Effects Of Various Inducers Research Articles

Poly(l-lactide)-Degrading Enzyme Production by Laceyella sacchari LP175 Under Solid State Fermentation Using Low Cost Agricultural Crops and Its Hydrolysis of Poly(l-lactide) Film

The aim of this study was to investigate the enhancement of poly(l-lactide) (PLLA)-degrading enzyme production by Laceyella sacchari LP175 under solid state fermentation (SSF) using low-cost agricultural crops as substrates and its hydrolysis of poly(l-lactide) film. Cassava chip, soybean meal and corncob were used to investigate the enzyme production using a statistical mixture design method. The effect of various inducers and SSF growth conditions parameters affecting PLLA-degrading enzyme production were also investigated. The fermentation of solid substrates was up-scale in a static tray bioreactor. The crude enzyme extracted from the fermented solid substrate was used to evaluate the biological degradation of PLLA film at 50 °C for 24 h. The results of substrate combination showed that 5 g of the substrate mixture, consisting of 4.3 g cassava chips and 0.7 g soybean meal, yielded 320 U/g dry solid. The addition of 0.1 g peptone to the mixture of solid materials increased enzyme production up to 456 U/g dry solid. The production of enzyme in a static tray bioreactor with optimized physical conditions yielded the maximum enzyme production, 472 U/g dry solid. The 20 mL reaction consisting of 8600 mg/L PLLA film was degraded by the crude enzyme extracted from the fermented solid substrate. Agricultural crops and wastes contain significant amounts of nutrients for microbial growth and products. This is the first reported of PLLA degrading enzyme production under SSF using low-cost substrates showing the possibility for application in large-scale biological recycling of bio-plastic as a future sustainable process.

The effect of various inducers and their combinations with copper on laccaseproduction of Trametes versicolor pellets in a repeated-batch process

The aim of this study was to increase laccase production in Trametes versicolor ATCC 200801 pellets by using various inducers and their combinations under repeated-batch conditions. Because copper is an effective inducer for laccase production, the effect of Cu on laccase production in this strain was tested first. The optimal Cu concentration for the highest laccase production was 0.5 mM. Following this preliminary study, the effect of other inducers [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), syringaldazine, guaiacol, and 2,5-xylidine] on laccase production was determined. Copper was determined to be the most efficient inducer among the inducers used. Therefore, the synergistic effect of each inducer with Cu on laccase production in this strain was investigated. While the maximum laccase activity was 0.60 U/mL in stock basal medium (SBM) alone, the highest enzyme activities detected were 4.76 and 2.87 U/mL in SBM + 0.05 mM ABTS and SBM + 1 mM 2,5-xylidine, respectively. Maximum laccase activities obtained from the combination of the inducers were 33.61 and 26.49 U/mL in SBM + 0.5 mM Cu + 0.5 mM 2,5-xylidine and SBM + 0.5 mM Cu + 0.05 mM ABTS, respectively. These were the most efficient combinations for laccase production.

Synthesis, evaluation, and metabolism of novel [6]-shogaol derivatives as potent Nrf2 activators

Oxidative stress is a central component of many chronic diseases. The Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2 p45-related factor 2 (Nrf2) system is a major regulatory pathway of cytoprotective genes against oxidative and electrophilic stress. Activation of the Nrf2 pathway plays crucial roles in the chemopreventive effects of various inducers. In this study, we developed a novel class of potent Nrf2 activators derived from ginger compound, [6]-shogaol (6S), using the Tg[glutathione S-transferase pi 1 (gstp1):green fluorescent protein (GFP)] transgenic zebrafish model. Investigation of structure–activity relationships of 6S derivatives indicates that the combination of an α,β-unsaturated carbonyl entity and a catechol moiety in one compound enhances the Tg(gstp1:GFP) fluorescence signal in zebrafish embryos. Chemical reaction and in vivo metabolism studies of the four most potent 6S derivatives showed that both α,β-unsaturated carbonyl entity and catechol moiety act as major active groups for conjugation with the sulfhydryl groups of the cysteine residues. In addition, we further demonstrated that 6S derivatives increased the expression of Nrf2 downstream target, heme oxygenase-1, in both a dose- and time-dependent manner. These results suggest that α,β-unsaturated carbonyl entity and catechol moiety of 6S derivatives may react with the cysteine residues of Keap1, disrupting the Keap1–Nrf2 complex, thereby liberating and activating Nrf2. Our findings of natural product-derived Nrf2 activators lead to design options of potent Nrf2 activators for further optimization.

