TY - JOUR
AU - Gollapudi, B. Bhaskar
AB - Abstract The heterozygous p53 knockout mouse is being used as a short-term alternative model for carcinogenicity screening of chemicals. In most cases, these mice develop tumors within 6 months of exposure to genotoxic carcinogens. The bladder and liver carcinogen, p-cresidine, is recommended as a positive control chemical for these assays. To evaluate early effects of p53 deficiency on bladder and liver histopathology and genotoxicity induced by p-cresidine, we treated 4-week-old heterozygous and nullizygous p53 male mice with p-cresidine by gavage (100, 200, 400, and 800 mg/kg/day) 5 days/week for 7 weeks. Tissue sections were prepared for hematoxylin-eosin staining and immunohistochemistry for PCNA protein or 3`-OH DNA fragments to assess cell proliferation and apoptosis, respectively. Blood and bone marrow were examined for methemoglobin and micronuclei in polychromatic erythrocytes (MN-PCE), respectively. Individual cell necrosis of the bladder transitional epithelium was evident in both p53 heterozygous and nullizygous mice at all doses. In addition, diffuse hyperplasia of the bladder epithelium was observed at 400 and 800 mg/kg in both genotypes. In the liver, both genotypes exhibited similar increases in hepatocyte apoptosis (10-fold increase) and cell proliferation (20-fold increase) at 800 mg/kg/day. Methemoglobin levels were increased 6-fold in both genotypes at 800 mg/kg. Background MN-PCE rates were similar in both genotypes and there were no treatment-related increases. Also, no point mutations were observed in codon 12 of the c-Ha-ras gene from urinary bladder DNA from p-cresidine treated p53 mice. These results suggest that loss of p53 allele(s) in mice does not influence the early markers of carcinogenic activity induced by subchronic treatment with p-cresidine. Increased tumor susceptibility associated with a reduction in p53 dosage may be dependent on neoplastic progression rather than initiation and promotional events elicited by p-cresidine. p53 mice, p53 function, genotoxicity, cell proliferation, cytotoxicity, p-cresidine, urinary bladder, liver, risk assessment, bioassay The identification of chemical carcinogens is an important public health concern and a major focus of scientific research in industry, academia and government. However, the cost and time required to conduct standard rodent bioassays can be significant. With new advances in biotechnology, there have been a number of transgenic mouse lines created that respond to carcinogen exposure in less than one year (Donehower et al., 1992; Leder et al.; 1990; Saitoh et al., 1990). The p53 knockout mouse, recently proposed by several regulatory agencies as an alternative in vivo model for carcinogenicity testing (International Conference on Harmonization [ICH], 1997), develops tumors in response to genotoxic carcinogens within 6 months of exposure (Dunnick et al., 1997; Finch et al., 1998; Tennant et al., 1995, 1996). The p53 tumor suppressor gene, “knocked out” in p53 mice by homologous recombination in embryonic stem cells (Donehower et al., 1992), normally inhibits tumor growth by regulating cell cycle progression and/or inducing apoptosis in response to DNA damage (Levine, 1997). It has been hypothesized that the presence of a single functional p53 allele in heterozygous mice enhances their susceptibility to tumor development because only one critical mutational event is necessary for loss of p53 function (Donehower et al., 1992; 1996; Knudson, 1971). The results obtained in this model using a limited number of chemicals indicated a good correlation to the carcinogenic response observed in the traditional 2-year rodent bioassay (Eastin et al., 1998; Tennant et al., 1995). An industrial chemical positive in the p53 mouse bioassay, p-cresidine (2-methoxy, 5-methylaniline), is a monocyclic aromatic amine used in the production of dyes and is a known liver and urinary bladder tumorigen in rodents and a possible human carcinogen (National Cancer Institute (NCI), 1979). Given in the diet at 0.25% or 0.5%, p-cresidine induced bladder tumors within 5 months after the start of treatment in p53 heterozygous knockout mice (Dunn et al., 1997; Sagartz et al., 1998; Tennant et al., 1996). However, within this same time frame, the induction of liver tumors by p-cresidine has been inconsistent (Sagartz et al., 1998). In addition, the mechanism(s) by which p-cresidine induces tumors in p53 mice, including any loss or alteration of p53 function, has not been elucidated. If the current paradigm for tumor suppressor function holds in p-cresidine-induced tumors, then loss of the other p53 allele would be necessary for the accelerated tumor development seen in p53 mice. Unfortunately, this is not the case since the majority of p-cresidine-induced bladder tumors do not demonstrate loss of heterozygosity (LOH) at the p53 locus (French, 1996). There is only limited evidence that p-cresidine is genotoxic in vivo (i.e., urinary bladder) and based on its genotoxicity in vitro, p-cresidine can be classified as a weak genotoxic