A novel 2,3-benzodiazepine-4-one derivative AMPA antagonist inhibits G2/M transition and induces apoptosis in human leukemia Jurkat T cell line.

It has been shown that the antagonism of glutamate receptors activity was able inhibit proliferation and induce apoptosis in several neuronal and non-neuronal cancer cell lines. In addition, it has been shown that glutamate might facilitate the spread and growth of leukemia T cells through interactions with AMPA receptors. The aim of the present study was to investigate the modulation of cell cycle elicited by a novel 2,3-benzodiazepine-4-one non-competitive AMPA antagonist derivative in the human leukemia Jurkat T cells. Our results indicated that the 1-(4-amino-3,5-dimethylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4h-2,3-benzodiazepin-4-one, named 1g, exerted a significant growth inhibition of leukemia Jurkat T cells in a time and dose dependent manner, arresting the transition of G2/M phase through activation of Myt-1. The molecule also induced apoptosis through the enhanced expression of the pro-apoptotic p53, and the inhibition of Bcl-2, and Bcl-xl, followed by the activation of caspase-3. The results suggested that compound 1g might act mostly as a cytostatic rather than cytotoxic compound. Although further studies are necessary, in order to identify others specific pathways involved in the activity of the present molecule, the presented results identified a novel molecule acting on specific G2/M checkpoint regulation pathway. Finally, our data suggest that compound 1g might be a good molecule for future development in the cancer research.

Carcinogenic potential of metal nanoparticles in BALB/3T3 cell transformation assay.

Metal-based nanoparticles (NPs), are currently used in many application fields including consumer products, pharmaceuticals, and biomedical treatments. In spite to their wide applications, an in-depth study of their potential toxic effects is still lacking. The aim of the present research was to investigate the potential initiator or promoter-like activity of different metallic NPs such as gold, iron, cobalt, and cerium using the Balb/3T3 two-stage transformation assay. The results indicated that all the selected metallic NPs, except for cobalt, when used as initiators did not induce any transformation in Balb/3T3 cell line. Moreover, Au and Fe3 O4 NPs, when used in place of the tumor promoter treatment TPA, increased significantly the number of Foci/dish as compared to the MCA treatment alone. The number of Foci/dish was 2.6 for Au NPs and 2.13 for Fe3 O4 ones, similar to those obtained by the positive control treatment (MCA + TPA), whereas 1.27 for MCA treatment alone. On the contrary, CeO2 NPs did not show any difference in the number of Foci/dish, as compared to MCA alone, but it decreased the number of foci by 65% in comparison to the positive control (MCA + TPA). As expected, cobalt NPs showed an increased cytotoxicity and only a few surviving cells were found at the time of analysis showing a number of Foci/dish of 0.13. For the first time, our data clearly showed that Au and Fe3 O4 NPs act as promoters in the two stage transformational assay, suggesting the importance to fully investigate the NPs carcinogenic potential with different models.

Lipid profile and cardiovascular risk factors among first-year Brazilian university students in São Paulo.

BACKGROUND/AIMS: The surveillance of cardiovascular risk factors has been recommended worldwide. The current study is aimed to estimate the prevalence of cardiovascular risk factors among first-year students from a public university in the city of Sao Paulo, Brazil. METHODS: A cross-sectional study of 56 first-year students, of both genders, was performed. Information about demographic characteristics, family history of chronic diseases, smoking, and physical activity was obtained by means of a standardised questionnaire. Anthropometrical parameters (BMI, waist circumference, body fat percentage), metabolic parameters (glycaemia, serum lipid profile), and dietary data (total energy intake, percentage of total energy from macronutrients, cholesterol and dietary fiber) were assessed. RESULTS: The risk of cardiovascular diseases was characterised by family history of cardiovascular diseases (44.6%), smoking (10.7%), physical inactivity (35.7%), borderline high total cholesterol and LDL-c levels (16.1% and 5.4, respectively), decreased HDL-c levels (8.9%), increased triglyceride levels (8.9%), and overweight and obesity (17.8% and 7.1%, respectively). The diet of the students was inadequate: it was high in fat and protein, and low in carbohydrate and dietary fibre. CONCLUSIONS: The prevalence of risk factors for cardiovascular diseases in young adults draws attention to the need to adopt preventive plans in the university setting.

