Chronic Microcystin Exposure Induces Hepatocyte Proliferation with Increased Expression of Mitotic and Cyclin-associated Genes in P53-deficient Mice

Abstract

Homozygous p53 deficient knockout mice were used to assess the role of p53 in tumor promotion by the protein phosphatase inhibitor and hepatic tumor promoter microcystin-LR (MCLR). More than 50% of human cancers bear mutations in the p53 gene, and in particular, p53 tumor suppressor gene mutations have been shown to play a major role in hepatocarcinogenesis. Trp53 homozygous (inactivated p53) and age-matched wild-type control mice were assigned to vehicle or MCLR-treated groups. MCLR or saline was administered daily for up to 28 days. RNA from the 28-day study was hybridized onto Mouse Genome GeneChip arrays. Selected RNA from 28 days and earlier time points was also processed for quantitative polymerase chain reaction (PCR). Livers from the 28-day, Trp53-deficient, MCLR group displayed greater hyperplastic and dysplastic changes morphologically and increases in Ki-67 and phosphohistone H3 (mitotic marker) immunoreactivity. Gene-expression analysis revealed significant increases in expression of cell-cycle regulation and cellular proliferation genes in the MCLR-treated, p53-deficient mutant mice compared to controls. These data suggest that regulation of the cell cycle by p53 is important in preventing the proliferative response associated with chronic, sublethal microcystin exposure, and therefore, conclude that p53 plays an important role in MCLR-induced tumor promotion.

Introduction

Microcystin-LR (MCLR) is a cyclic heptapeptide produced by the cyanobacteria (blue-green algae) Microcystis aeruginosa (Dawson, 1998). Toxic blooms of cyanobacteria have been associated with acute hepatotoxicity in various species of domestic animals and humans (Azevedo et al., 2002; Frazier et al., 1998; Puschner et al., 1998), while chronic exposure has been shown to have tumor-promoting activity in experimental studies (Falconer and Buckley, 1989; Hu et al., 2002; Ito et al., 1997; Nishiwaki-Matsushima et al., 1992; Zhou et al., 2002). In addition, epidemiologic studies in China have suggested that microcystins in contaminated water may play a role in the higher incidences of primary human hepatocellular carcinomas in that area (Ueno et al., 1996; Yu et al., 2001).

Microcystins inhibit the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A). Many PP1 and PP2A inhibitors, such as microcystin, okadaic acid, and nodularin, are classified as tumor promoters (Fujiki and Suganuma, 1999). The mechanisms of tumor promotion are unclear but most likely involve protein phosphatase inhibition leading to hyperphosphorylation of many cellular proteins (Fujiki and Suganuma 1999; Guy et al., 1992) and deregulation of cell-cycle control (Messner et al., 2001). Cell-cycle progression is controlled to a large extent by reversible phosphorylation of regulatory enzymes on their serine/threonine residues (Messner et al., 2001). Increases in oxidative stress have been related to chemical carcinogenesis (Elrick et al., 2005; Klaunig and Kamendulis, 2004). Accordingly, it has been proposed that microcystin-induced increases in oxidative stress, with resulting increases in reactive oxygen species, can cause DNA damage and that this may be associated with microcystin-induced liver carcinogenesis (Zegura et al., 2003). Studies in vitro and in vivo have found oxidative DNA damage in the form of 8-oxo-7,8 dihydro 2’-deoxyguanosine, associated with microcystin exposure (Bouaicha et al., 2005; Ding et al., 1999; Rao and Bhattacharya, 1996). Also, in a recent study, we have reported increases in transcripts related to liver cellular redox genes related to glutathione production and others related to intracellular protection from oxidant damage (Clark et al., 2007).

Few studies have examined the relationship between MCLR and p53. Recently, an increase in serine phosphorylation of p53 was identified following both lethal and sublethal MCLR exposure in mice (Guzman et al., 2003). In another study, it was also found that MCLR exposure resulted in a significant increase in the expression of p53 and bax, both in vitro and in vivo. From that, the researchers concluded that the expression of p53, bcl-2, and bax are involved in the regulation of MCLR–induced apoptosis (Fu et al., 2005).

The effect of microcystin on cell-cycle progression has been studied only recently. It was found that treatment of Chinese hamster ovary cells with high concentrations of MCLR resulted in changes in cell-cycle progression. In addition, a dose- and time-dependent increase in mitotic indices and increased numbers of abnormal polyploidy cells were identified after MCLR exposure. The authors concluded that the induction of cell-cycle arrest in mitosis was most likely associated with the MCLR-induced inhibition of PP1 and PP2A (Lankoff et al., 2003). Controlling the progression through the cell cycle is important in cell replication. Lack of control can result in cell death or neoplasia. Important regulatory proteins involved in the cell cycle are cyclin proteins and the cyclin-dependent kinases (cdks), which are intimately related with normal function of p53.

