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Biologically active aromatic amines derived from carcinogenic polycyclic aromatic hydrocarbons: synthesis and mutagenicity of aminobenzo[α]pyrenes
Mutation Research, 94 (1982) 13-21
13
Elsevier BiomedicalPress
Biologically active aromatic amines derived from carcinogenic polycyclic aromatic hydrocarbons: synthesis and mutagenicity of aminobenzo[a ]pyrenes Peter P. Fu, Robert H. Heflich, Daniel A. Casciano, Agnes Y. Huang, W. M. Trie, Fred F. Kadlubar and Frederick A. Beland National Cancerfor ToxicologicalResearch, Foodand Drug Administration, Department of Health and Human Sert~ices,Jefferson, A R 72079(U.S.A.)
(Received31 July 1981) (Accepted21 October 1981)
gumm~_ry
The mutagenicities of 6-aminobenzo[a]pyrene (6-NH2-BP), 4-, 11- and 12-NH 2BP, and two N,N-diacetyl derivatives (4- and 12-N(Ac)2-BP ) were compared to that of the parent compound, BP, and to the aromatic amine, 2-aminofluorene (AF), in the Ames' Salmonella typhimurium assay. In the presence of an $9 activating system all the compounds were mutagenic in strains TA100, TA98 and TA1538, but not in TA1535. The general order of mutagenic potency in TA98 and TA1538 was 4-NH 2-BP > 4-N(Ac)2 -BP > 12-NH 2 -BP > 12-N(Ac) 2 -BP > AF > 11-NH 2 -BP -~ BP > 6-NH2-BP; whereas in strain TA100, the order was 4-NH2-BP > 4-N(Ac)2-BP > BP > 12-NH~-BP ~- 12-N(Ac)2-BP ~- I 1-NH2-BP > 6-NH 2-BP -~ AF. Inclusion of the deacylase inhibitor, paraoxon, in the incubation decreased the mutagenicity of 4-N(Ac)2-BP but had no effect on its primary amine. These data suggest that, at least for this group of compounds, arylamines derived from carcinogenic polycyclic aromatic hydrocarbons are activated to potent mutagens primarily through S9mediated metabolism (e.g., N-oxidation) of the amine.
Mechanistic studies with aromatic amines have been a major area of research in chemical carcinogenesis. These investigations have focused primarily on arylamines derived from non-carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as fluorene, naphthalene, anthracene and phenanthrene (reviewed in Kriek and Westra, 1979; Kriek, 1979). Little is known about the biological activities of aromatic amines which are formed from carcinogenic PAHs, such as benzo[alpyrene (BP). With 0027-5107/82/0000-0000/$02.75 © ElsevierBiomedicalPress
14
compounds of this type, multiple bioactivation pathways are possible. Depending upon the location of the amine function, they may behave like carcinogenic PAHs, or alternatively, these compounds may be metabolically activated as arylamines. K-region ..,,.,~].~2 ~ ~i~,,~y~.~'7:2 '
8[ ~ ' ~ ~ K _ 7 6 $ BP
4-NH2-BP
II.NIt2oBp
region
NN,~ 6.NH2.Bp
|2°NN~-BP
Fig. I. Structures of BP and NH2-BPs used for mutagenicity studies.
Multiple routes of activation present, the possibility that amine derivatives of carcinogenic PAHs may have a spectrum of biological activity in vivo different from that of the parent PAH. A relatively high correlation has been found between mutations formed in the Ames' Salmonella typhimurium assay system and the carcinogenicity of a wide variety of compounds (reviewed in McCann et al., 1975). As a first step in an investigation of arylamines derived from carcinogenic PAHs, we have synthesized and compared the mutagenicities of 6-amino-BP (6-NH2-BP; Fig. 1), three K-region NH2oBPs (4-, 11- and 12-NH2-BP), and two N, Nodiacetyl derivatives (4- and 12-N(Ac)2-BP) in the Ames' tester strains TA1535, TA1538, TA98 and TAI00 to that of.the parent compound, BP, and the aromatic amine, 2-aminofluorene (AF). The N H 2-BPs were found to exhibit a broad range of mutagenic activity, which may be due to differences in their ability to undergo metabolic activation.
Materials and methods
Chemicals All solvents were reagent grade and purified prior to use. N-Bromosuccinimide, polyphosphoric acid, sodium cyanide, BP and AF were purchased from Aldrich Chemical Co. (Milwaukee, WI). P/roclor 1254 was purchased from the Monsanto Co. (St. Louis, MO). Nicotinamide adenine dinucleotide phosphate (NADP) and glucose
15 6-phosphate were obtained from Sigma Chemical Co. (St. Louis, MO), and glucose6-phosphate dehydrogenase was purchased from Calbiochem-Behring (LaJolla, CA).
Synthesis and identification of compounds l-Methylbenz[a]anthracene (1-methyl-BA), 12-methyl-BA and 5-methylchrysene were synthesized as described earlier (Fu and Harvey, 1974; Newman, 1976). 6-NHz-BP was prepared by reduction of the 6-nitro-BP according to the procedure of Fieser and Hershberg (1939). mH-NMR spectra were recorded with a Bruker WH270 spectrometer operated in the Fourier transform mode and melting-point determinations were obtained with an Engineering Ltd. electrothermal apparatus.
