RD-A125 034 CHEMICAL AND MOLECULAR BIOLOGICAL ASPECTS OF i/IALKYLHVDRAZINE-INDUCED CARCI..(U) OHIO STATE UNIVRESEARCH FOUNDATION COLUMBUS D T IdITIAK NOV 82

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FCHEMICAL AND MOLECULAR BIOLOGICAL ASPECTS OFALKYLHYDRAZINE-INDUCED CARCINOGENESIS IN

H CELLS IN VITRO

LDonald T. WitlakCollege of Pharmacy

For the Period[ December 1, 1981 - November 9, 1982N

-F AIR FORCE OFFICE OF SCIENTIFIC RESEARCHBolling Air Force BaseWashington, D.C. 20332

Contract No. F49620-80-C-0086

8 ei~utjLLJ November, 1982.

F 83 02 028 023

AZ' 7.

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WFOSR-TR- 83-0046 14. TITLE (and Subttels) S. TYPE Or REPORT S PERIOD COVERED

CHEMICAL AND MOLECULAR BIOLOGICAL ASPECTS OF InterimALKYLHYDRAZINE-INDUCED CARCINOGENESIS IN HUMAN Dec. 1, 1981 - Nov. 9, 1982

f CELLS IN VITRO IL PERFORMING OG. REPORT .USEcR7. AUTHOR(s) 9. CONTRACT OR GRANT NUMBERO)

Donald T. Witiak F49620-80-C-0086

9. PERFORMING ORGANIZATION NAME AND ADDRESS 0. PROGRAM E. MENT. PROJECT. TASK

The Ohio State University AREA S WORK UNIT NUMUERSResearch Foundation, 1314 Kinnear Road e, 3/:95-,Columbus, Ohio 43212 _ //_0_ _ _ _

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IS. SUPPLEMENTARY NOTES

IS. KEY WORDS (ContMu a, rtse iede add* it Mwesw and tdoif7d by bek 0mb0)

Methylhydrazines, methylazoxyethanol, DNA, human fibroblasts, tissue culture,DNA adducts-, DNA damage and repair.

30. ABSTRACT (CnUim. ao eese side if neessa, ai iditfiIJ, by block e)

See reverse side.

DD I ,0AT 1473 EDITIONo O1 INov es is ousSOaum &SECURITY CLASSIFICATION OF THIS PAGE (ON Done

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S.CUuITY CLASSICATION OF T1ra PAGC(fm, Dea SM.-e.

1. We have q ce;lop-d e a- .cation exchange HPLC technique.{whlch israpid and provides a high resolu-1on of pyrtmidtne oligonucleotides,methylated purines and a mthylated pyrimidine jf$tr-l4..?)

.Treatment of asynchronous cells at a low dose of l,l-DWjl 2-MH ortreatment of synchronized cells at a transforming dose of 1,1-DMH did notincrease the methylation of purine bases. However, a dose dependentincrease in incorporation of radiolabel into DNA is observed. The ratioof O0fteua/M'MeGua in 1,1-DU4 treated cell varied between 0.5 - 0.8. Inall experiments about 75-90% of the label was found in the ap ri T1c acidfraction. This may be due to a) methylation of pyrimidines, ) Centering the carbon pool and then being utilized in de novo nthesis of

" . pyrimidines or c) formation of phosphotriesters.

_fihe presence of detectable amount of phosphotriester oralkali labile-K alkylated bases in DNA due to methylation is tentatively ruled out since

sedimentation analysis of methylated DNA in alkaline sucrose gradient didnot show any single strand breaks even at concentrations as high as 5.0ram,

A priliminary labelling experiment with 14C-WutA employing a transformatiodose of HAMA showed distinctive labelling of 7-MeGUA and OANtua,

5. - Alkaline sucrose gradient analysis of DNA fro MAMA treated cell show adose dependent break in DNA (Fig. 6), investigations to determinewhether the damage it due to formation of methylated bases orphosphotriester is in progress./,,

5. An initial experiment to see the effect of 1, 1-D0H and 1,2-DNH onunscheduled DNA synthesis (UDS) revealed that both compounds at aconeentration of 0.026mM slightly increasdd the UDS but at atransforming dose (0.5mM), inhibition of DNA synthesis was morepronounced.

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6,4. A 5 .5C-jI<SECURItY CLAIeCATI i OF ?U'* PAGIMAN bW. IMew.

