Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

Exposure to Mycotoxin Citrinin Promotes Carcinogenic Potential of Human Renal Cells.

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.

Combined toxicity assessment of a naturally occurring toxin and a triazole fungicide on different biological processes through toxicogenomic data mining with mixtures.

Fungicides are widely used to prevent fungal growth and reduce mycotoxin contamination in food, which provides the opportunity for the co-occurrence of mycotoxins and fungicide residues in food and poses a greater risk to human health. To assess the combined effects of a naturally occurring mycotoxin, citrinin (CIT), and a widely used triazole fungicide, triadimefon (TAD) on different biological processes, the comparative toxicogenomics database was used to obtain phenotypes and response genes for CIT or TAD exposure. Then individual and combined exposure models were developed with zebrafish embryos, and the interaction between CIT and TAD was analyzed using the 2 × 2 factorial design approach to observe the toxic effects. Through data mining analysis, our results showed that CIT or TAD exposure is related to different biological phenotypes, such as cell death, regulation of antioxidant systems, and thyroid hormone metabolism. Our results also showed that CIT (4-day LC50 value of 12.7 mg/L) exposure possessed higher toxicity to zebrafish embryos compared with TAD (4-day LC50 value of 29.6 mg/L). Meanwhile, individual exposure to CIT and TAD altered the expression levels of biomarkers related to oxidative stress, inflammation, apoptosis and hypothalamic-pituitary-thyroid (HPT) axis. Notably, combined exposure to CIT and TAD induced changes in the mentioned biological processes and had an interactive effect on the expression of multiple biomarkers. In conclusion, we evaluated the toxic effects of CIT and TAD in isolation and combination by in-vivo experiments, which provide a new methodological basis and reference for future risk assessment and setting of safety limits for foodborne toxicants.

Oral exposure to citrinin significantly exacerbates the pathophysiology of a mouse model of imiquimod-induced psoriasis via direct activation of dendritic cell.

Citrinin, a mycotoxin produced by Penicillium citrinum and Penicillium verrucosum, mainly contaminates cereals. The aim of study was to investigate the novel immunoreactive effect of citrinin using a mouse model of psoriasis. A mouse model of psoriasis was generated by topical application of 5% imiquimod in female BALB/c mice. Standard rodent diet and rice samples with 3 ppm of citrinin were mixed to obtain a final citrinin concentration of 0.3 ppm, and a citrinin-contaminated diet was fed to mice daily. Skin thickness, scratching behavior, and trans epidermal water loss (TEWL) were monitored continuously during the imiquimod application. Immediately after the final imiquimod application, ear skin and auricular lymph node (LN) were sampled for further analysis. Only a slight increase was observed in skin thickness in the citrinin exposure group; however, citrinin exposure significantly exacerbated hyperkeratinization and inflammatory cell infiltration in histological evaluation. TEWL, which is representative of cutaneous barrier function, was significantly increased by citrinin exposure. In terms of immune function, the number of immune cells in LN (T cells and dendritic cells) and gene expression of interleukin (IL)-17 in skin tissue were significantly increased by citrinin exposure. Direct interaction of dendritic cells (DCs) in citrinin-induced psoriasis development was further examined by proinflammatory cytokine determination in THP-1 cells and murine bone marrow derived DCs. IL-6 and/or tumor necrosis factor α were significantly increased by citrinin exposure. Taken together, our results imply that oral exposure to citrinin exacerbates the symptoms of a mouse model of psoriasis via direct activation of DCs.

Citrinin inhibits the function of Leydig cells in male rats in prepuberty.

