The effect of dexamethasone on P450 activities in regenerating rat liver.

The aim of our study was to detect four P450s (CYP1A, CYP2B, CYP2E1, CYP3A) on the basis of selective enzyme activities and protein amount, and to investigate the effect of dexamethasone treatment during liver regeneration. Partial hepatectomy of rats resulted in the loss of CYP1A, CYP2B, CYP2E1, and CYP3A activities. The reduction of enzyme activities and the loss of enzyme protein of CYP2B1/2, CYP2E1, and CYP3A1/2 were the most pronounced. In the case of CYP1A1, only slight decrease was observed. Dexamethasone treatment seems to counteract this loss mainly in the first 12 h.

Hepatic regeneration induces transient acute phase reaction: systemic elevation of acute phase reactants and soluble cytokine receptors.

The growth factors present during liver regeneration partially overlap with the regulators of the hepatic acute phase response. We analysed the acute phase reaction and changes in soluble cytokine receptors after partial hepatectomy, when tissue injury inducing acute phase reaction and major reduction of liver mass occur simultaneously. Three acute phase proteins and mRNAs were determined by ELISA and northern blot hybridisation in rats. Serum levels of IL-6 and three soluble cytokine receptors (sTNF-alpha R I and II, sIL-6R) were detected by ELIBA or dot-blot assay. Time-course profiles of fibrinogen, alpha(2)-macroglobulin and haptoglobin proteins and mRNA are presented. Elevation of IL-6, soluble TNF-alpha receptors and soluble IL-6 receptor levels were also detected. The time-course of changes in haptoglobin concentration and elevation of soluble cytokine receptors is described by this in vivo experimental system. The results show good correlation with (post)transcriptional activation of immediate and delayed early gene products. These data suggest the involvement of both acute phase proteins and soluble cytokine receptors in the regulation of liver regeneration.

Elevated hepatic glucocorticoid receptor expression during liver regeneration in rats.

In rats within the first week of partial hepatectomy reconstruction of the normal histological structure of the liver already starts. To approach the possible role of endogenous glucocorticoids in the process of regeneration we measured the changes in the expression of steroid glucocorticoid receptor gene after various regeneration intervals. After partial hepatectomy, between 0.5 168 hours from the surgery, the gene expression (mRNA) of glucocorticoid receptor was determined by reverse transcription followed by PCR and normalized to that of glycerolphoshate dehydrogenase. Two peaks of glucocorticoid receptor mRNA were detected first, between 3 and 6 hours (first peak) and a second between 24 and 36 hours. Immunoreactive glucocorticoid receptor was detected by immunohistochemistry using monoclonal anti-glucocorticoid receptor. Three days after the surgery immunohistochemical studies showed substantially more immunoreactive GcR protein in the regenerated liver than in the controls. These semiquantitative data provide evidence suggesting elevation of glucocorticoid receptor expression during regeneration of liver at mRNA and protein levels.

Investigation on the formation of the hormone reception mechanism.

The formation of the hormone receptor mechanism is part of the embryonic development. The perinatal age seems to be critical for the maturation of this system, i.e. the development of the proper hormone selectivity, the number of the receptors, their sensitivity to the adequate hormone. Our team tried to get a closer view on this developmental period examining different parameters in different model systems. We were able to show that during the maturation of the hormonal system similar responses could be elicited by the later specific hormones or by the non-specific ones but structurally similar compounds. Also it became evident that one single hormonal treatment, applied during in the perinatal age, could influence the responsiveness to hormones of the adult animals.

Changes in sexual behavior of adult male and female rats neonatally treated with vitamin D3.

1. Neonatal treatment of rats with vitamin D3 resulted in a change of sexual behavior in adulthood. 2. 2.5 mg vitamin D3 completely inhibited the ejaculation of males without any apparent influence on sexual desire. 250 mg vitamin D3 influenced both the desire and ejaculation. 3. Sexual activity of females was depressed by both doses. 4. The experiments demonstrate that vitamin D3, a steroid in structure, given in the critical period of hormonal imprinting may influence steroid hormone-receptor commanded events for life, in a way similar to the effects exhibited by synthetic steroid hormone analogues and benzpyrene in earlier studies.

Effect of nifedipine treatment (imprinting) of rat feti and newborns on the responsiveness of adult rat's uterus. Extension of the imprinting theory.

1. The uterus of adult progeny of rats treated with nifedipine during the late phase of pregnancy react in vitro to oxytocin less and the contractility of ones treated with higher dose (100 micrograms) disappears. 2. There is a more pronounced deficiency or lack of responsiveness in five week old animals treated with nifedipine neonatally. 3. The experiments demonstrate that perinatal imprinting can be developed not only on hormone receptors and enzymes but on ion (Ca2+) channels of the plasma membrane. Consideration of this fact might have an importance in clinical aspects too.

Impact of single neonatal allylestrenol treatment on the estrus cycle of rats treated with FSH+LH or TSH.

