**2. Results**

### *2.1. Survival, Appearance and Weight*

No visible symptoms of sickness or discomfort were observed in Leprdb/J mice from either the 50 μg/kg or 100 μg/kg MC-LR exposed groups. However, early mortality was observed in 1/17 mice (95% survival) from the 50 μg/kg MC-LR exposed group and 3/17 mice (83% survival) from the 100 μg/kg MC-LR exposed group (Kaplan-Meier log-rank *p* = 0.07, Figure S1A). In each case the animals died overnight and there was no observed acute trauma (e.g., tracheal rupture resulting in immediate death) or other signs of improper gavage technique such as visible signs of discomfort or bloating in the time preceding death although we were not able to fully exclude mortality attributable to the gavage procedure in these animals. It should be noted that the Leprdb/J mice are more susceptible to comorbidities including compromised immune system [39]. No deaths were observed in either the vehicle exposed Leprdb/J group (*n* = 13) or in either of the vehicle or 100 μg/kg MC-LR exposed healthy background strain control C57Bl/6J (Wild Type - WT) mice study groups (*n* = 5 mice/group each). The body weight gain throughout the exposure in Leprdb/J MC-LR exposed groups was not significantly different from the respective vehicle exposed group (Figure 1A,B). The physical appearance (Figure S1B–D) as well as the weight of the livers (Figure 1C) were not significantly different from the MC-LR-exposed groups in either the Leprdb/J or WT mice (data not shown). Other organs including heart, lung, and kidney were collected from both the Leprdb/J and C57Bl/6J control and MC-LR-exposed groups. There was no significant difference in organ weight between WT C57Bl/6J control and C57Bl/6J MC-LR-exposed mice as well as no significant differences among Leprdb/J vehicle control and Leprdb/J MC-LR exposed mice. (Tables S1 and S2).

**Figure 1.** Effect of Microcystin-LR (MC-LR) on body and liver weights in both healthy (C57Bl/6J) and NAFLD (Leprdb/J) mice. (**A** and **B**) Total body weights. (A) C57Bl6/J (WT) mice exposed to vehicle (*n* = 5) or 100 μg/kg MC-LR (*n* = 5) and (B) Leprdb/J (db) mice exposed to vehicle (*n* = 13), 50 μg/kg MC-LR (*n* = 16), or 100 μg/kg MC-LR (*n* = 14). Mean and S.E.M. are indicated. (**C**) liver weights of animals from all groups. Individual, mean, and S.E.M. values indicated. NOTE: The green arrow over Week 1 represents the initiation of exposure to MC-LR or vehicle.

#### *2.2. Detection of MC-LR in Plasma and Urine*

To determine circulating levels and urinary excretion of MC-LR resulting from this exposure regimen, we collected plasma and 24-h urine samples at the end of the study prior to euthanasia. We then used Ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) and High-Performance Liquid chromatography–orbitrap mass

spectrometry (HPLC-orbitrap-MS) to detect and quantify the MC-LR levels in animals which met the sample volume requirements for the analysis. MC-LR was detected in the plasma (Figure 2A) and urine (Figure 2B) of exposed Leprdb/J mice. Significantly more MC-LR was detected in the 100 μg/kg MC-LR exposed Leprdb/J mice (*p* < 0.001 for plasma and *p* < 0.01 for urine, respectively) as compared to those exposed to 50 μg/kg MC-LR. Interestingly, the toxin was undetectable in the plasma of C57Bl6/J mice exposed to the toxin (Figure 2A), whereas the amounts detected in the 24-h urine excretion samples were significantly elevated (nearly 100-fold) as compared to the Leprdb/J mice exposed to the same amount of the toxin (Figure 2B).

