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2005 33: 86Toxicol PatholJames K. Loy and Roy L. Kerlin

Ingrid M. Pruimboom-Brees, Dominique J. J. E. Brees, Amy C. Shen, Mary Keener, Omar Francone, David E. Amacher, OxidationβUsing Laser Scanning Cytometry to Measure PPAR-Mediated Peroxisome Proliferation and

  

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Toxicologic Pathology, 33:86–91, 2005Copyright C© by the Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1080/01926230590881817

Using Laser Scanning Cytometry to Measure PPAR-MediatedPeroxisome Proliferation and β Oxidation

INGRID M. PRUIMBOOM-BREES, DOMINIQUE J. J. E. BREES, AMY C. SHEN, MARY KEENER, OMAR FRANCONE,DAVID E. AMACHER, JAMES K. LOY, AND ROY L. KERLIN

Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340-8014, USA

ABSTRACT

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry(IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissuelocalization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist werestained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes),Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal β oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) toevaluate peroxisomal β oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expressionin centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with thepalmitoyl CoA biochemical assay for peroxisomal β oxidation, and provided additional morphologic information about peroxisome proliferation andtissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity buthigher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologicobservations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

Keywords. Peroxisome; proliferation; β-oxidation; PTS1; Acyl CoA oxidase.

INTRODUCTION

The LSC is a microscope-based FC that can automati-cally measure fluorescence of cells on a microscopic slide(up to 5 different fluorochromes as well as light scatter)and may rapidly generate a huge database of biochemi-cal and morphologic information for thousands of cells ina typical sample (Kamentsky et al., 2001). Because themeasurements are microscope slide-based, the data gener-ated by laser scanning cytometry combines the advantagesof both FC and IHC by quantifying the total fluorescenceper cell, while maintaining the morphologic features of thetissue examined with respect to the fluorescent intensityand localization. The position of each cell on the slide isrecorded by the LSC so that any subpopulation of cellswith particular features can be selected (relocation step)and examined to confirm that data histograms are cor-rectly interpreted and allow correlation of cellular pheno-type (biochemical and/or morphological) with patterns oftissue injury. Furthermore, a feature referred to as maximumpixel intensity (MPI) analysis allows for the measurements ofpunctate, highly localized biochemical events in a subcellular

Address correspondence to: Dr. Ingrid M. Pruimboom-Brees, PfizerGlobal Research and Development, Groton Safety Sciences, Eastern PointRoad MS 8274–1219, Groton, Connecticut 06340–8014, USA; e-mail:ingrid m pruimboom-brees@groton.pfizer.com

Abbreviations: LSC: Laser Scanning Cytometry; FC: Flow cytometry;IHC: Immunohistochemistry; PPAR: Peroxisome Proliferator ActivatedReceptor; PTS-1: Peroxisome Targeting Signal-1; AOX: Acyl CoA Oxi-dase; MPI: Maximum Pixel Intensity; � Abs: Change in absorbance; KLH:keyhole limpet hemocyanine; FITC: Fluorescein Isothyocyanate; H&E:Hematoxylin and eosin; OWB: Optimax Wash Buffer; DAPI: 4′,6-diamino-2-phenylindole.

location that would be missed by flow cytometry integralanalysis.

We exploited these features of the LSC to develop tech-niques to sensitively and specifically evaluate peroxisomeproliferation and peroxisomal β oxidation in hepatocytesfrom rats exposed to a potent peroxisome proliferator (Moodyet al., 1992). Peroxisomes are cellular organelles contain-ing catalase (an inducible enzyme that ameliorates oxidativedamage by neutralizing H2O2) and flavin-containing oxidases(such as those that facilitate β oxidation of fatty acids). Tra-ditionally, IHC and immunoelectron microscopy (3′-methyl-2-methylamino-azobenzene reaction for catalase) have beenused to evaluate increases in peroxisomal number and vol-ume while biochemical methods (palmitoyl CoA and AOXmRNA assays) have been used to measure changes in per-oxisomal β oxidation (Lazarow et al., 1981; Amacher et al.,1997).

