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Frequent p53 and H-ras Mutations in Benzene- and Ethylene Oxide-Induced Mammary Gland Carcinomas from B6C3F1 Mice
1 Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709, USA Correspondence: Address correspondence to: Christopher D. Houle, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA; e-mail:houle{at}niehs.nih.gov
Benzene and ethylene oxide are multisite carcinogens in rodents and classified as human carcinogens by the National Toxicology Program. In 2-year mouse studies, both chemicals induced mammary carcinomas. We examined spontaneous, benzene-, and ethylene oxide-induced mouse mammary carcinomas for p53 protein expression, using immunohistochemistry, and p53 (exons 5–8) and H-ras (codon 61) mutations using cycle sequencing techniques. p53 protein expression was detected in 42% (8/19) of spontaneous, 43% (6/14) of benzene-, and 67% (8/12) of ethylene oxide-induced carcinomas. However, semiquantitative evaluation of p53 protein expression revealed that benzene- and ethylene oxide-induced carcinomas exhibited expression levels five- to six-fold higher than spontaneous carcinomas. p53 mutations were found in 58% (7/12) of spontaneous, 57% (8/14) of benzene-, and 67% (8/12) of ethylene oxide-induced carcinomas. H-ras mutations were identified in 26% (5/19) of spontaneous, 50% (7/14) of benzene-, and 33% (4/12) of ethylene oxide-induced carcinomas. When H-ras mutations were present, concurrent p53 mutations were identified in 40% (2/5) of spontaneous, 71% (5/7) of benzene-, and 75% (3/4) of ethylene oxide-induced carcinomas. Our results demonstrate that p53 and H-ras mutations are relatively common in control and chemically induced mouse mammary carcinomas although both chemicals can alter the mutational spectra and more commonly induce concurrent mutations.
Key Words: p53 • H-ras • benzene • ethylene oxide • mammary gland carcinoma • mice • carcinogen Abbreviations: EtO, ethylene oxide • PCR, polymerase chain reaction • AB, automation buffer • ROS, reactive oxygen species • DNA, deoxyribonucleic acid • IHC, immunohistochemistry • A, C, G, T, adenine, cytosine, guanine, thymine • NTP, National Toxicology Program
Benzene is used as an intermediate for the manufacturing of polymers, detergents, pesticides, drugs and other industrial chemicals and is also used as an additive in gasoline (Runion and Scott, 1985; NTP, 1986; Maltoni et al., 1989; Infante et al., 1990; Yardley-Jones et al., 1991). Benzene is a well-documented environmental pollutant that induces hematotoxicity and hematopoietic neoplasia in humans (IARC, 1994; NTP, 2004) and is a multisite carcinogen in rodents, including the development of mammary carcinomas in mice (Maltoni and Scarnato, 1979; Huff et al., 1989; Maltoni et al., 1989; NTP, 2004). Several studies indicate that metabolism of benzene to its reactive metabolites is a prerequisite for its cytotoxic, genotoxic, and carcinogenic properties (Gut et al., 1996; Snyder and Hedli, 1996; Valentine et al., 1996; Abernethy et al., 2004). It has long been recognized that several metabolites of benzene can bind covalently to proteins and nucleic acids (Creek et al., 1997), and certain metabolites can be oxidized by peroxidases resulting in the production of reactive oxygen species (ROS) and DNA reactive quinones (Hiraku and Kawanishi, 1996; Farris et al., 1997). Several studies suggest that p53 dysfunction is a potential factor in benzene-induced carcinogenesis (Donehower, 1996; Boley et al., 2002; Yoon et al., 2003; Hirabayashi et al., 2004). Ethylene oxide (EtO) is used as an intermediate in the production of several industrial chemicals and is also used as a gas sterilant for medical equipment (IARC, 1994; NTP, 2004; Recio et al., 2004). In humans, EtO exposure has been associated with the development of leukemia, lymphoma, pancreatic, stomach, and breast cancers (Bisanti et al., 1993; IARC, 1994; Steenland et al., 2003). In