by these two markers. Biochemical investigations indicate that DEHP alters the metabolic pattern in liver. An increase of the NADP-linked enzymes glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, extra-mitochondrial ICDH as well as an enhancement of NAD-dependent α-G3PDH and lactate dehydrogenase were found following DEHP administration. On the other hand the glycolytlc enzymes pyruvate kinase (PK) and enolase as well as the gluconeogenelic enzyme fructose-1,6.-bisphosphatase (FBPase) were significantly reduced. In protocol B (male rats) the reactions of PK, FBPase and malic enzyme were more altered after DEHP exposure than in protocol A, while the activity of G6PDH was more increased in protocol A. Most enzymes being involved in the carbohydrate metabolism are influenced by DEHP In a dose-dependent manner. There was no increase in senim FBPase activity in both male and female rats after DEHP treatment but a reduction of glutamate-oxalate-transaminase and glutainate-pyruvate-transaminase activities was observed. This and the fact that the control and DEHP-treated animals had similar weight gains indicate that DEHP does not exert a significant hepatotoxicity effect. [ABSTRACT FROM PUBLISHER]

Three rat liver foci bioassays have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic foci, and by the biochemical determination of alterations in enzyme activities of serum indicating hepatotoxicity. We studied the initiation/promotion schedules according to Oesterle and Deml (A), and according to Pereira (B, Broad Spectrum Protocol), and the initiation/selection protocol according to Tatematsu . (C), with diethyl nitrosamine (DEN), given as a single initiating dose of 10 and 30 mg/kg body wt respectively. With all schedules Sprague-Dawley rats, either females, 3 weeks old (A), or males, 6 weeks old (B, C) were used. For promotion polychiorinated biphenyls (A) or phenobarbital (B) were administered. Selection was performed with 2-acetylaminofluorene (C). The rats in schemes (B) and (C) underwent partial hepatectomy one day prior to initiation. The number and total area of foci deficient in adenoslne-5'-triphosphatase (ATPase) and positive in γ-glutamyttranspeptidase (GGTase) was evaluated. In the complete schedule with 30mg of DEN in system (A) foci incidence exceeded that of the other systems by about 7-fold (ATPase) and 2-fold (G.GTase) respectively. The tower dose of DEN and all control experiments resulted In a respective lower foci yield. With scheme (C), but not with schemes (A) and (B), e.g. serum fructose-1.6-bisphosphatase and alkaline phosphatase were increased, suggesting liver cell damage. Thus tested with DEN, scheme (A) is most sensitive and causes a low impairment of animals' welfare. [ABSTRACT FROM PUBLISHER]

Cyproterone acetate (CPA) is a synthetic steroid which is used in oral contraceptive and anti-androgen formulations. It has previously been classified as a tumor promoter in rat liver. Recent studies have shown that CPA induces DNA repair synthesis in isolated hepatocytes, and this implies that CPA is genotoxic. We studied the initiating activity of CPA by means of a rat liver foci bioassay, using weanling female Sprague-Dawley rats. The results show that CPA induces adenosine-triphosphatase-deficient and γ-glutamyltrans-peptidase-positive putative preneoplastic foci in a dose-dependent manner. This indicates that CPA has not only promoting but also initiating activity and may therefore act as a complete carcinogen in rat liver. [ABSTRACT FROM PUBLISHER]

Abstract: The potential of two asbestos substitute mineral fibres – rock (stone) wool RW1 and glass wool MMVF10 – to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous λ-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2mg/animal of fibres or with multiple doses of 2mg/animal administered weekly on four consecutive weeks (8mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RW1 fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2mg and 4×2mg, suggesting that any threshold for inflammation lies below the dose of 2mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres. [Copyright &y& Elsevier]

Abstract: In an attempt to examine the interaction of man-made mineral fibres with benzo[a]pyrene (B[a]P), homozygous λ-lacI transgenic F344 rats were intratracheally treated with rock (stone) wool RW1 and glass wool MMVF 10 fibres together with B[a]P. To analyze the induction of gene mutations by fibres and B[a]P in lung, single doses of 1 and 2mg fibres/animal or multiple doses of 2mg fibres/animal were administered weekly on 4 consecutive weeks (total dose 8mg/animal). B[a]P (10mg/animal) was administered either simultaneously with fibres (for single dose treatment with fibres) or together with the last fiber treatment (for multiple dose treatment with fibres). Animals were scarified 4 weeks after the last treatment. Benzo[a]pyrene administered simultaneously with RW1 fibres exhibited a strong synergistic effect on mutagenicity, the observed mutant frequency (MF) being more than three-fold higher than the net sum of the MF induced after separate administration of both agents. Our data suggest that DNA adducts induced by simultaneous B[a]P and fiber treatment lead to a strong increase in mutatant frequencies. [Copyright &y& Elsevier]

