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Airborne fine particulate matter (PM2.5) in air pollution has become a significant global public health concern related to allergic diseases. Previous research indicates that PM2.5 not only affects the respiratory system but may also induce systemic inflammation in various tissues. Moreover, its impact may vary among different populations, with potential consequences during pregnancy and in newborns. However, the precise mechanisms through which PM2.5 induces inflammatory reactions remain unclear. This study aims to explore potential pathways of inflammatory responses induced by PM2.5 through animal models and zebrafish embryo experiments. In this study, zebrafish embryo experiments were conducted to analyze the effects of PM2.5 on embryo development and survival, and mouse experimental models were employed to assess the impact of PM2.5 stimulation on various aspects of mice. Wild-type zebrafish embryos were exposed to a PM2.5 environment of 25-400 μg/mL starting at 6 h after fertilization (6 hpf). At 6 days post-fertilization, the survival rates of the 25, 50, 100, and 200 µg/mL groups were 100%, 80, 40%, and 40%, respectively. Zebrafish embryos stimulated with 25 μg/mL of PM2.5 still exhibited successful development and hatching. Additionally, zebrafish subjected to doses of 25-200 μg/mL displayed abnormalities such as spinal curvature and internal swelling after hatching, indicating a significant impact of PM2.5 stimulation on embryo development. In the mouse model, mice exposed to PM2.5 exhibited apparent respiratory overreaction, infiltration of inflammatory cells into the lungs, elevated levels of inflammatory response-related cytokines, and inflammation in various organs, including the liver, lungs, and uterus. Blood tests on experimental mice revealed increased expression of inflammatory and chemotactic cytokines, and GSEA indicated the induction of various inflammatory responses and an upregulation of the TNF-α/NFκB pathway by PM2.5. Our results provide insights into the harmful effects of PM2.5 on embryos and organs. The induced inflammatory responses by PM2.5 may be mediated through the TNF-α/NFκB pathway, leading to systemic organ inflammation. However, whether PM2.5-induced inflammatory responses in various organs and abnormal embryo development are generated through different pathways requires further study to comprehensively clarify and identify potential treatment and prevention methods.

Mycotoxin citrinin (CTN), commonly found in food and health supplements, may induce chromosomal instability. In this study, human renal proximal tubule epithelial cells (hRPTECs) that were exposed to CTN (10 and 20 μM) over 3 days exhibited numerical chromosomal aberrations. Short-term (3 days) and long-term (30 days) exposures to CTN significantly promoted mitotic spindle abnormalities, wound healing, cell migration, and anchorage-independent growth in human embryonic kidney 293 (HEK293) cells. Short-term exposure to 10 and 20 μM CTN increased the number of migrated cells on day 10 by 1.7 and 1.9 times, respectively. The number of anchorage-independent colonies increased from 2.2 ± 1.3 to 7.8 ± 0.6 after short-term exposure to 20 μM CTN and from 2.0 ± 1.0 to 12.0 ± 1.2 after long-term exposure. The transcriptomic profiles of CTN-treated HEK293 were subjected to over-representative analysis (ORA), gene set enrichment analysis (GSEA), and Ingenuity pathway analysis (IPA). Short-term exposure to CTN promoted the RTK/KRAS/RAF/MAPK cascade, while long-term exposure altered the extracellular matrix organization. Both short- and long-term CTN exposure activated cancer and cell cycle-related signaling pathways. These results demonstrate the carcinogenic potential of CTN in human cells and provide valuable insights into the cancer risk associated with CTN.