**Figure 2.** MC-LR determination in plasma and urine of both healthy (C57Bl/6J) and NAFLD (Leprdb/J) mice. (**A**) Plasma and (**B**) 24-h urine excretion levels of MC-LR in C57Bl/6J (WT) Leprdb/J (db) mice as assessed by Ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) and High-Performance Liquid chromatography–orbitrap mass spectrometry (HPLC-orbitrap-MS) at the end of the 4–week exposure protocol. For plasma analysis, WT/vehicle *n* = 5, WT/100 μg/kg MC-LR *n* = 5, db/vehicle *n* = 8, db/50 μg/kg MC-LR *n* = 9, db/100 μg/kg MC-LR *n* = 8. For 24-h urine excretion analysis, WT/vehicle *n* = 5, WT/100 μg/kg MC-LR *n* = 5, db/vehicle *n* = 7, db/50 μg/kg MC-LR *n* = 8, db/100 μg/kg MC-LR *n* = 8. The number of samples shown in the figure for the db mice vary due to insufficient amount of plasma or urine needed for the analysis. Significance calculated by unpaired Student's t-test for the indicated comparisons. \*\*, *p* < 0.01; \*\*\*, *p* < 0.001; \*\*\*\*, *p* < 0.0001.

#### *2.3. Blood Biochemistry*

To determine the levels of liver injury enzymes, namely alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as well as to study the factors of blood biochemistry, we conducted a comprehensive diagnostic analysis using whole blood collected via retro-orbital bleed at days 15 and 30 during the exposure. Results from the comprehensive diagnostic analysis are shown in Tables S3 and S4. In paired analysis of the Leprdb/J mice, the levels of ALT were non-specifically elevated (15 vs. 30-day time points) in the vehicle exposed mice (*p* < 0.001), 50 μg/kg MC-LR exposed mice (*p* < 0.001), as well as the 100 μg/kg MC-LR exposed mice (*p* < 0.05, Figure S2A). On the other hand, the levels of ALP were only significantly elevated (15 vs. 30-day time points) in the 50 μg/kg MC-LR exposed mice (*p* < 0.05) as well as in the 100 μg/kg MC-LR exposed mice (*p* < 0.01), but not in the vehicle exposed mice (Figure S2B). In the unpaired, between group analysis (Figure 3), the only significant change in blood chemistry between vehicle and MC-LR exposed mice was that ALP levels were elevated in the 100 μg/kg MC-LR exposed mice vs. vehicle (*p* < 0.01) at the 30-day time point (Figure 3B). All other blood biochemistry markers did not show any significant change between vehicle or MC-LR exposed groups for either the Leprdb/J mice (Figure 3, Table S3) or the C57Bl/6J mice (Figure 3, Table S4).

**Figure 3.** Effect of MC-LR on levels of liver injury enzymes in both healthy (C57Bl/6J) and NAFLD (Leprdb/J) mice. Circulating levels of Alkaline Phosphatase (ALP) (**A** and **B**) and Alanine aminotransferase (ALT) (**C** and **D**) were measured across all exposure groups at 15 days and 30 days after initial exposure. Significance between groups tested by unpaired Student's t-test for indicated comparisons. For 15-day analysis, WT/vehicle *n* = 5, WT/100 μg/kg MC-LR *n* = 5, db/vehicle *n* = 11, db/50 μg/kg MC-LR *n* = 13, db/100 μg/kg MC-LR *n* = 11. For 30-day analysis, WT/vehicle *n* = 5, WT/100 μg/kg MC-LR *n* = 5, db/vehicle *n* = 12, db/50 μg/kg MC-LR *n* = 13, db/100 μg/kg MC-LR *n* = 12, \*, *p* < 0.05, \*\*, *p* < 0.01; \*\*\*, *p* < 0.001; \*\*\*\*, *p* < 0.0001.