Effect of various physical parameters for the Production of the enzyme Xylanase from mixed culture of Bacillus polymyxa and Cellulomonas uda

In the last decade, Xylanases have extended their use from the paper and pulp industries to newer needs such as biofuel production. The enzyme xylanase was produced by utilizing cheaper substrates like grass extract and sugarcane sheath leaf extract supplanted with nutrient sources in Submerged Fermentation using a mixed culture of Bacillus polymyxa and Cellulomonas uda. A fermentation time of 6 days was required to obtain maximum xylanase activity.High activities of enzyme (14.662 IU/ml) were obtained with sugarcane sheath leaf extract as the major carbon source. The optimum pH and temperature for maximum production of xylanase were found to be 8 and 50°C respectively. Additionally the effect of various inducers were also studied and it was found that xylan(0.2%) could induce xylanase production resulting in an enzyme activity of 44.773 IU/ml.

Metabolism in Vitro of 3,4,3',4'- and 2,5,2',5'-Tetrachlorobiphenyl by Rat Liver Microsomes and Highly Purified Cytochrome P-450.

Metabolism of two polychlorinated biphenyls, 3,4,3',4'- and 2,5,2',5'-tetrachlorobiphenyl (TCB), was studied using rat liver microsomes and the four forms of cytochrome P-450 (P-450), P-450b, P-450e, P-450c and P-450d. At first, effects of various inducers of P-450 such as phenobarbital (PB), 3-methylcholanthrene (MC), isosafrole (ISF) and pregnenolone 16 alpha-carbonitrile on the formation of metabolites of these TCBs by liver microsomes were compared. 3,4,3',4'-TCB was significantly metabolized by liver microsomes from MC-treated rats to form two previously reported metabolites, 4-hydroxy-3,5,3',4'-TCB and 5-hydroxy-3,4,3',4'-TCB with a relative ratio of 2.5:1. Incubation with microsomes from untreated or PB-treated rats produced none of the metabolites. On the other hand, 2,5,2',5'-TCB was metabolized to 3-hydroxy-2,5,2',5'-TCB most easily by liver microsomes from PB-treated rats and at a moderate rate by liver microsomes from ISF-treated rats. Activities of microsomes from untreated or MC-treated rats to hydroxylate 2,5,2',5'-TCB were low or undetectable. When these TCB hydroxylase activities were examined with a reconstituted system consisting of each P-450, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, dilauroylphosphatidylcholine and NADPH-generating system, only P-450c catalyzed both the 4- and 5-hydroxylations of 3,4,3',4'-TCB at a ratio of 2.2:1. On the contrary, the hydroxylation of 2,5,2',5'-TCB proceeded efficiently with P-450b and P-450e, being more efficient with the former. P-450d did not show any catalytic activity toward 3,4,3',4'-TCB and 2,5,2',5'-TCB.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b 5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b 5. 7,8-BF pretreatment increased 7-ethoxyresorufin- O-deethylase (EROD) 3-fold and 7-pentoxy-resorufin- O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin- O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant K d is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The K d -value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.

Effects of various inducers on the expression of cytochromes P-450 IIC8, 9, 10 and IIIA in cultured adult human hepatocytes

Primary cultures of adult human hepatocytes were used to determine the capability of the human liver to respond to phenobarbital, 3-methylcholanthrene, troleandomycin and rifampicin, four compounds known to be potent inducers of hepatic cytochrome P-450 in various animal species. Both mRNA and corresponding protein of two major isoenzymes, that is, P-450 IIC8, 9, 10 and P-450 IIIA, were measured after daily exposure to the drugs for 3 days. Phenobarbital and rifampicin were found to increase the levels of P-450 IIC8, 9, 10 mRNA and protein while troleandomycin and 3-methylcholanthrene were ineffective. Different effects were obtained for P-450 IIIA. Both mRNA and related protein were markedly increased by troleandomycin and rifampicin and decreased by 3-methylcholanthrene. mRNAs were slightly increased by phenobarbital. The results demonstrate that human hepatocytes retain drug-inducible P-450 isoenzymes in primary culture and represent a unique approach to investigate regulation of human liver drug metabolizing enzymes.

Comparison of the effects of various inducers on 7-alkoxycoumarin O-dealkylase activities in liver microsomes.