carcinogen (Ashby et al., 1991; Dunkel et al., 1985; Jakubczak et al., 1996; Sasaki et al., 1998). The gavage dose of p-cresidine for inducing bladder tumors in p53 mice is quite high, i.e., 400 mg/kg/day. In addition, if there are non-genotoxic mechanisms involved in p-cresidine carcinogenicity, they have not been identified. Our objectives in this study were to (1) evaluate the role, if any, of the p53 gene in the toxicity of p-cresidine, (2) examine the response of early markers of carcinogenic activity (cell proliferation and apoptosis) associated with p-cresidine exposure in relation to p53 zygosity, and (3) determine the influence of p53 function on bone marrow (a non-target organ) and urinary bladder (target organ) genotoxicity induced by p-cresidine following subchronic exposure. These endpoints were measured to identify any differences in the carcinogenic potential of p-cresidine in p53 heterozygote and nullizygote mice. Also, by using p53 heterozygous and nullizygous mice, we might predict the role that p53 plays in mouse liver and bladder carcinogenesis. MATERIALS AND METHODS Treatment of Animals Three-week-old male heterozygous (+/–) and nullizygous (–/–) p53 knockout mice (C57Bl/6TacfBR-[ko]p53N5-T) were purchased from Taconic (Germantown, NY). The knockout mice lack a functional p53 allele because of the introduction of a null mutation by homologous recombination (Donehower et al., 1992). Mice were group-housed in clear plastic tubs with air filtered tops and Cell Sorb Plus bedding material (A&W Products Inc., New Philadelphia, OH) in rooms designed to maintain adequate environmental conditions (temperature, humidity, and photocycle). Mice were fed Purina Certified Rodent Chow #5002 (Purina Mills, Inc., St. Louis, MO) and municipal drinking water was provided ad libitum during the pre-study and study periods. After a 7-day acclimation period, male p53 mice were given p-cresidine (Aldrich Chemical Co., Milwaukee, WI, purity 99%) by gavage at 100, 200, 400, or 800 mg/kg/day, 5 days/week for 7 weeks. Control mice received corn oil vehicle. Sixty mice (12 per group) were randomly allocated to different dose groups without prior knowledge of their genotypes. Tail clippings (≈1cm from the distal end) were subsequently sent to the supplier for genotyping. After genotyping, the ratio of nullizygous/heterozygous animals in each treatment group was: controls, 6 Null (N)/6 Het (H); 100 mg/kg/day, 7N/5H; 200 mg/kg, 8N/4H; 400 mg/kg, 4N/8H; and 800 mg/kg, 8N/4H. All mice were sacrificed 24 h after the last gavage dose of vehicle or p-cresidine. All animals were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care (AAALAC) International. The Institutional Animal Care and Use Committee (IACUC) approved this study. Clinical Chemistry and Hematology Animals were euthanized and peripheral blood was taken by orbital eye bleeds. Methemoglobin was measured as an internal dosimeter of aromatic amine bioavailability and metabolic activation (Sabbioni, 1992). Methemoglobin levels were assayed spectrophotometrically similar to the Eveyln and Malloy (1938) method utilizing a Hitachi 914 Clinical Chemistry Analyzer (Boehringer-Mannheim, Indianapolis, IN). Triton-borate solution was used to eliminate the need for prolonged centrifugation. For hematology, blood samples were mixed with EDTA and blood smears prepared and stained with Wright's stain. Serum was harvested from clotted blood samples and clinical chemistry evaluations were performed using the Hitachi 914 Clinical Chemistry Analyzer. The following parameters were measured: alanine aminotransferase (ALT), aspartate aminotransferase (AST) and acid phosphatase (AP) activities, urea nitrogen (UN), and creatinine. Immunohistochemistry Hepatocyte mitotic and apoptotic indices were determined using commercially available immunohistochemical labeling methodologies. Briefly, liver tissues were fixed in 10% neutral phosphate-buffered formalin, dehydrated through a series of alcohol washes, embedded in paraffin and sectioned approximately 6 μm thick. After mounting on glass slides, tissues were probed with anti-PCNA (proliferating cell nuclear antigen) monoclonal mouse antibody (Dako Corp., Carpinteria, CA) for analysis of PCNA protein. After incubation with the primary antibody, tissues were probed with a biotinylated secondary antibody, followed by streptavidin-peroxidase conjugate as described by the manufacturer (HistoMouse-SP kit, Zymed Laboratories Inc., San Francisco, CA). Finally, the addition of peroxide-chromagen solution facilitated the identification of the antigen by a red precipitate. For apoptosis analysis using Apoptag Plus kit (Oncor, Gaithersburg, MD), 3`OH DNA termini were enzymatically labeled in situ with digoxigenin-nucleotides by terminal deoxynucleotidyl transferase (TdT). Tissues were then incubated with anti-digoxigenin antibody conjugated to peroxidase and apoptotic cells visualized by a localized brown stain after incubating with diaminobenzidine substrate. Proportion of labeled cells that were undergoing cell division or apoptosis were based upon a minimum