Inferred inactivation of the Cftr gene in the duodena of mice exposed to hexavalent chromium (Cr(VI)) in drinking water supports its tumor-suppressor status and implies its potential role in Cr(VI)-induced carcinogenesis of the small intestines.

Carcinogenicity of hexavalent chromium [Cr (VI)] has been supported by a number of epidemiological and animal studies; however, its carcinogenic mode of action is still incompletely understood. To identify mechanisms involved in cancer development, we analyzed gene expression data from duodena of mice exposed to Cr(VI) in drinking water. This analysis included (i) identification of upstream regulatory molecules that are likely responsible for the observed gene expression changes, (ii) identification of annotated gene expression data from public repositories that correlate with gene expression changes in duodena of Cr(VI)-exposed mice, and (iii) identification of hallmark and oncogenic signature gene sets relevant to these data. We identified the inactivated CFTR gene among the top scoring upstream regulators, and found positive correlations between the expression data from duodena of Cr(VI)-exposed mice and other datasets in public repositories associated with the inactivation of the CFTR gene. In addition, we found enrichment of signatures for oncogenic signaling, sustained cell proliferation, impaired apoptosis and tissue remodeling. Results of our computational study support the tumor-suppressor role of the CFTR gene. Furthermore, our results support human relevance of the Cr(VI)-mediated carcinogenesis observed in the small intestines of exposed mice and suggest possible groups that may be more vulnerable to the adverse outcomes associated with the inactivation of CFTR by hexavalent chromium or other agents. Lastly, our findings predict, for the first time, the role of CFTR inactivation in chemical carcinogenesis and expand the range of plausible mechanisms that may be operative in Cr(VI)-mediated carcinogenesis of intestinal and possibly other tissues.

Assessment of the mutagenic potential of hexavalent chromium in the duodenum of big blue® rats.

A cancer bioassay on hexavalent chromium Cr(VI) in drinking water reported increased incidences of duodenal tumors in B6C3F1 mice at exposures of 30-180ppm, and oral cavity tumors in F344 rats at 180ppm. A subsequent transgenic rodent (TGR) in vivo mutation assay in Big Blue® TgF344 rats found that exposure to 180ppm Cr(VI) in drinking water for 28days did not increase cII transgene mutant frequency (MF) in the oral cavity (Thompson et al., 2015). Herein, we extend our analysis to the duodenum of these same TgF344 rats. At study termination, duodenum chromium levels were below either the limit of detection or quantification in control rats, but were 24.6±3.8µg/g in Cr(VI)-treated rats. The MF in control (23.2×10-6) and Cr(VI)-treated rats (22.7×10-6) were nearly identical. In contrast, the MF in the duodenum of rats exposed to 1-ethyl-1-nitrosourea for six days (study days 1, 2, 3, 12, 19, 26) increased 24-fold to 557×10-6. These findings indicate that mutagenicity is unlikely an early initiating event in Cr(VI)-induced intestinal carcinogenesis.

Comparison of in vivo genotoxic and carcinogenic potency to augment mode of action analysis: Case study with hexavalent chromium.

Recent analyses-highlighted by the International Workshops on Genotoxicity Testing Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment-have identified a correlation between (log) estimates of a carcinogen's in vivo genotoxic potency and in vivo carcinogenic potency in typical laboratory animal models, even when the underlying data have not been matched for tissue, species, or strain. Such a correlation could have important implications for risk assessment, including informing the mode of action (MOA) of specific carcinogens. When in vivo genotoxic potency is weak relative to carcinogenic potency, MOAs other than genotoxicity (e.g., endocrine disruption or regenerative hyperplasia) may be operational. Herein, we review recent in vivo genotoxicity and carcinogenicity data for hexavalent chromium (Cr(VI)), following oral ingestion, in relevant tissues and species in the context of the aforementioned correlation. Potency estimates were generated using benchmark doses, or no-observable-adverse-effect-levels when data were not amenable to dose-response modeling. While the ratio between log values for carcinogenic and genotoxic potency was ≥1 for many compounds, the ratios for several Cr(VI) datasets (including in target tissue) were less than unity. In fact, the ratios for Cr(VI) clustered closely with ratios for chloroform and diethanolamine, two chemicals posited to have non-genotoxic MOAs. These findings suggest that genotoxicity may not play a major role in the cancers observed in rodents following exposure to high concentrations of Cr(VI) in drinking water-a finding consistent with recent MOA and adverse outcome pathway (AOP) analyses concerning Cr(VI). This semi-quantitative analysis, therefore, may be useful to augment traditional MOA and AOP analyses. More case examples will be needed to further explore the general applicability and validity of this approach for human health risk assessment.

