The sphingolipid degradation product trans-2-hexadecenal forms adducts with DNA

Article abstract of DOI:10.1016/j.bbrc.2012.06.012

Sphingosine 1-phosphate, a bioactive signaling molecule with diverse cellular functions, is irreversibly degraded by the endoplasmic reticulum enzyme sphingosine 1-phosphate lyase, generating trans-2-hexadecenal and phosphoethanolamine. We recently demonstrated that trans-2-hexadecenal causes cytoskeletal reorganization, detachment, and apoptosis in multiple cell types via a JNK-dependent pathway. These findings and the known chemistry of related α,β-unsaturated aldehydes raise the possibility that trans-2-hexadecenal may interact with additional cellular components. In this study, we show that it reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic 1,. N²-deoxyguanosine adducts 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8. R-hydroxy-6. R-tridecylpyrimido[1,2-a]purine-10(3. H)one and 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8. S-hydroxy-6. S-tridecylpyrimido[1,2-a]purine-10(3. H)one. Thus, our findings suggest that trans-2-hexadecenal produced endogenously by sphingosine 1-phosphate lyase can react directly with DNA forming aldehyde-derived DNA adducts with potentially mutagenic consequences.

Full text of DOI:10.1016/j.bbrc.2012.06.012

Biochemical and Biophysical Research Communications 424 (2012) 18–21
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
The sphingolipid degradation product trans-2-hexadecenal forms adducts with DNA
Pramod Upadhyaya a, Ashok Kumar b, Hoe-Sup Byun c, Robert Bittman c, Julie D. Saba b,
Stephen S. Hecht a,
⇑
a Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
b Center for Cancer Research, Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA
c Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, NY 11367, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2012
Available online 19 June 2012
Sphingosine 1-phosphate, a bioactive signaling molecule with diverse cellular functions, is irreversibly
degraded by the endoplasmic reticulum enzyme sphingosine 1-phosphate lyase, generating
trans-2-hexadecenal and phosphoethanolamine. We recently demonstrated that trans-2-hexadecenal
causes cytoskeletal reorganization, detachment, and apoptosis in multiple cell types via a JNK-dependent
Keywords:
trans-2-Hexadecenal
Sphingosine 1-phosphate
Sphingosine 1-phosphate lyase
1,N2-propanodeoxyguanosine adducts
DNA adducts
pathway. These findings and the known chemistry of related
a,b-unsaturated aldehydes raise the
possibility that trans-2-hexadecenal may interact with additional cellular components. In this study, we
show that it reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic
1,N2-deoxyguanosine adducts 3-(2-deoxy-b-
D
-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-
6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy-b- -erythro-pentofuranosyl)-5,6,7,8-tetra-
D
hydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one. Thus, our findings suggest that
trans-2-hexadecenal produced endogenously by sphingosine 1-phosphate lyase can react directly with
DNA forming aldehyde-derived DNA adducts with potentially mutagenic consequences.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Sphingosine 1-phosphate lyase (SPL) catalyzes the conversion
of sphingosine 1-phosphate (1) to trans-2-hexadecenal (2) and
phosphoethanolamine (3) (Scheme 1) [1]. The roles of 1 in cell
and animal physiology have been well described [2], but the bio-
logical consequences and fate of metabolic products of 1 such as
2 are not well understood. We have previously shown that 2 causes
cytoskeletal reorganization, detachment, and apoptosis in multiple
cell types via a JNK-dependent pathway, indicating that it may
interact with multiple cellular components including DNA [3].
2.1. HPLC systems
System 1 consisted of a Waters Corp. model 600 system control-
ler, two model 501 pumps, a model 440 UV detector (254 nm), and
an Agilent Technologies 1100 series auto-sampler. A Luna C18(2)
reversed-phase column (250 ꢀ 4.6 mm, 5
lm; Phenomenex) was
used for the separation. Elution was performed with a linear gradi-
ent from 85% 15 mM NH4OAc in CH3OH to 100% CH3OH over
30 min and held for 20 min. The flow rate was 0.7 ml/min.
