Nobuyuki Koga

High affinity of 2,3,4,7,8-pentachlorodibenzofuran to cytochrome P-450 in the hepatic microsomes of rats

The interaction of 2,3,4,7,8-pentachlorodibenzofuran (PenCDF) with cytochrome P-450 isozymes was studied in male Wistar rats using 14C-labeled PenCDF. Three forms of cytochrome P-450 isozymes, P-448 H, P-448 L and P-452, were purified to homogeneity from 14C-PenCDF-treated rat liver microsomes. The purified P-448 H contained 0.847 mole of PenCDF per mole of the hemoprotein, whereas the amounts of PenCDF bound to P-448 L and P-452 were far less than that to P-448 H. These results suggest that cytochrome P-450, particularly P-448 H, functions as the storage site of PenCDF in the rat liver.

Enzymic cis-trans isomerization of nitrofuran derivatives. Isomerizing activity of xanthine oxidase, lipoyl dehydrogenase, DT-diaphorase and liver microsomes

Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) supplemented with an electron donor could catalyze the cis-trans isomerization of 3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide, 3-(5-nitro-2-furyl)-2-phenylacrylamide and 3-(5-nitro-2-furyl)-2-(2-furyl)acrylonitrile. The direction of isomerization (cis leads to trans, cis in equilibrium trans or trans leads to cis) is dependent on the chemical structure of these nitrofuran derivatives. Lipoyl dehydrogenase (NADH:lipoamide oxidereductase, EC 1.6.4.3), DT-diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2) and liver microsomes could also catalyze the conversion of cis-3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide to its trans isomer in the presence of an appropriate electron donor. Such isomerizing activity of these enzymes is much higher than their nitro-reducing activity. In addition, the cis-trans isomerization of some nitrofuran derivatives was demonstrated with the liver slices and the small intestines of rats. A new cis-trans isomerization mechanism which is based on transfer of a single electron by an enzyme system to a nitrofuran derivative to give the radical-anion was proposed. This postulated mechanism was supported by the preliminary experiments using pulse radiolysis technique.

Metabolism in vivo of the tropane alkaloid, scopolamine, in several mammalian species

1. In vivo metabolism of scopolamine was studied in rats, mice, guinea pigs and rabbits. The structures of eight urinary metabolites including unchanged drug were elucidated by mass and nuclear magnetic resonance spectrometry. Determination of these metabolites was achieved by a g.l.c. method using a semi-capillary column. 2. The major metabolites in rats were the three phenolic metabolites, p-hydroxy-, m-hydroxy- and p-hydroxy-m-methoxy-scopolamine. 3. Significant intra-species difference of the metabolism was observed in rabbits. Tropic acid was the major metabolite in two rabbits out of three, while the other rabbit excreted mainly unchanged scopolamine, accompanied by five metabolites. Tropic acid was also the major metabolite in guinea pigs, but was of minor importance in mice. 4. The dehydrated metabolites, aposcopolamine and aponorscopolamine, were abundantly excreted in guinea pigs, moderately in mice, and least in rabbits and rats. 5. Excretion of glucuronide conjugates of scopolamine and norscopolamine were high in mice compared with other species. On the other hand, phenolic metabolites in rat urine; and tropic acid in rabbit and guinea pig urine, were excreted as the free forms. 6. These results indicate that scopolamine metabolism is highly species-specific.

Cis-trans isomerization of nitrofuran derivatives by xanthine oxidase

Enzymatic cis-trans isomerization of nitrofuran derivatives was 3-(5-Nitro-2-furyl)-2-(2-furyl)-demonstrated with milk xanthine oxidase. acrylamide (AF-2) and 3-(5-nitro-2-furyl)-2-(5-bromo-2-furyl)acrylamide (NFBFA) were mainly converted from the cis to the trans form by this enzyme supplemented with an electron donor. This enzymatic reaction was further characterized with respect to its cofactor requirements. Finally, a new cis-trans isomerization mechanism, which is based on transfer of a single electron by a nitroreductase such as xanthine oxidase to a nitrofuran derivative to give the anion free radical, was proposed.

Enhanced faecal excretion of 2,3,4,7,8-pentachlorodibenzofuran in rats by a long-term treatment with activated charcoal beads.

1. The effect of activated charcoal beads on the faecal excretion of 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), a causal agent of yusho, which accumulates in the body, was studied for 12 weeks in rats. 2. Diets supplemented with 1% and 5% activated charcoal beads stimulated faecal excretion of PenCDF about 2- and 4-fold, respectively. The concentration of PenCDF in liver, the major storage site, was decreased significantly and dose-dependently by the treatment. 3. The charcoal bead treatment decreased the extent of fatty liver, thymic atrophy, and induction of hepatic benzo[a]pyrene hydroxylase.