total count of 1000 cells or 10 separate microscopic fields (×300) from liver tissue sections from each animal. Our evaluation of apoptotic cells was solely based upon immunohistochemical staining. We have not tried to specifically identify the mode of action in necrotic cells (i.e., oncosis vs. apoptosis) based upon morphology. Cell Cycle Analysis The method for analyzing cell cycle stage in hepatocytes by PCNA staining was taken from Eldridge et al., 1993 and Foley et al., 1991. Briefly, cells in G0 phase are characterized by no immunochemical staining (Kurki et al., 1986), G1 hepatocytes have minimal nuclear staining 1+, hepatocytes in S-phase have more intense nuclear staining 2+ or 3+. Cells in G2 phase exhibit diffuse speckled nuclear and cytoplasmic staining 2+. In mitosis, the nucleoplasm and the cytoplasm (diffuse speckled staining 2+) coalesce with the loss of nuclear boundaries and the formation of mitotic figures. Histopathology Following euthanasia, liver, kidney, ureter and bladder were removed, preserved in 10% neutral phosphate-buffered formalin. Bladders were inflated with formalin prior to preservation. Tissues were processed with conventional techniques, stained with hematoxylin and eosin (H&E), and examined by a veterinary pathologist using light microscopy. Bone Marrow Micronucleus Assay Bone marrow was removed from both femurs by aspiration into fetal bovine serum. After centrifugation, the cell pellet was resuspended in a drop of serum and a smear was prepared on glass slides. After drying, slides were fixed in methanol and stained with Wright-Giemsa using an automatic slide stainer (Ames Hema-Tek, Miles Scientific, Naperville, IL). One thousand polychromatic erythrocytes (PCE) were examined from each animal. In order to determine the ratio of PCE to normochromatic erythrocytes (NCE) in the bone marrow, a total of approximately 200 erythrocytes from each animal were also examined. PCR-RFLP Analysis of c-Ha-ras The first two bases of codon 12 of the c-Ha-ras gene were analyzed for point mutations using a modified PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) procedure (Mitchell et al., 1998). Total bladder DNA (≈ 100 ng) was amplified by PCR (20 cycles) using primers (forward 5`-ACAGAATACAAGCTTGTGGTGGTGGGCCCT-3` and reverse 5`-CTTGCACCTCTCATACCCTGGTGGA-3`) that incorporate a Bst O1 enzyme (Promega Corp., Madison,WI) restriction site into wild type codon 12 ras PCR products (226 bp). Each PCR cycle was 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s (PCR primers and reagents, Life Technologies, Grand Island, NY). Following Bst O1 digestion at 60°C for 3 h, mutant alleles were selectively amplified in a second PCR reaction (25 cycles) that incorporated a second Bst O1 restriction site at the 3` end of the PCR product (140 bp), serving as a control for digestion efficiency. The same forward primer was used again in the second PCR reaction and a new reverse primer sequence was used 5`-AGCCCACCTCTGCCAGGTAG-3`. The final Bst O1 digestion yielded mutant ras PCR products (125 bp) and remaining wild type ras PCR products (95 bp) that were identified following electrophoresis on ethidium bromide stained 2.5% agarose gels. Mutant c-Ha-ras controls were generated as described by Mitchell et al., 1998. Statistical Analysis Treatment groups were compared to untreated controls using parametric ANOVA, followed by either the Dunnett's or Wilcoxon's test. A p value less than 0.05 was identified as statistically significant. RESULTS Body Weights P53 heterozygous and nullizygous mice given 800 mg/kg/day had approximately a 15% reduction in body weight relative to the controls (Table 1). However, this reduction was statistically significant only in the heterozygotes. Also, in the heterozygotes, there was a significant increase in body weight in the 200 mg/kg/day dose group relative to controls (Table 1). Relative liver weights were significantly increased in both p53 +/– and p53 –/– mice at 400 and 800 mg/kg (Table 1). Histopathology Individual necrosis of transitional epithelial cells of the urinary bladder epithelium (Fig. 1) was evident in all p53 +/– and p53 –/– dose groups and increased in severity with increasing doses of p-cresidine (Table 2). Treatment-related increases in diffuse hyperplasia of the bladder (Fig. 1) were also observed in both p53 genotypes given 400 or 800 mg/kg/day (Table 2). No focal areas of dysplasia were observed in the bladder epithelium of p53 mice treated with p-cresidine for 7 weeks. Hepatocyte hypertrophy and individual hepatocyte necrosis were also increased in both p53 genotypes at 800 mg/kg and in 400 mg/kg in heterozygotes (Table 2). Kidneys of several of the mice in both genotypes treated with 800 mg/kg had tubular degeneration (data not shown). Individual cell necrosis of ureter transitional epithelium was also evident in animals treated with 800 mg/kg (data not shown). Cell Proliferation and Apoptosis We assessed p-cresidine and genotype-related differences in hepatocyte proliferation and apoptosis using PCNA and 3`-OH DNA fragment immunohistochemistry, respectively. Significant