In this manuscript, we describe the evaluation of perox-isomal proliferation and peroxisomal enzyme induction byLSC, using 3 specific peroxisomal markers; Acyl coA oxi-dase, catalase, and peroxisome targeting signal-1. Catalaseis a well-established surrogate marker of peroxisomal β ox-idation and proliferation which in reality measures the de-gree of oxidative stress occasioned by production of per-oxide radicals during β oxidation (Nemali et al., 1988).Acyl CoA oxidase (AOX) is an inducible and rate-limitingenzyme of the β oxidation of medium, long, and verylong-chain fatty acids within peroxisomes and provides aspecific marker for peroxisomal enzyme induction (Osumiet al., 1980). Finally, all peroxisomal enzymes are directedto the peroxisomal matrix by virtue of a highly conservedCOOH-terminal tripeptide, serine-lysine-leucine, known asthe peroxisomal targeting signal type 1 (PTS-1). In contrast

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to proteins transported into the endoplasmic reticulum andmitochondria, posttranslational modification of peroxisomalproteins does not occur, allowing the use of probes such asPTS-1 to specifically target all peroxisome enzymes using im-munohistochemistry (Gould et al., 1990; Usuda et al., 1999;Otera et al., 2002).

MATERIAL AND METHODS

AnimalsSprague–Dawley rats (220–250 g) were exposed to a po-

tent PPAR agonist by oral gavage at doses of 1, 3, and10 mg/kg for 30 consecutive days. Rats were fasted overnightbefore necropsy. At necropsy, livers were weighed; sectionsof right and left liver lobes were collected, fixed in 10% neu-tral buffered formalin for 24 hours and embedded in paraffin.Fixed tissues were either processed for light microscopic ex-amination or for immunofluorescence and LSC. The remain-ing liver was snap frozen in liquid nitrogen for biochemicalanalysis. The treatment and handling of the study animalswas done in accordance with the Pfizer institutional animalcare and use guidelines.

Palmitoyl-CoA β-OxidationFrozen sections of liver were gradually thawed in Dul-

becco’s PBS. Following the addition of cold 0.25 M su-crose, samples were minced, homogenized, and centrifugedat 600g for 10 minutes in a refrigerated rotor. Supernatantswere frozen in an Ultralow freezer at –80◦C degrees centi-grade. On the day of assays, supernatants were thawed andassayed separately for protein content and palmitoyl CoA ac-tivity. Total protein was quantitated by the bicinchionic acidmethod. Following the addition of Triton X-100, each sam-ple was mixed and aliquots transferred to the Cobas samplecup with reaction mixture and 0.5 mM palmitoyl CoA sep-arately placed in the appropriate reagent cups. A 60-secondincubation at 37◦C was followed by 30 absorbance readingsat 340 nm taken every 10 seconds in order to determine thechange in absorbance (� Abs) per minute. For each sample,the resulting � Abs/min was multiplied by a calculation fac-tor F to yield µmol L−1 min−1. This value was then multipliedby the homogenate dilution factor and divided by 1,000 g/Lto yield the final sample palmitoyl CoA activity as µmolesNAD reduced/g liver x min.

AntibodiesAs previously described, PTS1 peptide NH2-

CRYHLKPLQSKL-COOH was synthesized both as afree peptide and conjugated to keyhole limpet hemocyanine(KLH) (Gould et al., 1990; Usuda et al., 1999). Rabbitpolyclonal antibodies were raised against the conjugatedPTS1 peptide and the serum with the highest titers wasused for all subsequent immunofluorescence experiments(antibody titers varied from 1:4,900 to 1:100,000). Thespecificity of PTS1 antibodies for the peroxisomal matrix hasbeen illustrated through immunoblot against peroxisomalproteins, immnunohistochemistry, and immunoelectronmicroscopy in multiple species and organs (Usuda et al.,1999). The antibody for AOX was purchased fromDr. Nobuteru Usuda and was prepared by immunizingrabbits with AOX purified from rat liver tissues. The

specificity of this antibody has been previously described(Osumi et al., 1980; Farioli-Vecchioli et al., 2001).

Other antibodies were purchased, including mouse anti-catalase (Sigma, St Louis, MO), goat anti-rabbit Alexa 488,and goat anti-mouse IgG fluorescein isothiocyanate (FITC)(Molecular probes Inc., Eugene, OR).

