T-2 toxin and deoxynivalenol (DON) exert distinct effects on stress granule formation depending on altered activity of SIRT1.

The T-2 toxin and deoxynivalenol (DON), as the most concerned members of trichothecenes, induce cellular stress responses and various toxic effects. Stress granules (SGs) are rapidly formed in response to stress and play an important role in the cellular stress response. However, it is not known whether T-2 toxin and DON induce SG formation. In this study, we found that T-2 toxin induces SG formation, while DON surprisingly suppresses SG formation. Meanwhile, we discovered that SIRT1 co-localized with SGs and regulated SG formation by controlling the acetylation level of the SG nucleator G3BP1. Upon T-2 toxin, the acetylation level of G3BP1 increased, but the opposite change was observed upon DON. Importantly, T-2 toxin and DON affect the activity of SIRT1 via changing NAD+ level in a different manner, though the mechanism remains to be clarified. These findings suggest that the distinct effects of T-2 toxin and DON on SG formation are caused by changes in the activity of SIRT1. Furthermore, we found that SGs increase the cell toxicity of T-2 toxin and DON. In conclusion, our results reveal the molecular regulation mechanism of TRIs on SG formation and provide novel insights into the toxicological mechanisms of TRIs.

Role of DNA methylation-related chromatin remodeling in aryl hydrocarbon receptor-dependent regulation of T-2 toxin highly inducible Cytochrome P450 1A4 gene.

Mycotoxins are toxic secondary metabolites produced by food-contaminating fungi, which lead to global epigenetic changes and cause toxicity to both farm animals and humans. However, whether mycotoxins induce gene-specific epigenetic alterations associated with inducible downstream gene expression is unclear as are the underlying regulatory mechanisms. Here, we found that T-2 toxin and its deacetylated metabolites but not deoxynivalenol (DON) or other representative mycotoxins highly induced the expression of cytochrome P450 1A4 (CYP1A4) in both Leghorn male hepatoma (LMH) cells and chicken primary hepatocytes, and this effect was related to the regulation of both aryl hydrocarbon receptor (AhR) and DNA methylation. We used methylation-sensitive restriction enzyme digestion-qPCR (MSRE-qPCR) and chromatin immunoprecipitation (ChIP) assays and found that the binding of DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) to highly methylated CpG island 3-2 at the enhancer of CYP1A4 was accompanied by the recruitment of the repressive histone modification marker H3K27me3, inducing a silent state. In turn, T-2 toxin stimulation enriched the binding of AhR to demethylated CpG island 3-2, which facilitated p300 and H3K9ac recruitment and ultimately generated an activated chromatin structure at the enhancer by increasing the active histone modification markers, including H3K4me3, H3K27ac, and H3K14ac. Interestingly, T-2 toxin-induced AhR activation also facilitated RNA polymerase II binding to CpG island 2, which may form a transcriptionally active chromatin structure at the promoter and ultimately transactivate CYP1A4. Our findings provide novel insights into the epigenetic regulation of T-2 toxin-induced gene expression.

Toxicokinetics and metabolism of deoxynivalenol in animals and humans.

Deoxynivalenol (DON) is the most widespread mycotoxin in food and feedstuffs, posing a persistent health threat to humans and farm animals. The susceptibilities of DON vary significantly among animals, following the order of pigs, mice/rats and poultry from the most to least susceptible. However, no study comprehensively disentangles factors shaping species-specific sensitivity. In this review, the toxicokinetics and metabolism of DON are summarized in animals and humans. Generally, DON is fast-absorbed and widely distributed in multiple organs. DON is first enriched in the plasma, liver and kidney and subsequently accumulates in the intestine. There are also key variations among animals. Pigs and humans are highly sensitive to DON, and they have similar absorption rates (1 h < tmax < 4 h), high bioavailability (> 55%) and long clearance time (2 h < t1/2 < 4 h). Also, both species lack detoxification microorganisms and mainly depend on liver glucuronidation and urine excretion. Mice and rats have similar toxicokinetics (tmax < 0.5 h, t1/2 < 1 h). However, a higher proportion of DON is excreted by feces as DOM-1 in rats than in mice, suggesting an important role of gut microbiota in rats. Poultry is least sensitive to DON due to their fast absorption rate (tmax < 1 h), low oral bioavailability (5-30%), broadly available detoxification gut microorganisms and short clearance time (t1/2 < 1 h). Aquatic animals have significantly slower plasma clearance of DON than land animals. Overall, studies on toxicokinetics provide valuable information for risk assessment, prevention and control of DON contamination.

