Hiroshi Kozuka

Effects of peroxisome proliferators on fatty acid-binding protein in rat liver.

The relationship between hepatic fatty acid-binding protein and peroxisomal beta-oxidation was studied. Rats were fed a diet containing p-chlorophenoxyisobutyric acid (clofibric acid), 2,2-(decamethylenedithio)-diethanol (tiadenol), di-(2-ethylhexyl)-phthalate (DEHP), di-(2-ethylhexyl)-adipate (DEHA) and acetylsalicylic acid. On the administration of these peroxisome proliferators, both [1-14C]oleic acid-binding capacity and content of fatty acid-binding protein were increased, in association with an increase in peroxisomal beta-oxidation activity. The order of the increase in binding capacity and content of fatty acid-binding protein was tiadenol greater than DEHP greater than or equal to clofibric acid greater than DEHA = acetylsalicylic acid. The order of the increase in peroxisomal beta-oxidation activity in liver was tiadenol greater than clofibric acid greater than or equal to DEHP greater than DEHA = acetylsalicylic acid. Linear regression analysis between the binding capacity or content of fatty acid-binding protein and peroxisomal beta-oxidation activity was highly significant.

Differential effects of altered hormonal state on the induction of ACYL-CoA hydrolases and peroxisomal β-oxidation by clofibric acid

Induction of hydrolase I, hydrolase II, peroxisomal beta-oxidation and hepatomegaly caused by clofibric acid (rho-chlorophenoxyisobutyric acid) administration was investigated in relation to alterations in hormonal state of glucocorticoid, thyroid hormone and insulin. (1) In adrenalectomized rats, the ability to induce hydrolase I was depressed effectively and little hepatomegaly was produced. Hydrolase II and peroxisomal beta-oxidation were induced to a similar extent, compared to those of intact rats. (2) In hypothyroid rats, induction of hydrolase I, hydrolase II, peroxisomal beta-oxidation and hepatomegaly was reduced. In hyperthyroid rats, the ability to induce hydrolase I, hydrolase II and peroxisomal beta-oxidation was depressed, although hepatomegaly was produced by the same or a greater extent as in intact rats. (3) In diabetic rats, marked reduction of ability to induce both hydrolase I and II was observed and induction of hepatomegaly was depressed slightly, although peroxisomal beta-oxidation was induced normally. Differences in the response of four parameters (hydrolase I, hydrolase II, peroxisomal beta-oxidation and liver size) to alterations in hormonal state suggest that the four biological responses to clofibric acid may each be mediated through distinct mechanism(s) other and regulated by distinct hormone(s).

Effects of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid on peroxisomal enzymes in rat liver

The effects of feeding 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on the level of peroxisomal enzymes in rat liver were studied. The concentration of triglyceride in serum was decreased and the activity of cyanide-insensitive palmitoyl-CoA oxidation, catalase and carnitine acetyltransferase increased. However, the extent of the increase in the activity of these enzymes by treatment with 2,4-D was less pronounced than that by 2,4,5-T treatment. The administration of 2,4-D or 2,4,5-T increased the concentration of polypeptide with a mol. wt of 80,000 in the light mitochondrial fractions of the liver from the rats.

Modification by clofibric acid of acyl composition of glycerolipids in rat liver. Possible involvement of fatty acid chain elongation and desaturation.

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) to rats induced a marked change in acyl composition of hepatic glycerolipids; a considerable increase in the proportion of octadecenoic acid (18:1) was accompanied by a marked decrease in the proportion of octadecadienoic acid (18:2). Among the glycerolipids, the changes in the proportions of 18:1 and 18:2 were the most marked in phosphatidylcholine. The change in the acyl composition of phosphatidylcholine paralleled the change in free fatty acid composition in microsomes. The treatment of rats with clofibric acid resulted in a 2.3-fold increase in activity of microsomal palmitoyl-CoA chain elongation and a 4.8-fold increase in activity of stearoyl-CoA desaturation. The activities of acyl-CoA synthetase, 1-acylglycerophosphate acyltransferase and 1-acylglycerophosphorylcholine acyltransferase in hepatic microsomes were increased approx. 3-, 1.7- and 3.6-times, respectively, by the treatment of rats with clofibric acid. These findings are discussed with respect to the role of fatty acid modification systems in the regulation of acyl composition of phosphatidylcholine.

