Influence of lithium on cell function in two different cell systems.

Lithium is widely used in the treatment and prophylaxis of bipolar psychiatric disorders. It accumulates in the thyroid gland and can cause goitre or thyroid dysfunction. The mechanisms of various effects of the lithium ion on thyroid cells have not been completely clarified. The aim of our work was to establish whether lithium, in the presence or absence of TSH, stimulates the synthesis of cAMP; as model systems we used a strain of rat thyroid follicular cells FRTL-5 and a line of Chinese hamster ovary fibroblasts with the human TSH receptor (CHO-R). Lithium at concentrations of 0.35 mM, 1 mM, 1.4 mM, 1.7 mM and 2 mM without TSH and at selected concentrations with TSH stimulation significantly increased cAMP synthesis in FRTL-5 and in CHO-R cells when compared with controls without lithium. These results are different from the published data, which have been unable to confirm the influence of lithium on cAMP synthesis or have even reported the inhibition of cAMP synthesis. However, in most published investigations only lithium in combination with TSH was tested. In conclusion, lithium was found to stimulate cAMP synthesis in FRTL-5 cells and in CHO-R cells.

Influence of lithium on growth and viability of thyroid follicular cells.

Lithium accumulates in the thyroid gland and can cause goiter or thyroid dysfunction. The aims of our work were: 1) to verify whether lithium stimulates proliferation of thyroid cells; as methods, the 3H-thymidine incorporation assay and the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) were used; as a model system the FRTL-5 (Fischer rat thyroid cells in low serum) cell line was selected, 2) to test whether lithium can have a cytotoxic effect on FRTL-5 cells, using the cytotoxicity assay with 51Cr release and the trypan blue exclusion method. Without TSH stimulation, lithium at 0.35-2 mM concentrations significantly increased the 3H-thymidine incorporation. A similar effect was observed in the case of the MTT assay: without TSH stimulation, lithium at 0.4-2 mM concentrations showed a significant stimulation of proliferation. Surprisingly, under TSH stimulation, lithium at the 2 mM concentration significantly inhibited proliferation of FRTL-5 cells. With the cytotoxicity assay, lithium was found to increase 51Cr release at 1.4-2 mM concentrations. Additionaly, the percentage of viable FRTL-5 cells at 0.35-2 mM concentrations of lithium was lower than in the controls without lithium. In conclusion, lithium was found to stimulate proliferation of FRTL-5 cells in conditions without TSH and, surprisingly, lithium in higher concentrations diminished proliferation of FRTL-5 cells under TSH stimulation. A cytotoxic effect of higher lithium concentrations was observed.

The influence of calcium on thyroid follicular cells FRTL-5 in vitro.

The present work is based on the results of in vivo experiments on rats, which had shown that hypercalcemia had led to morphological and biochemical hyperfunction of thyroid follicular cells. The regulation of the activity of follicular cells should directly, or indirectly via paracrine action of serotonin secreted from parafollicular cells, depend on the presence of calcium ions. The effect of calcium was studied on a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) using three methods: measuring the quantity of produced cAMP (cyclic adenosine 3',5'-monophosphate), measuring [3H]thymidine incorporation into cell DNA and transmission electron microscopy. Results show that calcium has no effect on cAMP production. Calcium at 1.3 mM, 3 mM, 10 mM, 20 mM and 30 mM concentrations increases [3H]thymidine incorporation into cell DNA when compared with controls without calcium. Calcium at the concentration of 30 mM has no effect on FRTL-5 cell morphology. TSH (thyrotropin) stimulates follicular cells; at higher extracellular concentrations (3 mM, 10 mM, 20 mM, 30 mM), calcium diminishes its effect, presumably by activation of a cAMP phosphodiesterase which disintegrates cAMP and/or by inhibition of adenyl cyclase.

The influence of serotonin on follicular cells FRTL-5 in vitro.

Serotonin is an aromatic monoamine found in parafollicular cells of rats, bats and in a human medullary thyroid carcinoma cell line. We have examined the hypothesis that serotonin stimulates follicular cells. The effect of serotonin in presence and absence of thyrotropin was studied in a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) by measuring [3H]thymidine incorporation into cell DNA (growth assay) and by transmission electron microscopy. The cell line was used to avoid the contamination with serotonin secreted from parafollicular cells. Results show that serotonin at 3 microM, 10 microM, 30 microM, and 100 microM concentrations increases [3H]thymidine incorporation into cell DNA. Serotonin at a 1000 microM concentration reduces sharply the [3H]thymidine incorporation into cell DNA. All cytoplasmic organelles completely disappear, only a thin fibrous membrane remains. The toxic effect of serotonin is observed in the nuclei, too. As expected, thyrotropin stimulates follicular cells. Serotonin does not have any influence on that stimulative effect of thyrotropin. Rough endoplasmic reticulum consists of round vesicles, several mitochondria are present in the cytoplasm. From the surface few pseudopodia extend into the culture medium.