Bacterial mutagenicity analysis Reversion to prototrophy using Salmonella typhimurium histidine auxotrophic strains TA1535, TA1538, TA98 and TA100 was measured essentially as described by Ames et al. (1975). The mutagen, dissolved in 0.1 ml glass-distilled dimethyl sulfoxide, was added to a tube containing 2.5 ml molten top agar (0.6% agar, 0.6% NaCl, 0.05 mM L-histidine, 0.05 mM biotin), 0.1 ml of the bacterial tester strain, and 0.5 ml of 0.15 M KC1 or 0.5 ml of $9 mix. The $9 mix consisted of 6 parts of Aroclor 1254-induced rat-liver homogenate (Ames et al., 1975) and 14 parts 33 mM KC1, 5 mM glucose 6-phosphate, 4raM NADP and 8 mM MgCl 2 in 100 mM sodium phosphate buffer, pH 7.4, containing 18 U / m l glucose-6-phosphate dehydrogenase. When paraoxon was used, 32/~1 of a 1-mM solution in dimethyl sulfoxide was added (final concentration = l0/~M) before the addition of the mutagen. The contents of the tube were mixed and poured into 100-mm petri dishes containing Vogel's minimum salts agar with glucose. After the agar had solidified, the plates were inverted and incubated at 37°C for 48h in the dark. Colonies were counted manually. When the total number of colonies exceeded approximately 500/dish, an estimate of the total was made from the number of colonies found in a measured fraction of the total surface area of the dish. Bacterial mutagenicity testing was performed in two stages. The initial experiments were carried out in TA1535, TA1538, TA98 and TA100, with and without $9 activation, over a wide range of doses (0.8, 4, 20, 100 /tg/dish), using 1 or 2 dishes/dose. (Because of limited quantities of compound, 6-NH2-BP was not tested at 100/~g/dish.) Those experiments where either the chemical, the bacterial strain, or the presence or absence of $9 did not result in reversion frequencies 2-fold greater than background were considered negative. The mean background (uninduced) reversion frequencies/plate were: TA1535, 32; TA1535 (+$9), 37; TA1538, 11; TA1538 (+$9), 30; TA98, 30; TA98 (+$9), 61; TA100, 194; TA100 (+$9), 213. Those combinations that resulted in a significant reversion frequency by this criterion were re-tested in triplicate using 4 doses per chemical. The initial experiments were used to choose doses that would give a linear dose-dependent relation. ship.
16
Results
Synthesis of K-region NH 2 -BPs and their N,N-diacetyl derivatives The general method for the synthesis of three K-region NH2-BPs required only 3 steps from available methyl-BAs or methylchrysene. The procedure can be illustrated by the synthesis of 11-NH 2-BP (Fig. 2). Bromination of 1-methyl-BA with N-bromosuccinimide in CCI 4 afforded l-bromomethyl-BA in 78% yield; mp 124125°C (benzene-hexane). Reaction of 1-bromomethyl-BA with sodium cyanide gave 1-cyanomethyl-BA in 66% yield; mp 129-131 °C (benzene-hexane). This compound underwent acid-catalyzed cyclization with polyphosphoric acid to give 1 I-NH2-BP (53% yield), apparently through enolization of the imino intermediate. The 270 MHz N M R spectrum of 11-NH 2-BP was entirely consistent with the assigned structure [in acetone-dr] 8 7.74-7.78 (m,H2.8,9), 7.88 (s,Hi2), 7.86-8.03 (m,H3,4,5,7), 8.36 (m,Hl), 8.61 (s,Hr), and 8 9.87 (m,Ht0). The peak at 9.87, assigned to H~0, was strongly displaced down field (8 -- 0.52) relative to H~o of BP, while the HI~ singlet at 8 7.88 was shifted up field (8 --- 0.40), which is clear evidence for the location of the amino substituent at C~ of BP~ Cyclization of 12-cyanomethyl-BA with polyphosphoric acid similarly afforded 12-NH2-BP in 52% yield. Its N,N-diacetyl derivative was prepared by refluxing in acetic anhydride with pyridine. 4-NH 2-BP (overall yield = 26%) and its N, N-diacetyl derivative were prepared in an analogous manner from 5-methylchrysene. Mutagenicity The mutagenicities of the NH 2- and N(Ac)2-BPs were compared to BP and the aromatic amine AF in Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1538. In the absence of $9 activation only 6-NH2-BP and high doses of AF induced revertants and only in the tester strains sensitive to frameshift mutagens, TA98 and TA1538 (data not shown). In the presence of $9, all the compounds were active in TA100, TA98 and TA1538, but none were mutagenic in TA1535. The trends observed with TA1538 were similar to those found with TA98, but the magnitude of the response was slightly lower. Therefore, only the data from TA98 and TA100 will be discussed in detail.
~
~ NaCN~ PPA_~ ~ -M I ETHYL-BA I-II,ROMOMETHYLB -A -C I YANOMETHYL-~A ~
NBS
11-NH2-BP
Fig. 2. Synthetic route from l-methyl-BA to 1 I-NH2-BP. NBS, N-bromosuccinimide: PPA, polyphosphoric acid.
17
8.l
I
i
~
6.
~
~
4
~
~ -
c)2-BP
?
0 0
~ I0
~' 20
~ 6"NH2"BP 40 Dose Mutagen (nMoles/Plate)
80
Fig. 3. ~eversions ~du~ed i~ ~. z~p~imuri,m~ t r ~ T~98 b~ ~P, ~F, &, 6-, 11- ~ d 1 2 - ~ - B P , ~ d 4~ d 12-~(~)z-~P ~ ~e presenceo~ rat-Gver 59. ~ c h poim repre~em~the m e ~ s t ~ d ~ d de~ation from data obt~ed from 3 s ~ a t e expe~me~ts ~ d i~ presented ~ u~duCed revert~t~ subtracted.
