Flow Cytometric Assessment of Peroxisome Proliferation from Frozen Liver of Fibrate-Treated Monkeys

Abstract

Multiple methods currently exist for the assessment of peroxisome proliferation, including gene expression, enzyme activity, immunolabeling coupled with image analysis, and electron microscopy. This study describes a novel flow cytometric method to efficiently quantify peroxisome proliferation in cells from frozen livers. Frozen livers from cynomolgus monkeys treated with ciprofibrate at doses of 0, 3, 30, 150, and 400 mg/kg/day for 15 days were mechanically disaggregated using an automated dispersion method. The resulting cell suspensions were labeled using an allophycocyanin (APC)-conjugated antibody directed against peroxisomal membrane protein 70 (PMP70). Statistically significant increases in mean fluorescence intensity were observed from animals dosed at 30, 150, and 400 mg/kg/day compared to control. Parallel comparisons using electron microscopy and immunofluorescence microscopy suggest that flow cytometry may be an alternative to electron microscopy in determinations of peroxisome proliferation. Flow cytometric analysis of freshly isolated hepatocytes and frozen liver from rats treated with fenofibrate at 200 mg/kg/day for 10 days showed the flow cytometric method could detect peroxisome proliferation in both species. The research described here demonstrates the feasibility of applying flow cytometry for the detection of peroxisome proliferation.

Peroxisomes are single membrane-bound cytoplasmic organelles found in nearly all eukaryotic cells. They contain numerous enzymes involved in diverse metabolic functions, including β-oxidation of the very long chain fatty acids, cholesterol metabolism, and the inactivation of reactive oxygen species such as H₂O₂ (Jakobs and Wanders 1991; Mannaerts and Van Velhoven 1993; Singh 1997; Masters 1998; Orbea et al. 1999). Isseman and Green (1990) demonstrated that a member of the nuclear hormone receptor family was activated by compounds that cause peroxisome proliferation, and named the gene peroxisome proliferator activated receptor (PPAR). The PPAR subfamily is now known to contain three subtypes, PPARα, PPARβ/δ, and PPAR γ (Lemberger, Desvergne, and Wahli 1996). Peroxisome proliferators form a broad group of chemicals, including both synthetic and naturally occurring compounds, such as hypolipidemic drugs, phthalate esters, industrial solvents, herbicides, some steroids, and food flavors. The hypolipidemic drugs ciprofibrate and fenofibrate, when administered to rats and mice, typically cause hepatomegaly, proliferation of peroxisomes in hepatic parenchymal cells, marked increases in the activities of enzymes related to peroxisomal β-oxidation of fatty acids, and eventually hepatocarcinogenesis (Reddy and Chu 1996). Peroxisome proliferators are considered to be nongenotoxic rodent hepatocarcinogenic agents. Proposed mechanisms for the hepatocarcinogenesis include oxidative stress, cell proliferation, and promotion of preneoplastic liver lesions (Reddy and Rao 1989; Dzhekova-Stojkova, Bogdanska, and Stojkova 2001). Rats and mice are extremely sensitive to PPARα agonists whereas guinea pigs and primates are relatively insensitive or nonresponsive at doses that produce clear effects in rodents (Short et al. 1987; Bentley et al. 1993). However, reports on hepatic peroxisome proliferation induction in nonhuman primates are contradictory. High doses of ciprofibrate and DL-040 have been reported to induce peroxisome proliferation in cynomolgus and rhesus monkeys (Reddy et al. 1984; Lalwani et al. 1985; Hoivik et al. 2004). To date, electron microscopy (EM) observation by immunogold labeling is considered the standard for peroxisome quantitation (Fahimi et al. 1996). Although the method is effective for accurate quantitation, it is costly and time-consuming. In this study, we used a new approach to measure peroxisome proliferation in frozen rat and primate livers as well as in freshly isolated hepatocytes by flow cytometry (FCM).

