The Dow Chemical Company, Health and Environmental Research Laboratory, 1803 Building, Midland, Michigan 48674
Received July 18, 1999; accepted January 11, 2000
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ABSTRACT |
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Key Words: p53 mice; p53 function; genotoxicity; cell proliferation; cytotoxicity; p-cresidine; urinary bladder; liver; risk assessment; bioassay.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 (x300) 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.
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RESULTS |
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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.
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DISCUSSION |
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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 34-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.
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NOTES |
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