THE MOLECULAR DOSIMETRY OF AN ENVIRONMENTAL CARCINOGEN 1,3-BUTADIENE, A MODEL COMPOUND FOR HUMAN RISK EXTRAPOLATIONS

Project leader: Kimmo Peltonen, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, FIN-00250 Helsinki, Finland,
tel. +358-9-4747216, e-mail: Kimmo.Peltonen@ttl.fi
 
 

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TIIVISTELMÄ SUOMEKSI

Researchers:
Tiina Anttinen-Klemetti Finnish Institute of Occupational Health (FIOH), Chemistry laboratory, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland, tel. +358-9-47472131
e-mail: Tiina.Anttinen-Klemetti@ttl.fi
Ulla Harju, Finnish Institute of Occupational Health (FIOH), Chemistry laboratory, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland,  tel. +358-9-474722586
e-mail: Ulla.Harju@ttl.fi
Pertti Koivisto, Finnish Institute of Occupational Health (FIOH), Chemistry laboratory, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland, tel. +358-9-47472443
e-mail: Pertti.Koivisto@ttl.fi
Marjo Rynö, Finnish Institute of Occupational Health (FIOH), Chemistry laboratory, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland, tel. +358-9-47472867
e-mail: Marjo.Ryno@ttl.fi

Financing SYTTY organisation:The Academy of Finland, The Finnish Work Environment Fund
Funding from SYTTY / Total funding of project (€): 348604 / 348604
Person-months of work funded by SYTTY / Total person-months of work: 62 / 128

KEY WORDS: 1,3-butadiene, metabolism, carcinogenesis, DNA adducts, risk extrapolation
 

EXTENDED ABSTRACT

1 Introduction

1,3-Butadiene (BD) is a high production volume chemical, with the worldwide production about 7,6 million tonnes. BD is used in the manufacture of polymers and copolymers, e.g., for car tires, synthetic rubbers, and latexes. BD is also generated during the combustion of fossil fuels and is thus present in the environment. In the early 1980s, inhalation exposure to BD was noticed to lead malignant tumors in mice, especially in the lung and heart of B6C3F1 mice. International Agency for Research on Cancer (IARC) classified BD as “a probable human carcinogen” and recently National Toxicology Program (US) upgraded BD to a class of “known to be a human carcinogen”.

BD is a prochiral compound that is oxidized to electrophilic epoxides with at least one stereogenic atom. The striking differences in the biological activities between oxirane enantiomers like benzo[a]pyrene diol epoxide and styrene oxide underline the importance of studies devoted to the determination of enantioselective DNA binding (FIGURE 1).

We have hypothesized that the enantioselective DNA binding has a key role in the observed sensitivity difference in tumorigenesis. In this project we have focused on enantioselective metabolism, enantioselective DNA adduct formation and structure specific DNA damage repair.

2 Methods

Only some of the used epoxides were commercially available and most of the reference compounds were synthesized within this project. Adducted DNA fragments were prepared in aqueous solutions by exposing nucleotides or oligonucleotides with carcinogens. Yields were usually low and mixture of products were formed, the fact that necessitated chromatographic purification and spectroscopic identification of the particular marker. All the animals were inhalation exposed for two weeks (6 h /day). The organs originated from the B6C3F1 strain of mice and F344 strain of rat were kindly provided by Professor W. Walker (Lovelace Respiratory Research Institute, New Mexico, USA). The wild type and the knock out mice (strain C58B1/6) were a kind gift from Dr R. Elder (Paterson Institute for Cancer Research, Manchester, UK). The oligonucleotides that have stereospecific modifications were from Professor T. Harris, Vanderbilt University, Tennessee, USA).

In 32P-postlabeling assay DNA is extracted, purified, quantified and digested to nucleotides. The adducted nucleotides are enriched with anion and reversed phase column chromatography. In the in-house modification of the assay isolated fraction of adducts and external standards were phosphorylated with radioactive phosphate (32P) using carrier free T4 polynucleotide kinase. Samples were transferred to polyethyleneimine TLC plates and eluted with ammonium formate (200 mM, pH 8). Exposing a film localized radioactive zones and spots were extracted from the plate. The fractions were analyzed with HPLC equipped with UV and radioactivity detectors (FIGURE 2).

