The influence of "selenium organicum" upon the hepatic function of carbon tetrachloride poisoned rats.

The hepatoprotective action of the Romanian preparation Orgasel containing selenium (Se) 5.01 mg/100 g autolysated yeast powder, was tested on adult Wistar rats poisoned with carbon tetrachloride (CCl4). The hepatotoxic agent (a 20% CCl4 solution in oil) was administered i.p. in a single dose of 0.3 ml CCl4 solution/100 g body weight, and the preparation tested (autolysate of seleniated yeast) was administered by gavage in 4 doses (of 100 mg Se powder/100 g animal each) along 2 days. After 48 hrs the animals were sacrificed, then their blood and liver were collected. The treatment with Orgasel significantly reduced the organs, morphological changes (fat liver degeneration, splenomegaly, testicle degeneration) induced by CCl4 poisoning in the rat, an effect found also at the biological parameters levels studied in plasma and liver. In the plasma, the high lipid peroxide concentrations, the increased activity of alkaline phosphatases, and the reduced antioxidative activity generated by CCl4 have been statistically significant brought to the normal range after Orgasel administration. At the liver this treatment significantly decreased the lipid peroxides production, the total lipids and cholesterol concentrations, and statistically significant increased the enzymes activity (alkaline phosphatases, GPT). The results obtained after Orgasel administration proved that this preparation has a global beneficial action upon the organism in the poisoned rat, as well as a strong antioxidative effect, confirming once again the essential role of Se in maintaining cells' integrity.

Redox capacity of the pineal gland in rats. Effect of castration.

The day/night cycle-induced effects, and the effect of castration on pineal oxidative potential in rats, were studied herein. Experiments were made in adult and castrated Wistar rats kept under normal light conditions during winter (on December and January). Castration was performed 72 hrs before sacrification. Groups of 6 intact or castrated animals were sacrificed at 4 hr-intervals during 24 hrs (the day/night cycle). Blood and pineal were then taken. Peroxides and total pineal antioxidants in plasma and pineal homogenate were assessed by chemiluminescence. The results obtained prove that photoperiod is involved in the organism oxidative potential, and that pineal is involved in the diurnal rhythm of this potential. Pineal peroxide and antioxidative concentrations show circadian variations with minimum and maximal values during the day or the night, which are also reflected at the plasma level. In the first half of the morning are registered increased peroxide and decreased antioxidative levels while at night the diagrams are reversed. As compared to the intact group, in the castrated one antioxidants and peroxides maintain their biorhythms but their concentrations are significantly changed. The diagram of pineal peroxides in the castrated group is situated above that of the intact one, with statistically significant differences only at midday (12:00). Taking into account the antioxidative characteristics of melatonin, one can suppose that maximum pineal antioxidative levels during the night might be due to maximum concentrations of nocturnal melatonin. The significant increase in peroxide concentration and the decrease in antioxidants after castration would partly explain the physiologic status of the elderly with decreased melatonin production and increased oxidative processes.

Total and free amino acids in the "Crinofizin" pineal extract.

In order to establish the chemical nature of Crinofizin, a protidic extract obtained from bovine pineal, the amino acids content was analysed using two analytic programs: one for biologic fluids, the other for protein hydrolysates. The results showed that the pineal extract contains besides the common protein amino acids reported earlier, others, rare in proteins, such as hydroxyproline, hydroxylysine and 1-methylhistidine as well as non-protein amino acids. From among the latter, the alpha-aminoadipic acid, beta- alanine, O-cysteine, are found only in a bound form, and are detected only by hydrolysis. Other amino acids, such as taurine, glutamine, citruline are present only in free form whereas the alpha-amino-n-butyric acid, GABA, ethanolamine and ornithine are present in both forms, free or bound. The paper discusses the physiologic significance of some amino acids.

Purification and physico-chemical characterization of the antigonadotropic fractions obtained by ultrafiltration from the pineal extract.

