High frequency of codon 61 K-ras A->T transversions in lung and Harderian gland neoplasms of B6C3F1 mice exposed to chloroprene (2-chloro-1,3-butadiene) for 2 years, and comparisons with the structurally related chemicals isoprene and 1,3-butadiene

R.C. Sills2, H.L. Hong, R.L. Melnick, G.A. Boorman and T.R. Devereux1

Environmental Toxicology Program and
1 Environmental Carcinogenesis Program, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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Chloroprene is the 2-chloro analog of 1,3-butadiene, a potent carcinogen in laboratory animals. Following 2 years of inhalation exposure to 12.8, 32 or 80 p.p.m. chloroprene, increased incidences of lung and Harderian gland (HG) neoplasms were observed in B6C3F1 mice at all exposure concentrations. The present study was designed to characterize genetic alterations in the K- and H-ras protooncogenes in chloroprene-induced lung and HG neoplasms. K-ras mutations were detected in 80% of chloroprene-induced lung neoplasms (37/46) compared with only 30% in spontaneous lung neoplasms (25/82). Both K- and H-ras codon 61 A->T transversions were identified in 100% of HG neoplasms (27/27) compared with a frequency of 56% (15/27) in spontaneous HG neoplasms. The predominant mutation in chloroprene-induced lung and HG neoplasms was an A->T transversion at K-ras codon 61. This mutation has not been detected in spontaneous lung tumors of B6C3F1 mice and was identified in only 7% of spontaneous HG neoplasms. In lung neoplasms, greater percentages (80 and 71%) of A->T transversions were observed at the lower exposures (12.8 and 32 p.p.m.), respectively, compared with 18% at the high exposure. In HG neoplasms, the percentage of A->T transversions was the same at all exposure concentrations. The chloroprene-induced ras mutation spectra was similar to that seen with isoprene, where the predominant base change was an A->T transversion at K-ras codon 61. This differed from 1,3-butadiene, where K-ras codon 13 G->C transitions and H-ras codon 61 A->G transitions were the predominant mutations. The major finding of K-ras A->T transversions in lung and Harderian gland neoplasms suggests that this mutation may be important for tumor induction by this class of carcinogens.

Abbreviations: H & E, hematoxylin and eosin; HG, Harderian gland; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SSCP, single-stranded conformational polymorphism analysis.


    Introduction
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Chloroprene (2-chloro-1,3-butadiene), isoprene (2-methyl-1,3-butadiene) and 1,3-butadiene are all carcinogenic in rats and mice following inhalation exposure (14). Chloroprene is used almost exclusively as an intermediate in the production of polychloroprene elastomer (neoprene). Polychloroprene is used in the manufacture of automotive rubber goods, wire and cable coatings, construction applications, fabric coatings, cements, sealants and adhesives (57). The National Occupational Exposure Survey estimated that ~17 000 workers were exposed to chloroprene during 1981–1983 (8). Correlations between exposure to chloroprene in the workplace and lung cancer risk have been reported (9,10).

The overall genotoxicity profile of chloroprene has been complicated by differences in the findings reported by a number of laboratories. In Salmonella typhimurium strains TA 100 and TA 1530, chloroprene was reported to be mutagenic, with and without metabolic activation with induced liver S9 enzymes (5,11,12). More recently, Zeiger et al. found no evidence of mutagenic activity in four strains of S.typhimurium, including strain TA 100 (1). Westphal et al. (13) attributed the reported mutagenic effects of chloroprene to be caused by decomposition products that accumulated in aged chloroprene samples. Similar differences in genotoxicity profiles were reported in mammalian systems in vivo. In some studies, chloroprene induced dominant lethal mutations in germ cells of male rats and mice, and chromosomal aberrations in bone marrow cells of C57BL/6 mice (5,14). However, in another in vivo study, Tice et al. demonstrated no effects on the frequencies of sister chromatid exchanges, chromosomal aberrations or micronucleated erythrocytes in bone marrow or peripheral blood in chloroprene-exposed B6C3F1 mice (15).

Chloroprene, isoprene and butadiene are multi-site carcinogens, and there is clear evidence of genotoxicity for butadiene and isoprene (16). For these reasons and because of conflicting evidence of mutagenicity for chloroprene (13,17), we decided to examine chloroprene-induced tumors for point mutations in the ras gene as evidence of in vivo genotoxicity. Specifically, lung and Harderian gland (HG) neoplasms of B6C3F1 mice were examined for mutations in K- and H-ras genes, because these two tissues are common sites of tumor induction by all three of these chemicals.

