Final Report: Mechanism of Carcinogenesis of Thia-PAHsEPA Grant Number: R826192
Title: Mechanism of Carcinogenesis of Thia-PAHs
Investigators: Kumar, Subodh , Sikka, Harish C.
Institution: The State University of New York at Buffalo
EPA Project Officer: Hahn, Intaek
Project Period: November 3, 1997 through November 2, 2000
Project Amount: $500,827
RFA: Exploratory Research - Human Health (1997) RFA Text | Recipients Lists
Research Category: Health Effects , Human Health , Health
Exposure to a wide variety of complex chemical mixtures such as in automobile exhaust, soot, coal tar and pitch, mineral oils, shale oil, coal-gasification residues, and cigarette smoke has been considered to be associated with an increased incidence of cancer in the human population. These complex mixtures contain a number of polycyclic aromatic hydrocarbons (PAHs) and their heterocyclic analogs (especially, aza-PAHs and thia-PAHs), many of which are carcinogens. To fully understand the risk that various carcinogenic chemicals in a complex mixture may present to human health, it is important that these chemicals be studied for their mechanism of carcinogenic action. Most of the mechanistic studies conducted in the past involve predominantly PAHs, and to some extent aza-PAHs. In contrast, thia-PAHs have been studied very little for their mechanism of action in spite of the observations that these thia-PAHs represent up to 50 percent of the total hydrocarbons in some of the crude oils and emissions from coal-fired residential furnaces. In addition, thia-PAHs also are known to be more persistent, bioaccumulated, and carcinogenic compared to their homocyclic analogs (PAHs). The objective of this research project was to investigate whether thia-PAHs are activated by the mechanism similar to that of PAHs, or follow a different metabolic activation pathway(s) due to the presence of sulfur heteroatom. The purpose of the proposed research was to identify the metabolic activation pathway(s) of benzo[b]phenanthro[2,3-d]thiophene (BPT), a model thia-PAH, known to be more carcinogenic than its PAH isoster dibenz[a,h]anthracene (DBA).
The project objectives were to: (1) study the metabolism of BPT by mouse liver microsomes from induced or uninduced mice and identify the metabolites formed; (2) characterize the DNA adducts produced in mouse skin treated with BPT; and (3) assess the mutagenicity of BPT and its metabolites.
The initial phase of the project was devoted to the synthesis of BPT, its various derivatives, and tritium-labeled BPT. These compounds are required for studying the metabolism of BPT, for quantifying and identifying the metabolites formed, and for studying their mutagenic activities. Synthesis of BPT was conducted using the published procedure. Tritiation of BPT using relatively convenient tritium exchange techniques did not produce desired tritiated BPT with stable specific activity. Therefore, an alternate procedure that involved bromination of BPT, and subsequent dehalogenation of the brominated compound with tritium gas in the presence of a catalyst (Pd) was employed. This approach produced the stable tritium-labeled BPT in desired quantities.
Synthesis of BPT-3,4-diol, a putative mutagenic/carcinogenic metabolite of BPT, did not proceed as expected. Oxidative photocyclization route, which has been widely applied to the synthesis of dihydrodiol metabolites of PAHs, led to the formation of a wrong isomer. The application of Suzuki cross coupling reaction that was developed in our laboratory for the synthesis of various dihydrodiol metabolites of PAHs allowed for the development of a new and efficient regiospecific synthesis of benzo[b]naphtha[2,1-d]thiophene and its dihydrodiol derivatives. However, the extension of this approach to the synthesis of BPT-3,4-diol was not very productive because the desired synthetic intermediate, which was chromatographically inseparable from the undesired isomer in practical quantities, was the minor product. Because of the importance of BPT-3,4-diol in understanding the mechanism of carcinogenesis of BPT, this effort was continued until a successful synthesis of BPT-3,4-diol was finally achieved. Again, the synthesis was not regiospecific but the desired intermediate, 3-methoxyBPT, needed for the synthesis of BPT-3,4-diol, was easily separable in desired quantities by column chromatography. A similar approach was investigated for the synthesis of 2-hydroxyBPT and BPT-1,2-diol, which are expected to be noncarcinogenic metabolites of BPT. Unfortunately, this approach produced only a small amount of 2-hydroxyBPT, which was not sufficient to develop a successful synthesis of BPT-1,2-diol. Additional BPT derivatives synthesized during this project for identifying potential metabolites of BPT were 3-hydroxyBPT, BPT-5,6-diol, BPT sulfoxide, BPT-sulfone, benzo[b]naphtha[2,3-d]thiophene, benzo[b]naphtha[2,3-d]thiophene sulfoxide, and benzo[b]naphtha[2,3-d]thiophene sulfone.