Reevaluation and Classification of Duodenal Lesions in B6C3F1 Mice and F344 Rats from 4 Studies of Hexavalent Chromium in Drinking Water.

Thirteen-week and 2-year drinking water studies conducted by the National Toxicology Program (NTP) reported that hexavalent chromium (Cr(VI)) induced diffuse epithelial hyperplasia in the duodenum of B6C3F1 mice but not F344 rats. In the 2-year study, Cr(VI) exposure was additionally associated with duodenal adenomas and carcinomas in mice only. Subsequent 13-week Cr(VI) studies conducted by another group demonstrated non-neoplastic duodenal lesions in B6C3F1 mice similar to those of the NTP study as well as mild duodenal hyperplasia in F344 rats. Because intestinal lesions in mice are the basis for proposed safety standards for Cr(VI), and the histopathology data are relevant to the mode of action, consistency (an important Hill criterion for causality) was assessed across the aforementioned studies. Two veterinary pathologists applied uniform diagnostic criteria to the duodenal lesions in rats and mice from the 4 repeated-dose studies. Comparable non-neoplastic intestinal lesions were evident in mice and rats from all 4 studies; however, the incidence and severity of intestinal lesions were greater in mice than rats. These findings demonstrate consistency across studies and species and highlight the importance of standardized nomenclature for intestinal pathology. The differences in the severity of non-neoplastic lesions also likely contribute to the differential tumor response.

Induction of duodenal mucosal tumors of intestinal epithelial cell origin showing frequent nuclear ß-catenin accumulation similar to the concurrently induced colorectal tumors in rats after treatment with azoxymethane.

Azoxymethane (AOM) is a potent carcinogen used for induction of colon tumors in rats and mice. It is also known that AOM treatment induces small bowel tumors in addition to colorectal tumors in rats. The present study examined the histogenesis of AOM-induced rat duodenal tumors in comparison with concurrently induced colorectal tumors by histochemical and immunohistochemical approaches. Duodenal and colorectal tumors were positive for both periodic acid-Schiff reaction and Alcian blue staining. Immunohistochemically, duodenal tumors were positive for intestinal epithelial markers such as cytokeratin (CK) 20 (100%) and mucin (MUC) 2 (91.7%) but negative for pancreaticobiliary markers such as CK7 (100%) and MUC1 (100%). All colorectal tumors were also negative for CK7 and MUC1 but positive for CK20. Eighty percent of colorectal tumors were positive for MUC2. In addition, nuclear accumulation of ß-catenin was found in duodenal tumors (70.8%), which was similar to colorectal tumors (90.0%). These results indicate that duodenal tumors induced by AOM treatment of rats were derived from intestinal epithelium. Similar to colorectal tumors, nuclear accumulation of ß-catenin indicates activation of Wnt signaling as a driving force for tumor progression in AOM-induced duodenal tumors.

Synchrotron-based imaging of chromium and γ-H2AX immunostaining in the duodenum following repeated exposure to Cr(VI) in drinking water.

Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodena from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum--the latter evidenced by lengthening of the crypt compartment by ∼2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. These findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia.

Duodenal mucosal risk markers in patients with familial adenomatous polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment and comparison with patient controls.

