Stucture of phosphoenolpyruvate carboxylase from maize leaves

AbstractPhosphoenolpyruvate carboxylase (PEPC) from maize leaves has an Mr of 400000. The native enzyme molecule is a homotetramer. The amino acid composition of PEPC is determined. The enzyme contains 8 half-cystine residues per subunit. The role of half-cystine residues and the steric arrangement of the enzyme protein molecule are discussed

Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2

Two compounds known to covalently bind to DNA after their activation with cytochromes P450 (CYPs), carcinogenic benzo(a)pyrene (BaP) and an antineoplastic agent ellipticine, were investigated for their potential to induce CYP and NADPH:CYP reductase (POR) enzymes in rodent livers, the main target organ for DNA adduct formation. Two animal models were used in the study: (i) rats as animals mimicking the fate of ellipticine in humans and (ii) mice, especially wild-type (WT) and hepatic POR null (HRN™) mouse lines. Ellipticine and BaP induce expression of CYP1A enzymes in livers of experimental models, which leads to increase in their enzymatic activity. In addition, both compounds are capable of generating DNA adducts, predominantly in livers of studied organisms. As determined by 32P postlabelling analysis, levels of ellipticine-derived DNA adducts formed in vivo in the livers of HRN™ mice were reduced (by up to 65%) relative to levels in WT mice, indicating that POR mediated CYP enzyme activity is important for the activation of ellipticine. In contrast to these results, 6.4 fold higher DNA binding of BaP was observed in the livers of HRN™ mice than in WT mice. This finding suggests a detoxication role of CYP1A in BaP metabolism in vivo. In in vitro experiments, DNA adduct formation in calf thymus DNA was up to 25 fold higher in incubations of ellipticine or BaP with microsomes from pretreated animals than with controls. This stimulation effect was attributed to induction of CYP1A1/2 enzymes, which are responsible for oxidative activation of both compounds to the metabolites generating major DNA adducts in vitro. Taken together, these results demonstrate that by inducing CYP1A1/2, ellipticine and BaP modulate their own enzymatic metabolic activation and detoxication, thereby modulating their either pharmacological (ellipticine) and/or genotoxic potential (both compounds)

Oxidation of the carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxy-naphthalene (Sudan I) by cytochromes P450 and peroxidases: a comparative study

Sudan I [1-(phenylazo)-2-hydroxynaphthalene, C.I. Solvent Yellow 14, CAS No: 842-07-9] is used as the compound employed in chemical industry and to color materials such as hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe and floor polishes and gasoline. Such a wide used could result in a considerable human exposure. Sudan I is known to cause developments of tumors in the liver or urinary bladder in rats, mice, and rabbits, and is considered a possible weak human carcinogen and mutagen. This carcinogen is also a potent contact allergen and sensitizer. Here, we compare the data concerning the Sudan I oxidative metabolism catalyzed by cytochrome P450 (CYP) and peroxidase enzymes, which has been investigated in our laboratory during the last two decades. These two types of enzymes are responsible both for Sudan I detoxication and activation. Among the Sudan I metabolites, C-hydroxylated derivatives and a dimer of Sudan I are suggested to be the detoxication metabolites formed by CYPs and peroxidases, respectively. Metabolic activation of Sudan I by both types of enzymes leads to formation of reactive species (the benzenediazonium ion by CYP and Sudan I radicals by peroxidase) that bind to DNA and RNA, generating covalent adducts in vitro and in vivo. Whereas the structure of the major adduct formed by the benzenediazonium ion in DNA has already been identified to be the 8-(phenylazo)guanine adduct, the structures of adducts formed by peroxidase, have not been characterized as yet. Biological significance of the DNA adducts of Sudan I activated with CYP and peroxidase enzymes and further aims of investigations in this field are discussed in this study

Mechanisms of Enzyme-Catalyzed Reduction of Two Carcinogenic Nitro-Aromatics, 3-Nitrobenzanthrone and Aristolochic Acid I: Experimental and Theoretical Approaches