#### *2.4. Liver Histology*

Histopathological analysis was performed using Hematoxylin & Eosin (H&E) and Periodic Acid-Schiff (PAS) staining of the liver sections. The slides were assessed by a pathologist, who was blinded to the group assignments, and histopathologic scoring was assessed based on the severity of fat infiltration, hepatic damage, glycogen content, and micro- and macro-vesicular lipid accumulation within the hepatocytes. As shown in Figure 4, significant hepatic injury was noted in both 50 μg/kg and 100 μg/kg MC-LR exposed Leprdb/J mice as compared to the vehicle control Leprdb/J mice. This was noted by the presence of fat vacuoles (Figure 4A, top panel, yellow arrows) as well as macro- and micro-vesicular fat infiltration and accumulation inside the hepatocytes (Figure 4A, top panels, red arrows). Furthermore, PAS staining demonstrated reduction in the glycogen content in Leprdb/J MC-LR exposed mice as compared to vehicle control, although this appeared to be more pronounced in the 50 μg/kg group (Figure 4A, bottom panels). Based on histopathologic scoring, liver injury was significantly increased in MC-LR exposed Leprdb/J mice as compared to the vehicle control Leprdb/J mice (*p* < 0.001 in 50 μg/kg and *p* < 0.05 in 100 μg/kg, Figure 4C). However, no significant changes were observed in the liver sections of MC-LR exposed C57Bl/6J mice compared to vehicle control C57Bl/6J mice (Figure 4B,C). The H&E and PAS derived injury scores for each individual animal are presented in Table S5.

**Figure 4.** *Cont*.

**Figure 4.** Histological analysis of hepatic injury in both healthy (C57Bl/6J) and NAFLD (Leprdb/J) mice. (A and B) Representative images of liver tissue sections from different exposure groups of Leprdb/J (db) mice (**A**) and C57Bl6/J (WT) mice (**B**) stained with Hematoxylin & Eosin (H&E) (top panels) and Periodic Acid-Schiff (PAS) (bottom panels) staining; yellow arrows indicate fat vacuoles, red arrows indicate fat infiltration inside hepatocytes; scale bar = 200 μm. (**C**) Composite liver injury score for C57Bl6/J mice (*n* = 5/group) and Leprdb/J mice receiving vehicle (*n* = 13), 50 μg/kg MC-LR (*n* = 16; \*\*\*, *p* < 0.0006 vs. vehicle) and 100 μg/kg MC-LR (*n* = 14; \*, *p* = 0.0023 vs. vehicle) as determined by Kruskal–Wallis ANOVA and Mann–Whitney test. No significant injury was determined in either the vehicle or 100 μg/kg MC-LR exposed C57Bl6/J mice.

#### *2.5. Genetic Analysis Hepatotoxicity and Oxidative Stress*

Since micro-vesicle lipid accumulation is associated with hepatic inflammation and oxidative stress, we assessed markers of inflammation and oxidative stress in the livers of Leprdb/J mice after microcystin exposure. Interestingly, we observed that several genetic markers associated with hepatotoxicity and oxidative stress response were significantly upregulated as compared to the respective db/vehicle control (Tables S6 and S7). Significantly (*p* < 0.05) upregulated markers of hepatotoxicity (*Cxcl12,* 170.3 fold upregulated; *Casp3*, 15.6 fold upregulated; *Cyp1a2*, 187.2 fold upregulated; *Rb1*, 2.5 fold upregulated), necrosis (*Fam214a*, 15.6 fold upregulated; *Mlxip1*, 97.1 fold upregulated; *Col4a1*, 33.2 fold upregulated), nongenotoxic hepatocarcinogenicity (*Ccng1*, 19.3 fold upregulated) as well as oxidative stress response (*Prdx2*, 2876.3 fold upregulated; *Gpx5*, 4.7 fold upregulated) were noted in the 50 μg/kg MC-LR exposed db mice vs. db/vehicle controls. Limited analysis of targeted genes associated with hepatotoxicity (*Cyp1a2*, *p* = 0.7; *Slc17a3*, *p* = 0.9), necrosis (*OSMR*, *p* = 0.9), steatosis (*CD36*, *p* = 0.5), phospholipidosis (*Serpina3n*, *p* = 0.07) and cholestasis (*Abcb4*, *p* = 0.6) were tested by RT-PCR in the C57Bl/6J groups and were not found to have any change in the gene expression between vehicle and MC-LR exposed groups.

#### *2.6. Phosphoproteomic Analysis*

Based on the above results and the fact that MC-LR is a known protein phosphatase inhibitor, we next examined phosphoproteomic pathways, which were affected in the livers of MC-LR exposed Leprdb/J mice, to better understand the possible disruptions in signaling pathways associated with the hepatotoxicity in this model. Phosphoproteomic analysis with mass spectrometry-based label-free quantification of TiO2 enriched samples was used to determine the phosphorylation sites and signaling pathways affected by MC-LR exposure.