BACKGROUND: Familial adenomatous polyposis (FAP) is a disease characterized by the development of hundreds to thousands of adenomatous polyps in the colorectum early in life. Virtually all patients with FAP will develop colorectal cancer before the age of 40 to 50 years, unless prophylactic colectomy is performed, which significantly improves their prognosis. The mortality pattern has changed and duodenal cancer now is one of the main cancer-related causes of death in these patients. Practically all patients with FAP develop premalignant duodenal adenomas, which may develop to duodenal cancer in approximately 3-7% of patients. Duodenal cancer in patients with FAP has a poor prognosis. The clinical challenge is to identify patients at high-risk for duodenal carcinoma. Chemoprevention would be desirable to avoid duodenectomy. The main goal of this study is to identify risk markers in normal duodenal mucosa of patients with FAP, that could help identify patients at increased risk for malignant transformation. METHODS: Messenger RNA (mRNA) levels of glutathione S-transferase A1 (GSTA1), glutathione S-transferase P1 (GSTP1), KIAA1199, E-cadherin, peroxisome proliferative activated receptor δ (PPARδ), caspase-3, cyclin D1, ß-catenin, and cyclooxygenase-2 (COX-2) were measured in duodenal mucosa, using the QuantiGene 2.0 Plex assay. Levels in normal appearing mucosa of patients with FAP (n = 37) were compared with levels in non-FAP patient controls (n = 16). In addition, levels before and after treatment with either celecoxib & ursodeoxycholic acid (UDCA, n = 14) or celecoxib & placebo (n = 13) were evaluated in patients with FAP. RESULTS: mRNA levels of glutathione S-transferase A1 (28.16% vs. 38.24%, p = 0.008) and caspase-3 (3.30% vs. 5.31%, p = 0.001) were significantly lower in patients with FAP vs. non-FAP patient controls, respectively. COX-2 mRNA levels in normal duodenal mucosa of patients with FAP were found to be unexpectedly low. None of the potential risk markers was influenced by celecoxib or celecoxib & UDCA. CONCLUSIONS: Protection against toxins and carcinogens (GSTA1) and apoptosis (caspase-3) is low in patients with FAP, which could contribute to increased susceptibility for malignant transformation of duodenal mucosa. TRIAL REGISTRATION: http://ClinicalTrials.gov number NCT00808743.

Lack of efficacy of blueberry in nutritional prevention of azoxymethane-initiated cancers of rat small intestine and colon.

BACKGROUND: Blueberries may lower relative risk for cancers of the gastrointestinal tract. Previous work indicated an inhibitory effect of consumed blueberry (BB) on formation of aberrant crypt foci (ACF) in colons of male Fisher F344 rats (inbred strain). However, effects of BB on colon tumors and in both genders are unknown. METHODS: We examined efficacy of BB in inhibition of azoxymethane (AOM)-induced colon ACF and intestine tumors in male and female Sprague-Dawley rats (outbred strain). Pregnant rats were fed a diet with or without 10% BB powder; progeny were weaned to the same diet as their dam and received AOM as young adults. RESULTS: Male and female rats on control diet had similar numbers of ACF at 6 weeks after AOM administration. BB increased (P < 0.05) ACF numbers within the distal colon of female but not male rats. There was a significant (P < 0.05) diet by gender interaction with respect to total colon ACF number. Colon and duodenum tumor incidences were less in females than males at 17 weeks after AOM. BB tended (0.1 > P > 0.05) to reduce overall gastrointestinal tract tumor incidence in males, however, tumor incidence in females was unaffected (P > 0.1) by BB. There was a tendency (0.1 > P > 0.05) for fewer adenocarcinomas (relative to total of adenomatous polyps plus adenocarcinomas) in colons of female than male tumor-bearing rats; in small intestine, this gender difference was significant (P < 0.05). BB favored (P < 0.05) fewer adenocarcinomas and more adenomatous polyps (as a proportion of total tumor number) in female rat small intestine. CONCLUSION: Results did not indicate robust cancer-preventive effects of BB. Blueberry influenced ACF occurrence in distal colon and tumor progression in duodenum, in gender-specific fashion. Data indicate the potential for slowing tumor progression (adenomatous polyp to adenocarcinoma) by BB.