The mutations induced in TA98 in the presence of $9 are shown in Fig. 3. A dose response was obtained, at least for the lower doses, for each compound examined. Throughout most of the dose range, the N, N-diacetyl derivatives were somewhat less mutagenic than their parent amines. At high concentrations, however, there was a decrease in mutagenicity for 4- and 12-NH2-BP, which was not found with the diacetyl derivatives. This may reflect a cytotoxic response to the primary amine because the highest concentration of 4-NHE-BP tested (20 nmoles/plate) was lethal to the bacteria (no background lawn). The 4-substituted analogues were clearly the most mutagenic in TA98. The general order of mutagenicity observed with these and the remaining compounds was: 4-NH2-BP > 4-N(Ac)2-BP > 12-NH 2-BP > 12-N(Ac)2-BP > AF > 11-NH 2-BP - BP > 6-NH 2-BP. In TA100, the 4-NH2-BP derivatives were again the most active (Fig. 4); and, as already observed at high doses in TA98, 4-NH 2-BP gave an apparent toxic response. Again, 4-N(Ac)2-BP proved somewhat less mutagenic than 4-NH2-BP but the two 12-substitutexi analogues were nearly equal in activity in this strain. In TA100, the general ranking of mutagenicity was: 4-NH~ -BP > 4-N(Ac)2-BP > BP > 12-NH 2-BP ~- 12-N(Ac)2-BP ~ I 1-NHE-BP > 6-NH2-BP ~- AF.
18 I
8'
t
7
4-NH2-BP
TA 100
6"
4-N{A¢}I-BP
tv~
i
~'4"
"
I o .~-~"-; 0
10
~
r~'''-''
20 40 Dose Mutagen (nMoles/Plate)
I
80
Fig. 4. Reversions induced in S. typhimurium strain TAI00 by BP, AF, 4-, 6-, l 1- and 12-NH2-BP and 4and 12-N(Ac)2-BP in the presence of rat-liver $9. Each point represents the mean-+ standard deviation from data obtained from 3 separate experiments and is presented with uninduced revertants subtracted.
To explore possible activation pathways for these N-substituted PAHs, the mutagenicities of 4-NH2-BP and 4-N(Ac)2-BP were examined in the presence of paraoxon, a deacylase inhibitor. (Fig. 5). In both strains, paraoxon had no effect on the overall activity of 4-NH2-BP. Inclusion of this inhibitor, however, decreased the mutagenicity of 4-N(Ac)2-BP by 75%.
Discussion
We hypothesized that, depending on the geometric location of the amino groups, an arylamine derived from a carcinogenic PAH would behave either like a carcinogenic PAH or a carcinogenic arylamine. For example, if the amino substituent is located in a region where detoxification through aryl-ring .hydroxylation would normally occur, then metabolism may be shifted toward regions where ring activation (i.e., epoxidation) to a mutagenic species could occur. Alternatively, if the amine moiety is situated at or near a position where metabolic activation takes place, then metabolism may be shifted toward detoxification pathways resulting in decreased
~9
2.5 ~-i
:
5 '"'
20
'
I
/~4-NH2-BP TA 98
4'
/
)c
10
/j~4-NH2-BP ÷ parooxon
/
~~..,~"~
~
..~
~ O0 ~¢
t
,
~.~,.
~
~
,
,
..
-~,-,,~
g
/ 4'
~
'
T/
0
4-NIAc)2-BP + paraoxon' , ,
2.5
,~ ,oo 4"NH2"BP + paraoxon
./
S 1O nMoles Mutagen/Plate
,~
4-.l~e,-.,
20
~8. ~. ~[~¢ ~ p~oxon on ~evers]ons ~du~ed ~n & ~ i ~ i ~ m str~ns T~9~ ~ d T ~ I ~ ~ 4-HH~-H~ ~ d 4-H(~¢)~-~P ]n the p~s~n~ o[ ra~-~ivc~Sg. ~eh ~o~nt ~ e ~ n t ~ the me~n~tand~d deviation o~ 3 d~shes~ the u ~ d u ¢ ~ reve~slo~[re~u~nCysuhtr~te& ~ss~yswere ~e~formedwith (~, ~ ) ~ d wi~out (~, A) ]0 ~M p~oxon ~, ~e top ~[ar.
