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.

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.

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.

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.

High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity.

Several features common to a Western lifestyle, including obesity and low levels of physical activity, are known risk factors for gastrointestinal cancers. There is substantial evidence suggesting that diet markedly affects the composition of the intestinal microbiota. Moreover, there is now unequivocal evidence linking dysbiosis to cancer development. However, the mechanisms by which high-fat diet (HFD)-mediated changes in the microbial community affect the severity of tumorigenesis in the gut remain to be determined. Here we demonstrate that an HFD promotes tumour progression in the small intestine of genetically susceptible, K-ras(G12Dint), mice independently of obesity. HFD consumption, in conjunction with K-ras mutation, mediated a shift in the composition of the gut microbiota, and this shift was associated with a decrease in Paneth-cell-mediated antimicrobial host defence that compromised dendritic cell recruitment and MHC class II molecule presentation in the gut-associated lymphoid tissues. When butyrate was administered to HFD-fed K-ras(G12Dint) mice, dendritic cell recruitment in the gut-associated lymphoid tissues was normalized, and tumour progression was attenuated. Importantly, deficiency in MYD88, a signalling adaptor for pattern recognition receptors and Toll-like receptors, blocked tumour progression. The transfer of faecal samples from HFD-fed mice with intestinal tumours to healthy adult K-ras(G12Dint) mice was sufficient to transmit disease in the absence of an HFD. Furthermore, treatment with antibiotics completely blocked HFD-induced tumour progression, suggesting that distinct shifts in the microbiota have a pivotal role in aggravating disease. Collectively, these data underscore the importance of the reciprocal interaction between host and environmental factors in selecting a microbiota that favours carcinogenesis, and they suggest that tumorigenesis is transmissible among genetically predisposed individuals.

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.

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.

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.

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.

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.

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.

Obesity promotes PhIP-induced small intestinal carcinogenesis in hCYP1A-db/db mice: involvement of mutations and DNA hypermethylation of Apc.

Obesity is associated with an increased risk of cancer. To study the promotion of dietary carcinogen-induced gastrointestinal cancer by obesity, we employed 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to induce intestinal tumorigenesis in CYP1A-humanized (hCYP1A) mice, in which mouse Cyp1a1/1a2 was replaced with human CYP1A1/1A2 Obesity was introduced in hCYP1A mice by breeding with Lepr(db/+) mice to establish the genetically induced obese hCYP1A-Lepr(db/db) mice or by feeding hCYP1A mice a high-fat diet. PhIP induced the formation of small intestinal tumors at the ages of weeks 28-40 in obese hCYP1A mice, but not in lean hCYP1A mice. No tumors were found in colon and other gastrointestinal organs in the lean or obese mice. Using immunohistochemistry (IHC), we found strong positive staining of NF-κB p65, pSTAT3 and COX2 as well as elevated levels of nuclear ß-catenin (Ctnnb1) in small intestinal tumors, but not in normal tissues. By sequencing Apc and Ctnnb1 genes, we found that most PhIP-induced small intestinal tumors in obese mice carried only a single heterozygous mutation in Apc By bisulfite-sequencing of CpG islands of Apc, we found DNA hypermethylation in a CpG cluster located in its transcription initiation site, which most likely caused the inactivation of the wild-type Apc allele. Our findings demonstrate that PhIP-induced small intestinal carcinogenesis in hCYP1A-db/db mice is promoted by obesity and involves Apc mutation and inactivation by DNA hypermethylation. This experimental result is consistent with the association of obesity and the increased incidence of small intestinal cancer in humans in recent decades.

Stromal Indian hedgehog signaling is required for intestinal adenoma formation in mice.

BACKGROUND & AIMS: Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. METHODS: Glioma-associated oncogene (Gli1)-CreERT2 and Patched (Ptch)-lacZ reporter mice were crossed with Apc(Min) mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc(Min) and Ptch-lacZ-Apc(Min) mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc (Apc(HET)) mice, which develop adenomas after administration of ß-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc(Min) mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). RESULTS: Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc(HET) mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc(HET) mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. CONCLUSIONS: Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice.

The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression.

Ly49E is a member of the Ly49 family of NK receptors and is distinct from other members of this family on the basis of its structural properties, expression pattern and ligand recognition. Importantly, Ly49E receptor expression is high on small intestinal and colonic intraepithelial lymphocytes (IELs). Intestinal IELs are regulators of the mucosal immune system and contribute to front-line defense at the mucosal barrier, including anti-tumor immune response. Whereas most Ly49 receptors have MHC class-I ligands, we showed that Ly49E is instead triggered by urokinase plasminogen activator (uPA). uPA has been extensively implicated in tumor development, where increased uPA expression correlates with poor prognosis. As such, we investigated the role of Ly49E receptor expression on intestinal IELs in the anti-tumor immune response. For this purpose, we compared Ly49E wild-type mice to Ly49E knockout mice in two established tumor models: ApcMin/+-mediated and azoxymethane-induced intestinal cancer. Our results indicate that Ly49E expression on IELs does not influence the development or progression of intestinal cancer.

Implications of dose-dependent target tissue absorption for linear and non-linear/threshold approaches in development of a cancer-based oral toxicity factor for hexavalent chromium.

