* Biomedical Research Laboratories, Discovery Research Center, and
Development Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Osaka 532-8686, Japan
Received August 2, 2004; accepted August 8, 2004
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ABSTRACT |
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Key Words: drug-induced phospholipidosis; DNA microarray; real-time PCR; HepG2 cells; toxicogenomics; in vitro screening test.
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INTRODUCTION |
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DNA microarray technology enables investigators to monitor and quantify the expression of thousands of genes simultaneously. Use of this technology in combination with conventional tools is rapidly contributing to our understanding of the mechanisms underlying cellular toxicity, and has emerged as the field of "toxicogenomics" (Aardema and MacGregor, 2002). DNA microarray technology has the potential to more comprehensively contribute to our understanding of toxicity than any available traditional approach, since toxic changes in cells generally result from alterations not just in a single or few molecules, but in many molecular cascades. It may also help to identify early, sensitive biomarkers of toxicity, since alterations in cellular molecules are thought to precede the toxic outcome. These markers could then be used to develop screening tests to predict the toxicity of particular compounds. In pharmaceutical research, such tests are an invaluable tool for cost-effectively selecting candidate drugs, prioritization and compound modification, especially in the early stages of their development.
The present study was undertaken to examine the molecular mechanisms that contribute to the development of phospholipidosis and to identify specific markers that might form the basis of an in vitro screening test. Specifically, we performed a large-scale gene expression analysis using DNA microarrays on human hepatoma HepG2 cells after they were treated with one of the following 12 compounds known to induce phospholipidosis: amiodarone (Lewis et al., 1990; Martin and Standing, 1988
; Reasor, 1989
), amitriptyline (Drenckhahn et al., 1976
), AY-9944 (Sakuragawa et al., 1977
), chlorcyclizine (Lullmann-Rauch and Stoermer, 1982
), chlorpromazine (Drenckhahn et al., 1976
; Lullmann-Rauch, 1974
), clomipramine (Lullmann-Rauch, 1974
; Lullmann-Rauch and Scheid, 1975
; Xia et al., 2000
), fluoxetine (Gonzalez-Rothi et al., 1995
), imipramine (Lullmann-Rauch, 1974
; Lullmann-Rauch and Scheid, 1975
), perhexiline (Hauw et al., 1980
; Pessayre et al., 1979
), tamoxifen (Drenckhahn et al., 1983
; Lullmann and Lullmann-Rauch, 1981
), thioridazine (Lullmann-Rauch, 1974
), and zimelidine (Bockhardt and Lullmann-Rauch, 1980
). HepG2 cells were used because they are widely accepted as a good model for in vitro toxicology studies. In vitro phospholipidosis was confirmed by electron microscopic observation of lamellar myelin-like bodies in the lysosomes of these cells.
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MATERIALS AND METHODS |
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Cell culture and drug treatment. The human hepatocellular carcinoma cell line (HepG2) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells in their log growth-phase were seeded (2 x 105 cells) in 24-well plates and incubated with 8.3 or 25 µmol/l of a particular compound in 0.25% dimethylsulfoxide (vehicle) in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 5% heat-inactivated fetal calf serum (FCS; Bio Whittaker, Walkersville, MD), 100 U/ml penicillin, and 100 g/ml streptomycin (Gibco BRL). Cells were incubated in a 37°C incubator in an atmosphere of 5% CO2 and 95% air, and were cultured for 72 h, 6 and 24 h, or 24 h, depending on whether they were being used for electron microscopy, DNA microarray analysis, or real-time PCR, respectively.
Transmission electron microscopy. Following incubation, cells were fixed in 1% glutaraldehyde for 2 h after which they were washed with sodium phosphate buffer and post-fixed with 2% osmium tetroxide for 2 h. They were then dehydrated in increasing concentrations of ethanol and embedded in epoxy resin (Quetol 812). Ultrathin sections (80 nm) were cut using an ultramicrotome (LKB-8800 Ultrotome), double stained with uranyl acetate and lead acetate, and observed in an electron microscope (H-300; Hitachi, Tokyo, Japan). The pathological changes indicative of phospholipidosis (formation of lamellar myelin-like bodies in lysosomes) were scored on a scale of 0 to 3 ( = none, + = slight, ++ = moderate, and +++ = severe) in a blinded fashion.
RNA preparation. Following incubation with a test compound, cells were harvested and stored at 80°C until their RNA was extracted using an RNeasy Mini Kit (QIAGEN, Hilden, Germany). For the microarray analysis, RNA was extracted from similarly treated cells that were harvested from six wells and pooled. The concentration and purity of their total RNA were determined by measuring absorbance at 260 and 280 nm with an Ultrospec 2000 spectrophotometer (Amersham Biosciences, Piscataway, NJ). The integrity of the purified total RNA was confirmed using an RNA 6000 Nano Assay kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Berlin, Germany). RNA samples were stored at 80°C until assayed.