The p53 tumor-suppressor gene plays a key role in the cell’s response to genotoxic stress and has been considered the “guardian of the genome.” Its protein product (p53) functions as a transcription factor that regulates the downstream genes involved in cell-cycle arrest, DNA repair, and apoptosis (Burns and El-Deiry, 1999; Smith et al., 2003). In response to a variety of cellular stresses, including DNA damage and hypoxia, p53-mediated pathways attempt to repair injury through cell-cycle arrest and DNA repair. If the cell damage is not repairable, then p53 promotes apoptosis (Smith et al., 2003). It has been well documented that p53 is involved in G1 and G2 cell-cycle arrest (Burns and El-Deiry, 1999). The G1 growth arrest that occurs in response to DNA damage is primarily mediated by p21WAF1/CIP1 cyclin-dependent kinase inhibitor. WAF1/CIP1 is a downstream p53-responsive target gene that encodes the p21 protein. The p21 protein binds to and inactivates cyclin D/Cdk4,6 and cyclin E/Cdk2 complexes, which results in pRB hypophosphorylation and cell-cycle arrest in G1 (Pietenpol and Stewart, 2002; Smith et al., 2003). The mechanisms of p53-mediated G2 arrest are mediated in part by the synthesis of 14-3-3 sigma, a protein that binds cdc25c and prevents it from translocating to the nucleus. Cdc25c is a phosphatase that acts on the G2/M cyclin-cdk complex, cyclin B-cdc2. Sequestration of cdc25c in the cytoplasm prevents it from activating cyclin B-cdc2 in the nucleus. This results in cell-cycle arrest in the G2 phase of the cell cycle (Harris and Levine, 2005). p53 also regulates G2 arrest through the transcriptional up-regulation of several other target genes, including GADD45 and WAF1/Cip1 (Burns and El-Deiry, 1999; Pietenpol and Stewart, 2002). Up-regulation of p21 can inhibit cdc2 directly, whereas Gadd45 causes the dissociation of cdc2 from cyclin B1. The repression of cyclin B1 and cdc2 genes by p53 enforces the cell-cycle arrest (Taylor and Stark, 2001).

The precise role of p53 in the chronic, sublethal hepatotoxicity associated with microcystin is largely unknown. The objective of this study was to compare the chronic, sublethal toxicity of MCLR in p53 knockout and wild-type mice and to examine the possible role of p53 in microcystin-induced tumorigenesis. Using global gene-expression profiling along with immunohistochemical techniques, an increase in hepatocyte proliferation and differences in hepatic cell-cycle control were identified in the microcystin-treated p53 knockout animals when compared to the wild-type animals.

Materials and Methods

Animal Use

Twenty-two male homozygous p53 knockout B6.129-Trp53^tm1BrdN4 mice and 24 aged-matched wild-type B6.129-Trp53^+,+N5 mice were obtained from Taconic, Inc. (Germantown, New York) and allowed to acclimate at the Purdue Laboratory Animal Care facility for 2 weeks or longer before use. Homozygous p53 knockout B6.129-Trp53^tmlBrdN4 mice are developmentally normal but are more susceptible to spontaneous tumors including thymic lymphomas (Donehower et al., 1992). A low incidence of thymic lymphomas was identified in this study. All experiments were approved by the Purdue Animal Care and Use Committee. Animals had free access to commercial laboratory chow and water and were maintained on a 12-hour light:dark cycle. Three or four mice were assigned to the various dosing groups (24-hour, 28-day control groups and 4-hour, 24-hour, 4-day, 14-day, and 28-day treatment groups) immediately before dosing and given free access to food and water for the duration of the study. MCLR (approximately 95% pure) from M. aeruginosa was purchased from Calbiochem, Inc. (San Diego, California). The MCLR was dissolved in sterile 0.9% NaCl and diluted before intraperitoneal administration. In a preliminary study, it was found that these p53 knockout mice responded the same as wild-type mice to the hepatotoxic effects of MCLR (data not shown). Forty μg of MCLR per kg of body weight or vehicle control were given daily by intraperitoneal injection. At 4 hours, 24 hours, 4 days, 14 days, and 28 days after the first MCLR dose, 3 or 4 mice from each group were anesthetized with carbon dioxide and exsanguinated. It has been shown that gene expression can change throughout the day; therefore, with the exception of the 4-hour group, all necropsies were performed at the same time each day (Boorman et al., 2005; Panda et al., 2002). Control mice were similarly anesthetized and exsanguinated at 24 hours and 28 days postdosing. Heparinized blood was collected for plasma chemistries from the caudal vena cava. The liver was removed, patted dry, and weighed. A section of liver was fixed in 10% neutral buffered formalin for light microscopy. An additional piece of liver from the left lateral liver lobe (Irwin et al., 2005) was placed in RNAlater (Ambion, Inc., Austin, Texas) for microarray analysis. The remainder was frozen in liquid nitrogen and stored at –80°C. Sections of lungs, kidneys, spleen, small intestine, pancreas, adrenal gland, and brain were also fixed by immersion in 10% neutral buffered formalin. Tissues were routinely processed, embedded in paraffin, cut at 4 to 6 μm, and stained with hematoxylin and eosin (H&E) for light microscopic examination.

