The role of oxidative stress in citreoviridin-induced DNA damage in human liver-derived HepG2 cells.

We hypothesize that citreoviridin (CIT) induces DNA damage in human liver-derived HepG2 cells through an oxidative stress mechanism and that N-acetyl-l-cysteine (NAC) protects against CIT-induced DNA damage in HepG2 cells. CIT-induced DNA damage in HepG2 cells was evaluated by alkaline single-cell gel electrophoresis assay. To elucidate the genotoxicity mechanisms, the level of oxidative DNA damage was tested by immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG); the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined; mitochondrial membrane potential and lysosomal membranes' permeability were detected; furthermore, protective effects of NAC on CIT-induced ROS formation and CIT-induced DNA damage were evaluated in HepG2 cells. A significant dose-dependent increment in DNA migration was observed at tested concentrations (2.50-10.00 µM) of CIT. The levels of ROS, 8-OHdG formation were increased by CIT, and significant depletion of GSH in HepG2 cells was induced by CIT. Destabilization of lysosome and mitochondria was also observed in cells treated with CIT. In addition, NAC significantly decreased CIT-induced ROS formation and CIT-induced DNA damage in HepG2 cells. The data indicate that CIT induces DNA damage in HepG2 cells, most likely through oxidative stress mechanisms; that NAC protects against DNA damage induced by CIT in HepG2 cells; and that depolarization of mitochondria and lysosomal protease leakage may play a role in CIT-induced DNA damage in HepG2 cells.

Citreoviridin enhances tumor necrosis factor-α-induced adhesion of human umbilical vein endothelial cells.

Endothelial adhesion plays an important role in the process of atherosclerosis, which is regulated by endothelial adhesion molecules and chemoattractant molecules. In some areas of China, citreoviridin (CIT) is considered a risk factor for the development of atherosclerosis. Here, we investigated the role of CIT in adhesion of human umbilical vein endothelial cells (HUVECs) together with the stimulation of tumor necrosis factor-α (TNF-α). Adhesion of HUVECs to monocytes was analyzed by coculture experiments using U937 cells labeled with 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester. The expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin was determined by Western blot and enzyme-linked immunosorbent assay (ELISA). The expression of monocyte chemoattractant protein-1 (MCP-1) was measured by reverse transcription polymerase chain reaction and ELISA. The activation of nuclear factor-κB (NF-κB) was assessed by Western blot and immunofluorescence staining. CIT markedly increased TNF-α-induced HUVECs adhesion to monocytes and the expression levels of ICAM-1, VCAM-1, E-selectin, and MCP-1. TNF-α-induced nuclear translocation of NF-κB in HUVECs was significantly elevated by CIT. Our study demonstrates that CIT upregulates TNF-α-induced endothelial adhesion via increasing activation of NF-κB, which results in the expression of ICAM-1, VCAM-1, E-selectin, and MCP-1. CIT plays a pivotal role in the process of endothelial cell adhesion and may thereby play an important role in the improvement of atherosclerosis in areas of China that have a high prevalence of CIT contamination and atherosclerosis.

The In Vivo and In Vitro Toxicokinetics of Citreoviridin Extracted from Penicillium citreonigrum.

Citreoviridin (CTVD), a mycotoxin called yellow rice toxin, is reported to be related to acute cardiac beriberi; however, its toxicokinetics remain unclear. The present study elucidated the toxicokinetics through in vivo experiments in swine and predicted the human toxicokinetics by comparing the findings to those from in vitro experiments. In vivo experiments revealed the high bioavailability of CTVD (116.4%) in swine. An intestinal permeability study using Caco-2 cells to estimate the toxicokinetics in humans showed that CTVD has a high permeability coefficient. When CTVD was incubated with hepatic S9 fraction from swine and humans, hydroxylation and methylation, desaturation, and dihydroxylation derivatives were produced as the predominant metabolites. The levels of these products produced using human S9 were higher than those obtained swine S9, while CTVD glucuronide was produced slowly in human S9 in comparison to swine S9. Furthermore, the elimination of CTVD by human S9 was significantly more rapid in comparison to that by swine S9. These results suggest that CTVD is easily absorbed in swine and that it remains in the body where it is slowly metabolized. In contrast, the absorption of CTVD in humans would be the same as that in swine, although its elimination would be faster.

Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC50s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

Developmental exposure of citreoviridin transiently affects hippocampal neurogenesis targeting multiple regulatory functions in mice.

To investigate the developmental exposure effect of citreoviridin (CIT) on postnatal hippocampal neurogenesis, pregnant ICR mice were dietary exposed to CIT at 0, 1, 3 and 10 ppm from gestation day 6 to postnatal day (PND) 21 on weaning. Offspring were maintained through PND 77 without CIT exposure. Male offspring were analyzed. At 10 ppm on PND 21, weak changes suggestive of neural stem cell reduction and progenitor cell proliferation were observed. Number of hilar CALB1+ interneurons reduced, suggesting an influence on neurogenesis. In contrast, number of hilar SST+ interneurons increased and Bdnf and Ntrk2 transcripts upregulated in the dentate gyrus, suggesting a facilitation of BDNF-TRKB signaling for progenitor cell proliferation. Transcript expression changes of an outside regulatory system suggested suppressed function of GABAergic interneurons, especially of PVALB+ interneurons for compensation on neural stem cell reduction. At ≥ 3 ppm, number of ARC+ mature granule cells increased, and at 10 ppm, number of hilar GRIA1+ cells increased and Gria2 and Gria3 upregulated, suggesting an operation of AMPA receptor membrane trafficking on the increase of ARC-mediated synaptic plasticity. On PND 77, all the transcript expression changes of the neurogenesis regulatory system except for Grin2d were inverted, suggesting an operation of a homeostatic mechanism on CIT-induced disruptive neurogenesis. Simultaneous downregulation of Grin2a and Grin2d suggests suppression of GABAergic interneuron function to adjust neurogenesis at the normal level. The no-observed-adverse-effect level of CIT for offspring neurogenesis was determined to be 1 ppm, translating to 0.13-0.51 mg/kg body weight/day of maternal oral exposure.

Occurrence of Penicillium brocae and Penicillium citreonigrum, which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin, Respectively, in Selected Commercially Available Rice Grains in Thailand.

Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 µg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains.

Citreoviridin induces ROS-dependent autophagic cell death in human liver HepG2 cells.

Citreoviridin (CIT) is one of toxic mycotoxins derived from fungal species in moldy cereals. Whether CIT exerts hepatotoxicity and the precise molecular mechanisms of CIT hepatotoxicity are not completely elucidated. In this study, the inhibitor of autophagosome formation, 3-methyladenine, protected the cells against CIT cytotoxicity, and the autophagy stimulator rapamycin further decreased the cell viability of CIT-treated HepG2 cells. Knockdown of Atg5 with Atg5 siRNA alleviated CIT-induced cell death. These finding suggested the hypothesis that autophagic cell death contributed to CIT-induced cytotoxicity in HepG2 cells. CIT increased the autophagosome number in HepG2 cells observed under a transmission electron microscope, and this effect was confirmed by the elevated LC3-II levels detected through Western blot. Reduction of P62 protein levels and the result of LC3 turnover assay indicated that the accumulation of autophagosomes in the CIT-treated HepG2 cells was due to increased formation rather than impaired degradation. The pretreatment of HepG2 cells with the ROS inhibitor NAC reduced autophagosome formation and reversed the CIT cytotoxicity, indicating that CIT-induced autophagic cell death was ROS-dependent. In summary, ROS-dependent autophagic cell death of HpeG2 cells described in this study may help to elucidate the underlying mechanism of CIT cytotoxicity.

Citreoviridin Enhances Atherogenesis in Hypercholesterolemic ApoE-Deficient Mice via Upregulating Inflammation and Endothelial Dysfunction.

