Regulation by growth hormone and glucocorticoid of testosterone metabolism in long-term cultures of hepatocytes from male and female rats.

The activities of 2-, 6 beta-, 7 alpha- and 16 alpha-testosterone hydroxylase and 5 alpha-testosterone reductase were measured in intact hepatocytes from male and female rats cultured for 8 days in a modified Waymouth medium supplemented with 0.1 or 1.0 microM dexamethasone with or without addition of 1 microgram/mL growth hormone. During culture of hepatocytes from female rats the activity of the male-specific 16 alpha-testosterone hydroxylase increased. This increase was significantly inhibited at day 8 by 1 microM dexamethasone as well as by growth hormone. Furthermore, in cultures of hepatocytes from male rats, the activity of the constitutive 16 alpha-testosterone hydroxylase was decreased by 1 microM dexamethasone as well as by growth hormone. The induction of 6 beta-testosterone hydroxylase by dexamethasone was suppressed by growth hormone in hepatocytes from both male and female rats, while the 7 alpha-testosterone hydroxylase activity was unaffected by culture time, hormone additions and gender. The decrease in female-specific 5 alpha-reductase activity with culture time in hepatocytes from female rats was significantly attenuated by growth hormone at 0.1 microM dexamethasone. The effects of growth hormone on testosterone hydroxylase activities in hepatocyte cultures from male and female rats are in accordance with the concept of growth hormone as a "feminization signal". The results suggest that the glucocorticoid-dependent expression of the male constitutive 16 alpha-hydroxylase requires periods of low levels of growth hormone.     

Progressive development of the osteoblast phenotype during differentiation of osteoprogenitor cells derived from fetal rat calvaria: model for in vitro bone formation

Osteoblasts are the primary cells responsible for bone formation and are thought to originate from mesenchymal osteoprogenitor cells within skeletal tissues. To elucidate the osteoblastic differentiation process, fetal rat calvariae (FRC) were enzymatically digested and fractionated to provide an osteoprogenitor-enriched cell population. The third fraction of cells from the five sequential digestions tested showed a significant osteogenic response to dexamethasone (Dex), a well-known differentiation hormone, which was demonstrated by high alkaline phosphatase activity early in culture and enhanced calcium deposition and bone nodule formation in late stage cultures. These data indicate that fraction three contains a large number of osteoprogenitor cells. During the osteoblastic differentiation of the third fraction of FRC cells, the formation of collagen cross-links (pyridinoline and deoxypyridinoline) was time-dependently accelerated with the accumulation of collagens, which coincided with an onset of mineralization of the cultures, i.e., calcium deposition and bone nodule formation. Moreover, noncollagenous matrix proteins, bone sialoprotein and osteocalcin, were also increased at both mRNA and protein level in Dex-treated cultures with advancing culture periods. Further examination for mRNA expression of bone morphogenetic proteins (BMPs) and TGF-beta1 revealed a notable elevation in BMP-6 mRNA expression on days 3 and 10, and no significant change in TGF-beta1 expression. These observations suggested that the progressive formation of collagen cross-links, production of noncollagenous proteins, and up regulation of BMP-6 mRNA play an important role in the osteoblastic differentiation process of osteoprogenitor cells isolated from FRC. This culture system provides us a suitable model for in vitro bone formation.     

4-tert-octylphenol regulates the differentiation of C3H10T1/2 cells into osteoblast and adipocyte lineages.

