Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification.

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

Comparative Analysis of the Physicochemical Properties and Metabolites of Farinose and Crisp Lotus Roots (Nelumbo nucifera Gaertn.) with Different Geographical Origins.

Lotus roots are widely consumed vegetables because of their great taste and abundant nutrients, but their quality varies with the environments and cultivar. This study systematically compared farinose (Elian No. 5) and crisp (Elian No. 6) lotus root cultivars from three geographical origins. Pasting and texture characteristics verified that Elian No. 5 possessed lower hardness and lower ability to withstand shear stress and heating during cooking compared with Elian No. 6. Untargeted metabolite profiling was first performed using ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with a Zeno trap. In total, 188 metabolites were identified based on the matching chemistry database. Multivariate analysis demonstrated that lotus roots from different cultivars and origins could be adequately distinguished. Sixty-one differential metabolites were identified among three Elian No. 5 samples, and 28 were identified among three Elian No. 6 samples. Isoscopoletin, scopoletin, and paprazine were the most differential metabolites between Elian No. 5 and Elian No. 6. These results can inform future research on the discrimination and utilization of lotus roots.

Facile fabrication of naphthalene-functionalized magnetic nanoparticles for efficient extraction of polycyclic aromatic hydrocarbons from environmental water and fish samples.

In this study, naphthalene-modified magnetic nanoparticles (Fe3O4@Nap) were simply prepared based on specific chelation interaction between phosphate groups and metal ions on Fe3O4 surface. The resultant Fe3O4@Nap were characterized by FTIR, BET, SEM, TEM, NAM, TGA, and VSM techniques. With Fe3O4@Nap as adsorbent, the polycyclic aromatic hydrocarbons (PAHs) were efficiently extracted by magnetic solid-phase extraction (MSPE) from environmental water and fish samples through the π-π interaction between modified naphthalene groups and PAHs, followed by their determination by GC-MS/MS. The key parameters influencing the extraction efficiency were investigated. Under the optimized conditions, the Fe3O4@Nap-based MSPE/GC-MS/MS method proposed in this paper was evaluated and applied for analyzing PAHs in environmental water and fish samples. And the proposed MSPE/GC-MS/MS method exhibited good linearities for water samples (in the range of 0.1-10 ng/mL, R2 >0.9945) and for fish samples (in the range of 1-100 ng/g, R2 > 0.9905). The limits of detection (LODs) for water and fish samples were 0.004-0.031 ng/mL and 0.07-0.28 ng/g, respectively. Additionally, this method exhibited desirable accuracy and precision. The PAH recovery values from water and fish samples ranged from 81.5% to 109.6% with inter- and intra-day relative standard deviations (RSDs) of less than 12.8%. The MSPE/GC-MS/MS method was successfully applied to the analysis of real environmental water and fish samples. Overall, the newly synthesized Fe3O4@Nap exhibited high sensitivity, specificity, reusability, repeatability, and it could efficiently extract PAHs from environmental water and fish samples by MSPE.

Genome Mining and Analysis of PKS Genes in Eurotium cristatum E1 Isolated from Fuzhuan Brick Tea.

Eurotium cristatum as the dominant fungi species of Fuzhuan brick tea in China, can produce multitudinous secondary metabolites (SMs) with various bioactivities. Polyketides are a very important class of SMs found in E. cristatum and have gained extensive attention in recent years due to their remarkable diversity of structures and multiple functions. Therefore, it is necessary to explore the polyketides produced by E. cristatum at the genomic level to enhance its application value. In this paper, 12 polyketide synthase (PKS) genes were found in the whole genome of E. cristatum E1 isolated from Fuzhuan brick tea. In addition, the qRT-PCR results further demonstrated that these genes were expressed. Moreover, metabolic analysis demonstrated E. cristatum E1 can produce a variety of polyketides, including citreorosein, emodin, physcion, isoaspergin, dihydroauroglaucin, iso-dihydroauroglaucin, aspergin, flavoglaucin and auroglaucin. Furthermore, based on genomic analysis, the putative secondary metabolites clusters for emodin and flavoglaucin were proposed. The results reported here will lay a good basis for systematically mining SMs resources of E. cristatum and broadening its application fields.

