Unusual metabolism of sulfur-containing amino acids in rats treated with DL-propargylglycine

S-(2-Hydroxy-2-carboxyethyl)homocysteine, S-(3-hydroxy-3-carboxy-n-propyl)-cysteine, N-acylated S-(beta-carboxyethyl)cysteine, and N-acylated S-(3-hydroxy-3-carboxy-n-propyl) cysteine were excreted in the urine after DL-propargylglycine treatment. Cystathionine was also accumulated in several tissues of DL-propargylglycine-treated rats. N-Monoacetylcystathione was found in the liver of rats and was also detected in the kidney and serum. Cystathionine gamma-lyase activity in liver decreased to about 4% of that of control rats 24 h after the DL-propargylglycine injection, and alanine aminotransferase activity decreased to about 35% of that of control rats. On the other hand, aspartate aminotransferase and cystathionine beta-synthese activity did not show significant changes from those of control rats. The ability of normal tissues to synthesize cystathionine utilizing cystathionine beta-synthase was 1.98 +/- 0.40 mumol/min/g in liver, 0.61 +/- 0.13 in kidney, and 0.18 +/- 0.015 in brain. The maximal contents of cystathionine in rat tissues and the administered amounts of DL-propargylglycine agreed well with the ability to synthesize cystathionine in each tissue.     

Placental transfer of halogenated benzenes (pentachloro-, pentachloronitro-, and hexabromo-) in rats.

The present study was carried out to provide information on the placental transfer of three organohalogens of environmental concern. Pentachloro-, pentachloronitro-, and hexabromobenzene were administered per os to rats daily on days 6 through 15 of gestation at level of 40, 100, and 200 mg/kg body weight. On day 22, the dams were killed and fetuses removed by caesarean section. Maternal brain, heart, kidney, liver, spleen and adipose tissue as well as whole fetus, fetal liver and fetal brain were analyzed for organohalogen residue by GLC. Pentachlorobenzene accumulated in the fetus to a greater extent than hexabromobenzene. In maternal tissues pentachlorobenzene accumulated to the greatest extent in adipose tissue, followed by liver, spleen, brain, heart and kidney. With hexabromobenzene, the greatest accumulation was observed in adipose tissue, followed by spleen, liver, heart, kidney and brain. Pentachloronitrobenzene was not detected (0.05 p.p.m.) in any maternal or fetal tissue.     

Placental permeability and energy metabolism enzymes in fetuses of lipemic rats

A model of maternal lipemia without hyperglycemia, in the rat, produced by high-fat feedings, was developed to study the effects of an abnormal maternal lipid homeostasis on placental transport of nutrients and possible alterations of key enzymes of energy metabolism in the liver and brain of the fetuses. Pregnant rats fed lower concentrations of fat served as controls. All studies were carried out in dams and fetuses one day prior to delivery. The dietary treatment of the dams and fetuses produced in the fetuses ketonemia as well as lipemia. Following a bolus of ¹⁴C-3-0-methyl-D-glucose to the dams, the levels of the tracer remained higher in the blood and brain of lipemic than in control fetuses. By contrast, there was a decrease in the fluxes of ¹⁴C--amino-isobutryic acid in the fetuses of lipemic dams as compared to controls. Among enzymes of energy metabolism, fetal liver glucose-6-phosphatase and succinic dehydrogenase were enhanced by lipemia. Fetal brain glucose-6-phosphatase was depressed. Thus, lipemia, as occuring in poorly controlled maternal diabetes, may be a factor in determining the access to the fetus of essential, neutral amino acids and alter the normal activity of energy metabolism enzymes in the fetus.     

Incorporation of orotic acid into nucleotides and RNA in mouse organs during 60 minutes.

Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP⁺) in the liver of adult rats increased 4–5-times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached the adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, is controlled by both glucagon and glucocorticoid.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats: in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP) in the liver of adult rats increased 4-5 times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, in controlled by both glucagon and glucocorticoid.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP⁺) in the liver of adult rats increased 4–5-times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached the adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, is controlled by both glucagon and glucocorticoid.     