N-Methyl-N-Nitrosourea (MNU): A positive control chemical for p53+/- mouse carcinogenicity studies.

This study evaluated the effects of a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in citrate buffer (pH 4.5) at a dose of 75 mg/kg in thirty male and thirty female p53+/- mice followed by a six-month observation period. Fifteen control mice per sex received a single intraperitoneal injection of citrate buffer. Fifty-six of sixty mice treated with MNU died or were sacrificed before the end of the observation period. Twenty-four males and twenty-seven females treated with MNU developed malignant lymphoma of the thymus; of these, twenty-three males and twenty-seven females had corresponding enlargement or masses in the thymus at necropsy. Lymphoblasts in thymic lymphomas stained positively for mouse CD3 antigen, indicating a T-cell lineage. One control female mouse had malignant lymphoma of the spleen that did not involve the thymus. Nine males and five females treated with MNU had adenomas or adenocarcinomas of the small intestine, whereas no intestinal neoplasms were observed in control mice. These findings support the use of a single dose of MNU as a positive control chemical in six-month p53+/- mouse carcinogenicity studies and suggest that examination of the thymus alone is sufficient to evaluate the validity of the model system.

Dietary whey protein lowers serum C-peptide concentration and duodenal SREBP-1c mRNA abundance, and reduces occurrence of duodenal tumors and colon aberrant crypt foci in azoxymethane-treated male rats.

We evaluated partially hydrolyzed whey protein (WPH) for inhibitory effects on the development of colon aberrant crypt foci (ACF) and intestinal tumors in azoxymethane (AOM)-treated rats. Pregnant Sprague-Dawley rats and their progeny were fed AIN-93G diets containing casein (CAS, control diet) or WPH as the sole protein source. Colons and small intestines from the male progeny were obtained at 6, 12, 20 and 23 weeks after AOM treatment. At 6 and 23 weeks, post-AOM, WPH-fed rats had fewer ACF than did CAS-fed rats. Intestinal tumors were most frequent at 23 weeks, post-AOM. At this time point, differences in colon tumor incidence with diet were not observed; however, WPH-fed rats had fewer tumors in the small intestine (7.6% vs. 26% incidence, P=.004). Partially hydrolized whey protein suppressed circulating C-peptide concentration (a stable indicator of steady-state insulin secretion) at all four time points relative to the corresponding CAS-fed animals. The relative mRNA abundance for the insulin-responsive, transcription factor gene, SREBP-1c, was reduced by WPH in the duodenum but not colon. Results indicate potential physiological linkages of dietary protein type with circulating C-peptide (and by inference insulin), local expression of SREBP-1c gene and propensity for small intestine tumorigenesis.

[Intramural small-bowel hematoma due to anticoagulant therapy: a radiologic study; with regard to two cases]. / Hématome intramural du grêle dû aux anticoagulants: une etude radiologique a propos de deux observations.

We report two cases of spontaneous small bowel hematoma in two patients receiving long-term anticoagulant therapy. Plain abdominal film, ultrasound, CT-scan and oral barium examination were performed. Abdominal ultrasonography and CT-scan are in most cases relevant for the correct diagnosis of intra-mural small bowel hematoma. The diagnosis was based on the acknowledgment by the patient of anticoagulant drug consumption. Early diagnosis is crucial because most patients are treated nonoperatively with good outcome.

Chemoprevention of 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine-induced colon carcinogenesis by 1-O-hexyl-2,3,5-trimethylhydroquinone after initiation with 1,2-dimethylhydrazine in F344 rats.

