• Journal of Preventive Medicine and Public Health
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• v.55 no.3
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• pp.280-288
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• 2022

Objectives: One of the most widely used pesticides today is chlorpyrifos (CPF). Cytochrome P450 (CYP)2B6, the most prominent catalyst in CPF bioactivation, is highly polymorphic. The objective of our study was to evaluate the role of CYP2B6^*6, which contains both 516G>T and 785A>G polymorphisms, in CPF toxicity, as represented by the concentration of 3,5,6-trichloro-2-pyridinol (TCPy), among vegetable farmers in Central Java, Indonesia, where CPF has been commonly used. Methods: A cross-sectional study was conducted among 132 vegetable farmers. Individual socio-demographic and occupational characteristics, as determinants of TCPy levels, were obtained using a structured interviewer-administered questionnaire and subsequently used to estimate the cumulative exposure level (CEL). TCPy levels were detected with liquid chromatography-mass spectrometry. CYP2B6^*6 gene polymorphisms were analyzed using a TaqMan^® SNP Genotyping Assay and Sanger sequencing. Linear regression analysis was performed to analyze the association between TCPy, as a biomarker of CPF exposure, and its determinants. Results: The prevalence of CYP2B6^*6 polymorphisms was 31% for ^*1/^*1, 51% for ^*1/^*6, and 18% for ^*6/^*6. TCPy concentrations were higher among participants with CYP2B6^*1/^*1 than among those with ^*1/^*6 or ^*6/^*6 genotypes. CYP2B6^*6 gene polymorphisms, smoking, CEL, body mass index, and spraying time were retained in the final linear regression model as determinants of TCPy. Conclusions: The results suggest that CYP2B6^*6 gene polymorphisms may play an important role in influencing susceptibility to CPF exposure. CYP2B6^*6 gene polymorphisms together with CEL, smoking habits, body mass index, and spraying time were the determinants of urinary TCPy concentrations, as a biomarker of CPF toxicity.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.11
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.956-964
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• 2017

Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steady-state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The $k_m$ and $k_{cat}$ values for compactin were $39.1{\pm}8.8{\mu}M$ and $1.12{\pm}0.09min^{-1}$, respectively. The $k_{cat}/K_m$ value for compactin ($0.029min^{-1}{\cdot}{\mu}M^{-1}$) was lower than that for diclofenac ($0.114min^{-1}{\cdot}{\mu}M^{-1}$). Spectroscopic analysis showed that CYP105D7 binds to compactin with a $K_d$ value of $17.5{\pm}3.6{\mu}M$. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

• Molecules and Cells
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• v.38 no.3
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• pp.236-242
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• 2015

Intake of caffeine during pregnancy can cause retardation of fetal development. Although the significant influence of caffeine on animal development is widely recognized, much remains unknown about its mode of action because of its pleiotropic effects on living organisms. In the present study, by using Caenorhabditis elegans as a model organism, the effects of caffeine on development were examined. Brood size, embryonic lethality, and percent larval development were investigated, and caffeine was found to inhibit the development of C. elegans at most of the stages in a dosage-dependent fashion. Upon treatment with 30 mM caffeine, the majority ($86.1{\pm}3.4%$) of the L1 larvae were irreversibly arrested without further development. In contrast, many of the late-stage larvae survived and grew to adults when exposed to the same 30 mM caffeine. These results suggest that early-stage larvae are more susceptible to caffeine than later-stage larvae. To understand the metabolic responses to caffeine treatment, the levels of expression of cytochrome P450 (cyp) genes were examined with or without caffeine treatment using comparative microarray, and it was found that the expression of 24 cyp genes was increased by more than 2-fold (p < 0.05). Among them, induction of the cyp-35A gene family was the most prominent. Interestingly, depletion of the cyp-35A family genes one-by-one or in combination through RNA interference resulted in partial rescue from early larval developmental arrest caused by caffeine treatment, suggesting that the high-level induction of cyp-35A family genes can be fatal to the development of early-stage larvae.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1059-1065
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• 2012

Biocatalytic transfer of oxygen in isolated cytochrome P450 or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. Cytochrome P450 CYP105P2 was isolated from Streptomyces peucetius that showed a high degree of amino acid identity with hydroxylases. Previously performed homology modeling, and subsequent docking of the model with flavone, displayed a reasonable docked structure. Therefore, in this study, in a pursuit to hydroxylate the flavone ring, CYP105P2 was co-expressed in a two-vector system with putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida for efficient electron transport. HPLC analysis of the isolated product, together with LC-MS analysis, showed a monohydroxylated flavone, which was further established by subsequent ESI/MS-MS. A successful 10.35% yield was achieved with the whole-cell bioconversion reaction in Escherichia coli. We verified that CYP105P2 is a potential bacterial hydroxylase.

