Effects of Phalloidin on Hepatic Gene Expression in Mice

Abstract

An attempt has been made to identify molecular markers of intrahepatic cholestasis in mice employing phalloidin as a cholestatic agent. Phalloidin was administered to BALB/c mice at three predetermined dose: 250 μg/kg, 500 μg/kg, and 1 mg/kg for 1, 3, and 7 days. Liver function was estimated to confirm cholestasis. Histopathological observations on liver were also made to confirm liver injury. Phalloidin at 1 mg/kg for 7 days was found to induce cholestasis. Therefore gene expression studies were confined to this group only. A total of 88 genes were found to be affected by phalloidin. These were the genes associated with cytoskeleton regulation as well as tight junction, focal adhesion, and ATP-binding cassette transporters. Such proteins obstruct the removal of bile components from hepatocytes to the bile canaliculus or blood. Phalloidin treatment did not affect the proteins responsible for cell maintenance or death. The authors show that phalloidin-induced intrahepatic cholestasis is manifested by disturbing the cytoskeleton. The set of genes up-regulated by phalloidin can be considered as molecular markers of intrahepatic cholestasis. The observations are further expected to be helpful in the management of cholestatic pharmaceuticals and associated problems of liver diseases in humans.

Phalloidin, a cyclic peptide found in the mushroom Amanita phalloides, causes intrahepatic cholestasis in experimental animals. Phalloidin targets liver as a consequence of extensive uptake into hepatocytes through sinusoidal transporters (Frimmer 1992). Bile formation is affected due to its influence on the functional integrity of sinusoidal transporters, canalicular exporters, cytoskeleton-dependent processes for transcytosis, and the contractile closure of the canalicular lumen.

This cholestatic agent is known to augment the synthesis of microfilaments (Elias et al. 1980; Vonk et al. 1982; Watanabe et al. 1983; Dubin et al. 1980; Dancker et al. 1975). It is also known to accelerate the polymerization of actin filaments in the cytoplasm close to bile canaliculi (Dancker et al. 1975; French and Davis 1975; Elias et al. 1980; Vonk et al. 1982; Collucio and Tilney 1984). Hepatocellular microfilaments are largely comprised of actin. They play an important role in maintaining cellular morphology and membrane elasticity so that bile components are discharged in accordance with repeated muscular contraction and slackness of bile canaliculi (Watanabe et al. 1991; Thibault, Claude, and Ballet 1992; Yamamoto et al. 1988).

Intrahepatic cholestasis, caused by an obstruction within the liver, is diagnosed at present using toxicological tests, i.e., bromosulphalein clearance or serum bilirubin levels. Recent developments in toxicogenomics have helped to develop molecular markers for different drugs and chemicals. However, no such attempt has been made to develop a molecular marker for cholestasis. Because phalloidin manifests its toxicity through intrahepatic cholestasis (Ishizaki et al. 1997, 2001), it was thought worthy to use this hepatotoxin for gene expression study. Microarray-based toxicological profile was developed in support with histopathological observations in liver and blood biochemistry. The overall objective of the study is to identify the molecular markers of cholestasis.

MATERIALS AND METHODS

Chemicals

Phalloidin and DMSO were purchased from Sigma-Aldrich, St. Louis, Missouri (USA). Phalloidin was dissolved in a 2% DMSO-saline solution. Trizol was purchased from Invitrogen Seoul (Korea).

Animals

Approximately 9-week-old BALB/c male mice (SLC, Japan) were kept in a 12-h light/dark cycle, under controlled temperature and humidity for 2 weeks prior to experiment in the animal room. The mice were fed standard food pellets. Their body weight ranged from 24 to 27 g (mean ± SD, 25.75 ± 0.97). Phalloidin dissolved in DMSO was intraperitoneally injected to five mice at three predetermined sublethal dose levels: 0.25, 0.5, and 1 mg/kg body weight. The control mice were administered with equal amount of saline. The mice were sacrificed after 24 h, 3 days, and 7 days of treatment by ether anesthesia. The initial and terminal body weights of each mouse were recorded. The mice were carefully operated to collect the blood and liver samples. Liver/body weight ratio was also calculated.