Microarray experiments. Microarray analysis was carried out using the GeneChip System (Affymetrix, Santa Clara, CA). The targets of GeneChip analysis were prepared as described in the Expression Analysis Technical Manual. Briefly, total RNA (5 µg) was converted into double-stranded cDNA using a Super Script Choice cDNA synthesis kit (Invitrogen, Carlsbad, CA) with an oligo(dT)24 primer containing a T7 polymerase promoter site at its 3' end (Amersham Biosciences). Biotin-labeled cRNA was generated from double-stranded cDNA using a BioArray HighYield RNA transcript labeling kit (Enzo Biochem, Farmingdale, NY) and was purified using a GeneChip Sample Cleanup Module (QIAGEN). Each cRNA sample (20 µg) was fragmented and hybridized with the Human Genome Array containing 22283 human specific probe sets (HG-U133A; Affymetrix, Santa Clara, CA) for 16 h at 45°C with rotation at 60 rpm. Each array was then washed and detected by consecutive exposure to phycoerythrin-streptavidin (Molecular Probes, Eugene, OR), biotinylated antibodies to streptavidin (Vector Laboratories, Burlingame, CA), and phycoerythrin-streptavidin, after which each array was washed again with a nonstringent wash buffer. All washing and staining procedures were performed with a Fluidics Station 400 (Affymetrix). The array was scanned using a confocal microscope scanner (Hewlett-Packard). To achieve wider signal dynamic range, each chip was scanned three times: before, after the first, and after the second signal amplification using an anti-streptavidin antibody.
The output fluorescence was captured using Affymetrix Microarray Analysis Suite 5.0 software (Affymetrix). This software qualitatively rates the abundance of mRNA of genes (detection call) as present, marginal, or absent, and can also perform comparative analyses between vehicle and drug treated samples at each time point to provide a change call (increased, decreased, marginally increased, marginally decreased, no change) and to generate a signal log ratio (SLR), i.e., the change in expression level for a transcript. This SLR was expressed as the log 2 ratio. A SLR of 1 is the same as a fold change of 2.
Probe sets that satisfied all of the following three conditions in two or three of the three scan data were extracted as up-regulated probe sets: (1) more than 0.6 in their SLR; (2) assigned as "increased"; and (3) determined as "present in the drug-treated data." Similarly, probe sets that satisfied the following three conditions in two or three of the three scan data were extracted as down-regulated genes: (1) less than 0.6 in their SLR; (2) assigned as "decreased"; and (3) determined as "present in vehicle-treated data."
All probe sets representing genes of interest for this study were functionally annotated by the NetAffx database (Affymetrix) and HumanPSD (Incyte, Beverly, MA).
Reverse transcription and real-time PCR analysis. Reverse transcription (RT) was performed using total RNA (1 µg) and oligo-dT oligonucleotide primer in 100 µl volumes using standard methods with MultiScribe Transcriptase in order to synthesize cDNA (TaqMan Reverse Transcription Reagent; Applied Biosystems).
Primers and TaqMan probes (SigmaGenosys, Hokkaido, Japan) used in this study were designed with Primer Express version 1.5 software (PE Applied Biosystems, Foster City, CA). The mRNA sequences used to design the primer and probe sets were obtained from the Affymetrix database (NetAffx). The TaqMan probes had 6-carboxyfluorescein (6-FAM) as the reporter dye and 6-carboxytetramethylrhodamine (6-TAMRA) as the quencher dye at their 5' and 3' ends, respectively. The primer and TaqMan probe sequences used in this assay are listed in Table 1.
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RESULTS |
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Real-Time PCR Analysis of Candidate Markers for Phospholipidosis and Selection of Phospholipidosis Markers
The relative expression levels of the candidate markers for phospholipidosis are shown in Table 6. The expression of five genes (PHYH; phytanoyl-CoA hydroxylase [Refsum disease], INHBE; activin beta E, P8; p8 protein [candidate of metastasis 1], ASNS; asparagine synthetase, AP1S1; adaptor-related protein complex 1, sigma 1 subunit) did not correspond well to the pathology score of the HepG2 cells. On the other hand, the following 12 genes were selected as phospholipidosis markers: ASAH1N-acylsphingosine amidohydrolase (acid ceramidase) 1; MGC4171hypothetical protein MGC4171; LSSlanosterol synthase (2,3-oxidosqualene-lanosterol cyclase); NR0B2nuclear receptor subfamily 0, group B, member 2; FABP1fatty acid binding protein 1, liver; HPNhepsin (transmembrane protease, serine 1); SERPINA3serine (or cysteine) proteinase inhibitor; clade A (alpha-1 antiproteinase, antitrypsin), member 3C10orf10chromosome 10 open reading frame 10; FLJ10055mdash;hypothetical protein FLJ10055 FRCP1likely ortholog of mouse fibronectin type III repeat containing protein 1; SLC2A3solute carrier family 2 (facilitated glucose transporter) member 3; and TAGLNtransgelin.