• Bulletin of the Korean Chemical Society
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• v.33 no.6
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• pp.1885-1889
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• 2012

Among 23 cytochrome P450s, CYP125A4 was proposed as a putative monooxygenase based on the high level of amino acid sequence homology (54% identity and 75% similarity) with the well characterized C27-steroid $Mycobacterium$ $tuberculosis$ CYP125A1. Utilizing MTBCYP125A1 as a template, homology modeling of SPCYP125A4 was conducted by Accelrys Discovery Studio 3.1 software. The modeled SPCYP125A4 structure with lowest energy value was subsequently assessed for its stereochemical quality and side-chain environment. The final model was generated by showing its active site through the molecular dynamics. The docking of steroids showed broad specificity of SPCYP125A4 with different orientation of ligand within active site facing the heme. One poses of C27-steroid with C26 facing the heme with distance of 3.734 ${\AA}$ from the Fe were predominant.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.139-143
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• 2005

Cytochrome P450 14${\alpha}$-sterol demethylase enzyme (CYP51) is the target a of azole type antifungals. The azole blocks the ergosterol synthesis and thereby inhibits fungal growth. A three-dimensional (3D) homology model of CYP51 from Candida albicans was constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using this model, the binding modes for the substrate (24-methylene-24, 25-dihydrolanosterol) and the known inhibitors (fluconazole, voriconazole, oxiconazole, miconazole) were predicted from docking. Virtual screening was performed employing Structure Based Focusing (SBF). In this procedure, the pharmacophore models for database search were generated from the protein-ligands interactions each other. The initial structure-based virtual screening selected 15 compounds from a commercial available 3D database of approximately 50,000 molecule library, Being evaluated by a cell-based assay, 5 compounds were further identified as the potent inhibitors of Candida albicans CYP51 (CACYP51) with low minimal inhibitory concentration (MIC) range. BMD-09-01${\sim}$BMD-09-04 MIC range was 0.5 ${\mu}$g/ml and BMD-09-05 was 1 ${\mu}$g/ml. These new inhibitors provide a basis for some non-azole antifungal rational design of new, and more efficacious antifungal agents.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.1145-1150
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• 2015

Associations of GSTT1, GSTM1 and CYP1A1 gene variants with risk of developing oral cancer were evaluated in this study. A case-control study was conducted in Pashtun population of Khyber Pakhtunkhwa province of Pakistan in which 200 hospital based oral cancer cases and 151 population based healthy controls exposed to similar environmental conditions were included. Sociodemographic data were obtained and blood samples were collected with informed consent for analysis. GSTM1 and GSTT1 were analysed through conventional PCR method while specific RT-PCR method was used to detect CYP1A1 polymorphisms. Results were analyzed for conditional logistic regression model by SPSS version 20. The study shows that patients with either GSTM1 or GSTT1 null genotypes have significantly higher risk of oral cancer (adjusted odds (OR): (3.019 (1.861-4.898) and 3.011(1.865-4.862), respectively), which further increased when either one or both null genes were present in combination (adjusted odds (OR): (3.627 (1.981-6.642 and 9.261 (4.495-19.079), respectively). CYP1A1 rs4646903 gene variants individually showed weak association OR: 1.121 (0.717-1.752); however, in the presence of GSTM1 and/or GSTT1 null genotypes further increasing the association (adjusted odds (ORs): 4.576 (2.038-10.273), 5.593 (2.530-12.362) and 16.10 (3.854-67.260 for GSTM/GSTT null and CYP1A1 wild type, GSTM/GSTT either null and CYP1A1 variant alleles, and all 3 gene polymorphisms combinations, respectively). Our findings suggest that presence of GSTM1 and/or GSTT1 null genotypes along with variant alleles of CYP1A1 may be the risk alleles for oral cancer susceptibility in Pashtun population.

