Azoxymethane-induced carcinogenesis-like model of mouse intestine and mouse embryonic stem cell-derived intestinal organoids.

BACKGROUND: Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. METHODS AND RESULTS: In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. CONCLUSION: We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.

Perfluorooctanesulfonic acid exposure leads to downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 2 expression and upregulation of markers associated with intestinal carcinogenesis in mouse intestinal tissues.

Perfluorooctanesulfonic acid (PFOS) is a widely recognized environment pollutant known for its high bioaccumulation potential and a long elimination half-life. Several studies have shown that PFOS can alter multiple biological pathways and negatively affect human health. Considering the direct exposure to the gastrointestinal (GI) tract to environmental pollutants, PFOS can potentially disrupt intestinal homeostasis. However, there is limited knowledge about the effect of PFOS exposure on normal intestinal tissues, and its contribution to GI-associated diseases remains to be determined. In this study, we examined the effect of PFOS exposure on the gene expression profile of intestinal tissues of C57BL/6 mice using RNAseq analysis. We found that PFOS exposure in drinking water significantly downregulates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme, in intestinal tissues of mice. We found that diets containing the soluble fibers inulin and pectin, which are known to be protective against PFOS exposure, were ineffective in reversing the downregulation of HMGCS2 expression in vivo. Analysis of intestinal tissues also demonstrated that PFOS exposure leads to upregulation of proteins implicated in colorectal carcinogenesis, including ß-catenin, c-MYC, mTOR and FASN. Consistent with the in vivo results, PFOS exposure leads to downregulation of HMGCS2 in mouse and human normal intestinal organoids in vitro. Furthermore, we show that shRNA-mediated knockdown of HMGCS2 in a human normal intestinal cell line resulted in increased cell proliferation and upregulation of key proliferation-associated proteins such as cyclin D, survivin, ERK1/2 and AKT, along with an increase in lipid accumulation. In summary, our results suggest that PFOS exposure may contribute to pathological changes in normal intestinal cells via downregulation of HMGCS2 expression and upregulation of pro-carcinogenic signaling pathways that may increase the risk of colorectal cancer development.

Intestinal carcinogenicity screening of environmental pollutants using organoid-based cell transformation assay.

The high incidence of colorectal cancer (CRC) is closely associated with environmental pollutant exposure. To identify potential intestinal carcinogens, we developed a cell transformation assay (CTA) using mouse adult stem cell-derived intestinal organoids (mASC-IOs) and assessed the transformation potential on 14 representative chemicals, including Cd, iPb, Cr-VI, iAs-III, Zn, Cu, PFOS, BPA, MEHP, AOM, DMH, MNNG, aspirin, and metformin. We optimized the experimental protocol based on cytotoxicity, amplification, and colony formation of chemical-treated mASC-IOs. In addition, we assessed the accuracy of in vitro study and the human tumor relevance through characterizing interdependence between cell-cell and cell-matrix adhesions, tumorigenicity, pathological feature of subcutaneous tumors, and CRC-related molecular signatures. Remarkably, the results of cell transformation in 14 chemicals showed a strong concordance with epidemiological findings (8/10) and in vivo mouse studies (12/14). In addition, we found that the increase in anchorage-independent growth was positively correlated with the tumorigenicity of tested chemicals. Through analyzing the dose-response relationship of anchorage-independent growth by benchmark dose (BMD) modeling, the potent intestinal carcinogens were identified, with their carcinogenic potency ranked from high to low as AOM, Cd, MEHP, Cr-VI, iAs-III, and DMH. Importantly, the activity of chemical-transformed mASC-IOs was associated with the degree of cellular differentiation of subcutaneous tumors, altered transcription of oncogenic genes, and activated pathways related to CRC development, including Apc, Trp53, Kras, Pik3ca, Smad4 genes, as well as WNT and BMP signaling pathways. Taken together, we successfully developed a mASC-IO-based CTA, which might serve as a potential alternative for intestinal carcinogenicity screening of chemicals.

Microbial metabolite trimethylamine-N-oxide induces intestinal carcinogenesis through inhibiting farnesoid X receptor signaling.