MATERIALS AND METHODS

Animal Treatment (Rat)

Male Sprague-Dawley rats (approximately 12 weeks old, body weight 200 to 450 g) were purchased from Charles River Laboratories (Raleigh, NC, USA). Food and water were provided ad libitum. LabDiet brand certified Rodent diet 5002 made by PM Nutrition International (Richmond, IN, USA) and municipal water with additional treatment by reverse osmosis were provided ad libitum. Animals were kept in rooms controlled for temperature (18°C to 26°C), humidity (30% to 70%) and 12-h light and dark cycle. The animals were acclimatized for 5 days before commencement of dosing. They were housed in solid polycarbonate cages with Bed-O’Cobs. All animals were cared for according to the US Department of Agriculture’s Animal Welfare Act and have been reviewed and approved by Glax-oSmithKline’s Institutional Animal Care and Use Committee (IACUC).

Fenofibrate (Sigma Chemical Company, USA) was formulated in 0.5% methylcellulose in water. A fresh dosing solution of fenofibrate was prepared daily. Rats were treated with vehicle (0.5% methylcellulose) or fenofibrate at 200 mg/kg/day for 10 consecutive days by oral gavage. After treatment, animals were fasted overnight and exsanguinated. Liver perfusion for isolation of hepatocytes was performed. Livers from animals treated in parallel were snap-frozen in liquid nitrogen and stored at –80°C. The frozen livers were stored for approximately 4 months prior to flow cytometric analysis.

Isolation of Primary Rat Hepatocytes

Isolated rat hepatocytes were obtained using a two-step collagenase method (Seglen 1973). Briefly, each rat was anesthesized and the abdominal cavity was opened. The liver was perfused with buffer and collagenase solution, removed, and teased apart to suspend cells. Cells were purified over a Percoll gradient. Hepatocytes were then washed, counted, adjusted to a concentration of approximately 1 × 10⁷ cells/ml, and resuspended in Dulbecco’s modified Eagle medium with 10% fetal bovine serum and placed on ice until fixation. These cells were not cultured.

Animal Treatment (Monkey)

The treatment of male cynomolgus monkeys (Macaca fascicularis) used as the source of livers in this paper has been described elsewhere (Hoivik et al. 2004), and these same animals described in Hoivik et al. (2004) were used for the present study. Briefly, ciprofibrate was dosed by oral gavage at 0, 3, 30, 150, and 400 mg/kg/day for 15 consecutive days using 0.5% hydroxypropyl methylcellulose as vehicle. A section of liver from the right lateral lobe, measuring approximately 8 × 8 × 4 mm, was harvested from each animal at necropsy, flash frozen, and stored at –80°C. The frozen livers were stored for approximately 3 years prior to flow cytometric analysis.

Frozen Liver Preparation for FCM

Sections of frozen livers from the rat and monkey were thawed by immersion in ice-cold Dulbecco’s phosphate buffer (D-PBS) with 5% fetal bovine serum (FBS). The thawed tissue was cut into four to five pieces approximately 2.5 mm³ in size and placed into a Medicon (BD Biosciences, San Jose, CA) wetted with 1.0 ml of 5% FBS in D-PBS. The Medicon is a polystyrene chamber containing an impeller and an immobile stainless steel screen with approximately 100 hexagonal holes, each surrounded by six microblades. The Medicon was inserted into the Medimachine (BD Biosciences) and was operated at 100 rpm for 15 s. The cell suspension was filtered using a Filcon (BD Biosciences), a disposable filter device, and the resulting cell suspension was washed twice with 5 ml of 5% FBS in D-PBS. The cells were counted with a hemocytometer and adjusted to a concentration of approximately 1 × 10⁷ cells/ml.

