nitrophenols and 4-nitrophenyl-sulfate

Morphological and functional changes have been investigated in the rat model of Crohn's disease. The inflammatory bowel disease was induced by indomethacin (1 × 10 mg/kg s.c. for 3 days). Morphological alterations were evaluated by macroscopic scoring system and on the base of histological changes in the small intestine. Functional activities were studied by determination of the intestinal and hepatic elimination of p-Nitrophenol (PNP) and its metabolites (PNP-glucuronide: PNP-G and PNP-sulfate: PNP-S) during the luminal perfusion of PNP. It was found that the indomethacin induced severe macroscopic changes (hyperaemia, petechia, bleeding, erosions, ulcerations) and significant histological alterations in the small intestine of rats which were definitely inhibited by mesalazine (1000 mg/kg by gastric tube for 3 days). Disappearance of PNP from the luminal perfusion solution was diminished by indomethacin which was corrected by administration of mesalazine. Significant depression was found in the luminal appearance of PNP metabolites by giving of indomethacin and these alterations could not be compensated by mesalazine.Hepatic elimination of PNP (biliary excretion of PNP and its metabolites) was decreased definitely by indomethacin which was - at least partly - compensated by mesalazine.The findings of the present study suggest that the indomethacin-induced inflammation in the small intestine represents a useful rat model of Crohn's disease. Morphological and functional alterations caused by indomethacin can be compensated by mesalazine.

Topics: Animals; Crohn Disease; Disease Models, Animal; Glucuronates; Indomethacin; Inflammation; Inflammatory Bowel Diseases; Intestine, Small; Liver; Male; Mesalamine; Nitrobenzenes; Nitrophenols; Rats; Rats, Wistar

Enzymes capable of modifying the sulfated polymeric molecule of fucoidan are mainly produced by different groups of marine organisms: invertebrates, bacteria, and also some fungi. We have discovered and identified a new strain of filamentous fungus Fusarium proliferatum LE1 (deposition number in Russian Collection of Agricultural Microorganisms is RCAM02409), which is a potential producer of fucoidan-degrading enzymes. The strain LE1 (RCAM02409) was identified on the basis of morphological characteristics and analysis of ITS sequences of ribosomal DNA. During submerged cultivation of F. proliferatum LE1 in the nutrient medium containing natural fucoidan sources (the mixture of brown algae Laminaria digitata and Fucus vesiculosus), enzymic activities of α-L-fucosidase and arylsulfatase were inducible. These enzymes hydrolyzed model substrates, para-nitrophenyl α-L-fucopyranoside and para-nitrophenyl sulfate, respectively. However, the α-L-fucosidase is appeared to be a secreted enzyme while the arylsulfatase was an intracellular one. No detectable fucoidanase activity was found during F. proliferatum LE1 growth in submerged culture or in a static one. Comparative screening for fucoidanase/arylsulfatase/α-L-fucosidase activities among several related Fusarium strains showed a uniqueness of F. proliferatum LE1 to produce arylsulfatase and α-L-fucosidase enzymes. Apart them, the strain was shown to produce other glycoside hydrolyses.

Topics: alpha-L-Fucosidase; Arylsulfatases; DNA, Ribosomal; Fucus; Fusarium; Laminaria; Nitrobenzenes; Nitrophenols; Polysaccharides; Sequence Analysis

In the extrahepatic drug metabolism the intestinal tract can play an important role. These experiments were designed to study the biotransformation of p-nitrophenol (PNP) in the small intestine in the rat. Various segments of the small intestine (proximal and distal jejunum, terminal ileum) were perfused with isotonic solution in vivo containing different concentrations of PNP (20-100-500-1000 μM) and the concentrations of metabolites (PNP-G: p-nitrophenol glucuronide, PNP-S: p-nitrophenol sulfate) were determined in the perfusion medium. It was found a decreasing tendency in the glucuronidation from the proximal to distal segment of the small intestine: e.g. 430 nmol, 240 nmol, and 100 nmol PNP-G appeared in the perfusion medium in the proximal, distal jejunum and in the terminal ileum, respectively, when 500 μM PNP was luminally perfused for 90 minutes. Similar ratio was found at the luminal perfusion of other PNP-concentrations, too. Luminal appearance of sulfoconjugate of PNP was considerably lower and no clear gradient tendency in the formation of PNP-S could be detected in the small intestine from the proximal to distal segment. Our results show that there are considerable differences in drug metabolism in various segments of the small intestine. We have found a gradient conjugating activity from proximal to distal segment of small intestine in the glucuronidation of PNP.