PURPOSE: Microbial dysbiosis is considered as a hallmark of colorectal cancer (CRC). Trimethylamine-N-oxide (TMAO) as a gut microbiota-dependent metabolite has recently been implicated in CRC development. Nevertheless, evidence relating TMAO to intestinal carcinogenesis remains largely unexplored. Herein, we aimed to examine the crucial role of TMAO in CRC progression. METHODS: Apcmin/+ mice were treated with TMAO or sterile PBS for 14 weeks. Intestinal tissues were isolated to evaluate the effects of TMAO on the malignant transformation of intestinal adenoma. The gut microbiota of mouse feces was detected by 16S rRNA sequencing analysis. HCT-116 cells were used to provide further evidence of TMAO on the progression of CRC. RESULTS: TMAO administration increased tumor cell and stem cell proliferation, and decreased apoptosis, accompanied by DNA damage and gut barrier impairment. Gut microbiota analysis revealed that TMAO induced changes in the intestinal microbial community structure, manifested as reduced beneficial bacteria. Mechanistically, TMAO bound to farnesoid X receptor (FXR), thereby inhibiting the FXR-fibroblast growth factor 15 (FGF15) axis and activating the Wnt/ß-catenin signaling pathway, whereas the FXR agonist GW4064 could blunt TMAO-induced Wnt/ß-catenin pathway activation. CONCLUSION: The microbial metabolite TMAO can enhance intestinal carcinogenesis by inhibiting the FXR-FGF15 pathway.

Intestinal Growth in Glucagon Receptor Knockout Mice Is Not Associated With the Formation of AOM/DSS-Induced Tumors.

Treatment with exogenous GLP-2 has been shown to accelerate the growth of intestinal adenomas and adenocarcinomas in experimental models of colonic neoplasia, however, the role of endogenous GLP-2 in tumor promotion is less well known. Mice with a global deletion of the glucagon receptor (Gcgr-/-) display an increase in circulating GLP-1 and GLP-2. Due to the intestinotrophic nature of GLP-2, we hypothesized that Gcgr-/- mice would be more susceptible to colonic dysplasia in a model of inflammation-induced colonic carcinogenesis. Female Gcgr-/- mice were first characterized for GLP-2 secretion and in a subsequent study they were given a single injection with the carcinogen azoxymethane (7.5 mg/kg) and treated with dextran sodium sulfate (DSS) (3%) for six days (n=19 and 9). A cohort of animals (n=4) received a colonoscopy 12 days following DSS treatment and all animals were sacrificed after six weeks. Disruption of glucagon receptor signaling led to increased GLP-2 secretion (p<0.0001) and an increased concentration of GLP-2 in the pancreas of Gcgr-/- mice, coinciding with an increase in small intestinal (p<0.0001) and colonic (p<0.05) weight. Increased villus height was recorded in the duodenum (p<0.001) and crypt depth was increased in the duodenum and jejunum (p<0.05 and p<0.05). Disruption of glucagon receptor signaling did not affect body weight during AOM/DSS treatment, neither did it affect the inflammatory score assessed during colonoscopy or the number of large and small adenomas present at the end of the study period. In conclusion, despite the increased endogenous GLP-2 secretion Gcgr-/- mice were not more susceptible to AOM/DSS-induced tumors.

Aristolochic acid I promoted clonal expansion but did not induce hepatocellular carcinoma in adult rats.

Aristolochic acid I (AAI) is a well-known nephrotoxic carcinogen, which is currently reported to be also associated with hepatocellular carcinoma (HCC). Whether AAI is a direct hepatocarcinogen remains controversial. In this study we investigated the association between AAI exposure and HCC in adult rats using a sensitive rat liver bioassay with several cofactors. Formation of glutathione S-transferase placental form-positive (GST-P+) foci was used as the marker for preneoplastic lesions/clonal expansion. We first conducted a medium-term (8 weeks) study to investigate whether AAI had any tumor-initiating or -promoting activity. Then a long-term (52 weeks) study was conducted to determine whether AAI can directly induce HCC. We showed that oral administration of single dose of AAI (20, 50, or 100 mg/kg) in combination with partial hepatectomy (PH) to stimulate liver proliferation did not induce typical GST-P+ foci in liver. In the 8-week study, only high dose of AAI (10 mg · kg-1 · d-1, 5 days a week for 6 weeks) in combination with PH significantly increased the number and area of GST-P+ foci initiated by diethylnitrosamine (DEN) in liver. Similarly, only high dose of AAI (10 mg· kg-1· d-1, 5 days a week for 52 weeks) in combination with PH significantly increased the number and area of hepatic GST-P+ foci in the 52-week study. No any nodules or HCC were observed in liver of any AAI-treated groups. In contrast, long-term administration of AAI (0.1, 1, 10 mg· kg-1· d-1) time- and dose-dependently caused death due to the occurrence of cancers in the forestomach, intestine, and/or kidney. Besides, AAI-DNA adducts accumulated in the forestomach, kidney, and liver in a time- and dose-dependent manner. Taken together, AAI promotes clonal expansion only in the high-dose group but did not induce any nodules or HCC in liver of adult rats till their deaths caused by cancers developed in the forestomach, intestine, and/or kidney. Findings from our animal studies will pave the way for further large-scale epidemiological investigation of the associations between AA and HCC.

