Urinary metabolites as biomarkers of acrylamide exposure in mice following dietary crisp bread administration or subcutaneous injection.

Heat-treated carbohydrate-rich foods may contain high levels of acrylamide (AA). Crisp bread is a significant dietary AA source in the Nordic countries. We studied whether urinary metabolites of AA could be candidate biomarkers of AA intake and internal dose in mice following dietary crisp bread administration or sc injection. The crisp bread was experimentally baked to contain three different concentrations of AA: 0.19, 1.02, and 2.65 mg/kg, giving dietary exposures to AA of 0.024 +/- 0.002, 0.14 +/- 0.02, and 0.29 +/- 0.04 mg/kg bodyweight (bw)/day (mean +/- SD), respectively. A linear relationship was found between dietary AA exposure and urinary AA metabolites. On average, 55% of the ingested dose was recovered as urinary AA metabolites, and the molar proportions between the urinary metabolites showed similar proportions for the different doses. Urine AA metabolites were measured after sc injection of AA at doses of 0.05, 0.5, 5, and 50 mg/kg bw, and the urinary recovery for the three lowest doses was 54%. With the highest dose, 80% was recovered in urine, and the changed pattern of urinary metabolites indicated saturation of the metabolic conversion of AA to glycidamide. These results indicate that urinary metabolites of AA are good biomarkers of AA intake and internal dose up to 5 mg/kg bw/day. After sc injection of [(14)C]AA, 92% of the radioactivity was found in the urine and 2% in feces, liver, blood, and intestinal content (6% was not detected), demonstrating that sc AA was highly systemically available, that the major part AA metabolites was excreted, and that a significant portion of urinary AA metabolites (most likely glyceramide) was not accounted for by the present analytical method. Since the urinary recovery of AA after crisp bread feeding and sc injection was practically identical, an indicative "bioavailability" of AA from crisp bread was suggested to be approximately complete.

Increased levels of PPARbeta/delta and cyclin D1 in flat dysplastic ACF and adenomas in Apc(Min/+) mice.

BACKGROUND: In Apc(Min/+) (Min; multiple intestinal neoplasia) mice two separate populations of aberrant crypt foci (ACF) develop in the colon after azoxymethane (AOM) exposure. ACF(Min), with a flat appearance, severe dysplasia and increased beta-catenin expression, are related to adenoma development, whereas classic ACF, with elevated structure, hyperplasia and normal beta-catenin level, are probably not. MATERIALS AND METHODS: The expressions of peroxisome proliferator-activated receptors (PPARs) beta/delta, cyclin D1 and beta-catenin in ACF, adenoma and normal tissue from AOM-treated Apc(Min/+) mice and a familial adenomatous polyposis (FAP) patient colon tumour were assessed by immunohistochemistry and immunoblotting. RESULTS: The flat ACF (ACF(Min)) displayed increased cytoplasmic levels of beta-catenin, and increased levels of cyclin D1 and PPARbeta/delta. In contrast, the expression in classic ACF resembled normal mucosa. Adenomas from Apc(Min/+) mice, as well as a FAP patient colon tumour, displayed increased nuclear and cytoplasmic levels of beta-catenin, and the same expression patterns of cyclin D1 and PPARbeta/delta as those found in flat ACF. CONCLUSION: In addition to activation of the Wnt signalling pathway in both flat ACF and in adenomas in Apc(Min/+) mice, the increased expression of PPARbeta/delta in these lesions could be a target for pro-inflammatory signals important for growth and reduced apoptosis.

Connexin43 is overexpressed in Apc(Min/+)-mice adenomas and colocalises with COX-2 in myofibroblasts.

