Complexing of Amino Acids to DNA by Chromate in Intact Cells Victoria Voitkun, Anatoly Zhitkovich, and Max Costa

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1 Complexing of Amino Acids to DNA by Chromte in ntct Cells Victori Voitkun, Antoly Zhitkovich, nd Mx Cost Nelson nstitute of Environmentl Medicine, New York University Medicl Center, New York, New York Using o-pthldildehyde (OPT) fluorescence, the mino cids ssocited with DNA were studied following exposure of intct Chinese hmster ovry cells to chromte. Rigorous extrction with EDTA, cid, or bse ws required to relese the mino cids cross-linked to the DNA isolted from control or chromte-treted cells by stndrd procedures (i.e., proteinse K, phenol, etc.). Amino cids resisting extrction from DNA were not studied since nlysis ws limited to those tht could be relesed by these procedures. There ws chromte dose-dependent increse in mino cids complexed with the DNA tht could be relesed by EDTA, cid, nd bse, nd these mino cids were seprted by HPLC nd identified. Substntil increses in cysteine, glutmine, glutmic cid, histidine, threonine, nd tyrosine were found s function of incresing concentrtions of chromte. There ws lso time-dependent increse in complexing of these mino cids to the DNA by chromte. The mino cids found complexed to DNA in intct cells by chromte were thought to originte from rections of free mino cids or smll peptides with the DNA rther thn being proteolytic products derived from lrger proteins tht were cross-linked to the DNA. This ws supported by number of experiments: ) free mino cids or bovine serum lbumin (BSA) were cross-linked by chromium to DNA in vitro nd the DNA ws isolted by stndrd procedures. With BSA, few mino cids were found cross-linked to the DNA, but with free mino cids, numerous mino cids were ssocited with DNA; b) when rdiolbelled threonine complexed to the DNA ws exmined in the bsence nd presence of protein synthesis inhibitor, there ws substntil stimultion of the threonine complex to DNA by chromte when protein synthesis ws inhibited with cyclohexmide; c) there were substntil increses in mino cids ssocited with DNA isolted without protese. Not only does the cross-linking of mino cids to DNA represent new type of lesion to study in intct cells but it my lso be useful biomrker of humn exposure to cross-linking gents. - Environ Helth Perspect (Suppl 3):5-55 (994). Key words: cysteine, histidine, glutmine, HPLC, DNA extrction ntroduction Even the most highly purified DNA hs residul mino cids ssocited with it tht cn be detected by extrction with strong cids, bses, or other rigorous extrction procedures (,). These residul mino cids re thought to hve been prt of the originl chromtin-scffold proteins or other tightly bound chromtin proteins (). A number of proteins re covlently cross-linked to the DNA nd these proteins re cross-linked through specific mino cid residues, which should be detected when mino cid-dna crosslinks re exmined. An exmple of one such protein is topoisomerse (3). Previous studies in our lbortory hve utilied DNA-protein cross-links s n indictor of exposure to vrious chem- This pper ws presented t the Second nterntionl Meeting on Moleculr Mechnisms of Metl Toxicity nd Crcinogenicity held -7 Jnury 993 in Mdonn di Cmpiglio, tly. This work ws supported by grnts ES 4895, ES 475, ES 55 nd ES 6 from the Ntionl nstitute of Environmentl Helth Sciences, nd by grnt CA 3343, Kpln Cncer Center. Address correspondence to Dr. Mx Cost, Nelson nstitute of Environmentl Medicine, New York University Medicl Center, 55 First Avenue, New York, NY. Telephone (94) /() Fx (94) 35-8/() icl gents, such s