Assessment of Mechanistic Data for Hexavalent Chromium-Induced Rodent Intestinal Cancer Using the Key Characteristics of Carcinogens.

Oral exposure to hexavalent chromium (Cr[VI]) induces intestinal tumors in mice. Mutagenic and nonmutagenic modes of action (MOAs) have been accepted by different regulatory bodies globally, the latter involving cytotoxicity-induced regenerative cell proliferation. However, concerns persist that all possible MOAs have not been fully considered. To address the potential for alternative MOAs, mechanistic data not represented in the existing two MOAs were evaluated. Relevant data were identified and organized by key characteristics of carcinogens (KCCs); literature related to epigenetics, immunosuppression, receptor-mediated effects, and immortalization were reviewed to identify potential key events associated with an alternative MOA. Over 200 references were screened for these four KCCs and further prioritized based on relevance to the research objective (ie, in vivo, oral exposure, gastrointestinal tissue). Minimal data were available specific to the intestine for these KCCs, and there was no evidence of any underlying mechanisms or key events that are not already represented in the two proposed MOAs. For example, while epigenetic dysregulation of DNA repair genes has been demonstrated, epigenetic effects were not measured in intestinal tissue, and it has been shown that Cr(VI) does not cause DNA damage in intestinal tissue. High-throughput screening data related to the KCCs were also evaluated, with activity generally limited to the two recognized MOAs. Collectively, no plausible alternative MOAs (or key events) were identified in addition to those previously proposed for Cr(VI) small intestine tumors.

Stroma-derived ANGPTL2 establishes an anti-tumor microenvironment during intestinal tumorigenesis.

Previous studies show that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) functions as a tumor promoter in some cancer contexts. However, we recently reported that host ANGPTL2 also shows tumor suppressive activity by enhancing dendritic cell-mediated CD8+ T cell anti-tumor immune responses in mouse kidney cancer and murine syngeneic models. However, mechanisms underlying ANGPTL2-mediated tumor suppression are complex and not well known. Here, we investigated ANGPTL2 tumor suppressive function in chemically-induced intestinal tumorigenesis. ANGPTL2 deficiency enhanced intestinal tumor growth in an experimental mouse colitis-associated colon cancer (CAC) model. Angptl2-deficient mice also showed a decrease not only in CD8+ T cell responses but in CD4+ T cell responses during intestinal tumorigenesis. Furthermore, we show that stroma-derived ANGPTL2 can activate the myeloid immune response. Notably, ANGPTL2 drove generation of immunostimulatory macrophages via the NF-κB pathway, accelerating CD4+ T helper 1 (Th1) cell activation. These findings overall provide novel insight into the complex mechanisms underlying ANGPTL2 anti-tumor function in cancer pathology.

An adverse outcome pathway for small intestinal tumors in mice involving chronic cytotoxicity and regenerative hyperplasia: a case study with hexavalent chromium, captan, and folpet.

Small intestinal (SI) tumors are relatively uncommon outcomes in rodent cancer bioassays, and limited information regarding chemical-induced SI tumorigenesis has been reported in the published literature. Herein, we propose a cytotoxicity-mediated adverse outcome pathway (AOP) for SI tumors by leveraging extensive target species- and site-specific molecular, cellular, and histological mode of action (MOA) research for three reference chemicals, the fungicides captan and folpet and the transition metal hexavalent chromium (Cr(VI)). The gut barrier functions through highly efficient homeostatic regulation of SI epithelial cell sloughing, regenerative proliferation, and repair, which involves the replacement of up to 1011 cells per day. This dynamic turnover in the SI provides a unique local environment for a cytotoxicity mediated AOP/MOA. Upon entering the duodenum, cytotoxicity to the villous epithelium is the molecular initiating event, as indicated by crypt elongation, villous atrophy/blunting, and other morphologic changes. Over time, the regenerative capacity of the gut epithelium to compensate declines as epithelial loss accelerates, especially at higher exposures. The first key event (KE), sustained regenerative crypt proliferation/hyperplasia, requires sufficient durations, likely exceeding 6 or 12 months, due to extensive repair capacity, to create more opportunities for the second KE, spontaneous mutation/transformation, ultimately leading to proximal SI tumors. Per OECD guidance, biological plausibility, essentiality, and empirical support were assessed using modified Bradford Hill considerations. The weight-of-evidence also included a lack of induced mutations in the duodenum after up to 90 days of Cr(VI) or captan exposure. The extensive evidence for this AOP, along with the knowledge that human exposures are orders of magnitude below those associated with KEs in this AOP, supports its use for regulatory applications, including hazard identification and risk assessment.

