Polyphenols as possible bioprotectors against cytotoxicity and DNA damage induced by ochratoxin A

The present study aimed to investigate the protective effects of luteolin (L), chlorogenic acid (ChlA) and caffeic acid (CafA) against cyto-genotoxic effects caused by OTA. Vero cells and rat lymphocytes were used and viability was measured by neutral red uptake, MTT and trypan blue dye exclusion method. L (50 and 100 μg/mL), ChlA (100 and 200 μg/mL) and CafA (10–50 μg/mL) reduced the damage induced by OTA (10 μg/mL) on both cells type shown a good protective effect. The comet and micronucleus tests in Balb/c mice were performed. ChlA (10 mg/kg bw) reduced OTA (0.85 mg/kg bw)-induced DNA damage on blood and bone marrow cells, CafA (10 mg/kg bw) showed protective effect only in blood cells and luteolin (2.5 mg/kg bw) failed to protect DNA integrity on cells. In conclusion, polyphenols tested reduced the toxicity caused by OTA on different target cells with good protective effect, being ChlA the compound that showed the best effects.     

Apoptosis and oxidative stress induced by ochratoxin A in rat kidney

Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of fungi. OTA induces a tubular-interstitial nephropathy in humans and in animals. It has been implicated as one of the aetiological agents involved in the development of endemic nephropathy. OTA-induced oxidative stress and apoptosis may play key roles in the development of chronic tubulointerstitial nephritis connected to the long-term exposure to this food contaminant. We studied the effects of low doses of OTA on kidney cells. Wistar rats were treated with 120 g OTA/kg bodyweight daily, for 10, 30 or 60 days. Toxin concentration in kidney was proportional to the time of exposure, and amounted to 547.2, 752.5 and 930.3 ng OTA/g kidney tissue after 10, 30 and 60 days, respectively. OTA treatment caused an increased number of cells undergoing apoptosis in both proximal and distal epithelial kidney cells. The apoptotic cells were visualised using the TUNEL assay and staining with haematoxylin and eosin in situ. The number of apoptotic cells in rats treated for 10, 30 and 60 days increased by 5-, 6.4- and 12.7-fold, respectively, compared with the control cells. However, DNA electrophoresis did not show characteristic fragmentation (DNA laddering). The oxidative stress was evident via increased malondialdehyde formation. The concentration of lipid peroxides showed an increase (36%), but the activity of superoxide dismutase decreased (26%) in 60-day treated rats. In spite of the observed biochemical and morphological changes in the kidney cells, renal functional status was preserved to the end of experiment. This study demonstrates that a combination of morphologic and biochemical markers can be used to monitor early cell death in OTA-induced renal injury. We have shown that the exposure to the relatively low OTA concentrations has activated apoptotic processes and oxidative damage in kidney cells.     

Comparative effect of ochratoxin A on inflammation and oxidative stress parameters in gut and kidney of piglets

Ochratoxin A (OTA) is a secondary metabolite produced by fungi of Aspergillus and Penicillium genra. OTA is mainly nephrotoxic but can also cause hepatotoxicity, mutagenicity, teratogenicity, neurotoxicity and immunotoxicity. As recent studies have highlighted the close relationship between gastrointestinal tract and kidney, as principal organs involved in absorption and respective excretion of xenobiotics, the aim of the present study was to analyze the effect of a subchronic exposure (30 days) to 0.05 mg/kg OTA on immune response and oxidative stress parameters at the level of intestine and kidney of young swine. The experiment was realised on twelve crossbred weaned piglets randomly allotted to both control group or toxin group fed 0.050 mg OTA/kg feed. Our results have shown that a subchronic intoxication with a low dose of OTA for 30 days affected the immune response and the anti-oxidant self-defense at gut and kidney level. The gene expression of both markers of signaling pathways involved in inflammation and inflammatory cytokines were affected in a much higher extent in the gut than in the kidney Of OTA intoxicated piglets.     