Effects of brief starvation on brain protease activity

Changes in the activity of proteases (cathepsin D and calpains) caused by 48-h food withdrawal were studied in the brain, liver, kidney, spleen, and heart of 3-, 12-, and 24-month-old Fischer rats. Cathepsin D activity was similar in brain, liver, and heart of control animals; in kidney it was 5-fold higher and in spleen about 10-fold higher. With age, activity increased in all organs tested except spleen. Brief starvation caused no change of cathepsin D activity in brain, but caused an increase in liver and a decrease in spleen. Neutral proteolytic activity in control was highest in the pons-medulla-cerebellum fraction of brain, and activity in liver and heart was below that in brain. Activity increased with age in brain and decreased in other organs. Brief starvation in young animals caused an increase in activity in brain, and a decrease in liver and spleen. Isolated calpain II activity was high in control brain. It increased with age in the cerebrum. Brief starvation resulted in a decrease in the brain. The results indicate that the protease content of the brain is altered with age and in malnutrition, with changes not being the same for all proteases, and changes in brain being different from those in other organs.Special issue dedicated to Dr. Louis Sokoloff.     

Alterations in the distribution of cholinesterase molecular forms in maternal and fetal brain following diisopropyl fluorophosphate treatment of pregnant rats

Previous work in this laboratory showed that during intoxication of rats with diisopropyl fluorophosphate at day 20 of pregnancy the recovery of ChE activity was faster in fetal than in maternal brain. In the present study the differences between recovery rates in dam and fetus brain were evaluated in terms of molecular forms and spontaneous reactivation. Using ultracentrifugation on sucrose gradient two molecular forms of ChE, namely 10S (tetrameric globular G₄ form) and 4S (monomeric G₁ form) were detected both in maternal and fetal brain of untreated rats. The ratios 10S/4S were about 5.0 and 0.75 for dams and 20-day fetuses, respectively. DFP administration (1.1 mg/kg sc) inducing at 90 min an about 80% inhibition of ChE in maternal brain caused a shift in its 10S/4S ratio to 1.63, and to 0.53 in fetal brain (in which overall inhibition was about 70%). This means that 10S forms were preferentially inhibited by DFP both in maternal and fetal brain. After 24 and 48 hr there was a negligible recovery of overall ChE in maternal brain with no shift in the ratio. On the other hand, complete recovery of ChE in fetal brain within 48 hr was accompanied by almost total normalization of the 10S/4S ratio. Rapid recovery of fetal ChE appeared not to depend on hydrolysis of DFP-inhibited ChE. In fact, maternal and fetal DFP-inhibited enzyme preparations following the addition of oximes (pralidoxime or obidoxime) in vitro showed similar rates of reactivation. The overall data indicate considerable differences in recovery rate of molecular forms between dams and fetuses, but not in reactivation by dephosphorylation.     

Hepatic insulin binding following in utero exposure to ethanol

Insulin binding to liver membranes has been studied in term fetuses of rats fed ethanol-containing liquid diet during pregnancy . Pair-fed and ad libitum-fed controls received liquid diet in which maltose-dextrins were substituted isocalorically for ethanol. Food consumption and body weigh gain of ethanol- imbibing dams were 35% and 70% less than their ad libitum counterparts respectively. Ethanol-fed rats also exhibited less gain in body weight than pair-fed controls despite isocalorically equivalent food intake. The number of live pups was not different among the various groups; however, liver weight of fetuses exposed to ethanol in utero was 47% less than those of the pups of ad libitum control dams and 28% less than those of the offspring of pair-fed control rats. Insulin binding to liver membranes of fetuses exposed to ethanol in utero was lower than that of ad libitum controls but was not significantly different from that of the pair-fed control animals. Average affinity profiles showed a reduction in K at all levels of receptor occupancy in the fetuses of ethanol-fed rats. For fetuses of the pair-fed group, K was reduced only at fractional occupancy below 20% but not at higher fractional occupancy. Because of the similarity of insulin binding in the fetuses of the ethanol-fed rats and their pair-fed counterparts, effects of ethanol on insulin binding cannot account for the reduced hepatic glycogen stores previously reported in term fetuses.     

