Darren N. Seril

Dietary iron supplementation enhances DSS-induced colitis and associated colorectal carcinoma development in mice.

Chronic ulcerative colitis (UC) patients frequently require iron supplementation to remedy anemia due to blood loss. However, the effect of iron supplementation on UC-associated carcinogenesis is unknown. In this study, the effect of an iron-enriched diet on dextran sulfate sodium-induced acute and chronic colitis in mice was assessed. In a short-term study, mice administered 1% DSS in the drinking fluid and an AIN76A diet containing increasing levels of iron exhibited dose-dependent increases in the severity of acute UC as compared to mice fed a control diet. A marked increase in iron deposition on the epithelial surface of the colon and in the inflamed areas and immunostaining for iNOS and nitrotyrosine were observed in the animals supplemented with diets containing different levels of iron. In a long-term carcinogenesis experiment, a twofold iron-enriched diet significantly increased colorectal tumor incidence (14/16, 88%) as compared with animals fed the control diet (3/16, 19%; P < 0.001). The present findings have implications for the management of human UC and suggest that dietary iron can enhance UC and its associated carcinogenesis by augmenting oxidative and nitrosative stress.

Inhibition of lung carcinogenesis and effects on angiogenesis and apoptosis in A/J mice by Oral administration of green tea

Oral administration of tea (Camellia sinensis) has been shown to inhibit the formation and growth of several tumor types in animal models. The present study investigated the effects of treatment with different concentrations of green tea on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in female A/J mice. Two days after a single dose of NNK (100 mg/kg body weight, i.p.), the mice were given 0.1, 0.2, 0.4, and 0.6% green tea solution (1, 2, 4, and 6 g of tea solids, respectively, dissolved in 1 l of water), 0.02% caffeine, or water as the sole source of drinking fluid until the termination of the experiment. Only the treatment with 0.6% tea preparation significantly reduced lung tumor multiplicity (mean ± SE, 6.07 ± 0.77 vs. 8.60 ± 0.50 tumors per mouse, P = 0.018). Treatment with 0.6% tea also inhibited angiogenesis, as indicated by the lower microvessel density (number of blood vessels/mm2) based on immunostaining for the von Willebrand factor antigen (81.9 ± 9.5 vs. 129.4 ± 8.2, P = 0.0018) and anti-CD31 antibody staining (465.3 ± 61.4 vs. 657.1 ± 43.6, P = 0.0012). Significantly lower vascular endothelial growth factor immunostaining scores were also observed in the 0.6% tea-treated group (0.98 ± 0.17 vs. 1.43 ± 0.07, P = 0.006). The apoptosis index was significantly higher in lung adenomas from 0.6% tea-treated mice based on morphological analysis of cell apoptosis (2.51 ± 0.18% vs. 1.57 ± 0.11%, P = 0.00005), and the result was further confirmed using the TUNEL method. Inhibition of angiogenesis and the induction of apoptosis by green tea may be closely related to the inhibition of pulmonary carcinogenesis.

Increased susceptibility of chronic ulcerative colitis-induced carcinoma development in DNA repair enzyme Ogg1 deficient mice †

Ogg1 DNA repair enzyme recognizes and excises oxidative stress‐caused 8‐hydroxyl‐deoxyguanosine (8‐OHdG) from GC base‐pairs. Ogg1 knockout mice are phenotypically normal, but exhibit elevated levels of 8‐OHdG in nuclear and mitochondrial DNA, as well as moderately elevated mutagenesis and spontaneous lung tumors and UV‐induced skin tumors. To elucidate the mechanistic role of inflammation‐caused oxidative stress in carcinogenesis, the development of chronic ulcerative colitis (UC)‐induced carcinoma in Ogg1 knockout mice was studied using a dextran sulfate sodium (DSS)‐induced UC model without the use of a carcinogen. Ogg1 (−/−), Ogg1 (+/−), and wild type C57BL/6 mice were subjected to long‐term, cyclic DSS treatment to induce chronic UC and carcinogenesis. In wild type C57BL/6 control mice after 15 cycles of DSS treatment, colorectal adenocarcinoma incidence was 24.1% (7/29 mice), with a tumor volume of 27.9 ± 5.2 mm3. Ogg1 (−/−) mice showed significantly increased adenocarcinoma development in the colon with a tumor incidence of 57.1% (12 of 21 mice, P < 0.05) and a tumor volume of 35.1 ± 6.1 mm3. Ogg1 mice (+/−) also exhibited significantly increased tumor development in the colon with a tumor incidence of 50.0% (13/26 mice) and a tumor volume of 29.1 ± 7.2 mm3. Histopathologic analyses revealed that colorectal tumors were well‐differentiated tubular adenocarcinomas or mucinous carcinoma and adjacent colonic mucosa showed mild to moderate chronic UC. Using immunohistochemical approaches, Ogg1 (−/−) and (+/−) mice exhibited similar numbers and staining intensities of macrophages in UC areas as seen in Ogg1 (+/+) mice, but markedly increased numbers and staining intensities of 8‐OHdG positive inflammatory and epithelial cells. These results provide important evidence on the relationship between inflammation‐caused oxidative stress, DNA repair enzyme Ogg1, and carcinogenesis.