chromte, nickel, formldehyde, UV light, etc. (4,5). We hve developed ssys for detection of DNA-protein complexes (4,6) nd proteins ssocited with DNA (5) hve been studied. This type of lesion hs been used s humn biomrker of exposure to cross-linking gents (7). However, the bility to extrct DNA in the presence of intct proteins is limited, nd most known procedures re generlly flwed with bckground problems. DNA isolted by stndrd proteinse K/phenol/chloroform extrction procedures, however, yielded highly purified DNA with miniml of bckground proteins. This DNA lso hd minimum of ssocited bckground mino cids (,8). n the present study, we hve ttempted to exmine whether residul mino cids ssocited with DNA isolted by the proteinse K method could be used to detect exposure to cross-linking gents. There re substntil increses in residul mino cids ssocited with DNA found in intct cells treted with cross-linking gent such s chromte. Direct exmintion of mino cids by fluorescent derivtives nd HPLC seprtion yielded n ccurte ssessment of the ctul mino cid crosslinked to the DNA. Mterils nd Methods Chinese hmster ovry cells (CHO) were cultured in -MEM t 37 C, supplemented with % fetl bovine serum nd ntibiotics in n tmosphere contining 95% ir nd 5% CO. Cells were cultivted in 5 mm dishes to pproximte 3% confluency. Subsequently, they were treted with vrious concentrtions of KCrO4 rnging from 5 to pm for 4 hr in complete medi. Cells were subsequently rinsed with phosphte buffered sline, scrped, collected by centrifugtion nd lysed in.5% SDS solution contining mm sodium chloride nd mm HEPES ph 8.. Cells were concentrted to bout to 3 million per ml. Cells were pssed through 5-guge needle nd 5 pg/ml of RNse ws dded nd the homogente ws incubted for 3 min t 37 C. Subsequently, 3 pg/ml of proteinse K ws dded nd the cells were incubted for 3.5 hr t 5 C. This homogente ws extrcted three times with phenol/chloroform/isomyl lcohol t rtio of 5:4: using equl volumes. The queous phse ws subsequently extrcted with chloroform/isomyl lcohol t 4: using equl volumes. The queous phse, which contined the DNA, ws mde. M in NCl two volumes of ethnol were Environmentl Helth Perspectives 5

2 VOTKUN ETAL. c 4, -5 = L control 6.BJM oc _ Jum Cr rescent dye o-pthldildehyde (OPT) (9). Sixty milligrms of OPT ws dissolved in ml of ethnol, nd 4 pl of -mercptoethnol nd ml of borte buffer, ph 9.5 were dded. Fifty microliters of mino cid solution ws rected with 5 pl of this OPT regent for exctly min. The rection ws stopped with 5 pl of 5 mm of NCH3 ph 4.5. Totl fluorescence ws either determined directly using Perkin- OTA Acid MOH Elmer fluorescent spectrophotometer (Norwlk, CT) or DNA smples were pre- Figure. Effect of chromte on complexing of mino cids to DNA. CHO cells were treted with 5 pm, cipitted with ethnol, the superntnt ws pm, or witlhout K CrO4 for hr in complete -miniml dried nd the residue dissolved in 5 id essentil rrnedi f-mem). DNA ws isolted by pro- ws injected in HPLC column. Amino teinse K/phenol/chloroform extrction s outlined in Mterils nd Methods. The DNA ws treted with mm EDTA for 4 hr t 37 C,.5 N HSO4 for min t 7 C or. N NOH for hr t 37 C. Smples of these extrcts were used directly to mesure fluorescence with o-phthldldehyde dye s outlined in Mterils nd Methods. dded nd the queous phse ws llowed to stnd overnight t - C. DNA ws collected by centrifugtion for min t,g nd the pellet ws rinsed in 7% ethnol. The finl DNA pellet ws dissolved in mm of HEPES buffer, ph 8.. DNA ws subsequently extrcted with either EDTA, NOH, or sulfuric cid s described in the figure legends. Amino cids ssocited with the DNA were quntitted by rection