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The association between mutagenicity and adduct formation of 1,2,7,8-diepoxyoctane and 1,2,5,6-diepoxycyclooctane
Chem.-Biol. Interactions, 20 (1978) 333--340 © Elsevier/North-Holland Scientific Publishers Ltd.
333
THE ASSOCIATION BETWEEN MUTAGENICITY AND ADDUCT FORMATION OF 1,2,7,8-DIEPOXYOCTANE AND 1,2,5,6-DIEPOXYCYCLOOCTANE
S.L. HUANG, D.N. RADER and C.-Y. LEE National Institutes of Health, National Institute of Environmental Health Sciences, Laboratory of Environmental Mutagenesis, Research Triangle Park, N.C. 27709 (U.S.A.) (Received September 9th, 1977) (Revision received November 8th, 1977) (Accepted December 1st, 1977)
SUMMARY
The mutagenicity of 1,2,5,6-diepoxycyclooctane (DECO) and 1,2,7,8diepoxyoctane (DEO) was investigated using diploid Chinese hamster lung cells. 6-thioguanine (6-TG) resistance was used as the marker for mutagenicity testing: DEO was found to be genetically active; DECO, on the contrary, totally inactive. DEO readily formed adducts with radiolabeled nucleotides, while DECO failed to do so, as demonstrated through thin-layer chromatography (TLC) and the shift of the ultraviolet absorption maximum in DEO/ nucleotide mixtures. The difference between the two compounds in chemical and genetic activities was attributed to their molecular conformations and the resulting differential flexibilities and adduct-forming abilities. Association between mutagenicity and adduct formation was conclusive.
INTRODUCTION
Epoxides have been identified as the probable ultimate carcinogenic or mutagenic form of many environmental agents. Many studies have demonstrated that the epoxides of polycyclic hydrocarbons are much more active biologically than the parent compounds or any other derivative tested to date [1--3]. In addition, studies of mono- and bifunctional epoxides have demonstrated their frequent mutagenicity and carcinogenicity in mice and rats [4--6], Neurospora crassa [7] and Drosophila melanogaster [8]. Diepoxides are more frequently carcinogenic than are monoepoxides [2,9], possibly as a result of the DNA cross-linking potential of the diepoxides and the subsequent miscoding of bases [10]. Molecular conformaAbbreviations: DECO, 1,2,5,6-diepoxycyclooctane; DEO, 1,2,7,8-diepoxyoctane; FCS, fetal calf serum; TLC, thin-layer chromatography; 6-TG, 6-thioguanine.
334 tion and flexibility also contribute to the carcinogenic/mutagenic potential of an epoxide [2]. In addition, several studies have shown that polyfunctional agents produce more chromosome breaks than monofunctional agents [11--131. In prior studies, it has been shown t h a t DEO was a p o t e n t mutagen induc ing point mutations and a high frequency of multilocus deletions in N. crassa [7]. Diepoxybutane and 1,2,4,5-diepoxypentane were found to be mutagenic in this same m u t a t i o n detection system [7]. The induction of point mutations in cultured mammalian cells makes possible studies in these cells of genetic events resulting from the action of mutagenic agents at the molecular level similar to previous studies with microorganisms. The frequency of spontaneous m u t a t i o n to 8-azaguanine and/or 6-TG resistance can be increased by physical and chemical mutagens [14--20]. The present studies deal with the mutagenicity of the bifunctional epoxide DEO and DECO as determined through the m u t a t i o n of cultured Chinese hamster cells to 6-TG resistance and the relationship between the mutagenicity of these compounds and their ability to form adducts with nucleotides. These compounds are structural analogue o f d i e p o x y b u t a n e which has been used in the curing of polymers for cross-linking textile fibres and in the prevention of microbial spoilage [21]. DEO and DECO are both carcinogenic compounds [2,6]. MATERIALS AND METHODS
Cell strains and culture media Chinese hamster lung cells were obtained from Americal Tissue Culture Collection. Culture medium consisted of 85 volumes of F10 (without h y p o x a n t h i n e ) and 15 volumes of fetal calf serum (FCS). Both F10 and FCS were obtained from Gibco. Penicillin and streptomycin were added to final concentrations of 100 units/ml and 0.1 mg/ml, respectively. TG-selective medium (FCS-F10-TG) consisted of FCS-F10 and 7.5 × 10 -s M 6-TG.