Our previous study demonstrated that myc, mitochondrial oxidative phosphorylation, mTOR, and stemness are independently responsible for chemoresistance in acute myeloid leukemia (AML) cells. This study aimed to identify potential mechanisms of chemoresistance of the "7 + 3" induction in AML by using a single-cell RNA sequencing (scRNA-seq) approach. In the present study, 13 untreated patients with de novo AML were enrolled and stratified into two groups: complete remission (CR; n = 8) and non-CR (n = 5). Single-cell RNA sequencing was used to analyze genetic profiles of 28,950 AML cells from these patients; results were validated using a previously published bulk RNA-seq dataset. Our study results showed chemoresistant AML cells had premature accumulation during early hematopoiesis. Hematopoietic stem cell-like cells from the non-CR group expressed more leukemic stem cell markers (CD9, CD82, IL3RA, and IL1RAP) than those from the CR group. Chemoresistant progenitor cells had impaired myeloid differentiation owing to early arrest of hematopoiesis. Notably, AML cells analyzed by scRNA-seq and bulk RNA-seq harbored a comparable myeloid lineage cell fraction, which internally validated our results. Using the TCGA database, our analysis demonstrated that patients with AML with higher expression of chemoresistant genetic markers (IL3RA and IL1RAP) had a worse overall survival (p < 0.01 for IL3RA; p < 0.05 for IL1RAP). In conclusion, AML cells responsive and resistant to the "7 + 3" induction were derived from a diverse cancerous hematopoietic stem cell population, as indicated by the specific genetic biomarkers obtained using scRNA-seq approach. Furthermore, arrest of hematopoiesis was shown to occur earlier in chemoresistant AML cells, furthering the current understanding of chemoresistance in AML., (© 2023 John Wiley & Sons Ltd.)

Citrinin (CTN) is a mycotoxin that is found as a contaminant in various types of food/feed grains and fermented food supplements. Previous studies have already established the nephrotoxicity and hepatotoxicity of CTN, but the neurotoxicity of CTN has not been clearly examined. In this study, CTN at 2-20 μM was first found to interfere with the neural ganglia formation and locomotive behavior of embryonic zebrafish, a vertebrate animal model, at 24 hpf and 6 dpf, respectively. Further exposure of human neuroblastoma SH-SY5Y cells to 10 and 20 μM CTN for 72 h indicated that pathways responsible for neuron differentiation and projection guidance were down-regulated while oxidative stress and electron transport chain pathways were up-regulated based on the enrichment results of GSEA in the transcriptomic profiling. PCR analysis verified that CTN significantly down-regulated the expression of marker genes involved in neuron differentiation and synaptic signaling. CTN at the doses impairing cellular neurite outgrowth did not trigger mitochondrial oxidative stress and dysfunction. The neurotoxic mechanisms of CTN provide new information that is valuable in the assessment of CTN-related health risk for the general public., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Allergic asthma, a chronic airway inflammatory disease, is a critical public health problem. Indoor house dust mites (HDMs) could cause allergic asthma. The prevalence of sensitization to Dermatophagoides microceras ( Der m ) was approximately 80% and is related to the immunoglobulin E crossing-reactivity of mites belonging to the same genus, Dermatophagoides pteronyssinus ( Der p ) and Dermatophagoides farina ( Der f ). However, studies on Der m are scant., Methods: We used integrated OMICs approaches to identify and characterize the group 2 mite allergen-like protein in Der m ( Der m 2 ). We established a Der m 2 -induced allergic asthma mouse model and treated the mice with a fungal immunomodulatory protein (FIP- fve ) isolated from Flammulina veluptipes to evaluate the allergenicity of Der m 2 and the immunomodulatory effects of FIP- fve ., Results: By performing de novo draft genome assembly and comparative genome analysis, we identified the putative 144-amino acid Der m 2 in silico and further confirmed its existence through liquid chromatography-tandem mass spectrometry. Der m 2 is a lipopolysaccharides (LPS)-binding protein. Thus, we examined the LPS-binding activity of recombinant Der m 2 by performing molecular docking analysis, co-immunoprecipitation (Co-IP), and a pull-down assay. Der m 2 elicited the production of pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in BEAS-2B cells, a human bronchial epithelial cell line, and induced airway hyperresponsiveness in mice. Furthermore, in mice sensitized with Der m 2 , the administration of FIP- fve in either the earlier stage or the late stage, FIP- fve alleviated allergic asthma by moderating airway inflammation and remodeling., Conclusions: Der m 2 induced inflammatory responses in cell and mouse models. FIP- fve alleviated inflammation in Der m 2 -induced asthma in mice by exerting an immunomodulatory effect., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hu, Wu, Wu, Yu, Ko, Lue and Liu.)