In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic λ-lacI (Big Blue™) rats were intratracheally instilled with single doses of 1 or 2 mg, or with four weekly doses of 2 mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 × 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect. [Copyright &y& Elsevier]

To study the suspected mechanism of the interaction between tobacco smoking and asbestos exposure in the modulation of cancer risk, the mutagenic potential of asbestos in combination with the tobacco smoke carcinogen benzo[a]pyrene (B[a]P) was examined in vivo in the rat lung. B[a]P was administered intratracheally in one set of experiments, or by two daily intraperitoneal injections in another set of experiments, to λlacI transgenic rats, together with 1, 2 or 4 × 2 mg amosite in one experiment. In the first experiment, the combined action of amosite and B[a]P caused a synergistic (superadditive) increase of mutation frequency in the lung, as compared to groups treated only with asbestos or B[a]P. In the second experiment, i.p. treatment with B[a]P did not significantly alter the mutation frequency induced by amosite, neither after 4 nor after 16 weeks of exposure. The B[a]P-DNA adduct levels were unaffected by amosite co-treatment in both experiments. We assume that the synergistic increase of mutation frequency after intratracheal treatment was due to the mitogenic activities of B[a]P and of amosite. In conclusion, our findings indicate that a weak and delayed mutagenic effect of amosite in rat lung observed in another study was strongly enhanced by the concomitant action of B[a]P. The striking enhancement effect of B[a]P may provide a basis for understanding the suspected synergism of smoking on asbestos carcinogenesis. [Copyright &y& Elsevier]

Objectives: The current study examined the association between alcohol use frequency (ie, days a week one consumes alcohol), sexual and gender identity, and bystander confidence to intervene in interpersonal violence (ie, bystander self-efficacy). Participants: Participants were 750 undergraduate students aged 18-25 (260 heterosexual men, 260 heterosexual women, 59 SM men [54 cisgender, 5 transgender men], and 171 SM women [169 cisgender, 2 transgender women]). Methods: Participants completed an online survey about alcohol and sexual behaviors. Results: Results indicated that (1) alcohol use frequency was positively associated with greater bystander self-efficacy, (2) heterosexual men, compared to heterosexual women, reported lower bystander self-efficacy, and (3) the association between alcohol use frequency and bystander self-efficacy was significant and positive among heterosexual, but not SM, women. Conclusions: Prevention efforts may benefit from targeting individuals who drink more frequently and ensuring that they have the skills to effectively intervene.

Sexual assault is a significant problem within the United States military. Bystander intervention skills training is recognized as a promising strategy for sexual assault prevention within both civilian and military populations. Sexual assault prevention programs which include training in bystander intervention teach individuals to notice situations that may pose a risk for harm and safely act to positively influence the outcome. This study examines correlates of bystander intervention attitudes and intentions among young adult active duty male soldiers (N = 282) between the ages of 18 and 24. Positive bystander intervention attitudes and intentions were associated with lower levels of rape myth acceptance, greater discomfort with sexism, lower likelihood of continuing an unwanted sexual advance after verbal resistance from a partner, greater likelihood of gaining verbal consent from a partner, and greater perceived peer approval for bystander intervention. In a multiple regression, perceived peer approval for bystander intervention and self-reported lower likelihood of continuing a sexual advance after verbal resistance from a partner emerged as significant predictors of positive bystander intervention attitudes and intentions (R 2 = .41). Given that perceptions of peer norms are modifiable, these findings highlight the importance of addressing peer norms in bystander intervention training programs for military personnel.

The present study examined the mediating role of sexual assertiveness in the relationship between psychological, physical, and sexual intimate partner violence (IPV) victimization and unprotected sex as a result of condom use resistance among sexually active young women attending community college. Women reported engagement in unprotected sex as a result of a partner's use of one of 32 forms of condom use resistance (e.g., physical force, deception, or other forms of coercion to avoid using a condom during intercourse). Women ages 18-24 years (N = 212) attending community college were recruited through paper advertisements to complete assessments of social and dating behavior in the campus computer laboratory. Only the women with a history of sexual intercourse (N = 178; 84% of the sample) were included in analyses. More frequent engagement in unprotected sex as a result of a partner's condom use resistance was associated with physical, psychological, and sexual IPV victimization. Sexual assertiveness mediated the relationship between physical IPV victimization and the frequency of unprotected sex as a result of condom use resistance. Efforts to prevent dating violence and enhance the sexual health of community college women may benefit from focusing on targeting sexual assertiveness as a protective factor.

