Molecular modeling and LC-MS-based metabolomics of a glutamine-valproic acid (Gln-VPA) derivative on HeLa cells.

Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8 mM) or DON (IC50 = 3.5 mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.

Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide, a Valproic Acid Aryl Derivative with Activity Against HeLa Cells.

BACKGROUND: Valproic acid (VPA) is an HDAC inhibitor (HDACI) with an anticancer activity, but is hepatotoxic. N-(2-hydroxyphenyl)-2-propylpentanamide (o-OH-VPA) is a VPA aryl derivative designed in silico as a selective inhibitor of HDAC8 with biological properties against HeLa, rhabdomyosarcoma and breast cancer cell cultures. OBJECTIVE: We studied the epigenetic mechanism of o-OH-VPA as an HDACI and evaluated whether it was toxic to normal cells. METHODS: HeLa cells and primary human fibroblasts were used for this study as carcinogenic and normal cells, respectively. Cell survival was evaluated by MTT assay, whereas viability and doubling time were determined by the Trypan-blue method. HDAC activity was tested using the colorimetric HDAC activity assay. The expression of p21 was analyzed by PCR and HDAC8 expression was also evaluated by real-time PCR. Cell cycle and caspase-3 activity were analyzed by flow cytometry and caspase-3 colorimetric assay, respectively. RESULTS: o-OH-VPA (IC50 = 0.1 mM) was fifty-eight times more effective than VPA (IC50 = 5.8 mM) to reduce HeLa cell survival. Furthermore, o-OH-VPA increased the doubling time of HeLa cells by 33% with respect to the control. o-OH-VPA acted as HDACI in HeLa cells without affecting the HDAC8 expression, arresting the cell cycle of HeLa cells in the G0/G1 phase due to the increase in p21 expression with the inhibition of caspase-3 activity without exhibiting toxicity toward normal cells. CONCLUSION: Our results revealed that o-OH-VPA is an HDACI with a selective effect against HeLa cells but without the known toxicity exerted by most pan-HDACIs on normal cells.

Docking Studies of Glutamine Valproic Acid Derivative (S)-5- amino-2-(heptan-4-ylamino)-5-oxopentanoic Acid (Gln-VPA) on HDAC8 with Biological Evaluation in HeLa Cells.

In this contribution, we focused on evaluating a novel compound developed by our group. This molecule, derived from glutamine (Gln) and valproic acid (VPA), denominated (S)- 5-amino-2-(heptan-4-ylamino)-5-oxopentanoic acid (Gln-VPA), was submitted to docking studies on histone deacetylase 8 (HDAC8) to explore its non-bonded interactions. The theoretical results were validated in HeLa cells as a cancer cell model and in human dermal fibroblasts as a normal cell model. The effects of Gln-VPA on HeLa and normal fibroblasts in terms of cell survival and the ability to inhibit HDAC activity in nude nuclear proteins and in nuclear proteins of whole cells treated for 24 h were analyzed. The HeLa cell cycle was analyzed after 24 and 48 h of treatment with Gln-VPA. The docking studies show that Gln-VPA can reach the catalytic site of HDAC8. Gln-VPA was organically synthesized with a purity greater than 97%, and its structure was validated using mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. Gln-VPA showed a similar effect to VPA as an HDAC inhibitor but with less toxicity to fibroblasts. Although Gln-VPA was less efficient than VPA in reducing the survival of HeLa cells, it could be studied for use as a cancer cell sensitizer.

Contamination of cheese by polycyclic aromatic hydrocarbons in traditional smoking. Influence of the position in the smokehouse on the contamination level of smoked cheese.

This paper sets out to determine the polycyclic aromatic hydrocarbon (PAH) contamination degree of a traditionally smoked cheese: Herreño cheese, which comes from one of the Canary Islands. Its PAH profile is thoroughly studied by means of gas chromatography-mass spectrometry in SIM mode, and compared with that of an unsmoked cheese. Furthermore, a parameter not previously studied is evaluated, namely the influence of the position of the individual cheeses in the smokehouse on their PAH contamination level. Heavy PAH, among which are included most of the carcinogens, are very scarce and their concentrations low. In fact, benz[a]anthracene, together with chrysene+triphenylene, are the only heavy PAH detected in all of the smoked samples studied. The concentration of benzo[a]pyrene, detected only in 1 of the samples, is below the limit established in Spain for the rind of smoked cheese. In contrast, high concentrations of light PAH have been found, especially of naphthalene and its alkyl derivatives, whose effect on human health is not yet well established. The results derived from the analysis of the PAH profile suggest the potential usefulness of certain ratios between some pairs of PAH (phenanthrene/anthracene, naphthalene/acenaphthylene) to provide information on the PAH contamination source. Furthermore, differences have been found, depending on the position of the cheeses in the smokehouse, those placed in the path followed by the smoke being more contaminated. Therefore, the findings of this study could help in improving the design of smokehouses, to decrease the PAH contamination degree of smoked cheese.