rodents, EtO is a multisite carcinogen capable of inducing mammary carcinomas in female mice (IARC, 1994; Snellings et al., 1984). EtO is a direct-acting carcinogen that reacts with nucleophilic molecules to form a variety of different DNA adducts and is a powerful mutagen and clastogen at all phylogenetic levels Segerback, 1990; Walker et al., 1992; IARC, 1994; Wu et al., 1999; Melnick, 2002). Inhaled EtO induces an increased frequency of lacI and/or Hprt mutations in mice, rats, and humans and 1 study in humans correlated EtO exposure with elevated ras and p53 expression (Sisk et al., 1997; Ember et al., 1998; Tates et al., 1999; van Sittert et al., 2000; Walker et al., 2000; Lorenti Garcia et al., 2001). p53 and ras are the 2 most commonly reported altered genes in human cancers and mutations in both genes often occur in the same cancer cell (Boukamp et al., 1995; Lengauer et al., 1997; Azzoli et al., 1998; Tuveson and Jacks, 1999). A number of lines of evidence support an interaction between p53 and ras in the process of tumorigenesis. Constitutively active ras/mapk signaling leads to elevated levels of p53 provoking cell cycle arrest in some cells (Lloyd et al., 1997; Sewing et al., 1997; Woods et al., 1997) and cellular senescence in others (Serrano et al., 1997). It is thought that the mechanisms leading to these cellular responses must be circumvented to induce tumorigenesis (Serrano et al., 1997; McMahon and Woods, 2001; Ferbeyre et al., 2002). In vitro studies have shown that the combination of mutant p53 and activated H-ras can efficiently transform primary-cultured fibroblasts, whereas p53 mutation by itself cannot (Zambetti et al., 1992; Kikuchi-Yanoshita et al., 1995). Likewise, oncogenic ras is unable to transform primary cells due to the induction of p53-mediated cell cycle arrest (Serrano et al., 1997; Palmero et al., 1998; Ferbeyre et al., 2000; Malumbres et al., 2000; Pearson et al., 2000;). In addition, MMTV-ras transgenic mice are highly susceptible to the development of mammary tumors and the incidence increases with the additional loss of p53 (Hundley et al., 1997). Based on the high frequency of p53 mutations and the frequent activation of ras signaling in human mammary cancers, we set out to determine how these genes might be involved, either individually or combined, in spontaneous, benzene-, and ethylene oxide-induced mouse mammary carcinogenesis. We additionally attempted to identify chemical-specific and universal alterations common to mouse mammary tumors in general.
Mammary Carcinomas The benzene study included 50 female B6C3F1 mice exposed to 0, 25, 50, or 100 mg/kg of benzene via gavage 5 days per week for 103 weeks (NTP, 1986; Huff et al., 1989). The ethylene oxide study included 50 female B6C3F1 mice exposed to 0, 50, or 100 ppm of ethylene oxide for 6 hours per day, 5 days per week for 102 weeks (NTP, 1987). Husbandry and experimental procedures complied with the requirements set forth by the Public Health Service’s Guide for the Care and Use of Laboratory Animals. Representative benzene- and ethylene oxide-induced mammary carcinomas from these studies were examined for p53 protein expression and for p53 (exons 5–8) and H-ras (codon 61) mutations. For comparison, a number of spontaneous mammary carcinomas selected from female control mice in National Toxicology Program (NTP) studies were analyzed for p53 and ras alterations. These NTP studies were conducted around the same time-period as the benzene and ethylene oxide studies and had similar collection procedures, fixation methods, and tissue processing techniques. All mammary carcinomas were fixed in 10% neutral-buffered formalin, routinely processed, and embedded in paraffin. The following tumor sets were used for analysis of p53 and H-ras alterations: 14 mammary carcinomas from benzene-exposed female mice, 12 mammary carcinomas from EtO-exposed female mice, and 19 spontaneous mammary carcinomas from control female mice.
