*4.3. Histological Studies*

Liver sections were collected and immediately fixed in 4% formalin solution for 24 h, dehydrated in 70% ethanol, embedded in a paraffin block, and cut with a microtome to yield sections of 4-micron thickness. Hematoxylin & Eosin (H&E) and Periodic Acid-Schiff (PAS) staining were then performed on these sections. Whole slides were scored for hepatic injury by a pathologist, who was blinded to the study group assignments. Hepatic injury was assessed in both H&E and PAS was scored on a semi-quantitative scale of 0 (none or least hepatic injury) to 4 (maximum hepatic injury) and a composite injury score was taken as the average of the H&E and PAS injury score for each liver. Hepatic injury was assessed according to established methods including pale coloration of the tissue, infiltration of microvesicular lipid accumulation inside the hepatocytes, reduced glycogen content as well as ballooned hepatocytes with marked macro- and/or micro-vesicular lipid infiltration [45]. Hepatic damage was characterized by pale coloration of the tissue, infiltration of microvesicular lipid accumulation inside the hepatocytes, reduced glycogen content as well as ballooned hepatocytes with marked macro- and micro-vesicular lipid infiltration. A detailed description of the observations can be found in Table S5.

### *4.4. Blood Chemistry*

Blood was collected via retro-orbital bleed in heparinized Micro-Hematocrit Capillary tubes (Cat no. 22-362-566, Fisher Scientific, Pittsburgh, PA, USA). Whole blood was then loaded onto Abaxis rotor with VetScan2 Chemistry Analyzer (Ref: 500-0038, Abaxis, Union City, CA, USA) with a comprehensive diagnostic profile for the quantitative analysis of Alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), amylase (AMY), globulin (GLOB), glucose (GLU), blood urea nitrogen (BUN) and total protein (TP).

#### *4.5. MC-LR Determination in Plasma and Urine*

Both plasma and 24-h urine samples were collected post MC-LR exposure. Blood was collected by cardiac puncture in K3-EDTA microtubes (Sarstedt, Newton, NC, USA) and separated plasma was stored at −80 ◦C until further use. MC-LR in urine samples was quantified using both Ultra High Pressure Liquid Chromatography–triple Quadrupole–tandem Mass Spectrometry (UHPLC-QqQ-MS/MS, Columbia, MD, USA) and High Pressure Liquid Chromatography (Shimadzu Technologies, Addison, IL

USA)–Orbitrap Fusion Mass Spectrometry (Thermo Fisher Scientific, San Jose, CA, USA) system, while plasma samples were analyzed using HPLC-Orbitrap Fusion MS as we have previously described [43].

#### *4.6. Genetic Analysis of Hepatotoxicity and Oxidative Stress*

For genetic analysis, each exposure group shows data from 3 arrays (i.e., biological triplicates) and each array used a pooled cDNA sample from 4 mice/group. Hence, the data is representative of 12 mice from each exposure group. The data was analyzed using Qiagen analysis software (Qiagen, Germantown, MD, USA) and the criteria for fold change is based on any changes that are at least 2-fold above the normalized value for the vehicle exposed group. The *p* value for significance was considered as *p* < 0.05. RNA was extracted from liver tissues and isolated using the QIAzol/Chloroform extraction method. Approximately 500 ng of extracted RNA was used to synthesize cDNA (QIAGEN's RT2 First Strand Kit (Qiagen, Germantown, MD, USA). The cDNA was then used to run RT2 ProfilerTM Mouse Hepatotoxicity Quantitative PCR Array (Cat No. PAMM-093Z, Qiagen, Germantown, MD, USA) as well as RT2 ProfilerTM Mouse Oxidative Stress and Antioxidant Defense Quantitative PCR Array (Cat No. PAMM-065Z, Qiagen, Germantown, MD, USA) performed utilizing a QIAGEN Rotor-Gene Q thermo-cycler. Because previous reports have shown hepatotoxic and oxidative stress mediated injury induced by MC-LR, we selected targeted arrays to assess several measures of hepatotoxicity using the RT<sup>2</sup> ProfilerTM Mouse Hepatotoxicity Quantitative PCR Array (Cat No. PAMM-093Z which assesses genetic markers of Hepatotoxicity—Cxcl12, Cyp1a2, Casp3, Rb1; Nongenotoxic hepatocarcinogenicity—Ccng1; Necrosis—Fam214a, Mlxipl; Steatosis—CD36 and Cholestasis—Abcb1A, Abcb4) as well as RT<sup>2</sup> ProfilerTM Mouse Oxidative Stress and Antioxidant Defense Quantitative PCR Array (Cat No. PAMM-065Z which assesses genetic markers of peroxiredoxins; glutathione peroxidases; superoxide dismutases; oxygen transporters and oxidative stress response). Both arrays use Actin b, β2-microglobulin, GAPDH, Gusb and Hsp90ab1 as the housekeeping genes. All RT-qPCR was performed utilizing a QIAGEN Rotor-Gene Q thermo-cycler.