Aflatoxin B 1 (AFB 1 ), a naturally occurring mycotoxin, is present in human placenta and cord blood. AFB 1 at concentrations found in contaminated food commodities (0.25 and 0.5 μM) did not alter the spontaneous movement, heart rate, hatchability, or morphology of embryonic zebrafish. However, around 86 % of 0.25 μM AFB 1 -treated embryos had livers of reduced size, and AFB 1 disrupted the hepatocyte structures, according to histological analysis. Additionally, AFB 1 treatment that begins at any stage before 72 h post-fertilization (hpf) effectively reduced the size of embryonic livers. In hepatic areas, AFB 1 suppressed the expression of Hhex and Prox1, which are two critical transcriptional factors for initiating hepatoblast specification. KEGG analysis based on transcriptome profiling indicated that p53 signaling and apoptosis are the only observed pathways in AFB 1 -treated embryos. AFB 1 at 0.5 μM significantly activated the expression of tp53, mdm2, puma, noxa, pidd1, and gadd45aa genes that are related to the p53 pathway and also that of baxa, casp 8 and casp 3a in the apoptotic process. TUNEL staining demonstrated that AFB 1 triggered the apoptosis of embryonic hepatocytes in a dose-dependent manner. These results indicate that the deficiency of both hhex and prox1 as well as hepatocyte apoptosis via the p53-Puma/Noxa-Bax axis may contribute to the embryonic liver shrinkage that is caused by AFB 1., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ochratoxin A (OTA), a mycotoxin widely found in foodstuffs, reportedly damages multiple brain regions in developing rodents, but the corresponding mechanisms have not been elucidated. In this study, zebrafish embryos at 6 h post fertilization (hpf) were exposed to various concentrations of OTA and the phenomenon associated with intracerebral hemorrhage was observed at 72 hpf. Exposure of embryos to OTA significantly increased their hemorrhagic rate in a dose-dependent manner. Large numbers of extravagated erythrocytes were observed in the midbrain/hindbrain areas of Tg(fli-1a:EGFP; gata1:DsRed) embryos following exposure to OTA. OTA also disrupted the vascular patterning, especially the arch-shaped central arteries (CtAs), in treated embryos. Histological analysis revealed a cavity-like pattern in their hindbrain ventricles, implying the possibility of cerebral edema. OTA-induced intracerebral hemorrhage and CtA vessel defects were partially reversed by the presence of miR-731 antagomir or the overexpression of prolactin receptor a (prlra); prlra is a downstream target of miR-731. These results suggest that exposure to OTA has a negative effect on cerebral vasculature development by interfering with the miR-731/PRLR axis in zebrafish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aflatoxin B 1 (AFB 1 ) is the major mycotoxin that contaminates aquafeeds and regarded as a causative agent in illnesses and the mortality of aquacultural species. However, the effects of AFB 1 on developing fish and associated toxic mechanism are still unknown. This study examines the behavioral changes, neuronal morphology and gene expression in zebrafish embryos and larvae upon exposure to aflatoxin solutions. Treatment of 6 h post fertilization (hpf) embryos with AFB 1 at 15-75 ng/mL significantly changed the swimming patterns of seven days post-fertilization (dpf) zebrafish larvae. Larvae in the 15 ng/mL group demonstrated a hypolocomotor activity in free swimming, but hyperlocomotion was observed in the larvae exposed to 30-75 ng/mL AFB 1 . AFB 1 at 75 ng/mL also significantly reduced the startle response of 7 dpf larvae after tapping stimulus. Exposure to AFB 1 resulted in an aberrant morphology of trigeminal ganglion and hindbrain neurons in transgenic embryos (HuC:eGFP); this finding was supported by acetylated alpha-tubulin staining in wild-type fish. Additionally, AFB 1 altered the levels of neurotoxic markers, including gfap and huC. The transcriptomic profile of AFB 1 -treated embryos revealed several differentially expressed genes that are related to neuroactivity and neurogenesis. PCR analysis verified that AFB 1 significantly down-regulated the expression of ngfa and atp1b1b genes and increased that of prtga gene. The results herein indicate the toxicological impacts of AFB 1 on the behaviors and neurodevelopment of fish in the early embryonic stage. Disruption of neural formation and synapse dysfunction may be responsible for the behavioral alteration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aristolochic acid I (AAI) is a phytocompound that is linked to the progressive renal disease and development of human urothelial carcinoma. The bladder cancer-associated protein (BLCAP) gene exhibits a tumor suppressor function in various tumors, including bladder carcinoma. This study evaluated the effect of AAI on BLCAP expression and its associated mechanism in human cells. Administering AAI to human embryonic kidney cells (HEK293), human proximal tubule epithelial cells (HK-2) and urinary bladder cancer cells (HT-1376) significantly reduced the expression of BLCAP mRNA and protein. AAI also effectively suppressed the luciferase activities driven by BLCAP promoters of various lengths in HEK293 cells. AAI significantly reduced both activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities in reporter assays, but further point mutations revealed that Ap-1 and NF-κB binding sites on the BLCAP promoter were not AAI-responsive elements. Application of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), reversed the decline of BLCAP expression that had been induced by AAI. However, AAI exposure did not alter hypermethylation of the BLCAP promoter, determined by methyl-specific polymerase chain reaction (PCR) and bisulfate sequencing. Knocking down BLCAP in HEK293 cell line enhanced the potential for cellular migration, invasion, and proliferation, along with the induction of a capacity for anchorage-independent growth. In conclusion, AAI down-regulated the expression of BLCAP gene and the deficiency in BLCAP expression contributed to the malignant transformation of human cells, implying that BLCAP may have a role in mediating AAI-associated carcinogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ochratoxin A (OTA) is a mycotoxin that is found in various food and feed products. The molecular mechanisms that are associated with OTA hepatotoxicity and teratogenicity have not been extensively elucidated in a developing organism. In this study, the transcriptomic profile of zebrafish embryos indicates that hemostasis and blood coagulation are the top two pathways affected by OTA. The treatment of embryos with OTA was able to decrease the expression of genes that encode coagulation factors and liver markers, including f7, f9b, cp and vtna. OTA also weakened the signal of liver-specific microRNA-122. OTA administration not only reduced the size of a developing embryonic liver, but also decreased the number of phosphorylated histone H3-positive cells by immunohistochemical staining. OTA suppressed the expression of hhex and prox1, two critical transcriptional factors during hepatoblast specification, in the developing liver, but did not alter the insulin signal in the pancreas. In vitro analysis with zebrafish liver (ZFL) cells indicated that OTA blocked the expression of f7, fgb and liver markers. In summary, OTA exposure resulted in the generation of small livers which led to deficiency of coagulation factors in embryonic zebrafish. Impairment of hhex and prox1 gene expression and hepatocyte proliferation contributed to the disruption of liver development mediated by OTA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mycotoxin ochratoxin A (OTA) frequently contaminates various food and feed products, including cereals, coffee and wine. While the nephrotoxicity and teratogenicity of OTA have been extensively documented, the molecular mechanisms associated with OTA toxicity remained poorly understood in a developing organism. We showed that zebrafish embryos exposed to OTA demonstrated incorrect heart looping and small heart chambers. OTA also impaired the renal morphology and reduced the glomerular filtration rate of the embryonic zebrafish. The treatment of embryos with OTA attenuated the expression of the prolactin receptor, a gene (PRLRa) that has a key role in organogenesis and osmoregulation in vertebrates. OTA not only inhibited the phosphorylation of STAT5 and AKT, but also down-regulated the level of serpina1 mRNA in a dose-dependent manner. On the other hand, the microRNA profiling based on RNA sequencing revealed the up-regulation of microRNA-731 (miR-731) in the OTA-treated embryos. Further in silico analysis predicted that PRLRa was a target gene of miR-731. AntagomiR-731 restored PRLRa levels that had been reduced by OTA and also recovered the pronephros morphology that was damaged by OTA. These observations suggest that the exposure to OTA adversely affected the organogenesis of zebrafish, and the modulation of miR-731 and the PRLR signaling cascade contributed to the abnormal renal development mediated by OTA.

Citrinin (CTN) is a fungal secondary metabolite that contaminates various foodstuffs and animal feeds; it also exhibits organotoxicity in several animal models. In this study, the zebrafish was used to elucidate the mechanism of CTN cardiotoxicity in developing embryos. Following CTN administration, the gross morphology of the embryonic heart was apparently altered, including heart malformation, pericardial edema, and red blood accumulation. Whole-mount immunostaining and histological analysis of ventricle and atrium indicated incorrect heart looping and reduced size of heart chambers. From the perspective of cardiac function, the heartbeat and blood flow rate of embryos were significantly decreased in the presence of CTN. CTN also modulated the expression of tbx2a and jun B genes, but not that of bmp4 and nkx2.5. Furthermore, the heart areas of CTN-exposed embryos demonstrated an elevated levels of aldh1a2 and cspg2 messenger RNA; these 2 cardiac-related genes are known to be involved in retinoic acid (RA) pathway as well as downstream targets of microRNA-138 (miR-138) in zebrafish. CTN treatment also downregulated the expression of miR-138. Moreover, overexpression of miR-138 was able to rescue the heart defects generated by CTN. These results support the notion that CTN exposure has a severe impact on heart development, affecting heart morphogenesis through the dysregulation of miR-138, RA signaling, and tbx2a.

