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Mutagenicity of some N- and O-acyl derivatives of N-hydroxy-2-aminofluorene in V79 chinese hamster cells
Cancer L,~tters. 6 (1979) 67--72 © Elsevier/North-Holland ScientificPublishers Ltd.
67
MUTAGENICITY OF SOME N- AND O-ACYL DERIVATIVES OF N-HYDROXY-2-AMINOFLUORENE IN V79 CHINESE HAMSTER CELLS
T. KUROKI* and H. BARTSCH
Unit o f Chemical Carcinogenesis, International Agency for Research on Cancer, 150 tours Albert T?~ornas, 69372 Lyon, Cedex 2 (France)
(Received 7 June 1978) (Revised version received 2 October 1978) (Accepted .q October 1978)
SUMMARY
Mutagenicity of 4 N- and O-acyl derivatives of N-hydroxy-2-aminofluorene (acyl : acetyl or myristoyl residue) was examined in V79 Chinese hamster cells in the absence of a metabolic activation system. N-Myristoyloxy-N-acetyl-2aminofluorene (N-MyO-AAF) was toxic and weakly mutagenic, inducing 8-azaguanine-resistaat (AZAr) mutants in V79 Chinese hamster cells in a concentration-dependent fashion; while N-acetoxy-N-myristoyl-2-aminofluorene (N-AcO-MyAF) and N - m y r i s t o y l o x y - N - m y r J s t o y l - 2 - a m i n o f l u o r e n e (N-MyOMyAF) were neither cytotoxic nor mutagenic. Under the same conditions, N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) was highly toxic and mutagenic. Neither of the 2 N-myristoyloxy derivatives was mutagenic in S a l m o n e l l a t y p h i m u r i u m . These esters have been reported to produce local turnouts at the site of their injection in rats, to be electrophilic towards methionine, and to induce unscheduled DNA synthesis in cultured human fibroblasts. In view of the fact that some of the esters were mutagenic in neither S. t y p h i m u r i u m nor V79 Chinese hamster ceils, our findings emphasize the need for multiple short-term tests in predicting potential carcinogenic activity of chemicals. INTRODUCTION Metabolic activation of 2,-acetylaminofluorene, a hepatocarcinogen in male *To whom correspondence is tn be addressed, Present address: Department of Cancer Cell Research, Institute of MedicalScience, University
of Tokyo, Shirokanedai, Minato-ky,Tokyo 108, Japan. Abbrev&tions: AZAr, 8-azaguanine-resistance;DMSO, dimetbylsulfoxide; N-AcO-AAF, N-aeetoxy.N.acetyl.2-aminofluvrene; N-AcO-MyAF, N-acetoxy-N-myristoyl-2-arninofluorene:, N-MyO-AAF,N.myristoyloxy-N.acetyl.2-aminofluorene; N-MyO-MyAF,N-myristoyloxy-N-myristoyl-2.aminofluorene.
68 rats, requires a 2-step activatiorL process, involving/V-hydroxylation and subsequent esterific-'ltion of the hydroxyl group by soluble sulfotransferases in the liver [9]. The resulting metabolite, N-sulfonoxy-N-acetyl-2.aminofluorene, binds covalently to Cellular constituents [3] ; such esters, e.g., N-AcO-AAF, are regarded:as ultimate carcinogens. A previous paper [ 1 ] described the carcinogenicity, electrophilicity and m utagenicity of such derivative~ of N:hydroxy-2aminofluorene in which a C~4 fatty acid chain (myristoyl residue) has been introduced at the N and/or O l~osition of the hydroxyl amino group. We now r e p o r t on the :mutagenicity of these lipophilic myristoyl derivatives in V79 Chinese hamster cells and compare these results with those on their carcinogenicity, electrophilicity, mutagenicity in S. typhimurium and DNA repair ~nduction acth4ty. The mutagenicity assay with V79 Chinese hamster cells has been shown to be efficient in detecting many carcinogens, both direct acting and those requiring metabolic activation [ 6,7,8 ]. MATERIALS AND METHODS
Chemicals The synt;hesis and characl;erization o f N-AcO-AAF, N-AcO-MyAF, N-MyO-AAF and N-MyO-MyAF (for structures se~: Fig. 1) b.ave previously been described []!]. DMSO (spectr0,grade) was a product of Merck Co., Darmstadt, G.F.R. 8-Azaguanine was purchased from PfaItz and Bauer Inc., Flushing, New York, USA.
