Consistent with the literature [13
], the mutagenic potency of the non-sulfur-containing parent PAH B[c
]P was considerably less than that of its 3,4-diol, and the 5,6-diol was barely mutagenic (). A similar effect was seen for P[4,3-b
]T and its diol derivatives, where the mutagenic potency of P[4,3-b
]T 8,9-diol was greater and that of P[4,3-b
]T 6,7-diol was less than that of their parent compound P[4,3-b
]T (). The pattern for P[3,4-b
]T and its diols also was similar, except that the 8,9-diol was more mutagenic than the parental compound in TA98 and TA104 but not in TA100. These results illustrate a generally consistent and large influence of the location of the diol on mutagenic potency relative to the parent compound.
In agreement with a previous study [11
], we found that the mutagenic potency of the thia-PAHs depended on the position of the sulfur heteroatom relative to the vicinal trans
-diol group. For example, the mutagenicity of P[3,4-b
]T, where the sulfur atom is distal to the angular benzo-ring, was similar to that of its parent compound B[c
]P in TA104 but much greater than that of its parent in TA100. In contrast, the mutagenicity of P[4,3-b
]T, where the sulfur atom is proximal to the angular benzo-ring, was much less than that of B[c
]P in both strains. This observation may reflect the greater steric availability of the sulfur atom for metabolic activation when the sulfur atom is distal rather than proximal to the angular benzo-ring. The K-region diols (6,7-diols) were the least mutagenic diols and were consistently less mutagenic than their respective parent compounds (). The sulfone required metabolic activation to be mutagenic, which raises a question as to the ultimate mutagenic form of these sulfur-PAHs. As shown previously for B[c
]P and B[c
]P 3,4-diol ([10
] and for the sulfone here and elsewhere [11
], metabolic activation was required for the mutagenicity of these compounds. The other seven compounds were mutagenic in the presence of S9 mix (); however, we were unable to test them in the absence of S9 mix due to limited availability of sample. Nonetheless, based on earlier studies showing that other sulfur-PAHs required S9 mix to be mutagenic [11
], it is likely that these seven sulfur-PAHs also require S9 mix for mutagenic activity.
The most mutagenic thia-PAH, the sulfone, produced the most abasic sites and was the only compound that induced mutation at primarily GC sites and a high level of frameshift mutation. In contrast, four of the five remaining compounds that were next in mutagenic potency (i.e., the parent PAH B[c]P, its 3,4-diol, and the 8,9 diols of the two phenanthrothiophenes) all produced only stable DNA adducts resulting in mutations primarily at AT sites. The exception, P[3,4-b]T, induced mutation only at GC sites. Our finding that B[c]P 3,4-diol, which was more mutagenic than the parent B[c]P in all strains of Salmonella, produced more stable DNA adducts and was more mutagenic than its parent compound B[c]P is consistent with a diol epoxide mechanism of mutagenicity for B[c]P in Salmonella. Because P[3,4-b]T sulfone induced only abasic sites, and more than twice the levels produced by P[3,4-b]T, then the sulfone is, apparently, not a major metabolite of P[3,4-b]T. In contrast, P[3,4-b]T 8,9-diol produced more stable DNA adducts than its parent P[3,4-b]T, consistent with metabolic activation of P[3,4-b]T to the diol and then to the diol epoxide in TA104. P[3,4-b]T 8,9-diol was, however, considerably less mutagenic in TA100 than its parent compound P[3,4-b]T, suggesting that unlike B[c]P, its sulfur analogue P[3,4-b]T is metabolically activated (or metabolized to mutagenic products) by several pathways.
A preliminary metabolism study [26
] involving incubation of selected compounds with rat liver microsomes found that the 6,7-diols (K-region diols) and 8,9-diols (diols with a bay-region double bond) were formed at considerably lower levels from P[3,4-b
]T and P[4,3-b
]T compared to the levels that the analogous 5,6-diol (K-region diol) and 3,4-diol (diol with a bay-region double bond) were formed from B[c
]P. A major (80–96% of the total metabolites), relatively nonpolar metabolite(s) was formed by both P[3,4-b
]T and P[4,3-b
The formation of extremely low or undetectable amounts of P[4,3-b
]T 8,9-diol [24
], which is 10–20 times more mutagenic for base-substitution mutation than the parent P[4,3-b
]T (), is consistent with the possible involvement of a dihydrodiol pathway in the metabolic activation of P[4,3-b
]T. Thus, P[4,3-b
]T 8,9-diol is a proximate (not ultimate) mutagenic form of P[4,3-b
]T. These mutagenicity data suggest the possibility that the ultimate mutagenic form is a diol expode formed from the 8,9-diol. P[3,4-b
]T, which is highly mutagenic in TA100, is also metabolized to a lower extent than B[c
]. However, in addition to low formation, this diol is also 7 times less mutagenic (in TA100) than its parental form (P[3,4-b
]T). These observations suggest that in contrast to P[4,3-b
]T is activated via a mechanism other than a diol epoxide pathway. Collectively, these data indicate that the two isomeric forms (P[3,4-b
]T and P[4,3-b
]T), which differ only in the position of the sulfur heteroatom, are metabolically activated by distinctly different pathways, with P[3,4-b
]T not involving a diol pathway but the P[4,3-b
]T likely involving a diol epoxide pathway.
The fact that the most mutagenic compound (the sulfone) produced the most abasic sites, whereas the less-mutagenic compounds produced more stable DNA adducts, illustrates the potential importance of aldehydic lesions in the mutagenesis of thia-PAHs, as has already been shown for other PAHs [27
]. The most common model for the conversion of an abasic site to a base substitution mutation (as produced by the sulfone in TA100 and TA104) involves the “A-rule,” in which dATP is incorporated preferentially opposite an abasic site by various DNA polymerases [28
]. Additional models have been proposed for the conversion of abasic sites into frameshift mutations [29
], which also were induced at a high level by the sulfone. Our data indicate that metabolism of the sulfone by S9 mix results in a highly reactive intermediate that results in mutations preferentially at guanine, and to a lesser extent to adenine (). Recent studies indicate that the mutations initiated by abasic lesions are a consequence of the nature of the lesion, the structure of neighboring nucleotides, and the type of DNA polymerase that performs translesion synthesis [28
All three benzo-ring dihydrodiols, as well as the parent compound B[c]P, induced stable DNA adducts, and they induced mutations preferentially at AT sites rather than GC sites (). These were the only compounds to exhibit this type of site specificity for mutation, and this study clearly links the structure of benzo-ring dihydrodiols to mutation induction preferentially at AT sites. In contrast, two of the K-region dihydrodiols, B[c]P 5,6-diol and P[3,4-b]T 6,7-diol, as well as the parent compounds P[3,4-b]T and P[4,3-b]T, induced mutations only at GC sites, and they were the only compounds that exhibited this site specificity for mutation (). P[3,4-b]T induced stable DNA adducts, but a limited amount of sample prevented our assessing P[4,3-b]T for induction of stable DNA adducts. Collectively, our results begin to define the structural features of these compounds that are associated with site specificity of mutation (and, presumably, site specificity of DNA damage), as well as the nature of the lesion (stable adducts or abasic sites).
Because of their persistence and multiple pathways of bioactivation, thia-PAHs may contribute substantially to the mutagenicity of complex mixtures in which they occur. Further studies are required to clarify their metabolic pathways and the types of DNA damage and mutations they induce. In particular, studies on the carcinogenicity of selected thia-PAHs are needed to relate emerging mechanistic data to overall health effects in vivo for this important class of environmental contaminant.