Cloning efficiency and staining o f clones 100 or 200 treated or untreated cells were inoculated into a 60 mm petri dish (p60) and were grown for 9 days. The dishes were then stained for colonies and the colony numbers were counted. This number was used to estimate cell survival in the calculation of mutation frequency. 2% methylene blue in citrate buffer (pH 6) was used to stain the cells. Cells were rinsed with 0.9% saline, fixed with 95% ethanol and stained with methylene blue.
Mutagenesis assays Two million cells in a 100 mm petri dish ( p l 0 0 ) were treated with die-
335 poxide dissolved in 0.05 ml of dimethylsulfoxide. After 60 min the medium was removed. The cells were rinsed twice with F10 and trypsinized. Samples of cells were inoculated into p l 0 0 ' s with l 0 s cells each. A total of 10--14 dishes were used to determine mutant frequency for each dose point. Cell survival was determined as indicated above. Selection for 6-TG-resistant cells started 48 h later and lasted for 9 days. The dishes were then stained with 2% methylene blue [22].
Formation o f adducts between epoxy compounds and nucleic acid bases Each of the 14C-radionucleotides AMP (528 mCi/mmol; final conc., 3.2 uM)~ GMP (495 mCi/mmol; final conc., 3.4/~M), CMP (404 mCi/mmol; final conc., 4.0 uM) and TMP (53.7 mCi/mmol, final conc., 31.0 #M) was incubated in 0.1 M phosphate buffer (pH 6.5) with DEO or DECO (previously dissolved in a small amount of DMSO) at 0.5 M for one, three, five and eight days. All reactions were conducted in the dark at reduced temperature (~4°C) to prevent possible dimer formation or decomposition of nucleotides. Adducts were separated by TLC on MN 300 cellulose plates (250 #M thick) I00
x 0
•
3 m
f0 0 3
G)
E _v {E
3 E
I
.I O-
a,
o 6. 5 dso
|
25.00 Dose × IO*M
50~DO
Fig. 1. DEO- and DECO-induced mutation frequencies in Chinese hamster lung cells to 6-TG resistance and relative survival as functions of dose. Each point is the mean o f two experiments. Induced mutation frequency: *, DEO; A, DECO. Relative survival: o, DEO; ~, DECO.
336 using an ethanol : 0.5 M a m m o n i u m acetate (9 : 4) solvent system, and detected using a Berthold TLC Scanner. Radiolabeled nucleotides were obtained from ICN, and were repurified in the same TLC system before use. DEO and DECO (97% purity) were purchased from Aldrich Chemical Company. The composition of the compounds were verified with proton NMR spectra by Dr. P. Albro. Infrared spectra of both compounds were also run in CC14 solution indicating that DEO contained no h y d r o x y l group and only a trace of h y d r o x y l was contained in DECO. In addition, 0.1 M AMP was incubated individually with 0.5 M DEO or DECO in 0.1 M phosphate buffer at pH 6.5 for 24 h at 37°C. In each case, the absorption spectrum was measured and monitored for shifts resulting from adduct formation. RESULTS AND DISCUSSION The frequencies of 6-TG-resist.ant colonies at different concentrations of DEO and DECO were calculated for separate experiments. Data from two experiments with DECO and two experiments with DEO are plotted in Fig. 1. DECO did not induce 6-TG resistance; DEO did induce 6-TG resistance. The frequencies induced by DEO were dose dependent. The slope for the linear dose-response relationship (in log transformation) was calculated to be 2 for the three higher doses. 6-TG resistance was induced by second order kinetics. This is in good agreement with the previous findings that DEO induces both gene mutations and multilocus deletions [7]. Exposure of nucleotides (AMP, GMP, CMP or TMP) to DEO resulted in alterations of physical properties. When AMP and DEO were incubated at 37°C in 0.1 M phosphate buffer (pH 6.5), a shift of the ultraviolet absorb-
o
2~,o
2,~o
~o
Wavelength (nM)
2~
30o
Fig. 2. Ultraviolet absorption spectrum of (a) AMP, (b) AMP + DEO and (c) AMP + DECO.
