Assessment of the Anti-Cancer Efficiency of Silver Moringa oleifera Leaves Nano-extract against Colon Cancer Induced Chemically in Rats.

BACKGROUND: Colorectal cancer (CRC) categorized as the most common type of gastrointestinal cancers affected both genders equally. Chemotherapeutic drugs became limited due to their deleterious side effects. Therefore, efficiency of M. oleifera leaves extract increased by incorporating silver nanoparticles (Ag-NPs) then studied against colon cancer induced by azoxymethane (AOM) in rats. METHODS: Different hematological and biochemical measurements in addition to specific tumor and inflammatory markers were quantified. Histopathological examination for Colonic tissues was performed. Native proteins and isoenzyme patterns were electrophoretically detected in addition to assaying expression of Tumor Protein P53 (TP53) and Adenomatous Polyposis Coli (APC) genes in colonic tissues. RESULTS: M. oleifera nano-extract restored levels of the hematological and biochemical measurements in addition to levels of tumor and inflammatory markers to normalcy in both of nano-extract simult- and post-treated groups. Also, it minimized severity of the histopathological alterations in the simult-treated group and prevented it completely in the post-treated group. The lowest similarity index (SI%) values were noticed with electrophoretic protein (SI=61.54%), lipid (SI=0.00%) and calcium (SI=75.00%) moieties of protein patterns, catalase (SI=85.71%), peroxidase (SI=85.71%), α-esterase (SI=50.00%) and ß-esterase (SI=50.00%) isoenzymes in addition to altering the relative quantities of total protein and isoenzyme bands in colon of cancer induced group. Moreover, levels of TP53 and APC gene expression increased significantly (P≤0.05) in colon cancer induced group. The nano-extract prevented the qualitative and quantitative alterations in the different electrophoretic patterns in addition to restoring levels of the gene expressions to normalcy in both of simult- and post-treated groups. CONCLUSION: M. oleifera nano-extract exhibited ameliorative effect against the biochemical, physiological and molecular alterations induced by AOM in nano-extract simult- and post-treated groups.
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Alternative Carcinogenicity Screening Assay Using Colon Cancer Stem Cells: A Quantitative PCR (qPCR)-Based Prediction System for Colon Carcinogenesis.

The carcinogenicity of chemicals in the environment is a major concern. Recently, numerous studies have attempted to develop methods for predicting carcinogenicity, including rodent and cell-based approaches. However, rodent carcinogenicity tests for evaluating the carcinogenic potential of a chemical to humans are time-consuming and costly. This study focused on the development of an alternative method for predicting carcinogenicity using quantitative PCR (qPCR) and colon cancer stem cells. A toxicogenomic method, mRNA profiling, is useful for predicting carcinogenicity. Using microarray analysis, we optimized 16 predictive gene sets from five carcinogens (azoxymethane, 3,2'-dimethyl-4-aminobiphenyl, N-ethyl-n-nitrosourea, metronidazole, 4-(n-methyl-n-nitrosamino)-1-(3-pyridyl)-1-butanone) used to treat colon cancer stem cell samples. The 16 genes were evaluated by qPCR using 23 positive and negative carcinogens in colon cancer stem cells. Among them, six genes could differentiate between positive and negative carcinogens with a p-value of < or =0.05. Our qPCR-based prediction system for colon carcinogenesis using colon cancer stem cells is cost- and time-efficient. Thus, this qPCR-based prediction system is an alternative to in vivo carcinogenicity screening assays.

Simultaneous Assessment of the Efficacy and Toxicity of Marine Mollusc-Derived Brominated Indoles in an In Vivo Model for Early Stage Colon Cancer.

The acute apoptotic response to genotoxic carcinogens animal model has been extensively used to assess the ability of drugs and natural products like dietary components to promote apoptosis in the colon and protect against colorectal cancer (CRC). This work aimed to use this model to identify the main chemopreventative agent in extracts from an Australian mollusc Dicathais orbita, while simultaneously providing information on their potential in vivo toxicity. After 2 weeks of daily oral gavage with bioactive extracts and purified brominated indoles, mice were injected with the chemical carcinogen azoxymethane (AOM; 10 mg/kg) and then killed 6 hours later. Efficacy was evaluated using immunohistochemical and hematoxylin staining, and toxicity was assessed via hematology, blood biochemistry, and liver histopathology. Comparison of saline- and AOM-injected controls revealed that potential toxic side effects can be interpreted from blood biochemistry and hematology using this short-term model, although AOM negatively affected the ability to detect histopathological effects in the liver. Purified 6-bromoisatin was identified as the main cancer preventive agent in the Muricidae extract, significantly enhancing apoptosis and reducing cell proliferation in the colonic crypts at 0.05 mg/g. There was no evidence of liver toxicity associated with 6-bromoisatin, whereas 0.1 mg/g of the brominated indole tyrindoleninone led to elevated aspartate aminotransferase levels and a reduction in red blood cells. As tyrindoleninone is converted to 6-bromoisatin by oxidation, this information will assist in the optimization and quality control of a chemopreventative nutraceutical from Muricidae. In conclusion, preliminary data on in vivo safety can be simultaneously collected when testing the efficacy of new natural products, such as 6-bromoisatin from Muricidae molluscs for early stage prevention of colon cancer.

