DOI10.5012/bkcs.2009.30.12.2949
Deguanylation of Guanine Based-Nucleosides and Calf Thymus DNA Induced by Halogenated Alkanes at the Physiological Condition
Jyoti Sherchan and Eung-Seok Lee*
CollegeofPharmacy, Yeungnam University, Kyongsan 712-749, Korea. *E-mail:eslee@ynu.ac.kr Received September4, 2009, Accepted October9, 2009
Massive deguanylation of guanine based-nucleoside s induced by halogenated alkanes at the physiological condition have been observed. For the study of deguanylation effects by the different substituents and/or functionality in haloge
nated alkanes, diverse kinds of halogenated alkanes were incubated with guanine based-nucleosides (ddG, dG and guanosine) for 48 h at the physiological condition (pH 7.4, 37 oC), which were analyzed by HPLC and further confirmed by LC-MS. Among the sixteen different halogenated alkanes, we observed massive deguanylation of nucleosides by 2-bromo-2-methylpropane, 2,3-dibromopropene, 2-bromopropane, bromoethane and 2-iodopropane. The order of deguanylation rate was highest in 2-bromo-2-methylpropane followed by 2,3-dibromopropene, 2-bromopropane, bro
moethane and 2-iodopropane. In addition, time and dose response relationship of deguanylation in guanine based- nucleosides induced by 2-bromo-2-methylpropane, 2,3-dibromopropene, 2-bromopropane, bromoethane and 2-iodo- propane at the physiological condition were investigated. Deguanylation of calf thymus DNA induced by halogenated alkanes was also investigated. These results suggest that the toxic effect of certain halogenated alkanes might be from the depurination of nucleosides.
KeyWords: Deguanylation, Depurination, Halogenatedalkanes, Guanine base-nucleosides, Calf thymusDNA
Introduction
Thedepurination or deguanylation of nucleicacids, which involve the release of purine orguanine bases, respectively, from nucleic acids by hydrolysis of theN-glycosidicbond (Fig.
1), gives rise to alterations ofthe cell genome.1,2 Although depu rination occursspontaneously under physiological conditions, the rate of depurinationisaccelerated atlowpH, high tempera ture, or by alkylation.3 Since the apurinic sitesresulting fromde purination haveshownlethality4,5 and basesubstitution errors,5 depurination (deguanylation) could be one of the promising novel mechanisms of toxicityinducedby theshort chain halo genatedalkanes.
HO、c N
k。왜
H성=H O
'NH2 HO
、
Nk。워
ho H1 h
O
'NH2 HO
O
'NH2
2',3'-Dideoxyguanosine
Guanosine 2'-Deoxyguanosine
InPBSsolution,
atpH7.4,37oC Halogenated alkanes
O
<N
项 쉇
N
H N NH2 Guanine
Figure 1. Scheme of deguanylation, the release of guanine bases from nucleic acids by hydrolysis of the N-glycosidic bond. Guanine based- nucleosides were incubated with 16 halogenated alkanes at the physio
logical condition (pH 7.4, 37 oC) for a certain period and analyzed by HPLC, LC-MS and UV.
Previously, we observed the massive depurination of nucleo sides such as 2',3'-dideoxyadenosine(ddA), 2’-deoxyadenosine (dA),2’,3’-dideoxyguanosine (ddG), 2'-deoxyguanosine (dG), and calf thymus DNA with an excessamount of 2-bromopro- pane(2-BP)atthe physiological condition.6 Also weobserved the massive deadenylation of adeninebased-nucleosides such as ddA, dA, and calfthymus DNA withexcess amount of halo genated alkanesatthe physiological condition.7 It would be very interesting to investigate deguanylation of guanine based- nucleosides inducedby halogenatedalkanesaccordingtothe difference of substituents and/or functionality in halogenated alkanes, which may provide valuable information forthe mecha nism of toxicity of halogenatedalkanes. Inaddition, it would also be interesting to compare the rate ofdepurination between guaninebased-nucleosides and adeninebased-nucleosides,as guanine hasbeen reportedtoreleasemore rapidly thanade- nine.3
In connectionwithprevious studies, diversekindsof haloge nated alkanes were incubatedwithguaninebased-nucleosides (ddG,dGandguanosine) for 48 h at the physiological condition (pH 7.4, 37 oC), which were analyzed by HPLC and further confirmed by LC-MS for the study of deguanylation effects by different substituents ofand/or functionality in halogenated alkanes. Short-chainhalogenated alkanes, which we utilizedfor the study of deguanylation of guaninebased-nucleosides,have been widely usedindustrially aschemical intermediates,extrac tionsolvents,degreasing compounds,copolymercross-linking agents, or havebeenreportedtobemutagenic and carcinoge- nic.8-17 Amongthesixteen differenthalogenatedalkanes, we observedmassivedeguanylation of nucleosides by 2-bromo- 2-methylpropane (2-B-2-MP),2,3-dibromopropene(2,3-dBPe), 2-bromopropane(2-BP),bromoethane (BE) and2-iodopropane (2-IP).
Maleii시s and Methods
Chemicals. lodomethane (99.5%),bromoethane (> 99%), iodoethane (99%), 1,2-dibromoethane (99+%), 1,2-dichloroe- thane(99.8%), 1-bromopropane(99%),2-bromopropane (99
%), 1-chloropropane (98%), 2-chloropropane(99+%), 1-iodo- propane(99%),2-iodopropane(2-IP, 99%), 1,2-dibromopro- pane(97%), 1,3-dibromopropane, 1,2,3 -tribromopropane (97
%), 2,3-dibromopropene(> 99%), 2' -deoxyguanosinehydrate (99%),guanosinehydrate (98%),calf-thymus DNA (deoxy ribonucleic acidsodium salt,fromcalf thymus), 5-fluorouracil (99%),phosphate buffered saline(pH 7.4)and ammonium ace
tate(99.995+%) werepurchasedfromSigma Aldrich Co. (ST.
