Melatonin stimulates glutathione peroxidase activity in human chorion.

In preeclampsia, placental production of lipid peroxides is abnormally increased, while placental glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities are decreased. Administration of melatonin, a powerful scavenger of oxygen free radicals, also may protect the placenta from free radical-induced damage by increasing the activity of antioxidant enzymes. To test this hypothesis we administered melatonin to pregnant women before they underwent voluntary interruption of pregnancy between 7 and 9 wk of gestation. Melatonin (6 mg) was administered orally at 12:00 hr, and samples of chorion and maternal blood were obtained at the time of the procedure, 1, 2 or 3 hr later. We measured the melatonin concentration in maternal serum and activities of GSH-Px and SOD and levels of melatonin in chorionic homogenates. Melatonin administration was reflected by markedly increased melatonin concentrations in maternal serum and in chorion, with peak levels achieved 1 hr after melatonin administration (serum, 46.87 +/- 10.87 nM/L; chorionic homogenate, 4.36 +/- 1.56 pmol/mg protein). Between 1 and 3 hr after melatonin administration, GSH-Px activity in chorionic homogenates increased significantly (P < 0.001), with peak levels occurring at 3 hr (51.68 +/- 3.22 mU/mg protein per min, 137.3% of GSH-Px activity in untreated control subjects). No significant changes in chorionic SOD activity occurred during the 3-hr post-administration period. These results indicate that exogenous melatonin increases GSH-Px activity in the chorion and thereby may protect indirectly against free radical injury. Melatonin could be useful in treating preeclampsia and possibly other clinical states involving excessive free radical production, such as intrauterine fetal growth retardation and fetal hypoxia.

Effects of short-term melatonin administration on lipoprotein metabolism in normolipidemic postmenopausal women.

OBJECTIVE: To investigate the effects of short-term administration of melatonin on lipoprotein metabolism in normolipidemic postmenopausal women. METHODS: Fifteen such women received 6.0 mg melatonin daily for 2 weeks. Blood was sampled before and after treatment. We measured concentrations of total cholesterol and total triglyceride in the plasma, as well as the levels of cholesterol, triglyceride, and protein in the very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Plasma apolipoprotein levels were determined by immunoturbidimetric assay. Activities of lipoprotein lipase, hepatic triglyceride lipase, and lecithin cholesterol acyltransferase were also determined by enzymatic analysis. RESULTS: Melatonin administration significantly increased the plasma levels of triglyceride by 27.2% (P < 0.05), of VLDL-cholesterol by 37.2% (P < 0.01), of VLDL-triglyceride by 62.2% (P < 0.001), and of VLDL-protein by 30.0% (P < 0.05). However, the plasma total cholesterol level and the concentration of lipid and protein in LDL and HDL were not significantly affected. Melatonin significantly increased the plasma levels of apolipoprotein C-II by 29.5% (P < 0.005), of C-III by 17.1% (P < 0.001), and of E by 7.6% (P < 0.05). The plasma levels of apolipoprotein A-I, A-II, and B were not altered. Melatonin significantly inhibited the activity of lipoprotein lipase by -14.1% (P < 0.05), but did not significantly affect the activities of hepatic triglyceride lipase or of lecithin cholesterol acyltransferase. CONCLUSIONS: Findings indicate that melatonin increases the plasma level of VLDL particles by inhibiting the activity of lipoprotein lipase, but may not affect the plasma levels of LDL and HDL particles in postmenopausal women with normolipidemia.

Melatonin protects fetal rat brain against oxidative mitochondrial damage.