Flow Cytometry

The cell suspension was fixed and permeabilized to allow antibody access to peroxisomes using the Fix and Perm kit from CALTAG Laboratories (Burlingame, CA) per the manufacturer’s protocol. Rabbit anti-PMP70 (Affinity Bioreagents, Golden, CO) was conjugated to allophycocyanin (APC) using a Zenon APC Rabbit IgG Labeling Kit (Molecular Probes, Eugene, OR) per the manufacturer’s product information sheets. The resulting antibody conjugate was immediately mixed at 1 μg/ml with 10 μl of D-PBS and with 5 μl of Zenon Component A. The mixture was incubated for 5 min at room temperature; 5 μl of Zenon Component B was added. The final mixture was incubated for another 5 min, and a 100-μl aliquot of cell suspension was added to the mixture and incubated for 30 min at room temperature. Subsequently, cells were washed twice with 0.05% NaN₃ + 0.5% bovine serum albumin (BSA) +0.1% Tween 20. After the final wash, cells were suspended in 0.5 ml of D-PBS +0.05% NaN₃+ 0.5% BSA for flow cytometric analysis. To control for nonspecific binding in each test, the Zenon-labeling system was used to prepare an APC-labeled negative control from a reagent grade IgG fraction of normal rabbit serum (Sigma, St. Louis, MO). For all staining procedures, a negative control was processed in parallel with each sample. Measurements of fluorescence intensity were performed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with an air-cooled argon-ion laser emitting at 488 nm and a red diode laser emitting at 633 nm. The fluorescence emitted by the APC–anti-PMP70 conjugate in peroxisomes was collected through a 661-nm band pass filter. A broad gate was established for data collection using a Cartesian plot of FSC (forward scatter) versus SSC (side scatter) to exclude cellular fragments and debris from the cell population. Fluorescence intensity distributions were recorded on a 4-decade logarithmic scale (Figure 1). The data from 10,000 events were acquired using CellQuest software (BD Biosciences). Before each data collection run, a quality control check was performed on the instrument using CaliBrite3 beads (BD Biosciences). List mode data analyses were accomplished using WinList version 3.0 software (Verity software, Topsham, ME).

In order to ensure that cells extracted from frozen liver remained intact, cells within corresponding data collection gates were sorted onto slides using a MoFlo cell sorting flow cytometer (Dako Cytomation, Fort Collins, CO) and examined using an Olympus LX81 inverted-fluorescent microscope (Opelco, Dulles, VA) fitted with 4′,6-diamidino-2-phenylindole (DAPI) and APC filter sets. After flow cytometric analysis, portions of remaining cell suspension were deposited on glass slides using a Cytospin cytocentrifuge (Shandon, Pittsburg, PA) run at 800 rpm for 8 min at room temperature. Slides were counterstained with Vectashield Mounting Medium containing DAPI (Vector Laboratories, Burlingame, CA). Visualization of all nuclei by DAPI staining provided a means of confirming the quality of the cells in the samples. The morphology and fluorescent staining was examined using an Olympus LX81 inverted-fluorescent microscope (Opelco) fitted with DAPI and APC filter sets.

Comparison of Mean Fluorescence Intensities

The population mean fluorescence intensity values from logarithmically amplified signals were used as data sources for normalization and comparison across samples. Mean fluorescence intensity values (linearized from WinList) of anti-PMP70-labeled cells were normalized by subtraction of mean fluorescence intensity values from corresponding cell samples treated with the negative (nonspecific antibody) control. This mean difference represents the magnitude of the difference between specific binding and nonspecific binding.

Electron Microscopy (EM) and Enzymatic Analysis for Monkey Liver

This paper compares results from FCM with those previously reported for EM and enzymatic analysis on liver of the same monkeys (Hoivik et al. 2004). Briefly, for EM, periportal thin section hepatocytes were stained with 5% methanolic uranyl acetate and Reynold’s lead citrate. Five random representative ultrastructural photomicrographs taken on the basis of a complete hepatocyte cross section were taken per monkey in each group. Peroxisomes were manually counted. The enzymatic analysis included carnitine acetyltransferase, catalase, enoyl–Coenzyme A (CoA) hydrate, and palmitoyl-CoA oxidase.

