Comparative metabolism of nitroaromatic compounds in freshwater, brackish water and marine decapod crustaceans

1. The metabolic pathways of p-nitroanisole, 4-nitro-m-cresol and methyl parathion in Malaysian prawns (Macrobrachium rosenbergii), ridgeback prawns (Sicyonia ingentis) and crayfish (Procambarus clarkii) were compared. 2. The Malaysian prawns and ridgeback prawns were shown to O-demethylate 29% and 11%, respectively, of an accumulated level of p-nitroanisole, while crayfish were able to O-demethylate 98% of the accumulated level of nitroanisole. 4-Nitro-m-cresol oxidation was not detected in either prawn species. In contrast, 5-hydroxy-2-nitrobenzaldehyde was the major metabolite formed from nitrocresol metabolism by crayfish. 3. Both prawn species readily dearylated methyl parathion to form p-nitrophenol and p-nitrophenyl conjugates. Crayfish displayed a similar trend in methyl parathion metabolism. 4. Nitroreduction was observed in the metabolism of 4-nitro-m-cresol by ridgeback prawns, which excreted 4-nitroso-m-cresol as a minor product. Reduction products were not observed in the metabolism of the three substrates by Malaysian prawns or crayfish. 5. Conjugation was overall the dominant detoxication pathway observed in the decapods. Malaysian prawns conjugated p-nitrophenol and 4-nitro-m-cresol to form the corresponding beta-D-glucosides and sulphate monoesters. Ridgeback prawns formed beta-D-glucosides in small quantities, preferring conjugation of p-nitrophenol and 4-nitro-m-cresol to form sulphates and unknown conjugates through a unique conjugation pathway. Crayfish conjugated the phenolic substrates to form exclusively the beta-glucosides. 6. The unknown conjugates formed by ridgeback prawns had chromatographic properties similar to the corresponding beta-D-glucosides but were refractory to the deconjugation by alpha- or beta-glucosidase, beta-galactosidase, aryl sulphatase and beta-glucuronidase. Phenolic conjugation ability appeared to follow the order of ridgeback prawns greater than Malaysian prawns greater than crayfish.     

The Metabolism of Foreign Compounds in the Cestode,Moniezia expansa,and the Nematode,Ascaris lumbricoides var suum

1. The ability of the cestode Moniezia expansa and the nematode Ascaris lumbricoides var suum to metabolize foreign compounds has been assessed.

2. Both species were unable to oxidase aldrin, aniline, biphenyl, butyl-benzene and nitrobenzene or to demethylate aminopyrine, and 4-nitroanisole.

3. M. expansa and A. lumbricoides var suum readily reduced 4-nitroanisole, nitrobenzene, 4-nitrobenzoic acid and 4-nitrophenol to the corresponding amines. Azobenzene, dimethylaminoazobenzene, and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene were also reduced.

4. Hydrolysis of esters, acetanilide, acetylsalicylic acid, aryl sulphates and aryl phosphates took place readily. However, β-glucuronides were not hydrolysed.

5. The following reactions were not detected in either species: phosphate, sulphate, β-glucuronide or β-glycoside conjugation of phenolic compounds; acetylation of amino compounds, or the formation of glycine conjugates with 4-aminobenzoic acid or benzoic acid.

6. Male nematodes showed a higher rate of drug metabolism than female nematodes.     

Stimulation of p-nitroanisole O-demethylation in perfused livers by xylitol and sorbitol

