Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox / 中国中药杂志

Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.

Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process

ABSTRACT Pyrene and benzo[a]pyrene (BaP) are high molecular weight polycyclic aromatic hydrocarbons (PAHs) recalcitrant to microbial attack. Although studies related to the microbial degradation of PAHs have been carried out in the last decades, little is known about degradation of these environmental pollutants by fungi from marine origin. Therefore, this study aimed to select one PAHs degrader among three marine-derived basidiomycete fungi and to study its pyrene detoxification/degradation. Marasmiellus sp. CBMAI 1062 showed higher levels of pyrene and BaP degradation and was subjected to studies related to pyrene degradation optimization using experimental design, acute toxicity, organic carbon removal (TOC), and metabolite evaluation. The experimental design resulted in an efficient pyrene degradation, reducing the experiment time while the PAH concentration applied in the assays was increased. The selected fungus was able to degrade almost 100% of pyrene (0.08 mg mL-1) after 48 h of incubation under saline condition, without generating toxic compounds and with a TOC reduction of 17%. Intermediate metabolites of pyrene degradation were identified, suggesting that the fungus degraded the compound via the cytochrome P450 system and epoxide hydrolases. These results highlight the relevance of marine-derived fungi in the field of PAH bioremediation, adding value to the blue biotechnology.

The use of aromatase inhibitors in boys with short stature: what to know before prescribing?

ABSTRACT Aromatase is a cytochrome P450 enzyme (CYP19A1 isoform) able to catalyze the conversion of androgens to estrogens. The aromatase gene mutations highlighted the action of estrogen as one of the main regulators of bone maturation and closure of bone plate. The use of aromatase inhibitors (AI) in boys with short stature has showed its capability to improve the predicted final height. Anastrozole (ANZ) and letrozole (LTZ) are nonsteroidal inhibitors able to bind reversibly to the heme group of cytochrome P450. In this review, we describe the pharmacokinetic profile of both drugs, discussing possible drug interactions between ANZ and LTZ with other drugs. AIs are triazolic compounds that can induce or suppress cytochrome P450 enzymes, interfering with metabolism of other compounds. Hydroxilation, N-dealkylation and glucoronidation are involved in the metabolism of AIs. Drug interactions can occur with azole antifungals, such as ketoconazole, by inhibiting CYP3A4 and by reducing the clearance of AIs. Antiepileptic drugs (lamotrigine, phenobarbital, and phenytoin) also inhibit aromatase. Concomitant use of phenobarbital or valproate has a synergistic effect on aromatase inhibition. Therefore, it is important to understand the pharmacokinetics of AIs, recognizing and avoiding possible drug interactions and offering a safer prescription profile of this class of aromatase inhibitors. Arch Endocrinol Metab. 2017;61(3):391-7.

Over-expression of CYP78A98, a cytochrome P450 gene from Jatropha curcas L, increases seed size of transgenic tobacco

Background: Jatropha curcas L. (further referred to as Jatropha), as a rapidly emerging biofuel crop, has attracted worldwide interest. However, Jatropha is still an undomesticated plant, the true potential of this shrub has not yet been fully realized. To explore the potential of Jatropha, breeding and domestication are needed. Seed size is one of the most important traits of seed yield and has been selected since the beginning of agriculture. Increasing the seed size is a main goal of Jatropha domestication for increasing the seed yield, but the genetic regulation of seed size in Jatropha has not been fully understood. Results: We cloned CYP78A98 gene from Jatropha,a homologue of CYP78A5 in Arabidopsis.Wefound that CYP78A98 was highly expressed in male flower, female flower, stem apex, leaf and developing seed. However, its transcripts were hardly detected in root and stem. CYP78A98 protein localized in endoplasmic reticulum (ER) and the hydrophobic domain at the N-terminus was essential for the correct protein localization. Furthermore, INNER NO OUTER promoter (pINO) drove specific overexpression of CYP78A98 in transgenic tobacco seeds resulted in increased seed size and weight, as well as improved seed protein and fatty acid content. Conclusions: The results indicated that CYP78A98 played a role in Jatropha seed size control. This may help us to better understand the genetic regulation of Jatropha seed development, and accelerate the breeding progress of Jatropha.