Citrinin, a mycotoxin existing in fruits, has nephrotoxicity, hepatotoxicity and embryotoxicity. The effects of citrinin on Leydig cell development in prepuberty remains unclear. Male Sprague-Dawley rats were gavaged with 0, 1, 2.5, and 5 mg/kg citrinin from postnatal days 21-28. Citrinin at 5 mg/kg significantly decreased serum testosterone levels, while increasing serum LH and FSH levels. Citrinin at 1-5 mg/kg markedly downregulated Hsd17b3 and HSD17B3 expression, while upregulating Srd5a1 (SRD5A1) and Akr1c14 (AKR1C14) expression at 2.5 and/or 5 mg/kg. Citrinin at 5 mg/kg also significantly increased PCNA-labeling index in Leydig cells. Citrinin at 5 mg/kg significantly raised testicular MDA amount, whiling at 2.5 and 5 mg/kg downregulating SOD1 and SOD2 expression. Citrinin at 5 mg/kg markedly decreased the ratio of Bcl2 to Bax, in consistent with the increased apoptosis in Leydig cells judged by TUNEL assay. Enzymatic assay revealed that citrinin inhibited rat testicular HSD3B1 activity at 100 µM and HSD17B3 activity at 10-100 µM. Citrinin at 50 µM and higher also induced reactive oxygen species (ROS) and apoptosis of R2C cell line. In conclusion, citrinin inhibits Leydig cell development at multiple levels via different mechanisms and oxidative stress partially plays a role.

Neurotoxicity of mycotoxin citrinin: Novel evidence in developing zebrafish and underlying mechanisms in human neuron cells.

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.

Mechanisms underlying citrinin-induced toxicity via oxidative stress and apoptosis-mediated by mitochondrial-dependent pathway in SH-SY5Y cells.

Citrinin (CIT) is a mycotoxin produced as a secondary product by the genera Aspergillus, Penicillium, Monascus, and other strains. CIT has the potential for contaminating animal feed and human food such as maize, wheat, rye, barley, oats, rice, cheese, and sake. Although CIT is primarily known as a nephrotoxic mycotoxin, it also affects other organs, including the liver and bone marrow, and its mechanisms of toxicity have not been clearly elucidated. There is a further lack of studies investigating the potential for CIT-induced neurotoxicity and its mechanisms. In the current study, SH-SY5Y human neuroblastoma cell line was treated with CIT for 24 h to evaluate various toxicological endpoints, such as reactive oxygen species (ROS) production and apoptosis induction. Results indicate that CIT has an IC50 value of 250.90 µM and cell proliferation decreased significantly at 50 and 100 µM CIT concentrations. These same concentrations also caused elevated ROS production (≥34.76%), apoptosis (≥9.43-fold) and calcium ion mobilization (≥36.52%) in the cells. Results show a significant decrease in the mitochondrial membrane potential (≥86.8%). We also found that CIT significantly upregulated the expression of some genes related to oxidative stress and apoptosis, while downregulating others. These results suggest that apoptosis and oxidative stress may be involved in the mechanisms underlying CIT-induced neurotoxicity.

Ochratoxin A and Citrinin Differentially Modulate Bovine Mammary Epithelial Cell Permeability and Innate Immune Function.

Frequent detection of mycotoxins ochratoxin A (OTA) and citrinin (CIT) in ruminant feed and feedstuff can be a potential threat to feed safety, animal performance and health. Ineffective biodegradation of these mycotoxins by rumen microflora following ingestion of contaminated feeds can lead to their circulatory transport to tissues such as mammary gland as the result of their biodistribution throughout the body. The bovine mammary epithelium plays a pivotal role in maintaining milk yield and composition and contributes to innate immune defense of the udder. The present study is the first to investigate individual effects of OTA and CIT on barrier and innate immune functions of the bovine mammary epithelium using a bovine mammary epithelial cell line (MAC-T). Results indicated that OTA and CIT exposure for 48 h significantly decreased cell viability in a concentration-dependent manner (p < 0.05). A decrease in transepithelial electrical resistance and increase in paracellular flux of FITC-40 kDa dextran was significantly induced by OTA treatment (p < 0.05), but not by CIT after 48 h exposure. qPCR was performed for assessment of expression of tight-junction proteins, Toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48 h of exposure. Both OTA and CIT markedly downregulated expression of claudin 3 and occludin (p < 0.05), whereas CIT did not affect zonula occludens-1 expression. Expression of TLR4 was significantly upregulated by OTA (p < 0.001) but downregulated by CIT (p < 0.05) at 48 h. Expression of IL-6, TNF-a and TGF-ß was significantly upregulated by OTA (p < 0.05), whereas IL-6 and TGF-ß expression was downregulated by CIT (p < 0.01). These results suggest that OTA and CIT could potentially differentially modulate barrier and innate immune functions of mammary epithelium. The present study not only throws light on the individual toxicity of each mycotoxin on bovine mammary epithelium but also lays the foundation for future studies on the combined effects of the two mycotoxins.