In vivo measurement of the shape of the tissue-refractive-index correlation function and its application to detection of colorectal field carcinogenesis.

Polarization-gated spectroscopy is an established method to depth-selectively interrogate the structural properties of biological tissue. We employ this method in vivo in the azoxymethane (AOM)-treated rat model to monitor the morphological changes that occur in the field of a tumor during early carcinogenesis. The results demonstrate a statistically significant change in the shape of the refractive-index correlation function for AOM-treated rats versus saline-treated controls. Since refractive index is linearly proportional to mass density, these refractive-index changes can be directly linked to alterations in the spatial distribution patterns of macromolecular density. Furthermore, we found that alterations in the shape of the refractive-index correlation function shape were an indicator of both present and future risk of tumor development. These results suggest that noninvasive measurement of the shape of the refractive-index correlation function could be a promising marker of early cancer development.

Toxicology and risk assessment of 5-Hydroxymethylfurfural in food.

5-Hydroxymethylfurfural (5-HMF) as a product of the Maillard reaction is found in many foods. Estimated intakes range between 4 and 30 mg per person and day, while an intake of up to 350 mg can result from, e.g., beverages made from dried plums. In vitro genotoxicity was positive when the metabolic preconditions for the formation of the reactive metabolite 5-sulphoxymethylfurfural were met. However, so far in vivo genotoxicity was negative. Results obtained in short-term model studies for 5-HMF on the induction of neoplastic changes in the intestinal tract were negative or cannot be reliably interpreted as "carcinogenic". In the only long-term carcinogenicity study in rats and mice no tumours or their precursory stages were induced by 5-HMF aside from liver adenomas in female mice, the relevance of which must be viewed as doubtful. Hence, no relevance for humans concerning carcinogenic and genotoxic effects can be derived. The remaining toxic potential is rather low. Various animal experiments reveal that no adverse effect levels are in the range of 80-100 mg/kg body weight and day. Safety margins are generally sufficient. However, 5-HMF exposure resulting from caramel colours used as food additives should be further evaluated.

Estimation of incidence and social cost of colon cancer due to nitrate in drinking water in the EU: a tentative cost-benefit assessment.

BACKGROUND: Presently, health costs associated with nitrate in drinking water are uncertain and not quantified. This limits proper evaluation of current policies and measures for solving or preventing nitrate pollution of drinking water resources. The cost for society associated with nitrate is also relevant for integrated assessment of EU nitrogen policies taking a perspective of welfare optimization. The overarching question is at which nitrogen mitigation level the social cost of measures, including their consequence for availability of food and energy, matches the social benefit of these measures for human health and biodiversity. METHODS: Epidemiological studies suggest colon cancer to be possibly associated with nitrate in drinking water. In this study risk increase for colon cancer is based on a case-control study for Iowa, which is extrapolated to assess the social cost for 11 EU member states by using data on cancer incidence, nitrogen leaching and drinking water supply in the EU. Health costs are provisionally compared with nitrate mitigation costs and social benefits of fertilizer use. RESULTS: For above median meat consumption the risk of colon cancer doubles when exposed to drinking water exceeding 25 mg/L of nitrate (NO3) for more than ten years. We estimate the associated increase of incidence of colon cancer from nitrate contamination of groundwater based drinking water in EU11 at 3%. This corresponds to a population-averaged health loss of 2.9 euro per capita or 0.7 euro per kg of nitrate-N leaching from fertilizer. CONCLUSIONS: Our cost estimates indicate that current measures to prevent exceedance of 50 mg/L NO3 are probably beneficial for society and that a stricter nitrate limit and additional measures may be justified. The present assessment of social cost is uncertain because it considers only one type of cancer, it is based on one epidemiological study in Iowa, and involves various assumptions regarding exposure. Our results highlight the need for improved epidemiological studies.