337 ance maximum from 257 to 263.5 nm was observed after 24-h incubation, as shown in Fig. 2. This shift is indicative of adduct formation, most probably (at this pH) by the attack of DEO on the base [23]. Attack at high pH, however, is usually centered on the ribose [24]. The time dependent formation of adducts between radionucleotides and DEO was also observed with TLC. All four nucleotides yielded reaction products with DEO. Fig. 3 shows a typical TLC scan of a DEO/TMP reaction mixture with at least one adduct present. Table I gives Rf values for the radionucleotides and their products with DEO in the TLC system used. When the same nucleotides were exposed to DECO, however, no effects were noted. No shift in the ultraviolet absorption maximum for AMP occurred (Fig. 2). In addition, no reaction products had formed between the nucleotides and DECO after 8 days of incubation. Control
|
~L.--&
~
L
,.
,,
Low dose
st
.
.
.
.
High dose
f
Origin
f Solvent front
Fig. 3. Typical TLC of two doses of DEO incubated with ['4C]TMP and ['4C]TMP alone for 5 days at 4°C. See text for experimental details.
338 TABLE I RESULTS OF TLC OF RADIOLABELED NUCLEOTIDES AND ADDUCTS FORMED BY INCUBATION WITH DEO AND DECO The existence o f a second adduct o f TMP and DEO is uncertain. Incubation in this case was for 5 days at 4°C. TLC was run on MN 300 cellulose plates (250 ~m), with a 0.5 M NH4Ac--EtOH (4 : 9) solvent system.
Compound
Nucleotide
Rf Nucleotide
DEO
DECO
AMP GMP CMP TMP No adducts were observed
Derivative
0.67 0.87 0.10 0.36 0.17 0.46 0.37 0.94, 0.72 with any of the four nucleotides
The observed d i c h o t o m y in effects on nucleotides is more probably a result of stability and conformational differences between the two molecules. DECO is much more rigid than DEO and is correspondingly ~ess accessible and reactive. Presumably, diepoxide-derived carcinogenesis results from the effects o f cross-linking between DNA strands on the genetic material [2]. A diepoxide must extend at least 80 nm in order to cross-link DNA [2,10] : DEO meets this requirement, DECO does not [25]. In general, cyclic diepoxides are much less carcinogenic than their straight-chain counterparts as a result of this differential flexibility of reactive centers [2]. Interestingly, our data demonstrate the greatly increased binding ability of DEO over DECO to individual nucleotides, where cross-linking considerations are minimized. Formation of second adduct is possible in our system and may have occurred in TMP/DEO reactions as pictured in Fig. 3. Molecular flexibility, therefore, seems as important as molecular size in affecting biochemical, and perhaps biological, activity. The mutagenic specificity of diepoxyoctanes closely parallels their carcinogenicity [2,6,7,8] and is apparently related to the c o m p o u n d ' s ability to bind to DNA. These findings provide a chemical basis for the observed genetic effects. REFERENCES 1 E. Huberman, L. Aspiras, C. Heidelberger, P. Grover and P. Sims, Mutagenicity to mammalian cells of epoxides and other derivatives of polycyclic hydrocarbons, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 3195. 2 B.L. Van Duuren, Carcinogenic epoxides, lactones, and halo-ethers and their mode of action, Ann. N.Y. Acad. Sci., 163 (1969) 633. 3 P.L. Grover, J.A. Forrester and P. Sims, Reactivity of the K-region epoxides of some
339
4
5 6
7 8
9 10 11
12 13
14 15 16 17 18 19 20 21 22 23