Citrinin-Induced Hepatotoxicity in Mice Is Regulated by the Ca2+/Endoplasmic Reticulum Stress Signaling Pathway.

Citrinin (CTN) is a mycotoxin found in crops and agricultural products and poses a serious threat to human and animal health. The aim of this study is to investigate the hepatotoxicity of CTN in mice and analyze its mechanisms from Ca2+-dependent endoplasmic reticulum (ER) stress perspective. We showed that CTN induced histopathological damage, caused ultrastructural changes in liver cells, and induced abnormal values of biochemical laboratory tests of some liver functions in mice. Treatment with CTN could induce nitric oxide (NO), malondialdehyde (MDA), and reactive oxygen species (ROS) accumulation in mice, accompanied with losses of activities of superoxide dismutase (SOD) and catalase (CAT), levels of glutathione (GSH), and capacities of total antioxidant (T-AOC), resulting in oxidative stress in mice. Furthermore, CTN treatment significantly increased Ca2+ accumulation, upregulated protein expressions of ER stress-mediated apoptosis signal protein (glucose regulated protein 78 (GRP78/BIP), C/EBP-homologous protein (CHOP), Caspase-12, and Caspase-3), and induced hepatocyte apoptosis. These adverse effects were counteracted by 4-phenylbutyric acid (4-PBA), an ER stress inhibitor. In summary, our results showed a possible underlying molecular mechanism for CTN that induced hepatocyte apoptosis in mice by the regulation of the Ca2+/ER stress signaling pathway.

Ochratoxin A potentiates citrinin accumulation in kidney and liver of rats.

Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3.

The role of ER stress and ATP/AMPK in oxidative stress meditated hepatotoxicity induced by citrinin.

Citrinin, a secondary metabolite, can pose serious risks to the environment and organisms, but its hepatotoxic mechanisms are still unclear. Histopathological and ultrastructural results showed that citrinin-induced liver injury in Kunming mice, and the mechanism of citrinin-induced hepatotoxicity was studied in L02 cells. Firstly, citrinin mades L02 cell cycle arrest in G2/M phase by inhibition of cyclin B1, cyclin D1, cyclin-dependent kinases 2 (CDK2), and CDK4 expression. Secondly, citrinin inhibits proliferation and promotes apoptosis of L02 cells via disruption of mitochondria membrane potential, increase Bax/Bcl-2 ration, activation of caspase-3, 9, and enhance lactate dehydrogenase (LDH) release. Then, citrinin inhibits superoxide dismutase (SOD) activity and increases the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS), resulting oxidative damage in L02 cells; upregulates the protein expression of binding immunoglobulin protein (Bip), C/EBP homologous protein (CHOP), PKR-like ER kinase (PERK) and activating transcription factor6 (ATF6), inducing ER stress in L02 cells; increases the phosphorylation of AMP-activated protein kinase (AMPK) and decreases the content of adenosine-triphosphate (ATP), activating AMPK pathway in L02 cells. Eventually, pretreatment with NAC, an ROS inhibitor, alleviates citrinin-induced cell cycle G2/M arrest and apoptosis by inhibiting ROS-mediated ER stress; pretreatment with 4-PBA, an ER stress inhibitor, reversed ER stress and p-AMPK; pretreatment with dorsomorphin, an AMPK inhibitor, decreases citrinin-induced cell cycle G2/M arrest and apoptosis. In summary, citrinin induces cell cycle arrest and apoptosis to aggravate liver injury by activating ROS-ER stress-AMPK signaling pathway.