Our objective was to investigate the effects of melatonin on the free radical-induced oxidative damage to mitochondria in fetal rat brain. Female Wistar rats on day 19 of pregnancy were used. Melatonin (10 mg/kg) or vehicle (control) was injected intraperitoneally 60 min prior to laparotomy for removal of the fetuses. The mitochondrial fraction was isolated from the fetal rat brain of each group. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were measured. As indicators of mitochondrial respiratory activity, we determined the respiratory control index (RCI) and the adenosine 5-diphosphate/oxygen (ADP/O) ratio in the presence and absence of 2.5 microM hypoxanthine and 0.02 units/mL xanthine oxidase. Mitochondrial lipid peroxidation was determined by measuring the concentration of thiobarbituric acid reactive substances in fetal brain mitochondria in the presence or absence of 2.5 microM hypoxanthine, 0.02 units/mL xanthine oxidase, and 50 microM FeSO4. The free radical-induced rates of inhibition of mitochondrial RCI and the ADP/O ratio were both significantly lower in the fetal rat brains treated with melatonin compared with those of the controls (RCI, 44.25 +/- 15.02% vs. 25.18 +/- 5.86%, P < 0.01; ADP/O ratio, 50.74 +/- 23.05% vs. 13.90 +/- 7.80%, P < 0.001). The mitochondrial lipid peroxidation induced by free radicals was significantly reduced in the melatonin-treated group compared with the controls (484.2 +/- 147.2%) vs. 337.6 +/- 61.0%, P < 0.01). Pretreatment with melatonin significantly increased the activity of GSH-Px (20.35 +/- 5.27 to 28.93 +/- 11.01 mU/min mg(-1) protein, P < 0.05) in fetal rat brain mitochondria, but the activity of SOD did not change significantly. Results indicate that the administration of melatonin to the pregnant rat may prevent the free radical-induced oxidative mitochondrial damage to fetal rat brain by a direct antioxidant effect and the activation of GSH-Px.

Melatonin inhibits oxidative modification of low-density lipoprotein particles in normolipidemic post-menopausal women.

In this study, we investigated the short-term effect of melatonin on the susceptibility of low-density lipoprotein (LDL) to oxidation in normolipidemic post-menopausal women. Fifteen post-menopausal women received 6.0 mg melatonin daily for 2 wk. Blood samples were obtained before and after the treatment and the plasma levels of total cholesterol, total triglyceride, high-density lipoprotein (HDL)-cholesterol, LDL-cholesterol, LDL-triglyceride, and LDL-apolipoprotein B were determined. LDL oxidation was performed by incubation with copper ions and was analyzed by monitoring the kinetics of conjugated diene formation and measuring the concentration of thiobarbituric-acid-reactive substances (TBARS). LDL-apolipoprotein B derivatization was analyzed by measuring trinitrobenzene sulfonic acid (TNBS) reactivity. Melatonin treatment significantly increased the plasma triglyceride levels (P<0.05), but did not significantly alter the plasma levels of total cholesterol, HDL-cholesterol, or LDL-lipids. The kinetics analysis of conjugated diene production revealed that melatonin treatment significantly prolonged the lag time of conjugated diene formation (from 64.71+/-11.89 to 70.15+/-10.52 min, P<0.05). The oxidation rate and the amount of conjugated diene, however, did not change significantly. The TBARS concentration was significantly reduced by melatonin treatment (from 49.31+/-7.57 to 38.69+/-23.90 nM/mg LDL, P<0.05). Furthermore, melatonin treatment significantly reduced the copper-induced decrease of TNBS reactivity (from 79.43+/-6.19 to 86.50+/-9.07% at 1 hr and from 71.03+/-6.74 to 76.31+/-4.99% at 2 hr, P<0.05). These results indicate that melatonin treatment may reduce LDL susceptibility to oxidative modification in normolipidemic post-menopausal women.

Melatonin increases activities of glutathione peroxidase and superoxide dismutase in fetal rat brain.

Melatonin is a powerful scavenger of oxygen free radicals. In humans, melatonin is rapidly transferred from the maternal to the fetal circulation. To investigate whether or not maternal melatonin administration can protect the fetal rat brain from radical-induced damage by increasing the activities of antioxidant enzymes, we administered melatonin to pregnant rats on day 20 of gestation. Melatonin (10 mg/kg) was injected intraperitoneally at daytime (14:00 hr) and, to remove the fetuses, a laparotomy was performed at 1, 2, or 3 hr after its administration. We measured the melatonin concentration in the maternal serum and in fetal brain homogenates and determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in fetal brain homogenates. Melatonin administration markedly increased melatonin concentrations in the maternal serum and fetal brain homogenates, with peak levels achieved 1 hr after melatonin administration (serum: 538.2+/-160.7 pM/mL; brain homogenates: 13.8+/-2.8 pM/mg protein). Between 1 and 3 hr after melatonin administration, GSH-Px activity in fetal brain homogenates increased significantly (P<0.01). Similarly, SOD activity increased significantly between 1 and 2 hr after melatonin administration (P<0.01). These results indicate that melatonin administration to the mother increases antioxidant enzyme activities in the fetal brain and may thereby provide indirect protection against free radical injury. Thus, melatonin may potentially be useful in the treatment of neurodegenerative conditions that may involve excessive free radical production, such as fetal hypoxia and preeclampsia.

