Dietary aflatoxin-induced stunting in a novel rat model: evidence for toxin-induced liver injury and hepatic growth hormone resistance

Abstract

Background: Despite a strong statistical correlation between dietary aflatoxin B1 (AFB1)-exposure and childhood stunting, the causal mechanism remains speculative. This issue is important because of emerging interest in reduction of human aflatoxin exposure to diminish the prevalence and complications of stunting. Pediatric liver diseases cause growth impairment, and AFB1 is hepatotoxic. Thus, liver injury might mediate AFB1-associated growth impairment. We have developed a rat model of dietary AFB1-induced stunting to investigate these questions.

Methods: Newly-weaned rats were given AFB1-supplemented- or control-diets from age 3-9 wk, and then euthanized for serum- and tissue-collection. Food intake and weight were serially assessed, with tibial-length determined at the experimental endpoint. Serum AFB1-adducts, hepatic gene and protein expression, and liver injury markers were quantified using established methodologies.

Results: AFB1-albumin adducts correlated with dietary toxin contamination, but such contamination did not affect food consumption. AFB1-exposed animals exhibited dose-dependent wasting and stunting, liver pathology, and suppression of hepatic targets of growth hormone (GH) signaling, but did not display increased mortality.

Conclusion: These data establish toxin-dependent liver injury and hepatic GH-resistance as candidate mechanisms by which AFB1-exposure causes growth impairment in this mammalian model. Interrogation of modifiers of stunting using this model could guide interventions in at-risk and affected children.

Figure 1. Dietary AFB1 Induced Stunting in Fischer Rats

(A) AFB1 Dose Dependent Wasting: Weight gain over time in newly weaned Fischer rats exposed to dietary AFB1 (● 0 ppm, ▼ 1 ppm, ■ 5 ppm, ◆ 10ppm, ▲ 20 ppm; *p<0.02 for area-under-the-growth-curves vs. 0, 1, 5, and 10 ppm; **p<0.01 vs. 0, 1 ppm; †p=0.02 vs. 0 ppm). (B) AFB1 Dose Dependent Stunting: Linear growth, assessed by tibial length, at the experimental endpoint (*p<0.005 vs. 0, 1, 5, 10 ppm; **p<0.03 vs. 0, 1 ppm; †p=0.03 vs. 0 ppm). (C) Food consumption (grams intake/rat/day) in control and toxin- exposed rats (● 0 ppm, ▼ 1 ppm, ■ 5 ppm, ◆ 10ppm, ▲ 20 ppm). (D) Serum AFB-albumin burden vs. dietary exposure (ANOVA p=0.03; very low level of adduct in controls likely reflects the allowable threshold of AFB1 contamination in PicoLab Rodent Diet 20).

Figure 2. Dietary AFB1 Induced Liver Injury in Rats

(A) AFB1-exposed rats demonstrate dose-dependent increases in bile duct hyperplasia (by H&E staining, image photographed at 50x magnification), fibrosis (by Sirius Red, 50x), and hepatocellular proliferation (by immunohistochemical detection of BrdU incorporation, 200x; 100 micron bar in lower left corner of left-most image in each series). (B) Quantification of serum ALT (left panel, ANOVA p=0.04); hepatocellular BrdU (middle panel, ANOVA p=0.02; *p<0.05 vs. control); serum glucose (black bars) and serum albumin (grey bars; right panel).

Figure 3. Effects of Dietary AFB1 on GH Signaling

(A) Hepatic mRNA expression of Igf1 (black bars; *p<0.01 vs. 0, 1, 5 ppm; **p<0.02 vs. 0, 1, 5 ppm; †p=0.04 vs. 0 ppm), Ghr (light grey bars; *p=0.02 vs. 0 ppm), Stat5a (dark grey bars; no significant differences), and Stat5b (white bars; *p=0.03 vs. 0 ppm) in Fischer rats exposed to dietary AFB1. (B) Protein immunoblot (upper panel) and quantification (lower panel) of hepatic expression of phosphorylated (Y694) and total STAT5 (*p=0.001 vs. 0 ppm control; β-actin (ACTB) and GAPDH shown as loading controls; phospho:total STAT5, black bars; total STAT5:GAPDH, grey bars).

Figure 4. Small Bowel Morphology in AFB1 Exposed Fischer Rats

(A) Representative H&E stained sections of proximal jejunum from AFB1-exposed Fischer rats (40x; 100 micron bar in lower left corner of left-most image). (B) Quantification of jejunal villous length in toxin-exposed rats.

Figure 5. Dietary AFB1 Induced Stunting in Fischer vs. Sprague-Dawley Rats

(A) Weight gain over time in newly weaned, male inbred Fischer and outbred Sprague-Dawley rats exposed to 10 ppm dietary AFB1 (● Fischer control, ○ Fischer 10 ppm, ▼ Sprague-Dawley control, ∆ Sprague-Dawley 10 ppm; *p<0.04 for area-under-the-growth-curves vs. corresponding control). (B) Serum AFB-albumin adduct levels in toxin-exposed Fischer vs. Sprague-Dawley rats (*p<0.01 vs. corresponding control).