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DISCUSSION |
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In this study, we examined the molecular mechanisms that contribute to the development of phospholipidosis and identified specific markers that might form the basis of an in vitro screening test. Specifically, we performed a large-scale gene expression analysis using DNA microarrays on human hepatoma HepG2 cells after they were treated with each of 12 compounds known to induce phospholipidosis. Probe sets that were up- or down-regulated by at least six compounds were identified as phospholipidosis-related genes, a large number of which were genes that played a role in lipid metabolism (Tables 4 and 5). Functional annotation and categorization of these genes suggested that the following four processes were involved in the induction of phospholipidosis (Fig. 4): (1) Inhibition of lysosomal phospholipase activitythis is generally regarded as the primary mechanism of induction, as confirmed by the up-regulation of phospholipid degradation-related genes such as N-acylsphingosine amidohydrolase 1 (ASAH1), sphingomyelin phosphodiesterase (SMPDL3A), and hypothetical protein MGC4171 (MGC4171). (2) Inhibition of lysosomal enzyme transport, as demonstrated by the down-regulation of genes involved in lysosomal enzyme transport such as adaptor-related protein complex 1 sigma 1 subunit (AP1S1). AP1S1 is responsible for the transport of newly synthesized lysosomal enzymes between the trans-golgi network and lysosomes (Zhu et al., 1999). (3) Enhanced phospholipid biosynthesis, which is supported by the up-regulation of fatty acid biosynthesis-related genes such as ELOVL family member 6 (ELOVL6) and stearoyl-CoA desaturase (SCD). (4) Enhanced cholesterol biosynthesis, as shown by the up-regulation of cholesterol biosynthesis-related genes such as 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), squalene epoxidase (SQLE), lanosterol synthase (LSS), and 7-dehydrocholesterol reductase (DHCR7).
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An important, practical goal of our study was to identify phospholipidosis specific gene markers that could be used for the establishment of an in vitro screening test. Using DNA microarray and real-time PCR analyses, we identified 12 phospholipidosis marker genes. The average fold change values of these gene markers were calculated and termed their phospholipidosis mRNA scores, the latter of which correlated well with the cells' pathological scores. Since these phospholipidosis markers included genes whose functions included phospholipid degradation, cholesterol biosynthesis, fatty acid transport, proteolysis and peptidolysis, and endopeptidase inhibition, among others, by monitoring these 12 marker genes, we were able to also monitor multiple intracellular events that were involved in the induction of phospholipidosis. Our results showed that the mRNA score was a good index of phospholipidosis induction potential for a given compound and as such, we went ahead to establish a novel in vitro real-time PCR screening test.
The in vitro screening test required a far lower amount of the test compound and shorter periods than did conventional in vivo toxicity studies, and was readily able to detect the phospholipidosis induction potential of multiple compounds. The assay also provided a more detailed ranking score than electron microscopy that can be useful in structure-activity relationship studies by sorting compounds in the order of their phospholipidosis induction potential. This rapid and sensitive system should facilitate the efficient screening of new compounds for their potential for inducing phospholipidosis at an early developmental stage. Finally, transferring our PCR-based screening system into a 96- or 384-well microplate-based mRNA measuring format such as Array Plate (High Throughput Genomics, Tucson, AZ) and QuantiGene branched DNA (Bayer Diagnostics, Tarrytown, NY), should improve its throughput and cost-efficiency.
The toxicogenomics approach used in this study should be helpful in the examination of the mode of action and identification of gene markers for other toxic conditions as well. The hope is that in the near future new toxic gene markers will be identified that will allow for the testing of multiple drug toxicities simply by measuring gene expression.
In conclusion, microarray analysis revealed that factors such as alterations in lysosomal function and cholesterol metabolism were involved in the induction of phospholipidosis. Furthermore, comprehensive gene expression analysis enabled us to identify biomarkers of this condition that we then used to develop a rapid and sensitive in vitro screening test for drug-induced phospholipidosis.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Biomedical Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-17-85 Juso-Honmachi Yodogawa-ku, Osaka 532-8686, Japan. Fax: +81-6-6300-6306. E-mail: Sawada_Hiroshi{at}takeda.co.jp
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