increases in the percent of hepatocytes staining positive for PCNA protein as compared to controls were limited to the 800 mg/kg p-cresidine dose group (Fig. 2). There was a 20-fold increase in PCNA staining of hepatocytes in the 800 mg/kg dose group compared to the control group in both p53 heterozygote and nullizygote mice (Fig. 2). In both genotypes, approximately 75% of the PCNA positive hepatocytes were in the G1 phase of the cell cycle (Fig. 3). Similarly, in only the high-dose group did both genotypes exhibit a 10-fold increase in apoptotic cells as compared to controls (Fig. 2). There were no differences in the background proliferation or apoptotic rates between control p53 +/– and –/– mice. Hematology and Clinical Chemistry Methemoglobin levels were elevated in both p53 genotypes given 200, 400, or 800 mg/kg/day (Fig. 4). These increases were statistically significant at 400- (3.5-fold) and 800 mg/kg (6.5-fold) p-cresidine groups in heterozygote mice and only at 800 mg/kg (6-fold) in nullizygote mice. Red blood cell counts, hemoglobin concentration, and hematocrit were all significantly lower than the controls in both p53 genotypes even at the lowest dose (100 mg/kg/day) of p-cresidine (Table 1). There was approximately a 3-fold increase in serum ALT values in mice treated with 800 mg/kg p-cresidine compared to the negative controls and this increases was statistically significant in the heterozygotes (Table 1). There were no treatment related differences in other serum chemistry parameters analyzed (data not shown). Genotoxicity The numbers of micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow were also compared between treatment and genotype groups (Table 3). Background micronuclei per 1000 PCE were similar in p53 heterozygotes (3.8 ± 2.1) and nullizygotes (2.7 ± 1.4) and there were no p-cresidine related increases in MN-PCE. Urinary bladder genotoxicity was measured by PCR-RFLP analysis of point mutations in codon 12 of the c-Ha-ras gene. Codon 12 of c-Ha-ras has been shown to be altered in mouse urinary bladder tumors (Yamamoto et al., 1995). However, with a sensitivity of one mutant in 10,000 normal ras alleles, no mutations in codon 12 were detected in bladder DNA from p-cresidine treated p53 +/– (Fig. 5) or –/– mice (n = 4, nullizygous data not shown). DISCUSSION These studies compared the subchronic effects of p-cresidine exposure in p53 heterozygote and nullizygote mice. Advantages of the p53 model include a shortened period for tumor development (6 months) and information concerning carcinogen mode of action (Gollapudi et al., 1998; Tennant et al., 1995). This increased tumor susceptibility to chemically induced tumors combined with the low background incidence of spontaneous tumors until 12 months of age make the p53 heterozygote mouse a potentially useful alternative to the 2-year bioassay (Donehower et al., 1996; Harvey et al., 1993; Tennant et al., 1995). Used in the production of dyes, p-cresidine is a liver and bladder carcinogen in the 2-year bioassay (NCI, 1979). It also induced bladder tumors in p53 heterozygote mice and is currently used as a positive control in the 6-month p53 mouse bioassay (Dunn et al., 1997; Sagartz et al., 1998). Although the in vivo genotoxicity data of p-cresidine is limited, its in vitro genotoxicity and rapid induction of bladder tumors in rats and mice suggest this carcinogen works through a genotoxic mode of action (Ashby et al., 1991; Dunkel et al., 1985; Jakubczak et al., 1996; Sasaki et al., 1998). Our objectives in this study were to characterize p-cresidine-induced cytotoxicity, cell proliferation, and genotoxicity in p53 heterozygous and nullizygous mice. The use of nullizygous mice will provide data on whether p53 function has any effect on these early indicators of carcinogenic activity. The organs most sensitive to p-cresidine exposure were the urinary bladder and blood. Even at the lowest dose, p-cresidine-induced cytotoxicity in a treatment-related manner in these tissues. Aniline and other structurally related monocyclic amines, including p-cresidine, commonly target red blood cells due to their reactivity with oxyhemoglobin, following hepatic N-oxidation (Ashby et al., 1991; Bus et al., 1987). In fact, this reactivity with hemoglobin can be used as a dosimeter for aromatic amine exposure (Sabbioni, 1992). Since there were no remarkable differences in methemoglobin levels between heterozygous and nullizygous p53 mice following p-cresidine treatment in our study, it can be concluded that the systemic bioavailability and metabolic activation of p-cresidine is similar in both genotypes. Transitional epithelial cell cytotoxicity of the urinary bladder was induced by p-cresidine at doses as low as 100 mg/kg/day in this study. This cytotoxicity may be associated with p-cresidine-induced genotoxicity as previously described (Jakubczak et al., 1996; Sasaki et al., 1998). Urinary bladder mucosa can metabolize N-oxidized arylamines to DNA reactive species (Badawi et al., 1995) and this may be responsible for the genotoxicity reported in this