Role of nitrite on nitration of 2′-deoxyguanosine by nitryl chloride

Article abstract of DOI:10.1002/jccs.200200043

Nitryl chloride and peroxynitrite are reactive nitrogen species generated by activated phagocytes against invading pathogens during infections and inflammation. In our previous report, formation of 8-nitroxanthine and 8-nitroguanine was observed in reaction of 2′-deoxyguanosine or calf thymus DNA with nitryl chloride generated by mixing hypochlorous acid (HOCl) with nitrite (NO₂^-). The present study investigates factors controlling the yields of 8-nitroxanthine and 8-nitroguanine formation in nitration of 2′-deoxyguanosine by nitryl chloride. We found that the yields of 8-nitroxanthine and 8-nitroguanine in reaction of 2′- deoxyguanosine with nitryl chloride were highly dependent on the ratio of NO₂^- versus HOCl concentration. The yields of 8-nitroxanthine and 8-nitroguanine reached a plateau when the ratio of NO ₂^- versus HOCl concentration was higher than 2. A possible mechanism was postulated to explain this observation. While 8-nitroguanine is not stable in the presence of peroxynitrite, 8-nitroxanthine is sensitive to HOCl. The stability of these two nitrated adducts might be a factor on their final yields in this reaction. Since HOCl is produced by neutrophils at sites of inflammation where the level of NO₂^- is elevated, it is conceivable that nitryl chloride contributes to DNA base nitration in vivo, forming 8-nitroxanthine and 8-nitroguanine.

Full text of DOI:10.1002/jccs.200200043

JournaloftheChineseChemicalSociety, 2002, 49, 275-281
275
Role of Nitrite on Nitrationof2¢-Deoxyguanosine by Nitryl Chloride
Hauh-Jyun Candy Chen* (
), Tze-Fan Wang (
) and Yuan-Mao Chen (
)
DepartmentofChemistry,NationalChungChengUniversity,Chia-Yi 621, Tai wan, R.O.C.
Nitrylchlorideandperoxynitritearereactivenitrogenspeciesgeneratedbyactivatedphagocytes against
invadingpathogensduringinfectionsandinflammation.Inourpreviousreport,format ion of 8-nitroxanthine
and 8-nitroguanine was ob served in re ac tion of 2¢-deoxyguanosine or calf thy mus DNA with nitryl chlo ride
-
gen er ated by mix ing hypochlorous acid (HOCl) with ni trite (NO2 ). Thepresentstudyinvestigatesfactors
con trol ling the yields of 8-nitroxanthine and 8-nitroguanine for ma tion in ni tra tion of 2¢-deoxyguanosine by
nitryl chlo ride. We found that the yields of 8-nitroxanthine and 8-nitroguanine in re ac tion of 2¢- deoxy-
-
guanosine with nitryl chlo ride were highly de pend ent on the ra tio of NO2 versusHOClconcentration.The
yields of 8-nitroxanthine and 8-nitroguanine reached a pla teau when the ra tio of NO2- versusHOClconcentra-
tion was higher than 2. A pos si ble mech a nism was pos tu lated to ex plain this ob ser va tion. While 8- nitro-
guanine is not sta ble in the pres ence of peroxynitrite, 8-nitroxanthine is sen si tive to HOCl. Thestabilityof
these two ni trated ad ducts might be a fac tor on their fi nal yields in this re ac tion. Since HOCl is pro duced by
-
neu tro phils at sites of in flam ma tion where the level of NO2 is el e vated, it is con ceiv able that nitryl chlo ride
contributestoDNAbasenitrationin vivo, form ing 8-nitroxanthine and 8-nitroguanine.