Xylitol and sorbitol, two sugar alcohols which readily enter into pathways of hepatic carbohydrate metabolism, stimulated p-nitroanisole O-demethylation in perfused livers from fasted, but not fed, phenobarbital-treated rats. The increase in mixed-function oxidation correlated well with the production of NADH from the metabolism of xylitol and sorbitol (half-maximal stimulation for both processes was observed with concentrations between 0.1 and 0.2 mM). p-Nitroanisole metabolism by isolated hepatic microsomes was unaffected by the addition of xylitol and sorbitol; however, when NADH was added to microsomes, or was generated from sorbitol, sorbitol dehydrogenase and NAD⁺, a synergistic increase in p-nitroanisole metabolism occurred. Ethanol (0.2 mM), which does not enter into pathways of carbohydrate metabolism, also caused an increase in the pyridine nucleotide redox state and stimululated p-nitroanisole O-demethylation in livers from fasted rats. In addition, sorbitol and xylitol stimulated p-nitrophenol conjugation in livers from fasted, phenobarbital-treated animals, probably by supplying substrate for increased UDP-glucuronic acid synthesis. The data indicate that sugars which influence the pyridine nucleotide redox state alter rates of mixed-function oxidation and conjugation in whole cells.     

The metabolism of phenol and substituted phenols in zebra fish

1. The metabolism of five phenols in zebra fish was studied after uptake from the medium. The results showed no qualitative differences to other Cyprinid fish species, only the oxidation rate seemed to be lower.

2. Phenyl glucuronide, phenyl sulphate, and quinol sulphate were identified as metabolites of phenol.

3. Identified metabolites of 2-cresol were 2-cresyl glucuronide, 2-cresyl sulphate, and 2-hydroxybenzoic acid in trace amounts.

4. Only the glucuronide and sulphate conjugates were detected as metabolites of 4-nitrophenol, 4-chlorophenol, and pentachlorophenol.     

Identification of the cytochrome P450 isoforms involved in the O-demethylation of 4-nitroanisole in human liver microsomes

1. 4-Nitroanisole is O-demethylated to 4-nitrophenol by human liver microsomes. Kinetic studies indicate that this metabolic route is mediated by two cytochrome P450 isoforms, one with a K_m = 2.1 μM and the other with a K_m = 220 μM. 2. Chemical inhibition and correlation studies in human liver microsomes indicate that the low K_m enzyme is CYP2A6 and the high K_m enzyme is CYP2E1 suggesting that 4-nitroanisole is not a general cytochrome P450 substrate. 3. Studies using expressed recombinant cytochrome P450s indicated that all the cytochrome P450s investigated metabolized 4-nitroanisole but CYP2A6 and CYP2E1 produced the highest rates. Kinetic studies with these two isoforms produced a K_m for CYP2A6 of 9 μM and 54 μM for CYP2E1. 4. The involvement of these two isoforms in the O-demethylation of 4-nitroanisole can be rationalized in terms of a hydrogen bond interaction with the nitro group and the active site of CYP2A6 and a hydrophobic interaction with the active site of CYP2E1.     

Dermal metabolism of 4-nitrophenol and 4-nitroanisole in single-pass perfused rabbit ears.

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.     

Different Results of the Salmonella umu Test Between Three Isomers of Phenylenediamine (PDA) Derivatives

A post-treatment assay of the umu test was performed to detect genotoxicity of 10 phenylenediamine (PDA) derivatives using Salmonella typhimurium TA1535/pSK1002 with/without S9 mix. Seven chemicals (o-PDA, 4-chloro-o-PDA, 4-nitro-o-PDA, p-PDA, 2-chloro-p-PDA, 2-nitro-p-PDA, and 2,5-diaminotoluene) showed positive results with S9 mix, but three chemicals (m-PDA, 4-chloro-m-PDA, and 2,4-diaminotoluene) were negative with and without S9 mix. Four of 7 chemicals (o-PDA, 4-chloro-o-PDA, 4-nitro-o-PDA, and 2-nitro-p-PDA) that gave positive results with S9 mix were also positive without S9 mix. These results indicate that the genotoxicity of PDA derivative possessing m-position amino substituents was not detected in the umu post-treatment assay using TA1535/pSK1002.     

