Application of flow cytometry in the study of apoptosis in neonatal rat cardiomyocytes.

Depending on the concentration, catecholamines activate various intracellular signaling pathways and can induce apoptosis in cardiac myocytes. Although 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) has been previously used to study mitochondria in intact cardiomyocytes, there have been no reports on the detection of apoptosis in neonatal cardiomyocytes in combination with flow cytometry and confocal microscopy. In our study, neonatal rat cardiomyocytes were exposed to norepinephrine (NE) and isoproterenol (ISO) in concentrations of 1 and 10 microM for 48 h. NE concentrations of 1 and 10 microM decreased the number of viable cardiomyocytes by 18% (*p < 0.05) and 24% (**p = 0.01), respectively. ISO in a concentration of 1 microM increased the number of viable cardiomyocytes by 13% while 10 microM decreased the number of viable cardiomyocytes by 43% (***p < 0.001). Apoptotic cells were detected by flow cytometry and confocal microscopy. NE in concentrations of 1 and 10 microM increased the percentage of apoptotic cells by 12.2% and 34.3%, respectively, while ISO alone in a concentration of 10 microM increased the percentage of apoptotic cells by 11.3%. The results demonstrated that these two methods are reliable and suitable for the detection and study of apoptosis in cultures of neonatal cardiomyocytes.

Aujeszky's disease virus production in disposable bioreactor.

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky's disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82x10(6) cells ml-1, the reactor was inoculated with 9 ml of gE- Bartha K-61 strain ADV suspension (10(5.9) TCID50) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10(7.0) TCID 50 ml-1, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.

New approach to the study of hormone-protein interaction using the microcalorimetric method.

Binding enthalpies of various hormones to bovine serum albumin (BSA) and human serum albumin (HSA) in 50 mM phosphate buffer, pH 7.4, at 37 degrees C have been determined by direct microcalorimetry. The observed enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to BSA were found to be -13.24 plus or minus 0.11 -10.31 plus or minus 0.02, -2.37 plus or minus 0.46, -17.64 plus or minus 0.32 and -17.14 plus or minus 0.36 kcal/mol of hormone, respectively. under the same experimental conditions the enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to HSA were found to be -23.94 plus or minus 0.32, -18.88 plus or minus 0.49, -11.14 plus or minus 0.02, -9.88 plus or minus 0.14 and -20.85 plus or minus 0.39 kcal/mol of hormone, respectively.

Environmental impact, healthful food and education in toxicology--trends in Croatia.

Following the informative regional development and environmental impacts in the Republic of Croatia, authors present the problems emphasized in environmental protection and health protection, and present the status of the contamination of food and drinking water in 1993. The level of education in the function of environmental protection in Croatia and its importance are discussed.

Application of anterior pituitary cell culture in toxicological research.

High density plating procedure was used to evaluate the effect of atrazine on anterior pituitary cells of rats in monolayer culture. Collagenase-dispersed pituitary cells plated in suspension with medium-199 and 10% foetal calf serum attached quantitatively to plastic surfaces within 24 hours. Electron microscopy showed subpopulations of different cell types. After prolonged cultivation, most cells established small colonies with extensive contacts among them. Cell-to-cell formation of aggregates was significant and the colonies manifested morphological changes. The cells retained their enzymatic activity, converting testosterone into 5 alpha-dihydrotestosterone by enzyme 5 alpha-reductase. Immunohistochemical techniques facilitated differentiation of gonadotrophs producing follicle stimulating hormone (FSH) and luteinising hormone (LH). Atrazine in concentrations of 5 to 50 micrograms/ml of medium was associated with a significant reduction in the number of viable cells within 72 hours. The results suggest that the pituitary cell culture may prove useful in toxicological testing of various toxic compounds and reduce or replace in vivo animal experiments.

Atrazine exposure decreases cell proliferation in Chinese Hamster Ovary (CHO-K1) cell line.

The aim of this study was to determine the toxic effect of atrazine at the ovarian cellular level. Chinese Hamster Ovary (CHO-K1) cell line was used to evaluate the degree of in vitro atrazine cytotoxicity and the morphological changes were followed during the cell death. Application of four bioassays confirmed that atrazine decreases ovarian cell proliferation and IC(50) were determined with each assay after 72 h of exposure. The level of apoptosis in atrazine treated cells was low.

Effects of coincubation of the pituitary and hypothalamus of intact and castrate male rats and the influence of LH-RH on pituitary 5 alpha-reductase activity1,2.