The aim of this study was to investigate whether 4-tert-octylphenol (OP) affects the differentiation of multipotent C3H10T1/2 cells, a cell line established from mouse embryonic connective tissue, into osteoblast and adipocyte lineages. Confluent C3H10T1/2 cells were incubated for 7 days with (OP-treated cultures) or without (control cultures) 15 microg/ml of OP. The 7-day treatment of confluent cells with OP decreased alkaline phosphatase activity by 81%, inhibited the expression of transforming growth factor beta2, and inhibited the morphological changes in cells to an osteoblastic appearance. These results indicate that the 7-day treatment of confluent C3H10T1/2 cells with OP inhibited their differentiation into osteoblasts. Since this treatment strongly induced the expression of peroxisome proliferator-activated receptor r (PPARr) but did not stimulate triacylglycerol (TG) accumulation in cells, C3H10T1/2 cells in the control and OP-treated cultures were incubated for 2 days with a hormone mixture (insulin [INS], dexamethasone, and 1-methyl-3-isobutylxanthine) and incubated for an additional 5 days with INS alone. The TG and adiponectin contents of the OP-treated cultures were 4.2 and 4.1 times higher, respectively, than those of the control cultures. There were many more Oil Red O-staining cells in the OP-treated cultures than in the control cultures. The expression of PPARr in the OP-treated cultures was higher than that in the control cultures. These results indicate that the OP-treated cultures contained a larger number of adipocytes than the control cultures. In conclusion, treatment of C3H10T1/2 cells with OP inhibited osteoblast differentiation, causing a lineage shift toward adipocytes.     

Expression and regulation of drug metabolizing enzymes in an immortalized rat hepatocyte cell line.

A hepatic cell line has been immortalized after simian vacuolating virus 40 infection of adult rat hepatocytes maintained in defined culture conditions. This cell line, designated SVHep B4, expressed nuclear large T antigen, exhibited an extended lifespan (50 subcultures) and had a hepatocyte-like morphology. Expression and regulation of drug metabolizing enzymes were studied in long-term cultures of SVHep B4 cells. Significant activities of phase I and phase II enzymes were detected. gamma-Glutamyltransferase, a marker often increased in neoplastic and dedifferentiated hepatocytes, showed a low activity whereas the hepatospecific enzyme tyrosine aminotransferase was expressed at levels similar to those in liver. Responsiveness of drug metabolizing enzymes to inducers was investigated with phenobarbital, dexamethasone and methylcholanthrene. IIB and IA subfamilies of cytochrome P450 were increased, respectively, by phenobarbital (170%) and methylcholanthrene (500%). Glucuronidation of 1-naphthol was increased by phenobarbital (140%) and 3-methylcholanthrene (160%). Phenobarbital, methylcholanthrene and dexamethasone were found to increase significantly gamma-glutamyltransferase while tyrosine aminotransferase activity was enhanced by dexamethasone. Stable expression and inducibility of drug metabolizing enzymes in long-term cultures of the SVHep B4 cell line demonstrate that immortalization of adult hepatocytes represents a promising tool for drug biotransformation studies in vitro.     

Monooxygenase and UDP-glucuronyltransferase activities in established cell culture

Cells in culture were investigated for the expression of monooxygenase and UDP-glucuronyltransferase activities as representatives of activating and inactivating pathways of drug metabolism. Most established cell lines express monooxygenase activity that is detected by the oxygenation of polycyclic hydrocarbons and appears to be a function of cytochrome P-448-dependent enzyme forms (Wiebel et al., 1977). In the hepatoma cell line, H-4-II-3, dexamethasone is capable of increasing the level of benzo(a)pyrene-monooxygenation about 10-fold and of potentiating its induction by benzo(a)anthracene. The enzyme activities elicited by dexamethasone and the polycyclic hydrocarbon did not significantly differ in their response to 7,8-benzoflavone, an inhibitor of cytochrome P-448-dependent monooxygenases. Observations on the pattern of benzo(a)pyrene metabolites formed in benz(a)anthracene-treated H-4-II-E and BHK21(C13) cells indicate that established cell cultures may contain different forms of monooxygenases of the cytochrome P-448 type. The majority of cell lines tested express UDP-glucuronyltransferase activity directed toward the substrate, 3-hydroxybenzo(a)pyrene. No clear correlation appears to exist between the presence and level of monooxygenase and glucuronyltransferase activities in the various cell lines, i.e., the cultures may express any one or both enzymes. The ratio of the two enzyme levels can be modified by selective induction. Thus, at present there is a choice of established cells in culture exhibiting widely differing ratios of activating and inactivating enzymes to analyse the metabolic pathways of selected classes of foreign compounds, such as polycyclic hydrocarbons, and to determine their toxicological significance. Further efforts are likely to yield cell lines that express the enzymic functions lacking in the cultures currently used and will be suitable to study the full spectrum of foreign compounds.     