Citrinin Mycotoxin Contamination in Food and Feed: Impact on Agriculture, Human Health, and Detection and Management Strategies.

Citrinin (CIT) is a mycotoxin produced by different species of Aspergillus, Penicillium, and Monascus. CIT can contaminate a wide range of foods and feeds at any time during the pre-harvest, harvest, and post-harvest stages. CIT can be usually found in beans, fruits, fruit and vegetable juices, herbs and spices, and dairy products, as well as red mold rice. CIT exerts nephrotoxic and genotoxic effects in both humans and animals, thereby raising concerns regarding the consumption of CIT-contaminated food and feed. Hence, to minimize the risk of CIT contamination in food and feed, understanding the incidence of CIT occurrence, its sources, and biosynthetic pathways could assist in the effective implementation of detection and mitigation measures. Therefore, this review aims to shed light on sources of CIT, its prevalence in food and feed, biosynthetic pathways, and genes involved, with a major focus on detection and management strategies to ensure the safety and security of food and feed.

Subchronic exposure of individual and combined ochratoxin A and citrinin selectively affects the expression of rat renal organic cation transporters.

Ochratoxin A (OTA) and citrinin (CIT) are nephrotoxins found co-occurring in various human/animal food/feed and recognized as a health threat. However, most studies investigate individual effects and neglect their combined nephrotoxic effects in mammals. Previous studies have indicated that organic anion/cation transporters (OATs/OCTs) localized in renal proximal tubules mediate the transport of OTA and CIT. Still, little is known about the in vivo effects of individual/combined OTA and CIT on protein localization/expression of OCTs, physiologically/pharmacologically important renal transporters. Here, we used Western blot and immunofluorescence microscopy to study the effects of subchronic (21-day) exposure to individual/combined OTA (0.125 and 0.250 mg kg-1 b.w.) and CIT (20 mg kg-1 b.w.) on protein localization/expression of organic cation transporters (rOct1/Slc22a1 and rOct2/Slc22a2) in kidneys of Wistar rats. Since the antioxidant resveratrol (RSV) has shown measurable protective effects against OTA- and CIT-related oxidative stress toxicity in vitro, we investigated the effects of an OTA + CIT + RSV combination on rOct1/2 localization/expression in the same model. Individual OTA induced a dose-dependent decrease of rOct1 but not rOct2 protein expression, whereas their localization pattern remained unchanged. Individual CIT did not affect the renal rOct1/2 protein localization/expression. Combined OTA + CIT exposure induced a significant decrease of rOct1 protein expression by an OTA250 dose, whereas oral co-administration of OTA + CIT + RSV resulted in a significant decrease of rOct1/2 protein expression. Thus, we revealed an OTA-related selective effect on the rOct1/2 protein expression and a non-specific adverse effect of RSV in the OTA + CIT + RSV combination on the renal organic cation transport system in rat.

Powerful toxic activity of citrinin, a fungal phytotoxin, and its mode of action in onion cells.

