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The covalently bound products of [3H]benzo[a]pyrene (BP) were determined in the DNA from the skin of mice and rats. The quality and the distribution of bound products was similar. The formation of products corresponding to 4,5-dihydro-4,5-epoxy benzo[a]pyrene (BPE) and to a further metabolite of 9-hydroxybenzo[a]pyrene bound to DNA were found in the 2 species at the 24 h end point.
The chemopreventive activity of chlorophyllin (CHL) was monitored by using 6-sulfooxymethylbenzo[a]pyrene (SMBP) which is an ultimate metabolite of benzo[a]pyrene (B[a]P). CHL was quite effective in reducing both cytotoxicity and mutagenicity for SMBP in dose dependent manner up to 12.5 mM CHL in Chinese hamster V79 cells. The inhibitory patterns of CHL for SMBP were also confirmed in Salmonella typhimurium strains TA98 and TA100. Mutation frequency caused by SMBP was diminished almost to a control level at a 50 nmol CHL. A similar but less effective prevention of CHL was indicated in the mammalian and the bacterial mutagenicity assays with 6-hydroxymethylbenzo[a]pyrene (HMBP). The inhibitory effect of CHL against assault of SMBP on V79 cells was found to be related to the reduced cellular uptake of SMBP and further the remarkably lowered DNA adducts.
6-Nitrobenzo[alpha]pyrene (6-NBaP) occurs in our environment. Since human exposure to environmental contaminants may occur via the inhalation route, we examined the metabolites of 6-NBaP formed in lung preparations, and compared the metabolite profile to that which was found with liver. The metabolites formed in both liver and lung preparations consisted of ring-hydroxylated 6-NBaP, 6-hydroxybenzo[a]pyrene (6-OHBaP) and small amounts of benzo[a]pyrene. In the lung experiments, 6-OHBaP most frequently oxidized to quinones. The mechanism for the formation of 6-OHBaP from 6-NBaP remains to be elucidated.
Hepatic microsomes were prepared from immature C57BL/6J mice 24 h after receiving intraperitoneal injections of either corn oil, benzo[e]pyrene (BeP, 50 mg/kg) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 4 x 10(-3) mg/kg). The capacity of these hepatic microsomes to bioactivate aflatoxin B1 (AFB1), 2-aminoanthracene (AA), benzo[a]pyrene (BaP), 3-methylcholanthrene (MC), 7,12-dimethylbenzanthracene (DMBA), BeP and pyrene (PY) was measured using strain TA100 in the Salmonella typhimurium/microsome reversion assay. BeP pretreatment of mice resulted in a 33% increase in mutagenic potency (MP) of AFB1 over the corn oil controls and a 70% increase in MP relative to TCDD-pretreated microsomes. With AA, BaP and DMBA as promutagens, BeP pretreatment reduced MP an average of 24%, while TCDD pretreatment increased MP of these 3 promutagens 263% compared to controls. Since the general effects of BeP and TCDD on murine hepatic cytochrome P-450 (P450)-mediated activities in this study were discordant, it appears that changes in P450 activity by BeP pretreatment are not mediated through the Ah receptor.
The syn- and anti-diastereoisomers of trans-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) were further metabolized by rat liver microsomes obtained from 3-methylcholanthrene(MC)-pretreated rats and NADPH to reactive intermediates, presumably 1,7,8- and 3,7,8-trihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (triol-epoxides), that bound to macromolecules or decomposed to products consistent with pentahydroxy derivatives of benzo[a]pyrene (BP-pentols). Three major metabolites of syn-BPDE and four major metabolites of anti-BPDE were isolated by high performance liquid chromatography and characterized by spectroscopic techniques. When fluorescence spectroscopy was employed all metabolites exhibited very similar spectral properties and showed substantial shifts in excitation and emission maxima to longer wavelengths when measured under alkaline conditions, consistent with the presence of a phenolic hydroxyl group. Furthermore, the spectral properties of the metabolites from syn- and anti-BPDE were similar to those of 1-hydroxypyrene. Previous data from this laboratory together with the data presented in this study thus strongly suggest that further metabolism of BPDE involves hydroxylation at the 1- and 3-positions to yield the corresponding triol-epoxides and various BP-pentols. The pentols could also be formed by incubating tetrols derived from syn- and anti-BPDE with microsomes and NADPH. However, the rate of formation of pentols from the BP-tetrols was much slower than the rate of further metabolism of BPDE. Accordingly, the major route of BP-pentol formation is likely to be via the intermediate formation of triol-epoxides. Isolated liver nuclei from MC-pretreated rats were also found to catalyze the activation of anti-BPDE in presence of NADPH to reactive intermediates. This resulted in a substantial increase in binding to histone and non-histone proteins, with a concomitant decrease in binding to DNA. No qualitative change in the distribution of DNA-bound products of anti-BPDE could be demonstrated as a result of the further metabolism of anti-BPDE.
