CYP19 Gene Research Articles

Transcriptome dynamics and metabolite analysis revealed the candidate genes and regulatory mechanism of ganoderic acid biosynthesis during liquid superficial-static culture of Ganoderma lucidum.

Ganoderic acid (GA), an important secondary metabolite of Ganoderma lucidum, exhibited many significant pharmacological activities. In this study, the biosynthetic mechanism of GAs was investigated by comparing metabolites and transcriptome dynamics during liquid superficial-static culture (LSSC) and submerged culture (SC). LSSC was a better method to produce GA because thirteen GAs were identified from mycelia by UPLC-QTOF-MS, and the content of all GAs was higher in LSSC than in SC. Ergosterol was accumulated during the SC process in G. lucidum. Transcriptome dynamics analysis revealed CYP5150L8 was the key gene regulating lanosterol flux into GA biosynthesis. Other sixteen CYP450 genes were significantly higher expressed during the culture time in LSSC and could be potential candidate genes associated with the biosynthesis of different GAs. In addition, six of the ten expressed genes in ergosterol biosynthetic pathway shown upregulated at some time points in SC. These results not only provide a fundamental information of the key genes in ergosterol and GA biosynthetic pathway, but also provide directions for future elucidating the regulatory mechanisms of GAs in G. lucidum and enabling us to promote the development and utilization of LSSC at the industrial level.

Genome mining and functional analysis of cytochrome P450 genes involved in insecticide resistance in Leucinodes orbonalis (Lepidoptera: Crambidae).

Genome-wide analysis of cytochrome P450 monooxygenase (CYP) genes from the advanced genome project of the Leucinodes orbonalis and the expression analysis provided significant information about the metabolism-mediated insecticide resistance. A total of 72 putative CYP genes were identified from the genome and transcriptome of L. orbonalis. The genes were classified under 30 families and 46 subfamilies based on the standard nomenclature. In the present study, a novel CYP gene, CYP324F1, was identified and it has not been reported from any other living system so far. Biochemical assays showed enhanced titers (5.81-18.5-fold) of O-demethylase of CYP in five field-collected populations. We selected 34 homologous CYP gene sequences, seemed to be involved in insecticide resistance for primer design and quantitative real-time PCR studies. Among the many overexpressed genes (>10 fold), the expression levels of CYP324F1 and CYP306A1 were prominent across all the field populations as compared with the susceptible iso-female line. Oral delivery of ds-CYP324F1 and ds-CYP306A1 directed against CYP324F1 and CYP306A1 to the larvae of one of the insecticide resistance populations caused reduced expression of these two transcripts in a dose-dependent manner (53.4%-85.0%). It appears that the increased titer of O-demethylase is the result of increased transcription level of CYP genes in resistant populations. The data provide insight for identifying the novel resistance management strategies against L. orbonalis.

Association of the CYP17 and CYP19 gene polymorphisms in women with polycystic ovary syndrome from Punjab, Pakistan

Background Polycystic ovarian syndrome (PCOS) is considered the most common multifactorial endocrinopathy. Genetic factors play an essential role in the pathogenesis of PCOS. CYP 17 enzyme is responsible for androgenesis, while CYP 19 enzyme works for androgen conversion into aromatic estrogen. Several studies have reported their association with PCOS. This study was aimed to investigate the association of cytochrome P450c17α gene (CYP17) 5′-untranslated region MspA1/(rs743572) genetic polymorphism and rs2414096 of cytochrome P450 or aromatase (CYP19) gene polymorphism with the susceptibility to PCOS in reproductive-age women from Punjab, Pakistan. Methods We performed a case-control association study was conducted, including 204 PCOS patients and 100 controls. Genotyping of SNP rs2414096 (CYP 19 gene) and P450c17α gene (CYP17) 5′-untranslated region MspA1 was performed on genomic DNA using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Statistical analysis was performed to find out the association of phenotypic and genotypic characters in etiopathology of PCOS. Results The genotype distribution for CYP 17 5′-UTR MspA1 (TT, TC, CC) revealed that the frequency of genotype TC is significantly higher in PCOS patients (54.9%) vs. controls (OR 4.97, 95% CI 2.75–8.33, p<.001) (12%). The genotype distribution for CYP 19 (GG, GA, AA) showed significantly higher frequency of GA (58.%) and AA (23.5%) in patients as compared to cases (18% and 8%, respectively) (OR 5.49, 95% CI 3.61–8.34, p<.001). Body mass index (BMI), waist, hip, infertility and family history of infertility, PCOS, diabetes, and hypertension were found significantly associated with PCOS. CYP 19 genotypes were found significantly associated with PCOS patients having paraclinical traits of infertility and family history of infertility, while CYP 17 genotypes showed no significant association with any paraclinical traits in PCOS patients. Conclusions This is the first study on PCOS genotypes from Pakistan and results suggest that CYP 17 5′-UTR MspA1 (rs743572) (genotype TC) and CYP 19 gene (rs2414096) (genotype GA) polymorphisms are significantly associated with susceptibility to PCOS in Pakistani women having the traits of infertility and family history of hypertension.