Research Progress on Abused Drugs Metabolic in vivo / 法医学杂志

Under the catalysis of a variety of metabolic enzymes in vivo, such as UDP-glucuronyl transferases, cytochrome P450, carboxylesterase, sulfotransferase, butyrylcholinesterase, catechol-O-methyl transferase and 6-morphine dehydrogenase, the drugs perform glucuronidation, hydrolysis, oxidation, sulfonation and other reactions, then translate into active or inactive metabolites, which are excreted through urination, bile or the other pathways at last. Different drugs own their different metabolic pathways. This paper introduces the studies about the metabolism of drugs in human and animal in recent years, such as morphine-like drugs, amphetamine, ketamine, cannabis and cocaine, and reviews the research progress about the sites of metabolism, metabolic enzymes, metabolites and physiological activity of those drugs metabolic in vivo.

Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development

Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring’s CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8– fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities’ alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development.

Modulatory effects of Azadirachta indica leaf extract on cutaneous and hepatic biochemical status during promotion phase of DMBA/TPA-induced skin tumorigenesis in mice.

The modulation in biochemical status of skin and hepatic tissue at the time point of commencement of promotion stage of skin carcinogenesis in mice and its intervention with aqueous Azadirachta indica leaf extract (AAILE) were investigated. 7,12-Dimethylbenz(a)anthracene (DMBA, 500 nmol/100 ul of acetone) was applied topically for 2 weeks (twice weekly), followed by phorbol-12-myristate-13-acetate (TPA, 1.7 nmol/100 ul) twice weekly for 6 weeks on the depilated skin of mice and AAILE was administered orally at a dose level of 300 mg/kg body wt thrice a week for 10 weeks. DMBA/TPA treatment upregulated the phase I enzymes in skin and hepatic tissue, as revealed by the increased cytochrome P450 (CYP) and cytochrome b5 (cyt b5) levels and aryl hydrocarbon hydroxylase (AHH) activity when compared to the control group and differentially modulated the activities of phase II enzymes like glutathione-s-transferase (GST), DT-diaphorase (DTD) and uridine diphosphate glucuronosyltransferase (UDP-GT). AAILE treatment decreased the DMBA/TPA-induced increase in cutaneous CYP level and enhanced the DTD and UDP-GT activities when compared with DMBA/TPA group. In the hepatic tissue of AAILE + DMBA/TPA group, an increase in UDP-GT activity was observed when compared to DMBA/TPA group. DMBA/TPA treatment did not alter the skin lipid peroxidation (LPO) level when compared to control group, however, in the animals that received AAILE treatment along with DMBA/TPA, a significant increase in LPO was observed when compared to control group. This was associated with a decrease in cutaneous reduced glutathione (GSH) level of AAILE + DMBA/TPA group. Enhanced LPO level was observed in the hepatic tissue of DMBA/TPA and AAILE + DMBA/TPA groups when compared to control group. However, no alteration was observed in their hepatic GSH levels. The micronuclei score in hepatic tissue did not exhibit significant inter-group differences. The results of the present study suggest that apart from skin, liver may be affected during DMBA/TPA-induced skin tumorigenesis. AAILE treatment has the ability to modulate these changes potentially influencing the process of tumor formation. These findings seem to be important to carcinogenesis and its intervention with anti-cancer agents.

Effects of cow ghee (clarified butter oil) & soybean oil on carcinogen-metabolizing enzymes in rats.

