Measurements of rat and mouse gastrointestinal pH, fluid and lymphoid tissue, and implications for in-vivo experiments

Abstract

To use rodent models effectively in in-vivo investigations on oral drug and vaccine delivery, the conditions in the gastrointestinal tract must be understood. Some fundamental information is currently unavailable or incomplete. We have investigated the pH, water content and lymphoid tissue distribution along the gastrointestinal tract, as well as the stomach volume, as these were critical to our investigations on pH-responsive drug delivery and colonic vaccination. The observed values were compared with those in man as an indication of the validity of the rodent model. The mouse stomach pH was 3.0 (fed) and 4.0 (fasted), and the corresponding values in the rat were 3.2 (fed) and 3.9 (fasted). The mean intestinal pH was lower than that in man (<pH 5.2 in the mouse; <pH 6.6 in the rat). This brings into question the use of rodents in investigations on enteric-coated drug carriers targeted to the large intestine/distal gut. The water content in the gastrointestinal tract in the fed and fasted mouse was 0.98+/-0.4 and 0.81+/-1.3 mL, respectively, and in the fed and fasted rat was 7.8+/-1.5 and 3.2+/-1.8 mL. When normalized for body weight, there was more water per kg body weight in the gastrointestinal tracts of the mouse and rat, than in man. The stomach capacity was found to be approximately 0.4 and 3.4 mL for mice and rats, respectively. The low fluid volume and stomach capacity have implications for the testing of solid dosage forms in these animal models. Substantial amounts of lymphoid tissue analogous to small intestinal Peyer's patches were measured in the rat and mouse colon, showing the feasibility of colonic vaccination, a route which might prove to have different applications to the more commonly studied oral vaccines. The existence of lymphoid tissue in the mouse and rat caecum has also been reported.

Carcinogenic mode of action of folpet in mice and evaluation of its relevance to humans

“…The localization of the lesions following dietary folpet administration corresponds to the potential for reaction with thiol groups and generation of thiophosgene (Bernard and Gordon, 2000;Milburn, 1997)� In the stomach, reactivity may be inhibited by the increased hydrolytic stability of folpet in acid media� No cytotoxicity is seen in the glandular stomach with a luminal pH of approximately 1-2 (Proctor et al�, 2007;McConnell et al�, 2009)� Some reactivity of folpet occurs in the forestomach, as there is an inflammatory and consequent regenerative process seen in the squamous epithelium� The luminal pH of the forestomach is approximately 5-6 (Proctor et al�, 2007;McConnell et al�, 2009)� Once the folpet enters the duodenum, the pH rises abruptly and significantly (approximately 5-6 in mouse, 6-7 in rat) because of the presence of the intestinal secretions and the presence of pancreatic secretions, rich in bicarbonate, entering the duodenum through the ampulla of Vater (McConnell et al�, 2009)� Thus, it is consistent with the chemical and physical properties of folpet that increased hydrolytic instability occurs with increased pH� Based on the pH of the forestomach, glandular stomach, and duodenum, the tumors occur, therefore, predominately in the duodenum� At this higher pH, nearly all of the dietary folpet reacts with thiol groups or is hydrolyzed before it can proceed beyond the duodenum� The folpet and the generated thiophosgene react with the duodenum mucosa� Little folpet or thiophosgene will be left by the time they reach the proximal jejunum, where occasional lesions were also observed� However, most of the changes are observed in the proximal 1�5 cm of the small intestine, which is where one would anticipate the rapid hydrolysis of the folpet and reactivity with the mucosa to occur� The reactivity of solubilized folpet and that of thiophosgene at neutral pH occurs in a matter of a few seconds� Systemic effects are unlikely because the folpet, if absorbed, would be rapidly degraded (half life of folpet: 4�9 s; half life of thiophosgene: 0�6 s)� The lesions in the forestomach and in the duodenum reflect contact toxicity that results in degradation of the parent substance�…”

“…The lack of effect on the glandular stomach mucosa is consistent with numerous other highly reactive substances administered directly into the gastric lumen by gavage, which also do not produce toxicity in the glandular stomach (see below)� The forestomach squamous mucosa appears to be much more susceptible to chemical reactivity whereas the glandular stomach mucosa is highly resistant� This may be due to the presence of a thick mucus layer on the glandular stomach as well as its normal, physiologic resistance to the extremely low pH (pH of 1�0-2�0) to which it is naturally exposed� Furthermore, folpet's hydrolytic stability increases at low pH, resulting in limited evolution of thiophosgene� In the forestomach, the pH of the contents approximates pH of 5 (Procter et al�, 2007;McConnell et al�, 2009); at this pH hydrolysis would increase with the concurrent generation of thiophosgene� As long as the folpet is present in the diet at sufficiently high doses, cytotoxicity of the duodenum will result� For folpet, the cytotoxicity is clearly a threshold phenomenon and is reversible� The necessity for continued exposure to folpet to produce cytotoxicity and the continued increased proliferation was noted in a reversibility study (Waterson, 1995)� Following 28 days of exposure, an additional 28 days without folpet administration resulted in complete recovery of the duodenal lesions� This is quite typical for chemicals having a cytotoxic mode of action� It is also typical for duodenal disorders involving blunting (hypertrophy) of the villi and chronic inflammation, in rodents or in humans (Noffsinger and Waxman, 2007)� Once the offending stimulus is removed, the villi are able to return to normal, usually within a matter of days (Potten and Loeffler, 1990;)� With this cytotoxicity in mice, there is consequent regenerative proliferation (Figure 7)� This is seen in the duodenal mucosa as measured both by an increase in bromodeoxyuridine (BrdU) or proliferating cell nuclear antigen (PCNA) labeling index and widening of the length of the stem cell portion of the small intestinal crypts, indicating an expansion of the target cell population that can evolve into tumors (Milburn, 1997;Waterson, 1995)� It is only the stem cell population of the small intestine that can actually evolve into a malignant tumor (Potten and Loeffler, 1990;)� Expansion of this stem cell population in the crypts represents an increase in the number of potential cells that can develop the spontaneous genetic errors that are necessary for the development of malignancy� This combination of an expanded number of cells with a higher proliferative rate than normal provides an ample background for which a tumorigenic response can evolve (Cohen and Ellwein, 1990;Greenfield et al�, 1984;Knudson, 1971;Moolgavkar and Knudson, 1981)� A similar process appears to be occurring in the mouse forestomach (see below)� It is also noted that there is a low incidence of spontaneously transformed cells resident in the crypt compartment, based on the incidence of duodenal tumors found in control mi...…”

Distillates (petroleum), hydrotreated light [MAK Value Documentation, 2016]

“…The stomach capacity of an adult female Wistar rat is 3.4 ml. The recommended maximum volume for gavage doses given to rats is 10 ml per kg body weight, which means 2 ml for a body weight of 200 g (McConnell et al 2008). The administration of up to 40 ml/kg body weight is technically feasible (Diehl et al 2001).…”

Prediction of in vivo developmental toxicity of all-trans-retinoic acid based on in vitro toxicity data and in silico physiologically based kinetic modeling

“…For the PBK model, it was assumed that the whole rat small intestine has the same absorption characteristics as this jejunum segment. The value for the small intestine surface area was calculated to be 94 cm 2 [radius of 0.18 cm (Zimmerman et al 2001) and a small intestine length of 83 cm (McConnell et al 2008)]. The surface area in this calculation does not need to include the increased surface area resulting from the presence of macro-and microvilli, since these are already present in the in situ perfused rat jejunum model.…”