Biochemical Analyses

Blood was collected from the inferior vena cava. Serum was separated by centrifugation. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities, and direct bilirubin (DBIL) were determined using an automated clinical chemistry analyzer (Fuji Dri-Chem 3500s; Fujifilm, Japan), at each time point. Data are expressed as the mean ± SD of five samples. The statistical significance between various experimental groups was tested using one-way analysis of variance (ANOVA) followed by the Dunnett test. p values less than .05 were considered to be significant.

Histopathology

Liver samples collected from treated and control mice were fixed in 10% neutral-buffered formalin, and embedded in paraffin. The sections (4 μm thick) were cut using RM2165 microtome (Leica, Germany), stained with hematoxylin and eosin (H&E), and examined under a light microscope (Nikon E400, Japan).

Isolation of RNA

The left lateral lobe of the liver was removed and processed for RNA extraction. For the cDNA microarray analysis, total RNA was extracted using the Trizol reagent (Invitrogen, USA) according to the instructions of the manufacturer and purified using RNeasy total RNA isolation kit (Qiagen, Germany). Total RNA was quantified by NanoDrop ND-1000 (NanoDrop, USA) and its integrity was assessed by 2100 Bioanalyzer (Agilent, Germany).

cDNA Microarray

For microarray analysis, fluorescent-labeled cDNA was prepared by reverse-transcription of total RNA in the presence of the coupled Cy3-dUTP or Cy5-dUTP (NEN) using Superscript II (Invitrogen, USA). Single-stranded cDNA probes were purified using a polymerase chain reaction (PCR) purification kit (Qiagen, Germany). Probes were resuspended in hybridization solution (50% formamide, 5× saline-sodium citrate buffer (SSC), 0.1% sodium dodecyl sulfate [SDS]). The Mouse oligo chip 10K (GenomicTree, Korea) was hybridized with the fluorescent-labeled cDNAs at 42°C in a humid chamber for 16 h.

Analysis of Fluorescence Spots

After proper washing, the slides were scanned using Axon GenePix 4000B (Axon Instrument, USA). Scanned images were calculated with GenePix 3.0 software (Axon Instrument) and analyzed with GeneSpring Software version 7.0 (Silicon Genetics, USA). The log gene expression ratios were normalized by LOWESS regression (Yang et al. 2001). Only genes that were expressed above twofold induction or below twofold repression after phalloidin treatment as compared with controls were considered for statistical analysis. Significant differences between control and treatment groups were determined using Student’s t test. p values <.05 were considered statistically significant.

Cluster Analysis and Data Annotation

Significant and differentially regulated genes as the result of phalloidin treatment were further analyzed using the Gene-Spring software. Hierarchical clustering was applied to identify identical groups and to identify phalloidin-related effects. The hierarchical clustering was created on the gene expression patterns. Information was also collected from different databases (Unigene from the National Center for Biotechnology Information [NCBI] or NetAffx Analysis Center).

RESULTS

Biological Observations

There was no significant difference in the weight of liver between phalloidin-treated (250, 500 μg/kg/day) and control groups. After 1 mg/kg/day phalloidin treatment for 7 days, the mice had a significantly higher liver weight than the time-matched vehicle-treated control mice. Apparently, this was not due to weight loss, but rather an increase in weight of the liver relative to the control animals. The percentage liver weight for the phalloidin-treated mice was 6.59% ± 0.28% versus 5.44% ± 0.22% for the control group and statistical differences were observed from time-matched control values p < .01 (Figure 1).

Histopathology

Hepatocytes near the centrilobular regions of the lobule were compared. No significant histopathological alterations were observed in the liver of mice treated with phalloidin for 1 and 3 days at a dose level of 250 and 500 μg/kg (Figure 2B and C ). However, phalloidin treatment for 7 days resulted in minimal to mild centrilobular hypertrophy in 1 mg/kg/day treated group. Light microscopy showed a decrease in the number of hepatocytes and an increase in cell size in the centrilobular region as compared to the centrilobular region of control mice. The severity of hepatic lesions was graded as minimal, mild, moderate, marked, and severe (Table 1 and Figure 2D ).