Louis, MO). 2’,3’-Dideoxyguanosine and 2-bromo-2-methyl- propane (> 97%)wereobtainedfrom Berry &AssociatesInc and Fluka, respectively.9-Methyladenine (9-MA)was obtained bysynthesis in the present lab. HPLC grade acetonitrile and methanolwaspurchased from World Science,Korea.All other chemicals,if not mentioned, were also obtained fromSigma Aldrich Co. (ST. Louis, MO).
Pi미iminaiy reactions. One mgof nucleoside(ddG,dG and guanosine)wasdissolved in 1mLphosphate buffered saline solution(PBS) in 5 mLvial.Ten 卩L (5 mg in 1mL PBS) of 5- flurouracil (5-FU) was added as an internal standard. It was then incubated with an excessamount (512 equivalents) of each of the sixteenhalogenatedalkaneslisted in Table 1 at 37 oC for 48 h, respectively. It wasanalyzedby HPLC and further con
firmed by LC-MS.All thereactions wererepeated for three times.
Timeresponse reaction. One mg ofnucleoside (ddG, dGand guanosine) was dissolved separatelyin 1mLphosphate buffered saline solution(PBS) in 5 mL vial.Ten 卩L (5 mg in 1 mLPBS) of 5-flurouracil(5-FU) was addedas aninternalstandard. It wasthen incubated with 512 equivalents of 2,3-dBPe,2-BP, BE and 2-IP for ddG and dG, 4 equivalents of 2-B-2-MP for ddG,32equivalents of 2-B-2-MP for dG, 512 equivalents of 2-B-2-MP for guanosine,respectively,at37 oC.About 10 卩L of sampleswere withdrawnat certain time intervals andanalyzed by HPLC until 100% deguanylationoccurred.Allthe reactions wererepeated for threetimes.
Dose response reaction. Onemg ofnucleoside (ddG, dG and guanosine) was dissolved separatelyin 1mLphosphate buffered saline solution(PBS) in 5 mL vial.Ten 卩L (5 mg in 1 mLPBS) of 5-fluorouracil (5-FU)wasadded as an internalstandard. It wasthen incubated with different amounts (0, 2, 4, 8, 16, 32, 64, 128, 256 and512 equivalents) of 2,3-dBPe,2-BP, BE and 2-IPat 37 oC fora time period inwhich 100% deguanylation occurred. For incubationwith2-B-2-MP, differentamounts (0, 1, 2, 3 and 4equivalents for ddG, 0, 2, 4, 8, 16 and 32equi valents for dG,0, 2,4, 8, 16, 32, 64, 128, 256 and 512 equivalents for guanosine) wereemployed. Again the samples were an alyzed byHPLC and repeated for threetimes.
Reactions with Clf-thymus DNA (ct-DNA). Twomg of ct- DNA was dissolved in 20 mL of PBSsolution and40 卩 L of9- methyl adenine (0.5 mg in 1 mLPBS) was added as aninternal standard and stirredtomix properly.One mL of theabove pre
pared solution ofct-DNA wastaken in 5 mL vial andincubated with 128 卩L of 2-B-2-MP, 2,3-dBPe, 2-BP,BEand 2-IPatthe
Table 1. List of halogenated alkanes with their chemical structure and molecular weight
No. Halogenated alkanes Structure Mol. wt.
1 Iodomethane (IM) 一I 141.94
2 Bromoethane (BE)
3 Iodoethane (IE)
4 1,2-dibromoethane (1,2-dBE)
5 1,2-dichloroethane (1,2-dCE)
6 1 -bromopropane (1 -BP)
7 2-bromopropane (2-BP)
8 1-chloropropane (1-CP)
9 2-chloropropane (2-CP)
10 1-iodopropane (1-IP)
11 2-iodopropane (2-IP)
108.97
155.97
187.86
98.96
122.99 /、、/Br
122.99
12 1,2-dibromopropane (1,2-dBP)
13 1,3-dibromopropane (1,3-dBP) Br /、/Br 201.89
14 2-bromo-2methylpropane (2-B-2-MP)
—L
137.03Br
15 1,2,3-tribromopropane (1,2,3-tBP) Br 人 Br Br
280
.78
16 2,3-dibromopropene (2,3-dBPe) k.Br Br I"-
8
physiologicalcondition for 48h as a preliminaryreaction. At the end of thereaction 300 卩 L of 1M HClwasadded and cen trifuged for 10 min at13,000rpm.Then it was analyzed by LC- MSunder theconditionmentioned below.
Timeresponse reaction was performed with128 卩L of 2-B-2- MP, 2,3-dBPe, 2-BP, BE and 2-IPat a timeinterval of 8h for 0, 8, 16, 24, 32, 40 and 48 hatthephysiological condition, res
pectively. At the end of the reaction 300 卩 L of 1M HCl was added and centrifuged for 10 min at13,000rpm. Thenit was analyzedby LC-MS underthecondition mentioned below.
Doseresponse reaction was performed with 2, 4, 8, 16, 32, 64 and 128 卩 L of 2-B-2-MP,2,3-dBPe,2-BP, BE and 2-IP for 48 h atthe physiologicalcondition,respectively.At the end of thereaction 300 卩L of 1 M HCl was added and centrifuged for 10 minat13,000rpm. Then itwasanalyzedbyLC-MS under
the condition mentionedbelow.
Calculation for deguanylationratio in nucleosides.Degu anylation ratio (DR, %) wascalculated onthe basis of thede creased amount of nucleosides in percentage bycomparing the integration value of thenucleosides in HPLC using the formula below:
A。 --- -- ---At ISo ISt
Deguanylationratio (%)=---x 100%
Ao ISo
where 'AJ is the initial amount ofnucleoside;‘At’ is the amount ofnucleoside after time,t ; ‘ISo’ is the initialamount ofinternal standard and ‘ISt’is the amount of nucleoside after time, t.