The effects of six inducers and malotilate on 7-alkoxycoumarin O-dealkylase activities in rat liver microsomes were examined. Phenobarbital (PB) (100 mg/kg) was administered intraperitoneally to rats for 6 days; 3-methylcholanthrene (3-MC) (40 mg/kg), beta-naphthoflavone (beta-NF) (40 mg/kg), isosafrole (150 mg/kg) and polychlorinated biphenyls (PCB) (100 mg/kg) were administered intraperitoneally for 3 days; isoniazid (INH) (50 mg/kg) was administered intraperitoneally for 10 days; and malotilate (500 mg/kg) was administered orally for 3 days. The O-dealkylase activities toward 7-methoxycoumarin (7-MC), 7-ethoxycoumarin (7-EC) and 7-propoxycoumarin (7-PC) were examined 24 hr after the final administration of the drugs. The ratios of 7-EC O-deethylase and 7-PC O-depropylase to 7-MC O-demethylase activity in the control and six inducer-treated groups were compared. The ratios in the groups treated with the six compounds, each of which induces a different form(s) of cytochrome P-450 (P-450), were clearly different from each other. Therefore, the measurement of 7-alkoxycoumarin O-dealkylase activities should be extremely useful for the routine determination of the molecular species of P-450. On the other hand, the ratio in the malotilate-treated group was different from that in any other inducer-treated group, so that there might be a possibility that malotilate induced a form(s) of P-450 that is different from any of the already known species.

Comparison of the Effects of Various Inducers on 7-Alkoxycoumarin O-Dealkylase Activities in Liver Microsomes

The effects of six inducers and malotilate on 7-alkoxycoumarin O-dealkylase activities in rat liver microsomes were examined. Phenobarbital (PB) (100 mg/kg) was administered intraperitoneally to rats for 6 days; 3-methylcholan-threne (3-MC) (40 mg/kg), β-naphthoflavone (β-NF) (40 mg/kg), isosafrole (150 mg/kg) and polychlorinated biphenyls (PCB) (100 mg/kg) were administered intraperitoneally for 3 days; isoniazid (INH) (50 mg/kg) was administered intraperitoneally for 10 days; and malotilate (500 mg/kg) was administered orally for 3 days. The O-dealkylase activities toward 7-methoxycoumarin (7-MC), 7-ethoxycoumarin (7-EC) and 7-propoxycoumarin (7-PC) were examined 24 hr after the final administration of the drugs. The ratios of 7-EC O-deethylase and 7-PC O-depropylase to 7-MC O-demethylase activity in the control and six inducer-treated groups were compared. The ratios in the groups treated with the six compounds, each of which induces a different form(s) of cytochrome P-450 (P-450), were clearly different from each other. Therefore, the measurement of 7-alkoxycoumarin O-dealkylase activities should be extremely useful for the routine determination of the molecular species of P-450. On the other hand, the ratio in the malotilate-treated group was different from that in any other inducer-treated group, so that there might be a possibility that malotilate induced a form(s) of P-450 that is different from any of the already known species.

Effects of various inducers on the activities of cytochrome P-450-mixed function oxidases and aflatoxin B1 activation in microsomes of hamster livers.

The effects of various inducers on the activities of drug-metabolizing enzymes including aflatoxin B1 activation were studied in Syrian golden hamsters. Activity for aflatoxin B1 was determined by aflatoxin B1-DNA adducts formation. The treatments of hamsters with 3-methylcholanthrene, alpha-naphthoflavone and benzo(a)pyrene elevated markedly the activity for aflatoxin B1 by 2460, 1380 and 450%, respectively. Phenobarbital induced slightly and isosafrole and ethanol did not induce the activity for aflatoxin B1. Pregnenolone-16 alpha-carbonitrile decreased aflatoxin B1 activation to 51% of that of the non-treated animals. These results were in good accordance with the induction rate of a form of cytochrome P-450 (P-450AFB) which has potent activity to aflatoxin B1. Characteristics in the induction of mixed function oxidases of hamsters by these inducers, especially in respect to benzo(a) pyrene metabolizing enzyme, seemed to differ from those of rats. These results suggest that the activity for aflatoxin B1 in hamster is inducible by 3-methylcholanthrene-type inducers and that hamster is a suitable animals model to study the mechanism of aflatoxin B1-hepatocarcinogenesis.