Citrinin (CTN) and patulin (PAT) are fungal secondary metabolites which are found in food and feed and showed organotoxicity in mature animals. In this study zebrafish embryos were applied to investigate the developmental toxicity of CTN and PAT on embryonic kidney. In the presence of CTN and PAT, the gross morphology of kidneys from embryos with green fluorescent kidney (wt1b:GFP) was not apparently altered. Histological analysis of CTN-treated embryos indicated cystic glomerular and tubular lesions; a disorganized arrangement of renal cells was also found in the PAT-treated group. From the view point of renal function, dextran clearance abilities of embryos exposed to CTN and PAT were significantly reduced. The damaged renal function caused by CTN could be partially rescued by the administration of pentoxifylline, suggesting the reduction of glomerular blood flow contributes to CTN-induced renal dysfunction. Additionally, CTN induced the expression of proinflammation genes, including COX2a, TNF-α and IL-1β, but failed to modify the levels and distribution of wt1a transcript and Na(+)/K(+)-ATPase protein. In summary, CTN and PAT caused profound nephrotoxicity in histological structure and biological function of zebrafish embryos; the inflammatory pathway and blood rheology may involve in CTN-induced renal impairment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aristolochic acid I (AAI) has been widely found in herbal remedies and linked to the development of nephropathy and urothelial carcinoma in humans. This study elucidated the mechanism of oxidative stress and DNA damage mediated by AAI in human cells. Treatment of human promyelocytic leukemia cells (HL-60) and human renal proximal tubular cells (HK-2) with AAI led to a dose-dependent increase of reactive oxygen species (ROS). AAI also elevated the levels of DNA strand breaks and 8-hydroxy guanosine in HL-60 and HK-2 cells. Antioxidants, including Tiron, N-acetyl-l-cysteine (NAC) and glutathione (GSH), effectively suppressed the AAI-induced ROS and AAI-elicited genotoxicity, indicating that AAI induced the DNA damage through oxidative stress. GSH depletion was also found in AAI-treated cultures and proceeded prior to ROS formation. Exposure of HL-60 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only MEK1/2 inhibitor, U0126, significantly decreased AAI-mediated ROS. Preincubation of cells with thiol-containing compounds (NAC and GSH) inhibited the caspase 3 activity triggered by AAI, but non-thiol Tiron did not show a similar effect. This study demonstrated that AAI treatment results in oxidative stress-related DNA damage through GSH depletion and ERK1/2 activation; AAI-induced apoptosis is associated with GSH loss, but is independent of ROS generation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aristolochic acid I (AAI) is a phytotoxin that has been found in various herbal remedies and linked to the development of human carcinogenesis. To investigate the playing role of AAI in the function of macrophages, lipopolysaccharide (LPS)-stimulated macrophage cells RAW264.7 were employed as a model to examine the effect of AAI on the expression of the inducible nitric oxide synthase (iNOS) gene. AAI reduced the expression of iNOS mRNA and protein, as well as the production of NO in LPS-stimulated macrophages. Treatment of transfected macrophages with AAI effectively suppressed the luciferase activities of the iNOS promoter which is activated by LPS. The results of promoter deletion and electrophoretic gel mobility shift assay (EMSA) indicated that the NF-κB binding site at nucleotides -86 to -76 was the major site that was most responsible for the inhibitory effect of AAI. Moreover, the presence of AAI substantially reduced the phosphorylation of the inhibitory κBα (IκBα) protein in LPS-stimulated cultures. AAI also down-regulated the LPS-induction of TNF-α, a NF-κB regulated gene. On the other hand, AAI did not modulate the luciferase activities of reporter construct that contained iNOS mRNA 3'-UTR. Taken together, the data herein suggest that in activated macrophages, AAI effectively down-regulated the expression of iNOS gene by interfering with the activation of NF-κB at the transcription level. The stability of iNOS mRNA was not the target of AAI inhibition., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Patulin (PAT) is a fungal secondary metabolite that exhibits potential cellular and animal toxicities. In this study, human promyelocytic leukemia (HL-60) cells were used to elucidate the mechanism and death mode associated with PAT. Morphological evidence of apoptosis, including membrane blebbing, nuclei fragmentation and DNA laddering formation was clearly observed 6h after exposure to PAT. The results of Western blotting indicated that PAT activated various processed caspases, and cleaved DFF45 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner; it also induced a time-dependent increase in caspase 3 and 9 catalytic activities. The apoptosis mediated by PAT in HL-60 was accompanied with cytochrome c release from mitochondria and Bcl-2 expression decrease. The presence of thiol-containing compounds with PAT dramatically reduced the caspase 3 activity that was triggered by PAT; the addition of antioxidants, including mannitol and Tiron, had a similar effect. However, the suppression of p53 protein expression by RNA interference (RNAi) in human embryonic kidney (HEK293) cells did not significantly modify PAT-elicited caspase 3 activity. These findings suggest that PAT-induced apoptosis is mediated through the mitochondrial pathway without the involvement of p53; the interaction with sulfhydryl groups of macromolecules by PAT and the subsequent generation of reactive oxygen species (ROS) plays a primary role in the apoptotic process.