In order to isolate the antigonadotropic factor from a pineal extract prepared from bovine pineals the authors first applied ultrafiltration, which yielded three fractions: F1 greater than 10 kDa, 1 kDa less than less than F2 less than 10 kDa, and F3 less than 1 kDa. Examination of the biologic activity showed that only F1 and F2 have a statistically significant antigonadotropic activity. Ion exchange chromatography of F1 yielded two subfractions: one, F11, which was not retained, and another one, F12, which was retained by the exchanger. Both subfractions showed biologic activity. While F11 is closer to F2 less than 10 kDa, F12 has a molecular weight around 13.7 kDa, as resulted after applying the SDS-PAGE procedure. The F1 fraction has a proteic content of 903 micrograms/ml, F11 = 308 micrograms/ml and F12 = 247 micrograms/ml. Amino acids assay showed that the F12 subfraction has the same composition as F1 and the F11 subfraction also has free amino acids which indicates a process of adsorption of small molecules on large molecules. The F2 less than 10 kDa fraction, biologically active, has a proteic content of 433 micrograms/ml and an amino acids composition similar to F11 except for cysteine and methionine. The F3 less than 1 kDa fraction with a proteic content of 79 micrograms/ml having an amino acid composition in which prevails the glutamic acid shows poor antigonadotropic activity. The results of this study, as well as the fact that the antigonadotropic activity is concentrated in fractions both above and under 10 kDa, suggests at least two hypotheses: either that the pineal antigonadotropic factor exists under various molecular forms in which the activity is preserved, or that the antigonadotropic factor is a peptide with strong aggregation properties as demonstrated in our study.

Melatonin involvement in oxidative processes.

The fact that the pineal gland, by its melatonin (MT) production, responds to environmental light variations (the day-night cycle), being also a modulator of the body adaptation to these conditions, may lead to the assumption of its involvement in the body oxidative processes. The redox capacity of melatonin was followed-up in vitro by the chemiluminescence phenomenon. The system generating chemiluminescence as well as free radicals was made up of luminol and H2O2. Incubation of melatonin in doses of 0.08-0.5 microM/ml with the generating system showed that in doses under 0.25 microM/ml melatonin has a pro-oxidative effect while in doses above this value it has an antioxidative effect. The diagram of the results shows the answer specific to a modulator. The study of the correlation between the dose of melatonin with highest pro-oxidative properties and the various peroxide concentrations in the generating system showed that melatonin gets antioxidative properties with the increase in peroxide concentrations (less than 8 mM/ml). In the presence of a hypothalamic homogenate, which is a stimulant of the chemiluminescence-generating system (PXI = 16), melatonin has a dose-dependent antioxidative effect. Similar results were also obtained by adding tryptophan--a free radicals acceptor (PXI = 0.1) and the substrate in melatonin synthesis to the reaction medium. Melatonin in low concentrations (greater than 0.1 microM/ml) has an antioxidative effect while in higher doses it has a dose-dependent pro-oxidative effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pineal treatment on rat HDL-cholesterol under basal conditions and under stimulation with HCG.

The effect of the pineal extract and that of two fractions, f1 and f2, one above, the other 10,000 daltons obtained by ultrafiltration of the extract, on HDL-cholesterol as well as on cholesterolemia and testosterone, under basal conditions and under exogenous HCG stimulation was followed up. Under basal conditions, the pineal extract induced a statistically significant decrease in the 3 biochemical indices, an effect which was also recorded with the two fractions, with the difference that the effect of f2 is more intense and significant as compared to f1, whereas the effect of the pineal extract appears more intense than that of the fractions. Under exogenous HCG stimulation, the pineal extract and less the fractions, partly inhibited the lowering effect of HCG on cholesterol and the rising effect of this hormone on testosterone. The role of the equilibrating agent of the pineal extract on HDL-cholesterol, cholesterolemia and testosterone, is discussed.

Decrease in high-density lipoprotein cholesterol after administration of melatonin-free pineal extract in the rat.