C시culation for deguanylation ratio in ct-DNA. Deguanyl- ation ratio wascalculated onthe basisof the increased amount of guanine compared to the internalstandard (IS)by comparing theintegration valuein EIC from LC-MS usingthe formula below:
Deguanylation ratio = guanine/ IS
Apparatus. HPLC analyses wereperformed using twoShi- madzu LC-10AT pumps gradient-controlled HPLC system equipped with Shimadzuphotodiodearray detector (Model SPD-M10A) and dual channel UV detection at 280 nm. Analytes were eluted with a 4.6 x 250 mm,5 卩mWaters XTerra® C18
reverse phase analytical column usingthe following HPLC condition:Isocratically with4%acetonitrile inwater with 50 mM ammonium formate atpH 6.9, 1mL/min flowrate and 10 卩L injection volume for guanine-basednucleosides.
ESI LC/MSanalyses were performedwith a Finnigan LCQ Advantage® LC-MS/MS spectrometry utilizing Xcalibur® program.The samples wereanalyzed using 2.1 x 150 mm, 3.5 卩m WatersXTerra® C18 reversephase analytical columnusing the following LC condition:Isocratically with 3% acetonitrile in water with 50 mM ammoniumformateat pH 6.9, 0.18mL/
min flow rate and 2 卩L injection volume. The mass spectrometer wasoperated in thepositivepolarity mode with ESI sourcetype.
Capillary voltage was controlled at 10V and 270oC andnitrogen gas wasusedas sheathgas.
Centrifugationwas done using Hanil Micro-12 (made in Korea) with maximum capacity 1.5 mLx 12, maximum speed 13,000rpm,maximum RCF 10,770 x g, andpower AC 110V, 60 Hz.
Statisticalanalysis.Allthe reactions were performed at least three times (n >3).Themean value 士standard error (SE) was determined for each test. Student’st-test was usedtocompare statisticalsignificance ofdata. The significantvalues at either P <0.05(*) or P <0.01(**)arerepresented by asterisks.
Results
The deguanylationeffect of thesixteenhalogenatedalkanes onguaninebased-nucleosides was analyzed by HPLC andfur
Table 2. Preliminary reaction of ddG, dG and guanosine with haloge
nated alkanes at the physiological condition for 48 h
No. halogenated alkanes DR (%) in ddG
DR (%) in dG
DR (%) in guanosine
1 Iodomethane * * *
2 Bromoethane 100.00 100.00 5.28
3 Iodoethane 6.30 0.84 3.49
4 1,2-dibromoethane 6.50 1.17 7.40
5 1,2-dichloroethane 4.80 -0.42 5.34
6 1-bromopropane 6.90 -0.18 6.70
7 2-bromopropane 100.00 100.00 13.66
8 1-chloropropane 5.40 -0.22 7.27
9 2-chloropropane 10.10 0.43 6.35
10 1-iodopropane 4.80 -0.08 5.96
11 2-iodopropane 100.00 100.00 7.30
12 1,2-dibromopropane 5.90 0.01 5.69
13 1,3-dibromopropane 2.00 2.00 -0.44
14 2-bromo-2methylpropane 100.00 100.00 100.00
15 1,2,3-tribromopropane 4.20 -3.49 4.69
16 2,3-dibromopropene 100.00 100.00 *
One mg of nucleoside(ddG, dG and guanosine) was dissolved in 1 mL phosphate buffered saline solution (PBS) in 5 mL vial. Twenty pL of 5- flurouracil(10 mg in 1 mL PBS) was added as an internalstandard. Itwas then incubated withexcessamount (512 equivalents) of halogenated alka
nes at the physiologicalcondition for48 h. It was analyzed by HPLCand LC-MS. *Only small amount of deguanyled product(guanine) was obser
ved with many adductsformation.
alkanes withchemical structure andmolecular weight, which weretreatedwithguanine based-nucleosides. From theprelimi nary reaction, it was found that 100%deguanylation occurred inddG and dG with excess amount (512equivalents) of 2-B-2- MP,2,3-dBPe, 2-BP, BE and 2-IP atthe physiological condition for48 h (Table 2), which is described in Figure2and 3.It is in teresting tonotice that 100%deguanylation occurred ingua
nosine withtreatment of excess amount (512equivalents) of 2-B-2-MP atthe physiological condition for48 h, whereas almost no change wasobserved for adenosineby treatment of all of thesixteen halogenatedalkanesat the samecondition.7 In addition, ddG, dG and guanosinewerenotaffected bythe otherhalogenatedalkanes.
Analysis of deguanylation of ddG inducedby2-B-2-MP, 2,3-dBPe,2-BP,BEand2-IPbyHPLC. Figure 2 showsthe HPLC chromatograms underthe chromatographic condition described in material andmethodsfor the analysis of deguanyl ation ofddGinducedby2-B-2-MP,2,3-dBPe,2-BP, BE and 2-IPat37 oC for48 h.
InFigure 2, chromatogram 1 indicates authentic guanine (Gua) at retention time of 4.87 min, andchromatogram 2indi cates themixture of ddG and 5-fuorouracil(5-FU) utilized as aninternal standardat retention times of 12.83 min and 4.12 min,respectively. Guanine, ddG and 5-fuorouracil werewell separatedfromthebiologicalbackgroundunderthedescribed chromatographic condition. Chromatogram3, 4, 5,6 and 7 indi catesthemixture afterincubationof ddG and excessamount (512equivalent)of 2-B-2-MP,2,3-dBPe, 2-BP, BE and 2-IP
Minutes
Figune 2. HPLC chromatogram of (1) guanine (Gua), (2) ddG + 5-FU, (3) ddG + 5-FU + 2-B-2-MP (48 h), (4) ddG + 5-FU + 2,3-dBPe (48 h) (5) ddG + 5-FU + 2-BP (48 h) (6) ddG + 5-FU + BE (48 h) (7) ddG + 5-FU + 2-IP (48 h). Retention time for 5-FU, Gua and ddG were 4.12, 4.87 and 12.83 min, respectively, under the HPLC condition mentioned in materials and methods.
