Rodent Forestomach Research Articles

No news from old drawings? Stomach anatomy in muroid rodents in relation to body size and ecology.

Muroid rodents mostly have a complex stomach: one part is lined with a cornified (nonglandular) epithelium, referred to as a “forestomach”, whereas the rest is lined with glandular epithelium. Numerous functions for the forestomach have been proposed. We collated a catalog of anatomical depictions of the stomach of 174 muroid species from which the respective nonglandular and glandular areas could be digitally measured, yielding a “stomach ratio” (nonglandular:glandular area) as a scale‐independent variable. Stomach ratios ranged from 0.13 to 20.15, and the coefficient of intraspecific variation if more than one picture was available for a species averaged at 29.7% (±21.5). We tested relationships of the ratio with body mass and various anatomical and ecological variables, including diet. There was a consistent phylogenetic signal, suggesting that closely related species share a similar anatomy. Apart from classifying stomachs into hemiglandular and discoglandular, no anatomical or ecological measure showed a consistent relationship to the stomach ratio. In particular, irrespective of statistical method or the source of dietary information, dietary proxies did not significantly correlate with the stomach ratio, except for a trend towards significance for invertivory (insectivory). Yet, even this relationship was not convincing: whereas highly insectivorous species had high but no low stomach ratios, herbivorous species had both low and high stomach ratios. Thus, the statistical effect is not due to a systematic increase in the relative forestomach size with invertivory. The most plausible hypotheses so far associate the muroid forestomach and its microbiome with a generic protective role against microbial or fungal toxins and diseases, without evident correlates of a peculiar need for this function under specific ecological conditions. Yet, this function remains to be confirmed. While providing a catalog of published depictions and hypotheses, this study highlights that the function of the muroid rodent forestomach remains enigmatic to date.

The role of ethyl acrylate induced GSH depletion in the rodent forestomach and its impact on MTD and in vivo genotoxicity in developing an adverse outcome pathway (AOP)

Adverse outcome pathways (AOP) and mode of action (MOA) frameworks help evaluate the toxicity findings of animal studies and their relevance to humans. To effectively use these tools to improve hazard identification and risk assessments for ethyl acrylate (EA), knowledge gaps in metabolism and genotoxicity were identified and addressed. For EA, hypothesized early key events relate to its irritation potential: concentration dependent irritation and cytotoxicity, progressing to regenerative proliferation and forestomach carcinogenicity after repeated oral bolus application in rodents. The current research quantitated glutathione (GSH) depletion to assess a kinetically-derived maximum tolerated dose (MTD) in the target tissue and used this information to conduct an in vivo genotoxicity study using current methods. In the mouse forestomach, gavage doses of EA caused GSH depletion to 47% of control at 20 mg/kg and 28% at 100 mg/kg. Cellular redox changes and histopathology support saturation of metabolism and an MTD of ∼50 mg/kg. No increases in point mutations or deletions occurred in the stomach or liver following a 28 day treatment of gpt delta transgenic mice at gavage doses up to 50 mg/kg/day. These results provide valuable information for evaluating AOP molecular initiating events or MOA key events for EA and other GSH depleting materials.

Toxicogenomic outcomes predictive of forestomach carcinogenesis following exposure to benzo(a)pyrene: Relevance to human cancer risk

Forestomach tumors are observed in mice exposed to environmental carcinogens. However, the relevance of this data to humans is controversial because humans lack a forestomach. We hypothesize that an understanding of early molecular changes after exposure to a carcinogen in the forestomach will provide mode-of-action information to evaluate the applicability of forestomach cancers to human cancer risk assessment. In the present study we exposed mice to benzo(a)pyrene (BaP), an environmental carcinogen commonly associated with tumors of the rodent forestomach. Toxicogenomic tools were used to profile gene expression response in the forestomach. Adult Muta™Mouse males were orally exposed to 25, 50, and 75mgBaP/kg-body-weight/day for 28 consecutive days. The forestomach was collected three days post-exposure. DNA microarrays, real-time RT-qPCR arrays, and protein analyses were employed to characterize responses in the forestomach. Microarray results showed altered expression of 414 genes across all treatment groups (±1.5 fold; false discovery rate adjusted P≤0.05). Significant downregulation of genes associated with phase II xenobiotic metabolism and increased expression of genes implicated in antigen processing and presentation, immune response, chemotaxis, and keratinocyte differentiation were observed in treated groups in a dose-dependent manner. A systematic comparison of the differentially expressed genes in the forestomach from the present study to differentially expressed genes identified in human diseases including human gastrointestinal tract cancers using the NextBio Human Disease Atlas showed significant commonalities between the two models. Our results provide molecular evidence supporting the use of the mouse forestomach model to evaluate chemically-induced gastrointestinal carcinogenesis in humans.

