[13C2]-acetaldehyde promotes unequivocal formation of 1, N 2-propano-2′-deoxyguanosine in human cells

Article abstract of DOI:10.1021/ja2004686

Acetaldehyde is an environmentally widespread genotoxic aldehyde present in tobacco smoke, vehicle exhaust and several food products. Endogenously, acetaldehyde is produced by the metabolic oxidation of ethanol by hepatic NAD-dependent alcohol dehydrogenase and during threonine catabolism. The formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2′-deoxyguanosine in DNA to form primarily N²-ethylidene- 2′-deoxyguanosine. The subsequent reaction of N²-ethylidenedGuo with another molecule of acetaldehyde gives rise to 1,N²-propano- 2′-deoxyguanosine (1,N²-propanodGuo), an adduct also found as a product of the crotonaldehyde reaction with dGuo. However, adducts resulting from the reaction of more than one molecule of acetaldehyde in vivo are still controversial. In this study, the unequivocal formation of 1,N ²-propanodGuo by acetaldehyde was assessed in human cells via treatment with [¹³C₂]-acetaldehyde. Detection of labeled 1,N²-propanodGuo was performed by HPLC/MS/MS. Upon acetaldehyde exposure (703 μM), increased levels of both 1,N²-etheno-2′- deoxyguanosine (1,N²-εdGuo), which is produced from α,β-unsaturated aldehydes formed during the lipid peroxidation process, and 1,N²-propanodGuo were observed. The unequivocal formation of 1,N²-propanodGuo in cells exposed to this aldehyde can be used to elucidate the mechanisms associated with acetaldehyde exposure and cancer risk.

Full text of DOI:10.1021/ja2004686

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[13C2]- Acetaldehyde Promotes Unequivocal Formation
of 1,N2-Propano-20-deoxyguanosine in Human Cells
Camila Carri~ao M. Garcia,† Josꢀe Pedro F. Angeli,† Flor^encio P. Freitas,† Osmar F. Gomes,†
Tiago F. de Oliveira,‡ Ana Paula M. Loureiro,‡ Paolo Di Mascio,† and Marisa H. G. Medeiros*,†
†Departamento de Bioquímica, Instituto de Química, ‡Departamento de Anꢀalises Clinicas e Toxicolꢀogicas,
Faculdade de Ci^encias Farmac^euticas, Universidade de S~ao Paulo, S~ao Paulo SP, Brazil
S Supporting Information
b
unstable Schiff base (3, Scheme 1).6 The subsequent reaction
ABSTRACT: Acetaldehyde is an environmentally wide-
spread genotoxic aldehyde present in tobacco smoke, ve-
hicle exhaust and several food products. Endogenously,
acetaldehyde is produced by the metabolic oxidation of
ethanol by hepatic NAD-dependent alcohol dehydrogenase
and during threonine catabolism. The formation of DNA
adducts has been regarded as a critical factor in the
mechanisms of acetaldehyde mutagenicity and carcinogen-
esis. Acetaldehyde reacts with 20-deoxyguanosine in DNA
to form primarily N2-ethylidene-20-deoxyguanosine. The
subsequent reaction of N2-ethylidenedGuo with another
molecule of acetaldehyde gives rise to 1,N2-propano-
20-deoxyguanosine (1,N2-propanodGuo), an adduct also
found as a product of the crotonaldehyde reaction with
dGuo. However, adducts resulting from the reaction of
more than one molecule of acetaldehyde in vivo are still
controversial. In this study, the unequivocal formation of 1,
N2-propanodGuo by acetaldehyde was assessed in human
cells via treatment with [13C2]-acetaldehyde. Detection of
labeled 1,N2-propanodGuo was performed by HPLC/MS/
MS. Upon acetaldehyde exposure (703 μM), increased
levels of both 1,N2-etheno-20-deoxyguanosine (1,N2-εdGuo),
which is produced from R,β-unsaturated aldehydes formed
during the lipid peroxidation process, and 1,N2-propanod-
Guo were observed. The unequivocal formation of 1,
N2-propanodGuo in cells exposed to this aldehyde can be