Blood Biochemistry

Present study showed no significant changes in AST (Figure 3A ). However, elevated levels of ALT were observed in the 1 mg/kg/day group at every time point (Figure 3B ). Levels of alkaline phosphatase (ALP) were markedly increased in a time-dependent manner but only in the high-dose group (Figure 3C ). Increased serum levels of direct bilirubin (DBIL) were found in the 1 mg/kg phalloidin-treated mice (Figure 3D ). Highest values were recorded only in the 1 mg/kg phalloidin-treated group after 7 days of treatment.

Gene Expression Profile

Microarray analysis was carried out to determine differences in hepatic gene expression between phalloidin- and vehicle- (2% DMSO-saline solution) treated mice. RNA integrity was intact (data not shown). Pooled liver samples from control animals and individual liver samples from treated animals were analyzed. Array based observations were made in triplicate. Gene expression was analyzed only in animals treated with 1 mg/kg of phalloidin for 1, 3, and 7 days.

A total of 3791 reliable genes were filtered from 10,000 probes by standard deviations of control strength after per-spot and per-chip normalization. Three hundred eight genes were sorted according to Student’s t test (p values from 0 to .05). One hundred sixty-five genes were selected based on the fold difference in at least three of nine conditions using ANOVA. A summary of these analyses is presented in Table 2. Those statistical screenings of gene expression with changes in DEGs (differentially expressed genes) corresponding to control or phalloidin-treated mice revealed that phenotypic alterations were more significant than time factors (Figure 4).

To further evaluate the microarray data, we also observed QT (quality threshold >0.95) clustering, which is an algorithm and groups genes into high quality clusters. A total of 45 genes were shown to have similar expression profiles. Hierarchical clustering of the genes and treatment time revealed a time-dependent up-regulation (Figure 5).

Table 3 lists genes that changed significantly (p < .05) in mice liver after 1 mg/kg phalloidin administration as compared to controls. Interestingly, many actin-binding proteins and other factors involving the polymerization of actin (regulator, actin-depolymerizing factor) were induced upon repeated phalloidin treatment. Actin, beta, cytoplasmic (Actb), Cofilin 1, nonmuscle (Cfl1), destrin (Dstn), homolog to alpha-fodrin (Spna2), and smooth muscle leiomodin (Lmod1) were found to be strongly induced by the phalloidin treatment. Furthermore, the list shows several membrane transporters such as the ATP-binding cassette, subfamily E, member 1 (Abce1), homolog to potential phospholipid-transporting ATPase (Atp11b), ATP-binding cassette, subfamily B, member 4 (Abcb4; Mdr/TAP), adaptor protein complex AP-2, mu1 (Ap2m1), seminal vesicle secretion 6 (Svs6), and glycosylphosphatidylinositol-specific phospholipase D1 (Gpld1). The list also shows changes in transcript levels of heat shock protein 1, beta (Hspcb), mpv17 transgene, kidney disease mutant (Mpv17), glutathione S-transferase, mu1 (Gstm1), flavin-containing monooxygenase 5 (Fmo5), uroporphyrinogen III synthase (Uros), and prolyl 4-hydroxylase, beta polypeptide (P4hb).

Analysis at the Pathway Level

After t test and fold difference filtering, 165 genes were found to be up- or down-regulated as the result of phalloidin treatment. By analyzing the lists of differentially regulated genes at the three time points, we found that most of the genes could be grouped within three main pathways, i.e., regulation of actin cytoskeleton, tight junction, and focal adhesion (Figure 5).

DISCUSSION

Several chemicals are known to cause cholestasis. They include chlorpromazine, cyclosporine A, 1,1-dichloroethylene, estrogens, manganese, and phalloidin. Cholestasis is characterized biochemically by elevated serum levels of compounds normally concentrated in bile, particularly bile acids and bilirubin. Present results on bilirubin show that high dose of phalloidin could cause cholestasis in mice. However, no marked elevation could be observed in the serum AST. Liver parenchyma has enormous functional reserves and in many experimental conditions no significant change is observed in their activity. An increase in alkaline phosphatase values after high-dose phalloidin treatment again indicated billiary obstruction. The histologic features of cholestasis can be very subtle and difficult to detect by light microscopy. However, centrilobular hypertrophy was observed after seven consecutive treatments with phalloidin. These observations confirm that 1 mg/kg/day treatment of mice with phalloidin induced cholestasis. The increased weight of liver further support these observations.

