Characterization of ANIT-Induced Toxicity using Precision-Cut Rat and Dog Liver Slices Cultured in a Dynamic Organ Roller System

Liver Slice Preparation

Rats were anesthetized with sodium pentobarbital (50 mg/kg), injected iv through the tail vein with 200 U heparin, and placed in a sterile biosafety hood, where all further work was done aseptically. After exposure of the abdominal cavity, the portal vein was perfused with ice-cold UW solution supplemented with 3 mM glutathione, 2 mM Glutamax I, 1x antibiotic-antimycotic solution, 30 μM L-ascorbic acid, 2 mM sodium pyruvate, 1 μM corticosterone, and 100 nM insulin (human). After the blood was cleared from the liver, it was quickly removed, and the lobes were separated with a scalpel and cored into cylinders 8 mm in diameter.

Beagle dog liver specimens were obtained during necropsy procedures from control animals in other ongoing studies. Dogs (Marshall Farms, Rose, NY) were quarantined for 14 days after arrival before the study began. On study completion, the dogs were necropsied following euthanasia with sodium pentobarbital (150 mg/kg iv). The liver tissue for liver slice studies (consisting of one or two lobes) was removed immediately after the abdominal cavity was opened. The lobes were immediately placed in ice-cold UW solution, supplemented with 3 mM glutathione, 2 mM Glutamax I, 1 × antibiotic-antimycotic solution, 30 μM L-ascorbic acid, 2 mM sodium pyruvate, 1 μM corticosterone, and 100 nM insulin (human). The vessel containing the lobes was placed in a biosafety hood where all further work was done aseptically.

The cores of either rat or dog liver were then sliced (using the Krumdieck slicer) in supplemented UW solution into ~200-μm-thick discs using a Krumdieck slicer (Alabama Research and Development, Munford, Alabama). Only the most uniform-shaped slices were selected for experiments; each contained approximately 3 mg protein. Using a sterile cotton swab, the slices were placed on sterile HATF paper inside the titanium inserts. All slice manipulations were done in ice-cold supplemented UW solution.

Slice Pre-incubation/Culture

The titanium inserts with the slices were placed in sterile scintillation vials, each containing 1.7 ml culture medium consisting of Waymouth’s MB 752/1 basal medium containing, per ml, 2 mg BSA, 0.084 mg gentamicin sulfate, 5 μg oleic acid, 5 μg linoleic acid, 0.5 μg DL-α-tocopherol, 7.9 μg D-thyroxine, 5 μg insulin, 5 μg transferrin, 5 ng selenium (ITS), 288 ng testosterone, 272 ng β-17estradiol, 39.3 ng dexamethasone, 30 ng glucagon, 0.02 U insulin, 0.2 μmol L-2-phosphate ascorbic acid, and 2 mM sodium pyruvate. The vials were then capped with sterilized open-end caps containing PTFE membrane filters, each held in place by a hole-punched Teflon liner to allow gas exchange with the external atmosphere. Vials were placed in the roller drum inside a humidified incubator at 37°C under 70% O₂/25% N₂/5% CO₂ or 75% O₂/20%N₂/5% air atmosphere (the latter environment proved slightly better than the former after trigas incubators were purchased). The roller drum containing the vials was rotated at 6–7 rpm. Following equilibration for 2–3 hrs, the zero time-point group was harvested. In a biosafety hood, the medium in the remaining vials was aspirated and replaced with designated warmed media with or without ANIT at designated concentrations. Medium containing ANIT was spiked using 1:1000 dilutions from a stock solution made up in ethanol. Control groups had ethanol at the same concentration. In the case of the highest concentration used (320 μM), ANIT was weighed and dissolved directly into the medium. The slice medium in each vial was replaced with fresh medium daily until the slices were harvested; for examination of the medium biomarker content, the collected medium (1 ml/vial) was pooled for respective slices and stored at 4°C until later biochemical analysis. After medium change, the vials were repositioned on the roller system in the incubator, and the incubation continued under the conditions described above until all groups (3–4 replicates/group) were harvested. Each group was comprised of 3–4 replicates (slices cultured in separate tubes). Experiments assessing the toxicity of ANIT were run for a total of 7 days, with cultures harvested on study days 0, 1, 3, 5, and 7.

Slice Harvest

At the indicated time points, slices and filter paper were removed from their titanium inserts and rinsed in PBS by briefly submerging the slice (still on its HATF paper) into a vessel containing ~20 ml PBS. The slices were bisected so that one-half could be used for biomarker analysis and the other for histology and morphological examination. For BrdU incorporation experiments, 20 μM BrdU was added to the medium and allowed to incubate with the slices for 18 hrs before their harvest.