Abstract: This review summarizes the results found in studies investigating the enzymatic activation of two genotoxic nitro-aromatics, an environmental pollutant and carcinogen 3-nitrobenzanthrone (3-NBA) and a natural plant nephrotoxin and carcinogen aristolochic acid I (AAI), to reactive species forming covalent DNA adducts. Experimental and theoretical approaches determined the reasons why human NAD(P)H:quinone oxidoreductase (NQO1) and cytochromes P450 (CYP) 1A1 and 1A2 have the potential to reductively activate both nitro-aromatics. The results also contributed to the elucidation of the molecular mechanisms of these reactions. The contribution of conjugation enzymes such as N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) to the activation of 3-NBA and AAI was also examined. The results indicated differences in the abilities of 3-NBA and AAI metabolites to be further activated by these conjugation enzymes. The formation of DNA adducts generated by both carcinogens during their reductive activation by the NOQ1 and CYP1A1/

Activation of carcinogens by peroxidase Horseradish peroxidase-mediated formation of benzenediazonium ion from a non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) and its binding to DNA

AbstractHorseradish peroxidase in the presence of hydrogen peroxide (HRP/H2O2) oxidizes a carcinogenic non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) to the ultimate carcinogen, which binds to calf thymus DNA. The principal product of Sudan I oxidation by the HRP/H2O2 system is the benzenediazonium ion. Minor products are hydroxy derivatives of Sudan I, in which the aromatic rings are hydroxylated. The principal oxidative product (the benzenediazonium ion) is responsible for the carcinogenicity of Sudan I, because this ion, formed from this azo dye, binds to DNA

The comparison of cytotoxicity of the anticancer drugs doxorubicin and ellipticine to human neuroblastoma cells

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the cytotoxicity of ellipticine to human neuroblastoma derived cell lines IMR-32 and UKF-NB-4 was investigated. Treatment of neuroblastoma cells with ellipticine was compared with that of these cancer cells with doxorubicin. The toxicity of ellipticine was essentially the same as that of doxorubicin to UKF-NB-4 cells, but doxorubicin is much more effective to inhibit the growth of the IMR-32 cell line than ellipticine. Hypoxic conditions used for the cell cultivation resulted in a decrease in ellipticine and/or doxorubicin toxicity to IMR-32 and UKF-NB-4 neuroblastoma cells

Ellipticine cytotoxicity to cancer cell lines — a comparative study

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. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. Here, a comparison of the toxicity of ellipticine to human breast adenocarcinoma MCF-7 cells, leukemia HL-60 and CCRF-CEM cells, neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cells and U87MG glioblastoma cells and mechanisms of its action to these cells were evaluated. Treatment of all cells tested with ellipticine resulted in inhibition of cell growth and proliferation. This effect was 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 CYP and peroxidase enzymes, in MCF-7, HL-60, CCRF-CEM, UKF-NB-3, UKF-NB-4 and U87MG cells, but not in neuroblastoma UKF-NB-3 cells. Therefore, DNA adduct formation in most cancer cell lines tested in this comparative study might be the predominant cause of their sensitivity to ellipticine treatment, whereas other mechanisms of ellipticine action also contribute to its cytotoxicity to neuroblastoma UKF-NB-3 cells

Genotoxic mechanisms for the carcinogenicity of the environmental pollutants and carcinogens o-anisidine and 2-nitroanisole follow from adducts generated by their metabolite N-(2-methoxyphenyl)-hydroxylamine with deoxyguanosine in DNA

An aromatic amine, o-anisidine (2-methoxyaniline) and its oxidative counterpart, 2-nitroanisole (2-methoxynitrobenzene), are the industrial and environmental pollutants causing tumors of the urinary bladder in rats and mice. Both carcinogens are activated to the same proximate carcinogenic metabolite, N-(2-methoxyphenyl)hydroxylamine, which spontaneously decomposes to nitrenium and/or carbenium ions responsible for formation of deoxyguanosine adducts in DNA in vitro and in vivo. In other words, generation of N-(2-methoxyphenyl)hydroxylamine is responsible for the genotoxic mechanisms of the o-anisidine and 2-nitroanisole carcinogenicity. Analogous enzymes of human and rat livers are capable of activating these carcinogens. Namely, human and rat cytochorme P4502E1 is the major enzyme oxidizing o-anisidine to N-(2-methoxyphenyl)hydroxylamine, while xanthine oxidase of both species reduces 2-nitroanisole to this metabolite. Likewise, O-demethylation of 2-nitroanisole, which is the detoxication pathway of its metabolism, is also catalyzed by the same human and rat enzyme, cytochorme P450 2E1. The results demonstrate that the rat is a suitable animal model mimicking the fate of both carcinogens in humans and suggest that both compounds are potential carcinogens also for humans