Methods for the rapid determination of benzo(a)pyrene rest mainly on the separation of the substance by thin-layer chromatography, followed by its spectrophotometric or fluorimetric estimation. Published papers seldom state the recovery results. It is shown here that, with the aid of radioactive benzo(a)pyrene tracer and liquid scintillation spectrometry, recovery becomes poorer and less reproducible as the sample decreases in size (to less than 1 mug benzo(a)pyrene). Coupling fluorimetry with liquid scintillation spectrometry provides an easy means of reducing this common source of error.
Alkaliphilic Mycobacterium sp. strain MHP-1, which can grow on pyrene as a sole carbon and energy source, was isolated from a soil sample. At the optimum pH for growth (pH 9), about 50% of pyrene (final concentration at 0.1% [w/v]) was degraded during 7 d of incubation, and 4,5-phenanthrenedioic acid, 4-phenanthroic acid and phthalic acid were identified as metabolic intermediates. Strain MHP-1 was found to possess aromatic-ring dioxygenase genes, which are highly homologous to the known nidAB genes from pyrene-degrading mycobacteria.
Non-K-region polycyclic aromatic hydrocarbon (PAH) o-quinones represent alternative metabolites of PAH trans-dihydro diol proximate carcinogens. These PAH o-quinones react readily with glutathione and N-acetyl-L-cysteine, and these adducts may be responsible for their detoxication. Reactions between benzo[a]pyrene-7,8-dione and either N-acetyl-L-cysteine or glutathione gave three predominant products which were purified by semipreparative reverse-phase high-pressure liquid chromatography and characterized by homonuclear two-dimensional correlation spectroscopy (COSY). The first product corresponded to a Michael type, 1,4-addition product isolated at the level of quinone oxidation. The second product converted to the first and is a presumptive 1,4-addition product isolated at the level of hydroquinone oxidation. The third product was 7,8-dihydroxybenzo[a]pyrene (a hydroquinone) and was formed as a result of the reductive potential of the thiol. Additional proof for the catechol structure was obtained by its conversion to its diacetate and its identity with authentic 7,8-diacetoxybenzo[a]pyrene. The structures of these adducts and intermediates confirm that thiol addition involves formation of the ketol and rearrangement to give a catechol followed by oxidation to yield the quinone adduct. No evidence was obtained for the formation of either bisphenol or bisglutathionyl adducts. The COSY spectra provide the first complete structure of a benzo[a]pyrenyl-peptide conjugate.
Resonance energy transfer from pyrene-fatty acid containing phospholipid derivatives to the heme of cytochrome c (cyt c) was used to observe the binding of this protein to liposomal membranes. Liposomes were formed of egg yolk phosphatidic acid (PA) and either egg yolk phosphatidylcholine or dipalmitoylphosphatidylcholine with 1 mol % of the fluorescent lipid. Binding of cyt c to liposomes was monitored by measuring the decrease either in the fluorescence intensity or in the lifetime of pyrene emission. The requirement for the presence of the acidic phospholipid in the membrane for the binding of cyt c could be reconfirmed. Below 5 mol % of phosphatidic acid in the membrane, no significant attachment of cyt c to liquid-crystalline bilayers was evident whereas upon increasing the concentration of PA further the association of cyt c progressively increased until a saturation was reached at about 30 mol % of phosphatidic acid. Addition of NaCl caused the fluorescence intensity and lifetimes to return to values observed in the absence of cyt c, thus revealing the dissociation of the protein from the membrane. The pyrene-labeled phosphatidic acid derivatives PPHPA and PPDPA were quenched more effectively than the corresponding phosphatidylcholines, apparently due to the direct involvement of the acidic head group in binding cyt c. When dipalmitoylphosphatidylcholine (DPPC) with 5 mol % of phosphatidic acid was used, no binding of cyt c to the liposomes above the phase transition temperature of the former lipid could be demonstrated whereas below the transition temperature (Tm) binding did take place.(ABSTRACT TRUNCATED AT 250 WORDS)
Hemoglobin plus H2O2 oxidizes trans-stilbene to trans-stilbene oxide, cis-stilbene to cis- and trans-stilbene oxide, and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to anti-trans-7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Oxidation of cis- and trans-stilbene to the corresponding cis- and trans-epoxides proceeds exclusively with incorporation of oxygen from the peroxide. Oxidation of cis-stilbene to the trans-epoxide, however, proceeds without detectable incorporation of oxygen from the peroxide and partial incorporation of oxygen from O2. The epoxidations in which stereochemistry is conserved thus appear to involve ferryl oxygen transfer, whereas the epoxidations in which stereochemistry is inverted are proposed to involve protein-mediated cooxidation [Ortiz de Montellano, P.R., & Catalano, C.E. (1985) J. Biol. Chem. 260, 9265-9271] and possibly electron abstraction-water addition. The epoxidation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene incorporates oxygen from H2O2 and H2O but not O2. The oxidation of this substrate is thus consistent with ferryl oxygen transfer and electron abstraction but not protein-mediated cooxidation.
