Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

Mutatton Research, 117 (1983) 31-40 Elsewer Biomedical Press

31

Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles R i c h a r d A. P e l r o y a, D o r o t h y L. S t e w a r t a, Y o s h i n o r i T o m i n a g a b,,, M a s a t o m o I w a o b,**, R a y m o n d N. Castle b., a n d M i l t o n L. L e e b a Btology Department, Pacific Northwest Laboratory, Operated by Battelle Memorial Institute, Rzchlana~ WA 99352 and b Department of Chemistry, Brtgham Young Umverstty, Provo, UT 84602 (U.S.A.) (Received 27 August 1981 (Revision received to September 1982) (Accepted 5 October 1982)

Summary The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene.Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.

The biological activity of the polycyclic aromatic sulfur heterocycles (PASH) is not well characterized. Limited data suggest that larger PASH are mutagenic (Karcher et al., 1981) and carcinogerfc in laboratory animals (Tilak, 1960). In practical terms, the genetic activity of PASH is of interest because these compounds

* Present address: Department of Chenustry, University of South Florida, Tampa, FL 33620 (U.S.A.) ** Present address: Faculty of Science, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852 (Japan). 0165-1218/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

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are significant constituents of synthetic fuels. For example, coal-derived materials and shale oils both contain a variety of 2-, 3-, 4- and 5-ring PASH (Wllley et al., 1981 ; Lee et al., 1980). A major difficulty m studying the genetic activity of PASH has been the lack of standards, particularly, of higher-molecular-weight compounds. As part of a systematic program of organic synthesis in one of our laboratories, a number of stable 3- and 4-ring PASH have been synthesized (Iwao et al., 1980; Tominaga et al., 1981). This present communication deals with the mutagenicity of the stable 3- and 4-ring PASH in the Ames test system.

Methods and Materials

Mutagenests assays Agar-plate mutagenicity assays were performed as described by Ames et al. (1975) using 4 of the Ames/Salmonella test strains: Salmonella typhtmurtum TA98, TA100, TA1535 and TA1537. The mutagen phenotype defining these strains was determined in each experiment as shown in Table 1. Dimethyl sulfoxide (DMSO) was the solvent used in the bioassay work reported. In addition to the use of the mutagens (Table 1) to check the phenotypes of the Ames/Salmonella strains, fixed concentrations of 2-aminoanthracene (2-AA), benzo[a]pyrene (BaP) and a complex basic fraction from an SRC-II heavy distillate (HD) were tested against the Aroclor-induced rat-liver ($9) homogenates used for metabolic activation. Optimum levels for $9 were thus established and monitored on a daily basis during the course of the Ames experiments reported herein. Negative controls included the Ames/Salmonella test strains, with and without microsomal enzymes ($9), and appropriate solvent controls. Mutagenic response (measured as revertant colonies) was counted electromcally using a Biotran II automated colony counter (New Brunswick Scientific Co., Inc., Edison, N J). The chemicals in this study were assayed for mutagenicity at concentrations of 2,

TABLE 1 M U T A G E N P H E N O T Y P E S O F A M E S T E S T E R S T R A I N S U S E D T O B I O A S S A Y P U R E POLYCYCLIC AROMATIC SULFUR HETEROCYCLES Compound

2-Anunoanthracene 9-Anunoacridme Nltrosoguamdine 7,9-Dlmethylbenz[ c l a c n d m e

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33 4, 8, 10, 20 and 50 btg or 20, 40, 80, 100, 200 and 500 #g/petri plate. Each concentration was assayed in duplicate. Stock solutions in DMSO, containing l0 000 /~g/ml of the various PASH, were stored at - 80°C when not in use. All thiophenes were synthesized as described elsewhere (Caruthers, 1953; Caruthers and Crowder, 1957; MacDowell and Wisowaty, 1972; Clarke et al., 1973; Iwao et al., 1980; Tominaga et al., 1981).