Figure 3. HPLC chromatogram of (1) Gua, (2) dG + 5-FU, (3) dG + 5- FU+2-B-2-MP (48 h), (4) dG + 5-FU+2,3-dBPe (48 h) (5) dG + 5-FU+
2-BP (48 h) (6) dG + 5-FU+BE (48 h) (7) dG + 5-FU+2-IP (48 h). Re
tention time for 5-FU, Gua and dG were 4.08, 4.82 and 8.38 min, respectively, under the HPLC condition mentioned in materials and methods.
Figune 4. HPLC chromatogram of (1) Gua, (2) guanosine (Guo) + 5-FU, (3) Guo + 5-FU + 2-B-2-MP (48 h), (4) Guo + 5-FU + 2,3-dBPe (48 h) (5) Guo + 5-FU + 2-BP (48 h) (6) Guo + 5-FU + BE (48 h) (7) Guo + 5-FU + 2-IP (48 h). Retention time for 5-FU, Gua and Guo were 4.06, 4.76 and 6.70 min, respectively, under the HPLC condition mentioned in materials and methods.
for48 h at the physiological condition, respectively, which indicatesthe peak of retention timeat 12.83min, which corres
ponds tothecomplete disappearance of ddG and a peak of reten tion timeat 4.87min,correspondingto thenewappearance of guanine.Theseresultsindicated that completedeguanylation occurredwhen ddG was incubated with excess amount of 2-B- 2-MP, 2,3-dBPe, 2-BP, BE, and 2-IPat37 oC for48 h.
Analysis of deguanylationofdG induced by 2-B-2-MP, 2,3- dBPe,2-BP,BE and 2-IP by HPLC. Figure3 showsthe HPLC chromatograms under the chromatographic conditiondescribed in material and methods, for theanalysis of deguanylation of dG inducedby2-B-2-MP,2,3-dBPe,2-BP, BEand 2-IPat 37 oC for48 h.
InFigure 3, chromatogram 1 indicates authentic guanine (Gua) at retentiontime of 4.82 min, andchromatogram 2indi catesthemixture of dG and 5-fluorouracil(5-FU) utilized as aninternal standard at retention times of 8.38 minand4.08 min, respectively.Guanine,dG, and 5-fuorouracil were well sepa ratedfrom the biologicalbackgroundunder the describedchro matographiccondition. Chromatogram 3,4,5,6,and 7 indicates the mixture afterincubationof dGandexcess amount(512 equi valent) of 2-B-2-MP,2,3-dBPe,2-BP, BEand 2-IP for 48 h at thephysiologicalcondition,respectively,whichindicatesthe peak of retention time at 8.38 min,whichcorresponds to the completedisappearance of dG and apeak ofretention timeat 4.82 min, correspondingtothe newappearance of guanine.
Theseresultsindicated that completedeguanylation occurred whendG was incubated with excess amount of 2-B-2-MP, 2,3-
dBPe, 2-BP,BE, and 2-IPat37 oC for48 h.
Analysis of deguanylation of guanosine induced by2-B-2- MP, 2,3-dBPe,2-BP,BE,and 2-IP by HPLC.Figure4shows the HPLCchromatogramsunder the chromatographic condition described in material and methods, for the analysis of deguanyl- ationof guanosine (Guo) inducedby 2-B-2-MP, 2,3-dBPe, 2-BP,BE, and 2-IPat 37 oC for 48 h.
InFigure 4, chromatogram 1 indicates authentic guanine (Gua) at retentiontime of 4.76 min, andchromatogram 2indi cates the mixture of guanosineand 5-fuorouracil (5-FU)utilized as aninternalstandardat retention times of 6.70min and4.06 min, respectively. Guanine, guanosine and 5-fluorouracil were wellseparated from the biological background underthe des
cribed chromatographic condition. Chromatogram3,4,5, 6,and 7 indicateschromatogram of themixture after incubation of guanosine and excess amount (512 equivalent)of2-B-2-MP, 2,3-
dBPe, 2-BP, BE, and 2-IP for48 hatthephysiological condition, respectively, which informs almost no change in amount of guanosine and noproductionof guanine at that con
dition by treatment of2,3-dBPe, 2-BP,BE, and 2-IP (chroma
togram 4, 5, 6 and7).However, in chromatogram 3, which cor
respondsto treatmentof 2-B-2-MP, the peakof guanosine com pletely disappeared and a peak ofretention time at4.76min correspondingto guanine newly appeared. The results indicated thatdeguanylation onlyoccurred when guanosine wasincubated withexcessamount of 2-B-2-MP for48 h atthe physiological condition.
In Figures 2, 3,and4,any change in amount of 5-fluorouracil during incubation of ddG,dG and guanosinewith 2-B-2-MP,
(c) 2-BP (汶)
o ra truo
ra든
r-으
一* (汶)
o ra truo
ra든
r-으
一* u 욿
든 그
善
9
7
5
3
1
(汶) o ra truo
ra든
r-으
一*
u 욿
든 그
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(c) 2-BP (汶) o ra truo
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**
Figure 6. Time response curves of dG with (a) 2-B-2-MP (b) 2,3-dBPe (c) 2-BP (d) BE and (e) 2-IP. Time response reactions were performed with 32 equivalents of 2-B-2-MP and 512 equivalents of 2,3-dBPe, 2-BP, BE and 2-IP respectively until the time at which 100% deguanyl- ation occurred. Then it was analyzed by HPLC following the condition mentioned in the materials and methods.
Time (h)
Figure 5. Time response curves of ddG with (a) 2-B-2-MP (b) 2,3-dBPe (c) 2-BP (d) BE and (e) 2-IP. Time response reactions were performed with 4 equivalents of 2-B-2-MP and 512 equivalents of 2,3-dBPe, 2- BP, BE and 2-IP respectively until the time at which 100% deguanyl- ation occurred. Then it was analyzed by HPLC following the condition mentioned in the materials and methods.
