Metabolism of melatonin by cytochrome P450s in rat liver mitochondria and microsomes

Abstract: In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O‐demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N¹‐acetyl‐N²‐formyl‐5‐methoxy‐kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N‐acetylserotonin, 2‐hydroxymelatonin, 6‐hydroxymelatonin and the same two unknown products. Eadie–Hofstee plots for 6‐hydroxylation and O‐demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower K_m value) and higher V_max for melatonin 6‐hydroxylation and O‐demethylation for both high‐affinity and low‐affinity components as compared with microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high‐ and low‐affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P450s metabolize melatonin principally by 6‐hydroxylation, with O‐demethylation representing a minor metabolic pathway.     

Serelaxin in acute heart failure: Most recent update on clinical and preclinical evidence

Heart failure continues to be a widely prevalent disease across the world, affecting millions of Americans annually. Acute heart failure (AHF) has a substantial effect on rising healthcare costs and is one of the major causes of morbidity and mortality. The search for new drugs for symptom relief and to improve long‐term outcomes in heart failure has led to development of serelaxin, a recombinant human relaxin‐2 hormone. Relaxin was discovered in pregnancy, but eventually found to have a number of other physiological actions, not only in pregnancy, but also in nonpregnant women and men. The actions of serelaxin are primarily via nitric oxide, leading to the observed vasodilatory effects, and increase in renal plasma flow. It has also been found to increase expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)‐2 and MMP‐9. The antifibrotic and antiinflammatory effects of the drug also play a role in heart failure. In Phase II studies, serelaxin has shown reduction in pulmonary arterial pressure, pulmonary capillary wedge pressure, and NT‐proBNP. The recently published results of the RELAX‐AHF, a phase III clinical trial on serelaxin, has opened new avenues into our understanding of its effects in heart failure. The trial showed improvement in short‐term dyspnea scores and 180‐day mortality, but, interestingly, failed to show any improvement of the secondary endpoints of death or readmission at 60 days. Ongoing Phase III trials like RELAX‐AHF‐2 and RELAX‐AHF‐ASIA would explain these data better and improve understanding of the use of serelaxin in clinical practice. This article summarizes the most updated published preclinical and clinical study data on serelaxin, including pharmacokinetic, pharmacodynamic, safety studies in hepatic, renal impaired patients, Phase II and Phase III trials.     

Ethanol-induced alterations in fatty acid-related lipids in serum and tissues in mice

Background: Chronic alcohol consumption is a major factor for several human diseases, and alcoholism is associated with a host of societal problems. One of the major alcohol‐induced metabolic changes is the increased NADH levels, which reduces glucose synthesis and increases fatty acid (FA) synthesis. Probably more important is the induction of FA synthesizing enzymes under the control of sterol regulatory element binding proteins (SREBP), plus increased malonyl‐CoA, which blocks FA entry to the mitochondria for oxidation. The changes in FA‐related lipids, particularly lysophospholipids and ceramides (Cers), in different tissues in ethanol‐fed mice have not been reported. Methods: We systematically determined the levels of FA‐related lipids, including FAs, phosphatidylcholines, phosphatidylethanolamines, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, sphingomyelins, and ceramides (Cers), in the serum and different tissues by high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS). The study was performed in C57BL/6J mice fed with Lieber‐DeCarli diet, in which ethanol was added to account for 27.5% of total calories. The serum and tissues were collected from these mice at the time of killing, and the results were compared to pair‐fed controls. Results: The important observation was that ethanol‐induced tissue‐specific changes, which were related to different FA chains. Several 22:6 FA, 18:0 FA, 18:0 to 18:3 FA‐containing lipids were significantly increased in the serum, liver, and skeletal muscle, respectively. In the kidney, all 22:6 FA‐containing lipids detected were increased. In addition, alterations in other lipids in tissues, except adipose tissue, were also observed. Conclusions: We found tissue‐specific alterations in the levels of FA‐related lipids after ethanol administration. The implications of these findings pertinent to human physiology/pathology warrant further investigation. More studies are needed to explore the mechanisms on the different effects of ethanol on certain lipids in different tissues.     