Biomarker Analyses

Each slice section designated for biochemical analysis was transferred into a 1.5 ml Eppendorf tube containing 0.5 ml PBS + 0.5% Triton X-100 on ice. The sections were then homogenized and briefly sonicated at ice-cold temperatures. The resulting lysates were centrifuged at 9000 × g for 5 min to remove particulate matter. The resulting supernatants were stored at 4°C until the experiment was completed and then sent on ice with the corresponding media by courier to a local laboratory (Quality Clinical Labs, Mountain View, California) for analysis with a Hitachi 911 clinical analyzer. Biomarker (AST, ALT, ALP, and GGT) levels measured from slice lysates were taken within the first 24 hrs and analyzed periodically thereafter (tests indicated they were stable at 4°C for a minimum of 18 days). A small fraction of the lysate was retained for protein analysis using a Pierce BCA protein assay kit (VWR International, West Chester, Pennsylvania) and BSA standards in PBS + 0.5% Triton X-100. A chemiluminescent ALP-specific substrate (Michigan Diagnostics, Troy, Michigan) was used to measure ALP activity in the medium. For these experiments, medium ALP activity was extrapolated from an ALP standard curve. All medium ALP assays were performed in 96-well microtiter plates.

Histology

The faces of slice sections designated for morphological examination were covered by lens paper (prewetted in 10% buffered formalin) and placed between 2 foam inserts. This “sandwich” was first placed in a histological cassette, fixed in 10% buffered formalin for 18–24 hrs, then transferred to 70% alcohol, and finally submitted to a local laboratory (Biopathology Lab, South San Francisco, California) for paraffin embedding. H&E, Periodic Acid Schiff (PAS), Masson’s Trichrome, and BrdU immunostaining were done on 4-μm-thick tissue sections cut from the paraffin blocks. BrdU immunostaining was done using a staining kit, according to the protocol provided by the manufacturer (Zymed Labs, South San Francisco, California).

Semi-Quantititative Histological Analysis

H&E-stained slice sections were thoroughly examined under low (25 × and 50 ×) and medium (100 × and 200×) magnification and assessed for HPC and BEC viability. Following examination, a viability percentage score was assigned to both HPC and BEC. In addition, glycogen deposition in HPCs was estimated on a scale of 0–4, where 0 was no glycogen, 1 was minimal, 2 was low, 3 was moderate, and 4 was high. BEC proliferation was evaluated on sections immunostained with BrdU. From each slice section, the five portal tracts with the best morphology containing small to medium-size bile ducts were selected and counted for BrdU-positive BEC using a Zeiss Axioskop 2 equipped with Axiocam digital camera and KS 300 interactive imaging software. Positive cells present in the stroma or lining the portal vessels were excluded. On the basis of these numbers, a mean BEC proliferation count for each slice was deduced.

Western Blot

Protein concentrations for the liver slice lysates were determined using BCA reagent (Pierce Biotechnology, Rockford, IL). A positive control for iNOS was prepared with RAW 264.7 cells (mouse macrophage) treated with 10 μg/ml LPS. Samples were prepared for Western analysis according to the Invitrogen (Carlsbad, CA) protocol: 20 μg protein per lane were prepared in 4X loading buffer, heated at 70°C for 10 min, then run in 4–12% NuPage gels (Invitrogen, Carlsbad, CA) at 150 volts for 2 hours 45 minutes. After protein transfer onto Immobilon membrane (Millipore, Bedford, MA) and blocking in 5% dry milk in PBS + 0.1% Tween-20, membranes were probed for iNOS using BD Transduction Laboratories antibody (#610332) and horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, SC2062, Santa Cruz, CA). ECL Plus (Amersham Biosciences, Piscataway, NJ) was used for chemiluminescent detection. Liver slices from a total of five animals were used for Western blotting experiments.

Statistics

Replicates from control and treatment groups among experiments were consolidated and compared using t-test analysis. Values were considered significantly different if p < 0.05. Treatment group means and standard deviations were calculated. Values were converted to percent of control, and standard deviations were determined as a percent of the ratio between the treated groups and the control.

Slice Culture

Rat liver slice cultures were well-maintained during experimentation for 7 days (Table 1). As seen previously with rat (Behrsing et al., 2005) and dog (Amin et al., 2005) liver slices, tissue AST and ALT levels diminish while ALP and GGT markers increase over time in culture. Histological results indicate good viability, and active glycogen accumulation is evident over the culture period. The viability of rat liver slices between days 3 to 7 ranged from 69% to 78% for HPC and from 87% to 90% for BEC.