System 2 used an Agilent Technologies 1100 capillary HPLC sys-
Previous studies have shown that a,b-unsaturated aldehydes such
as acrolein, crotonaldehyde (2-butenal), and 4-hydroxy-2-nonenal
react readily with deoxyguanosine (dG, 4) and DNA to produce cyc-
lic 1,N2-propanodeoxyguanosine adducts with a variety of muta-
genic properties [4–10]. Therefore, we hypothesized that 2 would
undergo a similar reaction (Scheme 1), potentially producing an
adduct such as 5, which could be central to some of the physiologic
effects of 2. We tested this hypothesis in the study reported here.
tem equipped with a 5
l
m, 150 ꢀ 0.5 mm ZorbaxSB-C18 column
eluted at 8 l/min with 70% 15 mM NH4OAc in CH3OH to 99%
l
CH3OH in 30 min and held for 15 min.
System 3 was the same as System 2 except that elution was
with 85% 15 mM NH4OAc in CH3OH to 95% CH3OH in 30 min and
held for an additional 25 min.
2.2. NMR spectra
These were recorded in DMSO-d6 using a Bruker Biospin (Bille-
rica, MA) spectrometer operating at 850 MHz. Resonance assign-
ments were made based on 1H–1H COSY and 1H–13C HMBC and
HSQC experiments acquired at 850 MHz.
⇑
Corresponding author. Address: Masonic Cancer Center, University of Minne-
sota, MMC 806, 420 Delaware St. SE, Minneapolis, MN 55455, USA. Fax: +1 612 626
5135.
E-mail address: hecht002@umn.edu (S.S. Hecht).
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2012.06.012

P. Upadhyaya et al. / Biochemical and Biophysical Research Communications 424 (2012) 18–21
19
Scheme 1. Conversion of sphingosine 1-phosphate (1) to trans-2-hexadecenal (2) and reaction of 2 with dG (4) to form adduct 5.
2.3. trans-2-Hexadecenal (2)
was worked up as described (9). The resulting G adduct was ana-
lyzed by LC–ESI-MS-MS-SRM using system 2, positive ion mode
m/z 390 ? m/z 346, 390 ? m/z 372, m/z 390 ? m/z 190, and m/z
390 ? m/z 152.
This compound was prepared as described previously [11].
2.4. Reaction of 2 with dG
2.6. Reaction of 2 with DNA
trans-2-Hexadecenal (2, 4.7 mg, 20 lmol) was allowed to react
with dG (4, 20 mg, 0.074 mmol) in 5 ml of 0.10 M phosphate buffer,
pH 7.0, at 50 °C for 72 h. Adduct 5, retention time 34 min, was puri-
fied using HPLC system 1 and analyzed by liquid chromatography-
electrospray ionization-tandem mass spectrometry-selected
reaction monitoring (LC–ESI-MS/MS-SRM) using system 2 and a
Thermo TSQ Quantum Discovery MAX instrument, in the positive
ion mode with N2 as the nebulizing and drying gas. MS parameters
were set as follows: spray voltage, 4 Kv; scan width, 0.4 amu;
sheath gas pressure, 20; capillary temperature 250 °C; collision en-
ergy 20 V (for m/z 390–372 and m/z 390–346) and 28 V for other
ions; and tube lens offset, 140 V. Data were acquired and processed
by Xcalibur software version 1.4. The mass transitions monitored
were m/z 506 ? 390, m/z 390 ? 346, m/z 390 ? 372, m/z
390 ? 190, and m/z 390 ? 152.