Vascular endothelial dysfunction and inflammatory response are early events during initiation and progression of atherosclerosis. In vitro studies have described that CIT markedly upregulates expressions of ICAM-1 and VCAM-1 of endothelial cells, which result from NF-κB activation induced by CIT. In order to determine whether it plays a role in atherogenesis in vivo, we conducted the study to investigate the effects of CIT on atherosclerotic plaque development and inflammatory response in apolipoprotein E deficient (apoE-/-) mice. Five-week-old apoE-/- mice were fed high-fat diets and treated with CIT for 15 weeks, followed by assay of atherosclerotic lesions. Nitric oxide (NO), vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were detected in serum. Levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), VEGF, and ET-1 in plaque areas of artery walls were examined. NF-κB p65 expression and NF-κB activation in aorta also were assessed. CIT treatment significantly augmented atherosclerotic plaques and increased expressions of ICAM-1, VCAM-1, VEGF and ET-1 in aorta. Mechanistic studies showed that activation of NF-κB was significantly elevated by CIT treatment, indicating the effect of CIT on atherosclerosis may be regulated by activation of NF-κB.

Teratogenic potential of the mycotoxin, citreoviridin, in rats.

Citreoviridin produced by the fungus Penicillium citreo-viride was administered by gavage to groups of 9-16 pregnant Fisher 344 rats either on days 8-11 (group A) or on days 12-15 (group B) of gestation. Doses of 0, 5, 10 or 15 mg/kg body weight were given daily in a constant volume of 1 ml/kg body weight in dimethylsulphoxide. Six rats in each high-dose group died during the dosing period. Compared with control groups, mean daily feed consumption was significantly reduced in the 10- and 15-mg/kg animals in both groups A and B. Weight gain during pregnancy in both groups was reduced with increasing dosage; dams in control groups gained an average of 75 g/rat compared with 30 g overall gain in group A, or 9 g overall gain in group B, both at a dose of 15 mg/kg. Male and female pup weights were reduced with increasing dosage for both groups A and B. The post-implantation foetal loss rate was significantly increased to 33% in group A high-dose animals. The main effect of citreoviridin on skeletal development in both groups A and B was one of retardation. No internal abnormalities were observed in group A pups. Some smaller than average pups from dams in group B that were treated with the high dose of citreoviridin had slightly dilated lateral ventricles of the brain and, in some cases, a palate defect. The foetotoxicity induced by citreoviridin was observed only at doses that also induced maternal toxicity.

Chronic exposure to cereal mycotoxin likely citreoviridin may be a trigger for Keshan disease mainly through oxidative stress mechanism.

The origin of Keshan disease (KD), an endemic cardiomyopathy particularly affected children and young women in China, has been a controversial and difficult problem in academics over the past decades. We hypothesize that mycotoxins likely citreoviridin may initiate KD mainly through oxidative stress mechanism by long-term consumption of mouldy cereals due to food shortage lifestyle. Dietary deficiency of selenium, proteins, Vitamin C, E, etc., may act as enhancing factors to exacerbate the pathological damage. This speculation can perfectly explain the features of KD and has received strong supporting evidences. If the hypothesis were to be confirmed, it is of very important value in monitoring and blocking the incidence of KD. Thus, future efforts are needed to investigate specific biomarkers of exposure to citreoviridin in KD cases.

Nematodetoxic aurovertin-type metabolites from a root-knot nematode parasitic fungus Pochonia chlamydosporia.

Chemical investigation of one fungal strain P. chlamydosporia YMF 1.00613 isolated from root knots of tobacco infected by Meloidogyne incognita led to the isolation and identification of four aurovertin-type metabolites, which include a new compound, aurovertin I (A1), and three known metabolites, aurovertins E, F and D (A2-A4). Their structures were established by spectroscopic studies such as 1D- and 2D-NMR and MS analysis. Aurovertin I (A1) is the first natural product with an aurovertin skeleton with one less carbon. Compounds A3 and A4 showed the toxicity to the worms of the free-living nematode Panagrellus redivevus with the LC(50) values 88.6 and 41.7 microg/mL at 48 h, respectively. All four aurovertins did not show obvious inhibitory effects on egg hatch of root knot nematode Meloidogyne incognita. The results suggested that the aurovertin-type metabolites produced by P. chlamydosporia might be one of the pathogenic factors involved in the suppression of nematodes.

