Santhanam Swaminathan

Xanthine oxidase-mediated mutagenicity of the bladder carcinogen 4-nitrobiphenyl

Xanthine oxidase catalyzed mutagenicity of 4-nitrobiphenyl (NBP), a dog-bladder carcinogen, was tested in Ames assay using Salmonella typhimurium TA98 strains. NBP was active as a mutagen in the parent strain TA98 which is proficient in nitroreductase, while it was inactive in the strain TA98NR which is deficient in nitroreductase. However, preincubation of NBP at 37 degrees C with NADH and commercial preparations of xanthine oxidase for 30 min resulted in a dose-dependent increase in the mutagenic activity in TA98NR. Allopurinol blocked the xanthine oxidase catalyzed mutagenicity of NBP in TA98NR and the extent of inhibition was dependent upon the concentration of the inhibitor. Rat-liver and dog-bladder cytosol preparations also enhanced the mutagenic activity of NBP in TA98NR in a dose-dependent manner. In addition, the cytosol-mediated activity was also inhibited by allopurinol, implying that the cytosolic enzyme activity might be due to xanthine oxidase. In vitro enzymatic reduction of NBP using bacterial cell lysates of TA98 and TA98NR revealed the major product of reduction to be 4-aminobiphenyl. The transient intermediates of reduction were not detected during the in vitro incubation. The reduction intermediate N-hydroxylaminobiphenyl showed direct and equal mutagenic activity in both TA98 and TA98NR, in contrast to NBP. These results suggest that N-hydroxylaminobiphenyl is generated during the preincubation of NBP with xanthine oxidase or cytosolic preparations and the former might account for the mutagenicity of NBP. Furthermore, the occurrence of such enzyme(s) in the target tissue for NBP carcinogenesis, support the hypothesis that metabolic activation of the bladder carcinogen NBP could occur within the target organ by virtue of its intrinsic metabolic potential.

Reductive metabolism of the carcinogen 4-(5-nitro-2-furyl)thiazole to 1-(4-thiazolyl)-3-cyano-1-propanone by rat liver subcellular fractions

Abstract The reductive metabolism of 4-(5-nitro-2-furyl)thiazole (NFT), a rat mammary gland and forestomach carcinogen, was examined in vitro using rat liver tissues. NFT was reduced by rat liver cytosol or microsomes on anaerobic incubation with NADPH. The stoichiometry of microsomal reduction revealed that about 3 moles of NADPH were used per mole of NFT. Gas chromatographic analysis of the reaction mixture showed a major peak with a retention time of about 4.0 min in contrast to NFT with a retention time of about 6.5 min. Catalytic hydrogenation of NFT with palladium and activated carbon yielded a product with a retention time of 4.0 min. The component corresponding with the above metabolite was isolated from chemically reduced NFT and identified as 1-(4-thiazolyl)-3-cyano-l-propanone. The metabolite derived from enzymatic reduction had chromatographic and spectral properties and a mass spectral fragmentation pattern similar to those obtained chemically. These data establish that the enzymatically derived product is identical to that obtained by chemical reduction and that it corresponds to 1(4thiazolyl)3cyano1propanone.

The Relationship Between DNA-Adduct Formation and Induction of Mutations in the Hypoxanthine-Guanine Phosphoribosyl Transferase (HGPRT) Locus in Human Uroepithelial Cells

Abstract To gain an insight on the mechanisms of mutagenesis and carcinogenesis of the human bladder carcinogen 4-aminobiphenyl (ABP) in the target uroepithelia, we analyzed the cellular DNA-adducts and mutational spectra in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene following exposure to N-hydroxy metabolites of ABP. [32P]-postlabelling analyses of DNA isolated from SV-40 immortalized human uroepithelial cells (HUC) exposed to N-hydroxy-4-aminobiphenyl derivatives revealed N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-ABP) as the major adduct and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-ABP) as a minor adduct. The adduct profiles of the HUC-DNA obtained at different periods after carcinogen exposure showed that dG-ABP was repaired rapidly while dA-ABP persisted in HUC cultures. DNA sequence analyses of thioguanine resistant mutants of HGPRT showed base deletions (G, bp. 599; A, bp. 608; and A,T bp. 672–673). These results imply that dG-ABP and dA-ABP are repaired at different rates a...

Human urinary bladder epithelial cells lacking wild-type p53 function are deficient in the repair of 4-aminobiphenyl–DNA adducts in genomic DNA