A high-quality reference genome sequence of Salvia miltiorrhiza provides insights into tanshinone synthesis in its red rhizomes.

Salvia miltiorrhiza Bunge, also known as red sage or Danshen, is an important traditional Chinese medicine (TCM) that has been used for thousands of years to treat cardiovascular and other diseases. It is also considered an important model TCM plant. Here, a high-quality reference genome of S. miltiorrhiza was generated by combining PacBio long-read sequencing and chromatin interaction mapping (Hi-C) technologies, resulting in the chromosome-scale assembly of a 594.75-Mb genome sequence with a contig N50 of 2.70 Mb. This assembly shows the highest level of continuity for a Danshen genome generated thus far. The S. miltiorrhiza genome contained 32,483 protein-coding genes, with a repetitive DNA content of approximately 64.84%. The high percentage of young LTRs suggests that multiple TE transposition bursts occurred recently in S. miltiorrhiza. Genes unique to secondary metabolism pathways were expanded in the S. miltiorrhiza genome. A new CYP450 gene cluster was identified in the phloem of red roots where active components were synthesized. This reference genome sequence will facilitate future studies aimed at the elucidation of the secondary metabolism synthesis pathway and the genetic improvement of S. miltiorrhiza.

Sequence variants of CYP345a1 and CYP6a14 gene regions in Tribolium castaneum (Coleoptera: Tenebrionidae) adults treated withthe novel characterızedBolanthus turcicus (Caryophyllaceae) extract.

In this study, various doses of plant extracts that obtained from Bolanthus turcicus was applied to an important storage pest Tribolium castaneum adults. Bolanthus turcicus is an endemic species and spreads on the Hasan Mountain above Karkın town (Turkey, Aksaray province). The plant species was collected from June to July with the field study to be carried out in this region. Obtained extract of plant was analyzed by gas chromatography mass spectrometry (GC-MS) method. The doses were defined during the study and the concentrations that kill 50% and 99% of the population were determined after applications. After 24 h, DNA was isolated from live and dead individuals that obtained from LC50 and LC99 concentration applications and analyzed for Cytochrome P450-mediated detoxification resistance genes, CYP345A1 and CYP6A14 gene regions, by polymerase chain reaction (PCR). CYP genes in insects are known to be rapidly regulated when exposed to insecticides. In the study, in order to screen for 206 bp and 353 bp fragments of CYP345A1 and CYP6A14 genes in T. castaneum adults were amplified using specific primers, respectively. DNA direct sequencing was performed on each template using the forward primer. When compared to the control, it is believed that mutation differences in live and dead individuals according to the sequencing results obtained from survival and dead adults, may allow these genes to play a protective role against the toxic effect of B. turcicus extract.

Polymorphism in the Androgen Biosynthesis Gene (CYP17), a Risk for Prostate Cancer: A Meta-Analysis.