The dose-related effects of citrinin (CTN) on various physiological, cytogenetic, biochemical, and anatomical parameters using Allium cepa L. bulbs as a test material were researched in the present study. The physiological parameters examinated were fresh weight, root length, root number, and germination percentage; the cytogenetic parameters were micronucleus (MN) frequency, chromosome aberration (CA), and mitotic index (MI); the biochemical parameters were catalase (CAT), superoxide dismutase (SOD) activities, malondialdehyde (MDA) level, and free proline contents. And the anatomical changes in root tip cells were investigated by cross-sections. For this aim, onion bulbs were splitted four groups as three applications and one control. The bulbs in the control group were treated with distilled water; the bulbs in the application groups were treated with 1 µM, 5 µM, and 10 µM doses of CTN for 7 days. CTN application caused a decrease in the physiological parameters compared to the control group. This treatment created an increase in the frequency of MN and CA, and a reduce in the MI. In addition, it induced a dose-dependent increase in CAT and SOD activities and MDA and proline contents compared to the control group. Moreover, after CTN application, anatomical changes such as flattened nucleus, cell wall thickening, and cell deformation were identified and it was found that these changes reached their maximum at 10 mg/L dose CTN. Concequently, CTN caused inhibitory effects and the Allium test material was found to be a useful bioindicator for monitoring these effects.

Citrinin impairs pig oocyte maturation by inducing oxidative stress and apoptosis.

Citrinin (CTN) is a polyketide mycotoxin produced by several strains of Penicillium, Monascus, and Aspergillus. While CTN poses various toxic effects on the female reproductive system in animals, its direct effects on germ cell development are unclear. This study aimed to evaluate the effects of increasing concentrations of CTN (0,20,40,80,100 µM) on porcine oocyte in vitro maturation. Our results indicate that CTN supplementation inhibited polar body extrusion in a dose-dependent manner. Actin and spindle assembly were also disrupted after treatment, indicating that CTN affects the cytoskeleton of porcine oocytes. Oxidative stress and apoptosis were observed under CTN treatment to explore the cause of meiotic maturation failure in porcine oocytes. The results showed that reactive oxygen species levels, cathepsin B activity, and caspase-3 activity were increased in the treated group, indicating that CTN induced oxidative stress and apoptosis. In conclusion, CTN exposure could reduce porcine oocyte maturation by affecting cytoskeletal dynamics, oxidative stress, and apoptosis.

Cytogenotoxic evaluation of the acetonitrile extract, citrinin and dicitrinin-A from Penicillium citrinum.

Endophytic fungi are promising sources of bioactive substances; however, their secondary metabolites are toxic to plants, animals, and humans. This study aimed toevaluate the toxic, cytotoxic, mutagenic and oxidant/antioxidant activities of acetonitrile extract (AEPc), citrinin (CIT) and dicitrinin-A (DIC-A) of Penicillium citrinum. For this, the test substances at 0.5; 1.0; 1.5 and 2 µg/mLwere exposed for 24 and 48 h in Artemia salina, and 48 h in Allium cepa test systems. The oxidant/antioxidant test was evaluated in pre-, co- and post-treatment with the stressor hydrogen peroxide (H2O2) in Saccharomyces cerevisiae. The results suggest that the AEPc, CIT and DIC-A at 0.5; 1.0; 1.5 and 2 µg/mL showed toxicity in A. saline, with LC50 (24 h) of 2.03 µg/mL, 1.71 µg/mL and 2.29 µg/mL, and LC50 (48 h) of 0.51 µg/mL, 0.54 µg/mL and 0.54 µg/mL, respectively.In A. cepa, the test substances also exerted cytotoxic and mutagenic effects. The AEPc, CIT and DIC-A at lower concentrations modulated the damage induced by H2O2 in the proficient and mutant strains of S. cerevisiae for cytoplasmic and mitochondrial superoxide dismutase. Moreover, the AEPc at 2 µg/mL and CIT at the two highest concentrations did not affect the H2O2-induced DNA damage in the test strains. In conclusion, AEPc, CIT and DIC-A of P. citrinum may exert their toxic, cytotoxic and mutagenic effects in the test systems possibly through oxidative stress induction pathway.

The individual and combined effects of ochratoxin A with citrinin and their metabolites (ochratoxin B, ochratoxin C, and dihydrocitrinone) on 2D/3D cell cultures, and zebrafish embryo models.