Occurrence of polycyclic aromatic hydrocarbons in artisanal Palmero cheese smoked with two types of vegetable matter.

Palmero cheese is a fresh smoked cheese from the Isle of Palma (Canary Islands), manufactured with goat's milk. To guarantee its safety, the occurrence of polycyclic aromatic hydrocarbons (PAH) in artisanal Palmero cheese smoked with 2 types of vegetable matter (almond shells and dry prickly pear) was studied. The determination of PAH includes extraction and clean-up steps, followed by separation, identification, and quantification of PAH by gas chromatography-mass spectrometry in selected ion-monitoring mode. The most abundant PAH are those with 2 and 3 aromatic rings. Although the highest total PAH concentrations corresponded to the cheeses smoked with almond shells, the degree of PAH contamination of the cheeses studied was lower than that found in other cheeses smoked in the traditional way. The nature of the vegetable material used for smoking seemed to have an influence on the type of PAH formed, especially on alkylderivatives and some light PAH. However, despite the artisanal, and consequently variable, production process of these cheeses, many similarities have been found among their PAH profiles. In fact, relatively constant relationships are observed between the concentrations of certain pairs of PAH. Benzo(a)pyrene was only present in 2 samples, and in much lower concentrations than the maximum allowed legal limits. Therefore, according to the results obtained, it appears that it is possible to obtain a safe product without renouncing the artisanal character or the sensory properties of this type of cheese.

Retinoic acid increases tissue and plasma contents of nerve growth factor and prevents neuropathy in diabetic mice.

BACKGROUND: Decreased production of nerve growth factor (NGF) may contribute to diabetic neuropathy; however, exogenous administration of NGF induces only a modest benefit. Retinoic acid (RA) promotes the endogenous expression of nerve growth factor and its receptor. We studied the effects of RA on diabetic neuropathy in mice with streptozotocin-induced diabetes. MATERIAL AND METHODS: One hundred and twenty National Institutes of Health (NIH) albino mice randomly separated into three groups (A, n = 30; B, n = 30; C, n = 60). Diabetes mellitus was induced with streptozotocin in groups A and B. Animals from group A received a subcutaneous injection of 25 microl of mineral oil daily for 90 days, while those from group B received a subcutaneous injection of 20 mg kg(-1) of all trans RA. Animals from group C were taken as controls. At the end of the experiment, blood glucose and NGF levels (both in serum and sciatic nerve) were measured. Two behavioural tests were conducted in a blind fashion to detect abnormalities of thermal and nociceptive thresholds. RESULTS: Contents of NGF in healthy untreated mice were 1490 +/- 190 pg mg(-1) in nerve and 113 +/- 67 pg mg(-1) in serum; in diabetic untreated mice the values were 697 +/- 219 pg mL(-1) in nerve and 55 +/- 41 pg mL(-1) in serum; and in diabetic mice treated with RA the values were 2432 +/- 80 pg mL(-1) in nerve and 235 +/- 133 pg mg(-1) in serum (P < 0.002). Ultrastructural evidence of nerve regeneration and sensitivity tests improved in diabetic mice treated with RA as compared with nontreated diabetic mice. CONCLUSION: Our findings indicate that administration of RA increases serum and nerve contents of NGF in diabetic mice and suggest a potential therapeutic role for retinoic acid in diabetic patients.

Chronic malnutrition does not influence the development of ethylnitrosourea-induced brain tumors.

There is controversy over the subject of malnutrition as a potential risk factor for cancer; we studied the effect of chronic malnutrition on the development of tumors in rats prenatally exposed to the carcinogenic ethylnitrosourea. Twelve pregnant Wistar rats were administered on the 19th day of gestation with a single i.v. dose of 30 mg/kg of ethylnitrosourea. Immediately after weaning, at 23 days of age, half of the offspring were nourished with a protein-deficient diet (less than 6%), which consisted mostly of a corn-based diet with high calorie and low fiber contents. In the adult age, 83 rats (74%) developed a tumor of the nervous system; in comparison with controls, we found no differences in time of development, site and histological characteristics of the tumors that developed in animals subjected to chronic malnutrition.

