Detection and relative quantification of amine oxidase gene (yobN) in Bacillus subtilis: application of real-time quantitative PCR.

Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we design two sets of primers for the detection and quantification of the amine oxidase gene (yobN) and endogenous (housekeeping) gene (gyrB) in Bacillus subtilis. Moreover, these sets can be used for relative quantification of yobN by real-time PCR (qPCR). We also tested the degradation of BAs by three bacterial strains (B. subtilis strains: IB1a, CCM 2216, CCM 2267) in a mineral medium over a two-day period. Their degradation abilities were verified by high performance liquid chromatography with UV detection (HPLC/UV). According to the results, two strains significantly (P < 0.05) reduced histamine, tyramine, putrescine, and cadaverine. Moreover, our results indicate that the degradation ability of B. subtilis strains could be limited by sporulation because the gene encoding amine oxidase (yobN) is no longer expressed in the spores.

Application of qPCR for multicopper oxidase gene (MCO) in biogenic amines degradation by Lactobacillus casei.

Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we designed two sets of primers for the detection and quantification of the multicopper oxidase gene (MCO), which encodes an enzyme involved in BAs degradation, and endogenous (glyceraldehyde-3-phosphate dehydrogenase) gene (GAPDH) in Lactobacillus casei group by real-time PCR (qPCR). We tested 15 Lactobacillus strains in the screening assays (thus, MCO gene possessing assay (PCR) and monitoring of BAs degradation by HPLC-UV), in which Lactobacillus casei CCDM 198 exhibited the best degradation abilities. For this strain, we monitored the expression of the target gene (MCO) in time (qPCR), the effect of redox treatments (cysteine, ascorbic acid) on the expression of the gene, and the ability to degrade BAs not only in a modified MRS medium (MRS/2) but also in a real food sample (milk). Moreover, decarboxylase activity (ability to form BAs) of this strain was excluded. According to the results, CCDM 198 significantly (P < 0.05) reduced BAs (putrescine, histamine, tyramine, cadaverine), up to 25% decline in 48 h. The highest level of relative expression of MCO (5.21 ± 0.14) was achieved in MRS/2 media with cysteine.

Occurrence of Biogenic Amines Producers in the Wastewater of the Dairy Industry.

Out of six samples of wastewater produced in the dairy industry, taken in 2017 at various places of dairy operations, 86 bacterial strains showing decarboxylase activity were isolated. From the wastewater samples, the species of genera Staphylococcus, Lactococcus, Enterococcus, Microbacterium, Kocuria, Acinetobacter, Pseudomonas, Aeromonas, Klebsiella and Enterobacter were identified by the MALDI-TOF MS and biochemical methods. The in vitro produced quantity of eight biogenic amines (BAs) was detected by the HPLC/UV-Vis method. All the isolated bacteria were able to produce four to eight BAs. Tyramine, putrescine and cadaverine belonged to the most frequently produced BAs. Of the isolated bacteria, 41% were able to produce BAs in amounts >100 mg L-1. Therefore, wastewater embodies a potential vector of transmission of decarboxylase positive microorganisms, which should be taken into consideration in hazard analyses within foodstuff safety control. The parameters of this wastewater (contents of nitrites, nitrates, phosphates, and proteins) were also monitored.

N-methyl-2-pyrrolidone-degrading bacteria from activated sludge.

N-methyl-2-pyrrolidone (NMP) is a widely used solvent for many organic compounds and a component found in a vast array of chemical preparations. For this research paper, NMP degrading bacteria were isolated from two samples of activated sludge. They pertained to both Gram-negative and Gram-positive members, and belong to the Pseudomonas, Paracoccus, Acinetobacter and Rhodococcus genera. All the strains utilized 300 mg/L of NMP as the only source of carbon, energy and nitrogen over several days, and they were shown to additionally be able to degrade N-acetylphenylalanine (NAP). The growth of all the isolated strains was recorded at different NMP concentrations, to a maximum of 20 g/L.

Effect of Selected Factors Influencing Biogenic Amines Degradation by Bacillus subtilis Isolated from Food.