Urinary excretion kinetics of p-nitrophenol following oral administration of parathion in the rabbit

The urinary excretion kinetics of p-nitrophenol were studied in rabbits following oral administration of parathion at a dose of 3 mg/kg. Elimination of p-nitrophenol began rapidly, and of the total amount excreted during the study period, 46% was excreted in the first 3 h; 85% was excreted at 6 h after administration of the pesticide. The mean maximum excretion rate of p-nitrophenol was 111.15±61.02 g/h reached in a time of 0.77±0.26 h. The formation and disappearance rate constants of the metabolite were 2.85±2.80 h^–1 and 0.80±0.28 h^–1, respectively. A linear relationship was observed between the plasma concentrations of parathion and the urinary excretion rate of p-nitrophenol.     

Interplay of metabolizing enzymes and transporter of xenobiotics

1.?Xenobiotics are metabolized and eliminated through the coordinated interplay of their metabolizing enzymes and transporters. However, these two activities in vitro are measured separately, with the addition of ATP as a pre-requisite.

2.?We propose a human renal cell-line model which integrates the sulfate and glutathione conjugation of xenobiotics with the efflux of their respective conjugates. Sulfation and glutathionylation represent two major Phase II detoxification of xenobiotics in man. The reactions are catalyzed, respectively, by phenolsulfotransferase and glutathione-S-transferase followed by extrusion of their respective conjugates.

3.?Using Ko-143, a specific inhibitor of breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) transporter, we identified this transporter to be responsible for the efflux of p-cresol sulfate, harmol sulfate and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene.

4.?The conjugation-cum-efflux was inhibited by oligomycin and uncouplers, which highlights the role of cellular mitochondria in providing ATP for the biosynthesis of their conjugating agents, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and reduced glutathione as well as for the transport function of BCRP.

5.?The human 786-O renal cell-line provides a “3-in-1” system linking ATP biosynthesis to metabolism of xenobiotics and their ultimate transport and elimination by BCRP; this integrated system was not apparent in other human cell-lines examined.     

Aryl ether O-dealkylase activity in the skin of untreated mice in vitro

1. O-Dealkylation of p-nitroanisole and p-nitrophenetole in 10000g supernatant preparations of mouse skin is detectable and quantifiable. The reaction is NADPH-dependent and is mediated by cytochrome P-450.

2. The rate of p-nitroanisole O-demethylation in mouse skin preparations is 2% of that in mouse liver preparations on a?mg protein basis, but only 0.23% on a?g of tissue basis.

3. For p-nitrophenetole O-deethylation, the cutaneous V _max is 3.8% of the hepatic V _max on a?mg protein basis. The K_m values for the reaction in these two tissues are in a 3 : 1 ratio, suggesting that there are no marked qualitative differences between cutaneous and hepatic cytochrome P-450.     

Effects and metabolic pathway of 4-dimethylaminophenol during kidney perfusion

1. Isolated rat kidneys were perfused (single pass) with 4 to 40μM soln. of 4-dimethylamino[¹⁴C]phenol (DMAP).

2. Nephrotoxicity was not detected at concn. of ¹⁴C-DMAP up to 18μM; higher conc. resulted in irreversible loss of physiological functions with simultaneous five-fold increase in covalently bound ¹⁴C.

3. At all concn., 85% of the post-renal ¹⁴C was due to unchanged DMAP, while 15% corresponded to DMAP conjugates. These conjugates were identified as DMAP-glucuronide (90% total), DMAP-sulphate and DMAP-thioethers.

4. All DMAP conjugates were conc. in the urine with urine/perfusate concn. ratios in the range 5–7 for the glucuronide, 50–100 for the sulphate, and 10–20 for the thioethers.

5. Rates of formation of metabolites vs DMAP concn. followed Michaelis-Menten kinetics for DMAP-sulphate (K_m 25μM, V_max2.2nmol/min per g) and DMAP-thioethers (K_m range 10–100μM, V_max 4nmol/min per g). DMAP-glucuronide formation rate increased linearly with DMAP concn. and showed a four-fold increase at toxic DMAP doses.

6. From the data for DMAP conjugation capacity, urine/perfusate concn. ratios of DMAP conjugates and microautoradiography, it is suggested that DMAP conjugation is located mainly in the proximal convoluted tubule, where deterioration of renal functions originates.     