Mu tagenicity assay The protocol for induction of A Z A r mutants in V79 Chinese hamster ceils was adapted from Chu et al. [2] and Huberman et al. [4]. The cells were grown in Eagle MEM {autociavable, from Flow Laboratories, Irvine, United Kingdom}, 8uppleraented with 10% fetal calf serum (Grand Island Biological 0
0
.....,/t>..,o,C:CH,8 N--Ac0-AAF
~_.~...,~.~/ ":)-,c,-Cc.,lo"C., N - MyO- AAF
O
O
.8.rc.,l,,c., 8
N - A cO- M'~A]L"
o N - M y 0 - M yAF
Fi,~{.1. Chernie',~lstruct-res of 4 hydro×amic esters.
69 Co., G r a n d Island, New Y o r k , U S A ) , a t 37°C in a n a t m o s p h e r e o f 5% CO= in air. T h e V 7 9 cells were p l a t e d at 100 c e l l s / 6 0 m m dish to d e t e r m i n e p l a t i n g e f f i c i e n c y (4 dishes for each p o i n t ) a n d a t 2 × 104 c e l l s / 6 0 m m dish t o i n d u c e A Z A r (8 dishes f o r each p o i n t ) . O n t h e f o l l o w i n g d a y , t h e cells, in 4 ml o f c u l t u r e m e d i u m , were t r e a t e d for 3 h w i t h t h e t e s t c o m p o u n d s , w h i c h were a d d e d as a s o l u t i o n o f 2 0 #l o f d i m e t h y l s u l f o x i d e ( D M S O ) u n d e r s u b d u e d light. A f t e r 2 d a y s ' expression period, 2 0 ~ g / m l o f 8 - a z a g u a n i n e were a d d e d . T h e m e d i u m c o n t a i n i n g 8-azaguanine was c h a n g e d every 2--3 days, for a t o t a l o f 4 times. C u l t u r e s were fixed a n d s t a i n e d w i t h G i e m s a a t 7 days, for p l a t i n g e f f i c i e n c y , a n d a t 12 days for A Z A r. T h e m u t a t i o n f r e q u e n c y was c a l c u l a t e d p e r 105 survivors, t a k i n g i n t o a c c o u n t t h e n u m b e r of cells plated a n d t h e p l a t i n g efficiency. T h e results r e p o r t e d were o b t a i n e d r e p r o d u c i b l y in 2--3 i n d e p e n d e n t e x p e r i m e n t s . The h e r i t a b i l i t y o f A Z A r a n d t h e sensitivity o f these c o l o n i e s t o a m e d i u m c o n t a i n i n g 10 p M h y p o x a n t h i n e , 3.2 ~M a m i n o p t e r i n e , 0.1 ~ M t h y m i d i n e a n d 100 ~M glycine was previously d e m o n s t r a t e d f o r 10 i s o l a t e d A Z A r colonies [ 8 ] . RESULTS
AND
DISCUSSION
Table 1 summarizes the data on the cytotoxicity and mutagenicity of N - A c O - A A F , N - A c O - M y A F , N - M y O - A A F a n d N - M y o - M y A F in V 7 9 Chinese TABLE 1 CYTOTOXICITY AND MUTAGENICITY OF HYDROXAMIC ESTERS IN V79 CHINESE HAMSTER CELLS Compound
Concentration
Plating efficiencya
Mutation frequency b
(~M) DMSO N-AcO-AAF
N-AeO-MyAF
(0.5%) 4 8 12 16 5 i0
N-MyO-AAF
N-MyO-MyAF
85.6 ± 56.8 ± 25.5± 6.7 ± 1.3± 89.9 ± lb0.4 ±
20
~
50 5 10 20 50 6.3 12.5 25.0 d
89.0± 73.1 ± 75.1± 71.5± 44.6 = 82.8 ~+ 83.5 ± 78.5 +-
~±
16.2 c 2.3 1.3 2.3 1.5 4.1 5.4
1.4 *. 1.8 c 8.8 + 3.3 86.8± 6.9 93.7-+ 42.0 350.4± 192.0 1.7 ± 1.2 3.0~+ i.i
6.6
0.6 ±
0.6
8.1 5.5 ~.8 3.7 3.9 1.1 2.4 7.8
0.6± 1.7 = 2,5± 7.0_* 15.5 ± 0.8 +0 0
0.8 1.1 1.2 2.3 4.3 0.8
a Obtained by plating 100 ceils, mean values ± S.E. of 4 dishes. b Number of AZA r colonies/10 ~ survivors, mean values ± S.E. of 8 dishes. c Mean values ± S.D. in 4 independent experiments. d Limit of solubility.
