Altered Hepatic Transport by Fetal Arsenite Exposure in Diet-Induced Fatty Liver Disease

Abstract

Non-alcoholic fatty liver disease can result in changes to drug metabolism and disposition potentiating adverse drug reactions. Furthermore, arsenite exposure during development compounds the severity of diet-induced fatty liver disease. This study examines the effects of arsenite potentiated diet-induced fatty liver disease on hepatic transport in male mice. Changes were detected for Mrp2/3/4 hepatic transporter gene expression as well as for Oatp1a4/2b1/1b2. Plasma concentrations of Mrp and Oatp substrates were increased in arsenic exposure groups compared with diet-only controls. In addition, murine embryonic hepatocytes and adult primary hepatocytes show significantly altered transporter expression after exposure to arsenite alone: a previously unreported phenomenon. These data indicate that developmental exposure to arsenite leads to changes in hepatic transport which could increase the risk for ADRs during fatty liver disease.

Keywords: Arsenic; Development; Fatty Liver Disease; Transport.

Figure 1. As (III) Potentiated Diet-induced NAFLD Disrupts Hepatic Transporter mRNA Expression

Transcript levels of select Mrp and Oatp transporter genes are shown as fold change compared to controls with error bars representing standard error. Analytical duplicates were used and a one-way ANOVA followed by a Dunnett’s multiple comparison test utilizing Prism6 (GraphPad) for comparisons between treatment groups and controls: the reported p-value is the multiplicity adjusted p-value corrected for multiple comparisons. (*** = p ≤ 0.001; ** = p ≤ 0.01 ; * = p ≤ 0.05 compared to controls). n=5.

Figure 2. Hepatic Transporter Protein Levels

Mrp3, Mrp4, Oatp1a1, Oatp1a4, and Oatp1b2 protein levels were examined relative to Erk2 in liver lysate. Results representative from at least 2 analytical duplicates were subjected to densitometry analyses. The detected protein levels are shown as fold change compared to controls with error bars representing standard error. A one-way ANOVA followed by a Dunnett’s multiple comparison test utilizing Prism6 (GraphPad) for comparisons between treatment groups and controls was performed on representative blots from analytical replicates. (p ≤ 0.1 = † compared to controls). n=4.

Figure 3. Localization of Mrp3 and Abcg2 with As (III) Potentiated Diet-induced NAFLD

Liver sections were probed with Abcg2 and Mrp3. Mislocalization of Abcg2 to the canalicular membrane and Mrp3 to the sinusoidal membrane has not been reported in NALFD models and is not observed. H&E stained sections demonstrate the presence of NAFLD in IU and IU+13W treatment groups.

Figure 4. Effects of Intralipid and As (III) on Transporter Gene Expression in Embryonic Liver Culture

mRNA levels of select Mrp and Oatp transporter genes found to be statistically different than control with described treatments are shown as fold change compared to control. The box extends from 25^th to 75^th percentile and whiskers show the entire distribution: the horizontal line is the median and the + is the mean. Analytical duplicates were used and a one-way ANOVA followed by a Tukey’s multiple comparison test utilizing Prism6 (GraphPad) for comparisons between groups: the reported p-value is the multiplicity adjusted p-value corrected for multiple comparisons. (**** = p ≤ 0.0001; *** = p ≤ 0.001; ** = p ≤ 0.01 ; * = p ≤ 0.05 compared to controls). n=4.

Figure 5. Effects of Intralipid and As (III) on Transporter Gene Expression in Primary Hepatocytes

mRNA levels of select Mrp and Oatp transporter genes found to be statistically different in the indicated treatments compared to control. Fold change compared to control is shown for each target. The line is the mean and the error bars are the standard deviation. Analytical duplicates were used and a one-way ANOVA followed by a Tukey’s multiple comparison test utilizing Prism6 (GraphPad) for comparisons between groups: the reported p-value is the multiplicity adjusted p-value corrected for multiple comparisons. (**** = p ≤ 0.0001; *** = p ≤ 0.001; ** = p ≤ 0.01 ; * = p ≤ 0.05 compared to controls). Representative results are shown from a primary hepatocyte isolation with each treatment condition examined in triplicate. n=3.