Despite the prevalence of sexual assault on college campuses, few interventions aimed at decreasing college men's proclivity to perpetrate sexual aggression have been developed and tested. This article details the theoretical framework, content, and piloting of a sexual assault prevention program for college men who engage in heavy drinking, a high-risk group who may be particularly well positioned to intervene as proactive bystanders in drinking environments. In an open trial, male facilitators delivered the three-session Sexual Assault and Alcohol Feedback and Education (SAFE) program to 25 heavy drinking college men. Session 1 was a 90-min review of personalized normative feedback regarding alcohol use, sexual activity, alcohol-related sexual consequences, understanding of consent, and engagement in bystander intervention, delivered individually in a motivational interviewing style. Session 2 was a 2½-hr group-based sexual assault prevention workshop focusing on social norms, empathy, masculinity, consent, and bystander intervention. Session 3 was a 90-min booster group session that reviewed previous topics and included the active practice of bystander intervention skills. Analyses of postsession assessments of utility, therapeutic alliance, and satisfaction and examination of alcohol use and sexual assault-related outcomes from baseline to the 2-month assessment support the preliminary feasibility and acceptability of the SAFE program.

Current efforts to reduce sexual violence in college campuses underscore the role of engaging men in prosocial bystander behavior. The current study implemented an online survey to explore associations between engaging in heavy drinking and attitudes toward bystander intervention among a sample of college men (N = 242). Correlates of sexual aggression were also explored as mediators of the hypothesized relationship between engaging in heavy drinking and attitudes toward bystander intervention. Data indicated that men who engaged in two or more episodes of heavy drinking over the past month reported lower prosocial bystander attitudes compared with men who did not engage in such behavior. The association between engaging in heavy drinking and lower prosocial bystander attitudes was mediated by men's perception of their peers' approval for sexual aggression, their own comfort with sexism, and engagement in coercive sexual behavior. Implications for sexual assault prevention are discussed., (© The Author(s) 2015.)

Nitric oxide (NO) induces relaxation of colonic smooth muscle cells predominantly by cGMP/cGMP-dependent protein kinase I (cGKI)-induced phosphorylation of the inositol 1,4,5-trisphosphate receptor (IP(3)R)-associated cGMP kinase substrate (IRAG), to block store-dependent calcium signaling. In the present study we analyzed the structure and function of the human IRAG/MRVI1 gene. We describe four unique first exon variants transcribed from individual promoters in diverse human tissues. Tissue-specific alternative splicing with exon skipping and alternative splice donor and acceptor site usage further increases diversity of IRAG mRNA variants that encode for NH(2)- and COOH-terminally truncated proteins. At the functional level, COOH-terminally truncated IRAG variants lacking both the cGKI phosphorylation and the IP(3)RI interaction site counteract cGMP-mediated inhibition of calcium transients and relaxation of human colonic smooth muscle cells. Since COOH-terminally truncated IRAG mRNA isoforms are widely expressed in human tissues, our results point to an important role of IRAG variants as negative modulators of nitric oxide/cGKI-dependent signaling. The complexity of alternative splicing of the IRAG gene impressively demonstrates how posttranscriptional processing generates functionally distinct proteins from a single gene.

APC(min/+) mice, carrying a nonsense mutation in the adenomatous polyposis coli (APC) gene, appear as a perfect model to study development or therapy of intestinal neoplasia. We tested whether the flavonoid flavone is able to affect adenoma development in APC(min/+) mice. Tumor sizes were significantly increased by flavone selectively in small intestine. This was associated with reduced cell numbers displaying cleaved caspase-3 and enhanced expression of phosphoglycoprotein (P-gp). However, according to great variability in P-gp expression in all parts of mice intestines, an association between expression of P-gp and inhibition of apoptosis was demonstrated in human Caco-2 colorectal cancer cells.

The cholinergic neurotransmitter system and prolonged glucocorticoid-induced stress can affect cognitive functions in opposite ways. While pharmacological enhancement of cholinergic neurotransmission is known to induce neuroprotective effects, chronic glucocorticoids impair cognitive functions. Up to now, there is no consensus as to whether a subchronic stress period of several days would affect cognitive function. The goal of this study was to investigate whether or not repeated applications of physostigmine over 3 days lead to protective effects on rat spatial cognitive abilities in contrast to the deteriorating effect on rat cognitive function after corticosterone treatment. Furthermore, we wanted to investigate in what extent this cognition-modulating effect is associated with rat cerebral acetylcholinergic system. Male adult rats (n = 40) were randomly divided into four groups with n = 10 per group: (I) placebo-, (II) corticosterone- (15 mg/day), (III) physostigmine- (0.014 mg/day), and (IV) physostigmine + corticosterone-treated rats. Body mass and plasma corticosterone concentrations were measured. Psychometric investigations were conducted using a Morris water maze before and after a subchronic treatment. In cerebral tissue, ACh and acetylcholinesterase (AChE) content and ACh receptor density were determined. Tissue corticosterone concentration was measured in cerebral cortex, hippocampus, and adrenal glands. In corticosterone-treated rats, reduced spatial cognitive abilities were associated with a significant increase in plasma (+25%) and cerebral corticosterone levels (+350%) parallelled by a significant reduction in adrenal gland concentrations (-84%) as compared to placebo. Repeated physostigmine injections improved rats' spatial memory and increased cerebral ACh and AChE content (p < 0.05). When physostigmine was administered at the same time as corticosterone (group IV), it was not able to reverse the corticosterone effect. A significant correlation was detected between cerebral AChE and corticosterone concentrations as well as between AChE and psychometric parameters. We conclude that subchronic exogenous corticosterone administration induces memory dysfunction whereas physostigmine exerts cognitive-enhancing effects if given for 3 days. An apparently existing interaction between glucocorticoid excess and ACh metabolism is discussed.