Topics: Animals; Glucuronates; Inactivation, Metabolic; Intestine, Small; Male; Nitrobenzenes; Nitrophenols; Rats; Rats, Wistar

Both phosphoryl and sulfuryl transfers are ubiquitous in biology, being involved in a wide range of processes, ranging from cell division to apoptosis. Additionally, it is becoming increasingly clear that enzymes that can catalyze phosphoryl transfer can often cross-catalyze sulfuryl transfer (and vice versa). However, while there have been extensive experimental and theoretical studies performed on phosphoryl transfer, the body of available research on sulfuryl transfer is comparatively much smaller. The present work presents a direct theoretical comparison of p-nitrophenyl phosphate and sulfate monoester hydrolysis, both of which are considered prototype systems for probing phosphoryl and sulfuryl transfer, respectively. Specifically, free energy surfaces have been generated using density functional theory, by initial geometry optimization in PCM using the 6-31+G* basis set and the B3LYP density functional, followed by single-point calculations using the larger 6-311+G** basis set and the COSMO continuum model. The resulting surfaces have been then used to identify the relevant transition states, either by further unconstrained geometry optimization or from the surface itself where possible. Additionally, configurational entropies were evaluated using a combination of the quasiharmonic approximation and the restraint release approach and added to the calculated activation barriers as a correction. Finally, the overall activation entropy was estimated by approximating the solvent contribution to the total activation entropy using the Langevin dipoles solvation model. We have reproduced both the experimentally observed activation barriers and the observed trend in the activation entropies with reasonable accuracy, as well as providing a comparison of calculated and observed (15)N and (18)O kinetic isotope effects. We demonstrate that, counterintuitively, the hydrolysis of the p-nitrophenyl sulfate proceeds through a more expansive pathway than its phosphate analogue. Additionally, we show that the solvation effects upon moving from the ground state to the transition state are quite different for both reactions, suggesting that the enzymes that catalyze these reactions would need active sites with quite different electrostatic preorganization for the efficient catalysis of either reaction (despite which many enzymes can catalyze both phosphoryl and sulfuryl transfer). We believe that such a comparative study is an important foundation for understanding the

Topics: Catalysis; Enzymes; Hydrolysis; Kinetics; Models, Molecular; Models, Theoretical; Nitrobenzenes; Nitrophenols; Organophosphorus Compounds; Solutions; Sulfur; Water

Kinetic isotope effects and medium effects have been measured for sulfuryl-transfer reactions of the sulfate ester p-nitrophenyl sulfate (pNPS). The results are compared to those from previous studies of phosphoryl transfer, a reaction with mechanistic similarities. The N-15 and the bridge O-18 isotope effects for the reaction of the pNPS anion are very similar to those of the p-nitrophenyl phosphate (pNPP) dianion. This indicates that in the transition states for both reactions the leaving group bears nearly a full negative charge resulting from a large degree of bond cleavage to the leaving group. The nonbridge O-18 isotope effects support the notion that the sulfuryl group resembles SO(3) in the transition state. The reaction of the neutral pNPS species in acid solution is mechanistically similar to the reaction of the pNPP monoanion. In both cases proton transfer from a nonbridge oxygen atom to the leaving group is largely complete in the transition state. Despite their mechanistic similarities, the phosphoryl- and sulfuryl-transfer reactions differ markedly in their response to medium effects. Increasing proportions of the aprotic solvent DMSO to aqueous solutions of pNPP cause dramatic rate accelerations of up to 6 orders of magnitude, but only a 50-fold rate increase is observed for pNPS. Similarly, phosphoryl transfer from the pNPP dianion to tert-amyl alcohol is 9000-fold faster than the aqueous reaction, while the sulfuryl transfer from the pNPS anion is some 40-fold slower. The enthalpic and entropic contributions to these differing medium effects have been measured and compared.

Topics: Anions; Dimethyl Sulfoxide; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Nitrobenzenes; Nitrophenols; Organophosphorus Compounds; Oxygen Isotopes; Pentanols; Solvents; Water

Effect of hyperglycemia on the intestinal elimination of p-nitrophenol has been investigated in rats. Hyperglycemia was produced by a continuous i.v. infusion of glucose, p-nitrophenol was used as a model compound for the investigation of intestinal metabolism and excretion of drugs. Intestinal conjugation of p-nitrophenol with glucuronic acid did not change significantly in hyperglycemic rats, however, formation of sulfoconjugate of p-nitrophenol was enhanced by hyperglycemia. Sum of metabolites (p-nitrophenol glucuronide and sulfate) appeared in the intestinal lumen in hyperglycemic rats was similar to the total luminal appearance of these metabolites of control rats.