Dose-dependent changes in target tissue absorption have important implications for determining the most defensible approach for developing a cancer-based oral toxicity factor for hexavalent chromium (CrVI). For example, mouse target tissue absorption per unit dose is an estimated 10-fold lower at the CrVI dose corresponding to the federal maximum contaminant level (MCL) than at the USEPA draft oral slope factor (SFo) point of departure dose. This decreasing target tissue absorption as doses decrease to lower, more environmentally-relevant doses is inconsistent with linear low-dose extrapolation. The shape of the dose-response curve accounting for this toxicokinetic phenomenon would clearly be non-linear. Furthermore, these dose-dependent differences in absorption indicate that the magnitude of risk overestimation by a linear low-dose extrapolation approach (e.g., SFo) increases and is likely to span one or perhaps more orders of magnitude as it is used to predict risk at progressively lower, more environmentally-relevant doses. An additional apparent implication is that no single SFo can reliably predict risk across potential environmental doses (e.g., doses corresponding to water concentrations⩽the federal MCL). A non-linear approach, consistent with available mode of action data, is most scientifically defensible for derivation of an oral toxicity factor for CrVI-induced carcinogenesis.

Agent Orange exposure and cancer incidence in Korean Vietnam veterans: a prospective cohort study.

BACKGROUND: During the Vietnam War, US and allied military sprayed approximately 77 million liters of tactical herbicides including Agent Orange, contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. To the authors' knowledge, few studies to date have examined the association between Agent Orange exposure and cancer incidence among Korean veterans who were exposed to Agent Orange during the Vietnam War. METHODS: An Agent Orange exposure index, based on the proximity of the veteran's military unit to the area that was sprayed with Agent Orange, was developed using a geographic information system-based model. Cancer incidence was followed for 180,251 Vietnam veterans from 1992 through 2003. RESULTS: After adjustment for age and military rank, high exposure to Agent Orange was found to significantly increase the risk of all cancers combined (adjusted hazards ratio [aHR], 1.08). Risks for cancers of the mouth (aHR, 2.54), salivary glands (aHR, 6.96), stomach (aHR, 1.14), and small intestine (aHR, 2.30) were found to be significantly higher in the high-exposure group compared with the low-exposure group. Risks for cancers of all sites combined (aHR, 1.02) and for cancers of the salivary glands (aHR, 1.47), stomach (aHR, 1.03), small intestine (aHR, 1.24), and liver (aHR, 1.02) were elevated with a 1-unit increase in the exposure index. CONCLUSIONS: Exposure to Agent Orange several decades earlier may increase the risk of cancers in all sites combined, as well as several specific cancers, among Korean veterans of the Vietnam War, including some cancers that were not found to be clearly associated with exposure to Agent Orange in previous cohort studies primarily based on Western populations.

In utero exposure to perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) did not increase body weight or intestinal tumorigenesis in multiple intestinal neoplasia (Min/+) mice.

We examined whether perfluorooctanoate (PFOA) or perfluorooctane sulfonate (PFOS) had obesogenic effects and if they increased spontaneous intestinal tumorigenesis in the mouse model C57BL/6J-Min/+ (multiple intestinal neoplasia) after in utero exposure. The dams were exposed to PFOA or PFOS (0.01, 0.1 or 3.0mg/kg bw/day) by po gavage on GD1-17. The Min/+ and wild-type offspring were terminated at week 11 for examination of intestinal tumorigenesis or at week 20 for obesogenic effect, respectively. Body weights of the dams and pups were recorded throughout life. Food intake was determined at week 6 and 10. Blood glucose (non-fasted) was measured at week 6 and 11. No obesogenic effect of PFOA or PFOS was observed up to 20 weeks of age. PFOA or PFOS did not increase the incidence or number of tumors in the small intestine or colon of the Min/+ mice or affect their location along the intestines. Feed intake was not affected. There were some indications of toxicity of PFOA, but not of PFOS. There was lower survival of pups after 3.0mg/kg PFOA, lower body weight in pups after 3.0 and possibly 0.1mg/kg PFOA, and increased relative liver weight after 0.01 and possibly 0.1mg/kg PFOA. Plasma glucose was lower after 0.01 and 0.1mg/kg PFOA. In conclusion, exposure to PFOA and PFOS in utero with the doses used did not have obesogenic effect on either Min/+ or wild-type mice, at least not up to 11 or 20 weeks of age, nor increased intestinal tumorigenesis in Min/+ mice.

A chronic oral reference dose for hexavalent chromium-induced intestinal cancer.

High concentrations of hexavalent chromium [Cr(VI)] in drinking water induce villous cytotoxicity and compensatory crypt hyperplasia in the small intestines of mice (but not rats). Lifetime exposure to such cytotoxic concentrations increases intestinal neoplasms in mice, suggesting that the mode of action for Cr(VI)-induced intestinal tumors involves chronic wounding and compensatory cell proliferation of the intestine. Therefore, we developed a chronic oral reference dose (RfD) designed to be protective of intestinal damage and thus intestinal cancer. A physiologically based pharmacokinetic model for chromium in mice was used to estimate the amount of Cr(VI) entering each intestinal tissue section (duodenum, jejunum and ileum) from the lumen per day (normalized to intestinal tissue weight). These internal dose metrics, together with corresponding incidences for diffuse hyperplasia, were used to derive points of departure using benchmark dose modeling and constrained nonlinear regression. Both modeling techniques resulted in similar points of departure, which were subsequently converted to human equivalent doses using a human physiologically based pharmacokinetic model. Applying appropriate uncertainty factors, an RfD of 0.006 mg kg(-1) day(-1) was derived for diffuse hyperplasia-an effect that precedes tumor formation. This RfD is protective of both noncancer and cancer effects in the small intestine and corresponds to a safe drinking water equivalent level of 210 µg l(-1). This concentration is higher than the current federal maximum contaminant level for total Cr (100 µg l(-1)) and well above levels of Cr(VI) in US drinking water supplies (typically ≤ 5 µg l(-1)).

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.

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.