Physico-chemical characterization of bilirubin-10-sulfonate and comparison of its acid-base behavior with unconjugated bilirubin.

Unconjugated bilirubin (UCB) is the end-product of heme catabolism in the intravascular compartment. Although beneficial for human health when mildly elevated in the body, when present at greater than a critical threshold concentration, UCB exerts toxic effects that are related to its physico-chemical properties, particularly affecting the central nervous system. The aim of the present study was to characterize bilirubin-10-sulfonate (ranarubin), a naturally occurring bile pigment, including determination of its mixed acidity constants (pKa*). Thanks to the presence of the sulfonic acid moiety, this compound is more polar compared to UCB, which might theoretically solve the problem with an accurate determination of the UCB pKa* values of its propionic acid carboxylic groups. Bilirubin-10-sulfonate was synthesized by modification of a previously described procedure; and its properties were studied by mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared (IR), and circular dichroism (CD) spectroscopy. Determination of pKa* values of bilirubin-10-sulfonate and UCB was performed by capillary electrophoresis with low pigment concentrations in polar buffers. The identity of the synthesized bilirubin-10-sulfonate was confirmed by MS, and the pigment was further characterized by NMR, IR, and CD spectroscopy. The pKa values of carboxylic acid moieties of bilirubin-10-sulfonate were determined to be 5.02, whereas those of UCB were determined to be 9.01. The physico-chemical properties of bilirubin-10-sulfonate were partially characterized with low pKa* values compared to those of UCB, indicating that bilirubin-10-sulfonate cannot be used as a surrogate pigment for UCB chemical studies. In addition, using a different methodological approach, the pKa* values of UCB were found to be in a mildly alkaline region, confirming the conclusions of a recent critical re-evaluation of this specific issue.

Phytoremediation of carbamazepine and its metabolite 10,11-epoxycarbamazepine by C3 and C4 plants.

The anticonvulsant drug carbamazepine is considered as an indicator of sewage water pollution: however, its uptake by plants and effect on metabolism have not been sufficiently documented, let alone its metabolite (10,11-epoxycarbamazepine). In a model system of sterile, hydroponically cultivated Zea mays (as C4 plant) and Helianthus annuus (as C3 plant), the uptake and effect of carbamazepine and 10,11-epoxycarbamazepine were studied in comparison with those of acetaminophen and ibuprofen. Ibuprofen and acetaminophen were effectively extracted from drug-supplemented media by both plants, while the uptake of more hydrophobic carbamazepine was much lower. On the other hand, the carbamazepine metabolite, 10,11-epoxycarbamazepine, was, unlike sunflower, willingly taken up by maize plants (after 96 h 88 % of the initial concentration) and effectively stored in maize tissues. In addition, the effect of the studied pharmaceuticals on the plant metabolism (enzymes of Hatch-Slack cycle, peroxidases) was followed. The activity of bound peroxidases, which could cause xylem vessel lignification and reduction of xenobiotic uptake, was at the level of control plants in maize leaves contrary to sunflower. Therefore, our results indicate that maize has the potential to remove 10,11-epoxycarbamazepine from contaminated soils.

Medicinal applications of perfluoroalkylated chain-containing compounds.

Compounds with polyfluorinated molecular fragments possess unique properties associated with the presence of a large number of fluorine atoms that affect lipophilicity and conformational rigidity of the parent molecule along with other effects. The aim of this review is to provide an overview of synthesized compounds possessing perfluoroalkylated or polyfluorinated chains that have been tested for bioactivity or as potential drug candidates for the treatment of various diseases. As far as the length of the perfluoroalkylated chain is concerned the focus is centered on the compound bearing perfluoroethyl or tetrafluoroethyl as well as longer chains. The perfluoroalkylated compounds discussed are classified according to their biological activity.

Using phytoremediation technologies to upgrade waste water treatment in Europe.

