nitrophenols and 4-nitroanisole

Our previous studies showed that administration of a subtoxic dose of acetaminophen (APAP) to female rats increased generation of carbon monoxide from dichloromethane, a metabolic reaction catalyzed mainly by cytochrome P450 (CYP) 2E1. In this study we examined the changes in metabolism and toxicity of APAP upon repeated administration. An intraperitoneal dose of APAP (500 mg/kg) alone did not increase aspartate aminotransferase, alanine aminotransferase, or sorbitol dehydrogenase activity in serum, but was significantly hepatotoxic when the rats had been pretreated with an identical dose of APAP 18 h earlier. The concentrations and disappearance of APAP and its metabolites in plasma were monitored for 8 h after the treatment. APAP pretreatment reduced the elevation of APAP-sulfate, but increased APAP-cysteine concentrations in plasma. APAP or APAP-glucuronide concentrations were not altered. Administration of a single dose of APAP 18 h before sacrifice increased microsomal CYP activities measured with p-nitrophenol, p-nitroanisole, and aminopyrine as probes. Expression of CYP2E1, CYP3A, and CYP1A proteins in the liver was also elevated significantly. The results suggest that administration of APAP at a subtoxic dose may result in an induction of hepatic CYP enzymes, thereby altering metabolism and toxicological consequences of various chemical substances that are substrates for the same enzyme system.

Topics: Acetaminophen; Alanine Transaminase; Aminopyrine; Analgesics, Non-Narcotic; Animals; Anisoles; Aspartate Aminotransferases; Cysteine; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Injections, Intraperitoneal; L-Iditol 2-Dehydrogenase; Liver; Microsomes, Liver; Nitrophenols; Rats; Rats, Sprague-Dawley

Different sensitivity of the xenobiotic biotransformation system to phenobarbital treatment in female and male rat hepatocytes is shown. Hepatocytes isolated from females had less cytochrome P-450 content and metabolized the xenobiotic p-nitroanisole (p-NA) more slowly than hepatocytes from males. Phenobarbital treatment increased the cytochrome P-450 amount and the p-nitrophenol (p-NPh) formation rate in hepatocytes from female and male rats to the same extent. However, in control and female induced cells the main form of p-NPh was so conjugates, while induction of male rats reactions of conjugation were depressed and free p-NPh accumulated. The sex differences in the response of xenobiotic biotransformation system to an inducing treatment should be taken into account into transplantation and prescription of drug treatment courses.

Topics: Animals; Anisoles; Biotransformation; Cells, Cultured; Cytochrome P-450 Enzyme System; Female; Liver; Male; Nitrophenols; Phenobarbital; Rats; Rats, Wistar; Sex Characteristics; Xenobiotics

1. Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism. Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures. Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro. 2. The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells. Light microscopical examination of the cultures indicated that the cells remained viable over 1 month. As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity. 3. An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis. After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values. 4. Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital. Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected. Almost identical metabolism was found, comparing day 3 with 16 of culture. 5. We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.

Topics: Acetaminophen; Adult; Aged; Anisoles; Biotransformation; Cell Separation; Cell Survival; Cells, Cultured; Collagen; Culture Media; Cytochrome P-450 Enzyme System; DNA; Extracellular Matrix; Female; Gels; Glucuronates; Humans; Liver; Nitrophenols; Sulfates