HISTOPATHOLOGY AND IMMUNOFLUORESCENCE

Serial 5-µm liver sections were cut from the paraffinblocks, deparaffinized, and rehydrated. For light microscopicexamination, sections were stained with hematoxylin andeosin (H&E). For AOX and PTS1 immunofluorescence only,proteolytic digestion was first performed by incubating theslides for 12 minutes at 37◦C in Pepsin (Dako Corporation,Carpeinteria, CA). For all staining, slides were then washedin OptiMax wash buffer (OWB)(Biogenex, San Ramon, CA)3 times for 5 minutes. Nonspecific binding sites were blockedby the addition of 5% normal donkey serum (AOX andPTS1) (Jackson Immuno Research Laboratories Inc., WestGrove, PA) or 5% normal goat serum (catalase) (VectorLaboratories Inc., Burlingame, CA) in OWB for 30 min-utes at room temperature. Sections were then incubatedat room temperature with either of the following antibod-ies, dilutions, and times: rabbit anti-AOX (1:3,000, 1 hour),rabbit anti-PTS1 (1:500, 2 hours), and mouse anti-catalase(1:250, 3 hours). Sections were washed in OWB 3 timesfor 5 minutes at room temperature. Secondary goat anti-rabbit Alexa 488 IgG (AOX and PTS1, dilution 1:200) orgoat anti-mouse IgG FITC (catalase, dilution 1:200) con-jugated antibodies were added and slides were placed for30 minutes at 37◦ (AOX and PTS1) or at room temper-ature (catalase). Slides were washed 3 times for 5 min-utes in OWB, rinsed in distilled water, and coverslippedin Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector laboratories, Burlingame, CA).Negative control slides were obtained by omitting the incu-bation with the primary antibody.

Analysis of Cell Fluorescence by LSCSlides were inspected under an epifluorescence micro-

scope (Olympus BX50, Olympus America Inc., Melville,NY) using a mercury lamp illumination. Fluorescence of aminimum of 20,000 hepatocytes (in previous unpublishedstudies, we have determined this sample size to be the min-imum to produce statistically significant and reproducibleresults) per section of liver was measured using the LSC(CompuCyte Corp., Cambridge, MA) at 20X magnification.Briefly, FITC and DNA-associated DAPI fluorescence wereexcited with an argon ion laser (488 nm) and a violet diodelaser (400 nm), and emission detection measured using thestandard band pass 515–545 nm and long pass 460–485 nmfilters, respectively. Individual hepatocytes were identifiedwith nuclear DAPI fluorescence, and hepatocellular perime-ters were selected at 10 pixels outside the nuclear contouron the basis of the FITC fluorescence (contour analysis).For each hepatocytes, the values of green and blue fluores-cence intensities were measured and automatically processedto generate integral analysis (sum of all the fluorescent dotswithin each cell), MPI (maximal fluorescence intensity or“brightness” for every one of the fluorescent dots in the

88 PRUIMBOOM-BREES ET AL. TOXICOLOGIC PATHOLOGY

data set), XY coordinates of individual hepatocytes. Negativecontrol slides were used to determine the level of backgroundfluorescence and define gating prior to analysis. FITC-labeledhepatocytes were mapped to visualize tissue architecture (X-Y plot) and examined visually within the section (relocated)to confirm that data scattergrams were correctly interpreted.Fluorescein isothiocyanate or Alexa 488 MPI versus FITC-integral scattergram was displayed for each scan.

RESULTS

Animals and HistopathologyAt necropsy, group mean absolute and relative (to body

weight) liver weights were increased at ≥1 mg/kg PPARagonist (1.93–2.51x control mean) in both sexes and weregenerally similar across dose groups. Microscopically, theincreases in liver weight correlated with mild-to-moderatehypertrophy of centrilobular hepatocytes.

Palmitoyl-CoA β-OxidationAs shown in Table 1, exposure of both males and females

to ≥1 mg/kg PPAR agonist for 30 days resulted in significantelevation of palmitoyl-CoA β oxidation without change inliver protein content compared to control rats. Values weregenerally higher in males than in females. Results were simi-lar or greater in magnitude to values obtained for concurrentlyprocessed samples from rats dosed with 300 mg/kg/day clofi-brate in a previous, unrelated study.

ImmunofluorescenceIn control rats, occasional centrilobular or midzonal hep-

atocytes of normal size showed intense AOX, catalase, andPTS1 expression that was characterized by a cytoplasmic,0.5–1 µm in diameter, granular fluorescence consistent withperoxisomes (Figure 1a). In PPAR-exposed rats, there was a

FIGURE 1.—Immunohistochemistry for catalase using Alexa 488 FITC. (A) Control rat. Within occasional centrilobular or midzonal hepatocytes, peroxisomes(arrow) were stained with catalase. Bar = 20 µm. (B) PPAR-treated rat. Centrilobular to midzonal hepatocytes are enlarged due to an increased number of catalase-positive peroxisomes. Bar = 40 µm.

TABLE 1.—Palmitoyl CoA β oxidation and protein content in liver ho-mogenates prepared from rats dosed with PPAR agonist.

