Study of hepatotoxins influence in vitro on basic biochemical indicators of liver functional state

An antimicrobial drug of the fluoroquinolone group, ciprofloxacin, is widely used in Ukraine. However, some researchers have noted the probable hepatotoxicity of this drug with prolonged use or use of high doses of ciprofloxacin. The aim of this study was to compare the effects of carbon tetrachloride, as a classical model of hepatocyte injury, with the effects of ciprofloxacin. The aim of the study was to investigate the biochemical parameters of the liver when simulating toxic damage to hepatocytes with carbon tetrachloride or ciprofloxacin. Materials and methods. The study was carried out on isolated rat hepatocytes, in whose culture medium carbon tetrachloride or ciprofloxacin was added. After incubation in the supernatant and cell homogenate, the activities of marker enzymes of cytolysis were determined: ALT, AST, γ-GTP, LF, LDH, DC and MDA. Results. The introduction of ciprofloxacin into the culture of hepatocytes at a concentration of LC50 caused changes in biochemical parameters similar to those caused by carbon tetrachloride. Thus, an increase in ALT, AST, γ-GTP, LF, LDH, DC and MDA was observed when CCl4 or ciprofloxacin was added to the culture. Conclusion. Incubation of rat hepatocytes with carbon tetrachloride or ciprofloxacin caused an increase in the level of enzymes and lipid peroxidation products. These parameters are indicators of gross changes in cells, which are the result of impaired keto acid formation, impaired redox reactions, impaired glycogen production

Rapid Separation of Human Hemoglobin on a Large Scale From Non-clarified Bacterial Cell Homogenates Using Molecularly Imprinted Composite Cryogels

The production of a macroporous hydrogel column, known as cryogel, has been scaled up (up to 150 mL) in this work for the purification of human hemoglobin from non-clarified bacterial homogenates. Composite cryogels were synthesized in the presence of adult hemoglobin (HbA) to form a molecularly imprinted polymer (MIP)network where the affinity sites for the targeted molecule were placed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The MIP composite cryogel column was first evaluated in a well-defined protein mixture. It showed high selectivity toward HbA in spite of the presence of serum albumin. Also, when examined in complex non-clarified E. coli cell homogenates, the column showed excellent chromatographic behavior. The binding capacity of a 50 mL column was thus found to be 0.88 and 1.2 mg/g, from a protein mixture and non-clarified cell homogenate suspension, respectively. The recovery and purification of the 50 mL column for separation of HbA from cell suspension were evaluated to be 79 and 58%, respectively. The MIP affinity cryogel also displayed binding and selectivity toward fetal Hb (HbF) under the same operational conditions.

Commensal Bacterium Rothia aeria Degrades and Detoxifies Gluten via a Highly Effective Subtilisin Enzyme

Celiac disease is characterized by a chronic immune-mediated inflammation of the small intestine, triggered by gluten contained in wheat, barley, and rye. Rothia aeria, a gram-positive natural colonizer of the oral cavity and the upper digestive tract is able to degrade and detoxify gluten in vitro. The objective of this study was to assess gluten-degrading activity of live and dead R. aeria bacteria in vitro, and to isolate the R. aeria gluten-degrading enzyme. Methods: After an overnight fast, Balb/c mouse were fed a 1 g pellet of standard chow containing 50% wheat (and 4% gliadin) with or without 1.6 × 107 live R. aeria bacteria. After 2 h, in vivo gluten degradation was assessed in gastric contents by SDS-PAGE and immunoblotting, and immunogenic epitope neutralization was assessed with the R5 gliadin ELISA assay. R. aeria enzyme isolation and identification was accomplished by separating proteins in the bacterial cell homogenate by C18 chromatography followed by gliadin zymography and mass spectrometric analysis of excised bands. Results: In mice fed with R. aeria, gliadins and immunogenic epitopes were reduced by 20% and 33%, respectively, as compared to gluten digested in control mice. Killing of R. aeria bacteria in ethanol did not abolish enzyme activity associated with the bacteria. The gluten degrading enzyme was identified as BAV86562.1, here identified as a member of the subtilisin family. Conclusion: This study shows the potential of R. aeria to be used as a first probiotic for gluten digestion in vivo, either as live or dead bacteria, or, alternatively, for using the purified R. aeria enzyme, to benefit the gluten-intolerant patient population.