INTRODUCTION
DNA dam age caused by peroxynitrite (ONO2-), formed
dityrosine,21 oxidationandnitrationofhumanlow-densityli-
·
po pro tein (LDL), an in duced lipid peroxidation.21,22 A NO2
-
radicalmaybeformedviatheone-electronoxidationofNO2
bytherapidreactionofsuperoxideandnitricoxideproduced
ininflamedtissues,1-3 is be lieved to play an im por tant role in
themultistagecarcinogenesisassociatedwithchronicinfec-
tions and in flam ma tion.4,5 Peroxynitrite re acts with DNA
mainly by mod i fy ing the gua nine base, form ing xanthine,
8-nitroguanine (8NG) and 8-oxoguanine.6-8 For ma tion of
apurinic sites in duced GC to TA transversion mu ta tions,9 the
same mu ta tions in duced by peroxynitrite.4 It is well doc u-
mented that peroxynitrite can be formed underoxidative
stress in sev eral dis ease states10-14 as well as from cig a rette
smoking.15,16 Anotherendogenousnitratingspeciesisnitryl
bycompoundIorcompoundIIofmyeloperoxidase.17,18 In re-
ac tion of the myeloperoxidase/H2O2/Cl-/NO2 sys tem with
-
2¢-deoxyguanosine (dG), 8NG and 8-nitro-2¢-deoxyguano-
sine were formed.19
In our pre vi ous re port, for ma tion of 8-nitroxanthine
(8NX) and 8NG (Scheme I) was ob served in re ac tion of dG or
calf thy mus DNA with nitryl chlo ride gen er ated by mix ing
NO2- with HOCl.23 Thisstudyexaminesfactorsimportantto
-
8NXand8NGformation, suchasreactiontime, ratioofNO2
ver sus HOCl, and con cen tra tions of nitryl chlo ride and 2¢-
·
chlo ride (NO2Cl) or ni tro gen di ox ide rad i cal (NO2 ) pro-
Scheme I Struc tures of 8-nitroxanthine and
duced in ac ti vated phagocytes pro moted by chronic in fec-
tions and inflammation.17,18 It is be lieved that myelo per-
oxidaseuseshydrogenperoxide(H2O2)generatedbyneutro-
phils to ox i dize the chlo ride ion and pro duce hypochlorous
acid (HOCl), a bac te ri cidal toxin. This toxin re acts with ni-
8-nitroguanine
O
O
N
N
HN
HN
H2N
NO2
NO2
N
-
·
O
N
N
H
N
trite (NO2 ), the pri mary me tab o lite of NO , form ing NO2Cl
that is ca pa ble of ni trat ing gua nine19 andtyrosine.20 Nitryl
chlo ride also leads to for ma tion of chlo ri nated ty ro sine and
H
H
8-nitroxanthine
(8NX)
8-nitroguanine
(8NG)
* Cor re sponding au thor. Fax: +886-5-272-1040; E-mail: chehjc@ccunix.ccu.edu.tw
Abbreviations: N2O4, dinitrogen tetroxide; dG, 2¢-deoxyguanosine; H2O2, hy dro gen per ox ide; HOCl, hypochlorous acid; NO2 , ni-
-
·
+
trite; NO2 , ni tro gen di ox ide rad i cal; 8NG, 8-nitroguanine; NO2 , nitronium ion; 8NX, 8-nitroxanthine; NO2Cl, nitryl chlo ride;
-
ONO2 , peroxynitrite; NaNO2,sodiumnitrite.

276 J. Chin. Chem. Soc., Vol. 49, No. 2, 2002
Chen et al.
deoxyguanosine. Since both 8NX and 8NG are rap idly re-
moved from DNA once they are formed, these two ni trated
DNA bases might be im por tant sources of apurinic le sions in
DNAderivedfromreactivenitrogenspeciesproducedin vivo
or from ex og e nous sources. There fore, 8NX and 8NG might
be used as biomarkers for DNA ni tra tion to in ves ti gate their
roleincancerdevelopment.
dif fer ent time in ter vals up to 2 hours. Levels of 8NX and
8NG were not a func tion of the in cu ba tion time. They reach
the max i mum at around 10 min and de clined dur ing pro-
longedincubationandstabilized(Fig. 2). Theseresultsindi-
cated that ni trated ad ducts formed can be de com posed by the
excessreagentsremaininginthereactionmixture,whichwas
evidencedbyexaminingtheirstabilityasdescribedinalater
section.Nosignificantchangeinyieldsof8NXand8NGwas
-
observedafter1h,suggestingthattheexcessHOClandNO2
RESULTSANDDISCUSSION
80
60
40
20
0
-
Nitration of xanthine with peroxynitrite or NO2 /HOCl
mixture
Reactivenitrogenspecies,includingperoxynitriteand
nitrylchloride,arecapableofnitratingproteintyrosine,lead-
ing to the for ma tion of 3-nitrotyrosine in hu man plate lets.