Interaction between cadmium and zinc, copper, or lead in relation to the collagen and mineral content of embryonic chick bone in tissue culture

Abstract To investigate the interaction between Cd and Zn, Cu, or Pb in relation to the content of collagen and mineral in bone, femurs obtained from 9-day-old chick embryos were cultivated for 6 days by the Rollertube method. Zn prevented a decrease in collagen content caused by Cd, although Cu and Pb showed no such protective effect. Neither Zn, Cu, nor Pb prevented the decrease in mineral content caused by Cd. In the Cd-treated bone, the Cd content in the presence of Zn was larger than that in the absence of Zn. The Cd content in the cytosol fraction from Cd plus Zn-treated bones was larger than that from Cd-treated ones. When the cytosol fraction was applied to a Sephadex G-75 column, three Cd-containing peaks were observed. The first small peak (HM fraction) was eluted near the void volume, the second large peak (MT fraction) was metallothionein-like protein (MW = about 10,000), and the third peak contained low-molecular-weight (about 2500) compounds. The Cd content in the MT fraction from Cd plus Zn-treated bones was larger than that from Cd-treated ones. From these results it was concluded that a metallothionein-like protein, induced by Zn, served as a Cd scavenger and contributed to the protection against Cd-induced toxicity. On the other hand, when the 100,000 g precipitate from cultured bone was solubilized and applied to a Sephadex G-75 column, one peak was observed in the position of the HM fraction. The activity of alkaline phosphatase in the HM fraction from Cd-treated bones was inhibited, but this inhibition was not observed in Cd plus Zn-treated ones. This finding suggests that competition between Cd and Zn in the HM fraction contributes, at least partly, to the protection against Cd-induced toxicity.

Selective increase in acylation of 1-acylglycerophosphorylcholinein livers of rats and mice by peroxisome proliferators

Rats were fed a diet containing p-chlorophenoxyisobutyric acid (clofibric acid). Activity of microsomal 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase in liver was increased approx. 3-fold by the treatment with clofibric acid. The treatment of rats with clofibric acid did not increase activity of microsomal 2-acyl-GPC acyltransferase. Feeding a diet containing 2,2-(decamethylenedithio)diethanol (tiadenol), di(2-ethylhexyl)phthalate or acetylsalicylic acid also resulted in a selective increase in the activity of 1-acyl-GPC acyltransferase in rat liver. Treatment with clofibric acid increased the activity of 1-acyl-GPC acyltransferase in liver of mouse as well as rat, but did not change the activity in liver of guinea-pig. The relative rate of acylation of 1-acyl-GPC with various acyl-CoAs by hepatic microsomes was not changed by the treatment of rats with clofibric acid.

Induction of hepatic long-chain acyl-CoA hydrolase by clofibric acid administration.

The induction of hepatic long-chain acyl-CoA hydrolase in the cytosolic fraction by administration of clofibric acid (p-chlorophenoxyisobutyric acid) was compared in rats, mice and guinea-pigs. In rats, two long-chain acyl-CoA hydrolases were induced by the administration of clofibric acid. In mice, only one long-chain acyl-CoA hydrolase was induced, and this hydrolase had properties similar to those of the lower-molecular-weight hydrolase induced in the hepatic cytosol of rats. In hepatic cytosol of guinea-pig, no hydrolase was induced by the administration of clofibric acid.

Species Difference of Liver Cytosolic Fatty Acid-Binding Protein in Rat, Mouse and Guinea Pig

Binding properties of liver cytosolic protein for oleic acid, palmitoyl-CoA and bromosulphophthalein (BSP) were compared for rat, mouse and guinea pig. Hepatic cytosol of rat, mouse and guinea pig contained proteins with a molecular weight of ca. 12,000 and had an affinity for [1-14C]-oleic acid. The concentration of fatty acid-binding protein (FABP) was almost the same in livers of the animals of the 3 species and was ca. 50 μg/mg cytosolic protein. Electrophoretic studies revealed that FABP from hepatic cytosol of rat, mouse and guinea pig, purified with affinity chromatography, are distinct from one another in terms of their charge. FABP of rat liver was capable of binding any 3 ligands-oleic acid, palmitoyl-CoA and BSP—at relatively high binding capacity. FABP of mouse liver also bound oleic acid and palmitoyl-CoA to a great extent, but its binding capacity for BSP was only one-third that of rat liver. FABP of guinea pig liver bound less oleic acid and palmitoyl-CoA than rat liver, whereas it had almost the same binding capacity for BSP as rat liver.

Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds

1. Metabolites produced by the incubation of 2,6-dinitrotoluene (2,6-DNT) with Salmonella typhimurium strains TA 98, TA 98/1,8-DNP6 and TA 98NR were examined. Mutagenicities of bacterial products and related compounds were also examined in the Ames assay using TA 98 and TA 100. 2. 2,6-DNT was converted to 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotoluene and 2-amino-6-nitrotoluene, with concurrent spontaneous formation of 2,2-dimethyl-3,3-dinitroazoxybenzene, in the incubation with TA 98 and TA 98/1,8-DNP6. Capacity of TA 98NR to reduce 2,6-DNT was much lower than that of TA 98 and TA 98/1,8-DNP6. 3. Bacterial products, including 2,2-dimethyl-3,3-dinitroazoxybenzene, showed no mutagenic activity in the Ames assay. 4. Results indicate that the lack of mutagenic activity of 2,6-DNT is not due to low reductive metabolism of 2,6-DNT by bacteria, but due to the lack of mutagenic activity of the bacterial reductive products of 2,6-DNT.

Metabolism of 2,6-Dinitrotoluene in Male Wistar Rat

1. Unchanged 2,6-dinitrotoluene (2,6-DNT), 2-amino-6-nitrotoluene, 2,6-dinitrobenzyl alcohol, 2-amino-6-nitrobenzyl alcohol, conjugated 2,6-dinitrobenzyl alcohol and conjugated 2-amino-6-nitrobenzyl alcohol were detected in urine of male Wistar rats dosed with 2,6-DNT. The major metabolite was conjugated 2,6-dinitrobenzyl alcohol, which accounted for about 1.5% of the dose. 2. Unchanged 2,6-DNT, 2-amino-6-nitrotoluene, 2,6-dinitrobenzyl alcohol, and conjugates of 2,6-dinitrobenzyl alcohol, 2-amino-6-nitrotoluene and 2,6-dinitrobenzaldehyde were detected in the bile of rats dosed with 2,6-DNT. The major metabolite was conjugated 2,6-dinitrobenzyl alcohol, which accounted for 30% of the dose. Conjugates of 2,6-dinitrobenzyl alcohol (major) and 2,6-dinitrobenzaldehyde (minor) were common biliary metabolites in rats dosed with 2,6-dinitrobenzyl alcohol or 2,6-dinitrobenzaldehyde. 3. 2,6-Dinitrobenzyl alcohol and 2,6-dinitrobenzaldehyde were detected by incubating bile from rats given 2,6-DNT with rat intestinal contents under N2. 4. Incubation of 2,6-DNT with hepatic microsomal preparations gave 2,6-dinitrobenzyl alcohol. Incubation of 2,6-dinitrobenzyl alcohol with microsomal plus cytosol preparations gave 2,6-dinitrobenzaldehyde. Incubation of 2,6-dinitrobenzaldehyde with cytosol preparations gave 2,6-dinitrobenzyl alcohol and 2,6-dinitrobenzoic acid. The activities of 2,6-DNT oxidation to 2,6-dinitrobenzyl alcohol, 2,6-dinitrobenzyl alcohol oxidation to 2,6-dinitrobenzaldehyde, 2,6-dinitrobenzaldehyde oxidation to 2,6-dinitrobenzoic acid, and 2,6-dinitrobenzaldehyde reduction to 2,6-dinitrobenzyl alcohol were 22.0, 4.7, 1.3, and 23.3 nmol formed/g liver per min, respectively. 5. These results indicate that 2,6-dinitrobenzaldehyde, an intermediary metabolite of 2,6-DNT in male Wistar rats, is produced either by oxidation of 2,6-DNT in the liver, or by oxidation of 2,6-dinitrobenzyl alcohol formed by hydrolysis of 2,6-dinitrobenzyl alcohol conjugates excreted in the bile, and further indicate that enterohepatic circulation of 2,6-dinitrobenzyl alcohol and 2,6-dinitrobenzaldehyde occurs. This result, together with previous findings, shows that there are metabolic differences, including the biliary excretion of a diol glucuronide of 2,6-dinitrobenzaldehyde and the lack of urinary excretion of 2,6-dinitrobenzoic acid, between 2,4-DNT and 2,6-DNT in male Wistar rat.