Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA involves attack at the N7-position of guanine moieties

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE) with DNA prelabelled with [14C] and [3H]-purine precursors has indicated that in addition to the N2-position of guanine previously reported [10--12] reaction also involves the N7-position of guanine. The hydrocarbon-N7-guanine product was not detected earlier because it is lost from the DNA very readily at pH 7. The same N7-product was obtained by reaction of anti-BPDE with guanine in dimethylformamide.     

Comparison of six rabbit liver cytochrome P-450 isozymes in formation of a reactive metabolite of acetaminophen

This laboratory has recently reported the isolation of an ethanol-inducible form of rabbit liver microsomal cytochrome P-450, designated isozyme 3a. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, all of which are present in significant amounts in the liver microsomes from rabbits chronically administered ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective, and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a (or a homologous enzyme in other species) may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.     

Interaction of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with relaxed circular pBR322 DNA

The interaction of (+/-)-BPDE (1) with DNA at neutral pH was investigated by the application of relaxed circular pBR322 DNA. (+/-)-BPDE causes a rapid positive supercoiling of this DNA followed by a slower spontaneous relaxation. The results indicate that there are two clearly discernible types of chemical interactions between 1 and DNA, a rapid intercalative covalent binding and a slower strand breakage. The implications of these findings are discussed.     

A spectrofluorimetric investigation of calf thymus DNA modified by BP diolepoxide and 1-pyrenyloxirane

7β,8α-Dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP diolepoxide, $\text{1}$) and 1-pyrenyloxirane ($\text{2}$) bind chemically to calf thymus DNA. The fluorescence efficiency of pyrenyl groups in mutagen modified DNA varies appreciably with its conformation and decreases in the order: pyrenees, modified denatured DNA and modified native DNA. A particularly interesting observation is that the fluorescence efficiency of mutagen modified DNA intensifies substantially upon denaturation. Our results suggest that the pyrenyl groups in mutagen modified DNA are intercalated between the base pairs of DNA. Since both $\text{1}$ and $\text{2}$ are powerful frame-shifting mutagens for S. typhimurium TA-98, the intercalative covalent binding of these compounds to DNA may provide a molecular basis for their mutagenic activity.     

Bakuchalcone,a dihydrofuranochalcone from the seeds of Psoralea corylifolia

A new dihydrofuranochalcone has been identified in seeds of Psoralea corylifolia and its structure confirmed by synthesis.     

Simulating structural and thermodynamic properties of carcinogen-damaged DNA

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.     

Alkaloid and lignan constituents of Cinnamosma madagascariensis

A new lignan glycoside, 5-methoxy-9-β-xylopyranosyl-(-)- isolariciresinol and two indole alkaloids have been characterised from the bark of Cinnamosma madagascariensis.     

Fluorescence study of DNA-complexes formed after metabolic activation of benzo(a)pyrene derivatives

Benzo(a)pyrene derivatives (1-, 2-, 3-, 7-, and 9-hydroxy-benzo(a)pyrene and trans-9,10-dihydro-9,10-dihydroxy-, -4,5-dihydro-4,5-dihydroxy-, and -7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene) were metabolized by liver microsomes isolated from 3-methylcholanthrene-treated rats in the presence of calf thymus DNA. The isolated DNA was then assayed by fluorescence for bound metabolic products. Only 2-hydroxy-benzo(a)pyrene, 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene yielded detectable amounts of DNA-bound products. In contrast to the product(s) from 9-hydroxy-benzo(a)pyrene, the metabolites of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene, both strong carcinogens, had similar excitation spectra and gave considerably increased fluorescence intensities when the DNA was denatured. These data indicate structural similarities in the DNA complexes formed after metabolic activation of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene.     

Stereoselective metabolism of the optical isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene to bay-region diol epoxides by rat liver microsomes