Peroxynitrite has been shown to be a better ni trat ing agent
than nitryl chlo ride in this sys tem.24,25Inreactionofxanthine
withvariousamountsofperoxynitrite, reversedphaseHPLC
analysisrevealedthatformationof8NXreachedamaximum
when the con cen tra tion of peroxynitrite was 10 times in ex-
cess of xanthine and it de clined with a large ex cess (50 times)
of peroxynitrite. When an equal con cen tra tion of HOCl and
NaNO2 was mixed in the pres ence of xanthine, the yield of
8NXwasoptimumattheconcentrationofnitrylchloridebe-
ing 5 times of xanthine and it de creased with in creas ing con-
cen tra tion of nitryl chlo ride. No 8NX was de tected in the
pres ence of 50 times of nitryl chlo ride. In ter est ingly, ex cess
NO2- ap peared to in crease the yield of 8NX. When the con-
centrationofNO2- was twice that of HOCl, the yields of 8NX
increasewithincreasingconcentrationsofHOCl.Thenitrat-
0
10
20
30
40
50
[HOCl] / [Xan] or [peroxynitrite] / [Xan]
-
Fig. 1. Ef fect of NO2 /HOCl ra tio on for ma tion of
8NX in xanthine reaction with various
amounts of nitryl chlo ride or peroxynitrite.
-
-
[NO2 ]/[HOCl] = 2 (¨ ); [NO2 ]/[HOCl] = 1
(o); peroxynitrite (D). Re sults are ex pressed as
meansofatleastduplicateexperiments.
3 .5
3 .0
2 .5
2 .0
1 .5
-
ing abil ity for the NO2 /HOCl mix ture could be higher than
peroxynitrite in the pres ence of ex cess ni trite. The yield of
8NX
8NG
-
8NX (51%) in the re ac tion with the con cen tra tions of NO2
and HOCl be ing 100 and 50 times in ex cess of xanthine, re-
spec tively, ex ceeded that (17%) in re ac tion with a 50 mo lar
equiv a lent of peroxynitrite (Fig. 1). The re ac tion yields of
8NX ap pear to cor re late with the sta bil ity of 8NX in the pres-
enceofexcessreactivespecies. Detailswillbediscussedina
latersection.
1 .0
0 .5
0 .0
0
20
40
6 0
80
100
12 0
1 40
time (min)
Fig. 2. Ef fect of in cu ba tion time on for ma tion of 8NX
(¨ ) and 8NG (o) in re ac tion of dG with nitryl
chlo ride. To a so lu tion con tain ing dG (2.0 mM)
in 0.1 M po tas sium phos phate buffer (pH 7.0)
-
Ef fect of in cu ba tion time, NO2 , nitryl chloride, and dG
con cen tra tions on 8NX and 8NG for ma tion in re ac tion
of dG with NO2-/HOCl mixture
Ourearlierworkshowedformationof8NXand8NGin
re ac tion of dG or DNA with nitryl chlo ride gen er ated from
mixingNO2 with HOCl.23 In this study, a time-dependent
-
and NO2 (200 mM) was added NaOCl (100
mM) at room temperature, quenched with
methione (100 mM) and an a lyzed by HPLC as
de scribed in Ma te rials and Methods. Data are
ex pressed as means of at least du pli cate ex per i-
ments.