Data analysis using Maxquant identified 9616 phosphorylation sites in total. A moderated t-test indicated that 10 phosphorylation sites were affected by 50 μg/kg microcystin exposure compared to vehicle control samples (q < 0.1, *n* = 8, Table 1, Figure S3A,B). Similar comparison was done, to indicate changes in phosphorylation sites, between 100 μg/kg microcystin exposure and Vehicle control samples (Table 1, Figure S3C,D). An alternative analysis using linear regression of site intensity vs microcystin dose identified 25 sites with a linear relationship between dose and intensity (*q* < 0.1, *n* = 7, Table 1).

**Table 1.** Effect of microcystin on phosphorylation sites in Leprdb/J liver protein. Number of sites affected by microcystin exposure in Leprdb/J livers using moderated t-tests or linear regression. Results were corrected for multiple testing, *n* = 8 for 50 μg/kg and *n* = 7 for 100 μg/kg microcystin.


Enrichment analysis of Gene Ontology (GO) biological processes, using Platform for Integrative Analysis of Omics (PIANO, v2.0.2., Bioconductor, Package for R, Sweden) software [40], was used to identify molecular pathways that were affected by microcystin exposure. Here we tested whether the mean t-statistic for each pathway was different from 0 indicating that the process components taken together were affected by MC-LR exposure. Exposure to 50 μg/kg MC-LR resulted in decreased phosphosite abundance of 91 pathways which represented 16 biological processes including urogenital system development, regulation of T cell proliferation and response to oxidative stress as well as positive regulation of cell cycle proliferation. The 16 processes selected to reduce redundancy are shown in Table 2. No GO processes were hyperphosphorylated in response to 50 μg/kg MC-LR (PIANO, FDR < 0.1). An analysis to identify specific classes of kinases that were affected by microcystin exposure failed to identify any significant kinase enrichment (PIANO, FDR < 0.1). Pathways in the Reactome pathway database [41] were also tested for changes in response to microcystin exposure. All GO and Reactome results are shown in Tables S8–S10.

In order to identify which microcystin-affected systems were the most sensitive to MC-LR exposure, fuzzy c-means clustering was used to identify common phosphorylation site abundance profiles across the MC-LR exposure groups. Among them, 4259 sites were assigned to one of 6 clusters (Figure S4). Biological process categories that were identified as affected by 50 μg/kg microcystin at an FDR < 0.03 were tested for enrichment in the c-means clusters using a Fisher's exact test. The results of this enrichment analysis demonstrated significant enrichment of 11 biological processes (Fishers exact test, *p* < 0.02) including several processes related to immune cell function as well as kidney/urogenital system development (Table 3). Cluster 3 contains sites that were dephosphorylated in both the 50 and 100 μg/kg samples compared to controls. It had the greatest number of enriched categories including several processes related to immune cell function as well as kidney/urogenital system development, suggesting that proteins in those categories are dephosphorylated by MC-LR exposure. Equivalent analysis using pathways that were affected by 100 μg/kg microcystin (FDR < 0.03) did not identify any cluster-enriched pathways. Cluster enrichment of Reactome pathways were also conducted as previously described [41] and are shown in Tables S8–S10. Analysis using the UniProt database was performed to summarize all the proteins with their phosphorylation sites affected by either 50 or 100 μg/kg MC-LR exposure (Table S11).



To reduce redundancy categories were clustered and one pathway per cluster listed. This indicates the size of the cluster.