polycyclic hydrocarbons towards the nucleic acids and proteins of BHK 21 cells, Biochem. Pharmacol., 20 (1971) 1297. B.L. Van Duuren, L. Langseth, L. Orris, G. Teebor, N. Nelson and M. Knschner, Carcinogenicity o f epoxides, lactones, and peroxy compounds, IV. Tumor response in epithelial and connective tissue in mice and rats, J. Natl. Cancer Inst., 37 (1966) 825. B.L. Van Duuren, L. Langseth, L. Orris, M. Baden and M. Kuschner, Carcinogenicity of epoxides, lactones, and peroxy compounds. V. Subcutaneous injection in rats, J. Natl. Cancer Inst., 39 (1967) 1213. B.L. Van Duuren, L. Langseth, B.M. Goldschmidt and L. Orris, Carcinogenicity of epoxides, lactones, and peroxy compounds. VI. Structure and carcinogenic activity, J. Natl. Cancer Inst., 39 (1967) 1217. T. Ong and F.J. de Serres, Mutation induction by difunctional alkylating agents in Neurospora crassa, Genetics, 80 (1975) 475. Y. Nakao and C. Auerbach, Test of a possible correlation between cross-linking and chromosome breaking abilities of chemical mutagens, Z. Vererbungsl., 92 (1961) 457. C.S. Wiel, N. Condra, C . Haun and J.A. Striegel, Experimental carcinogenicity and acute toxicity of representative epoxides, Am. Ind. Hyg. Ass. J., (1963) 305. P.D. Lawley and P. Brookes, Interstrand cross-linking of DNA by difunctional alkylating agents, J. Mol. Biol., 25 (1967) 143. O.G. Fahmy and M.J. Fahmy, Cytogenetic analysis of the action of carcinogens and tumor inhibitors in Drosophila melanogaster. X. The nature of the mutations induced by the mesyloxy esters in relation to molecular cross-linking, Genetics, 46 (1960) 447. M.M. Nawar, C.F. Konzak and R.A. Nilan, Comparative studies of the biological effectiveness of nitrogen mustards, ethyleneimine and X-rays, Murat. Res., 11 (1971) 339. G. Obe, Chemical constitution and genetic action, V. Comparative investigation of the action of ethyleneimines on human leukocyte chromosomes, Mutat. Res., 6 (1968) 467. C.F. Arlett and S.A. Harcourt, Expression time and spontaneous mutability in the estimation of induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light, Murat. Res., 16 (1972) 301. C.F. Arlett and J. Patter, Mutation to 8-azaguanine resistance induced by gammaradiation in a Chinese hamster cell line, Murat. Res., 13 (1971) 59. B.A. Bridges and J. Huckle, Mutagenesis of cultured mammalian cells by X-radiation and ultraviolet light, Murat. Res., 10 (1970) 141. E.H.Y. Chu, Mammalian cell genetics. III. Characterization of X-ray-induced forward mutations in Chinese hamster cell cultures, Mutat. Res., 11 (1971) 23. E.H.Y. Chu and H.V. Mailing, Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. U.S.A., 61 (1968) 1306. M.E. Duncan and P. Brooks, The induction of azaguanine-resistant mutants in cultured Chinese hamster cells by reactive derivatives of carcinogenic hydrocarbons, Murat. Res., 21 (1973) 107. S.H. Orkin and J.W. Littlefield, Nitrosoguanidine mutagenesis in synchronized hamster cells, Exp. Cell Res., 66 (1971) 69. P.G. Stecher (Ed.), The Merck Index, 8th edn., Rahway, N.J., Merck & Co, 1968 p. 418. S.L. Huang, Dilution of hypoxanthine-guanine phosphoribosyl transferase and other factors affecting the frequency of 6-thioguanine resistance in Chinese hamster lung cells, Mutat. Res., 44 (1977} 119. P. Zappeli, A. Rossodivita and L. Re, Synthesis of coenzymically active soluble and insoluble macromolecularized NAD ÷ derivatives, Eur. J. Biochem., 54 (1975) 475.