Systemic iron supplementation replenishes iron stores without enhancing colon carcinogenesis in murine models of ulcerative colitis: comparison with iron-enriched diet.

Ulcerative colitis (UC) patients frequently require iron supplementation to remedy anemia. The impact of systemic iron supplementation (intraperitoneal injection) on UC-associated carcinogenesis was assessed in mice subjected to cyclic dextran sulfate sodium (DSS) treatment and compared with dietary iron enrichment. Systemic iron supplementation, but not a twofold iron diet, remedied iron deficiency as indicated by the histochemical detection of splenic iron stores. A twofold iron diet, but not systemic iron, increased iron accumulation in colonic luminal contents, at the colonic mucosal surface, and in superficial epithelial cells. Colitis-associated colorectal tumor incidence after 15 DSS cycles was not affected by systemic iron (2/28; 7.1%) compared to nonsupplemented controls (4/28; 14.1%) but was significantly increased by the twofold iron diet (24/33; 72.7%) (P < 0.001). Mechanistic study revealed that systemic iron had no effect on DSS-induced inflammation, or colonic iNOS and COX-2 protein levels, compared to controls. Systemic iron supplementation for 16 weeks replenished splenic iron in a spontaneous colitis model (interleukin-2-deficient mice) and significantly reduced colonic inflammation compared to interleukin-2 (−/−) controls without increasing hyperplastic lesions. These results suggest that iron supplemented systemically could be used to remedy anemia in UC patients without exacerbating inflammation or enhancing colon cancer risk. These findings need to be verified in clinical studies.

Immunohistochemical studies on Waf1p21, p16, pRb and p53 in human esophageal carcinomas and neighboring epithelia from a high-risk area in northern China

To better understand the roles of p53 and cell cycle–regulating protein alterations in human esophageal carcinogenesis, we investigated immunohistochemically the distribution patterns of Waf1p21, pRb, p16 and p53 in 22 cases of surgically resected esophageal cancer as well as in the neighboring non‐cancerous squamous epithelia. Waf1p21 protein was detected in 13 of the 20 cases of well‐differentiated squamous‐cell carcinoma (SCC), where the Waf1p21‐positive cells were located mainly in the interior layers of the cancer nests. Conversely, p53‐positive cells were found mostly in the peripheral layers. Cells containing both Waf1p21‐ and p53‐positive immunostaining were not observed in a double‐immunostaining experiment. p16 was detected in both the nucleus and cytoplasm in 3 of the 22 cases of SCC. All of these p16‐positive cancers showed an absence of pRb immunostaining; this result is consistent with the idea that expression of p16 is regulated negatively by pRb. Eleven of the 22 esophageal SCCs (50%) showed extensive pRb immunostaining cells, and the remaining 11 cases displayed a few pRb‐positive cells or an absence of pRb immunostaining. In a majority of the morphologically normal squamous‐cell epithelia samples, immunostaining of Waf1p21 and pRb was found in most of the cells in the parabasal layers (proliferation compartment), where PCNA‐positive cells also resided. In the pre‐cancerous lesions, Waf1p21 and pRb were detected in cells surrounding the top of the lesioned region, p16‐positive cells were scattered in the basal cell hyperplastic and dysplastic lesions and p53‐positive cells existed in 2 distinct patterns: “scattered” and “focal”. Int. J. Cancer 72:746–751, 1997.

Colorectal Carcinoma Development in Inducible Nitric Oxide Synthase-Deficient Mice With Dextran Sulfate Sodium-Induced Ulcerative Colitis

The overproduction of reactive oxygen and nitrogen species (RONS) may play an important role in ulcerative colitis (UC)‐associated carcinogenesis. In order to study the role of nitric oxide (NO) in UC‐associated colorectal carcinogenesis, the development of colorectal carcinoma was studied using the DSS‐induced and iron‐enhanced model of chronic UC in inducible nitric oxide synthase (iNOS)‐deficient mice. Female wild‐type C57BL/6 (iNOS+/+) and iNOS−/− mice were administered 1% DSS (w/v) through the drinking fluid for 15 DSS cycles and fed twofold iron‐enriched diet. Colorectal inflammation and mucosal ulceration of moderate severity were observed in both iNOS+/+ and iNOS−/− mice. Similar tumor incidence and multiplicity in the colon were observed that 15 out of 23 (65.2%) iNOS+/+ mice developed colorectal tumors with a tumor multiplicity of 1.47 ± 0.17 (mean ± SE) after 15 DSS cycles, and 13 out of 19 (68.4%) iNOS−/− mice developed colorectal tumors with a tumor multiplicity of 2.08 ± 0.21. Histopathologically, the tumors were confirmed to be well‐differentiated adenocarcinomas. Nitrotyrosine, an indicator of peroxynitrite‐caused protein modification, was detectable by immunohistochemistry in inflammatory cells and epithelial cells of the colon in iNOS+/+ and iNOS−/− mice, and no difference in staining intensity was observed between the two groups. Immunostaining for endothelial NOS (eNOS) was observed in lamina propria macrophages and colonic blood vessels, and eNOS protein levels were increased in the inflamed colon. These results show that there is no difference in UC‐associated cancer development in iNOS+/+ and iNOS−/− mice, and suggest that in the absence of iNOS, other factors, such as eNOS, may play a role in nitrosative stress and UC‐associated carcinogenesis in this model system.