Effects of various pretreatments on the acute nephrotoxic potential of styrene in fischer-344 rats

The effects of various inducers and inhibitors of hepatic microsomal mixed-function oxidase (MFO) system and diethylmaleate treatment on styrene-induced acute nephrotoxicity in male Fischer-344 rats were studied. Groups of rats were pretreated with either 3-methylcholanthrene (15 mg/kg, i.p., 3 days), or phenobartial (80 mg/kg, i.p., 3 days), or SKF525-A (50 mg/kg, i.p., 1 h), or piperonyl butoxide (300 mg/kg, i.p., 2 h), or diethylmaleate (400 mg/kg, i.p., 90 min) prior to an i.p. administration of styrene (0, 0.6 and 0.9 g/kg) in corn oil. The uptake of p- aminohippurate (PAH) by renal cortical slices, the morphology of renal cortices, as well as urinary excretion of N- acetyl-β- D-glucosaminidase (NAG) and γ-glutamyl transpeptidase (γ-GT) of control and pretreated rats were examined 24 h after styrene. The inducers and inhibitors of MFO system failed to modify further the acute nephrotoxicity of styrene. On the other hand, diethylmaleate pretreatment not only reduced further the uptake of PAH, but also produced further significant increase in the urinary excretion of NAG and γ-GT observed at the higher dose of styrene. Similarly, ultrastructural studies showed a moderate increase in the severity of kidney damage induced at the higher dose of styrene due to pretreatment with diethylmaleate. These data suggest that tissue glutathione concentrations and hence, corresponding conjugating activity might be important determinants of styrene nephrotoxicity The results further indicate that a metabolic activation system not involving certain cytochrome P-450 might be responsible in styrene-induced nephrotoxicity.

In vitro metabolism of diethylstilbestrol by hepatic, renal and uterine microsomes of rats and hamsters : Effects of different inducers

In order to elucidate possible differences in the metabolism of the synthetic estrogen diethylstilbestrol (DES) by target and non-target tissues for DES carcinogenicity, the biotransformation of [ 14C]DES has been studied in vitro with hepatic and renal microsomes of male and female hamsters and rats, and from hamster and rat uterus. Of these tissues, only the male hamster kidney is susceptible to the carcinogenic effect of DES. Moreover, the effect of various inducers on the in vitro metabolism of DES has been investigated. It was found that male hamster kidney microsomes produced a markedly different pattern of DES metabolites as compared to renal microsomes from female hamster or male and female rats. Pretreatment with phenobarbital markedly increased oxidative DES metabolism by renal microsomes from female rat but not from male rat. Diethylstilbestrol metabolism by hepatic microsomes was different between hamster and rat, but was not sex-dependent and could not be significantly affected by pretreatment with phenobarbital, DES, 3-methylcholanthrene and 7:8-benzoflavone. The differences in DES metabolism between target and non-target organs and its modulation by inducers may help to gain further insight into the mechanism of DES tumorigenesis.

The effect of induction of hemoglobin synthesis in cultured friend cells on the number of initiation sites for replication and transcription

It was found that differentiation of murine erythroleukemia cells can be induced by 5-fluorodeoxyuridine (FudR), amethopterin and α-aminoisobutyrate. Each of these compounds is believed to delay the onset of DNA synthesis. Since relief of the FudR block to DNA synthesis by addition of thymidine can increase the number of initiation sites for replication (Taylor, 1977), the effect of various inducers and inhibitors of differentiation of Friend cells upon the relative number of initiation sites for replication and transcription was investigated. Very efficient inducers of hemoglobin synthesis, hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (DMSO), increase the number of initiation sites for transcription and HMBA also increases the number of functional initiation sites for replication. Two other compounds that induce differentiation of Friend cells, low levels of actinomycin D and FudR, did not increase the number of initiation sites for transcription. Compounds that prevent induction of hemoglobin synthesis by HMBA and DMSO include 5-bromodeoxyuridine (BrdU) and novobiocin. Both of these compounds were found to decrease the number of functional initiation sites for transcription and it is known that both compounds reduce the number of initiation sites for replication. The relation between initiation of replication and transcription, and its effect upon differentiation of erythroleukemia cells is discussed.

In vitro covalent binding of acrylonitrile to rat liver proteins

The irreversible protein binding of [3- 14C]acrylonitrile (ACN) was evaluated in the presence of microsomes, cytosolic and post-mitochondrial fractions of rat liver. Spontaneous binding was measured after incubation of ACN in the presence of the various liver fractions at 4°C and 37°C respectively and in a comparative study performed at 37°C in the presence of S9 or bovine serum albumin (BSA). The effect of various inducers and inhibitors was investigated. A spontaneous binding resulting from a direct alkylation by ACN seemed to play an important role however, binding resulting from the microsomal activation of ACN into reactive intermediate(s) was also detected.