The work was focused on the effect of the pineal extract on HDL-cholesterol alongside the effect of the same extract on serum cholesterol and testosterone in the rat under basal conditions. HDL-cholesterol is utilized in the rat, preferentially by the Leydig cells for steroidogenesis. Administration of melatonin-free pineal extract, in doses of 2 ml/day/animal along 3, 6 and 12 days caused a statistically significant decrease in HDL-cholesterol of 19, 21 and 20%, respectively. The same depressor effect occurred 4 hr following a unique injection of pineal extract (-25%, p less than 0.001). Cholesterolemia decreased significantly alongside with HDL-cholesterol (14, 18, and 16% after 3, 6 and 12 days and 18% after 4 hr). The effect of the pineal extract on testosterone tested in only two experiments was to depress it significantly by 43% after 4 hr and 45% after 6 days of treatment. The decrease in testosterone level following the decrease of its major precursor, cholesterol, is discussed.

The antigonadotropic activity and amino acid composition of a total and dialyzed melatonin-free pineal extract.

The antigonadotropic activity of a melatonin-free pineal extract before and after a 24 h dialysis was assessed by the mouse uterine-weight test. In both cases, the uterus-stimulating effect of HCG was inhibited, showing that after dialysis the extract not only retains its effect but that the latter is even higher. The amino acids content established by an amino acid analyzer was the same in two batches of extract obtained from two different stores of pineal powder. The analyzer also showed that the glutamic acid is the prevailing component as a free and bound amino acid in the total extract. It was also found that methionine is absent from the dialyzed extract. Qualitative analysis of the pineal extract revealed 16 amino acids and a concentration of proteins of 1.90-1.96 mg/ml. Quantitatively, the glutamic and aspartic acids, proline and glycine prevail over the other constituents. The glutamic acid concentration was over two times higher than the latter three.

Effect of melatonin-free pineal extract on acid phosphatase activity in the testosterone-treated, castrated rat.

The effect of a melatonin-free pineal extract on acid phosphatase activity in the prostate of castrated and testosterone-treated rat was studied. Prostatic acid phosphatases in the mature rat are lysosomal and secretory, the former being inhibited by tartrate. Castration induces a decrease in the secretory phosphatases and an increase in the lysosomal ones with a subsequent increase in the tartrate-induced inhibition index. The specific activity of total acid phosphatase also rises. Administration of the pineal extract in the first 4 post-surgical days did not have a significant effect, but when it was prolonged to 7 days, it partially inhibited the effect of castration on prostatic acid phosphatase. Administration of testosterone to the castrated rat in the first 4 post-castration days, partially compensated the prostatic alterations, owing to the fact that the prostate is under the control of androgenic hormones. Administration of both pineal extract and testosterone in the first 4 post-castration days partially inhibited the effect of testosterone. The equillibrating role of the melatonin-free pineal extract and its effect in relation to the amount of time lapsed from surgery is discussed.

Effect of melatonin-free pineal extract on sialic acid in hemicastrated and testosterone-treated rats.

Administration of a melatonin-free pineal extract to hemicastrated rats in doses of 2 ml/day/animal induced a statistically significant decrease in their serum and testicular sialic acid after 3 days but not after 30 days of treatment. Unilaterally castrated rats receiving testosterone (6 injections) showed 10 days post-castration a statistically significant decrease in sialic acid in their serum and testes but not in the epididymis. Twenty-one days post-castration, time in which 11 testosterone injections were given, the sialic acid was low in the epididymis as well. Concomitant administration of testosterone and pineal extract caused a total reduction in the serum and partial reduction in the testes, of the lowering effect of testosterone on sialic acid. The return to the normal values of the serum sialic acid, to almost normal values of the testicular sialic acid and the increase over the normal values of the epididymal sialic acid shows the equilibrating role played by the pineal extract on the sialic acid in the hemicastrated rat treated with testosterone. The role of the melatonin-free pineal extract on sialic acid, a biochemical index involved in reproduction, is discussed.

Comparative study of the effects of melatonin-free pineal extract and melatonin on biochemical and hormonal parameters in the rats.