Background & objectives: Our previous study showed that cow ghee relative to soybean oil had a protective effect against carcinogen induced mammary cancer in rats. The objective of this study was to elucidate its biochemical mechanism. Methods: Two groups of 21 day old rats (20 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). Five animals from each group were sacrificed at 0 day and at 5, 21 and 44 wk for analysis of phase I and phase II pathways enzymes of carcinogen metabolism. Results: Dietary cow ghee relative to soybean oil decreased the activities of cytochrome P450 (CYP) enzymes, CYP1A1, CYP1A2, CYP1B1 and CYP2B1, responsible for activation of carcinogen in liver. Carcinogen detoxification activities of uridinediphospho-glucuronosyl transferase (UDPGT) and quinone reductase (QR) in liver, and γ-glutamyltranspeptidase (GGTP) and QR in mammary tissue were significantly higher in cow ghee fed rats than in soybean oil fed rats. The hepatic GGTP activity decreased on soybean oil diet; while in cow ghee group it remained unaffected. Interpretation & conclusions: Our findings show that dietary cow ghee compared to soybean oil downregulates the enzyme activities responsible for carcinogen activation in liver and upregulates carcinogen detoxification activities in liver and mammary tissues.

Reversible Dysphasia and Statins

This paper presents a case of reversible dysphasia occurring in a patient prescribed atorvastatin in combination with indapamide. A milder dysphasia recurred with the prescription of rosuvastatin and was documented on clinical examination. This resolved following cessation of rosuvastatin. The case highlights both a need for a wider understanding of potential drug interactions through the CYP 450 system and for an increased awareness, questioning and reporting of drug side-effects.

Pediatric Obesity and Non-alcoholic Fatty Liver Disease: Is It Real Problematic? / 대한소화기학회지

No abstract available.

Targeting CYP450 modulation to decrease the risk of induced cataract in the experimental model.

Background: Diabetes is one of the major causes of cataract. Some drugs prescribed for the treatment of diabetes are the modulators of CYP450, which may alter the risk of cataract. Objective: To study the effect of CYP450 modulation in galactosemic cataract. Materials and Methods: Male Sprague-Dawley suckling rats were allotted to four groups (n = 6), as follows: Group 1: Normal control, Group 2: Galactose control, Group 3: CYP450 inhibitor pretreated and Group 4: CYP450 inducer pretreated. Cataract was induced in animals of all groups except group 1 by feeding them galactose (50%), 21 days after parturition. From the eighteenth day of life, CYP450 inhibitor (nifedipine; 8.1 mg/kg) and CYP450 inducer (pioglitazone; 3.8 mg/kg) were given orally to groups 3 and 4, respectively. The maturation pattern of the cataract was observed by an operating microscope, every third day. Biochemical changes in the lenses of all groups, for example, CYP450 activity expressed as µM NADPH oxidized / unit time, alterations in the levels of total proteins, soluble proteins, and reduced glutathione (GSH) following the induction of cataract, were estimated. Results: The microscopic examination of the lenses indicated that CYP450 inhibitor pre-treatment delayed (fourteenth day) the occurrence of cataract, while CYP450 inducer pretreatment demonstrated an early (ninth day) cataract as compared to galactose control rats (twelfth day). A significant decrease and increase in CYP450 activity was observed with the CYP450 inhibitor and inducer pre-treatment, respectively. There was no alteration in the GSH level, but a significant increase in total and soluble protein was found in groups 3 and 4 as compared to group 2. Conclusion: CYP450 may have a role in the initiation of cataract without any effect on the maturation pattern, as revealed by the delayed occurrence of cataract with the CYP450 inhibitor and an early onset of cataract with the CYP450 inducer.

Drugs impact on CYP-450 enzyme family: A pharmacogenetical study of response variation.