Modern food technology research has researched possible approaches to reducing the concentration of biogenic amines in food and thereby enhance and guarantee food safety. Applying adjunct cultures that can metabolise biogenic amines is a potential approach to reach the latter mentioned goal. Therefore, this study aims to study the crucial factors that could determine the decrease in biogenic amines concentration (histamine, tyramine, phenylethylamine, putrescine and cadaverine) in foodstuffs using Bacillus subtilis DEPE IB1 isolated from gouda-type cheese. The combined effects of cultivation temperature (8 °C, 23 °C and 30 °C) and the initial pH of the medium (5.0, 6.0, 7.0 and 8.0) under aerobic and also anaerobic conditions resulted in the decrease of the tested biogenic amines concentration during the cultivation time (another factor tested). Bacillus subtilis was cultivated (in vitro) in a medium supplemented with biogenic amines, and their degradation was detected using the high-performance liquid chromatography equipped with UV-detector. The course of biogenic amines degradation by Bacillus subtilis DEPE IB1 was significantly influenced by cultivation temperature and also the initial pH of the medium (p < 0.05). At the end of the cultivation, the concentration of all of the monitored biogenic amines was significantly reduced by 65-85% (p < 0.05). Therefore, this strain could be used for preventive purposes and contributes to food safety enhance.

Effect of methotrexate on inflammatory cells redistribution in experimental adjuvant arthritis.

The aim of this study was to evaluate the morphological changes in the spleen, the thymus and the knee joints of rats with experimental adjuvant arthritis induced by Mycobacterium butyricum in the incomplete Freund's adjuvant and the effect of treatment with methotrexate (MTX). Particular attention was aimed on the redistribution of granulocytes in the tissues during the inflammatory process. Clinical parameters, e.g., joint edema, body weight and of gamma glutamyl transferase (GGT) activity as an inflammatory marker, have also been determined. Induction of adjuvant arthritis caused a significant decrease in granulocyte number in the spleen and vice versa a significant increase in the knee joints, but without significant changes in the thymus. Treatment with methotrexate reversed this phenomenon by increasing the granulocyte number in the spleen and decreasing it in knee joints. MTX decreased the joint edema as well as the activity of GGT in the spleen, modified the size of the white pulp of the spleen and increased the cortex/medulla ratio in the thymus. The observed changes support the anti-inflammatory and immunomodulatory properties of MTX supporting its use as the first-line medication in patients with rheumatoid arthritis.

Changes in the Quality Attributes of Selected Long-Life Food at Four Different Temperatures over Prolonged Storage.

This study reports the development of selected indicators affecting changes in food quality and safety of selected long-life canned (Szeged goulash, canned chicken meat, pork pâté, canned tuna fish) and dehydrated (instant goulash soup) food during a two-year storage experiment at four different temperatures. The storage temperatures were selected to represent Arctic (−18 °C), temperate (5 °C), subtropical (25 °C) and tropical (40 °C) climatic zones where such food is likely to be stored during, for example, humanitarian and military missions. Microorganism amounts below the detection limit (p < 0.05), regardless of the storage temperature (p ≥ 0.05), were monitored in canned samples. The contents of dry matter, fat and proteins did not change during storage, regardless of the storage temperature (p ≥ 0.05). During the 24-month storage, all food showed an increase in the level of ammonia (p < 0.05) and the TBARS-value (p < 0.05), whereas the rate of increase in both parameters was significantly higher at higher storage temperatures (p < 0.05). The losses of individual amino acids during storage ranged from 5% rel. calculated on the amino acid contents in Month "0" up to 15% rel. (p < 0.05). With storage temperatures above the freezing point, the hardness values decreased with the increase in the storage temperature (p < 0.05) and prolongation of the storage period (p < 0.05). Moreover, with temperatures of −18 °C, the development of hardness, measured as the "decrease rate", was significantly higher compared to the absolute values.

Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases.

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined. The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification. Hepatocytes and microsomes formed silybin A-7-O-ß-D-glucuronides, B-7-O-ß-D-glucuronides, A-20-O-ß-D-glucuronides and B-20-O-ß-D-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation. The predominant formation of 7-O-ß-D-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes.

