Cells Synthesis of Protein and Nucleic Acid by Murine ...

Effects of 4-Nitrobenzofurazans and Their N-Oxides on Synthesis of Protein and Nucleic Acid by Murine Leukemia Cells David Kessel and James G. Belton Cancer Res 1975;35:3735-3740. Published online December 1, 1975.

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[CANCER

RESEARCH

35, 3735-3740,

December

1975]

Effects of 4-Nitrobenzofurazans and Their N-Oxides on Synthesis of Protein and Nucleic Acid by Murine Leukemia Cells' David Kessel2and James G. Belton Departments of Oncology and Pharmacology, Wayne State University School of Medicine. and the Michigan Cancer Foundation, Detroit, Michigan 4820! [D. K.], and the Laboratories, Medical Research Council of Ireland, Trinity College, Dublin [J. G. B.]

SUMMARY A series of benzo-2, I ,3-oxadiazoles (benzofunazans) and their N-oxides (benzofuroxans) inhibit incorporation of precursors into nucleic acids and protein by munine leu kemia cells. At slightly higher levels, substantial single- and double-strand DNA breakage was observed. At still higher concentrations, inhibition of phosphorylation of uridine and thymidine was found. Structure-activity relationships show that only compounds bearing appropriate substitutions at positions 4 and 7 were effective inhibitors of biosynthetic pathways. Such compounds appear to interact with a wide variety of biological systems and may be useful in elucidat ing modes of macromolecule synthesis. INTRODUCTION The 4-nitro derivatives of benzofuroxans and benzofura zans (Chart I) inhibit DNA and RNA synthesis in mamma han cells (9, 11, 12, 21). In some systems, this activity is enhanced by appropriate substitution on position 7 of the molecule (1 1). These drugs react with both thiol and amino groups (10, 21); biological consequences ofsuch interactions are being explored. Recent data show that certain ben zofuroxan derivatives are potent vasodilators (8) and mono amine oxidase inhibitors (4, 5), indicating a wide range of interactions between these compounds and compounds of biological importance. In this study, we have examined effects of selected compounds on uptake and incorporation of labeled precur sons into nucleic acids and protein, using murine leukemia cells in culture. Drug effects on preformed DNA were also measured. MATERIALS

AND METHODS

Compounds used in this study were prepared by published methods (3, 11), and structures and purity were determined as described therein. Procedures used to measure thymidine uptake have been described ( 17, 18), as have methods used to detect inhibition of synthesis of nucleic acids and protein â€˜Supported in part by Grants CA-16053 and CA-07l77 from the National Cancer Institute, NIH, and by the Irish Cancer Society. 2 To

whom

requests

for

reprints

should

be

addressed,

Memorial Center, 4160 John R Street, Detroit. Mich. 48201. Received March 20, 1975; accepted September 9, 1975.

DECEMBER

at

Darling

(15, 16) and drug-induced alteration of sedimentation patterns of DNA (2, 13, 20). Ll210 cells were grown in sealed flasks using Eagle's minimum essential medium supplemented with 10% fetal calf serum. L5 l78Y cells were grown in Fisher's medium containing 10% horse serum. Exponentially growing cells were collected and suspended in N-2-hydnoxyethylipiperazine-N'-2-ethanesulfonic acid buffered growth medium (pH 7.4) at a density of 7 x 106 cells/mi. This medium was prepared with an equimolar concentration of N-2-hydnoxyethylpiperazine-N'-2-ethane sulfonic acid, replacing NaHCO3 in the minimum Eagle's medium formulation. The suspensions were warmed to 37Â° for 20 mm, divided into 1-mI portions, and mixed with 5 zl of specified drug dissolved in N,N'-dimethylformamide. Ten mm later, the suspensions were treated as follows. For measurement of drug effects on thymidine and unidine uptake and phosphorylation, the suspensions were mixed with 5 sl of [2-14C]thymidine (50 MCi/@zmole) or [14Cjunidine (60 @Ci4tmole) for 10 mm. Final level of nucleoside was 50 zM (0.2 zCi/ml). The cell suspension was then mixed with I ml of 0.9% NaCI solution containing 20 mM Persantin. The cells were collected by centnifugation and washed with 0.9% NaCI solution containing 10 mM Pensantin. Under these conditions, free nucleoside was not lost ( 18); and intracellular radioactivity represented nucleo side, nucleotides, and incorporation of labeled precursor into nucleic acid. When cell suspensions were washed with 0.9% NaCI solution without addition of Persantin, free nucleoside was washed from the cells (18, 19), and intracellular nucleotide pools plus nucleic acid were measured. For measurement of drug effects on incorporation of thymidine into DNA, unidine into RNA, and leucine into protein, the cells were washed with 0.3 M HCIO4 after incubation with labeled precursors. [2- 14C]Thymidine, [2'4C]unidmne, and L-[U14CJleucine (all 50 to 75 MCi/@smole) were used at final concentrations of 100 sM, 0.2 zCi/ml of cell suspension. These macromolecules were localized in acid-insoluble material. In other studies, cells were treated with drugs plus labeled precursors simultaneously, and cells were collected at intervals. The temporal sequence of inhibition of RNA, DNA, and protein synthesis was measured by determining extent ofincorporation oflabel into acid-insoluble material. When the cells were treated with drugs for 10 mm at 37Â° and then resuspended in fresh medium, we found that inhibition of macromolecule synthesis was not thereby