tissue by Jakubczak et al. (1996) and Sasaki et al. (1998). Induction of bladder mucosa genotoxicity and hyperplasia together may be necessary for bladder tumor induction by p-cresidine. Treatment-related increases in urinary bladder hyperplasia were limited to the 400 and 800 mg/kg/day dose groups in both p53 +/– and p53 –/– mice. However, at 400 mg/kg/day, one animal from both genotype groups did not demonstrate diffuse hyperplasia of the bladder epithelium. This data strengthens the notion that a minimum dose of 400 mg/kg/day is necessary for a carcinogenic response in p53 mice treated with p-cresidine. P53 protein did not affect the severity of diffuse hyperplasia or cell necrosis induced by p-cresidine in the urinary bladder. Although p53 has been implicated in bladder carcinogenesis in rodents and humans (Fujimoto et al., 1992; Yamamoto et al., 1995), the lack of apparent p53 involvement after 7 weeks of p-cresidine treatment in mice may be the result of its effects on tumor progression vs. initiation and promotion of preneoplastic lesions. Other studies have demonstrated a lack of p53 involvement in the initiation and promotional stage of carcinogenesis (Kemp et al., 1993). In the study by Kemp et al., p53 nullizygous mice actually displayed a reduced incidence of papillomas compared to heterozygous mice induced by a combined treatment of dimethylbenzanthracene (DMBA) and TPA. However, the latency for malignant conversion to carcinoma was reduced in p53 –/– mice and the percentage of papillomas converting to carcinomas was significantly higher in p53 –/– mice compared to p53 +/– mice. Genetic alterations in the p53 gene may have an earlier effect on the cancer process in other models of skin carcinogenesis, including UV-induced skin cancer, where UV-induced p53 mutations have been observed in normal sun-exposed skin biopsies (Ouhtit et al., 1997). Furthermore, there is a strong correlation among specific Chinese populations exposed to aflatoxin B1 in the incidence of hepatocelluar carcinoma and a mutational hot spot in the p53 gene (Shimizu et al., 1999). However, in our study we did not observe any atypical lesions in bladder epithelium after 7 weeks of treatment and hence could not determine the effect, if any, of p53 on bladder tumor development. Significant increases in p-cresidine-induced hepatic cytotoxicity and cell proliferation were only observed at dose levels that reduced animal body weight. This may suggest that liver tumor induction by p-cresidine may only occur at relatively high-dose levels. Also, the lack of p53 protein in nullizygote mice did not affect hepatocyte cell cycle progression or the rate of apoptosis as compared to heterozygote mice with one functional allele following p-cresidine exposure. Other studies have also demonstrated a similar apoptotic response in heterozygote and nullizygote mouse hepatocytes in response to irradiation or chemical exposure (Bellamy et al., 1997; Unger et al., 1998). It is believed that apoptosis may proceed through p53 independent pathways in murine hepatocytes and may explain, in part, the lack of p53 involvement in mouse liver tumors (Kress et al., 1992). Similarly, cell cycle progression may also occur independent of p53 expression as evidenced in mRNA and protein analyses of regenerating liver and hepatocellular carcinomas (Albrecht et al., 1997; Qin et al., 1998). Since cell cycle progression was measured by PCNA staining in our study, it can be concluded that p53 expression in mouse liver does not alter hepatic PCNA protein levels. In another p53 mouse study PCNA expression was shown to be 3–4-fold lower in irradiated tumors that contained a wild type p53 allele compared to tumors that lacked functional p53 protein (Venkatachalam et al., 1998). The lack of p53 involvement in PCNA expression in hepatocytes may be a tissue specific phenomenon or a result of unique effects elicited by p-cresidine-induced toxicity. We have selected a cytogenetic end point (i.e., micronucleus indution) to assess the influence of p53 status on the incidence of clastogenicity in bone marrow, since loss of p53 function has been associated with chromosomal instability in other systems or models of carcinogenesis (Bischoff et al., 1990; Bouffler et al., 1995; Donehower et al., 1995). Although bone marrow is not a target tissue for p-cresidine-induced tumors, it is generally considered to be an excellent surrogate tissue for determining chemically induced cytogenetic damage. A number of clastogenic carcinogens are capable of inducing micronuclei in bone marrow cells of mice, even though this tissue is seldom a target for tumor induction with such materials. In the present study, p-cresidine treatment or p53 status did not have an effect on the incidence of micronuclei in polychromatic erythrocytes (PCE) in bone marrow. This was surprising since loss of p53 function has been associated with chromosomal instability in other test systems or models of carcinogenesis, including the p53 mouse (Bischoff et al., 1990; Bouffler et al., 1995; Donehower et al., 1995). Bouffler et al. demonstrated