trans-2-Hexadecenal (2, 2.4 mg, 10 lmol) was allowed to react
with calf thymus DNA (10 mg) in 5 ml of 0.10 M phosphate buffer,
pH 7.0, for 24 h at 37 °C. The DNA was precipitated by addition of
ethanol and washed with 70% and 95% ethanol. After the DNA
(0.5 mg) was dissolved in 1 ml of Tris buffer, pH 7.0, enzyme
hydrolysis was carried out as described [12]. The hydrolysate
was loaded onto a Strata-X polymeric sorbent solid-phase extrac-
tion cartridge (33 lm, 30 mg/1 ml) that was previously activated
with 1 ml of CH3OH and 1 ml of H2O. The cartridge was washed
with 2 ml of H2O, and the analyte was eluted with 2 ml of 100%
CH3OH. The eluant was collected in a 2 ml silanized vial, and the
solvents were removed on a Speedvac. The residue was taken up
in 300
to dryness. The residue was dissolved in 20
NH4OAc in CH3OH, and 8 l was analyzed by LC–ESI-MS/MS-SRM
ll of CH3OH, transferred to an insert vial, and concentrated
l
l of 70% aqueous
To enable collection of sufficient material for NMR analysis, a
larger scale reaction with 56.4 mg (0.24 mmol) of 2 and 240 mg
(0.89 mmol) of 4 was performed for 72 h at 50 °C. The reaction
was carried out in 25 ml of 25 mM potassium phosphate buffer,
l
using system 3, positive ion mode, m/z 506 ? 390. Incubation of
1 with calf thymus DNA for 72 h gave a higher yield of 5.
pH 9.0, since Michael addition of dG to a,b-unsaturated aldehydes
appears to proceed at higher rates at alkaline pH [9]. Adduct 5 was
collected using system 1 (<0.5% yield). 1H NMR (850 MHz, DMSO-
d6) d 7.912 and 7.913 (2s, 1H, C2-H), 7.82 (d, 1H, N5-H), 6.65 (m,
1H, C8-OH), 6.2 (s, 1H, C8-H), 6.1 (1H, t, J = 6.3 Hz, 10-H), 5.28 (m,
1H, 30-OH), 4.92 (m, 1H, 50-OH), 4.36 (br s, 1H, 30-H), 3.79 (m, 1H,
40-H), 3.54 (m, 1H, C6-H), 3.5–3.49 (m, 2H, 50-H2), 2.5 (m, 1H, 20-
H), 2.18 (m, 1H, 20-H), 2.06 (1H, d, J = 14 Hz, C7H), 1.4 (m, 3H,
C7-H + C9-H2), 1.2 (br s, 20H, alkyl chain CH2), 0.87 (t, 3H, CH3);
13C NMR (850 MHz, DMSO-d6) exocyclic carbons 44.81(C6), 34.56
(C7), 69.77 (C8); guanine carbons 135.75 (C2), 156.08 (C = O); alkyl
chain 31.77 (CH2-Gua), 14.45 (CH3); deoxyribose 62.14 (C-50),
87.92 (C-40), 71.18 (C-30), and 82.71 (C-10); UV (90% CH3OH/10%
15 mM NH4OAc) kmax 204, 254, 275 (sh) nm: ESI-HRMS (m/z)
[M + H]+ calcd for C26H43N5O5Na, 528.3156; found 528.3169.
2.7. Acid hydrolysis of the calf thymus DNA adduct
In brief, 0.5 mg of DNA from reaction of 2 and calf thymus DNA
was dissolved in 1 ml of HCl (0.10 N), heated at 90 °C for 60 min,
and then was worked up as described under ‘‘Acid hydrolysis of
5’’ (Section 2.5). The resulting G adduct was analyzed by LC–ESI-
MS-MS-SRM using system 2 (m/z 390 ? 346, m/z 390 ? 372, m/z
390 ? 190, and m/z 390 ? 152).
2.5. Acid hydrolysis of 5
In brief, 50 lg of 5 was dissolved in 1 ml of HCl (0.10 N) and the
mixture was heated at 90 °C for 60 min. The mixture was cooled,
neutralized with 1 N NaOH, and applied to a Strata-X polymeric
sorbent solid-phase extraction cartridge (33 lm, 30 mg/1 ml, Phe-
nomenex). The cartridge was sequentially eluted with 1 ml of H2O,
Scheme 2. Rationalization of the MS fragmentation of 5, as seen in Fig. 1.
1 ml of 10% methanol, and 2 ml of methanol, and then the product
dR = deoxyribose.