Scope: Colorectal cancer is a major cause of cancer deaths worldwide with the need for improved therapeutics and adjuvants., Methods and Results: We here tested whether the secondary plant compound flavone affects the development of aberrant crypt foci and microadenomas triggered in C57BL/6J mice by 1,2-dimethylhydrazine. Ten weeks after the last 1,2-dimethylhydrazine injection, flavone was applied at 400 mg/kg body weight over 4 wk by gavage. Flavone was found to increase apoptosis and to reduce the rate of proliferation and aberrant crypt formation. More importantly, development of microadenomas was completely suppressed by flavone. Proteome analysis by 2-DE with mass spectrometric identification of regulated proteins suggests a downregulation of tricarboxylic acid cycle activity in colonocytes with compensation by increased FADH(2) production via a partial beta-oxidation of long-chain fatty acids to meet energy demands. Transcriptome analysis, using a Gene Chip expression array with 24,000 gene probes confirmed the proteome data and moreover revealed the increased expression of various solute transporters, suggesting increased substrate supply to be used for tricarboxylic acid cycle-independent energy production., Conclusion: In conclusion, changes in the levels of proteins from intermediary metabolism or their encoding mRNAs are linked to flavone-induced apoptosis and the prevention of microadenoma formation in transformed colonocytes of mice.

Colon cancer patients frequently show increased levels of serum insulin-like growth factor-binding protein-2 (IGFBP-2), however, the pathogenetic relevance of this phenomenon for colorectal cancer is unclear. Therefore, we have used IGFBP-2 transgenic animals which overexpress IGFBP-2 systemically and locally in the intestine to study its role in chemically induced colorectal carcinogenesis. Mice received intraperitoneal injections of 1,2-dimethylhydrazine (DMH) (40 mg/kg body weight) once a week for 6 weeks to selectively induce aberrant crypt foci (ACF) and tumors in the colon. While tumor incidence was comparable in transgenic and control mice, the volume of adenomas in IGFBP-2 transgenic mice was reduced more than 2-fold. Furthermore, serum IGFBP-2 levels negatively correlated with tumor volume in the IGFBP-2 transgenic group. Histological examination showed that IGFBP-2 transgenic mice developed significantly less dysplastic ACF with a high potential to progress to advanced stages. The reduced tumor volume in IGFBP-2 transgenic animals was due to significantly reduced proliferative capacity, evidenced by a lower proportion of cells positive for Ki67. Our results demonstrate for the first time in an experimental model that IGFBP-2 overabundance prior to the onset and during colorectal carcinogenesis reduces tumor growth by inhibition of cell proliferation., ((c) 2008 Wiley-Liss, Inc.)

Propylene oxide (PO) concentrations >or=300 ppm induced cell proliferation and tumors in rat nasal respiratory epithelium (NRE). Cell proliferation was suggested to result from depletion of glutathione (GSH) in NRE. In order to substantiate this hypothesis, cell proliferation - measured by bromodeoxyuridine incorporation into DNA of the epithelium lining middle septum, dorsal medial meatus, and medial and lateral surfaces of the nasoturbinate in transverse nasal sections taken immediately posterior to the upper incisor teeth - and water-soluble non-protein thiol (NPSH) in NRE were determined after exposing male Fischer 344 rats to 50 ppm, 100 ppm, 200 ppm, or 300 ppm PO (6 h/day, 3 days). Both parameters were also investigated after treating rats for 3 days with diethylmaleate (DEM; 2 x 250 mg/kg/day or 500 + 150 mg/kg/day) or buthionine sulfoximine (BSO; 500 mg/kg/day). Exposure to 50 ppm PO and treatment with 2 x2 50 mg/kg/day DEM resulted in NPSH levels approximating 50% and 80% of the level in untreated controls, respectively. Cell proliferation did not increase. After exposures to >or= 100 ppm PO or treatment with BSO or 500 + 150 mg/kg/day DEM, NPSH was depleted to

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 to be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.