The aim of this study is to investigate the chemopreventive effects of the synthetic phenolic antioxidant 1-O-hexyl-2,3, 5-trimethylhydroquinone (HTHQ) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colon carcinogenesis in rats after initiation with 1,2-dimethylhydrazine (DMH) in male F344 rats. Groups of 20-22, 6-week-old male F344 rats were given four subcutaneous injections of 40 mg/kg body wt of DMH during the initial 4 weeks. They were then maintained on powdered basal diet containing 0.03% PhIP alone, PhIP together with 0.5 or 0.125% HTHQ, 0.5 or 0.125% HTHQ alone or basal diet for 32 weeks. A small number (1.1 +/- 1.1/rat) of colon tumors were induced by DMH treatment alone. After initiation with DMH, the number of colon tumors was greatly increased to 8.3 +/- 5.6 by the administration of PhIP. Additional treatment with HTHQ dose-dependently decreased the multiplicity of colon adenocarcinomas to 4.9 +/- 2.8 and 2.6 +/- 1.4 with 0.125 and 0.5%, respectively. This treatment similarly reduced atypical hyperplasias of the ventral prostate. Furthermore, HTHQ significantly reduced the multiplicity of duodenal adenocarcinomas induced by DMH + PhIP or DMH alone. Immunohistochemically, HTHQ was revealed to suppress PhIP-DNA adduct formation in the epithelial cells of the colon and prostate in a separate 2 weeks experiment. The present results clearly showed that HTHQ has chemopreventive potential for PhIP-associated colon and prostate carcinogenesis. The observed inhibition may largely be due to interference with PhIP-DNA adduct formation. In addition, HTHQ has been demonstrated to inhibit duodenal carcinogenesis in the post-initiation stage.

17Beta-estradiol modulates gastroduodenal preneoplastic alterations in rats exposed to the carcinogen N-methyl-N'-nitro-nitrosoguanidine.

Gastric cancers are a significant cause of morbidity worldwide. Epidemiological studies and animal models show that males have higher incidences of gastric cancers compared with females, suggesting that sex hormones may modulate gastric cancer risk. An animal model of the initiation phase of gastric cancer was used to determine the effects of systemic estrogen administration on morphological progression of preneoplastic lesions and to define cell populations at which estrogens may act. Preneoplastic progression in antral and duodenal mucosa was examined in male rats that received the chemical carcinogen, N-methyl-N'-nitro-nitrosoguanidine (MNNG), during treatment with implants containing 17beta-estradiol or oil vehicle. Histopathological changes in antral and duodenal gland morphology, numbers of proliferating cells and apoptotic bodies, and antral gastrin cell numbers and protein storage levels were determined 4 weeks later. With MNNG treatment, duodenal villous heights were significantly decreased, and epithelial cells displayed histological features of hyperplasia and dysplasia. Antral glands showed epithelial hyperplasia and dysplasia, increased mucosal height, and decreased mucin levels. Antral gastrin storage protein levels were decreased by MNNG. Systemic treatment with 17beta-estradiol significantly reversed MNNG-induced alterations in duodenal gland heights while increasing mucin and gastrin levels in antral glands. Cell proliferation and apoptosis rates were not significantly different between groups. The present results indicate that systemic 17beta-estradiol treatment influences antral and duodenal gland differentiation during the initiation phase of chemical gastroduodenal carcinogenesis in male rats. These results explain, in part, a potential pathway through which protective effects of estrogens on chemical carcinogenesis are mediated in the upper gastrointestinal tract.

Administration of an unconjugated bile acid increases duodenal tumors in a murine model of familial adenomatous polyposis.