Natural endogenous ligands for benzodiazepine receptors in hepatic encephalopathy.

Benzodiazepines of natural origin (NBZDs) have been found in human blood and brains as well as in medicinal plants and foods. In plasma and brain tissue there are i.e. diazepam and nordiazepam equal to commercial drugs but there are also other benzodiazepine-like compounds termed "endozepines", which act as agonists at the benzodiazepine receptors of central type (CBR). A synthetic pathway for the production of NBZDs has not yet been found, but it has been suggested that micro-organisms may synthesize molecules with benzodiazepine-like structures. Hence NBZDs could be of both endogenous and exogenous source and be considered as natural anxyolitic and sedative. Interestingly there are also natural compounds, such as the polypeptide Diazepam Binding Inhibitor (DBI) acting as an "inversive agonist" implicated in fair and panic disorders. It has been suggested that NBZDs may play a role in the pathogenesis of hepatic encephalopathy (HE). Multidirectional studies evaluated NBZDs levels (1) in the blood of normal subjects, of cirrhotic with or without HE and in commercial benzodiazepine consumers; (2) in the blood of cirrhotic treated or not with a non-absorbable antibiotic; (3) in several constituents of our diet. In conclusion, NBZDs increase sometime in cirrhotics with or without HE but they reach concentrations not higher than those found in commercial benzodiazepines consumers. Hence NBZDs must be considered as occasional precipitating factor of HE and benzodiazepine antagonists only symptomatic drugs. The finding that NBZDs may be in part synthesized by intestinal bacterial flora and in part constituent of our diet underlines the importance to feed cirrhotic patients with selected food.

Clinical evaluation of piroxicam-FDDF and azithromycin in the prevention of complications associated with impacted lower third molar extraction.

Combined treatments with non-steroidal anti-inflammatory drugs and antibiotics may offer significant benefits in the prevention of pain and infections associated with oral surgery. In this study, piroxicam and azithromycin were administered to patients undergoing dental extraction to examine the efficacy of piroxicam in the prevention of post-operative pain and inflammatory complications, either in the absence or in the presence of a concomitant antibiotic treatment. Thirty patients were randomly assigned to three groups and treated for 3 days, before impacted lower third molar removal, as follows: (1) sublingual piroxicam-FDDF (fast dissolving dosage formulation) 20 mg/day; (2) oral azithromycin 500 mg/day; (3) piroxicam-FDDF 20 mg/day plus azithromycin 500 mg/day. Oral acetaminophen (500 mg tablets) was allowed as rescue analgesic medication. Pain intensity was evaluated on a 100-mm visual-analogue scale after dental extraction (day 1), and at days 2, 3, 7 after surgery. Edema and trismus were estimated at days 2 and 7. At days 1 and 2, pain intensity was significantly lower in patients treated with piroxicam-FDDF, either alone (p < 0.05) or in combination with azithromycin (p < 0.05), than in patients administered with azithromycin alone. A higher acetaminophen consumption was also recorded in the latter group (p < 0.01). Pain intensity values did not differ among treatment groups at days 3 and 7. At day 2, the facial edema was significantly less intense in patients exposed to piroxicam-FDDF alone, as compared to patients treated with azithromycin, either alone (p < 0.05) or in combination with piroxicam-FDDF (p < 0.05). No significant differences were detected when comparing groups for trismus at days 2 and 7. The present results indicate that, when given alone in the pre-operative period, piroxicam-FDDF effectively counteracts post-surgical pain and inflammatory reactions in oral tissues. Upon combined treatment with piroxicam-FDDF and azithromycin, the macrolide antibiotic may reduce the influence of piroxicam on post-operative inflammation, without affecting its beneficial effect on surgical pain.

Nuclear location-dependent role of peripheral benzodiazepine receptor (PBR) in hepatic tumoral cell lines proliferation.