2,3-
dBPe, 2-BP, BE,and2-IPwasnot observed,which indicates the concentration of5-fluorouracil was consistently well main
tainedand 5-fluorouracil wasnot affectedby2-B-2-MP, 2,3- dBPe, 2-BP,BE, 2-IP, or nucleosides.
Analysisof time response deguanylation of ddG induced by 2-B-2-MP, 2,3-dBPe, 2-BP, BE, and2-IP. Figure 5 아lows time responsecurves of deguanylation rate of ddG inducedby 512 doseequivalent of 2-B-2-MP, 2,3-dBPe, 2-BP,BE, and 2-IP accordingto time.Figure5(a) indicatestime response curve of deguanylation after incubationof ddG and 512 doseequi valent of 2-B-2-MP atthe physiological condition at a time in
terval of 15 min. Deguanylationbegin tooccur at 15 min,and increase until 45min in a time dependent manner. Complete de guanylation occurred at60min. In Figure 5(b), corresponding to treatmentwith 2,3-dBPe,deguanylation begin tooccur at 30 min, and drasticallyincrease until 80min ina timedependent
manner. Complete deguanylation occurred at100 min. InFigure 5(c),corresponding totreatment with 2-BP, deguanylation begin to occurat 3 h, and drastically increase until 5 h in a time depen dent manner. Complete deguanylation occurred at6 h. InFigure 5(d),corresponding to treatment with BE,deguanylation begin to occurat 12 h, anddrasticallyincrease until 18 h in a timede pendent manner.Completedeguanylation occurred at21 h. In Figure 5(e), corresponding to treatment with2-IP, deguanyl- ation begin tooccurat12 h, and drasticallyincrease until 18 h in a time dependentmanner. Complete deguanylationoccurred at21h. Compared todeguanylation rates in ddGamong2-B-2- MP,2,3-dBPe,2-BP,BE, and 2-IP, the orderof deguanylation mte was observed in2-B-2-MP > 2,3-dBPe >2-BP> BE = 2-IP.
Especially,thedeguanylation rates of 2-B-2-MP and 2,3-dBPe were muchfaster than that of 2-BP, BE and 2-IP.
An지ysis of time responsedeguanylation of dG induced by
Figure 7. Time response curves of guanosine with 2-B-2-MP. Time res
ponse reactions were performed with the 512 equivalents of 2-B-2-MP at a time interval of 6 h until the time at which 100% deguanylation occurred. Then it was analyzed by HPLC following the condition mentioned in the materials and methods.
2-B-2-MP, 2,3-dBPe, 2-BP, BE, and2-IP. Figure 6 아lows time response curves of deguanylationrate of dGinduced by 512 dose equivalent of 2-B-2-MP, 2,3-dBPe, 2-BP, BE and 2-IP according to time.Figure6(a)indicatestimeresponse curve of deguanylation after incubation ofdG and512 doseequivalent of 2-B-2-MPat thephysiological condition at a timeinterval of10 min. Deguanylation begintooccurat10min, anddrasti
callyincrease until 40min in a timedependent manner. Com pletedeguanylation occurred at 60min.In Figure6(b), corres
pondingto treatment with 2,3-dBPe,deguanylation begin to occur at1h, and drastically increase until6 h ina timedependent manner. Complete deguanylation occurredat7 h. InFigure 6(c), corresponding to treatmentwith2-BP, deguanylationbeginto occur at 6 h, and drastically increase until 15 h in a time depen dent manner. Complete deguanylation occurred at 21 h. In Figure 6(d), correspondingtotreatment with BE,deguanylation begin to occurat 12 h, and drasticallyincrease until 30 hin a time dependent manner. Complete deguanylationoccurred at36 h.
In Figure 6(e), correspondingtotreatment with 2-IP, deguanyl- ation beginto occur at 12 h, and drasticallyincrease until 30h ina timedependent manner. Completedeguanylation occurred at 42 h. Comparedtodeguanylationrates indGamong2-B- 2-MP, 2,3-dBPe,2-BP, BE,and 2-IP, the order ofdeguanylation mtewas observed in 2-B-2-MP >2,3-dBPe >2-BP > BE = 2-IP.
Especially, deguanylation rate of 2-B-2-MPwasmuch faster than that of2-BP,BE, and 2-IP. Comparing deguanylation rates of ddG and dG, therate of ddGwas faster thandG.Inaddition, the rate of deguanylation of guanine based-nucleosides was generallyfaster than deadenylation of adeninebased-nucleo- sides.7
Analysis oftime iesponse deguanylation of guanosinein
ducedby 2-B-2-MP. Figure 7 shows timeresponse curve of de guanylation rate of guanosineinducedby 512 doseequivalent of 2-B-2-MP atthe physiologicalconditionat a time interval of 6 h.Deguanylation begintooccur within6 h,and drastically increase until 12 h in a time dependent manner. Complete degu anylation occurredat24h.Therate of deguanylation of gua
nosinewasmuch lowerthan that of ddGor dG.
Figure 8. Dose response curves of ddG with (a) 2-B-2-MP (b) 2,3-dBPe (c) 2-BP (d) BE and (e) 2-IP. Dose response reactions were performed with the 0, 2, 4, 8, 16, 32, 64, 128, 256 and 512 equivalents of haloge
nated alkanesfor a fixed time at which 100% deguanylation occurred.
Then it was analyzed by HPLC under the condition mentioned in the materials and methods.