Abstract 4792: Analysis of the relationship between experimentally induced rodent forestomach hyperplasia and tumorigenesis.

Abstract Hyperplasia is an increase in cell number resulting from physiological processes such as irritation, or pathological processes like carcinogenesis. The difficulty in differentiating reactive versus preneoplastic (or dedicated) hyperplasia has been the subject of debate in the literature, especially in regard to prediction of human health risks following chronic chemical exposure. Squamous hyperplasia and neoplasia are frequently detected in the rodent forestomach (FS) following chronic oral administration of chemicals evaluated in rodent cancer bioassays. Molecular level mechanistic details related to FS hyperplasia progression are not available for a wide range of chemicals, despite the increasing need for this level of mechanistic understanding to support evaluations for human health risk. In order to facilitate further discussion on the use of rodent FS lesions in the estimation of cancer and noncancer reference values, a comparison of chemical hyperplasia versus tumor induction was performed. FS lesion classification and incidence data were obtained from rat and mouse gavage bioassays on over 50 chemicals from National Toxicology Program (NTP) 2-year bioassays that reported dose-related increases in FS hyperplastic or neoplastic lesions in one or more rodent sex/strain combinations tested. Chemical effects were tabulated and sorted by strain, sex, genotoxicity assay results and irritation effects, and the relationship between FS hyperplasia and FS tumor (e.g., squamous cell papilloma or carcinoma) induction was analyzed using grouped animal data. When considering chemicals with positive results in genotoxicity assays, FS tumors positively correlated with FS hyperplasia incidence in both rats and mice. Increased incidence of FS hyperplasia was not correlated with FS tumors in animals exposed to chemicals that were negative for genotoxicity and irritation. The potential results of this analysis may be useful in the evaluation of chemicals with limited chronic bioassay data by informing predictions of long-term tumor formation based on chemical properties observable during short-term animal or in vitro studies (e.g., genotoxicity, irritation). The views expressed in this abstract are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency. Citation Format: Jason M. Fritz, Maureen R. Gwinn. Analysis of the relationship between experimentally induced rodent forestomach hyperplasia and tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4792. doi:10.1158/1538-7445.AM2013-4792

Transcriptional and Metabolomic Consequences of luxS Inactivation Reveal a Metabolic Rather than Quorum-Sensing Role for LuxS in Lactobacillus reuteri 100-23

Autoinducer-2 (AI-2)-mediated quorum sensing has been extensively studied in relation to the regulation of microbial behavior. There are, however, two potential roles for the AI-2 synthase (LuxS). The first is in the production of AI-2 and the second is as an enzyme in the activated methyl cycle, where it catalyzes the conversion of S-ribosylhomocysteine to homocysteine. The by-product of the reaction catalyzed by LuxS is (S)-4,5-dihydroxy-2,3-pentanedione, which spontaneously forms the furanones known collectively as AI-2. The mammalian gut contains a complex collection of bacterial species so a method of interspecies communication might influence community structure and function. Lactobacillus reuteri 100-23 is an autochthonous inhabitant of the rodent forestomach, where it adheres to the nonsecretory epithelium, forming a biofilm. Microarray comparisons of gene expression profiles of the L. reuteri 100-23 wild type and a luxS mutant under different culture conditions revealed altered transcription of genes encoding proteins associated with cysteine biosynthesis/oxidative stress response, urease activity, and sortase-dependent proteins. Metabolomic analysis showed that the luxS mutation affected cellular levels of fermentation products, fatty acids and amino acids. Cell density-dependent changes (log phase versus stationary phase growth) in gene transcription were not detected, indicating that AI-2 was unlikely to be involved in gene regulation mediated by quorum sensing in L. reuteri 100-23.

Mode-of-Action Framework for Evaluating the Relevance of Rodent Forestomach Tumors in Cancer Risk Assessment

Studies have shown that a majority of known human carcinogens also cause cancer in laboratory animals. The converse, however, is not as well established-known animal carcinogens are not equally predictive of human carcinogenicity. A particularly controversial aspect of interspecies extrapolation is application of rodent forestomach tumor data for predicting cancer risk in humans, given that a human counterpart for the rodent forestomach does not exist. Proliferative lesions in the rodent forestomach may result from a combination of factors related to route-specific tissue irritation and/or unnatural dosing regimens and are less likely to be relevant in evaluating human carcinogenic potential, particularly when tumors are exclusive to the forestomach. We review the comparative functional anatomy, physiology, tumor biology, tissue concordance, and historical regulatory practices in the use of forestomach tumors for cancer risk assessment, examining specific chemical examples. We also propose a standardized mode-of-action approach that combines multiple risk characterization criteria, including relevance to human exposure conditions, physiologically based toxicokinetics, genotoxicity, and comparative/mechanistic toxicology. Forestomach tumors associated with chronic irritation of the forestomach epithelium, particularly those induced by repeated oral gavage dosing, should not form the basis for carcinogenic classification or quantitative cancer potency estimates for humans. Genotoxic chemicals and those that cause tumors at multiple sites, at doses at or below the maximum tolerated dose, and in the absence of forestomach irritation, are more likely to be relevant human carcinogens. Cancer risk assessment that utilizes forestomach tumor data should consider relevant human exposures, systemic bioavailability, tissue dosimetry and concordance.