Follow-up of tumor development in the colons of living rats and implications for chemoprevention trials: assessment of aspirin-difluoromethylornithine combination.

We aimed to establish a reliable procedure allowing the follow-up of tumor development by computed tomographic (CT) colonography in an animal model of colon carcinogenesis in order to assess the chemopreventive efficacy of aspirin and difluoromethylornithine (DFMO) given in combination. Fischer rats received an intraperitoneal injection (25 mg/kg) of dimethylhydrazine (DMH) once a week for two weeks in order to initiate colon carcinogenesis. Five months after the last injection of DMH, a first CT colonography was performed and rats were then randomly separated into two groups (control and experimental). The experimental group received a 0.1% mixture of aspirin and DFMO in drinking water. CT colonography was performed at 6, 7 and 8 months. Data showed a precise correlation between location and size of tumors found at autopsy and those detected by CT colonography at 8 months. All tumors were also detected on the CT views obtained previously. Animals of the aspirin/DFMO group exhibited an inactivation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, and a two-fold reduction in the prostaglandin E2 content of the colonic mucosa (p<0.01). In rats with tumors at the start of the aspirin/DFMO treatment, a significant slow-down of tumor development was observed. In contrast, in rats where no tumors were detected at the start of the treatment, tumor formation was inhibited. Our data show that CT colonography represents a reliable method to assess in a living animal the efficacy of chemopreventive agents.

Quantitative assessment of azoxymethane-induced aberrant crypt foci in inbred mice.

Heritable differences in tumor susceptibility are observed in mice after repetitive exposures to the organotropic colon carcinogen azoxymethane (AOM). The following study was undertaken to determine whether early morphological alterations within the colonic epithelium correlate with subsequent cancer risk. A/J and SWR/J (susceptible) and AKR/J (resistant) mice were injected once a week with AOM at a dose of 10 mg/kg, i.p., for a total of 6 weeks. Four weeks after the last injection, methylene blue-stained whole-mount colons were examined for the presence of colonic epithelial lesions referred to as aberrant crypt foci (ACF). Putative lesions identified under low magnification were further characterized by H&E staining of corresponding sections. AOM produced a treatment-related increase in ACFs in each of the mouse lines examined. The tumor-susceptible SWR/J and A/J mice developed on average between three- and sixfold more ACFs in the distal colon (32 and 15/cm of colon, respectively) than the resistant AKR/J mice (5/cm colon). The size distribution of ACFs was further analyzed in each of the strains. In SWR/J and A/J, 20-35% of lesions were classified as large ACFs, consisting of 5 or more aberrant crypts per focus. This is in striking contrast to the size distribution of lesions identified in the AKR/J colons, where fewer than 5% of grossly identified lesions were classified as large. In fact, the majority (> 80%) of ACFs in AKR/J mice consisted of only 1-2 aberrant crypts@focus. In addition, there was no evidence of dysplasia in any of the AKR/J lesions examined, whereas the lesions in susceptible mice were dysplastic (adenomas). Our data indicate that tumorigenic response is associated with the extent and multiplicity of ACFs that form within the colonic epithelium at an early time point after carcinogen exposure. These studies further support the use of this morphological biomarker as a short-term endpoint of colon tumorigenesis.

Microsatellite instability is infrequent in azoxymethane-induced rat intestinal tumors: An assessment by capillary electrophoresis.

A rat model of colon cancer in which tumors are induced by azoxymethane (AOM) is frequently used to study putative environmental agents that may modify the risk of human colon cancer development. In order to evaluate the usefulness of this model for human risk assessment, a comparison of the molecular changes associated with tumorigenesis in the rat model with those in human colon cancer is desirable. Microsatellite instability (MSI), an alteration in length of short repetitive DNA sequences associated with defective DNA mismatch repair, is an important molecular characteristic of many human colon tumors. Intestinal tumors were induced in male Fischer 344 rats injected with 15 mg/kg body wt AOM in four weekly doses. Thirteen intestinal tumors were examined for MSI at 10 different microsatellite loci, using a capillary electrophoresis (CE) method for accurate assessment of DNA length. This method was shown to have a resolution of 1 bp for a 140-bp PCR product and to be capable of detecting one mutant sequence within a background of 10 wild-type sequences. The CE method also readily distinguished a known MSI-positive human tumor sample from its matching control sample. Among the 13 rat intestinal tumors examined, only one had MSI, which was present at only a single locus. We conclude that, unlike sporadic human colon tumors in which 15-30% of tumors have MSI (usually at multiple loci), MSI is very rare in AOM-induced rat intestinal tumors.