70 hamster cells i]~ the absence of a metabolic activation system. Concentrations of N-AcO-AAF above 8 ~M were highly to~:ic and mutagenie, inducing A ZA r colonies in V79 Chinese hamster cells; this is in accord with published data [5]. N-MyO-AAF also showed toxicity and mutagenicity in V79 cells, but to a lesser extent: a concentration o£ 50/aM reduced surviving cell fractions to 60% and induced 15 AZA r colonies per 10 s survivors, a mutation frequency 10 times higher than that of the solvent control (Table 1). A linear region in the dose-response curve was obtained with concentrations of N-MyO-AAF up to 50/JM. Neither mutagenicity nor cytotoxicity was observed with N-AcO-MyAF or N-MyO-MyAF when they were tested at concentrations o f up to 50 t~M or 25 uM, respectively (Table 1). Experiments using hig~,er concentrations were hampered by the limited solubility of these compounds. In Table 2, the carcinogenicity, electrophil~city, mutagenicity in S. typhimurium strains TA98 and TA1538 and in V79 Chinese hamster ceils, and DNA repair induction activity of the 4 hydroxarnic esters ~ e compared qualitatively. After their subcutaneous injection into rats, N-AcO-MyAF, N-MyO-AAF and N-MyO-MyAF produced local sarcomas within 5-'7 months, the tumour incidence in 18 animals being 90~100%. In the same experiment, N-AcO-AAF was tess carcinogenic than any of the other estecs; t~us the presence of 1 or 2 myristoyl groups in a molecule considerably increvses its carcinogenicity [1 ]. All of the esters listed in Table 2 showed electrophilic reactivity towards methionine, N-AcO-AAF being the most and N-MyO-MyAF the least reactive [ 1 ]. Each of these ester~ also induced unscheduled incorporation of tritiated thymidine in non-dividing, cultured, human fibroblasts and thus caused DNA lesions that led to repair synthesis. Ma=c:iraal repair synthesis (expressed as grains/nucleus) was iLndueed by N-AcO-MyAF and N-MyO-MyAF at concentrations greater than 100 uM [ 1 ] , while N-AcO-AAF and N-MyOTABLE 2 COMPARATIVE CARCINOGENIC~TY,ELECTROPHILICITY, MUTAGENICITYIN S. typhit,~um TABLE 2
COMPARATIVE CARCINOGENICITY,ELECTROPHILICITY, MUTAGENICITY~N S. TYPHIMURIUM AND IN V79 CHINESE IIt.~vISTERCELLS, AND DNA REPAIR INDUCTION ACTIVITY OF 4 HYDROXAlVtlCESTERS~ Ester
CarcinogenicElectrophilic Mutagenicin in rats to methionine S. typhimurium
Unscheduled DNA synthesis in V79 human fibroblasts
N-AcO-AAF N-AcO-MyAF N-MyO-AAF N-MyO-MyAF
+ + + +
+
+ + + +
+ + -
÷
+ + + +
a Data on carcinogenieity, electrophilieity and mutagenicity in S. typhiraurium are from Bartsch et al. [ 1 J.