Monooxygenase and UDP-glucuronyltransferase activities in established cell cultures

Cells in culture were investigated for the expression of monooxygenase and UDP-glucuronyltransferase activities as representatives of activating and inactivating pathways of drug metabolism. Most established cell lines express monooxygenase activity that is detected by the oxygenation of polycyclic hydrocarbons and appears to be a function of cytochrome P-448-dependent enzyme forms (Wiebel et al., 1977). In the hepatoma cell line, H-4-II-E, dexamethasone is capable of increasing the level of benzo(a)pyrene-monooxygenation about 10-fold and of potentiating its induction by benz(a)anthracene. The enzyme activities elicited by dexamethasone and the polycyclic hydrocarbon did not significantly differ in their response to 7,8-benzoflavone, an inhibitor of cytochrome P-448-dependent monooxygenases. Observations on the pattern of benzo(a)pyrene metabolites formed in benz(a)anthracene-treated H-4-II-E and BHK21(C13) cells indicate that established cell cultures may contain different forms of monooxygenases of the cytochrome P-448 type.The majority of cell lines tested express UDP-glucuronyltransferase activity directed toward the substrate, 3-hydroxybenzo(a)pyrene. No clear correlation appears to exist between the presence and level of monooxygenase and glucuronyltransferase activities in the various cell lines, i.e., the cultures may express any one or both enzymes. The ratio of the two enzyme levels can be modified by selective induction. Thus, at present there is a choice of established cells in culture exhibiting widely differing ratios of activating and inactivating enzymes to analyse the metabolic pathways of selected classes of foreign compounds, such as polycyclic hydrocarbons, and to determine their toxicological significance. Further efforts are likely to yield cell lines that express the enzymic functions lacking in the cultures currently used and will be suitable to study the full spectrum of foreign compounds.     

The effects of dexamethasone on palate mesenchymal cell phospholipase activity

Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with [3H]arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity.     

Dexamethasone induces a down regulation of rat mast cell protease II content in rat basophilic leukaemia cells.

Corticosteroids are widely used to treat severe allergic and inflammatory disease in which mast cells have been implicated as playing an important role. It has been previously shown that in vivo treatment with large bolus doses of corticosteroid can induce a down regulation of the number of IMMC in the rat. Previous in vitro studies of the RBL cell line have shown that culture in the presence of high doses of dexamethasone can induce an increase in cellular histamine content similar to that induced by other agents known to reduce the rate of cell division such as 5-hydroxyurea or sodium butyrate. In our studies the rat mucosal mast cell like cell line RBL-2H3 was cultured in the continuous presence of dexamethasone for periods of up to 4 weeks. Cell-associated histamine levels were found not to increase significantly at the doses of the corticosteroid we used. Of greater interest was the observation that levels of the mucosal mast cell specific protease RMCPII were dramatically reduced in these dexamethasone-treated cultures even at doses that might be considered to be physiologically relevant. This effect could be observed at 3 days after dexamethasone treatment and a continued reduction of RMCPII content was noted up to 4 weeks, at which time RMCPII levels were less than 5% of the control values in 10(-7) M dexamethasone treated cells. 5-Hydroxyurea did not reduce cellular RMCPII content even at a concentration which substantially reduced the rate of cell division and significantly increased cell associated histamine content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroendocrine effects of l-tryptophan and dexamethasone

An infusion of l-tryptophan was administered twice to five healthy male volunteers, once after pretreatment with dexamethasone 1 mg the previous evening and once after no dexamethasone. Cortisol, prolactin, and growth hormone levels were measured, and the responses to l-tryptophan were compared with those seen after an infusion of l-threonine. l-tryptophan did not produce cortisol secretion after dexamethasone, but prolactin and growth hormone responses were noticed. The results demonstrate a stimulatory effect of l-tryptophan on prolactin and growth hormone secretion, and the former is facilitated by pretreatment with dexamethasone.     