1975

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D. Kessel and J. G. Belton

incorporation of labeled thymidine into DNA by 50% were generally equally effective as inhibitors of RNA and protein synthesis, but the precise nature ofthe inhibition varied. The data shown in Chant 3 describe effects of I and 3 @tM levels of Drug b (Chart 2) on incorporation of labeled unidine, thymidine, and leucine into macromolecules by Ll2lO cells. Incorporation of leucine ceased after about 5 mm, while incorporation of nucleosides continued, although at a reduced rate. In other studies, we determined that inhibition of protein synthesis with cycloheximide (10 @sg/ml)did not produce an effect on nucleic acid synthesis similar to that shown in Chart 3; the immediate inhibition of leucine incorporation into protein by cycloheximide resulted in incorporation of 3000 15Mand incubated for 10 mm at 37Â°.The cells were unidine and thymidine into nucleic acid at 65 to 70% of con then collected and suspended in 0.9% NaC1 solution; a trol values for at least 10 mm. Drug-induced Degradation of DNA. To assess effects of l00-@l portion containing 5 x l0@ cells was used for further analysis. This solution was pipetted onto a layer of benzofurazans and benzofuroxans on DNA, we incubated 0.2 ml of detergent (I % sodium dodecyl sulfate or cells containing labeled DNA with various levels of drugs. Sarkosyl) on top of a 4.5-mi 5 to 20% sucrose gradient in a DNA damage could be detected on both neutral and alkaline sucrose gradients; the threshold required for both 1.5- x 2-inch polyallomer tube. At the bottom of the tube was a shelf of 80% sucrose. All solutions were made up in types of damage appeared to be essentially the same, but 100 m@iNaCI, 10 m@iEDTA, and 10 mM Tris at pH 7.4 substantially more damage was detected on the alkaline (20). The gradients were kept at room temperature for 20 gradients. Typical patterns are shown on Chart 4; levels mm, chilled at 5Â°,and spun at that temperature for 60 mm required to produce such alterations in the sedimentation pattern are reported in Tables I and 2. A 3-fold increase in at 30,000 rpm in the 50. 1 rotor of the Beckman/Spinco moved the peak of radioactivity on ultracentnifuge. Gradient were fractionated from the top drug concentration into scintillation vials; no acid-soluble radioactivity was neutral gradients to Fractions 3 to 4; the rather poorly defined alkaline gradient patterns were similarly altered present. with peaks of radioactivity moved from Fractions 3 and 7 to Drug effects on sedimentation of DNA through alkaline Fractions 2 and 5. When drug levels were reduced 3-fold sucrose gradients were carried out as described above, below those reported in Tables I and 2, alterations in the except that the top layer consisted of 0.2 ml of I N NaOH, and the gradients were made up in 0.3 N NaOH, 0.7 M neutral sedimentation pattern were barely discernible, with radioactivity appearing in Fractions 13 to 14; alkaline NaCI, and 10 mM EDTA. gradients showed radioactivity in Fractions 12 to 14. Studies on L5178Y Cells. Gradient studies of DNA from RESULTS these cells were generally more subject to variation than were experiments using L12l0 cells; so the latter line was Drug-induced Inhibition of Nucleoside Uptake and generally used. Data obtained from L5178Y cells show this Phosphorylation. No compound tested, at levels of 3 mM, line to be more sensitive to inhibitory effects of these agents altered uptake of thymidine or unidine (data not shown). than is Ll210. Where the data differ substantially, the Phosphorylation of intracellular unidine and thymidine was L5178Y results are shown (Tables 1 and 2). inhibited by some compounds; the data are summarized in Tables I and 2. No inhibition of phosphorylation was found when cells were exposed to these compounds at 0-27Â°. DISCUSSION However, at 37Â°, inhibition of phosphorylation could be These data show a variety of interactions between measured. These results were not altered when cells were mammalian cells and benzofurazans and their N-oxides, treated with drugs at 37Â° and then suspended in fresh resulting in inhibition of phosphorylation of nucleosides, in medium, with phosphorylation measured at 27Â°. This inhibition of synthesis of nucleic acids and protein, and in procedure was therefore generally used, since any incorpo degradation of performed DNA. These interactions were ration of nucleoside substrates into DNA and RNA was irreversible over time intervals used here at a temperature of thereby prevented. We report here drug levels that inhibited 37Â°. We did not find any drug-induced inhibition of thymidine and uridine phosphonylation by 50% under nucleoside transport. Although inhibition of phosphoryla standard conditions. Drug structures are shown in Charts tion of nucleosides would alter rates of nucleoside incorpo land 2. Drug-induced Inhibition of DNA, RNA, and Protein ration into nucleic acid, we found that drug levels that Synthesis. At levels below those required for inhibition of clearly inhibited the latter were substantially below levels required for inhibition of phosphorylation. The single phosphorylation of thymidine and unidine, certain com exception was fumoxanobenzofumoxan (Table 2). Further pounds inhibited incorporation of precursors into nucleic more, degradation of DNA was found at drug levels similar acids and protein (Tables I and 2). Drug levels that inhibited

