Enhanced demethylation and denitrosation of N-nitrosodimethylamine by human liver microsomes from alcoholics.

The metabolism of N-nitrosodimethylamine (NDMA) was investigated in incubations with human liver microsomes from alcoholics and control patients who suffered from other diseases, but had a histological normal liver. All of the microsomal samples studied were able to metabolize NDMA at various concentrations to both formaldehyde and nitrite. Analysis of the liver microsomes from alcoholics revealed that both enzymatic activities--formaldehyde and nitrite formation--were enhanced several times as compared to the control patients. The results point to the fact that alcoholics metabolize NDMA at a higher rate probably due to the induction of one or more ethanol-inducible human liver cytochromes (cyt.) P450. The question if alcoholics therefore possess a higher risk for carcinogenic events is discussed.

The oxidation of the calcium probe quin2 and its analogs by prostaglandin H synthase.

Quin2 and its analogs BAPTA, 5,5'-dimethyl BAPTA, 5,5'-difluoro BAPTA, fura-2, and indo-1 were developed to measure intracellular calcium concentrations. In this study we investigated whether quin2 and its analogs are susceptible to peroxidase-catalyzed oxidation. The hydroperoxidase activity of prostaglandin H synthase, like other peroxidases, is capable of oxidizing a wide variety of substrates. It was found that quin2 and its analogs served as reducing cofactors for the hydroperoxidase activity of prostaglandin H synthase, undergoing oxidation in the process. Furthermore, arachidonic acid metabolism was stimulated. Oxidation of quin2 and its analogs resulted in the formation of a carbon-centered radical, as could be detected by ESR, and in the formation of formaldehyde. Quin2 fluorescence decreased upon addition of arachidonic acid and prostaglandin H synthase. Furthermore, addition of calcium no longer resulted in an increase in quin2 fluorescence, as was observed prior to the addition of arachidonic acid and the enzyme. This indicates that one or more of the -N-CH2-COOH groups, which are responsible for the binding of calcium, were oxidized by the hydroperoxidase. Since prostaglandin H synthase is present in many cellular systems in which calcium concentrations are modulated, oxidation of the calcium probe might not only affect the measurement of intracellular calcium but could activate arachidonic acid metabolism as well.

Physiological regulation of Paracoccus denitrificans methanol dehydrogenase synthesis and activity.

An enzyme-linked immunosorbent assay and a whole-cell activity assay were developed which allowed detection of methanol dehydrogenase (MDH) of Paracoccus denitrificans with increased sensitivity. By these methods, it was shown that MDH was not induced by its natural substrate, methanol. Relief from a catabolite repression-like mechanism seemed responsible for low-level MDH synthesis, while product induction was the hypothesized mechanism for synthesis of high amounts of MDH. In the latter process, formaldehyde may play an important role as effector. For a variety of culture conditions, inconsistencies were observed in the relation between amounts of MDH protein synthesized and enzyme activities measured in vitro. Regulation of pyrrolo-quinoline-quinone biosynthesis or a modulation of its incorporation and stability in MDH may constitute an overriding mechanism to ensure a correct tuning between metabolic rates of methanol consumption and the required methanol oxidation rates.

Generation of formaldehyde by N-demethylation of antipyrine. Detection of formaldehyde in bile by 13C-NMR spectroscopy.

The importance of NMR spectroscopy as a tool to investigate metabolic events in vitro and in vivo becomes more and more evident. Particularly 13C-NMR spectroscopy is able to deliver a wide range of information regarding the chemistry of xenobiotics in vivo. We studied the N-demethylation of N-methyl-13C-labelled antipyrine using an isolated perfused rat liver with a fluorocarbon suspension (FC 43) as oxygen carrier. Bile was collected in different fractions during the experiment. On the vascular side metabolite formation was monitored by continuous flow NMR spectroscopy. In bile the metabolic events were detected by standard NMR techniques. The bile spectra exhibit, among others, a signal at 84.2 ppm, indicating formaldehyde hydrate derived from the N-methyl group of antipyrine by an oxidative metabolic pathway. Neither formaldehyde hydrate nor other oxidation products could be detected in the vascular perfusate. The biliary excretion of considerable amounts of formaldehyde during the N-demethylation of antipyrine might have toxicological consequences for the intra- and extrahepatic bile ducts.

Activities of serine hydroxymethyltransferase in murine tissues and tumours.