植物雌激素金雀异黄酮通过p38MAPK通路促进骨髓间充质干细胞向成骨细胞分化

目的研究丝裂原激活的蛋白激酶(m itogen-activatedprote in k inases,MAPKs)的亚型p38 MAPK在植物雌激素金雀异黄酮(Gen iste in)促进小鼠骨髓间充质干细胞(bone m ar-row-derived m esenchym al stem cells,BMSCs)向成骨细胞分化过程中的作用。方法BMSCs用无酚红-αMEM(含经活性碳吸附的10%FBS、β-磷酸甘油、维生素C)培养,先用Gen iste in处理细胞,观察BMSCs向成骨细胞分化情况,然后用SB203580(p38 MAPK通路阻断剂)以及PD98059(p44/42MAPK通路阻断剂)阻断相应的通路后,再用Gen iste in处理细胞,观察BMSCs向成骨细胞分化情况,并同时观察上述处理后MAPK通路的变化。测定碱性磷酸酶(ALP)活性和钙(Ca)沉积量反映BMSCs向成骨细胞分化状况,用W esternb lot来检测MAPK通路是否激活。结果Gen iste in(0.01,0.1,1μmol.L-1)剂量依赖性增加小鼠BMSCs细胞内ALP活性和细胞外Ca沉积量,并同时引起p38MAPK通路的激活和p44/42MAPK通路的抑制。SB203580预处理能减弱Gen iste in刺激引起的p38MAPK通路的激活并同时阻止Gen iste in诱导的BMSCs向成骨细胞分化。结论Gen iste in在0.01~1μmol.L-1剂量范围内可通过p38MAPK通路促进小鼠BMSCs向成骨细胞分化。     

Cells isolated from bone-marrow and lungs of allergic BALB/C mice and cultured in the presence of IL-5 are respectively resistant and susceptible to apoptosis induced by dexamethasone

We have previously reported that, in IL-5-stimulated bone-marrow cultures, dexamethasone upregulates eosinophil differentiation and protects developing eosinophils from apoptosis induced by a variety of agents. Recently developed procedures for the isolation of hemopoietic cells from allergic murine lungs have enabled us to evaluate how these cells respond to dexamethasone in IL-5-stimulated cultures, when compared with bone-marrow-derived cells isolated from the same donors, and whether differences in response patterns were linked to apoptosis. Ovalbumin challenge of sensitized mice increased significantly the numbers of mature leukocytes as well as hemopoietic cells recovered from digested lung fragments, relative to saline-challenged, sensitized controls. Both mature eosinophils and cells capable of differentiating into eosinophils in the presence of IL-5 were present in lungs from sensitized mice 24 h after airway challenge. Dexamethasone strongly inhibited eosinophil differentiation in IL-5-stimulated cultures of lung hemopoietic cells. By contrast, dexamethasone enhanced eosinophil differentiation in cultures of allergic bone-marrow cells, in identical conditions. Hemopoietic cells from lungs and bone-marrow were respectively susceptible and resistant to induction of apoptosis by dexamethasone. The dexamethasone-sensitive step was the response to IL-5 in culture, while accumulation of IL-5 responsive cells in allergen-challenged lungs was dexamethasone-resistant. Cells from lungs and bone-marrow, cultured for 3 days with IL-5 in the absence of dexamethasone, did not respond to a subsequent exposure to dexamethasone in the presence of IL-5. These findings confirm that IL-5-responsive hemopoietic cells found in challenged, sensitized murine lungs differ from those in bone-marrow, with respect to the cellular responses induced by dexamethasone, including apoptosis.     