with the fluo cids were seprted on Adsorbosphere OPA-US column (Altech, Dierfield, L) in NCH3 buffer, ph 5.7, contining 3% -propnol ginst % methnol contining.5% -propnol for 4 min t flow rte of.5 ml/min (grd: -8% methnol for 4 min). A specil procedure ws utilied to determine cysteine content ssocited with the DNA. DNA purified from cells ws used for lkyltion of cysteine residues to convert them to S-crboxymethyl cysteine (CMC). Dithiothreitol ws dded to the DNA to finl concentrtion of mm. Nitrogen ws blown over the surfce of the solution nd the rection mixture ws incubted t 37C for hr. Then 4 mm iodcetic cid nd. N NOH were dded to the solution nd nitrogen ws - Asp - Cys 3 - Glu 4 - Asn 5 - Ser 6- Gln 7 - Thr 8 - His 9-Al - Arg 3 -Tyr - Met 3 - Vl 4 - Phe 5- lleu - Leu 7 - Lys Figure HPLC profiles showing the seprtion of individul mino cids complexed to the DNA. DNA from control (dsh lne) or CHO cells (solid lne) exposed to pm, potssium chromte ws treted with sodium hydroxide s outlined in the legend of Figure. DNA ws then precipitted with volumes of ethnol nd the supemtnt ws used for the rection of cysteine lkyltion nd mino cid derivtion s outlined in Mterils nd Methods. 7 gin blown over the surfce of the solution. Relese of mino cids from the DNA nd lkyltion of relesed cysteine residues were llowed to proceed in the drk t 37 C for hr. DTT nd iodocette were not ble to gree to relese cysteine residues from the DNA. Tretment of the DNA with NOH ws required to render thiol groups of cysteine ccessible to the regents for lkyltion tht llowed us to detect cysteine residues bound to DNA following Cr-tretment of CHO cells. Experiments to determine complexing of proteins or mino cids with DNA in vitro were performed using clf thymus DNA, stndrd mino cids, nd bovine serum lbumin. One hundred microgrms per milliliter DNA ws incubted with.5 mg/ml BSA or 5,uM ech of mino cids in the presence or bsence of gm CrC3 for 4 hr t 37 C s indicted in Figure 6. DNA ws purified with the proteinse K/phenol/chloroform procedures s described for CHO cells, except tht proteinse K ws not pplied to the smples of DNA incubted with mino cids. Results Figure shows the totl fluorescent mino cids tht could be extrcted from the DNA in untreted or cells treted with 5 or pm chromte. Note the increse of fluorescent derivtives of DNA ssocited mino cids tht ws dose-dependent following tretment of cells with incresing concentrtions of chromte. The mino cids ssocited with DNA were seprted by HPLC to resolve the individul mino cids contributing to the overll fluorescence. Figure shows the seprtion of mino cids present in control or chromtetreted DNA. A number of NOHextrctble mino cids were incresed in their cross-linking compred with control DNA. Prticulrly striking ws the increse in crboxymethylcystiene, glutmic cid, glutmine, nd histidine. Figures 3 nd 4 quntitte nd compre number of the mino cid peks tht were resolved by HPLC. Note tht the consistency of the method is illustrted by the smll stndrd-error brs nd note tht NOH ws the most effective gent in relesing mino cids. Figure 3 is quntittive comprison from both control nd chromte-treted cells, while Figure 4 illustrtes the cross-linked mino cids reltive to control. Glutmic cid nd glutthione were more strikingly ssocited with DNA fter chromte tretment. Other mino cids were lso incresed in their ssoci- 5 Environmentl Helth Perspectives