increased chromosomal aberrations in p53 heterozygous and nullizygous bone marrow cells as compared to bone marrow from p53 wild type mice. However, there was not a significant difference in chromosome damage between p53 heterozygous and nullizygous mice, suggesting a p53 gene dosage effect. Several other studies support this hypothesis (Marty et al., 1999; Venkatachalam et al., 1998) and may help explain the lack of p53-dependent differences in chromosomal damage seen in our study. In addition, p-cresidine had no effect on micronuclei incidence in bone marrow PCE from either p53 genotype. This data, using a prolonged treatment regimen, extends earlier studies by Ashby et al. (1991) where p-cresidine was negative in the bone marrow micronucleus assay in both CBA and B6C3F1 mice after a single or 3 consecutive doses of p-cresidine. We also analyzed urinary bladder DNA for codon 12 mutations in the c-Ha-ras gene to evaluate any genotype-related differences in genotoxicity of p-cresidine in the primary tumor target tissue. Mutations in codon 12 are common among ras alterations found in bladder cancer (Orntoft and Wolf, 1998; Yamamoto et al., 1995). DNA strand breaks and point mutations induced by p-cresidine in mouse urinary bladder have been described previously (Jakubczak et al., 1996; Sasaki et al., 1998). Although other aromatic amines like dimethylbenzidine induce codon 12 c-Ha-ras gene mutations in non-hepatic rodent tissues (Reynolds et al., 1990), we did not see any treatment-related increases in mutation frequency at codon 12 of this gene in mouse bladder DNA of either p53 genotype. In conclusion, p53 function does not affect hepatic cytotoxicity or cell proliferation induced by p-cresidine. Also, after 7 weeks of continuous exposure to p-cresidine, the extent of cell necrosis and hyperplasia in the urinary bladder was indistinguishable between heterozygote and nullizygote mice. P53 involvement in mouse tumorigenesis may be more important in facilitating the malignant progression of neoplastic foci rather than in the initiation or promotion stage, and this effect may occur with simply a reduction in gene dosage. TABLE 1 Select Clinical Parameters in Mice Treated with p-Cresidine Treatment (mg/kg/day)  Genotype (n)  Body wt (g)  Liver wta (g/100g)  ALT (mu/ml)  RBC (106/μL)  HGB (g/dL)  HCT (%)   Note. Values represent mean ± standard deviations. The number of animals in each group is indicated within parenthesis.  aRepresents relative liver weights normalized for body weight. Alanine transaminase (ALT) values are expressed in milliunits of enzyme activity per milliliter of serum. Red blood cell (RBC) count, hemoglobin (HGB) concentration, and hematocrit (HCT) values are given as 106 cells per microliter of blood, grams per deciliter of blood, and volume percent of erythrocytes in whole blood, respectively.  b,cRepresent means statistically different (p = 0.05) from control using parametric ANOVA followed by the Dunnett's and Wilcoxon's tests, respectively.  Control  p53 +/– (6)  24.0 ± 1.8  5.71 ± 0.42  68.0 ± 42.0  9.81 ± 0.41  15.1 ± 0.7  49.5 ± 2.2     p53 –/– (6)  24.6 ± 3.5  4.94 ± 0.29  109.0 ± 107.0  9.82 ± 0.24  15.3 ± 0.3  49.9 ± 1.2   100  p53 +/– (5)  26.3 ± 1.0  5.78 ± 0.47  63.0 ± 29.0  8.87 ± 0.50b  13.7 ± 0.5b  44.7 ± 2.8b    p53 –/– (7)  23.5 ± 2.3  5.13 ± 0.32  78.0 ± 25.0  9.05 ± 0.74b  13.6 ± 1.4b  45.3 ± 4.6c  200  p53 +/– (4)  27.3 ± 0.7b  6.18 ± 0.57  76.0 ± 85.0  8.73 ± 0.34b  13.5 ± 0.7b  45.1 ± 2.0b    p53 –/– (7)  23.9 ± 1.8  5.38 ± 0.35  77.0 ± 40.0  9.03 ± 0.18b  13.9 ± 0.8  47.0 ± 1.2c  400  p53 +/– (8)  23.7 ± 2.2  6.99 ± 0.35b  71.0 ± 23.0  8.39 ± 0.23b  13.4 ± 0.7b  42.8 ± 1.7b    p53 –/– (4)  22.1 ± 3.4  5.70 ± 0.12b  149.0 ± 113.0  8.57 ± 0.29b  13.7 ± 0.7  44.7 ± 1.7c  800  p53 +/– (4)  20.0 ± 2.1b  7.65 ± 0.34b  197.0 ± 76.0b  7.55 ± 0.21b  11.3 ± 0.3b  35.4 ± 1.2b    p53 –/– (6)  21.2 ± 1.8  6.55 ± 0.45b  354.0 ± 163.0  7.69 ± 0.74b  11.8 ± 1.4b  37.0 ± 5.4c  Treatment (mg/kg/day)  Genotype (n)  Body wt (g)  Liver wta (g/100g)  ALT (mu/ml)  RBC (106/μL)  HGB (g/dL)  HCT (%)   Note. Values represent mean ± standard deviations. The number of animals in each group is indicated within parenthesis.  aRepresents relative liver weights normalized for body weight. Alanine transaminase (ALT) values are expressed in milliunits of enzyme activity per milliliter of serum. Red blood cell (RBC) count, hemoglobin (HGB) concentration, and hematocrit (HCT) values are given as 106 cells per microliter of blood, grams per deciliter of blood, and volume percent of erythrocytes in whole blood, respectively.  b,cRepresent means statistically different (p = 0.05) from control using parametric ANOVA followed by the Dunnett's and Wilcoxon's tests, respectively.  