A Mechanistic Interpretation of the Oncogenicity of Chlorothalonil in Rodents and an Assessment of Human Relevance

Chronic dietary treatment of rodents with the fungicide chlorothalonil causes an increased incidence of papillomas and carcinomas of the forestomach squamous epithelium (rats and mice, both sexes) and adenomas and carcinomas of the renal proximal tubule epithelium (rats, both sexes; mice, males only); the product elicits no tumorigenic response in dogs. As a result, chlorothalonil is classified by EPA as a Group B2 “probable human carcinogen.” However, chlorothalonil is not genotoxic and there is strong evidence that both the forestomach and renal tumors observed in rodents result from cytotoxicity followed by compensatory cell proliferation and hyperplasia. In the case of the forestomach, cytotoxicity results from sustained irritation of the squamous epithelium by chlorothalonil leading to inflammation, ulceration, and restorative hyperplasia. Cytotoxicity in the renal tubular epithelium is associated with formation of di- and trithiols that arise through the action of renal β-lyase on cysteine S-conjugates derived from the corresponding glutathione conjugates of chlorothalonil. Renal cytotoxicity and cell necrosis in rodents result from the ability of the di- and trithiols to inhibit kidney mitochondrial respiration and disrupt cellular integrity. There is strong evidence that this mechanism is not operative in other species such as dogs and monkeys. The progression from cytotoxicity to hyperplasia to neoplasia is becoming increasingly well-recognized as a threshold-based mechanism of carcinogenesis. Unless exposure is excessively prolonged or intense, the cytotoxic effects will be fully reversible. Furthermore, the effects observed in rodents are not appropriate for evaluating the potential human cancer risk from chlorothalonil. Humans do not possess an organ equivalent to the rodent forestomach and the rat is a poor model for evaluating potential human risk for the renal tumorigenicity of chlorothalonil. Humans are likely to be very much less sensitive than rats to the nephrotoxic effects of chlorothalonil. In view of the fact that the tumorigenic effects of chlorothalonil are mediated through a well-understood, nongenotoxic, threshold-based mechanisms of little or no relevance to humans, chlorothalonil should be a prime candidate for re-review under EPA's new risk assessment guidelines. Expert committees in both Europe and Canada have concluded that human risks to chlorothalonil should be evaluated by means of the NOEL/safety factor approach usually employed for noncarcinogenic materials.

Propionic acid and the phenomenon of rodent forestomach tumorigenesis: A review

Propionic acid (PA) is widely used as an antifungal agent in food. It is present naturally at low levels in dairy products and occurs ubiquitously, together with other short-chain fatty acids (SCFA), in the gastro-intestinal tract of humans and other mammals as an end-product of the microbial digestion of carbohydrates. It has significant physiological activity in animals. PA is irritant but produces no acute systemic effects and has no demonstrable genotoxic potential. Rats receiving PA at 4% in the diet develop hyperplastic lesions and tumours of the forestomach. Such changes are also reported to have been produced by a variety of other chemicals, mechanical and chemical irritants, parasites, and even various dietary insufficiencies or imbalances. Evidence suggests that repeated or persistent damage to cells of the forestomach epithelium and associated proliferative responses may be a common factor in rodent forestomach tumorigenesis. Although humans do not have a forestomach, the mouth, pharynx and oesophagus are lined with similar epithelium and thus are potential target organs. However, food contact time is considerably less in these organs than in the rodent forestomach. On current evidence, PA cannot be considered a carcinogenic risk to humans; nevertheless, to determine a safe level of dietary exposure it is suggested that no-observable-effect levels should be determined for epithelial cell damage and proliferative responses in the squamous mucosa of laboratory animals, particularly in those parts of the gastro-intestinal tract having a counterpart in humans, such as the pharynx and oesophagus.

