The metabolic activation of pentachlorophenol to chloranil as a potent inhibitor of human and rat placental 3ß-hydroxysteroid dehydrogenases.

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3ß-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3ß-HSD1, with an IC50 value of 29.83 µM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 µM, while chloranil markedly reduced progesterone production at ≥1 µM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3ß-HSD4, with IC50 values of 27.94 and 23.42 µM, respectively. Dithiothreitol (DTT) alone significantly increased human 3ß-HSD1 activity. Chloranil not PCP mediated inhibition of human 3ß-HSD1 activity was completely reversed by DTT and that of rat 3ß-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3ß-HSDs. The difference in the amino acid residue Cys83 in human 3ß-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3ß-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3ß-HSD1.

Role of tetrachloro-1,4-benzoquinone reductase in phenylalanine hydroxylation system and pentachlorophenol degradation in Bacillus cereus AOA-CPS1.

This study reports a ≅12.5 kDa protein tetrachloro-1,4-benzoquinone reductase (CpsD) from Bacillus cereus strain AOA-CPS1 (BcAOA). CpsD is purified to homogeneity with a total yield of 35% and specific activity of 160 U·mg-1 of protein. CpsD showed optimal activity at pH 7.5 and 40 °C. The enzyme was found to be functionally stable between pH 7.0-7.5 and temperature between 30 °C and 35 °C. CpsD activity was enhanced by Fe2+ and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 µmol·s-1, 94 µmol, 0.029 s-1 and 3.13 × 10-4 s-1·µmol-1, respectively, for substrate tetrachloro-1,4-benzoquinone. The bioinformatics analysis indicated that CpsD belongs to the PCD/DCoH superfamily, with specific conserved protein domains of pterin-4α-carbinolamine  dehydratase (PCD). This study proposed that CpsD catalysed the reduction of tetrachloro-1,4-benzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in BcAOA PheOHS using a probable Yin-Yang mechanism. The study also concluded that CpsD may play a catalytic and regulatory role in BcAOA PheOHS and pentachlorophenol degradation pathway.

Metabolic pathway elucidation towards time- and dose-dependent electrophoretic screening of stable oxidative phenolic compounds.

This study demonstrates an untested link between model phenolic compounds and the formation/electrophoretic separation of stable urinary metabolites. Sterically encumbered carbonyl groups were examined, and mass determination was used to confirm the presence and stability of two oxidative metabolites of pentachlorophenol: tetrachloro-1,2-benzoquinone and tetrachloro-1,4-dihydroquinone. Subsequently, baseline resolved separation of pentachlorophenol and the two oxidative metabolites was demonstrated under the following conditions: 75 mM sodium tetraborate buffer (pH = 8.5) with 5 % methanol and 50 mM SDS, +10.0 kV running voltage, injection time = 5.0 s, effective capillary length = 55 cm, and run temperature = 20 °C. Results not only provide key metabolic inferences for pentachlorophenol, they also exhibit improvements in the ability to separate and detect changes in urinary metabolites in response to phenolic-related exposure.

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol.

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s(-1)) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

Molecular complexes of ketaconazole and oxatomide with p-chloranil: spectroscopic and spectrofluorimetric studies.

The molecular complexes of the donors ketaconazole (KTZ) and oxatomide (OXA) drugs with 2,3,5,6-tetrachloro-1,4-benzoquinone (p-chloranil, p-CHL) have been investigated spectroscopically (UV-vis, FT-IR and 1H NMR) and spectrofluorimetrically in different solvents and temperatures. The stoichiometry of the complexes was found to be 1:1. The data are discussed in terms of formation constant, molar extinction coefficient, oscillator strength, dipole moment, ionization potential, dissociation energy and thermodynamic parameters. The results indicated that the formation of molecular complex is spontaneous and endothermic. The fluorescence quenching studies indicated that the interaction of the donors is spontaneous and the fluorescence quenching increased with an increase in the intensity of complexation with the acceptor.

Effects of methyl substituent on the charge-transfer complexations of dicarbazolylalkanes with p-chloranil, tetracyanoethylene and tetracyanoquinodimethane.