Immunoblot Analysis of PMP70

The immunoblot results from monkeys treated with ciprofibrate that are discussed in this paper were previously reported by Colton et al. (2004). The immunoblot methods used in this study for the rat samples were described in detail in Colton et al. (2004). Briefly, proteins from rat liver total homogenate were separated by 4% to 12% gradient sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane, and incubated with antibody against PMP70. The signal was detected with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Immunohistochemistry and Image Analysis (Quantum Dots)

This paper also compares the results from FCM to those previously reported for immunohistochemistry and image analysis from monkeys treated with ciprofibrate (Colton et al. 2004). Again, the primates used in Colton et al. (2004) were the same animals as in Hoivik et al. (2004). Briefly, an antibody to the 70-kDa peroxisomal membrane protein (PMP70) coupled with the fluorescent nanocrystal, Quantum Dots was labeled on 5-μm sections of formalin-fixed, paraffin-embedded primate livers. Image analysis was performed using Image-Pro Plus version 4.5 (Media Cybermetics, Silver Spring, MD, USA). Several animals for each dose group were analyzed and multiple images per slide were examined. A total of 50 images were quantitated.

Statistical Analysis

Data are presented as mean ± standard deviation of the mean. For comparison of mean fluorescence intensity between treatment groups and the vehicle control group, a one-way analysis of variance followed by the Dunnett’s multiple comparison test was performed. A probability level of p < 0.05 was considered as the criterion of significance. Statistical analysis was carried out using JMP software, version 5.1 (SAS, Cary, NC).

RESULTS

Fresh and Frozen Livers from Fenofibrate-Treated Rats

To quantitate peroxisome proliferation, we examined the expression of PMP70 using APC-conjugated anti-PMP70 using flow cytometry. PMP70 is one of the major components of the peroxisomal membrane (Ackers, Johnson, and Haasch 2000; Kamijo et al. 1990). As measured by flow cytometry, fenofibrate caused a 3.83-fold increase in the normalized (corrected for non-specific binding) mean fluorescence intensity from freshly isolated hepatocytes from rats treated with 200 mg/kg/day fenofibrate compared to the vehicle control, in good agreement in the frozen rat liver samples obtained from animals in the same treatment group, a 4.11-fold increase in the mean fluorescence intensity was observed. The variability around the fold-changes among animals ranged from 2.97 to 4.70 (two animals) and from 3.74 to 4.59 (four animals) from freshly isolated hepatocytes and frozen rat liver samples, respectively. As measured by immunoblot analysis, the PMP70 protein level in frozen liver was 5.6-fold higher in animals receiving fenofibrate compared to control (Figure 2). As measured by EM, a 4.07-fold increase in peroxisome numbers was observed in rats treated with fenofibrate for 10 days (unpublished data).

Frozen Livers from Ciprofibrate-Treated Monkeys

As measured by flow cytometry, treatment of monkeys with ciprofibrate resulted in 1.3-, 2.4-, 3.8-, and 3-fold increases in the mean fluorescence intensity of cell-associated PMP70 from animals treated with 3, 30, 150, and 400 mg/kg/day of ciprofibrate, respectively. A statistically significant increase when compared with concurrent control occurred at dose levels of 30, 150, and 400 mg/kg/day. The 150 mg/kg/day dose group had a greater-fold increase than the 400 mg/kg/day dose group (Figure 3).

In order to visually confirm that the fluorescent staining was consistent with peroxisome proliferation, slides were prepared from an aliquot of the cytometry sample material. The cells, based on morphologic appearance, consisted primarily of hepatocytes rather than nonparenchymal cells. PMP70 fluorescence demonstrated that the signal was localized to punctate regions inside the cells (Figure 4). This staining pattern is consistent with peroxisome localization.

DISCUSSION

Studies have been published demonstrating that frozen colorectal surgical specimens and tumor from patients with prostate or breast cancer disaggregated with the Medimachine provided a reliable and practical technique to produce single-cell suspensions for flow cytometric analysis (Brockhoff et al. 1999; Nap et al. 2001). Analysis of cells extracted from frozen tissues requires maintenance of intact cells in the samples. We investigated the preservation of whole cells isolated from frozen liver after mechanical sample preparation using the Medimachine (Figure 5). Microscopy of cells gated on the basis of the forward and side scatter light shows that the Medimachine produces relatively well-preserved cells (Figure 6). To our knowledge, this is the first report describing the use of the Medimachine for producing intact cell samples for FCM from frozen livers.

