Xiaozhe Qi

MicroRNA profiling of rats with ochratoxin A nephrotoxicity

BackgroundNephrotoxicity is the most prominent one among the various toxicities of ochratoxin A (OTA). MicroRNAs (miRNAs) are small non-coding RNAs that have an impact on a wide range of biological processes by regulating gene expression at post-transcriptional level or protein systhesis level. The objective of this study is to analyze miRNA profiling in the kidneys of rats gavaged with OTA.ResultsTo profile miRNAs in the kidneys of rats with OTA nephrotoxicity, high-throughput sequencing and bioinformatics approaches were applied to analyze the miRNAs in the kidney of rats following OTA treatment. A total of 409 known miRNAs and 8 novel miRNAs were identified in the kidney and the levels of the novel miRNAs were varied in response to different doses of OTA. Expression of miR-129, miR-130a, miR-130b, miR-141, miR-218b and miR-3588 were uniquely suppressed in mid dose but then elevated in high dose, with opposite expression to their target genes. The expression pattern was closely related with the “MAPK signaling pathway”. Dicer1 and Drosha were significantly suppressed, indicating an impairment of miRNA biogenesis in response to OTA.ConclusionsThe abrogation of miRNA maturation process suggests a new target of OTA toxicity. Moreover, the identification of the differentially expressed miRNAs provides us a molecular insight into the nephrtoxicity of OTA.

Subchronic feeding study of stacked trait genetically-modified soybean (3Ø5423 × 40-3-2) in Sprague-Dawley rats.

The genetically-modified (GM) soybean 3Ø5423 × 40-3-2 expresses siRNA for the fatty acid desaturase-2 enzyme which results in higher concentrations of oleic acid (18:1) relative to linoleic acid (18:2) compared with non-GM soybeans. It also expresses the CP4 EPSPS protein for tolerance to glyphosate. In this study, three different dietary concentrations (7.5%, 15% and 30% wt/wt) of 3Ø5423 × 40-3-2 or non-GM soybeans were fed to Sprague-Dawley rats for 90 days during which in-life nutritional and growth performance variables were evaluated followed by analysis of standard clinical chemistry, hematology and organ variables. Compared with rats fed the non-GM control diet, some statistically significant differences were observed in rats fed the 3Ø5423 × 40-3-2 diet. However the differences were not considered treatment-related and commonly fell within the normal ranges of the control group consuming the commercial diet. These results demonstrated that the GM soybean 3Ø5423 × 40-3-2 is as safe as non-GM soybeans.

Combination of metagenomics and culture-based methods to study the interaction between ochratoxin a and gut microbiota.

Gut microbiota represent an important bridge between environmental substances and host metabolism. Here we reported a comprehensive study of gut microbiota interaction with ochratoxin A (OTA), a major food-contaminating mycotoxin, using the combination of metagenomics and culture-based methods. Rats were given OTA (0, 70, or 210 μg/kg body weight) by gavage and fecal samples were collected at day 0 and day 28. Bacterial genomic DNA was extracted from the fecal samples and both 16S rRNA and shotgun sequencing (two main methods of metagenomics) were performed. The results indicated OTA treatment decreased the within-subject diversity of the gut microbiota, and the relative abundance of Lactobacillus increased considerably. Changes in functional genes of gut microbiota including signal transduction, carbohydrate transport, transposase, amino acid transport system, and mismatch repair were observed. To further understand the biological sense of increased Lactobacillus, Lactobacillus selective medium was used to isolate Lactobacillus species from fecal samples, and a strain with 99.8% 16S rRNA similarity with Lactobacillus plantarum strain PFK2 was obtained. Thin-layer chromatography showed that this strain could absorb but not degrade OTA, which was in agreement with the result in metagenomics that no genes related to OTA degradation increased. In conclusion, combination of metagenomics and culture-based methods can be a new strategy to study intestinal toxicity of toxins and find applicable bacterial strains for detoxification. When it comes to OTA, this kind of mycotoxin can cause compositional and functional changes of gut microbiota, and Lactobacillus are key genus to detoxify OTA in vivo.