Series of 1,n-dicarbazolylalkanes and 1,n-di(3-methylcarbazolyl)alkanes (where n=1-5) were synthesized and the molar extinction coefficients, equilibrium constants, enthalpies, and entropies of their charge-transfer (CT) complexes with the π-acceptors p-chloranil, tetracyanoethylene, and tetracyanoquinodimethane were investigated. 1,n-Di(3-methylcarbazolyl)alkanes formed CT complexes with higher equilibrium constants, more negative enthalpies and entropies than 1,n-dicarbazolylalkanes. Vibrational spectra of CT complexes of one of the donor molecules (1,4-dicarbazolylbutane) with all three acceptors were compared.

Blockade of LTB4-induced chemotaxis by bioactive molecules interfering with the BLT2-Galphai interaction.

BLT2, a low-affinity leukotriene B4 (LTB4) receptor, is a member of the G-protein coupled receptor (GPCR) family and is involved in the pathogenesis of inflammatory diseases such as asthma. Despite its clinical implications, however, no pharmacological inhibitors are available. In the present study, we screened for small molecules that interfere with the interaction between the third intracellular loop region of BLT2 (BLT2iL3) and the Galphai3 protein subunit (Galphai3), using a high-throughput screening (HTS) assay with a library of 1040 FDA-approved drugs and bioactive compounds. We identified two small molecules-purpurin [1,2,4-trihydroxy-9,10-anthraquinone; IC50 = 1.6 microM for BLT2] and chloranil [tetrachloro-1,4-benzoquinone; IC50 = 0.42 microM for BLT2]-as specific BLT2-blocking agents. We found that blockade of the BLT2iL3-Galphai3 interaction by these small molecules inhibited the BLT2-downstream signaling cascade. For example, BLT2-signaling to phosphoinositide-3 kinase (PI3K)/Akt phosphorylation was completely abolished by these molecules. Furthermore, we observed that these small molecules blocked LTB4-induced chemotaxis by inhibiting the BLT2-PI3K/Akt-downstream, Rac1-reactive oxygen species-dependent pathway. Taken together, our results show that purpurin and chloranil interfere with the interaction between BLT2iL3 and Galphai3 and thus block the biological functions of BLT2 (e.g., chemotaxis). The present findings suggest a potential application of purpurin and chloranil as pharmacological therapeutic agents against BLT2-associated inflammatory human diseases.

Nucleobase-dependent reactivity of a quinone metabolite of pentachlorophenol.

Pentachlorophenol (PCP) is a possible human carcinogen detected widely in the environment. A quinone metabolite of PCP, tetrachloro-1,4-benzoquinone (Cl4BQ), is a reactive electrophile with the capacity to damage DNA by forming bulky covalent DNA adducts. These quinone adducts may contribute to chlorophenol carcinogenesis, but their structures, occurrence, and biological consequences are not known. Previous studies have indicated that several DNA adducts are formed in vivo in rats exposed to Cl4BQ, but these adducts were not identified structurally. In the present study, we have elucidated the structure of new agent-specific DNA adducts resulting from the reaction of dGuo, dCyd, and Thd with Cl4BQ. These have been characterized chemically by liquid chromatography-electrospray ionization mass spectrometry, HPLC, UV, and NMR analysis. Two dGuo adducts and one dCyd adduct resulting from the reaction of double-stranded DNA with Cl4BQ have been identified. The results indicate that, in the structural context of DNA, Cl4BQ reacts most readily with dGuo compared to the other DNA bases and that the mode of Cl4BQ reactivity is dependent on the base structure; i.e., multiple types of adducts are formed. Finally, DNA adducts consistent with Cl4BQ reactions are observed when DNA or dGuo is treated with PCP and a peroxidase-based bioactivating system.

Characterization of a deoxyguanosine adduct of tetrachlorobenzoquinone: dichlorobenzoquinone-1,N2-etheno-2'-deoxyguanosine.