Components detected by means of solid-phase microextraction and gas chromatography/mass spectrometry in the headspace of artisan fresh goat cheese smoked by traditional methods.

The study of the headspace components of fresh smoked goat cheese, was carried out by means of solid-phase microextraction using a polyacrylate fiber followed by gas chromatography/mass spectrometry. The samples studied were six artisan Palmero cheeses manufactured following traditional methods and smoked using pine needles. The cheese regions studied were exterior, interior, and a cross section. In total, more than 320 components were detected, the exterior region being the richest in components, among which were acids, alcohols, esters, hydrocarbons, aldehydes, ketones, furan and pyran derivatives, terpenes and sesquiterpenes, nitrogen derivatives, phenol, guaiacol and syringol derivatives, ethers, and others. In addition to typical cheese components, typical smoke components were also detected; these latter were present especially in the headspace of the exterior region and only those in significant concentrations in the exterior region were also detected in the interior. The main components were acids and phenolic derivatives. These latter compounds play an important role in the flavor of this cheese, and their relative proportions together with the presence of specific smoke components derived from pine leaves may be considered of interest in order to distinguish this cheese from others smoked with different vegetable matter.

Paradoxical effect of aspirin on the growth of C6 rat glioma and on time of development of ENU-induced tumors of the nervous system.

PURPOSE: Administration of acetylsalicylic acid (ASA), an inhibitor of the synthesis of prostaglandins and thrombzoxanes, decreases the incidence of colorectal cancer and other neoplasms and inhibits in vitro some tumor growth. We studied the effect of various doses of ASA on the growth of C6 glioma implanted in rats as well as the effect of chronic administration of ASA on time of development and incidence of tumors of the central nervous system (CNS) induced by prenatal exposure to ethylnitrosourea (ENU). METHODS: In a controlled study, various doses of ASA, 12.5, 25, 50, 100, 200, 300, and 400 mg/kg per day, were administered to Wistar rats in whom a subcutaneous C6 glioma had been transplanted. Changes in tumor size, histologic characteristics, mitotic index, cell proliferation, and vascular density were studied. In a parallel experiment, we administered ASA (70 mg/kg per day) to rats who were prenatally exposed to ENU; treatment started on day 50 of age, and continued until the end of the experiment at day 400. The time of tumor development as well as incidence, localization, and histological diagnosis were compared with matched controls. RESULTS: A paradoxical effect of ASA administration was observed on the dynamics of cell proliferation of C6 glioma. When high ASA doses were administered (200 or 400 mg/kg per day), tumor volume, cell proliferation, vascular density, and mitotic index increased. In contrast, when low doses were administered (12.5 or 25 mg/kg per day) the tumor size diminished. In the second experiment, localization and incidence of CNS tumors induced by ENU were similar in animals treated with ASA and in controls; however, in rats treated with ASA the time of tumor development was shortened. CONCLUSIONS: The growth-promoting effects of high doses of ASA found in the present study in both transplanted and chemically-induced brain tumors, might be due to the blockage of autocrine inhibitory factors dependent on the cyclooxygenase pathway or by increased vascular permeability and blood supply to the tumor due to inhibition of platelet aggregation. In contrast, the inhibition of tumor growth obtained with low ASA doses in transplanted glioma might be due to different mechanisms such as the induction of apoptosis.

Clomipramine increases the striatal concentrations of dopamine and homovanillic acid in rats chronically consuming alcohol.

OBJECTIVE: There have been many studies on the relationships between the dopaminergic system, alcoholism and antidepressant drugs; the information, however, is controversial. The purpose of this study was to examine the effect of clomipramine (CMI), a tricyclic antidepressant, on the striatal concentration of dopamine (DA) and homovanillic acid (HVA), and their turnover, in a rat model of chronic alcohol ingestion. METHOD: After 10 months of exposure to either water (expW) or alcohol (expA), female Wistar rats (N = 60) were randomly assigned to one of the following six groups: (1) (expW) control group (C), normal diet; (2) (expA) alcohol administration group (A), drinking water was replaced by commercial brandy that was 38% ethanol; (3) (expW) clomipramine group (CMI), received intraperitoneal injections over 4 months; (4) (expA) alcohol administration + clomipramine group (ACMI); (5) (expA) alcohol abstinence group (AA); and (6) (expA) alcohol abstinence + clomipramine group (AACMI). Each group consisted of 10 animals. RESULTS: Chronic alcohol intake decreased striatal dopamine concentration (p < .001), whereas clomipramine administration produced a significant increase (p < .001) when administered in both control rats and rats exposed to long-term alcohol administration. The increase in the ACMI group was significantly different (p < .001) when it was compared with the CMI group. In the AA and AACMI groups, a significant decrease of striatal dopamine was observed (p < .001). During long-term alcohol administration, homovanillic acid decreased (p < .005). HVA/DA ratio increased in AA and AACMI groups (p < .001). CONCLUSIONS: These findings show that clomipramine produces favorable changes in dopaminergic systems altered by chronic alcohol administration. Results also provide evidence to support further prospective studies of potential therapeutic effects of antidepressant drugs in alcoholism.