Rat Slice ANIT Toxicity

Initial screening experiments were conducted using rat slices to determine the responsiveness of the slice model to ANIT exposure. Due to the negligible proliferation response in control tissue, day 1 BrdU was difficult to assess. However, by day 3, BrdU staining showed an increased number of proliferating BECs in ANIT-treated vs. control groups. Further experimentation also examined both slice histology and biochemical biomarkers corresponding to HPC and BEC, following exposure to a range of ANIT concentrations from day 1 until day 7 (Figures 1, and 2). Interestingly, ALP values measured in the ANIT treated tissue were found elevated at the earliest time point (Figure 1).

Initially, ranges of concentrations (1–8, 20, and 80–100 μM) were used to determine the ANIT concentrations that produced the best effects on tissue. Results from treated rat slices indicate that application of ANIT at the lowest concentration range (1–8 μM) caused a statistically significant increase in slice content of AST and ALT at all time points with the exception of day 1. HPC appeared normal except after prolonged application of ANIT; in that case, 20 and 80–100 μM ANIT treatments caused a slight decrease in viability by day 7 (87 ± 3% and 86 ± 6% of control values, respectively). Lower AST, ALT, and glycogen values are seen at day 7 only with the application of 80–100 μM ANIT (Figure 1). BEC viability was not affected at any concentration or time point. Biochemical markers for BEC were also found to be elevated at days 1, 3, and 5, but the increase diminished by day 7. An increase in the number of BrdU positive BEC cells was noted at days 3 (largest increase) and 5 with the application of 20 and 80–100 μM ANIT (Figure 1). Western blot analysis to determine iNOS levels in rat slice lysates showed that ANIT exposure increased the production of iNOS at all concentrations within 12 hrs (Figure 3). However by day 3, iNOS dropped to undetectable levels at all concentrations. The highest level of iNOS expression was seen at 24 hr at a concentration of 20 μM ANIT and this was followed by a small decrease in expression at the highest concentration (80 μM).

Dog Slice ANIT Toxicity

Experiments were conducted using dog tissue to compare species response to ANIT toxicity. Dog liver slices were cultured for 7 days, and average control cultures indicate slightly lower baseline (day 0) AST values, but higher ALT values (Table 2) compared to rat liver slices. ALP and GGT baseline values were approximately the same as rat values, but slice ALP was found to increase more and GGT less than rat slice values. Although a similar trend in biomarker level fluctuation was found when comparing 7 day cultures of rat liver slices, dog slices appeared to retain a higher overall viability of HPC and BEC (Table 1 and Table 2). Glycogen and BrdU staining of control slices appeared comparable for both rat and dog slices.

When exposed to a range of concentrations (4, 20, 80, and 320 μM), dog slices were less responsive to the toxic effects of ANIT. Although lower concentrations of ANIT (1–8 and 20 μM) caused an elevation of AST and/or ALT in rat slices at days 3, 5, and 7, this increase was not observed with dog slices at any concentration or time point (Figure 4). The highest concentration of ANIT applied (320 μM) tended to decrease ALT and glycogen values at several time points, suggesting HPC toxicity. Histological assessment indicated that HPC and BEC viability is unchanged except at the highest ANIT concentration of 320 μM (87%, 74%, and 74% of control for HPC, and 95%, 87%, and 84% of control for BEC, at days 3, 5, and 7, respectively). Although elevations of BEC biochemical parameters are noted with rat tissue on all days assayed (1, 3, 5, and 7), only slight elevations are noted with dog slices, and neither ALP nor GGT elevations were found to be statistically significant (p < 0.05). However, BrdU staining indicated increased BEC proliferation at days 5 and 7 (Figure 5), although it was less pronounced than that seen with rat slices at day 3 (Figure 1, Figure 4). Since biochemical biomarkers of slices did not reflect the histological observations of increased proliferation, slice medium was examined in an attempt to explain histological findings.

Dog Medium

Measurement of medium from ANIT-treated dog slices indicated that although no histological indication of HPC viability loss was noted, an increase of AST and ALT activity was found at days 5 and 7 (Figure 6). Previous work indicated that a direct correlation exists between the levels of ALP in the medium and the level of observed proliferation (Behrsing et al., 2005). Results from ALP measurements indicate an initial reduction of ALP release by day 3 (in a concentration-dependent manner), but increasing values over time. By day 7, average medium ALP values from all treatment concentrations are well above control values.