The success rate of liver transplantation has improved markedly during the last few years and, although this patient population receives multiple drug therapies, the effect of liver transplantation on drug metabolism has been studied very little. Therefore, the purpose of this study was to assess the metabolism of model drug substrates after liver transplantation in the rat. Rat livers were stored for 4 hr in cold Euro-Collins solution, transplanted orthotopically, and then perfused 2 hr later with oxygenated Krebs-Henseleit buffer, using a nonrecirculating system. Rates of monooxygenation of the model compound p-nitroanisole, conjugation of p-nitrophenol, and uptake of oxygen were measured. All parameters studied were elevated significantly, by nearly 2-fold, by transplantation. Specifically, monooxygenation was increased from 2.9 +/- 0.2 to 5.1 +/- 0.4 mumol/g/hr, conjugation was elevated from 3.3 +/- 0.6 to 7.7 +/- 0.1 mumol/g/hr, and O2 uptake was stimulated from basal values of 114 to 197 mumol/g/hr. Transplantation did not, however, alter rates of monooxygenation and conjugation in isolated microsomes supplemented with excess cofactor. When donor rats were pretreated with the Kupffer cell toxicant gadolinium chloride (10 mg/kg, intravenously) 30 hr before liver storage, the elevation after transplantation in all parameters studied was prevented. Depletion of carbohydrate reserves by fasting of donor rats did not prevent stimulation of monooxygenation and conjugation. On the other hand, urea synthesis from ammonium chloride, a process dependent on mitochondrial NADPH, was increased and monooxygenation was diminished after transplantation, suggesting the involvement of mitochondria in this phenomenon. Indeed, mitochondria isolated 2 hr postoperatively exhibited significantly elevated respiratory control ratios and higher state 3 rates of respiration. Taken together, these data support the hypothesis that Kupffer cells, activated by transplantation, release mediators that stimulate mitochondria in parenchymal cells and enhance drug metabolism by increasing cofactor supply (e.g., NADPH for monooxygenation and UDP-glucuronic acid for glucuronidation).

Topics: Ammonium Chloride; Animals; Anisoles; Carbohydrate Metabolism; Female; Gadolinium; Kupffer Cells; Liver; Liver Transplantation; Mitochondria, Liver; Nitrophenols; Oxidation-Reduction; Oxygen; Oxygen Consumption; Perfusion; Rats; Rats, Inbred Strains; Time Factors

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

Topics: Animals; Anisoles; Glucuronates; In Vitro Techniques; Kinetics; Liver; Male; Nitrophenols; Perfusion; Polychlorinated Biphenyls; Salmonidae; Trout

The conjugative metabolism of p-nitroanisole (pNA) and p-nitrophenol (pNP) was studied in isolated hepatocytes of male and female rats rendered diabetic with streptozotocin. Hepatocytes of male diabetic rats formed more of the glucuronide conjugate from pNA than controls and a tendency toward higher sulfate conjugate production was observed. By summation of conjugated metabolites and unconjugated phenol, it was determined that O-demethylation of pNA was also increased, possibly accounting for the increased production of conjugated metabolites. Production of the sulfate conjugate directly from pNP was inhibited in male diabetic hepatocytes when the substrate concentration was 50 microM but was not altered at other substrate concentrations. Glucuronidation was increased in hepatocytes of diabetic rats when pNP was used as substrate at concentrations of 50 and 100 microM, whereas there was no difference from control at 25 microM pNP. Treatment of diabetic rats with insulin returned glucuronidation to control values. The effect of diabetes on glucuronidation in hepatocytes was not due to increases in microsomal glucuronyltransferase activity. In contrast to males, no differences were observed between hepatocytes of control and diabetic female rats in the capacity to form conjugated metabolites from pNA or pNP. It is consistent with previous reports that alterations in carbohydrate metabolism in hepatocytes of male diabetic rats led to accelerated formation of uridine diphosphate glucuronic acid, resulting in greater rates of glucuronidation.

Topics: Animals; Anisoles; Diabetes Mellitus, Experimental; Female; Glucuronates; Glucuronosyltransferase; In Vitro Techniques; Liver; Male; Microsomes, Liver; Nitrophenols; Rats; Sex Factors; Sulfates

Topics: Animals; Anisoles; Carbohydrates; Female; Fructose; Glucose; Glucuronates; Kinetics; Liver; Mixed Function Oxygenases; Nitrophenols; Nutritional Physiological Phenomena; Oxidoreductases; Rats