The specific activity of the enzyme serine hydroxymethyltransferase (EC 2.1.2.1) was determined in various murine, rat and human tumour cell lines. The activities of the enzyme were also investigated in tissues of non-tumour bearing DBA/2 mice and BALB/c mice bearing the PC6 ascites tumour. The highest enzyme activity in the murine tissues was found in the liver and then the kidneys. The enzyme was present in all the tissues assayed. The activities of enzyme found in the tumours varied considerably, with the PC6 ascites, Walker 256 and Lewis lung cells, being the highest.

Renal microsomal N-nitrosodimethylamine demethylase determined by a sensitive radiometric method.

Renal microsomal N-nitrosodimethylamine (NDMA) N-demethylation was measured in several species by a sensitive radiometric method using [14C]NDMA as a substrate and 2,4-dinitrophenylhydrazine (DNPH) as a trapping agent of the [14C]formaldehyde formed. The activities were the highest in mice and lowest in hamsters. The activities in rats could not be detected. Among several strains of mice studied the DDY strain was the highest in its activities. Although nicotinamide adenine dinucleotide supported NDMA demethylation by about 32% of nicotinamide adenine dinucleotide phosphate in kidney and only 16% in liver, other properties (pH profiles, km values, and effects of inhibitors) exhibited almost similar results in liver as compared to kidney.

Toxicity and association of polycyanoacrylate nanoparticles with hepatocytes.

The degradation of polybutylcyanoacrylate (PBC) and polyhexylcyanoacrylate (PHC) nanoparticles, together with their association with and toxicity towards isolated hepatocytes, were determined. Nanoparticles were not taken up by rat hepatocytes at a significant level. The LD50S of PBC and PHC nanoparticles towards hepatocytes were 0.4 mg/2 x 10(6) cells and greater than 1 mg/2 x 10(6) cells respectively. This hepatocyte toxicity cannot be attributed solely to the formaldehyde formed during degradation.

Formaldehyde generation from methenamine salts in spinal cord injury.

To achieve effective suppression of bacteriuria in spinal cord injured (SCI) patients, methenamine mandelate and methenamine hippurate are commonly given with ascorbic acid. Since the effectiveness of ascorbic acid as a urinary acidifier has been challenged and as it also has been suggested that methenamine salts do not produce effective urine formaldehyde concentrations in patients with indwelling urethral catheters, we studied two groups of SCI patients to determine (1) the effect of ascorbic acid on urine pH and formaldehyde concentration when administered with methenamine salts; (2) the effect of an indwelling urethral catheter versus intermittent catheterization on formaldehyde concentration in the urine of SCI patients taking methenamine salts; and (3) the relative urine formaldehyde concentrations produced by treatment with methenamine mandelate and methenamine hippurate in SCI patients. Methenamine mandelate produced significantly higher urine formaldehyde concentrations than did methenamine hippurate, especially among patients with intermittent catheterization. Ascorbic acid produced a significant effect on urine pH but not on formaldehyde concentration.

Chicken resistance to dimethylnitrosamine acute effects on the liver: a comparative study with other species.

Dimethylnitrosamine (DMN)-induced liver damage, as measured by the increase in plasma isocitrate dehydrogenase as well as by histologic assessment of necrosis, was marked after DMN ip administration (70 mg/kg) in males of all noninbred species tested (BALB/c mouse, Sprague-Dawley rat, Syrian golden hamster, general purpose guinea pig) but not in the noninbred White Leghorn chicken. At 1 and 3 hours after DMN injection, liver DMN levels were not lower in the chicken as compared to levels in the other species. Furthermore, in all species except the chicken, significant decreases were found at 3 hours as compared to 1 hour after DMN administration. DMN metabolism to CO2 and to formaldehyde, as well as covalent binding of DMN-reactive metabolites to either proteins or nucleic acid, was measured with the use of liver slices, microsomes, and/or 9,000 X g supernatants. Results indicated that chicken liver had a very low capacity for metabolism and activation (29-3,166 times lower than comparable data in mice or hamsters).

Hepatic aminopyrine N-demethylase system: effect of cyanide on microsomal N-demethylase activity.