Control  p53 +/– (6)  24.0 ± 1.8  5.71 ± 0.42  68.0 ± 42.0  9.81 ± 0.41  15.1 ± 0.7  49.5 ± 2.2     p53 –/– (6)  24.6 ± 3.5  4.94 ± 0.29  109.0 ± 107.0  9.82 ± 0.24  15.3 ± 0.3  49.9 ± 1.2   100  p53 +/– (5)  26.3 ± 1.0  5.78 ± 0.47  63.0 ± 29.0  8.87 ± 0.50b  13.7 ± 0.5b  44.7 ± 2.8b    p53 –/– (7)  23.5 ± 2.3  5.13 ± 0.32  78.0 ± 25.0  9.05 ± 0.74b  13.6 ± 1.4b  45.3 ± 4.6c  200  p53 +/– (4)  27.3 ± 0.7b  6.18 ± 0.57  76.0 ± 85.0  8.73 ± 0.34b  13.5 ± 0.7b  45.1 ± 2.0b    p53 –/– (7)  23.9 ± 1.8  5.38 ± 0.35  77.0 ± 40.0  9.03 ± 0.18b  13.9 ± 0.8  47.0 ± 1.2c  400  p53 +/– (8)  23.7 ± 2.2  6.99 ± 0.35b  71.0 ± 23.0  8.39 ± 0.23b  13.4 ± 0.7b  42.8 ± 1.7b    p53 –/– (4)  22.1 ± 3.4  5.70 ± 0.12b  149.0 ± 113.0  8.57 ± 0.29b  13.7 ± 0.7  44.7 ± 1.7c  800  p53 +/– (4)  20.0 ± 2.1b  7.65 ± 0.34b  197.0 ± 76.0b  7.55 ± 0.21b  11.3 ± 0.3b  35.4 ± 1.2b    p53 –/– (6)  21.2 ± 1.8  6.55 ± 0.45b  354.0 ± 163.0  7.69 ± 0.74b  11.8 ± 1.4b  37.0 ± 5.4c  View Large TABLE 2 Histopathology in p53 Knockout Mice Treated with p   Heterozygous (mg/kg/day)  Nullizygous (mg/kg/day)     0  100  200  400  800  0  100  200  400  800   Notes. Values represent the number of animals positive for the end point listed. For diffuse hyperplasia, very slight and slight signify 4–6 and 6–8 epithelial cells thick, respectively. For individual cell necrosis in the bladder, very slight and slight signify 2–4 and ≥ 5 necrotic epithelial cells per high power field (×400), respectively. Histopathology was not done on bladders used for DNA isolation. Four bladders from each treatment group (2 Null and 2 Het) were used for DNA isolation and subsequent PCR-RFLP analysis of c-Ha-ras point mutations. In addition, 2 nullizygous mice (one in each of the 200 and 800 mg/kg dose groups) did not survive the 7-week exposure period and therefore their bladders were not examined.  Urinary bladder                       Number of animals examined  4  3  2  6  2  4  5  5  2  5    Hyperplasia, transitional epithelium                        Very slight  1  0  1  4  1  0  0  0  1  2     Slight  0  0  0  1  1  0  0  0  0  3    Necrosis, individual cell transitional epithelium                         Very slight  0  3  2  5  1  0  3  4  1  1     Slight  0  0  0  1  1  0  0  0  1  4   Liver                       Number of animals examined  6  5  4  8  4  6  7  7  4  7    Hypertrophy, hepatocyte  0  0  0  6  4  0  0  0  0  6    Necrosis, individual cell, hepatocyte                         Very slight  1  2  2  5  0  3  3  3  2  0     Slight  0  0  0  1  4  0  0  0  0  6    Pigment laden macrophages                        Very slight  0  0  0  0  4  0  0  0  0  5    Heterozygous (mg/kg/day)  Nullizygous (mg/kg/day)     0  100  200  400  800  0  100  200  400  800   Notes. Values represent the number of animals positive for the end point listed. For diffuse hyperplasia, very slight and slight signify 4–6 and 6–8 epithelial cells thick, respectively. For individual cell necrosis in the bladder, very slight and slight signify 2–4 and ≥ 5 necrotic epithelial cells per high power field (×400), respectively. Histopathology was not done on bladders used for DNA isolation. Four bladders from each treatment group (2 Null and 2 Het) were used for DNA isolation and subsequent PCR-RFLP analysis of c-Ha-ras point mutations. In addition, 2 nullizygous mice (one in each of the 200 and 800 mg/kg dose groups) did not survive the 7-week exposure period and therefore their bladders were not examined.  Urinary bladder                       Number of animals examined  4  3  2  6  2  4  5  5  2  5    Hyperplasia, transitional epithelium                        Very slight  1  0  1  4  1  0  0  0  1  2     Slight  0  0  0  1  1  0  0  0  0  3    Necrosis, individual cell transitional epithelium                         Very slight  0  3  2  5  1  0  3  4  1  1     Slight  0  0  0  1  1  0  0  0  1  4   Liver                       Number of animals examined  6  5  4  8  4  6  7  7  4  7    Hypertrophy, hepatocyte  0  0  0  6  4  0  0  0  0  6    Necrosis, individual cell, hepatocyte                         Very slight  1  2  2  5  0  3  3  3  2  0     Slight  0  0  0  1  4  0  0  0  0  6    Pigment laden macrophages                        Very slight  0  0  0  0  4  0  0  0  0  5  View Large TABLE 3 Incidence of MN-PCE in Control and p-Cresidine Treated p53 Mice Treatment (mg/kg/day)  Zygoisty  n  MN-PCE  %PCE   Note. Values represent the mean ± SD. n represents the number of animals per group. MN-PCE represents micronucleated polychromatic erythrocytes per 1000 PCE. % PCE represents the percentage of polychromatic erythrocytes to monochromatic erythrocytes.  0  Hetero  6  3.83 ± 2.14  45.8 ± 8.8   0  Null  6  2.67 ± 1.37  48.8 ± 8.3   800  Hetero  4  1.75 ± 0.96  56.5 ± 8.3   800  Null  6  2.50 ± 2.81  55.6 ± 5.9  Treatment (mg/kg/day)  Zygoisty  n  MN-PCE  %PCE   Note. Values represent the mean ± SD. n represents the number of animals per group. MN-PCE represents micronucleated polychromatic erythrocytes per 1000 PCE. % PCE represents the percentage of polychromatic erythrocytes to monochromatic erythrocytes.  