1. The disposition of an aromatic amine and three aromatic nitro compounds was investigated in the sea urchin, Strongylocentrotus purpuratus. 2. The sea urchin rapidly eliminated injected compounds. The elimination rate constants decreased in the order p-toluidine greater than p-nitroanisole = p-nitrophenol greater than p-nitrotoluene. The fraction of total injected compound eliminated in 8 h was lowest for p-nitrophenol less than p-toluidine less than p-nitrotoluene less than p-nitroanisole. 3. Biotransformation for the sea urchin was primarily reduction of the nitro group followed by acetylation of the amine. 4. Other animals, starfish (Pisaster ochraceus), sea cucumber (Cucumaria miniata), gum boot chiton (Cryptochiton stelleri) and mussels (Mytilus californianus), injected with p-nitroanisole exhibited a trend toward oxidative biotransformation. 5. Elimination of parent compound was the major pathway for reducing body burden of xenobiotics for the invertebrates studied. 6. p-Toluidine oxidizes during analysis and was thus not suitable for studying biotransformation.

Topics: Amines; Animals; Anisoles; Biotransformation; Bivalvia; Invertebrates; Mollusca; Nitro Compounds; Nitrophenols; Sea Cucumbers; Sea Urchins; Species Specificity; Starfish; Toluene; Toluidines; Water Pollutants; Water Pollutants, Chemical

Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and "malic" enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for "malic" enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes.

Topics: 6-Aminonicotinamide; Adenine Nucleotides; Adenosine Triphosphate; Animals; Anisoles; Female; Fructose; Liver; NADH, NADPH Oxidoreductases; NADP; NADPH Dehydrogenase; NADPH-Ferrihemoprotein Reductase; Nitroanisole O-Demethylase; Nitrophenols; Oxidation-Reduction; Perfusion; Rats

Phase-sensitive ac polarography was applied to the simultaneous quantitative determination of 4-nitroanisole, 4-nitrophenol, and 4-nitrocatechol in alkaline solutions. Certain experimental precautions are necessary to determine each compound in the presence of the other two. Thus, 4-nitrocatechol is determined indirectly by forming a yellow ratio chelate with cupric ions, wheras 4-nitroansole is determined directly by the reduction waves of the nitro group. For the determination of 4-nitrophenol, the interferency by the simultaneously present 4-nitrocatechol must be eliminated by masking it by the addition of magnesium ions. The method described permits a qualitative and quantitative analysis of all three compounds in one solution since linear calibration curves are obtained.

Topics: Anisoles; Catechols; Copper; Hydrogen-Ion Concentration; Magnesium; Methods; Nitro Compounds; Nitrophenols; Polarography

Phase sensitive alternating current polarography was introduced for the simultaneous determination of p-nitroanisole and its metabolites p-nitrophenol and p-nitrocatechol in kinetic studies with rat liver microsomes. The substrate p-nitroanisole disappears rather rapidly while p-nitrophenol is formed. First traces of a second oxidation product, p-nitrocatechol, can be detected only after a few minutes after the initiation of the reaction. This suggest that O-demethylation of p-nitroanisole is the primary reaction which is followed by aromatic ortho hydroxylation of p-nitrophenol. After incubation times longer than 15 minutes, appreciable amounts of p-nitrocatechol are found which shows optical absorption characteristics similar to those of p-nitrophenol (absorption maximum at 440 nm). It is concluded from these kinetic experiments that optical determination of the primary metabolite during the initial reaction phase constitutes a reliable measure of microsomal O-demethylation activity. Phenobarbital induction differentially increases O-demethylation and ring-ortho-hydroxylation activities. From this and respective inhibition studies it is concluded that possibly multiple forms of cytochrome P-450 are involved in the metabolism of either p-nitroanisole or p-nitrophenol.

Topics: Animals; Anisoles; Catechols; Dealkylation; Hydroxylation; In Vitro Techniques; Kinetics; Male; Metyrapone; Microsomes, Liver; Mixed Function Oxygenases; Nitrophenols; Phenobarbital; Polarography; Proadifen; Proteins; Rats; Time Factors