Distribution of the [3H]-label from low doses of radioactive ochratoxin a ingested by rats,and evidence for DNA single-strand breaks caused in liver and kidneys

The distribution of a single low dose of [³H]ochratoxin A (OTA) in different tissues of male Wistar rats, after administration by intubation, was investigated after 5 h, 24 h and 48 h. This dose corresponds to concentrations encountered in naturally contaminated feed (4 ppm). The distribution of [³H]-label varied with the time elapsed after administration; at 5 h the highest specific label was found in the stomach contents and in decreasing order in: intestinal contents, lung, liver, kidney, heart, fat, intestine, testes, and the lowest in muscles, spleen and brain. With exception of brain, fat, stomach and lung, all tissues showed maximum levels at 24 h, after which time the label decreased steadily, whereas in fat it increased.After a 12-week feeding experiment, with doses of 288.8 g/kg corresponding to an intake of 4 ppm in feed each 48 h, the DNA in liver and kidneys was investigated for damage. By the alkaline elution method combined with micro-spectrofluorimetric determinations of DNA, evidence for DNA single-strand breaks was obtained. These findings support reports on the carcinogenic action of OTA.     

Acute kidney damage by PM2.5 exposure in a rat model

PM_2.5 exposure is associated with a glomerular filtration rate (GFR) reduction, and renal tissue damage. The goal of this study was demonstrate the acute effect of PM_2.5 on the kidney. Male rats were acutely exposed to PM_2.5 or filtered air. Blood pressure was mesure and early kidney biomarkers were evaluated in serum and urine samples, and also IL-1β, IL-6 and TNFα were determined. Oxidative biomarkers, angiotensin/bradykinin-related proteins, KIM-1, IL-6 and histology were determined. Blood pressure, GFR, and early kidney damage biomarkers increase together with oxidative biomarkers and angiotensin/bradykinin endocrine-related proteins increased after exposure to PM_2.5. Urinary IL-6 increased after exposure to PM_2.5, whereas in kidney cortex decreased. Histological changes were observed and accompanied by the induction of KIM-1. Acute exposure to PM_2.5 not decline kidney function. However, it can induce early kidney damage biomarkers, oxidative stress, inflammation and angiotensin mediators, which perhabs culminates in a lose of renal function.     

Protective effect of N-acetylcysteine against DNA damage and S-phase arrest induced by ochratoxin A in human embryonic kidney cells (HEK-293)

N-acetylcysteine (NAC) has recently gained particular interest as a beneficial antioxidant. This study investigated the protective effects of NAC against ochratoxin A (OTA)-induced DNA damage and S-phase arrest in human embryonic kidney cells (HEK-293). OTA exposure results in nephrotoxicity, hepatotoxicity as well as immunotoxicity; and, in the present study, the toxicity of OTA toward HEK-293 cells was explored by analyzing the involvement of the oxidative pathway. It was found that OTA treatment led to oxidative damage; meanwhile, OTA treatment induced significant DNA damage and S-phase arrest by down-regulating cyclin A2, cyclin E1, and CDK2 expression. However, NAC pretreatment alleviated OTA-induced ROS overproduction, the loss of mitochondrial membrane potential (ΔΨ_m), and the decrease in superoxide dismutase (SOD) activity. NAC pretreatment was also discovered to attenuate OTA-induced DNA damage using the comet assay and by determining the expression of γ-H2AX. In addition, NAC pretreatment partly ameliorated OTA-induced S-phase arrest by preventing the down-regulation of cyclin A2, cyclin E1 and CDK2 expression in HEK-293 cells. All of these results demonstrated that oxidative damage was involved in OTA-induced DNA damage and cell cycle arrest in HEK-293 cells. Therefore, NAC has the potential to reverse the DNA damage and S-phase arrest induced by OTA.     