Ochratoxin A and citrinin are nephrotoxic mycotoxins produced by Aspergillus, Penicillium, and/or Monascus species. The combined effects of ochratoxin A and citrinin have been examined in more studies; however, only limited data are available regarding the co-exposure to their metabolites. In this investigation, the individual toxic effects of ochratoxin A, ochratoxin B, ochratoxin C, citrinin, and dihydrocitrinone were tested as well as the combinations of ochratoxin A with the latter mycotoxins were examined on 2D and 3D cell cultures, and on zebrafish embryos. Our results demonstrate that even subtoxic concentrations of certain mycotoxins can increase the toxic impact of ochratoxin A. In addition, typically additive effects or synergism were observed as the combined effects of mycotoxins tested. These observations highlight that different cell lines (e.g. MDBK vs. MDCK), cell cultures (e.g. 2D vs. 3D), and models (e.g. in vitro vs. in vivo) can show different (sometimes opposite) impacts. Mycotoxin combinations considerably increased miR-731 levels in zebrafish embryos, which is an early marker of the toxicity on kidney development. These results underline that the co-exposure to mycotoxins (and/or mycotoxin metabolites) should be seriously considered, since even the barely toxic mycotoxins (or metabolites) in combinations can cause significant toxicity.

Exposure to citrinin induces DNA damage, autophagy, and mitochondria dysfunction during first cleavage of mouse embryos.

Citrinin (CTN) is a mycotoxin, which is isolated from Penicillium citrinum and widely existed in the contaminated feeds. It is reported that CTN is toxic to heart, liver, and reproductive system. Previous studies indicated that CTN induced apoptosis in oocytes and embryos. In this study, we reported the potential causes of CTN on embryo development. Our results showed that 40 µM CTN exposure significantly reduced the first cleavage of mouse embryos, showing with the low rate of 2-cell embryos. We found that CTN induced DNA damage, showing the higher positive γH2A.X signals. Autophagy was occurred since more LC3 positive autophagosomes were found in the cytoplasm. This could be confirmed by the enhanced lysosome function, since higher accumulated lysosome distribution were found and LAMP2 was also increased under CTN exposure. Besides, we showed that mitochondria distribution was disturbed, indicating that CTN could disrupt mitochondria function, which could be the possible reason for the oxidative stress and apoptosis in CTN-exposed embryos. In conclusion, our study showed that CTN exposure had adverse effects on the early embryo development during first cleavage through its effects on the induction of DNA damage, autophagy, and mitochondria dysfunction.

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A, Ochratoxin α, Citrinin and Dihydrocitrinone.

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.

Evaluation of citrinin-induced toxic effects on mouse Sertoli cells.

Penicillium citrinum-derived mycotoxin citrinin (CTN) is known to be a toxic agent for humans and animals. Previous studies have shown that CTN leads to toxicity in many biological systems; however, a limited number of studies have been performed to demonstrate the harmful effects of CTN on the male reproductive system. In the present study, the effects of CTN on cytotoxicity and apoptosis were examined in Sertoli cells as a model. Sertoli cells were treated with eight different CTN concentrations (from 0 up to 200 µM, for 6-72 h). Toxic potential of CTN was estimated by measuring metabolic activity (MTT test), DNA synthesis (BrdU test), and cell membrane damage (LDH test) as well as apoptosis and necrosis (via staining with propidium iodide and Hoechst 33342). The results showed that CTN significantly decreased the cell viability and cell proliferation, increased cytotoxicity, apoptosis, and necrosis in a concentration-dependent manner. Furthermore, CTN showed cytotoxicity in Sertoli cells with an IC50 value of 116.5 µM for 24 h. In conclusion, these findings clearly showed that, CTN affects Sertoli cells even at low concentrations. Thus, as a result of the damage of CTN shown in Sertoli cells, it can be deduced that CTN may also have detrimental effects on the testes.