Gene polymorphism is one of the few factors that increases the risk of prostate cancer. T to C substitution in the 5’ promoter region of the CYP17 gene is hypothesized to increase the rate of gene transcription, increase androgen production, and thereby increase the risk of prostate cancer. Nevertheless, the inconsistencies originating from studies on CYP17 polymorphism and prostate cancer prompted this meta-analysis, to decipher the association between CYP17 polymorphism and prostate cancer. Most case-control studies addressing CYP17 polymorphism and prostate cancer were exhaustively searched from Web of Science, Google Scholar, and PubMed. The various genotype distributions as well as the minor allele distributions were retrieved. Pooled odds ratios (ORs) with their 95% CI and estimates of the Hardy–Weinberg Equilibrium were calculated. Analyses were performed using the RevMan v.5.3 software and SPSS v.21. There was high-pooled heterogeneity (I2 = 87.0%, OR = .42, CI [.39, .45], and p < .001) among the A2 versus A1 allele. With the per-allele model (A2 versus A1), ethnicity was a major risk factor to prostate cancer, with Asians recording the highest risk (OR = 12.61, 95% CI [8.77, 18.12]). From the genotype models, A1/A1 versus A2/A2 (OR = 3.02, 95% CI [2.65, 3.44]) and A1/A2 versus A2/A2 (OR = 4.39, 95% CI [3.86, 5.00]) were all significantly associated with prostate cancer. Although some genotype models were associated with the risk of prostate cancer, we should be mindful when interpreting the results of this study because of the limited number of studies and the small sample size used.

CYP19 gene rs2414096 variant and differential genetic risk of polycystic ovary syndrome: a systematic review and meta-analysis

Background Previously, many studies investigated the association between CYP19 rs2414096(G > A) and susceptibility to develop PCOS. However, results had been inconsistent. Therefore, our systematic review and meta-analysis aimed to identify the association between CYP19 rs2414096 and PCOS risk. Methods: A systematic literature search was done from database PubMed, Google Scholar, Science Direct, and Cochrane Library up to July 15 2020 and statistical analysis was performed by RevMan5.3. Results: A total of seven studies comprised of 1414 PCOS cases and 1276 controls were included in the current meta-analysis. The pooled analysis showed that overall, there is a significant association between CYP19 rs2414096(G > A) and risk of PCOS (OR = 0.74, 95% CI= 0.62–0.88, p = .0008). In dominant model, GG + AG vs GG and recessive genetic model AA vs AG + GG found a significant association (OR = 1.60,95% CI = 1.10–2.31, p = .01; OR = 0.65,95% CI = 0.45–0.93, p = .02) respectively which indicates that GG phenotype might be risk factor for PCOS development. In stratified subgroup analysis, there was significant association between CYP19 rs2414096 polymorphism and PCOS risk for non-Indian population only while no association was found with Indian population. Conclusion: Present meta-analysis studies indicate that CYP19 rs2414096 is associated with PCOS risk and important in pathogenesis of PCOS for many populations but for Indian population more studies are required as Indian population comprises of various subpopulations genetically isolated since long.

Effect of genistein, daidzein and coumestrol on sex-related genes expression in Russian sturgeon (Acipenser gueldenstaedtii)

Phytoestrogens are compounds of estrogenic properties, present in high concentrations in sturgeon aquafeeds due to common utilization of the soybean and its derivatives for fish feed production. The mechanism of those compounds and their effect on sex-related gene expression is poorly understood. Therefore, this study aimed to examine the expression of the amh, ar, cyp19, dmrt1, erα, erβ, foxl2, sox9, star, vasa and vtg genes in the gonads, the brain and the liver of one-year-old Russian sturgeons exposed for 265 days to daidzein, genistein and coumestrol administrated as feed additives in the concentration of 10,000, 500 and 10 mg kg−1 feed, respectively. In the brain tissue the most significant effect of the studied compounds was observed. With the exception of a few females from Genistein and Coumestrol groups, the expression patterns of erα, vtg, amh, foxl2, sox9, cyp19 and ar in the brain were altered to the greatest extent in the individuals from experimental groups in comparison to those fed a diet without phytoestrogen additives. The most notable difference observed in the gonads was a significant down-regulation of the sex-related gene expression in the individuals fed with phytoestrogen-supplemented feed, which suggests that the studied phytoestrogens may inhibit molecular pathways responsible for sturgeon sex differentiation. Additionally, in the gonads, regardless of the studied group, the expression of foxl2 and cyp19 was observed to be female-biased, further establishing the significance of those factors during female development. In the liver, the daidzein additive was observed to have the strongest altering effect on the expression patterns of estrogen and androgen receptors which may influence estrogen- and androgen-regulatory pathways. The abovementioned findings suggested that phytoestrogens can cause significant changes in the expression profiles of sex-related genes resulting in molecular pathway alterations, which can ultimately lead to sex differentiation disturbances in sturgeons.