Immunohistochemistry p53 protein expression was graded semiquantitatively using the quickscore method (Detre et al., 1995). This method independently grades the proportion of cells stained using a 6-point scale (1 = 0–4%; 2 = 5–19%; 3 = 20–39%; 4 = 40–59%; 5 = 60–79%; 6 = 80–100%) and the intensity of the stain using a scale of 0 to 3 (0 = no expression; 1 = weak; 2 = intermediate; 3 = strong). The 2 grades are multiplied together to arrive at the quickscore that can range from a minimum of 0 to a maximum of 18.
DNA Isolation and Amplification The second exon of H-ras was amplified using nested primers as previously described (Devereux et al., 1991). In addition, PCR amplification of exons 5–8 of the p53 gene was done using PCR sequencing primers and annealing temperature profiles as reported previously (Lambertini et al., 2005). Positive and no-DNA controls for both H-ras and p53 mutations were run with all sets of reactions. Amplified DNA was electrophoresed on 1% agarose minigels with ethidium bromide to test for proper product size and purity. The amplification bands were cut from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.
Cycle Sequencing
Both benzene and EtO induce mammary carcinomas in female B6C3F1 mice (NTP, 1986, 1987; Huff et al., 1989) (Table 1). These chemically induced mammary neoplasms exhibited a variety of histologic patterns, although no specific pattern was associated with a particular chemical or dose group. The same variety of histologic patterns was observed in spontaneous mammary tumors. Most tumors displayed glandular patterns with occasional solid regions and some tumors had spindle cells streaming around adjacent glands. A few exhibited varying degrees of squamous differentiation and were diagnosed as adenosquamous carcinomas.
Benzene-Induced Mammary Carcinomas The incidence of altered p53 protein expression, p53 mutations, and H-ras mutations in benzene-induced mammary carcinomas is presented in Table 2. There were no clear differences in the frequency or pattern of p53 or H-ras mutations between the various benzene exposure levels. p53 protein expression (Figure 1a) was detected in 43% (6/14) of the tumors and was localized to the nucleus. The average quickscore for all benzene-induced tumors was 3.57. p53 mutations (Figure 2A) were detected in 57% (8/14) of the tumors and 3 of these exhibited 2 or more mutations per tumor. Mutations were identified at codons 147, 149, 150, 151, 199, 203, 207, 210, 212, 244, and 290. Overall, we found 14 base pair substitutions including double mutations at codons 150 (exon 5) and 290 (exon 8). Of the 12 mutant codons, 9 were missense mutations and 3 were silent mutations. Of the 14 p53 mutations, 71% involved either guanine or adenine bases. These mutations were most common in exons 5 (5/14) and 6 (6/14), which accounted for 79% of all p53 mutations.
Codon 61 H-ras mutations (Figure 3A) were detected in 50% (7/14) of the benzene-induced mammary tumors and all were missense mutations. Six of the 7 mutations (86%) involved the second base (A), whereas just 1 involved the first base (C). Five of the 7 H-ras mutations were A to G transitions (71%) resulting in an amino acid change from glutamine to arginine.
Ethylene Oxide-Induced Mammary Carcinomas The incidence of altered p53 protein expression, p53 mutations, and H-ras mutations in ethylene oxide-induced mammary carcinomas is presented in Table 3. p53 protein expression (Figure 1b) was detected in 67% (8/12) of the ethylene oxide-induced mammary carcinomas and was localized to the nucleus. The average quickscore for all EtO-induced tumors was 3.83. p53 mutations (Figure 2B) were detected in 67% (8/12) of the carcinomas with 5 of the 8 having 2 or more mutations. Mutations were identified at codons 188, 189, 191, 198, 241, 246, 252, 255, and 264. Overall, we found 14 base substitutions including 8 silent mutations, 5 missense mutations, and 1 nonsense mutation. Of the 14 mutations, 9 (64%) involved guanine bases. p53 mutations were most common in exon 7 (7/14), 57% of which occurred at codon 241.