Assessment of mutations in Ki-ras and p53 in colon cancers from azoxymethane- and dimethylhydrazine-treated rats.

Mutations in the Ki-ras oncogene and the p53 tumor suppressor gene are known to occur at high frequencies in human colon cancers. We measured the frequency of mutations in these two genes in colon adenocarcinomas obtained from a widely used experimental model of human colon carcinogenesis: F344 rats treated with the carcinogens azoxymethane (AOM) or dimethylhydrazine (DMH). We detected codon 12 mutations in Ki-ras in approximately 60% of colon adenocarcinomas induced by either carcinogen. We characterized the rat p53 intron-exon junctions to construct primers for polymerase chain reaction amplification of this gene. We discovered that the rat p53 gene was structurally different from the human p53 gene, as the rat gene was missing one intron between exons 6 and 7. Both single-stranded DNA conformational polymorphism analysis and direct DNA sequencing of the highly conserved regions of rat exons 5-7 were conducted because the corresponding human regions (exons 5-8) have been reported as being mutated most frequently in human colon cancers. Using these methods, we were unable to identify any p53 mutations in the highly conserved regions of exons 5-7 in either AOM- or DMH-induced colon adenocarcinomas. These data confirm that Ki-ras was mutated in most colon cancers in AOM- or DMH-treated rats but indicate that molecular alterations in the p53 gene, if they occur in this animal model, are different from most p53 mutations in human colon cancers.

Assessment of the nucleolar organizer regions by automated image analysis in benign and malignant colonic tumours and adjacent tissues in rats.

An automated computer image analysis technique was used to study the morphological parameters of argyrophilic nucleolar organizer regions (AgNORs) in chemically induced rat colonic tumours of different grades. Different nuclear parameters were of different diagnostic value. For distinguishing tumorous tissue from normal tissue all the parameters studied were capable of serving as diagnostic markers. Malignant processes could, however, be more reliably detected by means of the area of the nucleus, nuclear shape factor, area of AgNOR and ratio of AgNOR area to nuclear area. In macroscopically normal tissue adjacent to a tumour, the values of all the AgNOR parameters studied were similar to those in tumorous tissues. It can be concluded that the initial stages in tumorigenesis are accompanied by changes in all of the nucleus and AgNOR parameters, but as malignancy develops, only some of these parameters continue to change. Close correlations between nuclear parameters in malignant tissue suggest that for diagnostic purposes only nuclear and AgNOR areas should be used.

DNA adduct formation and assessment of aberrant crypt foci in vivo in the rat colon mucosa after treatment with N-methyl-N-nitrosourea.

N-Nitroso-compound DNA adduct formation in vivo and occurrence of aberrant crypt foci (ACF) were studied in the rat colon mucosa after a single, local treatment with a carcinogen, N-methyl-N-nitrosourea (MNU), using a simple surgical approach. A segment of F344 rat colon was ligated to make a pouch and injected with MNU solution. For the study of DNA adduct formation, the solution contained 50 microCi of [3H]MNU. The results demonstrated that similar ranges of carcinogen dose, i.e. 0.15 x 10(-2) - 1.5 x 10(-2) M MNU, could induce both DNA adduct formation and appearance of ACF in the rat colon with both parameters showing a nearly linear dose dependence. HPLC analysis revealed the DNA adducts to include both 7-methylguanine (7-mGua) and O6-methylguanine (O6-mGua) with the 7-mGua/O6-mGua ratio being 8.2-11.3:1 in the system used. Assessment of ACF development from 4 to 16 weeks after MNU treatment at a dose of 7.5 x 10(-2) M showed the numbers to increase up to the 8th week, followed by a decrease at weeks 12 and 16, when 40% of the ACF counted at the peak time point were still present. The percentage of large ACF (> or = 4 crypts/ACF) significantly increased with time. These results indicate a clear relation between DNA adducts and preneoplastic lesions, i.e. ACF. In conclusion, DNA adduct formation and ACF can be efficiently and simply detected in vivo by using the method described in the present paper.

An assessment of the risk of neoplasia in long-term ileal reservoirs using the DMH rodent model.

