Male and female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 4 weeks of age and were cared for in the Department of Laboratory Animal Resources (DLAR) at the University of South Carolina’s School of Medicine. Mice (n = 4–5/group/sex/experiment) were randomized upon arrival to the DLAR to prevent litter biases. Mice were housed 4–5 per cage in a low-stress environment (22 °C, 50% humidity, low noise) and maintained on a 12:12-h light-dark cycle. All mice were handled only by the primary investigator (ATS). Mice were habituated to the AIN-76A diet (BioServ, Frenchtown, NJ, USA; catalog#:F1515) for 6 weeks prior to any experimental procedure (i.e. until 10 weeks of age). Experiments were initiated at 10 weeks of age and at average body weights of 27.57 ± 0.37 g for male and 19.73 ± 0.27 g for female mice. Food and water were provided ad libitum throughout the course of the study. All methods were performed in accordance with the American Association for Laboratory Animal Science and the Institutional Animal Care and Usage Committee at the University of South Carolina.
Emodin was purchased from Nanjing Zelang Medical Technology Co., Ltd., (Nanjing, China). Emodin was independently analyzed by the Mass Spectrometry Center at the University of South Carolina prior to the initiation of the experimental study. Liquid Chromatography -Ultraviolet-Mass Spectrometry (LC-UV-MS) and Nuclear Magnetic Resonance (NMR) was performed to confirm the purity and molecular structure of emodin.
Emodin dosing and time course analysis for pharmacokinetic studies
At 10 weeks of age, emodin was delivered intraperitoneally (I.P.) or by oral gavage (P.O.) at 20 mg/kg or 40 mg/kg doses. For the I.P. study, emodin, dissolved in dimethyl sulfoxide (DMSO) (Spectrum Chemical, New Brunswick, NJ, USA; catalog#: BS580) was made in a large batch, aliquoted, and stored at -20 °C until used for injections. It was subsequently diluted in phosphate-buffered saline (PBS) (VWR, Suwanee, GA, USA; catalog# MRGF-6235-010Q) (1% DMSO) and administered to mice I.P. at doses of 20 mg/kg (0.5 mg/mL) or 40 mg/kg (1 mg/mL). To deliver emodin P.O., we utilized a 20G 30-mm flexible plastic tubal oral gavage needle (Instech, Plymouth Meeting, PA, USA; catalog#: FTP-20). To prevent aspiration of the emodin bolus, mice were briefly anesthetized with 2% isoflurane (provided by DLAR) prior to gavage and held upright until consciousness was regained. Emodin was prepared fresh on the day of administration. Briefly, emodin was mixed in pure propylene glycol (VWR, Suwanee, GA, USA; catalog# 97061) for 6–8 h at room temperature while protected from light. The emodin solution was delivered to mice at 20 mg/kg (6 mg/mL) and 40 mg/kg (12 mg/mL) doses. We used 1% DMSO in PBS (I.P.) or pure propylene glycol (P.O.) for vehicle controls.
Plasma emodin content was analyzed at three specific time points (t = 1 h, 4 h, and 12 h) after treating male and female wildtype (WT) C57BL/6 J mice with I.P. or P.O. administered emodin. Emodin was given at 20 mg/kg or 40 mg/kg; n = 4 mice were used per dosage, per timepoint, and per sex. Only a single dose of emodin was administered for the pharmacokinetic study. Vehicle treated mice were used to analyze empty plasma extracts.