Inhibition of p-nitrophenol glucuronidation by calcium mobilizing hormones

1. Vasopressin and phenylephrine markedly inhibited the glucuronidation of p-nitro-phenol in isolated murine hepatocytes.

2. After longer preincubation of hepatocytes in the presence of vasopressin or phenylephrine the rate of conjugation began to return to the control values indicating the reversibility of the inhibition caused by these agents.

3. The inhibitory effect of both agents was dependent on the Ca²⁺ filled state of the intracellular stores.

4. The inhibition caused by the α1 receptor agonist phenylephrine was receptor mediated because it could be prevented by the addition of α1 antagonist prazosin.

5. The data support the theory that the maintenance of the intralumenal Ca²⁺ concentration is necessary for the optimal activity of p-nitrophenol UDP-glucuronosyl-transferase.     

Excretion and metabolism of phenol, 4-nitrophenol and 2-methylphenol by the frogs Rana temporaria and Xenopus laevis

1. Rana and Xenopus excrete 90–95% dose, and metabolize 50–65% dose of phenol, 4-nitrophenol and 2-methylphenol within 24 h, to about the same extent.

2. Kinetic data for the excretion of phenols from both species fit a two-compartment model. The elimination constants of Rana and Xenopus are not significantly different.

3. Metabolism is mostly conjugation by glucuronidation and sulphation of the original phenols. Additionally, oxidations leading to dihydroxyphenols and benzoic acid from 2-methylphenol, and reduction of 4-nitrophenol occur, followed by conjugation.

4. There is an important difference between the metabolite patterns of Rana and Xenopus in that the latter is unable to glucuronidate phenols. As the amount of metabolites produced is similar in both species, Xenopus compensates for its inability to glucuronidate by increasing other metabolites.     

The metabolism of foreign compounds in the cestode, Moniezia expansa, and the nematode, Ascaris lumbricoides var suum.

1. The ability of the cestode Moniezia expansa and the nematode Ascaris lumbricoides var suum to metabolize foreign compounds has been assessed. 2. Both species were unable to oxidase aldrin, aniline, biphenyl, butylbenzene and nitrobenzene or to demethylate aminopyrine, and 4-nitroanisole. 3. M. expansa and A. lumbricoides var suum readily induced 4-nitroanisole, nitrobenzene, 4-nitrobenzoic acid and 4-nitrophenol to the corresponding amines. Azobenzene, dimethylaminoazobenzene, and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene were also reduced. 4. Hydrolysis of esters, acetanilide, acetylsalicylic acid, aryl sulphates and aryl phosphates took place readily. However, beta-glucuronides were not hydrolysed. 5. The following reactions were not detected in either species: phosphate, sulphate, beta-glucuronide or beta-glycoside conjugation of phenolic compounds; acetylation of amino compounds, or the formation of glycine conjugates with 4-aminobenzoic acid or benzoic acid. 6. Male nematodes showed a higher rate of drug metabolism than female nematodes.     

Biochemical characteristics of insect microsomes. N- and O-demethylation

The N-demethylation of N,N-dimethyl-p-nitrophenol carbamate and the O-demethylation of p-nitroanisole by the microsomes from housefly abdomens were studied.     

Acute toxicity of the insecticide methyl parathion and its hydrolytic product p-nitrophenol to the native Australian cladoceran Daphnia carinata

The toxicity of an organophosphorus (OP) insecticide, methyl parathion (MP), and its hydrolysis product, p-nitrophenol (PNP), to the native Australian cladoceran species, Daphnia carinata, was assessed. Both MP and PNP were stable in cladoceran water during the test period. D. carinata was sensitive to both MP and PNP; however, the parent compound was more toxic than its metabolite. This is the first study that demonstrated the acute toxicity of MP and PNP towards an Australian daphnid species. The present investigation emphasizes the need for including the native taxa as non-target test organisms while evaluating the toxicity of environmental pollutants.