Ras mutations in chemically induced tumors are considered to be indicators of in vivo mutagenesis (18). Previous studies have shown that ras proto-oncogenes are activated by point mutations induced by various classes of carcinogens (19,20). The patterns of mutation observed in the ras gene are often specific for a chemical or class of chemicals (21), and may be consistent with DNA adduct profiles of the chemical (22). For example, topical application of dibenz[a,j]anthracene to mice induced exclusively A->T transversions at codon 61 of c-Ha-ras in skin tumors, which suggests an important role for the deoxyadenosine adduct in the tumorigenesis of this polycyclic aromatic hydrocarbon (23,24). Recently, in studies where both isoprene- and butadiene-induced HG neoplasms were evaluated for ras mutations, there was targeting of adenine bases with a predominance of A->T transversions and A->G transitions for the respective chemicals (25,26). The targeting of adenine bases is consistent with the hypothesis that adducts play a role in the mutagenesis of these chemicals. Specific guanine and adenine adducts of 1,3-butadiene have been identified and may contribute to the carcinogenicity of this chemical (27,28).

The objectives of our study were to (1) examine the K- and H-ras mutation frequency and spectrum in chloroprene-induced lung and HG neoplasms to determine whether there were any signature mutations that would provide clues of a chemically induced effect, (2) examine the dose–response for K-ras mutations in chloroprene-induced lung and HG neoplasms, (3) examine the K-ras mutation profile in isoprene-induced lung neoplasms (an analysis that has not been conducted previously) and (4) compare the mutation frequency and spectra for K- and H-ras mutations in lung and HG neoplasms induced by chloroprene with the results from tumors induced by the structurally related chemicals isoprene and 1,3-butadiene.


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 Materials and methods
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Lung and HG neoplasms
Male and female B6C3F1 mice were exposed to 0, 12.8, 32 or 80 p.p.m. chloroprene by inhalation for 6 h/day, 5 days/week for 2 years. At necropsy, tissues were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin, sectioned to a thickness of 5 µm and stained with hematoxylin and eosin (H & E). Subsequently, five unstained serial sections, 10 µm thick, were prepared from paraffin blocks containing alveolar/ bronchiolar adenomas or carcinomas, and HG adenomas or carcinomas. HGs are bilateral orbital secretory glands that are located posterior to the eye (29). Benign and malignant neoplasms of the HG are often associated with multiple site and multiple species carcinogens (30).

In order to isolate adequate amounts of DNA, neoplasms >1 mm in diameter were identified for analysis. This included 46 lung neoplasms and 27 HG neoplasms from chloroprene-exposed mice and six lung neoplasms and two HG neoplasms from control mice. In addition, 11 paraffin-embedded lung neoplasms from mice exposed to 2200 or 7000 p.p.m. isoprene by inhalation for 6 h/day, 5 days/week for 26 weeks followed by a 26 week recovery period were examined (2).

DNA isolation
The DNA isolation procedure has been described previously (31,32). The paraffin-embedded tissue was deparaffinized and rehydrated before digesting with proteinase K (33). DNA was extracted with phenol and chloroform and precipitated with ethanol.

ras mutation identification
DNA was amplified by the polymerase chain reaction (PCR) (32,34) and details of the use of nested primers have been described previously (19,20,35).

For identification of H-ras mutations at codon 61, most of exon 2 surrounding codon 61 was amplified followed by non-radioactive restriction fragment length polymorphism (RFLP) analysis (36). The sense and antisense amplification primer sequences were 5'-GACATCTTAGACACAGCAGTT-3' and 5'-TAGCCATAGGTGGCTCACCT-3', respectively. Restriction sites for MSEI, XbaI or TaqI enzyme (New England Biolabs, Beverly, MA) are created by the presence of a C->A, A->T or A->G mutation in the first or second base of codon 61. By using this technique, we detected codon 61 AAA, CTA and CGA mutations by MSEI, XbaI and TaqI digestion, respectively; the normal sequence (CAA) of codon 61 is not cut by these enzymes. Following restriction digestion, the samples were mixed with bromophenol blue dye and loaded onto a 6% acrylamide TBE gel (8 cmx8 cmx1 mm, 15 wells; Novex, San Diego, CA). The gel was run at 100 V for 1 h at room temperature in a Novex gel electrophoresis unit. Restricted PCR products were stained with ethidium bromide.

For analysis of K-ras mutations, non-radioactive single-stranded conformational polymorphism analysis (SSCP) was performed. A mixture consisting of 5 µl of PCR product, 0.6 µl of 1 M methylmercury hydroxide, 1 µl of 15% w/v Ficoll (mol. wt 400 000) loading buffer containing 0.25 bromophenol blue and 0.25% xylene cyanol, and 13.4 µl of 1x TBE buffer (Novex) was prepared to yield a total volume of 20 µl (37). This mixture was denatured at 85°C for 5 min prior to loading on a 20% polyacrylamide TBE gel (Novex). The buffer chamber was filled with 1.5x TBE buffer. The gel was run at 300 V at 5°C until the xylene cyanol light blue dye reached the bottom of the gel. Positive controls for K-ras mutations and one undenatured DNA control (without methylmercury hydroxide and no heat) were run with unknown samples. Gels were stained with ethidium bromide. For identification of K-ras mutations at codon 61, restriction digestion was performed with XbaI prior to SSCP analysis. This combination assay enhanced visualization of a K-ras codon 61 CTA mutation that is not always detectable by SSCP analysis alone.