Covalent binding in vitro of polychlorinated biphenyls to microsomal macromolecules: Involvement of metabolic activation by a cytochrome P-450-linked mono-oxygenase system

Incubation of 14C-labeled polychlorinated biphenyls (PCBs) with rat, mouse or rabbit liver microsomes in the presence of an NADPH-generating system and molecular oxygen caused covalent binding of radioactive metabolites of PCBs to microsomal macromolecules. The binding was more pronounced with liver microsomes from animals pretreated with inducers of the microsomal mono-oxygenase system. The order of induction effect of the inducers used was KC-500 (a PCB preparation containing 55% chlorine) ≥ phenobarbital (PB) ⪢ 3-methylcholanthrene (3-MC) in rats, PB > KC-500 > 3-MC in mice, and PB > KC-500 in rabbits. [ 14C]KC-300 (a PCB preparation containing 42% chlorine) was more effective than [ 14C]KC-500 as substrate for all the microsomal preparations. The binding reaction was dependent on both NADPH and oxygen, sensitive to carbon monoxide, glutathione, cysteine, hexobarbital, and aniline, and enhanced by EDTA, which inhibits lipid peroxidation. The addition of NADH, which was by itself a very poor electron donor, caused a synergistic increase of the NADPH-dependent binding of PCBs. It is concluded that the conversion of PCBs to active metabolites by the cytochrome P-450-linked mono-oxygenase system is prerequisite to the binding reaction. A survey of the effects of various inducers suggested that a cytochrome P-450 having a high aminopyrine N-demethylation activity is mainly responsible for the metabolic activation of PCBs in liver microsomes. Kidney and lung microsomes from untreated rats were virtually devoid of the PCB-binding capacity, but in kidney microsomes this capacity could be induced by pretreatment with 3-MC or KC-500, though not with PB.

Effect of various inducers on electron transport system associated with drug metabolism by liver microsomes

The metabolism of a wide variety of drugs and other compounds is catalyzed by liver microsomal enzyme systems involving cytochrome P-450 and NADPH-cytochrome c reductase. The sequence of events in these reactions is presumably as follows: The drug substrate reacts with the oxidized form of cytochrome P-450 to form a complex which is reduced either directly by NADPH-cytochrome c reductase or indirectly via an unidentified electron carrier. The reduced cytochrome P-450-substrate complex then reacts with oxygen to form an “active oxygen” complex, which decomposes with the formation of the oxidized drug and oxidized cytochrome P-450. Since the rate of reduction of the cytochrome P-450-substrate is considerably slower than the rate of oxidation of the complex under steady-state conditions, most of the cytochrome P-450-substrate complex is in the oxidized form. The rate of electron flux through the system, however, may be modified by altering the amount of cytochrome P-450-substrate complex, or the rate at which it is reduced. Inducers of drug metabolism may be classified according to their effects on the various components of the system. Phenobarbital-like inducers cause increases in NADPH-cytochrome c reductase and cytochrome P-450, and thus increases in the amount of cytochrome P-450-substrate complex and its rate of reduction. Polycyclic hydrocarbons, such as methylcholanthrene, induce the formation of a variant of cytochrome P-450, which has different affinities for the various drug substrates than does the usual form. This kind of inducer does not increase either NADPH-cytochrome c reductase or the rate of cytochrome P-450 reduction; indeed in some experiments they decrease the rate of cytochrome P-450 reduction per cytochrome P-450. In female rats, spironolactone and perhaps other anabolic steroids increase NADPH-cytochrome c reductase activity and the rate of cytochrome P-450-substrate reduction, but have little or no effect on the amount of cytochrome P-450. The relationship between the electron flux and drug metabolism in liver microsomes depends on the substrate. For example, the demethylation of ethylmorphine and NADPH oxidation (or cytochrome P-450 reduction) approaches the theoretical 1:1 relationship, whereas, the rate of hydroxylation of hexobarbital may be considerably less than the substrate induced rate of NADPH oxidation (and cytochrome P-450 reduction).

Induction and Inhibition of In Vivo Glucuronidation of Apomorphine in Mice

The effect of various inducers and inhibitors of drug-metabolizing enzyme systems was studied on the kinetics of apomorphine disappearance and of the appearance of apomorphine glucuronides. The generally claimed inducers, phenobarbital and testosterone, and inhibitors, β-diethylaminoethyl diphenylpropyl-acetate and actinomycin D, were found to have no significant effect on the metabolism of apomorphine. Estradiol, however, produced a significant inhibition of the metabolism. Apomorphine pretreatment for 3 days exhibited a definite induction of its own metabolism.