DNA Methylation of Steroidogenic Enzymes in Benign Adrenocortical Tumors: New Insights in Aldosterone-Producing Adenomas.

DNA methylation has been identified among putative regulatory mechanisms for CYP11B2 expression in primary aldosteronism. The objective of this work is to investigate DNA methylation and expression of genes encoding steroidogenic enzymes in benign adrenocortical tumors. This cross-sectional study took place at university hospitals. We collected fresh-frozen tissues from patients with benign adrenocortical adenomas (n = 48) (nonfunctioning n = 9, autonomous cortisol secretion n = 9, Cushing syndrome n = 17, aldosterone-producing [APA] n = 13) and adrenal cortex adjacent to APA (n = 12). We collected formalin-fixed, paraffin-embedded (FFPE) specimens of paired APA and concurrent aldosterone-producing cell clusters (APCCs) (n = 6). DNA methylation levels were evaluated by quantitative bisulfite next-generation sequencing in fresh-frozen tissues (CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, HSD3B1, HSD3B2, NR5A1, STAR, and TSPO) and FFPE APA/APCC paired samples (CYP11B2). CYP11B1, CYP11B2, CYP17, CYP21, and STAR gene expressions were examined by quantitative real-time polymerase chain reaction. The main outcome measure was DNA methylation. CYP11B2 methylation levels were significantly lower in APA than in other adrenal tissues (P < .001). Methylation levels of the remaining genes were comparable among groups. Overall, CYP11B2 expression and DNA methylation were negatively correlated (ρ = -0.379; P = .003). In FFPE-paired APA/APCC samples, CYP11B2 methylation level was significantly lower in APA than in concurrent APCCs (P = .028). DNA methylation plays a regulatory role for CYP11B2 expression and may contribute to aldosterone hypersecretion in APA. Lower CYP11B2 methylation levels in APA than in APCCs may suggest an APCC-to-APA switch via progressive CYP11B2 demethylation. Conversely, DNA methylation seems not to be relevant in regulating the expression of genes encoding steroidogenic enzymes other than CYP11B2.

First pregnancy after in vitro culture of early antral follicles in goats: Positive effects of anethole on follicle development and steroidogenesis.

This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.

Detoxification mechanism of Aconiti Lateralis Radix Praeparata combined with dried Rehmanniae Radix based on metabolic enzymes in liver

The enzymes CYP1 A2 and CYP3 A4 were measured by building a &quot;Cocktail&quot; probe drug and the incubation system of liver microsomes. The compatibility of Aconiti Lateralis Radix Praeparata combined with dried Rehmanniae Radix on CYP450 enzyme protein and gene expression was explored from the level of protein and molecular biology. It explored the molecular mechanism of compatibility detoxication of Aconiti Lateralis Radix Praeparata to provide scientific support for clinical safe and effective application of Aconiti Lateralis Radix Praeparata. The CYP450 enzyme activity was determined by using &quot;Cocktail&quot; probe drugs. The content of CYP450 enzyme was measured by CO reduction of differential spectrum method. The mRNA expression of CYP1 A2 and CYP3 A4 enzyme was detected by RT-PCR technology. Compared with the blank group, the CYP1 A2 and CYP3 A4 enzyme activity and mRNA expression were increased in the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group with significant differences(P&lt;0.05), while the CYP3 A4 enzyme activity and mRNA expression were no influence in the Aconiti Lateralis Radix Praeparata group. The CYP3 A4 enzyme activity and mRNA expression were increased in the dried Rehmanniae Radix and the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group, and there were significant differences(P&lt;0.05). The content of CYP450 enzyme was decreased in the Aconiti Lateralis Radix Praeparata group, contributed to extremely significant difference(P&lt;0.01). The content of CYP450 enzyme was increased in the dried Rehmanniae Radix and the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group, and there were significant differences(P&lt;0.05). The CYP1 A2 and CYP3 A4 enzyme activity and gene expression were enhanced after dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata. The metabolism of toxic ingredients of Aconiti Lateralis Radix Praeparata was accelerated to reach an effect of detoxication. The detoxication mechanism of compatibility of Aconiti Lateralis Radix Praeparata was verified from the viewpoint of liver metabolic enzymes.