Direct sequencing
Direct sequencing of the amplified first or second exon of the K-ras gene was performed as described by Tindall and Stankowski (38) using previously described sequencing primers (19).


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Analyses of both H- and K-ras mutations were performed using DNA from lung and HG neoplasms from mice exposed to chloroprene. Similarly, K-ras mutation analysis was performed with DNA from isoprene-induced lung neoplasms. Examples of the sequencing data are shown in Figure 1Go. Point mutations were not detected in DNA isolated from the non-tumor regions of the lung or HG in control or chloroprene/isoprene-exposed mice.



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Fig. 1. Identification of K-ras mutations at codon 61 in chloroprene-induced lung neoplasms from B6C3F1 mice by direct sequencing of amplified exon 2. Sequencing panels are from left to right: normal K-ras codon 61 sequence CAA and mutated codon 61 sequences CTA and CGA. The normal K-ras sequence is also present in the neoplasms, except for the fourth panel in which only mutant CTA appeared. The variable density of bands representing mutations at codon 61 may be caused by allelic imbalances on chromosome 6.

 
Lung neoplasms
A higher frequency (80%) of K-ras mutations was detected in chloroprene-induced lung neoplasms than in spontaneous lung neoplasms of control B6C3F1 mice (30%) (Table IGo). The predominant mutation in chloroprene-induced neoplasms consisted of an A->T transversion (CAA->CTA) at K-ras codon 61 (22/46). This pattern of ras mutations was similar to what was found in isoprene-induced lung neoplasms where 10 of 11 neoplasms also had K-ras codon 61 A->T transversions. However, this A->T transversion at K-ras codon 61 was not detected in nine butadiene-induced lung neoplasms examined previously by Goodrow et al. (39), or in 82 spontaneous lung neoplasms (Table IGo). A number of rare point mutations, which have not been detected in spontaneous lung neoplasms, were detected at codon 12 (CGT, CTT and ATT) in chloroprene-induced lung neoplasms.


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Table I. Pattern of K-ras mutations in lung neoplasms from B6C3F1 mice
 
Although the majority of lung neoplasms from chloroprene-exposed mice had K-ras codon 61 A->T transversions, they appeared in an inverse dose–response relationship. Eighty percent (8/10) and 71% (10/13) of the lung neoplasms from the low and mid dose groups, respectively, had this mutation, while it was detected in only 18% (4/22) of the neoplasms from the high dose group (Figure 2Go).



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Fig. 2. Comparison of dose–response for lung neoplasms versus K-ras CTA mutations in lung neoplasms following chloroprene exposure in B6C3F1 mice for 2 years. Note the inverse relationship between chemical exposure and K-ras codon 61 CTA mutations; the higher frequencies of K-ras CTA mutations are at the 12.8 and 32 p.p.m. exposures.

 
The frequency and pattern of mutations in the K-ras gene were also examined to determine if a specific mutation was associated with the morphological pattern (papillary, solid or mixed) or type (benign or malignant) of lung neoplasm. No consistent morphological pattern or type of neoplasm was associated with specific K-ras mutations (data not shown).

HG neoplasms
All chloroprene-induced and isoprene-induced (26) HG neoplasms contained K- and H-ras mutations. This contrasted with the patterns of ras mutations seen in butadiene-exposed mice (25) or in spontaneous HG neoplasms (Table IIGo). The predominant mutation in chloroprene-induced HG neoplasms was an A->T transversion (CAA->CTA) at K-ras codon 61 (25/27). This transversion was considered to be a chemical-specific effect since it has been detected infrequently in spontaneous HG neoplasms (25,26). A similar pattern was seen in isoprene-exposed mice where K-ras codon 61 A->T transversions were identified in 15/30 HG neoplasms (26). One difference between the mutation spectra of chloroprene and isoprene was the higher frequency of C->A (CAA->AAA) transversions at H-ras codon 61 (8/30) in isoprene-induced HG neoplasms. This pattern of ras mutations was not apparent in butadiene-induced HG neoplasms or in spontaneous HG neoplasms (Table IIGo). Three of the 12 butadiene-induced HG neoplasms that were examined also had an A->T transversion in the second base of K-ras codon 61 (Table IIGo). A similar frequency of K-ras codon 61 A->T transversions was detected in chloroprene-induced HG neoplasms from the low (100%), mid (80%) and high (100) exposure groups (data not shown).


