Kazumi Sugihara

Variation of Hepatic Methotrexate 7-Hydroxylase Activity in Animals and Humans

This study deals with individual and species variations in the converting activity of methotrexate (MTX) to 7‐hydroxymethotrexate in animals and humans. When MTX 7‐hydroxylase was assayed in six human liver cytosols, a 48‐fold range of intersubject variation of the activity was observed. The variations were correlated to the concentrations of aldehyde oxidase activity in human subjects assayed with benzaldehyde as a substrate. Species differences of liver MTX 7‐hydroxylase activity were also observed. The activity was highest in rabbits, followed by rats, hamsters, and monkeys but was undetectable in dogs. Strain differences of MTX 7‐hydroxylase activity based on aldehyde oxidase activity were also observed in rats and mice. The results suggest that aldehyde oxidase functions as MTX 7‐hydroxylase in livers of animals and humans, and the observed differences of MTX 7‐hydroxylase activity are due to variations in the amount of aldehyde oxidase present.

DIFFERENCES IN ALDEHYDE OXIDASE ACTIVITY IN CYTOSOLIC PREPARATIONS OF HUMAN AND MONKEY LIVER

This study presents data showing individual differences in aldehyde oxidase activity in human and monkey liver cytosols. When assayed with benzaldehyde as a substrate, a significant inter‐subject variation in the activity was found in the human liver preparations. When assayed with N1‐methylnicotinamide as a substrate, the inter‐subject variation of the activity was also observed, but to a lesser extent compared with that of the activity with benzaldehyde. Similarly, variations in aldehyde oxidase activity were found in the monkey liver preparations when assayed with benzaldehyde or N1‐methylnicotinamide. The present study suggested that at least two isozymes of aldehyde oxidase exist in the human liver preparations.

The role of mammalian intestinal bacteria in the reductive metabolism of zonisamide

Zonisamide (1,2‐benzisoxazole‐3‐methanesulphonamide), a new anticonvulsant, is mainly metabolized to 2‐sulphamoylacetylphenol by reduction of the benzisoxazole ring. Recent studies have shown that mammalian liver enzymes are responsible for the reduction of zonisamide. Because intestinal bacteria can also mediate the reduction of xenobiotics, this study was designed to evaluate the role of intestinal bacteria in in‐vivo reductive metabolism of zonisamide.

ESTIMATION OF ALDEHYDE OXIDASE ACTIVITY IN VIVO FROM CONVERSION RATIO OF N1-METHYLNICOTINAMIDE TO PYRIDONES, AND INTRASPECIES VARIATION OF THE ENZYME ACTIVITY IN RATS

The in vivo conversion ratio of N1-methylnicotinamide (NMN) to N1-methyl-2-pyridone-5-carboxamide (2-PY) and N1-methyl-4-pyridone-3-carboxamide (4-PY) as a parameter for the estimation of aldehyde oxidase level in rats was examined. NMN and its pyridones (2-PY and 4-PY) are usually detected in the urine of rats. When we measured the ratio of the amount of pyridones to the total amount of NMN and pyridones (RP value) in the urine of rats, marked intraspecies variations were observed. The variation in RP value among strains was closely related to the differences of liver aldehyde oxidase activity measured with NMN as a substrate. RP values after administration of NMN to different strains of rats confirmed the existence of strain differences of aldehyde oxidase activity in vivo. We demonstrated that measurements of NMN and its pyridones usually excreted in the urine can be used to predict the in vivo level of aldehyde oxidase.

Developmental Changes of Aldehyde Oxidase Activity in Young Japanese Children

Aldehyde oxidase (AO) plays an important role in metabolizing many drugs, so AO activity in individual patients may be a useful parameter for dose adjustment to avoid severe toxicity. In this study, we investigated the developmental changes of AO activity in 101 children. Urine was collected in the morning, and AO activity was assessed in terms of the ratio of pyridone formation from N1‐methylnicotinamide, an AO substrate. Significant correlations were found between AO activity and various growth indices (age, body weight, body surface area, and liver volume). Age showed the moderate correlation (r2=0.506). AO activity rapidly increased with increase of the subjects age up to about 1 year. These findings suggest that the AO activity begins to increase soon after birth. Because AO activity is immature in children below 1 year of age, dose adjustment based on individual AO activity should be made for such patients.

Participation of liver aldehyde oxidase in reductive metabolism of hydroxamic acids to amides

The liver enzyme responsible for the reduction of aromatic and heterocyclic hydroxamic acids to the corresponding amides was investigated with salicylhydroxamic acid, benzohydroxamic acid, anthranilhydroxamic acid, and nicotinohydroxamic acid. Rabbit liver cytosol exhibited significant reductase activities toward the hydroxamic acids under anaerobic conditions when supplemented with an electron donor of aldehyde oxidase. Similarly, rabbit liver aldehyde oxidase reduced these compounds to amides in the presence of its own electron donor, indicating that the reductase activities observed in the liver cytosol are due mainly to the cytosolic molybdoflavin enzyme. Furthermore, a significant reduction of salicylhydroxamic acid and nicotinohydroxamic acid was also observed, when an electron donor of aldehyde oxidase was added, with liver cytosols from hamsters, guinea pigs, rats, and mice. The cytosolic reductase activities toward salicylhydroxamic acid were markedly inhibited by menadione, an inhibitor of aldehyde oxidase.