Diversity and evolution of cytochrome P450s of Jacobaea vulgaris and Jacobaea aquatica

BackgroundCollectively, plants produce a huge variety of secondary metabolites (SMs) which are involved in the adaptation of plants to biotic and abiotic stresses. The most characteristic feature of SMs is their striking inter- and intraspecific chemical diversity. Cytochrome P450 monooxygenases (CYPs) often play an important role in the biosynthesis of SMs and thus in the evolution of chemical diversity. Here we studied the diversity and evolution of CYPs of two Jacobaea species which contain a characteristic group of SMs namely the pyrrolizidine alkaloids (PAs).ResultsWe retrieved CYPs from RNA-seq data of J. vulgaris and J. aquatica, resulting in 221 and 157 full-length CYP genes, respectively. The analyses of conserved motifs confirmed that Jacobaea CYP proteins share conserved motifs including the heme-binding signature, the PERF motif, the K-helix and the I-helix. KEGG annotation revealed that the CYPs assigned as being SM metabolic pathway genes were all from the CYP71 clan but no CYPs were assigned as being involved in alkaloid pathways. Phylogenetic analyses of full-length CYPs were conducted for the six largest CYP families of Jacobaea (CYP71, CYP76, CYP706, CYP82, CYP93 and CYP72) and were compared with CYPs of two other members of the Asteraceae, Helianthus annuus and Lactuca sativa, and with Arabidopsis thaliana. The phylogenetic trees showed strong lineage specific diversification of CYPs, implying that the evolution of CYPs has been very fast even within the Asteraceae family. Only in the closely related species J. vulgaris and J. aquatica, CYPs were found often in pairs, confirming a close relationship in the evolutionary history.ConclusionsThis study discovered 378 full-length CYPs in Jacobaea species, which can be used for future exploration of their functions, including possible involvement in PA biosynthesis and PA diversity.

Cytochrome P450 1 enzymes in black rockfish, Sebastes schlegelii: Molecular characterization and expression patterns after exposure to benzo[a]pyrene

Cytochrome P450 (CYP) enzymes play important roles in the detoxification and bioactivation of environmental contaminants and are involved in the responses to pollution in fish. In this study, we cloned four new CYP1 genes, CYP1A, CYP1B, CYP1C1, and CYP1C2, from black rockfish (Sebastes schlegelii), a dominant and economically important fish species in Korea. This species is at a significant risk of exposure to petrohydrocarbons, such as benzo[a]pyrene(B[a]P), due to frequent oil spills along the Korean coast. Quantitative PCR analysis of CYP1 gene transcription in 12 organs of the fish revealed tissue-specific expression patterns. CYP1A was significantly expressed in the liver, heart, kidneys, and muscle, and CYP1B was significantly expressed in the gills, muscle, and heart. CYP1C1 and CYP1C2 showed similar tissue expression patterns, with the highest levels in the muscle. Furthermore, exposure to an aryl hydrocarbon receptor (AHR) agonist, B[a]P, at 2, 20, and 200 mg/kg body weight showed significant dysregulation of the CYP1A, CYP1B, CYP1C1, and CYP1C2 expression levels in the gills, liver, kidneys, and spleen. The mRNA expression levels of CYP1A and CYP1B were upregulated by 450- and 17-fold, respectively, in the spleen. Compared with their levels in the control, CYP1C1 increased by 45-fold, while CYP1C2 remained unchanged in the gills, indicating differential effects of the polycyclic aromatic hydrocarbon on CYP1 expression in different fish organs. The results suggested that expression profiles of inducible CYP1 enzymes in S. schlegelii might be used as indicators for assessing aquatic contamination by AHR agonists. Determination of the basal and induced expression levels, as well as substrate specificity, of the four CYP1 enzymes may contribute to a better understanding of their roles in the metabolization of toxicants or drugs.