Dexamethasone-induced growth hormone release: A dose-response study

Glucocorticoids have recently been shown to acutely stimulate growth hormone secretion in man. The aim of our study was to investigate the acute effects of three graded doses of dexamethasone. Six healthy male volunteers received in random order placebo, 1, 2 or 4 mg of oral dexamethasone. Mean stimulated growth hormone levels were significantly higher after 2 and 4 mg of DEX than after 1 mg or placebo. We conclude that dexamethasone used in lower doses than previously reported appears to stimulate growth hormone release in a dose dependent manner.     

Dexamethasone affects Fas- and serum deprivation-induced cell death of human osteoblastic cells through survivin regulation

Glucocorticoid-induced bone loss is the most prevalent form of secondary osteoporosis. Such loss could be due to the alteration of osteoclast and osteoblast lifespan through regulated apoptosis. The current study investigated the effect of dexamethasone on Fas- and starvation-induced apoptosis of mature osteoblasts and their precursors. Using the human osteoblastic hFOB1.19 and the MG63 osteosarcoma cell lines, we found that sub-lethal doses of dexamethasone act on pre-osteoblasts but not on mature cells by increasing their susceptibility to apoptosis. Apoptosis occurs in a caspase-dependent manner as both DNA fragmentation and mitochondrial transmembrane potential dissipation (ΔΨm) are inhibited by the pan-caspase inhibitor zVAD. The increased susceptibility of osteoblast precursors to apoptosis could be due to dexamethasonemediated down-regulation of survivin expression. Dexamethasone can up-regulate survivin, and to a lesser extent Bcl-2, in mature cells but not in pre-osteoblasts. In addition, it can induce FLIP over-expression in osteosarcoma cells. All these effects are inhibited by the glucocorticoid antagonist RU486, indicating that dexamethasone action is specific and, furthermore, that it depends on glucocorticoid receptor. Finally, we have found that survivin and Bcl-2 are essential for pre- and mature osteoblast survival as their silencing is sufficient to induce spontaneous apoptosis in both cell types. In conclusion, our data outline a new molecular mechanism of glucocorticoid-mediated bone loss due to the enhanced apoptosis of precursors compared to mature osteoblasts. Furthermore, the data suggest a mechanism of dexamethasone-induced resistance of osteosarcoma cells to Fas- and stress-induced apoptosis.     

高浓度他克莫司抑制人骨髓间质干细胞增殖及向成骨细胞分化(英文)

目的探讨他克莫司(FK506)对人骨髓间质干细胞(hBMSCs)增殖及向成骨细胞分化的影响。方法 FK506 0.001~5μmo.lL-1处理hBMSCs细胞中,雌二醇0.01μmol·L-1或咖啡因100μmol·L-1为阳性对照组,作用24 h后用BrdU掺入法检测细胞增殖,在促成骨细胞分化液中作用8 d后用比色法检测碱性磷酸酶(ALP)活性,作用12 d后用邻甲酚酞络合法检测钙沉积量;通过检测磷酸盐释放量间接反映钙调神经磷酸酶(CaN)活性,Western印迹法检测核心结合因子α1亚基(Cbfα1)表达。结果与DMSO对照组相比,FK506 0.001~0.01μmol.L-1促进细胞增殖,但对ALP活性及钙沉积量无影响;FK506 0.5~5μmo·lL-1则呈浓度依赖性地抑制细胞增殖,显著抑制ALP活性及减少钙沉积量(P<0.05)。此外,FK5060.1~5μmo·lL-1呈浓度依赖性地降低CaN活性,与相同浓度FK506呈浓度依赖性地下调Cbfα1的表达效应相一致。结论高浓度FK506可通过CaN/Cbfα1通路抑制hBMSCs增殖及向成骨细胞成骨分化。     

Resveratrol prevents CsA inhibition of proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells through an ER/NO/cGMP pathway.