A melatonin-free pineal extract administered to rats in doses of 2 ml/day/animal along 12 days, induced a statistically significant drop in urinary 17 KS, cholesterolemia, hepatic, adrenal and testicular cholesterol. The dose of 1 microgram melatonin/day/animal did not alter these biochemical indices. This dose administered for 3 days did not affect the serum level and testicular concentration of testosterone, unlike the pineal extract which lowered significantly serum and testicular testosterone. Unlike the pineal extract administration of melatonin in doses of 10 micrograms/day/animal along 3 days had no effect on DNA, RNA and testicular proteins. Administration of the pineal extract lowered significantly serum LH and FSH after 3 days whereas melatonin in doses of 50 micrograms/day/animal did not have a similar effect. Furthermore, while the pineal extract inhibited the post-castration elevation of LH and FSH, melatonin in doses of 50 micrograms/day/animal had no effect. The same difference was noted with rats under GnRH stimulation: the pineal extract inhibited the pituitary response to GnRH whereas melatonin did not. The authors conclude that the effects produced by the pineal extract are not mediated by melatonin.

Decrease in testicular testosterone synthesis by melatonin-free pineal extract in the rat.

Administration of 2 ml of melatonin-free pineal extract (30 mg bovine pineal powder per ml) per day per animal for 12 days caused, under basal conditions, a decrease of 31% in the testicular testosterone and 33% in the esterified cholesterol, a fraction used in testosterone biosynthesis, but did not change free cholesterol. Under stimulation with 50 UI HCG for 3 days, testicular testosterone was found to be very high, esterified cholesterol low and free cholesterol unchanged. Simultaneous administration of HCG and pineal extract showed that the latter inhibits partly the testicular response to HCG. The decrease in testosterone after pineal extract administration under exogenous HCG stimulation correlates with an increase in the esterified cholesterol fraction. The effect of the melatonin-free pineal extract on testicular testosterone synthesis in the rat is discussed.

Effect of pineal treatment on testicular biochemical parameters under basal conditions and after stimulation with HCG in the rat.

The effect of a melatonin-free pineal extract on sialic and ascorbic acid, nucleic acids (DNA RNA), proteins, total, free and esterified cholesterol in the rat testis under basal conditions and after stimulation with HCG was followed up. The pineal extract induced a decrease in sialic and ascorbic acid, total and esterified cholesterol, but it increased the proteins and RNA after 3 days of treatment, under basal conditions. The decrease in RNA and proteins was noticed after 30 days of treatment. Administration of HCG increased the sialic and ascorbic acids and decreased the total and esterified cholesterol, proteins and DNA without affecting RNA and free cholesterol in the testicular tissue. Concomitant administration of HCG and pineal extract showed that the pineal treatment corrected partially and sometimes completely the increasing or decreasing effect produced by HCG on the biochemical indices studied. The decrease in testicular esterified cholesterol the latter associated with androgen hormone synthesis, brings into discussion the role of the pineal gland in testicular testosteronogenesis, as the same pineal extract also caused a decrease in testosterone. The authors also discuss the regulating role of the pineal gland on some testicular biochemical indices.

Effect of melatonin-free pineal extract on cholesterol in basal and HCG treated rats.

The effect of a melatonin-free pineal extract on total, free and esterified testicular and serum cholesterol in the rat under basal conditions and after stimulation with exogenous HCG was followed up. Administration of a 2 ml daily dose from this extract to the rat under basal conditions along 3, 12 and 30 days reduced the esterified fraction of testicular cholesterol, more markedly in the 12-day experiment. The total free and esterified serum cholesterol also diminished significantly in the 3 and 12-day experiments. Under HCG stimulation the pineal extract inhibited the lowering effect of HCG on the esterified fraction in the testis and on the free and esterified fraction at serum level. Under both these experimental conditions, the pineal extract affected the esterified cholesterol at testicular level, a fraction used in androgen hormones production, and the free and esterified serum cholesterol. The auhors comment on the inhibiting role of the pineal extract exerted on testicular synthesis of cholesterol and testosterone under basal conditions as well as the regulating role of the pineal gland under conditions of exogenous HCG stimulation.

Effect of pineal treatment on serum and testicular sialic and nucleic acids in basal and HCG treated rats.