Ochratoxin A induces rat renal carcinogenicity with limited induction of oxidative stress responses.

Ochratoxin A (OTA) has displayed nephrotoxicity and renal carcinogenicity in mammals, however, no clear mechanisms have been identified detailing the relationship between oxidative stress and these toxicities. This study was performed to clarify the relationship between oxidative stress and the renal carcinogenicity induced by OTA. Rats were treated with 70 or 210 μg/kg b.w. OTA for 4 or 13 weeks. In the rats administrated with OTA for 13 weeks, the kidney was damaged seriously. Cytoplasmic vacuolization was observed in the outer stripe of the outer medulla. Karyomegaly was prominent in the tubular epithelium. Kidney injury molecule-1 (Kim-1) was detected in the outer stripe of the outer medulla in both low- and high-dose groups. OTA increased the mRNA levels of clusterin in rat kidneys. Interestingly, OTA did not significantly alter the oxidative stress level in rat liver and kidney. Yet, some indications related to proliferation and carcinogenicity were observed. A dose-related increase in proliferating cell nuclear antigen (PCNA) was observed at 4 weeks in both liver and kidney, but at 13 weeks, only in the kidney. OTA down-regulated reactive oxygen species (ROS) and up-regulated vimentin and lipocalin 2 in rat kidney at 13 weeks. The p53 gene was decreased in both liver and kidney at 13 weeks. These results suggest that OTA caused apparent kidney damage within 13 weeks but exerted limited effect on oxidative stress parameters. It implies that cell proliferation is the proposed mode of action for OTA-induced renal carcinogenicity.

Subchronic toxicity study in vivo and allergenicity study in vitro for genetically modified rice that expresses pharmaceutical protein (human serum albumin)

Genetically modified (GM) crops that express pharmaceutical proteins have become an important focus of recent genetic engineering research. Food safety assessment is necessary for the commercial development of these crops. Subchronic toxicity study in vivo and allergenicity study in vitro were designed to evaluate the food safety of the rice variety expressing human serum albumin (HSA). Animals were fed rodent diets containing 12.5%, 25.0% and 50.0% GM or non-GM rice for 90 days. The composition analysis of the GM rice demonstrated several significant differences. However, most of the differences remained within the ranges reported in the literature. In the animal study, a range of indexes including clinical observation, feed efficiency, hematology, serum chemistry, organ weights and histopathology were examined. Random changes unrelated to the GM rice exposure, within the range of historical control values and not associated with any signs of illness were observed. The results of heat stability and in vitro digestion of HSA indicated no evidence of potential allergenicity of the protein. Overall, the results of these studies suggest that the GM rice appears to be safe as a dietary ingredient when it is used at up to 50% in the diet on a subchronic basis.

Safety assessment of genetically modified rice expressing human serum albumin from urine metabonomics and fecal bacterial profile

The genetically modified (GM) rice expressing human serum albumin (HSA) is used for non-food purposes; however, its food safety assessment should be conducted due to the probability of accidental mixture with conventional food. In this research, Sprague Dawley rats were fed diets containing 50% (wt/wt) GM rice expressing HSA or non-GM rice for 90 days. Urine metabolites were detected by (1)H NMR to examine the changes of the metabolites in the dynamic process of metabolism. Fecal bacterial profiles were detected by denaturing gradient gel electrophoresis to reflect intestinal health. Additionally, short chain fatty acids and fecal enzymes were investigated. The results showed that compared with rats fed the non-GM rice, some significant differences were observed in rats fed with the GM rice; however, these changes were not significantly different from the control diet group. Additionally, the gut microbiota was associated with blood indexes and urine metabolites. In conclusion, the GM rice diet is as safe as the traditional daily diet. Furthermore, urine metabonomics and fecal bacterial profiles provide a non-invasive food safety assessment rat model for genetically modified crops that are used for non-food/feed purposes. Fecal bacterial profiles have the potential for predicting the change of blood indexes in future.

