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3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the 32P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA–DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,O-acetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA–DNA adduct formation increased 10- to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs.

12 Tables, 3 Figure from Environmental Pollutant and Potent Mutagen 3-Nitrobenzanthrone Forms DNA Adducts after Reduction by NAD(P)H:Quinone Oxidoreductase and Conjugation by Acetyltransferases and Sulfotransferases in Human Hepatic Cytosols

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo., Highlights • The anticancer drug ellipticine is activated by cytochrome P450 (CYP) enzymes. • Cytochrome b5 (Cyb5) and NADPH:CYP oxidoreductase (POR) modulate CYP enzyme activity. • Ellipticine bioactivation was investigated in mouse lines with hepatic deletions of Cyb5 and/or POR. • POR strongly contributed to ellipticine bioactivation in vivo. • Cyb5 contributed little to ellipticine bioactivation in vivo contrasting previous in vitro studies.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the (32)P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/–) or TP53(–/–) were treated for up to 48 h with 60 μM cisplatin, 50 μM etoposide or 5 μM ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60–80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(–/–) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5 μM BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(–/–) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.

The term "endocrine disruptor" (ED) is used for compounds that mimic or antagonize the effects of endogenous hormones. Synthetic estrogen 17α-ethinylestradiol (EE2) and a human carcinogen benzo[a]pyrene (BaP) are assigned as exogenous endocrine disruptors and an estrogenic hormone estradiol is a natural endogenous disruptor. Here, the potency of these three disruptors administered to rats individually and in combination to induce expression of cytochrome P450 (CYP) enzymes involved in their own metabolism (CYP1A1, 2C and 3A) in vivo was investigated.Changes in CYP protein expression after exposure of rats to BaP, EE2 or estradiol were analyzed by Western blotting. Using the HPLC method, CYP1A1, 2C and 3A specific activities in hepatic microsomes isolated from exposed rats were analyzed.Whereas exposure to BaP induces expression of CYP1A1 protein and its marker activity (Sudan I oxidation) in liver, kidney and lung of rats, no significant induction of this CYP and its enzyme activity was produced by EE2 and estradiol. Treatment of BaP in combination with EE2 and/or estradiol decreased the BaP-mediated CYP1A1 induction in liver of exposed rats. BaP also induces CYP2C11 protein in rat liver and kidney, but does not increase its enzyme activity measured as testosterone 16α-hydroxylation. The enzyme activity of another enzyme of the 2C subfamily, CYP2C6, diclofenac 4'-hydroxylation, is even decreased by BaP. The CYP2C11 protein expression and/or its activity are also increased in liver of rats treated with EE2 and estradiol, but its expression is significantly decreased in lung. The CYP2C6 activity is also elevated by treatment of rats with EE2 and estradiol administered individually as well as in their combination. Whereas only a slight increase in CYP3A protein expression was found by BaP in rat liver, its enzyme activity, testosterone 6β-hydroxyalation, increased significantly in this organ. In contrast, no effect or even a decrease in CYP3A expression and its enzyme activity was produced by EE2 and estradiol in rats exposed to these compounds.

Ellipticine is an anticancer agent that functions through multiple mechanisms participating in cell cycle arrest and initiation of apoptosis. This drug forms covalent DNA adducts after its enzymatic activation with cytochrome P450 (CYP), which is one of the most important ellipticine DNA-damaging mechanisms of its cytotoxic effects. The improvements of cancer treatment are the major challenge in oncology research. Nanotransporters (nanoparticles) are promising approaches to target tumor cells, frequently leading to improve drug therapeutic index. Ellipticine has already been prepared in nanoparticle forms. However, since its anticancer efficiency depends on the CYP3A4-mediated metabolism in cancer cells, the aim of our research is to develop nanoparticles containing this enzyme that can be transported to tumor cells, thereby potentiating ellipticine cytotoxicity.The CYP3A4 enzyme encapsulated into two nanoparticle forms, liposomes and microsomes, was tested to activate ellipticine to its reactive species forming covalent DNA adducts. Ellipticine-derived DNA adducts were determined by the 32P-postlabeling method.The CYP3A4 enzyme both in the liposome and microsome nanoparticle forms was efficient to activate ellipticine to species forming DNA adducts. Two DNA adducts, which are formed from ellipticine metabolites 12-hydroxy- and 13-hydroxyellipticine generated by its oxidation by CYP3A4, were formed by both CYP3A4 nanoparticle systems. A higher effectiveness of CYP3A4 in microsomal than in liposomal nanoparticles to form ellipticine-DNA adducts was found.Further testing in a suitable cancer cell model is encouraged to investigate whether the DNA-damaging effects of ellipticine after its activation by CYP3A4 nanoparticle forms are appropriate for active targeting of this enzyme to specific cancer cells.

Metallothioneins (MTs) are involved in heavy metal detoxification in a wide range of living organisms. Currently, it is well known that MTs play substantial role in many pathophysiological processes, including carcinogenesis, and they can serve as diagnostic biomarkers. In order to increase the applicability of MT in cancer diagnostics, an easy-to-use and rapid method for its detection is required. Hence, the aim of this study was to develop a fully automated and high-throughput assay for the estimation of MT levels. Here, we report the optimal conditions for the isolation of MTs from rabbit liver and their characterization using MALDI-TOF MS. In addition, we described a two-step assay, which started with an isolation of the protein using functionalized paramagnetic particles and finished with their electrochemical analysis. The designed easy-to-use, cost-effective, error-free and fully automated procedure for the isolation of MT coupled with a simple analytical detection method can provide a prototype for the construction of a diagnostic instrument, which would be appropriate for the monitoring of carcinogenesis or MT-related chemoresistance of tumors.

Factors affecting continuous 2,4-DNT degradation by an immobilized mixed microbial culture were investigated including the cell adaptation to this toxic substrate, 4-NT co-degradation, packing material porosity and substrate mass loading. Experiments were carried out in two packed bed reactors, with poraver (porous glass) and expanded slate as packing materials, using a concurrent water-air flow with ample oxygen. Running the system as a batch reactor with re-circulated medium showed that the immobilized cells adapted to higher 2,4-DNT concentrations yielding higher substrate biodegradation rates. The 2,4-DNT removal rate further increased, up to 180-265 mg L(-1)d(-1), when the immobilized biomass cultivation was switched to a continuous mode. The type of the packing material influenced the 2,4-DNT removal rate, apparently due to the difference in biofilm development. Significant changes in the biofilm composition were observed compared to the original inoculum despite poor biofilm growth.