High-iron diet: foe or feat in ulcerative colitis and ulcerative colitis-associated carcinogenesis.

Anemia associated with long-standing chronic inflammation and iron deficiency, and the increased risk for the development of dysplasia and carcinoma, are two of the most common complications in patients with ulcerative colitis (UC). Because of iron and nutrition deficiency, UC patients are encouraged to consume a high-protein and high-iron diet. The crucial clinical question is the effect of a high-iron diet on inflammation activity and inflammation-driven carcinogenesis. Is a high-iron diet a foe or a feat in UC and UC-associated carcinogenesis? This review updates the progress and information on (1) iron nutrition and iron-deficiency anemia in patients with UC, (2) experimental evidence of the exacerbating effect of a high-iron diet on UC and its associated carcinogenesis and the difference between a high-iron diet and parental iron supplementation, (3) the clinical efficacy of, and concerns about, oral and intravenous iron supplements in patients with inflammatory bowel disease and iron deficiency anemia, and (4) the clinical implications of long-term iron supplements and management of UC. These experimental findings from animal models provide evidence to warrant further consideration and clinical studies of iron nutrition, inflammation activity, and cancer development.

Mycobacterium avium Subspecies paratuberculosis in Crohn's disease: the puzzle continues.

The article by Dr Thomas Julius Borody in this issue is a review of a topic that has long been debated: the role of Mycobacterium avium subspecies paratuberculosis (MAP) in the etiopathogenesis of Crohn’s disease.1 Dr Borody addresses the key elements of the MAP argument in a point-by-point manner. However, the debate is likely to continue despite the long-established role of MAP in the causation of Johne disease (a chronic, intestinal inflammatory condition in ruminants similar to Crohn’s disease), advances in the methodology of detecting MAP in human tissue samples, and a better understanding of the response of MAP to immunosuppressive and antitumor necrosis factor (TNF)-a therapy. The primary counterpoints to the MAP theory persist, however, and revolve around the challenge of establishing a causal relationship between MAP infection and the evolution of the heterogenous inflammatory processes observed in Crohn’s disease, and the absence of convincing data establishing a role for anti-MAP therapy in the treatment of Crohn’s disease. As discussed in the article, Koch postulates have been satisfied implicating MAP as the causative factor of Johne disease. It has been argued that Koch criteria have been met implicating MAP in Crohn’s disease as well, predicated on the finding that MAP isolated from humans results in intestinal inflammation when administered orally to goats. Johne disease has many similarities to Crohn’s disease: it is a diarrheal illness characterized by chronic enteritis and granulomatous lesions.2 However, Crohn’s disease has a diverse presentation, with fistulizing and fibrostenosing disease and multiple extraintestinal manifestations. It would be more difficult to directly link MAP infection with the extraintestinal findings of Crohn’s disease, which are likely due to dysregulated immunity and autoimmunity.3 The suggestion of a causal relationship with MAP infection is inherently limited to correlative evidence. As summarized by the meta-analysis by Feller et al,4 studies have consistently shown that the prevalence of MAP is higher in the tissues of patients with Crohn’s disease as compared with controls, or even compared with those with ulcerative colitis. Given the finding of MAP in the tissues of individuals without Crohn’s disease, it is clear that MAP infection alone is not sufficient to cause the disease. If MAP is in fact a causative factor, it is likely that it does so in the setting of a susceptible individual. This would in part reconcile the finding that farmer’s exposed to cattle with Johne disease do not exhibit an inclination to developing Crohn’s disease, the inability to detect an association between Crohn’s disease and the consumption of MAP-containing foods, and the lack of vertical transmission of Crohn’s disease through MAP-containing breast milk.2,5 It would be important to learn whether there is a correlation between MAP infection, the presence of Crohn’s disease susceptibility genes such as NOD2 (which has been shown to take part in the clearance of intracellular MAP), and the occurrence of the disease. There have been reports of an increased prevalence of MAP infection in individuals diagnosed with Crohn’s disease at a young age in comparison with children without the disease, but this does not give us an idea of causation either. A prospective study would have the potential to reveal a temporal relationship between MAP infection and the onset of Crohn’s disease, but such a study would be technically unfeasible. Given the lack of information regarding cause-and-effect between the presence of MAP and the onset of disease, the higher frequency of MAP in tissues of patients with Crohn’s disease compared with nondiseased controls can be interpreted in another way. Perhaps the higher prevalence of MAP represents an increased susceptibility of the compromised intestinal mucosa of patients with Crohn’s disease to colonization by a bacterium that is ubiquitous in the environment.