Benzodiazepine-like Molecules Research Articles

Presence of benzodiazepine-like molecules in food and their implication in the nutrition of cirrhotic patients

Benzodiazepine (BDZ)-like compounds, present in trace amounts in normal subjects increase in the blood of liver cirrhotic patients. The origin of these compounds is still unknown but they are present in medicinal plants and foods. Herein we report the detection of BDZ-like molecules in fruits, vegetables, cereals, meat, milk and cheeses and in different cultivars of potatoes, tomatoes and carrots. The extracted food was separated by HPLC purification and the collected fractions were tested by radioreceptor binding assay in order to evaluate their ability to selectively bind the central benzodiazepine receptors. The mean value was 14.80 ng of diazepam equivalent (DE)/g in fruits, 4.34 ng DE/g in vegetables, 6.35 in cereals and 4.09 in meat. BDZ-like compounds are poorly present in cheeses and completely absent in olive and seeds oil. From these findings it is possible to select food with low amount of BDZ-like molecules useful for cirrhotic diet in order to prevent hepatic encephalopathy.

Brain Benzodiazepine-like Molecules and Stress-anxiety Response

Benzodiazepines(BZDs) are among the most widely prescribed drugs in the world. They are potent anxiolytic, antiepileptic, hypnotic, and muscle relaxing agents. There is an emerging model of the role of several neural systems in anxiety and their relation to the mechanism of action of BZDs. It has been postulated that BZD drugs exert their anxiolytic action by regulating GABAergic transmission in limbic areas such as the amygdala, in the posterior hypothalamus, and in the raphe nuclei. The involvement of the amygdala in the behaviors triggered by fear and stress has been suggested by many previous studies. In this review, reports about regulatory effects of endogenous BZD receptor ligands on the perception of anxiety and memory consolidation were summerized. These findings further support the contention that BZD receptor ligands modulate memory consolidation of averse learning tasks by influencing the level of stress and/or anxiety that accompanies a learning experience. The findings suggest that the decrease in the limbic levels of BZD-like molecules seen after the various behavioral procedures represent a general response to stress and/or anxiety, since it occurs in proportion to the level of stress and/or anxiety that accompany these tasks. In addition, these findings further support the hypothesis that the /BZD receptor complex in limbic structures plays a pivotal role in the stress and anxiety.

IMMUNOCHEMICAL EVALUATION OF BENZODIAZEPINE-LIKE MOLECULES IN PLANTS

The concentration of benzodiazepine-like molecules were evaluated in 32 potato cultivars and 16 tomato cultivars and found to be quite variable, ranging from 0.1 - 3 ng/g of dry matter. The temperature stability of BZD-like molecules was tested in potato cooked in different ways, and more than 86% of BZD (TDx® equivalent) content was still found after cooking. Immunological methods performed with the polyclonal and monoclonal antibodies made it possible to evaluate very low concentrations of synthetic BZD substances (sensitivity of about 60 pg/ml). The variability determined open plenty of opportunities for promising breeding programs. A description is given of preliminary food evaluation and the new immunological methods developed.

Naturally occurring benzodiazepines and benzodiazepine-like molecules in brain.

Great progress has been made in the last five years in demonstrating the presence of benzodiazepines (BZDs) in mammalian tissues, in beginning studies on the origin of these natural compounds and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and the biosynthetic pathways responsible for the presence of BZDs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BZDs are of natural origin; (2) mammalian brain contains BZDs in concentrations ranging between 5.10(-10) to 10(-8) M; (3) BZDs and BZD-like molecules are unevenly distributed in brain; the highest concentration is found in limbic structures (4) dietary source of BZDs might be a plausible explanation for their occurrence in animal tissues, including man; (5) the formation of BZDs-like molecules in brain is a possibility, experimentally supported; (6) BZDs like molecules including diazepam and N desmethyldiazepam are elevated in hepatic encephalopathy; (7) natural BZDs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral and molecular biological techniques available to the neuroscientist will hopefully continue to yield new and exciting information concerning the biological roles that BZDs might play in the normal and pathological functioning of the brain.