Cadmium,iron and zinc interaction and hematological parameters in rat dams and their offspring

The effects of cadmium (Cd) were evaluated in offspring exposed from birth until weaning (neonatal day 0–21) and 4 weeks after exposure cessation focusing on iron (Fe) and zinc (Zn) levels in organs and hematological parameters. Wistar female rats were administered 50 mg Cd/L in drinking water (Cd-exposed) for 4 weeks before mating and during 3 weeks of gestation plus 3 weeks of lactation. Controls were supplied drinking water. At birth, part of Cd-exposed dams’ litters was cross-fostered to control dams (CCd group) and their control litters were cross-fostered to Cd-exposed dams (CdC group). This procedure enabled to discern the effects of gestational, lactational and gestational plus lactational Cd exposure until weaning in F₁ offspring. Elements were analyzed by atomic absorption spectrometry; hematological parameters manually; and histopathological changes by light microscopy. Gestational plus lactational exposure in Cd-exposed dams and their offspring increased Cd and decreased Fe levels, increased Zn in dams and decreased Zn and body weights in 11- and 21-day pups. In 21-day weanling pups, decreased red blood cell (RBC) count, hemoglobin and hematocrit values and increased reticulocytes in peripheral blood were also found with concomitant histopathological finding of extramedullary hematopoiesis in the liver. In cross-fostered pups with gestational exposure (CCd pups), Fe in the liver decreased on day 11 and Zn increased in the kidney on day 21 whereas in pups with lactational exposure (CdC pups) Zn in the brain decreased on day 11 and Fe decreased in the liver and brain on day 21. Regardless of exposure cessation at weaning, in offspring with gestational plus lactational exposure (Cd-exposed) body weights, kidney and brain Fe levels and RBC and hemoglobin remained decreased in blood until puberty. Furthermore Zn levels increased in the liver, kidney and brain. It was concluded that gestational plus lactational Cd exposure caused decreases in Fe and Zn levels and hematotoxic effects in F₁ offspring more pronouncedly than exposure during either gestational or lactational period alone and the adverse effects of maternally mediated Cd exposure continued after exposure cessation into adulthood.     

Time-dependent changes of amino acids in the serum, liver, brain and urine of rats administered with theanine.

Time-dependent changes of theanine (gamma-glutamylethylamide) and other amino acids in various tissues of rats were investigated during the 24 hrs after theanine administration. When theanine (4 g/kg of body weight) was intragastrically administered to rats, the concentrations of theanine in the serum, liver and brain were significantly increased 1 hr after its administration, and thereafter gradually decreased, but reached the maximum level in the brain after 5 hrs. Theanine in these tissues had completely disappeared 24 hrs after its administration. In contrast, the administration of theanine resulted in the concentrations of theanine, urea, ethylamine and glutamic acid in the urine being significantly enhanced. These results suggest that theanine might be degraded via glutamic acid.     

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.     

Incorporation and intraparticulate hydrolysis of 131 I-albumin by liver and kidney of an amphibian (Bufo arenarum)

After a single injection of formaldehyde-treated 131 I-albumin into the heart, the incorporation of the labelled protein by liver (% of total injected radioactivity/% of body weight of the organ) was far greater than in other organs. In kidney and spleen it was respectively six and three times greater than in lungs, intestine, testis and fatty body. No radioactivity was found in brain. The radioactivity in liver and kidney reached a peak 30 minutes after the injection, and quickly decreased during the following four hours. In the 27,000 g × ten minute particles recovered from liver homogenates of animals sacrificed at various times after injection, the rate of 131 I-albumin hydrolysis in vitro and the percentage of trichloroacetic acid soluble radioactivity at zero time of incubation showed different stages of intraparticulate hydrolytic activity. The incorporation and intraparticulate hydrolysis in toad kidney was very low if compared with that of toad liver or mouse kidney; however the catheptic specific activity in toad kidney doubles that of mouse kidney. Isolated toad liver was perfused with total blood, containing 131 I-albumin, for five hours at 22°C in a special chamber. In this conditions, 16% of the labelled albumin was hydrolyzed by the liver.     

Analysis of metopirone-induced hypertrophy of adrenals in fetal rats encephalectomized in utero

Pregnant rats were treated with 30 mg metopirone (M) each day for 2 days and autopsied on the third day in various gestational periods (Days 18-20, 19-21, and 20-22). Control rats were treated with saline alone (S). The adrenals of intact fetuses in M-treated dams were significantly heavier than those of intact fetuses in S-treated dams in every experimental period. In both M- and S-treated dams, the adrenals of encephalectomized (E) fetuses were lighter than those of intact littermates. However, in the experimental period of Days 18-20 and 19-21, the adrenals of E fetuses in M-treated dams were slightly but significantly heavier than those of similar E fetuses in S-treated dams. In contrast, in the experimental period Days 20-22, there was no significant difference in the weight of adrenals of E fetuses of M- and S-treated dams. These changes in fetal adrenal weight were reflected histologically in parallel changes in the size of adrenocortical cells. The observations suggest that the fetal adrenal hypertrophy following maternal treatment with metopirone can occur to some extent independent of the fetal brain, but that close to the end of gestation the hypertrophy occurs completely under the control of the fetal brain.     

Toxicological Study of Sodium Selenite on Fetal Development and DNA Fragmentation in Liver Cells of Pregnant Rats

The present study was carried out to evaluate the effects of sodium selenite on fetal development and DNA in liver of rats. Pregnant rats were divided into three groups: control group, group treated orally with 5 μg Se/kg body wt. and group treated orally with 10 μg Se/kg body wt. Dams were treated orally with sodium selenite from day 7 to 19 of gestation. Sodium selenite treatment revealed decrease in maternal body weight, reduction in fetal weight, length and number of viable fetuses, increased number of resorbed fetuses and post-implantation loss at the two doses tested. Fetal skeleton showed signs of developmental delay in skull and limbs of the treated groups. Sodium selenite treatment revealed significant reduction of placental and liver weights in treated dams. Sodium selenite-induced oxidative stress in liver tissue of rats as evidenced by increase in lipid peroxidation and glutathione peroxidase activity, while catalase was significantly decreased. Also, increase in DNA fragmentation, marked reduction of hepatic DNA content, and many histopathological changes in the liver were recorded. The results demonstrated that treatment of pregnant rats with sodium selenite at the toxic dosages chosen showed maternal and fetal toxicity that may be concerned with hepatic oxidative stress accompanied with DNA fragmentation and depletion of total DNA content.     

Stimulation of ornithine decarboxylase activity in rat tissues by growth hormone and by serum growth factor from rats infested with spargana of Spirometra mansonoides

Ornithine decarboxylase activity was studied in heart, kidney, liver, thymus, lung, spleen, skeletal muscle and fat of hypophysectomized rats after growth hormone treatment. A marked increase in enzyme activity was observed in kidney and liver, and a significant increase in heart and thymus at 4 h after injection of growth hormone. The kidney was the most responsive organ with an increase in activity of about 100 fold. The enzyme activity in kidney responded to a dose of 10 μg of growth hormone. Daily injection for 12 days raised activity only in the heart. Infestation for 6–13 days with spargana of Spirometra mansonoides, which also causes growth of hypophysectomized rats, increased enzyme activity in the heart and thymus. Intravenous injection of serum of hypophysectomized rats infested with spargana of Spirometra mansonoides caused a significant increase in the enzyme activity in liver and kidney after 4 h. Growth hormone and the serum growth factor of sparganosis seem to share the characteristic of causing an early increase in ornithine decarboxylase activity in rat tissues. The marked response in kidney and liver raises the possibility that these organs are the primary targets of both substances.     

Period of sensitivity to androgens of the Wolff duct of the rat fetus]

The external and internal (lumen) diameter and the height of the epithelium of the Wolffian ducts of control rat fetuses were measured between 14 days 8 hrs and 21 days 8 hrs at the level of the gonad and in the genital folds. In males, at the level of the gonad, these ducts display a transitory increase in diameter and lumen at 16 days 8 hrs of fetal age. This increase, which is absent in females, occurs just before the development of the epididymides and might reflect the endocrine activity of the fetal testis. In the females, the first involutive changes appear at both levels at 17 days 8 hrs of age (decrease in diameter by reduction of the lumen and height of the epithelium). After injections of androgens to pregnant rats or directly into the fetuses in utero, the Wolffian ducts can be maintained in female fetuses only if submitted to androgens before and until 16 days 16 hrs. If the treatment starts once the involutive changes have appeared (17 days 8 hrs) inconstant persistence is obtained. The portions of ducts still present on day 18 cannot be maintained by androgens any more. Even if injected at 15 days 8 hrs, exogenous androgens do not hasten or anticipate the formation of Wolfian derivatives (epididymides and seminal vesicles) in males or in females.     

Prolonged inhibition of brain nitric oxide synthase by short-term systemic administration of nitro-l-arginine methyl ester

We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-l-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [³H]l-arginine into [³H]l-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.