Strain differences of the ability to hydroxylate methotrexate in rats.

Converting activity of methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX) was examined using eight strains of rats. Marked variability of the activity was found in liver cytosols from the rats. The highest activity was observed with Sea:SD rats, followed by LEW/Sea and Jcl:Wistar rats. The lowest activity was observed with WKA/Sea rats. The difference in the activity between Sea:SD and WKA/Sea strains was 104-fold. The variation was correlated to the strain difference of benzaldehyde oxidase activity in the rats. The cytosolic 7-hydroxylase activities in other tissues of Sea:SD rats were much higher than those of WKA/Sea, similarly to the case in liver. The liver microsomes of Sea:SD rats exhibited no 7-hydroxylase activity toward MTX even in the presence of NADPH. The cytosolic 7-hydroxylating activity of the livers of Sea:SD rats was inhibited by menadione, beta-estradiol, chlorpromazine and disulfiram, inhibitors of aldehyde oxidase, but not oxypurinol, an inhibitor of xanthine oxidase. The purified aldehyde oxidase from the livers of Sea:SD rats exhibited a significant 7-hydroxylating activity toward MTX. However, xanthine oxidase had no ability to hydroxylate MTX. These facts suggest that MTX hydroxylating activity in rats is predominantly due to aldehyde oxidase, and the strain differences are due to the variations of the flavoenzyme level.

Involvement of Molybdenum Hydroxylases in Reductive Metabolism of Nitro Polycyclic Aromatic Hydrocarbons in Mammalian Skin

Molybdenum hydroxylases, aldehyde oxidase and xanthine oxidoreductase, were shown to be involved in the nitroreduction of 2-nitrofluorene (NF), 1-nitropyrene, and 4-nitrobiphenyl, environmental pollutants, in the skin of various mammalian species. NF was reduced to 2-aminofluorene by hamster skin cytosol in the presence of 2-hydroxypyrimidine, 4-hydroxypyrimidine, N1-methylnicotinamide, or benzaldehyde, but not hypoxanthine or xanthine. Inhibitors of aldehyde oxidase markedly inhibited these nitroreductase activities, but oxypurinol, an inhibitor of xanthine oxidoreductase, had little effect. In DEAE column chromatography of hamster skin cytosol, the major fraction exhibiting nitroreductase activity also showed aldehyde oxidase activity. 2-Hydroxypyrimidine-linked nitroreductase activities of skin cytosol from rabbits and guinea pigs were also inhibited by an inhibitor of aldehyde oxidase. In contrast, nitroreductase activities of skin cytosols of rats and mice were markedly inhibited by oxypurinol. When aldehyde oxidase activity was estimated in skin cytosol of various mammals using benzaldehyde oxidase activity as a marker, considerable variability of the activity was found. The highest activity was observed with hamsters, and the lowest activity with rats. On the other hand, the highest xanthine oxidoreductase activity was observed with rats, and the lowest activity with rabbits. These skin cytosols of various mammals also exhibited significant 2-hydroxypyrimidine-linked nitroreductase activities toward 1-nitropyrene and 4-nitrobiphenyl catalyzed by aldehyde oxidase and xanthine oxidoreductase. Thus, NF was mainly reduced by aldehyde oxidase and xanthine oxidoreductase in skins of animals. However, the contributions of these two molybdenum hydroxylases were considerably different among animal species.

Non-enzymatic reduction of aliphatic tertiary amine N-oxides mediated by the haem moiety of cytochrome P450

1. The mechanism of reduction of aliphatic tertiary amine N-oxides to tertiary amines in liver microsomes was examined and a novel type of reduction by cytochrome P450 was found. 2. Rat liver microsomes exhibited a significant N-oxide reductase activity toward brucine N-oxide and imipramine N-oxide in the presence of both NAD(P)H and FAD under anaerobic conditions. These N-oxide reductase activities were inhibited by carbon monoxide or air. However, the activities were not abolished by boiling the microsomes; indeed, in the case of brucine N-oxide, the activity was enhanced. 3. The activity toward brucine N-oxide was also observed after the conversion of cytochrome P450 to cytochrome P420. Cytochrome P4502B1 alone exhibited the reductase activity in the presence of both NAD(P)H and FAD. After the removal of haem from cytochrome P4502B1, the activity was observed in the haem moiety, but not in the cytochrome P450 apoprotein. 4. Photochemically reduced FAD was effective in the reduction in place of NAD(P)H and FAD. 5. The N-oxide reduction appears to proceed non-enzymatically, catalysed by the haem group of cytochrome P450 in the presence of a reduced flavin.

S‐(‐)‐nicotine‐1′‐N‐oxide reductase activity of rat liver aldehyde oxidase

In the present study, a rat liver cytosolic enzyme responsible for reduction of S‐(‐)‐nicotine‐1′‐N‐oxide to S‐(‐)‐nicotine was investigated. We found that aldehyde oxidase (EC 1.2.3.1) can function as a S‐(‐)‐nicotine‐1′‐N‐oxide reductase in the presence of its electron donor such as 2‐hydroxypyrimidine. The apparent Km and Vmax values of the enzyme for the N‐oxide were 0.24 mM and 30.3 nmol/10 min/mg protein, respectively.