Coordinated regulation of melatonin synthesis and degradation genes in rice leaves in response to cadmium treatment

We investigated the expression patterns of genes involved in melatonin synthesis and degradation in rice leaves upon cadmium (Cd) treatment and the subcellular localization sites of melatonin 2‐hydroxylase (M2H) proteins. The Cd‐induced synthesis of melatonin coincided with the increased expression of melatonin biosynthetic genes including tryptophan decarboxylase (TDC), tryptamine 5‐hydroxylase (T5H), and N‐acetylserotonin methyltransferase (ASMT). However, the expression of serotonin N‐acetyltransferase (SNAT), the penultimate gene in melatonin biosynthesis, was downregulated, suggesting that melatonin synthesis was counter‐regulated by SNAT. Notably, the induction of melatonin biosynthetic gene expression was coupled with the induction of four M2H genes involved in melatonin degradation, which suggests that genes for melatonin synthesis and degradation are coordinately regulated. The induced M2H gene expression was correlated with enhanced M2H enzyme activity. Three of the M2H proteins were localized to the cytoplasm and one M2H protein was localized to chloroplasts, indicating that melatonin degradation occurs both in the cytoplasm and in chloroplasts. The biological activity of 2‐hydroxymelatonin in the induction of the plant defense gene expression was 50% less than that of melatonin, which indicates that 2‐hydroxymelatonin may be a metabolite of melatonin. Overall, our data demonstrate that melatonin synthesis occurs in parallel with melatonin degradation in both chloroplasts and cytoplasm, and the resulting melatonin metabolite 2‐hydroxymelatonin also acts as a signaling molecule for defense gene induction.     

Mitochondria and chloroplasts as the original sites of melatonin synthesis: a hypothesis related to melatonin's primary function and evolution in eukaryotes

Mitochondria and chloroplasts are major sources of free radical generation in living organisms. Because of this, these organelles require strong protection from free radicals and associated oxidative stress. Melatonin is a potent free radical scavenger and antioxidant. It meets the criteria as a mitochondrial and chloroplast antioxidant. Evidence has emerged to show that both mitochondria and chloroplasts may have the capacity to synthesize and metabolize melatonin. The activity of arylalkylamine N‐acetyltransferase (AANAT), the reported rate‐limiting enzyme in melatonin synthesis, has been identified in mitochondria, and high levels of melatonin have also been found in this organelle. From an evolutionary point of view, the precursor of mitochondria probably is the purple nonsulfur bacterium, particularly, Rhodospirillum rubrum, and chloroplasts are probably the descendents of cyanobacteria. These bacterial species were endosymbionts of host proto‐eukaryotes and gradually transformed into cellular organelles, that is, mitochondria and chloroplasts, respectively, thereby giving rise to eukaryotic cells. Of special importance, both purple nonsulfur bacteria (R. rubrum) and cyanobacteria synthesize melatonin. The enzyme activities required for melatonin synthesis have also been detected in these primitive species. It is our hypothesis that mitochondria and chloroplasts are the original sites of melatonin synthesis in the early stage of endosymbiotic organisms; this synthetic capacity was carried into host eukaryotes by the above‐mentioned bacteria. Moreover, their melatonin biosynthetic capacities have been preserved during evolution. In most, if not in all cells, mitochondria and chloroplasts may continue to be the primary sites of melatonin generation. Melatonin production in other cellular compartments may have derived from mitochondria and chloroplasts. On the basis of this hypothesis, it is also possible to explain why plants typically have higher melatonin levels than do animals. In plants, both chloroplasts and mitochondria likely synthesize melatonin, while animal cells contain only mitochondria. The high levels of melatonin produced by mitochondria and chloroplasts are used to protect these important cellular organelles against oxidative stress and preserve their physiological functions. The superior beneficial effects of melatonin in both mitochondria and chloroplasts have been frequently reported.     