This study was undertaken to determine if the construction of an ileal reservoir induces mucosal changes that can potentiate the effect of a chemical carcinogen (1,2-dimethylhydrazine) on ileal mucosa. Animals were divided into three groups: 1) sham operation (n = 19), 2) total colectomy with ileorectal anastomosis (n = 20), 3) total colectomy with an ileal reservoir made of terminal ileum sutured to the rectum (n = 20). An adaptation period of 12 weeks was allowed to promote fecal stasis and the histologic changes before exposure to weekly subcutaneous injections of DMH (25 mg/kg) for 16 weeks. Sodium butyrate was added to the diet as a tumor promotor. All animals were sacrificed one month later. Fecal stasis, along with enlargement, occurred in all the reservoirs (mean dimensions, 74 X 58 X 43 mm). Their mean volume was 88 +/- 14 ml. The histologic changes in the ileal reservoirs were: chronic inflammation (14/20), villous atrophy (14/20), and atrophy of the glands (8/20). In group 3, five carcinomas were seen. There were three in the duodenum and two in the reservoirs. In contrast, 21 carcinomas were detected in the control groups. There were 17 in the colon, 3 in the jejunum, and 1 in the ileum. No significant difference in the number of carcinomas was seen in the ileum with and without reservoir. Although it is possible to induce carcinomas in ileal reservoirs, the incidence remained significantly less than in the colon. In conclusion, the histologic changes induced by the construction of an ileal reservoir do not increase the risk of malignant transformation in the DMH model for intestinal carcinogenesis.

Relationship between dietary fiber and cancer: metabolic, physiologic, and cellular mechanisms.

The relationships between fiber consumption and human cancer rates have been examined, together with an analysis of the effects of individual dietary fibers on the experimental induction of large bowel cancer. The human epidemiology indicates an inverse correlation between high fiber consumption and lower colon cancer rates. Cereal fiber sources show the most consistent negative correlation. However, human case-control studies in general fail to confirm any protective effect due to dietary fiber. Case-control studies indicate that if any source of dietary fiber is possibly antineoplastic then it is probably vegetables. These results may mean that purified fibers alone do not inhibit tumor development, whereas it is likely that some other factors present in vegetables are antineoplastic. Experiments in laboratory animals, using chemical induction of large bowel cancer, have in general shown a protective effect with supplements of poorly fermentable fibers such as wheat bran or cellulose. In contrast, a number of fermentable fiber supplements including pectin, corn bran, oat bran, undegraded carageenan, agar, psyllium, guar gum, and alfalfa have been shown to enhance tumor development. Possible mechanisms by which fibers may inhibit colon tumorigenesis include dilution and adsorption of any carcinogens and/or promoters contained within the intestinal lumen, the modulation of colonic microbial metabolic activity, and biological modification of intestinal epithelial cells. Dietary fibers not only bind carcinogens, bile acids, and other potential toxins but also essential nutrients, such as minerals, which can inhibit the carcinogenic process. Fermentation of fibers within the large bowel results in the production of short chain fatty acids, which in vivo stimulate cell proliferation, while butyrate appears to be antineoplastic in vitro. Evidence suggests that if dietary fibers stimulate cell proliferation during the stage of initiation, then this may lead to tumor enhancement. Fermentation also lowers luminal pH, which in turn modifies colonic microbial metabolic acidity, and is associated with increased epithelial cell proliferation and colon carcinogenesis. Because dietary fibers differ in their physiochemical properties it has been difficult to identify a single mechanism by which fibers modify colon carcinogenesis. Clearly, more metabolic and physiological studies are needed to fully define the mechanisms by which certain fibers inhibit while others enhance experimental colon carcinogenesis.

The assessment of response of murine transplantable colon tumors to combination chemotherapy.

The mouse adenocarcinoma of the colon (MAC) system, which has been shown to be a good model for human colorectal carcinoma in terms of its chemosensitivity, was tested with two modified human protocols (MeCCNU + 5 FU, BCNU + 5 FU) in an attempt to evaluate its suitability as a model for developing new regimens of combination chemotherapy for treating patients with colorectal carcinoma. This attempted evaluation raised problems regarding, firstly, the length of time available before tumours became too large in control and non-responding hosts to maintain adequate mobility and, secondly the assessment of response to the drugs. The commencement of drug administration 3 days after transplantation and the assessment of response by measuring delay in time for tumor growth to reach a given volume, with the results analysed by Gehan's (generalised Wilcoxon) test, gave a workable method of evaluation. This method is presented as being suitable for use in the study of transplantable solid tumor lines as models for combination chemotherapy.