Integrated assessment of biomarker responses and comparative toxicity pathway in snails Bellamya aeruginosa exposed to tributyltin and triphenyltin in sediments

BackgroundSediments function as a secondary and significant source of tributyltin (TBT) and triphenyltin (TPT) in aquatic ecosystems and may pose a potential threat on benthic organisms and human health. The subchronic toxicity of sediment-associated TBT or TPT to snails Bellamya aeruginosa at environmentally relevant concentrations was investigated in this study. Multiple endpoints at the biochemical [ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), protein carbonyl content (PCC) and lipid peroxidation (LPO)] and transcriptomic levels were examined.ResultsTBT or TPT in sediment could induce antioxidant enzymes’ activities and result in oxidative damage in the hepatopancreas of B. aeruginosa after 28-day exposure. A transcriptomic profile of B. aeruginosa exposed to TBT and TPT was reported. CYP genes and EROD activity were sensible and reliable biomarkers for toxicity assessment of TBT or TPT in sediments. Comparative pathway analysis revealed the alteration of steroid hormone biosynthesis and retinol metabolism in B. aeruginosa after 90-day exposure to sediment-associated TBT at the concentration of 2000 ng/g dw, which might affect both reproduction and lipogenesis functions. The ubiquitin proteasome system and immune system might be the toxicity target in B. aeruginosa after exposure to sediment-associated TPT for 90 days.ConclusionsThe results offered new mechanisms underlying the toxicity of sediment-associated tributyltin and triphenyltin.

Establishment of RNA interfered hairy root system of two CYP450 genes in Arnebia euchroma and its influence

In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β'-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75&gt;RNAi-CYP76B100&gt;RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. RESULTS:: of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.

Quantitative analysis of PAH compounds in DWH crude oil and their effects on Caenorhabditis elegans germ cell apoptosis, associated with CYP450s upregulation

The Deepwater Horizon (DWH) oil spill marked the largest environmental oil spill in human history, where it was estimated a large amount of the polycyclic aromatic hydrocarbons (PAHs) were released with crude oil into the environment. In this study, common PAH compounds were quantitatively determined in crude oil from the DWH spill by gas chromatography-mass spectroscopy (GC–MS). Twelve PAH compounds were identified and quantified from a 100× dilution of DWH crude oil: naphthalene (7800 ng/mL), acenaphthylene (590 ng/mL), acenaphtehen (540 ng/mL), fluorene (2550 ng/mL), phenanthrene (2910 ng/mL), anthracene (840 ng/mL), fluoranthene (490 ng/mL), pyrene (290 ng/mL), benzo(k) fluoranthene (1050 ng/mL), benzo(b)fluoranthene (1360 ng/mL), dibenz(a,h)anthracene (2560 ng/mL), and benzo(g, h, i) perylene (630 ng/mL). Toxicity assays using the nematode, Caenorhabditis elegans (C. elegans), indicated a single PAH compound naphthalene, exposure increased C. elegans germ cell apoptosis which may adversely affect progeny reproduction. The number of apoptotic germ cells significantly increased from 1.4 to 2.5 when worms were treated with 10 μg/mL of naphthalene and from 1.3 to 2.5 and 3.5 cells in presence of 1 μg/mL and 5 μg/mL of benzo(a)pyrene, respectively. Five CYP450 genes (CYP14A3, CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were significantly upregulated following 500× dilution of dispersed crude oil exposure (p < 0.05). These results suggest that CYP450s may play a role in bioactivation of PAHs in crude oil, resulting in DNA damage related germ cell apoptosis.

Single Nucleotide Polymorphisms in the Promoter of CYP19 Gene in Cattle Bred in Iraq

The present study was undertaken to characterize the genetic diversity of the aromatase cytochrome P450 (CYP19) gene in 34 cows (15 local, 14 Holstein, and 5 Crosses) in Iraq. The objectives of the present study are to detect SNPs (mutations) in promoter p1.1 of the CYP19 gene in cattle bred in Iraq using sequencing techniques. We identified five single-nucleotide polymorphisms (SNP) loci of the CYP19 gene that were detected, namely G933T, G994C, A1044G, A1062T, and C1468A. The results showed the presence of 3, 4, and 2 polymorphic sites leading to the construction of 4, 5, and 3 different haplotypes for Holstein, local, and crosses respectively. Haplotype diversity were 0.791, 0.752, and 0.700 respectively. While nucleotide diversity was 0.0017, 0.0022, and 0.0013 respectively. Besides, we carried out a phylogenetic analysis of these sequences to address the evolutionary relationship between the animal species. These fragments were assigned in the GenBank database under the accession numbers: LC490756, LC490757, LC491437, LC491438, LC491439, LC491588, and LC491589.