-
for ma tion of 8NX and 8NG from re ac tion of dG with nitryl
chlo ride was an a lyzed af ter quench ing with methionine at

Role of Ni trite on Ni tra tion of 2¢-Deoxyguanosine by Nitryl Chlo ride
J. Chin. Chem. Soc., Vol. 49, No. 2, 2002 277
-
were converted to NO3 and Cl-, the stable end products
(Scheme II).
log i cal pH with a sec ond-order rate con stant of 7.4´ 103 M-1
s-1.20 It is con ceiv able that for ma tion of nitryl chlo ride by
mix ing equal amounts of NaNO2 and HOCl is not very ef fi-
cient. Ac cord ingly, the unreacted HOCl is re spon si ble for
Scheme II For ma tion of nitryl chlo ride from HOCl
-
low yield of 8NX when the ra tio of [NO2 ] ver sus [HOCl] is
-
and NO2
less than 2. How ever, prior to the proof of this pos tu la tion,
the con cen tra tion of nitryl chlo ride stated in the text de notes
-
2
-
-
-
+
HOCl + NO
NO Cl + OH
NO + Cl + H
2
3
-
theconcentrationofNO2 or HOCl, which ever is smaller, as-
sumingtheircompleteconversiontonitrylchloride.
AnotherpossibilityforNO2- affectingyieldofnitration
istheinvolvementofNO2+ as the ni trat ing agent. Nitryl chlo-
ride can be con verted to the more re ac tive ni trat ing spe cies,
.
+
-
N O
2
NO
NO
2
4
2
·
such as nitrogendioxideradical(NO2 ) or nitronium ion
NO
2
+
(NO2 ) (Scheme II). Since HOCl is a poor one-electron ox i-
-
diz ing agent, but a strong two-electron ox i dant with the re-
duc tion po ten tial be ing + 0.2 V and + 1.1 V, re spec tively,26
The ef fect of the rel a tive con cen tra tion of NO2 versus
HOCl on the yields of 8NX and 8NG was in ves ti gated. Since
the re ac tiv ity of nucleoside (dG) with the NO2 /HOCl mix-
ture was much lower than the nucleobases, xanthine or gua-
nine,23 HOCl in the con cen tra tion of 50 times that of dG was
used,whileconcentrationsofnitritewerevaried.Inreaction
-
-
oxidationofNO2 to NO2+ may be fa vor able over its con ver-
·
-
sion to NO2 .20 In the pres ence of ex cess NO2 , re duc tion of
NO2+ from NO2- stoichiometrically re sults in two NO2· (NO2
+ NO2 ® 2 NO2 ). It is also fea si ble that the highly un sta ble
+
-
·
+
-
-
NO2 re acts with ex cess NO2 form ing the rel a tively sta ble
dinitrogen tetroxide (N2O4) with a sec ond-order rate con stant
of 4.5 ´ 108 M -1 s-1, thenitratingagentthatcanslowlydecom-
pose into two NO2 (k = 6.9 ´ 103 s-1)27 (SchemeII). Reaction
of the HOCl/NO2 mix ture with dG, the yield of 8NX or 8NG
reachedaplateauwhenitwasplottedasafunctionoftheratio
-
-
of [NO2 ]/[HOCl]. A ra tio of [NO2 ]/[HOCl] higher than 2 did
notsignificantlyaffecttheyieldsofthenitratedadducts8NX
and 8NG (Fig. 3). In re ac tion of dG with the con cen tra tion of
HOCl be ing twice that of NO2 ([NO2 ]/[HOCl] = 0.5), no
8NX or 8NG was de tected. It there fore ap pears to be im por-
·
·
of NO2 with tyrosyl rad i cals in pro teins form ing 3- nitro-
-
-
tyrosine was in a near dif fu sion con trolled rate (k = 3 ´ 109
M-1 s-1).28 Combination of NO2 with the dG(-H)· or the
·
-
diazoniumionradicalintermediateI23 resultsinformationof
tantthattheconcentrationofNO2 ex ceeds HOCl to form ni-
-
8NG and 8NX, re spec tively, and thus ac counts for the in-
trated bases. Hypochlorous acid re acts with NO2 at phys i o-
-
creaseintheyieldofnitrationduetoincreaseoftheNO2 con-
5
4
3
2
1
0
cen tra tion. So far, the rate con stants in these bi mo lec u lar
pathwayshavenotbeeninvestigated.