Flowerlike Ni-NiO composite as magnetic solid-phase extraction sorbent for analysis of carbendazim and thiabendazole in edible vegetable oils by liquid chromatography-mass spectrometry.

A rapid, selective, and sensitive method was developed for the detection of carbendazim and thiabendazole in edible vegetable oil. Two benzimidazole analytes were pre-concentrated by magnetic solid phase extraction (MSPE) using flowerlike Ni-NiO composite as sorbents and followed by LC-MS/MS analysis. The flowerlike Ni-NiO composite sorbent displayed a high affinity towards benzimidazole analytes due to the reversible coordination interaction between the Ni(Ⅱ) ion and the electron-donating imidazole group. In comparison to the previous methods, this procedure is less time-consuming and simpler during sample preparation. The parameters affecting the extraction efficiency were optimized in detail. The method was validated according to SANTE/12682/2019. The limits of detection were in the range of 0.001-0.003 mg•kg-1. The recoveries ranged from 89.3% to 110.7% with inter-day and inter-day precision less than 10.9%. The results indicate that flowerlike Ni-NiO composite might be a promising alternative for MSPE of benzimidazole compounds in foods.

Dispersive liquid-liquid microextraction based on a new hydrophobic deep eutectic solvent for the determination of phenolic compounds in environmental water samples.

Dispersive liquid-liquid microextraction has garnered increasing attention in sample preparation due to its rapid and efficient extraction process. In this study, a new terpineol-based hydrophobic deep eutectic solvent was firstly synthesized by mixing α-terpineol with 1-octanoic acid, and then applied to analysis of phenols from water samples by dispersive liquid-liquid microextraction combined with high-performance liquid chromatography and diode array detection. Infrared spectroscopy indicated that hydrogen bonding was responsible for the formation of deep eutectic solvent between α-terpineol and 1-octanoic acid. After optimization of several parameters, such as the type and volume of deep eutectic solvent and the disperser, pH and ionic strength of sample solution, the developed method exhibited excellent extraction performance to the phenols with the enrichment factors from 27 to 32. Good linearity was acquired ranging from 5 to 5000 µg/L, and detection of limits of the proposed method for the phenols ranged from 0.15 to 0.38 µg/L. The recoveries measured by spiked samples at three concentration levels ranged from 81.6 to 99.3%, and precision was found with intra- and inter-day relative standard deviations less than 8.7 and 9.2%, respectively. Finally, the proposed method was successfully applied to the determination of the phenols in environmental water samples.

Multifunctionalized magnetic mesoporous silica as an efficient mixed-mode sorbent for extraction of phenoxy carboxylic acid herbicides from water samples followed by liquid chromatography-mass spectrometry in tandem.

In current study, a rapid and sensitive method for simultaneous determination of 8 phenoxy carboxylic acid (PCA) herbicides in environmental water samples was established based on an octyl and amino functionalized magnetic mesoporous silica (mOAS). The mOAS sorbent was fabricated by simple modification of octyl and aminopropyl groups onto the surface of magnetic mesoporous silica. Various techniques were employed to characterize the physical and chemical properties of the mOAS sorbent. The mOAS sorbent showed good extraction capacity and selectivity for PCA herbicides owing to the mixed-mode hydrophobic and ionic exchange interaction mechanisms. Then a one-factor-at-a-time approach was utilized for the optimization of extraction, washing and eluting conditions. Under the optimum conditions, wide linearity ranges with squared correlation coefficients ranging from 0.9888 to 0.9966 were obtained, and the method detection of limits (LODs) were in the range of 0.005-0.02 ng/mL. Furthermore, recoveries ranged from 86.9 to 114.6% with intra- and inter-day relative standard deviation (RSD) of 0.8-11.0% at three concentration levels spiked in blank water sample. Finally, the as-proposed method was successfully applied to extract 8 PCA herbicides in real water samples followed by determination with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Results showed that the mOAS as adsorbent for magnetic solid phase extraction (MSPE) of acidic herbicides is an efficient alternative with simplicity and rapidity.