Pentachlorophenol (PCP), a widespread environmental pollutant that is possibly carcinogenic to humans, is metabolically oxidized to tetrachloroquinone. DNA adducts attributable to tetrachloroquinone have been observed previously in vitro and detected in vivo. In addition, an unidentified adduct in these studies coeluted with the product of the reaction of deoxyguanosine (dG) and tetrachlorobenzoquinone (Cl4BQ). We have synthesized, isolated, purified, and characterized the predominant adduct formed from the reaction of dG and Cl4BQ. The preparation of a 13C-labeled version of this adduct facilitated its structural characterization. On the basis of 1H NMR, 13C NMR, MS, IR, UV, and cyclic voltammetry, we propose that the adduct is a dichlorobenzoquinone nucleoside in which two chlorine atoms in Cl4BQ have been displaced by reaction at the 1- and N2-positions of dG. The 1H and 13C NMR chemical shifts are consistent with the dichlorobenzoquinone assignment. In contrast, under standard analytical conditions, LC-MS data are consistent with a reduced hydroquinone structure, similar to what may be expected based on results from other chloroquinones. Data from the present study indicate that this reduction could be occurring in the electrospray ionization source and that the initial product of the reaction of dG and Cl4BQ is a dichlorobenzoquinone. The results of this study contribute to the hypothesis that direct reactions between chlorophenols and DNA may play a role in the toxic effects of chlorophenols and indicate a potential difference in reactivity and biological influence between PCP and other less substituted chlorophenols or phenols.

A previously unrecognized step in pentachlorophenol degradation in Sphingobium chlorophenolicum is catalyzed by tetrachlorobenzoquinone reductase (PcpD).

The first step in the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum has been believed for more than a decade to be conversion of PCP to tetrachlorohydroquinone. We show here that PCP is actually converted to tetrachlorobenzoquinone, which is subsequently reduced to tetrachlorohydroquinone by PcpD, a protein that had previously been suggested to be a PCP hydroxylase reductase. pcpD is immediately downstream of pcpB, the gene encoding PCP hydroxylase (PCP monooxygenase). Expression of PcpD is induced in the presence of PCP. A mutant strain lacking functional PcpD has an impaired ability to remove PCP from the medium. In contrast, the mutant strain removes tetrachlorophenol from the medium at the same rate as does the wild-type strain. These data suggest that PcpD catalyzes a step necessary for degradation of PCP, but not for degradation of tetrachlorophenol. Based upon the known mechanisms of flavin monooxygenases such as PCP hydroxylase, hydroxylation of PCP should produce tetrachlorobenzoquinone, while hydroxylation of tetrachlorophenol should produce tetrachlorohydroquinone. Thus, we proposed and verified experimentally that PcpD is a tetrachlorobenzoquinone reductase that catalyzes the NADPH-dependent reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone.

Metal-independent production of hydroxyl radicals by halogenated quinones and hydrogen peroxide: an ESR spin trapping study.

The metal-independent production of hydroxyl radicals (*OH) from H(2)O(2) and tetrachloro-1,4-benzoquinone (TCBQ), a carcinogenic metabolite of the widely used wood-preservative pentachlorophenol, was studied by electron spin resonance methods. When incubated with the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), TCBQ and H(2)O(2) produced the DMPO/*OH adduct. The formation of DMPO/*OH was markedly inhibited by the *OH scavenging agents dimethyl sulfoxide (DMSO), ethanol, formate, and azide, with the concomitant formation of the characteristic DMPO spin trapping adducts with *CH(3), *CH(CH(3))OH, *COO(-), and *N(3), respectively. The formation of DMPO/*OH and DMPO/*CH(3) from TCBQ and H(2)O(2) in the absence and presence, respectively, of DMSO was inhibited by the trihydroxamate compound desferrioxamine, accompanied by the formation of the desferrioxamine-nitroxide radical. In contrast, DMPO/*OH and DMPO/*CH(3) formation from TCBQ and H(2)O(2) was not affected by the nonhydroxamate iron chelators bathophenanthroline disulfonate, ferrozine, and ferene, as well as the copper-specific chelator bathocuproine disulfonate. A comparative study with ferrous iron and H(2)O(2), the classic Fenton system, strongly supports our conclusion that *OH is produced by TCBQ and H(2)O(2) through a metal-independent mechanism. Metal-independent production of *OH from H(2)O(2) was also observed with several other halogenated quinones.