An시ysis of dose responsedeguanylation of ddGinduced by 2-B-2-MP,2,3-dBPe,2-BP, BE and 2-IP. Figure 8 shows dose response curves of deguanylation rates of ddGinduced by 2-B- 2-MP,2,3-dBPe,2-BP, BE,and 2-IP accordingtodose.Figure 8(a) indicatesdose responsecurve of deguanylation after in
cubationof ddG and differentdose equivalents of 2-B-2-MP at the physiological condition for 24 h. Deguanylation begin to occur at1 doseequivalent of 2-B-2-MP,and drasticallyincrease until 3 doseequivalent of 2-B-2-MP in a dosedependentman ner. Completedeguanylation occurredat 4doseequivalent of 2-B-2-MP. In Figure8(b), corresponding to treatment with 2,3- dBPe,deguanylation begintooccur at 32dose equivalent, and drasticallyincrease until 128doseequivalent in a dosedepen dent manner. Completedeguanylationoccurred at 256 dose equivalent.InFigure7(c),correspondingto treatmentwith 2- BP,deguanylation begin tooccurat 16 doseequivalent, and
(a) 2-B-2-MP (b) 2,3-dBPe
Dose equivalents Dose equivalents
E
5
5
5
5
5
5 3
9
7
5
3
1
-
u 욿
든 그
善
0
Dose equivalents Dose eq니valents
Figure 9. Dose response curves of dG with (a) 2-B-2-MP (b) 2,3-dBPe (c) 2-BP (d) BE and (e) 2-IP. Dose response reactions were performed with the 0, 2, 4, 8, 16, 32, 64, 128, 256 and 512 equivalents of haloge
nated alkanes for a fixed time at which 100% deguanylation occurred.
Then it was analyzed by HPLC under the condition mentioned in the materials and methods.
drasticallyincrease until128 doseequivalent in a dose depen
dent manner. Complete deguanylationoccurred at 256 dose equivalent. In Figure 7(d), corresponding to treatment withBE, deguanylation begin tooccur at16dose equivalent, and drasti cally increase until 64 dose equivalent in dose dependentman ner. Completedeguanylation occurredat64doseequivalent.
In Figure 7(e), corresponding totreatment with 2-IP, deguanyl- ation begin to occur at 64 doseequivalent, and drasticallyin crease until 256doseequivalent ina dosedependent manner.
Complete deguanylation occurredat 256 dose equivalent.Com paredto deguanylation rates in ddG among 2-B-2-MP, 2,3- dBPe,2-BP, BE, and2-IP, the order of deguanylation rate was observed in 2-B-2-MP>2-IP =2-BP=2,3-dBPe> BE.
An시ysisof doseresponsedeguanylation of dG induced by 2-B-2-MP, 2,3-dBPe, 2-BP, BE, and2-IP.Figure 9 showsdose response curves of deguanylation rates of dGinduced by 2-B-2-
Figure 10. Dose response curves of guanosine with 2-B-2-MP. Dose response reactions were performed with the 0, 2, 4, 8, 16, 32, 64, 128, 256 and 512 equivalents of 2-B-2-MP for 36 h. Then it was analyzed by HPLC underthe condition mentioned in the materials and methods.
MP, 2,3-dBPe, 2-BP, BE, and 2-IPaccording todose. Figure 9(a) indicates thedoseresponsecurveofdeguanylation after incubation of dG anddifferent doseequivalents of 2-B-2-MP atthe physiological condition for24 h. Deguanylation begin to occur at2 dose equivalent of 2-B-2-MP,and drasticallyincrease until 16dose equivalent of 2-B-2-MP in a dose dependentman ner. Complete deguanylationoccurred at 32 dose equivalent of 2-B-2-MP. In Figure9(b), corresponding to treatment with 2,3- dBPe, deguanylationbegin tooccur at 8 doseequivalent, and drasticallyincrease until 32 dose equivalent in a dose dependent manner. Completedeguanylationoccurredat 64 dose equiva
lent.In Figure 8(c),correspondingto treatment with2-BP,degu- anylation begin to occur at2 doseequivalent, anddrastically in
crease until 16 dose equivalent in a dose dependentmanner.
Complete deguanylation occurredat 32 doseequivalent. In Fig ure8(d), corresponding to treatmentwith BE, deguanylation beginto occur at 16doseequivalent, and drasticallyincrease until 64 dose equivalentin a dose dependentmanner.Complete deguanylation occurred at128doseequivalent.InFigure 7(e), correspondingto treatment with 2-IP,deguanylationbegin to occur at2 dose equivalent, and drastically increase until16 dose equivalent ina dosedependentmanner. Completedegu- anylationoccurred at64 dose equivalent. Compared to deguanyl- ation ratesin ddAamong2-B-2-MP,2,3-dBPe,2-BP, BE,and 2-IP, the order of deguanylationratewasobserved in 2-B-2- MP >2-IP m2-BPm2,3-dBPe> BE.
An지ysis of dose response deguanylation ofguanosine ind ucedby 2-B-2-MP. Figure10 shows dose response curves of the deguanylation rate of guanosineinduced by 2-B-2-MP accord ing todoseat thephysiologicalcondition for 36h. Deguanyl- ation begin to occurat8 dose equivalent of 2-B-2-MP, and drasticallyincrease until 256doseequivalent of 2-B-2-MP in a dose dependent manner.Complete deguanylationoccurredat 512 doseequivalent of 2-B-2-MP.Therate of deguanylation of guanosinewas muchlowerthan that of ddGor dG.