The expression of gap junction proteins, connexins, in the intestine and their role in tumorigenesis are poorly characterised. Truncating mutations in the tumour suppressor gene adenomatous polyposis coli (APC) are early and important events, both in inheritable (familial adenomatous polyposis, FAP) and spontaneous forms of intestinal cancer. Multiple intestinal neoplasia (Min) mice, a FAP model with inherited heterozygous mutation in Apc, spontaneously develop numerous intestinal adenomas. We recently reported reduced expression of connexin32 in Paneth cells of Min-mice. We further examine the expression of connexin43 (Cx43) and other connexins as a function of heterozygous and homozygous Apc mutation in normal intestinal tissues and adenomas of Min-mice. Qualitative analysis of connexin mRNA in intestine revealed a similar expression pattern in Min- and wild-type (wt) mice. Connexin26 and connexin40 proteins were found in equal amounts in Min and wt epithelia of large and small intestine, respectively. Interestingly, the connexin43 level was increased in the stroma of Min-mice adenomas, in close proximity to epithelial cells with nuclear beta-catenin staining. Cx43 and COX-2 were located to the same areas of the adenomas, and immunostaining exhibited coexpression in the myofibroblasts. Prostaglandin E2 induces Cx43 expression and COX-2 is the rate-limiting enzyme in the prostaglandin synthesis. However, the COX-2-specific inhibitor, celecoxib, did not reduce Cx43 expression. Although both Cx43 and COX-2 are target genes for beta-catenin, they were overexpressed in stromal cells but not in epithelial tumour cells. We hypothesise that gap junctions may be of importance in the transfer of signals between epithelium and stroma.

Truncated mouse adenomatous polyposis coli reduces connexin32 content and increases matrilysin secretion from Paneth cells.

Heterozygous mutations in adenomatous polyposis coli (APC) is an early event in inheritable and sporadic colon cancer development. We recently found reduced connexin (Cx43) expression in intestinal cell lines with heterozygous Apc mutation. In this study we investigated Cx expression and the role of one mutated Apc allele in epithelia of multiple intestinal neoplasia (Min) mouse intestines by immunohistochemistry. Cx43 was not expressed in intestinal epithelia of Min and wild-type mice. Cx32 was specifically expressed in enterochromaffin cells in both mice types, and in Paneth cells of wild-type mice. In contrast, Min mice had nearly undetectable level of Cx32 in Paneth cells. Isolated small intestinal crypts from Min mice had markedly increased secretion of both lysozyme and matrilysin compared with wild-type mice. Absence of matrilysin in Min mice reduces adenoma development. Reduced Cx32 and increased matrilysin secretion from Paneth cells could be important to neoplastic development in the intestine.

Cells heterozygous for the ApcMin mutation have decreased gap junctional intercellular communication and connexin43 level, and reduced microtubule polymerization.

Mutations in the tumour suppressor gene adenomatous polyposis coli (Apc) are early and critical events in the development of colon cancer. In the absence of functional Apc, beta-catenin is not degraded in the cytoplasm and can be transported to the nucleus and turn on transcription of several genes, including the gap junction protein connexin43. Apc also stabilizes microtubules and regulates microtubule polymerization. Changes in Wnt signalling and microtubule function are reported to affect the connexin level. To study the effect of heterozygous Apc mutation we examined gap junctional intercellular communication (GJIC) in IMCE (Immorto-Min colonic epithelium) cells with one mutated Apc allele and in YAMC (Young adult mouse colon) cells with normal Apc function. IMCE cells had only half the GJIC level compared with YAMC cells. RT-PCR showed that both YAMC and IMCE cells express a common complement of seven connexin genes (Cx26, Cx31, Cx39, Cx40, Cx43, Cx45 and Cx50), with an additional Cx29 gene expression in YAMC cells. We found that the Cx43 level was correspondingly lower in IMCE cells as detected by western blotting and immunofluorescence. There were no differences in the level or localization of beta-catenin and the downstream gene E-cadherin between the cells, indicating no activation of the Wnt-signalling pathway in cells with one mutated Apc allele. We also examined the microtubule polymerization rate, and IMCE cells had markedly slower microtubule polymerization than YAMC cells. Hence, it appears that mutation in one Apc allele is sufficient to affect microtubule function, while inactivation of both wild-type Apc alleles may be necessary for activation of Wnt signalling. Reduction in GJIC and Cx43 level in IMCE cells may be caused by reduced Cx43 transport as a result of alterations in microtubule function.