3 PRELMNARY CHARACTERZATON OFAMNO ACD-DNA ADDUCTS 4 < Us CM E <: 8 4 E c! Ec E c S S S < E e - oop o EDTA i Asp Olu Am Se Oln His bw Ae Aug Tyr Mt Vl Pes Acid -A A As OluASAO Gin ghi Thr As AM Tyr NO Vol Ph* -o NOH JuA A A].V j -A m_- u- Asp im Ass Sw in HN Th ANe A Tr Mm Vl Pi Figure 3. Quntittion of mino cids ssocited with DNA. DNA ws treted with EDTA, cid, or sodium hydroxide s described in Figure. The mino cids were seprted with HPLC nd severl mino cid stndrds were used to identify nd quntitte unknown peks. tion with DNA (Figures 3,4). There ws no prticulrly different pttern in the individul mino cids extrcted by either EDTA or cid or bse. Figure 5 exmines the increse in number of mino cids (EDTA extrcted) including histidine, methionine, nd threonine exhibiting dose-dependency in their ssocition with DNA following chromte tretment of intct cells. The bottom portion of Figure 5 shows the mino cids most ffected in this complexing with DNA. For cysteine, s well s glutmine, there ws 35- to 6-fold increse in crosslinking tht ws dose-dependent. Even 5 pm chromte ws found to substntilly increse cysteine, glutmine, nd glutmic cid complexing with the DNA. Figure 6 shows time-dependence in the complexing of mino cids with DNA. Reltively long incubtion periods were # l! S! S.. 5 MM uiucr - luspmor EDTA Asp s Am S_. Nh T_ Al M VWse P.e w J X Acid NOH Aspl Ami Set.Hi Thu Mm Au W MM ~Pisl Figure 4. Amino cids ssocited with DNA following tretment with chromte. CHO cells were treted with 5 or,um potssium chromte for hr in complete oc- MEM. Smples were prepred s described in Figure nd mino cids were seprted by HPLC s described in Mterils nd Methods. The rtio of ech mino cid mount extrcted from the DNA fter Cr tretment to the mount of mino cid relesed from control DNA ws determined. required to produce substntil increses in mino cid DNA complexes (- hr). To evlute whether the extrction procedure mesures mino cid residues from protein-dna cross-links or cross-linking of free mino cids with DNA, we rected DNA with BSA in the bsence or presence of trivlent Cr() under conditions in which we hve shown tht BSA could be cross-linked to the DNA Cr (). We lso rected DNA with mino cids in the bsence nd presence of trivlent Cr. Figure 7 illustrtes tht when BSA ws cross-linked to the DNA by Cr, there ws little increse of mino cids bove bckground. However, when free mino cids were cross-linked with DNA by Cr(), there ws substntil increse of mino cids complexed with DNA bove the. 'D co p 3 4D 'O. 6 Cys 5.-o 45.-o 'o,, gln 5 - v,,, -,,,.,# 5. 5.,/,- glu 4 6 Cr ( um ) 4 6 Cr (pm ) so Figure 5. Concentrtion dependence in the ssocition of mino cids with DNA. CHO cells were treted with pm potssium chromte for 4, 8,, nd hr in complete medi. Smples of mino cids were prepred s described in Mterils nd Methods. bckground. When protein synthesis ws inhibited by cyclohexmide, more rdiolbeled threonine ws ssocited with DNA following chromte tretment thn when it ws not inhibited (Figure 8). These results suggest tht free mino cids rect redily with DNA in intct cells to produce DNA-mino cid complexes. Additionlly, we used recently developed nonprotese method to isolte cellulr DNA following chromte tretment (6). Protein-DNA complexes were seprted by precipittion with K+- SDS of DNA mechniclly frgmented to homogeneous sies while DNA not precipitted by the K+- SDS hd little or no DNA-protein cross-links. Exmintion of this soluble DNA reveled substntil increses in the mount of mino cid DNA complexes in chromte-treted cells compred to controls (dt not shown). Discussion Rdiolbeled mino cids complexed with DNA following tretment of intct cells with Ni nd Cr compounds hve been studied previously (). Cysteine, histidine, tyrosine, threonine, nd methionine were incresed in their ssocition with DNA in dose-dependent mnner (). The present study grees with the previous ones in finding these mino cids complexed with DNA; however, the results lso demonstrte tht glutmine nd glutmic cid were incresed substntilly in their ssocition with DNA by chromte but Volume, Supplement 3, September