Metallothioneins (MTs) are a family of ubiquitous, biologically interesting proteins that have been isolated and studied in a wide variety of organisms, including prokaryotes, plants, invertebrates and vertebrates. Due to the property of MTs being metal-inducible and their high affinity to metal ions, homeostasis of heavy-metal levels is probably their most important biological function. MTs are also involved in other important biochemical pathways, including scavenging of reactive oxygen species, activation of transcription factors and participation in carcinogenesis. Detection and quantification of MTs are not simple due to the unique primary structure and their relatively low molecular mass. Analytical methods are based on: a) detection of bound metal ion; b) detection of free ‐SH groups; c) protein mobility in electrical field; and, d) interaction with different types of sorbent. This review highlights techniques used for detection and determination of MTs with discussion of the advantages and the disadvantages of particular approaches.

Sarcosine (N-methylglycine) was previously delineated as a substantial oncometabolite of prostate cancer (PCa) and its metabolism seems to be significantly involved in PCa development and behavior.We focused on investigation whether the exposure of prostate cells (PNT1A, 22Rv1, and PC-3) to sarcosine-related amino acids (glycine, dimethylglycine, and sarcosine) affects their aggressiveness (cell mobility and division rates, using real-time cell based assay). The effect of supplementation on expression of glycine-N-methyltransferase (GNMT) mRNA was examined using qRT-PCR. Finally, post-treatment amino acids patterns were determined with consequent statistical processing using the Ward's method, factorial ANOVA and principal component analysis (P 0.05).The highest migration induced sarcosine and glycine in metastatic PC-3 cells (a decrease in relative free area about 53% and 73%). The highest cell division was achieved after treatment of 22Rv1 and PC-3 cells with sarcosine (time required for division decreased by 65% or 45%, when compared to untreated cells). qRT-PCR revealed also significant effects on expression of GNMT. Finally, amino acid profiling shown specific amino acid patterns for each cell line. In both, treated and untreated PC-3 cells significantly higher levels of serine, glutamic acid, and aspartate, linked with prostate cancer progression were found.Sarcosine-related amino acids can exceptionally affect the behavior of benign and malignant prostate cells.

The improvements of cancer treatment are the major challenge in oncology research. Nanocarriers are one of the promising approaches to selectively target tumor cells, frequently leading to improve drug therapeutic index. Ellipticine is an anticancer agent that functions through multiple mechanisms. Here, the toxic effects of an anticancer drug ellipticine encapsulated in a micellar nanotransporter and free ellipticine on human HL-60 leukemia cells and formation of ellipticine-derived DNA adducts by both forms of the drug in these cells were investigated.The toxicity of modified ellipticine on cells was compared to that of free ellipticine using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide cytotoxicity assay. 32P-postlabeling was utilized to determine ellipticine-DNA adducts in treated cells.The comparison of efficiencies of free ellipticine and ellipticine-micelles [the poly(ethylene oxide)-block-poly(allyl glycidyl ether) block copolymer] to form ellipticine-derived DNA adducts in leukemia HL-60 cells and to act as cytotoxic agent on these cells was performed. Exposure of HL-60 cells to ellipticine in micelles resulted in formation of ellipticine-DNA adducts and caused the cytotoxic effect on these cells. The influence of ellipticine in micelles on HL-60 cells was very similar to that of free ellipticine. The ellipticine half maximal inhibition concentration was determined as 1.3±0.3 µmol.L(-1) and 1.4±0.3 µmol.L(-1) for ellipticine and ellipticine in micelles, respectively. Likewise, the levels of ellipticine-DNA adducts generated in HL-60 cells by both forms of ellipticine were analogous.The results found in this work demonstrate similar cytotoxicity and DNA-damaging effects of ellipticine and its micellar form on leukemia HL-60 cells in vitro.

Dihydromyricetin (DHM) is a flavonoid, which has been shown to antagonize effects of ethanol intoxication. As a potential pharmacological agent, its biological interactions with enzymes metabolizing foreign compounds should be tested. Thus, the aim of this study was to analyze the influence of DHM on the induction and metabolic activity of selected cytochromes P450 (CYPs).After flavonoid administration by oral gavage to stomach the CYP expression at protein and mRNA levels was determined in rat liver and small intestine. The effects of flavonoids on CYP1A1/2, CYP1A2 or CYP2B1/2 enzyme activities in microsomes were measured using marker activities of these enzymes. Flavonoid-mediated inhibition of recombinant CYP1A2 was also assayed with luciferin-ME substrate. The flavonoid interaction with aryl hydrocarbon receptor (AhR) was assayed by reporter luciferase activity in Hep2G cells.The value of half maximal inhibitory concentration of DHM for CYP1A1/2, CYP1A2, and CYP2B1 were determined to be 4.1, 14.2, and 98.5 mmol.L(-1), respectively. With the exception of a weak induction of CYP2B1 and CYP1A2 in the middle part of small intestine and in the liver, respectively, DHM did not affect the CYP expression at protein levels. On the contrary, real-time PCR revealed elevated expression of CYP1A1 and CYP1A2 mRNA in proximal part of the small intestine while decreased in the middle part. In the study utilizing the HepG2 cells, DHM showed only an additive effect on the benzo[a]pyrene-mediated activation of Ah receptor.Dihydromyricetin doesn't significantly interfere with metabolic activity of CYP1A1/2 and CYP2B1 enzymes.