reversed, so that removal of drug did not alter the course of inhibition. If cells were subsequently incubated with labeled thymidine or unidine at 22Â°,no incorporation into nucleic acid occurred, and drug effects on nucleoside uptake versus incorporation could be delineated. Drug-induced alteration on the rate of sedimentation of DNA through alkaline and neutral sucrose gradients in a centrifugal field was measured using cell labeled with [methyl- â€˜4C]thymidine (55 sCi/mmole). For this purpose, 10-mi portions of suspension containing 10' cells were in cubated with 25 .tl (1 @zCi)of labeled thymidine for 12 hr. The cells were collected and suspended in fresh medium for 4 hr to chase the label into DNA. The cells were again suspended in fresh medium containing drugs at 0. 1 to

3736

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VOL. 35

Benzofurazans Table I of thymidine phosphorylation, DNA synthesis, and degradation of DNA by benzofurazans Cells were incubated at 37Â°with drugs as described in the text. Drug levels required for 50% inhibition of thymidine phosphorylation, for incorporation of thymidine into DNA, and for DNA degradation are shown. Relevant structural features of drugs are shown in Chart I (left). For selected drugs, structures of the corresponding N-oxides are shown in Chart 2. Inhibition

ofThymidine foralteration level (@M)required

DrugstructureDrug

profileR'R'HHInactiveInactiveInactiveNO,H3033NO,Cl10045NO,N(C,H5OH),20040100NO,N(CH,),700(l25)@300(50)300NO,Sâ€”C,H,3033I,,C,H,S02Â°NC,H,O1500( phosphorylationDNA synthesisDNA

(50)IC,CH,C,H,SO,SC4H4NO17033Id,CH,C,HSSO:SO,C,HSCH,80055I,, NO,N,C4H,CH,2000(200)650(40)500

SO,C,H5CH,C,H,SInactiveInactiveInactive a Numbers

in

parentheses.

data

obtained

with

the

L5178Y

cell

line.

b I to â€˜e' benzofurazan analogs of structures shown in Chart 2. Table 2 of thymidine phosphorylation, DNA synthesis, and degradation of DNA by benzofuroxans Cells were treated as described in the legend to Table I . The parent compound is shown in Chart I (right). Structures of selected compounds are shown in Chart 2. Inhibition

ofThymidine level (NM)required

for alteration

Drug structureDrug

profileR'R2HHInactiveInactiveInactiveNO2H3033NO,Cl9544NO,N(C,H,OH),302.55NO,N(CH,),502535NO2SC,H,3033II,,C,H,SO,'NC,HIO3000(1500)b250(50)350(50) phosphorylationDNA synthesisDNA