The effect of a melatonin-free pineal extract, two pineal fractions, obtained by ultrafiltration one above, the other under 10,000 daltons molecular weight, melatonin and the partly purified gonadotropin inhibiting urinary substance (GIS) on the serum and testicular sialic acid, testicular nucleic acids and proteins in the rat under basal conditions and after stimulation with exogeonus HCG, was investigated. Under basal condition the pineal extract induced a statistically significant decrease in serum and testicular sialic acid after 3 and 12 days of treatment. The effect of the extract on the sialic acid was also present under the action of the two fractions after a 12-day treatment. Conversely, after a 30-day treatment the pineal extract induced an opposite effect, i.e. a statistically significant increase in the serum and testicular sialic acid. Melatonin failed to induce any significant changes after a 3-day trial. GIS induced an increase only in serum sialic acid after a 6-day trial. Under exogenous HCG stimulation the following results were obtained: doses of HCG (25,50 and 75 i.u.) given along 3 days caused a significant increase in sialic acid; the pineal extract inhibited only partly the stimulating effect of HCG on the sialic acid; melatonin did not produce comparable effects and GIS caused an elevation only at serum level. As concerns the nucleic acids and proteins, under basal conditions the pineal extract caused the following effects: a significant increase in RNA and proteins after 3 days, a slightly increased level after 12 days and a significant decrease after 30 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of serum and testicular sialic acid by melatonin-free pineal extract in rats.

The effect of a melatonin-free pineal extract on sialic acid in the rat was studied. Administration of 2 ml extract/day/animal for 3 and 12 days induced the following effects: a) a statistically significant decrease in testicular concentration and content of sialic acid after 3 days (by 14 and 16% respectively) and b) a statistically significant decrease both in testicular concentration and content and serum level of sialic acid after 12 days (by 13, 12 and 9% respectively). The involvement of the pineal extract in the synthesis and secretion of testicular sialic acid is discussed.

Inhibition of HCG induced testosterone increase by melatonin-free pineal extract in the rat.

The effect of a pineal treatment on rat testosterone under conditions of exogenous HCG stimulation was followed-up. Administration of 2 ml/day/animal of a melatonin-free pineal extract for 3 days lowered by 23% (p less than 0.01) the serum level and by 32% (p less than 0.02) the testis content in testosterone, both values being increased by concomitant administration of 50 UI HCG per animal. In other words, the melatonin-free pineal extract inhibited partially the testis response to HCG. The antiandrogenic action of the pineal extract is discussed.

Effect of urinary gonadotropin-inhibiting substance on rat prolactin.

Administration of the partially purified urinary gonadotropin inhibiting substance (GIS) to the rat induced the following effects on prolactin: a) under basal conditions, 4 hrs after a single dose, or after 3 days of treatment, serum prolactin decreased by 36%, 35% respectively (p less than 0.05) and was associated with a nonsignificant decrease in pituitary content and concentration of this substance; b) under endogenous stimulation (castration) the 49% (p less than 0.05) decrease in serum prolactin was associated with a decrease to the limit of statistical significance of its pituitary content and concentration; c) under exogenous stimulation with TRH, the significant decrease in serum prolactin was associated with a nonsignificant decrease in its pituitary content and concentration. The action of lowering prolactin serum in relation to the similar effect of the pineal extract is discussed with reference to the possible pineal origin of GIS.

Effect of melatonin-free pineal extract on prolactin in castrated and thyrotropin releasing hormone-stimulated rats.

Administration of a melatonin-free pineal extract to the rat induced the following effects on prolactin: a) under basal conditions, after 4 hrs from a single injection or after 3 days of treatment, there was a decrease of 34% and 28% respectively, in serum prolactin, with no change in the pituitary content of this hormone; b) when prolactin was stimulated by castration the decrease of the serum level of this hormone (-37%) was accompanied by a decrease in its pituitary content (-33%) and concentration (-35%); c) under conditions of exogenous TRH stimulation the decrease in serum prolactin (-26%) was accompanied by a decrease in pituitary prolactin content (-27%) and concentration (-21%), the latter two without statistical significance. The role of the pineal gland on prolactin is discussed.

The effect of pineal treatment and pinealectomy on prolactin in rats.

Administration of melatonin-free pineal extract in doses of 2 ml/day/animal along 3 or 7 days caused a statistically significant decrease in the rat serum prolactin level by 28 and 25% respectively, as against the controls. Pinealectomy caused a statistically significant increase in the serum prolactin level by 34% as against the intact controls and by 30% as against the sham-operated ones. The authors discuss their completely oposite results and the role of the pineal gland on prolactin.