The yields of 8NX and 8NG also de pend on the con cen-
trationofthenitratingspeciesrelativetothatofdG.Usingthe
-
sys tem giv ing the op ti mum yield of ni tra tion (i.e. [NO2 ]/
[HOCl] = 2) with a fixed con cen tra tion of dG (0.96 mM),
the yields of 8NX in creased with in creas ing nitryl chlo ride
con cen tra tions, i.e. the con cen tra tion of HOCl, in a dose-
dependent man ner with a rapid rise at high con cen tra tions.
Con versely, the yields of 8NG in creased at lower con cen tra-
tions of the nitryl chlo ride to a cer tain ex tent (up to 0.29%
0
1
2
3
4
5
6
7
-
ratio of [NO2 ]/[HOCl]
-
with [NO2 ]/[HOCl]/[dG] = 25:12.5:1) and re mained con-
Fig. 3. Ef fect of [NO2]/[HOCl] ra tio on for ma tion of
8NX (¨ ) and 8NG (o) in re ac tion of dG (2.0
mM)withnitrylchloride. Toasolutioncontain-
ingdG(2.0mM)withvaryingconcentrationsof
stant at higher con cen tra tions. At HOCl con cen tra tions £ 12
mM, theyieldsof8NGexceededthoseof8NX. Thesituation
was re versed with HOCl con cen tra tions ³ 24 mM, in which
theformationof8NXwasmuchmorefavoredover8NG(Fig.
4). Thus, the pref er ence in for ma tion of 8NX or 8NG is a
functionoftheconcentrationofthenitratingspecies.
-
NO2 in 0.1 M po tas sium phos phate buffer (pH
7.0) was added NaOCl (0.1 M) with stir ring at
room tem per a ture for 5 min. Re sults are ex-
pressedasmeansoftriplicateexperiments.
WealsoexaminedtheeffectofdGconcentrationinthe

278 J. Chin. Chem. Soc., Vol. 49, No. 2, 2002
Chen et al.
yields of 8NX and 8NG since the con cen tra tions of DNAin
vivovarywithincellularcompartmentssuchasthenucleusor
mi to chon dria. With a fixed con cen tra tion of nitryl chlo ride
sum of the two nitrated adducts increased 0.04% per
millimolar HOCl ac cord ing to Fig. 4. It is prob a bly due to the
fact that nitryl chlo ride is a re ac tive spe cies, pre sum ably
lead ing to for ma tion of the non-reactive ni trite and chlo ride
ions (Scheme II). Ac cord ingly, the steady-state level of nitryl
chloride is much lower than that from the presumed
-
gen er ated from [NO2 ]/[HOCl] = 2, the yields of 8NX and
8NG in creased lin early with in creas ing dG con cen tra tion
rang ing from 0.36 to 2.0 mM (Fig. 5). The slope of this graph
showed that the yields of 8NX in creased 1.66% per milli-
molar dG while the yields of 8NG re mained con stant, but the
-
stoichiometricformationfromNO2 andHOCl.Collectively,
theseresultsindicatethattheconcentrationofdGisthedomi-
nat ing fac tor in the yield of ni tra tion over the con cen tra tion
of nitryl chlo ride and the lat ter de ter mines which ni trated
adduct, 8NX or 8NG, is formed pref er en tially over the other.
It is there fore highly con ceiv able that both 8NX and 8NG are
formed in the vi cin ity of in flamed tis suesin vivo.
2.0
y = 0 .03 84 x
R2
0.99 3 5
1.5
=
The in vivo concentrationsofNO2- have been re ported
to be at submicromolar lev els in plasma,29 100 mMinrespira-
tory tract ep i the lial cells,30 and up to 210 mM in sa liva31 in
1.0
0.5
0.0
-
healthy in di vid u als. Di etary in take of ni trate (NO3 )-rich
-
foods can cause a dra matic in crease in the level of NO2 in the
mouth which is then in gested to the stom ach.32 These lev els
areelevatedduringtheinflammatoryprocesses.Ontheother
hand, the con cen tra tion of HOCl can reach 100 mM in the vi-
cinityofactivatedneutrophils.33 There fore, nitryl chlo ride
0
10
2 0
3 0
4 0
5 0
[HOCl] (mM)
Fig. 4. Formationof8NX( ¨ ) and 8NG (o) in re ac tion
of dG with var i ous amounts of nitryl chlo ride.