Characterization of metabolic activation of pentachlorophenol to quinones and semiquinones in rodent liver.

Pentachlorophenol (PCP), a widely used biocide, induces liver tumors in mice but not in rats. Metabolic activation of PCP to chlorinated quinones and semiquinones in liver cytosol from Sprague-Dawley rats and B6C3F1 mice was investigated in vitro (1) with microsomes in the presence of either beta-nicotinamide adenine dinucleotide phosphate (NADPH) or cumene hydroperoxide (CHP), (2) with CHP in the absence of microsomes, and (3) with horseradish peroxidase (HRP) and H2O2. Mono-S- and multi-S-substituted adducts of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) and Cl4-1,2-BQ and their corresponding semiquinones [i.e. tetrachloro-1,4-benzosemiquinone (Cl4-1,4-SQ) and tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ)] were measured by gas chromatography-mass spectrometry (GC-MS). Qualitatively, the metabolites of PCP were the same in both rats and mice for all activation systems. Induction of PCP metabolism by either 3MC or PB-treated microsomes was observed in NADPH- but not in CHP-supported systems. In rats, the amount of induction was comparable with either 3MC or PB. 3MC was a stronger inducer than PB in mice and also induced a greater amount of metabolism than in rats. This suggests that induction of specific P450 isozymes may play a role in the toxicity of PCP to mice. Both HRP/H2O2 and CHP led to production of the full spectrum of chlorinated quinones and semiquinones, confirming the direct oxidation of PCP. CHP (with or without microsomes) converted PCP into much greater quantities of quinones and semiquinones than did microsomal P450/NADPH or HRP/H2O2 in both species. This implies that, under conditions of oxidative stress, endogenous lipid hydroperoxides may increase PCP metabolism sufficiently to enhance the toxicity and carcinogenicity of PCP.

Room temperature solution studies of complexation between o-chloranil and a series of anilines by spectrophotometric method.

Electron donor-acceptor (EDA) complex formation between o-chloranil and a series of anilines has been studied in CCl4 medium. In all the cases, EDA complexes are formed instantaneously on mixing the donor and acceptor solutions. N,N-dimethylaniline and N,N-dimethyl-p-toluidine form stable EDA complexes with o-chloranil while the other complexes decay slowly into secondary products. The kinetics of all these reactions has been studied by UV-VIS absorption spectrophotometric method and the rate constants of the reactions and formation constants of the EDA complexes have been determined. The charge transfer (CT) transition energies of the complexes are found to change systematically with change in the number and position of the methyl groups in the donor molecules (methylanilines). From an analysis of this variation, the electron affinity of o-chloranil has been found to be 2.54 eV. A perturbational inductive effect Hückel parameter hMe has been found from this trend and the value obtained (-0.27) is very close to that (-0.3) obtained by Lepley (J. Am. Chem. Soc., 86 (1964) 2545) from a study of tetracyano ethylene (TCNE)-methylbenzene complexes.

Dose-specific production of chlorinated quinone and semiquinone adducts in rodent livers following administration of pentachlorophenol.