Assessment of self-reported self-esteem in healthy and diabetic children and adolescents in Greece

Background: The aim of the present study was to evaluate any possible negative effect of diabetes on the self‐esteem of children and adolescents with diabetes. Methods: Self‐esteem was evaluated using the Culture‐Free Self‐Esteem Inventory (CFSEI‐2) in 144 patients with Type 1 diabetes mellitus (T1DM; 7–18 years of age) treated in a diabetes center and compared against that of 136 healthy children and adolescents. Results: Self‐esteem was correlated with age (P = 0.017), but not with diabetes (P = 0.886). The median CFSEI‐2 score for both healthy and diabetic children was 22. There was no significant correlation between self‐esteem and sex, body mass index (BMI), physical exercise, HbA1c or parental educational level. According to Spearman’s rank correlation coefficient (r_s), there was a significant association between age and self‐esteem (r_s = ?0.15). Conversely, although BMI (r_s = ?0.09) and treatment duration (r_s = ?0.107) had a slight negative effect on self‐esteem and the duration of physical exercise (r_s = 0.11) and parental education (r_s = 0.07) seemed to have a positive effect, the associations did not reach statistical significance. Conclusions: Self‐esteem in diabetic children is mainly affected by their age, level of physical activity and level of family support. These findings emphasize the need to discriminate between glycemic control and diabetic adjustment.     

Comparison of the efficacy and tolerability of simvastatin and atorvastatin in the treatment of hypercholesterolemia

BACKGROUND: Simvastatin and atorvastatin are effective statins for treating hypercholesterolemia. HYPOTHESIS: The study was undertaken to compare the efficacy and tolerability of simvastatin 20 and 40 mg/day and atorvastatin 10 and 20 mg/day. METHODS: In this multinational, open-label, crossover study, 258 patients with primary hypercholesterolemia were randomized after 4 weeks of diet plus placebo to once-daily administration of a starting dose sequence of simvastatin (20 mg) or atorvastatin (10 mg), or a higher dose sequence of simvastatin (40 mg) or atorvastatin (20 mg) for 6 weeks. Patients were then switched after a 1-week washout to the corresponding starting or higher dose of the alternate drug for a second 6-week period. The primary endpoint was the mean percent change from baseline to Week 6 in low-density lipoprotein (LDL) cholesterol; percent changes from baseline in total cholesterol, high-density lipoprotein cholesterol, triglycerides, and apolipoprotein B were also compared. Safety was assessed through adverse experiences and laboratory measurements. RESULTS: Both statins produced statistically significant improvements in all measured plasma lipids and lipoproteins. The main treatment comparison showed no statistically significant difference in changes in LDL cholesterol and triglycerides, whereby the overall effects were comparable when doses of 20 mg and 40 mg of simvastatin were compared with atorvastatin 10 mg and 20 mg. The mean percent reductions for LDL cholesterol from baseline to Week 6 ranged from 35-42% for the entire study cohort. An LDL cholesterol level < or = 130 mg/dl (3.4 mmol/l) was achieved in approximately 70% of patients treated with both drugs in this study. Simvastatin and atorvastatin were well tolerated at the doses studied. CONCLUSION: In patients with hypercholesterolemia, the most commonly used doses of simvastatin and atorvastatin produced similar changes in LDL cholesterol and achieved an LDL cholesterol level < or = 130 mg/dl (3.4 mmol/l) in a similar number of patients. Both statins were well tolerated.     

Melatonin and its metabolites ameliorate ultraviolet B‐induced damage in human epidermal keratinocytes

We investigated the protective effects of melatonin and its metabolites: 6‐hydroxymelatonin (6‐OHM), N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), N‐acetylserotonin (NAS), and 5‐methoxytryptamine (5‐MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm²) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50–60%) when UVB‐exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose‐dependent increase in viability of UVB‐irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot‐blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB‐induced pyrimidine photoproducts (6‐4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser‐15 but not at Ser‐46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6‐OHM, AFMK, 5‐MT, which are endogenously produced in keratinocytes, protect these cells against UVB‐induced oxidative stress and DNA damage.     

Serum concentration of complement components of the lectin pathway in maintenance hemodialysis patients, and relatively higher levels of L-Ficolin and MASP-2 in Mannose-binding lectin deficiency