The arachidonic acid dependent oxidation of benzo[a]pyrene to a mixture of 3,6-, 1,6-, and 6,12-quinones has been studied by using enzyme preparations from sheep seminal vesicles. Maximal oxidation is observed at 100 microM benzo[a]pyrene and 150 microM arachidonic acid. The arachidonic acid dependent oxidation is peroxidatic and utilizes prostaglandin G2 (PGG2), generated in situ from arachidonate, as the hydroperoxide substrate. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid is equivalent to PGG2 as a hydroperoxide substrate, but hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide are much poorer substrates. Arachidonic acid dependent benzo[a]pyrene oxidation by microsomal and solubilized enzyme preparations is markedly.
The conformational properties of GpU modified with the reactive derivative of benzo[a]pyrene, (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, has been investigated utilizing circular dichroism spectroscopy. Binding of this carcinogen to the N2 of G residues in GpU resulted in the formation of four compounds (I to IV) representing two pairs of diastereoisomers. The molar ellipticity values of the modified dimers were approximately twofold higher than those of the modified guanosine monomers. These values were decreased appreciably when the spectra of the dimers were obtained at 80 degrees C or in methanol rather than at 25 degrees C in water, suggesting that under the latter conditions there is a stacking interaction between the carcinogen and the neighboring uridine residue. Based on these results, a conformation is proposed for modified GpU. It includes insertion of the benzo[a]pyrene moiety, by rotation of the modified guanine residue about its glycoside bond, coplanar to the neighboring uridine and perpendicular to the phosphodiester backbone.
The conformation of calf thymus DNA modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, which binds covalently mainly to the 2-amino group of guanosine residues, was studied. With samples in which 1.5 or 2.2% of the bases were modified, there was a slight decrease in Tm during heat denaturation and a slight increase in susceptibility to the single strand specific nuclease S1. In a DNA sample in which 4.5% of the bases were modified, there was an appreciable decrease in Tm and a marked increase in susceptibility to S1 nuclease. The kinetics of the reaction of the modified DNAs with formaldehyde provided evidence for locally destabilized regions ranging from 1 to 7 base plates, depending on the extent of modification. Alkaline and neutral sucrose gradient analyses revealed no evidence for strand breakage in the 1.5 and 2.2% modified samples, although single-strand breaks were found in the 4.5% modified samples. Taken together, these results suggest that DNA molecules containing a covalently bound benzo[a]pyrene derivative have an altered conformation characterized by small localized regions which are destabilized and easily denatured. The conformational changes associated with the covalent binding of the benzo[a]pyrene derivative to native DNA appear to be different from, and less marked, than those associated with the covalent binding of N-2-acetylaminofluorene to native DNA.
The purpose of the present study was to determine the effects of covalent binding to DNA of a reactive derivative of benzo[a]pyrene on template activity during in vitro transcription with RNA polymerase. Calf thymus deoxyribonucleic acid, modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, was transcribed with Escherichia coli DNA-dependent RNA polymerase. With increasing levels of modification, there was a progressive inhibition of transcription. The inhibition was much greater under conditions where continuous reinitiation of transcription occurred than under conditions where only one RNA chain was synthesized per initiation site. This suggested that the modified sites block the movement of polymerase along the template and prevent recycling of the enzyme. Consistent with this interpretation were analyses of RNA transcripts on sucrose density gradients which showed a progressive decrease in average RNA chain length as the extent of template modification increased. In contrast to the inhibitory effect on chain elongation, evidence was obtained that the modified DNA had an increase in the number of initiation sites for transcription. These results are consistent with separate physical studies indicating that modification of DNA by this benzo[a]pyrene derivative can induce small localized regions of denaturation.