• Korean Journal of Agricultural and Forest Meteorology
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• v.16 no.4
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• pp.316-326
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• 2014

Determination of the late marginal heading date (LMHD), which would allow estimation of the late marginal seeding date and the late marginal transplanting date, would help identification of potential double cropping areas and, as a result, establishment of cropping systems. The objective of this study was to determine the LMHD at 51 sites in Korea. For these sites, weather data were obtained from 1971 to 2000 and from 1981 to 2010, which represent past and current normal climate conditions, respectively. To examine crop productivity on the LMHD, climatic yield potential (CYP) was determined to represent the potential yield under a given climate condition. The LMHD was calculated using accumulated temperature for 40 days with threshold values of $760^{\circ}C$, $800^{\circ}C$, $840^{\circ}C$ and $880^{\circ}C$. The value of CYP on a given LMHD was determined using mean temperature and sunshine duration for 40 days from the LMHD. The value of CYP on the LMHD was divided by the maximum value of CYP (CYPmax) in a season to represent the relative yield on the LMHD compared with the potential yield in the season. Our results indicated that the LMHD was delayed at most sites under current normal conditions compared with past conditions. Spatial variation of the LMHD differed by the threshold temperature. Overall, the minimum value of CYP/CYPmax was 81.8% under all of given conditions. In most cases, the value of CYP/CYPmax was >90%, which suggested that yield could be comparable to the potential yield even though heading would have occurred on the LMHD. When the LMHD could be scheduled later without considerable reduction in yield, the late marginal transplanting date could also be delayed accordingly, which would facilitate doublecropping in many areas in Korea. Yield could be affected by sudden change of temperature during a grain filling period. Yet, CYP was calculated using mean temperature and sunshine duration for 40 days after heading. Thus, the value of CYP/CYPmax may not represent actual yield potential due to change of the LMHD, which suggested that further study would be merited to take into account the effect of weather events during grain filling periods on yield using crop growth model and field experiments.

• Korean Journal of Veterinary Service
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• v.26 no.4
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• pp.345-359
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• 2003

A number of toxicants have been incriminated as a causing hepatic disease. Among many detrimental injury, alcohol has been noted for hepatitis, fatty liver, fibrosis, and hepatic cirrhosis. The purpose of this study was to develop animal model for hepatic fibrosis in pigs fed ethanol, and to search for a new anti-fibrogenic agent via this model. Twelve male Landrace pigs were divided into 3 groups of 4 animals each. Group 1, 2 and 3 were fed with active ceramic water only, ceramic water + liquid diet containing 15% ethanol and normal tap water + liquid diet containing 15% ethanol for 12 weeks, respectively. At week 12, all pigs were immediately sacrificed for collection each tissue and blood. Serologically, serum ALT and AST levels were significantly reversed in group 2, as compared to group 3. They were normal range in pigs of group 1. Microscopically, macrovesicular lipid droplets and moderate hepatocellular necrosis were evident in the tap water + ethanol fed group 3. However, the active ceramic water treated group 1 showed normal architecture. Moreover, in group 2, mild fatty changes and necrosis were observed in hepatocytes. Collagen fibers were increased in spaces surrounding periportal and interlobular connective tissues in the group 3 of tap water + ethanol, but collagen synthesis and its thickness of fibrotic septa connecting portal tracts were markedly reduced in the group 2 of ceramic water + ethanol. Myofibroblasts were detected mainly in the interlobular connective tissues of pig liver of group 3 treated ethanol and tap water. Few to no myofibroblasts were observed in groups 1 and 2. CYP2E1 was not or rarely detected in group 1 fed ceramic water. However, group 2 showed slightly activation of CYP2E1 in the area of pericentral vein, while CYP2E1 was significantly activated in group 3 fed tap water and ethanol. Based on the above data, we believe that we have developed a unique alcohol induced fibrosis model in pig, which will be useful in developing anti-fibrotic agents and drugs. Furthermore, the active ceramic water used in our study had an inhibitory and may be protective against ethanol induced hepatic toxicity and fibrosis.