Cytochromes P450 (CYPs) are heme enzymes oxygenating a broad range of substrates. Their activity is dependent on the presence of a suitable electron donor (eukaryotic NADPH:CYP oxidoreductase or cytochrome b5). The Escherichia naturally contain no CYPs and no NADPH:CYP oxidoreductase, however it was reported that some CYPs heterologously expressed in E. coli may exist in the ferrous form. A small bacterial flavoprotein, flavodoxin is considered to be responsible for reduction some of these CYPs.The reduction state of several human CYPs expressed in the intact living E. coli cells was examined. In addition, molecular dynamics and steered molecular dynamics simulations were performed to predict and compare affinity of flavodoxin toward selected CYPs.We determined the reduction state of five human CYPs heterologously expressed in E. coli. The computationally predicted stabilities of CYP-flavodoxin complexes correlate with the percentage of reduced CYPs in bacterial cells. The mean electron transfer distance within optimized complexes was also related to the percentage of reduced CYPs.Depending on the resting state, the CYPs heterologously expressed in E. coli could be divided into two groups; CYP2C8, 2C9, 3A4 are in E. coli present mainly in the oxidized form; while CYP1A1, 1A2, 2A6, 2A13, 2B6, 2D6 are found predominantly in the reduced form. We found a significant correlation between the stability of CYP-flavodoxin complexes and the percentage of reduced CYPs in bacteria. Hence, the naturally expressed flavodoxin is probably responsible for reduction of a larger group of human CYPs in bacterial cells.

Cytochromes P450 (CYP) are monooxygenases, which metabolize mostly hydrophobic endogenous and exogenous compounds. CYPs without any clear connection to metabolism are called "orphans". Interestingly, these "orphan" CYPs are over-expressed in tumor tissues. Thus, the main aim of the paper is the development of antibodies for immunodetection of these CYPs as potential malignancy markers.Unique sequences of CYP2S1 and 2W1 were selected and peptides synthesized. Chickens were immunized with peptides bound to hemocyanin (KLH). The antibodies were isolated from egg yolks and their reactivity was tested by ELISA. Antibodies were further affinity purified on immobilized peptides. Western blots containing CYP2S1 and 2W1 standards were developed with purified antibodies.Using unique peptide immunogens of CYP2S1 and 2W1 the antibodies were developed. As judged from ELISA all chickens produced specific antibodies against the respective peptides. Both affinity purified antibodies against CYP2S1 peptide recognized the CYP2S1 standard on Western blots, but only one of four anti-peptide antibodies against CYP2W1 reacted with CYP2W1 standard. The antibodies were used for the detection of CYPs in cancer cell lines and human tissues samples. Although both CYPs were frequently co-expressed in cancer cells, CYP2S1 was solely induced in the cell line BxPC3, while CYP2W1 was predominantly present in cell lines MCF7 and HeLa. Our data show that anti-peptide antibodies are an indispensable tool for detection of homologous CYPs.The anti-peptide antibodies successfully recognized CYP2S1 and 2W1 in the cancer cell lines and tissue samples.

17α-Ethinylestradiol (EE2) is an endocrine disruptor that is an ingredient of oral contraceptives. Here, EE2 metabolism catalyzed by cytochromes P450 (CYP) was studied. Two model organisms, rat and ligninolytic fungus Pleurotus ostreatus, were used.To resolve the role of rat and/or fungal CYPs in EE2 oxidation, microsomes were incubated with EE2 and NADPH or cumene hydroperoxide. Using Supersomes™, we examined which of rat CYPs oxidize EE2.EE2 is effectively degraded by P. ostreatus in vivo. In vitro, EE2 is metabolized by CYPs by the NADPH-dependent and organic hydroperoxide-dependent mechanisms. Rat hepatic microsomes metabolize EE2 in the presence of NADPH to three products; two of them are hydroxylated EE2 derivatives. Using rat Supersomes™ we found that EE2 is hydroxylated by several rat CYPs, among them CYP2C6 and 2C11 are most efficient in 2-hydroxy-EE2 formation, while CYP2A and 3A catalyze EE2 hydroxylation to the second product. On the contrary, the products of the NADPH-dependent hydroxylating reactions were not detected in Pleurotus ostreatus. During the reaction of EE2 in microsomes isolated from rat and P. ostreatus in the presence of the alternate oxidant, cumene hydroperoxide, another metabolite, different from the above mentioned products, is generated. Rat CYP1A1 is the most efficient enzyme catalyzing formation of this EE2 product.The results suggest that CYPs play a role in EE2 metabolism in rat and P. ostreatus. To our knowledge this is the first finding describing ligninolythic fungal metabolism of EE2 by CYP in the presence of cumene hydroperoxide.

Chicken Antibiotics- Superior Alternatives for Conventional Immunoglobulins

The tumour suppressor p53 is one of the most important cancer genes. Previous findings have shown that p53 expression can influence DNA adduct formation of the environmental carcinogen benzo[a]pyrene (BaP) in human cells, indicating a role for p53 in the cytochrome P450 (CYP) 1A1-mediated biotransformation of BaP in vitro. We investigated the potential role of p53 in xenobiotic metabolism in vivo by treating Trp53(+/+), Trp53(+/–) and Trp53(−/−) mice with BaP. BaP-DNA adduct levels, as measured by 32P-postlabelling analysis, were significantly higher in liver and kidney of Trp53(−/−) mice than of Trp53(+/+) mice. Complementarily, significantly higher amounts of BaP metabolites were also formed ex vivo in hepatic microsomes from BaP-pretreated Trp53(−/−) mice. Bypass of the need for metabolic activation by treating mice with BaP-7,8-dihydrodiol-9,10-epoxide resulted in similar adduct levels in liver and kidney in all mouse lines, confirming that the influence of p53 is on the biotransformation of the parent compound. Higher BaP-DNA adduct levels in the livers of Trp53(−/−) mice correlated with higher CYP1A protein levels and increased CYP1A enzyme activity in these animals. Our study demonstrates a role for p53 in the metabolism of BaP in vivo, confirming previous in vitro results on a novel role for p53 in CYP1A1-mediated BaP metabolism. However, our results also suggest that the mechanisms involved in the altered expression and activity of the CYP1A1 enzyme by p53 in vitro and in vivo are different. Electronic supplementary material The online version of this article (doi:10.1007/s00204-015-1531-8) contains supplementary material, which is available to authorized users.