71 AAF were effective at concentrations of 20 and 50 uM, respectively [1,10] ; only the latter 2 compounds were mutagenic in V79 cells. Both of the N-acetoxy derivatives, N-AcO-AAF and N-AcO-MyAF, were mutagenic per se for S. typhimurium strains TA98 and TA1538, whereas neither of the N-myristoyloxy derivatives, N-MyO-AAF and N-Myo-MyAF, produced any revertants [1]. N-AcO-AAF and N-MyO-AAF were both mutagenic in V79 Chinese hamster cells, but the latter was negative in S. typhimurium. It remains to be investigated whether the lack of mutagenicity of some of these esters can be attributed to their high lipophilicity, which may prevent their approach to the DNA of target cells. The data show a general, qualitative correspondence between the induction of unscheduled DNA synthesis, the electrophilicity and the carcinogenicity of these esters. However, correlations of these activities with mutagenicity were poor in view of the lack of mutagenicity of N-AcO-MyAF and N-MyO-MyAF in V79 Chinese hamster cells and of N-MyO-AAF and N-MyO-AAF in S. typhimurium. Furthermore, there was no good quantitative correlation between the carcinogenic activity of these esters and their effects in vitro: although N-AcO-AAF was the least active carcinogen, it was the most active in assays for electrophilicity, mutagenicity in V79 Chinese hamster cells and induction of DNA repair synthesis. In view of these false-negative results (carcinogenic chemicals that are not detected as mutagens}, obtained in 2 mutagenic assays, the need for raultiple short-term tests in the prediction of potential carcinogenic activity is further stressed. ACKNOWLEDGEMENTS The esters were kindly supplied by Professors J.A. and E.C. Miller, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin, USA. V79 Chinese hamster cells were obtained from Dr. E. Huberman, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA. The authors thank Miss J. Mitchell for secretarial assistance, Mrs E. Heseltine for editorial aid, and Drs J.A. Miller, E.C. Miller, L. Tomatis and R. Montesano for the critical reading of this manuscript. This work was supported in part by the US NCI Contract No. 1CP-55630. REFERENCES 1 Bartsch, H., Malaveille,C., Stich, H.F., Miller, E.C. and Miller, J.A. (1977) Comparative electrophilicity, mutagenieity, DNA repair induction activity and careinogenieity of some N- and O-acyl derivativesof N-hydroxy-2-amir~ofluorene.Cancer Res., 37, 1461-1467. 2 Chu, E.H.Y. and Mailing,H.V. (1965) Mammaliancell genetics. IL Chemical induction of specific locus mutations in Chinesehamster cells in vitro. Proc. Natl. Aead. Sci. USA, 61, 1306--1312. 3 DeBaun,J.R., Miller, E.C. and Miller, J.A. (1970)N.Hydroxy-2-aeetyiaminofluorene sulfotransferase its probable roJe in carcinogenesisand in protein-(methie-s-yl) binding in rat liver. Cancer Re$., 30, 577--595.
72 4 Huberman, E., Aspiras, L., Heidelberger, C., Grover, P,L. and Sims, P. (1971) Mutagenicity ~o mammalian cells of epoxides and other derivatives of polycyctic hydro~ carbons~ Proc. Natl. Acad. Sci. USA, 68, 3195--3199. 5 Huberman, E., Donovan, P.J. mad DiPaolo, J.A. (1972) Mutation and tra.n~formation of etfltured mammalian cells by N-aceto×y-N-2-fluorenylaeetamide. J. Nat]. Cancer Inst.~ 48,837--840. 6 Huberman, E. and Bachs, L. (1974) Cell-medlated mutagenesis of mammalian cells with chemical carcinogens. Int. J. Cancer, 13,326--333. 7 Krahn, D.F. and HeidelberFjer, C. (1977) Liver homogenate-mediated mutagenesis in Chinese hamster V79 celts by polycyclic hydrocarbons and aflatoxins. Murat. Res., 46, 27--44. 8 Kuroki, T., Drevon, C. and Montesano, R. ('t977) Microsome-mediated mutagenesis in V79 Chine.,~ehamster cells by various nitrosamines. Cancer Res., 37, 1044--1050. 9 Miller, J.A. (1970) Carcinogenesis by chemicals: an overview. G.H.A. Clowes Memorial Lecture. Cancer Res., 30,559--576. 10 Stieh, H.F., San~ R.H.C., Miller, J.A. and Miller, E.C. (1972) Various levets of DNA repair synthesis in xeroderma pigmentosum celts exposed to the carcinogens N-hydroxyand h'-aeetoxy-g-acetylaminofluorene.Nature New Biol., 238, 9--10.
