The bacterial mutagenic response (Ames-assay, Salmonella typhimurium strain TA98 ± S9-mix) of a series of monocyclopentafused polycyclic aromatic hydrocarbons (CP-PAHs) identified in combustion exhausts, viz. cyclopenta[cd]pyrene (1), acephenanthrylene (2), aceanthrylene (3) and cyclopenta[hi]chrysene (4), is re-evaluated. The mutagenic effects are compared with those exerted by the corresponding partially hydrogenated derivatives, 3,4-dihydrocyclopenta[cd]pyrene (5), 4,5-dihydroacephenanthrylene (6), 1,2-dihydroaceanthrylene (7) and 4,5-dihydrocyclopenta[hi]chrysene (8). It is shown that the olefinic bond of the externally fused five-membered ring of 1, 3 and 4 is of importance for a positive mutagenic response. In contrast, whilst CP-PAH 2 is found inactive, its dihydro analogue (6) shows a weak metabolism-dependent response. The importance of epoxide formation at the external olefinic bond in the five-membered ring is substantiated by the bacterial mutagenic response of independently synthesized cyclopenta[cd]pyrene-3,4-epoxide (9), acephenanthrylene-4,5-epoxide (10), aceanthrylene-1,2-epoxide (11) and cyclopenta[hi]chrysene-4,5-epoxide (12). Their role as ultimate, active mutagenic forms, when CP-PAHs 1, 3 and 4 exhibit a positive mutagenic response, is confirmed. Semi-empirical Austin Model 1 (AM1) calculations on the formation of the CP-arene oxides (9-12) and their conversion into the monohydroxy-carbocations (9a-12a and 9b-12b) via epoxide-ring opening support our results. For 2 and 4, which also possess a bay-region besides an annelated cyclopenta moiety, the calculations rationalize that epoxidation at the olefinic bond of the cyclopenta moiety is favoured.

Cyclopenta[cd]pyrene (1) and its congeners dicyclopenta [cd,mn]-(2), dicyclopenta [cd,fg]-(3), dicyclopenta [cd,jk]pyrene (4), which were all identified as constituents of combustion exhausts, as well as their partially hydrogenated derivatives 3,4-dihydrocyclopenta[cd]-(5), 1,2,4,5-tetrahydrodicyclopenta[cd,mn]-(6), 5,6,7,8-tetrahydrodicyclopenta[cd,fg]- (7) and 1,2,6,7-tetrahydrodicyclopenta[cd,jk]pyrene (8), were assayed for mutagenicity in the Salmonella typhimurium strain TA98 using different concentrations of microsomal protein in the metabolic activation system (S9-mix, with S9-fraction from liver of Aroclor-1254-treated rats: 2, 4 and 10% (v/v), respectively). Whereas a positive mutagenic response is found for 1-4 in the presence of S9-mix, 5-8 exert no mutagenicity either with or without S9-mix. Since for 1-4 the highest response is observed with S9-mix 2% (v/v) instead of the standard 4% (v/v), a one-step activation pathway, i.e. epoxidation of the five-membered ring olefinic bonds, appears to be operational. Surprisingly, 3 and, to a lesser extent, 2 (11.7 versus 4.2 His revertants/nmol) also give a positive response in the absence of S9-mix. Hence, 2 and 3 are expected to contribute to the direct-acting mutagenicity of the non-polar fraction of combustion exhausts. Presumably for the direct-acting mutagenicity one-electron transfer processes play a role in bioactivation. The experimental observations are supported by semi-empirical AM1 calculations on the possible ultimate metabolites, i.e. mono-epoxides (2a-4a), cis-di-epoxides (2b-4b) and trans-di-epoxides (2c-4c) and the related mono-hydroxy carbocations (2d-4d and 2e-4e), and the radical anions 1 •− -4 •− .

To rationalize the high bacterial mutagenic response recently found for the (di-) cyclopenta-fused pyrene congeners, viz. cyclopenta[cd]-(1), dicyclopenta[cd,mn]-(2), dicyclopenta[cd,fg]-(3) and dicyclopenta [cd,jk]pyrene (4), in the presence of a metabolic activation mixture (S9-mix), their (di-)epoxides at the externally fused unsaturated five-membered rings were previously proposed as the ultimate mutagenic active forms. In this study, cyclopenta[cd]pyrene-3,4-epoxide (5) and the novel dicyclopenta[cd,mn]pyrene-1,2,4,5-di-epoxide (6), dicyclopenta[cd,fg]pyrene-5,6,7,8-di-epoxide (7) and dicyclopenta[cd,jk]pyrene-1,2,6,7-di-epoxide (8) were synthesised from 1 to 4, respectively, and subsequently assayed for bacterial mutagenicity in the standard microsomal/histidine reverse mutation assay (Ames-assay with Salmonella typhimurium strain TA98). The di-epoxides 6-8 are present as a mixture of their cis-and trans-stereo-isomers in a close to 1:1 ratio ( 1 H NMR spectroscopy and ab initio IGLO/III//RHF/6-31G** calculations). The direct-acting mutagenic activity and the strong cytotoxicity exerted by 5-8 both in the absence or presence of an exogenous metabolic activation system (±S9-mix) demonstrate that the ultimate mutagenic active forms are the proposed (di-)epoxides of 1-4.

Complete 1 H and 13 C NMR assignments of the (di-)cyclopenta-fused pyrene congeners, cyclopenta[cd]-(2), dicyclopenta[cd, fg]-(3), dicyclopenta[cd,jk]-(4) and dicyclopenta[cd,mn]pyrene (5), respectively, are achieved using two-dimensional (2D) NMR spectroscopy. The experimental 13 C chemical shift assignments are compared with computed ab initio CTOCD-PZ2/6-31G** 13 C chemical shifts; a satisfactory agreement is found. Substituent-induced chemical shifts in the pyrene core induced by annelation of cyclopenta moieties are discussed. Effects of dicyclopenta topology on electronic structure are illustrated for 3-5.

Cyclopenta [cd]fluoranthene (1) and 3-ethynylfluoranthene (2) have both recently been identified in combustion exhausts. In this study, their mutagenic activities were compared to that of fluoranthene (3), one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in combustion exhausts, in the Salmonella/microsome reversion assay (Ames assay) using S. typhimurium strain TA98. The mutagenicity of 1 was modest in comparison to other active cyclopenta PAHs. Unexpectedly, 2 was mutagenic both with and without exogenous metabolic activation (rat liver S9). Furthermore, cyclopenta[cd]fluoranthene-3,4-epoxide (6) was synthesized in order to evaluate its role as the ultimate mutagenic active form of 1. The epoxide 6 was a direct-acting mutagen. In addition, a pyrolysate containing a mixture of 1 (85%), 2 (2%), and 3 (13%) obtained by flash vacuum thermolysis of 3-(1-chloroethenyl)fluoranthene (2a) at 1,050°C was also mutagenic, but a significant mutagenic response was detected only in the presence of S9 activation. The results of this study indicate that 1 and 2 can contribute to the mutagenic activity of combustion exhausts. Environ. Mol. Mutagen. 44: 304 -312, 2004.