mutagenicity. In addition, the geometric location of the amine will dictate whether or not it may undergo activation (i.e., N-oxidation) and participate in mutation induction. The hepatic activation of BP proceeds through two pathways to yield the DNA binding and the presumably mutagenic metabolites, 7,8-dihydroxy-9,10-epoxy7,8,9,10-tetrahydro-BP and an epoxide derivative of 9-hydroxy-BP (Shen et al., 1980); while hepatic detoxification appears to occur primarily through 3-hydroxy-BP
20 formation. This suggests that the extensive mutagenicity observed with 4-NH2-BP could be due to an inhibition of metabolism in the 3-position (detoxification) because of a peri interaction with the 4-substituent. Metabolism could then be shifted toward the terminal benzo ring where activation to mutagenic species (diol-epoxide) could occur. Analogous reasoning may be invoked to explain the lower mutagenicities of 6- and 11-NH 2-BP; metabolic oxidation would be inhibited in the 7,8-position by the 6-amine and in the 9,10-position by the 11-amino function. As a result, metabolism could shift from activation regions to detoxification by 3-phenol formation. The possible involvement of amine N-oxidation in the activation sequence also needs to be considered. Figs. 3 and 4 indicate that BP is approximately 2-3-fold more mutagenic in strain TA100 than strain TA98. AF, on the other hand, is much more mutagenic in strain TA98 than TA100. The characteristic of being a good frameshift mutagen (i.e., more mutagenic, in strain TA98) is shared by many, but not all, mutagenic aromatic amines (Scribner et al., 1979), whereas aromatic hydrocarbons tend to be more mutagenic in strain TA100 (Bartsch et al., 1980), which detects both frameshift and base-pair substitution mutagens (Belser et al., 1981). A comparison of each of the NH 2-BPs indicates that they induce nearly equal numbers of revertants in TA98 and TA100. Therefore, each of the derivatives is a better frameshift mutagen (or at least relatively more mutagenic in strain TA98) than the parent compound, BP. This result is consistent with the mutagenicity displayed by compounds whose metabolites are derived from activation of the amine portion of the molecule. As a test of this possibility, the mutagenicities of 4-N(Ac)2-BP and 4-NH2-BP were determined in the presence of the deacylase inhibitor, paraoxon (Schut et al., 1978). A substantial body of evidence indicates that arylamines and amides are bioactivated to mutagenic species through N-hydroxylation (reviewed in Miller, 1978; Kriek and Westra, 1979; Kriek, 1979). Diacetyl substitution of the amine nitrogen of 4-NH2-BP would be expected to block hydroxylation of this atom. Therefore, if the mutagenicity displayed by this compound proceeds by way of a hydroxamic acid or hydroxylamine intermediate, paraoxon should decrease the mutagenicity by blocking deacetylation of the diacetylamine and, indirectly, hydroxylation of the nitrogen. Fig. 5 indicates that paraoxon does, indeed, reduce the mutagenicity of 4-N(Ac)2-BP while it has virtually noeffect on the mutagenicity of 4-NH 2-BP. The residual mutagenicity observed with the paraoxon-diacetylamine incubations may be due to incomplete inhibition of deacetylation. Alternatively, some activation may occur through epoxidation of the hydrocarbon moiety. Elucidation of the exact mechanism will depend upon more detailed metabolic and nucleic-acid-binding experiments. Nevertheless, these studies indicate that the aminobenzo[a]pyrenes can be metabolized to potent mutagenic species which may have potential carcinogenic activity.
21
Acknowledgements We gratefully acknowledge the excellent technical assistance of J.R. Cunningham a n d G . L . W h i t e in c o n d u c t i n g t h e A m e s ' tests. W e also t h a n k D . W . M i l l e r for o b t a i n i n g t h e N M R s p e c t r a a n d C . M . P h i f e r for t y p i n g t h e m a n u s c r i p t .
References Ames, B.N., I. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 3 I, 347-364. Bartsch, H., C. Malaveille, A.-M. Camus, G. Martel-Planche, G. Brun, A. Hautefeuille, N. Sabadie, A. Barbin, T. Kuroki, C. Drevon, C. Piccoli and R. Montesano (1980) Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems, Mutation Res., 76, 1-50. Belser Jr., W.L., S.D. Shaffer, R.D. Bliss, P.M. Hynds, L. Yamamoto, J.N. Pitts Jr. and J.A. Winer (1981) A standardized procedure for quantification of the Ames Salmonella/mammalian-microsome mutagenicity test, Environ. Mutagen., 3, 123-139. Fieser, L.F., and E.B. Hershberg (1939) The orientation of 3,4-benzpyrene in substitution reactions, J. Am. Chem. Soc., 61, 1565-1574. Fu, P.P., and R.G. Harvey (1974) A convenient dehydrogenation reagent: Trityl trifluoroacetate generated in situ, Tetrahedron Left., 3217-3220. Kriek, E. (1979) Aromatic amines and related compounds as carcinogenic hazards to man, in: P. Emmelot and E. Kriek (Eds.), Environmental Carcinogenesis, Elsevier Biomedical Press, Amsterdam, pp. 143-164. Kriek, E., and J.G. Westra (1979) Metabolic activation of aromatic amines and amides and interactions with nucleic acids, in: P.L. Grover (Ed.), Chemical Carcinogens and DNA, Vol. II, CRC Press, Boca Raton, FL, pp. 1-28. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Miller, E.C. (1978) Some current perspectives on chemical carcinogenesis in humans and experimental animals: Presidential address, Cancer Res., 38, 1479-1496. Newman, M.S. (1976) Carcinogenic activity of benz[a]anthracenes, in: R.I. Freudenthal and P.W. Jones (Eds.), Carcinogenesis, Vol. I, Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis, Raven, New York, pp. 203-207. Schut, H.A.J., P.J. Wirth and S.S. Thorgeirsson (1978) Mutagenic activation of N-hydroxy-2acetylaminofluorene in the Salmonella test system: The role of deacetylation by liver and kidney fractions from mouse and rat, Mol. Pharmacol., 14, 682-692. Scribner, J.D., S.R. Fisk and N.K. Scribner (1979) Mechanisms of action of carcinogenic aromatic amines: An investigation using mutagenesis in bacteria, Chem.-Biol. Interact., 29, I 1-25. Shen, A.L., W.E. Fahl and C.R. Jefcoate (1980) Metabolism of benzo[a]pyrene by isolated hepatocytes and factors affecting covalent binding of benzo[a]pyrene metabolites to DNA in hepatocyte and microsomal systems, Arch. Biochem. Biophys., 204, 511-523.