67
MUTAGENICITY OF SOME N- AND O-ACYL DERIVATIVES OF N-HYDROXY-2-AMINOFLUORENE IN V79 CHINESE HAMSTER CELLS
T. KUROKI* and H. BARTSCH
Unit o f Chemical Carcinogenesis, International Agency for Research on Cancer, 150 tours Albert T?~ornas, 69372 Lyon, Cedex 2 (France)
(Received 7 June 1978) (Revised version received 2 October 1978) (Accepted .q October 1978)
SUMMARY
Mutagenicity of 4 N- and O-acyl derivatives of N-hydroxy-2-aminofluorene (acyl : acetyl or myristoyl residue) was examined in V79 Chinese hamster cells in the absence of a metabolic activation system. N-Myristoyloxy-N-acetyl-2aminofluorene (N-MyO-AAF) was toxic and weakly mutagenic, inducing 8-azaguanine-resistaat (AZAr) mutants in V79 Chinese hamster cells in a concentration-dependent fashion; while N-acetoxy-N-myristoyl-2-aminofluorene (N-AcO-MyAF) and N - m y r i s t o y l o x y - N - m y r J s t o y l - 2 - a m i n o f l u o r e n e (N-MyOMyAF) were neither cytotoxic nor mutagenic. Under the same conditions, N-acetoxy-N-acetyl-2-aminofluorene (N-AcO-AAF) was highly toxic and mutagenic. Neither of the 2 N-myristoyloxy derivatives was mutagenic in S a l m o n e l l a t y p h i m u r i u m . These esters have been reported to produce local turnouts at the site of their injection in rats, to be electrophilic towards methionine, and to induce unscheduled DNA synthesis in cultured human fibroblasts. In view of the fact that some of the esters were mutagenic in neither S. t y p h i m u r i u m nor V79 Chinese hamster ceils, our findings emphasize the need for multiple short-term tests in predicting potential carcinogenic activity of chemicals. INTRODUCTION Metabolic activation of 2,-acetylaminofluorene, a hepatocarcinogen in male *To whom correspondence is tn be addressed, Present address: Department of Cancer Cell Research, Institute of MedicalScience, University
of Tokyo, Shirokanedai, Minato-ky,Tokyo 108, Japan. Abbrev&tions: AZAr, 8-azaguanine-resistance;DMSO, dimetbylsulfoxide; N-AcO-AAF, N-aeetoxy.N.acetyl.2-aminofluvrene; N-AcO-MyAF, N-acetoxy-N-myristoyl-2-arninofluorene:, N-MyO-AAF,N.myristoyloxy-N.acetyl.2-aminofluorene; N-MyO-MyAF,N-myristoyloxy-N-myristoyl-2.aminofluorene.
68 rats, requires a 2-step activatiorL process, involving/V-hydroxylation and subsequent esterific-'ltion of the hydroxyl group by soluble sulfotransferases in the liver [9]. The resulting metabolite, N-sulfonoxy-N-acetyl-2.aminofluorene, binds covalently to Cellular constituents [3] ; such esters, e.g., N-AcO-AAF, are regarded:as ultimate carcinogens. A previous paper [ 1 ] described the carcinogenicity, electrophilicity and m utagenicity of such derivative~ of N:hydroxy-2aminofluorene in which a C~4 fatty acid chain (myristoyl residue) has been introduced at the N and/or O l~osition of the hydroxyl amino group. We now r e p o r t on the :mutagenicity of these lipophilic myristoyl derivatives in V79 Chinese hamster cells and compare these results with those on their carcinogenicity, electrophilicity, mutagenicity in S. typhimurium and DNA repair ~nduction acth4ty. The mutagenicity assay with V79 Chinese hamster cells has been shown to be efficient in detecting many carcinogens, both direct acting and those requiring metabolic activation [ 6,7,8 ]. MATERIALS AND METHODS
Chemicals The synt;hesis and characl;erization o f N-AcO-AAF, N-AcO-MyAF, N-MyO-AAF and N-MyO-MyAF (for structures se~: Fig. 1) b.ave previously been described []!]. DMSO (spectr0,grade) was a product of Merck Co., Darmstadt, G.F.R. 8-Azaguanine was purchased from PfaItz and Bauer Inc., Flushing, New York, USA.