used to elucidate the mechanisms associated with acetal-
dehyde exposure and cancer risk.
of adduct 3 with a second molecule of AA leads to the formation
of the (6S,8S) and (6R,8R) diastereomers of 1,N2-propanodGuo
adducts7 (5, Scheme 1). In addition, 1,N2-propanodGuo forma-
tion is catalyzed by polyamines and histones.8 1,N2-propanod-
Guo is also formed in the reaction of crotonaldehyde with dGuo
and DNA.9 Regarding its presence in DNA, 1,N2-propanodGuo
adduct exists in equilibrium between the closed and open forms.
The open form is favored in double-stranded DNA, whereas the
closed form predominates in single-stranded DNA.10
1,N2-PropanodGuo promotes DNA miscoding in human
cells, mainly through GfT transversions, and can inhibit DNA
synthesis.11 The ring-opened form can lead to an interstrand
cross-link when the adduct is formed in a 50-CpG sequence.12
Cross-linking events involving DNA and proteins occur after
reaction of the ring-opened free aldehyde with peptides.13
The base modification induced by AA is well documented. For
example, the reduced form of N2-ethylidenedGuo (4, Scheme 1),
the product of the reaction of one molecule of AA with dGuo, has
been detected in vivo.14 However, the formation of 1,N2-propa-
nodGuo from AA in vivo is controversial because it requires two
successive reactions of AA. Its presence is mainly credited to the
reaction of dGuo with the crotonaldehyde generated during lipid
peroxidation (Scheme 1); however, the formation of protein
adducts involving more than one molecule of AA was observed in
patients with alcohol-induced liver disease.15
Additionally, epoxidized R,β-unsaturated aldehydes, which
are end products of lipid peroxidation, can generate ethano
or etheno derivatives upon reaction with DNA.16 1,N2-εdGuo
(2, Scheme 1), for example, is derived from the reaction between
trans,trans-2,4-decadienal (DDE) and dGuo17 (Scheme 1). Some
of these DNA lesions have proved to be highly mutagenic18 and
are considered possible pathways for oxidative stress-related
carcinogenesis.
cetaldehyde (AA), a proven mutagenic and carcinogenic
A
compound, is formed endogenously as the main product of
ethanol oxidation and has been measured in liver and saliva
after ingestion of ethanol.1 In addition, small amounts of AA are
produced endogenously during the catabolism of threonine.2 AA
is widespread in the environment, including foods and beverages,
cigarette smoke, ethanol metabolism, and fuel combustion (both
alcohol and diesel).3 AA genotoxicity and carcinogenicity are
highlighted in populations that are deficient in aldehyde dehy-
drogenase, a condition which results in a higher risk of esopha-
geal cancer after alcohol consumption compared to populations
with the fully active enzyme.4 The formation of DNA adducts has
been regarded as a critical factor in the mechanism of AA toxicity.5
AA reacts with the 20-deoxyguanosine (dGuo, 1, Scheme 1) in
DNA to primarily form N2-ethylidene-20-deoxyguanosine, an
In the present study, the formation of exocyclic DNA adducts
was investigated in cells treated with AA. Because of the volatility
of AA, its actual concentration in the cell medium was estimated
through conjugation with 2,4-dinitrophenylhydrazine (DNPH).
The resulting hydrazones were analyzed by HPLC with UV
detection (λ = 360 nm). The effective concentrations in the stock
solutions made in water and the concentrations measured in
the culture cell media presented high AA evaporation rates.
Nevertheless, AA can also react promptly with cell media
Received: January 17, 2011
Published: May 23, 2011
r
2011 American Chemical Society
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|