The effect of sodium perchlorate and ionizing irradiation on the thyroid parenchymal and pituitary thyrotropic cells.

In a 46-week-experiment on 72 female mice the influence of peroral 1.2% sodium perchlorate application, total ionizing irradiation with 8 Gy on 5 consecutive days and their interaction on the pituitary-thyroid axis was studied by histological and stereological methods. It was observed that perchlorate alone caused long-term and strong hypothyroidism with hypertrophic and hyperplastic thyroid epithelial cells as well as pituitary thyrotropic cells. When only irradiation was used, no uniform changes in the structure and function of these cells could be detected. The interaction of perchlorate and irradiation showed similar effects as thyrostatics alone, with some exceptions; paradoxically, minor hyperplasia of thyrotropic as well as of parafollicular cells was observed. A high percentage of the follicular cell carcinoma was found after perchlorate application and after its combination with irradiation. No medullary carcinoma was found.

The effect of chronic hypercalcemia or hypocalcemia on the follicular and parafollicular cells in rat thyroid gland.

In three experiments of 30 weeks' duration, 93 adult female Wistar rats received controlled amounts of calcium with food and water, to produce a state of either hypercalcemia or hypocalcemia. A systematic stereological analysis of the thyroid glands and a radioimmunological analysis of thyroxine, triiodothyronine, and thyrotropine were performed. In the hypercalcemic rats, a reactive hyperplasia of the parafollicular cells was established; this was accompanied by morphological and biochemical signs of hyperfunction of the follicular cells, despite a reduced central stimulation by thyrotropin. In the hypocalcemic animals, no quantitative morphological changes in the parafollicular cells were observed; however, morphological and biochemical signs of hypofunction of the follicular cells were obvious, despite stronger central stimulation by thyrotropin. It is concluded that the extrinsic regulation of follicular cells by the blood calcium level is stronger than the intrinsic regulation by hypothalamo-hypophyseal hormones.

The interdependence of the follicular, parafollicular, and mast cells in the mammalian thyroid gland: a review and a synthesis.

The aim of this paper is to summarize some of our quantitative descriptive and experimental studies, to discuss them in view of the literature data, and to present a synthesis of the topic. The results of stereological analysis of some tissue components of the rat thyroid gland have been compared with the results of topological studies on the parafollicular cells of various mammalian species. Localization of the parafollicular cells in the central regions of the thyroid gland lobes, where the follicular cell activity seems to be greater than in the periphery of the lobes, has led to the hypothesis that the parafollicular cells regulate (stimulate and/or suppress) the activity of the follicular cells. Long-term application and antithyroid drugs to mice and rats has shown that excessive concentrations of thyrotropin provoke hyperplasia of both the follicular cells and the intrathyroid mast cells and, transiently, of the parafollicular cells. This and some of the literature data are congruent with the hypothesis that the parafollicular and mast cells also stimulate the follicular cells by their paracrine secretions. Long-term application of antithyroid drugs to mice and rats has shown that excessive concentrations of cular cells but also probably stimulation of the follicular cells, as judged by the stereological measurements. The biological meaning of the spatial integration of follicular and parafollicular cells seems to be a functional coordination of both epithelial cell lines, supported by intrathyroid mast cells.

Evaluation of the admission procedure and academic performance on the Medical Faculty in Ljubljana, Yugoslavia.

The data about the applicants and medical students who matriculated at the Medical Faculty of Ljubljana during the period from 1962-63 to 1969-70 by admission procedure were reviewed. A higher proportion of women than men was accepted, but men went on from year to year more regularly (P less than 0.05). Women graduated significantly later (P less than 0.05). More than half the students came from Ljubljana and its surrounding area. Academic success was correlated with general success in secondary school and with the raw scores at the admission examinations. Pearson's correlation coefficients were calculated and their values varied greatly between men and women, as well as among single cohorts. The multiple regression analysis showed that the best predictor for academic performance was the average success in secondary school (gymnasium) and in addition, the raw scores in biology and foreign language obtained at the admission examination. The results also showed the standardized regression coefficients beta and these variables should therefore be retained in the admission procedure in future. The cumulated coefficient of determination could explain about 11% to 15% of the variability of dependent variables--i.e., average academic success (mean mark of all examinations) and average academic success standardized to the duration of study. The psychological test was of the least importance and could be omitted in future admission procedures. The mean mark in mathematics in secondary school and the mean mark in somatology (the study of the anatomy and physiology of the body) at the admission examination correlated highly with other admission criteria and could also be omitted in future.

Carcinogenesis after sublethal ionizing irradiation and regular avoidance irritation.