Mu tagenicity assay The protocol for induction of A Z A r mutants in V79 Chinese hamster ceils was adapted from Chu et al. [2] and Huberman et al. [4]. The cells were grown in Eagle MEM {autociavable, from Flow Laboratories, Irvine, United Kingdom}, 8uppleraented with 10% fetal calf serum (Grand Island Biological 0
0
.....,/t>..,o,C:CH,8 N--Ac0-AAF
~_.~...,~.~/ ":)-,c,-Cc.,lo"C., N - MyO- AAF
O
O
.8.rc.,l,,c., 8
N - A cO- M'~A]L"
o N - M y 0 - M yAF
Fi,~{.1. Chernie',~lstruct-res of 4 hydro×amic esters.
69 Co., G r a n d Island, New Y o r k , U S A ) , a t 37°C in a n a t m o s p h e r e o f 5% CO= in air. T h e V 7 9 cells were p l a t e d at 100 c e l l s / 6 0 m m dish to d e t e r m i n e p l a t i n g e f f i c i e n c y (4 dishes for each p o i n t ) a n d a t 2 × 104 c e l l s / 6 0 m m dish t o i n d u c e A Z A r (8 dishes f o r each p o i n t ) . O n t h e f o l l o w i n g d a y , t h e cells, in 4 ml o f c u l t u r e m e d i u m , were t r e a t e d for 3 h w i t h t h e t e s t c o m p o u n d s , w h i c h were a d d e d as a s o l u t i o n o f 2 0 #l o f d i m e t h y l s u l f o x i d e ( D M S O ) u n d e r s u b d u e d light. A f t e r 2 d a y s ' expression period, 2 0 ~ g / m l o f 8 - a z a g u a n i n e were a d d e d . T h e m e d i u m c o n t a i n i n g 8-azaguanine was c h a n g e d every 2--3 days, for a t o t a l o f 4 times. C u l t u r e s were fixed a n d s t a i n e d w i t h G i e m s a a t 7 days, for p l a t i n g e f f i c i e n c y , a n d a t 12 days for A Z A r. T h e m u t a t i o n f r e q u e n c y was c a l c u l a t e d p e r 105 survivors, t a k i n g i n t o a c c o u n t t h e n u m b e r of cells plated a n d t h e p l a t i n g efficiency. T h e results r e p o r t e d were o b t a i n e d r e p r o d u c i b l y in 2--3 i n d e p e n d e n t e x p e r i m e n t s . The h e r i t a b i l i t y o f A Z A r a n d t h e sensitivity o f these c o l o n i e s t o a m e d i u m c o n t a i n i n g 10 p M h y p o x a n t h i n e , 3.2 ~M a m i n o p t e r i n e , 0.1 ~ M t h y m i d i n e a n d 100 ~M glycine was previously d e m o n s t r a t e d f o r 10 i s o l a t e d A Z A r colonies [ 8 ] . RESULTS
AND
DISCUSSION
Table 1 summarizes the data on the cytotoxicity and mutagenicity of N - A c O - A A F , N - A c O - M y A F , N - M y O - A A F a n d N - M y o - M y A F in V 7 9 Chinese TABLE 1 CYTOTOXICITY AND MUTAGENICITY OF HYDROXAMIC ESTERS IN V79 CHINESE HAMSTER CELLS Compound
Concentration
Plating efficiencya
Mutation frequency b
(~M) DMSO N-AcO-AAF
N-AeO-MyAF
(0.5%) 4 8 12 16 5 i0
N-MyO-AAF
N-MyO-MyAF
85.6 ± 56.8 ± 25.5± 6.7 ± 1.3± 89.9 ± lb0.4 ±
20
~
50 5 10 20 50 6.3 12.5 25.0 d
89.0± 73.1 ± 75.1± 71.5± 44.6 = 82.8 ~+ 83.5 ± 78.5 +-
~±
16.2 c 2.3 1.3 2.3 1.5 4.1 5.4
1.4 *. 1.8 c 8.8 + 3.3 86.8± 6.9 93.7-+ 42.0 350.4± 192.0 1.7 ± 1.2 3.0~+ i.i
6.6
0.6 ±
0.6
8.1 5.5 ~.8 3.7 3.9 1.1 2.4 7.8
0.6± 1.7 = 2,5± 7.0_* 15.5 ± 0.8 +0 0
0.8 1.1 1.2 2.3 4.3 0.8
a Obtained by plating 100 ceils, mean values ± S.E. of 4 dishes. b Number of AZA r colonies/10 ~ survivors, mean values ± S.E. of 8 dishes. c Mean values ± S.D. in 4 independent experiments. d Limit of solubility.