PROGRESS REPORT TO

AM FORCE OFFICE OF ITII

RESEARCH DIRECTORATE OF IWE SClENCES

Waelngtom, D.C. 23332

CHEMICAL AND MOLECULAR IOLOGICAL ASPECTS OP

ALKYLHYDRAZINE-DNDUCED CARCINOGEVESU IN

HUMAN CELIA IN VITRO

SDember 1, 1931 - Novmber 9,IM

N~~ rySt ' . Donald T. Wtlak, MhD.The Ohio Sate UniveMity3College of Flmrmae

Dist-. .' .,i 2. , fWttlthAvImMmA " " . "'' C0lUMblB, Oio 43 19

Chier, Tachnical Information DivIslou

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* Table of Camtmb

L Syathetie Preu

BL Blalcglol Prapm

A. Imhtel 2

B. Ru~to 2

L Deeamtof an HPLC Teuhiques

2. methlto of DNA In Cultwed Humo

C. Conebnlom 3

D. Refernm 4

L. Tables and FIgure 5

P Methylazoxymethanol acetate (MAMA) was prepared following essentially themethod of Matsumoto et all which consisted of oxidation of l,2-dimethylhydrazine(I,2-DMH) In two steps to azoxymethane followed by further conversion bybromination and aeetylation to MAMA. Modifications have been made in theisolation proedures enabling one to carry out the synthesis on a fairly small scale.(A typical batch in our laboratory employed 0.34 mmol of 1,2-DMH.2HCI).

H Hi) Amberlite IRA-93H3 CN-NCH3 2HCI H3C1 = NCH3

ii) Yellow HIgOiii) m-Cl PBA 80-85%

i) NBS/CCI4 0) H3 C* - NCH2OCOCH3ii) AgOAc

(22% overall)

I1l,2-DMH generated in situ from its dihydrochloride with a weakly basic ion

exchanger was stirred with yellow HgO. The azomethane thus liberated wastrapped in CH 2 CI2 containing m-chloroperozybenzoic acid. In view of the smallamounts involved it was highly inconvenient to isolate the product, azozymethane,free of solid organic acids by distillation. Isolation was facilitated by passing theCH2 C12 solution over basic alumina which rendered the product totally free ofacids and in high yields (80-85% as estimated by NMR analysis of product +CH2 C12; Ut 2 yields 36%).

Further conversion of azoxymethane to MAMA was accomplished in 2 stepsby bromination followed by acetylation, according to a published proedure*wlthoutisolation of the intermediate bromossoxymethane because of its reportedinstability. MAMA was purified by chromatopaphy over a silica gel column orpreparative TLC. Overall yields of MAMA from I,2-DMH.2HCI were about 22%.Several attempts at improving yields in the last two steps employing differentconditions as well as reagents fufted to afford better results.

Synthesis of high sp. activity W4 C-labelied IZ-DMH.2HCI has been describedin the previous report. Attempts to prepare labelled MAMA using this materialhave not given satisfactory yields in preliminary runs.

p

2

As stated earlier, studies by Milo et l 3 have revealed that in humanfibroblast cells in culture, hydrazine, l,l-dlmethylhydrazine (l,l-DMH) andmethylazoxymethanol acetate (MAMA) induced neoplastic transformation.Monomethyl hydrazine elicited only cellular toxicity. One approach to theunderstanding of differences between toxicity and carcinogenicity in terms ofalkylation of DNA is to (a) analyse alkylated bases, and (b) study DNA damage andrepair in cells treated with closely related analogues, but with different oneogenicpotentials.

With this in mind we have undertaken the analyses of DNA in humanfibroblast cells exposed to 14C-labelled 1,1-DMH, 1,2-DMH and MAMA. The alkylhydrazines used in our studies are of high specific activity ( 110 mCi/mmole) andwere synthesized by methodology developed in our laboratories.

B. Reslts

. Development of an HPLC technique for separation of apurinic acid andmethylated purines and pyrimidines.

We have recently developed a cation exchange HPLC technique which is rapidand provides a high resolution of pyrimidine ollgonucleotides, methylated purinesand pyrimidines obtained by acid hydrolysis or neutral-thermal hydrolysis ofalkylated DNA. Fig. I shows a typical HPLC profile of 10 purine and pyrimidinestandards. This method is far superior to separation on Ssphadex G-10 in that a)resolution is considerably improved and b) lution time Is short.

2. Methylatlon of DNA in cultured human fibroblast cells.

Experiments were carried out using higher concentrations (0.166 mM) of 1,1-and ,2-DMH. Additionally the offet of transforming and non-transforming doses(0.5 mM and 0.026 mM respectively) of I,-DMH were investigated in synchronizedcells. A 6 fold increase in concentration of ,1- and ,2-DMH resulted in a 4 foldincrease in 14C incorporation into asynchronous cells. Utilization of a 19 foldincrease in ,1-DMH concentration as well as synchronized cells only increased theincorporation of label by 3 fold (Table 1).

Results of mild acid hydrolysis of alkylated DNA revealed that high

alkylhydrazine concentrations did a) not lead to a detectable increase inmethylation of ade.ne (position 3) or guanine (position 0-6 and N-T), b) increaseIncorporation of 14C-label into unmodified adenine and guanine. When theconcentration of 1,1-and l,2-DMH were increased 6-fold in asynchronous cells, therewas a 3-8 fold Increase (Tablo 2) in 14C Incorporaton into these unmodified bases.Such treatment resulted in a concomitant decrease of 5-19 fold in methylatedpurine. Treatment of synchronized cells with a transforming dose of l,1-DMHresulted in 90% inoporatlon of radiolabel into the apurinie acids (Table 3). Atypical HPLC profile of hydrolysed alkylated DNA isolated from ,1-DMH treatedCOs is shown in Figure 2. Interestingly, there ae major peaks of radioactivitywhich do not co-elute with known major purine standards. Identification of thesesubstances represent a significant oal of our work.