Cyanide, an inhibitor of many hemoproteins, was shown to affect a number of microsomal drug-metabolizing activities catalyzed by cytochrome P-450. The N-demethylation reaction of aminopyrine was inhibited noncompetitively by this inhibitor in microsomal preparations from rats. The binding reaction of aminopyrine with microsomal cytochrome P-450 was also modified by cyanide, and an abnormal aminopyrine-induced difference spectrum of microsomes by cyanide, and an abnormal aminopyrine-induced difference spectrum of microsomes appeared when cyanide was added to the reaction mixture. Partial dissociation of cytochrome P-450. Cyanide complex by aminopyrine was observed by spectrophotometrical and epr spectroscopic methods. These results suggest that aminopyrine and cyanide reciprocally affect binding with cytochrome P-450 and modification by cyanide of aminopyrine binding reaction with the hemoprotein produces an inhibition of N-demethylase activity.

Formaldehyde formation as a metabolite of methoxyflurane.

The production of formaldehyde as a metabolite of enzymatic biotransformation of methoxyflurane was observed in vitro in hepatic microsomal preparations derived from Japanese monkeys, rabbits and Wistar strain rats. Production rates of formaldehyde were 0.80-2.50 n moles/mg protein/min in the induced group which was pretreated with phenobarbitone, and 0.15-0.68 n moles/mg protein/min in the non-induced group. This reaction needed the presence of NADPH and oxygen. Formaldehyde production was almost completely inhibited by addition of 25% CO. Further degradation of formaldehyde did not occur in microsomal suspension. No detectable species difference was observed among the animals used in this study. These results indicate that the biotransformation of methoxyflurane is preceded by interaction with cytochrome p-450 in hepatic microsomes in the presence of NADPH and oxygen.

[Methanol metabolism by Pseudomonas oleovorans]. / Metabolizm metanola u Pseudomonas oleovorans.

A typical facultative methylotroph Pseudomonas oleovorans oxidizes methanol to formaldehyde by a specific dehydrogenase which is active towards phenazine metosulphate. Direct oxidation of formalydehyde to CO2 via formiate is a minor pathway because the activities of dehydrogenases of formaldehyde and formiate are lwo. Most formaldehyde molecules are involved in the hexulose phosphate cycle, which is confirmed by a high activity of hexulose phosphate synthase. Formaldehyde is oxidized to CO2 in the dissimilation branch of the cycle providing energy for biosynthesis; this confirmed by higher levels of dehydrogenases of glucose-6-phosphate and 6-phosphogluconate during the methylotrophous growth of the cells. The acceptor of formaldehyde (ribulose-5-phosphate) is regenerated and pyruvate is synthesized in the assimilation branch of the hexulose phosphate cycle. Aldolase of 2-keto-3-deoxy-6-phosphogluconate plays an important role in this process. Further metabolism of trioses involves reactions of the tricarboxylic acid cycle which performs mainly an anabolic function due to complete repression of alpha-ketoglutarate dehydrogenase during the methylotrophous growth. The carbon of methanol is partially assimilated as CO2 by the carboxylation of pyruvate or phosphoenolpyruvate. NH+4 is assimilated by the reductive amination of alpha-ketoglutarate.

Hepatic aminopyrine N-demethylase system: further studies of assay procedure.

Rat hepatic aminopyrine N-demethylase activity was measured by detecting the amount of formaldehyde produced from aminopyrine. Some optimal conditions for the N-demethylation were determined using both isolated microsomes and whole homogenates, and the standard assay method is described. Formaldehyde production from the substrate by microsomal enzyme system was linear to the amount of microsomes added during 3 min reaction time, whereas long-time incubation caused a decrease in the apparent activity of aminopyrine N-demethylation. The N-demethylase activity observed in normal rat liver homogenate was quite similar to that in microsomes when the activity was expressed on the basis of cytochrome P-450 as molecular activity. Pretreatment of animals with typical inducers, phenobarbital and 3-methylcholanthrene, resulted in alteration of the aminopyrine N-demethylase system, which was detectable in both microsomes and whole homogenates.

Generation of formaldehyde from methenamine: effect of pH and concentration, and antibacterial effect.

Using an in vitro system that simulates the dynamics of the urinary tract, we have shown that concentrations of formaldehyde >/= 25 mug/ml can be achieved in urine containing >/= 0.6 mg of methenamine per ml at pH /= 1 mg/ml at pH

Regulation of one-carbon biosynthesis and utilization in Escherichia coli.

The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10-methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.