Plasma Chemistries

Whole blood for clinical chemistry determinations was collected from the caudal vena cava using heparinized syringes, and samples were placed on ice. The plasma was collected following centrifugation and stored at –20°C until further examination. Concentrations of total bilirubin, glucose, cholesterol, triglycerides, total protein, albumin, and the activities of alkaline phosphatase(ALP), gamma glutamyltransferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinasein plasma were determined using standard methods on a Hitachi 917 Automatic Analyzer (Roche Diagnostics Corporation, Indianapolis, Indiana).

Immunohistochemistry

A monoclonal antibody against mouse Ki-67 antigen (LabVision, Fremont, California) was used to evaluate hepatic proliferation in both control and treated animals. Tissue sections were placed on slides and deparaffinized in xylene. The slides were then rehydrated in a graded ethanol series to distilled water. After a brief rinse in distilled water, the tissue sections were placed in 3.0% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. For the recovery of antigenicity, specimens in 10 mM sodium citrate buffer (pH 6.0) were pretreated in a steamer at 121°C for 20 minutes. Tissue sections were incubated for 10 minutes with a protein block (DakoCytomation California Inc., Carpinteria, California). The primary antibody was a rabbit monoclonal antimouse Ki-67 antigen (LabVision, 1:100 dilution, 60 minutes). Rabbit IgG was used as a negative isotype control. The Envision+ (DakoCytomation California Inc., Carpinteria, California) rabbit detection system was used for detection. For chromogenic development, the slides were incubated in diaminobenzidine (DAB) containing 0.02% hydrogen peroxide for 10 minutes. Finally, the tissue sections were rinsed in deionized water, counterstained with hematoxylin, coverslipped, and examined microscopically. Duplicate negative control sections were incubated with rabbit IgG instead of the primary Ki-67 antibody. The crypt cells in the sections of small intestines served as internal positive controls. To quantitate the number of Ki-67 positive cells, 10 low-magnification (10×) fields were photographed, and the number of Ki-67 positive cells were counted using Image Pro Plus (Media Cybernetics, Inc., Silver Spring, Maryland) morphometry software.

An antibody against human phospho-histone H3 antigen (Upstate, Waltham, Massachusetts) was used to evaluate the mitotic index in both control and treated animals. Tissue sections were placed on slides, and the immunohistochemistry reactions were performed on the Ventana Discovery staining module. After loading the slides on the Ventana automated stainer, they were deparaffinized and rehydrated. The slides were then conditioned (antigen retrieval) with the Ventana CCl for 60 minutes at 100°C and blocked for 10 minutes with 4% goat serum. The primary antibody was a rabbit polyclonal antimouse phospho-histone H3 (Upstate, Waltham, Massachusetts, 1:500 dilution, 60 minutes). Rabbit IgG (R&D System, Minneapolis, Minnesota, 1:500, 60 minutes) was used as a negative isotype control. A biotinylated goat-anti rabbit (DakoCytomation California Inc., Carpinteria, California) was used as the secondary antibody (10 minutes). Before applying the strep-avidin horseradish peroxidase (Ventana), a biotin block was performed. For chromogenic development, the slides were incubated in DAB containing 0.02% hydrogen peroxide for 8 minutes. Finally, the tissue sections were rinsed in deionized water, counterstained with hematoxylin, coverslipped, and examined microscopically. To quantitate the number of phospho-histone H3 positive cells, 10 low-magnification (10×) fields were photographed, and the number of phospho-histone H3 positive cells were counted using Image Pro Plus morphometry software.