µmoles NAD reduced Protein contentper gram per minute (mg/mL)

Treatmentgroup Males Females Males Females

ControlMean 0.24 0.14 17.9 12.997SD 0.8 0.05 4.43 0.78

1 mg/kgMean 6.813∗ 4.486∗ 18.53 15.407SD 0.72 0.48 2.88 0.24

3 mg/kgMean 5.834∗ 4.186∗ 17.59 13.147SD 1.13 0.4 3.97 0.29

10 mg/kgMean 8.417∗ 4.517∗ 14.89 13.603SD 1.31 0.34 2.29 1.42

∗Significant elevations are indicated with an asterisk. p < 0.05.

significant increase in immunofluorescence in centrilobularhepatocytes that sometimes extended out to the periportal ar-eas in high-dose animals (Figure 1b). The hepatocytes wereenlarged due to increased number of prominent fluorescentperoxisomes, and this observation correlated with light mi-croscopic observation of centrilobular hypertrophy. Negativecontrol sections incubated with only the secondary antibodywere devoid of staining, indicating that the reaction was spe-cific for the primary antibody.

Analysis of Cell Fluorescence by LSCThe FITC or Alexa 488-MPI data versus FITC or Alexa

488 integral scattergrams indicated that there was a signifi-cant dose-related increase in the percentage of AOX, cata-lase, and PTS-1 positive hepatocytes in all PPAR treatedrats compared to control rats (Table 2). In control rats,the mapping (X-Y plot), scattergrams and relocation gal-leries (Figures 2 and 3) indicated that the rare hepatocytes

Vol. 33, No. 1, 2005 LASER SCANNING CYTOMETRY AND PEROXISOMES 89

TABLE 2.—Summary data comparing the results from the biochemical assay and laser scanning cytometry.

µmoles NAD PTS1 percentage AOX percentage Catalase percentagereduced/g/min “positive” hepatocytes “positive” hepatocytes “positive hepatocytes”

Treatmentgroup Males Females Males Females Males Females Males Females

ControlMean 0.24 0.14 0.86 0.88 0.02 0.24 0.7 0.48

1 mg/kgMean 6.813∗ 4.486∗ 4.02∗ 5.38∗ 21.34∗ 16.18∗ 44.52∗ 5.66∗

3 mg/kgMean 5.834∗ 4.186∗ 3.42∗ 5.08∗ 30.56∗ 27.7∗ 61.5∗ 14.34∗

10 mg/kgMean 8.417∗ 4.517∗ 16.96∗ 9.12∗ 59.86∗ 30.26∗ 59.125∗ 16.88∗

∗Significant elevations are indicated with an asterisk. p < 0.05.

with very high MPI and integral values were randomly lo-cated in the hepatic lobule or adjacent to centrilobular veinsand contained a low number of fluorescent peroxisomes.In PPAR-treated rats, the large numbers of hepatocyteswith very high MPI and integral values were centrilob-ular to midzonal. These hepatocytes were also enlargedby numerous and prominent fluorescent peroxisomes. Incontrol rats, the LSC data are consistent with occasionalindividual hepatocytes presenting high levels of peroxi-somal enzymes activity but no peroxisomal proliferation,whereas in PPAR-treated rats, the LSC data are consis-tent with PPAR-mediated induction of peroxisomal enzymes(β oxidation and antioxidant pathways) and peroxisomeproliferation.

The results from LSC analysis were also comparable tothe palmitoyl CoA assay. For example, there was almostno increase in palmitoyl CoA activity (µmoles NAD re-

FIGURE 2.—Laser scanning cytometry analysis (performed using the 20X objective) of AOX immunofluorescence. Mapping (X-Y plots) and scattergrams (AlexaFluor 488-MPI versus FITC integral) are represented for a control (a), low-dose (b), mid-dose (c), and high-dose rat (d). In the low-dose animal, 7.3% (5.8% + 1.5%)of hepatocytes have high integral and/or MPI values associated with AOX immunofluorescence and these hepatocytes surround centrilobular veins. In the high-dosegroup, 68.8% (53.1% + 15.7%) of hepatocytes have high MPI and integral values and the AOX immunofluorescence extend further into the midzonal areas.

duced/g/min) or in the percentage of PTS1 positive hepa-tocytes in rats at 1 and 3 mg/kg. Also, females were approxi-mately half as responsive to PPAR agonists as males for boththe biochemical assay and the LSC analysis. PTS1 analysis,compared to catalase and AOX, provided the best correlationwith the biochemical assay. This result was expected dueto the fact that PTS1 antibodies bind to all peroxisomal pro-teins instead of only one enzyme (such as AOX and catalase),thereby generating a stronger fluorescent signal.