Colorectal cancer is the second leading cause of cancer deaths worldwide with diet playing a prominent role in disease initiation and progression. Flavonoids are secondary plant compounds that are suggested as protective ingredients of a diet rich in fruits and vegetables. We here tested whether flavone, a flavonoid that proved to be an effective apoptosis inducer in colon cancer cells in culture, can affect the development of aberrant crypt foci (ACFs) in C57BL/6J mice in vivo when preneoplastic lesions were induced by the carcinogen 1,2-dimethylhydrazine (DMH). Flavone applied at either a low dose (15 mg/kg body wt per day) or a high dose (400 mg/kg body wt per day) reduced the numbers of ACFs significantly, independent of whether it was supplied simultaneously with the carcinogen (blocking group) or subsequent to the tumor induction phase (suppressing group). Proteome analysis performed in colonic tissue samples revealed that flavone treatment increased the expression of a number of Krebs cycle enzymes in the suppressing group and this was associated with reduced crypt multiplicity. It suggests that mitochondrial substrate oxidation is increased by flavone in colonic cells in vivo as already observed in HT-29 cells in vitro as the prime mechanism underlying tumor cell apoptosis induction by flavone. In conclusion, flavone reduces the number of ACFs in DMH-treated mice at doses that can be achieved for flavonoids by a diet rich in fruits and vegetables. Moreover, reduction in crypt multiplicity by flavone is most probably due to the preservation of a normal oxidative metabolism.

Flavonoids are secondary plant metabolites included in our diet but are also provided in a growing number of supplements. They are suggested to interact with intestinal transport systems including phospho-glycoprotein (P-gp) which mediates the efflux of a variety of xenobiotics back into the gut lumen. In human intestinal Caco-2 cells, we tested the effects of 14 different flavonoids on P-gp expression in vitro. Protein expression levels were quantified by Western blotting, flow cytometry, and real-time PCR. Except apigenin, all flavonoids at concentrations of 10 microM increased P-gp expression in Western blotting experiments when cells were exposed to the compounds over 4 wk. Flavone was one of the most effective P-gp inducers in Caco-2 cells and its effects were, therefore, also assessed for changes in P-gp in vivo in the gastrointestinal tract of C57BL/6 mice. P-gp expression was significantly increased by flavone (400 mg/kg body weight x day over 4 wk) in the small intestine but not in the colon which displayed intrinsically the highest expression level. In conclusion, the increase in P-gp expression caused by flavonoids in intestinal epithelial cells in vitro and also in vivo may serve as an adaptation and defense mechanism limiting the entry of lipophilic xenobiotics into the organism.

In colorectal cancer insulin-like growth factor II (IGF-II) is frequently overexpressed. To evaluate, whether IGF-II affects different stages of tumorigenesis, we induced neoplastic alterations in the colon of wild-type and IGF-II transgenic mice using 1,2-dimethylhydrazine (DMH). Aberrant crypt foci (ACF) served as markers of early lesions in the colonic mucosa, whereas adenomas and carcinomas characterized the endpoints of tumor development. DMH-treatment led initially to significantly more ACF in IGF-II transgenic than in wild-type mice. This increase in ACF was especially prominent for those consisting of > or =three aberrant crypts (AC). Nevertheless, adenomas and adenocarcinomas of the colon, present after 34 weeks in both genetic groups, were not found at different frequency. Tumor volumes, however, were significantly higher in IGF-II transgenic mice and correlated with serum IGF-II levels. Immunohistochemical staining for markers of proliferation and apoptosis revealed increased cell proliferation rates in tumors of IGF-II transgenic mice without significant affection of apoptosis. Increased proliferation was accompanied by elevated localization of beta-catenin in the cytosol and cell nuclei and reduced appearance at the inner plasma membrane. In conclusion, we provide evidence that IGF-II, via activation of the beta-catenin signaling cascade, promotes growth of ACF and tumors without affecting tumor numbers.