**Table 3.** Identification of the clusters of pathways affected in 50 and 100 μg/kg MC-LR versus vehicle exposed Leprdb/J mice using Reactome database: Reactomepathways in liver were identified as affected by 50 as well as 100 μg/kg MC-LR versus db/vehicle (False Discovery Rate, FDR<0.2) and enriched in a c-means cluster(Fisher's exact test *p* < 0.02). Results were summarized from *n* = 7–8 mice/group; 50 μg/kg MC-LR group (*<sup>n</sup>* = 8), 100 μg/kg MC-LR group (*<sup>n</sup>* = 7), Vehicle (*<sup>n</sup>* = 8),all proteomics

 for

#### **3. Discussion**

In the current study, we used Leprdb/J mice, a well-established genetic model for NAFLD [42], to examine the effect of prolonged low dose oral exposure to MC-LR in mice with pre-existing liver disease. The dosages used in the current study approximated the 40 μg/kg NOAEL [1] albeit with a reduced dosing regimen (every other day vs. daily) and total study duration (4 weeks vs. 13 weeks) [1,2]. This chronic low dose regimen resulted in significant increases in both circulating plasma levels of MC-LR as well as increased 24 h urinary excretion of MC-LR in the Leprdb/J mice, as measured by UHPLC-QqQ-MS/MS and HPLC-orbitrap MS as we have described [43]. Interestingly, while there was no mortality or clinically observable MC-LR induced liver injury in the non-NALFD wild type C57Bl/6J group in our study, we did note a decreasing, albeit non-significant trend in survival in the MC-LR exposed Leprdb/J mice, despite similar body and liver weights, as well as the gross morphology of the livers measured in the surviving mice. Additionally, non-NALFD wild type C57Bl/6J had undetectable circulating plasma levels of MC-LR and a highly significant increase in the 24-h urine excretion of the toxin as compared to the Leprdb/J mice. This finding may be an indication of differing metabolic and excretory mechanisms between the healthy vs. NALFD diseased models and is likely relevant to the increased pathology and gene expression associated with hepatotoxicity and oxidative stress in the Leprdb/J mice.

Similarly, we noted in the Leprdb/J NAFLD model that while ALT levels were elevated non-specifically, ALP levels had a modest but significant increase specifically in the MC-LR exposed Leprdb/J mice. Neither ALT or ALP levels were significantly elevated in the wild type C57Bl/6J mice exposed to MC-LR at the highest dose studied, 100 μg/kg. While ALT is a marker of both acute or chronic liver injury, ALP is a marker of cholestatic predominance [44]. This indicates that ALT may not be a clinically useful marker of MC-LR induced liver injury in the setting of NAFLD. On the other hand, the fact that ALP levels were significantly elevated in MC-LR exposed Leprdb/J mice suggests that MC-LR may exacerbate liver damage in a cholestatic and obstructive pattern.

There are different patterns of hepatic-injury including hepatocyte-degeneration, intracellular fat accumulation, apoptosis, inflammation, regeneration and fibrosis [45]. Indeed, our histological analysis confirmed that hepatic injury in the form of hepatic micro-vesicular lipid accumulation, ballooned hepatocytes and reduced glycogen content was significantly higher in the MC-LR exposed Leprdb/J mice. This finding indicates that exposure to MC-LR affects lipid metabolism and leads to increased fat accumulation in the livers of NAFLD mice and supports the observation that MC-LR exposure may promote cholestasis in this setting. Recent studies have investigated MC-LR toxicity in the setting of pre-existing NAFLD [18,28,38]. Clarke et al. studied the acute effects of MC-LR by a single-dose intravenous (IV) injection (20 μg/kg) and a single-dose of intraperitoneal (IP) injection (60 μg/kg) in two separate rat models of NAFLD: a methionine and choline deficient (MCD) diet and a high fat/high cholesterol (HFHC) diet [18]. Observed findings were informative but different between the two NAFLD models. While plasma MC-LR area under the concentration-time curve (AUC) was doubled and biliary clearance (Clbil) was unchanged in the MCD rats, AUC was unchanged and CLbil was doubled in the HFHC rats. In addition, while liver pathology decreased in the MCD rats due to decreased binding to PP2A, hepatic inflammation, plasma cholesterol, proteinuria, and urinary KIM1 were increased in HFHC rats. These results reveal that pre-existing NAFLD potentially affects subject susceptibility to MC-LR toxicity, but there is variation to exact manifestations due to differences in NAFLD induction. Our current study provides a robust genetic model of NAFLD in mice that helps further the investigation of MC-LR toxicity in pre-existing NAFLD. In addition, our study provides a chronic exposure model by gavage, a more physiologically relevant method of exposure, mimicking a more typical exposure route in animals and humans.