Intestinal carcinogenesis involves the successive accumulation of multiple genetic defects until cellular transformation to an invasive phenotype occurs. This process is modulated by many epigenetic factors. Unconjugated bile acids are tumor promoters whose presence in intestinal tissues is regulated by dietary factors. We studied the role of the unconjugated bile acid, chenodeoxycholate, in an animal model of familial adenomatous polyposis. Mice susceptible to intestinal tumors as a result of a germline mutation in Apc (Min/+ mice) were given a 10 week dietary treatment with 0.5% chenodeoxycholate. Following this, the mice were examined to determine tumor number, enterocyte proliferation, apoptosis and beta-catenin expression. Intestinal tissue prostaglandin E2 (PGE2) levels were also assessed. Administration of chenodeoxycholate in the diet increased duodenal tumor number in Min/+ mice. Promotion of duodenal tumor formation was accompanied by increased beta-catenin expression in duodenal cells, as well as increased PGE2 in duodenal tissue. These data suggest that unconjugated bile acids contribute to periampullary tumor formation in the setting of an Apc mutation.

[The role of enterochromaffin-like cells in physiology and pathology of stomach and duodenum]. / Rola komórek enterochromafinopodobnych w fizjologii i patologii zoladka i dwunastnicy.

During recent years, gastric ECL cells have attracted much attention, mainly due to the fact that mice and rats were found to develop gastric carcinoids following lifelong treatment with blockers of acid secretion. We present the structure and functions of ECL cells and their influence on physiology and pathology of stomach and duodenum. We describe interactions of enzymes and hormones in histamine-stimulated gastric output.

Inhibitory effects of rebamipide on ENNG-induced duodenal carcinogenesis in mice: a possible strategy for chemoprevention of gastrointestinal cancers.

Rebamipide is a potent antioxidative agent; it increases gastric mucosal PGE2 production and thus protects the gastric mucosa. We hypothesized that the mechanisms of ulcer formation could be extended to carcinogenesis and that an increase in gastric mucosal protection may result in a decrease in gastric carcinogenesis. Therefore, we assessed the inhibitory effects of rebamipide on N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) -induced carcinogenesis in mice. The percentage of tumor-bearing mice in three treatment groups--ENNG + rebamipide 20 mg, ENNG + rebamipide 50 mg, and ENNG alone--was 55%, 42%, and 67%, respectively. The incidence of tumorigenesis tended to decrease with increasing doses of rebamipide. The difference between ENNG + rebamipide 50 mg and ENNG alone was statistically significant (P < 0.05). These results suggest that rebamipide may strengthen the host defense mechanisms related to carcinogenesis in the digestive tract.

Inhibition of MNNG-induced gastroduodenal carcinoma in rats by synchronous application of wine or 11% ethanol.

There are divergent opinions on the effect of ethanol in the carcinogenesis of gastroduodenal tumors. The effect of the synchronous application of 11% ethanol or wine (11% ethanol) and N-methyl-N'-nitro-N-nitrosoguanidine (100 micrograms/ml, MNNG) in a drinking solution on the incidence of gastroduodenal tumors was evaluated. Sixty outbred male Wistar rats were distributed among three groups. The animals drank MNNG and ethanol or wine for six months and consumed the same quantity of MNNG. Then they consumed a normal diet until the 13th month, when the experiment was terminated. The stomach and duodenum were examined histologically. In the stomach, 15 tumors (2 squamous paillomas, 4 squamous carcinomas, 1 sarcoma, and 8 adenocarcinomas) and 4 cases of dysplasia were found; in the duodenum, there were four cases of adenocarcinoma. There were 6 cases of multiple tumors. Incidence of forestomach tumors did not differ among the groups, whereas the incidence of glandular stomach carcinoma and duodenal carcinoma was significantly lower in the groups treated with 11% ethanol or wine than in the control group. MNNG was not inactivated by ethanol in the drinking solutions. We concluded that the inhibitory effect on gastroduodenal carcinogenesis is the result of 11% ethanol ingestion and its protective action on the mucosa and not of the wine's nonethanol components.

Initiated stem cells in murine intestinal carcinogenesis: prolonged survival, control by NK cells, and progression.