13
Elsevier BiomedicalPress
Biologically active aromatic amines derived from carcinogenic polycyclic aromatic hydrocarbons: synthesis and mutagenicity of aminobenzo[a ]pyrenes Peter P. Fu, Robert H. Heflich, Daniel A. Casciano, Agnes Y. Huang, W. M. Trie, Fred F. Kadlubar and Frederick A. Beland National Cancerfor ToxicologicalResearch, Foodand Drug Administration, Department of Health and Human Sert~ices,Jefferson, A R 72079(U.S.A.)
(Received31 July 1981) (Accepted21 October 1981)
gumm~_ry
The mutagenicities of 6-aminobenzo[a]pyrene (6-NH2-BP), 4-, 11- and 12-NH 2BP, and two N,N-diacetyl derivatives (4- and 12-N(Ac)2-BP ) were compared to that of the parent compound, BP, and to the aromatic amine, 2-aminofluorene (AF), in the Ames' Salmonella typhimurium assay. In the presence of an $9 activating system all the compounds were mutagenic in strains TA100, TA98 and TA1538, but not in TA1535. The general order of mutagenic potency in TA98 and TA1538 was 4-NH 2-BP > 4-N(Ac)2 -BP > 12-NH 2 -BP > 12-N(Ac) 2 -BP > AF > 11-NH 2 -BP -~ BP > 6-NH2-BP; whereas in strain TA100, the order was 4-NH2-BP > 4-N(Ac)2-BP > BP > 12-NH~-BP ~- 12-N(Ac)2-BP ~- I 1-NH2-BP > 6-NH 2-BP -~ AF. Inclusion of the deacylase inhibitor, paraoxon, in the incubation decreased the mutagenicity of 4-N(Ac)2-BP but had no effect on its primary amine. These data suggest that, at least for this group of compounds, arylamines derived from carcinogenic polycyclic aromatic hydrocarbons are activated to potent mutagens primarily through S9mediated metabolism (e.g., N-oxidation) of the amine.
Mechanistic studies with aromatic amines have been a major area of research in chemical carcinogenesis. These investigations have focused primarily on arylamines derived from non-carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as fluorene, naphthalene, anthracene and phenanthrene (reviewed in Kriek and Westra, 1979; Kriek, 1979). Little is known about the biological activities of aromatic amines which are formed from carcinogenic PAHs, such as benzo[alpyrene (BP). With 0027-5107/82/0000-0000/$02.75 © ElsevierBiomedicalPress
14
compounds of this type, multiple bioactivation pathways are possible. Depending upon the location of the amine function, they may behave like carcinogenic PAHs, or alternatively, these compounds may be metabolically activated as arylamines. K-region ..,,.,~].~2 ~ ~i~,,~y~.~'7:2 '
8[ ~ ' ~ ~ K _ 7 6 $ BP
4-NH2-BP
II.NIt2oBp
region
NN,~ 6.NH2.Bp
|2°NN~-BP
Fig. I. Structures of BP and NH2-BPs used for mutagenicity studies.
Multiple routes of activation present, the possibility that amine derivatives of carcinogenic PAHs may have a spectrum of biological activity in vivo different from that of the parent PAH. A relatively high correlation has been found between mutations formed in the Ames' Salmonella typhimurium assay system and the carcinogenicity of a wide variety of compounds (reviewed in McCann et al., 1975). As a first step in an investigation of arylamines derived from carcinogenic PAHs, we have synthesized and compared the mutagenicities of 6-amino-BP (6-NH2-BP; Fig. 1), three K-region NH2oBPs (4-, 11- and 12-NH2-BP), and two N, Nodiacetyl derivatives (4- and 12-N(Ac)2-BP) in the Ames' tester strains TA1535, TA1538, TA98 and TAI00 to that of.the parent compound, BP, and the aromatic amine, 2-aminofluorene (AF). The N H 2-BPs were found to exhibit a broad range of mutagenic activity, which may be due to differences in their ability to undergo metabolic activation.
Materials and methods
Chemicals All solvents were reagent grade and purified prior to use. N-Bromosuccinimide, polyphosphoric acid, sodium cyanide, BP and AF were purchased from Aldrich Chemical Co. (Milwaukee, WI). P/roclor 1254 was purchased from the Monsanto Co. (St. Louis, MO). Nicotinamide adenine dinucleotide phosphate (NADP) and glucose
15 6-phosphate were obtained from Sigma Chemical Co. (St. Louis, MO), and glucose6-phosphate dehydrogenase was purchased from Calbiochem-Behring (LaJolla, CA).
Synthesis and identification of compounds l-Methylbenz[a]anthracene (1-methyl-BA), 12-methyl-BA and 5-methylchrysene were synthesized as described earlier (Fu and Harvey, 1974; Newman, 1976). 6-NHz-BP was prepared by reduction of the 6-nitro-BP according to the procedure of Fieser and Hershberg (1939). mH-NMR spectra were recorded with a Bruker WH270 spectrometer operated in the Fourier transform mode and melting-point determinations were obtained with an Engineering Ltd. electrothermal apparatus.