RNA Isolation

At necropsy, liver sections of approximately 500 mg from the left lateral liver lobe were excised and stored in RNAlater (Ambion, Inc., Austin, Texas) as per manufacturer’s recommendations. Total RNA was isolated from approximately 100 mg of liver tissue by homogenization using guanidium isothio-cyanatephenol (RNA-Stat 60, Tel-Test, Inc., Friendswood, Texas). RNA was further purified using RNeasy columns (Qiagen, Valencia, California) according to package instructions. RNA quality was assessed spectrophotometrically using a Gene Quant II instrument (Amersham Biosciences Corp., Piscataway, New Jersey).

cDNA Synthesis

First-strand cDNA was synthesized from 5 μg of each RNA sample using Superscript II RT (Invitrogen Corp., Carlsbad, California). Second-strand cDNA was then synthesized using DNA polymerase I (Invitrogen Corp., Carlsbad, California). The double-stranded cDNA was purified using Genechip Sample Cleanup Module (Affymetrix Inc., Santa Clara, California). In vitro transcription reaction was with MEGAscript T7 RNA poly-merase (Ambion, Inc., Austin, Texas), and the cDNA was linearly amplified and labeled with biotinylated ribonucleotides (Affymetrix Inc., Santa Clara, California). Labeled RNA was further purified using RNeasy columns (Qiagen, Valencia, California) and fragmented. Fifteen micrograms of labeled and fragmented cRNA was then hybridized onto Mouse Genome 430A 2.0 GeneChip arrays (Affymetrix Inc., Santa Clara, California) for 16 hours at 45 °C. Labeling, hybridization, staining, and washing were performed according to manufacturer’s protocols (Affymetrix Expression Analysis Technical Manual). The arrays were scanned with an Affymetrix Gene Chip Scanner, and images were quantified usingMAS 5.1 software (MicroArray Suite, Affymetrix). Data analysis and mining were performed using Affymetrix Microarray suite (MAS4.0) and data mining tool (DMT 2.0) software.

Quantitative Polymerase Chain Reaction (PCR)

The gene transcripts for RT-PCR analysis were selected from the biological pathways shown to be altered by chronic MCLR exposure. Representative gene transcripts from actin-organization, cell-cycle, apoptotic, cellular redox, and cell-signaling pathways were examined by RT-PCR to confirm our microarray data and also to further examine the mechanisms of chronic MCLR toxicity. Oligonucleotide primers used for PCR confirmation studies were identified using the PrimerBank Web site (http://pga.mgh.harvard.edu/primerbank/index.html). Oligonucleotide sequences are shown in Table 1. Total RNA was isolated as described above and treated with DNase I for 30 minutes at 37°C. DNase I was inactivated (DNAfree, Ambion Inc., Austin, Texas), and the RNA was used to generate first-strand cDNA (High Capacity cDNA Archive Kit, Applied Biosystems, Foster City, California) for use in quantitative (SybrGreen) PCR analysis. Quantitative real-time PCR was performed using the ABS 7700 sequence detector. Each sample was assayed in duplicate, and relative quantitation was performed using the ΔΔC_t method (ABS user bulletin number 2) using beta-actin as the normalization gene. The primer/probe sets were determined to have the same amplification efficiencies as the beta-actin control sets (data not shown). Primer concentrations and cycle number were optimized to ensure that reactions were analyzed in the linear phase of amplification.

Statistics

Liver percent body weight and plasma chemistries were statistically analyzed using the Student’s t-test with a statistical significance at p ≤ .05. For the microarray data, an ANOVA analysis was used to compare the treatment groups of interest with p ≤ .05.

Results

Gross Pathologic Findings

Similar gross findings were identified in both the mutant and wild-type treated groups. A loss in body weight was observed throughout the time course of the study in microcystin-treated animals. The drop in body weight was most prominent in the 14-day and 28-day treatment groups (approximately 9% and 12%, respectively). The livers from the control, 4-hour treated, 24-hour treated, and 4-day treated groups appeared grossly normal. The livers from both the wild-type and p53 mutant mice in the 14-day and 28-day MCLR-treated groups were pale and moderately enlarged. Statistically significant increases in liver to body-weight ratios were seen in the 14-day and 28-day treated groups (Table 2).

Plasma Chemistry Data

No statistically significant differences were identified in plasma chemistry values between the p53 mutant and wild-type control or MCLR-treated groups. However, in both mutant and wild-type mice, there was a statistically significant increase in plasma alanine aminotransferase (ALT) and aspartate amino-transferase (AST) activities identified in the 14-day and 28-day MCLR treated groups compared to saline-treated controls. No changes were identified in ALT or AST activities in the 4-hour or 24-hour treated groups. The plasma ALT activity began to increase in the 4-day treated group and continued to increase until the end of the study at 28 days, at which time there was a greater than 20-fold increase in activity over control in both the mutant and wild-type MCLR-treated groups. Statistically significant decreases in total protein (15% to 20%), albumin (20% to 30%), and glucose (40% to 50%) levels were also identified in both 14-day and 28-day treatment groups. An increase in total bilirubin was also identified in the 28-day treated groups, although no evidence of cholestasis was identified histologically. No changes were seen in plasma gamma-glutamyltransferase activity throughout the study.