The effect of the tumor suppressor gene TP53 on repair of genomic DNA damage was examined in human urinary bladder transitional cell carcinoma (TCC) cell lines. Utilizing TCC10 containing wild-type p53 (wt-p53) as the parental line, an isogenic set of cell lines was derived by retroviral infection that expressed a transdominant mutant p53 (Arg --> His at codon 273, TDM273-TCC10), or the human papilloma virus 16-E6 oncoprotein (E6-TCC10). 32P-postlabeling analyses were performed on DNA from TCC cultures obtained after treatment with N-hydroxy-4-aminobiphenyl (N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP). The major adduct was identified as N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) with all three chemicals. The amount of adducts in urothelial DNA ranged between 0.1 and 20 per 10(6) nucleotides, N-OAc-AABP yielding the highest levels, followed by N-OH-ABP and N-OH-AABP. To determine, if the functional status of p53 affects the rate of repair of dG-C8-ABP in genomic DNA, TCC10 and the TDM273-TCC10 and E6-TCC10 isotypes were exposed to N-OH-AABP for 12h and the DNA damage was allowed to repair up to 24h. The adduct levels were quantified and compared between the TCC10 isotypes. The amounts of dG-C8-ABP that remained in genomic DNA from E6-TCC10 and TDM273-TCC10 were approximately two-fold higher, as compared to the parental TCC10. At the dose used for DNA repair studies, N-OH-AABP or N-OAc-AABP did not induce apoptosis in TCC10. However, N-OAc-AABP at high doses (>5 microM) induced apoptosis, as evidenced by DNA fragmentation analyses. Furthermore, N-OAc-AABP-mediated apoptosis was independent of the functional status of wt-p53, since both E6-TCC10 and the parental TCC10 exhibited DNA fragmentation following treatment. These results suggest that p53 might modulate the repair of DNA adducts generated from the human bladder carcinogen ABP in its target human uroepithelial cells. This implies that in p53 null cells the unrepaired DNA damage could cause accumulation of mutation, which might contribute to increased genomic instability and neoplastic progression.

Deacetylation of 4-(5-acetylamino-2-furyl)tiazole and formation of 1-(4-thiazolyl)-3-cyano-1-propanone by rat liver tissues☆

Abstract The reductive metabolism of the rat carcinogen 4-(5-nitro-2furyl)thiazole (NFT) to 1-4-thiazolyl)-3-cyano-1-propanone (TCP) is reported. Formation of TCP from NFT involved furan ring fission. This could have occurred through involvement of either aminofuran or N -hydroxylaminofuran as precursors. To examine if 4-(5-amino-2-furyl)thiazole is a precursor for TCP, a stable model compound, 4-(5-acetylamino-2-furyl)thiazole (AAFT), was prepared and subjected to enzymatic deacetylation, using rat liver tissue homogenates. AAFT was synthesized by catalytic hydrogenation of NFT with 5% palladium on activated carbon, followed by acetylation with acetic anhydride. AAFT, a white crystalline powder, melted at 168–170°, had an extinction coefficient of 17.9 mM −1 cm −1 at 293 nm in ethyl acetate, and exhibited spectroscopic and mass spectral characteristics consistent with the assigned structure. Incubation with rat liver 10,000 g supernatant preparations resulted in the biotransformation of AAFT as evidenced by a decrease in absorption at 290 nm. Incubation of 14 C-labeled AAFT followed by extraction with chloroform-diethyl ether (1:1) resulted in the recovery of a major portion (56%) of the radioactivity in the organic phase when the label was at the 2-position of the thiazole ring, while the major amount (82%) of radioactivity was recovered in the aqueous phase when the 1- 14 C-acetyl group was labeled. The radioactivity from the aqueous phase was extractable into the organic phase following acidification to pH 1, an observation consistent with deacetylation. Furthermore, the deacetylation product exhibited a mass spectrum, and retention times in gas and high pressure liquid chromatography, similar to those of synthetic TCP. These data establish 4-(5-amino-2-furyl)thiazole, derived from AAFT by deacetylation, as a precursor for TCP.

CHARACTERIZATION OF CYTOCHROME P450-DEPENDENT AND GLUTATHIONE TRANSFERASE ACTIVITIES IN SV40-IMMORTALIZED UROEPITHELIAL CELL LINES: POSSIBLE ROLE IN TRANSFORMATION AND TUMOR FORMATION

An in vitro/in vivo transformation system has been developed as a model for bladder tumorigenesis. SV40-immortalized human uroepithelial cells are exposed to putative carcinogens and then implanted into athymic nude mice to testfortumorigenesis.Studieswith4-aminobiphenyl(4-ABP)demonstratedthat one cell line, SV-HUC-PC, was sensitive to chemical-induced transformation and another line, SV-HUC-BC, was refractory.Weare currently testing this system as a model to identify occupational carcinogens and develop biomarkers of exposure and effects of exposure. As part of this study, we examined P450- dependent metabolism, glutathione transferase, and the effects of chemicals on deoxyribonucleic acid(DNA)synthesisandrepair inSV-HUC-PC andSV-HUCBC. Activities for CYP1A1/1A2, CYP3A, and CYP2B1/2B2 were estimated by determining o -dealkylation of ethoxy-, benzoxy-, and pentoxy-resorufin, respectively. Coumarin hydroxylase and p -nitrophenol hydroxylase were used to estimate CYP2A and CYP2E1, respectively. SV-HUC-PC microsomes had fivefold greaterCYP1A1/1A2activityandtwofoldhigherCYP3AactivitythanSV-HUCBC. CYP2B1/2B2 and CYP2A activities and glutathione transferase were not different between the two cell lines. DNA synthesis and repair, by BrdU incorporation, was not different between the two lines when N-methyl-N-nitroN-nitrosoguanidine (MNNG) or other reactive metabolites were tested; however, SV-HUC-PC was more sensitive to n -nitrosodimethylamine, 4-ABP, and 4,4-methylene bis (2-chloroaniline) (MOCA). The data demonstrate that, while these cells have retained form-specific P450 activities, SV-HUC-PC has greater CYP1A1/1A2 and CYP3A activities.