Rapid and Sensitive Detection of Aflatoxin B1, B2, G1 and G2 in Vegetable Oils Using Bare Fe3O4 as Magnetic Sorbents Coupled with High-Performance Liquid Chromatography with Fluorescence Detection.

This paper reports a simple, sensitive and reliable method for the simultaneous detection of aflatoxin B1, B2, G1 and G2 in vegetable oils. Aflatoxins were extracted by magnetic solid phase extraction followed by high-performance liquid chromatography, then postcolumn photochemical derivatization and finally detected by fluorescence detector. Vegetable oil samples were first diluted with hexane and then commercial bare Fe3O4 nanoparticles were directly employed as sorbents to extract aflatoxins from complex vegetable oil samples, which significantly simplified the procedure of sample preparation and largely improved the sample analysis throughput. The effects of various parameters such as the amount of sorbent, loading, washing and eluting conditions were carefully optimized to improve the extraction efficiencies of aflatoxins. Under the optimal conditions, the limits of detection of four aflatoxins ranged from 0.01 µg/kg to 0.16 µg/kg, and squared regression coefficients (R2) >0.9990 were obtained within the linear range of 0.1-20 µg/kg (except for aflatoxin G2 with 0.5-20 µg/kg). Furthermore, the recoveries spiked at four concentration levels in a blank vegetable oil sample were from 82.6 to 106.2%, with inter- and intraday relative standard deviations <9.8%, indicating good accuracy and precision of the proposed method.

Acidic conditions induce the accumulation of orange Monascus pigments during liquid-state fermentation of Monascus ruber M7.

The influence of pH on the biosynthesis of orange Monascus pigments (OMPs) in Monascus ruber M7 was investigated. Under acidic fermentation conditions, pigment mixtures predominantly rich in OMPs were obtained. HPLC analysis revealed the presence of four orange components (O1-O4) and four yellow components (Y1-Y4) in the mixtures, and the dominant ones were O1 and O3, which accounted for 56.0% to 75.9% of the total pigments in the pH range 3-6. Subsequently, O1 and O3 were identified by LC-DAD-ESI/MS as Rubropunctatin and Monascorubrin, respectively. The yield of OMPs was observed to be inversely dependent on pH. At pH 3, large amounts of OMPs with high purity (79.1%) were accumulated. A real-time quantitative PCR analysis revealed that the expression of genes related to the biosynthesis of OMPs in M. ruber M7 was upregulated at acidic pH as compared to neutral pH, and the variation in the level of expression of these genes with pH was consistent with the production of OMPs. These results indicated that the large accumulation of OMPs under acidic condition involved the acidic pH-induced transcription of genes related to the biosynthesis of OMPs. These results would contribute towards the development of an efficient technology for large-scale production of OMPs.

Cyclic peptide: a safe and effective alternative to synthetic aflatoxin B1-competitive antigens.

Aflatoxin B1 (AFB1) is one of the major mycotoxins, which naturally occurs in food and agricultural products. In this study, a cyclic peptide (CVPSKPGLC) mimicking AFB1 was used to develop a biotinylated peptide-based immunoassay (bp-ELISA) for AFB1 determination. This cyclic peptide was isolated from a commercially available phage-displayed random 7-mer cyclic peptide library, and then synthesized chemically. Instead of phage particles, the peptide was biotinylated and used to detect AFB1 by bp-ELISA, with an IC50 of 0.92 ng/mL, which was approximately 60-fold better than that of phage ELISA. Good recoveries (83-102%) were obtained in spiked rice and corn samples, which were further validated by high-performance liquid chromatography-fluorescence detector. As better sensitivities (0.92-1.21 ng/mL) were obtained by bp-ELISA even using selected anti-AFB1 antibodies prepared previously in laboratory, this cyclic peptide is suitable as a substitute for synthetic competitive AFB1 antigens in food contamination monitoring. Graphical abstract.

