Although BD is a known mutagen and carcinogen present ubiquitously in urban air, the exact mechanisms of its mutagenic and carcinogenic activity remain to be established. Furthermore, human BD exposure risk assessment is complicated by large interspecies differences in sensitivity to BD-induced cancer. BD is a potent carcinogen in mice, but is only a weak carcinogen in rats. B6C3F1 mice develop tumors at BD exposure concentrations three orders of magnitude lower than those that cause cancer in Sprague-Dawley rats (19
). It has been proposed that the carcinogenic potency of BD in a given organism can be predicted from the relative amount of the diepoxide metabolite, DEB, generated upon metabolic activation (27
). However, in vivo
formation and repair of bifunctional DEB-DNA adducts in laboratory rats and mice exposed to BD have not been previously investigated.
We have developed sensitive and specific HPLC-ESI-MS/MS methods which, for the first time, quantify the formation of DEB-specific DNA adducts in vivo
following inhalation exposure to BD. Our results for dose-dependent formation of bis
-N7G-BD and N7G-N1A-BD/N7G-N6
A-BD in tissues of laboratory mice and rats exposed to 0–625 ppm BD by inhalation ( and ) reveal remarkable interspecies differences between adduct levels. For example, the concentrations of bis
-N7G-BD adducts in mouse liver following 10 day exposure to 625 ppm BD were 10-fold greater than in rats exposed at the same conditions (). Both bis
-N7G-BD and N7G-N1A-BD/N7G-N6
A-BD reach a plateau in rat after inhalation exposures above 62.5 ppm, possibly a result of enzymatic saturation, P450 2E1 inactivation through phosphorylation (35
), or suicide inhibition of the protein by covalent binding of epoxide products to the active site (36
). These results are consistent with a greater sensitivity of laboratory mice to BD-mediated carcinogenesis (8
), suggesting that DEB-induced bifunctional DNA lesions play an important role in BD-mediated cancer.
Our results are in accord with previously published dose response data for DEB-specific hemoglobin adducts (pyr
-Val), which revealed that DEB adduct levels in mice were 4–10× greater than levels in rats exposed to the same conditions (37
). The dose response curves for both DEB-induced hemoglobin adducts (37
) and DNA-DNA cross-links ( and of the current manuscript) in the rat are supralinear, providing evidence for the saturation of metabolic activation pathways in this species following exposure to 62.5 ppm BD (38
). In contrast, the dose response curves for the formation of DEB-specific DNA adducts in the mouse are curvilinear between 6.25 to 625 ppm BD, and do not show any signs of metabolic saturation ( and ).
Taken together, these results suggest that the interspecies differences in the carcinogenic potency of BD in rats and mice are related to metabolic differences (11
). Mice form 5-fold more DEB than rats per unit of BD exposure (39
), which is reflected in a higher efficiency for the formation of DEB-globin adducts (37
) and DEB-DNA adducts in this species (). As demonstrated in , the number of bis
-N7G-BD adducts per BD exposure level in mice is greatest following a low BD exposure, but exceeds the efficiency of DEB-mediated DNA cross-linking in the rat at all BD exposures examined.
Bis-N7G-BD adduct levels in mouse liver DNA per unit dose of 1,3-butadiene. ND, not detected.
In addition to significant differences in sensitivity to BD carcinogenesis, rats and mice also differ in tissue specificity of BD-mediated tumor formation. In long-term inhalation studies, mice developed tumors of the lung, lymphatic system, liver, forestomach, and heart, while rats exhibited tumors in different tissues, including pancreas, testis, mammary gland, and thyroid gland (7
). Our results presented in indicate that these differences are not a result of tissue-dependent generation/accumulation of DEB. In both species, DEB-DNA adduct levels were highest in the liver (), consistent with the high activity of cytochrome P450 enzymes which activate BD to DEB in this tissue. Bis
-N7G-BD concentrations in extrahepatic tissues (lung, kidney, brain, and thymus) were 1.5–3-fold lower than those in liver DNA, but similar to one another. These results are consistent with a model in which DEB is formed primarily in the liver and is transported throughout the body to reach other tissues (22
Several recent studies suggested that there may be gender differences in cancer susceptibility and BD metabolism in laboratory animals. In carcinogenesis studies on BD, female mice develop tumors at lower BD concentrations than males (15
). Furthermore, female rats contained higher blood DEB concentrations than male rats following a single 6 h exposure to 62.5 ppm BD (22
). Female rats also had 3–4 fold greater levels of pyr
-Val hemoglobin adducts than male rats following a 90 day exposure to 1000 ppm BD (24
). Our results presented here are consistent with these earlier findings. Bis
-N7G-BD adduct amounts in female rats and mice exposed to 625 ppm BD are 2–2.5-fold higher than in males (). A similar trend is observed following lower BD exposure (200 ppm, ). In contrast, human epidemiology studies comparing male and female industry workers exposed to BD observed no significant difference in gene mutations (e.g., HPRT
mutant frequencies) or sister chromatid exchanges between male and female workers (40
). Future studies of DNA and protein adducts in occupationally exposed humans are needed to determine whether gender differences in biomarker levels of BD exposure and metabolism are observed in humans.