In Vivo Formation of Benzodiazepine-like Molecules in Mammalian Brain

The possible cerebral formation of benzodiazepine (BDZ)-like molecules was evaluated in rats bilaterally microinjected into either the lateral ventricles (ICV) or the hippocampus (IH), with 10-70 μCi of different radiolabeled amino acids. After 3, or 24 h, the rats were sacrificed and BDZ-like molecules from total brain or hippocampus were purified by reversed phase HPLC. At 24 h, but not at 3 h of the ICV or IH microinjections with [ 3H] tryptophan, a peak of radioactivity containing material that inhibited the binding of [ 3H] flunitrazepam to both the BDZ receptor and the anti-BDZ inonoclonal antibody MAb 21-7F9 was obtained. This active labeled fraction eluting just before diazepam also bound directly and specifically to the BDZ receptor and to MAb 21-7F9. No peak of radioactivity containing BDZ-like material was obtained when [ 3H] phenylalanine, [ 14C] glycine, [ 14C] methionine or [ 14C] tyrosine alone or in different combinations were micro-injected into the hippocampus. The present results, together with our previous findings on the in vitro production of BDZ-like molecules in rat brain homogenates or slices (11), strongly suggest that the mammalian brain is capable of synthesizing low molecular weight substances possessing BDZ-like activity.

Effect of various training procedures on performance in an elevated plus-maze: Possible relation with brain regional levels of benzodiazepine-like molecules

Rats submitted to one, two, or seven sessions of exploration to a new environment (habituation) or exposed to an inhibitory avoidance training showed different degrees of anxiety, evaluated by the elevated plus-maze test. Also, the brain regional levels of benzodiazepine (BDZ)-like molecules in rats submitted to one, two, or seven sessions of habituation were differentially decreased with respect to nontrained rats. The percentage of time spent in the open arms of the elevated plus-maze for each group correlates with the data of decrease in the BDZ-like immunoreactivity in amygdala ( r = 0.77, p < 0.0005), hippocampus ( r = 0.68, p < 0.0005), and septum ( r = 0.57, p < 0.005). These results suggest that the limbic system responds to anxiogenic experiences by changing the BDZ-like molecule levels in relation to the degree of anxiety and/or stress that accompany these experiences.

Production of benzodiazepine-like compounds in bovine rumen

The presence of benzodiazepine-like molecules was detected radioimmunologically in bovine rumen contents and in incubates of ruminal contents with homogenates of several common grasses. A similar production was found “in vivo” in samples obtained from a grazing cow with a rumen cannula.

Formation of benzodiazepine-like molecules in rat brain

The possible biosynthetic origin of benzodiazepine-like molecules was investigated in mammalian tissue. Rat brain homogenates or cortical slices incubated under physiological conditions showed a 4 to 7 fold increase in the content of BZD-like compounds as compared with control non incubated or boiled tissue. The quantitative analysis was performed by a radioimmunoassay with a specific monoclonal antibody. The active fraction eluting just before diazepam exhibited a Mr lower than 1300 and inhibited the [3H]flunitrazepam binding to the central benzodiazepine receptor. No activity was measured in the absence of tissue. These data suggest that under our experimental conditions, low molecular weight substances similar to benzodiazepines are formed in rat brain.

Habituation and inhibitory avoidance training alter brain regional levels of benzodiazepine-like molecules and are affected by intracerebral flumazenil microinjection.

The effects of habituation and inhibitory avoidance training on the rat brain regional levels of benzodiazepine (BZD)-like molecules and on central type BZD binding sites were examined. BZD-like immunoreactivity was decreased by 26-50% in the amygdala, cerebral cortex and septum of rats sacrificed immediately after stepping-down from the platform of an inhibitory avoidance apparatus (non-trained group) as compared to naive controls. Rats submitted to a second step-down session 20 h later (habituated group) have significantly lower BZD-like immunoreactivity in the septum (-60%) as compared to non-trained animals. Rats exposed to an inhibitory avoidance training, i.e. stepping-down and receiving a footshock (trained group), showed a significant reduction in the content of BZD-like molecules in cerebral cortex (-44%), amygdala (-68%), septum (-80%) and hippocampus (-82%) as compared to non-trained rats. In addition, the density of central type BZD binding sites was slightly increased in the hippocampus and septum of trained rats. No changes were observed in the apparent dissociation constant. No changes were observed in parallel measurements of [3H]-L-quinuclidinyl benzylate binding constants at cholinergic muscarinic binding sites. The immediate posttraining intrahippocampal bilateral injection of the central type BZD receptor antagonist flumazenil (10 nmol/hippocampus), enhanced the retention of habituation but not when injected in the amygdala or septum. In contrast, retention of the inhibitory avoidance task was significantly increased by flumazenil administered bilaterally into any of the 3 brain structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Naturally occurring benzodiazepines in human milk