A Comprehensive Analysis of the Small GTPases Ypt7 Involved in the Regulation of Fungal Development and Secondary Metabolism in Monascus ruber M7.

Ypts (yeast protein transports),also called as ras-associated binding GTPases (Rab), are the largest group of the small GTPases family, which have been extensively studied in model eukaryotic cells and play a pivotal role in membane trafficking, while this study showed potential regulation role of Ypts in fungi. One of Ypts, Ypt7 may be involved in fungal development and secondary metabolism, but the exact mechanism still exists a controversy. In current study, the functions of a Monascus ypt7 homologous gene (mrypt7) from Monascus ruber M7 was investigated by combination of gene-deletion (Δmrypt7), overexpression (M7::PtrpC-mrypt7) and transcriptome analysis. Results showed that the radial growth rate of Δmrypt7 was significantly slower than M. ruber M7, little conidia and ascospores can be observed in Δmrypt7, but the yield of intracellular secondary metabolites was dramatically increased. Simultaneously, the mrypt7 overexpression strain possessed similar capacity for sporulation and secondary metabolism observed in M. ruber M7. Transcriptome results further illustrated that mrypt7 could coordinate with numerous genes involved in the vegetative growth, conidiogenesis, secondary metabolism biosynthesis and transportation of M. ruber M7. Combined with the similar effect of Ypt7 homologs on other fungi, we propose that Ypt7 works more like a global regulatory factor in fungi. To our knowledge, it is the first time to investigate Ypt7 functions in Monascus. It could also improve the understanding of Ypt7 functions in fungi.

Rapid and sensitive detection of the phenoxy acid herbicides in environmental water samples by magnetic solid-phase extraction combined with liquid chromatography-tandem mass spectrometry.

Phenoxy acid herbicides are widely used herbicides that play an important role in improving the yield and quality of crops. However, some research has shown that this kind of herbicide is poisonous to human and animals. In this study, a rapid and sensitive method was developed for the detection of seven phenoxy acid herbicides in water samples based on magnetic solid-phase extraction followed by liquid chromatography and tandem mass spectrometry. Magnetic amino-functionalized multiwalled carbon nanotubes were prepared by mixing bare magnetic Fe3 O4 nanoparticles with commercial amino-functionalized multiwalled carbon nanotubes in water. Then the amino-functionalized multiwalled carbon nanotubes were used to enrich phenoxy acid herbicides from water samples based on hydrophobic and ionic interactions. The effects of experimental variables on the extraction efficiency have been studied in detail. Under the optimized conditions, the method validation was performed. Good linearities for seven phenoxy acid herbicides were obtained with squared regression coefficients ranging from 0.9971 to 0.9989. The limits of detection ranged from 0.01 to 0.02 µg/L. The method recoveries of seven phenoxy acid herbicides spiked at three concentration levels in a blank sample were from 92.3 to 103.2%, with inter- and intraday relative standard deviations less than 12.6%.

Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi.

Monascus azaphilone pigments (MonAzPs) are very widely used as food colorants, but their biosynthetic pathway has remained poorly characterized for more than half a century. In this study, the individual steps of MonAzPs biosynthesis in Monascus ruber M7 were elucidated by a combination of targeted gene knockouts, heterologous gene expression, and in vitro chemical and enzymatic reactions. This study describes the first rational engineering of MonAzPs biosynthesis and provides a roadmap for future pathway engineering efforts directed towards the selective production of the most valuable pigments and serves as a model for the biosynthesis of fungal azaphilones in general.

NAD+-dependent HDAC inhibitor stimulates Monascus pigment production but inhibit citrinin.