CH,C,H4SO,502C,H4CH,34023II,, SO,C,H,CH,C,H,SInactiveInactiveInactivelI,,Furoxanobenzo

60(15)60(15)50(15)furoxan a II

to II,,

a Numbers

structures

shown

in parentheses,

in Chart

2.

data on L5178Y

to those required for inhibition of incorporation of labeled thymidine into DNA. We conclude that the observed inhibition of thymidine and unidine incorporation into nucleic acid is not merely a reflection of kinase inhibition. Inhibition of DNA synthesis and DNA damage detecta ble on sucrose gradients was also observed with a related compound, 4-nitroquinoline N-oxide (2, 13). The drug camptothecin, causing mainly single-strand DNA breaks (1) along with a few double-strand breaks (19), is also a potent inhibitor of nucleic acid synthesis with protein synthesis affected more slowly (17). The present data suggest that a given level of drug causes appearance of many

DECEMBER

cells; other data obtained

with L1210.

single-strand breaks along with DNA molecule and that coincidence of breaks on each strand causes the apparent double-strand breakage seen on neutral sucrose gradients. This pattern is substantially different than the campto thecin-induced damage but closely parallels results found with nitnoquinoline N-oxide (2). The latter drug, causing both double- and single-strand DNA damage, is postulated to cause scission of proposed protein segments of the DNA structure (13). The benzofurazans (Chart I, Structure I) and ben zofuroxans (Chart I , Structure II) under study, have as substituents a leaving group (R2) and an activating group

1975

3737


D. Kessel and J. G. Belton (R'). They react with thiols (Chart 5), amines, and other nucleophiles to form adducts, known as Meisenheimer complexes, when R' = NO2. Compounds with good leaving groups, (i.e., Cl or RSO2) yield only the substitution product, but for those with poor leaving groups (i.e., H on R2N) the adduct complex can usually be isolated (3). The relationship between the structure of the molecule to its ability to affect nucleic acid synthesis is fairly consistent. Of benzofuroxans (Table 1) with a powerful activating group (Chart 1, Structure I!, R' = NO2), the only inactive compounds observed were those with a very poor leaving group (R2 = NR2). When R2 = N(CH2CH2OH)2, the decreased activity is possibly due to a change in water solubility. The monosubstituted compound (R2 = H) repre sents a special case. When the activating group is less powerful (Chart I, Structure 1, R' = RSO2), compounds with R2 = NR2 and even R2 = SR are inactive, and a good leaving group (R2 = RSO2) is needed to confer activity. The pynidyl-(N-oxide) thio- compound (Chart 2, Drug c) may be more active because the leaving group is more efficient than the simple arylthio- group.

R1

In the benzofuroxan series (Table 2), a nitro group (Chart I, Structure 1!, R' = NO2) confers activity on all corn pounds examined, even those where R2 = NR2. For the sulfones (Chart 1, Structure II, R' = RSO2), the situation is unchanged from the benzofurazans. Other compounds of the general formulae shown in Chart C

d

a b

NO2

o.s.o

c:_)\@e

CH3

e

R1

f

â€˜3 Â®,0

\-

0

R2

Chart 2. Structures of benzofuroxans. The corresponding benzofura

I Chart I . Structural features of benzofurazans (I) and benzofuroxans (II). If the activating group R' is considered to be on position 4 ofthe ring

system, the leavinggroup, R2, is a 7-substituent.

zans lack the N-oxide grouping as shown in Chart I (left). Compound names are: II,, 4-(phenylsulfonyl)-7-piperidinobenzofuroxan; â€œb' 4@ nitro-7-(N-methylpiperazinyl)benzofuroxan; II@. 4-(p-tolylsulfonyl)-7-(2pyridylthio N-oxide)benzofuroxan; â€œd.4,7-bis(p-tolylsulfonyl)benzofu

roxan; II, , 4-(p-tolylthio)-7-(phenylsulfonyl)benzofuroxan; obenzofuroxan.