Physiological and biochemical mechanisms leading to cholestasis have been studied in the past. Compounds can cause cholestasis by several mechanisms. Chlorpromazine impairs bile acid uptake and canalicular contractility (Farrell 1994). Multiple mechanisms have been documented for estrogens (Vore 1991). Concentration of active form of chemicals or drugs at confined region are also a plausible factor of billiary cholestasis (Liebler 1994). Phalloidin and microcystin disrupt the integrity of hepatocyte cytoskeleton by affecting proteins that are vital to its dynamic nature. Their toxicity does not depend on their biotransformation and is exclusive for hepatocytes. Phalloidin tightly binds to actin filaments. It alters the actin-rich web of cytoskeleton adjacent to the canalicular membrane; the actin web becomes accentuated and the canalicular lumen is dilated (Phillips, Poucell, and Oda 1986). Molecular mechanism of this effect remains unknown. To examine this mechanism it seemed necessary to make a transcriptomic analysis. Gene expression profile indicated that fairly large percentage of genes belongs to the molecular regulation of actin polymerization system. In present study, six genes (Cfl1, Dstn, Capns, Lmod1, Spna2, Actb) correlated with polymerization of actin filaments were up-regulated. Cofilin is a member of the actin depolymerization factor (ADF) protein family. Destrin (Dstn) also constitutes a depolymerizing factor of F-actin and contains a sequence that is nearly identical with putative nuclear transport signal sequence of cofilin (Hotulainen et al. 2005). Capns1 is a Ca²⁺-activated cysteine protease that regulates cell adhesion (Arthur, Lireer, and Elce 1998; Sorimachi, Ishiura, and Suzuki 1997). Lmod1 is a member of the tropomodulin family of actin-binding proteins and is highly expressed in smooth muscles (Conley 2001; Conley et al. 2001). Tropomodulins stabilize actin filaments. Spna2 and Actb are the structural constituents of the cytoskeleton and account for general flexibility and elasticity of the cytoskeleton. Moreover, filamentous proteins Spna2 and Actb play an important role in membrane organization. Thus, up-regulation of these genes suggests an adaptive mechanism occurring against the factors contributing to cholestasis. Effects of obstructive cholestasis (bile duct ligation) on a wider range of gene expression using microarray technology were studied by Denk et al. (2006) in male C57BL/6J mice. After several days of bile duct legation (BDL), 265 genes were up-regulated in the liver. Metabolism-related genes represented the largest functional group.

Effect of phalloidin on protein involved in billiary transport was also investigated. Genes related with ATP transport, i.e., Abcb4, Atp11b, and Abce1 were found to be up-regulated. Bile acids and organic anions are excreted into the bile by canalicular ATP-binding cassette (ABC) transporters located at the canalicular membrane (Takikawa 2002). In normal liver these transporter participate in diverse cellular processes including the excretion of xenobiotics (Lecureur et al. 2000; Rippin et al. 2001; Trauner and Boyer 2003). It is again an adaptive mechanism that helps in maintaining a low intracellular level of toxic compounds (Ros et al. 2003). Schrenk et al. (1993) have shown that mdr7, a member of the multidrug resistant (mdr) gene family, was focused to be altered in both obstructive and alpha-naphtylisocyanate–induced cholestasis in rat liver. Twenty-four hours after bile duct ligation, gene encoding plasminogen activator inhibitor 7 (PAI-7) was found to be up-regulated. The authors concluded that inhibiting PAI-7 might attenuate liver injury in cholestatic liver diseases (Wang et al. 2005).

Furthermore, Gstm1 encoding glutathione S-transferase was also found to be up-regulated after 7 days of phalloidin treatment. This observation reflects that phalloidin is detoxified by glutathione. Glutathione is a driving force in the bile salt independent bile flow within bile canaliculi.

In summary, up-regulation of genes suggests alterations in microfilaments around the bile canaliculi. Glycoproteins do play a major role in cholestatic liver injury. However, more information is needed from other cholestatic agents such as cyclosporine A, chlorpromazine, manganese, and estrogens. Because pharmaceuticals cause serious complications that often lead to termination of therapy molecular markers of cholestasis need to be identified.