William M. Baird

Effects of synthetic and naturally occurring flavonoids on benzo[a]pyrene metabolism by hepatic microsomes prepared from rats treated with cytochrome P-450 inducers

Activity-directed fractionation of Trifolium pratense resulted in isolation of the isoflavone biochanin A, a potent inhibitor of metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) in cells in culture. To determine the structural features required for maximal inhibition of cytochrome P-450 mediated metabolism of B[a]P, the inhibitory potencies of 23 flavonoids on metabolism of B[a]P to water-soluble derivatives were examined in liver S-9 homogenate from rats induced with Aroclor 1254. Flavones were much more efficient inhibitors than their corresponding isoflavone or flavanone analogs. Most flavonols were as effective inhibitors as their flavone analogs with the exception of kaempferide. Flavones with two hydroxyl or two methoxyl groups at positions 5 and 7 were the most active. Although all eight flavonoids tested effectively inhibited B[a]P metabolism by beta-naphthoflavone-induced microsomes, none were very effective inhibitors of B[a]P metabolism by phenobarbitol-induced microsomes, and only three were effective inhibitors of B[a]P metabolism by microsomes from non-induced rats. These results indicate that flavones or flavonols that contain free 5- and 7-hydroxyls are potent inhibitors of P-450 induced by beta-naphthoflavone (P-450IA1 and/or P-450IA2) and may potentially be useful as chemopreventive agents against hydrocarbon-induced carcinogenesis.

Photochemical covalent binding of urocanic acid to polynucleic acids

Photolysis of E-[ring-2-14C]urocanic acid (UA) with native or denatured calf thymus DNA leads to covalent binding of the radiolabel to the nucleic acid. A similar observation is made upon photolysis of the labeled UA with the polyribonucleotides, in which case a strong preference is observed for binding to poly[U]. DNA or poly[U], which had been reacted with UA and purified by dialysis and multiple precipitations, releases UA upon further irradiation with 254 nm light (as expected for cyclobutane adducts). Quantum efficiencies for binding of the UA to native DNA have been measured at 308 and 266 nm and are 0.30 x 10(-5) and 1.3 x 10(-4), respectively, at comparable reactant concentrations. The large increase at the shorter wavelength (where DNA absorption is more competitive) is taken as evidence for the primary role of a DNA excited state in initiating the binding reaction(s).

Identification of Dibenzo[a,l]pyrene-DNA Adducts Formed in Cells in Culture and in Mouse Skin

Abstract The DNA adducts formed from the potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in cultures of Sencar mouse embryo cells and a mouse keratinocyte cell line and in Sencar mouse epidermis in vivo were analyzed by 35S−phosphorothioate postlabeling, immobilized boronate chromatography and reverse-phase HPLC. The results demonstrate that the major DNA adducts result from the activation of DB[a,l]P to DB[a,l]P-11,12-diol-13,14-epoxide. Adducts from both the syn− and anti−isomers of this diol epoxide are formed in cells in culture, but in mouse epidermis only anti−isomer adducts are detected.

UROCANIC ACID PHOTOBIOLOGY. PHOTOCHEMICAL BINDING TO CALF THYMUS DNA

Abstract— Urocanic acid (UA) has previously been shown to photochemically react with N, N‐dimethylthymine and with phage G4 single‐stranded DNA. In this study, E‐[ring‐2‐14C]‐U A and calf thymus DNA have been irradiated with UV light (Λ > 270 nm) in buffered, aqueous solutions. Rc‐isolation of the DNA indicates covalent binding of UA at levels of up to 80 nmol UA/mg DNA. Binding is observed for both native and heat denatured DNA. Equilibrium dialysis studies give no evidence for complexation of UA to either form of DNA in the dark. Enzymatic cleavage of the UA bound DNA and analysis by HPLC shows peaks for l4C‐labelled products with retention volumes identical to those of a marker mixture prepared by irradiating UA with [3H‐methyl]thymidine. Photolysis of the DNA before irradiation with U A leads to the formation of a second product with a retention volume corresponding to that for a deoxyadenosine/UA marker.