     

Kinetic properties of UDP-glucuronosyltransferase(S) in different membranes of rat liver cells

1. Glucuronidation of 4-nitrophenol, borneol and morphine occurred in rough and smooth endoplasmic reticulum, Golgi apparatus and plasma membranes of rat liver cells.

2. In all fractions, prior fixation of either substrate (UDP-glucuronic acid or the aglycone) enhanced the affinity for the second substrate.

3. Whatever the membrane, glucuronidation of 4-nitrophenol was characterized by high V_max and high affinity for UDP-glucuronic acid. On the other hand, glucuronidation of borneol exhibited a lower V_max and a lower affinity for UDP-glucuronic acid.

4. In the endoplasmic reticulum, conjugation of morphine had a low V_max but the enzyme had high affinities for both UDP-glucuronic acid and the aglycone.     

Action of styrene and its metabolites styrene oxide and styrene glycol on activities of xenobiotic biotransformation enzymes in rat liver in vivo

The effects of the administration of styrene and its metabolites, styrene oxide and styrene glycol, on drug metabolizing enzymes of rat liver were studied. Styrene (three or six daily doses of 500 g/kg) doubled the activities of microsomal p-nitroanisole O-demethylase, epoxide hydrase (styrene oxide as substrate), and UDP glucuronosyltransferase (p-nitrophenol) when measured in microsomes pretreated in vitro with digitonin or trypsin. On the other hand, glycine conjugation, the cytochrome P-450 content, and activities of NADPH cytochrome c reductase and benzpyrene hydroxylase, were less affected by styrene. Styrene oxide was more toxic to rats than styrene or styrene glycol and it also caused (one dose of 375 mg/kg) a significant decrease in the activities of benzpyrene hydroxylase and p-nitroanisole O-demethylase and in the cytochrome P-450 content. Epoxide hydratase NADPH cytochrome c reductase, and UDP glucuronosyltransferase were more resistant towards styrene oxide. Styrene glycol did not significantly alter the activities of drug metabolizing enzymes. The current data show that styrene causes an increase in the activities of some enzymes involved in its own metabolism.     

A review of the methods of chemical synthesis of sulphate and glucuronide conjugates

1. Methods for the synthesis of drug conjugates with sulphuric acid have been reviewed.

2. Some analytical methods are presented for the analysis of sulphate conjugates.

3. The synthesis of several types of N, O and C β-D-glucuronides is reviewed. Different β-coupling reactions of protected glucuronides are presented.

4. Application of n.m.r. and mass spectrometry to the analysis of β-D-glucuronides is discussed.     

Glutathione-dependent bioactivation of xenobiotics

Glutathione conjugation has been identified as an important detoxication reaction. However, in recent years several glutathione-dependent bioactivation reactions have been identified. Current knowledge on the mechanisms and the possible biological importance of these reactions are discussed.

1. Dichloromethane is metabolized by glutathione conjugation to formaldehyde via S-(chloromethyl)glutathione. Both compounds are reactive intermediates and may be responsible for the dichloromethane-induced tumorigenesis in sensitive species.

2. Vicinal dihaloalkanes are transformed by glutathione S-transferase-catalyzed reactions to mutagenic and nephrotoxic S-(2-haloethyl)glutathione S-conjugates. Electrophilic episulphonium ions are the ultimate reactive intermediates formed.

3. Several polychlorinated alkenes are bioactivated in a complex, glutathione-dependent pathway. The first step is hepatic glutathione S-conjugate formation followed by cleavage to the corresponding cysteine S-conjugates, and, after translocation to the kidney, metabolism by renal cysteine conjugate β-lyase. β-Lyase-dependent metabolism of halovinyl cysteine S-conjugates yields electrophilic thioketenes, whose covalent binding to cellular macromolecules is responsible for the observed toxicity of the parent compounds.

4. Finally, hepatic glutathione conjugate formation with hydroquinones and aminophenols yields conjugates that are directed to γ-glutamyltransferase-rich tissues, such as the kidney, where they undergo alkylation or redox cycling reactions, or both, that cause organ-selective damage.