Early indicators of potential neoplasia produced in the rat forestomach by non-genotoxic agents: the importance of induced cellular proliferation

Forestomach neoplasia induced by the apparently non-genotoxic carcinogens, butylated hydroxyanisole and propionic acid, appears to arise by way of sustained high levels of cellular proliferation. Several other inducers of enhanced cellular proliferation, or the consequential indicidence of hyperplastic lesions, have been identified in the rodent forestomach bu the requisite carcinogenicity bioassays remain undone. In other tissues, such as the male rat kidney, the rodent thyroid follicuklar cell and the bladder epithelium, there is also evidence supporting the concept that sustained enhanced cellular proliferation may be an important early marker for non-genotoxic carcinogens. This reaction is, howerver, not likely to be the only marker necessary for the identification of non-genotoxic carcinogens.

Studies on the effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors on the rodent forestomach

A novel class of hypocholesterolaemic agents, HMG CoA reductase inhibitors, was shown to cause mucosal thickening in the rodent (mouse and rat) forestomach after subacute/subchronic oral administration. These changes were characterized histologically by acanthosis and hyperkeratosis of the squamous epithelium with submucosal oedema and occasionally cellular infiltration. This drug-induced hyperplastic response was both dose and time dependent, did not occur after subcutaneous administration, and was confined entirely to the rodent forestomach (not observed in any other area of the gastro-intestinal tract). The forestomach hyperplastic response correlated with the pharmacological potency of HMG CoA reductase inhibitors of similar structure (observed to varying degrees with all HMG CoA reductase inhibitors examined to date).

Butylated hydroxyanisole (BHA) does not cause forestomach hyperplasia by inhibiting the release of gastric mucus

High doses of BHA cause hyperplasia and subsequent neoplasia in the rodent forestomach and can inhibit gastric prostaglandin (PG) synthesis in vitro. This paper examines the hypothesis that BHA induced forestomach hyperplasia occurs in response to a reduction of gastric mucus, with consequent irritation of the forestomach. This could result from inhibition of the formation of the PG's which mediate the synthesis and release of protective mucus. Groups of 10 rats received 0 or 2% BHA in the diet for 1 or 3 weeks and a positive control group was fed a diet containing indomethacin (3.5 mg/kg), a potent inhibitor of PG synthesis. After 1 week BHA caused focal erosion and ulceration of the forestomach consistent with an irritant effect, but 2 weeks later the epithelium was healed, thickened and markedly hyperplastic. Histochemical staining for mucus showed that the development of forestomach hyperplasia was associated with increased amounts of gastric and duodenal mucus and increased numbers of serotonergic-cells in the gastric and duodenal epithelium. In contrast, indomethacin caused a marked reduction in both gastric and Brunner's gland mucus. Neither BHA nor indomethacin exerted an effect on one specific type of mucus (viz: neutral, acidic or mixed) in the stomach. These results do not support the hypothesis that forestomach hyperplasia arises from an inhibition of either the synthesis or release of gastric mucus. It is possible that the increased numbers of serotonergic-cells are related to the initial ulcerative, or subsequent hyperplastic response.

THE SIGNIFICANCE OF INDUCED FORESTOMACH TUMORS

Scientific interest in forestomach carcinogenesis has been greatly heightened by the observation that butylated hydroxyanisole (BHA) is carcinogenic to rats at this site (1). BHA is one of only three synthetic phenolic antioxidants that are permitted to be added to food in Canada. These chemicals inhibit oxidative spoilage of food (rancidity) during transportation and storage and are thus of importance to the functioning of the modern centralized food industry. The demonstration that high levels of BHA incorporated into the food of male and female rats resulted in papillomas and carcinomas of the forestomach led to an international research effort focused on the forestomach and designed to outline the way in which these tumors arose and determine whether they are relevant to humans (2). This report addresses the development of cancerous lesions in the rodent forestomach, the nature of the chemicals that induce these lesions, and their interactions with the animal body. Presently available evidence indicates there are at least two different types of chemical carcinogen that act on the forestomach and that these may have very different degrees of relevance to the human population.

Safety evaluation of butylated hydroxyanisole from the perspective of effects on forestomach and oesophageal squamous epithelium

Butylated hydroxyanisole (BHA) induces tumours of the squamous epithelium of the forestomach of rodents, but not at other sites. Although humans do not have squamous epithelium in their stomach the likelihood that BHA will induce tumours of the squamous epithelium of the oesophagus needs to be considered. Studies in several species indicate that the forestomach epithelium is very responsive to hyperplastic and neoplastic change induced by BHA, but the oesophageal epithelium is not responsive. The lack of effect in the oesophagus is likely to be due to the fact that the rapid speed of transit through the oesophagus limits the exposure time of the oesophageal mucosa to the food contents. Conversely, as the rodent's forestomach has storage function, exposure of the squamous epithelium of the forestomach would be continuous. The fact that the no-observed-effect level for hyperplasia of the oesophageal mucosa is several hundred times the acceptable daily intake for BHA supports the view that BHA would not be a human carcinogen at food additive levels of use.