4 VOTKUN ETAL..9.9 t gin glu rg sn time (h) C _ +.,L._A met thr his ~~~~~~~~~~~~~,- tyr,,, + 4 G 8 *A,Ntime (h) Rgure 6. Time dependence in the ssocition of mino cids with DNA. CHO cells were treted with 5,, 5, 5, or pm, potssium chromte for hr in complete -MEM. The mino cids were extrcted with. N NOH ( hr t 37 C) nd were seprted with (Mterils nd Methods). The dt presented re the rtio of mino cid mount relesed from the DNA of Cr-treted CHO cells compred to control. these mino cids were not found to be incresed when rdiolbeled mino cids were used. The reson for this is presently unknown but my hve to do with rdiolbeling problems. Most of the cysteine ssocited with DNA using rdiolbeled mino cids ws found to originte from glutthione cross-links induced by chromte (). This ws demonstrted by inhibiting protein synthesis with cycloheximide, which ctully stimulted the mount of cysteine ssocited with DNA (). Thus, the cysteine-dna cross-links do not represent residul proteins ssocited with the DNA, but most probbly represent ctul glutthione-dna dducts. n fct, most cysteine in proteins is generlly not ccessible for cross-linking in intct cells. The fct tht glutmine nd glutmic cid were found to be highly cross-linked to the DNA in the present study when nonrdioctive method ws used my be due to the high levels of glutmine tht were dded in the tissue-culture medi. n fct, using rdioctive glutmine, the specific ctivity of glutmine would be so low tht glutmine DNA cross-linking my not be detected by these rdioctive methods. These results suggest tht, in fct, the residul mino cids ssocited with DNA did not, in lrge prt, originte from protein cross-linked with DNA, but originted 5 m ML + m 5 DU -. AGM Figure 7. HPLC seprtion of mino cids ssocited with DNA following in vitro cross-linking with bovine serum lbumin nd mino cids. Clf thymus DNA ws incubted with mino cids (D, E) or BSA (B, C) in the presence (C, E) or bsence (A, B, C) of,um CrC3. DNA ws purified s described in Mterils nd Methods. Amino cids were extrcted with. N NOH ( hr t 37 C) nd nlyed by HPLC (Mterils nd Methods). The peks re designted numbers ccording to the numbering of peks in Figure. from free mino cid pools or smll peptides tht were cross-linked to the DNA in intct cells. This ws certinly the cse with glutthione tht ws found complexed with the DNA (). Most of the cysteine tht ws found complexed with the DNA originted from glutthione (). Experiments conducted here lso suggest tht when lbumin ws cross-linked to the DNA, there ws little residul mino cid complexed with DNA; however, when free mino cids were cross-linked to DNA nd the DNA ws isolted by stndrd methods, there ws n bundnce of mino cid. Further, when protein synthesis ws inhibited in intct cells, there ws more rdiolbeled mino cid cross-linked to the DNA, suggesting tht free mino cids do, in fct, rect with the DNA. Although there my be some contribution from residul protein-dna cross-links, substntil percentge of the mino cids ssocited with DNA probbly originted from the rection of free mino cids with DNA. n generl, free mino cids re chemiclly more rective thn mino cids compexed s peptides. We previously studied the chemistry of the cross-linking of mino cids to the DNA nd demonstrted in the cse of chromte tht trivlent Cr ws involved in t 3 t Control C ex os uolar Figure 8. Effect of cycloheximide on the crosslinking of threonine to the DNA by chromte. ) Cells were incubted with rdiolbeled threonine (3H-Thr) 4 hr before exposure. 