340 24
25
L. Sundberg and J. Poranth, Preparation of absorbents for biospecifie affinity chromatography. I. Attachment of group-containing ligands to insoluble polymers by means of bifunctional oxiranes, J. Chromatogr., 90 (1974) 87. Z. Smith and D.A. Kohl, Electron-diffraction study of the structure of 1,2,7,8diepoxyoctane, J. Chem. Phys., 60 (1974) 49'20.
333
THE ASSOCIATION BETWEEN MUTAGENICITY AND ADDUCT FORMATION OF 1,2,7,8-DIEPOXYOCTANE AND 1,2,5,6-DIEPOXYCYCLOOCTANE
S.L. HUANG, D.N. RADER and C.-Y. LEE National Institutes of Health, National Institute of Environmental Health Sciences, Laboratory of Environmental Mutagenesis, Research Triangle Park, N.C. 27709 (U.S.A.) (Received September 9th, 1977) (Revision received November 8th, 1977) (Accepted December 1st, 1977)
SUMMARY
The mutagenicity of 1,2,5,6-diepoxycyclooctane (DECO) and 1,2,7,8diepoxyoctane (DEO) was investigated using diploid Chinese hamster lung cells. 6-thioguanine (6-TG) resistance was used as the marker for mutagenicity testing: DEO was found to be genetically active; DECO, on the contrary, totally inactive. DEO readily formed adducts with radiolabeled nucleotides, while DECO failed to do so, as demonstrated through thin-layer chromatography (TLC) and the shift of the ultraviolet absorption maximum in DEO/ nucleotide mixtures. The difference between the two compounds in chemical and genetic activities was attributed to their molecular conformations and the resulting differential flexibilities and adduct-forming abilities. Association between mutagenicity and adduct formation was conclusive.
INTRODUCTION
Epoxides have been identified as the probable ultimate carcinogenic or mutagenic form of many environmental agents. Many studies have demonstrated that the epoxides of polycyclic hydrocarbons are much more active biologically than the parent compounds or any other derivative tested to date [1--3]. In addition, studies of mono- and bifunctional epoxides have demonstrated their frequent mutagenicity and carcinogenicity in mice and rats [4--6], Neurospora crassa [7] and Drosophila melanogaster [8]. Diepoxides are more frequently carcinogenic than are monoepoxides [2,9], possibly as a result of the DNA cross-linking potential of the diepoxides and the subsequent miscoding of bases [10]. Molecular conformaAbbreviations: DECO, 1,2,5,6-diepoxycyclooctane; DEO, 1,2,7,8-diepoxyoctane; FCS, fetal calf serum; TLC, thin-layer chromatography; 6-TG, 6-thioguanine.
334 tion and flexibility also contribute to the carcinogenic/mutagenic potential of an epoxide [2]. In addition, several studies have shown that polyfunctional agents produce more chromosome breaks than monofunctional agents [11--131. In prior studies, it has been shown t h a t DEO was a p o t e n t mutagen induc ing point mutations and a high frequency of multilocus deletions in N. crassa [7]. Diepoxybutane and 1,2,4,5-diepoxypentane were found to be mutagenic in this same m u t a t i o n detection system [7]. The induction of point mutations in cultured mammalian cells makes possible studies in these cells of genetic events resulting from the action of mutagenic agents at the molecular level similar to previous studies with microorganisms. The frequency of spontaneous m u t a t i o n to 8-azaguanine and/or 6-TG resistance can be increased by physical and chemical mutagens [14--20]. The present studies deal with the mutagenicity of the bifunctional epoxide DEO and DECO as determined through the m u t a t i o n of cultured Chinese hamster cells to 6-TG resistance and the relationship between the mutagenicity of these compounds and their ability to form adducts with nucleotides. These compounds are structural analogue o f d i e p o x y b u t a n e which has been used in the curing of polymers for cross-linking textile fibres and in the prevention of microbial spoilage [21]. DEO and DECO are both carcinogenic compounds [2,6]. MATERIALS AND METHODS
Cell strains and culture media Chinese hamster lung cells were obtained from Americal Tissue Culture Collection. Culture medium consisted of 85 volumes of F10 (without h y p o x a n t h i n e ) and 15 volumes of fetal calf serum (FCS). Both F10 and FCS were obtained from Gibco. Penicillin and streptomycin were added to final concentrations of 100 units/ml and 0.1 mg/ml, respectively. TG-selective medium (FCS-F10-TG) consisted of FCS-F10 and 7.5 × 10 -s M 6-TG.