The presence of benzodiazepine-like molecules was detected radioimmunologically in the plasma and milk of 12 women and in the plasma of 9 men. All subjects were non-users of benzodiazepines. The concentration of these biological materials expressed as diazepam equivalents per mL amounted to 2.54 ± 0.74 ng in male plasma; to 2.20 ± 0.35 ng in female plasma and to 1.91 ± 0.54 ng in milk. Further investigation of the active compounds in milk permitted the unequivocal identification of diazepam, both free and bound to a presumably protein carrier and, at least, three more benzodiazepine-like molecules. Their origin either from dietary sources or as a result of endogenous biosynthesis is still unclear.

Benzodiazepines and benzodiazepine-like molecules in brain: A. L. De Blas (Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City. Kansas City, MO 64110-2499, U.S.A.)

Benzodiazepines and benzodiazepine-like molecules in brain: A. L. De Blas (Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City. Kansas City, MO 64110-2499, U.S.A.)

Formation of benzodiazepine-like molecules in rat brain: M. Piva, 1 J. Medina, 2 A. De Blas 3 and C. Peña1 ( 1Instituto de Química y Físicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina; 2Instituto de Biología Celular, UBA; 3Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, U.S.A.)

Formation of benzodiazepine-like molecules in rat brain: M. Piva, 1 J. Medina, 2 A. De Blas 3 and C. Peña1 ( 1Instituto de Química y Físicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina; 2Instituto de Biología Celular, UBA; 3Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, U.S.A.)

Benzodiazepine-like molecules, as well as other ligands for the brain benzodiazepine receptors, are relatively common constituents of plants

The presence of benzodiazepine (BZD)-like molecules as well as of other substances with affinity for the brain BZD-receptors was explored in eight non-flowering plants known to contain biflavonoids, three flowering plants used as sedatives in folkloric medicine and one plant extensively used in Argentina, Uruguay, Brazil and Paraguay as a tea substitute. All the plants examined contained substances which bound to the central BZD-receptors and the majority of them also had BZD-like compounds detected by their specific interaction with a monoclonal antibody against BZDs. In various cases this last type of compound was present in amounts which exceeded trace levels (0.5-1.0 ng/g). The biological or clinical significance for humans of all these substances should be explored.

Presence of benzodiazepine-like molecules in mammalian brain and milk

From bovine cerebral cortex extracts, used to isolate n-butyl beta carboline-3-carboxylate (8), fractions active in displacing [3H] flunitrazepam binding were purified and shown to contain benzodiazepine-like molecules. These were recognized by UV spectra, retention time in HPLC, and interaction with a specific monoclonal antibody. Such molecules were localized in synaptic vesicles and cytosol of synaptosomes. Similar molecules were also found in cow milk. The possible dietary origin of these benzodiazepine-like molecules is discussed.

Endogenous benzodiazepine-like molecules in the human, rat and bovine brains studied with a monoclonal antibody to benzodiazepines

The anti-benzodiazepine monoclonal antibody 21-7F9 has been used for the identification and study of endogenous benzodiazepine-like molecules in the human, rat and bovine brains. A sandwich radioimmunoassay has been designed for the quantification of the membrane-bound endogenous benzodiazepine-like molecules. The localization of these molecules is not restricted to the brain tissue. They are also present in kidney, liver and spleen as well as in the neuroblastoma × glioma NG108-15 hybrid cells line. Immunoblots show benzodiazepine-like immunoreactivity in the membrane proteins of all these tissues. The membrane-bound benzodiazepine-like molecules are resistant to limited proteolysis of the membranes. Moreover, this treatment increases the binding of the monoclonal antibody 21-7F9 to the membranes, probably by exposing sites that normally are not accessible to the antibody. Immunocyto experiments show that benzodiazepine-like molecules are also present in samples of human cerebella that have been stored in paraffin since 1940, 15 years before the first chemical synthesis of benzodiazepines. The results indicate that the cerebellar benzodiazepine-like molecules recognized by the antibody are the product of biological (not chemical) synthesis. Benzodiazepine-like immunoreactivity has also been detected in NG108-15 cells that have been cultured for 3 months ib serum-free medium. These results suggest that the cells could biosynthesize benzodiazepine-like molecules.