Toasolutioncontaining2¢-dG (0.96 mM) in 0.1
M po tas sium phos phate buffer (pH 7.0) was
added with vary ing con cen tra tions of NO2- and
-
gen er ated by mix ing HOCl and NO2 is likely to play an im-
por tant role in nonenzymatic DNA nitrative dam age. Ni trite
in hib its the kill ing of E. coli by HOCl by re mov ing these
toxic ni trat ing spe cies and thus pro tects the cells,34,35 which
sug gests that ni tra tion of biomolecules in E. coli does not en-
dangertheimmediatesurvivalofthecells.Whetheraccumu-
latednitratedbiomoleculesarerelatedtocancerformationin
thelongterminhigherorganismsremainsunanswered.
-
HOCl ([NO2 ]/[HOCl] = 2) with stirring at
room tem per a ture for 5 min, fol lowed by HPLC
analysisasdescribedinMaterialsandMethods.
The sum of 8NX and 8NG (D) are ex pressed by
lin ear re gres sion. Data are pre sented as means
ofatleastduplicateexperiments.
Stability of 8NX and 8NG in the presence of reactive
species
4 .0
In re ac tions with xanthine or dG, lev els of 8NX and/or
8NG de creased sub stan tially when a large ex cess of nitryl
chlo ride was gen er ated by mix ing equal con cen tra tions of
3 .0
2 .0
-
8NX
8NG
HOCl with NO2 (see Figs. 1 and 3). There fore, the sta bil ity
of 8NX and 8NG in the pres ence of ex cess re ac tive spe cies
was ex am ined. When pure 8NX was mixed with a 25 times
mo lar ex cess of nitryl chlo ride gen er ated by mix ing equal
1 .0
0 .0
-
amounts of HOCl with NO2 , 8NX was com pletely de com-
0
0.5
1
1.5
2
posed as sup ported by the re versed phase HPLC anal y sis. On
the other hand, 63% of 8NX sur vived in a mix ture of NO2
[dG] (mM)
-
and HOCl 50 and 25 times in ex cess, re spec tively (Ta ble 1).
HOCl also ef fec tively de stroyed 8NX, as ev i denced by the
com plete de com po si tion of 8NX when HOCl of 25 times in
excesswasaddedtoasolutionofisolated8NXatneutralpH.
It is con ceiv able that mix ing equal con cen tra tions of NO2
and HOCl does not form nitryl chlo ride ef fi ciently and that
Fig. 5. Therelativeformationof8NX(¨ ) vs. 8NG (o)
was a func tion of dG con cen tra tion in its re ac-
-
tion with nitryl chlo ride. The ra tio of [NO2 ]/
[HOCl]/[dG] was 100/50/1. Results are ex-
pressed as means of at least du pli cate ex per i-
ments.
-

Role of Ni trite on Ni tra tion of 2¢-Deoxyguanosine by Nitryl Chlo ride
J. Chin. Chem. Soc., Vol. 49, No. 2, 2002 279
Table 1. Percentage Survival Rate of 8NX or 8NG Reaction
with Excess Reactive Species
alsobeusedtoexaminetheprotectiveeffectofantioxidants
againstreactivenitrogenspecies-inducednitrationofDNA39
andthuscontributetounderstandingthecorrelationbetween
these ni trated DNA ad ducts and in flam ma tion-induced can-
cer and other dis eases.
Reaction
yield of 8NX
&
8NGa
+
+
%
0
63%
0%
8NX NaNO2 HOCl (1:25:25)
8NX + NaNO2 + HOCl (1:50:25)
8NX + HOCl (1:25)
8NX ONO2- (1:25)
69
+
%
8NX + H2O2 (1:25)
8NG + NaNO2 + HOCl (1:25:25)
95%
1.8%
EXPERIMENTAL SECTION
94%
+
+
%
%
95
98%
0.3%
%
97
8NG NaNO2 HOCl (1:50:25)
8NG + HOCl (1:25)
2.4
N.D.b
N.D.