PBR is involved in numerous biological functions, including steroid biosynthesis, mitochondrial oxidative phosphorylation and cell proliferation. The presence of PBR at the perinuclear/nuclear subcellular level has been demonstrated in aggressive breast cancer cell lines and human glioma cells where it seems to be involved in cell proliferation. In our study we investigated the presence of perinuclear/nuclear PBR in different hepatic tumor cell lines with regard to binding to [3H] PK 11195 and protein analysis. The results obtained by saturation binding experiments and scatchard analysis of perinuclear/nuclear PBR density in parallel with the results on the growth curves of the cell lines tested, indicate that the perinuclear/nuclear PBR density correlates inversely with cell doubling time. Moreover, the cell line with high perinuclear/nuclear PBR proliferated in response to PBR ligand, whereas that with low perinuclear/nuclear PBR did not. Our results reinforce the idea that the subcellular localisation of PBR defines its function and that this receptor could be a possible target for new strategies against cancer.

Antiproliferative effects of Ceratonia siliqua L. on mouse hepatocellular carcinoma cell line.

Extracts from pods and leaves of carob (Ceratonia siliqua L.) were tested for their ability to inhibit cell proliferation of mouse hepatocellular carcinoma cell line (T1). The two extracts showed a marked alteration of T1 cell proliferation in a dose-related fashion reaching the maximal effect at 1 mg/ml. Moreover, we demonstrated that leaf and pod extracts were able to induce apoptosis in T1 cell lines after 24-h treatment mediating a direct activation of the caspase 3 pathway. HPLC analysis revealed the presence of gallic acid, (-) epigallocatechin-3-gallate and (-) epicatechin-3-gallate in pod and leaf extracts, compounds well known to exert antiproliferative effects. Their concentration reached 6.28 mg/g in carob leaves and 1.36 mg/g in carob pods extract. The discovery that carob pod and leaf extracts contained antiproliferative agents could be of practical importance in the development of functional foods and/or chemopreventive drugs.

Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver.

The peripheral benzodiazepine receptor system triggers intracellular metabolic events and has been associated with cell proliferation. Its endogenous ligand, the diazepam binding inhibitor, contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. The present study was undertaken to verify whether this system is altered in tumors sited in the liver. Peripheral benzodiazepine receptors and diazepam binding inhibitor were studied using immunocytochemistry and in situ hybridization in 9 human tumors sited in the liver, in liver hyperplasia, cirrhotic nodular regeneration, intestinal adenocarcinoma and in surrounding non-tumoral tissue. Immunocytochemical staining and in situ hybridization demonstrated that peripheral benzodiazepine receptors and diazepam binding inhibitor were more prominently expressed in neoplastic cells than in non-tumoral tissue. They were present in the same cells, suggesting that diazepam binding inhibitor may act in an intracrine manner in these cells. Higher peripheral benzodiazepine receptors and diazepam binding inhibitor expression in tumor cells suggest an implication of this system in the metabolism of neoplastic cells. Furthermore the evaluation of peripheral benzodiazepine receptor and diazepam binding inhibitor expression might be useful in evaluating malignancy and in diagnostic approaches of tumors in liver tissue.

Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma.

Increased number of peripheral benzodiazepine receptors (PBRs) have been found in some tumors outside the liver. The present study was to verify whether the PBR system is altered in hepatocellular carcinoma (HCC). The levels of endogenous benzodiazepine-like compounds (BZDs), measured by radioreceptor binding technique after HPLC purification and the endogenous ligand for PBRs, termed diazepam binding inhibitor (DBI), measured by radioimmunoassay utilizing a specific antibody for human DBI, were studied in the blood of 15 normal subjects, 12 liver cirrhosis and 10 patients with HCC. The levels of BZDs in serum were increased hundred fold in liver cirrhosis patients and slightly elevated in HCC patients. DBI was found to be increased in HCC patients. The binding recognition sites for PBRs (Bmax) were increased 4 to 7 fold in HCC tissue in comparison with that found in non-tumoral liver tissue (NTLT). On the contrary the concentrations of DBI were found to be significantly decreased in HCC tissue in comparison with the respective NTLT. These results seem to suggest an implication of PBRs and of their putative endogenous ligands in the metabolism of these neoplastic cells and possibly in their proliferation. The up-regulation of PBRs found in HCC tissue seems to indicate an increased functional activity of these receptors and opens up the possibility of new pharmacological and diagnostic approaches while the changes in the circulating endogenous ligands for the above receptors might be envisaged as early markers of tumorigenesis in liver cirrhosis.