As an alternative therapeutic approach for the prevention and treatment of bacterial infections with P. aeruginosa of cystic fibrosis (CF) patients, chicken yolk antibodies (IgY) could be used. The most significant advantage of IgY, in contrast to mammalian IgG, consists in the fact, that when bound to the antigen, they usually do not induce inflammatory reaction. In addition, the simplicity of egg production and the ease of IgY preparation makes this kind of antibody an excellent tool for passive immunization. Thus, the aim of our project was to study the effect of IgY and its Fab fragment on the potential induction of pro-inflammatory reactions in lung epithelial cells.Chicken IgY were prepared from pooled egg yolks. Fab fragmens of IgY were purified from the papain digest of IgY using DEAE-Sephacel ion exchange chromatography. Their purity was verified by SDS electrophoresis in polyacrylamide gel. Immortalized human cell lines, CuFi (CF patient) and NuLi (healthy subject), and A549 (human adenocarcinoma cells) were exposed to IgY, Fab, OVA, LPS (positive control), PBS (negative control), and human and goat IgG for 24 hours. The concentration of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and GM-CSF were determined in cell media using the BioPlex method, which enables the quantification of multiple analytes simultaneously in one sample.Our results show that i) the Fab fragment induced levels of some proinflammatory cytokines, when compared to the PBS control, whereas ii) chicken IgYs did not induce any notable production of pro-inflammatory cytokines in contrast to intense effect of LPS on TNF-α and GM-CSF. In summary, our data show that levels of all cytokines are comparable with physiological values in human serum except of IL-1β, which concentration in cell medium was markedly elevated by Fab fragment.The present data indicate that IgY are not inflammatory for lung cells and thus they are possibly applicable for prevention of airway bacterial infections.

Flavanol dihydromyricetin (DHM) has been shown to counteract acute ethanol (EtOH) intoxication and reduce excessive EtOH consumption. Since this flavonoid is being considered for human use, the in vivo study of DHM interactions with the cytochrome P450 (CYP) multienzyme system in the respect of metabolic activation of a model food-born carcinogen, benzo[a]pyrene (BaP), is of high importance. Flavonoids of known properties, alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF) were included into the study to compare their and DHM effects on BaP-DNA adduct formation. METH0 DS: The flavonoids were administered by oral gavage either 72 hrs prior or simultaneously with a single dose of BaP to experimental rats. The expression of CYP1A1/2 enzymes was examined based on the enzymatic activity with a marker substrate, 7-ethoxyresorufin, and on Western blots. The nuclease P1 version of the 32P-postlabeling assay was used to detect and quantify covalent DNA adducts formed by BaP.Treatment of rats with a single dose of DHM or ANF prior to or simultaneously with BaP did not produce an increase in levels of CYP1A1 and in formation of BaP-DNA adducts in liver. BNF, a known inducer of CYP1A1, showed a synergistic effect on BaP-mediated CYP1A1 induction and BaP activation in liver. Contrary to that, in small intestine the stimulatory effect of BNF on both parameters was not detected. Animal pre-treatment with DHM or ANF before BaP administration resulted in a significant elevation of BaP-DNA adducts, namely in the distal part of small intestine, while the CYP1A1 mediated 7-ethoxyresorufin-O-deethylation (EROD) was decreased markedly. It is important to note that under all regimens of animal treatment, DHM or ANF produced the higher inhibitory effect on the BaP-DNA adduct formation and BaP-induced EROD activity of CYP1A1 when administered simultaneously than sequentially with BaP. Our data show that DHM or ANF did not enhance the BaP-activation leading to BaP-mediated genotoxicity (the formation of BaP-DNA adducts) in rat liver, however, in small intestine the pretreatment of rats with these flavonoids may enhance BaP genotoxicity.The data indicate that the intake of DHM prior to or simultaneously with the administration of BaP may increase the risk of a BaP-induced tumorigenesis in small intestine.

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA-AAI adduct induces AT to TA transversions in the tumor-suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin-layer chromatography 32P-postlabeling and mass spectrometric analysis we report the detection of dA-AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA-AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA-AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA-associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long-term persistence of dA-AAI provides a useful long-term biomarker of exposure and attests to the role of AA in human urothelial malignancy.

OBJECTIVES:The aim of this study was to investigate a role of cytochrome P450 (CYP) and peroxidase in ellipticine oxidative activation in two mouse strains differing in expression of NADPH:CYP reductase (POR) [the HRN (Hepatic Cytochrome P450 Reductase Null) mice, in which POR is deleted in hepatocytes and its wild-type (WT) counterpart], and in levels of CYP1A1/2 and cytochrome b5 that were modulated by treatment of these mouse models with a CYP1A inducer, benzo[a]pyrene (BaP).METHODS:Ellipticine-DNA adducts were detected by 32P-postlabeling. HPLC was employed for the separation and characterization of ellipticine metabolites.RESULTS:Hepatic microsomes of HRN and WT mice activate ellipticine to form ellipticine-derived DNA adducts. A 2.2- and 10.4-fold increase in amounts of ellipticine-derived DNA adducts formed by liver microsomes was caused by exposure of HRN and WT mice to BaP, respectively. The results found and utilization of NADPH and arachidonic acid, cofactors of CYP- and cyclooxygenase (COX)-dependent enzyme systems, respectively, as well as inhibitors of CYP1A1/2 and 3A, demonstrate that the CYP1A and 3A enzymes play a major role in ellipticine activation in liver microsomes. In addition, the COX enzyme is important in ellipticine activation in liver of HRN mice.CONCLUSION:The CYP1A and 3A enzymes activate ellipticine mainly in liver of WT mice, whereas peroxidase COX plays this role in liver of HRN mice. Treatment of mice with BaP increases an impact of CYP1A on ellipticine activation. A pattern of expression levels of these enzymes plays a crucial role in their impact on this process.

³²P-Postlabeling analysis is an ultrasensitive method for the detection and quantitation of DNA adducts and covalent modifications of the DNA. It consists of four main steps: (1) enzymatic digestion of DNA to 3'-monophosphate nucleosides; (2) enrichment of the adducts; (3) 5'OH-labeling of the adducts by T4 polynucleotide kinase-catalyzed transfer of ³²P-orthophosphate from [γ-³²P]ATP; and (4) chromatographic or electrophoretic separation of labeled adducts and detection and quantification by means of their radioactive decay. The assay requires only micrograms of DNA and is capable of detecting adduct levels as low as one adduct in 10⁹-10¹⁰ normal nucleotides. It is applicable to a wide range of investigations in human, animal, and in vitro studies including monitoring exposure to environmental or occupational carcinogens, determining whether a chemical or a complex mixture has genotoxic properties, elucidation of the toxicological pathways of carcinogens, and monitoring DNA repair.