Double stranded DNA (ds DNA) was synthesized by annealing the singlestranded DNA (ss DNA) with the complementary strand. Ds DNA were exposed with the bifunctional diepoxybutane. Modified ds DNA was analyzed with denaturing HPLC (DHPLC) and DHPLC/MS/MS.

3 Results

A new method to study the mechanisms of the formation of intra and interstrand cross-links in DNA was developed. The method is based on the on-line denaturation of the ds DNA to sd DNA and a subsequent analysis of the formed fragments with HPLC/MS/MS. It was demonstrated that ds DNA oligonucleotide is by far much better model for DNA alkylation than ss DNA oligonucleotide or denatured DNA. DHPLC is a new tool to denature ds DNA for spectroscopic analysis like MS and MS/MS. DHPLC/MS with multiple charged technique is a versatile method to measure alkylation and identification of the alkylation site (FIGURE 3). In the model monoadducts were detected in both strands, one, two and three interstrand cross-link was detected in a G rich strand. Tentatively a hot spot was identified at the codon 13 with the interstrand cross-link formed to two adjacent guanine bases. One interstrand cross-link was detected which was located at the codon 11 in the sixth base from the 5’ end of the complementary strand. Based on the data presented, it is obvious that adduct formation is not a random process in ds DNA, but the adduct formation is highly dependent on the concentration of the alkylator.

A species difference was seen in the BMO derived adduct formation in rat and mice lung. In B6C3F1 mice most of the BMO derived adducts was formed from the S-enantiomer. Adducts in F344 rats were formed from both enantiomers. The analysis of the EBD derived adducts also indicate that S-BMO enantiomer was the precursor of the major adducts formed, which has the RR and RS configuration, respectively.  In a quantitative point of view 98% of all adducts from the electrophilic epoxymetabolites were derived from the RR and RS diastereomers of butadiene diolepoxide. The total number of adducts were not statistically different between the species in any of the organs studied so far.

A specific marker of DEB derived DNA adducts formed in vivo is needed to give some light to the mechanism of the observed species difference in the sensitivity to get malignant tumors. A new analytical method was used in the analysis of the specific marker of DEB. The analysis was carried out at high pH that allowed the separation of the particular adducts and the method was optimized in in vitro experiments.  In vivo analyses indicate that the level of DEB monoadduct was less than 20 % of the level of BMO adducts. The level of BMO derived adducts was only about 2 % of all the addutcs formed. Clearly, most of adducts in mouse lung were formed from BDE.

BMO and BDE derived adducts were also analyzed in APNG knock out (alkylpurine-DNA-N-glycosylase) and in a corresponding wild type mice (C57B1/6). Surprisingly, adduct levels were the same in both types of animals. However, the level of BD derived adducts were lower compared to the adduct levels of exposed B6C3F1 mice. Intensity of the endogenously formed guanine N7 methyl and hydroxyethyl adducts was however on the same level in both strains of mice.

4 Conclusions

By using guanine N7 adducts as a marker of the biologically relevant dose we have demonstrated that 1,3-butadiene is metabolized to three epoxides in various organs in vivo (FIGURE 1). Major adducts were derived from BDE diastereomers and the lowest level of adducts were derived from DEB. Because specific adducts indicate also the level of the particular metabolite in the organ tissue we were able to conclude that in mice BD is metabolized clearly in a stereospecific manner but much less stereoselectivity was observed in rat. In mice BD is first oxidized mainly to S-BMO and after hydrolysis further oxidized to RR- and RS diastereomers of BDE. Our in vitro studies indicate intra and interstrand cross-link formation, but we were not yet able to demonstrate cross-link formation in vivo. In vitro data suggest a low level of cross-link formation. In vivo model to detect cross-link formation in nuclear DNA is clearly the challenge of the future.

We were able to demonstrate that if risk extrapolation is based on animal experiments with prochiral chemicals or pollutants the stereochemical processes present in vivo ought to have a high priority in interpretation of the results.

Figure1. The metabolism and DNA adduct formation of 1,3-butadiene. Stereogenic atoms are labeled with an asterix.

Figure 2.  HPLC analysis of four stereoisomers of 32P-postlabeled guanine N7 adducts of butadiene monoepoxide. The upper chromatogram is from the radioactivity detector and the lower chromatogram is from the UV detector demonstrating the usefulness of the internal standard.

Figure 3.  The mass spectrum of the ds DNA with 16 bases (the upper spectrum) and the butadienediepoxide alkylated ds DNA (the lower spectrum).
 
 

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