Steady-state and time-resolved spectroscopic properties of bichromophoric species containing [Ru(bpy)(3)](2+) and pyrene (pyr) units linked together by flexible poly(ethylene glycol) chains of variable length, [Ru(bpy)(2)(bpy-pyr)](PF(6))(2) (1) and [Ru(bpy)(2)(bpy-O6-pyr)](PF(6))(2) (2), have been investigated in acetonitrile solvent. The complexes were designed with the aim of examining the intercomponent energy-transfer processes taking place after light absorption at the two chromophores and the influence of the distance separation between them; in the case of complex 2, the linking chain in the extended conformation is as long as 21 A. Direct excitation of the pyrene unit (lambda(exc) = 410 nm) results in singlet-to-singlet energy transfer (an antenna effect) to the Ru-based component, (1)pyr --> (1)MLCT, which we analyze in terms of the Förster mechanism taking place with unit efficiency. Analysis of the time-resolved pyrene fluorescence reveals that the actual center-to-center distance separation (d(cc)) between the photoactive centers changes according to a Gaussian distribution, with an average d(cc) = 13.6 A (distribution width, a = 2.8 A) and 12 A (a = 10.2 A), for 1 and 2, respectively; this is ascribed to folding of the poly(ethylene glycol) linking chain. In O(2)-free solvent at room temperature, after population of the (1)MLCT level (which takes place either because of direct excitation by using lambda(exc) > 355 nm or via the "antenna" effect) and subsequent intersystem crossing localized at the Ru center, (1)MLCT --> (3)MLCT, a triplet-triplet thermal equilibration is established which involves the physically separated centers, (3)MLCT <--> (3)pyr, with K(eq) = 11 (the energy gap between the two levels is 480 cm(-1), as determined from luminescence data obtained at 77 K). As a consequence of this equilibrium, the (3)MLCT luminescence lifetime becomes tau(Ru) approximately 9 micros both in 1 and 2, i.e., 1 order of magnitude longer than for the unsubstituted [Ru(bpy)(3)](2+) luminophore. In air-equilibrated solvent, diffusional quenching by O(2) effectively depletes the (3)pyr level and only the forward (3)MLCT --> (3)pyr energy transfer step is observed with k(en) = 4 x 10(8) and 2 x 10(8) s(-1) for 1 and 2, respectively. As briefly discussed, reasons for the high rate constants observed for the various triplet-triplet steps may be traced back to the folding properties of the linking chains.
6-O-(2-sulfonato-6-naphthyl)-gamma-cyclodextrin (1) and 6-deoxy-(pyrene-1-carboxamido)-beta-cyclodextrin (2) were prepared. Homodimerizations of 1 and 2 and heteroassociation between 1 and 2 were investigated by (1)H NMR, circular dichroism, and fluorescence spectroscopic methods. The compounds 1 and 2 form head-to-head dimers with dimerization constants of 140 +/- 50 and 270 +/- 70 M(-)(1), respectively. We also determined the association constants of 1 with beta-CD as 270 +/- 20 M(-)(1) and 2 with gamma-CD as 100 +/- 30 M(-)(1) from fluorescence and circular dichroism titration data, respectively. The heteroassociation between 1 and 2 was manifested in increased circular dichroism ellipticities of 2, downfield shift of the H-2 proton of the pyrene group of 2, and upfield shift of the H-5 proton of the naphthyl group of 1 upon mixing 1 and 2. The analysis of circular dichroism titration data of 2 with 1 gave the association constant as 9300 +/- 1600 M(-)(1). The NMR and circular dichroism spectra suggested that the naphthyl group of 1 is deeply included into the beta-CD cavity of 2, while the pyrene group of 2 is partially inserted in the gamma-CD cavity of 1 in the complex. The energy-minimized structure from molecular modeling of the complex supports this. We believe that the facile heteroassociation of two cyclodextrin derivatives having different sizes of cavity and pendant group could be utilized as a useful strategy for assembling functionalized CDs for various applications.
Two types of double-armed lariat ether derivatives having pyrene moieties at each end of two sidearms, (3x + 1)-crown-x derivatives 1 (x = 5), 2 (x = 6), and 3 (x = 4) (type A) and 3y-crown-y derivatives, 6 (y = 5) and 7 (y = 6) (type B), were synthesized, and their complexation behavior toward alkali metal and alkaline earth metal cations was examined by fluorescence spectroscopy. Pyrene excimer emission decreased accompanied by an increase in monomer emission upon metal ion complexation. This finding is ascribed to the change of the spatial distance of two pyrene rings by movement suppression of both the crown ring and one of the two sidearms based on complexation with the metal cation. The selectivity for alkaline earth metal cations was highly dependent on the fitness of the host cavity and the guest size. Although most of the fluorophores did not respond to alkali metal cations, only trans-7a containing an 18-crown-6 ring showed K(+) selectivity.
This report deals with the synthesis and the spectroscopic properties of two second generation (G2) dendrons with site-specific incorporated phenyl pyrene derivatives as solvatochromic fluorescent probes. The generations that do not carry the probe are equipped with volume dummies, pyrene moieties that do not show a solvatochromic effect. Two complementary G2 phenylene alkylene dendrons were synthesized using Suzuki-Miyaura cross coupling. Most of the reactions used in the 10-step sequence generating the target compounds proceeded in good yields. The incorporated probes can be selectively photoexcited and show solvatochromic shifts that are of the same magnitude as for the free probes in a homogeneous solvent environment. In addition to the charge-transfer fluorescence, a broad emission band is observed that is assigned to an intramolecular exciplex formation between the aryl pyrene chromophores.