Monascus species are edible fungi due to the production of food colorant and other beneficial compounds. Hence, it has been an attractive thesis to improve their productivities. Increasing numbers of investigations revealed that regulating the activities of histone deacetylases can significantly perturb secondary metabolites (SM) production at a global level. In this study, dihydrocoumarin (DHC, an inhibitor of the Sirtuin family of NAD+-dependent deacetylases) was used to treat Monascus ruber for evaluating its effects on organism growth and SM production. The results revealed that the variation trends of colonial sizes, biomass and mycotoxin were in a dose-dependent manner. Generally, they decreased with the increased DHC concentrations in the designed range. But the variation trend of pigment was different. Comparison of SM profile, three new peaks occurred to the mycelia extractions from DHC-treated strain corresponding to molecular weights 402, 416 and 444, respectively. These three compounds were identified as Monasfluol B, Monascus azaphilone C and acetyl-monasfluol B (a new Monascus chemical pigment structure). In short, DHC can stimulate M. ruber strain to produce more pigment-like polyketides but inhibition of mycotoxin (citrinin).

Effects of an alternative oxidase gene on conidia viability under external stresses in Monascus ruber M7.

Monascus species can produce natural edible pigments and many other bioactive metabolites. In this study, mraox gene (Monascus ruber alternative oxidase) was isolated, sequenced, and replaced in order to investigate the function in resistance of conidia to stressful conditions. The derived protein of the mraox gene consisted of 350 amino acids with a conserved ferritin-like diiron-binding domain at the C-terminus, sharing a high homolog with alternative oxidase proteins in other filamentous fungi. Deletion of mraox gene repressed the conidia germination rate (CGR) when conidia were exposed to H2 O2 , high temperature (40 and 50 °C) and alkerline buffer (pH8.0), but CGR of mraox-deleted strain was not decreased when the conidia were treated with NaCl, acid buffer (citric acid-dibasic sodium phosphate buffer, pH3) compared to that of the wild-type strain, suggesting that mraox gene is partially responsible for the resistance of conidia to stressful conditions in M. ruber.

mrskn7, a putative response regulator gene of Monascus ruber M7, is involved in oxidative stress response, development, and mycotoxin production.

Skn7, a response regulator (RR), is associated with oxidative stress adaptation, hypo-osmotic stress response, fungicide sensitivity, cell wall biosynthesis, cell cycle regulation, sexual mating, and sporulation in many filamentous fungi and yeasts. In this study a Skn7-like protein gene mrskn7 (Monascus ruber skn7) was isolated, sequenced, and disrupted to investigate its function in M. ruber Bioinformatics predicted that the deduced protein encoded by mrskn7 contained the conserved DNA-binding and signal-receiver domains similar to the Skn7-like protein structure in other filamentous fungi. The Δmrskn7 strain produced fewer conidia and less mycotoxin, demonstrated increased sensitivity to peroxide but the same level of osmotic resistance to NaCl and glycerol with the wild-type. Additionally, cleistothecia observed at different time point showed a different morphology between the wild-type and the Δmrskn7 strain, suggesting the involvement of mrskn7 in sexual development of M. ruber These results indicated that mrskn7 plays important roles in asexual and sexual development, the production of mycotoxin as well as regulation of oxidative stress signal in M. ruber.

Identification and role analysis of an intermediate produced by a polygenic mutant of Monascus pigments cluster in Monascus ruber M7.

Monascus pigments (Mps) are a group of azaphilonic secondary metabolites produced by Monascus spp. via a polyketide pathway. A mutant deleted an about 30 kb region of Mps gene cluster from Monascus ruber M7 was isolated previously, which produces a high amount of a light yellow pigment. The current study revealed that the mutant named ΔMpigJ-R lost proximate eight genes of the Mps gene cluster in M. ruber M7 through genetic analysis at DNA and RNA levels. The produced light yellow material was identified as a benzaldehyde derivative named as 6-(4-hydroxy-2-oxopentyl)-3-methyl-2, 4-dioxocyclohexane carb-aldehyde (M7PKS-1) by FT-IR, NMR, and MS. The sodium acetate-1-(13)C feeding experiment indicated that M7PKS-1 was a product produced from polyketide pathway. Finally, the feeding of M7PKS-1 helped to induce and regain Mps production of the mutants (ΔMpigA and ΔMpigE) which were previously unable to biosynthesize Mps and proved that M7PKS-1 was an initial intermediate of Mps. The results in this study provide a line of action to unveil Monascus pigments biosynthesis pathway.