Deoxynivalenol induces caspase-3/GSDME-dependent pyroptosis and inflammation in mouse liver and HepaRG cells.

Deoxynivalenol (DON), a frequent food and feed contaminant, poses a severe threat to human and livestock health. Some studies have demonstrated that DON could induce liver damage and cell death. However, novel cell death styles and detailed mechanisms to explain DON-induced liver inflammatory injury are still lacking. Here, we found both chronic and subacute oral administration of DON (3 mg/kg for 4 weeks and 4 mg/kg for 8 days) induced mouse liver inflammatory injury and activated caspase-3, PARP and gasdermin E (GSDME), which were inhibited by caspase-3 inhibitor Z-DEVD and Ac-DEVD. In vitro, HepaRG cells showed typical pyroptotic characteristics after 32 and 64 µM DON exposure for 24 h, including balloon-like bubbling emerging, release of lactate dehydrogenase (LDH), secretion of IL-1ß and IL-6 and activation of caspase-3 and GSDME. Furthermore, knocking down GSDME and inhibiting caspases activity by Z-VAD and Z-DEVD dramatically blocked DON-induced pyroptotic characteristics, while over-expressed GSDME prompted that. These data demonstrate that caspase-3/GSDME pathway plays a key factor in DON-induced pyroptosis and inflammation in liver. Interestingly, knocking down GSDME could inhibit DON-induced pyroptosis but prompt DON-induced apoptosis, while opposite results were obtained when over-expressed GSDME, indicating the critical role of GSDME in DON-induced crosstalk between apoptosis and pyroptosis. Taken together, our data determine DON-induced caspase-3/GSDME-dependent pyroptosis in liver and its role in DON-induced liver inflammatory injury, which provide a novel mechanistic view into DON-induced hepatotoxicity and may offer a new target to reduce latent harm of DON to both humans and animals.

Phosphorylation-mediated interaction between human E26 transcription factor 1 and specific protein 1 is required for tumor cell migration.

Transcription factors, human E26 transcription factor 1 (Ets1) and specific protein 1 (Sp1), are known to induce gene expression in tumorigenicity. High Ets1 expression is often associated with colorectal tumorigenesis. In this study, we discover that metastasis and clone formation in SW480 cells mainly depend on the direct interaction between Ets1 and Sp1 instead of high Ets1 expression. The interaction domains are further addressed to be the segment at Sp1(626-708) and the segment at Ets1(244-331). In addition, the phosphorylation inhibition of Ets1 at Tyr283 by either downregulation of Src kinase or Src family inhibitor treatment decreases the interaction between Sp1 and Ets1 and suppresses SW480 migration. Either administration or overexpression of the peptides harboring the interaction segment strongly inhibits the colony formation and migration of SW480 cells. Our findings suggest that the interaction between Ets1 and Sp1 rather than Ets1 alone promotes transformation in SW480 cells and provide new insight into the Ets1 and Sp1 interaction as an antitumour target in SW480 cells.

Influenza A virus protein PA-X suppresses host Ankrd17-mediated immune responses.

Influenza A virus (IAV) PA-X is a critical ribonuclease protein involved in host cell shutoff but its role in modulating the host immune response to IAV infection remains to be addressed. In this study, host cellular proteins that directly interact with PA-X were screened to investigate the biological function of PA-X in the pathogenesis of IAV infection. The protein ankyrin repeat domain 17 (Ankrd17), a positive regulator of inflammatory responses via the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, was identified as a specific PA-X binding partner that preferred PA-X to the PA protein. The N-terminal ankyrin repeats of Ankrd17 are the key domain for the interaction with PA-X rather than PA, which is required for the function of Ankrd17 in elevating the host immune response. Using Ankrd17 knockout and overexpression, we confirmed that PA-X significantly affected the Ankrd17-mediated response to infection in host cells. Our data therefore reveal a novel function for PA-X in the regulation of innate immune pathways via the interaction between PA-X and Ankrd17.