Cytochrome P450 (CYP) 1A1 is the most important enzyme in both activation and detoxification of carcinogenic benzo[a]pyrene (BaP), in combination with microsomal epoxide hydrolase (mEH). To evaluate metabolism of BaP in human, identification of a suitable animal model that mimics the metabolic fate of BaP in human is of great importance. The aim of this work was to compare BaP oxidation by human CYP1A1 and CYP1A1 of one animal model, rat. Investigation of the effect of cytochrome b5 on BaP oxidation by CYP1A1 was another target of this study.High performance liquid chromatography (HPLC) was employed for separation of BaP metabolites formed by enzymatic systems. Their structures were identified by mass- and NMR-spectrometry.Human hepatic microsomes oxidized BaP to BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione and BaP-3-ol. The same metabolites were generated by rat liver microsomes, but BaP-9-ol and a metabolite Mx, the structure of which has not been identified as yet, were also formed in these microsomes. Human CYP1A1 expressed with NADPH:CYP reductase (POR) in Supersomes™ oxidized BaP to the same metabolites as microsomes, but BaP-4,5-dihydrodiol has not been detected. Rat recombinant CYP1A1 in this SupersomesTM system oxidized BaP to BaP-9,10-dihydrodiol, a metabolite Mx, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol and BaP-3-ol. Addition of cytochrome b5 to rat and human recombinant CYP1A1 systems led to a more than 2-fold increase in BaP oxidation.The results show similarities between human and rat CYP1A1 in BaP oxidation and demonstrate rats as a suitable model mimicking BaP oxidation in human.

The aim of this study was to prove that the incidence of the more unusual and largely under-researched cardiac dysfunction, i.e. diastolic, is more frequent in patients with alcoholic cirrhosis. Comparison of the incidence of left ventricular diastolic dysfunction in medical-ward patients with no prior history of cardiovascular disease to that of the patients with hepatic cirrhosis caused by alcohol abuse was carried out. The study is original from the point of view of examination of patients with cirrhosis of solely alcoholic aetiology in one Central-European university hospital.Three methods of echocardiographic examination were used: (i) pulse Doppler echocardiography to assess blood flow through the mitral valve and in the pulmonary veins, (ii) tissue Doppler imaging (TDI) to assess mitral annular motion, and (iii) colour M-mode Doppler echocardiography to assess blood flow from the left atrium into the left ventricle.The results found confirmed that the incidence of left ventricular diastolic dysfunction in patients with alcohol-related liver cirrhosis, classified as Child-Pugh grade A and B, was significantly higher than in the controls without any prior liver disease. Furthermore, our research team has newly noticed how the severity of diastolic dysfunction affects the morbidity and mortality of patients undergoing such treatments as the transjugular intrahepatic portosystemic shunt (TIPS), liver transplantation and other surgical interventions resulting from different indications.The high incidence of diastolic dysfunction in cirrhotic alcoholics should not be underestimated. Examination of diastolic dysfunction should be a standard procedure for making clinical decisions about these patients.

The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Human cytochrome P450 (CYP) 1A1 and 1A2 enzymes were found to be responsible for the AAI reductive activation to form AAI-DNA adducts, while its structurally related analogue, CYP1B1 is almost without such activity. However, knowledge of the differences in mechanistic details of CYP1A1-, 1A2-, and 1B1- mediated reduction is still lacking. Therefore, this feature is the aim of the present study.Molecular modeling capable of evaluating interactions of AAI with the active site of human CYP1A1, 1A2 and 1B1 under the reductive conditions was used. In silico docking, employing soft-soft (flexible) docking procedure was used to study the interactions of AAI with the active sites of these human enzymes.The predicted binding free energies and distances between an AAI ligand and a heme cofactor are similar for all CYPs evaluated. AAI also binds to the active sites of CYP1A1, 1A2 and 1B1 in similar orientations. The carboxylic group of AAI is in the binding position situated directly above heme iron. This ligand orientation is in CYP1A1/1A2 further stabilized by two hydrogen bonds; one between an oxygen atom of the AAI nitro-group and the hydroxyl group of Ser122/Thr124; and the second bond between an oxygen atom of dioxolane ring of AAI and the hydroxyl group of Thr497/Thr498. For the CYP1B1:AAI complex, however, any hydrogen bonding of the nitro-group of AAI is prevented as Ser122/Thr124 residues are in CYP1B1 protein replaced by hydrophobic residue Ala133.The experimental observations indicate that CYP1B1 is more than 10× less efficient in reductive activation of AAI than CYP1A2. The docking simulation however predicts the binding pose and binding energy of AAI in the CYP1B1 pocket to be analogous to that found in CYP1A1/2. We believe that the hydroxyl group of S122/T124 residue, with its polar hydrogen placed close to the nitro group of the substrate (AAI), is mechanistically important, for example it could provide a proton required for the stepwise reduction process. The absence of a suitable proton donor in the AAI-CYP1B1 binary complex could be the key difference, as the nitro group is in this complex surrounded only by the hydrophobic residues with potential hydrogen donors not closer than 5 Å.

Etoposide (Vepesid, VP-16), an inhibitor of topoisomerase II, is a chemotherapeutic drug commonly used for treatment of different types of malignant diseases. By inhibiting the topoisomerase II enzyme activity in cancer cells, this drug leads to DNA damage and subsequently to cell death. In this study, we investigated the effect of this anticancer drug alone and in combination with a histone deacetylase (HDAC) inhibitor, valproic acid (VPA), on a human UKF-NB-4 neuroblastoma cell line.The effects of etoposide and VPA on UKF-NB-4 cells were tested under the normoxic and also the hypoxic (1% O2) cultivation conditions. The cytotoxicity of etoposide and VPA to a UKF-NB-4 neuroblastoma cell line was evaluated with MTT assay. Apoptosis of the cells was analyzed by flow cytometry using an Annexin V and propidium iodide binding method. The effect of etoposide and VPA on the cell cycle distribution was determined by flow cytometric analysis using propidium iodide staining.The results of the study demonstrate that UKF-NB-4 neuroblastoma cells are sensitive both to etoposide and to VPA. They also indicate that the impact of VPA on cytotoxicity of etoposide in these tumor cells varies depending on the sequence of cultivation of the cells with the drugs. As a suitable sequence of cultivation, with a high rate of suppression of neuroblastoma cell growth was found the preincubation of the cells with etoposide, which was followed by their cultivation with VPA. In contrast, the reversed combination (preincubation of the cells with VPA before their treating with etoposide) did not give any increase in etoposide cytotoxicity. The effect of such combined treatment can be explained by measuring the cell cycle distribution, which shows that both etoposide and VPA change the cell cycle phase distribution.Etoposide and VPA were found as cycle phase specific drugs that are cytotoxic to human UKF-NB-4 neuroblastoma cells used either as single drugs or both together. However, whereas VPA might sensitize the cells to etoposide, inappropriate sequence of cultivation of the cells with VPA can decrease the etoposide cytotoxic efficacy. The results found here warrant further studies of combined treatment of neuroblastoma cells with etoposide with HDAC inhibitors and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.