Bacterial mutagenicity analysis Reversion to prototrophy using Salmonella typhimurium histidine auxotrophic strains TA1535, TA1538, TA98 and TA100 was measured essentially as described by Ames et al. (1975). The mutagen, dissolved in 0.1 ml glass-distilled dimethyl sulfoxide, was added to a tube containing 2.5 ml molten top agar (0.6% agar, 0.6% NaCl, 0.05 mM L-histidine, 0.05 mM biotin), 0.1 ml of the bacterial tester strain, and 0.5 ml of 0.15 M KC1 or 0.5 ml of $9 mix. The $9 mix consisted of 6 parts of Aroclor 1254-induced rat-liver homogenate (Ames et al., 1975) and 14 parts 33 mM KC1, 5 mM glucose 6-phosphate, 4raM NADP and 8 mM MgCl 2 in 100 mM sodium phosphate buffer, pH 7.4, containing 18 U / m l glucose-6-phosphate dehydrogenase. When paraoxon was used, 32/~1 of a 1-mM solution in dimethyl sulfoxide was added (final concentration = l0/~M) before the addition of the mutagen. The contents of the tube were mixed and poured into 100-mm petri dishes containing Vogel's minimum salts agar with glucose. After the agar had solidified, the plates were inverted and incubated at 37°C for 48h in the dark. Colonies were counted manually. When the total number of colonies exceeded approximately 500/dish, an estimate of the total was made from the number of colonies found in a measured fraction of the total surface area of the dish. Bacterial mutagenicity testing was performed in two stages. The initial experiments were carried out in TA1535, TA1538, TA98 and TA100, with and without $9 activation, over a wide range of doses (0.8, 4, 20, 100 /tg/dish), using 1 or 2 dishes/dose. (Because of limited quantities of compound, 6-NH2-BP was not tested at 100/~g/dish.) Those experiments where either the chemical, the bacterial strain, or the presence or absence of $9 did not result in reversion frequencies 2-fold greater than background were considered negative. The mean background (uninduced) reversion frequencies/plate were: TA1535, 32; TA1535 (+$9), 37; TA1538, 11; TA1538 (+$9), 30; TA98, 30; TA98 (+$9), 61; TA100, 194; TA100 (+$9), 213. Those combinations that resulted in a significant reversion frequency by this criterion were re-tested in triplicate using 4 doses per chemical. The initial experiments were used to choose doses that would give a linear dose-dependent relation. ship.
16
Results
Synthesis of K-region NH 2 -BPs and their N,N-diacetyl derivatives The general method for the synthesis of three K-region NH2-BPs required only 3 steps from available methyl-BAs or methylchrysene. The procedure can be illustrated by the synthesis of 11-NH 2-BP (Fig. 2). Bromination of 1-methyl-BA with N-bromosuccinimide in CCI 4 afforded l-bromomethyl-BA in 78% yield; mp 124125°C (benzene-hexane). Reaction of 1-bromomethyl-BA with sodium cyanide gave 1-cyanomethyl-BA in 66% yield; mp 129-131 °C (benzene-hexane). This compound underwent acid-catalyzed cyclization with polyphosphoric acid to give 1 I-NH2-BP (53% yield), apparently through enolization of the imino intermediate. The 270 MHz N M R spectrum of 11-NH 2-BP was entirely consistent with the assigned structure [in acetone-dr] 8 7.74-7.78 (m,H2.8,9), 7.88 (s,Hi2), 7.86-8.03 (m,H3,4,5,7), 8.36 (m,Hl), 8.61 (s,Hr), and 8 9.87 (m,Ht0). The peak at 9.87, assigned to H~0, was strongly displaced down field (8 -- 0.52) relative to H~o of BP, while the HI~ singlet at 8 7.88 was shifted up field (8 --- 0.40), which is clear evidence for the location of the amino substituent at C~ of BP~ Cyclization of 12-cyanomethyl-BA with polyphosphoric acid similarly afforded 12-NH2-BP in 52% yield. Its N,N-diacetyl derivative was prepared by refluxing in acetic anhydride with pyridine. 4-NH 2-BP (overall yield = 26%) and its N, N-diacetyl derivative were prepared in an analogous manner from 5-methylchrysene. Mutagenicity The mutagenicities of the NH 2- and N(Ac)2-BPs were compared to BP and the aromatic amine AF in Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1538. In the absence of $9 activation only 6-NH2-BP and high doses of AF induced revertants and only in the tester strains sensitive to frameshift mutagens, TA98 and TA1538 (data not shown). In the presence of $9, all the compounds were active in TA100, TA98 and TA1538, but none were mutagenic in TA1535. The trends observed with TA1538 were similar to those found with TA98, but the magnitude of the response was slightly lower. Therefore, only the data from TA98 and TA100 will be discussed in detail.
~
~ NaCN~ PPA_~ ~ -M I ETHYL-BA I-II,ROMOMETHYLB -A -C I YANOMETHYL-~A ~
NBS
11-NH2-BP
Fig. 2. Synthetic route from l-methyl-BA to 1 I-NH2-BP. NBS, N-bromosuccinimide: PPA, polyphosphoric acid.
17
8.l
I
i
~
6.
~
~
4
~
~ -
c)2-BP
?
0 0
~ I0
~' 20
~ 6"NH2"BP 40 Dose Mutagen (nMoles/Plate)
80
Fig. 3. ~eversions ~du~ed i~ ~. z~p~imuri,m~ t r ~ T~98 b~ ~P, ~F, &, 6-, 11- ~ d 1 2 - ~ - B P , ~ d 4~ d 12-~(~)z-~P ~ ~e presenceo~ rat-Gver 59. ~ c h poim repre~em~the m e ~ s t ~ d ~ d de~ation from data obt~ed from 3 s ~ a t e expe~me~ts ~ d i~ presented ~ u~duCed revert~t~ subtracted.