Histopathology

Liver histology from the control mice in both groups showed normal hepatic cord pattern, hepatic lobules, normal nonparenchymal cells, and normal hepatocytes (Figures 1A and 1B). Histopathologic changes in the livers of treated mice in both the mutant and wild-type groups were not apparent until after 4 days of MCLR treatment. The 4-day MCLR-treated groups (both mutant and wild-type) had only subtle centrilobular changes that were most consistent with hepatocellular hypertrophy. These changes were more prominent in the 14-day MCLR treatment group and were much more pronounced in the 28-day MCLR treatment group. In the 14-day treated groups (both mutant and wild-type), hepatocellular hypertrophy was primarily centrilobular to midzonal in location. The enlarged hepatocytes had moderate karyomegaly and a loss of cytosolic vacuolation (loss of glycogen accumulation) compared to control mice. The areas of hypertrophy were sharply demarcated from the surrounding normal periportal areas. Livers from the 28-day MCLR-treatment groups (both mutant and wild-type) were characterized by marked hepatocytomegaly and karyomegaly that appeared worse in the centrilobular areas but that did appear to involve the entire hepatic lobule (Figures 1C and 1D). Many multinucleated hepatocytes were noted that contained as many as 10 giant nuclei per cell (Figure 1C and 1D). These dysplastic and hyperplastic changes were much more prominent in the treated mutant animals at both 14 and 28 days (Figure 1D). Inflammatory foci consisting primarily of neutrophils and lymphocytes were identified throughout the hepatic parenchyma. These foci appeared to be associated with necrotic hepatocytes.

Immunohistochemistry

A marker of cell proliferation, Ki-67, and a mitotic marker, phosphohistone H3, were used to evaluate and compare the proliferative response in the livers of MCLR-treated animals from both mutant and wild-type mice compared to controls (Figures 1E to 1H). In the 14-day mutant MCLR-treated group, very few nuclei had positive immunoreactivity for Ki-67 (data not shown). However, in mutant animals treated with MCLR for 28 days, many nuclei had positive immunoreactivity for Ki-67 (Figure 1F). Only low numbers of nuclei had positive immunoreactivity for Ki-67 in the 28-day wild-type MCLR-treated group (Figure 1E). Figure 2 illustrates the effect of MCLR treatment on the number of proliferative (Ki-67 positive) cells identified in the livers of saline or MCLR-treated animals. Note the increase in proliferative nuclei following microcystin treatment in both the p53 mutant and wild type animals. Also note the statistically significant increase in the numbers of proliferative nuclei in the livers of p53 mutant MCLR-treated animals compared to the wild-type MCLR-treated animals. A similar pattern was identified using the phospho-histone H3 antibody, mitotic marker (Figures 1G and 1H). There was a statistically significant increase in the number of mitotic cells in the mutant 28-day treatment group when compared to the wild-type group treated for 28 days (Figure 3). The histologic examination of H&E-stained liver sections also revealed increased numbers of mitotic hepatocytes, which correspond with the increased numbers of hepatocytes stained with phospho-histone H3 antibody.

Microarray Analysis

Figure 4 is a Star Tree image illustrating the biological pathways affected by chronic microcystin exposure for 28 days in both the wild-type and mutant animals. The pathways most affected by MCLR in both treated groups compared to saline controls include cell organization, cell death, and the response to stimulus pathways (Figure 4). However, the major differences in gene expression between the mutant and wild-type MCLR-treated animals occurred in the cell-cycle regulation groups (Figure 4). No major differences between the mutant and wild-type MCLR-treated mice were identified in the apoptotic, redox, or cell-signaling pathways, although many of these are significantly different from their saline controls (data not shown). Selected genes associated with cell-cycle control, actin filament organization, apoptosis, redox, and cellular-signaling pathways are listed in Table 3. The fold changes between the wild-type treated/wild-type control and the mutant treated/mutant control are listed in Table 3 along with the fold changes between the two treated groups.