Citreoviridin levels in Eupenicillium ochrosalmoneum-infested maize kernels at harvest.

Citreoviridin contents were measured in eight bulk samples of maize kernels collected from eight fields immediately following harvest in southern Georgia. Citreoviridin contamination in six of the bulk samples ranged from 19 to 2,790 micrograms/kg. In hand-picked samples the toxin was concentrated in a few kernels (pick-outs), the contents of which were stained a bright lemon yellow (range, 53,800 to 759,900 micrograms/kg). The citreoviridin-producing fungus Eupenicillium ochrosalmoneum Scott & Stolk was isolated from each of these pick-out kernels. Citreoviridin was not detected in bulk samples from two of the fields. Aflatoxins were also present in all of the bulk samples (total aflatoxin B1 and B2; range, 7 to 360 micrograms/kg), including those not containing citreoviridin. In Biotron-grown maize ears that were inoculated with E. ochrosalmoneum through a wound made with a toothpick, citreoviridin was concentrated primarily in the wounded and fungus-rotted kernels (range, 142,000 to 2,780,000 micrograms/kg). Samples of uninjured kernels immediately adjacent to the wounded kernel (first circle) had less than 4,000 micrograms of citreoviridin per kg, while the mean concentration of toxin in kernel samples representing the next row removed (second circle) and all remaining kernels from the ear was less than 45 micrograms/kg. Animal toxicosis has not been linked to citreoviridin-contaminated maize.

The toxicology of mycotoxins.

Mycotoxin problems are one of great concern to health scientists. Toxic fungal metabolites such as aflatoxins, trichothecenes, zearalenone and others are contaminated in our environments and induce various diseases. In this manuscript, the author will summarize the recent advances on toxicology of mycotoxins in special references to toxicological characters, cytotoxicity, genotoxicity (mutagenicity and carcinogenicity), metabolism, and biochemical mode of action. Interaction of mycotoxins with cellular components will be reviewed in order to clarify the toxicological characteristics of mycotoxins such as aflatoxins, trichothecenes, zearalenone, toxic peptides, and anthraquinoid mycotoxins.

Toxicity of citreoviridin.

The mycotoxin citreoviridin (CIT) isolated from Penicillium citreoviride was studied to elucidate the mechanism of its toxic actions. In CF#1 mice, near lethal doses of CIT decreased motor activities, body temperature and had cataleptic effects. Male mice appeared to be more susceptible to CIT and had lower subcutaneous (sc) LD50 values and longer CIT-induced hypothermia and hypokinesia. In CIT-treated mice the weights and histology of liver, kidneys and adrenals were normal one week after sc treatment, except for the increased adrenal weights in female mice. Single doses of CIT (sc), given on either day 4 or 5 of pregnancy (perinidation period), had no adverse effect on the rates of pregnancy, implantation of ova and embryonal resorptions in those mice examined on day 12 of pregnancy. CIT (40 mg/kg ip) produced a brief electro-encephalographic (EEG) activation, cardiac sinus arrhythmias and tachypnea in the rabbit. Intravenous (iv) lethal doses of CIT (greater than or equal to 5 mg/kg) caused an EEG activation followed by high voltage delta waves, increased the T wave in the electrocardiogram (ECG) and depressed the respiratory amplitude. The death caused by iv CIT started with the respiratory arrest, followed by isoelectric EEG and ECG was the last to stop. In urethane-anesthetized rabbits CIT decreased the blood pressure, and in succession it lowered, flattened and inverted the T wave of ECG suggesting heart ischemia. These observations indicated that the toxic effects of CIT resulted from respiratory and cardiovascular failures (apnea, delta EEG waves, sinus arrhythmia, hypotension) leading to central nervous system depression due to systemic hypoxia.