Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat following gonadectomy.

The activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase of rat liver were determined at different time points after gonadectomy on day 75 of life. Following testectomy the activities in male rats assume female values. However this change is relatively slow, 10--14 days being necessary for significant trends in individual activities to develop, and 40--60 days before the final level of activity is reached. The changes in enzyme activities after ovariectomy are only slight. The change in microsomal 5 alpha-reductase activity following gonadectomy of male rats is biphasic, the activity increasing initially to the normal female level before falling to the intermediate "neonatally androgen-imprinted" level. The reaction of 17 beta-hydroxysteroid dehydrogenase activity to testectomy and ovariectomy indicates that in the course of several years, during which we have investigated the behaviour of this enzyme in Chbb/THOM rats, the regulation of its activity has changed from one of oestrogen dependency to one of androgen dependency.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Neonatal phenobarbital administration results in increased cytochrome P450-dependent monooxygenase activity in adult male and female rats

The effects of neonatal exposure to phenobarbital during the first five days after birth on the enzymatic activity of the adult male and female rat liver P₄₅₀-dependent monooxygenase system were investigated. Although liver weight per 100 grams of body weight and total hepatic microsomal protein content were not altered in adult rats treated neonatally with phenobarbital, both sexes did show significant increases in cytochrome P₄₅₀ content, cytochrome P₄₅₀ reductase activity, cytochrome c reductase activity, ethoxycoumarin-O-deethylase activity and in the activity of a specific glucuronyl-transferase. Several of these activities were increased to a larger extent in the females, suggesting that females may be more sensitive to this phenomenon.     

Neonatal Handling, Sweet Food Ingestion and Ectonucleotidase Activities in Nucleus Accumbens at Different Ages

Neonatal handled rats ingest more sweet food than non-handled ones, but it was documented only after puberty. Here, we studied the purinergic system in the nucleus accumbens, a possible target for the alteration in the preference for palatable food. We measured the ATP, ADP and AMP hydrolysis mediated by ectonucleotidases in synaptosomes of the nucleus accumbens in periadolescent and adult rats from different neonatal environments: non-handled and handled (10 min/day, first 10 days of life). Before adolescence, we found a decreased ingestion of sweet food in the neonatally handled group, with no effect on ATP, ADP or AMP hydrolysis. In adults, we found a greater ingestion of sweet food in the neonatally handled group, with no effect on ATPase or ADPase activities, but a decreased AMP hydrolysis. The nucleus accumbens is a site of intensive interaction between the adenosinergic and dopaminergic systems. Therefore, adenosine may modulate accumbens’ dopamine neurotransmission differently in neonatally handled rats.     

Water Concentration and Activity Effects on Aminoacylase in Aqueous/Organic One-Liquid-Phase Systems

Aminoacylase has been employed as a model system to study its catalytic properties at low water concentrations/water activities with different water-miscible organic cosolvents. Cosolvents assayed were alcohols and polyols with pure logarithm of the partition coefficient (log P) values, on the standard water/octanol system, ranging between -5.2 and 0.24.

Experimental hydrolysis equilibrium constants (K_app), at a constant water concentration, decreased with the fall in log P of the cosolvent, as well as with reduction of the water concentration/water activity, as would be expected. The enzyme hydrolytic and synthetic activities, measured at a constant water concentration/water activity value, followed a sigmoidal dependence on log P of the cosolvent employed when the water concentration or water activity values were lower than 50% (w/w) or 0.66, respectively. This became a hyperbolic relationship at higher water concentration/water activity values. A linear relationship between the logarithm of the limiting water activity necessary to maintain enzyme activity and log P was obtained. Both hydrolytic and synthetic activities were suppressed for water activities higher than 0.66 and cosolvents with log P lower than -1.6.     

ACETYL- AND BUTYRYLCHOLINESTERASE ACTIVITY OF SELECTED BRAIN AREAS IN DEVELOPING RATS AFTER NEONATAL X-IRRADIATION*

Acetylcholinesterase and butyrylcholinesterase activities in sensori-motor cortex, hypothalamus, cerebellum, and brain stem were compared in normally developing Long-Evans rats and after neonatal whole-body exposure to 450 r X-radiation. Enzyme activities were measured on three postnatal days: day 10, when brain is still immature; day 24, when it has reached functional and morphological maturity; and day 64, after sexual maturation. In controls, acetylcholinesterase and butyrycholinesterase activities increased with age in all areas, especially between 10 and 24 days; e.g., in sensori-motor cortex acetylcholinesterase activity increased 60 per cent from 10 to 24 days and 12 per cent from 24 to 64 days. At all ages acetylcholinesterase activity was highest in the brain stern, followed in decreasing order by the hypothalamus, cerebellum, and sensori-motor cortex. Butyrylcholinesterase activity was higher in subcortical than in cortical areas. In neonatally irradiated rats, acetylcholinesterase activity was significantly decreased in the ontogenetically newer structures at 10, but not at 64, days; in the hypothalamus, it remained normal at 10 days but was significantly decreased at 24 and 64 days. Butyrylcholinesterase activity was significantly decreased in some areas 1 week after radiation but returned to normal at 24 days. Total esterase activity in whole blood was signtficantly decreased at 10 days in irradiated rats but returned to control levels by the end of the experiment. The greatest post-radiation decline in acetylcholinesterase activity (60 per cent below controls) did not result in spontaneous gross behaviour alterations, but may be related to disturbances in functional brain maturation evidenced by specific tests. If the role of acetycholine as a central neurotransmitter is accepted, these data suggest that radiation alters acetycholine/acetylcholinesterase ratios and thereby cholinergie synaptic transmission.     

Effects of nutritional deficiencies during neonatal and post-weaning period on rat intestinal phytase and phosphatase activities

Studies were made of the effects of pre- and post-weaning undernutrition and/or protein deficiency on intestinal phytase and phosphatase activities in albino rats and reversibility of the same by subsequent dietary rehabilitation. Neonatal undernutrition induced by rearing the pups in litters of 16 caused a marked decrease in alkaline phytase activity (as compared to those reared in litters of 8), while acid phytase activity decreased to a lesser extent and acid and alkaline phosphatase activities did not change. When neonatally undernourished rats were subsequently continued on a 4 or a 20% protein diet in restricted amounts (2.5 g/day) for 6 weeks the decreases in the alkaline phytase activity but not in that of acid phytase were further aggravated. Acid and alkaline phosphatases were not influenced by these treatments either. On dietary rehabilitation of these rats for subsequent 6 weeks on a 20% protein diet (ad libitum) acid and alkaline phytase activities of intestine recovered partially. These studies indicate the importance of alkaline phytase activity as a marker of intestinal maturation and is also suggestive of interrelationships between nutrition, intestinal development and its alkaline phytase activity.     

Role of immunity in age-related resistance to paralysis after murine leukemia virus infection.

Resistance to the paralytic effects of a wild mouse (Cas-Br-M) murine leukemia virus infection develops with age and is complete by 10 days of age in susceptible NFS mice. The possibility that cell-mediated immunity plays a significant role in this resistance was suggested by the observation that treatment of 10-day-old mice with antithymocyte serum rendered them susceptible to paralysis. By comparison, mice rendered incapable of generating a humoral immune response by treatment from birth to 1 month of age with anti-immunoglobulin M serum did not develop paralysis after challenge with virus at day 10. Transfer of unseparated and T-cell-enriched populations of Cas-Br-M murine leukemia virus-immune spleen cells protected neonatally infected NFS recipients from paralysis; transfer of Cas-Br-M murine leukemia virus-immune populations enriched for B cells delayed the onset but did not ultimately protect neonatally infected NFS mice from paralysis. Transfer of naive adult spleen cells had no protective effect in neonatally infected NFS mice. High-level virus replication occurred in the spleens and brains of all mice that developed paralysis regardless of treatment; low-level virus replication in spleen and barely detectable replication in brain occurred in mice that remained clinically normal. These studies suggest that the age-acquired resistance to the paralytic effect of Cas-Br-M murine leukemia virus infection is immunologically mediated and that T cells may play a major role.     

Does depression of NK activity cause lymphadenopathy in lpr mice?

B6-lpr/lpr mice develop massive T cell lymphoproliferation, as associated with autoimmune disease. We found a reduced NK activity in the spleen of B6-lpr/lpr mice. Neonatal thymectomy markedly retarded the development of lymphoproliferation and the development of autoantibodies in the B6-lpr/lpr mice. These animals had a higher level of NK activity in the spleen. When the neonatally thymectomized B6-lpr/lpr mice were given anti-asialo GM1 serum (30 microliter) four times at 6-day intervals, initiated at the 8th-10th postnatal week, these mice developed lymphoproliferative disorders and splenomegaly, concomitantly with depression of NK activity. It is therefore tempting to speculate that NK cells are involved in the regulation of the occurrence of lymphoproliferative disorders.     

Regulation of urine marking in male and female mice: effects of sex steroids

Effects of sex steroids on urine-marking activity were studied in male, female, and neonatally androgenized female mice. Urine marking was estimated by suspending ceramic tubes that were connected in a horizontal row with a steel rod into the home cage of an isolated mouse. Intact males showed high marking activity, which was diminished after castration. Both testosterone propionate (TP) and estradiol benzoate (EB) were effective in restoring the marking activity of castrated males, while 5-alpha-dihydrotesterone (DHT) did not have any stimulative effects. Intact normal females showed quite low marking activity and ovariectomy further depressed it. TP and DHT enhanced the marking of ovariectomized females, but EB restored the activity only to the preovariectomy level. In intact females which were neonatally androgenized, the marking activity was much higher than that of normal females. The pattern of the change induced by gonadectomy and hormone treatment in these females resembled that in males. Thus, ovariectomy reduced the activity and both TP and EB restored the level. These results indicate that the sexual dimorphism in the urine marking in mice is primarily determined by hormonal environment during early postnatal age. Hormonal control of scent marking is discussed in relation to the studies in other rodents.     

Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment

The effects of neonatal exposure to diethylstilbestrol (DES) on hepatic activation/detoxication enzyme levels in the adult rat were investigated. Neonatal exposure of male rats to DES (DES males) decreased the endogenous levels of UDP-glucuronyltransferase as compared to control males. Female rats exposed neonatally to DES (DES females) had higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult animals treated neonatally with DES also had altered metabolic potential following exposure to 3-methylcholanthrene and phenobarbital. The DES males treated in adulthood with 3-methylcholanthrene had higher benzo(a)pyrene hydroxylase activities and lower UDP-glucuronyltransferase activity levels than did control males treated in adulthood with 3-methylcholanthrene. The DES males exposed in adulthood to phenobarbital had reduced cytochrome P-450 and glutathione transferase activity levels as compared with respective controls. The DES females treated in adulthood with 3-methylcholanthrene had lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels than control females receiving 3-methylcholanthrene. The DES females challenged in adulthood with phenobarbital also had decreased benzo(a)pyrene hydroxylase, epoxide hydrolase, UDP- glucuronyltransferase, and glutathione transferase activity levels as compared with respective controls. Our results demonstrated that neonatal exposure to DES changed the endogenous levels of specific hepatic enzymes and altered the metabolic response of these adult animals to a carcinogen and a drug.     

EFFECTS OF UNDER NUTRITION AND PROTEIN DEFICIENCY ON GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN RAT BRAIN

Abstract— Studies were made on the effects of undernutrition at different ages during the neonatal period and of the comparative effects of postweaning protein and calorie deficiencies in neonatally undernourished or normally reared animals. Neonatal undernutrition resulted in deficits in body wt, brain wt and the activities of brain glutamate dehydrogenase and glutamate decarboxylase. Percentage deficits in brain wt were maximum in the first week of life but those in brain enzymes were greater in the second week. Rehabilitation of neonatally undernourished animals reversed the deficits in brain wt and brain enzymes. Post-weaning protein deficiency produced similar deficits in brain enzymes in both neonatally undernourished and normally reared animals. With post-weaning undernutrition, however, these deficits were found only in animals subjected to neonatal undernutrition as well.     

Neonatal effect of clomiphene and o,p'-DDT on hepatic oxidative metabolism of male rats.

The hepatic oxidative metabolism of dehydroepiandrosterone (DHA) and aminopyrine has been investigated in adult rats of both sexes treated neonatally with either clomiphene citrate (100 mug on day 3) or o,p'-DDT (1 mg daily on days 2-3). The rates of 16 alpha-, 7 alpha- and 7 beta-hydroxylase activities of DHA and the N-demethylase activities of aminopyrine in hepatic microsomes of normal males were significantly (p less than 0.05) higher than those of females. Treatment with clomiphene citrate reduced significantly (p less than 0.05) the 16 alpha-hydroxylase activities of males and appeared to suppress the 7 alpha-hydroxylase and N-demethylase activities to a lesser extent. Neonatal o,p'-DDT also reduced these hepatic oxidative activities. Neither treatment affected the 7 beta-hydroxylase activities. On the other hand, no significant differences in these activities were observed between the control and the treated females, all of which had persistent vaginal estrus. Therefore, it appears that treatment of male neonates with these weak estrogenic compounds antagonizes the androgen-mediated expression of the DHA-16alpha-hydroxylase activity in liver.     

Palatine tonsils and immunity. VI. Effect of thymosin and palatine tonsil extract on the immunologic reactivity of neonatally thymectomized and intact mice]

Experiments were conducted on Wistar rats, intact and neonatally thymectomized CBA mice; a study was made of the immunological activity of the extracts of the thymus and the palatine tonsils of calves obtained by the method of Goldstein et al (1966). The extract of the palatine tonsils obtained proved to contain a small amount of thymosin at whose expense an insignificant restoration of immunological reactivity occurs in its administration neonatally to thymectomized mice.     

Effects of neonatal estrogen treatment of female guinea pigs on mounting behavior in adulthood

Female guinea pigs were treated with 200 micrograms estradiol dipropionate (ED) daily from Days 1 to 15 of life. As adults they all ovulated. After gonadectomy and estrogen-progesterone treatment in adulthood, lordosis behavior was evident and showed no decrements (compared with neonatally oil-treated controls) except for a marginal decrease in maximum duration scores. However, testosterone propionate treatment in adulthood significantly increased mounting behavior in neonatally ED-treated females compared to the controls. On the other hand, estrogen-progesterone treatment in adulthood stimulated mounting in the neonatally oil-treated group, but not in the neonatally estrogenized group. The results suggest that at least some aspects of steroid-dependent behaviors can be permanently influenced by estrogen treatment after the presumed prenatal critical period for sexual differentiation has been completed in guinea pigs.     

Influence of age and sex on hepatotoxic and hypothermic effects of estrogole in mice

Some physiological effects of estragole under a single intraperitoneal injection in oil solution were studied in GR mice; elevation of blood aminotransferases activity, body temperature and animals lethality were registered. At a dose of 600 mg/kg, estragole killed 100% sucklings of both sexes and 90% adult females but no any adult male. The males aquire resistance to estragole at the time of maturation. Exogenous testosterone administered at the dose of 50 mg/kg 4-2 days earlier increases the resistance of female mice to estragole up to its level in males. However, neonatally androgenized females are as sensitive to the toxic action of estragole as the males. At the dose of 900 mg/kg, estragole defeats adult males as well: significant elevation of aminotransferase activities in their blood is indicative of this. In this case, the enzyme activity reaches its peak after 2-3 days, not at the 1st day as in the case of carbon tetrachloride administration. We have discovered a strong hypothermic effect of estragole which appears to be unrelated to its hepatotoxicity and testosteron level.     

Specific and sensitive plate assay for bacterial lipases

A plate assay to detect bacterial lipase (EC 3.1.1.3) in a medium containing trioleoylglycerol and the fluorescent dye rhodamine B is presented. Substrate hydrolysis causes the formation of orange fluorescent halos around bacterial colonies visible upon UV irradiation. The logarithm of lipase activity from cell-free culture supernatants is linearly correlated with the diameter of halos, thereby allowing quantitation of lipase activities ranging from 1 to 30 nkat.     

Specific and sensitive plate assay for bacterial lipases.

A plate assay to detect bacterial lipase (EC 3.1.1.3) in a medium containing trioleoylglycerol and the fluorescent dye rhodamine B is presented. Substrate hydrolysis causes the formation of orange fluorescent halos around bacterial colonies visible upon UV irradiation. The logarithm of lipase activity from cell-free culture supernatants is linearly correlated with the diameter of halos, thereby allowing quantitation of lipase activities ranging from 1 to 30 nkat.     

DIFFERENCES IN RHYTHMS OF ENZYMATIC ACTIVITY OF MATERNAL AND FETAL BLOOD

Blood specimens were obtained at different daily times from the umbilical cord and brachial vein from 53 women within 10 minutes after delivery. Enzyme activities were measured in the white blood cells (WBCs) and serum of each sample. For each enzyme, the results were grouped according to sampling (delivery) times and arrayed to form a 24h time series. Separate time series were generated for maternal and fetal enzymes. Analysis of variance (ANOVA) and cosine best-fit analyses were applied to elucidate significant differences between enzyme activity patterns of mothers and fetuses with regard to time dependency, number of peaks, and acrophases. These and previously documented results indicate that not all mothers and fetuses have rhythms that are concordant. For some enzymes of fetuses, the activity rhythms differ in phase and shape of the time series pattern from those of the mothers; for other enzymes, the activity rhythms develop after birth. (Chronobiology International, 17(2), 221–228, 2000)     

Differences in rhythms of enzymatic activity of maternal and fetal blood

Blood specimens were obtained at different daily times from the umbilical cord and brachial vein from 53 women within 10 minutes after delivery. Enzyme activities were measured in the white blood cells (WBCs) and serum of each sample. For each enzyme, the results were grouped according to sampling (delivery) times and arrayed to form a 24h time series. Separate time series were generated for maternal and fetal enzymes. Analysis of variance (ANOVA) and cosine best-fit analyses were applied to elucidate significant differences between enzyme activity patterns of mothers and fetuses with regard to time dependency, number of peaks, and acrophases. These and previously documented results indicate that not all mothers and fetuses have rhythms that are concordant. For some enzymes of fetuses, the activity rhythms differ in phase and shape of the time series pattern from those of the mothers; for other enzymes, the activity rhythms develop after birth. (Chronobiology International, 17(2), 221-228, 2000)