Preparation of the hver homogenate ($9) fractions Young-adult male Sprague-Dawley rats were used for the preparation of Aroclor-induced $9 homogenates. Rats (250 g) were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before sacrifice to induce hepatic monoxygenases. On the fifth day of induction, rats were decapitated; livers removed under aseptic conditions, and placed in sterile beakers with 0-4°C KCI solution (0.15 M). The livers were washed several times in cold sterile KC1, then placed in a preweighed cold sterile blender jar. The appropriate volume of KC1 (3 m l / g wet liver) was added and blended for 30-60 sec and then homogenized in a Tekemar tissue homogenizer. The crude homogenate were centrifuged for 10 min at 9000 g. The supernatant ($9 fraction) was decanted and saved in 50-ml aliquots at - 80°C. Each 50-ml aliquot was then filter-sterlized and divided into 2-ml quantities and stored at -80°C. As required, sufficient $9 fraction was thawed (at room temperature) and then kept on ice for each experiment. For each batch of $9, the level of activity was titrated with reference promutagens (in this study, 2-AA and BaP) to find the optimum amount of $9 for general screening.

Results and discussion

3- and 4-ring PASH compounds were tested for mutagenicity in the Ames standard plate incorporation test (Ames et al., 1975) and, when sufficient material was available, in a variant of the Ames test based on a liquid pre-incubation step (Yahagi et al., 1975). All possible 3- and 4-ring isomers were synthesized and tested for biological activity, except for those compounds in which ring fusion is on side c of the thiophene ring. Fusion at this position reduces resonance stabilization of the thiophene ring portion of the molecule rendering it less stable and most likely unable to survive coal liquefaction or shale oil retorting processes. The mutagenicity for the 3-ring PASH compounds is shown in Table 2. Both linear and nonlinear condensed structures were treated. Of these compounds, only naphtho[1,2-b]thiophene was active, inducing an estimated 22 revertants/~tg against TA100 by the standard plate incorporation method. Much less mutagenicity was demonstrated against TA98 (ca. 2 revertants//~g). No mutagenesis was induced against TA 1535 or TA 1537 (data not shown). All mutagenesis was indirect, requiring metabolic activation. Naphtho[2,1-b]thiophene, which differs from the genetically active [1,2-b]isomer only in the position of the sulfur atom, showed no activity in either the standard or

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35 pre-incubation Ames tests. The 2 linear structures, naphtho[2,3-b]thiophene and dibenzothiophene were inactive against the 4 Ames tester strains used in this study, and the TA98 and TA100 in the Ames pre-incubation test (Table 2). 13 four-ring PASH were also tested for mutagenicity, as shown by the data in Table 3. Of the 13, 7 were mutagenically active. 4 of these compounds induced mutation in both TA98 and TA100. As for the 3-ring PASH, TA100 was more sensitive to induced mutation than TA98. With the exception of one compound, anthra[2,3-b]thiophene, no mutagenesis was detected with either TA1535 or TA1537. Anthra[2,3-b]thiophene induced mutagenesis in all 4 Ames tester strains used in this study. However, only data for TA98 and TA100 are shown in Table 3. All mutagenesis with the 4-ring PASH required metabolic activation. In quantitative terms, phenanthro[3,4-b]thiophene was the most mutagenic of the 4-ring PASH. This compound induced an estimated 195 revertants of TA100/#g and 29 revertants of TA98/#g in the Ames standard plate incorporation test. In comparative terms, phenanthro[3,4-b]thiophene was about as active in these Ames tests as BaP. The results of the pre-incubation tests with TA98 and TA100 were inconclusive because of extensive killing. The 3 anthrathiophenes were next in potency. For example, anthra[2,1-b]thiophene induced approximately 7 revertants of TA100/#g and less than one revertant of TA98/#g in the standard Ames test. Anthra[1,2-b]thiophene, and anthra[2,3b]thiophene (differing from the [2,1-b] isomer only in the position of the sulfur atom) induced about 3 revertants of TA100/#g and less than 0.5 revertants of TA98/#g. The TA98 responded to the anthrathiophenes tested in the pre-incubation assay better than TA100, but was less active in the standard Ames test. The least mutagenically active of the 4-ring PASH, induced mutation only in the Ames pre-incubation assay. Phenanthrol[3,2-b]thiophene induced approximately 1 revertan t/# g tested in both TA 100 and TA98. Benzo[ b ]naphtho[2,1 -b ]thiophene was active only against TA100 and induced mutations at the rate of approximately 1 revertant/gg. Although the data are limited, they suggest that both the aromatic ring system and the position of the sulfur heteroatom in the thiophene ring influence the mutagenicity of the 3- and 4-ring PASH in the Ames assay. For example, both anthrathiophenes were of roughly the same mutagenicity although the positions of their sulfur atoms were different relative to the anthracene portion of the molecule. Thus the aromatic portion of these compounds may play a more important role than the thiophene ring in determining genotoxicity. The mutagenicity of the two most active of the PASH compounds studied was apparently determined largely by the position of the sulfur atom. For example, the highly active phenanthro[3,4-b]thiophene (i.e., 195 revertants of TA100/#g) differed from the genetically inactive phenanthro[4,3-b]thiophene only in the location of its sulfur atom. Otherwise, both compounds had identical geometry, e.g., an aromatic phenanthrene fused to a thiophene ring. In exactly this same way, the 3-ring naphtho[1,2-b]thiophene (i.e., 22 revertants of TA100/#g) differed from the inactive naphtho[1,2-b]thiophene only in the position of the sulfur atom.

