Integrated Transcriptional and Proteomic Analysis with In Vitro Biochemical Assay Reveal the Important Role of CYP3A46 in T-2 Toxin Hydroxylation in Porcine Primary Hepatocytes

Wang, Jianshe; Jiang, Jun; Zhang, Hongxia; Wang, Junping; Cai, Hua; Li, Cheng; Li, Kangbai; Liu, Jing; Guo, Xuejiang; Zou, Guangxun; Wang, Dazhi; Deng, Yiqun; Dai, Jiayin

doi:10.1074/mcp.m111.008748

Cited by 41 publications

(30 citation statements)

References 60 publications

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“…In pigs, after T-2 toxin exposure, the mRNA levels of CYP1A2 were not significantly induced, but those of CYP3A22 and CYP3A46 were markedly induced [10,11]. Furthermore, in vitro catalysis assays suggested that the two CYP3As could metabolize T-2 to form 3′OH-T-2.…”

Section: Resultsmentioning

confidence: 99%

“…The 0.1 μg/mL T-2 dose was selected for treatment based on our previous MTT assay [10,11]. After exposure to different levels of T-2 toxin for 48 h, the IC 50 of T-2 toxin for pig hepatocytes was determined to be 0.124 μg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…T-2 toxin incubation with the S9 fractions from HeLa-CYP1A4 and HeLa-CYP1A5 and the detection of its metabolites were performed as described previously [11]. To study the metabolism of 7-ethoxyresorufin by S9 fractions, only the toxin was replaced by 7-ethoxyresorufin in the reaction system.…”

Section: Methodsmentioning

confidence: 99%

“…In pigs, some CYP3As have been reported to transform T-2 into its hydroxylation products, but until now, the specific CYP subfamilies in chickens that transform T-2 toxin into its hydroxylation products have not been reported [10,11]. Herein, we investigated which cytochrome P450 isoforms in chicken were involved in T-2 metabolism.…”

Section: Introductionmentioning

confidence: 99%

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Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3'OH-T-2

Shang

Jiang

Deng

2013

IJMS

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The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. We report that T-2 toxin significantly induces CYP1A4 and CYP1A5 expression in chicken embryonic hepatocyte cells. The enzyme activity assays of CYP1A4 and CYP1A5 heterologously expressed in HeLa cells indicate that only CYP1A5 metabolizes T-2 to 3′OH-T-2 by the 3′-hydroxylation of isovaleryl groups. In vitro enzyme assays of recombinant CYP1A5 expressed in DH5α further confirm that CYP1A5 can convert T-2 into TC-1 (3′OH-T-2). Therefore, CYP1A5 is critical for the metabolism of trichothecene mycotoxin in chickens.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3'OH-T-2

Shang

Jiang

Deng

2013

IJMS

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“…CYP3A46 may have relevance for protecting the gut against T-2 toxin [30], the mold byproduct of Fusarium spp fungus, that, among other effects, causes vomit. Interestingly, SCGB1A1 is known for its anti-inflammatory properties and for the predominant localization in Clara cells of distal conducting pulmonary airways [31], [32].…”

Section: Discussionmentioning

confidence: 99%

Differential Gene Expression in the Oxyntic and Pyloric Mucosa of the Young Pig

et al. 2014

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The stomach is often considered a single compartment, although morphological differences among specific areas are well known. Oxyntic mucosa (OXY) and pyloric mucosa (PYL, in other species called antral mucosa) are primarily equipped for acid secretion and gastrin production, respectively, while it is not yet clear how the remainder of genes expressed differs in these areas. Here, the differential gene expression between OXY and PYL mucosa was assessed in seven starter pigs. Total RNA expression was analyzed by whole genome Affymetrix Porcine Gene 1.1_ST array strips. Exploratory functional analysis of gene expression values was done by Gene Set Enrichment Analysis, comparing OXY and PYL. Normalized enrichment scores (NESs) were calculated for each gene (statistical significance defined when False Discovery Rate % <25 and P-values of NES<0.05). Expression values were selected for a set of 44 genes and the effect of point of gastric sample was tested by analysis of variance with the procedure for repeated measures. In OXY, HYDROGEN ION TRANSMEMBRANE TRANSPORTER ACTIVITY gene set was the most enriched set compared to PYL, including the two genes for H+/K+-ATPase. Pathways related to mitochondrial activity and feeding behavior were also enriched (primarily cholecystokinin receptors and ghrelin). Aquaporin 4 was the top-ranking gene. In PYL, two gene sets were enriched compared with OXY: LYMPHOCYTE ACTIVATION and LIPID RAFT, a gene set involved in cholesterol-rich microdomains of the plasma membrane. The single most differentially expressed genes were gastrin and secretoglobin 1A, member 1, presumably located in the epithelial line, to inactivate inflammatory mediators. Several genes related to mucosal integrity, immune response, detoxification and epithelium renewal were also enriched in PYL (P<0.05). The data indicate that there is significant differential gene expression between OXY of the young pig and PYL and further functional studies are needed to confirm their physiological importance.

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A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

Liu

Dai

Bones

et al. 2015

Biotechnology Progress

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A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by upregulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial-and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

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Integrated Transcriptional and Proteomic Analysis with In Vitro Biochemical Assay Reveal the Important Role of CYP3A46 in T-2 Toxin Hydroxylation in Porcine Primary Hepatocytes

Cited by 41 publications

References 60 publications

Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3'OH-T-2

Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3'OH-T-2

Differential Gene Expression in the Oxyntic and Pyloric Mucosa of the Young Pig

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

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