Weekly injections of dimethylhydrazine (DMH) (25 mg/kg), or azoxymethane (AOM) (8 mg/kg) to young adult male CDI mice for 1-2 months produced generalized intestinal crypt hyperplasia, which we measured in duodenum in terms of number of interphase and mitotic cells present in crypts. As shown earlier, the crypts expanded because of the presence of a hyperproliferative "initiated" crypt subpopulation which was also sensitive to natural killer (NK) cells. Hyperplasia was thus present as long as NK activity was suppressed by the carcinogen treatment. After interruption of the treatment for periods of 1, 2, 3, 6 and 10 months in the various groups, hyperplasia soon regressed as a result of elimination of the subpopulation by the recovering NK cells. When NK activity was once again eliminated during the terminal days of these "interruption periods" (by injections of anti-asialo GM-I antibody, alpha AGM-I), the original hyperplasia was fully reconstituted, apparently from stem cells of the subpopulation which survived up to 10 months in their crypt base location. These "initiated stem cells" represented, then, the original carcinogenic insult during the pre-cancerous period. They also appeared to be the source of the eventual neoplasia, as treating the animals with mutagens during the interruption periods produced specific changes in crypt base histology: new "crypt base basophilic" (CBB) cells appeared which produced large accumulations as well as microscopic tumors when NK activity was suppressed (by alpha AGM-I). Some of the initiated stem cells were apparently transformed into neoplastic ones which remained under NK control, the NK cells preventing the establishment of their progeny. Further experiments indicated that, although the initiated stem cells are not eliminated by normal NK activity, activated NK cells can kill them, thereby eliminating the potential source of neoplasia.

Inhibitory effects of dietary curcumin on forestomach, duodenal, and colon carcinogenesis in mice.

Curcumin (diferuloylmethane), a yellow pigment that is obtained from the rhizomes of Curcuma longa Linn., is a major component of turmeric and is commonly used as a spice and food-coloring agent. The inhibitory effects of feeding commercial grade curcumin (77% curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin) in AIN 76A diet on carcinogen-induced tumorigenesis in the forestomach, duodenum, and colon of mice were evaluated. Administration p.o. of commercial grade curcumin in the diet inhibited benzo(a)pyrene-induced forestomach tumorigenesis in A/J mice, N-ethyl-N'-nitro-N-nitrosoguanidine-induced duodenal tumorigenesis in C57BL/6 mice, and azoxymethane (AOM)-induced colon tumorigenesis in CF-1 mice. Dietary commercial grade curcumin was given to mice at: (a) 2 weeks before, during, and for 1 week after carcinogen administration (during the initiation period); (b) 1 week after carcinogen treatment until the end of the experiment (during the postinitiation period); or (c) during both the initiation and postinitiation periods. Feeding 0.5-2.0% commercial grade curcumin in the diet decreased the number of benzo(a)pyrene-induced forestomach tumors per mouse by 51-53% when administered during the initiation period and 47-67% when administered during the postinitiation period. Feeding 0.5-2.0% commercial grade curcumin in the diet decreased the number of N-ethyl-N'-nitro-N-nitrosoguanidine-induced duodenal tumors per mouse by 47-77% when administered during the postinitiation period. Administration of 0.5-4.0% commercial grade curcumin in the diet both during the initiation and postinitation periods decreased the number of AOM-induced colon tumors per mouse by 51-62%. Administration of 2% commercial grade curcumin in the diet inhibited the number of AOM-induced colon tumors per mouse by 66% when fed during the initiation period and 25% when fed during the postinitiation period. The ability of commercial grade curcumin to inhibit AOM-induced colon tumorigenesis is comparable to that of pure curcumin (purity greater than 98%). Administration of pure or commercial grade curcumin in the diet to AOM-treated mice resulted in development of colon tumors which were generally smaller in number and size as compared to the control group of AOM-treated mice. These results indicate that not only did curcumin inhibit the number of tumors per mouse and the percentage of mice with tumors but it also reduced tumor size. Histopathological examination of the tumors showed that dietary curcumin inhibited the number of papillomas and squamous cell carcinomas of the forestomach as well as the number of adenomas and adenocarcinomas of the duodenum and colon.