The difference in the staining pattern and relative quality of cells between Figures 4 and 6 (both were from cynomolgus monkey liver samples) is due to different methods of slide preparation. Photomicrographs from Figure 4 were taken from cells prepared by cytocentrifugation, a thin-layer preparation that retains better cytomorphologic details, whereas a picture from Figure 6 was obtained from cells sorted directly onto a microscopic slide using a MoFlow cell sorter. In regards to size differences of nuclei, pictures from Figure 4 were taken with high magnification to capture a close-up PMP70 signal, whereas picture from Figure 6 was taken with lower magnification for a wider field of view in order to demonstrate the overall population of cells.

In this study, we investigated the ability of FCM to quantitate peroxisomes in the liver of rats and primates treated with fibrates, which cause peroxisome proliferation in both species. We used an antibody to PMP70, which is a peroxisome membrane protein, for peroxisome quantitation. The FCM mean fluorescence intensity in rats treated with fenofibrate was determined using both freshly isolated hepatocytes and frozen livers. The fold-increase in peroxisomes was nearly identical for freshly isolated hepatocytes and frozen livers, indicating that the freezing process does not affect the peroxisome quantitation. In addition, quantitation of rat peroxisomes by electron microscopy and by Western blot analysis of PMP70, both showed a very similar fold-change increase compared to flow cytometry.

The primate liver samples used in this study were from the same animals that were used by Hoivik et al. (2004), and liver EM and enzymatic analysis are described in that manuscript. EM analysis from ciprofibrate-treated monkeys showed that peroxisome numbers were increased 1.6-, 2.5-, and 2.8-fold increase for doses of 30, 150, and 400 mg/kg/day, respectively.

Livers from the same primates from Hoivik et al. (2004) were also examined in Colton et al. (2004). From Colton et al. (2004), results obtained from immunohistochemistry and image analysis (Quantum Dots) demonstrated a statistically significant increase in PMP70 staining at 150 and 400 mg/kg/day. In addition, immunoblot analysis of PMP70, again from the same monkeys, showed a statistically significant increase in PMP70 protein content at 30, 150, and 400 mg/kg/day compared to controls.

Four independent methods of analysis of livers from the same primates dosed with ciprofibrate produced similar results in regard to quantitation of peroxisome proliferation. Flow cytometry, electron microscopy (Hoivik et al. 2004), immunohistochemistry with PMP70 (Colton et al. 2004), and immunoblot analysis of PMP70 (Colton et al. 2004) all showed an increase in peroxisome proliferation at dose levels of 30, 150, and 400 mg/kg/day, and statistically significant increases at dose levels of 150, and 400 mg/kg/day. A statistically significant increase in peroxisome proliferation at 30 mg/kg/day was observed only for the FCM and immunoblot analysis methods. This finding suggests that the FCM and immunoblot methods might be more sensitive than the EM and Quantum Dots methods for detecting peroxisome proliferation. Antibody-based methods for detection of PMP70, such as FCM, Western blots, and immunohistochemistry, can detect PMP70 protein before the protein is incorporated into functional peroxisomes. Detection of PMP70 in microperoxisomes, which are common in mammalian cells but not in functional peroxisomes, could cause different dose-response curves when comparing EM with antibody-based methods because only fully formed peroxisomes are counted in EM. It is possible that PMP70 antibody-based methods are more a reflection of β-oxidation potential of the peroxisome in the liver than the peroxisome number (Tanaka et al. 2002; Colton et al. 2004).

It is highly unlikely that different methods will agree precisely. However, comparisons of these methods suggest that the flow cytometry method is a viable alternative to EM.

In summary, our data demonstrate the feasibility of a novel flow cytometry method for quantitation of peroxisome proliferation from cells extracted from frozen livers in both rats and monkeys. FCM analysis of frozen livers has the advantages of easy preparation, objective analyses, quantitative reading and fast results. These features suggest that FCM may serve as an attractive alternative to EM for peroxisome quantitation analysis. Moreover, the ability to use frozen archival tissue is a significant advantage.