The purpose of this study was to investigate the in vitro effects of resveratrol (RSVL) and cyclosporin A (CsA) on proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures. Application of RSVL (10(-8) -10(-6) mol l(-1)) resulted in a dose-dependent increase in [3H]-thymidine incorporation, alkaline phosphatase (ALP) activity and calcium deposition of BMSCs cultures, which was accompanied with the increase of NO production and cGMP content. Concurrent treatment with the estrogen receptor antagonist ICI182,780 (10(-7) mol l(-1)) or the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (6 x 10(-3) mol l(-1)) abolished the RSVL (10(-6) mol l(-1))-induced increase in NO production and cGMP content and eliminated the RSVL-induced increase in proliferation and osteoblastic differentiation of BMSCs. In contrast, CsA (10(-6) -10(-5) mol l(-1)) dose-dependently decreased [3H]-thymidine incorporation, ALP activity and calcium deposition of BMSCs cultures, which was accompanied with the reduction of NO production in the conditioned media. Concurrent treatment with RSVL (10(-6) mol l(-1)) significantly reversed the CsA (3 x 10(-6) mol l(-1))-mediated decrease in NO production and restored the proliferation and differentiation potential of BMSCs. Our data suggest that (1) the NO/cGMP pathway may play an important role in both RSVL-induced and CsA-inhibited proliferation and osteoblastic differentiation of mouse BMSCs, and (2) RSVL may act through an ER/NO/cGMP pathway to reverse the inhibitory effect of CsA on BMSC cultures. Taken together, the data suggest that RSVL may prevent osteoporosis induced by CsA.     

克罗米酚对骨髓间质干细胞增殖和向成骨细胞分化的影响

目的　在离体条件下研究克罗米酚(CLO)对小鼠骨髓间质干细胞(BMSCs)增殖和向成骨细胞分化的影响。方法　在含经活性炭吸附的 10%血清的无酚红α MEM中加入β 磷酸甘油和维生素C诱导小鼠BMSCs向成骨细胞分化,同时加入 0 1nmol·L-1至 10nmol·L-1CLO处理细胞。用细胞计数来反映细胞增殖情况;测定碱性磷酸酶 (ALP)活性与钙的沉积量反映细胞向成骨细胞分化状态;用试剂盒来检测一氧化氮 (NO)的产物。结果　CLO ( 0 1 ~10nmol·L-1 )呈剂量依赖性地增加小鼠BMSCs的数目,ALP活性和钙的沉积量,同时培养基中NO代谢产物明显增加。分别加入雌激素受体 (ER)拮抗剂ICI182, 780 (0 1μmol·L-1 )及一氧化氮合酶抑制剂L NAME( 6mmol·L-1 )均可阻止CLO促进BMSCs的增殖及向成骨细胞分化的作用,并取消NO代谢产物的增加。结论　CLO在 0 1 ~10nmol·L-1剂量范围内具有雌激素样受体激活效应,可通过ER/NO途径促进小鼠骨髓间质干细胞的增殖及向成骨细胞分化。     

Prolonged treatment with glucocorticoid dexamethasone suppresses melatonin production by the chick pineal gland and retina

The chick pineal gland and retina synthesize melatonin in a circadian rhythm with high levels during the night. The rhythmic changes in the hormone production result predominantly from the fluctuation in the activity of serotonin N-acetyltransferase (AA-NAT), a penultimate and key regulatory enzyme in melatonin biosynthesis. The aim of this study was to analyze the effects of an acute and prolonged in vivo treatment with a glucocorticoid dexamethasone (4 mg/kg, ip) on the nocturnal increase in AA-NAT activity in chick pineal gland and retina. In acute experiments, dexamethasone (single dose)-injected chicks were killed after 2 h, while in prolonged experiments the glucocorticoid was given once daily for 7 days and the animals were killed 26-32 h after the last injection. Acute administration of dexamethasone did not affect AA-NAT activity in the chick pineal gland and retina. In the pineal glands and retinas of chicks that were treated with dexamethasone for one week and then killed at the end of the light phase of the 12:12 h light-dark cycle, AA-NAT activity was significantly higher than the enzyme activity found in tissues isolated from the vehicle-treated (control) animals. In addition to that, the nocturnal increase in pineal and, to a lower extent, retinal AA-NAT activity was significantly lower in dexamethasone-treated birds when compared with the respective control groups. It is suggested that prolonged treatment of animals with dexamethasone reduces the amplitude of the rhythmic melatonin production, a phenomenon which may affect chronobiological processes being under control of this hormone.     

