Miroslav Šulc

Role of Cytochromes P450 1A1/2 in Detoxication and Activation of Carcinogenic Aristolochic Acid I: Studies with the Hepatic NADPH:Cytochrome P450 Reductase Null (HRN) Mouse Model

Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

α-Naphthoflavone acts as activator and reversible or irreversible inhibitor of rabbit microsomal CYP3A6

This report describes the effect of alpha-naphthoflavone (alpha-NF), a known substrate, inhibitor and activator of several cytochromes P450 (CYP), on rabbit CYP3A6. Hepatic microsomes of rabbit pretreated with rifampicine (RIF), enriched with CYP3A6, as well as purified CYP3A6 reconstituted with isolated NADPH:CYP reductase were used as enzymatic systems in this study. The data from difference spectroscopy experiments showed that alpha-NF does yield a type I binding spectrum. This compound is oxidized by microsomal CYP3A6 into two metabolites (5,6-epoxide and trans-7,8-dihydrodiol). While alpha-NF is a substrate of CYP3A6, it also acts as an enzyme modulator. Under the conditions used, stimulation of 17beta-estradiol 2-hydroxylation by alpha-NF was observed. In contrast, this compound reversibly inhibited N-demethylation of erythromycin and tamoxifen, competitively with respect to these substrates, having the K(i) values of 51.5 and 18.0 microM, respectively. Moreover, alpha-NF was found to be an effective inactivator of progesterone and testosterone 6beta-hydroxylation catalyzed by CYP3A6 in RIF-microsomes. In addition, time- and concentration-dependent inactivation of human CYP3A4-mediated 6beta-hydroxylation of testosterone by alpha-NF, was determined. The inactivation of CYP3A6 followed pseudo-first-order kinetics and was dependent on both NADPH and alpha-NF. The concentrations required for half-maximal inactivation (K(i)) were 80.1 and 108.5 microM and the times required for half of the enzyme to be inactivated were 10.0 and 11.9 min for 6beta-hydroxylation of progesterone and testosterone, respectively. The loss of the enzyme activity was not recovered following dialysis, while 90% of the ability to form a reduced CO complex remained. This indicates the binding of alpha-NF to a CYP apoprotein molecule rather than to a heme moiety. Protection from inactivation was seen in the presence of all tested CYP3A substrates. Progesterone and testosterone protected CYP3A6 against inactivation competitively with respect to inactivator, erythromycin non-competitively and 17beta-estradiol showed a mixed type of protection. Here, we described for the first time that alpha-NF is capable of irreversible inhibition of microsomal rabbit CYP3A6 and human CYP3A4. The obtained results strongly suggest that the CYP3A active center contains at least two and probably three distinct binding sites for substrates.

14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3).

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the alpha-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G(alpha) subunit. Since RGS proteins interact with G(alpha) subunits through their RGS domains, it is reasonable to assume that the 14-3-3 protein can either sterically occlude the G(alpha) interaction surface of RGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein binding affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two single-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Our measurements revealed that the 14-3-3 protein binding induces structural changes in both the N-terminal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolated RGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 protein in a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 was solved at 2.3A resolution. The data obtained from the resolution of the structure of the RGS domain suggest that the 14-3-3 protein-induced conformational change affects the region within the G(alpha)-interacting portion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity of RGS3.

Chemopreventive compounds—View from the other side

Increasing attention is being paid to the possibility of applying chemopreventive agents for the protection of individuals from cancer risk. The beneficial potential of chemoprotective compounds is usually well documented by extensive experimental data. To assure the desired effect, these compounds are frequently concentrated to produce dietary supplements for human use. The additive and synergistic effects of other food constituents are, however, frequently ignored. Even natural chemopreventive compounds have to be considered as xenobiotics. Thus, as much attention has to be paid to their testing prior to their wide application as is usual in drug development for human treatment. Unfortunately, much of the research in this area is solely based on simplified in vitro systems that cannot take into account the complexity of biotransformation processes, e.g. chemopreventive compound-drug interaction, effect on metabolism of endogenic compounds. Hence, the predicted chemopreventive potential is not attained in respect of cancer prevention; moreover, the administration of high doses of chemopreventive compounds might be even detrimental for the human health.