Effects of synthetic and naturally occurring flavonoids on metabolic activation of benzo[a]pyrene in hamster embryo cell cultures

Biochanin A, an isoflavone, has previously been shown to inhibit the metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) to metabolites that bind to DNA in hamster embryo cells and are mutagenic in Chinese hamster V79 cells. To determine the structural features required for this activity and to attempt to find more effective inhibitors, a series of synthetic and naturally occurring flavonids were tested for their ability to modulate B[a]P metabolism in hamster embryo cell cultures. The observed structure-activity relationships indicate that the structural features of flavonoids important for effective inhibition of B[a]P metabolism in hamster embryo cells are the presence of two hydroxyl, two methoxyl, or methyl and hydroxyl substituents at the 5- and 7-positions and a 2,3-double bond. Flavones are slightly better inhibitors of B[a]P metabolism than the corresponding isoflavones. A substituent at the 4-position is not essential for inhibition of B bdP metabolism. The presence of a hydroxyl group at position 3 slightly enhances activity. Apigenin, acacetin and kaempferide are effective inhibitors of B[a]P-induced mutagenesis in a hamster embryo cell-mediated V79 cell mutation assay. However, apigenin is cytotoxic at the inhibitory dose, whereas acacetin and kaempferide are not. These results suggest that acacetin and kaempferide are promising candidates for in vivo testing as potential chemopreventive agents.

Temperature-induced alterations in the metabolic activation of benzo[a]pyrene to DNA-binding metabolites in the Bluegill fry cell line BF-2

Abstract Many carcinogenic chemicals such as the environmental pollutant benzo[ a ]pyrene (B[ a ]P) require metabolic activation to reactive DNA-binding intermediates in order to induce cancer formation. To determine whether the temperature of incubation affects the proportion of B[ a ]P metabolized to DNA-binding metabolites as well as the amount of B[ a ]P metabolized, cultures of the Bluegill ( Lepomis macrochirus ) fry cell line BF-2 maintained at 23°C and 35°C were exposed to [ 3 H]B[ a ]P. Exposure at 35°C resulted in an increase in the amount of B[ a ]P metabolized by 61% at 24 h compared with cultures incubated at 23°C. The amount of B[ a ]P-9,10-diol present in the medium was similar at both temperatures, but the amount of water-soluble metabolites was greater in the cultures maintained at 35°C. The amount of B[ a ]P bound to DNA in cultures exposed to B[ a ]P at 35°C was 97% greater at 24 h and 61% greater at 48 h than in cultures exposed at 23°C. The increase in the major B[ a ]P-deoxyribonucleoside adduct, (+)- anti -B[ a ]P-7,8-diol-9,10-epoxide (B[ a ]PDE; the isomer with the epoxide and benzylic hydroxyl on opposite faces of the molecule)-deoxyguanosine at 35°C, was 314% and 100% at 24 h and 48 h, respectively. There was no significant change in the amount of syn -B[ a ]PDE (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule)-deoxyguanosine. These results indicate that although the amount of B[ a ]P metabolized by BF-2 cell cultures is 60% greater at 35°C than at 23°C, the amount of B[ a ]P bound to DNA through the (+)- anti -B[ a ]PDE increased by more than 300%. Thus increased temperature can increase the proportion of B[ a ]P metabolized to an ultimate carcinogenic metabolite in BF-2 cells in culture.

PHOTOLYTIC COVALENT BINDING OF INDOLEACRYLIC ACID TO DNA

Abstract— E‐β‐IndoI‐3‐ylacrylic acid (IA), radiolabeled at the 2 position with l+C, and calf thymus DNA have been irradiated with UV light (Λ > 280 nm) in phosphate buffer (pH 7.0). Re‐isolation of the DNA indicates covalent binding of IA at levels of up to 295 nmol IA/mg native DNA (0.097 IA/ base). Binding is observed for both native and heat denatured DNA, but is more efficient with the latter. Quantum efficiencies of 2.60 times 10−5 and 2.30 times 10 −4 mol IA bound to native DNA/mol photon absorbed have been measured at 308 and 266 nm, respectively. Studies with the four polyribonucleotides indicate a strong preference for binding to poly[U]. Photolysis of either untreated or enzymatically degraded labeled native DNA with 254 nm light leads to the reformation of IA, and a 2 + 2 photocy‐cloadduct of IA and thymidine has been isolated and characterized and matched by HPLC to a DNA derived adduct. Equilibrium dialysis studies provide no evidence for preassociation of IA to DNA.