LC-MS depurination analysisof calf thymus DNAinduced by2-B-2-MP.Figure 11 shows LC-MSdepurination analysis ofcalf thymus DNA induced by 128pL of 2-B-2-MP for 48 hat the physiological condition under theLC-MSconditionmen-
(a) RT： 0.00 - 24.99 SM： 7B TIC
NL： 3.95E7 TICF： MS ct-DNA+
2b2mp(48h)-3
NL：
으
」--
3000000
2000000
1000000-
0
5.47
14.85 15.55
1000000-
500000-
0
3.64E6 m/z=
135.50
136.50 F MS ct-DNA+
j8：81 ,i.9..88_2_i.13_22：43 … 2b2mp(48h)-3
EIC 150
2.51
5.83 7.91 8.43
1.44 18.48 20.07 20.74
10.96
3.43
11.57
12：81 13.48 15.64
1.19E6 m/z=
149.50
150.50 F： MS ct-DNA+
2b2mp(48h)-3 NL： 2000000■三
1500000三
1000000三
500000■三
2.18E6 m/z=
151：50
152.50 F： MS ct-DNA+
o = 1：92 2：14
0 2
, 5.43 6.06 8.88 10.38 1 1.29 I I I I I T I I J I I I ' I il l
4 6 8 10 12
13.70 14.46 16.19 17.74 20.44 21.90 23.08 2b2mp(48h)-3 14 16 1 1 1 1
18 1 1 1 1
20 1 1 1 1 1 1 1 22 24 Time (min)
①은 unqv흉
으糸
①- (b)
100
80
60
40
20
0
100
80
60
40—
20
0
100
80—
60-
NL： 2.56E6 ct-DNA+
2b2mp(48h)-3#354 RT： 3.43 AV： 1 SB： 1 0.00 T： + c ESI Full ms [100.00-400.00]
124.07 ... . .1
N
(‘
N H Adenine Mol.Wt.: 135.13 176.79
150.15
302.92
295.19 324.96
,I , " L ,, J I I “ ，，丨 ， 353.89. J u 390.95 I」m.
NL： 2.99E6 ct-DNA+
2b2mp(48h)-3#564 RT： 5.47 AV： 1 SB： 1 0.00 T： + c ESI Full ms [100.00-400.00]
352.70
174.14 178.79 218.02 23^7 260.08 274.79 306：70 328.20 || , 390.16
N
NL： 1.02E6 ct-DNA+
2b2mp(48h)-3#1130 RT： 10.96 AV： 1 SB： 1 0.00 T： + c ESI Full ms [100.00-400.00]
40
20
0—2 100
130.93
150
9-methyladenine Mol. Wt.: 149.15
227.16 242：62 172.09 196.97
I it J... 111 ii I j i.i nJ I lb 1. 11. Li j. 11. iih J .1 ii. I ..I |. I rip 1.^ I IVH |l H7 H. |.l I. |inT| 1. I rp.
) 200
. 261.44 292.59 305.90 Ii. 1.1 1.11.1 , .I 1.1 lli.ii. ,i111 ,
"I... ...
250 300
390.84
330.32 361.81
J .!I ! I I .II.I . I. I. . I ill .I. I] I. . ... ^|. .1. j. I. I |7. .. j
350 400
m/z
Figure 11. LC-MS analysis of Ct-DNA with 128 卩L 2-B-2-MP for 48 h at the physiological condition under the LC-MS condition mentioned in materials and methods. (a) LC-MS chromatogram. RT for Ade, Gua and 9-MA (internal standard) are 5.47, 3.43 and 10.96 min, respectively.
(b) ESI MS spectrogram. [M+H]+ peaks for Ade, Gua and 9-MA are 136.13, 152.11 and 150.15, respectively.
tioned in materials and methods. Figure 11(a) shows LC-MS chromatogram of calf thymus DNA treated by 2-B -2-MP under theabovecondition.Inthe totalion chromatogram(TIC), it is
difficulttoidentifypeaks of corresponding adenine,guanine, and9-methyl adenine as an internal standard because of the for
mationof relatively smallamountsof products along with many
Figure 12. Time response curves of Ct-DNA for deguanylationwith 2-B-2-MP, 2,3-dBPe, 2-BP, BE and 2-IP. Time response reactions were performed with the 128 卩L of halogenated alkanes at a time interval of 8 h until 48 h.
Figure 13. Dose response curves of Ct-DNA for deguanylationwith 2-B-2-MP, 2,3-dBPe, 2-BP, BE and 2-IP Dose response reactions were performed with the 2, 4, 8, 16, 32, 64 and 128 卩L of halogenated alkanes for 48 h.
impurities.However, in theextracted ion chromatogram (EIC), thepeak of retention time at 5.47 correspondingto adenine (EIC 136), thepeakof retention time at 10.96corresponding to9-methyl adenine(EIC 150), and the peak ofretention time at 3.43correspondingtoguanine (EIC 152), werewell separated anddisplayed ina single peak in each EIC chromatogram, whichwasutilized for theanalysis of time and dose response relationship.Figure11(b)shows ESI MSspectrograms atEIC 152, EIC 136,and EIC 150, whichcorresponds to guanine, adenine, and9-methyl adenine, respectively. The [M+H]+ peaks for adenine,guanine,and 9-methyl adenine are 136.13, 152.11, and 150.15, respectively. Withthe same methods, LC/MS analyses of other haloalkaneswere performed, and timeand dose response deguanylationrelationships inducedby halo
alkaneswereadditionally performed.
Analysis of time response deguanylation of calfthymusDNA induced by2-B-2-MP,2,3-dBPe,2-BP,BE,and 2-IP. Figure 12 showstime response curves of thedeguanylation rate of calf thymus DNA inducedby2-B-2-MP,2,3-dBPe,2-BP, BE, and 2-IP,accordingto time. The formation of guanine from calf thymus DNA by treatment with 2-B-2-MP, 2,3-dBPe, 2-BP, BE, and 2-IPwasobservedin a timeresponsemanner. Com paredto deguanylationrates in calf thymus DNAamong2-B-2- MP,2,3-dBPe,2-BP,BE, and 2-IP, the orderof deguanylation mte was observed in2-B-2-MP >2-BP >2,3-dBPe =2-IP >BE.
Comparedto the previouslyreportedresults,7 the rate of degu- anylation in calfthymusDNA was much lowerthan that of de adenylation.