Diazepam binding inhibitor and total cholesterol plasma levels in cirrhosis and hepatocellular carcinoma.

Cholesterol is used by cells for biosynthetic processes and for steroid synthesis. Although the role of cholesterol in tumorigenesis is not clear it is known that steroids are important factors in human carcinogenesis. A polypeptide, diazepam binding inhibitor (DBI), which is an endogenous ligand for peripheral benzodiazepine receptors enhances steroidigenesis by promoting cholesterol delivery to the inner mitochondrial membrane which represents the rate-limiting step of steroid biosynthesis. We have assayed the total cholesterol (TC) and the DBI plasma concentrations in patients with liver cirrhosis complicated by hepatocellular carcinoma (HCC) in comparison with those of uncomplicated liver cirrhosis. TC and DBI levels have been studied in 73 cirrhotic patients and in 23 patients with HCC. Both TC and DBI levels were higher in HCC patients when compared with age, sex and Child-Pugh class matched cirrhotic controls. The values (mean+/-S.D.) in patients in Child-Pugh class B and C with and without HCC were respectively 128+/-30 mg/dl vs. 106+/-27 mg/dl (P < 0.01) and 2.05+/-0.78 pmol/ml vs. 0.78+/-0.84 pmol/ml (P < 0.0001). The data may be the result of the metabolic influence of tumors that enhances steroid biosynthesis during tumor proliferation.

N-acetylaspartylglutamate stimulates metabotropic glutamate receptor 3 to regulate expression of the GABA(A) alpha6 subunit in cerebellar granule cells.

We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 microM) results in the transient increase in content of GABA(A) alpha6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine and (2S)-alpha-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in alpha6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, alpha2-adrenergic, muscarinic, and GABA(B) receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in alpha6 levels induced by 30 microM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the beta-adrenergic receptor agonist isoproterenol. The increase in alpha6 mRNA content is followed by a fourfold increase in alpha6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional alpha6-containing GABA(A) receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA(A) alpha6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA(A) receptor subunit composition.

Hepatic encephalopathy in liver transplant recipients precipitated by benzodiazepines present in transfused blood.

The observation that there are episodes of encephalopathy in liver cirrhosis patients after orthotopic liver transplantation, despite a well functioning graft and despite the lack of cerebral complications, prompted us to investigate the potential role of circulating benzodiazepine-like compounds in these episodes. The plasma levels of benzodiazepines were examined in 14 liver cirrhotic patients before and after transplantation. The benzodiazepines in the fluids infused during surgery and in individual bags of blood administered after surgery to 4 of these patients were also assayed. Herein we report that benzodiazepines accumulating in the blood of some transplanted patients appear to derive from blood transfusions utilized during surgery. The analysis of the types of benzodiazepines present in the blood utilized for transfusions suggests the use of commercial benzodiazepines by the donors. These compounds seem to be able to precipitate hepatic encephalopathy in patients with preexisting encephalopathy. Hence we suggest not using benzodiazepine consumers as blood donors, at least for patients with encephalopathy undergoing to liver transplantation.

[Relationship between exposure to environmental toxins and motor neuron disease: a case report]. / Rapporto tra l'esposizione a fattori tossici ambientali e malattia del neurone di moto: osservazioni su un caso.

A case is reported of amyotrophic lateral sclerosis with occupational exposure to solvents and metals. The environmental toxin theory of motorneuron disease, including aspects of epidemiological, clinical, experimental and individual susceptibility is discussed. An overall evaluation of animal and human data was made using a methodological approach developed by the International Agency for Research on Cancer and a Scandinavian group of experts. It is concluded that there is a probable linkage between metals/solvents exposure and motorneuron disease.