Pharmacogenetics is the study of genetic basis in the individual response to drugs. A thorough knowledge of this will lead to a future where tailor-made drugs, suiting an individual, can be used. Scandinavian countries have been known for wide usage of pharmacogenetics and the most widely used application is for genotyping CYP2D6 in treating psychiatric illness. The CYP-450 enzyme, a super family of microsomal drug-metabolizing enzymes, is the most important of enzymes that catalyzes phase-I drug metabolism reaction. CYP2D6 is a member of this family and it has been most intensively studied and the best example of pharmacogenetics variation in drug metabolism. Neuro-transmitter and drug acting CNS viz. codeine, dextromethorphan, metoprolol and tryptyline etc. are well metabolized by this enzyme. Thus, CYP2D6 is one of the most important and responsible enzymes which regulates bioavailability and metabolism of drug. Presently 75 alleles of CYP2D6 have been described which are responsible for variance of metabolism and toxicity of drugs. Thus, by determining variance of CYP2D6 using molecular approaches viz., PCR, real-time PCR, DNA micro-array and molecular docking can determine the adverse effects, drug toxicity, bioavailability and therapeutic potential of new drug.

Comparative docking studies of CYP1b1 and its PCG-associated mutant forms.

Molecular docking has been used to compare and contrast the binding modes of oestradiol with the wild-type and some disease-associated mutant forms of the human CYP1b1 protein.The receptor structures used for docking were derived from molecular dynamics simulations of homology-modelled structures. Earlier studies involving molecular dynamics and principal component analysis indicated that mutations could have a disruptive effect on function,by destabilizing the native properties of the functionally important regions, especially those of the haem-binding and substrate-binding regions,which constitute the site of catalytic activity of the enzyme.In order to gain more insights into the possible differences in substrate-binding and catalysis between the wild-type and mutant proteins,molecular docking studies were carried out. Mutants showed altered protein -ligand interactions compared with the wild-type as a consequence of changes in the geometry of the substrate-binding region and in the position of haem relative to the active site. An important difference in ligand -protein interactions between the wild-type and mutants is the presence of stacking interaction with phenyl residues in the wild-type,which is either completely absent or considerably weaker in mutants.The present study revealed essential differences in the interactions between ligand and protein in wild-type and disease mutants,and helped in understanding the deleterious nature of disease mutations at the level of molecular function.

Lead hepatotoxicity & potential health effects.

Occupational and environmental exposures to lead (Pb), one of the toxic metal pollutants, is of global concern. Health risks are increasingly associated with environmental exposures to Pb emissions from, for example, the widespread use of leaded gasoline in developing countries. Exposure occurs mainly through the respiratory and gastrointestinal systems, and the ingested and absorbed Pb is stored primarily in soft tissues and bone. Autopsy studies of Pb-exposed patients have shown a large amount (approximately 33%) of the absorbed Pb in soft tissue stored in liver. In addition to neuronal encephalopathy observed in persons after exposure to very high concentrations of Pb, gastrointestinal colic (abdominal pain, constipation, intestinal paralysis) is a consistent early symptom of Pb poisoning in humans. Such severe gastrointestinal effects are consistently observed in patients with a blood Pb range of 30 to 80 microg/dl. Ingestion of Pb is one of the primary causes of its hepatotoxic effects. Hepatocarcinogenic effects of Pb reported in animal toxicology studies have led to new research into the biochemical and molecular aspects of Pb toxicology. Gains in the molecular understanding of Pb effects on hepatic drug metabolizing enzymes, cholesterol metabolism, oxidative stress, and hepatic hyperplasia suggest a potential role for Pb in damaging extrahepatic systems, including the cardiovascular system. This review also discusses the therapeutic potential of chelation therapy in treating Pb-induced hepatotoxicity in animals.

Cytochrome P450 1A activity in liver and fixed wavelength fluorescence detection of polycyclic aromatic hydrocarbons in the bile of tongue-fish (Cynoglossus acrolepidotus, Bleeker) in relation to petroleum hydrocarbons in the eastern Gulf of Thailand.