SP600125, a specific inhibitor of c-Jun-N-Terminal kinase (JNK), was reported as a ligand and antagonist of aryl hydrocarbon receptor (AhR) [Joiakim A, Mathieu PA, Palermo C, Gasiewicz TA, Reiners Jr JJ. The Jun N terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor. Drug Metab Dispos 2003;31(11):1279-82]. Here we show that SP600125 is not an antagonist but a partial agonist of human AhR. SP600125 significantly induced CYP1A1 and CYP1A2 mRNAs in primary human hepatocytes and CYP1A1 mRNA in human hepatoma cells HepG2. This effect was abolished by resveratrol, an antagonist of AhR. Consistent with the recent report, SP600125 dose-dependently inhibited CYP1A1 and CYP1A2 genes induction by a prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human hepatocytes. Moreover, SP600125 displayed typical behavior of a partial agonist in HepG2 cells transiently transfected with a reporter plasmid containing two inverted repeats of the dioxin responsive element or with a plasmid containing 5'-flanking region of human CYP1A1 gene. SP600125 transactivated the reporter plasmids with EC(50) of 0.005 and 1.89 microM, respectively. On the other hand, TCDD-dependent transactivation of the reporter plasmids was inhibited by SP600125 with IC(50) values of 1.54 and 2.63 microM, respectively. We also tested, whether the effects of SP600125 are due to metabolism. Using liquid chromatography/mass spectrometry approach, we observed formation of two minor monohydroxylated metabolites of SP600125 in human hepatocytes, human liver microsomes but not in HepG2 cells. These data imply that biotransformation is not responsible for the effects of SP600125 on AhR signaling. In conclusion, we demonstrate that SP600125 is a partial agonist of human AhR, which induces CYP1A genes.

SB203580, a pharmacological inhibitor of p38 MAP kinase transduction pathway activates ERK and JNK MAP kinases in primary cultures of human hepatocytes.

Mitogen-activated protein kinases (MAPKs) were extensively studied in cancer-derived cell lines; however, studies in non-transformed human cells are scarce. In the current paper, we studied the effect of SB203580, a pharmacological inhibitor of p38 MAPK, on activation and inhibition of p38 MAPK transduction partway in primary human hepatocytes (in vitro model of differentiated cells) in comparison with several tumor cell lines (proliferating non-differentiated in vitro model). In addition, we analyzed the effect of SB203580 on extracellular-regulated protein kinase (ERK) and c-jun-N-terminal kinase (JNK) pathways both in primary human hepatocytes and tumor cell lines employing primary antibodies detecting phosphorylated kinases. We show that SB203580 activates ERK and JNK in primary cultures of human hepatocytes. The levels of ERK-P(Thr202/Tyr204), JNK-P(Thr183/Tyr185) and c-Jun-P(Ser63/73), a target down-stream protein of JNK, were increased by SB203580. In contrast, SB203580 activated ERK but not JNK in HepG2, HL-60, Saos-2 and HaCaT human cancer cell lines. We tested, whether the effects of SB203580 are due to metabolism. Using liquid chromatography/mass spectrometry, we found one minor metabolite in human liver microsomes but not in HepG2 cells. These data imply that biotransformation could be responsible for the effects of SB203580 in human hepatocytes. This study is the first report on the effects of MAPK activators (sorbitol, anisomycin, EGF) and MAPK inhibitors in primary human hepatocytes. We observed differential effects of these compounds in primary human hepatocytes and in cancer cells, implying the cell-type specificity and the essential differences between the role and function of MAPKs in normal and cancer cells.

Interspecies comparison of the glucuronidation processes in the man, monkey, pig, dog and rat.

OBJECTIVES: The study of interspecies differences in glucuronidation processes in the man, monkey, pig, dog and rat using liver microsomal fraction. The study is focused on determination of the enzyme activity of UGT1A6 (having also a toxicological importance) in microsomes of different species. METHODS: For determination of glucuronides formed, an HPLC method with UV detection and LC-MS characterization was used. p-Nitrophenol and 4-methylumbelliferon and silybin were chosen as model substrates. RESULTS: The data presented in this paper show an overall similarity in kinetic parameters of the UGT1A6 with p-nitrophenol and 4-methylumbelliferon for man, pig and monkey. The pattern of silybin glucuronides formed in monkey and dog samples are relatively close to this of the man. CONCLUSIONS: For studies of glucuronidation of xenobiotics where the role UGT1A6 is expected, the use of pig and monkey microsomes should be considered. As an optimal model for study of silybin glucuronidation, both the rhesus monkey and dog (Beagle) seem to be the best models. To elucidate the role of the UGT forms involved in metabolism of silybin, the experiments with recombinant UGT enzymes are needed.