Real-time PCR

Real-time PCR (RT-PCR) was used to confirm the gene-expression changes of selected genes identified with the gene chip in the 28-day treated group. RT-PCR was also used with selected genes to examine the gene-expression changes that occurred over time with MCLR treatment in both treated groups. Ki-67 was markedly different between the MCLR-treated wild-type and mutant animals. The expression of Ki-67 increased with time in both groups; however, the mutant animals had an approximately 7-fold increase in expression at 28 days compared to the MCLR-treated wild-type mice (Table 4). This is similar to what was identified with Ki-67 immunohistochemistry, in which the increase was approximately 4-fold to 5-fold. An increase in gene expression over time was identified for jun and cystatin B in both groups (Table 4). An increase in expression was also seen in the redox genes gss, gstm3, and nqo1 in both groups (Table 4). Cyclin G1, a p-53 dependent cyclin, increased with treatment over time in both groups; however, much stronger expression was identified in the wild-type animals, as would be expected (Table 4). Expression of the cdk inhibitor, p21, also increased over time in both groups. At 28 days, the expression was much stronger in the mutant group (Table 4). The mitotic cyclins and kinase followed a similar pattern of expression. Cdc2a, cyclin A2, cyclin B1, cyclin B2, and cdc20 expression was only slightly increased in both the mutant and treated animals in the 4-hour, 1-day, and 4-day treatment groups. However, at 14 days, the expression increased higher in the mutant animals, and at 28 days, there was a marked increase in expression in the mutant animals (Table 4).

Discussion

The pathogenesis of chronic, sublethal microcystin hepatotoxicity is still largely unknown. However, it has been documented that prolonged, sublethal exposure to microcystin can result in an increase in hepatic oxidative stress, apoptosis/necrosis, and even hepatic neoplasia (Guzman and Solter, 1999; Ito et al., 1997). In this study, MCLR treatment in both wild-type and p53-deficient knockout mice resulted in treatment-related decreases in body weight, increases in plasma enzyme activities related to liver damage, and decreases in plasma concentrations of albumin and glucose with related decreases in hepatic expression of genes related to albumin and glucose production as we previously described in wild-type mice alone (Clark et al., 2007). Although the p53 tumor-suppressor protein plays a critical role in regulating these events, it has not been previously investigated, and its role with regard to MCLR-induced toxicity is not known. Using p53 homozygous knockout mice, we were able to better define the role of p53 in chronic, sublethal microcystin hepatotoxicity, and through the combined use of histology, immunohistochemistry, RT-PCR, and gene arrays, we found that the major difference between the MCLR-treated, p53-deficient mutant, and wild-type animals involved regulation of the cell-cycle pathway (Figure 4 and Table 3).

Histologically and immunohistochemically, there were major differences identified between the mutant and wild-type MCLR-treated animals at 28 days. In the livers of mutant mice lacking functional p53, treated with MCLR for 28 days, an increased proliferative response and more dysplastic changes were identified following microcystin exposure. The proliferative response was characterized by statistically significant increases in the nuclear Ki-67 immunoreactivity (p ≤ .01) and in the numbers of mitotic cells (phospho-histone H3 positive, p ≤ .05) in the livers of mutant mice treated with MCLR when compared to the wild-type mice treated with MCLR. A statistically significant (p ≤ .007) increase in Ki-67 mRNA expression in the p53-deficient treated animals corresponded with the immunohistochemistry findings (Table 3 and Figure 2). Although hepatic neoplasms were not identified histologically in this 4-week study and toxicant-induced hepatocyte proliferation does not in all cases induce hepatic cancer (Melnick and Huff, 1993; Yang et al., 2007), it has been reported in another study that mice receiving 100 intraperitoneal injections of sublethal doses of microcystin (20 μg/kg) for 28 weeks developed neoplastic nodules in their livers (Ito et al., 1997). However, the dysplastic and hyperplastic changes that we identified were similar to those found in Balb/c mice given repeated sublethal intraperitoneal doses (45 μg/kg/day) of microcystin for 7 days (Guzman and Solter, 2002). As in our study, these changes consisted of marked hepatocytomegaly and karyomegaly with increased numbers of large multinucleated cells. In Chinese hamster ovary cells exposed to MCLR, similar cell-cycle changes were also identified, resulting in an increase in the numbers of mitotic and polyploid cells (Lankoff et al., 2003). It has been suggested that microcystin exposure may cause hepatocytomegaly and karyomegaly by inhibiting or blocking progression through mitosis (Guzman and Solter, 2002). In our current study, these changes were much more severe in the p53-deficient mutant mice, suggesting a role of p53 in limiting these proliferative dysplastic/hyperplastic changes in MCLR-treated wild-type animals.