Localization of benzodiazepine-like molecules in the rat brain. A light and electron microscopy immunocytochemistry study with an anti-benzodiazepine monoclonal antibody

The anti-benzodiazepine (BZD) monoclonal antibody 21-7F9 was used with light and electron microscopy immunocytochemistry techniques for studying the distribution of BZD-like molecules in the rat brain. With light microscopy, BZD-like immunoreactivity was found throughout the brain, mainly in neurons and occasionally in some glial cells (in periventricular areas, as well as in some perivascular astrocytes). Despite the fact that in the cerebellum the GABAergic neurons exhibit BZD-like immunoreactivity, co-localization of these two molecules is not exact, since there are also BZD-like positive neurons that are non-GABAergic (e.g. cerebellar granule cells, some neocortical and hippocampal pyramidal cells). Ultrastructural study of the cerebellar cortex disclosed that all neuronal categories were immunoreactive, as were some astrocytes within the granular layer. The reaction product was concentrated in neuronal perikarya and dendritic processes. Axons and axon terminals remained mostly unlabeled. The absence of immunoprecipitate within cytoplasmic organelles (Golgi apparatus, mitochondria, lumen of endoplasmic reticulum) and its presence at the cytoplasmic face of the cell membranes strongly suggests that endogenous BZD-like molecules are present in both the soluble cytoplasm (hyaloplasm), and also in association with both external and internal cell membranes. The results suggest that the brain BZD-like molecules might be functionally involved in either the modulation of GABA neurotransmission and/or the biotransformation, accumulation and elimination of benzodiazepines and the benzodiazepine-like molecules in the brain.

Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

An endogenous brain substance that binds to the central-type benzodiazepine receptors with agonist properties is present in both rat and bovine brains. This substance has been purified to homogeneity from bovine brain by immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reversed-phase HPLC steps. The purified substance was characterized as the benzodiazepine N-desmethyldiazepam (nordiazepam). The techniques used for the identification were mass spectrometry, HPLC, spectrophotometry, benzodiazepine receptor binding, and immunological techniques. Benzodiazepine-like immunoreactivity was also found in all the human brains tested, including six brains that had been stored in paraffin since 1940, fifteen years before the first synthesis of benzodiazepines. These results show that benzodiazepine-like molecules of natural origin--and possibly benzodiazepines themselves--are present in human and other mammalian brains.

I. Demonstration and purification of an endogenous benzodiazepine from the mammalian brain with a monoclonal antibody to benzodiazepines

Four hybridoma lines secreting monoclonal antibodies to benzodiapines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Roll-6896, and Ro5-3438 (the K d values are in the 10 −9 M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (β-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistent. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter γ-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam.

Demonstration of benzodiazepine-like molecules in the mammalian brain with a monoclonal antibody to benzodiazepines.

An anti-benzodiazepine monoclonal antibody has been used to demonstrate the existence of benzodiazepine-like molecules in the brain. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. Small benzodiazepine-like molecules were isolated from the brain soluble fraction by immunoaffinity chromatography. They block the binding of agonists, inverse agonists, and antagonists to the neuronal-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified endogenous molecules. The results indicate that the immunoaffinity-purified molecules behave like the neuronal-type benzodiazepine receptor agonists. The purified molecules, however, do not inhibit the binding of tritiated Ro 5-4864 to the "peripheral-type" benzodiazepine receptor. The results demonstrate the existence of benzodiazepine-like molecules in the brain that bind to the benzodiazepine receptor. These molecules are different from the endogenous benzodiazepine receptor ligands reported by others.

Rebound insomnia: a new clinical syndrome.

Rebound insomnia followed the withdrawal of three benzodiazepine hypnotic drugs, each of which had been administered in a single nightly dose for only short-term periods. The intense worsening of sleep is attributed to the short duration of the action of these drugs. A hypothesis involving benzodiazepine receptors in the brain is proposed in which there is a delay or lag in replacement of endogenous benzodiazepine-like molecules after the abrupt withdrawal of exogenous drugs.