Other studies have investigated the potential of NOX2 as a mediator of MC-LR toxicity in the setting of pre-existing NAFLD [28,38]. Albadrani et al. have shown that in mice with pre-existing NAFLD, intraperitonial exposure of MC-LR potentially activates hepatic Kupffer cells and stellate cells through the NOX2 pathway, causing an exacerbation of pre-existing liver pathology through increased CD68 and increased pro-inflammatory cytokines. Sarkar et al. has shown that intraperitonial exposure of MC-LR in NAFLD mice alters the gut microbiome, leading to inflammatory processes within the intestines. Again, while these studies have provided invaluable insight using intraperitoneal-delivered MC-LR in diet induced NAFLD mice, our study builds upon these results by utilizing a genetic model of NAFLD with a physiologically relevant oral exposure of MC-LR.

MC-LR is a potent inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), which are responsible for dephosphorylating serine and threonine residues on regulatory and structural proteins. The inhibition of PP1 and PP2A leads to increased protein phosphorylation, which may alter the structure of the cell cytoskeleton [46]. It has been demonstrated that oxidative stress may contribute to the toxicity of MC-LR and that the formation of excessive free radical species from oxidative lipid alterations causes an increase in lipid peroxidation in mice serum [11,46]. Since PP2A regulates several mitogen-activated protein kinases (MAPKs), its inhibition by MC-LR may have secondary effects on downstream MAPK signaling pathways involved in essential cellular processes [47]. Additionally, many inflammatory and immune responses are regulated by several MAPK signaling pathways, making the release of pro-inflammatory cytokines a consequence of MAPK disruptions caused by MC-LR [48].

Our genetic and phosphoproteomic analysis of livers from MC-LR exposed NAFLD mice is in agreement with the aforementioned studies. In the present study, we observed that genes associated with hepatotoxicity (*Cxcl12, Casp3, Cyp1a2, Rb1*), necrosis (*Fam214a, Mlxipl, Col4a1*) and nongenotoxic hepatocarcinogenicity (*Ccng1*) were significantly upregulated in the mice that were exposed to MC-LR. Oxidative stress is another mechanism associated with MC-LR toxicity [49]. Many in vitro studies have shown that exposure to MC-LR can cause oxidative stress in cells, further leading to apoptosis [50–53]. Our study shows that exposure to MC-LR significantly upregulated the expression of oxidative stress response genes such as *Prdx2 and Gpx5*. As mentioned earlier, limited analysis of targeted genes associated with hepatotoxicity (*Cyp1a2*, *Slc17a3*), necrosis (*OSMR*), steatosis (*CD36*), phospholipidosis (*Serpina3n*) and cholestasis (*Abcb4*) were tested for the C57Bl/6J groups and were not found to show any change in gene expression between vehicle and MC-LR exposed groups.

Our phosphoproteomic analysis revealed that MC-LR exposure significantly affected Reactome pathways related to immune function, urogenital system development, T cell proliferation, cellular response to oxidative stress, and cell cycle regulation pathways. Studies have shown that MC-LR exposure in mice increases the levels of superoxide dismutase and catalase as well as increases the expression of *TNF-*α*, NF*κ*B, IL-6* and *IL-1*β in HepG2 cells exposed with MC-LR [53,54]. Another study showed an increase in transcription of IL-8, a chemokine involved in the recruitment of monocytes and neutrophils during inflammation, in fish that were exposed to MC-LR [55]. In the present study MC-LR exposure regulated pathways related to Toll-like receptor (TLR) 2 and 6 signaling as well as those related to cell cycle and cell division regulatory functions. TLR2 activation has been shown to activate the PI3K/AKT/NF-kB pathway in immunological response to MC-LR in testicular cells [56]. These studies performed in various in vivo and in vitro models, complement our findings about the various pathways that are affected by exposure with MC-LR.