Photochemical covalent binding of p-methoxycinnamic acid to calf thymus DNA

Abstract Irradiation of E- p -[α- 14 C]methoxycinnamic acid and calf thymus DNA with uv light leads to incorporation of the radiolabel into the DNA. Sephadex chromatography confirms that the label incorporation is due to covalent bond formation. Photobinding is more efficient with denatured vs native DNA and with higher frequency light, e.g., overall quantum efficiencies at 308 nm for denatured and native DNA are 1.1 and 0.6 × 10 −5 , respectively, vs 22.0 and 8.0 × 10 −5 for 266 nm, respectively (values for 0.7 m M pMCA and ca. 0.7 mg/ml DNA). A comparative study with several polyribonucleotides shows the relative selectivity for binding to be poly(C) > poly (A) ⪢ poly(G). Photolysis of labeled DNA with 254-nm light leads to the release of both isomers of pMCA, evidence that at least a portion of the covalent binding is due to the formation of cyclobutane adducts. Equilibrium dialysis studies give no evidence for preassociation of pMCA and DNA.

Distribution of urethane and its binding to dna, rna, and protein in sencar and balb/c mice following oral and dermal administration

Urethane produces threefold more skin papillomas when administered orally than dermally in SENCAR mice, a strain susceptible to tumorigenesis. To better understand the relation of distribution to the initiation stage, [14C]urethane (0.10 mg/kg, 2.5 muCi/25 g) was administered orally and dermally to male SENCAR and BALB/c mice. Absorption of urethane was greater in the first hour in SENCAR mice by both routes, as indicated by more label in the liver, lung, and stomach than found in these tissues in BALB/c mice. These differences were not observed at later time periods after oral administration. Following dermal application, higher levels were maintained in the liver, lungs, and stomach through 48 h in the SENCAR mice when compared to BALB/c mice. Binding of [14C]urethane (0.062 mg/g body weight, 20 microCi/20 g body weight) to DNA, RNA, and protein 6 h after oral administration varied with tissue (liver greater than stomach greater than skin = lung) but did not differ with strain. Binding to DNA in skin, lung, and stomach, RNA in stomach, and protein in stomach and liver after 48 h were significantly higher in SENCAR mice than in BALB/c mice. Dermal application of [14C]urethane resulted in severalfold higher binding to liver DNA of SENCAR mice than BALB/c mice, but DNA binding was comparable in other tissues after 6 h. At 48 h after dermal application, significantly higher levels of [14C]urethane remained bound to skin DNA, RNA, and protein in BALB/c mice, although all values were lower than at 6 h after treatment. Differences in the distribution and binding of urethane probably do not account for the discrepancies in tumor sensitivity. Liver DNA hydrolysates were examined after 48 h. Thin-layer chromatography showed little incorporation of the 14C into the normal deoxyribonucleotide or deoxyribonucleoside bases, and no modified bases were apparent. Radioactivity was present in the fraction that remained at the origin and was consistent with a dinucleotide fragment resistant to phosphodiesterase cleavage, such as a phosphotriester.

UROCANIC ACID PHOTOBIOLOGY. PHOTOCHEMICAL BINDING OF UROCANIC ACID TO BOVINE SERUM ALBUMIN

Abstract— The photolysis of E‐[ring‐2‐14C]urocanic acid with bovine serum albumin (BSA) leads to incorporation of radiolabel into the protein. Levels up to 68 nmol mg−1 BSA have been observed if the irradiation is carried out in an inert atmosphere. Oxygen reduces the level slightly. It appears from gel‐filtration chromatography that the label incorporation is covalent in nature.