Environmental relevant concentration of λ-cyhalothrin and 3-phenoxybenzoic acid caused endocrine-disrupting effects on male lizards (Eremias argus)

In the present study, the endocrine toxicity of LCT and PBA was investigated through exposure to Eremias argus for two weeks under environmental relevant concentration. RNA-sequencing identified 4442 and 4653 differentially expressed genes in lizard liver after LCT and PBA exposure. Four differentially expressed genes (hsd17β, ar, sult, ugt) related with hypothalamic-pituitary-gonadal axis were quantified by qPCR. The expression of genes associated with HPG axis in different tissues differed significantly. In LCT treatment group, ar, cyp17 and hsd3β genes involved in testosterone synthesis and transportation were significantly decreased in lizard testes, and the spermatogensis was inhibited in the testes, which indicated the anti-androgenic activity of LCT. After PBA exposure, the genes related with estradiol synthesis, transportation and metabolism, such as hsd17β, erα, ugt in lizard liver were important biomarkers and the significant decrease of estradiol level was highly correlated with hsd17β, erα, ugt gene expressions. The relative high binding affinity of PBA with ERα further demonstrated the anti-estrogenic activity of PBA. Our results elucidate the different toxic mechanism of LCT and PBA on lizard endocrine system at environmental relevant concentration. Pyrethroids metabolism may cause more seriously toxicity rather than detoxification.

Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology.

Cytochrome P450s (CYPs) are important enzymes in the secondary metabolism of plants and have been recognized as key players in bioengineering and synthetic biology. Previously reported CYP76AH1 and CYP76AH3, having greater than 80% sequence homology, played a continuous catalytic role in the biosynthesis of tanshinones in Salvia miltiorrhiza. Homology modeling indicates that four sites might be responsible for differences in catalytic activity between the two enzymes. A series of modeling-based mutational variants of CYP76AH1 were designed to integrate the functions of the two CYPs. The mutant CYP76AH1D301E,V479F, which integrated the functions of CYP76AH1 and CYP76AH3, was found to efficiently catalyze C11 and C12 hydroxylation and C7 oxidation of miltiradiene substrates. Integration and utilization of CYP76AH1D301E,V479F by synthetic biology methods allowed the robust production of 11-hydroxy ferruginol, sugiol, and 11-hydroxy sugiol in yeast. The functionally integrated CYP gene after active site modifications improves catalytic efficiency by reducing the transfer of intermediate metabolites between component proteins. This provides a synthetic biology reference for improving the catalytic efficiencies of systems that produce plant natural products in microorganisms.

Cytochrome P450s regulates aloperine-induced pathological changes in mouse liver and kidney

Aloperine is a major active component in Sophora alopecuroides L that plays diverse pharmacological properties. Recent studies have indicated the potential effect of aloperine against hypertension and cancers. However, possible toxicity of aloperine has not been carefully studied in vivo. The aim of this study was to assess the effect of intraperitoneal aloperine injection on mouse liver and kidney tissues and to investigate the role of CYP450 genes in aloperine-induced toxicity. 72 BALB/c mice were randomly divided into four groups: vehicle control group (normal saline), low-dose group (4 mg/kg), medium-dose group (8 mg/kg), and high-dose group (16 mg/kg). 18 mice in each group were intraperitoneally injected with aloperine daily for 4 weeks, and were then kept for another 1 or 4 weeks without aloperine treatment. Serum was colleted for analysis of serum biochemical indexes including ALT, AST, BUN and CRE. The liver and kidney were collected for analysis of histopathologic changes and CYP450 expression and activity. Vacuolization of cytoplasm in liver cells, swelling in kidney tubular cells, increased levels of ALT, AST, BUN, and CRE, and alteration in the expression and activity of CYP450 were observed in the high-dose group after 4 weeks of treatment. However, all aloperine-induced damages were recovered to a certain degree after maintained without aloperine for 1 week, and fully recovered after maintained without aloperine for 4 weeks. These findings suggested that aloperine regulated the expression of CYP450, which was possibly involved in aloperine-induced reversible toxicity in mouse liver and kidney tissues.