Inhibition of EZH2 and activation of ERRγ synergistically suppresses gastric cancer by inhibiting FOXM1 signaling pathway.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related mortality worldwide, because of the low efficacy of current therapeutic strategies. Estrogen-related receptor γ (ERRγ) was previously showed as a suppressor of GC. However, the mechanism and effective therapeutic method based on ERRγ is yet to be developed. METHODS: The expression levels of ERRγ, EZH2, and FOXM1 were detected by immunohistochemistry, qRT-PCR, and western blot. The regulatory mechanisms of ERRγ and FOXM1 were analyzed by ChIP, EMSA, and siRNA. The effects of EZH2 inhibitor (GSK126) or/and ERRγ agonist (DY131) on the tumorigenesis of gastric cancer cell lines were examined by cell proliferation, transwell migration, wound healing, and colony formation assays. Meanwhile, the inhibitory effects of GSK126 or/and DY131 on tumor growth were analyzed by xenograft tumor growth assay. RESULTS: The expression of ERRγ was suppressed in tumor tissues of GC patients and positively correlated with prognosis, as opposed to that of EZH2 and FOXM1. EZH2 transcriptionally suppressed ERRγ via H3K27me3, which subsequently activated the expression of master oncogene FOXM1. The combination of GSK126 and DY131 synergistically activated ERRγ expression, which subsequently inhibited the expression of FOXM1 and its regulated pathways. Synergistic combination of GSK126 and DY131 significantly inhibited the tumorigenesis of GC cell lines and suppressed the growth of GC xenograft. CONCLUSION: The FOXM1 signaling pathway underlying the ERRγ-mediated gastric cancer suppression was identified. Furthermore, combined treatment with EZH2 inhibitor and ERRγ agonist synergistically suppressed GC progression by inhibiting this signaling pathway, suggesting its high potential in treating GC patients.

Derived habitats of indoor microbes are associated with asthma symptoms in Chinese university dormitories.

Increasing evidence from the home environment indicates that indoor microbiome exposure is associated with asthma development. However, indoor microbiome composition can be highly diverse and dynamic, and thus current studies fail to produce consistent results. Chinese university dormitories are special high-density dwellings with similar building and occupants characteristics, which facilitate to disentangle the complex interactions between microbes, environmental characteristics and asthma. Settled air dust and floor dust was collected from 87 dormitory rooms in Shanxi University. Bacterial communities were characterized by 16 S rRNA amplicon sequencing. Students (n = 357) were surveyed for asthma symptoms and measured for fractional exhaled nitric oxide (FeNO). Asthma was not associated with the overall bacterial richness but associated with specific phylogenetic classes. Taxa richness and abundance in Clostridia, including Ruminococcus, Blautia, Clostridium and Subdoligranulum, were positively associated with asthma (p < 0.05), and these taxa were mainly derived from the human gut. Taxa richness in Alphaproteobacteria and Actinobacteria were marginally protectively associated with asthma, and these taxa were mainly derived from the outdoor environment. Bacterial richness and abundance were not associated with FeNO levels. Building age was associated with overall bacterial community variation in air and floor dust (p < 0.05), but not associated with the asthma-related microorganisms. Our data shows that taxa from different phylogenetic classes and derived habitats have different health effects, indicating the importance of incorporating phylogenetic and ecological concepts in revealing patterns in the microbiome asthma association analysis.

Quantitative proteomics implicates YggT in streptomycin resistance in Salmonella enterica serovar Enteritidis.