Accelerated Biodegradation of Agriculture Film Based on Aromatic-Aliphatic Copolyester in Soil under Mesophilic Conditions.

A study was conducted on the biodegradation of aromatic-aliphatic copolyester-based agricultural film in soil at 25 °C. The polymer is known to be biodegradable under composting conditions although rather recalcitrant under mesophilic conditions. The material investigated comprised of the copolyester filled with approximately 25% of starch containing biodegradable plasticizers, and its behavior was compared to the corresponding material without the filler. Mineralization followed by CO2 production merely reached the point of about 6% after 100 days of incubation in the pure copolyester film, whereas the value of around 53% was recorded for the filled copolyester film, which exceeded the readily biodegradable starch filler content in the material by more than 20% and could be accounted for biodegradation of the copolyester. It was suggested that the accelerated copolyester biodegradation in the starch-filled material was most likely explained by the increase in the active surface area of the material available for the microbial attack after biodegradation of the filler. The results were supported by changes in molecular weight distributions of the copolyester and observations made by several microscopic techniques. These findings encourage further development of biodegradable agricultural films based on this material.

Phase II drug metabolizing enzymes.

BACKGROUND: Phase II biotransformation reactions (also 'conjugation reactions') generally serve as a detoxifying step in drug metabolism. Phase II drug metabolising enzymes are mainly transferases. This review covers the major phase II enzymes: UDP-glucuronosyltransferases, sulfotransferases, N-acetyltransferases, glutathione S-transferases and methyltransferases (mainly thiopurine S-methyl transferase and catechol O-methyl transferase). The focus is on the presence of various forms, on tissue and cellular distribution, on the respective substrates, on genetic polymorphism and finally on the interspecies differences in these enzymes. METHODS AND RESULTS: A literature search using the following databases PubMed, Science Direct and EBSCO for the years, 1969-2010. CONCLUSIONS: Phase II drug metabolizing enzymes play an important role in biotransformation of endogenous compounds and xenobiotics to more easily excretable forms as well as in the metabolic inactivation of pharmacologically active compounds. Reduced metabolising capacity of Phase II enzymes can lead to toxic effects of clinically used drugs. Gene polymorphism/ lack of these enzymes may often play a role in several forms of cancer.

Effect of acetylcholinesterase oxime-type reactivators K-48 and HI-6 on human liver microsomal cytochromes P450 in vitro.

Substances K-48 and HI-6, oxime-type acetylcholinesterase (AChE) reactivators, were tested for their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The compounds were shown to bind to microsomal cytochromes P450 with spectral binding constants of 0.25+/-0.05 microM (K-48) and 0.54+/-0.15 microM (HI-6). To find which cytochrome P450 from the human liver microsomal fraction interacts with these compounds, an inhibition of enzyme activities specific for nine individual CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) was studied. The results have shown no prominent inhibition of individual CYP activities with both compounds except the CYP2E1 activity and the HI-6 reactivator. However, the inhibition of this activity was less than 50% which makes the possible drug interactions highly unlikely. Hence, the interaction of K-48 and HI-6 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be clinically significant and both compounds could be taken in this respect as antidotal drugs with low risk of drug interactions.

Silybin is metabolized by cytochrome P450 2C8 in vitro.

Silybin (a flavonolignan, the main component of silymarin, an extract from the seeds of Silybum marianum) has been used to date mostly as a hepatoprotectant. However, it also has other interesting activities, e.g., anticancer and hypocholesterolemic effects. It is also known that silybin can inhibit the activities of the cytochrome P450 (P450) enzymes. In this study, a weak interaction of silybin with human microsomal CYP2E1, 2A6, 2B6, 2C19, and 2D6 (IC(50) > or = 250 microM) was found; a moderate inhibition was observed for CYP1A2 and 2C8. The most prominent inhibition effect was found with CYP3A4 and CYP2C9 (IC(50) < or = 50 microM). Using mass spectometry detection, production of O-demethylated (the main metabolite) as well as hydroxylated derivatives of silybin formed by P450 enzymes was detected. The effect of different P450 inhibitors on the formation of O-demethylated product was also studied. In particular, a relatively specific inhibitor of CYP2C8 (quercetin) markedly inhibited the formation of this metabolite. With the help of recombinant enzymes (bactosomes), it was confirmed that the CYP2C8 enzyme is responsible for the reaction leading to O-demethylated silybin.