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Table II. Pattern of ras mutations in HG neoplasms from B6C3F1 mice
 
The frequency and pattern of mutations in the K-ras gene were also examined to determine if a specific mutation was associated with the morphological pattern of HG neoplasms or type of neoplasm. No consistent morphologic pattern or type of neoplasm was associated with specific K-ras mutations (data not shown).


    Discussion
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 Materials and methods
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A major finding in the chloroprene study was the high proportion of K-ras codon 61 second base A->T transversions in both lung and HG neoplasms, which is consistent with results following isoprene exposure (this study and ref. 26). Because of the structural similarities of chloroprene, isoprene and butadiene, we predicted that neoplasms from the three studies would have similar mutation profiles. In fact, we reexamined 12 HG neoplasms from the butadiene study that had not been analyzed for K-ras codon 61 mutations and found three neoplasms with A->T transversions. Interestingly, an increased frequency of A->T transversions was recently reported in human TK6 lymphoblastoid cells following exposure to the 1,3-butadiene metabolite 1,2,3,4-diepoxybutane (40). In addition, a high frequency of mutations at A:T base pairs was observed at the hprt locus in splenic T cells of butadiene-exposed B6C3F1 mice (41) and in transgenic mice carrying the lacI gene as a mutational target (42). The finding of similar ras mutation profiles, with a predominance of mutations at A:T base pairs, suggests that these mutations may be important in the mechanism of carcinogenesis for these structurally related chemicals (Figure 3Go).



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Fig. 3. Schematic showing the mutational profiles for the structurally related chemicals chloroprene, isoprene and butadiene. A->T transversions represent the most common mutation detected within this chemical class.

 
The identification of a large number of ras mutations at A:T base pairs in the various neoplasms after exposure to chloroprene, isoprene (26) and butadiene (26) suggests an interaction of the metabolic intermediates with DNA to form adenine adducts (24,43). The generation of apurinic sites and misreplication at these sites may represent a critical step in the initiation of the carcinogenic process (44). Mutagenic epoxide intermediates have been identified for both 1,3-butadiene (45) and isoprene (46). The oxidation of chloroprene to epoxide intermediates (2-chloro-1,2-epoxybutene-3 and 2-chloro-3,4-epoxybutene-1) was suggested to occur based on the detection of alkylated 4-(4-nitrobenzyl) pyridine in incubations of chloroprene and mouse liver microsomes (12). This oxidative intermediate of chloroprene may be DNA reactive and account for the mutagenic effects of this compound.

The finding of an inverse relationship between chemical dose and K-ras codon 61 CTA mutation frequency in lung neoplasms, but a similar high frequency of K-ras codon 61 CTA mutations in HG neoplasms irrespective of dose, could be explained by a number of factors. In the lung, the higher frequency of K-ras CTA mutations at the lower doses may be due, in part, to saturation of one or more metabolic pathways at the higher doses (47). Alternatively, the tumor responses at higher doses may be due to non-ras mechanisms of genotoxicity (48). Chromosomal alterations have been observed in bone marrow cells of C57BL/6 mice exposed to chloroprene (5,14). Dose-dependent differences in the mutation spectra in the lung and HG may be due to differences in covalent binding of metabolites or adducts to DNA, or due to differences in the removal of possible adducts from DNA (49). By understanding the dose–response for each mutation, and comparing these data with the dose–response for tumor induction, it may be possible to identify the mutations that are most important for risk assessment purposes. Our results suggest that, in the lung, K-ras CTA mutations predominate at low levels of chloroprene exposure. This finding may be valuable in future evaluations of human lung tumor samples from exposed workers. The detection of K-ras CTA mutations could provide a link in relating chloroprene exposures to cancer risk.

Although chloroprene did not produce point mutations in the Salmonella assay (1), and was negative for cytogenetic damage in the bone marrow evaluated for chromosomal aberrations and negative for micronucleated erythrocytes (15,16), our inhalation study underscores the fact that mutagenic models should be used cautiously in predicting carcinogenesis. Based on the finding of a predominant codon 61 K-ras A->T transversion in both lung and HG neoplasms following exposure of mice to chloroprene, we would classify chloroprene as genotoxic, like its structurally related chemicals isoprene and butadiene. It is likely that chloroprene is bioactivated to epoxide intermediates in the lung and HG in a manner similar to that of isoprene and 1,3-butadiene. Our data also illustrate that organ systems unique to the mouse, such as the HG, provide mechanistic data similar to that in the lung, and point out the value of responses in these organ systems to an overall understanding of the carcinogenicity of environmental chemicals.


    Acknowledgments
 
The authors thank Dr Errol Zeiger and Dr Richard D.Irwin for their critical review of this manuscript.


    Notes
 
2 To whom correspondence should be addressed Email: sills{at}niehs.nih.gov Back


    References
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 

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Received April 29, 1998; revised October 12, 1998; accepted October 21, 1998.