Mechanisms for stimulated fecal excretion of 2,3,4,7,8-pentachlorodibenzofuran in rats by treatment with squalane and liquid paraffin

Abstract Squalane and liquid paraffin stimulated the fecal excretion of 2,3,4,7,8-pentachlorodibenzofuran (PenCDF) about 3- and 2-fold, respectively, for 3 weeks in rats administered with 1 mg/kg of PenCDF. Squalane also enhanced the fecal excretion of PenCDF for 12 weeks in rats administered with 0.2 mg/kg of it. The accumulated PenCDF in rat body was considered to be mainly excreted through the intestinal wall into the lumen. The absorption of PenCDF was reduced by dissolving it in squalane or liquid paraffin. Thus these agents appear to stimulate the fecal excretion of PenCDF by the inhibition of the re-absorption of PenCDF which has been eliminated into the intestinal lumen.

Metabolic fate of fenetylline in rat and man

1. Metabolic fate of 7-[2-(alpha-methylphenylethylamino)ethyl]theophylline hydrochloride (fenetylline) was investigated in male Sprague-Dawley rats and three male volunteers. 2. Six metabolites were identified in the rat urine as amphetamine(AP), p-hydroxy-AP, acetylaminoethyl-theophylline(TP), aminoethyl-TP, hydroxyethyl-TP and carboxymethyl-TP by comparison of their spectral properties and h.p.l.c. and g.l.c. characteristics with those of authentic samples. All these metabolites was also detected in the urine of humans receiving fenetylline. 3. Quantification of these metabolites using h.p.l.c. and g.l.c. showed that carboxymethyl-TP, p-hydroxy-AP and acetylaminoethyl-TP were the major metabolites in 0-24 h rat urine at 13.7%, 11.2% and 9.3% of dose, respectively. In men, carboxymethyl-TP(39-43% dose) and AP(23-33% dose) were the major metabolites in 0-48 h urine. 4. These results suggest that fenetylline metabolism proceeds via oxidative cleavage at two different sites to produce aminoethyl-TP and AP, respectively. The pathway producing AP predominates, in both man and rat, but is more predominant in the former.

Toxicological evaluation of sulfur-containing metabolites of 2,5,2′,5′-tetrachlorobiphenyl in rats

Abstract Sulfur-containing metabolites of 2,5,2′,5′-tetrachlorobiphenyl (TCB), 4-methylthio-TCB (MT-TCB), 4-methylsulfoxyl TCB (MSX-TCB) and 4-methylsulfonyl TCB (MS-TCB) were examined for their acute toxicities, hepatic enzyme inducing activities, accumulation in the liver and lung, and excretion to the feces in rats. TCB and MT-TCB suppressed body weight and recovery of body weight gain was delayed in the MT-TCB-treated rats. MT-TCB and MS-TCB caused an increase in total liver lipid and only MT-TCB brought about an atrophy of the thymus. Treatment with MT-TCB increased cytochrome P-450 content and benzphetamine N-demethylase activity. The same enzymes were also induced by treatment with MSX-TCB. Although TCB administered was excreted mostly as hydroxylated TCB, a part was excreted as unchanged and a very small portion as the sulfur-containing metabolites. MT-TCB, MSX-TCB and MS-TCB were excreted from the MT-TCB- and MSX-TCB-treated rats. The MS-TCB-treated rats excreted only MS-TCB. The same compounds as found in the feces were identified in the liver and lung of the rats treated with those compounds except in the liver of TCB-treated rats. These results indicate that sulfur-containing metabolites, especially MT-TCB, were more important than their parent compound, TCB, from a toxicological point of view.

Unique features of subcellular distribution of 2,3,4,7,8-pentachlorodibenzofuran in rat liver

Abstract Among various polychlorinated dibenzofurans, 2,3,4,7,8-pentachlorodibenzofuran(PenCDF) is recognized to be one of the most important congeners as the causal agents of Yusho, because of its very high toxicity and persistency in the liver. We have already found that this PenCDF has a high affinity to the endoplasmic reticulum of the rat liver, in which PenCDF is bound rather tightly, but with non-covalent binding to P-448 H, an isozyme with a high spin form of cytochrome P-450 induced specifically by treatment with PenCDF. Molar ratio of PenCDF to P-448 H was 1 to 1. We have therefore studied to learn how PenCDF absorbed is transported and incorporated selectively into the liver cells, using 14C-PenCDF. As the results, we found that the 14C-PenCDF added to rat plasma was bound partly to lipoproteins, but mostly to albumin. This binding of PenCDF to albumin also enhanced strongly the cellular uptake of 14C-PenCDF. These findings indicated that albumin is very effective carrier in the blood to facilitate uptake of 14C-PenCDF into the liver cells. Examination of subcellular distribution of 14C-PenCDF incorporated into the liver slices proved that 14C-PenCDF was localized to the endoplasmic reticulum, in which P-448 H was not yet induced. This means that PenCDF has also some affinity to endoplasmic reticulum without P-448 H, although it binds specifically to P-448 H after the induction occurs. These findings strongly suggested that cytochrome P-450, especially P-448 H, might function as the storage site of PenCDF, explaining the high persistency and long-lasting biological effects of PenCDF in rats.