In addition, there were no mutations in exon 5 and all of the mutations from exon 8 were localized to codon 264. The exposure level of ethylene oxide also appeared to have an influence on the incidence of p53 mutations and protein expression. Three of the 7 tumors from the 50-ppm dose group exhibited increased p53 protein expression, whereas all 5 of the tumors from the 100-ppm dose group exhibited p53 protein expression. Similarly, of the 7 tumors from the 50-ppm dose group 3 p53 mutations were found as compared to 9 mutations in the 5 tumors from the 100-ppm dose group. Codon 61 H-ras mutations (Figure 3B) were detected in 33% (4/12) of the ethylene oxide-induced mammary carcinomas. All of these were missense mutations and in every tumor the mutation was localized to the second base (A), although 1 tumor had a double mutation that also involved the first base (C). Overall, there were 2 amino acid changes from glutamine to arginine (A to G) and 2 from glutamine to leucine (A to T).
Spontaneous Mammary Carcinomas
Codon 61 H-ras mutations were detected in 26% (5/19) of the spontaneous mammary carcinomas (Figure 3C). All 5 of these were missense mutations, the majority of which resulted in amino acid changes of glutamine to lysine (C to A). Figures 4 and 5 are group summaries of the genetic alterations showing the mutational spectra or base preferences between spontaneous, benzene-, and ethylene oxide-induced mammary carcinomas.
The present study documents the incidence and spectrum of p53 (exons 5–8) and H-ras (codon 61) mutations in benzene- and ethylene oxide-induced mouse mammary carcinomas and compares them with those found in spontaneous mouse mammary carcinomas. The hypothesis was that the chemically induced tumors would exhibit a higher incidence and different spectrum of mutations and would more commonly exhibit concurrent alteration of both genes. Our results demonstrate that p53 and H-ras mutations are relatively common in mouse mammary carcinomas, although concurrent H-ras and p53 mutations were more common in the chemically induced tumors. Concurrent mutations occurred in 71% (5/7) and 75% (3/4) of the benzene- and ethylene oxide-induced mammary tumors, respectively, as compared to only 40% (2/5) of the spontaneous mammary carcinomas. Furthermore, the chemically induced tumors exhibited a distinct shift in the p53 and H-ras mutational spectra as compared to spontaneous tumors suggesting that benzene and EtO exposure induces mammary specific genetic alterations predisposing female mice to mammary tumor development. The difference in mutation profiles between spontaneous and chemically induced neoplasms suggests that different mechanisms are likely involved. A comparison of the alterations noted in spontaneous and chemically induced mammary carcinomas revealed several notable differences (Figures 4 and 5). H-ras mutations in chemically induced tumors most frequently involved the second base of codon 61, whereas mutations in spontaneous tumors usually involved the first base. In spontaneous tumors, the majority of the H-ras mutations resulted in amino acid changes of glutamine to lysine (C to A), whereas 10 of the 11 H-ras mutations in the chemically induced mammary tumors resulted in amino acid changes from glutamine to either leucine (A to T) or arginine (A to G). An additional difference between spontaneous and the EtO-induced neoplasms was the location of the p53 mutations. Interestingly, the EtO-induced neoplasms exhibited p53 mutations in exons 6, 7, and 8 but not in exon 5, whereas the spontaneous tumors had the majority of their p53 mutations in exon 5, but completely lacked mutations in exon 6. Another striking difference with the p53 gene was the shift from predominantly C to T mutations in spontaneous tumors to a relatively high number of mutations involving guanine and adenine in benzene-induced tumors and guanine in EtO-induced tumors. Overall, these mutational data suggest that purine bases (guanine and adenine) serve as the primary targets for mutation by both chemicals, whereas mutation involving pyrimidine bases (mostly cytosine) appears to be a more common spontaneous event. G to A mutations following EtO exposure have been described by others and are frequently attributed to the most common EtO DNA adduct, N7-(2-hydroxyethyl)guanine (van Sittert et al., 2000). Although benzene itself is not highly reactive with DNA, several benzene metabolites can form DNA adducts