OBJECTIVES: To identify proteins that may be associated with antibiotic resistance in the multidrug-resistant Salmonella enterica D14, by constructing proteomic profiles using mass spectrometry-based label-free quantitative proteomics (LFQP). RESULTS: D14 was cultured with four antibiotics (ampicillin, nalidixic acid, streptomycin, and tetracycline) separately. Subsequently, the findings from an equal combination of the four cultures were compared with the profile of sensitive S. enterica 104. 2255 proteins, including 149 differentially up-regulated proteins, were identified. Many of these up-regulated proteins were associated with flagellar assembly and chemotaxis, two-component system, amino acid metabolism, ß-lactam resistance, and transmembrane transport. A subset of 10 genes was evaluated via quantitative real-time PCR (qPCR), followed by the construction of cheR, fliS, fliA, arnA, and yggT deletion mutants. Only the yggT-deleted D14 mutant showed decrease in streptomycin resistance, whereas the other deletions had no effect. Furthermore, complementation of yggT and the overexpression of yggT in S. enterica ATCC 14028 increased the streptomycin resistance. Additionally, spot dilution assay results confirmed that Salmonella strains, harboring yggT, exhibited an advantage in the presence of streptomycin. CONCLUSIONS: The above proteomic and mutagenic analyses revealed that yggT is involved in streptomycin resistance in S. enterica.

Cereulide Exposure Caused Cytopathogenic Damages of Liver and Kidney in Mice.

Cereulide is one of the main food-borne toxins for vomiting synthesized by Bacillus cereus, and it widely contaminates meat, eggs, milk, and starchy foods. However, the toxicological effects and mechanisms of the long-time exposure of cereulide in vivo remain unknown. In this study, oral administration of 50 and 200 µg/kg body weight cereulide in the mice for 28 days caused oxidative stress in liver and kidney tissues and induce abnormal expression of inflammatory factors. In pathogenesis, cereulide exposure activated endoplasmic reticulum stress (ER stress) via the pathways of inositol-requiring enzyme 1α (IRE1α)/Xbox binding protein (XBP1) and PRKR-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (eIF2α), and consequently led to the apoptosis and tissue damages in mouse liver and kidney. In vitro, we confirmed that the accumulation of reactive oxygen species (ROS) caused by cereulide is the main factor leading to ER stress in HepaRG and HEK293T cells. Supplementation of sodium butyrate (NaB) inhibited the activations of IRE1α/XBP1 and PERK/eIF2α pathways caused by cereulide exposure in mice, and reduced the cell apoptosis in liver and kidney. In conclusion, this study provides a new insight in understanding the toxicological mechanism and prevention of cereulide exposure.

T-2 Toxin Induces Oxidative Stress at Low Doses via Atf3ΔZip2a/2b-Mediated Ubiquitination and Degradation of Nrf2.

T-2 toxin is mainly produced by Fusarium species, which is an extremely toxic mycotoxin to humans and animals. It is well known that T-2 toxin induces oxidative stress, but the molecular mechanism is still unknown. In this study, we found that T-2 toxin significantly promoted reactive oxygen species (ROS) accumulation in MCF-7 cells at low doses which maintains cell viability at least 80%. Further analysis showed that T-2 toxin downregulated the expression of the master regulator of antioxidant defense gene, nuclear factor erythroid 2-related factor (Nrf2), and its targeted antioxidant genes. Overexpression of Nrf2 or its target gene heme oxygenase 1 (HO1) significantly blocked the ROS accumulation in MCF-7 cells under T-2 toxin treatment. Moreover, we found that T-2 toxin downregulated the antioxidant genes via inducing the expression of ATF3ΔZip2a/2b. Importantly, overexpression of ATF3ΔZip2a/2b promoted the ubiquitination and degradation of Nrf2. Altogether, our results demonstrated that T-2 toxin-induced ROS accumulation via ATF3ΔZip2a/2b mediated ubiquitination and degradation of Nrf2, which provided a new insight into the mechanism of T-2 toxin-induced oxidative stress.

Identification of NOVA family proteins as novel ß-catenin RNA-binding proteins that promote epithelial-mesenchymal transition.

The NOVA (neuro-oncological ventral antigen) protein family, composed of two paralogs, NOVA1 and NOVA2, consists of RNA-binding proteins involving in processes such as alternative splicing and transport of some target mRNAs. The function of NOVA has been well studied, and increasing evidence has shown that NOVA proteins may be important contributors to carcinogenesis. However, the molecular mechanisms underlying the roles of NOVA proteins in carcinogenesis remain to be determined. Here, we have identified both NOVA1 and NOVA2 as novel ß-catenin RNA-binding proteins. The NOVA1/NOVA2 heterodimer positively regulates ß-catenin expression by enhancing ß-catenin mRNA stability. Furthermore, we demonstrated that NOVA1 and NOVA2 promote epithelial-mesenchymal transition via ß-catenin in breast cancer cells, as NOVA-induced upregulation of epithelial and mesenchymal marker expression was attenuated by restoring ß-catenin expression. Our results advance the current understanding of ß-catenin post-transcriptional regulation and shed light on the role of NOVA proteins in cancer, suggesting that NOVA proteins are potential therapeutic targets in breast cancer.