The mutations induced in TA98 in the presence of $9 are shown in Fig. 3. A dose response was obtained, at least for the lower doses, for each compound examined. Throughout most of the dose range, the N, N-diacetyl derivatives were somewhat less mutagenic than their parent amines. At high concentrations, however, there was a decrease in mutagenicity for 4- and 12-NH2-BP, which was not found with the diacetyl derivatives. This may reflect a cytotoxic response to the primary amine because the highest concentration of 4-NHE-BP tested (20 nmoles/plate) was lethal to the bacteria (no background lawn). The 4-substituted analogues were clearly the most mutagenic in TA98. The general order of mutagenicity observed with these and the remaining compounds was: 4-NH2-BP > 4-N(Ac)2-BP > 12-NH 2-BP > 12-N(Ac)2-BP > AF > 11-NH 2-BP - BP > 6-NH 2-BP. In TA100, the 4-NH2-BP derivatives were again the most active (Fig. 4); and, as already observed at high doses in TA98, 4-NH 2-BP gave an apparent toxic response. Again, 4-N(Ac)2-BP proved somewhat less mutagenic than 4-NH2-BP but the two 12-substitutexi analogues were nearly equal in activity in this strain. In TA100, the general ranking of mutagenicity was: 4-NH~ -BP > 4-N(Ac)2-BP > BP > 12-NH 2-BP ~- 12-N(Ac)2-BP ~ I 1-NHE-BP > 6-NH2-BP ~- AF.
18 I
8'
t
7
4-NH2-BP
TA 100
6"
4-N{A¢}I-BP
tv~
i
~'4"
"
I o .~-~"-; 0
10
~
r~'''-''
20 40 Dose Mutagen (nMoles/Plate)
I
80
Fig. 4. Reversions induced in S. typhimurium strain TAI00 by BP, AF, 4-, 6-, l 1- and 12-NH2-BP and 4and 12-N(Ac)2-BP in the presence of rat-liver $9. Each point represents the mean-+ standard deviation from data obtained from 3 separate experiments and is presented with uninduced revertants subtracted.
To explore possible activation pathways for these N-substituted PAHs, the mutagenicities of 4-NH2-BP and 4-N(Ac)2-BP were examined in the presence of paraoxon, a deacylase inhibitor. (Fig. 5). In both strains, paraoxon had no effect on the overall activity of 4-NH2-BP. Inclusion of this inhibitor, however, decreased the mutagenicity of 4-N(Ac)2-BP by 75%.
Discussion
We hypothesized that, depending on the geometric location of the amino groups, an arylamine derived from a carcinogenic PAH would behave either like a carcinogenic PAH or a carcinogenic arylamine. For example, if the amino substituent is located in a region where detoxification through aryl-ring .hydroxylation would normally occur, then metabolism may be shifted toward regions where ring activation (i.e., epoxidation) to a mutagenic species could occur. Alternatively, if the amine moiety is situated at or near a position where metabolic activation takes place, then metabolism may be shifted toward detoxification pathways resulting in decreased
~9
2.5 ~-i
:
5 '"'
20
'
I
/~4-NH2-BP TA 98
4'
/
)c
10
/j~4-NH2-BP ÷ parooxon
/
~~..,~"~
~
..~
~ O0 ~¢
t
,
~.~,.
~
~
,
,
..
-~,-,,~
g
/ 4'
~
'
T/
0
4-NIAc)2-BP + paraoxon' , ,
2.5
,~ ,oo 4"NH2"BP + paraoxon
./
S 1O nMoles Mutagen/Plate
,~
4-.l~e,-.,
20
~8. ~. ~[~¢ ~ p~oxon on ~evers]ons ~du~ed ~n & ~ i ~ i ~ m str~ns T~9~ ~ d T ~ I ~ ~ 4-HH~-H~ ~ d 4-H(~¢)~-~P ]n the p~s~n~ o[ ra~-~ivc~Sg. ~eh ~o~nt ~ e ~ n t ~ the me~n~tand~d deviation o~ 3 d~shes~ the u ~ d u ¢ ~ reve~slo~[re~u~nCysuhtr~te& ~ss~yswere ~e~formedwith (~, ~ ) ~ d wi~out (~, A) ]0 ~M p~oxon ~, ~e top ~[ar.
