**Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease**

**Apurva Lad 1, Robin C. Su 1, Joshua D. Breidenbach 1, Paul M. Stemmer 2, Nicholas J. Carruthers 2, Nayeli K. Sanchez 1, Fatimah K. Khalaf 1, Shungang Zhang 1, Andrew L. Kleinhenz 1, Prabhatchandra Dube 1, Chrysan J. Mohammed 1, Judy A. Westrick 3, Erin L. Crawford 1, Dilrukshika Palagama 4, David Baliu-Rodriguez 4, Dragan Isailovic 4, Bruce Levison 5, Nikolai Modyanov 6, Amira F. Gohara 7, Deepak Malhotra 1, Steven T. Haller 1,\* and David J. Kennedy 1,\***


Received: 2 July 2019; Accepted: 19 August 2019; Published: 23 August 2019

**Abstract:** Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 μg/kg, 100 μg/kg MC-LR or vehicle every 48 h for 4 weeks (*n* = 15–17 mice/group). Early mortality was observed in both the 50 μg/kg (1/17, 6%), and 100 μg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.

**Keywords:** Microcystin-LR; Non-alcoholic Fatty Liver Disease; No Observed Adverse Effect Level; Leprdb/J mice; hepatotoxicity; oxidative stress; TiO2 enriched phosphopeptides

**Key Contribution:** This is the first study to investigate the effect of the permissible levels of the cyanotoxin on the susceptibility to MC-LR toxicity in a model of a pre-existing liver condition.

#### **1. Introduction**

Cyanobacteria are a natural component of the freshwater phytoplankton, but their overgrowth has increasingly been recognized as a potential health hazard due to the release of toxins by selected species of algae, such as *Microcystis aeruginosa*, *Raphidiopsis mediterranea, Cylindrospermopsin raciborskii*, *Anabaena circinalis, Planktothrix rubescens and Planktothrix agardhii* [1–5]. Their ability to adapt and flourish in a wide variety of climate conditions, including extremes, contributes to their successful occurrence in a variety of ecosystems. An extensive study done by Mantzouki et al. in 2018, showed that global warming has direct and indirect effects on the temperature and nutrient gradient in the freshwater systems, thus, affecting the distribution of the cyanotoxins [5]. Therefore, changing climatic conditions and increased eutrophication of freshwater ecosystems are two main factors that favor the growth of cyanobacterial blooms and allow them to flourish, increasing the duration and intensity on a global scale [2,6].

Toxin production is a common characteristic of many of the cyanobacterial species. Of the many toxins produced by cyanobacteria, microcystins (MCs) are among the most widely distributed, and microcystin-LR (MC-LR) is one of the most commonly produced [7]. MC-LR is a cyclic heptapeptide characterized structurally by leucine and arginine amino acids at positions 2 and 4 within a cyclo-(D-alanine-1-X2-D-MeAsp3-Y4-Adda 5-d-glutamate 6-Mdha7) structure [8]. It is usually taken up into the cells through organic anion transporting polypeptides (OATPs) [9]. MC-LR then exhibits its deleterious effects by inhibiting the activity of protein phosphatases 1 (PP1) and 2A (PP2A) and by increasing the production of reactive oxygen species (ROS) [10,11]. As a result, there is a hyperphosphorylation and pro-oxidative state induced by MC-LR, which leads to alterations in the cytoskeleton, OATP expression, mitogen-activated protein kinase (MAPK) activity, glycogen storage, and mitochondrial structure and function in addition to DNA damage, cellular disruption, endoplasmic reticulum dysfunction, inflammation and tumor growth [12–20].

MC-LR has become a global health concern due to its potent toxicity in humans and animals alike. In addition to its well-known hepatotoxic effects, MCs also cause reproductive, developmental and, immune toxicities [9,21–28]. Humans can be exposed to MC-LR most commonly through oral ingestion of contaminated water, or through recreational activities like swimming or boating in contaminated waters, as well as intake of contaminated fish or algal supplements [29,30]. Once ingested, the toxin is absorbed into the tissues, especially into hepatic tissue via the bile acid carrier system [1].

The current guidelines for safe exposure to the toxin in humans have been extrapolated from experiments performed in healthy animal models. In a 13-week study performed by Fawell et al., the No Observed Adverse Effect Level (NOAEL) was 40 μg/kg and the Low Observed Adverse Effect Level (LOAEL) was 200 μg/kg of body weight [1]. In accordance with these findings, the World Health Organization (WHO) established the provisional guideline value of 1 μg/L of MC-LR in drinking water. In 2015, as part of the Safe Drinking Water Act, the United States Environmental Protection Agency developed a Health Advisory for microcystins of 0.3 μg/L for bottle-fed infants and pre-school children and 1.6 μg/L for School-age children and adults [31]. Nevertheless, the effects of MC-LR in the setting of pre-existing liver disease remain unknown.

Non-alcoholic Fatty Liver Disease (NAFLD) is one of the most common liver conditions in the United States. The incidence of NAFLD is increasing concurrent with the rise in obesity and diabetes [32–34]. NAFLD is characterized by the presence of steatosis/ inflammation with ≥ 5% fat infiltration into the liver and potentially elevated liver enzymes [35]. In the past two decades, the prevalence of this condition has nearly doubled, affecting about 20–30% of the population in the Western countries [36]. Given the increase in the occurrence of both NAFLD and cyanobacteria blooms globally, we evaluated the effects of MC-LR in the setting of pre-existing liver disease using the well-established Leprdb/J murine model of NAFLD [37]. Given NAFLD's growing prevalence worldwide, several studies have recently investigated MC-LR's effects in pre-existing NAFLD [18,28,38]. These studies have provided valuable insight, alerting us to the fact that individuals with pre-existing NAFLD may be more susceptible to MC-LR's toxic effects. However, these studies utilize intraperitoneal (IP) and

intravenous (IV) methods of MC-LR exposure. In our current study, we provide additional insight by using a robust genetic model of NAFLD and expose mice to MC-LR by gavage, an exposure route more relevant to environmental methods of exposure. We tested the hypothesis that chronic low dose oral exposure to MC-LR at levels below the NOAEL established in healthy animal models would exacerbate the hepatotoxicity seen in a NAFLD murine model.
