Post-doctoral Research
Following my doctoral research, I wanted to get more experience with “smaller” DNA
and protein adducts. Therefore, I joined Dr. Swenberg’s laboratory and became interested
in butadiene, a four carbon carcinogen. Butadiene (BD) carcinogenesis and mutagenesis
are species, tissue and concentration dependent. BD is oxidized mainly by P450 2E1 to
epoxybutene (EB), diepoxybutane (DEB) and epoxybutane-diol (EB-diol). All these
epoxides are known to form DNA and protein adducts, however, the DNA adducts
responsible for mutations at AT sites had not been identified. It was well known that N1-
adenine adducts in general rearrange to the N6-adenine or deaminate to N1-inosine
adducts. Interestingly, N1-(1-hydroxybutenl-2-ly)-inosine (HB-dI) adducts had been shown
to be highly mutagenic in site directed mutagenesis studies.

Therefore, it was hypothesized that N1 adenine and N1 inosine adducts may be
important in BD mutagenesis by producing mutations at AT sites. I was awarded an
individual NRSA grant by NIEHS, to determine whether N6-adenine and promutagenic N1-
inosine adducts are formed in vivo and to correlate DNA adduct levels with mutations at AT
sites. An LC-MS/MS method was established for detection of HB (EB derived) and
trihydroxybutyl-adducts (THB-adducts, EB-diol derived) adducts. Following method
development, DNA from lung and liver of mice and rats exposed to 1000 ppm BD by
inhalation for 90 days or to 62.5 ppm BD for 20 days were analyzed for HB and THB
adenine and inosine adducts. None of the samples had detectable amounts of any of the
adducts of interest. Co-injection with 1 fmol standard confirmed the ability to detect N1-HBdIno
in these samples when 10 µg DNA equivalents were analyzed. A manuscript is in
preparation describing the synthesis of standard compounds, method validation and
sample analysis. These data demonstrated that amounts are below 6 adducts per 108
nucleotides and suggest that these adducts either do not form, are only present at low
levels, or are rapidly repaired.

Parallel to the DNA adduct studies, efforts were directed towards the analysis of
hemoglobin adducts as surrogate markers for the internal formation of the individual BD
derived epoxides. Protein adducts, while not causally linked to cancer, are important tools
for translational research because they allow cross species comparisons of internal
exposure without invasive samples. Previously, the formation of HB-Val and THB-Val
adducts as markers for EB and EB-diol, respectively, were investigated using a modified
Edman degradation. DEB, however, forms a cyclic pyrrolidine complex with the NH2 group
of the N-terminal valine and is therefore unsuitable for this methodology. A major gap in BD
research was the lack of a DEB specific protein adduct because it became apparent that
DEB was the most mutagenic and potentially most carcinogenic metabolite. To fill this gap
an assay was established based on tryptic hydrolysis and immunoaffinity (IA) enrichment
prior to quantitation by LC-MS/MS. With this method it was shown that mice, the most
susceptible species, form much more DEB-specific protein adducts compared to rats at the
same BD exposure (2,3). Further it was shown that the formation of DEB is linear in mice
and saturated at exposures > 62.5 ppm BD in rats. This is strong evidence that the
formation of DEB may be critical in BD tumorigenesis. To investigate whether DEB is
formed in humans, we are currently analyzing globin from humans exposed to BD at the
workplace. These studies have greatly aided species comparison and future results will be
used to improve cancer risk assessment.

A long term goal in our laboratory is to combine the immunoaffinity enrichment to
allow analysis of all three BD derived protein adducts. Therefore, HB-valine containing 11-
mer human peptides were synthesized, antibodies were raised, IA columns were prepared
and method validation is underway (4). Similar efforts are ongoing for the THB-Val adduct
assay. A parallel approach was to redesign this assay by generating antibodies against the
normal C-terminus. These antibodies will be used to enrich all N-terminal heptapetides from
globin digests, which then can be analyzed for corresponding alkylations. For this,
antibodies were raised, IA columns were prepared and preliminary data were generated
using standard peptides. This assay may allow the simultaneous quantification of several
alkylation products of interest to elucidate the relative significance of a single carcinogen in
mixtures. The later approach has also the potential to identify until now unknown adducts
and to be a universal tool for analysis of reactive metabolites derived from carcinogens or
drugs in vivo.