II, , furoxan

I00

Chart 3. Effects of I (â€¢)or 3 (@) MM levels of Drug b (Chart 2) on incorporation of leucine, uridine, and thymidine into macromolecules by Ll210 cells. Drug and radioactive precursor were added simul taneously, and incorporation or radioac

tivity into acid-solublematerial was mea

0

L. __

C

0

50

U

0

sured at specified times. 100%, incorpora

tion of precursor by controls (x ) during a 15-mm incubation.

Time

(mm) CANCER RESEARCH

3738


VOL. 35

@

@}@N

Benzofurazans thine and quanine and certain derivatives thereof (7). The latter have been postulated to act via free-radical inter mediates (6, 7), a mechanism of action which is unlikely for compounds studied here. Regardless of the mode of ac tion, it seems likely that the 4-nitrobenzofunazans and their N-oxides must be considered as potential carcinogens. Use of these agents as vasodilators (8) on monoamine oxidase inhibitors (4, 5) must be explored with the drug-DNA in teractions and their potenti@l effects in mind.

30@

20@

0

0

0

ACKNOWLEDGMENTS IO@ Helpful discussions with Dr. R. S. McElhinney, Council of Ireland, were much appreciated. Gwynne Blahnik provided excellent technical assistance.

,

0@ -@ i I I I 1@ I I I I I I I (TOP) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Chart 4. Typical sedimentation profiles of DNA through sucrose gradients. Profiles of untreated (â€¢)and drug-treated (0) DNA on neutral

sucrose and drug-treated (0) DNA on alkaline sucrose are shown. Not shown is the control sedimentation profile on control DNA through the alkaline gradient; peak fractions were in Tubes I 2 and I 3. Drug treatment consisted of 20 mm exposure at 37Â° to Drug b (Chart 2). Bottom of gradient, Fraction 15.

NO2

NO2

@7Z@ @)

r@@N\

II â€œ>@z:@'â€”@' N

RS

x0

X@eNO@

Adduct

Drug

RS Substitution Product

Chart 5. Postulated reaction of benzofurazans (and benzofuroxans) with nucleophiles (e.g., thio and amino groups). The adduct can be isolated with X a poor leaving group; only the substitution product is isolated if X is a good leaving group.

1 have been tested with results consistent with these generalizations. The particular selection of R' and R2 will depend on the particular cell line under study and on other factors. For example, Compound b (Chart 2) could be synthesized in radioactive form with a labeled carbon atom in the methyl group on the piperazine ring. Since this compound has a poor leaving group, studies on drug-DNA binding could readily be carried out. Some drugs that are poor inhibitors of DNA synthesis in Ll210 cells are evidently quite potent in L5178Y cells, suggesting that transport barriers to uptake of certain molecular configurations may play a role here. In addition to degrading DNA and inhibiting synthesis of nucleic acid, these drugs at high levels affect kinases. Ef fects on RNA processing have not been examined. The me lated nitnoquinoline N-oxide, which also degrades DNA (2, 13), is a potent carcinogen (14) as are N-oxides of xan

DECEMBER

REFERENCES I. Abelson, H. T., and Penman, S. Induction Cellular DNA by Camptothecin. Biochem.

FRACTION NUMBER

Medical Research Smith and Joanne

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50:

1048-1054,

of Alkali Labile Links in Biophys. Res. Commun.,

1973.

2. Andoh, T., and Ide, T. Strand Scission and Rejoining of DNA in Cultured Mammalian Cells Induced by 4-Nitroquinoline I-Oxide. Cancer Res.,32: 1230-1235,1972. 3. Belton, J. G., and Novel, A. N-S Oxygen Migration in 2,l,2-Benzox adiazole Systems. Proc. Roy. Irish Acad., 74B: 185- 192, 1974. 4. Bolt, A. G., Ghosh, P. B., and Sleigh, M. i. Benzo-2,l,5Oxadiazolesâ€”A Novel Class of Heterocyclic Monoamine Oxidase Inhibitors. Biochem. Pharmacol., 23: 1963- 1968, 1974. 5. Bolt, A. G., and Sleigh, M. J. Furoxanobenzofuroxan, a Selective Monamine Oxidase Inhibitor. Biochem. Pharmacol., 23: 1969-1977, 1974. 6. Brown, G. B. Purine N-Oxides and Cancer. Progr. Nucleic Acid Res.