20
P. Upadhyaya et al. / Biochemical and Biophysical Research Communications 424 (2012) 18–21
3. Results and discussion
trans-2-Hexadecenal (2) was allowed to react with dG (4), and
the products were analyzed by LC–ESI-MS/MS-SRM. The transi-
tions monitored were m/z 506 ? 390 [BH]+, m/z 390 ? 346 [BH–
CH2CHOH]+, m/z 390 ? 372 [BH–H2O]+, m/z 390 ? 190 [BH–
(CH3(CH2)12 + OH)]+, and m/z 390 ? 152 [GH]+ (Scheme 2). As
shown in Fig. 1, all of these transitions were observed and are con-
sistent with the expected product, 5.
The UV spectrum and the 850 MHz 1H NMR and 13C NMR spec-
tra of 5 are completely consistent with those of other exocyclic
1,N2-propano-dG adducts produced by the reaction of
a,b-unsat-
urated aldehydes with dG [9,13,14] and all of the assignments
were confirmed by 1H–1H COSY and 1H–13C HMBC and HSQC
experiments (data not shown).
Previous studies demonstrated that adducts such as 5 are mix-
tures of 6S, 8S-and 6R, 8R-diastereomers in which the alkyl and hy-
droxy groups are trans to each other [4–10]. Importantly, the
850 MHz 1H NMR data obtained in this study allowed us to observe
2 singlets at 7.912 and 7.913 ppm, corresponding to the protons at
the 2-position of each of the two diastereomers. Observation of
these distinct signals has not been reported previously in studies
using lower field strength NMR instruments.
Acid hydrolysis of adduct 5 provided the corresponding G ad-
duct, as determined by LC–ESI-MS/MS-SRM analysis for the transi-
tions m/z 390 ? 346, m/z 390 ? 372, m/z 390 ? 190, and m/z
390 ? 152. The results are virtually identical to those shown in
Fig. 1, except that the retention time was 39.6 min. All fragments
are consistent with Scheme 2, starting with the G adduct.
Reaction of 2 with calf thymus DNA, followed by enzymatic
hydrolysis and LC–ESI-MS/MS-SRM analysis, produced the chro-
matogram illustrated in Fig. 2, which shows the presence of 5 in
the hydrolysate. Acid hydrolysis of this DNA produced the G ad-
duct, which was identified by LC–ESI-MS/MS-SRM analysis and
co-injection with the standard G adduct.
Fig. 2. LC–ESI-MS/MS-SRM analysis of an enzymatic hydrolysate of calf thymus
DNA that had been reacted with 2. The shaded peak is adduct 5.
ducts may have some special properties because of the combina-
tion of lipophilic and hydrophilic residues in the same molecule.
The results of this study support our hypothesis that 2, an
endogenous
a,b-unsaturated aldehyde produced by the action of
SPL on 1, as shown in Scheme 1, reacts with DNA and may lead
to potentially mutagenic consequences or perhaps trigger a previ-
ously unrecognized DNA damage response. A variety of mutagenic
properties of structurally related dG adducts derived from acrolein,
crotonaldehyde, and 4-hydroxynonenal have been observed, and
the ultimate biological properties of these adducts are influenced
by DNA sequence context effects, chromatin condensation, DNA re-
pair mechanisms, cross-linking, and other factors [10]. It will be
important to determine the biological properties of adduct 5 and
analyze human DNA samples for its occurrence, as many previous
studies have shown the presence of related endogenous DNA ad-
ducts such as those formed from acrolein and crotonaldehyde in
DNA from human liver, lung, leukocytes, and other tissues [7,15–
19].
Collectively, these results establish the structures of the prod-
ucts of the reaction of 2 with dG or DNA as a mixture of 3-(2-
deoxy-b-
xy-6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy-
b- -erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-
D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydro-
D
tridecylpyrimido[1,2-a]purine-10(3H)one (5). While these are the
expected products based on literature precedent [4–10], we note
that there are seven more carbons in the alkyl side chain than
any previously reported structures of this type. These amphilic ad-
Acknowledgments
This study was supported by NIH grants CA-77528 and
CA-129438 (JDS), CA-81301 (SSH), and HL-083187 (RB). Mass spec-
trometry was carried out in the Analytical Biochemistry Shared Re-
source of the Masonic Cancer Center, supported in part by NIH
grant CA-77598. We thank Todd Rappe, University of Minnesota
NMR Center, for acquiring the NMR spectra.
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