Deoxynivalenol globally affects the selection of 3' splice sites in human cells by suppressing the splicing factors, U2AF1 and SF1.

Deoxynivalenol (DON) is one of the most abundant mycotoxins and has adverse effects on several biological processes, posing risks of protein synthesis-disrupting effects and ribotoxic response. Therefore, chronic exposure to DON would fundamentally reshape the global expression pattern. Whether DON causes toxic effects on mRNA splicing, a fundamental biological process, remains unclear. In this study, we found that administration of the relative low dosage of DON dramatically changed the alternative splicing of pre-mRNA in HepG2 cells. The overall number of transcripts with aberrant selection of 3' splice sites was significantly increased in DON-exposed HepG2 cells. This effect was further confirmed in two other human cell lines, HEK293 and Caco-2, suggesting that this DON-induced alteration in splicing patterns was universal in human cells. Among these DON-induced changes in alternative splicing, the expression levels of two related splicing factors, SF1 and U2AF1, which are essential for 3' splice site recognitions, were strongly suppressed. Overexpression of either of the two splicing factors strongly alleviated the DON-induced aberrant selection of 3' splice sites. Moreover, SF1 was required for human cell proliferation in DON exposure, and the restoration of SF1 expression partially reinstated the proliferation potential for DON-treated cells. In conclusion, our study suggests that DON, even at a low dosage, has great potential to change gene expression globally by affecting not only protein synthesis but also mRNA processing in human cells.

Shotgun metagenomics of dust microbiome from flight deck and cabin in civil aviation aircraft.

Microbial exposure is related to the health of passengers on commercial aircraft, but no studies characterized the microbial composition at the species level and identified their ecological determinants. We collected vacuum dust from floor and seat surfaces in flight decks and cabins of 18 aircraft, and amplification-free shotgun metagenomics was conducted to characterize the microbial composition. In total, 7437 microbial taxa were identified. The relative abundance for bacteria, eukaryote, viruses, and archaea was 96.9%, 1.8%, 0.3%, and 0.03%, respectively. The top bacterial species mainly derived from outdoor air and human skin included Sphingomonas, Corynebacterium, Micrococcus luteus, Variovorax paradoxus, Paracoccus dentrificans, and Propionibacterium acnes. The abundance of NIAID-defined pathogens was low, accounted for only 0.23% of total microbes. The microbial species and functional composition were structured by the indoor surface type (R2  = 0.38, Adonis), followed by the manufacturer of the aircraft (R2  = 0.12) and flight duration (R2  = 0.07). Indoor surfaces affected species derived from different habitats; the abundance of dry skin and desiccated species was higher on textile surfaces, whereas the abundance of moist and oily skin species was higher on leather surfaces. The growth rates for most microbes were stopped and almost stopped.

Associations between respiratory infections and bacterial microbiome in student dormitories in Northern China.

Recent studies reveal that the microbial diversity and composition in the respiratory tract are related to the susceptibility, development, and progression of respiratory infections. Indoor microorganisms can transmit into the respiratory tract through breathing, but their role in infections is unclear. Here, we present the first association study between the indoor microbiome and respiratory infections. In total, 357 students living in 86 dormitory rooms in Shanxi University were randomly selected to survey symptoms of infections. Settled air dust was collected to characterize bacterial compositions by 16S rRNA sequencing. The overall microbial richness was not associated with respiratory infections, but microorganisms from specific phylogenetic classes showed various associations. Taxa richness and abundance of Actinobacteria were protectively associated with infections (P < .05). The abundance of several genera in Gammaproteobacteria, including Haemophilus, Klebsiella, Buttiauxella, and Raoultella, was positively associated with infections (P < .005). The role of these microorganisms was consistent with previous human microbiota studies. Building age was associated with the overall microbial composition variation in dormitories and negatively associated with three potential risk genera in Proteobacteria (P < .05). The weight of vacuum dust was positively associated with a protective genus, Micrococcus in Actinobacteria (P < .05).

