The water-soluble and protein-bound metabolites of benzo(a)pyrene formed by rat liver

An optimal incubation system to study benzo(a)pyrene metabolism to its polar derivatives is described. The system using the 20,000g supernatant enzymes of rat liver converts up to 80 per cent of micromolar concentrations of [¹⁴C]benzo(a)pyrene to water-soluble products of which about 10 per cent become covalently bound to protein, are trichloroacetic acid-precipitable, not extractable into organic solvents and are stable to polyacrylamide gel electrophoretic and gel filtration separation techniques.Four (I–IV) discrete[¹⁴C]benzo(a)pyrene-protein complexes have been purified by ion exchange Sephadex gel filtration from post-incubation cytosols. Protein I has the highest binding specificity, a molecular weight estimated at 44,500, migrates as a dimer in sodium dodecyl sulphate polyacrylamide gel, and has glutathione S-transferase activity. This binding protein is believed to be at least similar to, if not identical with, ligandin. Proteins II–IV are also active as glutathione S-transferases. This enzymic function of the binding proteins is markedly reduced by the covalent attachment of the metabolised benzo(a)pyrene, so that the de-activating conjugation with GSH of reactive metabolites is inhibited. The carcinogen, under these conditions hampers its own detoxification reaction.Certain metabolic inhibitors reduce specific binding of the carcinogen to protein I and enhance the binding to the other proteins. The less specific binding to proteins II–IV may be important to the carcinogenic process.     

[3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel,Potent, and Selective Phosphatidylinositol-3-kinase β Inhibitors

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Eugenol,a Component of Holy Basil (Tulsi) and Common Spice Clove,Inhibits the Interaction Between SARS-CoV-2 Spike S1 and ACE2 to Induce Therapeutic Responses

Journal of Neuroimmune Pharmacology - Spike S1 of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells to enter the cell and...     

6-Nitrobenzo[a]pyrene can be denitrated during mammalian metabolism

Nitropolycyclic aromatic hydrocarbons (nitroarenes), including 6-nitrobenzo[a]pyrene (6-NBap), occur in our environment and are mutagenic in bacterial mutagenesis assays. The mutagenicity of 6-NBaP is enhanced when rat liver S9 is added. To investigate the cause of this increased activity, the metabolism of 6-NBaP was carried out with a total rat liver homogenate obtained from 3-methylcholanthrene- (MC-) induced rats, a 9000 X g supernatant enzyme, and with both unwashed and washed microsomes. Ring-hydroxylated 6-NBaP was detected. On the basis of retention times for known standards in a high-performance liquid chromatographic system, benzo[a]pyrene (BaP) and 6-acetoxy-BaP (6-OAcBaP) were isolated as products. BaP was further characterized via ultraviolet (UV) and mass spectra and 6-OAcBaP by UV, mass, and nuclear magnetic resonance (NMR) spectra. 6-HydroxyBaP (6-OHBaP) was also detected by UV and mass spectra. It is suggested that BaP is formed via a nitroanion radical of 6-NBaP and undergoes metabolism, while the 6-OHBaP is acetylated to form 6-OAcBaP. The acetyl donor remains to be identified.     

Identification of the alpha1L-adrenoceptor in rat cerebral cortex and possible relationship between alpha1L- and alpha1A-adrenoceptors

BACKGROUND AND PURPOSE: In addition to alpha1A, alpha1B and alpha1D-adrenoceptors (ARs), putative alpha1L-ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the alpha1A-AR and clarify its pharmacological profile using a radioligand binding assay. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed in intact tissue segments and crude membrane preparations of rat cerebral cortex. Intact tissue binding assays were also conducted in rat tail artery. KEY RESULTS: [3H]-silodosin at subnanomolar concentrations specifically bound to intact tissue segments and membrane preparations of rat cerebral cortex at the same density (approximately 150 fmol mg(-1) total tissue protein). The binding sites in intact segments consisted of alpha1A and alpha1L-ARs that had different affinities for prazosin, while the binding sites in membranes showed an alpha1A-AR-like profile having single high affinity for prazosin. [3H]-prazosin also bound at subnanomolar concentrations to alpha1A and alpha1B-ARs but not alpha1L-ARs in cerebral cortex; the binding densities being approximately 200 and 290 fmol mg(-1) protein in the segments and the membranes, respectively. In the segments of tail artery, [3H]-silodosin only recognized alpha1A-ARs, whereas [3H]-prazosin bound to alpha1A and alpha1B-ARs. CONCLUSIONS AND IMPLICATIONS: The present study clearly reveals the presence of alpha1L-ARs as a pharmacologically distinct entity from alpha1A and alpha1B-ARs in intact tissue segments of rat cerebral cortex but not tail artery. However, the alpha1L-ARs disappeared after tissue homogenization, suggesting their decomposition and/or their pharmacological profile changes to that of alpha1A-ARs.     

Different affinities of native alpha1B-adrenoceptors for ketanserin between intact tissue segments and membrane preparations

The pharmacological profiles of alpha1-adrenoceptors for ketanserin, prazosin, silodosin, and BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride) were examined under different assay conditions. Among the tested antagonists and alpha1-adrenoceptors subtypes, ketanserin showed significantly lower affinity for the alpha1B-adrenoceptor subtype in intact tissue sampled from the rat tail artery, thoracic aorta, and cerebral cortex (functional pKB and binding pKi were approximately 6), than in cerebral cortex membrane preparations or whole cell and membrane preparations of alpha1B-adrenoceptor transfected human embryonic kidney 293T (HEK 293T) cells (pKi was approximately 8). In these tissues and cells, however, ketanserin showed a similar affinity (pKi = approximately 8) for alpha1A- and alpha1D-adrenoceptors even though the assays were conducted under different conditions. In contrast, the affinities of alpha1A-, alpha1B-, and alpha1D-adrenoceptors for prazosin, silodosin, and BMY 7378 did not significantly change under different assay conditions and in different tissues. The present study reveals that the pharmacological profiles of native alpha 1B-adrenoceptors for ketanserin is strongly influenced by the assay conditions and suggest that antagonist affinity is not necessarily constant.     

Flavonoids of Korean Citrus aurantium L. Induce Apoptosis via Intrinsic Pathway in Human Hepatoblastoma HepG2 Cells

Korean Citrus aurantium L. has long been used as a medicinal herb for its anti‐inflammatory, antioxidant, and anticancer properties. The present study investigates the anticancer role of flavonoids extracted from C. aurantium on human hepatoblastoma cell, HepG2. The Citrus flavonoids inhibit the proliferation of HepG2 cells in a dose‐dependent manner. This result was consistent with the in vivo xenograft results. Apoptosis was detected by cell morphology, cell cycle analysis, and immunoblot. Flavonoids decreased the level of pAkt and other downstream targets of phosphoinositide‐3‐kinase/Akt pathway – P‐4EBP1 and P‐p70S6K. The expressions of cleaved caspase 3, Bax, and Bak were increased, while those of Bcl‐2 and Bcl‐xL were decreased with an increase in the expression of Bax/Bcl‐xL ratio in treated cells. Loss of mitochondrial membrane potential was also observed in flavonoid‐treated HepG2 cells. It was also observed that the P‐p38 protein level was increased both dose and time dependently in flavonoid‐treated cells. Collectively, these results suggest that flavonoid extracted from Citrus inhibits HepG2 cell proliferation by inducing apoptosis via an intrinsic pathway. These findings suggest that flavonoids extracted from C. aurantium L. are potential chemotherapeutic agents against liver cancer. Copyright © 2015 John Wiley & Sons, Ltd.