Cloning efficiency and staining o f clones 100 or 200 treated or untreated cells were inoculated into a 60 mm petri dish (p60) and were grown for 9 days. The dishes were then stained for colonies and the colony numbers were counted. This number was used to estimate cell survival in the calculation of mutation frequency. 2% methylene blue in citrate buffer (pH 6) was used to stain the cells. Cells were rinsed with 0.9% saline, fixed with 95% ethanol and stained with methylene blue.
Mutagenesis assays Two million cells in a 100 mm petri dish ( p l 0 0 ) were treated with die-
335 poxide dissolved in 0.05 ml of dimethylsulfoxide. After 60 min the medium was removed. The cells were rinsed twice with F10 and trypsinized. Samples of cells were inoculated into p l 0 0 ' s with l 0 s cells each. A total of 10--14 dishes were used to determine mutant frequency for each dose point. Cell survival was determined as indicated above. Selection for 6-TG-resistant cells started 48 h later and lasted for 9 days. The dishes were then stained with 2% methylene blue [22].
Formation o f adducts between epoxy compounds and nucleic acid bases Each of the 14C-radionucleotides AMP (528 mCi/mmol; final conc., 3.2 uM)~ GMP (495 mCi/mmol; final conc., 3.4/~M), CMP (404 mCi/mmol; final conc., 4.0 uM) and TMP (53.7 mCi/mmol, final conc., 31.0 #M) was incubated in 0.1 M phosphate buffer (pH 6.5) with DEO or DECO (previously dissolved in a small amount of DMSO) at 0.5 M for one, three, five and eight days. All reactions were conducted in the dark at reduced temperature (~4°C) to prevent possible dimer formation or decomposition of nucleotides. Adducts were separated by TLC on MN 300 cellulose plates (250 #M thick) I00
x 0
•
3 m
f0 0 3
G)
E _v {E
3 E
I
.I O-
a,
o 6. 5 dso
|
25.00 Dose × IO*M
50~DO
Fig. 1. DEO- and DECO-induced mutation frequencies in Chinese hamster lung cells to 6-TG resistance and relative survival as functions of dose. Each point is the mean o f two experiments. Induced mutation frequency: *, DEO; A, DECO. Relative survival: o, DEO; ~, DECO.
336 using an ethanol : 0.5 M a m m o n i u m acetate (9 : 4) solvent system, and detected using a Berthold TLC Scanner. Radiolabeled nucleotides were obtained from ICN, and were repurified in the same TLC system before use. DEO and DECO (97% purity) were purchased from Aldrich Chemical Company. The composition of the compounds were verified with proton NMR spectra by Dr. P. Albro. Infrared spectra of both compounds were also run in CC14 solution indicating that DEO contained no h y d r o x y l group and only a trace of h y d r o x y l was contained in DECO. In addition, 0.1 M AMP was incubated individually with 0.5 M DEO or DECO in 0.1 M phosphate buffer at pH 6.5 for 24 h at 37°C. In each case, the absorption spectrum was measured and monitored for shifts resulting from adduct formation. RESULTS AND DISCUSSION The frequencies of 6-TG-resist.ant colonies at different concentrations of DEO and DECO were calculated for separate experiments. Data from two experiments with DECO and two experiments with DEO are plotted in Fig. 1. DECO did not induce 6-TG resistance; DEO did induce 6-TG resistance. The frequencies induced by DEO were dose dependent. The slope for the linear dose-response relationship (in log transformation) was calculated to be 2 for the three higher doses. 6-TG resistance was induced by second order kinetics. This is in good agreement with the previous findings that DEO induces both gene mutations and multilocus deletions [7]. Exposure of nucleotides (AMP, GMP, CMP or TMP) to DEO resulted in alterations of physical properties. When AMP and DEO were incubated at 37°C in 0.1 M phosphate buffer (pH 6.5), a shift of the ultraviolet absorb-
o
2~,o
2,~o
~o
Wavelength (nM)
2~
30o
Fig. 2. Ultraviolet absorption spectrum of (a) AMP, (b) AMP + DEO and (c) AMP + DECO.