Mannose-binding lectin (MBL), L-ficolin and MBL associated serine protease-2 (MASP-2) are molecules involved in initiation of the lectin pathway (LP) in the complement system. Although MBL deficiency is observed in almost 10% of healthy people, studies of associations between MBL deficiency and end-stage renal disease (ESRD) remain rare. The objective of the present study is to clarify the significance of the LP in maintenance hemodialysis (HD) patients, especially in terms of MBL levels. Two hundred and forty-four HD patients who had been followed up for 74±84months and 199 healthy controls were included in this study. Measurements of serum concentrations of MBL, L-ficolin, and MASP-2 were performed. Low serum MBL levels (<0.1μg/mL) in the patients were confirmed by examination of a point mutation in the Mbl-2 gene. Seventeen HD patients (7%) and 20 healthy controls (10%) had MBL deficiency. During the follow-up period, 99 patients died. There was no significant difference in the frequency of deaths by infectious diseases between MBL deficient and non-deficient patients. In both patients and healthy controls with MBL deficiency, the serum concentration of L-ficolin tended to be high, and that of MASP-2 was significantly high (P<0.05). MBL deficiency is not a risk factor for HD induction or life-threatening infections. It is postulated that the elevation of concentration of the two components of the LP, L-ficolin and MASP-2, may compensate for the insufficient activity of the LP in MBL deficiency.     

Modulation of sphingosine‐1‐phosphate and apolipoprotein M levels in the plasma,liver and kidneys in streptozotocin‐induced diabetic mice

Aims/Introduction

Sphingosine-1-phosphate (S1P), a multifunctional bioactive lipid mediator, is involved in various diseases. Apolipoprotein M (ApoM) carries S1P on high-density lipoprotein and modulates S1P metabolism to increase the total S1P mass in the body. Both S1P and ApoM are involved in diabetes.

Materials and Methods

The present study examined the modulation of S1P and ApoM levels in the plasma, liver and kidneys in streptozotocin-induced diabetes (STZ) mice, and the effects of insulin on the S1P and ApoM levels in the plasma and liver in STZ mice and normal mice. We also examined the effects of insulin and glucose on the ApoM levels in HepG2 cells.

Results

In STZ mice, both the plasma S1P and ApoM levels were higher than those in control mice. The hepatic S1P and ApoM contents were also elevated. The hepatic S1P and ApoM contents were reduced by insulin treatment, whereas high-dose insulin decreased the plasma S1P and ApoM levels. In mice without streptozotocin treatment, the administration of insulin decreased the plasma S1P and ApoM levels, and the hepatic content of ApoM, whereas the hepatic level of S1P was not altered. Treatment with insulin and incubation under a low glucose level decreased the ApoM levels in HepG2 cells. Regarding the kidney, the renal levels of S1P and ApoM were increased in STZ mice, and insulin treatment partially restored this increment.

Conclusions

In STZ mice, the levels of S1P and ApoM in the plasma, liver, and kidneys were increased. Insulin treatment somehow reversed this modulation in STZ mice.     

Halofuginone inhibits multiple myeloma growth in vitro and in vivo and enhances cytotoxicity of conventional and novel agents

Multiple Myeloma (MM), a malignancy of plasma cells, remains incurable despite the use of conventional and novel therapies. Halofuginone (HF), a synthetic derivative of quinazolinone alkaloid, has recently been shown to have anti-cancer activity in various preclinical settings. This study demonstrated the anti-tumour activity of HF against a panel of human MM cell lines and primary patient-derived MM cells, regardless of their sensitivity to conventional therapy or novel agents. HF showed anti-MM activity in vivo using a myeloma xenograft mouse model. HF suppressed proliferation of myeloma cells alone and when co-cultured with bone marrow stromal cells. Similarly, HF induced apoptosis in MM cells even in the presence of insulin-like growth factor 1 or interleukin 6. Importantly, HF, even at high doses, did not induce cytotoxicity against CD40 activated peripheral blood mononuclear cells from normal donors. HF treatment induced accumulation of cells in the G(0) /G(1) cell cycle and induction of apoptotic cell death associated with depletion of mitochondrial membrane potential; cleavage of poly (ADP-ribose) polymerase and caspases-3, 8 and 9 as well as down-regulation of anti-apoptotic proteins including Mcl-1 and X-IAP. Multiplex analysis of phosphorylation of diverse components of signalling cascades revealed that HF induced changes in P38MAPK activation; increased phosphorylation of c-jun, c-jun NH(2)-terminal kinase (JNK), p53 and Hsp-27. Importantly, HF triggered synergistic cytotoxicity in combination with lenalidomide, melphalan, dexamethasone, and doxorubicin. Taken together, these preclinical studies provide the preclinical framework for future clinical studies of HF in MM.