Patulin (PAT), a mycotoxin mainly produced by Penicillium and Aspergillus, is found in various foods and feeds. In the present study, its effects on oxidative stress in various mammalian cell lines were investigated. When cell-permeating fluorescent dyes were used as indicators of the generation of reactive oxygen species (ROS), we found that PAT treatment directly increased intracellular oxidative stress in human embryonic kidney (HEK293) and human promyelocytic leukemia (HL-60) cells. Lipid peroxidation levels were also significantly increased in HL-60 cells and mouse kidney homogenates treated with PAT. Suppression of CuZn-superoxide dismutase (SOD) expression in mammalian cells by small interfering RNA resulted in an increase in PAT-mediated membrane damage, while overexpression of human CuZn-SOD or catalase led to a reduction in damage, indicating the involvement of ROS in PAT toxicity. Pretreatment of HEK293 cells with Tiron, a free radical scavenger, reduced the phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 elicited by PAT. The ERK1/2 signaling pathway inhibitor, U0126, also significantly decreased the levels of ROS associated with PAT treatment. These findings indicate that PAT treatment results in the ROS production in mammalian cells, and ROS partially contributes to PAT-induced cytotoxicity. Activation of ERK1/2 signaling pathway is correlated with PAT-mediated ROS.

Patulin (PAT), a mycotoxin mainly produced by Penicillium and Aspergillus, is frequently detected in moldy fruits and fruit products. Exposure of human embryonic kidney (HEK293) cells to PAT led to a dose- and time-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), p38 kinase and c-Jun N-terminal kinase (JNK). The phosphorylated forms of MAPK kinase 4 (MKK4), c-Jun, and ATF-2 were also seen in PAT-treated cultures. The cell death caused by PAT was significantly reduced by the p38 kinase inhibitor, SB203580, but not by the JNK inhibitor, SP600125. Neither p38 kinase nor JNK played a role in the PAT-induced DNA damage. In PAT-treated cells, inactivation of double-stranded RNA-activated protein kinase R (PKR) by the inhibitor, adenine, markedly suppressed JNK and ERK phosphorylation. Treatment of HEK293 cells with PAT-cysteine adduct, a chemical derivative of PAT, showed no effect on MAPK signaling pathways, cell viability, or DNA integrity. These results indicate that PAT causes rapid activation of p38 kinase and JNK in HEK293 cells, but only the p38 kinase signaling pathway contributes to the PAT-induced cell death. PKR also plays a role in PAT-mediated MAPK activation.