The cytochrome P450 (P450) and cytochrome b5 are membrane hemoproteins composing together with flavoprotein NADPH:P450 reductase a mixed function oxidase (MFO) system. The knowledge of the interaction between P450 and its redox partners within a MFO system is fundamental to understand P450 reaction mechanism, an electron transport from its redox partner and also detoxification of xenobiotics and/or metabolism of endogenous substrates with all positive or negative aspects for organisms.The chemical cross-linking by soluble carbodiimide (EDC) in combination with the liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) has been employed to characterize the contact surface regions involved in the transient interaction between two catalytic domains of P450 2B4 and cytochrome b5.The cross-linking reaction was accomplished in an equimolar catalytic complex of P450 2B4:cytochrome b5 and the covalent hetero-dimers detected on SDS-PAGE electrophoresis were analyzed (after in gel trypsin digestion) using LC-HRMS to identify cross-linked amino-acid residues. The computed in silico models of P450 2B4:cytochrome b5 complex using amino-acids participating in cross-links (Asp134, Lys139, Glu424 and Glu439 located on a proximal surface of P450 2B4) suggest interpretation that two different types of cytochrome b5 orientations are present in the studied interaction within a MFO system: the first allowing potential cytochrome b5 electron donation to P450, the second one inducing cytochrome b5 modulation of P450 structural changes.The results demonstrated the capability of the used experimental approach to map the interaction between P450 and cytochrome b5 suggesting the formation of multi-meric structures within a MFO system as interpretation of the two observed mutual orientations.

Of several enzymes metabolizing xenobiotics, cytochrome P450 (CYP) and peroxidase enzymes seem to be most important. One of the major challenges in studies investigating metabolism of xenobiotics is to resolve which of these two groups of enzymes is predominant to metabolize individual xenobiotic compounds. Utilization of selective inhibitors of CYP and peroxidase enzymes might be a useful tool to identify the contribution of these enzymes to metabolism of xenobiotics in samples, where both types of enzymes are present. The aim of this study was to investigate specificities of several known CYP inhibitors to these enzymes; whether they inhibit only the CYP enzymes and do not inhibit peroxidases.Since the oxidation of o-anisidine catalyzed by a model peroxidase used, horseradish peroxidase (HRP), is a two-substrate reaction, the inhibition potential of tested chemicals was studied with respect to both peroxidase substrates, o-anisidine and hydrogen peroxide. Initial velocities of o-anisidine oxidation by HRP under various conditions were determined spectrophotometrically.The CYP inhibitors metyrapone, troleandomycine, disulfiram, sulfaphenazole, quinidine and 1-aminobenzotriazole do not inhibit o-anisidine oxidation catalyzed by HRP. In contrast, ketoconazole, diethyldithiocarbamate, ellipticine, α-naphtoflavone, proadifen SKF525A, piperonylbutoxide, were found to inhibit not only the CYPs, but also the HRP-mediated oxidation of o-anisidine. Interestingly, α-naphtoflavone inhibits oxidation of o-anisidine by HRP with respect to H2O2, but not with respect to o-anisidine. Diethyldithiocarbamate is the most potent peroxidase inhibitor of o-anisidine oxidation with Ki with respect to o-anisidine of 10 μM and Ki with respect to H2O2 of 60 μM, being even the better peroxidase inhibitor than the classical "peroxidase inhibitor" - propyl gallate (Ki with respect to o-anisidine of 60 μM and Ki with respect to H2O2 of 750 μM).The results of the present study demonstrate that 1-aminobenzotriazole, a potent inhibitor of various CYP enzymes, seems to be the best candidate suitable for utilization in studies evaluating participation of CYP enzymes in metabolism of xenobiotics in various complex biological materials containing both CYP and peroxidase enzymes. Moreover, precaution to prevent misinterpretation of results is necessary in cases when proadifen SKF525A, piperonylbutoxide, diethyldithiocarbamate, ketoconazole, α-naphtoflavone and ellipticine are used in similar studies (as CYP inhibitors in various complex biological materials containing both CYP and peroxidase enzymes), since these chemicals can except of CYP enzymes inhibit also peroxidase-mediated reactions.

Ellipticine and doxorubicin are antineoplastic agents, whose action is based mainly on DNA damage such as intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts. The key target to resolve which of these mechanisms are responsible for ellipticine and doxorubicin anticancer effects is the development of suitable methods for identifying their individual DNA-damaging effects. Here, the (32)P-postlabeling method was tested to detect covalent DNA adducts formed by ellipticine and doxorubicin.The standard procedure of (32)P-postlabeling assay, this procedure under ATP-deficient conditions, the version using extraction of adducts with n-butanol and the nuclease P1 enrichment version were used to analyze ellipticineand/ or doxorubicin-derived DNA adducts.Two covalent ellipticine-derived DNA adducts, which are associated with cytotoxicity of ellipticine to human UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines, were detected by the (32)P-postlabeling method. These adducts are identical to those formed by the ellipticine metabolites, 13-hydroxy- and 12-hydroxyellipticine. In contrast, no covalent adducts formed by doxorubicin in DNA of these neuroblastoma cells and in DNA incubated with this drug and formaldehyde in vitro were detectable by the (32)P-postlabeling assay.The results presented in this paper are the first to demonstrate that in contrast to covalent DNA adducts formed by ellipticine, the adducts generated by formaldehyde-mediated covalent binding of doxorubicin to DNA are not detectable by the (32)P-postlabeling assay. No DNA adducts were, detectable either in vitro, in incubations of DNA with doxorubicin or in DNA of neuroblastoma cells treated with this drug. The results also suggest that covalent binding of ellipticine to DNA of UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines is the predominant mechanism responsible for the cytotoxicity of this drug. To understand the mechanisms of doxorubicin anticancer effects on neuroblastoma cells, development of novel methods for identifying covalent doxorubicin-derived DNA adducts is the major challenge for further research.