This investigation was conducted in an area of oil spill along the east coast of Thailand to examine the relations among cytochrome P450 1A activity in liver and PAHs in the bile of the tonguefish and petroleum hydrocarbons in the sediments. PAH sediment concentrations in the reference and oil spill areas were 5.03 +/- 0.42 and 0.21 +/- 0.043 microg(-1) dry weight respectively Cytochrome activity in fish liver from oil spill area was 45.40 +/- 3.50 pmoles/ min/mg protein, almost threefold higher than that from the reference sites. Flourescense detection in bile metabolites at the oil spill area, 69.8 +/- 9.9 flourescense unit was significantly higher than that at the reference sites, 22.9 +/- 5.5 and 22.2 +/- 3.5 fluorescence unit. A strong correlation was found among cytochrome P450 1A activity in liver, PAH of bile metabolites and petroleum hydrocarbons. Both cytochrome and bile metabolites activity decreased seaward varying to the distance from the oil polluted area. We concluded that both detections in tonguefish can be regarded as a complementary biomarkers for the exposure of PAHs in tropical marine environments.

Impediment of diethylnitrosamine induced hepatotoxicity in male Balb/c mice by pretreatment with aqueous Azadirachta indica leaf extract.

Considering the hepatoprotective properties of Azadirachta indica, the present study was designed to evaluate its preventive effects against diethylnitrosamine (NDEA) induced hepatotoxicity in male Balb/c mice. Exposure of NDEA caused a significant increase in micronucleated cell score, lipid peroxidation levels (LPO) and activity of lactate dehydrogenase (LDH). A significant decrease in reduced glutathione (GSH) contents and activity of glutathione-S-transferase (GST) was also observed upon NDEA treatment, whereas their activities of cytochrome P450 and cytochrome b5 showed non-significant alterations. Aqueous A. indica leaf extract (AAILE) pretreatment showed protective effects against NDEA induced toxicity by decreasing the frequency of micronucleated cell, levels of LPO and LDH activity. Also, a decreased activity of GST, cytochrome P450 and an increased activity of cytochrome b5, GSH contents was observed when AAILE pretreated mice were injected with NDEA. Only AAILE treatment caused a noticeable decrease in the frequency of micronuclei, activity of cytochrome P450 and cytochrome b5, but a significant increase in the activity of GST and GSH contents, whereas, non significant alterations were observed in the activity of LDH and levels of LPO. Significance of these observations with respect to hepatoprotective efficacy of A. indica has been discussed in the present manuscript.

Environmental contaminants in pathogenesis of breast cancer.

This review is an attempt to comprehend the diverse groups of environmental chemical contaminants with a potential for pathogenesis of breast cancer, their probable sources and the possible mechanisms by which these environmental contaminants act and interplay with other risk factors. Estrogens are closely related to the pathogenesis of breast cancer. Oxidative catabolism of estrogen, mediated by various cytochrome P450 enzymes, generates reactive free radicals that can cause oxidative damage. The same enzymes of estrogenic metabolic pathways catalyze biological activation of several environmental (xenobiotic) chemicals. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals. Breast tissue can be a target of several xenobiotic agents. DNA-reactive metabolites of different xenobiotic compounds have been detected in breast tissue. Many phase I and II xenobiotic metabolizing enzymes are expressed in both normal and cancerous breast tissues. These enzymes play a significant role in the activation/detoxification of xenobiotic and endogenous compounds including estrogens. More than 30 carcinogenic chemicals are present in tobacco smoke; many of them are fat-soluble, resistant to metabolism and can be stored in breast adipose tissue. Similarly, pesticides are also known to cause oxidative stress; while some act as endocrine disruptor, some are shown to suppress apoptosis in estrogen sensitive cell lines. Reports have shown an association of smoking (both active and passive) and pesticides with breast cancer risk. However, the issues have remained controversial. Different mutagenic substances that are generated in the cooking process e.g., heterocyclic amines and polycyclic aromatic hydrocarbons (PAHs) can be a threat to breast tissue. PAHs and dioxins exert their adverse effects through the aryl hydrocarbon receptor (AhR), which activates several genes involved in the metabolisms of xenobiotic compounds and endogenous estrogens. These chemicals also induce AhR-dependent mitochondrial dysfunction. Many of the environmental pollutants suppress the immune system, which are implicated to risk. A better understanding about the biological effects of different environmental carcinogenic compounds and determination of their impact on rising incidence of breast cancer will be beneficial in improving preventive policy against breast cancer.