The anti-androgen and progestagen cyproterone acetate (CPA) is known to cause liver tumors in rats. The drug has been identified recently as a mutagen in the liver of female transgenic lambdalacI (Big Blue) rats at high doses after an expression time of 6 weeks. A dose of 50 mg CPA/kg BW, however, did not increase the mutation frequency (MF) of controls indicating a no-effect level of mutagenicity [Carcinogenesis 19 (1998) 241]. The present study was performed to assess the existence of a no-effect level of mutagenicity. In order to figure out conditions of maximum response, the time course of the MF was determined after administration of a single dose of 100 mg CPA/kg BW to female Big Blue rats. The MF showed a strong initial rise to a maximum 2 weeks after CPA administration accompanied by a corresponding increase of cell proliferation and of DNA adduct levels. Thereafter, the MF decreased within further 2 weeks to one third of the maximum level which was maintained for another 4 weeks. The DNA adduct levels decreased only by 15% during this time period suggesting that mutated hepatocytes were eliminated predominantly. A dose dependence curve determined at a fixation time of 2 weeks revealed a no-effect level of 5 mg CPA/kg BW for mutagenicity. In conclusion, our findings indicate that the length of the observation period may be a critical determinant for the outcome of a mutagenesis study in rat liver. Furthermore, the existence of a no-effect level for the mutagenicity of CPA in rat liver was confirmed. However, it has to be clarified whether the dose of 5 mg CPA/kg BW corresponding to the "transient" type of mutations or the previous dose of 50 mg CPA/kg BW related to a "permanent" type of mutations is more relevant for the assessment of the genotoxic risk.

In order to probe for the existence of a no-effect levels for mutations induced by multiple dose treatment with cyproterone acetate (CPA), female lacI-transgenic Big Blue rats were treated daily for 3 weeks with oral doses of 5.0, 1.0 or 0.2mg/kg CPA b.w., respectively. The dose of 5mg/kg CPA b.w. ineffective as a single dose (see part I) increased the mutation frequency by 2.5-fold. Daily treatment with 1.0 and with 0.2 mg/kg CPA b.w. for 3 weeks, however, was not effective indicating that the 1 mg dose represents a no-effect level for multiple dose treatment. The finding that a total dose of 21 x 5 = 105 mg/kg CPA b.w. caused 50% less DNA adducts, but a mutation frequency three-fold higher (data extrapolated from part I) than observed after daily treatment with 5mg/kg CPA b.w. for 21 days points to a crucial role of cell proliferation in mutagenesis by CPA. Our present results, in combination with previous findings, offer a basis to estimate the risk to develop mutations in human liver following treatment with CPA. Previous studies revealed that CPA-DNA adducts are formed in human hepatocytes at lower levels than in those of female rats [Mutat. Res. 395 (1997) 179; Cancer Res. 56 (1996) 4391]. Moreover, an in vitro-study indicated that human hepatocytes in culture do not respond to the mitogenic effect of CPA, while rat hepatocytes did [Cancer Res. 51 (1991) 1143]. We conclude that the risk of humans to develop mutations under treatment with CPA is substantially lower than in the female rat.

Nitric oxide (NO)-mediated relaxation of colonic smooth muscle is crucial for the maintenance of human gut function. The molecular mechanisms of NO-dependent smooth muscle relaxation involve cyclic GMP-mediated inhibition of store-dependent calcium signaling. Recently, IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate) has been characterized as a novel target molecule of cGMP-dependent protein kinase (cGKI) mediating NO-/cGMP-dependent inhibition of inositol 1,4,5-trisphosphate (InsP(3))-dependent calcium release in transfected COS cells. The aim of the present study was to characterize IRAG expression and its functional role in NO-dependent signaling in human colonic smooth muscle. Reverse transcriptase-PCR revealed IRAG mRNA expression in human colon, rectum, and cultured colonic smooth muscle cells. In cultured human colonic smooth muscle cells, bradykinin (BK) elicited InsP(3)-dependent calcium transients that were repeatable and independent of extracellular calcium. The NO donor sodium nitroprusside and the specific cGK activator 8-(4-chlorophenylthio)guanosine-3',5'-cyclic-monophosphate (8-pCPT-cGMP) significantly inhibited BK-induced increase in intracellular calcium. Cells transfected with antisense oligonucleotides raised against IRAG (IRAG-AS) showed strongly decreased IRAG protein expression. In these cells, sodium nitroprusside and 8-pCPT-cGMP both failed to modulate BK-induced calcium transients. Thus, endogenous IRAG appears to be essentially involved in the NO/cGK-dependent inhibition of InsP(3)-dependent Ca(2+)-signaling in colonic smooth muscle.

A rat liver foci bioassay (RLFB) based on an initiation-promotion protocol employing preneoplastic foci of altered hepatocytes (FAH) as an endpoint, was prevalidated in 5 different laboratories. FAH were identified by immunohistochemical demonstration of glutathione-S-transferase (placental form, GSTP) and by staining with hematoxilin/eosin (H&E), and their area fraction was quantified morphometrically. The four model hepatocarcinogens N-nitrosomorpholine, 2-acetylaminofluoren, phenobarbital, and clofibrate were selected according to characteristic differences in their presumed mode of action, and tested in a total of 1,600 male and female rats at 2 different dose levels. The chemicals were found to differ characteristically in their potency and dose-response relationship to induce FAH when given alone or when administered following initiation with diethylnitrosamine. The interlaboratory variation was small for results obtained with the GSTP-stain and somewhat larger with respect to H&E. The assessment of the carcinogenic potential of the four chemicals by the different laboratories was in the same range and the nature of their dose-response relationships did not differ essentially between laboratories. Our results suggest that this RLFB is a sensitive bioassay, providing potentially valuable information for risk assessment including the classification of carcinogenic chemicals according to their mode of action.