Inactivation of the global regulator LaeA in Monascus ruber results in a species-dependent response in sporulation and secondary metabolism.

The nuclear regulator LaeA has been proven to globally govern fungal development and secondary metabolism, but its function may be species-dependent, even though its amino acid sequences are well conserved in numerous fungi. Herein we identified the LaeA in Monascus ruber M7 (MrLaeA), and verified its role to mediate growth, sporulation and secondary metabolism. Results showed that the radial growth rate of the selected MrlaeA knock-out mutant (MrΔlaeA-22) was significantly faster than that of the parental strain M. ruber M7, and growth was accompanied by the formation of an abnormal colony phenotype with more abundant aerial hyphae. Interestingly, conidia production of the MrΔlaeA-22 strain was about thrice that of M. ruber M7, but ascospores were not observed in the MrΔlaeA-22 strain. Additionally, compared to M. ruber M7, MrΔlaeA-22 exhibited drastically reduced production of multiple secondary metabolites, especially those of the six well-known Monascus pigments and citrinin. Simultaneously, the selected MrlaeA complementation strain (MrΔlaeA::laeA-45) nearly recovered the capacity for sporulation and secondary metabolism observed in the parental strain. These results demonstrate that MrLaeA regulates not only secondary metabolism, but also asexual and sexual differentiation in M. ruber, but some of its regulation appears to differ from other fungi.

Insights into Monascus biology at the genetic level.

The genus of Monascus was nominated by van Tieghem in 1884, but its fermented product-red mold rice (RMR), namely red yeast rice, has been used as folk medicines, food colorants, and fermentation starters for more than thousands of years in oriental countries. Nowadays, RMR is widely developed as food supplements around the world due to its functional compounds such as monacolin K (MK, also called lovastatin) and γ-aminobutyric acid. But the usage of RMR also incurs controversy resulting from contamination of citrinin (a kind of mycotoxin) produced by some Monascus strains. In the past decade, it has made great progress to Monascus spp. at the genetic level with the application of molecular biology techniques to restrain the citrinin production and increase the yields of MK and pigment in RMR, as well as aid Monascus classification and phylogenesis. Up to now, hundreds of papers about Monascus molecular biology (MMB) have been published in the international primary journals. However, to our knowledge, there is no MMB review issued until now. In this review, current understanding of Monascus spp. from the view of molecular biology will be covered and insights into research areas that need to be further investigated will also be discussed.

Waterborne exposure to microcystin-LR alters thyroid hormone levels and gene transcription in the hypothalamic-pituitary-thyroid axis in zebrafish larvae.

Microcystin-leucine-arginine (MCLR) is the most toxic and the most commonly encountered variant of microcystins (MCs) in aquatic environment, and it has the potential for disrupting thyroid hormone homeostasis, but the molecular mechanisms underlying this process have not yet been clarified. In the present study, we observed body growth retardation associated with decreased levels of thyroid hormones (THs) in zebrafish larvae, highlighting the interferences of MCLR with the growth of fish larvae. To further our understanding of mechanisms of MCLR-induced endocrine toxicity, quantitative real-time PCR analysis was performed on hypothalamic-pituitary-thyroid (HPT) axis related genes of developing zebrafish embryos exposed to 100, 300 and 500 µg L(-1) MCLR until 96 h post-fertilization. The expression of several genes in the HPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid receptors (TRα and TRß) and iodothyronine deiodinases (Dio1 and Dio2) was examined using quantitatively real-time PCR. The gene expression levels of CRF, TSH, NIS and TG were significantly induced after exposure to 500 µg L(-1) MCLR. The transcription of TRs gene was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of Deio1 and Deio2 gene expression, respectively, were observed upon exposure to MCLR. The above results indicated that MCLR could alter gene expression in the HPT axis which might subsequently contribute to MCLR-induced thyroid disruption.