To identify the various metabolites of BPT and to study the effect of various cytochrome P-450 inducers in their formation, the metabolism of [G-3H]BPT was investigated by liver microsomes from control and induced mice. Liver microsomes from DBA-, 3-methylcholanthrene (3-MC)-, Aroclor-1254-, phenobarbital (PB)-treated, and control mice metabolized BPT at 1.469, 0.565, 1.538, 0.121, and 0.022 nmole/mg protein/minute, respectively. Highest metabolism rate of BPT was observed with DBA (a cytochrome P-4501A1 inducer) and Aroclor-1254 (a mixed cytochrome P-450 inducer).
Liver microsomes from mice treated with various cytochrome P-450 inducers metabolized BPT to BPT phenols, BPT sulfoxide, BPT sulfone, and BPT-3,4-diol. A number of additional metabolites also were formed. The formation of some of these metabolites was inhibited in the presence of the epoxide hydrolase inhibitor TCPO, suggesting that these metabolites are BPT diols or their derivatives. Among identifiable metabolites, BPT-3,4-diol was produced as a major metabolite (15-27 percent) of BPT with all liver microsomes, except liver microsomes from PB-induced mice, which produced BPT sulfoxide (30 percent) as a major metabolite of BPT. BPT sulfone was the minor metabolite (0-6 percent) of BPT in all cases. These data suggest that different cytochrome P-450 isozymes are involved in sulfoxidation vs. 3,4-diol formation from BPT.
These studies also indicated that the incubation of BPT with calf-thymus DNA in the presence of liver microsomes from DBA-treated female mice produced at least seven adducts. Under similar condition, BPT-3,4-diol produced five distinct adducts, all of which co-chromatographed with the corresponding five of seven adducts produced by BPT. These five adducts represented nearly 80 percent of the total BPT-DNA adducts. In contrast, BPT sulfoxide produced only minor adducts that appeared to co-migrate with some of the adducts produced by BPT or BPT-3,4-diol. BPT sulfone did not produce any detectable DNA adduct(s).
In mouse skin, BPT produced six detectable DNA adducts. Five of these adducts co-migrated with the five DNA adducts produced in vitro. On the other hand, only two major DNA adducts, which represented nearly 40 percent of total adducts detected in BPT-treated mouse skin, were formed in mouse skin treated with BPT-3,4-diol. The formation of additional adducts from BPT-3,4-diol in vitro compared to those formed in vivo suggests that certain activating enzymes, required to produce reactive metabolites responsible for forming these additional adducts, are inducible in the liver of mice treated with DBA but not in mouse skin treated with BPT-3,4-diol. However, a qualitative similarity noted between BPT-DNA adducts produced in vitro and in vivo suggests that the enzymes involved in the formation of reactive metabolites responsible for producing BPT-DNA adducts are inducible by DBA and BPT in mouse liver and skin, respectively. Thus, these observations suggest that the majority of stable BPT-DNA adducts formed in mouse skin is the result of activation of BPT via BPT-3,4-diol.
The data from the mutagenicity studies have demonstrated that BPT sulfoxide is considerably more mutagenic than BPT-3,4-diol (a metabolic precursor to the bay-region diol epoxide of BPT) or BPT, and suggest a possibility for the first time that BPT is metabolically activated via the formation of both BPT-3,4-diol and BPT sulfoxide.
The data showed that BPT-3,4-diol, a precursor to the bay-region diol epoxide of BPT, is less mutagenic than BPT sulfoxide suggesting for the first time that BPT is metabolically activated to mutagenic products via BPT sulfoxide. However, the potential involvement of BPT sulfoxide in the carcinogenicity of BPT in mouse skin is not evident from the observation that the majority of the stable DNA adducts produced in vivo (mouse skin) from BPT are derived from BPT-3,4-diol rather than BPT sulfoxide. Therefore, to conclude on the importance of BPT sulfoxide in the metabolic activation of BPT and possibly other thia-PAHs, further studies are needed to demonstrate: (1) whether mutagenic BPT sulfoxide is a carcinogenic metabolite of BPT; and (2) the mechanism by which BPT sulfoxide exhibits mutagenic/carcinogenic activity.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
|Other project views:||All 8 publications||3 publications in selected types||All 3 journal articles|
||Fertuck KC, Kumar S, Sikka HC, Matthews JB, Zacharewski TR. Interaction of PAH-related compounds with the and isoforms of estrogen receptor. Toxicology Letters 2001;121(3):167-177.||
||Kumar S, Kim TY. An improved and regiospecific synthesis of trans-3,4-dihydrodiol metabolite of benzo[b]naphtho[2,1-d]thiophene. Journal of Organic Chemistry 2000;65(12):3883-3884.||
||Kumar S. Synthesis of trans-3,4-dihydroxy-3,4-dihydrophenanthro[3,2-b]benzothiophene, a potentially carcinogenic metabolite of sulfur heterocycle phenanthro[3,2-b]benzo-thiophene. Journal of the Chemical Society, Perkin Transactions 2001;9:1018-1023.||