The fact that we did not note significant liver injury in the C57Bl/6J mice in the current study is in contrast with other studies where damage to mice was observed after oral exposure of MC-LR at similar doses of MC-LR by Zhang et al. and He et al. [6,57]. However, it should be noted that Zhang et al. gavaged the mice every 24 h for 28 days while in our study the mice were only gavaged every 48 h. Therefore, the total dosing in these mice was doubled compared to our study and this may account for the differences observed. In the He et al. study, the researchers used the Balb/c strain, whereas we used the C57Bl/6J because this strain is a better background control for the Leprdb/J mice. Importantly strain differences exist between Balb/c and C57Bl/6J strains where Balb/c mice have been shown to be more susceptible and C57Bl/6J mice less susceptible to hepatic injury [58,59].

MC-LR has been observed in freshwater environments around the world at varying levels. In the USA, 1000 μg/L of MC-LR was detected in the Lake Erie region [60]. In August 2014, elevated microcystin levels in the US city of Toledo, Ohio, led to a drinking water crisis when the city was left without drinking water for >2 days [61]. MC-LR not only accumulates within the water, but also accumulates within the aquatic ecosystem as extensively reviewed by Pham et al. [62]. MC-LR within the aquatic food chain, as well as within aquatic environments, pose potential risk of exposure in humans. In fact, human cases of exposure have been extensively reviewed by Svircev et al. [63]. A study in Australia reported MC-LR levels measured at 1 and 12 μg/L on two separate occasions within recreational locations (lakes and rivers in southern Queensland and in the Myall Lakes area of New South Wales), with surveyed individuals reporting gastrointestinal and respiratory symptoms [64]. In China, levels of MC-LR in Lake Chaohu have been reported to range from 2.2–3.9 μg/L [29], with measurable liver damage found in fishermen that work on the lake. In Sweden, 121 individuals from three villages reported abdominal pain, nausea, vomiting, diarrhea, fever, headaches, and muscle pain after their drinking water became discolored [63]. It was later found that the raw lake water contained 1 μg/L of MC-LR.

Taken together, our study provides insight into the potential increase in susceptibility of individuals with pre-existing liver conditions such as NAFLD, to the harmful effects of MC-LR exposure. By using exposure levels below the established NOAEL value of 40 μg/kg, our study reveals that MC-LR can exacerbate the liver damage in the setting of pre-existing liver disease. Such liver damage can be attributed to excess micro-vesicular lipid accumulation in the liver, upregulation of genes associated with hepatotoxicity and oxidative stress as well as changes in phosphorylation of the biological pathways related to cell cycle and immune response regulation. The results of this study suggest a need to review the preventative guidelines for safe exposure to MC-LR in at-risk settings, such as those with a pre-existing NAFLD. Indeed, we have recently demonstrated that MC-LR not only prolongs, but also worsens the severity of pre-existing colitis, supporting the notion that the effects of cyanotoxins such as MC-LR may be magnified in certain at-risk populations [65]. We have also demonstrated that the levels of the cyanotoxin can be detected in both plasma and urine samples of mice that were gavaged with low doses of the toxin, using UHPLC-QqQ-MS/MS and HPLC-orbitrap-MS methods [43]. This suggests that mass spectrometric analysis can be used in a clinical setting to assess the exposure to the cyanotoxins in humans. Further studies are warranted to examine the mechanistic basis of MC-LR hepatotoxicity in conditions such as NAFLD in order to assess diagnostic and therapeutic strategies for these potentially vulnerable populations. Additionally, epidemiological observations are needed to provide clinical context for the potential effects of cyanotoxins such as MC-LR in vulnerable patient populations.

#### *Limitations*

An important limitation of the current study was the fact that, because of the premature deaths in the MC-LR exposed NAFLD groups, not all animals were available for histopathological, biochemical, and phosphoproteomic examination. This may have affected the significance and dose dependence of the histopathological, biochemical, and phosphoproteomic changes we reported. It should also be noted that the doses used in this study are likely above doses to which humans, including those with NALFD, are exposed. Another limitation of the current study is that we were not able to measure MC-LR levels in the livers of the exposed mice nor did we perform analysis of activities or expression of PP1 and PP2A. However, despite these limitations, our study is the first to report the effects of chronic low dose oral exposure to MC-LR in a model of NAFLD. Our study suggests that in settings of pre-existing liver disease such as NAFLD, there may be an increase in the susceptibility to cyanotoxin toxicity.