Neonatal allylestrenol treatment administered to female rats significantly increases the duration of estrus phase in the sexual cycle. Treatment with follicle stimulating hormone (FSH) + luteinizing hormone (LH) in adulthood prolongs the duration of estrus even on its own; the effect, however, is more pronounced in those animals who were treated (imprinted) with allylestrenol neonatally. When administered to the control animals, the chemically related thyreotrop hormone (TSH) is either indifferent or it even decreases the estrus index. In animals having received neonatal allylestrenol treatment, however, TSH administration increases significantly the duration of the estrus phase. Either with or without FSH+LH treatment, the ratio of estrogenic to gestagenic phase increases following neonatal allylestrenol treatment. The experiments call attention to the potential functional risks inherent in neonatal allylestrenol treatment. The actual risks, however, seem to be smaller than the effects seen at the receptor level.

Impact of a single insulin treatment (imprinting) applied during liver regeneration on hepatic insulin receptor development, blood glucose level and liver function parameters in adult rats.

Rats subjected to partial hepatectomy (surgical removal of two thirds of the liver) showed no appreciable change in serum cholesterol, bilirubin, albumin, total protein and A/G values at 2, 5, 12 and 21 days after the intervention. The enzyme activities characteristic of liver damage (GOT, GPT, LDH, AP) were high in the control group and low in the insulin-imprinted group at 2 days, tended to normalize in both groups at 5 days and changed slightly at 12 days. The blood glucose level was markedly decreased in the control group and to a lesser degree also in the experimental group at 2 and 5 days of sampling. Insulin treatment (loading) performed at 2 and 5 days accounted for a drop of blood glucose which was followed by normalization within 2 h. Starving value and response to insulin loading uniformly fell into the physiological range at 21 days, whereas at 12 days no normalization occurred in either group within 2 h of insulin loading, although the starving value was physiological. The binding capacity of the insulin receptor was markedly low in the control group as long as 12 days, and tended to normalize by 21 days. In the insulin-imprinted group the binding capacity increased over the control at 2 and 5 days and normalized by 12 days.

Effect of contraceptive treatment on thymic glucocorticoid receptors and hepatic microsomal enzyme system of adult rats.

Contraceptive steroid treatment accounted for about a 30 per cent decrease in the number of thymic glucocorticoid receptors of adult rats. Neonatal allylestrenol treatment had no influence on that treatment. The activity of the hepatic microsomal (PSMO) enzyme system was not changed by the contraceptive treatment. It appears that contraceptive treatment may account for overlaps on receptors in adulthood.

Impact of neonatal benzpyrene imprinting on liver microsomal enzyme induction by benzpyrene and phenobarbital in adulthood.

A single neonatal treatment of rats with benzpyrene accounted for a durable induction of the liver microsomal enzyme (PSMO) system. Neonatal treatment with benzpyrene enhanced the inducing action of phenobarbital administrated in adulthood, but did not change the effect of benzpyrene treatment in the adult age. It follows that the imprinting and inducing effects of the neonatal benzpyrene treatment took different trends.

Hormonal imprinting in adults: insulin exposure during regeneration alters the later binding capacity of the hepatic insulin receptors.

Insulin treatment following subtotal hepatectomy caused a long lasting change in the binding capacity of hepatic insulin receptors. The insulin binding increased in females and decreased in males in the insulin treated animals (during regeneration) fourteen days after the operation. The tendency of changes was very similar to those which had been caused by neonatal insulin treatment.

Influence of pituitary hormones (hCG, TSH, Pr, GH) on testosterone level and on the functional activity of the Leydig cell in rat fetuses.

One day after the cessation of treatment the Leydig cells of the fetuses of pregnant rats, treated between the 11th and 15th or the 16th and 20th days of gestation, reacted to pituitary hormones. This finding indicates that both the receptors and the postreceptor mechanisms were in operative state. The effect of the thyrotropic hormone (TSH) overlaps the effect of related gonadotropic hormone (hCG), although this effect becomes smaller from the 21st day. The parameters investigated - the spectrocyto-fluorimetrically measured RNA-DNA ratio and the plasma testosterone level - ran generally in parallel. Similarly to the above-mentioned hormones, prolactin also increased the testosterone level (though to lesser degree than hCG and TSH did), however, while it increased the RNA level but at the age of 16 days, it decreased it the age of 21 days. Somatotropin (GH) also increased somewhat the testosterone level; however, the effects of the two related hormones (Pr and GH) fell far beyond the effect of either TSH or hCG.

Influence of neonatal treatment with porcine insulin (hormonal imprinting) on the receptor binding of insulins of porcine, rat and human origin.

A single neonatal insulin treatment in the male rat decreases, whereas in the female rat it increases, the number of insulin receptors detected at the age of one month. No difference could be observed in affinity. The extent of the binding of rat, porcine and human insulin is different (the binding of human insulin is the most pronounced), meanwhile the alterations evoked by neonatal treatment are basically identical.

Effect of the absence of neonatal testosterone imprinting on the activity of the microsomal enzyme system and on the dexamethasone binding of the thymus in adulthood.