Pituitary 5 alpha-reductase activity in intact male rats increases after the pituitary is incubated with the hypothalamus. Incubating the pituitary of castrate rats with the hypothalamus of intact rats relatively inhibits pituitary 5alpha-reductase activity. Coincubation of the pituitary and hypothalamus of castrate rats, or the pituitary of intact with the hypothalamus of castrate males, does not elicit changes in pituitary 5alpha-reductase activity. Different amounts of LH-RH in the incubation medium can modify 5alpha-reductase activity, i.e., activate it in the intact pituitary and inhibit it in the castrate pituitary. Hypothalamus from intact rats, which according to SHIN et al. [1974] is 'rich' in LH-RH, induces changes in pituitary 5alpha-reductase activity. The LH-RH-'poor' hypothalamus of castrate rats does not cause changes in this enzyme activity. The results suggest that there is a very close relationship between LH-RH and 5alpha-reductase activity in the pituitary.

Incorporation of 14C-amino acids into rat hypothalamus in vitro.

Biosynthesis of peptides in vitro in male rat hypothalamic fragments wasinvestigated by incubating the tissue with 14C-labelled leucine, proline and glycine. Depending on the amino acid used, 4 or 6 labelled peptides were extracted from the tissue fragments with organic solvent mixture. The incorporation of the label into peptides was time dependent. LH-RH and oxytocin thus synthesized were identified by thin layer chromatography and high voltage electrophoresis using cold LH-RH and oxytocin as standards.

Androgen hydroxysteroid dehydrogenases under the influence of pyridoxine derivatives.

The metabolism of testosterone in the rat ventral prostate, anterior pituitary, basal hypothalamus and amygdala was studied in vitro under the influence of vitamin B6 compounds. The influence of these compounds on the activity of 5 alpha-reductase (5 alpha-R), 3 alpha- and 17 beta-hydroxysteroid dehydrogenase (3 alpha-HSD, 17 beta-HSD) was determined for all the examined tissues. Pyridoxine hydrochloride significantly increased the activity of 5 alpha-R, 3 alpha- and 17 beta-HSD, but pyridoxal hydrochloride had an inhibitory influence on 5 alpha-R and showed no effect on 3 alpha-HSD activity at the prostate level. Male rat anterior pituitary, basal hypothalamus or amygdala incubated with pyridoxal phosphate and pyridoxal hydrochloride showed modified enzymatic activities. Pyridoxal hydrochloride showed an inhibitory effect on 5 alpha-R in the rat pituitary and basal hypothalamus as well as in the rat prostate.

Effects of atrazine on reproductive performance in the rat.

The effects of subacute exposure to atrazine on body weight, ovarian cycling, conception rate and the size of litters in Fischer strain rats were studied. An atrazine dose of 120 mg kg-1 body wt given p.o. daily for 7 days to both female and male rats caused statistically significant loss of body weight in both sexes during the period of treatment. After the cessation of treatment the body weight of female rats remained below control body weights at least for 2 weeks. In male rats the difference in body weights narrowed with time and reached control level 5 days after the last dose. Atrazine caused a significant increase in the relative weights of pituitary and prostate. Atrazine also transiently prolonged the oestrous cycle, characterized by extended vaginal dioestrus. As a result of disturbed ovarian cycling, the rate of successful mating decreased in the first week after treatment when both sexes were exposed or exposed females were mated with an unexposed male. No similar effect was observed when only the males were exposed. The size of litters did not differ significantly between experimental groups.

Binding of testosterone, dihydrotestosterone and progesterone to rat prostate cytosol: application of microcalorimetry.

The binding capacity of rat prostate cytosol for dihydrotestosterone, testosterone and progesterone was studied in vitro. The thermodynamic parameters obtained by microcalorimetry point to the importance of the observation of energy changes occuring during the hormone-receptor complex formation.

[Induction of ovulation by means of synthetic LH-RH following unsuccessful stimulation with clomide]. / Induktion der Ovulation durch synthetisches LH-RH nach erfolgloser Stimulation mit Clomid

Synthetic LH-RH was used to induce ovulation and pregnancy in 6 women with suspected hypothalamic anovulation. The women had been previously treated with Clomid, but according to indirect evidence this drug either failed to induce ovulation or the women had insufficient luteal phase. After treatment with LH-RH one pregnancy occurred and in 3 more cases indirect evidence indicated that ovulation had occurred.

Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence.