The content and activity of cytochrome P-450 in long-term culture of hepatocytes from male and female rats

The content of cytochrome P-450 and the capacity for O-demethylation have been measured in cultures of hepatocytes from male and female rats for a period of 21 days. The effect of dexamethasone, insulin, glucagon, phenobarbital and hemin was investigated. In hepatocytes from female rats the content of cytochrome P-450 was unchanged after one day of culture. From day 1 to day 3 the content of cytochrome P-450 decreased by 65% and only the combined addition of dexamethasone, phenobarbital and hemin diminished the fall. After the initial fall, addition of 0.1 microM dexamethasone resulted in a stable value. Addition of 1 microM dexamethasone or 1 mM phenobarbital gave rise to an induction of cytochrome P-450 (285%). The high level of cytochrome P-450 was maintained for 3 weeks. In hepatocytes from male rats the content of cytochrome P-450 decreased by 40% after one day of culture. From day 1 to day 3 the content decreased by 45% and the decrease continued irrespective of the presence of hormones and/or phenobarbital. The O-demethylase activity in cultures of hepatocytes from female rats correlated to the cytochrome P-450 content independent of medium composition and age of the cultures, whereas no correlation was found in cultures from male rats. The present study demonstrates that hepatocytes from female rats in cultures retain O-demethylase activity for at least 3 weeks and that, with the experimental conditions used, the response to the hormones and inducers is different for hepatocytes from male and female rats.     

Effects of juvenile hormone and related insect growth regulators on mammalian cell proliferation: A comparative study

The effects of the synthetic C₁₈ juvenile hormone, methoprene (ZR-0515), and of the compound ZR-0619 on morphology, mitotic activity, and pattern of growth of a subline of L-929 cells were compared. The cells grew as monolayers for 24 hr in a standard medium, then for another 24 or 48 hr under one of the following conditions: (1) in the standard medium, (2) in standard medium with the addition of one of the compounds tested (20, 50, 100 μg/ml), or (3) in the presence of solvent at the appropriate concentrations (0.1, 0.25, 0.5%). The susceptibility of the cells to the compounds under examination was compared by analysis of variance of the data for mitotic indices and for the frequency of occurrence of monokaryocytes and polykaryocyts in the cultures. We found that the solvent itself did not influence either cell morphology or mitotic activity of the cells until it was at 0.5% concentration whereas juvenile hormone and either of its analogs suppressed this activity significantly at all three concentrations. At both lower concentrations applied, their officacy was similar, since the fall of the mitotic indices for the cells of the media with the hormone or its analogs did not differ significantly until their concentration was 100 μg/ml, when most of the cells died out in the media with the hormone, but grow in the media with either of the analogs, although their morphology and growth patterns were more or less affected. The number of monokaryocytes and polykaryocytes did not change in cultures of the cells under our experimental conditions.     

Induction of G1 cell cycle arrest and apoptosis by berberine in bladder cancer cells

The dried root of Saussurea lappa Clarke (Compositae) has been used as a traditional medicine. Dehydrocostus lactone is one of the main bioactive constituents of this medicinal plant. In the present study, the protective effect of dehydrocostus lactone against antimycin A (an inhibitor of mitochondrial complex III)-induced cytotoxicity was investigated in osteoblastic MC3T3-E1 cells. Pre-treatment with dehydrocostus lactone prior to antimycin A exposure significantly prevented mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, intracellular calcium elevation and potassium loss, and reactive oxygen species production induced by antimycin A. These results suggest that dehydrocostus lactone protects osteoblastic MC3T3-E1 cells from antimycin A-induced cell damage through the improved mitochondrial function.