Thyroxine and triiodothyronine influence cultured throid cells: alterations of morphology and mucosubstances.

Further experimentation with the steer thyroid cell line indicates that the formation of "follicles" is enhanced by exposure to 8.9 X 10(-7) M thyroxine (T4) or triiodothyronine (T3) for 96 hr. The production of mucosubstances by the cultured thyroid cells is also increased by treating with T4 or T3, effects demonstrable after staining with PAS-alcian blue at pH 2.5. It is suggested that these in vitro effects bear on morphological organization and synthetic activity of the thyroid gland as well as on autoregulation and intraglandular homeostasis that may occur in situ.     

Formation and metabolism of 3',5'-diiodothyronine and 3,5-diiodothyronine by cultured monkey hepatocarcinoma cells.

Cultured monkey hepatocarcinoma cells were incubated with [3',5'-125I] diiodo-L--thyronine and with [3,5-125I] diiodo-L-thyronine. In both instances monodeiodination as well as sulfoconjugation took place. [3.-125I] iodothyronine and [3',5'-125I] diiodothyronine were identified as metabolites of [3'-5'-125I]-L-thyroxine in the cells, but neither [3-125I]-iodothyronine nor [3,5-125I] diiodothyronine was detected after incubation of the cells with ]3,5-125I]-L-thyroxine. No products of diphenyl ether splitting were observed in the medium after incubation of the cells with either [3,5-125I] diiodo-L-thyronine or [3,5-125I]-L-thyroxine.     

Presence of triiodothyronine, no detectable thyroxine and reverse triiodothyronine in human milk.

Thyroxine (T4), triiodothyronine (T3) and reverse triodothyronine (rT3) concentrations in human milk were measured by radioimmunoassay in 114 samples obtained from 1 week to 8 months postpartum. Several assay systems applied for the determination of serum thyroid hormone concentration were proved to be unsuitable for human milk, and the method of separating free and antibody-bound hormone by polyethylene glycol was also inappropriate for milk specimens, which tended to give a falsely high value. The binding of finity of T4 to milk was lower than that to serum protein, on which 8-anilino-1-naphthalene sulfonic acid showed no remarkable effect. In spite of the high sensitivity of 100 pg/tub in T4 assay system, no immunoassayable T4 was detected in all samples with or without ethanol extraction and trypsin hydrolysates of milk. In contrast, T3 was present in a measurable amount in most of the samples, the mean +/- SD value of which was 10 +/- 9 ng/100 ml, and those in colostrum were significantly higher than those in matured milk (P less than 0.01), whereas rT3 was not detectable in 76 samples tested. These results indicate that permeability of thyroid hormones through the mammary gland is different between T4 and T3 as well as in placental transport, and human milk can not be a source of thyroxine supply for the breast-fed infant.     

Sequential events of apoptosis induced by zearalenone in cultured hepatocarcinoma cells

Zearalenone (ZEA) is a fungal metabolite that can contaminate feed and foodstuffs and can cause serious health problems for animals as well as for humans. The present investigation was conducted to determine the chronological succession of the main events that characterise ZEA-induced toxicity in human hepatocarcinoma cells. To this aim, we have monitored the effects of ZEA on (1) cell viability, (2) heat-shock protein expression, (3) oxidative damage, (4) DNA fragmentation, (5) the cell cycle and (6) the cell-death-signalling pathway. Our results demonstrated that ZEA reduced cell viability in a time- and dose-dependent manner. When we exposed HepG2 cells to 100 μM ZEA (80% of cells are viable) for different treatment times (2, 4, 8, 24, 30, 48 and 60 h), we demonstrated an induction of Hsp70 protein, an increase in reactive oxygen species (ROS) generation, DNA fragmentation and cell-cycle arrest. These events begin after only 2 h of mycotoxin exposure and are earlier than those implicated in the execution of apoptosis. However, significant apoptotic cell death was observed after at least 30 h of ZEA exposure as a consequence of increased Bax expression, decreased Bcl-2 expression and mitochondrial membrane potential (Δψm)-released cytochrome c and activated caspase-3 and caspase-9.     

Reversible inactivation of phenylalanine hydroxylase by catecholamines in cultured hepatoma cells.