Synthesis and in vitro characterization of the genotoxic, mutagenic and cell-transforming potential of nitrosylated heme.

Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis.

Effects of chronic low-dose aspirin treatment on tumor prevention in three mouse models of intestinal tumorigenesis.

Although early detection and treatment of colorectal cancer (CRC) have improved, it remains a significant health-care problem with high morbidity and mortality. Data indicate that long-term intake of low-dose aspirin reduces the risk of CRC; however, the mechanisms underlying this chemopreventive effect are still unclear. Different mouse models for inflammation-associated, sporadic, and hereditary CRC were applied to assess the efficacy and mechanism of low-dose aspirin on tumor prevention. An initial dosing study performed in healthy mice indicates that aspirin at a dose of 25 mg/kg/d has a similar pharmacodynamic effect as low-dose aspirin treatment in human subjects (100 mg/d). Chronic low-dose aspirin treatment suppresses colitis-associated and to a lesser extent spontaneous tumorigenesis in mice. Aspirin's antitumor effect is most pronounced in a preventive approach when aspirin administration starts before the tumor-initiating genotoxic event and continues for the duration of the experiment. These effects are not associated with alterations in cell proliferation, apoptosis, or activation of signaling pathways involved in CRC. Aspirin-induced reduction in tumor burden is accompanied by inhibition of thromboxane B2 formation, indicating reduced platelet activation. Aspirin treatment also results in decreased colonic prostaglandin E2 formation and tumor angiogenesis. With respect to colitis-triggered tumorigenesis, aspirin administration is associated with a reduction in inflammatory activity in the colon, as indicated by decreased levels of pro-inflammatory mediators, and tumor-associated iNOS-positive macrophages. Our results suggest that low-dose aspirin represents an effective antitumor agent in the context of colon tumorigenesis primarily due to its well-established cyclooxygenase inhibition effects.

HISTOLOGICAL CHANGES IN SPLEEN UNDER CONDITIONS OF TOXIC CARCINOGENESIS.

Under development of dimethylhydrazine-induced adenocarcinomatosis of the large intestine in white outbred male rats morphological changes of the structural components of the spleen were studied. It was found, that the progression of experimental carcinogenesis is accompanied by severe violations of the morphological state of all structural components of the spleen, manifested by destructively degenerative changes of the stroma, red and white pulp and significant vascular disorders. The severity of the pathomorphological changes in the spleen increases directly proportionally to the increase of the duration of the oncogenic factor impact.

Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17-dependent ligand release.

High fat diet is implicated in the elevated deoxycholic acid (DCA) in the intestine and correlated with increased colon cancer risk. However, the potential mechanisms of intestinal carcinogenesis by DCA remain unclarified. Here, we investigated the carcinogenic effects and mechanisms of DCA using the intestinal tumour cells and Apcmin/+ mice model. We found that DCA could activate epidermal growth factor receptor (EGFR) and promote the release of EGFR ligand amphiregulin (AREG), but not HB-EGF or TGF-α in intestinal tumour cells. Moreover, ADAM-17 was required in DCA-induced promotion of shedding of AREG and activation of EGFR/Akt signalling pathway. DCA significantly increased the multiplicity of intestinal tumours and accelerated adenoma-carcinoma sequence in Apcmin/+ mice. ADAM-17/EGFR signalling axis was also activated in intestinal tumours of DCA-treated Apcmin/+ mice, whereas no significant change occurred in tumour adjacent tissues after DCA exposure. Conclusively, DCA activated EGFR and promoted intestinal carcinogenesis by ADAM17-dependent ligand release.

A Novel Modification of the AOM/DSS Model for Inducing Intestinal Adenomas in Mice.