N.D.
Materials
8NG + ONO2- (1:25)
2¢-Deoxyguanosine was from Sigma Chem i cal Co. (St.
Louis, MO). Gua nine, so dium ni trite, and so dium hy po-
chloride (NaOCl) were ob tained from Aldrich Chem i cal Co.
(Mil wau kee, WI). All re agents are re agent grade or above.
Hydrogen peroxide was purchased from Acros Organic
Chem i cal Co. (Geel, Bel gium) and quan ti fied based on the
absorbance at 240 nm ( e= 43.6 M-1cm -1 ).40 Peroxynitrite was
syn the sized ac cord ing to the pre vi ously de scribed pro ce-
duresusingiso-amylnitriteandhydrogenperoxide41 and was
stored at -80 °C. The con cen tra tion of peroxynitrite was de-
ter mined by the absorbance at 302 nm in 1 N NaOH (e = 1670
M-1cm-1).42 The con cen tra tion of NaOCl was de ter mined by
+
8NG H2O2 (1:25)
a Values are means of at least duplicated experiments.
b Not detectable.
the unreacted HOCl is re spon si ble for the de creased yield of
-
8NX. When the ra tio of con cen tra tion for NO2 ver sus HOCl
-
was greater than 2 in re ac tions of dG with mix tures of NO2
-
and HOCl (Fig. 3), ex cess NO2 led the equi lib rium to ward
nitrylchlorideformation(SchemeII)andtherewasanegligi-
bleamountoffreeHOCl.Theseobservationsalsorationalize
the fact that the yield of 8NX in xanthine re ac tion with the
-1
the absorbance at 292 nm (pH 12, e = 350 M cm-1).43 Stan-
-
NO2 /HOCl mix ture was much higher when the amount of
dard 8NG and 8NX were syn the sized as de scribed pre vi-
ously.23
NO2- exceededHOCl(Fig.1).Collectively,theseresultssug-
gest that the pres ence of ex cess NO2- in creases the flux of the
nitratingspeciesandprotects8NXfromdecomposition,al-
thoughthedecompositionproductsarenotidentifiedatthis
stage.
HPLCChromatography
HPLC chro ma tog ra phy was per formed by a Hitachi
L-7000 pump sys tem with D-7000 in ter face, a Rheodyne in-
jec tor, and a L-7450A photodiode ar ray (PDA) de tec tor. (1)
Sys tem 1. A Prod igy ODS (3) 250 mm ´ 4.6 mm, 5 mm col-
umn (Phenomenex, Torrance, CA) was used with the fol low-
ing isocratic con di tions: 50 mM am mo nium formate buffer
(pH = 4.0) at a flow rate of 1.0 mL/min. (2) Sys tem 2.A Prod-
igy ODS (3) 250 mm ´ 4.6 mm, 5 mm col umn was used with
the fol low ing isocratic con di tions: 50 mM am mo nium for-
mate (pH = 5.5) buffer at a flow rate of 1.0 mL/min.
In the presence of other re ac tive spe cies, 8NX was
some what sta ble with peroxynitrite (69% sur vived) but it
merely de com posed (95% re mained) with H2O2, abiologi-
callyimportantoxidant.Ontheotherhand,8NGwasstablein
thepresenceofH 2O2 or HOCl and it was not af fected by nitryl
-
chlo ride gen er ated from HOCl with equal or ex cess NO2 .