Predicting Intestinal and Hepatic First-Pass Metabolism of Orally Administered Testosterone Undecanoate

The bioavailability of orally administered drugs could be impacted by intestinal and hepatic first-pass metabolism. Testosterone undecanoate (TU), an orally administered ester prodrug of testosterone, is significantly subjected to first-pass metabolism. However, the individual contribution of intestinal and hepatic first-pass metabolism is not well determined. Therefore, the aim of the current study was to predict the metabolic contribution of each site. The hydrolysis–time profiles of TU incubation in human liver microsomes and Caco-2 cell homogenate were used to predict hepatic and intestinal first-pass metabolism, respectively. The in vitro half-life (t1/2 inv) for the hydrolysis of TU in microsomal mixtures was 28.31 ± 3.51 min. By applying the “well-stirred” model, the fraction of TU that could escape hepatic first-pass metabolism (FH) was predicted as 0.915 ± 0.009. The incubation of TU in Caco-2 cell homogenate yielded t1/2 inv of 109.28 ± 21.42 min, which was applied in a “Q gut” model to estimate the fraction of TU that would escape intestinal first-pass metabolism (FG) as 0.114 ± 0.02. Accordingly, only 11% of the absorbed fraction of TU could escape intestinal metabolism, while 91% can pass through hepatic metabolism. Hence, compared to the liver, the intestinal wall is the main site where TU is significantly metabolised during first-pass effect.

Chemical Properties of Human Amniotic Membrane for Potential Opthalmological Use

Human amniotic membrane (hAM) allows the exchange of water and organic and inorganic substances between the amniotic fluid, the fetus, and the maternal circulation. Our paper is aimed at determining the chemical composition of the amniotic membrane and at comparing it with the chemical structure of the tears, in order to identify possible uses of the amniotic membrane in eye surgery. In order to determine the chemical composition of hAM we sampled 7 pieces of fresh amniotic membrane, which were processed to create cell homogenate. The chemical tests run on the amniotic membrane specimens in our research revealed the following values: mean glucose concentration = 3mg/100mL and mean total protein concentration = 0.07g/100mL; the electrolyte concentration was: Na+ =152 mEq/L; K+=5.74 mEq/L; Cl- =131.6 mEq/L, and pH=7.2, whereas the total antioxidant capacity (TAC) = 1.1�0.1 mmol/L. These values are similar to those determined by other authors for the amniotic membrane, which resemble those reported for the chemical composition of tear and aqueous humor. To conclude with, the human amniotic membrane is a useful biological material in ophthalmological transplant.

Molecular mechanisms involved in the adaptive regulation of the colonic thiamin pyrophosphate uptake process

A considerable amount of the thiamin generated by gut microbiota exists in the form of thiamin pyrophosphate (TPP). We have previously shown that human colonocytes possess an efficient carrier-mediated uptake process for TPP that involves the SLC44A4 system and this uptake process is adaptively regulated by prevailing extracellular TPP level. Little is known about the molecular mechanisms that mediate this adaptive regulation. We addressed this issue using human-derived colonic epithelial NCM460 cells and mouse colonoids as models. Maintaining NCM460 cells in the presence of a high level of TPP (1 mM) for short (2 days)- and long-term (9 days) periods was found to lead to a significant reduction in [3H] TPP uptake compared with cells maintained in its absence. Short-term exposure showed no changes in level of expression of SLC44A4 protein in total cell homogenate (although there was a decreased expression in the membrane fraction), mRNA, and promoter activity. However, a significant reduction in the level of expression of the SLC44A4 protein, mRNA, and promoter activity was observed upon long-term maintenance with the substrate. Similar changes in Slc44a4 mRNA expression were observed when mouse colonoids were maintained with TPP for short- and long-term periods. Expression of the transcription factors ELF3 and CREB-1 (which drive the SLC44A4 promoter) following long-term exposure was unchanged, but their binding affinity to the promoter was decreased and specific histone modifications were also observed. These studies demonstrate that, depending on the period of exposure, different mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level.