GOAL, SCOPE AND BACKGROUND: One of the burning problems of our industrial society is the high consumption of water and the high demand for clean drinking water. Numerous approaches have been taken to reduce water consumption, but in the long run it seems only possible to recycle waste water into high quality water. It seems timely to discuss alternative water remediation technologies that are fit for industrial as well as less developed countries to ensure a high quality of drinking water throughout Europe. MAIN FEATURES: The present paper discusses a range of phytoremediation technologies to be applied in a modular approach to integrate and improve the performance of existing wastewater treatment, especially towards the emerging micro pollutants, i.e. organic chemicals and pharmaceuticals. This topic is of global relevance for the EU. RESULTS: Existing technologies for waste water treatment do not sufficiently address increasing pollution situation, especially with the growing use of organic pollutants in the private household and health sector. Although some crude chemical approaches exist, such as advanced oxidation steps, most waste water treatment plants will not be able to adopt them. The same is true for membrane technologies. DISCUSSION: Incredible progress has been made during recent years, thus providing us with membranes of longevity and stability and, at the same time, high filtration capacity. However, these systems are expensive and delicate in operation, so that the majority of communities will not be able to afford them. Combinations of different phytoremediation technologies seem to be most promising to solve this burning problem. CONCLUSIONS: To quantify the occurrence and the distribution of micropollutants, to evaluate their effects, and to prevent them from passing through wastewater collection and treatment systems into rivers, lakes and ground water bodies represents an urgent task for applied environmental sciences in the coming years. RECOMMENDATIONS: Public acceptance of green technologies is generally higher than that of industrial processes. The EU should stimulate research to upgrade existing waste water treatment by implementing phytoremediation modules and demonstrating their reliability to the public.

Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions.

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu359-Lys373), peptide II (Leu30-Arg48), peptide III (Phe127-Arg140), and peptide IV (Arg434-Arg443). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg133, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.

Oxidative detoxication of carcinogenic 2-nitroanisole by human, rat and rabbit cytochrome P450.

OBJECTIVES: The detoxifying metabolism of a potent rodent carcinogen, 2-nitroanisole (2-NA) by human, rabbit and rat cytochromes P450 (P450) was investigated. Comparison between P450s of experimental animals and humans is essential for the extrapolation of animal carcinogenicity data to the human situation and to assess health risk. METHODS: HPLC with UV detection was employed for the separation and characterization of 2-NA metabolites formed by hepatic microsomes, human recombinant P450s and purified rat and rabbit P450s. RESULTS: An O-demethylated metabolite of 2-NA, 2-nitrophenol (2-NP), and two oxidation products of this metabolite [2,5-dihydroxynitrobenzene (2,5-DNB) and 2,6-dihydroxynitrobenzene (2,6-DNB)] were generated by microsomes and P450s from the species investigated, but at different levels. All the metabolites are detoxication products. 2-NP is the major metabolite generated by rabbit and rat microsomes, but 2,5-DNB is the predominant product in human microsomes. Using human recombinant P450s and purified rodent P450s, we found that human P450 2E1, 1A1 and 2B6 as well as orthologous animal P450s were the most efficient enzymes oxidizing 2-NA to 2-NP, while P450 2E1 and 1A1 were the most effective in the formation of 2,5-DNB and 2,6-DNB. In human hepatic microsomes, 2-NA was oxidized mainly by P4502E1. 2-NA and its reductive metabolite o-anisidine induced rat hepatic and renal P450 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1), thus modifying their own detoxication and/or activation pathways. CONCLUSIONS: The data demonstrated the participation of orthologous P450s in 2-NA oxidation by all species and indicated that the rat and rabbit might serve as suitable models to mimic 2-NA oxidation in man.

Structural requirements for inhibitors of cytochromes P450 2B: assessment of the enzyme interaction with diamondoids.

The series of diamondoids: adamantane, diamantane, triamantane, 2-isopropenyl-2-methyladamantane and 3-isopropenyl-3-methyldiamantane (3-IPMDIA), were employed to elucidate the molecular basis of their interaction with the active site of cytochromes P450 (CYP) of a 2B subfamily. These potent inhibitors of CYP2B enzymes were docked into the homology model of CYP2B4. Apparent dissociation constants calculated for the complexes of CYP2B4 with docked diamandoids agreed closely with the experimental data showing inhibition potency of the compounds and their binding affinity to CYP2B4. Superimposed structures of docked diamondoids mapped binding site residues. As they are mainly non-polar residues, the hydrophobicity plays the major role in the binding of diamondoids. Overlapping structure of diamondoids defined an elliptical binding cavity (5.9A inner diameter, 7.9A length) forming an angle of approximately 43 degrees with the heme plane. CYP2B specific diamondoids, namely 3-IPMDIA, showing the highest binding affinity, should be considered for a potential clinical use.