Healthy and neonatally castrated male rats were treated with testosterone twice perinatally, while other groups were treated with testosterone also in adulthood or received testosterone only in adulthood. Castration resulted in a moderate (but in some instances significant) decrease of PSMO (polysubstrate monooxygenase) level measured in adulthood. The decrease could partially be compensated by perinatal testosterone treatment. Further testosterone treatment administered in adulthood did not result in further alteration when compared either with the controls or with the neonatally treated animals. However, since in the controls the second testosterone treatment (following the neonatal one), had a decreasing effect, therefore the testosterone treatment administered in adulthood was responsible for the disappearance of the difference between the castrated animals and the controls treated both perinatally and in adulthood. On the basis of these findings it seems likely that the perinatal presence of testosterone plays a major role in the development of enzymatic imprinting and thus, in securing the capability of the liver to split testosterone in adulthood. Since testosterone influences the glycocorticoid receptors of the thymus (presumably by its overlapping effect), so the amount of free glycocorticoid receptors is always higher in the animals castrated neonatally than in the controls. Conversely, neonatal testosterone treatment somewhat increases the number of receptors detectable in adulthood.

Hormonal imprinting of the microsomal enzyme system in adults. Microsomal activity change in response to estrogen (DES, AE) treatment during liver regeneration.

Estrogen (diethylstilbestrol-DES or allylestrenol-AE) treatment applied to rats of both sexes during liver regeneration following subtotal hepatectomy had a long lasting influence on the inducibility by phenobarbital of the hepatic microsomal enzyme system of the females. The enzyme activities of the DES-treated females differed hardly from the baseline two weeks after treatment, but increased almost two-fold over control on induction with phenobarbital 5 and 7 weeks later. The AE-treated females showed a smaller although yet significant, enzyme activity increase only at 7 weeks. The influence of estrogens was negligible, and inhibitory rather than stimulatory, in the males. It appears that, in appropriate conditions, enzyme imprinting can also be induced in adult organisms, since, in all probability, availability for imprinting depends not so much on the age of the organism, as on the developmental state of the target cell.

Effect of a single neonatal treatment with steroid hormone or steroid-like molecules on myocardial ouabain binding in the adult rat.

A single neonatal treatment of rats with vitamin D3, gibberellin, allylestrenol or diethylstilbestrol (DES) influenced the ouabain binding capacity of myocardial Na, K-dependent ATP-ase. Of the active molecules tested, vitamin D3, DES and gibberellin had appreciable impact on myocardial ouabain receptors, enhancing and depressing their activity, respectively. The thymic dexamethasone and uterine estrogen receptors did not alter their binding capacity in response to neonatal exposure to vitamin D3 or gibberellin.

Influence of hormonal imprinting on hormone level. Permanent receptor damaging effect of pituitary hormones administered to newly-hatched cockerels.

The overlapping effect of TSH and FSH on the gonad and on the thyroid gland can be demonstrated in cockerels even at the age of five weeks. These hormones influence the secretion of testosterone in a similar way and to a similar extent, while on the thyroxine level the influence of the specific hormone is more pronounced. Neonatal FSH and TSH treatment considerably decreased the basal testosterone level measured at the age of five weeks. Neonatal FSH treatment increased the basal T4 level while TSH treatment decreased it. The effect of TSH treatment administered at the age of five weeks in increasing the testosterone level was weakened after neonatal pretreatment with any iodine hormone. The effect of TSH treatment could only be inhibited by neonatal FSH pretreatment. Neonatal pretreatment with any of the trophormones caused a diminution of the T4 level augmenting of FSH and TSH administered at the age of five weeks.

A single neonatal treatment with methylcholanthrene or benzo(a)pyrene alters microsomal enzyme activity for life.

A single neonatal exposure to methylcholanthrene or benzo(a)-pyrene altered the hepatic microsomal enzyme activity of rats for a long time. Methylcholanthrene depressed, whereas benzo(a)pyrene enhanced the activity of the microsomal enzyme system. The former had a greater influence on males than on females, whereas the latter acted practically uniformly on both sexes.

Hormonal imprinting by steroids: a single neonatal treatment with diethylstilbestrol or allylestrenol gives rise to a lasting decrease in the number of rat uterine receptors.

Binding of hormone by the uterine receptors of 6 week old rats treated with diethylstilbestrol (DES) or allylestrenol (AE) in neonatal age differed considerably from the controls. Both pretreatments accounted for a decrease in the number of Type II binding sites for estradiol without altering receptor affinity. It follows that steroids, too, are able to induce a hormonal imprinting during the critical stage of receptor maturation.

Influence of the neonatal (clomiphene) inhibition of estradiol imprinting on gonadal development and gonadal and thyroid hormone production later in life.

Treatment of newborn rats with clomiphene citrate during the first 5 days of life gave rise to a marked decrease in body mass and to a still greater decrease in gonadal mass. A decrease was also observed in the testicular diameter of the males. The females showed a 43% increase in their estradiol levels over the control and an increase in the sensitivity to gonadotropins. Thyroxine level, which was also determined in view of the known gonadotropin-thyrotropin overlap, showed no change 6 weeks after pretreatment with clomiphene, while the thyroid gland responded to gonadotropin in the same manner as to thyrotropin.