#### **4. Materials and Methods**

#### *4.1. Mice*

Eight-week-old male B6.BKS(D)-Leprdb/J (JAX Stock No. 000697, B6 db) mice (hereafter referred to as Leprdb/J mice) on the C57Bl/6J background and wild type C57Bl/6J (JAX Stock No. 000664, Black 6) healthy background strain control mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA) and maintained in the Department of Laboratory Animal Research at University of Toledo. All the mice were specific-pathogen free status and were housed in plastic cages (five mice per cage) and fed ad libitum on balanced rodent diet (Teklad global 16% protein diet, Envigo, Indianapolis, IN, USA) and water. The mice were kept in a well-ventilated room maintained at 23 ± 1 ◦C on a 12-h light and dark cycle. The mice were allowed to acclimatize for a week, before the beginning of the study. All the protocols were approved by the University of Toledo Institutional Animal Care and Use Committee (IACUC protocol number 108663, approval date February 9, 2016) and conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

#### *4.2. Exposure and Experimental Design*

At 10-weeks of age, Leprdb/J mice were randomly divided into three groups with 13–17 mice per group (Figure 5). Group 1 was the Leprdb/J vehicle control group which was exposed to water with volume equivalent to that of the MC-LR exposed mice (300 μL). Group 2 and 3 consisted of Leprdb/J mice that were exposed to 50 μg/kg and 100 μg/kg of body weight of MC-LR (Item No. 10007188, Cayman Chemicals, Ann Arbor, MI, USA) respectively. MC-LR or vehicle control was administered to the mice via gavage every 48 h for a period of 4 weeks (15 total administrations over 4 weeks). These doses approximate the currently accepted NOAEL (40 μg/kg) established over a 13 week study [1]. The toxin was freshly prepared by dissolving it in purified water at a working concentration of 0.5 mg/mL. The mice were weighed once every week. Blood samples were collected at day 15 (midpoint) and 30 (endpoint) via retro-orbital bleeding (after isoflurane anesthesia) for analyzing blood chemistry, and at study termination, via intracardiac puncture after euthanasia. At the end of the study, mice were placed in metabolic cages for a 24-h urine collection immediately following the final dose of MC-LR or vehicle. 24-h urine samples were collected and stored at −80 ◦C. Mice were then removed from metabolic cages and euthanized for final blood and organ collection after another 24 h (i.e., a total of 48 h after last MC-LR/vehicle gavage). Blood samples were collected at day 15 (midpoint) and 30 (endpoint) via retro-orbital bleeding (after isoflurane anesthesia) for analyzing blood chemistry and at study termination via intracardiac puncture after euthanasia.

The animals were humanely euthanized and flushed with 10 ml of 1X Phosphate Buffered Saline (PBS) (Fisher Scientific, Pittsburgh, PA, USA). Liver tissues from individual animals were collected and weighed. Part of the liver tissue was frozen in OCT embedding media (Fisher Scientific, Pittsburgh, PA, USA) and another section was fixed with 4% *w*/*v* Formalin (Fisher Scientific, Pittsburgh, PA, USA) for histological purposes. The remainder of the tissue was snap frozen in liquid nitrogen and then stored at −80 ◦C until further use for experimental purposes. In a parallel study, wild type C57Bl/6J mice were randomly divided into 2 groups: a vehicle control and 100 μg/kg MC-LR exposed group, which were exposed as detailed above.

**Figure 5.** Schematic representation of the study design used to study the effect of low doses of MC-LR on healthy C57Bl/6J and NAFLD LepRdb/J mice. Ten-week-old mice were exposed with vehicle, 50 μg/kg or 100 μg/kg of MC-LR for a period of 4 weeks (15 total administrations) every 48 h. The mice in the vehicle control group were exposed with an equivalent volume of purified water.