70 hamster cells i]~ the absence of a metabolic activation system. Concentrations of N-AcO-AAF above 8 ~M were highly to~:ic and mutagenie, inducing A ZA r colonies in V79 Chinese hamster cells; this is in accord with published data [5]. N-MyO-AAF also showed toxicity and mutagenicity in V79 cells, but to a lesser extent: a concentration o£ 50/aM reduced surviving cell fractions to 60% and induced 15 AZA r colonies per 10 s survivors, a mutation frequency 10 times higher than that of the solvent control (Table 1). A linear region in the dose-response curve was obtained with concentrations of N-MyO-AAF up to 50/JM. Neither mutagenicity nor cytotoxicity was observed with N-AcO-MyAF or N-MyO-MyAF when they were tested at concentrations o f up to 50 t~M or 25 uM, respectively (Table 1). Experiments using hig~,er concentrations were hampered by the limited solubility of these compounds. In Table 2, the carcinogenicity, electrophil~city, mutagenicity in S. typhimurium strains TA98 and TA1538 and in V79 Chinese hamster ceils, and DNA repair induction activity of the 4 hydroxarnic esters ~ e compared qualitatively. After their subcutaneous injection into rats, N-AcO-MyAF, N-MyO-AAF and N-MyO-MyAF produced local sarcomas within 5-'7 months, the tumour incidence in 18 animals being 90~100%. In the same experiment, N-AcO-AAF was tess carcinogenic than any of the other estecs; t~us the presence of 1 or 2 myristoyl groups in a molecule considerably increvses its carcinogenicity [1 ]. All of the esters listed in Table 2 showed electrophilic reactivity towards methionine, N-AcO-AAF being the most and N-MyO-MyAF the least reactive [ 1 ]. Each of these ester~ also induced unscheduled incorporation of tritiated thymidine in non-dividing, cultured, human fibroblasts and thus caused DNA lesions that led to repair synthesis. Ma=c:iraal repair synthesis (expressed as grains/nucleus) was iLndueed by N-AcO-MyAF and N-MyO-MyAF at concentrations greater than 100 uM [ 1 ] , while N-AcO-AAF and N-MyOTABLE 2 COMPARATIVE CARCINOGENIC~TY,ELECTROPHILICITY, MUTAGENICITYIN S. typhit,~um TABLE 2
COMPARATIVE CARCINOGENICITY,ELECTROPHILICITY, MUTAGENICITY~N S. TYPHIMURIUM AND IN V79 CHINESE IIt.~vISTERCELLS, AND DNA REPAIR INDUCTION ACTIVITY OF 4 HYDROXAlVtlCESTERS~ Ester
CarcinogenicElectrophilic Mutagenicin in rats to methionine S. typhimurium
Unscheduled DNA synthesis in V79 human fibroblasts
N-AcO-AAF N-AcO-MyAF N-MyO-AAF N-MyO-MyAF
+ + + +
+
+ + + +
+ + -
÷
+ + + +
a Data on carcinogenieity, electrophilieity and mutagenicity in S. typhiraurium are from Bartsch et al. [ 1 J.