Gene-expression analysis verified major differences in the hepatic expression of cell-cycle regulatory genes between the p53-deficient mutant and wild-type MCLR-treated animals. p53-deficient animals treated with microcystin for 28 days had up-regulation and greater than 2-fold increase in the expression of mitotic genes in the livers, many of which are involved in the formation of the mitotic cyclin/cdc2 complexes. These include the cyclin-dependent kinase cdc2a along with the mitotic cyclins, cyclin A2, cyclin B1, and cyclin B2, similar to those that increased following okadaic acid treatment in vitro. It is well known that p53 regulates the G2/M transition by regulating mitotic cyclin/cdc2 complexes (Stark and Taylor, 2004; Taylor and Stark, 2001). It has been shown that p53 can repress the transcription of cyclin A2, cyclin B1, and cyclin B2 and inhibit progression into mitosis (Badie et al., 2000; Krause et al., 2000; Stark and Taylor, 2004). The p53 protein can also repress the transcription of cdc2 (Stark and Taylor, 2004). In our study, the data indicate that the lack of functional p53 in the knockout microcystin-treated animals prevented the transcriptional repression of the mitotic cyclins and cyclin-dependent kinases, allowing the increased expression of these genes, which then resulted in increased numbers of proliferating cells entering mitosis, some of which could have had DNA damage.

The mechanisms of mitotic arrest and tumor promotion by protein phosphatase inhibitors, such as MCLR, have been examined in vitro using okadaic acid, the prototypical protein phosphatase inhibitor. Okadaic acid exposure has been shown to cause mitotic arrest in many different in vitro systems (Lerga et al., 1999; Traore et al., 2001; You and Bird, 1995). Treatment of transformed HeLa cells with okadaic acid has resulted in an increase in the levels of genes involved in cell-cycle regulation. These genes included cdk2, cyclin A, cyclin B, and Rb (You and Bird, 1995), which were also increased in this present study. The role of p53 in okadaic acid–induced apoptosis is unclear. Okadaic acid exposure may result in p53-dependent or p53-independent apoptosis, depending on the cell line tested and the concentration of okadaic acid. It has also been suggested that p53 may play a role in the G2/M cell-cycle block associated with okadaic acid exposure in A1-5 rat embryonic fibroblasts (Milczarek et al., 1999). In these fibroblasts, this okadaic acid–induced block correlated with an increase in p53 phosphorylation and an increase in p21 expression at the mRNA level. These authors suggested that wild-type p53 blocks the proliferative effect of okadaic acid through p21 protein-mediated growth arrest (Milczarek et al., 1999).

The p53 protein also controls the G2/M transition by enhancing the transcription of 3 cdc2 inhibitors, gadd45, p21, and 14-3-3 sigma (Taylor and Stark, 2001). We did not identify any statistical differences in the expression of gadd45a, gadd45b, or 14-3-3 sigma between the mutant and wild-type MCLR-treated animals. However, we did identify differences in the expression of p21 (cdkn1a). The p21 protein is a cyclin-dependent kinase inhibitor whose expression is under the control of p53. It functions to inhibit cyclin-cdk complexes involved in G1 and G2/M transition (el-Deiry, 1998). At 28 days posttreatment with microcystin, we identified a greater than 2-fold decrease in the expression of p21 in the mutant animals when compared to the wild-type animals in the microarray analysis. Although there is a greater fold-change increase in p21 expression between the mutant control animals and the mutant treated animals than the wild-type control animals and wild-type treated animals, this difference is caused by the extremely low basal expression of p21 in the mutant control animals, as would be expected. These findings are consistent with p53 causing the up-regulation of p21 in the wild-type animals. However, a modest increase in p21 expression in the mutant treated animals that must be p53-independent, similar to that previously reported (Sukata et al., 2000), is also seen. The high level of expression of p21 in the wild-type MCLR-treated animals is most likely secondary to an increase in p53. This high level of expression is important in blocking the transition from G2 to M in cells damaged by microcystin exposure. In the mutant MCLR-treated animals, the low level of p21 expression may not be sufficient to prevent this G2/M block, resulting in microcystin-damaged cells entering mitosis.