Ultrastructural changes in limb distal nerves of rats with alcoholism and/or malnutrition before and after dietary correction.

To study peripheral nerves changes in chronic alcoholism and in malnutrition, we examined ultrastructurally the distal nerve branches of the digits of rats treated with four different dietary schemes, as follows: controls (n = 22), fed standard rodent diet plus water ad libitum; alcoholism (n = 12), fed the standard diet and 2-12% ethanol in drinking water; malnutrition (n = 21), fed with corn tortillas instead of standard diet; and alcoholism and malnutrition combined (n = 22). After 10 months under these conditions, a proportion of animals from each group were sacrificed. The remaining rats of the malnutrition and alcoholism alone groups were deferred a standard diet. The combined alcohol + malnutrition group was subdivided into standard diet, malnutrition and ethanol. After a further 4 months under these new conditions, the animals were sacrificed. Ultrastructural examination of limb distal nerve branches showed that glycogen-like particles were more common in malnourished animals, whereas remyelinating axons were more numerous in ethanol-treated rats. Bands of regeneration were present in both groups, but were more common in animals treated with ethanol. These features decreased significantly when the respective nutritional factor was reversed. The results confirm that ethanol plays a definitive role in the development of alcoholic neuropathy and that malnutrition accentuates, the histopathological abnormalities.

Effects of chronic inhalation of volatile solvents, malnutrition and their interaction on seizures induced by pentylenetetrazole in rats.

Chronic inhalation of volatile solvents induce severe brain damage. In humans, intense exposure to volatile solvents for recreational purposes is frequently associated with chronic malnutrition. We studied in rats the effects of chronic inhalation of volatile solvents and malnutrition, alone and combined, on the seizures induced by pentylenetetrazole. Animals were subjected to 14 months of either normal rodent diet or malnutrition induced by a diet based on corn derivatives; some animals were subjected for the last 4 months to daily inhalation of volatile solvents (paint thinner). Afterwards, a trial of pentylenetetrazole-induced seizures was conducted in all animals. When compared with controls, malnutrition, chronic inhalation of volatile solvents and their combination greatly reduced the threshold for both, the forebrain and the brain stem components of seizures. However, an expected lowering of the threshold when malnutrition and solvent inhalation were combined was not observed when compared with each condition alone. It is possible that malnutrition plus solvents exposure induce severe brain damage that interferes with the brain structures involved in the propagation of epileptic seizures.

Chronic malnutrition caused by a corn-based diet lowers the threshold for pentylenetetrazol-induced seizures in rats.

The incidence of epilepsy is high in developing countries where malnutrition is prevalent. Although malnutrition is not a direct cause of seizures, chronic malnutrition may predispose the brain to seizures. In large undernourished human groups from Latin America, the most common sources of food are corn and corn derivatives. We used a rat model of chronic malnutrition, in which corn tortillas were the only solid food intake, to study the possible influence of malnutrition at late stages of brain development on the dynamics of experimental seizures induced by pentylenetetrazole (PTZ). The threshold and does of PTZ required to produce seizures were greatly reduced in malnourished rats. The model of malnutrition used in the study imitates a form of malnutrition common among large numbers of humans. Our results suggest that chronic malnutrition early in life induces changes that lower the seizure threshold and leave the brain more susceptible to seizures. Whether this observation relates to the high incidence of epilepsy in underdeveloped countries remains to be determined.

Reversibility of the alterations induced by chronic alcoholism and malnutrition in rats after alcohol withdrawal and proper nutrition.