71 AAF were effective at concentrations of 20 and 50 uM, respectively [1,10] ; only the latter 2 compounds were mutagenic in V79 cells. Both of the N-acetoxy derivatives, N-AcO-AAF and N-AcO-MyAF, were mutagenic per se for S. typhimurium strains TA98 and TA1538, whereas neither of the N-myristoyloxy derivatives, N-MyO-AAF and N-Myo-MyAF, produced any revertants [1]. N-AcO-AAF and N-MyO-AAF were both mutagenic in V79 Chinese hamster cells, but the latter was negative in S. typhimurium. It remains to be investigated whether the lack of mutagenicity of some of these esters can be attributed to their high lipophilicity, which may prevent their approach to the DNA of target cells. The data show a general, qualitative correspondence between the induction of unscheduled DNA synthesis, the electrophilicity and the carcinogenicity of these esters. However, correlations of these activities with mutagenicity were poor in view of the lack of mutagenicity of N-AcO-MyAF and N-MyO-MyAF in V79 Chinese hamster cells and of N-MyO-AAF and N-MyO-AAF in S. typhimurium. Furthermore, there was no good quantitative correlation between the carcinogenic activity of these esters and their effects in vitro: although N-AcO-AAF was the least active carcinogen, it was the most active in assays for electrophilicity, mutagenicity in V79 Chinese hamster cells and induction of DNA repair synthesis. In view of these false-negative results (carcinogenic chemicals that are not detected as mutagens}, obtained in 2 mutagenic assays, the need for raultiple short-term tests in the prediction of potential carcinogenic activity is further stressed. ACKNOWLEDGEMENTS The esters were kindly supplied by Professors J.A. and E.C. Miller, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin, USA. V79 Chinese hamster cells were obtained from Dr. E. Huberman, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA. The authors thank Miss J. Mitchell for secretarial assistance, Mrs E. Heseltine for editorial aid, and Drs J.A. Miller, E.C. Miller, L. Tomatis and R. Montesano for the critical reading of this manuscript. This work was supported in part by the US NCI Contract No. 1CP-55630. REFERENCES 1 Bartsch, H., Malaveille,C., Stich, H.F., Miller, E.C. and Miller, J.A. (1977) Comparative electrophilicity, mutagenieity, DNA repair induction activity and careinogenieity of some N- and O-acyl derivativesof N-hydroxy-2-amir~ofluorene.Cancer Res., 37, 1461-1467. 2 Chu, E.H.Y. and Mailing,H.V. (1965) Mammaliancell genetics. IL Chemical induction of specific locus mutations in Chinesehamster cells in vitro. Proc. Natl. Aead. Sci. USA, 61, 1306--1312. 3 DeBaun,J.R., Miller, E.C. and Miller, J.A. (1970)N.Hydroxy-2-aeetyiaminofluorene sulfotransferase its probable roJe in carcinogenesisand in protein-(methie-s-yl) binding in rat liver. Cancer Re$., 30, 577--595.
72 4 Huberman, E., Aspiras, L., Heidelberger, C., Grover, P,L. and Sims, P. (1971) Mutagenicity ~o mammalian cells of epoxides and other derivatives of polycyctic hydro~ carbons~ Proc. Natl. Acad. Sci. USA, 68, 3195--3199. 5 Huberman, E., Donovan, P.J. mad DiPaolo, J.A. (1972) Mutation and tra.n~formation of etfltured mammalian cells by N-aceto×y-N-2-fluorenylaeetamide. J. Nat]. Cancer Inst.~ 48,837--840. 6 Huberman, E. and Bachs, L. (1974) Cell-medlated mutagenesis of mammalian cells with chemical carcinogens. Int. J. Cancer, 13,326--333. 7 Krahn, D.F. and HeidelberFjer, C. (1977) Liver homogenate-mediated mutagenesis in Chinese hamster V79 celts by polycyclic hydrocarbons and aflatoxins. Murat. Res., 46, 27--44. 8 Kuroki, T., Drevon, C. and Montesano, R. ('t977) Microsome-mediated mutagenesis in V79 Chine.,~ehamster cells by various nitrosamines. Cancer Res., 37, 1044--1050. 9 Miller, J.A. (1970) Carcinogenesis by chemicals: an overview. G.H.A. Clowes Memorial Lecture. Cancer Res., 30,559--576. 10 Stieh, H.F., San~ R.H.C., Miller, J.A. and Miller, E.C. (1972) Various levets of DNA repair synthesis in xeroderma pigmentosum celts exposed to the carcinogens N-hydroxyand h'-aeetoxy-g-acetylaminofluorene.Nature New Biol., 238, 9--10.