The expression of the insulin-like growth factor II (IGF-II) gene (Igf2) in rodents is completely abrogated in almost all adult tissues. A prominent exception are neoplasms in which IGF-II frequently serves as an autocrine growth factor. We have investigated the potential role of Igf2 expression during liver carcinogenesis. After application of diethylnitrosamine (DEN) preneoplastic foci and adenomas emerged in liver tissue of wild-type and phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II transgenic mice. Surprisingly, number and size of preneoplastic foci were not significantly increased in PEPCK-IGF-II mice as compared with wild-type animals. In situ preparation showed that early adenomas expressed Igf2 transcripts. Reverse transcriptase polymerase chain reaction (RT-PCR) and restriction enzyme analysis confirmed that DEN treatment had indeed reactivated the hepatic expression of murine Igf2 in control mice in a dose-dependent manner. This re-expression of Igf2 persisted for at least 18 months. Species-specific RT-PCR analyses also revealed the presence of murine Igf2 mRNAs in some PEPCK-IGF-II mice. A similar reactivation of Igf2 was detected in bovine growth hormone transgenic mice which develop hepatocellular neoplasms with high frequency. Our results suggest that reactivation of Igf2 is an early event during hepatocarcinogenesis in mice. Its appearance in two independent animal models suggests that Igf2 may be important at pivotal checkpoints of hepatocarcinogenesis.

1. CPA does not only induce the formation of DNA adducts but also of mutations in female rat liver. 2. The mutation frequency exhibited a characteristic time course. Within a period of 3 days post administration, a tremendous increase was noted, which remained at a high level until 2 weeks post exposure. Thereafter, most mutation-carrying cells were eliminated within a period of 2 weeks leaving a cell population remaining at a constant level for another 4 weeks. Thus, the length of the observation period post exposure, i. e. the manifestation time, seems to be a critical factor for the strength of the mutagenic response. The highest as observed between 1 and 2 weeks post exposure. Correspondingly, the dose response curve recorded 2 weeks post exposure showed a higher mutagenic response than the curve after 6 weeks of exposure recorded previously. 3. When CPA-induced mutations were recorded as a function of the dose, mutation frequencies at the lower dose range were found that did not differ from those of controls. The non-effective dose recorded 2 weeks post exposure was much lower than that recorded after 6 weeks of exposure indicating that it is a function of the manifestation time. Since DNA adducts were formed in high amounts at the non-effective doses, we assume that the mitogenic activity required for the conversion of DNA adducts into mutations was not sufficiently strong. The liver of adult animals exhibits a very low endogeneous proliferation rate, which is not likely to contribute significantly to the expression of mutations. We conclude that it is the mitogenic activity of CPA itself, which stimulates the expression of mutations.

The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.

Micro- and macronodular experimental cirrhosis-like liver lesion was induced in female rats by administration of 0.03% thioacetamide (TAA) in drinking water for 3 or 6 months. The activity of gamma-glutamyltranspeptidase (GGT) and the distribution pattern of this enzyme within the liver structure were investigated 14 d after withdrawal of TAA in comparison to neonatal and adult normal liver. GGT activity was extremely high at birth. Chronic TAA administration led to a strong increase in hepatic GGT activity in dependence on duration of TAA administration in comparison to adult controls. In accordance to these results we observed by enzyme-histochemistry a small to moderate hepatocellular GGT activity after 3 months of TAA treatment. GGT activity was also demonstrable in epithelia of proliferated ductuli biliferi of single enlarged portal tracts. After 6 months of TAA administration the hepatocellular GGT activity was moderate to strong. It was demonstrable both in parenchymal (preneo-plastic) nodules and in cholangiocellular/cholangioductular proliferates. A GGT activity of mesenchymal cells was not demonstrable. We conclude that the increased hepatic GGT activity after chronic TAA administration can be correlated with the process of development of preneoplastic nodules. A relation between increased GGT activity and the process of cirrhogenesis does not seem to be probable in this animal cirrhosis model.

The rat liver foci bioassay designed as short-term screening test system in vivo is a reliable procedure for the detection of the carcinogenic potential of chemicals. The use of tumor prestages as markers is favorable in many respects: 1. There is a convincing correlation between foci and tumors. 2. The sensitivity is by a factor of more than 1,000 higher compared to the chronic carcinogenicity study. 3. It is the only system available so far for detecting liver tumor promotors. 4. There is promising prospect that carcinogenic agents with extrahepatic target organs can be detected.

