Efficient synthesis of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons from o-quinones and catechols

Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants and suspected human lung carcinogens. In patients with non-small cell lung carcinoma, differential display shows that aldo-keto reductase (AKR1C) transcripts are dramatically overexpressed. However, whether AKR1C isoforms contribute to the carcinogenic process and oxidize potent PAH trans-dihydrodiols (proximate carcinogens) to reactive and redox active o-quinones is unknown; nor is it known whether these reactions occur in human lungs. We now show that four homogeneous human recombinant aldoketo reductases (AKR1C1-AKR1C4) are regioselective and oxidize only the relevant non-K region trans-dihydrodiols. However, these enzymes are not stereo-selective, since they oxidized 100% of these racemic substrates. The highest utilization ratios (V max /K m ) were observed for some of the most potent proximate carcinogens known (e.g. 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) and benzo[g]chrysene-11,12-diol). In vitro, DMBA-3,4-diol was oxidized by AKR1C4 to the highly reactive 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBA-3,4-dione), which was trapped in situ as its mono-and bis-thioether conjugates, which arise from the sequential 1,6-and 1,4-Michael addition of thiol nucleophiles. Human multiple tissue expression array analysis showed that AKR1C isoform transcripts were highly expressed in the human lung carcinoma cell line A549. Isoform-specific reverse transcriptase-PCR showed that AKR1C1, AKR1C2, and AKR1C3 transcripts were all expressed. Western blot analysis and functional assays confirmed high expression of AKR1C protein and enzyme activity in these lung cells. A549 cell lysates were found to convert DMBA-3,4-diol to the corresponding o-quinone. In trapping experiments, LC/MS analysis identified peaks in the cell lysates that corresponded to the synthetically prepared mono-and bis-thioether conjugates of DMBA-3,4-dione. This quinone is one of the most electrophilic and redox-active o-quinones produced by AKRs. Its unique ability to form bis-thioether conjugates parallels the formation of bis-and tris-glutathionyl conjugates of hydroquinone, which display end organ toxicity. The ability to measure DMBA-3,4-dione formation in A549 cells implicates the AKR pathway in the metabolic activation of PAH in human lung. PAHs1 are ubiquitous environmental pollutants and are tobacco carcinogens implicated in the causation of human lung cancer. PAHs are metabolically activated to exert their deleterious effects. Three principal pathways have been proposed for PAH activation and are shown for the representative compound BP (Fig. 1).The first pathway involves the formation of radical cations catalyzed by P450 peroxidases (1). Radical cations form N-7 guanine-depurinating DNA adducts, a process that can lead to G to T transversions in ras (2, 3).In the second pathway, PAHs are activated by members of the CYP superfamily to form an arene oxide on the terminal benzo-ring; subsequent hydrolysis by epoxide hydrolase results in the formation of non-K reg...

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“…Synthesis of PAH Metabolites-NP-1,2-diol was synthesized by reducing naphthalene-1,2-dione with sodium borohydride in ethanol (22). (Fig.…”

Section: Methodsmentioning

confidence: 99%

Activation of Polycyclic Aromatic Hydrocarbontrans-Dihydrodiol Proximate Carcinogens by Human Aldo-keto Reductase (AKR1C) Enzymes and Their Functional Overexpression in Human Lung Carcinoma (A549) Cells

Palackal¹,

Lee²,

Harvey³

et al. 2002

Journal of Biological Chemistry

215

214

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“…The bacterial mutagenicity of the pure enantiomers of trans-dihydrodiols of DBA obtained by preparative HPLC of the synthetic racemic compounds (22,23) on chiral stationary phases as depicted in Figure 2 was determined by measuring the reversion rate of Salmonella typhimurium TA100 from histidine dependence to histidine prototrophy (14,24). The enantiomeric trans-dihydrodiols were metabolically activated using the same hepatic microsomal system employed to determine their regio- (Table 1) and stereoselective (Table 2) formation from DBA, and they exhibited the mutagenic effects presented in Figure 3.…”

Section: Bacterial Mutagenicity Of the Pure Enantiomers Of Trans-dihymentioning

confidence: 99%

Stereoselective Metabolism of Dibenz(a,h)anthracene to trans-Dihydrodiols and Their Activation to Bacterial Mutagens