The possible roles of bis
-N7G-BD and N7G-N1A-BD/N7G-N6
A-BD lesions investigated in the present work in the genotoxicity of DEB remain to be established. Several laboratories, including our group, have shown that bis
-N7G-BD cross-links are formed preferentially within the 5'-GNC sequence context (31
), with nucleosome structure having little effect on sequence selectivity of DEB-DNA cross-link formation (43
). Interestingly, while the S,S
stereoisomer of DEB produces a greater number of interstrand cross-links, all three DEB stereoisomers target 5'-GNC trinucleotides (44
). Large local distortions of the DNA helix are required to accommodate 1,3-interstrand bis
-N7G-BD lesions because the four-carbon tether length (6 Å) is much shorter than the spacing between the distal N7-dG atoms in 5'-GNC sequences of canonical B-DNA (8.9 Å). In addition, meso
DEB is capable of inducing 1,2-intrastrand bis
-N7G-BD lesions (31
). Molecular dynamics simulations predict that the base stacking and hydrogen bonding interactions in the vicinity of 1,3-interstrand and 1,2-intrastrand bis
-N7G-BD cross-links are disrupted as a result of twisting of the cross-linked residues with respect to the base-pairing plane (31
). 3'-Exonuclease activity of E. coli
Polymerase I is blocked one nucleotide ahead of the interstrand bis
-N7G-BD lesions (31
), consistent with an induced structural change in the vicinity of the cross-link. These structural changes may be important for the recognition of bis
-N7G-BD adducts by DNA repair enzymes.
Another factor that must be considered when evaluating possible contributions of bis
-N7G-BD and N7G-N1A-BD cross-links to BD-mediated mutagenesis and cancer is differences in their hydrolytic stability. All N7-alkylguanine adducts are hydrolytically labile because of the intrinsic destabilization of the glycosidic bond when the N7 position of guanine is alkylated (45
). Both glycosidic bonds of bis
-N7G-BD cross-links can be hydrolyzed, with a half-life in double stranded DNA of 147 h (interstrand) and 35 h (intrastrand) (31
). In contrast, spontaneous depurination of the N7-guanine portion of N7G-N1A and N7G-N6
A DEB cross-links () results in hydrolytically stable adenine adducts containing a butanediol cross-link to free guanine in one DNA strand and an abasic site (Ab) in the other (Supplement S-3
). If produced in vivo,
semi-depurinated interstrand cross-links may be important to DEB mutagenesis, because repair synthesis on either strand must proceed past the damaged nucleobase in the opposite strand.
In addition to interspecies differences in BD metabolism, the concentrations of DEB-DNA adduct levels in tissues of rats and mice can be affected by variations in DNA repair pathways. Experiments examining the potential roles of nucleotide excision repair and base excision repair pathways in the removal of bis-N7G-BD and N7G-N1A-BD adducts are currently in progress in our laboratory.
In summary, our results presented herein provide additional support to the hypothesis that DEB is the key metabolite largely responsible for the interspecies differences in sensitivity to BD-induced cancer (37
). Our study provides a greater molecular detail of the consequences of interspecies metabolic differences by quantifying DNA-DNA lesions specific for DEB. This work also has implications for human risk assessment. While the mouse model is sometimes preferred because mice are the more sensitive species to BD-induced cancer, studies in liver microsomes suggest that mice form DEB at faster rates than both rats and humans (11
). In addition, human microsomes have higher epoxide hydrolase activity than rats and mice, and therefore hydrolyze DEB more efficiently than both rat and mouse microsomes (11
). Therefore, if DEB is responsible for BD-induced cancer in the mouse, a rat model of sensitivity may be more appropriate for setting limits of human exposure.