Aflatoxin B1 Induces Neurotoxicity through Reactive Oxygen Species Generation, DNA Damage, Apoptosis, and S-Phase Cell Cycle Arrest.

Aflatoxin B1 (AFB1) is a mycotoxin widely distributed in a variety of food commodities and exhibits strong toxicity toward multiple tissues and organs. However, little is known about its neurotoxicity and the associated mechanism. In this study, we observed that brain integrity was markedly damaged in mice after intragastric administration of AFB1 (300 µg/kg/day for 30 days). The toxicity of AFB1 on neuronal cells and the underlying mechanisms were then investigated in the neuroblastoma cell line IMR-32. A cell viability assay showed that the IC50 values of AFB1 on IMR-32 cells were 6.18 µg/mL and 5.87 µg/mL after treatment for 24 h and 48 h, respectively. ROS levels in IMR-32 cells increased significantly in a time- and AFB1 concentration-dependent manner, which was associated with the upregulation of NOX2, and downregulation of OXR1, SOD1, and SOD2. Substantial DNA damage associated with the downregulation of PARP1, BRCA2, and RAD51 was also observed. Furthermore, AFB1 significantly induced S-phase arrest, which is associated with the upregulation of CDKN1A, CDKN2C, and CDKN2D. Finally, AFB1 induced apoptosis involving CASP3 and BAX. Taken together, AFB1 manifests a wide range of cytotoxicity on neuronal cells including ROS accumulation, DNA damage, S-phase arrest, and apoptosis-all of which are key factors for understanding the neurotoxicology of AFB1.

Deoxynivalenol Exposure Suppresses Adipogenesis by Inhibiting the Expression of Peroxisome Proliferator-Activated Receptor Gamma 2 (PPARγ2) in 3T3-L1 Cells.

Deoxynivalenol (DON)-a type B trichothecene mycotoxin, mainly produced by the secondary metabolism of Fusarium-has toxic effects on animals and humans. Although DON's toxicity in many organs including the adrenal glands, thymus, stomach, spleen, and colon has been addressed, its effects on adipocytes have not been investigated. In this study, 3T3-L1 cells were chosen as the cell model and treated with less toxic doses of DON (100 ng/mL) for 7 days. An inhibition of adipogenesis and decrease in triglycerides (TGs) were observed. DON exposure significantly downregulated the expression of PPARγ2 and C/EBPα, along with that of other adipogenic marker genes in 3T3-L1 cells and BALB/c mice. The anti-adipogenesis effect of DON and the downregulation of the expression of adipogenic marker genes were effectively reversed by PPARγ2 overexpression. The repression of PPARγ2's expression is the pivotal event during DON exposure regarding adipogenesis. DON exposure specifically decreased the di-/trimethylation levels of Histone 3 at lysine 4 in 3T3-L1 cells, therefore weakening the enrichment of H3K4me2 and H3K4me3 at the Pparγ2 promoter and suppressing its expression. Conclusively, DON exposure inhibited PPARγ2 expression via decreasing H3K4 methylation, downregulated the expression of PPARγ2-regulated adipogenic marker genes, and consequently suppressed the intermediate and late stages of adipogenesis. Our results broaden the current understanding of DON's toxic effects and provide a reference for addressing the toxicological mechanism of DON's interference with lipid homeostasis.

Coordinated Transcriptional Regulation of Cytochrome P450 3As by Nuclear Transcription Factor Y and Specificity Protein 1.

