Antioxidant Therapy Significantly Attenuates Hepatotoxicity following Low Dose Exposure to Microcystin-LR in a Murine Model of Diet-Induced Non-Alcoholic Fatty Liver Disease

Abstract

We have previously shown in a murine model of Non-alcoholic Fatty Liver Disease (NAFLD) that chronic, low-dose exposure to the Harmful Algal Bloom cyanotoxin microcystin-LR (MC-LR), resulted in significant hepatotoxicity including micro-vesicular lipid accumulation, impaired toxin metabolism as well as dysregulation of the key signaling pathways involved in inflammation, immune response and oxidative stress. On this background we hypothesized that augmentation of hepatic drug metabolism pathways with targeted antioxidant therapies would improve MC-LR metabolism and reduce hepatic injury in NAFLD mice exposed to MC-LR. We chose N-acetylcysteine (NAC, 40 mM), a known antioxidant that augments the glutathione detoxification pathway and a novel peptide (pNaKtide, 25 mg/kg) which is targeted to interrupting a specific Src-kinase mediated pro-oxidant amplification mechanism. Histological analysis showed significant increase in hepatic inflammation in NAFLD mice exposed to MC-LR which was attenuated on treatment with both NAC and pNaKtide (both p ≤ 0.05). Oxidative stress, as measured by 8-OHDG levels in urine and protein carbonylation in liver sections, was also significantly downregulated upon treatment with both antioxidants after MC-LR exposure. Genetic analysis of key drug transporters including Abcb1a, Phase I enzyme-Cyp3a11 and Phase II metabolic enzymes-Pkm (Pyruvate kinase, muscle), Pklr (Pyruvate kinase, liver, and red blood cell) and Gad1 (Glutamic acid decarboxylase) was significantly altered by MC-LR exposure as compared to the non-exposed control group (all p ≤ 0.05). These changes were significantly attenuated with both pNaKtide and NAC treatment. These results suggest that MC-LR metabolism and detoxification is significantly impaired in the setting of NAFLD, and that these pathways can potentially be reversed with targeted antioxidant treatment.

Keywords: microcystin-LR (MC-LR); n-acetylcysteine (NAC); non-alcoholic fatty liver disease (NAFLD); pNaKtide.

Figure 1

Overview of the study designs to study the effect of antioxidants on low dose exposure to MC-LR with (a) choline deficient high fat diet–induced NAFLD and (b) healthy C57Bl/6J mice. (a) Six-week-old mice were fed on CDHFD for six weeks to induce NAFLD. During weeks 5 and 6, mice were orally gavaged with 100 µg/kg MC-LR every 24 h. Mice in the antioxidant groups were given an I.P. injection of 25 mg/kg pNaKtide once a week (2 doses) or 40 mM NAC in drinking water every day during weeks 5 and 6. (b) In a parallel study the mice were fed a standard diet and gavaged with MC-LR during weeks 5 and 6.

Figure 2

Antioxidant treatment reduces MC-LR induced increases in total body fat: NMR spectroscopy-based analysis revealed reduction in the total body fat content of mice that were treated with the antioxidant pNaKtide after MC-LR exposure in a diet-induced model of NAFLD. * p ≤ 0.05, ** p ≤ 0.01.

Figure 3

Targeted antioxidant treatment with pNaKtide and NAC significantly reduces MC-LR induced lobular inflammation and apoptotic hepatocytes: (a) H&E staining of the liver tissues from diet-induced NAFLD mice treated with antioxidants showed reduced inflammatory foci (circled in green) as compared to those exposed to MC-LR alone (scale bar, 100 μm); (b) Quantification of these inflammatory foci showed significant reduction of infiltrating immune cells upon treatment with antioxidants. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 3

Targeted antioxidant treatment with pNaKtide and NAC significantly reduces MC-LR induced lobular inflammation and apoptotic hepatocytes: (a) H&E staining of the liver tissues from diet-induced NAFLD mice treated with antioxidants showed reduced inflammatory foci (circled in green) as compared to those exposed to MC-LR alone (scale bar, 100 μm); (b) Quantification of these inflammatory foci showed significant reduction of infiltrating immune cells upon treatment with antioxidants. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4

Targeted antioxidant therapy with pNaKtide and NAC significantly downregulates markers of hepatotoxicity. Quantitative PCR (qPCR) analysis of markers of hepatotoxicity such as (a,b) CD40 and Itgam, also known as MAC1, that are known markers of inflammation and (c) MMP2, a known marker of fibrosis, revealed significant upregulation on exposure to MC-LR which was significantly downregulated with antioxidant treatment (n = 4). ns—not significant, * p ≤ 0.05, **** p ≤ 0.0001.

Figure 5

Oxidative stress is significantly reduced after antioxidant treatment. 8-OHDG levels in urine were elevated on exposure to MC-LR and reduced after treatment with antioxidants. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 6

Exposure to MC-LR significantly reduces Glutathione-S-transferase (GST) activity. GST activity was significantly reduced in CDHFD mice exposed to MC-LR but was restored on treatment with targeted antioxidant therapy using pNaKtide or NAC. Treatment of CDHFD mice with antioxidant alone did not alter the enzyme activity. * p ≤ 0.05, *** p ≤ 0.001.

Figure 7

Presence of NAFLD affects MC-LR metabolism. Mass spectrometric analysis of the urine samples revealed that antioxidant therapy with pNaKtide and NAC in the setting of diet-induced NAFLD improved hepatic metabolism of MC-LR to favor detoxified MC-LR-Cysteine metabolite. * p ≤ 0.05.

Figure 8

Diet-induced NAFLD mice exposed to low levels of MC-LR affects the expression of Phase I and II enzymes. Quantitative PCR (qPCR) analysis of key drug transporters as well as Phase I and II enzymes revealed significant fold regulation on exposure to MC-LR (a) which was reversed by antioxidant treatment using pNaKtide (b) and NAC (c). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.