Valproic acid (VPA) and trichostatin A (TSA) exert antitumor activity as histone deacetylase inhibitors, whereas ellipticine action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of cytochrome P450 (CYP)- and peroxidase-mediated covalent DNA adducts. This is the first report on the molecular mechanism of combined treatment of human neuroblastoma UKF-NB-3 and UKF-NB-4 cells with these compounds.HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites formed by microsomes and peroxidases. Covalent DNA modifications by ellipticine in neuroblastoma cells and in incubations with microsomes and peroxidases were detected by 32P-postlabeling. Expression of CYP enzymes, peroxidases and cytochrome b5 was examined by Western blot.The cytotoxicity of ellipticine to neuroblastomas was increased by pre-treating these cells with VPA or TSA. A higher sensitivity of cells to ellipticine correlated with an increase in formation of covalent ellipticine-derived DNA adducts in these cells. To evaluate the mechanisms of this finding, we investigated the modulation by VPA and TSA of CYP- and peroxidase-mediated ellipticine-derived DNA adduct formation in vitro. The effects of ellipticine in the presence of VPA and TSA on expression of CYPs and peroxidases relevant for ellipticine activation and levels of cytochrome b5 and P-glycoprotein in neuroblastoma cells were also investigated. Based on these studies, we attribute most of the enhancing effects of VPA and TSA on ellipticine cytotoxicity to enhanced ellipticine-DNA adduct formation caused by an increase in levels of cytochrome b5, CYP3A4 and CYP1A1 in neuroblastoma cells. A lower sensitivity of UKF-NB-4 cells to combined effects of ellipticine with VPA and TSA than of UKF-NB-3 cells is also attributable to high levels of P-glycoprotein expressed in this cell line.The results found here warrant further studies and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.

Comamonas testosteroni Pb50 is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This bacterial strain is capable of utilizing phenol as the sole carbon and energy source. Although examples are known in which the C. testosteroni utilizes phenol for growth or metabolism, much less information are known on the nature of the phenol-oxidizing enzymes in this microorganism. Therefore, the occurrence and cellular location of phenol hydroxylase (EC 1.14.13.7), the enzyme participating in the first step of phenol degradation, catalyzing its hydroxylation to catechol in a bacterial Comamonas testosteroni Pb50 strain grown in the presence of phenol as a sole carbon and energy source are the aims of this study.Combination of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300 was used for isolation of phenol hydroxylase detectable in the medium in which C. testosteroni was cultivated. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephacryl S-300 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (phenol consumption and/or catechol formation).Whereas low activity of phenol hydroxylase was detected in cytosol isolated from C. testosteroni, more than 16-fold higher activity of this enzyme was found in the medium in which C. testosteroni was cultivated. The presence of phenol hydroxylase extracellular activity suggests that this microorganism may secrete the enzyme into the extracellular medium. Using the procedure consisting of fractionation with polyethylene glycol 6000 and gel permeation chromatography on columns of Sepharose 4B and Sephacryl S-300, the enzyme was isolated from the medium to homogeneity. The formation of catechol mediated by purified phenol hydroxylase is strictly dependent on the presence of NADPH, which indicates that this enzyme is the NADPH-dependent phenol hydroxylase. The enzyme is a homotetramer having a molecular mass of 240 000, consisting of four subunits having a molecular mass of 60 000. The optimum pH of the enzyme for the phenol oxidation is pH 7.6.The results are the first report showing isolation and partial characterization of extracellular NADPH-dependent phenol hydroxylase of a bacterial C. testosteroni Pb50 strain capable of oxidizing phenol to catechol. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by bacteria.

The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model.DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC.Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed.Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.

N-(2-methoxyphenyl)hydroxylamine is a human metabolite of two industrial and environmental pollutants and bladder carcinogens 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole). Metabolism of N-(2-methoxyphenyl)hydroxylamine by rat hepatic microsomes and identification of the major microsomal enzymes participating in this process are aims of this study.HPLC with UV detection was employed for the separation of N-(2-methoxyphenyl)hydroxylamine metabolites. Inducers and inhibitors of microsomal enzymes and rat recombinant CYPs were used to characterize the enzymes participating in N-(2-methoxyphenyl)hydroxylamine metabolism.N-(2-methoxyphenyl)hydroxylamine is metabolized by rat hepatic microsomes predominantly to o-anisidine, the parent carcinogen from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome b5 reductase and hepatic microsomes of rats pre-treated with specific inducers of CYPs and NADPH:CYP reductase were used to characterize rat liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute most of N-(2-methoxyphenyl)hydroxylamine metabolism to o-anisidine in rat liver to CYP2C, followed by CYP2E1, 2D and 2A. Among recombinant rat CYP enzymes tested in this study, rat CYP2C11 and 2E1, followed by CYP2A2, 2D1/2, 2C12, 3A1/2 and 1A1/2 were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine.The results found in this study, the first report on the reduction of N-(2-methoxyphenyl)hydroxylamine by rat CYP enzymes, demonstrate that CYP2C, followed by CYP2E1, 2D and 2A are the major enzymes participating in this process in rat liver.

Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. The target of this study was to investigate a role of CYP and peroxidase enzymes in ellipticine oxidative activation in rats, a suitable model mimicking the fate of ellipticine in humans, in details. The contribution of pulmonary and renal CYP- and peroxidase enzymes to ellipticine metabolic activation is investigated and compared with that found in the liver.Ellipticine oxidation and DNA adduct formation in vitro were investigated using microsomes isolated from liver, lung and kidney of rats, either control (untreated) or treated i.p. with a single dose of 40 mg of ellipticine per kg of body weight. HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites. Inhibitors of CYPs and cyclooxygenase (prostaglandin H synthase, COX) were used to characterize the enzymes participating in ellipticine oxidative activation in rat liver, lung and kidney. Ellipticine-derived DNA adducts were detected by 32P-postlabeling.Using α-naphthoflavone, furafylline and ketoconazole, inhibitors of CYP1A, 1A2 and 3A, respectively, we found that the CYP1A and 3A enzymes play a major role in ellipticine activation to species forming DNA adducts in liver microsomes. Because of lower expression of these enzymes in lungs and kidneys, even after their induction by ellipticine, they play a minor role in ellipticine activation in these extrahepatic tissues. Arachidonic acid, a cofactor of COX, increased ellipticine activation in the microsomes of extrahepatic tissues. In addition, indomethacin, an inhibitor of COX, efficiently inhibited formation of ellipticine-derived DNA adduct in these microsomes. Based on these results, we attribute the higher activation of ellipticine in lung and kidney microsomes to COX than to CYP enzymes.The results demonstrate that whereas CYP enzymes of 1A and 3A subfamilies are the major enzymes activating ellipticine in rat livers, peroxidase COX plays a significant role in this process in lungs and kidneys.

