Dietary freeze-dried black raspberry's effect on cellular antioxidant status during reflux-induced esophagitis in rats

Abstract

Introduction: Esophageal cancer consists of two distinct types, esophageal adenocarcinoma (EAC) and squamous cell carcinoma, both of which differ significantly in their etiology. Freeze-dried black raspberry (BRB) has been consistent in its ability to modulate the biomarkers and reduce the incidence of carcinogen-induced squamous cell carcinoma in rats. In our previous studies in the esophagoduodenal anastomosis (EDA) model, we have shown that the early modulation of manganese superoxide dismutase (MnSOD) significantly correlates with the development of reflux-induced EAC in rats. In this study we looked at the short-term effects of a BRB-supplemented diet on the modulation of antioxidant enzymes in reflux-induced esophagitis.

Methods: Male SD rats (8 wk old; n = 3-5) were randomized into three groups--sham-operated, fed control AIN-93M diet (SH-CD), EDA operated and fed either control diet (EDA-CD) or 2.5% (w/w) BRB diet (EDA-BRB). The effect of both reflux and dietary supplementation was analyzed 2 and 4 wk after EDA surgery.

Results: Animals in the EDA groups had significantly lower weight gain and diet intake compared to SH-CD (P < 0.05). The sham-operated animals received an average esophagitis score of 0.1 ± 0.1; this increased significantly in EDA-CD animals to 1.8 ± 0.14 (P < 0.001 versus SH-CD) and in EDA-BRB group to 1.7 ± 0.06 (P < 0.001 versus SH-CD), with BE changes also present. However, dietary supplementation of BRB did not alter or ameliorate the grade of esophagitis or the induction of BE. BRB diet caused a 43% increase in MnSOD levels compared to EDA-CD (0.73 ± 0.16; P = 0.09); however, this effect was not statistically significant and at 4 wk, EDA-CD (0.58 ± 0.12) showed an increase in MnSOD expression compared to SH-CD (0.34 ± 0.01).

Conclusions: In conclusion, our data suggest that dietary BRB does not increase the levels of cellular antioxidant enzymes or reduce the levels of lipid peroxidation compared to a control diet, in a short-term study of gastroesophageal reflux induction in the EDA animal model. However, it remains to be tested whether this is indicative of its ineffectiveness to inhibit reflux-induced EAC incidence over the long term.

Figure 1. Comparison of weight gain (A) and diet intake (B) in rats that underwent EDA surgery

EDA surgery to induce mixed-reflux was performed in 8 weeks-old male SD rats as described in Materials and Methods. (A) Weight gain and (B) diet intake were measured weekly after surgery and then compared using either two-way ANOVA (weight gain) or one-way ANOVA (diet intake). #- significantly different from SH-CD (p<0.05).

Figure 2. Histopathological changes observed in the esophageal epithelium 4 weeks after reflux-induction

Representative H&E stained sections of esophageal mucosa (200X magnification) from (A) SH-CD –normal mucosa (B) EDA-PD and (C) EDA-BRB. Esophagitis (denoted by triangular arrowhead) and Barrett’s esophagus (blunt arrow head) can be visualized in both EDA-operated groups (B and C).

Figure 3. Effect of dietary BRB on antioxidant enzyme expression levels in the esophageal mucosa in the presence of reflux

The enzymes (A) Manganese superodxide dismutase (MnSOD), (B) Catalase and (C) Glutathione peroxidase (GPX) in the esophageal mucosa 2 and 4 weeks after EDA surgery were measured using immunoblot and normalized to tissue β-actin levels (D). SH-CD - sham operated on control diet; EDA-CD – EDA-operated on control diet and EDA-BRB – EDA-operated on BRB diet.

Figure 4. Effect of dietary BRB on SOD enzyme activities in the esophageal mucosa in the presence of reflux

SOD enzyme activities in the esophageal mucosa 2 (A) and 4 weeks (B) after EDA surgery were measured using an SOD assay kit according to attached instructions. Cu/ZnSOD activity was inhibited using KCN (10 μM) and the MnSOD was measured alongside total SOD activity. Cu/ZnSOD activity was then derived by subtracting MnSOD from total SOD activity.

Figure 5. Effect of dietary BRB on lipid peroxidation of the esophageal mucosa in the presence reflux

MDA concentration (nmol/ml) was measured in the esophageal mucosa 2 and 4 weeks after EDA surgery using a commercially available TBARS assay kit as described in Materials and Methods.