and 2 of these are known to induce G to A mutations (Gaskell et al., 2005). G to C transversions were the second most common p53 mutations in EtO-induced tumors but were rare in benzene-induced and spontaneous tumors. Similar mutations were found in 1,3-butadiene-induced mammary tumors and it was speculated that epoxide derived N7 guanine adducts were the cause (Zhuang et al., 2002). As mentioned previously, these adducts are also common following EtO exposure suggesting that a similar mechanism may be involved. Alternatively, ROS-induced mutations should be considered given that G to C transversions are one of the more common mutations generated by singlet oxygen (Jackson and Loeb, 2001). In benzene-induced tumors, A to G transitions were the most common H-ras mutations and the second most common p53 mutations. H-ras A to G transitions were also one of the more common mutations identified in EtO-induced tumors. One major mechanism capable of inducing A to G mutations is adenine adduct formation (You et al., 1992; Routledge et al., 1993). Previous studies have shown that EtO is capable of forming N1 and N3 adenine adducts (van Sittert et al., 2000). In addition, malonaldehyde, a naturally occurring product of lipid peroxidation, can react with DNA to form adducts to deoxyadenosine and deoxyguanosine (Marnett, 1999). Considering the high lipid content of mammary tissue and the potential for redox cycling related to benzene metabolism (Avogbe et al., 2005) it is possible that this mechanism is involved in benzene-induced mammary tumorigenesis. A to T transversions were one of the most common H-ras mutations found in EtO-induced mammary carcinomas. Previous studies have demonstrated that EtO exposure was capable of inducing A to T mutations (Recio et al., 2004). A number of other chemicals are also known to induce these mutations specifically in H-ras codon 61 (Vousden et al., 1986; Wiseman et al., 1986; Sills et al., 1999). As with A to G mutations, one potential mechanism for inducing A to T mutations would be through the formation of adenine adducts, particularly those that lead to depurination with insertion of an adenine opposite the apurinic site (Sagher and Strauss, 1983). Our study showed that C to T transitions were particularly common p53 mutations in spontaneous mammary carcinomas. One mechanism for the formation of these mutations is deamination of 5-methylcytosine (Steinberg and Gorman, 1992). The prevalence of this mutation may be related to the presence of a number of methylated CpG sites within the p53 gene. Transition mutations at methylated CpG sites are common in many cancers (Pfeifer, 2000), and p53 has a number of potentially methylated CpG sites. Additional studies would be necessary to determine if this mechanism was related to the p53 mutations detected in this study. EtO can also form N3 cytosine adducts (Li et al., 1992) that could, through hydrolytic deamination, result in the C to T mutations seen in some of the EtO-induced tumors. In addition, oxidative damage to cytosine results in at least 40 modified species some of which have the potential to induce C to T mutations (Jackson and Loeb, 2001; Lee et al., 2002). C to A mutations were particularly common in the H-ras gene from spontaneous mammary tumors. Other investigators have demonstrated a high incidence of H-ras C to A mutations in spontaneous mouse liver tumors suggesting that these are likely spontaneously derived (Rumsby et al., 1991; Goodrow et al., 1994; Parsons et al., 2002). Endogenous mutagens such as ROS are also capable of causing these mutations suggesting a possible mechanism whereby these mutations may arise spontaneously over time. EtO-induced mammary carcinomas exhibited a high incidence of silent p53 mutations, which may suggest an overall increase in the general mutation rate. Others have suggested that the p53 gene can become hypermutable under certain conditions (Strauss, 1997). Although the exact mechanism for this finding is uncertain, it has been shown that silent mutations are not necessarily benign in that they have the potential to create or destroy splice sites (Hongyo et al., 1995; Strauss, 1997). In addition, although the amino acid remains unchanged, it is possible that these mutations may alter translation efficiency (Strauss, 1997). An additional consideration for this finding would be rare polymorphisms. Only the EtO-induced tumors exhibited a clear dose-related response in relation