Identification of a genotoxic mechanism for 2-nitroanisole carcinogenicity and of its carcinogenic potential for humans.

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated in this study. Here we have used two independent methods, (32)P-post-labeling and (3)H-labeled 2-NA, to show that 2-NA binds covalently to DNA in vitro after reductive activation by human hepatic cytosol and xanthine oxidase (XO). We also investigated the capacity of 2-NA to form DNA adducts in vivo. Male Wistar rats were treated i.p. with 2-NA (0.15 mg/kg body wt daily for 5 days) and DNA from several organs was analyzed by (32)P-post-labeling. Two 2-NA-specific DNA adducts, identical to those found in DNA incubated with 2-NA and human hepatic cytosol or XO in vitro, were detected in the urinary bladder (3.4 adducts/10(7) nt), the target organ, and, to a lesser extent, in liver, kidney and spleen. The two DNA adducts found in rat tissues in vivo were identified as deoxyguanosine adducts derived from a 2-NA reductive metabolite, N-(2-methoxyphenyl)hydroxylamine. This reactive metabolite of 2-NA was identified in incubations with human hepatic cytosol, besides 2-methoxyaniline (o-anisidine). The results of our study, the first report on the potential of human cytosolic enzymes to contribute to the activation of 2-NA by nitroreduction, strongly suggest a carcinogenic potency of this rodent carcinogen for humans.

Phytoremediation of polyaromatic hydrocarbons, anilines and phenols.

Phytoremediation technologies based on the combined action of plants and the microbial communities that they support within the rhizosphere hold promise in the remediation of land and waterways contaminated with hydrocarbons but they have not yet been adopted in large-scale remediation strategies. In this review plant and microbial degradative capacities, viewed as a continuum, have been dissected in order to identify where bottle-necks and limitations exist. Phenols, anilines and polyaromatic hydrocarbons (PAHs) were selected as the target classes of molecule for consideration, in part because of their common patterns of distribution, but also because of the urgent need to develop techniques to overcome their toxicity to human health. Depending on the chemical and physical properties of the pollutant, the emerging picture suggests that plants will draw pollutants including PAHs into the plant rhizosphere to varying extents via the transpiration stream. Mycorrhizosphere-bacteria and -fungi may play a crucial role in establishing plants in degraded ecosystems. Within the rhizosphere, microbial degradative activities prevail in order to extract energy and carbon skeletons from the pollutants for microbial cell growth. There has been little systematic analysis of the changing dynamics of pollutant degradation within the rhizosphere; however, the importance of plants in supplying oxygen and nutrients to the rhizosphere via fine roots, and of the beneficial effect of microorganisms on plant root growth is stressed. In addition to their role in supporting rhizospheric degradative activities, plants may possess a limited capacity to transport some of the more mobile pollutants into roots and shoots via fine roots. In those situations where uptake does occur (i.e. only limited microbial activity in the rhizosphere) there is good evidence that the pollutant may be metabolised. However, plant uptake is frequently associated with the inhibition of plant growth and an increasing tendency to oxidant stress. Pollutant tolerance seems to correlate with the ability to deposit large quantities of pollutant metabolites in the 'bound' residue fraction of plant cell walls compared to the vacuole. In this regard, particular attention is paid to the activities of peroxidases, laccases, cytochromes P450, glucosyltransferases and ABC transporters. However, despite the seemingly large diversity of these proteins, direct proof of their participation in the metabolism of industrial aromatic pollutants is surprisingly scarce and little is known about their control in the overall metabolic scheme. Little is known about the bioavailability of bound metabolites; however, there may be a need to prevent their movement into wildlife food chains. In this regard, the application to harvested plants of composting techniques based on the degradative capacity of white-rot fungi merits attention.