Phenylalanine hydroxylase in Reuber H4 hepatoma cell cultures can be rapidly inactivated by the addition of epinephrine, norepinephrine, dopamine, or 3,4-dihydroxyphenylalanine, in order of decreasing effectiveness, to the culture medium. The enzyme was 50% inactivated in 1 hour by 25 muM (R)-epinephrine or 45 muM (R)-norepinephrine in the medium. High concentrations of epinephrine caused a 70% inactivation in 15 min. Phenylalanine hydroxylase appears to be reversibly inactivated by epinephrine within the cells; since washing the compound off the cell cultures resulted in a rapid restoration of enzyme activity (40% in 1 hour), cycloheximide had little effect on the initial rate of recovery of enzyme activity and the same amount of phenylalanine hydroxylase antigen per cell was isolated from treated and normal cultures. Both (S)- and (R)-epinephrine inactivated the enzyme, and 0.1 mM desmethylimipramine, an inhibitor of amine transport, significantly decreased the effect of epinephrine on the hydroxylase activity. The possibility, suggested by the above results, that epinephrine might be directly inactivating phenylalanine hydroxylase within the cells was supported by the finding that purified rat liver phenylalanine hydroxylase would be 50% inactivated by 1.5 muM epinephrine in 10 min.     

Thyroxine and triiodothyronine in milk of cows, goats, sheep, and guinea pigs

Milk was collected for the first 21 days of lactation twice daily from dairy cows and once daily from goats, sheep, and guinea pigs. Thyroxine (T4) and triiodothyronine (T3) were extracted from 100 microliter of milk using acidified ethanol. T4 and T3 were reconstituted in 100 microliter buffer and measured by radioimmunoassay. Concentrations (ng/ml) of T4 and T3 for milk of cows, goats, sheep, and guinea pigs, respectively, were: 0.97 and 0.94, 1.24 and 0.52, 0.99 and 0.79, and 1.41 and 0.53. T4 concentration for guinea pig milk was significantly higher than for cow and sheep milk, but not for goat milk (P less than 0.05). T3 was found in higher concentration in milk of cows and sheep than in milk of goats and guinea pigs (P less than 0.05). Species differences in conversion of T4 to T3 in mammary gland cells are suggested. Summations of T4 and T3 concentrations in milk indicated no differences among the four species. Regression analyses of changes in milk production, T4 and T3 concentrations, total T4 and T3 in milk per day, and ratios of T4 to T3 revealed variations in patterns. Concentrations of T4 or T3 tended to decrease as lactation progressed over 21 days. Total T3 tended to increase, and the ratio of T4 to T3 tended to decrease. Amounts of T4 and T3 available to offspring from milk were calculated to be minor sources (4 to 7%) of total requirements for maintenance of metabolic functions.     

Effects of propylthiouracil and methylmercaptoimidazol on metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells.

Monkey hepatocarcinoma cell monolayer cultures (NCLP-6E) metabolized thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine by phenolic and nonphenolic ring deiodinations and sulfation of the deiodinated products, as shown in previous work with this system. The effects of the antithyroid drugs, propylthiouracil (PTU) and methylmercaptoimidazole (MMI), on these processes was investigated. PTU, at 0.1 and 1 mM, inhibited only phenolic ring deiodination. MMI at 1 mM had no effect, but 32 mM inhibited deiodination of both rings as well as sulfation. The findings suggest that the increased serum rT3 level caused by PTU in vivo is the result of decreased rT3 deiodination, in contrast to the increased rT3 production which is caused by starvation.     

Activation and inactivation of thyroxine by cultured rat hepatoma cells

The metabolism of thyroxine, 3,3′,5-triiodothyronine and 3,3′,5′-triiodothyronine was investigated in rat hepatoma cell cultures (R117-21B). These iodothyronines were labeled with ¹²⁵I in the phenolic ring and the metabolites were analyzed by ion-exchange column chromatography.When thyroxine was incubated with the cells at 37°C, its glucuronide was the major product and a little increase in ¹²⁵I^? was detected. Although 3,3′,5-triiodothyronine was not observed in the incubation medium, this metabolite was clearly identified in the ethanol extract obtained from the cell homogenates after 24 h incubation.This cell line also metabolized labeled 3,3′,5-triiodothyronine added to culture medium. After 24 h incubation, 3,3′,5-triiodothyronine glucuronide was the major metabolite and iodothyronine sulfates were also formed. The sulfates contained, 3,3′,5-triiodothyronine and 3,3′-diiodothyronine sulfates and an unknown component.In the metabolism of 3,3′,5′-triiodothyronine, the cells were very active in carrying out glucuronidation and phenolic ring deiodination, and this metabolism yielded 3,3′,5′-triiodothyronine and 3,3′-diiodothyronine glucuronides. The iodide fraction contained a small amount of 3,3′-diiodothyronine sulfate.These results show that the R117-21B rat hepatoma cells metabolize the thyroid hormones and their analogs by phenolic and nonphenolic ring deiodinations, by glucuronidation and by sulfation.     