Platt¹,

Schollmeier²,

Frank³

et al. 1990

Environmental Health Perspectives

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Dibenz(a,h)anthracene (DBA), a carcinogenic, polycyclic aromatic hydrocarbon ubiquitous in the environment, is metabolized by the hepatic microsomal fraction of immature Sprague-Dawley rats pretreated with Aroclor 1254 to 27 ethyl acetate-extractable metabolites. More than half of these metabolites (51%) consisted of trans-1,2-; -3,4-; and -5,6-dihydrodiols including their identified secondary metabolites. The three trans-dihydrodiols (4.9, 15.8, and 0.6% of total metabolic conversion) were highly enriched in their R,R enantiomers (85, 71, and 98%) as determined by high performance liquid chromatography on suitable chiral stationary phases. This is explained on the basis of the stereoselective epoxidation of DBA by cytochrome P-450c (induced by Aroclor 1254) followed by regioselective hydration catalyzed by microsomal epoxide hydrolase. Determination of the bacterial mutagenicity by measuring the reversion rate of histidine-dependent Salmonella typhimurium TA100 to histidine prototrophy revealed marked differences in the mutagenicity of the enantiomers of the trans-dihydrodiols of DBA when activated by the same metabolizing system as used in the metabolism studies. In the case of trans-1,2-and -5,6-dihydrodiol, the S,S enantiomers were converted to more mutagenic metabolites than their corresponding optical antipodes, whereas in the case of trans-3,4-dihydrodiol it was the R,R enantiomer that produced the stronger mutagens. Therefore, both regio-and stereoselectivity of the metabolizing enzymes attribute to the dominant role of trans-3,4-dihydrodiol in the mutagenicity of DBA.

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“…We investigated whether human CYP1A1 could be reconstituted into vesicle membranes effectively enough to study the effects of membrane properties and/or distinct phospholipids on CYP1A1 activity. We characterized the impact of negatively charged and nonbilayer lipids on CYP1A1-dependent stereoselective epoxidation by using both (optically pure) enantiomeric For the preparation of (+)-7,8-diol and (-)-7,8-diol, racemic 7,8-diol was synthesized [20] and chromatographically separated into the enantiomers using a chiral stationary phase [21]. Structure, assignment of absolute configuration and optical purity of the two enantiomers were confirmed by UV, CD and measurements of the rotation.…”

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confidence: 99%

“…UV spectra were recorded with a Shimadzu (Japan) UV 2401 PC, CD spectra were recorded with a Jasco J-720, and specific rotations with a Perkin-Elmer 241-MC automatic polarimeter. The specific rotations [a 20 D in acetone were )395°and +417°for (-)-7,8-diol and (+)-7,8-diol, respectively. These are nearly identical with the data reported for these compounds when optically pure [22].…”

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confidence: 99%

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

Kisselev¹,

Schwarz²,

Platt³

et al. 2002

Eur J Biochem

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Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo [a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmolAEmin )1 AEpmol )1 CYP1A1 of the enantiomeric promutagens (-)-and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both V max and K m values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1. ((-)-7,8-diol) and then metabolized by CYP1A1 to the ultimately genotoxic r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (so-called diolepoxide-2 or antidiolepoxide, DE2) [1][2][3]. The last reaction appeared to be highly stereoselective as no or much less of the less carcinogenic r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydo-B[a]P (diolepoxide-1 or syn-diolepoxide, DE1) was produced from (-)-7,8-diol [4,5]. Racemic (+/-)-7,8-diol is mainly converted by human CYP1A1 to the DE2 [6,7].CYP1A1-dependent activity can be reconstituted by mixing the basic components of the monooxygenase system, i.e. purified CYP1A1, NADPH-cytochrome P450 reductase, which transfers electrons from NADPH to P450, and dilaurylglycerophosphocholine (Lau 2 PtdCho) [8][9][10]. However, this micellar system is not appropriate for studying the interactions between the components of the system as some of its properties are unlike those of the endoplasmic reticulum membrane, the natural environment of the microsomal monooxygenase system. This membrane is a bilayer, and it is highly probable that CYP1A1 and NADPH-cytochrome P450 reductase exhibit other important protein-lipid and protein-protein interactions there. Indeed, reconstitution systems using bilayer vesicles yielded higher rates of activity with rabbit liver CYP...

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Efficient synthesis of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons from o-quinones and catechols

Abstract: being recrystallized from CHC13/CH30H/C8H8 to give 8: 0.12 g (25.9%); mp 194-195 °C; NMR (CD3CN/D20) 8 3.2 (m, 2

Cited by 98 publications

References 1 publication

Activation of Polycyclic Aromatic Hydrocarbontrans-Dihydrodiol Proximate Carcinogens by Human Aldo-keto Reductase (AKR1C) Enzymes and Their Functional Overexpression in Human Lung Carcinoma (A549) Cells

Activation of Polycyclic Aromatic Hydrocarbontrans-Dihydrodiol Proximate Carcinogens by Human Aldo-keto Reductase (AKR1C) Enzymes and Their Functional Overexpression in Human Lung Carcinoma (A549) Cells

Stereoselective Metabolism of Dibenz(a,h)anthracene to trans-Dihydrodiols and Their Activation to Bacterial Mutagens

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

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