mutagenicity. In addition, the geometric location of the amine will dictate whether or not it may undergo activation (i.e., N-oxidation) and participate in mutation induction. The hepatic activation of BP proceeds through two pathways to yield the DNA binding and the presumably mutagenic metabolites, 7,8-dihydroxy-9,10-epoxy7,8,9,10-tetrahydro-BP and an epoxide derivative of 9-hydroxy-BP (Shen et al., 1980); while hepatic detoxification appears to occur primarily through 3-hydroxy-BP
20 formation. This suggests that the extensive mutagenicity observed with 4-NH2-BP could be due to an inhibition of metabolism in the 3-position (detoxification) because of a peri interaction with the 4-substituent. Metabolism could then be shifted toward the terminal benzo ring where activation to mutagenic species (diol-epoxide) could occur. Analogous reasoning may be invoked to explain the lower mutagenicities of 6- and 11-NH 2-BP; metabolic oxidation would be inhibited in the 7,8-position by the 6-amine and in the 9,10-position by the 11-amino function. As a result, metabolism could shift from activation regions to detoxification by 3-phenol formation. The possible involvement of amine N-oxidation in the activation sequence also needs to be considered. Figs. 3 and 4 indicate that BP is approximately 2-3-fold more mutagenic in strain TA100 than strain TA98. AF, on the other hand, is much more mutagenic in strain TA98 than TA100. The characteristic of being a good frameshift mutagen (i.e., more mutagenic, in strain TA98) is shared by many, but not all, mutagenic aromatic amines (Scribner et al., 1979), whereas aromatic hydrocarbons tend to be more mutagenic in strain TA100 (Bartsch et al., 1980), which detects both frameshift and base-pair substitution mutagens (Belser et al., 1981). A comparison of each of the NH 2-BPs indicates that they induce nearly equal numbers of revertants in TA98 and TA100. Therefore, each of the derivatives is a better frameshift mutagen (or at least relatively more mutagenic in strain TA98) than the parent compound, BP. This result is consistent with the mutagenicity displayed by compounds whose metabolites are derived from activation of the amine portion of the molecule. As a test of this possibility, the mutagenicities of 4-N(Ac)2-BP and 4-NH2-BP were determined in the presence of the deacylase inhibitor, paraoxon (Schut et al., 1978). A substantial body of evidence indicates that arylamines and amides are bioactivated to mutagenic species through N-hydroxylation (reviewed in Miller, 1978; Kriek and Westra, 1979; Kriek, 1979). Diacetyl substitution of the amine nitrogen of 4-NH2-BP would be expected to block hydroxylation of this atom. Therefore, if the mutagenicity displayed by this compound proceeds by way of a hydroxamic acid or hydroxylamine intermediate, paraoxon should decrease the mutagenicity by blocking deacetylation of the diacetylamine and, indirectly, hydroxylation of the nitrogen. Fig. 5 indicates that paraoxon does, indeed, reduce the mutagenicity of 4-N(Ac)2-BP while it has virtually noeffect on the mutagenicity of 4-NH 2-BP. The residual mutagenicity observed with the paraoxon-diacetylamine incubations may be due to incomplete inhibition of deacetylation. Alternatively, some activation may occur through epoxidation of the hydrocarbon moiety. Elucidation of the exact mechanism will depend upon more detailed metabolic and nucleic-acid-binding experiments. Nevertheless, these studies indicate that the aminobenzo[a]pyrenes can be metabolized to potent mutagenic species which may have potential carcinogenic activity.
21
Acknowledgements We gratefully acknowledge the excellent technical assistance of J.R. Cunningham a n d G . L . W h i t e in c o n d u c t i n g t h e A m e s ' tests. W e also t h a n k D . W . M i l l e r for o b t a i n i n g t h e N M R s p e c t r a a n d C . M . P h i f e r for t y p i n g t h e m a n u s c r i p t .
References Ames, B.N., I. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 3 I, 347-364. Bartsch, H., C. Malaveille, A.-M. Camus, G. Martel-Planche, G. Brun, A. Hautefeuille, N. Sabadie, A. Barbin, T. Kuroki, C. Drevon, C. Piccoli and R. Montesano (1980) Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems, Mutation Res., 76, 1-50. Belser Jr., W.L., S.D. Shaffer, R.D. Bliss, P.M. Hynds, L. Yamamoto, J.N. Pitts Jr. and J.A. Winer (1981) A standardized procedure for quantification of the Ames Salmonella/mammalian-microsome mutagenicity test, Environ. Mutagen., 3, 123-139. Fieser, L.F., and E.B. Hershberg (1939) The orientation of 3,4-benzpyrene in substitution reactions, J. Am. Chem. Soc., 61, 1565-1574. Fu, P.P., and R.G. Harvey (1974) A convenient dehydrogenation reagent: Trityl trifluoroacetate generated in situ, Tetrahedron Left., 3217-3220. Kriek, E. (1979) Aromatic amines and related compounds as carcinogenic hazards to man, in: P. Emmelot and E. Kriek (Eds.), Environmental Carcinogenesis, Elsevier Biomedical Press, Amsterdam, pp. 143-164. Kriek, E., and J.G. Westra (1979) Metabolic activation of aromatic amines and amides and interactions with nucleic acids, in: P.L. Grover (Ed.), Chemical Carcinogens and DNA, Vol. II, CRC Press, Boca Raton, FL, pp. 1-28. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Miller, E.C. (1978) Some current perspectives on chemical carcinogenesis in humans and experimental animals: Presidential address, Cancer Res., 38, 1479-1496. Newman, M.S. (1976) Carcinogenic activity of benz[a]anthracenes, in: R.I. Freudenthal and P.W. Jones (Eds.), Carcinogenesis, Vol. I, Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis, Raven, New York, pp. 203-207. Schut, H.A.J., P.J. Wirth and S.S. Thorgeirsson (1978) Mutagenic activation of N-hydroxy-2acetylaminofluorene in the Salmonella test system: The role of deacetylation by liver and kidney fractions from mouse and rat, Mol. Pharmacol., 14, 682-692. Scribner, J.D., S.R. Fisk and N.K. Scribner (1979) Mechanisms of action of carcinogenic aromatic amines: An investigation using mutagenesis in bacteria, Chem.-Biol. Interact., 29, I 1-25. Shen, A.L., W.E. Fahl and C.R. Jefcoate (1980) Metabolism of benzo[a]pyrene by isolated hepatocytes and factors affecting covalent binding of benzo[a]pyrene metabolites to DNA in hepatocyte and microsomal systems, Arch. Biochem. Biophys., 204, 511-523.