160 mice of the BALB/C strain of both sexes, aged 3 months, were divided into four equal groups out of which two were regularly irritated by a combination of an optical signal and electrical stroke. After one month of irritation one nonirritated and one irritated group were whole body irradiated with a single dose of 6.65 Gy (1.83 Gy/min), the other two groups were sham irradiated. The mice lived until their spontaneous death or one year after irradiation, respectively, when the rest of the animals were sacrificed. The appearance of malignant tumors was noted. Irradiation shortened the survival time while the irritation had an appeasing, compensatory effect, more expressed in the males than in the females. After irradiation the number and assortment of the tumors increased and the latent period essentially shortened. In the irritated animals the number and assortment of the malignant tumors were reduced and a tendency for lengthening of the latent time period was seen; these differences, however, were not statistically significant. In spite of some differences the response in both sexes to irradiation and irritation or their combination was similar.

Histological and stereological analysis of microinvasive carcinoma of the uterine cervix.

To estimate the size of microinvasive carcinoma, stereological analysis proved to be an appropriate objective method. The difference between reticular and droplike pattern of invasion was presented by calculation of the absolute surface and the factor of deformity. Zeiss's light microscope at 100 X magnification and Weibel's multipurpose test system were used for the investigation of 14 cases of microinvasive carcinoma. High significant correlation was established between the pattern of invasion, the presence of vascular spread, and the stereological findings, indicating that in microcarcinoma with reticular pattern of invasion increasing values of the absolute surface and the factor of deformity could signify a greater degree of tumour invasiveness.

Stress and radiation carcinogenesis in mice.

In the present experiment irritation consisting of a combination of an optic signal followed by a mild electroshock administered at regular intervals was started in 2 groups of animals at the age of 3 months. At 4 months of age, one of the irritated and one of the nonirritated groups were exposed to whole-body gamma irradiation at 20 daily doses of 0.5 Gy (50 rad(, 1.4 Gy/min (140 rad/min), while the other 2 groups were sham-irradiated. The animals were autopsied and the specimens were microscopically studied for the presence of malignant tumors. Malignant tumors involving particularly the testes and lungs and leukosis were found in 29% of males, whereas in females the tumor incidence with mammary, pulmonary and ovarian involvement and leukosis was 39%. The irradiation decreased the minimum latency time in the irritated males and both female groups. In males, the irritation lowered the cumulative prevalence of malignant tumors, a significant decrease being noted at the age of 15 months. In females, it was increased, with a significant rise observed to occur at the end of the experiment. The opposite effects of irritation on the radiation carcinogenesis in males and females can be attributed to the irradiation-induced specific alterations of the gonads in females and, in part, to a longer survival time observed in the irradiated females.

Stress and Ehrlich ascites tumor in mouse.

The aim of the study was to investigate the interaction of the psychosomatic stress and the Ehrlich ascites tumor (EAT) growth in mice. The stressor consisted of combination of a light signal followed by a mild electric shock. The first experiment was performed on CBA mice irritated for 0, 2 and 4 weeks respectively, prior to intraperitoneal transplantation of the EAT. In the second study, mice of BALBc strain were used. Stress was applied 4 weeks before the tumor transplantation and continued throught the experiment. Both the irritated and the nonirritated animals were subjected to either intraperitoneal or subcutaneous inoculation of the EAT. In both experiments, mice were left to live until their spontaneous death. In the first experiment, after a 2-week irritation the experimental animals showed a significantly longer survival time as compared to the controls. Longer or shorter duration of the irritation had no significant effect on the results obtained. Results yielded by the second experiment showed no significant difference in the time of survival of the irritated and nonirritated animals after the i. p. transplantation, whereas after the s. c. inoculation of the EAT, the irritation significantly increased the survival period. The EAT in irritated mice was observed to have invaded the vitals later and less frequently than in the nonirritated animals. Quantitative histological analysis of some endocrine and lymphatic organs revealed signs of stress in the experimental animals. The EAT transplant, per se, had a stressogenic effect too.

"Genital dyscrinism" as a cause of subfertility in mice of the CBA strain.

Following four generations of inbred mating (brother-sister) in three direct lineages of CBA strain mice, sterility appeared which from that generation forward became more frequent. The genital organs in animals of both sexes were altered. There was a noticeable occurrence of cysts in the ovaries of female animals already following the third month; in mice approximately one year of age this condition was followed by cystic glandular hyperplasia of the endometrium, sometimes complicated by disturbances in blood circulation, inflammation or even malignancy. In some female animals, manifesting, due to cysts, completely degenerated ovaries, the rest of the genital system was severely atrophic. Male animals frequently showed severe atrophy of the seminiferous epithelium along with preserved interstitial cells and hypertrophic seminal vesicles. These pathological changes represent an independent nosologic unit, for which the label "genital dyscrinism" has been proposed. The authors have considered an endocrine mechanism as the possible cause of these pathological changes which are presumed to be genetically conditioned.