Treatment of adult female rats with a single dose of benzo[a]pyrene (BaP) fails to initiate preneoplastic enzyme-altered islands in their livers. Treatment with polychlorinated biphenyls (PCBs) at a dose which strongly induces aryl hydrocarbon(BaP)hydroxylase prior to BaP application and followed by promotion with PCBs causes the appearance of about 9 adenosine-5'-triphosphatase-deficient and 7 gamma-glutamyltranspeptidase-positive islands/cm2 after 12 weeks. PCB-pretreatment or promotion alone did not increase the BaP-dependent formation of islands above that of the PCB-treated controls (2-3 islands/cm2). The results suggest that upon alteration of its metabolism BaP causes the formation of preneoplastic lesions in the liver which become manifest by promotion.

The promoting effect of Clophen A 50, a commercial mixture of polychlorinated biphenyls (PCBs) on preneoplastic islands, initiated by diethylnitrosamine (DEN), was studied in male and female Sprague-Dawley rats. The islands were identified histochemically by loss of adenosine-5'-triphosphatase (ATPase) and/or emergence of gamma-glutamyltranspeptidase (GGTase). Treatment with 12 X 8 mg DEN/kg body wt./day initiated a similar number and total area of islands in males and females. Additional weekly application of Clophen A 50 (50 or 100 mg/kg body wt./week, for 7 weeks) enhanced the number of ATPase-deficient islands 3-fold in males and 9-fold in females. The total area was increased 4-fold in males and 15-fold in females. Number and area of GGTase-positive islands were similarly enhanced. The emergence of a small number of islands after application of Clophen A 50 alone may indicate a weak carcinogenic potency. PCB treatment caused an increase in liver weight, which amounted to approximately 55% in males and 20% in females compared to controls. This increase is partly due to cell hypertrophy, as indicated by determination of cell size. The mitogenic activity of Clophen A 50 was evaluated by measurement of the mitotic index of unaltered hepatocytes at 24, 48 h, and 7 days after application of a single dose (100/mg/kg body wt.) of Clophen A 50. The mitotic index in control animals of both sexes was approximately 0.3%, and was enhanced approximately 8-fold in males, 24 h after PCB treatment. In females only a slight, non-significant increase was observed. The results indicate that the sex-dependent promoting effect of Clophen A 50 is independent from its mitogenic action.

The nuclear morphology of preneoplastic and unaltered hepatocytes in diethylnitrosamine-treated rats was investigated. Two-micrometer-thick sections of methacrylate-embedded liver were scanned with a TV camera and evaluated in a computer using multivariate analysis methods. The preneoplastic cell populations (islands) were distinguished from unaltered hepatocytes by histochemical demonstration of glycogen storage in specimens from starved animals. After the hemalaun-stained liver sections were scanned randomly, the sections were stained for glycogen, and the previously registered cells were identified visually using a scanning stage for relocation. This objective identification of unaltered and preneoplastic hepatocytes formed the basis for the selection of a training set for feature evaluation and supervised classification. Image analysis for quantitative nuclear morphology was applied to the hemalaun-stained cells. The results showed that condensed chromatin was reduced and nuclear area was increased in the nuclei of glycogen-storing cells. Differences in the nuclear structure were also found. A multivariate analysis including seven features gave a correct classification result of about 82%.

The effect of co-administration of diethylnitrosamine (DEN) and Clophen A 50, a commercial mixture of polychlorinated biphenyls (PCB), on pre-neoplastic enzyme-altered islands in livers of female Sprague-Dawley rats was studied. The islands were identified by the loss of adenosine-5'-triphosphatase (ATPase), emergence of gamma-glutamyltranspeptidase (GGTase) and glycogen storage after fasting. DEN was given p.o. (0.4 or 4 mg/kg body wt respectively) twice a week for 11 consecutive weeks. Clophen A 50 (1 or 5 mg/kg body wt respectively) was given alternatively three times a week for 11 weeks. Four groups of rats each received either DEN or PCBs in the respective doses. Control animals were treated with the vehicle or remained untreated. All animals were killed at week 12. In rats treated with 4 mg DEN/kg body wt approximately 80 ATPase-deficient islands/cm2 were observed. Additional treatment with Clophen A 50 enhanced the island number 3-fold. Treatment with 0.4 mg/kg body wt DEN induced 17 islands/cm2. Additional application of Clophen A 50 enhanced the island number approximately 3-fold. The total island area was enhanced to the same extent in both groups. The island incidence in PCB-treated rats and controls was below 1/cm2 with all markers tested. The results indicate that PCBs may exhibit a co-carcinogenic activity.