Solid phase extraction and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis
Whole blood was collected in ethylenediaminetetraacetic (EDTA) (VWR, Suwanee, GA, USA; catalog#: 454428) coated tubes from the inferior vena cava and centrifuged at 4000 RPM for 10 min. Plasma was aliquoted and stored at -20 °C until solid phase extractions. For free emodin quantification, 50 μl of plasma was mixed with equal volume of 0.2 M sodium acetate buffer (Honeywell, Charlotte, NC, USA; catalog#: 71190) with 1% ascorbic acid (VWR, Suwanee, GA, USA; catalog#: BDH9242) (pH 5.0). For emodin glucuronide quantification, 50 μl of plasma was mixed with half the volume of 0.2 M sodium acetate buffer with 1% ascorbic acid and 1000 units of β-glucuronidase (Millipore Sigma, Burlington, MA, USA; catalog# G2174). D4-emodin (Santa Cruz Biotechnology, Dallas, TX, USA; catalog# 218302) (1 ng/μl) was used as an internal standard. Both tubes were then incubated at 37 °C for 2 h. After incubation, the mixture was extracted with 600 μl ethyl acetate (Fisher Scientific, Waltham, MA USA; catalog #: E195–4) three times. The ethyl acetate layer was evaporated under N2 gas to dryness and reconstituted in 5% ammonia water. Solid phase extraction was performed using a vacuum manifold (Millipore Sigma, Burlington, MA, USA; Supelco catalog#: SU57250-U) with Oasis MCX cartridges (Fisher Scientific, Waltham, MA USA; catalog #:186000254) and was eluted with 3 mL 5% formic acid-methanol (Fisher Scientific, Waltham, MA USA; catalog #s: A117–50 and A452–4). After elution, the eluent was evaporated under N2 gas to dryness and reconstituted in 400 μl 5% ammonia methanol (Fisher Scientific, Waltham, MA USA; catalog #s: A669–212 and A452–4).
Emodin samples were analyzed and quantified by LC-MS/MS using electrospray ionization in negative ion mode. Chromatorgraphic separation was performed on a Waters Acquity UPLC system using a binary solvent gradient. Solvent A was water containing 0.1% formic acid and solvent B was methanol. The LC column was a Waters XBridge C18 reversed phase column (2.1 mm X 100 mm containing 3.5um particles) running at a flow rate of 0.2 mL/min. The solvent gradient started at 50%B, ramped to 95%B over 10 min and was maintained at 95%B until 14 min. The gradient then returned to initial conditions. The mass spectrometer was a Waters Premier XE triple quadrupole instrument. Data was collected in multiple reaction monitoring (MRM) mode. Two precursor/product ion pairs were monitored; one pair for emodin (269 Da > 225 Da) and one pair for the internal standard deuterium labeled emodin (273 Da > 229 Da). According to the standard curve of emodin in 5% ammonia methanol, the concentration of emodin was calculated relative to the D4-emodin internal standard. Total emodin was measured from the tube containing the β-glucuronidase, free emodin was measured from the tube without β-glucuronidase, and glucuronidated emodin was calculated as the difference between total emodin and free emodin.
Emodin diets for sub-chronic toxicity study
We analyzed the effect of emodin feeding over a 12-week period. Emodin was infused into the AIN-76A diet at three different concentrations; 170 mg/kg, 340 mg/kg, and 680 mg/kg. These concentrations were based on established average food intake of C57BL/6 mice and translate to a daily ingested dose per body weight of ~ 20 mg/kg, 40 mg/kg, and 80 mg/kg, respectively – doses that we have found to have efficacy in our cancer and cancer therapy studies. Briefly, mice were purchased at 4 weeks of age, kept on an AIN-76A diet until 10 weeks of age, and then separated into experimental groups and started on their respective diets as follows: at 10 weeks of age male and female mice were randomized into 4 groups consisting of n = 5 sex/diet including control (AIN), 20 mg/kg, 40 mg/kg, and 80 mg/kg. Mice were maintained on their treatment diets for 12 weeks and were given food and water ad libitum.
Body weights and body composition
Body weight, food, and water consumption were monitored on a weekly basis for the duration of the study. Body composition was assessed after 12 weeks of emodin diet using dual-energy X-ray absorptiometry (DEXA) (Lunar PIXImus, Madison, WI, USA). Briefly, mice were placed under gas anesthesia (isoflurane, 2%) and were assessed for bone mineral density (BMD), lean mass, fat mass, and body fat percentage. Lean mass (%) was calculated as percent lean weight and bone mineral content (BMC) of total body weight.
