title: Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system.
abstract: Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogen for benzo[a]pyrene (BP). We have demonstrated previously that the interaction between sulfite, the physiological form of sulfur dioxide, and (+/-) -7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenic form of BP, results in an enhanced mutagenic effect in Salmonella typhimurium strains TA98 and TA100. We report here that this same co-mutagenic effect of sulfite occurs in a mammalian cell line. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE, a concentration on the linear portion of the dose-response, resulted in a four-fold increase in mutations at the hprt locus relative to the spontaneous rate. When V79 cells were exposed to 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE, the mutation rate increased by 73% and 210%, respectively, over that elicited by anti-BPDE alone. Sulfite itself was moderately cytotoxic, but caused no increase in mutation over the spontaneous rate. Characterization of the dose- and time-dependance of this enhancement of diol epoxide mutagenicity by sulfite closely resembled the effects seen previously in the bacterial system. In particular, enhancement by sulfite was evident when sulfite was added to the cells between 60 min and 1 min prior to the addition of the diol epoxide. Concurrent addition of sulfite and the diol epoxide attenuated the enhancement, and the effect was lost altogether when sulfite was added 10 min after the diol epoxide. The specificity of this effect of sulfite was shown by comparison with sulfate, which at concentrations of either 1 or 10 mM exhibited modest cytotoxicity, but neither was directly mutagenic nor able to enhance the mutagenic effect of anti-BPDE. Binding studies with labeled anti-BPDE showed that the addition of 10 mM sulfite increased binding of anti-BPDE to DNA by over 43%, corresponding to the observed increase in mutant frequency. Interestingly, this difference in level of DNA modification was not apparent after 30 min to 2 h exposures, but only emerged at the 4 h time point. The 4 h point was routinely used for all mutagenicity studies. Binding of anti-BPDE-derived materials to cellular RNA was not altered by 10 mM sulfite. The emergence of increased DNA modification at the latest time point suggests either a more prolonged period of active DNA binding than would occur with diol epoxide, or a difference in the ability to recognize and clear specific DNA adducts. Both possibilities are discussed in regard to the observed formation of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a] pyrene-10c-sulfonate (BPT-10-sulfonate) in those incubations. BPT-10-sulfonate is a relatively stable BP derivative which retains the ability to covalently modify DNA. The role of this derivative in the enhancement of diol epoxide mutagenicity by sulfite is strongly suggested by these data.
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City 66160-7417, USA.
authors.forename: G A
authors.initials: GA
authors.lastname: Reed
authors.forename: B C
authors.initials: BC
authors.lastname: Jones
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism; D004357:Drug Synergism; D005978:Glutathione / Q000502:physiology; D009153:Mutagens / Q000633:toxicity*; D013447:Sulfites / Q000633:toxicity*
chemical_list: D009153:Mutagens; D013447:Sulfites; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D005978:Glutathione