Further, I have been awarded a pilot grant from the Center of Environmental Health
and Susceptibility (CEHS) at UNC to study post-translational modifications (PTMs) in P450,
as potential molecular mechanisms responsible for observed species and concentration
dependency in BD metabolism and carcinogenesis. The initial attempt to isolate P450 2E1
from liver tissues by immuno-precipitation was challenging, however, we recently confirmed
the identity of the isolated protein as P450 2E1 by mass spectrometry sequencing, with >
35 % sequence coverage. Parallel to this, we identified potential binding sites of EB in
human P450 2E1 in vitro (5). In this study, 6 binding sites were identified, with 5 of them
being theoretically important in enzyme activity. In preliminary studies it was shown that
pre-incubation of P450 2E1 with EB at 1:1 ratio for 30 min, reduces its activity for
p-nitrophenol hydroxylation by 60%. This suggest a mechanism based inactivation of P450
2E1 by EB and supports the hypothesis that the biphasic dosimetry may be caused by
inactivation of P450 2E1 through covalent binding of EB. The hypothesis has been
confirmed in in vitro experiments and methods are in place to analyze tissues from BD
treated rodents for these modifications.

Publications

1. Boysen G, Georgieva NI, Upton PB and Swenberg JA, N-Terminal Globin Adducts as
Biomarkers for Formation of Butadiene Derived Epoxides Chemico-Biological Interactions,
2006, In Press

2. Georgieva N.I Boysen G, Jayaraj K and Swenberg JA, Quantitative Analysis of N-terminal
Valine Peptide Adducts Specific for 1,2-Epoxy-3-butene, Chemico-Biological Interactions,
2006, In Press

3. Boysen G, Scarlett CO, Temple B, Combs TP, Brooks NL, Borchers CH and Swenberg JA,
Identification of covalent modifications in P450 2E1 by 1,2-Epoxy-3-butene in vitro Chemico-
Biological Interactions, 2006, In Press

4. Ye W, Sangaiah R, Degen DE, Gold A, Jayaraj K, Koshlap KM, Boysen G, Williams J, Tomer
KB, and Ball LM, A 2-Iminohydantoin from the Oxidation of Guanine, Chemical Research in
Toxicolog,2006, Web Released

5. Stout MD, Jeong Y-C, Boysen G, Li Y, Sangaiah R, Ball LM, Gold,A and Swenberg JA
LC/MS/MS Method for the Quantitation of trans-2-Hexenal-Derived Exocyclic 1,N2-
Propanodeoxyguanosine in DNA, Chemical Research in Toxicology, 2006, Web Released

6. Li Y, Warren JT, Boysen G, Gilbert LI, Gold A, Sangaiah R, Ball LM, Swenberg JA. Profiling of
ecdysteroids in complex biological samples using liquid chromatography/ion trap mass
spectrometry.Rapid Commun Mass Spectrom. 2006;20(2):185-92.

7. Wu Y, Pradhan P , Havener J, Boysen G , Swenberg JA Campbell SL, and Chaney SG NMR
solution structure of an oxaliplatin 1,2-d(GG) intrastrand cross-link in a DNA dodecamer
duplex. J.Mol.Biol., 2004, 341, 1251-1269

8. Boysen G., Georgieva N.I., Upton P.B., Li Y., Walker V.E., and Swenberg J. A. Analysis of
Diepoxide-Specific Cyclic N terminal Globin Adducts in Mice and Rats after Inhalation
Exposure to 1,3-Butadiene. Cancer Research 2004, 64(23):8517-20