337 ance maximum from 257 to 263.5 nm was observed after 24-h incubation, as shown in Fig. 2. This shift is indicative of adduct formation, most probably (at this pH) by the attack of DEO on the base [23]. Attack at high pH, however, is usually centered on the ribose [24]. The time dependent formation of adducts between radionucleotides and DEO was also observed with TLC. All four nucleotides yielded reaction products with DEO. Fig. 3 shows a typical TLC scan of a DEO/TMP reaction mixture with at least one adduct present. Table I gives Rf values for the radionucleotides and their products with DEO in the TLC system used. When the same nucleotides were exposed to DECO, however, no effects were noted. No shift in the ultraviolet absorption maximum for AMP occurred (Fig. 2). In addition, no reaction products had formed between the nucleotides and DECO after 8 days of incubation. Control
|
~L.--&
~
L
,.
,,
Low dose
st
.
.
.
.
High dose
f
Origin
f Solvent front
Fig. 3. Typical TLC of two doses of DEO incubated with ['4C]TMP and ['4C]TMP alone for 5 days at 4°C. See text for experimental details.
338 TABLE I RESULTS OF TLC OF RADIOLABELED NUCLEOTIDES AND ADDUCTS FORMED BY INCUBATION WITH DEO AND DECO The existence o f a second adduct o f TMP and DEO is uncertain. Incubation in this case was for 5 days at 4°C. TLC was run on MN 300 cellulose plates (250 ~m), with a 0.5 M NH4Ac--EtOH (4 : 9) solvent system.
Compound
Nucleotide
Rf Nucleotide
DEO
DECO
AMP GMP CMP TMP No adducts were observed
Derivative
0.67 0.87 0.10 0.36 0.17 0.46 0.37 0.94, 0.72 with any of the four nucleotides
The observed d i c h o t o m y in effects on nucleotides is more probably a result of stability and conformational differences between the two molecules. DECO is much more rigid than DEO and is correspondingly ~ess accessible and reactive. Presumably, diepoxide-derived carcinogenesis results from the effects o f cross-linking between DNA strands on the genetic material [2]. A diepoxide must extend at least 80 nm in order to cross-link DNA [2,10] : DEO meets this requirement, DECO does not [25]. In general, cyclic diepoxides are much less carcinogenic than their straight-chain counterparts as a result of this differential flexibility of reactive centers [2]. Interestingly, our data demonstrate the greatly increased binding ability of DEO over DECO to individual nucleotides, where cross-linking considerations are minimized. Formation of second adduct is possible in our system and may have occurred in TMP/DEO reactions as pictured in Fig. 3. Molecular flexibility, therefore, seems as important as molecular size in affecting biochemical, and perhaps biological, activity. The mutagenic specificity of diepoxyoctanes closely parallels their carcinogenicity [2,6,7,8] and is apparently related to the c o m p o u n d ' s ability to bind to DNA. These findings provide a chemical basis for the observed genetic effects. REFERENCES 1 E. Huberman, L. Aspiras, C. Heidelberger, P. Grover and P. Sims, Mutagenicity to mammalian cells of epoxides and other derivatives of polycyclic hydrocarbons, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 3195. 2 B.L. Van Duuren, Carcinogenic epoxides, lactones, and halo-ethers and their mode of action, Ann. N.Y. Acad. Sci., 163 (1969) 633. 3 P.L. Grover, J.A. Forrester and P. Sims, Reactivity of the K-region epoxides of some
339
4
5 6
7 8
9 10 11
12 13
14 15 16 17 18 19 20 21 22 23
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