0  Hetero  6  3.83 ± 2.14  45.8 ± 8.8   0  Null  6  2.67 ± 1.37  48.8 ± 8.3   800  Hetero  4  1.75 ± 0.96  56.5 ± 8.3   800  Null  6  2.50 ± 2.81  55.6 ± 5.9  View Large FIG. 1. View largeDownload slide Urinary bladder from control and p-cresidine treated p53 nullizygous mice (H&E, ×140). (A) Normal transitional epithelium (TE), connective tissue (CT), and muscularis (M) from a control male bladder. (B) Hyperplastic transitional epithelium demonstrating individual cell necrosis (arrow) from a male given 800 mg/kg p-cresidine for 7 weeks. FIG. 1. View largeDownload slide Urinary bladder from control and p-cresidine treated p53 nullizygous mice (H&E, ×140). (A) Normal transitional epithelium (TE), connective tissue (CT), and muscularis (M) from a control male bladder. (B) Hyperplastic transitional epithelium demonstrating individual cell necrosis (arrow) from a male given 800 mg/kg p-cresidine for 7 weeks. FIG. 2. View largeDownload slide Hepatic PCNA and free 3`OH DNA fragment labeling indices in control and p-cresidine treated heterozygous and nullizygous p53 mice. (A) Bar graph illustrates the percentage of hepatocytes containing immunoreactive PCNA protein in control and p-cresidine treated (800 mg/kg) p53 mice. Values represent the mean ± SD of 4–6 individual animals. (B) Percentage of hepatocytes containing immunoreactive labeled nucleotides (apoptotic cells) from the same mice. *Denotes statistically significant from control (p < 0.05). FIG. 2. View largeDownload slide Hepatic PCNA and free 3`OH DNA fragment labeling indices in control and p-cresidine treated heterozygous and nullizygous p53 mice. (A) Bar graph illustrates the percentage of hepatocytes containing immunoreactive PCNA protein in control and p-cresidine treated (800 mg/kg) p53 mice. Values represent the mean ± SD of 4–6 individual animals. (B) Percentage of hepatocytes containing immunoreactive labeled nucleotides (apoptotic cells) from the same mice. *Denotes statistically significant from control (p < 0.05). FIG. 3. View largeDownload slide Hepatic immunohistochemical staining for PCNA; G1, S, G2, and M phases of the cell cycle in p53 mice treated with p-cresidine. (A) Photomicrographs depicting labeled hepatocytes in each phase of the cell cycle, ×920. (B) Proportion of labeled hepatocytes in each phase of the cell cycle from p53 +/– and –/– mice (n = 4 and n = 6 for heterozygous and homozygous mice, respectively) treated with 800 mg/kg/day p-cresidine for 7 weeks. Data from both genotypes is included in the same graph because the proportion of cells in each phase of the cell cycle was not affected by p53 allele status. FIG. 3. View largeDownload slide Hepatic immunohistochemical staining for PCNA; G1, S, G2, and M phases of the cell cycle in p53 mice treated with p-cresidine. (A) Photomicrographs depicting labeled hepatocytes in each phase of the cell cycle, ×920. (B) Proportion of labeled hepatocytes in each phase of the cell cycle from p53 +/– and –/– mice (n = 4 and n = 6 for heterozygous and homozygous mice, respectively) treated with 800 mg/kg/day p-cresidine for 7 weeks. Data from both genotypes is included in the same graph because the proportion of cells in each phase of the cell cycle was not affected by p53 allele status. FIG. 4. View largeDownload slide Methemoglobin levels in control and p-cresidine treated p53 heterozygous and nullizygous mice. Bar graph illustrates the mean ± SD of measurements from 4–8 individual animals. *Denotes statistically significant from control (p < 0.05). FIG. 4. View largeDownload slide Methemoglobin levels in control and p-cresidine treated p53 heterozygous and nullizygous mice. Bar graph illustrates the mean ± SD of measurements from 4–8 individual animals. *Denotes statistically significant from control (p < 0.05). FIG. 5. View largeDownload slide PCR-RFLP analysis of wild type and mutant c-Ha-ras alleles in control and p-cresidine treated p53 +/– mice. Ethidium bromide-stained agarose gels illustrating the electrophoretic separation of wild type and mutant ras PCR products digested with Bst O1. (A) Lane 1, 20 bp DNA ladder; lanes 2–5, digested PCR products that were generated from p53 +/– genomic DNA that contained increasing amounts (attograms) of ras mutant standard; lane 6, undigested PCR product. (B) Lane 1, undigested PCR product; lanes 2 and 3, digested products from control p53 +/– DNA and p53 +/– DNA containing 3 attograms of ras mutant standard, respectively; lane 4, DNA ladder; lanes 5 and 6, digested PCR products from urinary bladder DNA from 11-week-old control p53 +/– mice (n = 2); lanes 7 and 8, products from urinary bladder DNA from 11-week-old p53 +/– mice (n = 2) treated with p-cresidine for 7 weeks. FIG. 5. View largeDownload slide PCR-RFLP analysis of wild type and mutant c-Ha-ras alleles in control and p-cresidine treated p53 +/– mice. Ethidium bromide-stained agarose gels illustrating the electrophoretic separation of wild type and mutant ras PCR products digested with Bst O1. 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TI - Evaluation of Cytotoxicity, Cell Proliferation, and Genotoxicity Induced by p-Cresidine in Hetero- and Nullizygous Transgenic p53 Mice
JF - Toxicological Sciences
DO - 10.1093/toxsci/55.2.361
DA - 2000-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/evaluation-of-cytotoxicity-cell-proliferation-and-genotoxicity-induced-4MTXR1gAzY
SP - 361
EP - 369
VL - 55
IS - 2
DP - DeepDyve
ER -