Mol. Biol., 8: 209-255, 1968. 7. Brown, G. B., Teller, M. N., Smullyan, I., Birdsall, N. i. M., Lee, T-C., Parham, J. C., and Stoorher, G. Correlations between Onco

genic and Chemical Properties of Several Derivativesof 3-Hydroxy xanthine and 3-Hydroxyguanine. Cancer Res., 33: 1113-1118, 1973. 8. Ghosh, P. B., and Everitt, B. J. Furazanobenzofuroxan, Furazanoben zothiadiazole, and Their N-Oxides. A New Class of Vasodilator Drugs. i. Med. Chem., 117: 203-206, 1974. 9. Ghosh, P. B., Ternai, B., and Whitehouse, M. W. Potential Anti Leukemic and Immunosuppressive Drugs. 3. Effects of Homocyclic Ring Substitution on the In Vitro Drug Activity of 4-Nitrobenzo-2,l,2Oxdiazoles (4-Nitrobenzofurazans) and Their N-Oxides (4-Nitroben zofuroxans). J. Med. Chem., 15: 255-260, 1972. 10. Ghosh, P. B., and Whitehouse, M. W. 7-Chloro-4-Nitrobenzo-2-

Oxa- 1,3-Diazole: A New Fluorogenic Reagent for Amino Acids and Other Amines. Biochem. J., 108: 155-156, 1968. II. Ghosh, P. B., and Whitehouse, M. W. Potential Anti-Leukemic and Immunosuppressive Drugs Preparation and In Vitro Pharmacological Activity of Some Benzo-2, I ,3-Oxadiazoles (Benzofurazans) and Their N-Oxides (Benzofuroxans). J. Med. Chem., 11: 305-31 1, 1968. 12. Ghosh, P. B., and Whitehouse, M. W. Potential Anti-Leukemic and Immunosuppressive Drugs. II. Further Studies with Benzo-2,l,3Oxadiazoles (Benzofurazans) and Their N-Oxides (Benzofuroxans). J.

Med. Chem., 12: 505-507, 1969. 13. Ide, T., and Andoh, T. Scissions Mammalian Cells Induced by Repair. Cancer Res., 32: 123514. Kawazoe, Y., Araki, M., and

of Proteins Linking DNA in Cultured 4-Nitroquinoline-l-Oxide and Their 1242, 1972. Huang, G-F. Chemical Aspects of

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D. Kessel and J. G. Belton Carcinogenesis by 4-Nitroquinoline-l-Oxide. In: W. Nakahara, S. Takayama, T. Sugimura, and S. Odashima (eds), Topics in Chemical Carcinogenesis. pp. 1â€” I 3. Baltimore: University Park Press, 1972. I 5. Kessel, D. Some Determinants of Camptothecin Responsiveness in Leukemia Ll210 Cells. Cancer Res., 31: 1883-1887, 1971. 16. Kessel, D., Bosmann, H. B., and Lohr, K. Camptothecin Effects on

DNA Synthesis in Murine Leukemia Cells. Biochim. Biophys. Acta, 269: 210-216, 1972. 17. Kessel, D., and Dodd, D. C. Effects of Persantin on Several Transport Systems of Murine Leukemias. Biochim. Biophys. Acta, 288: 190-194, 1972. 18. Kessel. D., and Hall, T. C. Effects of Persantin on Deoxycytidine

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Transport by Murine 88-94, 1970.

Leukemia

Cells. Biochim.

Biophys. Acta, 211:

19. Spataro, A., and Kessel, D. Studies on Camptothecin-Induced Degra dation and Apparent Reaggregation of DNA from L12l0 Cells. Biochem. Biophys. Res. Commun., 48: 643-648, 1972. 20. Tereshima, T., and Tsubio, A. Mammalian Cell DNA Isolated with Minimal Shearing, a Sensitive System for Detecting Strand Breaks by Radiation. Biochim. Biophys. Acta, 174: 309-314, 1969. 21. Whitehouse, M. W., and Ghosh, P. B. 4-Nitrobenzofurazans and 4-Nitrobenzofuroxans: New Class of Thiol-Neutralizing Agents and Potent Inhibitors of Nucleic Acid Synthesis in Leukocytes. Biochem. Pharmacol., 17: 158-161, 1968.

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