BACKGROUND/AIM: Our aim was to develop an animal model of the precancerous stages of colitis-associated carcinogenesis by modifying the established azoxymethane/dextran sulfate sodium (AOM/DSS) protocol. MATERIALS AND METHODS: Six mice were treated with varying cycles of DSS following AOM administration as above (group 1: three mice received three 5-day cycles of 3.0% DSS and group 2: three mice received three 7-day cycles of 2.5% DSS; every cycle was followed by a 2-week rest period) and were sacrificed on day 84 of the experiment. By contrast, three female C57BL6 mice (group 3) were treated with a single intraperitoneal dose (10 mg/kg of body weight) of AOM followed by three 5-day cycles of oral 2.5% DSS, with each cycle interrupted by a 2-week rest period. The mice of this group were sacrificed at 60 days. RESULTS: In groups 1 and 2, cancer was noted in five out of the six mice. In group 3, adenomas with dysplastic lesions were noted in all of the mice, but none had developed adenocarcinoma. CONCLUSION: Our results suggest that the administration of three 5-day cycles of 2.5% DSS following an initial dose of AOM may successfully induce adenoma formation without the concurrent presence of carcinoma in female C57BL6 mice that are sacrificed on experimental day 60. In turn, this modification of the widely used AOM/DSS protocol may constitute a novel approach for investigating colitis-related colonic adenomas.

Mbd2 enables tumourigenesis within the intestine while preventing tumour-promoting inflammation.

Epigenetic regulation plays a key role in the link between inflammation and cancer. Here we examine Mbd2, which mediates epigenetic transcriptional silencing by binding to methylated DNA. In separate studies the Mbd2-/- mouse has been shown (1) to be resistant to intestinal tumourigenesis and (2) to have an enhanced inflammatory/immune response, observations that are inconsistent with the links between inflammation and cancer. To clarify its role in tumourigenesis and inflammation, we used constitutive and conditional models of Mbd2 deletion to explore its epithelial and non-epithelial roles in the intestine. Using a conditional model, we found that suppression of intestinal tumourigenesis is due primarily to the absence of Mbd2 within the epithelia. Next, we demonstrated, using the DSS colitis model, that non-epithelial roles of Mbd2 are key in preventing the transition from acute to tumour-promoting chronic inflammation. Combining models revealed that prior to inflammation the altered Mbd2-/- immune response plays a role in intestinal tumour suppression. However, following inflammation the intestine converts from tumour suppressive to tumour promoting. To summarise, in the intestine the normal function of Mbd2 is exploited by cancer cells to enable tumourigenesis, while in the immune system it plays a key role in preventing tumour-enabling inflammation. Which role is dominant depends on the inflammation status of the intestine. As environmental interactions within the intestine can alter DNA methylation patterns, we propose that Mbd2 plays a key role in determining whether these interactions are anti- or pro-tumourigenic and this makes it a useful new epigenetic model for inflammation-associated carcinogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Severe Intestinal Inflammation in the Small Intestine of Mice Induced by Controllable Deletion of Claudin-7.

BACKGROUND: As a potential tumor suppressor gene, Claudin-7 (Cldn7), which is a component of tight junctions, may play an important role in colorectal cancer occurrence and development. AIMS: To generate a knockout mouse model of inducible conditional Cldn7 in the intestine and analyze the phenotype of the mice after induction with tamoxifen. METHODS: We constructed Cldn7-flox transgenic mice and crossed them with Villin-CreERT2 mice. The Cldn7 inducible conditional knockout mice appeared normal and were well developed at birth. We induced Cldn7 gene deletion by injecting different dosages of tamoxifen into the mice and then conducted a further phenotypic analysis. RESULTS: After induction for 5 days in succession at a dose of 200 µl tamoxifen in sunflower oil at 10 mg/ml per mouse every time, the mice appeared dehydrated, had a lower temperature, and displayed inactivity or death. The results of hematoxylin-eosin staining showed that the intestines of the Cldn7 inducible conditional knockout mice had severe intestinal defects that included epithelial cell sloughing, necrosis, inflammation and hyperplasia. Owing to the death of ICKO mice, we adjusted the dose of tamoxifen to a dose of 100 µl in sunflower oil at 10 mg/ml per mouse (aged more than 8 weeks old) every 4 days. And we could induce atypical hyperplasia and adenoma in the intestine. Immunofluorescent staining indicated that the intestinal epithelial structure was destroyed. Electron microscopy experimental analysis indicated that the intercellular gap along the basolateral membrane of Cldn7 inducible conditional knockout mice in the intestine was increased and that contact between the cells and matrix was loosened. CONCLUSIONS: We generated a model of intestinal Cldn7 inducible conditional knockout mice. Intestinal Cldn7 deletion induced by tamoxifen initiated inflammation and hyperplasia in mice.