Patulin (PAT), a mycotoxin produced by certain species of Penicillium and Aspergillus, is often detectable in moldy fruits and their derivative products. PAT led to a concentration-dependent and time-dependent increase in phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human embryonic kidney (HEK293) cells, human peripheral blood mononuclear cells (PBMCs), and Madin-Darby canine kidney (MDCK) cells. Exposure of HEK293 cells to concentrations above 5 microM PAT for 30 min induced ERK1/2 phosphorylation; activation of ERK1/2 was also observed after 24 h incubation with 0.05 microM of PAT. Treatment of human PBMCs for 30 min with 30 microM PAT dramatically increased the phosphorylated ERK1/2 levels. Both MEK1/2 inhibitors, U0126 and PD98059, suppressed ERK1/2 activation in either HEK293 or MDCK cells. In HEK293 cells, U0126-mediated inhibition of PAT-induced ERK1/2 phosphorylation resulted in a significant decrease in levels of DNA damage, expressed as tail moment values, in the single cell gel electrophoresis assay. Conversely, U0126 did not affect cell viability, lactate dehydrogenase release, and the DNA synthesis rate in PAT-treated cultures. Exposure of HEK293 cells for 90 min to 15 microM PAT elevated the levels of early growth response gene-1 (egr-1) mRNA, but not of c-fos, fosB, and junB mRNAs. These results indicate that in human cells, PAT causes a rapid and persistent activation of ERK1/2 and this signaling pathway plays an important role in mediating PAT-induced DNA damage and egr-1 gene expression.

Monascus purpureus and its fermentation products have been used in food coloring and meat preservation in Asia for centuries and have also been recently used as dietary supplements because of their cholesterol-lowering ability. However, the presence of the mycotoxin citrinin (CTN), a secondary metabolite of Monascus species, in fermentation products is a potential threat to public health. In the present study, HPLC was used to analyze CTN levels in lipid and aqueous extracts of commercialized Monascus products. CTN was detected in lipid extracts of all examined samples at concentrations varying between 0.28 and 6.29 microg/g, but was not found in aqueous extracts. When human embryonic kidney cells (HEK293) were incubated for 72 h with Monascus extracts, the concentrations causing 50% cell death by all lipid extracts were in the range of 1.8-4.7 mg/mL, whereas aqueous extracts showed a lower cytotoxicity. Incubation of HEK293 cells with 60 microM pure CTN for 72 h caused cell viability to fall to 50% of control levels. In addition, coadministration of pure CTN and lipid extracts from Monascus samples significantly enhanced CTN cytotoxicity for HEK293 cells using the MTT assay. These results provide the first information about the cytotoxic effects of various Monascus samples and CTN-Monascus mixtures on a human cell line.

Polyclonal antibodies for domoic acid were generated from rabbits after the animals had been immunized with either domoic acid-keyhole limpet hemocyanin (KLH) or domoic acid-bovine serum albumin (BSA). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of domoic acid in blue mussels and clams. The antibody titers in the serum of rabbits immunized with domoic acid-KLH were considerably higher than those in rabbits immunized with domoic acid-BSA. The antibodies from the rabbits immunized with domoic acid-KLH were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of domoic acid-horseradish peroxidase to the antibodies by domoic acid and a domoic acid analogue, kainic acid, were found to be 0.75 and 200 ng/mL, respectively. In the presence of blue mussel matrix, the detection limit of domoic acid was <25 ng/g. The overall analytical recovery of domoic acid (25-500 ng/g) added to the blue mussels and then extracted with 50% aqueous methanol in the cdELISA was found to be 81.1%. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method. Analysis of domoic acid in shellfish samples showed that 10 of the 15 shellfish examined were contaminated with domoic acid at levels of <50 ng/g.

Mycotoxins are fungal secondary metabolites with very diversified toxic effects in humans and animals. In the present study, patulin (PAT) and citrinin (CTN), two prevalent mycotoxins, were evaluated for their genotoxic effects and oxidative damage to mammalian cells, including Chinese hamster ovary cells (CHO-K1), human peripheral blood lymphocytes, and human embryonic kidney cells (HEK293). PAT, but not CTN, caused a significant dose-dependent increase in sister chromatid exchange (SCE) frequency in both CHO-K1 and human lymphocytes. PAT also elevated the levels of DNA gap and break in treated CHO-K1. In the single cell gel electrophoresis (SCGE) assay, exposure of HEK293 to concentrations above 15 microM of PAT induced DNA strand breaks; the tail moment values also greatly increased after posttreatment with formamidopyrimidine-DNA glycosylase (Fpg). This suggests that in human cells PAT is a potent clastogen with the ability to cause oxidative damage to DNA. However, no significant change in the tail moment values in CTN-treated cultures was found, suggesting that CTN is not genotoxic to HEK293. Incubation of HEK293 with CTN increased the mRNA level of heat shock protein 70 (HSP70), but not that of human 8-hydroxyguanine DNA glycosylase 1 (hOGG1). PAT treatment did not modulate the expression of either HSP70 or hOGG1 mRNA.