OBJECTIVE: The specific influence of malnutrition on the cellular damage induced by alcoholism has been poorly defined; therefore, the possible degree of improvement that could be obtained after the institution of proper nutrition and after alcohol withdrawal remains unclear. We studied both conditions in several combinations, either after chronic alcoholism, chronic malnutrition or both, and after reversal of each condition or of both. METHOD: Using experimental models of alcoholism and malnutrition, we studied the alterations induced by each condition and on the combination of both in five histological locations and in 10 hematological and biochemical parameters; afterwards, we studied the possible restorative effect on each parameter after 4 months of alcohol withdrawal or proper nutrition or both. RESULTS: Our results showed that chronic alcoholism induced a mean of 31% deviation from values obtained in controls, malnutrition induced 17%, and alcoholism and malnutrition combined induced 52%. Upon the removal of the offending condition, alcohol withdrawal brought 13% improvement and proper nutrition 5% improvement; in the animals with alcoholism and malnutrition, alcohol withdrawal and proper nutrition brought 26% improvement, alcohol withdrawal but continuation of malnutrition 10% improvement, and continuation of alcoholism but proper nutrition led to a further 8% worsening from the alterations already found during alcoholism and malnutrition. CONCLUSIONS: Multivariate statistical comparisons demonstrated that chronic malnutrition produced an important, independent and difficult to reverse effect on the damage induced by alcoholism in several histological and physiological parameters.

Determination of boldine in drug formulations by first-derivative synchronous spectrofluorimetry.

A simple and sensitive method is proposed for the determination of boldine by first-derivative synchronous spectrofluorimetry based on its native fluorescence in 0.1 N sulphuric acid. The constant wavelength difference (delta lambda = lambda em--lambda ex) chosen to optimize the determination is 90 nm. The linear concentration range of application is between 2.0 and 50.0 micrograms l-1 of boldine, the detection limit 0.4 microgram l-1 and the relative standard deviation at a concentration of 25 microgram l-1 was 2.1%. The method has been satisfactorily applied to the determination of boldine in pharmaceutical formulations.

Detrimental effects of malnutrition on the damage induced by alcoholism: a study of animal models that simulate chronic alcoholism and malnutrition of large human groups.

The specific influence of malnutrition on the pathophysiological changes induced by chronic alcoholism is controversial; most studies are inconclusive because they have been made in chronic alcoholics that develop malnutrition as a complication of alcoholism. However, in vast human groups, alcoholism evolves in individuals that belong to a chronically undernourished population. In an attempt to simulate real-life conditions of malnutrition-alcoholism in humans, we studied in rats: (1) the effects of chronic malnutrition with tortilla, a corn bread that constitutes the main diet of large human groups; (2) the effects of chronic alcoholism with commercial brandy; and (3) the effects of chronic alcoholism and malnutrition combined. The damage induced either by alcoholism or that induced by malnutrition alone were of similar degree, whereas the combination of malnutrition and alcoholism led to a worsening of some parameters studied: body weight, leucocyte count and, more remarkably, in the tissue damage of several areas of the central nervous system.

Immunodiagnosis of human cysticercosis in cerebrospinal fluid. Antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium.

Tapeworm antigens from Taenia crassiceps performed as well as those antigens from Taenia solium in an enzyme-linked immunosorbent assay for the detection of Cysticercus antibodies in 96 cerebrospinal fluid samples from patients with neurocysticercosis and in 96 CSF samples from patients with other varied neurological ailments. Thus, this manageable murine model of experimental cysticercosis solved the problem of antigen supply for clinical and epidemiological applications, and it provided an immediate means of abundant production of antigens for the wide distribution and standardization of immunodiagnostic tests for cysticercosis.

Effect of chronic stress on ENU-induced tumors.

The relation of stress to cancer is the subject of considerable controversy. We studied the possible influence of chronic stress on the time of development and frequency of tumors induced in rats after a single exposure to ethylnitrosourea during prenatal life. Time of development, localization, incidence, type, and size of tumors were similar in stressed rats and in controls. Our results in this paradigm do not support the hypothesis that chronic stress exerts a potentiating effect on carcinogenesis.

Freezing of infested pork muscle kills cysticerci.

A method for culturing cysticerci that allows successful evagination and growth of scolexes from metacestodes of Taenia solium was used to study the survival of cysticerci subjected to low temperatures. Refrigeration of pork muscle infested with cysticerci at temperatures above 0 degrees C did not affect the parasites' survival in culture. Conversely, freezing of meat prevented survival of cysts. A practical procedure to kill cysticerci is the storage of pork muscle for four days at -5 degrees C, three days at -15 degrees C, or one day at -24 degrees C. These simple measures would help prevent the most frequent parasitosis of man's central nervous system.