3H-Thr ws removed when cells were treted with potssium chromte for hr. ) After 7 hr of Crexposure of non lbeled CHO cells, rdioctive threonine ws dded nd Cr tretment ws continued for n dditionl 9 hr. 3) After 6 hr of Cr exposure of nonlbeled CHO cells, 5 pmj cycloheximide ws dded to the medi; then in hr, H-Thr ws dded nd cells were incubted for n dditonl 9 hr. DNA ws isolted nd mino cids were extrcted with sodium hydroxide s previously described. Rdioctivity corresponded threonine HPLC pek ws collected nd counted using Beckmn scintilltion counter. the cross-link, since DNA-bound cysteine could be extrcted with EDTA (). Trivlent Cr is the only form of chromte tht cn be chelted by EDTA, nd we re not studying mino cid cross-links except those involving trivlent Cr with EDTA. However, we hve lso observed in previous studies tht mino cids cn be crosslinked to the DNA by oxidtive mechnisms tht do not ctully involve trivlent Cr (). The biologic significnce of the mino cids cross-linked to the DNA is not currently understood; however, these were thought to be mino cid residues rising from chromtin-scffold proteins (-3). We show here tht peptides nd free mino cid cross-linking in intct cells must lso be considered s possible mjor origin of these mino cid cross-links. The fct tht one cn demonstrte such lrge increse in mino cids ssocited with DNA following tretment with cross-linking gents indictes tht these mino cid-dna complexes my be substntil impediment to DNA repliction tht could possibly led to muttions nd my be responsible for crcinogenic effects of chromte. Complexes of DNA nd mino cids probbly enhnce the stbility of the Cr bound to the DNA. An dditionl utilition of this lesion s potentil biomrker of humn exposure to cross-linking gents is possible since mino cids ssocited with DNA cn be quntitted with high precision. 54 Environmentl Helth Perspectives

5 PREUMNARY CHARACTERZATON OFAMNO ACD-DNA ADDUCTS REFERENCES. Ginfrnceschi GL, Brr D, Boss F, Coderoni S, Pprelli M, Venni F, Cicconi F, Amici D. Smll peptides controlling trnscription in vitro re bound to chromtin DNA. Biochem Biophys Act 69:38-48 (98).. Juodk B, Pfuit M, Werner D. Chemicl nd enymtic nlysis of covlent bonds between peptides nd chromosoml DNA. Nucleic Acid Res 9: (99). 3. Sutcliffe JA, Goot TD, Brrett JF. Biochemicl chrcteristics nd physiologicl significnce of mjor DNA topoisomerses. Antimicrob Agents Chemother 33:7-33 (989). 4. Miller CA, Cost M. Anlysis of proteins cross-linked to DNA fter tretment of cells with formldehyde, chromte, nd cisdimminechloropltinum(). Mol Toxicol :-6 (989). 5. Cost M. Anlysis of DNA-protein complexes induced by chemicl crcinogens. J Cell Biochem 44:7-35 (99). 6. Zhitkovich A, Cost M. A simple, sensitive ssy to detect DNAprotein cross-links in intct cells nd in vivo. Crcinogenesis 3: (99). 7. Cost M, Zhitkovich A, Toniolo P. DNA-protein cross-links in welders: moleculr implictions. Cncer Res 53: (993). 8. Neuer B, Plgens U, Werner D. Phosphodiester bonds between polypeptides nd chromosoml DNA. J Mol Biol 4:3-35 (983). 9. Lindroth P, Mopper K. High performnce liquid chromtogrphic determintion of subpicomole mounts of mino cids by precolumn fluorescence derivtition with o-phtldildehyde. Anl Chem 5:67-74 (979).. Slnikow K, Zhitkovich A, Cost M. Anlysis of the binding sites of chromium to DNA nd protein in vitro nd in intct cells. Crcinogenesis 3: (99).. Lin X, Zhung Z, Cost M. Anlysis of residul mino cid-dna cross-links induced in intct cells by nickel nd chromium compounds. Crcinogenesis 3: (99). Volume, Supplement 3, September