Analysis of dose responsedeguanylationof calf thymus DNA induced by 2-B-2-MP, 2,3-dBPe, 2-BP, BE,and 2-IP.Figu^13 shows doseresponsecurves of deguanylationrate of calf thy
mus DNAinduced by 2-B-2-MP, 2,3-dBPe, 2-BP,BE, and 2-IP, according to dose. The formation of guanine from calf thymus DNA by treatment with 2-B-2-MP,2,3-dBPe,2-BP,BE, and 2-IPwasobserved ina doseresponse manner for48 h. Com paredto deguanylationrates in calf thymus DNAamong2-B-2- MP,2,3-dBPe,2-BP,BE, and 2-IP, the orderof deguanylation mte was observed in2-B-2-MP >2-BP= 2,3-dBPe > 2-IP ^ BE.
Alsotherate of deguanylation in calf thymus DNA was much lowerthan that of deadenylation.
Discussion
Deguanylationratios(%) in guaninebased-nucleosides were calculated onthe basis of the decreased amount ofnucleosides in percentage by comparingthe integration value of the nucleo sides in HPLCusing the formula mentioned in materials and methods. Since the solubility of guanineis relatively low at phy siologicalcondition, the guanine formed after deguanylation precipitates in a pH 7.4 buffer solution,whichdecreasesthe accuracy of deguanylationratios, if weapply theincreasing amount of deguanylatedproduct (guanine) for the determination of deguanylation ratios. Therefore,weappliedthedecreasing amounts ofnucleosides for the determination of deguanylation ratios. Meanwhile, deguanylationratios in calf thymus DNA were calculatedon thebasis of theincreasedamount ofguanine bycomparingthe integration value between increasedguanine and the internalstandard in EIC from LC-MS/MS using the for
mula mentioned in materials and methods. Sinceguanine is soluble in acidic condition, 1 MaqueousHCl was addedtodis solve the precipitated guanine before analysis.
In Figures 2, 3, and 4, chromatogram1 showsthepeakof authenticguanineas a reference, chromatogram 2shows that of guaninebased-nucleosides, ddG, dG, orguanosine,along withthepeak of internalstandard, and chromatogram3, 4, 5, 6, and 7showthe peaks of products formedafterincubation of ddG, dG,or guanosine with 2-B-2-MP, 2,3-dBPe,2-BP,BE, and 2-IP for 48 h, respectively.It is evident that thepeaksof ddG and dGcompletelydisappeared and the peaksof guanine haveappeared after incubation with 2-B-2-MP, 2,3-dBPe, 2-BP, BE,or2-IP for48 h (chromatogram3, 4, 5, 6, and 7in Figures 2and3). However, almost no change of chromatogram was
BE,and 2-IP for 48 h (chromatogram 4, 5,6,and7 inFigure 4).
Meanwhile, in chromatogram 3 in Figure 4, which corresponds totreatment of 2-B-2-MP and guanosine, thepeak of guanosine completely disappeared, and the peak of guanine appeared, which indicates that deguanylationonly occurred when guano sine wasincubated with excess amountof 2-B-2-MP for 48 h atthephysiological condition, whereasalmostno deadenylation was observedfor adenosineby treatment of 2-B-2-MPat the samecondition.7
Time anddose response reaction with ddG and dGby2-B- 2-MP, 2,3-dBPe, 2-BP, BE, and 2-IP, and also with guanosine by 2-B-2-MP indicate that deguanylation increased in a time and dose dependent manner(Figures 5,6, 7, 8,9,and 10). According totime (Figures 5and6), the orderof deguanylation rate among halogenatedalkanes was observed as 2-B-2-MP > 2,3-dBPe >
2-BP > BE- 2-IP in both ddGand dG. Especially, deguanylation rate of 2-B-2-MP wasmuch faster than that of 2-BP,BE,and 2-IP. Comparing deguanylation rates of ddG,dG, andguano
sine, the rateof ddG wasfaster than dG,followed byguano- sine.18-20According to dose (Figures 8 and 9), the order of degu- anylation rate among halogenated alkanes was observed as 2-B-2-MP>2-IP =2-BP=2,3-dBPe> BEin both ddAand dA.
Similary, deguanylation rate of 2-B-2-MP was much faster than thatof2-BP, BEand 2-IP, which indicatedthat the rateof degu- anylation in tertiary halide was highest, followed bysecondary and primaryhalide.
Time and doseresponse reaction with calf thymus DNA by 2-B-2-MP, 2,3-dBPe, 2-BP,BE, and 2-IP indicated thatdegu- anylationincreased in a time and dosedependentmanner (Fi gures 12 and 13). The orderof deguanylationratewas observed in 2-B-2-MP > 2-BP > 2,3-dBPe = 2-IP > BE in timeresponse tactions,and2-B-2-MP> 2-BP = 2,3-dBPe > 2-IP ^ BE in dose responsereactions. Comparingrates ofdeadenylation and degu- anylationin calfthymusDNA,therate of deadenylationwas much higher than that of deguanylation.7 This resultmaybe ex plainedby the fact that guanine is bound tocytosinewith three H-bonds whereas adenine is boundto thymine with two H-bonds in doublestrandedDNA,whichmakes adenine easier to detach from thechain.3
Con이usion
Amongthe sixteenhalogenatedalkanes, we observed that five halogenatedalkanes (2-B-2-MP, 2,3-dBPe, 2-BP,BE and 2-IP) induced deguanylation of ddGand dG, and 2-B-2-MP also induceddeguanylation of guanosine as a probable mechanism of toxicity. 2-B-2-MPshowed thehighestdeguanylation rate compared to the other halogenated alkanes,whichindicates
that tertiaryalkylhalides displaygreater reactivity than secon daryalkyl halides, followed by primary alkylhalides.It was also observedthat ddG showedthehighest reactivity, followed by dGand guanosine.Although theexact mechanism of degu- anylationis not known, ourresults show that the hydroxyl group in thesugar moiety of the nucleosidesplays an important role regarding the rate of deguanylation.The study which aims toelucidate themechanism ofdeguanylation inducedbyhalo genatedalkanes is in progress.
Acknowledgments.This work wassupportedby BasicSci enceResearch Program through the NationalResearch Foun dationof Korea(NRF) funded bytheMinistry of Education, Science and Technology(R11-2007-040-02004-0).
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