V 3

The lack of increase in DNA methylation with increasing hydrazineconcentration may reflect isotope dilution or DNA repair. Sedimentation onalkaline sucrose gradients should be reflective of methylation and could serve todifferentiate reasons for this apparent lack of increase. In fact, results of theseexperiments (Figures 3 and 4) showed no visible difference in the sedimentationprofile of DNA from untreated and treated (various concentrations from 0.026-50mM of 1,1- or 1,2-DMH). An initial experiment on unscheduled DNA synthesis(UDS) in 1,1 and 1,2-DNA treated cells done in presence and absence of microsomalextract induced a slight enhancement of repair synthesis at O0.26mM concentrationand an inhibition at 0.5 mM concentration.

A DNA labelling experiment using 14 C-MAMA at a transforming dose insynchronized cells showed a significant amount of radiolabel in methylated bases(Figure 5). Further quantitation is in progress.

Analysis of MAMA-treated cellular DNA, in contrast to I,1 or 1,2-DMH-treated DNA, sedimented more slowly than the control DNA (Fig. 6). These dataindicates that the DNA damage is due to either alclali labile alkylated bases,phosphotriesters or both. Investigations are underway to determine whichmechanism results in single strand breaks.

3. Conclusion

Under conditions of varying concentration, treatment of asynchronized orsynchronized cells with 1,1-or 1,2-DMH did not markedly affect methylation ofpurine bases. Using higher concentrations of thaw hydrazines resulted in aconcentration-dependent increase in incorporation of label into DNA.

The ratio of 06 to N7-methylguanine ranged between 0.5-0.8 in II-DMH and0.4-0.5 in 1,2-DMH-treated cellular DNA..

In all experiments 75-90% of the radiolabel was found in the apurinic acidfraction. This was shown not to be attributable to phosphotriester formation sinceno single strand breaks could be detected by alkaline sucrose density gradientsedimentation of treated DNA.

Further investigations planned using MAMA (a potent carcinogen) shouldprovide more detailed information about events leading to chemicaltransformation. Use of restriction enzyme analysis of DNA treated withcarcinogenic and non-carcinogenic structurally related analogues should reveal

S:. molecular differences reflective of carcinogenicity vs. gvaotoxicity.

'We

4

Referume

L Horisberger, M. and Matusmoto, H. (1968), Studies on methylazoxymethanol;Synthesis of 14C and 3H labelled methylazozymethyl acetate, J. LabelledCompds. 4, 164-170.

2. Matsumoto, H., Nagahama, T. and Larson, H.O. (1965), Studies onmethylazoxymethanol, the aglycone of eyeasim A synthesis ofmethylazozymethyl acetate, Biochem. J., 95, 13C-14C.

3. Milo, G.E. and Blakeslee, J.R. (1977), Hydrazine and UDMH inducedneoplastic transformation and feline sarcoma virus induced cocareinogeniceffect on human diploid cells in vitro. In Ninth Annual Symposium ofEnvironmental Toxicology, pp. 112-120.

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P Does dependent incorporation of 14 C 1,2,-DMH and 1,I-DMH into DNA of humanforeskin fibroblast eells.

Asynchronized cellsa Synchronized eellsb

Cone. 1,2-DMH II-DMH LI -DMH

r mM DPM/mg DNA DPM/mg DNA DPM/mg DNA

- 0.026 35,0600 31,800 80,617

0.166 1,53,671 1929,648

0.5 - 2,52,861

a Calls in 150 emm culture dishes at 70-80/ conflueney were treated for 24 hrs.with 1,1- or 1,2-DMH.

b Cells blocked in GI phase by arginine and glutamine deprivation were treated10 hrs after release with 1,1 DMH for 12 hrs (0.026 mM) or 4 hrs (0.5 mM).

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6

Table 2

Methylation of DNA from randomly proliferating human foreskin fibroblast cellstreated with alkylhydrazines.

l,I-DMH 1,2-DMH

Praction .:.mM 0.166mM 0.026mM 0.166mM

Apurinic acid 87.56a 76.24 87.9 61.6

3.MeAde 3.18 0.64 2.9 0.5

7 MeGua L27 0.23 2.7 0.17

" 06 MeGua L09 0.09 L33 0.07

Guanine 2.99 9.20 2.45 16.5

Adenine 3.8 11.6 2.60 21.1

06 Gua/N 7 MeGua 0.86 0.4 0.49 0.42

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Table 3

Methylation of DNA from synchronized human foreskin fibroblast cells treatedwith i,1-DMH

Fraction 0.166mM 0.5mM

Apurinic acid 85.7& 0.1

3-MeAde 1.3 0.26

7-MeGua 0.5 0.24

06-MeGua 0.3 0.18

Guanine 7.6 2.3

Adenine 5.6 1.7

O6 MeGua/N 7 MeGua 0.6 0.75

a Percentage of adduets

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