The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP).The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin.The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate.Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.

3-Aminobenzanthrone (3-ABA) is the main human metabolite of carcinogenic environmental pollutant 3-nitrobenzanthrone (3-NBA). Understanding which cytochrome P450 (CYP) enzymes are involved in metabolism of this toxicant is important in the assessment of individual susceptibility. Characterization of 3-ABA metabolites formed by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major rat and human CYPs participating in this process are aims of this study.HPLC with UV detection was employed for the separation and characterization of 3-ABA metabolites. Inducers and inhibitors of CYPs and rat and human recombinant CYPs were used to characterize the enzymes participating in 3-ABA oxidation.Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize rat liver CYPs metabolizing 3-ABA (measured as consumption of 3-ABA). Kinetics of these reactions catalyzed by rat hepatic microsomes was also evaluated. Based on these studies, we attribute most of 3-ABA metabolism in rat liver to CYP1A and 3A. Among recombinant rat and human CYP enzymes tested in this study, rat CYP3A2 and human CYP3A4/5, followed by CYP1A1 of both organisms were the most effective enzymes converting 3-ABA. Rat hepatic CYP enzymes oxidize 3-ABA up to three metabolites. Two of them were identified to be the products formed by oxidation of 3-ABA on its amino group back to the parent compound from which 3-ABA is generated in organisms, 3-NBA. Namely, N-hydroxylation metabolite, N-hydroxy-3-ABA and 3-NBA were identified to be these 3-ABA oxidation products. These metabolites are formed by CYPs of a 1A subfamily. Another 3-ABA metabolite, whose structure remains to be characterized, is generated not only by CYP1A but also by other CYP enzymes, predominantly by CYPs of a 3A subfamily.The results found in this study, the first report on the metabolism of 3-ABA by human and rat CYPs, clearly demonstrate that CYPs of 3A and 1A subfamilies are the major enzymes metabolizing 3-ABA.

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of activity of several cytochromes P450 (CYP). To elucidate the mechanism of such modulations it is necessary to evaluate not only the effect of native cyt b5, but also that of apo-cyt b5. To prepare apo-cyt b5, heme transfer from native cyt b5 to a protein with higher affinity toward the heme, the horse heart apo-myoglobin, was utilized.Butanone extraction was employed to prepare apo-myoglobin. Apo-cyt b5 was separated from myoglobin by chromatography on DEAE-Sepharose. Mass spectrometry was utilized to characterize proteins eluted from DEAE- Sepharose.The prepared apo-myoglobin was incubated with the cyt b5 at pH 4.2 that is the optimal pH for heme transfer from cyt b5 into apo-myoglobin. The apo-cyt b5 protein was separated from myoglobin present in the reaction mixture by chromatography on a column of DEAE-Sepharose. Using such a procedure, 16% yield of apo-cyt b5 that did not contain any heme in its molecule was obtained from the native rabbit cyt b5. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native cyt b5. The prepared apo-cyt b5 reconstituted with heme can receive electrons from NADPH:CYP reductase.A biologically active apo-cyt b5 was prepared using transfer of heme from cyt b5 to horse heart apo-myoglobin by the procedure described here.

2-Nitrophenol (2-NP) is the major detoxification metabolite of an important industrial pollutant and a potent carcinogen, 2-nitroanisole (2-NA). Characterization of the products of 2-NP metabolism by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major CYP enzymes participating in this process are aims of this study.HPLC with UV detection was employed for the separation and characterization of 2-NP metabolites. Inducers and inhibitors of CYPs and rat recombinant CYPs were used to characterize the enzymes participating in 2-NP oxidation.Rat hepatic microsomes oxidize 2-NP to its hydroxylated metabolite, 2,5-dihydroxynitrobenzene (2,5-DNB). No nitroreductive metabolism leading to the formation of o-aminophenol was evident when using rat hepatic microsomes. Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize CYPs oxidizing 2-NP in rat livers. Based on these studies, we attribute most of 2-NP oxidation in rat liver to CYP2E1 and 3A, followed by CYP2D and 2C. Among recombinant rat CYP enzymes tested in this study, CYP2E1 and 2C11 were the most effective enzymes oxidizing 2-NP. Oxidation of 2-NP by rat CYP2E1 exhibits the Michaelis-Menten kinetics, having the Km value of 0.35 mM.The results found in this study, the first report on the metabolism of 2-NP by rat hepatic microsomes and rat CYP enzymes, demonstrate that CYP2E1 is the major enzyme oxidizing this compound in rat liver.

Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action with promising brain tumor specificity. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP) - and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. The toxicity of ellipticine to U87MG glioblastoma cells and mechanisms of its action to these cells are aims of this study.Ellipticine metabolites formed in U87MG cells were analyzed using HPLC. Covalent DNA modifications by ellipticine were detected by 32P-postlabeling. CYP enzyme expression was examined by QPCR and Western blot.U87MG glioblastoma cell proliferation was efficiently inhibited by ellipticine. This effect might be associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP1A1, 1B1 and 3A4, lactoperoxidase and cyclooxygenase 1, the enzymes expressed in U87MG cells. Moreover, by inducing CYP1B1, 3A4 and 1A1 enzymes in U87MG cells, ellipticine increases its own enzymatic activation, thereby enhancing its own genotoxic and pharmacological potential in these cells. Ellipticine concentration used for U87MG cell treatment is extremely important for its pharmacological effects, as its metabolite profiles differed substantially predicting ellipticine to be either detoxified or activated.The results found in this study are the first report showing cytotoxicity and DNA adduct formation by ellipticine in glioblastomas.