Identification of a genotoxic mechanism for the carcinogenicity of the environmental pollutant and suspected human carcinogen o-anisidine.

2‐methoxyaniline (o‐anisidine) is an industrial and environmental pollutant and a bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated with 2 independent methods, 32P‐postlabeling and 14C‐labeled o‐anisidine, to show that o‐anisidine binds covalently to DNA in vitro after its activation by human hepatic microsomes. We also investigated the capacity of o‐anisidine to form DNA adducts in vivo. Rats were treated i.p. with o‐anisidine (0.15 mg/kg daily for 5 days) and DNA from several organs was analyzed by 32P‐postlabeling. Two o‐anisidine‐DNA adducts, identical to those found in DNA incubated with o‐anisidine and human microsomes in vitro, were detected in urinary bladder (4.1 adducts per 107 nucleotides), the target organ, and, to a lesser extent, in liver, kidney and spleen. These DNA adducts were identified as deoxyguanosine adducts derived from a metabolite of o‐anisidine, N‐(2‐methoxyphenyl)hydroxylamine. This metabolite was identified in incubations with human microsomes. With 9 human hepatic microsomal preparations, we identified the specific CYP catalyzing the formation of the o‐anisidine metabolites by correlation studies and by examining the effects of CYP inhibitors. On the basis of these analyses, oxidation of o‐anisidine was attributed mainly to CYP2E1. Using recombinant human CYP (in Supersomes) and purified CYPs, the participation of CYP2E1 in o‐anisidine oxidation was confirmed. In Supersomes, CYP1A2 was even more efficient in oxidizing o‐anisidine than CYP2E1, followed by CYP2B6, 1A1, 2A6, 2D6 and 3A4. The results, the first report on the potential of the human microsomal CYP enzymes to activate o‐anisidine, strongly suggest a carcinogenic potential of this rodent carcinogen for humans.

Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b5, and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼ 1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR.

Role of individual phosphorylation sites for the 14-3-3-protein-dependent activation of yeast neutral trehalase Nth1

Trehalases are important highly conserved enzymes found in a wide variety of organisms and are responsible for the hydrolysis of trehalose that serves as a carbon and energy source as well as a universal stress protectant. Emerging evidence indicates that the enzymatic activity of the neutral trehalase Nth1 in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through an unknown mechanism. In the present study, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. We determined four residues that are phosphorylated by PKA (protein kinase A) in vitro within the disordered N-terminal segment of Nth1. Sedimentation analysis and enzyme kinetics measurements show that both yeast 14-3-3 isoforms form a stable complex with phosphorylated Nth1 and significantly enhance its enzymatic activity. The 14-3-3-dependent activation of Nth1 is significantly more potent compared with Ca2+-dependent activation. Limited proteolysis confirmed that the 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis in conjunction with the enzyme activity measurements in vitro and the activation studies of mutant forms in vivo suggest that Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1.

Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

Background: Ameloblastin plays a key role in the complex biomineralization process that forms tooth enamel, the hardest tissue of the body. Results: Ameloblastin self-associates into ribbon-like supramolecular structures via a short segment encoded by exon 5. Conclusion: Ameloblastin self-association may be essential for correct structural organization and mineralization of the enamel in vivo. Significance: The results provide molecular insight into biology of tooth enamel formation. Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.

Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.

Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method

Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method A simple and sensitive method was developed to separate the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), and six of its oxidation metabolites generated by rat hepatic microsomes enriched with cytochrome P450 (CYP) 1A1, by high pressure liquid chromatography (HPLC). The HPLC method, using an acetonitrile/water gradient as mobile phase and UV detection, provided appropriate separation and detection of both mono- and di-hydroxylated metabolites of BaP as well as BaP diones formed by rat hepatic microsomes and the parental BaP. In this enzymatic system, 3-hydroxy BaP, 9-hydroxy BaP, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-9,10-dihydrodiol and BaP-dione were generated. Among them the mono-hydroxylated BaP metabolite, 3-hydroxy BaP followed by di-hydroxylated BaP products, BaP-7,8-dihydrodiol and BaP-9,10-dihydrodiol, predominated, while BaP-dione was a minor metabolite. This HPLC method will be useful for further defining the roles of the CYP1A1 enzyme with both in vitro and in vivo models in understanding its real role in activation and detoxification of BaP.