To verify gene-expression changes that we identified with the microarray chip and to examine some of these pathways over the time course of the study, RT-PCR was performed on selected genes (Table 1). Interestingly, the expression of mitotic cyclins—cyclin A2, cyclin B1, and cyclin B2—and cyclin-dependent kinase, cdc2a, followed a similar pattern of expression over time. The majority of these genes exhibited little or no increase in expression at 4 hours, 24 hours, or 4 days (Table 4). These expression patterns were similar in both the wild-type and p53-deficient, MCLR-treated animals up to 4 days postdosing; however, there was a slight increase in the expression of these genes in the p53-deficient animals. At 14 days, the expression of these genes began to increase until at 28 days, there was a marked increase in the expression of these mitotic genes in the p53-deficient, MCLR-treated animals that was markedly higher than that in the wild-type MCLR-treated animals. Interestingly, this pattern of expression of mitotic genes in both the mutant and wild-type animals followed the expression pattern of the proliferative marker, Ki-67. Based on these findings, we speculate that dysregulation of the mitotic cell-cycle pathway may be involved in the proliferative response associated with microcystin exposure in the p53-deficient animals. An increase in the expression of other mitotic genes required for the progression through mitosis was also found in the p53-deficient MCLR-treated animals. Genes involved in cytokinesis (kif20a, kif23, stk6, cenpa, ect2, incenp), spindle formation (stathmin1), and chromatin condensation (top2a, ki-67) were up-regulated in microcystin-treated, p53-deficient, mutant animals. The expression of these genes in the wild-type animals was not affected or only mildly affected following microcystin exposure. These data also support the role of p53 in the transcriptional repression of genes required for G2/M transition and the progression through mitosis. Again, a lack of transcriptional repression by p53 results in the up-regulation of these genes and inappropriate entry into mitosis.

Since p53 is also involved in the induction of apoptosis and in the response to oxidative stress, we also examined the differences in gene expression between the MCLR-treated, p53-deficient, and wild-type animals in these pathways. Only a small increase in the expression of bax was found in the wild-type MCLR-treated animals, and no changes were identified in the mutant MCLR-treated animals. This is expected since bax is a p53-target gene. The expression of the cysteine proteases, cystatin B and calpastatin, were also similar between the 2 groups, suggesting no differences in the apoptotic response. Although an increase in the expression of caspase 4 was identified in the p53-deficient mutant, MCLR-treated animals, the significance of this finding is unknown. Similar to the apoptotic pathway, no major differences in the cell-signaling, actin, or cellular morphogenesis pathways were identified between the p53-deficient mutant and wild-type MCLR-treated groups.

Microcystin treatment resulted in the increased expression of genes involved in glutathione metabolism and other genes involved in redox reactions in both the p53-deficient mutant and wild-type groups. Interestingly, in the wild-type MCLR-treated animals alone, we identified an increase in the expression of gamma-glutamylcysteine synthetase (gclc), the rate-limiting enzyme involved in glutathione synthesis. This lack of increase in gclc expression in the p53-deficient animals may have resulted in the decreases in the expression of gpx3, gpx4, gstm3, and gstm1. Also, a decrease in the expression of 2 glutamate transporters, slc7a11 and slc1a4, was identified in the p53-deficient, MCLR-treated animals when compared to the wild-type MCLR-treated animals, suggesting that p53-deficient animals may be more susceptible to the oxidant damage that is associated with MCLR exposure. In addition, expression of nqo1 was also decreased in the p53-deficent, MCLR-treated groups versus the wild-type MCLR-treated groups. Nqo1, NAD(P)H:quinone oxidoreductase, is a key enzyme involved in the defense against reactive oxygen species and is an inhibitor of neoplasia (Nioi and Hayes, 2004). It is known that nqo1 is required for the stabilization of the p53 protein in response to DNA-damaging stimuli; however, it has not been reported that p53 may cause an increase in the expression of nqo1. In this current study, we identified a significant difference in the expression of nqo1 between wild-type and p53-deficient mutant animals following microcystin exposure, suggesting that p53 may play a role in regulating the transcription of nqo1.

To examine the role of p53 during chronic, sublethal micro-cystin exposure, we treated p53-deficient mutant and p53 wild-type mice with 40 μg MCLR/kg intraperitoneally for up to 28 days. Under these conditions, lack of functional p53 resulted in an increase in hepatocellular proliferation. Histologically, there was increased hepatocyte hypertrophy and dysplasia, which was further characterized immunohistochemically as increased proliferation and mitosis via staining with Ki-67 and phospho-histone H3 in MCLR-treated, p53-deficient mice. With gene-expression analysis, we were able to identify major differences in the expression of genes involved in the regulation of mitosis between the p53-deficient and wild-type MCLR-treated animals. In addition, exposure to MCLR resulted in up-regulation of the majority of the cyclins and cdks involved in the G2/M transition in the p53-deficient animals. Finally, as we reported previously (Clark et al., 2007), the results of this present study reaffirm that in both p53-deficient and wild-type mice, the apoptotic, actin-filament organization, cell-signaling, redox, and cell-cycle pathways are all important in the response of the liver to chronic, sublethal MCLR exposure. In conclusion, our results indicate that p53 is important in regulating cell-cycle progression following oxidative stress associated with chronic, prolonged microcystin exposure. The lack of functional p53 may result in inappropriate cell-cycle progression leading to an increase in proliferation, which may be a contributing factor to the increase in hepatocellular carcinoma that has been reported with longer exposure to MCLR.