The inhibitory influence of atrazine and deethylatrazine on testosterone metabolism in male rat anterior pituitary and hypothalamus were studied under in vivo and in vitro experimental conditions. In vivo strong influence of atrazine (12 mg/100 g by wt. daily during 7 days) on 5 alpha-R, 3 alpha- and 17 beta-HSD activities was detected in the anterior pituitary. This dose provokes a significant increase in the weight of the pituitary gland, with hyperemia and hypertrophy of chromophobic cells with vacuolar degeneration. In vivo treatment of male rats with the same dose of deethylatrazine markedly inhibited 5 alpha-R activity in the anterior pituitary. The rate of 5 alpha-R activity inhibition in the anterior pituitary was the same after in vivo treatment with atrazine (37.3%) as with deethylatrazine (33.9%). This could suggest that the mechanism of inhibition of deethylatrazine is similar to that of atrazine. In vitro atrazine or deethylatrazine addition into the incubation medium significantly (P less than 0.01) inhibited 5 alpha-R, 3 alpha- and 17 beta-HSD activities in the anterior pituitary. The inhibition of 5 alpha-R activity was marked more by atrazine than deethylatrazine, while 3 alpha- and 17 beta-HSD activities were inhibited at the same rate. In vivo treatment with the same dose of atrazine or deethylatrazine (12 mg/100 g by wt daily 7 days) significantly inhibited (P less than 0.01) 5 alpha-R and 17 beta-HSD at the male rat hypothalamic level. 3 alpha-HSD activity inhibition was not significant for either compound. The in vitro addition of deethylatrazine was much more effective (P less than 0.01) in inhibiting 5 alpha-R, 3 alpha- and 17 beta-HSD in male rat hypothalamus than atrazine. In spite of this, deethylatrazine seems to be less toxic in in vivo experiments due to its higher polarity and faster biodegradation.

Influence of Ethynodiol Diacetate on the Formation of A-Homo-3Oxa-5?-Pregnane-4,20-Dione in Female Rats.

OBJECTIVE: To give more insight in the progesterone metabolism in rat after the treatment with the progestin ethynodiol diacetate. METHODS: Urinary excretion of the metabolites of subcutaneously administred (4-14C)-progesterone was studied in female rats. After an acid hydrolysis and extraction of urine the metabolites were analysed by thin layer chromatography and by gas chromatography-mass spectrometry. RESULTS: The most of radioactivity was excreted during the first 24 h, and total of 8.36 % has been recovered within four days. The excreted metabolites in urine were found as glucuronides and free steroids (80.72 %), and 19.28 % were determined as sulphates. Among detected metabolites, 5alpha-pregnane-3,20-dione, 3alpha-hydroxy-5alpha-pregnan-20-one and A-homo-3-oxa-5alpha-pregnane-4,20-dione were determined in the urinary extracts. The last one has not yet been identified before in rat urine. CONCLUSIONS: Consecutive injections of progestin ethynodiol diacetate (6 mg/kg b.w. daily) to adult female rats during 10 days (short-term treatment), or during 70 days (long-term treatment), starting on the 21st day of life, caused significant differences in the amounts of excreted 3alpha-hydroxy-5alpha-pregnan-20-one and A-homo-3-oxa-5alpha-pregnane-4,20-dione. Significant increase in the weights of pituitary, liver and kidneys were noted in rats treated with ethynodiol diacetate. The short-term treatment caused an increase, while after the long-term treatment a decrease of the ovarian weight was observed.

Disorders of male rat reproductive tract under the influence of atrazine.

The effects of atrazine exposure on testicular sperm number, epididymal sperm number and motility and alpha-glucosidase activity in the epididymis were studied in Fischer rats. Histological changes in the testicular tissue were followed by light and electron microscopy. Groups of adult animals were treated i.p. with 60 and 120 mg atrazine kg(-1) body wt. twice a week over 60 days. The results indicate a decrease in the body weight and relative weights of pituitary and ventral prostate vs control, measured on the last day of treatment in both treated groups. Testicular sperm number (expressed as number of sperm per 500 Sertoli cells) in atrazine-treated groups increased with the treatment time due to the reduced sperm motility. Therefore atrazine treatment provoked a significant decrease in sperm number and motility in epididymis, measured after the last day of treatment. alpha-Glucosidase activity in the epididymis, after the last day of treatment, showed a decrease in both treated groups vs control values. Histological analysis of testicular tissue from treated rats showed the cell disorganization and cell clusters together with spermatocytes. Electron microscopy presented differently vacuolated cytoplasm, collagen fibre was reduced, Leydig cells were of irregular shape with unequal form and cisternae of rough endoplasmic reticulum were accentuated and softly widened. In Sertoli cell cytoplasm, atrazine treatment provoked degenerative changes. According to the results obtained, it is evident that atrazine exerted morphological changes and a toxic effect on sperm and their motility.

Indirect influence of s-triazines on rat gonadotropic mechanism at early postnatal period.

Daily s.c. injections of atrazine and deethylatrazine to rat mothers during pregnancy only or during pregnancy and lactation influenced the pituitary-gonadal axis of male and female offsprings. In female and male offspring, slow maturation of gonadotropic system is evident and as a consequence modified male and female pituitary 5 alpha-R activity is present. The number of specific steroid-hormone receptor sites at the offsprings' gonads is unchanged if the mothers were treated only during pregnancy, but 5 alpha-DHT prostate receptors are strongly inhibited in offspring of mothers treated with atrazine and deethylatrazine during pregnancy and lactation.