Topics: Animals; Glucuronates; Hyperglycemia; Intestinal Mucosa; Nitrobenzenes; Nitrophenols; Rats; Reference Values

1. 4-Nitrophenol and 4-nitroanisole were applied either dermally or arterially to isolated rabbit ears perfused under single-pass conditions with protein-free buffer solution. 2. 4-Nitroanisole yielded only phase II metabolites of 4-nitrophenol. 3. The apparent Vmax values for 4-nitrophenol glucuronidation and sulphation were about 20 pmol and 10 pmol/min per cm2, respectively. 4. The difference in apparent Km between dermal and arterial drug application is a measure of first-pass metabolism by the epidermal layer. 5. The amount of 4-nitrophenyl conjugate detected after 4-nitroanisole administration was assumed to represent O-dealkylation of 4-nitroanisole; the capacity of this reaction was one order of magnitude lower than the direct conjugation of 4-nitrophenol.

Topics: Administration, Cutaneous; Animals; Anisoles; Ear; Glucuronates; Glycoconjugates; In Vitro Techniques; Infusions, Intra-Arterial; Kinetics; Nitrobenzenes; Nitrophenols; Perfusion; Rabbits; Skin

p-Nitrophenol (pNP) and its conjugated metabolites, generated in a perfused rat liver preparation, are readily separated and quantitated in serum perfusate and bile samples using a reverse-phase high-performance liquid chromatographic method. Serum perfusate samples can be analyzed following protein precipitation with acetonitrile: following protein precipitation with 1.5 M perchloric acid (1 part to 2 parts serum) there was degradation of pNP sulfate to pNP when samples were stored at room temperature. pNP can also be analyzed in blood perfusate samples following extraction with a number of organic solvents including ethyl acetate or isobutanol-methylene chloride (4:1, v/v). Rat liver perfusions at a constant input concentration of 40 microM demonstrated a high hepatic extraction ratio of pNP (mean of 0.90) due to the formation of the sulfate and glucuronide conjugates; no pNP glucoside was detected in perfusate or bile samples.

Topics: Animals; Bile; Butanols; Chromatography, High Pressure Liquid; Glucuronates; Liver; Male; Methylene Chloride; Nitrobenzenes; Nitrophenols; Perfusion; Rats; Rats, Inbred Strains; Solvents

Single-pass perfusion in situ of mouse livers with the organophosphate paraoxon resulted in formation of p-nitrophenol (PNP), p-nitrophenyl sulfate (PNPS), and p-nitrophenyl-beta-D-glucuronide (PNPG). Following initiation of perfusion of paraoxon steady--state conditions were achieved in 15-25 min, at which time the extraction ratio was 0.55 (S.D. = 0.05). This suggests the capacity of mouse liver to biotransform paraoxon is not as great as previously reported. At all concentrations of paraoxon examined the amount of PNPS produced exceeded that of PNPG. However, as the concentration of paraoxon increased the relative proportion of PNP to PNPS and PNPG increased, indicating the capacity of liver to biotransform paraoxon exceeded the capacity to biotransform PNP. Single-pass perfusion in situ of mouse livers with PNP resulted in production of PNPS and PNPG. As with paraoxon, steady-state conditions were achieved in 15-25 min. The extraction ratio of PNP, as well as the metabolic profile, changed markedly with varying concentrations of PNP. At PNP reservoir concentrations of 4 microM or less the extraction ratio of PNP was 1, with all PNP metabolized to PNPS. As PNP concentrations increased (up to 75 microM) both unchanged PNP and PNPG appeared in the effluent. Thus the hepatic biotransformation of PNP was clearly dependent on substrate concentration.

Topics: Animals; Biotransformation; Glucuronates; Glutathione; In Vitro Techniques; Liver; Male; Mice; Nitrobenzenes; Nitrophenols; Paraoxon

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.

Topics: 4-Aminobenzoic Acid; Animals; Cytochrome P-450 Enzyme System; Glucuronates; In Vitro Techniques; Liver; Male; Methylcholanthrene; Nitrobenzenes; Nitrophenols; Phenobarbital; Rats; Rats, Inbred Strains; Sulfates