Oral trivalent bismuth ions decrease, and trivalent indium or ruthenium ions increase, intestinal tumor burden in ApcΔ14/+ mice.

Immature forms of the peptide hormone gastrin have been implicated in the development of colorectal cancer (CRC). The biological activity of glycine-extended gastrin (Ggly) is dependent on the binding of Fe3+ ions in vitro and in vivo. The aim of the present study was to determine the effect of blocking Fe3+ ion binding to Ggly, using Bi3+, In3+ or Ru3+ ions, on the development of intestinal tumors in APCΔ14/+ mice. APCΔ14/+ mice were treated orally with Bi3+, In3+ or Ru3+ ions for up to 60 days, serum trace metals were analyzed by inductively coupled plasma mass spectrometry, and the incidence and size of intestinal tumors were assessed. Bi3+ treatment significantly decreased the number of tumors larger than 3 mm in male mice. In3+ or Ru3+ treatment significantly increased the tumor burden in all animals and In3+ increased the number of tumors larger than 3 mm or 5 mm in male mice alone. The fact that binding of In3+ or Ru3+ ions to Ggly was orders of magnitude stronger than the binding of Bi3+ ions implies that the inhibitory effect of Bi3+ ions is not a consequence of a reduction in Ggly activity. However, further testing of higher doses of Bi3+ ions for longer periods as an oral treatment for intestinal tumors is warranted.

Integration of mechanistic and pharmacokinetic information to derive oral reference dose and margin-of-exposure values for hexavalent chromium.

The current US Environmental Protection Agency (EPA) reference dose (RfD) for oral exposure to chromium, 0.003 mg kg-1  day-1 , is based on a no-observable-adverse-effect-level from a 1958 bioassay of rats exposed to ≤25 ppm hexavalent chromium [Cr(VI)] in drinking water. EPA characterizes the confidence in this RfD as "low." A more recent cancer bioassay indicates that Cr(VI) in drinking water is carcinogenic to mice at ≥30 ppm. To assess whether the existing RfD is health protective, neoplastic and non-neoplastic lesions from the 2 year cancer bioassay were modeled in a three-step process. First, a rodent physiological-based pharmacokinetic (PBPK) model was used to estimate internal dose metrics relevant to each lesion. Second, benchmark dose modeling was conducted on each lesion using the internal dose metrics. Third, a human PBPK model was used to estimate the daily mg kg-1 dose that would produce the same internal dose metric in both normal and susceptible humans. Mechanistic research into the mode of action for Cr(VI)-induced intestinal tumors in mice supports a threshold mechanism involving intestinal wounding and chronic regenerative hyperplasia. As such, an RfD was developed using incidence data for the precursor lesion diffuse epithelial hyperplasia. This RfD was compared to RfDs for other non-cancer endpoints; all RfD values ranged 0.003-0.02 mg kg-1  day-1 . The lowest of these values is identical to EPA's existing RfD value. Although the RfD value remains 0.003 mg kg-1  day-1 , the confidence is greatly improved due to the use of a 2-year bioassay, mechanistic data, PBPK models and benchmark dose modeling.

Glucagon like peptide-2 and neoplasia; a systematic review.

INTRODUCTION: Glucagon like peptide-2 is synthesized from enteroendocrine L cells primarily located in the ileum and large intestine. GLP-2 stimulates crypt cell proliferation, increases intestinal blood flow, enhances gut barrier function, induces mucosal healing, and exerts an anti-apoptotic effect. Due to these effects GLP-2 is used in the treatment of short bowel syndrome (SBS). Areas covered: The aim of this systematic review was to provide information on the potential risk of intestinal neoplasia in patients receiving treatment with GLP-2. The literature search was performed independently by two authors in the following databases; Pubmed, Embase, Scopus, Web of Science and Cochrane. Expert commentary: This systematic review indicated that treatment with GLP-2(1-33) up to 30 months in humans without any known pre-existing cancer did not confer an increased risk of intestinal neoplasia in patients or animals. However, due to the small amount of patients studied it is premature to reach any final conclusions about GLP-2 - induced neoplasia. GLP-2(1-33) treatment in animals with a pre-induced cancer showed that GLP-2(1-33) may promote growth of existing neoplasia.