How ever, 8NG al most com pletely de com posed with the ad-
di tion of peroxynitrite (Ta ble 1). This re sult was con sis tent
withpreviousreports.36,37
Since 8NG is not re paired by the en zyme formami do-
pyrimidine glycosylase36,38 and that for ma tion of 8NX and
8NG can re sult in the mutagenic apurinic sites in DNA, they
might be important DNA lesions. Because of the labile
glycosidic link ages of 8NX and 8NG in DNA, the ex is tence
ofthesetwoadductsinbiologicalfluidsishighlypossible.If
theyarepresentinhighenoughlevelsfordetection,measure-
mentof8NXand8NGintissueDNAandinbiologicalfluids
should give in sights into the roles of these ad ducts in car-
cinogenesis. These ni trated DNA ad ducts should be use ful
biomarkers for en dog e nous ni tra tion of DNA. They might
-
Reaction of Xanthine with Peroxynitrite or NO2 /HOCl
Mixture
A. with peroxynitrite
To a so lu tion of xanthine (0.34 mg, 2.25mmol) in 0.5 N
HCl (0.2 mL) was added 11 mL of peroxynitrite (200 mM in
0.67 N NaOH and 50 mM am mo nium formate) and 989 mL of
0.67 N NaOH con tain ing 50 mM am mo nium formate with
vig or ous stir ring at room tem per a ture for 2 min. Fi nal pH of
the re ac tion was 7.0 with a fi nal vol ume of 1.2 mL. For re ac-
tions with higher amounts of peroxynitrite, the vol ume of
peroxynitrite was in creased and that for the NaOH so lu tion

280 J. Chin. Chem. Soc., Vol. 49, No. 2, 2002
Chen et al.
wasdecreasedaccordinglywiththefinalvolumeunchanged.
B. with NO2 /HOCl mixture
mmol) in 1.083 mL of 0.2 M po tas sium phos phate buffer (pH
7.0) was added 27 mL of H2O2 (200 mM) with stir ring. Fi nal
pH of the re ac tion mix ture was 7.0. The en tire re ac tion mix-
ture was an a lyzed by HPLC us ing sys tem 2 at 376 and 393
nm.
-
To a so lu tion of xanthine (0.34 mg, 2.25 mmol) in 0.5 N
HCl (0.2 mL) was added 828 mL of a so lu tion of NaNO2 (3.7
mMin0.4Mpotassiumphosphatebuffer, pH12.8), followed
by 172 mL of a so lu tion of NaOCl (13.1 mM in 0.4 M po tas-
siumphosphatebuffer, pH12.8)withstirring. FinalpHofthe
reactionmixtureswas7.0.Forreactionswithhigheramounts
of nitryl chlo ride, the con cen tra tions of NaNO2 and NaOCl
wereincreasedaccordinglywithafinalvolumeof1.2mL. A
120 mL aliquot of the reaction mixture was analyzed by
HPLC us ing sys tem 1 at 376 nm.
ACKNOWLEDGMENT
This work was sup ported by grants from the Na tional
Sci ence Coun cil of Tai wan and from Na tional Chung Cheng
University(toH.-J.C. C.).
Reaction of dG Re ac tion with Nitryl Chlo ride
Typically, to a so lu tion con tain ing 2¢-dG and NaNO2
dissolvedin0.5mLofpotassiumphosphatebuffer(finalcon-
cen tra tion 0.1 M, pH 7.0) was added a so lu tion con tain ing
NaOCl (0.24 M) with stir ring at room tem per a ture for 5 min.
The re ac tion mix ture was ad justed to pH 4.0 or 5.5 and an
aliquotofthereactionmixturewasanalyzedbyHPLCusing
sys tem 1 at 376 nm or us ing sys tem 2 at 393 nm.
Received October 19, 2001.
Key Words
DNA; 2¢-Deoxyguanosine; Hypochlorous acid;
Nitration; Ni trite; 8-Nitroguanine;
8-Nitroxanthine; Nitryl chloride; Peroxynitrite.
Reaction of 8NX or 8NG with Excess Nitryl Chloride
To a so lu tion con tain ing 8NX or 8NG (43.5mg, 0.22
mmol) in 1.0 mL of 0.2 M po tas sium phos phate buffer (pH
7.0) was added 87 mL of a so lu tion of NaNO2 (63 mM or 126
mM) and 23mL ofa solutionofNaOCl (0.24M)withstirring.
FinalpHofthereactionmixturewas7.0. Theentirereaction
mix ture was an a lyzed by HPLC us ing sys tem 2 at 376 and
393 nm.
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