Formation of endothelin by cultured airway epithelial cells

Immunoreactivity to endothelin was detected in conditioned culture medium from both canine and porcine tracheal epithelial cells. Gel permeation chromatography and fast protein liquid chromatography were used to confirm the identity of the endothelin. The two peaks demonstrated on fast protein liquid chromatography co-eluted with endothelin 1 and endothelin 3 respectively.     

Synthesis of phosphatidylserine in carrot cells cultured under carbon-source starvation

When carrot suspension cells were cultured on medium containing no carbon source (starvation), the levels of phosphatidylserine (PS) increased transiently 3-4 d after the initiation of starvation while levels of most other phospholipid (PL) species decreased. We previously reported that fatty acids of these PLs served as an alternative carbon source during starvation. The present study showed that cells possess two different biosynthetic pathways involving phosphatidylcholine (PC)/phosphatidylethanolamine (PE) exchange enzymes and PS synthase to synthesize PS. These activities peaked similarly 4 d after the initiation of starvation and coincided with the peak of PS level. The synthesis of serine was also significantly activated during starvation. The activity of phosphoserine aminotransferase (PSAT) which is involved in serine synthesis increased with a time course similar to that of the increase in the PS level. These observations suggest that the increase in PS level plays an important role in membranes which are degraded during starvation.     

Manganese uptake and release by cultured human hepatocarcinoma (Hep-G2) cells

The liver is the primary organ involved in manganese (Mn) homeostasis. The human hepato-carcinoma cell line, Hep-G2, shows many liver specific functions. Consequently, Hep-G2 cells were investigated as a possible model of hepatic metabolism of Mn. Initial experiments showed that the concentration of Mn in the diet, or culture medium, similarly affected the retention of Mn by isolated rat hepatocytes and Hep-G2 cells. Manganese uptake by Hep-G2 cells suggested that uptake was followed by release from the cell. Uptake was saturable and half-maximal at 2.0 μmol Mn/L, and was inhibited by iodoacetate, vanadate, cold, and bepridil. The cations Fe²⁺, Cu²⁺, Ni²⁺, Cd²⁺, and Zn²⁺ decreased Mn uptake. Uptake was dependent on Calcium (Ca) concentration in a manner that resembled saturation kinetics. Cells that were pulsed with⁵⁴Mn and then placed into nonradioactive medium quickly released a large portion of their internalized Mn. Release of internalized Mn could be inhibited by low temperature, nocodozole, quinacrine and sodium azide. These data show that Hep-G2 cells are a potentially good model of hepatic Mn metabolism. Mn is taken up by a facilitated process that may be related to Ca uptake. Release apparently is an active, controlled process, that may involve microtubules and lysosomes. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Uptake and metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells. Effects of potassium cyanide and dinitrophenol

Cultured monkey hepatocarcinoma cell (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic trnasformation of the hormones to deiodinated products and their sulfates. When hepatocarcinoma cell monolayers were studied at hormone concentrations below 10(-10) M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3'-diiodothyronine and 3'-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol. Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 micrometer triidothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3'-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.     

Effect of insulin-induced hypoglycemia on the serum concentrations of thyroxine,triiodothyronine and reverse triiodothyronine

The effect of insulin-induced hypoglycemia on serum thyroid hormone concentrations was studied in nine healthy individuals. Before, during and after the hypoglycemia blood samples were taken for measurement of the concentrations of glucose, thyroxine (T₄), triiodothyronine (T₃), reverse triiodothyronine (rT₃), catecholamines and pituitary hormones.There was no change in the mean serum T₄ level (± the standard error of the mean) of 67 ± 2 μg/l. However, the T₃ concentrations rose from a mean basal level of 1.86 ± 0.06 μg/l to a mean peak of 2.51 ± 0.21 μg/l (P < 0.01) at 45 minutes after the insulin injection, and the rT₃ concentrations fell from a mean basal level of 0.184 ± 0.008 μg/l to a mean nadir of 0.171 ± 0.022 μg/l (not a significant change). The mean peak epinephrine level was 545 ± 103 ng/l and it occurred between 30 and 45 minutes after the insulin injection; the mean peak norepinephrine level was 584 ± 114 ng/l and it occurred between 30 and 90 minutes after the injection. The growth hormone levels reached a mean peak of 26.1 ± 4.8 μg/l and the plasma cortisol levels rose to 215 ± 9 μg/l. The mean basal prolactin level was 8.5 ± 0.9 μg/l; in five subjects there was a rise to a mean peak of 50.6 ± 14.6 μg/l, whereas in the remaining four no significant increase occurred. No correlation was found between the changes in the serum T₃ concentration and any of the other factors studied.It was concluded that acute hypoglycemia is associated with a rapid increase in the serum T₃ concentration.