to the level of p53 protein expression and the number of p53 mutations. Furthermore, there appears to be a better correlation between p53 protein expression and concurrent p53 mutation in the high dose group. Interestingly, the tumor response in vivo did not show a dose-related response, suggesting that p53 mutational analysis may be a more sensitive method for identifying dose response with this chemical. Benzene-induced tumors exhibited a wider range of mutations as compared to EtO-induced and spontaneous tumors. Figure 5 illustrates this point by showing the base preference for p53 mutations in the 3 respective groups. The spontaneous and EtO-induced neoplasms exhibited a clear preference for cytosine and guanine bases, respectively. In the benzene-induced neoplasms, both guanine and adenine bases were common targets. In addition, mutations in the benzene-induced neoplasms were more evenly distributed between all 4 bases as compared to those identified in the spontaneous or EtO-induced neoplasms. This is perhaps not surprising considering the complex nature of benzene metabolism and the potential generation of a number of different genotoxic metabolites. This may suggest that benzene-induced mutations are generated through a number of different mechanisms or that the predominant mechanism results in widespread random mutations such as those that might occur with some forms of oxidative damage (Jackson and Loeb, 2001). The percentage of tumors exhibiting p53 protein expression was similar between spontaneous and benzene-induced mammary tumors (42% and 43%, respectively) but was slightly higher for EtO-induced tumors (67%). However, the overall p53 protein expression levels, as measured by the quickscore, were much higher in benzene- (3.57) and EtO-induced (3.83) tumors as compared to spontaneous tumors (0.63). Also, although EtO-induced tumors more frequently exhibited p53 protein expression, the benzene-induced tumors tended to exhibit higher intensity staining when present. Interestingly, p53 protein expression in the EtO-induced tumors was often most intense in cells with a spindle cell morphology although the significance of this finding is uncertain. In certain instances, we noted discordance between p53 protein expression and p53 mutation. This has been reported in other tumor types although the exact mechanisms are not always understood (Greenblatt et al., 1994). One possible consideration is that not all p53 mutations result in enhanced p53 protein stability. Another would be related to sampling variation particularly when many of these alterations are not universal changes present throughout the entire tumor. Additional sample variation could occur due to differing proportions of stroma and tumors cells. The presence of p53 protein expression without p53 mutation could be due to p53 mutations outside the regions examined or possibly due to alteration of other proteins downstream of p53 (Greenblatt et al., 1994). In conclusion, our results show that, like human breast cancers, multiple genetic pathways are altered in mouse mammary carcinomas. We demonstrate that p53 and H-ras mutations are relatively common in mouse mammary carcinomas although both benzene and ethylene oxide are capable of shifting the mutational spectra of both genes. This chemically induced shift in mutational spectra likely underlies the mammary carcinogenic properties of benzene and ethylene oxide in the mouse. Moreover, we show that concurrent H-ras and p53 mutations are more common in the benzene-and ethylene oxide-induced tumors suggesting a potential interaction between these genes in the process of chemically induced mammary carcinogenesis.
The authors thank Dr. Kimberly McAllister and Dr. Yong-baek Kim for their critical reviews of the manuscript. We also thank the National Toxicology Program and all those from the many labs that contributed to these studies. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
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Abstract
Introduction
-33P dideoxynucleotide terminators (A, C, G, and T) into the sequencing products. The amplification primers also served as the sequencing primers. The sequencing reaction products were analyzed by electrophorectic separation on an 8% acrylimade gel containing 8M urea. Gels were dried and exposed to X-ray films overnight. 
TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair-deficient cells. Nucleic Acids Res, 30, 3566-73
