Negligible colon cancer risk from food-borne acrylamide exposure in male F344 rats and nude (nu/nu) mice-bearing human colon tumor xenografts

Abstract

Acrylamide, a possible human carcinogen, is formed in certain carbohydrate-rich foods processed at high temperature. We evaluated if dietary acrylamide, at doses (0.5, 1.0 or 2.0 mg/kg diet) reflecting upper levels found in human foods, modulated colon tumorigenesis in two rodent models. Male F344 rats were randomized to receive diets without (control) or with acrylamide. 2-weeks later, rats in each group received two weekly subcutaneous injections of either azoxymethane (AOM) or saline, and were killed 20 weeks post-injections; colons were assessed for tumors. Male athymic nude (nu/nu) mice bearing HT-29 human colon adenocarcinoma cells-derived tumor xenografts received diets without (control) or with acrylamide; tumor growth was monitored and mice were killed 4 weeks later. In the F344 rat study, no tumors were found in the colons of the saline-injected rats. However, the colon tumor incidence was 54.2% and 66.7% in the control and the 2 mg/kg acrylamide-treated AOM-injected groups, respectively. While tumor multiplicity was similar across all diet groups, tumor size and burden were higher in the 2 mg/kg acrylamide group compared to the AOM control. These results suggest that acrylamide by itself is not a "complete carcinogen", but acts as a "co-carcinogen" by exacerbating the effects of AOM. The nude mouse study indicated no differences in the growth of human colon tumor xenografts between acrylamide-treated and control mice, suggesting that acrylamide does not aid in the progression of established tumors. Hence, food-borne acrylamide at levels comparable to those found in human foods is neither an independent carcinogen nor a tumor promoter in the colon. However, our results characterize a potential hazard of acrylamide as a colon co-carcinogen in association with known and possibly other environmental tumor initiators/promoters.

Figure 1. Experimental design of (a) the F344 rat study and (b) the nude (nu/nu) mouse- human colon tumor xenograft study.

Numbers in boxes depict the time (in weeks) after the animals arrived at our housing facility. In both studies, a one-week acclimatization period was maintained before specific interventions. Diets were based on AIN-93G semi-synthetic formulation and the four experimental diets differed from one another in the level of acrylamide added: 0 (control), 0.5, 1.0 and 2.0 mg/kg diet. All animals remained on the respective experimental diets until necropsy.

Figure 2. Body weight gain of F344 rats.

Body weight (mean ± SE, g) per week of F344 rats injected with (a) saline or (b) AOM and treated with acrylamide at 0 (control), 0.5, 1.0 and 2.0 mg/kg diet.

Figure 3. Histopathological identification of lesions (5 µm thick, H&E-stained sections) in the colon of AOM-injected F344 rats.

One of the five tumor types were observed in the colons. Tubular adenoma (TA) is shown in panel (a), with dysplastic tubules forming the polypoid adenoma (black arrow) and non-neoplastic glands in the stalk of the tumor (red arrow). Adenocarcinoma with invasion to submucosa (AC-2) is shown in panel (b), featuring neoplastic glandular structures (black arrow) in submucosa with a clear muscularis mucosa (black donut) and scirrhous inflammatory response to tumor (red arrow). Mucinous adenocarcinoma with invasion to submucosa (AC-3) is shown in panel (c), characterised by tumor cells forming glandular structures (red arrow) in submucosa with a clear muscularis mucosa (black donut) and signet-ring cells distended by mucin (black arrow). Mucinous adenocarcinoma invading tunica muscularis (AC-4) is shown in panel (d), characterised by tumor cells invading tunica muscularis (black arrow) and replacing normal mucosa (black donut) and an intact serosa (red arrow). Mucinous adenocarcinoma invading serosa (AC-5) is shown in panel (e), with tumor cells in vascular structures in tunica muscularis (black arrow), mucous and debris in dilated tumor gland (black donut), and a serosa (red arrow). Tumor type as a percentage in each diet group is depicted in panel (f).

Figure 4. Body weight gain of nude (nu/nu) mice.

Body weights (mean ± SE, g) of nude (nu/nu) mice injected with HT-29 human colon cancer cells treated with acrylamide at 0 (control), 0.5, 1.0 and 2.0 mg/kg diet.

Figure 5. Tumor xenograft characteristics in nude (nu/nu) mice.

Effect of dietary acrylamide at 0 (control), 0.5, 1.0 and 2.0 mg/kg diet on growth (mean ± SE, %) shown in panel (a) and wet weights (mean ± SE, g) shown in panel (b) of HT-29 human colon tumor xenografts in nude (nu/nu) mice. Tumor growth was calculated based on the volume of each tumor recorded at different time points from the beginning of the acrylamide diets until necropsy, and wet weights were recorded at necropsy.

Figure 6. Histology, proliferation and apoptosis of tumor xenograft characteristics in nude (nu/nu) mice.

Fixed HT-29 human colon tumor xenografts from nude (nu/nu) mice were sectioned at 5 µm. Panel (a) shows a representative H&E section of the tumor xenografts as a typical undifferentiated carcinoma with viable solid sheets of tumor cells (area marked by black dotted line) and clearly delineated subcutis margin (red arrow); key characteristics include grey distending tumor cell cytoplasm (black arrow) and the necrotic area (black donut). Representative immunohistochemical staining of PCNA is shown in panel (b) with proliferating cells seen as dark brown stained cells. TUNEL staining is shown in panel (c) with apoptotic cells observed as dark spots (red arrow). The bar graph in panel (d) shows the ratio of the indices (mean ± SE) of proliferating (PCNA-positive) and apoptotic (TUNEL-positive) cells for each diet: acrylamide at 0 (control), 0.5, 1.0 and 2.0 mg/kg diet.