Subcellular localization of protein phosphatase type 1 isotypes in mouse osteoblastic cells.

The cytolocalization of protein phosphatase type 1 catalytic subunits in exponentially growing mouse osteoblastic MC3T3-E1 cells was determined. Formaldehyde-fixed and alcohol-permeabilized cultured cells were reacted with the PP1 alpha, PP1 delta, PP1 gamma 1, and PP1 gamma 2 antibodies using immunohistochemical methods. With PP1 alpha antibody intense staining occurred in the nuclei, while with PP1 delta antibody nucleolus-like bodies were intensely stained. PP1 gamma 1 localized in the perinuclear region and in the nucleus of the cultured cells, with the staining reaction of the former being much stronger than that in the latter. An immunoreaction did not occur in the cells interacted with PP1 gamma 2 antibody or with the normal rabbit serum. Proteins were prepared from the exponentially growing cells and subconfluent cells. Cellular fractionation was also done with the exponentially growing cells and proteins were prepared from each fraction. Each protein preparation was subjected to SDS-PAGE followed by Western blot analysis with the antibodies. PP1 alpha recognized the 38 kDa proteins mainly present in the nucleus, whereas PP1 delta interacted with the proteins in the nucleolar fraction whose molecular weight was estimated as 37 kDa. PP1 gamma 1 antibody recognized a band corresponding to an estimated molecular weight of 36 kDa mainly in the cytosolic fraction. PP1 gamma 2 antibody and the normal rabbit serum did not interact with any proteins prepared from the cultured cells. Our observations show that four different isozymes of protein phosphatases occupy distinct compartments in MC3T3-E1 cells. This differential distribution suggests that these isozymes may play different roles in cellular functions.

Overexpression of mitochondrial genes in alloplasmic common wheat with a cytoplasm of wheatgrass (Agropyron trichophorum) showing depressed vigor and male sterility.

An alloplasmic hybrid (nucleus-cytoplasm hybrid) of common wheat (Triticum aestivum) with a cytoplasm of wheatgrass (Agropyron trichophorum) shows highly depressed vigor and complete male sterility. The presence of one short-arm telocentric homeologous group 1 chromosome (telosome) of the cytoplasm donor, however, restores normal vigor and male fertility of the hybrid. To study role(s) of the telosome on vigor/fertility restoration, mitochondrial genome organization and gene expression were compared among seedlings of the alloplasmic line showing depressed vigor, the corresponding restored line having a pair of the telosomes, and a euplasmic nuclear donor as control. No differences were detected in the mitochondrial genome structure between the depressed line and the restored line. Northern blot analysis using ten mitochondrial genes as probes showed no differences in transcript size and number between the depressed and restored lines, although clear differences were found in size of the major transcripts of two genes (cob and orf25) between the alloplasmic lines and the euplasmic control. Steady-state transcript levels were higher in the depressed line than in the other lines for all the mitochondrial genes analyzed including rrn18&5 when the same amount of mitochondrial RNA was loaded. The amount of rrn18&5 transcript in the total cellular RNA, however, did not differ among the lines. Run-on transcription analysis demonstrated markedly elevated transcriptional activities of all the mitochondrial genes analyzed in the depressed line based on unit amount of mitochondrial DNA, RNA and protein. The presence of Agropyron telosomes apparently normalized the level of mitochondrial transcription. These observations suggest either direct or indirect association of the observed mitochondrial gene overexpression with the depressed vigor and male sterility of the alloplasmic hybrid.