Discovery of systematic responses and potential biomarkers induced by ochratoxin A using metabolomics

Ochratoxin A (OTA) is known to be nephrotoxic and hepatotoxic in rodents when exposed orally. To understand the systematic responses to OTA exposure, GC-MS- and 1H-NMR-based metabolomic techniques together with histopathological assessments were applied to analyse the urine and plasma of OTA-exposed rats. It was found that OTA exposure caused significant elevation of amino acids (alanine, glycine, leucine etc.), pentose (ribose, glucitol, xylitol etc.) and nucleic acid metabolites (pseudouridine, adenosine, uridine). Moreover, myo-inositol, trimethylamine-oxide (TMAO), pseudouridine and leucine were identified as potential biomarkers for OTA toxicity. The primary pathways included the pentose phosphate pathway (PPP), the Krebs cycle (TCA), the creatine pathway and gluconeogenesis. The activated PPP was attributed to the high requirements for nicotinamide adenine dinucleotide phosphate (NADPH), which is involved in OTA metabolism through cytochrome P450. The elevated gluconeogenesis and TCA suggest that energy metabolism was involved. The up-regulated synthesis of creatinine reveals the elevated catabolism of proteins. These findings provide an overview of systematic responses to OTA exposure and metabolomic insight into the toxicological mechanism of OTA. Graphical Abstract

Limited Link between Oxidative Stress and Ochratoxin A—Induced Renal Injury in an Acute Toxicity Rat Model

Ochratoxin A (OTA) displays nephrotoxicity and hepatotoxicity. However, in the acute toxicity rat model, there is no evidence on the relationship between OTA and nephrotoxicity and hepatotoxicity. Based on this, the integrated analysis of physiological status, damage biomarkers, oxidative stress, and DNA damage were performed. After OTA treatment, the body weight decreased and AST, ALP, TP, and BUN levels in serum increased. Hydropic degeneration, swelling, vacuolization, and partial drop occurred in proximal tubule epithelial cells. PCNA and Kim-1 were dose-dependently increased in the kidney, but Cox-2 expression and proliferation were not found in the liver. In OTA-treated kidneys, the mRNA expressions of Kim-1, Cox-2, Lcn2, and Clu were dose-dependently increased. The mRNA expressions of Vim and Cox-2 were decreased in OTA-treated livers. Some oxidative stress indicators were altered in the kidneys (ROS and SOD) and livers (SOD and GSH). DNA damage and oxidative DNA damage were not found. In conclusion, there is a limited link between oxidative stress and OTA-induced renal injury in an acute toxicity rat model.

Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes

Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A‐induced toxicity was investigated using Hepatoblastoma Cell Line HepG‐2 cells. Disruption of cholesterol‐containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time‐dependent manner. Isobaric tags for relative and absolute quantitation‐based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology “transport”, “cell adhesion” and “vesicle‐mediated transport”. In conclusion, lipid rafts play a key role in OTA‐induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts.

Dynamic changes of global DNA methylation and hypermethylation of cell adhesion-related genes in rat kidneys in response to ochratoxin A

Ochratoxin A (OTA), which is found in a variety of food products, is associated with the development of nephrotoxicity and carcinogenicity in rats and has raised public health concerns. A previous study in our laboratory indicated that OTA exposure induced cytotoxicity by decreasing global DNA methylation in vitro. However, the relationship between OTA-induced nephrotoxicity and DNA methylation changes in vivo remains unclear. The object of this study was to investigate whether OTA can change global DNA methylation or alter the expression of several critical tumour-related genes by inducing methylation modifications before carcinogenesis. We focused on the mechanism of action of OTA in regard to DNA methylation, including the expression of DNA methyltransferases and the regulation of specific cell signalling pathways. Dynamic and dose-dependent changes of global DNA methylation were observed during OTA-induced nephrotoxicity and probably associated with the expression of DNA methyltransferase 1. 13-week e...