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40 Acknowledgement T h i s s t u d y w a s s u p p o r t e d b y the U . S . D e p a r t m e n t o f E n e r g y , O f f i c e o f H e a l t h and Environmental Research, Contract No. DE-AC02-79EV10237.

References Ames, N., T. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian rmcrosome mutagenicity test, Mutation Res., 31,347-364. Caruthers, W. (1953) 6 : 7-Benzotbaonaphthen, J. Chem. Soc., 4186-4187 Caruthers, W., and J. Crowder (1957) Ultrawolet absorption spectra of some condensed thlophene derivatives, J. Chem. Soc., 1932-1933. Clarke, J , D. Gregory and R. Scrowston (1973) Naphtho[ 1,2-b]thlophene, I. Preparation and electrophdlc substitution, J. Chem. Soc., Perkin Trans., 2956-2960. Iwao, M., M.Lee and P. Castle (1980) Synthesis of phenanthro[b]thlophenes, J Heterocycl. Chem, 17, 1259-1264. Karcher, W., A. Nelen, R. Depaus, J. van E1jk, P. Glaude and J. Jacob (1981) New results m detection, identification and mutagenic testing of heterocyclic polycyclic aromatic hydrocarbons, in: W. Cooke and A. Dennis (Eds.), Proc. 5th Syrup. Polynuclear Aromatic Hydrocarbons, Battelle, Columbus. Lee, M., C Wdley, R. Castle and C. White (1980) Separation and identification of sulfur heterocycles in coal-derived products, in: A. B.l~rseth and A. Denms (Eds.) Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects, Battelle, Columbus, pp. 59-73. MacDowell, D., and J. Wlsowaty (1972) Thlophene analogs of anthraqumone, J. Org Chem., 37, 1712-1717 Tdak, B. (1960) Carcinogenesis by thaophene lsosters of polycychc hydrocarbons. Synthesis of condensed thiophenes, Tetrahedron, 9, 76-95 Tommaga, Y, M. Lee and R. Castle (1982) Synthesis of anthra[b]ttuophenes and benzo[b]naphtho[d]thlophenes, J. Heterocyel. Chem., in press. Wdey, C, M. Iwao, R. Castle and M Lee (1981) Determination of sulfur heterocycles in coal hqmds and shale ods, Anal Chem., 52, 499-407. Yahagl, T., M Tagawa, Y. Semo,T. Matsustuma, T. Nagao, T. Sugimura and Y. Hashlmoto (1975) Cancer Lett., 1, 91-96