Enantiomerically pure isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene have been obtained by chromatographic separation of their diastereomeric bis esters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. Liver microsomes from control rats, as well as rats treated with phenobarbital or 3-methylcholanthrene, metabolize these dihydrodiols to a pair of diastereomerically related bay-region 1,2-diol-3,4-epoxides in which the benzylic hydroxyl group and the epoxide oxygen are either cis (isomer-1) or trans (isomer-2) to each other. In general, diol epoxide-1 was the major metabolite of the (+)-(1S,2S)-dihydrodiol, whereas diol epoxide-2 was the major metabolite of the (?)-(1R-2R)-dihydrodiol. The extent of this stereoselectivity is dependent on the source of the microsomes and is greatest for liver microsomes from 3-methylcholanthrene-treated rats; the ratio of diol epoxide-1 relative to diol epoxide-2 was 5.6 : 1 with the (+)-enantiomer as substrate and 1 : 5.5 with the (?)-enantiomer as substrate. For a given microsomal preparation, rates of metabolism were independent of the enantiomer composition of the substrate. Relative to microsomes from control animals, treatment of rats with 3-methylcholanthrene enhanced rates of metabolism by about 40%, whereas treatment with phenobarbital decreased rates to a similar extent when the amounts of metabolites formed per nanomole of cytochrome P?450 were compared. The failure of treatment by 3-methylcholanthrene to enhance markedly the rate of metabolism of a polycyclic aromatic hydrocarbon substrate is unusual.     

Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture

In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 μg/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 μg/ml of 7,8 benzoflavone(BF).Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones.One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the ±(trans)-7α, 8β-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (±)-7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro-BaP (diolepoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8.The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.     

A new type of biological chemiluminescence: The microsomal chemiluminescence of benzo[a]pyrene arises from the diol epoxide product of the 7,8-dihydrodiol

The chemiluminescence, CL, accompanying the metabolism of the carcinogen benzo[a]pyrene, BP, by the aryl hydrocarbon hydroxylase system is a new type of low intensity biological chemiluminescence. It is the result of spontaneous oxygenation of a specific reactive metabolic intermediate; not inhibitable by superoxide dismutase or catalase. The reactive metabolite is the strongly mutagenic 7,8-dihydrodiol-9,10-epoxide, produced enzymatically from the 7,8-dihydrodiol precursor. Hydroxylation of benzo[a]pyrene at the 3 position does not lead to chemiluminescent emission; the CL quantum yields of BP and 3-OH-BP are the same. The CL quantum yields of microsomal metabolism of (?) 7,8-diol-BP and the racemic 7,8-diol-BP are identical. The kinetics of CL of the latter show a much faster initial reaction rate, correlating with the greater reactivity of diol epoxide I formed from (+) 7,8-diol-BP. CL may therefore be used to follow the pathways and the rates of production of the mutagenic diol epoxides of BP.     

Isoflavonoids from Myroxylon balsamum

Myroxylon balsamum (Leguminosae-Lotoideae) trunk wood contains a series of biogenetically related flavonoids, including the novel (±) -7-hydroxy-4′-methoxyisoflavanone, (±)-7,3′-dihydroxy-4′-methoxyisoflavanone and 2-(2′,4′-dihydroxyphenyl)-5,6-dimethoxybenzofuran.     

S1 nuclease sensitivity to cis- and trans-diamminedichloroplatinum(II) modified DNAS: influence of (G+C) content and nucleotide sequence

The sensitivity of S1 nuclease to cis- and trans-(NH3)2PtCl2 modified DNAs is examined as a function of the level of cis- and trans-(NH3)2PtCl2 bound, the % (G+C) content in DNA from different sources and the sequence dependence in poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). The extent of DNA digested increases with increasing levels of either isomer and is inversely influenced by the % (G+C) content of the DNA. However, the difference in the extent of digestion between the cis-and trans-(NH3)2PtCl2 modified DNAs at equivalent levels of bound isomer follows the order, calf-thymus greater than M. lysodeikticus greater than poly(dG-dC).poly(dG-dC). While there is virtually no difference in the digestion profiles for poly(dG-dC).poly(dG-dC) modified with the two isomers, there is a striking difference in the extent of digestion between cis- and trans-(NH3)2PtCl2 modified poly(dG).poly(dC). These results are discussed in light of the possible modes of binding for cis-(NH3)2PtCl2, previously reported findings on modified DNA and possible implications for modifications in cellular chromatin.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.     

Resolution and Synthesis of (S)-1-(2-Naphthyl)ethanol with Immobilized Pea Protein: As a New Biocatalyst

(S)-1-(2-Naphthyl)ethanol was yielded by immobilized pea (Pisum sativum L.) protein (IPP) from (R, S) 2-naphthyl ethanol (>99% ee, yield; about 50%), in which the (R)-enantiomer was selectively oxidized to 2-acetonaphthone. IPP could be reused consecutively at least three times without any decrease of yield and optical purity.     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolusaureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 10⁴ times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with n_H values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme.