Evolution of the activity of UGT1A1 throughout the development and adult life in a rat.

Biliary excretion is the main route of disposal of bilirubin and impaired excretion results in jaundice, a well recognisable symptom of liver disease. Conjugation of bilirubin in the liver is essential for its clearance. The glucuronidation of bilirubin is catalysed by the microsomal UDP-glucuronosyltransferase UGT1A1. Patients with Crigler-Najjar syndrome type 1 and Gunn rats, mutant strain of the Wistar rats, bear an autosomal recessive disorder resulting in hyperbilirubinemia. The aim of this work is to add new data about activity of UGT1A1 during the perinatal period and adult life. The results showed that activity of UGT1A1 is detectable from day 22 of the gestation. After birth, activity of UGT1A1 gradually increases and reaches the levels of adult life. Furthermore, bilirubin azopigments have been separated and characterized by thin layer chromatography. We have found that concentration of samples by evaporation and ulterior storing at -20 degrees C seemed to be suitable for the maintenance of samples.

Hepatic proliferation in Gunn rats transplanted with hepatocytes: effect of retrorsine and tri-iodothyronine.

Hepatocyte transplantation would offer an attractive alternative to liver transplantation in the treatment of inborn errors of liver metabolism. However, a major problem in most transplantation studies to date has been the limited growth of transplanted cells in the recipient organ. We performed a strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting liver cells in conjunction with repeated administration of triiodothyronine, an inducer of hepatocyte proliferation in rats. In the present study, foetal and adult syngeneic hepatocyte transplantation into spleen was performed in retrorsine-treated hyperbilirubinemic Gunn rats. In parallel, repeated injections of triiodothyronine were given to recipients. Rats were sacrificed at 1, 7, 30 and 90 days after transplantation and blood and bile samples were taken to assess the functionality of transplanted cells. The proliferative activity of transplanted hepatocytes was evaluated using proliferating cell nuclear antigen labelling index. In summary, both adult and foetal hepatocyte transplantation were effective in correcting a metabolic abnormality in Gunn rats for as long as 3 months. The RS/T3 model, as a measure to increase graft function, could represent an important advance to future clinical application of hepatocyte transplantation.

Intracellular calcium alterations and free radical formation evaluated by flow cytometry in endotoxin-treated rat liver Kupffer and endothelial cells.

During endotoxic shock, the liver exerts an endotoxin (lipopolysaccharide, LPS) clearance function with the participation of both sinusoidal (mainly Kupffer and endothelial cells) and parenchymal cells. In order to determine the specificity and diversity of response of each liver cell type, the effect of Escherichia coli 0111:B4 endotoxin (LPS) on intracellular Ca2+ content and reactive oxygen metabolite production in rat liver Kupffer, endothelial and parenchymal cells, was evaluated by flow cytometry during short treatment times (from 0-2 min) with a low dose of LPS (10 micrograms/ml). Concerning sinusoidal cells, LPS produced a significant increase of intracellular Ca2+ in both endothelial and Kupffer cells. The LPS effect on Kupffer cells was higher than on endothelial cells. When intracellular reactive oxygen metabolite production was evaluated in LPS-treated sinusoidal cells, a fast and significant increase of Kupffer cells in activated state (cells with a high reactive oxygen intermediate production) was observed. However, endothelial cells did not show LPS-induced changes in their intracellular reactive oxygen metabolite content. All these results support a rapid activation of liver Kupffer cells by endotoxin consistent with the major role of this cellular type as active first line of defense during endotoxic shock. The liver endothelial cells are also involved in the first steps of the cell damage showing intracellular Ca2+ alterations. Liver parenchymal cells did not show any response at these experimental conditions (short treatment time and low LPS dose) indicating that longer treatment times are needed for LPS binding and action in agreement with previous studies.

Stimulation of phagocytic function in mouse macrophages by neurotensin and neuromedin N.

The neuropeptides neurotensin and neuromedin N (from 10(-12) M to 10(-9) M) have been shown in this study to stimulate significantly in vitro several steps of the phagocytic process: adherence to substrate, chemotaxis, ingestion of inert particles (latex beads) and production of superoxide anion measured by nitroblue tetrazolium reduction in resting peritoneal macrophages from BALB/c mice. A dose-response relationship was observed, with a maximal stimulation of the phagocytic process at 10(-11) M. The two neuropeptides induced no change of intracellular cyclic AMP in murine macrophages. Moreover, adherence and chemotaxis decreased significantly in the presence of EGTA (1 mM), a chelator of extracellular Ca2+, or ryanodine (0.5 mM), a blocker of a Ca(2+)-gated channel from the endoplasmic reticulum, in both controls and samples with the addition of neurotensin or neuromedin N. These results suggest that there is no relation between the cAMP messenger system and the phagocytic process stimulation in murine peritoneal macrophages by neurotensin or neuromedin N. In addition, the results observed with EGTA and ryanodine could indicate that these two neuropeptides produce their effects through an increase of intracellular Ca2+ concentration.

Modulation by neurotensin and neuromedin N of adherence and chemotaxis capacity of murine lymphocytes.

The action of neurotensin and related peptides has not been yet studied on lymphocytes, although there are studies indicating the stimulative action of neurotensin, a peptide first isolated from bovine hypothalamus, on different functions of phagocytic immune cells. The present study demonstrates that neurotensin and a related peptide, neuromedin N, increased significantly the adherence and chemotaxis capacity of murine peritoneal lymphocytes, when they were incubated in the presence of neuropeptide concentrations between 10(-9) M and 10(-12) M. With respect to their adherence capacity, neuromedin N showed a slightly higher stimulation than neurotensin at a shorter time. However, both neuropeptides stimulated the chemotaxis capacity in a similar percentage. The study of the action mechanisms of these neuropeptides showed that intracellular cAMP levels were not modified by neurotensin or neuromedin N, but using an extracellular calcium chelator, EGTA (1 mM), and a blocker of calcium channels in endoplasmic reticulum, ryanodine (0.5 mM), we observed that neurotensin and neuromedin N could produce their effects through an augmentation of the intracellular Ca2+ concentration. As adherence and chemotaxis are initial processes of immune response, the results show that both neuropeptides may be physiological modulators of the lymphocyte function.

Effect of metyrapone administration in pregnant rats on monoamine concentration in fetal brain.

Studies performed in our laboratory indicate that the adrenal deprivation during gestation can greatly influence the fetal catecholamines development in several cerebral areas. The present study was undertaken to determine whether the administration of metyrapone to pregnant rats affects the content of monoamines in fetal brain at term. To test wether the content of monoamines in fetal brain is regulated, at least in part, by endogenous glucocorticoids, pregnant rats were injected for 5 days prior to delivery with metyrapone, an adrenal 11-beta-steroid hidroxylase inhibitor which crosses the placenta and blocks endogenous glucocorticoid synthesis, or saline. On day 21 of gestation, delivery of all animals was accomplished by cesarean section. The encephalons were extracted and immediately dissected in metencephalon, mesencephalon, diencephalon and telencephalon. Monoamine determination was carried out using HPLC-ED. The results obtained indicate that the metyrapone treatment increases both DA and 5-HT and their metabolites in the brain studied areas.

Effect of maternal adrenal deprivation on the content of catecholamines in fetal brain.

Previous studies performed in our laboratory showed the importance of the effects that the absence of maternal adrenal hormones have on fetal brain. In the present study we investigated the effect of adrenal deprivation during gestation on the fetal catecholamines development in several cerebral areas. Fetuses from both control and adrenalectomized mothers from the first day of gestation were removed on the 20th embryonary day. Plasma corticosterone levels were significantly lower in the maternal serum of adrenalectomized rats, while the contents were non significantly higher in the adrenalectomized-mothers group of fetuses. Catecholamine contents in diencephalon, metencephalon, mesencephalon and telencephalon were measured by HPLC-ED. The results obtained showed that when the development of the catecholaminergic systems was previous enough to the fetal adrenal function, and under maternal adrenal deprivation conditions, the lack of corticosterone promotes an increase in the level of the catecholamines, as observed in the diencephalic NA, the earlier in maturational process. In those areas where the maturation starts at the same time than the fetal adrenal hypersecretion, no changes were observed. In the cortex, where both DA and NA develop later, the corticosterone produces an inhibition in the proliferation of the catecholaminergic neurons, showing decreased telencephalic levels of both catecholamines.

Liver gene expression and increase in albumin synthesis by fetal hepatocytes transplanted into analbuminemic rats.

This report describes the evolution of hepatocytes isolated from 21-day fetuses and transplanted into spleens of Nagase analbuminemic rats which have negligible serum albumin levels due to a mutation affecting albumin mRNA processing. Albumin and alpha-fetoprotein expression, in addition to other parameters related to cellular proliferation status (thymidine kinase and proliferating cell nuclear antigen expression) were studied as indicative of the behavior and evolution of the cells. In recipient rats, only a few clusters of hepatocytes could be observed in the red pulp of the spleen 24 h after transplantation. The fetal hepatocytes migrated to the liver and could be seen in portal branches immediately after transplantation. Fifteen days later, albumin mRNA was detected in recipient livers and was expressed throughout the entire 3-month study. Alpha-fetoprotein was not detected. Cell proliferation was not relevant, although 3 months after transplantation, the proliferation rates appeared to show a tendency to increase. These data demonstrate that fetal hepatocytes transplanted into spleen migrate to liver, settle there and acquire an adult phenotype free of malignant transformation. Our study is a first step towards the thorough understanding of fetal hepatocyte transplantation. The next steps will involve in-depth studies of the possibilities of genetic manipulation to achieve a high degree of repopulation/expression, employing the least possible number of donor cells, and of how the cells reach the liver parenchyma, overcoming the endothelial barrier.

Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation.

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.

Influence of maternal adrenalectomy and glucocorticoid administration on the development of rat cerebral cortex.

In order to determine the incidence of maternal glucocorticoids on morphological parameters in fetal development, we performed optic and electron microscopic analysis of the cerebral cortex of fetuses of 16 and 20 days of gestation, from control (C) and pregnant rats bilaterally adrenalectomized on day 1 of gestation (ADX). We also studied fetuses 20 days old from pregnant rats betamethasone-injected on days 15, 16 and 17 (BET), and adrenalectomized on day 1 and betamethasone-injected on days 15, 16 and 17 (ADX+BET). Absence of maternal glucocorticoids during gestation caused, in fetuses 16 and 20 days old, a marked increase of cellular density, laxity of tissue and lower cellular maturation in comparison with the control group. Beta-methasone injected into sham-operated animals (BET) caused a slight advance in relation to controls in developmental parameters such as cellular density, maturation and synapse formation. Betamethasone injection into adrenalectomized animals prevented the lower degree of maturation characteristic of the adrenalectomized group, although an increase of cellular density could be detected. The cerebral cortex from fetuses of 16 days of gestation from adrenalectomized mothers also showed an increase of cellular density as compared with the control group. These results show that glucocorticoids participate in prenatal rat brain in control mechanisms of cellular division and maturation.

Physiological changes in antioxidant defences in fetal and neonatal rat liver.

This study describes the physiological changes in the activities of the hepatic antioxidant enzymes superoxide dismutase isoenzymes (Cu/Zn-and Mn-superoxide dismutase) and catalase, in the glutathione content and in the lipid peroxidation levels in fetal (20 and 21 days of gestation) and neonatal rat liver (Days 1, 8, 15, and 22 post partum). The catalase and superoxide dismutase activities decreased before birth and increased after birth. The oxidized:reduced glutathione (GSSG:GSH) ratio declined before birth, but it increased between Days 1 and 15 post partum and then remained stable. Finally, newborn rat liver from the 1st day of life shows the highest susceptibility to lipid peroxidation. These results suggest that the changes in antioxidant defences could be related mainly to the beginning of diet intake after birth, which entails a higher hepatic metabolism rate, as well as a higher oxygen consumption.

Effects of endogenous and exogenous glucocorticoids on liver differentiation.

The effects of maternal bilateral adrenalectomy on day 1 of gestation and betamethasone treatment on fetal liver development were compared, in terms of biochemical and morphological parameters. For fetuses 20 days old (E20), absence of maternal glucocorticoids during gestation caused an increase in the number of nuclei in whole livers, and a significantly decrease of both body weight and protein content per nucleus, in comparison with the control group (C). Betamethasone injection on days 15, 16 and 17 of gestation into adrenalectomized pregnant rats (ADX + BET) did not completely prevent these effects. The electron microscopic analysis of the ADX fetal liver (E20) showed some hepatocyte lesions such as loss of cytoplasmic organelles, increase in hematopoietic cell number as well as a lower cellular maturation in comparison with the control group. The fetal liver from ADX + BET mothers 20 days after gestation displayed a noticeable involution of the hematopoietic component in spite of its relatively immature stage. However, there was no significant change in the degree of fetal hepatocyte lesions. Therefore, supply of maternal glucocorticoids from the beginning of gestation is essential for maintenance of the integral structure of the rat fetal hepatic parenchyma, for the correct maturation of the blood strains and for the beginning of involution of the hematopoietic tissue at the end of gestation.

Lipid metabolism in pregnancy. Perfusion of liver in pregnant rats.

Livers of pregnant or nonpregnant rats were perfused, and incorporation of 14C-acetate into lipids was studied. Total lipids were extracted from samples of perfusion medium, taken at the entrance and exit from the liver, at different time intervals, and from the liver homogenates at the end of the experiment. Time-course of the incorporation of 14C-acetate into different lipid classes from perfusion medium and liver tissue was compared for pregnant and nonpregnant rats. Radioactivity incorporation into circulant FFA and TG was higher in the control rats than in the pregnant animals. Contrariwise, there was a high incorporation of labelled fatty acids into phosphoglycerides in pregnant rats. There was an increase of unsaturated fatty acids of large carbon chain (24 : 1 and 22 : 6) in the perfusion media of pregnant liver. Moreover, the percentages of labelled fatty acids 16 : 1, 18 : 1 and 18 : 0 were higher in pregnant rats than in controls.