DISCUSSION

In this manuscript, we described the validation of the LSCfor the measurement of peroxisomal enzyme induction andperoxisome proliferation in rats. The LSC provided quali-tative and quantitative information on the expression of 3critical peroxisomal markers of the β oxidation and antioxi-dant pathways, AOX, catalase, and PTS1. Acyl CoA oxidase

90 PRUIMBOOM-BREES ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 3.—Laser scanning cytometry analysis (performed with the ×20 objective) of PTS-1 immunofluorescence (Alexa 488 FITC): relocation gallery. (A) Fromthe scattergram (FITC-MPI versus FITC integral), 12 hepatocytes with high MPI and integral values were selected. (B) Because each hepatocyte is given an Xand Y spatial coordinate, the computer relocates to each of the 12 selected hepatocytes and provides a standardized photograph of the region where each of thesehepatocytes reside within the liver section.

expression, compared to catalase (oxidative stress enzyme),had the advantage of being more specific for peroxisomalfatty acid β oxidation. We also have shown in a recent tox-icology study with a known peroxisome inducer that AOXexpression also occurred at earlier time points (unpublisheddata). In this study, AOX and PTS1 were already increased 24hours after treatment, whereas catalase was only increased at96 hours, likely in order to remove the toxic species gen-erated by the perosisomal oxidases. The LSC analysis ofPTS1 expression seems to be more sensitive than catalaseand AOX, probably because PTS1 antibodies bind to all per-oxisomal proteins instead of only one enzyme. This sensi-tivity is especially advantageous for the detection of perox-isomal induction in non-rodent species, in control animals,and in organs other than the liver where basal expressionof peroxisomal enzymes can be so low that it would notbe detected by the biochemical assay and light microscopicevaluation would be unlikely to be successful in detectingan effect (data unpublished). Because it is based on im-munostaining, the LSC analysis is also more sensitive thanbiochemical assays requiring whole-tissue measurements inwhich many hepatocytes would not exhibit induction. To-gether with the published information on immunoelectronmicroscopy, the hepatocellular cytoplasmic granular stain-ing associated with the three antibodies and the predilectionof the staining for centrilobular areas supported the speci-ficity of these antibodies for the peroxisomal matrix sincelittle or no leakage of the antigen (visualized as a diffuse cyto-plasmic staining) occurred during the immunohistochemistryprocedure.

Quantification of peroxisomal β oxidation by LSC directlycorrelated with the palmitoyl-CoA biochemical assay. How-ever, when compared to the biochemical assay, the LSC anal-ysis provided additional morphologic information on perox-isome proliferation and tissue pattern of activation. In toxi-cologic studies, the biochemical assay requires the sampling,

during necropsy, of frozen tissue sections followed by severaldays of organelle isolation and protein measurements. On theother hand, measuring peroxisomal induction/proliferationby LSC can be completed within 48 hours (1 day of im-munohistochemistry, 1 day of LSC analysis) and does notrequire special processing during necropsy other than theroutine formalin fixed, paraffin embedded sections. Perox-isome analysis can therefore be performed retrospectivelyfrom archival formalin-fixed tissues or prospectively whenthe liver is a known toxicologic target. Also, when the expres-sion of a marker is very limited to occasional cells, LSC beingimmunohistochemistry-based, becomes a very sensitive toolcompared to protein or gene expression profiling because thelater measures whole-tissue levels in which many cells do notexhibit induction.

In conclusion, quantification of peroxisomal β oxidationby LSC was sensitive, specific and relatively high through-put compared to the palmitoyl CoA biochemical assay. Inaddition, the LSC analysis provided further information onperoxisome proliferation, tissue patterns of expression, cor-relation with hepatocellular hypertrophy observed using lightmicroscopy, and can be performed on archival material froma variety of organs and species.

ACKNOWLEDGMENTS

We gratefully thank C. Aldinger, M. Haghpassand (De-partment of Cardiovascular and Metabolic Disease, Pfizer),S. Boldt (Molecular and Investigative Toxicology, Safety Sci-ences Groton, Pfizer) as well as J. Wolfgang, A. Jackowski,and A. Opsahl (Department of Pathology, Safety SciencesGroton, Pfizer) for their support during the validation studies.

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