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Scheme 1. Representative Formation of DNA Adducts from
the Lipid Peroxidation End Products, 1,N2-εdGuo (2) and 1,
N2-PropanodGuo (5), and from Acetaldehyde, N2-Ethylide-
nedGuo (3) and 1,N2-PropanodGuo (5)
Figure 2. Quantification by HPLC/MS/MS of 1,N2-εdGuo/107dGuo
(A) and 1,N2-propanodGuo/107dGuo (B) in 100 μg of cell-extracted
DNA after treatment with AA at 155 and 703 μM. (*) p < 0.05 compared
to control; (**) p < 0.01 compared to control, (***) p < 0.001 compared
to control and 155 μ M.
Figure 1 shows that crotonaldehyde was not detected under
our experimental conditions.
Exocyclic DNA adducts were analyzed by HPLC/MS/MS
using an API 4000 QTRAP mass spectrometer (Applied
Biosystems) as described by Garcia et al.19 The adducts were
detected by multiple reaction monitoring (MRM). Briefly, the
DNA hydrolysates containing 100 fmol of internal standards
were injected into the HPLC/ESI/MS/MS system. The m/z
292f176 (1,N2-εdGuo), 297f181 ([15N5]1,N2-εdGuo),
338f222 (1,N2-propanodGuo) and 343f227 ([15N5]1,N2-
propanodGuo) transitions were monitored with a dwell time of
280 ms. All other mass spectrometer parameters were adjusted
for acquisition of the best [M þ H]þ/[M þ H ꢀ 2-D-erythro-
pentose]þ transition.
IMR-90 cellsweretreated withAA(155 μM or703μM) for 3 h.
Cell viability as measured by the MTT assay was higher than
70% after AA treatment (data not shown). DNA was extracted,
hydrolyzed, and analyzed as already described, with minor
modifications.19 Using this highly sensitive methodology, a
significant increase in the levels of both 1,N2-propanodGuo and
1,N2-εdGuo was observed (Figure 2).
In the present experimental approach, 1,N2-εdGuo could
only arise from aldehydes formed endogenously, whereas 1,
N2-propanodGuo could arise from exogenous AA or endo-
genous aldehydes produced during the lipid peroxidation
process. In fact, AA was able to induce lipid peroxidation in
cells, as measured by malondialdehyde production (Support-
ing Information).
According to the DNA adduct quantification (Figure 2), after
treatment with 703 μM of aldehyde, the increase in levels of
etheno adducts compared to control cells was lower (about 5
times, Figure 2A) than the increase in propano adducts (20
times, Figure 2B). Moreover, formation of the propano adduct
seems to present a more direct correlation with increasing AA
concentration than does formation of the etheno adduct, sup-
porting the relevance of this lesion as biomarker of AA exposure.
In contrast, upon treatment with 155 μM aldehyde, the relative
increase in levels of the etheno adduct was higher than that
observed for the propano adduct, showing that adducts are
preferentially formed via the lipid peroxidation pathway at this
concentration.
Figure 1. Analysis of DNPH derivatives in cell supernatant. Cells
were treated with 10 mM [13C2]-acetaldehyde for 3 h; subsequently,
cell media aliquots were taken and derivatized (1:1) with 20 mM
DNPH. HPLC/ESI/MS/MS analyses, carried out in the negative
mode of the corresponding derivatives, are the following: (A) [13C2]-
Acetaldehyde (225/163). (B) [12C2 ]-Acetaldehyde (223/163). (C)
[13C4]-Crotonaldehyde (253/172). (D) [12C4]-Crotonaldehyde
(249/172).
components and with protein from the cell membrane, therefore
decreasing its availability (Supporting Information).
In order to verify if crotonaldehyde arising from aldol con-
densation is formed in the cell media, cells were treated with
703 μM [13C2]-AA for 3 h, and subsequently, cell media aliquots
were taken and derivatized (1:1) with 20 mM DNPH. The
resulting hydrazones were analyzed by HPLC/ESI/MS/MS.
The unequivocal formation of 1,N2-propanodGuo in a two-
step reaction with exogenously added AA was assessed by
treating cells with [13C2]-AA and detecting the labeled adduct
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Scheme 2. Reaction of dGuo with Two Molecules of [13C2]-
Acetaldehyde
unfavorable and N2-ethylidenedGuo should be formed more
promptly. Therefore, it is important to state that N2-ethylide-
nedGuo would be the main adduct formed under human
exposure conditions. Nevertheless, more studies are warranted
in order to fully comprehend this phenomenon.
In conclusion, the application of our highly sensitive HPLC/
MS/MS method, in combination with [13C2]-AA, showed direct
and unequivocal AA-induced formation of 1,N2-propanodGuo in
living cells. In addition, higher levels of endogenously generated
1,N2-εdGuo could be detected after AA exposure. The adducts
detected here could play a role in the genotoxicity of AA, an
ubiquitous environmental pollutant and alcohol metabolite.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental section and Re-
b
sults: Determination of acetaldehyde concentrations, Malondial-
dehyde determination and Assessment of [13C4]1,N2-propanodGuo
generated by AA carryover during DNA extraction. This material
is available free of charge via the Internet at http://pubs.acs.org
’ AUTHOR INFORMATION
Corresponding Author
mhgdmede@iq.usp.br
’ ACKNOWLEDGMENT
This work was supported by the following Brazilian research
funding institutions: FAPESP (Fundac-~ao de Amparo ꢁa Pesquisa
do Estado de S~ao Paulo), CNPq (Conselho Nacional para o
Desenvolvimento Científico e Tecnolꢀogico), CAPES (Coordenac-~ao
de Aperfeic-oamento de Pessoal de Nível Superior), INCT de
Processos Redox em Biomedicina - Redoxoma, and Prꢀo-Reitoria
de Pesquisa da USP.
Figure 3. Representative chromatogram obtained by HPLC/MS/MS
of hydrolyzed DNA from cells treated with 703 μM [13C2]-acetalde-
hyde. (A) Chromatographic detection of normal basal levels of 1,N2-
propanodGuo (m/z 338/222). (B) Detection of [13C4]-labeled 1,N2-
propanodGuo (m/z 342/226) by the addition of two [13C2]-AA.
(Scheme 2). The adducts were detected by HPLC/MS/MS
using an API 4000 QTRAP mass spectrometer (Applied
Biosystems) in the MRM mode. Briefly, the DNA hydrolysates
were injected into the HPLC/ESI/MS/MS system. Then, the
m/z 338f222 (1,N2-propanodGuo) and 342f226 ([13C4]1,
N2-propanodGuo) transitions were monitored. The formation of
a molecule 4 amu over the expected weight of 1,N2-propanodGuo
confirmed that two molecules of labeled AA had been incorpo-
rated. In order to exclude possible artifactual adduct formation
due to carryover of AA during DNA extraction, 1,N2-propanod-
Guo was measured in DNA extracted immediately (0 h) and 3 h
after AA treatment. 1,N2-PropanodGuo formation at 0 h was not
detected in our analytical platform (limit of detection 10 fmol)
(Supporting Information). Thus, the resulting 1,N2-propanod-
Guo molecule was due to treatment with labeled AA and was not
due to nonspecific lipid peroxidation product or artifactual
formation (Scheme 2 and Figure 3).
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