Effects of maternal bilateral adrenalectomy and betamethasone administration on fetal rat encephalic development.

The present study examined the effects of maternal bilateral adrenalectomy and betamethasone treatment on fetal encephalic development, in terms of fetal body weight, brain weight, DNA, protein and lipid content and morphological development. Both influenced the developmental time patterns of fetal brain and cerebellum. Fetuses of adrenalectomized rats had decreased body weights, whereas brain weight was not affected. Maternal adrenalectomy produces in fetal brain a decreased number of cells and increased cell size, while betamethasone treatment of adrenalectomized rats increased cell number, which was not different from control values; cell size remained lower than in control fetuses. Lipid content was increased in the fetuses of betamethasone-treated rats. In terms of morphological development, laminated structures (hippocampus and brain and cerebellar cortex) were the ones most affected.

Glucose and ketone bodies production in hepatocytes isolated from fetuses at term--I. Effect of maternal fasting.

The effect produced by maternal fasting on glucose and ketone bodies production has been studied in hepatocytes isolated from fetal rat. Maternal fasting produces a decrease in the weight of fetal liver. Maternal fasting produces a decrease in glucose production, both from endogenous substrates and adding lactate (10 mM) to the incubation medium. Maternal fasting produces an increase in ketone bodies production, both from endogenous substrates and adding acetate (5 mM) to the incubation medium.

Glucose and ketone bodies production in hepatocytes isolated from fetuses at term--II. Effect of maternal adrenalectomy.

Glucose and ketone bodies production has been studied in hepatocytes isolated from fetuses at term of fed and fasted adrenalectomized mothers. Maternal adrenalectomy diminishes the fetal liver weight. This effect is increased when the adrenalectomized pregnant rat is fasted for the last 2 days of gestation. Maternal adrenalectomy diminishes glucose production in hepatocytes isolated from fetuses at term. This diminution is markedly greater when the adrenalectomized pregnant rat is fasted for the last 48 hr of gestation. Maternal adrenalectomy diminishes ketone bodies production in hepatocytes isolated from fetuses at term.

Effects of maternal adrenalectomy and glucocorticoid administration on the development of rat hippocampus.

Optic and electron microscopic analysis were performed on the hippocampus of fetuses of 20 days gestation, from pregnant rats bilaterally adrenalectomized on day 1 of gestation (ADX) and control (C), in an attempt to determine the incidence of maternal glucocorticoids on morphological parameters in fetal development. In addition, we studied 20 day old fetuses from pregnant rats betamethasone-injected on days 15, 16 and 17 (BET), and adrenalectomized on day 1 and betamethasone-injected on days 15, 16 and 17 (ADX + BET). The adrenalectomy led to a decreased total cell number and a marked depletion of the dentate gyrus of the hippocampus, and also to widespread indifferentiation, both in the pyramidal cell layer of the Ammon's horn and the dentate gyrus, as well as a decreased cell number in CA3 stratum pyramidale. No differences in cell number were found in CA1. So, the ADX effect is in relation to the neurogenic gradient. The main effect of the exogenous glucocorticoid treatment was increased maturation in relation to the control group. Betamethasone injection in adrenalectomized animals prevented the lower degree of maturation of the adrenalectomized group but not the impaired cell number. These results show that glucocorticoids participate in prenatal hippocampus in control mechanisms of cellular division and in maturation.

Expression of bilirubin UDP-glucuronosyltransferase (bUGT) throughout fetal development: intrasplenic transplantation into Gunn rats to correct enzymatic deficiency.

The aim of this work was to determine the pattern of expression of hepatic bilirubin UDP-glucuronosyltransferase throughout fetal development in rats, with the purpose of using fetal hepatocytes at the most appropiate stage of development for transplantation into Gunn rats lacking bilirubin UDP-glucuronosyltransferase activity and then assessing the therapeutic capacity of the implants. The results show that at day 13 of gestational life there is already bilirubin UDP-glucuronosyltransferase gene expression. Twenty-one-day fetal hepatocyte transplantation was also performed into the spleens of hyperbilirubinemic Gunn rats, when alpha-fetoprotein mRNA is still detectable. At 15, 30, and 90 days after transplantation, a mild decrease in total bilirubin serum levels was observed. An increase in bile conjugated bilirubin also was observed at 30 and 90 days. These data suggest the favorable evolution of transplanted cells and show its feasibility for therapy.