After 12 weeks of dietary treatment, mice were euthanized by isoflurane overdose following a 4 h fast. Blood was collected from the inferior vena cava and placed in EDTA coated lavender top tubes for plasma collection and blood panel analysis. Spleen, liver, kidney, heart, colon, and ileum were harvested and fixed overnight in 10% neutral buffered formalin (VWR, Suwanee, GA, USA; catalog#:16004–128) and subsequently embedded in paraffin blocks. Colon and ileum were cleaned with PBS and swiss rolled prior to fixation. Liver, epididymal fat, mesenteric fat, and spleen weight were determined from freshly excised tissue prior to fixation. Colon, entire small intestine, and tibial length were also measured using calipers during tissue collection.
Blood panel analysis
A complete blood panel analysis was performed using the VetScan HMT (Abaxis, Union City, CA, USA) for determination of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), neutrophils (NEU), platelets (PLT), red blood cells (RBC), hematocrit (HCT), and hemoglobin (Hb). Neutrophil/lymphocyte ratio (NLR) was calculated from obtained values.
Plasma markers of toxicity
Plasma was analyzed for common markers of major organ toxicity/physiological impairment including alanine transaminase (ALT) (Cayman Chemicals, Ann Arbor, MI, USA; catalog#: 700260), aspartate transaminase (AST) (Cayman Chemicals, Ann Arbor, MI, USA; catalog#: 701640), and creatinine (Cayman Chemicals, Ann Arbor, MI, USA; catalog#: 700460) and according to manufacturer’s instructions.
All tissues collected were stained with hematoxylin and eosin (H&E) (Fisher HealthCare, Irvine, CA, USA; catalog#: 245–651 and 245–827) as previously described by our group . To analyze the presence of fibrosis, trichrome staining was performed in heart, liver, kidney, and spleen as previously described . Goblet cells were identified in the colon by Alcian blue (Alfa Aesar, Haverhill, MA, USA: catalog#: J6012209) staining and counterstained by nuclear fast red as previously described . Histological sections of the small intestine were examined for findings of inflammation (atrophy of the villi, infiltration of the lamina propria by inflammatory cells). Sections of the colon were evaluated for findings of colitis (destruction of the colonic mucosa, decreased number of goblet cells, and infiltration of the lamina propria by inflammatory cells) and/or dysplasia, graded as low or high dysplasia. Kidney, spleen and heart were evaluated with H&E staining for architectural and cellular abnormalities and for the presence of fibrosis (kidney and heart) with trichrome staining. For the histological examination of the liver specimens we used the scoring system designed by the Pathology Committee of nonalcoholic steatohepatitis (NASH) Clinical Research Network, which addresses the full spectrum of lesions of NAFLD . alpha-Smooth Muscle Actin (α-SMA) (Cell Signaling, Danvers, MA, USA: #D4K97) was detected by immunohistochemical analysis in liver specimens using heat-based antigen retrieval rodent decloaker (Biocare, Pacheco, CA, USA: #RD913). Positivity was detected with goat anti-rabbit HRP-conjugated IgG secondary antibody (Abcam, Cambridge, UK: ab6721) and developed with DAB chromogen (Biocare, Pacheco, CA, USA: #BDB2004L). All histological analyses were performed blindly by a certified pathologist (I.C.).
Analysis was performed using commercial software (SigmaStat V3.5, SPSS, Chicago, IL). All toxicity data were analyzed using a one-way analysis of variance (ANOVA). To examine sex differences in the pharmacokinetic data we performed a paired t-test at each time point. Any data that were not normally distributed or did not display equal variance were logarithmically transformed so that those criteria were met. Statistical significance was set with an alpha value of p < 0.05. Data are presented as mean ± standard error of mean (SEM).