The cytochrome P450 3A subfamily plays vital roles in the metabolism of endogenous chemicals and xenobiotics. Understanding the basal expression of CYP3A in humans and pigs is crucial for drug evaluation. In this study, we demonstrated that the basal transcriptional regulation of CYP3A genes in hepatocytes is evolutionarily conserved between humans and pigs. The basal expression of CYP3A genes is transactivated by two cis-acting elements, the CCAAT and GC boxes, located a constant distance apart in the proximal promoter region of six CYP3A genes. Mutation analysis of these two cis-acting elements suggested that they play important roles in mediating basal expression, but to different extents because of the nucleotide variations in the elements. Two transcription factors, nuclear transcription factor Y (NF-Y) and specificity protein 1 (Sp1), directly bind to these cis-acting elements in CYP3A proximal promoters in HepG2 cells and porcine hepatocytes. Furthermore, changing the distance between the NF-Y and Sp1 binding sites resulted in decreases in the promoter activity of CYP3A genes. Conclusively, our results show that human and porcine CYP3A genes are regulated by NF-Y and Sp1 in a coordinated manner, and that the distance between these two cis-acting elements is crucial for constitutive CYP3A expression.

T-2 toxin induces the expression of porcine CYP3A22 via the upregulation of the transcription factor, NF-Y.

BACKGROUND: T-2 toxin is one of the major pollutants in crops and feedstuffs. CYP3A22, one of hCYP3A4 homologs, detoxifies T-2 toxin in pigs. We investigated the mechanisms of expression activation of CYP3A22 under basal and induced conditions. METHODS: Based on MatInspector analysis, several mutations in the CYP3A22 promoter were assayed by dual luciferase reporter to identify the function of cis elements in the region. EMSA experiments were used to assess the binding of transcription factors to the cis elements. The mRNA and protein levels of CYP3A22 and the transcription factors were measured by RT-qPCR and Western blot. The enhancement of NF-Y binding to the CYP3A22 promoter was assayed by ChIP. RESULTS: As predicted, two cis DNA elements in the CYP3A22 promoter, a CCAAT box and GC box, were confirmed to be crucial in the activation of CYP3A22 transcription. These two DNA motifs recruited two transcription factors, NF-Y and Sp1, which are involved in the activation of the basal transcription of CYP3A22. More interestingly, CYP3A22 expression was induced in porcine primary hepatocytes by the treatment with 0.1µg/mL T-2 toxin. This induction of transcription by T-2 toxin was dominantly regulated by the binding of NF-Y to the CCAAT box, rather than GC box, which recruits Sp1 and functions only in the constitutive expression of CYP3A22. CONCLUSIONS: Our study reveals the regulatory mechanisms of both basal and inducible transactivation of CYP3A22 in pigs. In particular, we identified that the mechanism by which T-2 toxin induces CYP3A22 expression is mediated by the upregulation of NF-YA. GENERAL SIGNIFICANCE: Although porcine CYP3A22 is homologous to hCYP3A4, the regulation of basal and induced expression of CYP3A22 occurred via distinct mechanisms. This may account for the variety of CYP3A expression in animals and humans.

Role of Specificity Protein 1, Hepatocyte Nuclear Factor 1α, and Pregnane X Receptor in the Basal and Rifampicin-Induced Transcriptional Regulation of Porcine Cytochrome P450 3A46.

Cytochrome P450 (CYP) 3A46, one of human CYP3A4 homologs, functions as a key enzyme in the metabolism of xenobiotics in pigs. However, the regulatory mechanism for the transcriptional activation of CYP3A46 in porcine liver remains unknown. In this study, we confirmed that CYP3A46 is constitutively expressed in porcine primary hepatocytes, and its expression was significantly induced by rifampicin (RIF) instead of dexamethasone. We further found that a proximal GC box and a distal hepatocyte nuclear factor 1 (HNF1) binding site within the 5'-flanking region of CYP3A46 are the important cis-regulatory elements involved in regulating the constitutive expression of CYP3A46, via recruiting specificity protein 1 (Sp1) and HNF1α, respectively. Furthermore, we revealed that HNF1α and pregnane X receptor (PXR) activate the RIF-mediated transcription of CYP3A46 by binding to the distal HNF1 binding site and the proximal direct repeats of AGGTCA separated by 4 bases motif, respectively. Meanwhile, HNF1α is also involved in regulating RIF-induced expression of CYP3A4 through a novel distal HNF1 binding site identified in the xenobiotic-responsive enhancer module. In summary, our data demonstrate that several transcription factors, including Sp1, HNF1α, and PXR, function in the basal and RIF-mediated transcriptional regulation of CYP3A46 by binding to their related cis-regulatory elements in the proximal promoter and distal enhancer.