Production of chlorinated quinoid metabolites was investigated in the livers of Sprague-Dawley rats and B6C3F1 mice following single oral administration of pentachlorophenol (PCP) (0-40 mg/kg body weight) and in male Fischer 344 rats, following chronic ingestion of PCP at 1,000 ppm in the diet for 6 months (equivalent to 60 mg PCP/kg body weight/day). Analyses of the rates of adduction in the livers of Sprague-Dawley rats and B6C3F1 mice suggested that the production of tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ) adducts was proportionally greater at low doses of PCP (less than 4-10 mg/kg body weight) and was 40-fold greater in rats than in mice. Production of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) adducts, on the other hand, was proportionally greater at high doses of PCP [greater than 60-230 mg/kg body weight] and was 2- to 11-fold greater in mice than in rats over the entire range of dosages. A mathematical model employed these data to predict the rates of daily adduct production and steady state levels of PCP-derived quinone and semiquinone adducts in rats and mice. To evaluate predictions of the model, levels of PCP-derived adducts at steady state were investigated in the livers of male Fischer 344 rats chronically ingesting 60 mg PCP/kg body weight/day. Levels of total Cl4-1,4-BQ-derived adducts in liver cytosolic proteins (Cp) (22.0 nmol/g) and in liver nuclear proteins (Np) (3.07 nmol/g) were comparable to those of model predictions (15.0 and 3.02 nmol/g for Cp and Np, respectively). Overall, these results suggest that species differences in the metabolism of PCP to semiquinones and quinones were, in part, responsible for the production of liver tumors in mice but not rats in chronic bioassays.

Dosimetry of chlorinated quinone metabolites of pentachlorophenol in the livers of rats and mice based upon measurement of protein adducts.

The dosimetry of chlorinated quinones arising from metabolism of pentachlorophenol (PCP), in the livers of male Sprague-Dawley rats and B6C3F1 mice was investigated via measurements of cysteinyl protein adducts and estimates of the second-order reaction rate constants between the quinones and the proteins. We had previously shown that adducts of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) and tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ) were observed at the highest levels in the livers of Sprague-Dawley rats to which PCP had been administered by gavage (5-40 mg/kg body wt) (Biomarkers 1, 232-243, 1996). In the current study we observed that adducts of Cl4-1,4-BQ and tetrachloro-1,2-benzoquinone (CL4-1,2-BQ) were predominant in the livers of B6C3F1 mice receiving 20 mg PCP/kg body wt. The second-order rate constants, representing in vitro reactions between Cl4-1,2-BQ and Cl4-1,4-BQ and various cysteine residues of hepatic proteins of liver cytosol and liver nuclei, were estimated to be 0.012-1.96 L(g protein)(-1) hr(-1) in rats and 0.082-1.67 L(g protein)(-1) hr(-1) in mice. The estimated tissue doses of the quinones to liver cytosol decreased in the order rat Cl4-1,4-BQ > mouse Cl4-1,4-BQ > mouse Cl4-1,2-BQ and to liver nuclei in the order mouse Cl4-1,2-BQ > mouse Cl4-1,4-BQ > rat Cl4-1,4-BQ. The corresponding doses of Cl4-1,2-SQ could not be inferred due to our inability to estimate the second-order rate constants. After aggregating the estimated contributions of all quinone species, mice had a fourfold greater dose to liver nuclei than rats, whereas rats had a threefold greater dose to liver cytosol. The increased nuclear dose to mouse liver compared to that of the rat suggests that the mouse is at greater risk to hepatic DNA damage from PCP-derived quinones. Investigation of the time course of levels of unconjugated tetrachlorohydroquinone (Cl4HQ) in the livers indicated that about 0.4% of Cl4HQ was oxidized to Cl4-1,4-BQ in both rats and mice.

Identification of multiple target sites for a glutathione conjugate on glutathione-S-transferase by matrix-assisted laser desorption/ionization mass spectrometry.

A mass spectrometric method providing qualitative site-specific information regarding covalent modification of proteins is described. The method involves comparison of unmodified and modified proteins by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) peptide mapping in combination with site-specific mutagenesis of possible target amino acids. The approach is demonstrated through the mapping of glutathione-S-transferases (GSH transferases) before and after inhibition with the glutathione conjugate 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GSTCBQ). The results demonstrate the utility of site-specific mutagenesis in combination with MALDI MS peptide mapping. Evidence is presented that three residues in or near the active site, including the hydroxyl groups of Tyr6 and Tyr115 and the sulphydryl group of Cys114, are target sites for GSTCBQ. Although only one GSTCBQ molecule per active site was detected, it appears to be distributed among all three target sites. In addition, MALDI MS peptide mapping covered 81% of the cDNA deduced amino acid sequence for GSH transferase and site-directed mutagenesis corresponding to a single amino acid substitution were verified.

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species.

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.