Effects of pentoxifylline and alpha lipoic acid on methotrexate-induced damage in liver and kidney of rats

The aim of the current study was to investigate the probable protective effects of Pentoxifylline (PTX) and Alpha Lipoic Acid (ALA), which display anti-oxidative efficacy against hepatotoxicity and nephrotoxicity, those being the major side effects of Methotrexate (MTX). Rats were divided into four groups: a control group; MTX (20 mg/kg/day) group; MTX + PTX (20 mg/kg/day + 50 mg/kg/day) group; and an MTX + ALA (20 mg/kg/day + 100 mg/kg/day) group. At the end of the experiment, biochemical, histochemical and immunohistochemical analyses were performed on liver and kidney tissues of rats. We determined Glutathione Peroxidase (GSH-Px), Superoxide Dismutase (SOD), Catalase (CAT), Malondialdehyde (MDA), Nitric Oxide (NO) and Xanthine Oxidase (XO) levels in the liver and kidney. Moreover, Gamma Glutamyl Transferase (GGT), Direct Bilirubin (DBil), Blood Urea Nitrogen (BUN), and urea levels were measured in the serum. The histochemical evaluation revealed a significant decrease in MTX caused damage in the PTX- and ALA-treated groups (especially in ALA group). On the other hand, the immune staining of iNOS and TNF-α were observed most densely in the MTX group, while the density decreased in the PTX- and ALA-administered groups. We determined increased GGT, BUN, urea and levels of CAT, MDA, NO, and XO values in both groups, while GSH-Px (an increase in liver tissue) and DBil levels were decreased in the group that received MTX. However, we determined decreased SOD levels in liver tissue. In the PTX and ALA groups, the levels of GGT, BUN and urea as well as the levels of CAT, MDA, NO and XO decreased (SOD increased in the liver tissue), and the levels of GSH-Px and DBil increased. In conclusion, it can be stated that, although ALA is more effective in preventing the toxic effects of MTX on the liver and kidney, PTX also has a preventive effect. As a result, we can readily suggest that ALA and PTX can have protective effects by decreasing MDA, NO, BUN and urea values as antioxidants against MTX-induced damage in liver and kidney of rats.     

In vitro gene expression data supporting a DNA non-reactive genotoxic mechanism for ochratoxin A

Ochratoxin A (OTA) is a mycotoxin often found in cereals and agricultural products. There is unequivocal evidence of renal carcinogenicity of OTA in male rats, although the mechanism of action is unknown. At present, available data support an epigenetic mechanism (DNA non-reactive) resulting from oxidative stress and cytotoxicity, because a direct OTA interaction with DNA has not been demonstrated. Genotoxic mechanism (DNA-reactive vs. DNA non-reactive) may have implications on human risk assessment. Therefore, the aim of the present work was to identify biological pathways modulated by OTA in vitro in a human renal cell line (HK-2) to contribute to the elucidation of the mechanism of OTA toxicity. For that purpose, cells were exposed to 50 microM OTA during 6 and 24 h, and gene expression profiles were analyzed using Affymetrix Human Genome U133 A 2.0 Gene Chips. Under the same experimental conditions, genotoxicity was evaluated by the modified comet assay using FPG and Endo III to detect oxidative DNA damage, and intracellular ROS level by the H(2)DCF assay. After 6 h, with slight cytotoxicity (83% survival), genes involved in mitochondrial electron transport chain were up-regulated; and after 24 h, with a more pronounced cytotoxicity (51% survival), genes implicated in oxidative stress response were also up-regulated. Increase in intracellular ROS level and oxidative DNA damage was evident at both exposure times being more pronounced with high cytotoxicity. On the contrary, up-regulation of genes implicated in DNA damage response, as cell cycle control or apoptosis, was not detected at any exposure time. In conclusion, these results support a DNA non-reactive mechanism of OTA genotoxicity.     

Grafting of gallic acid onto chitosan nano particles enhances antioxidant activities in vitro and protects against ochratoxin A toxicity in catfish (Clarias gariepinus)

This study aimed to prepare and characterize enzymatic modified chitosan nanoparticles (CSNPs) with gallic acid (GA) or octyl gallate (OG) to optimize its potential in human application and to evaluate their protective role against ochrtoxin A (OTA) toxicity in catfish. The modified CSNPs have average size around 90 nm with positive charge and high scavenging activity especially GA-CSNPs. In the in vivo study, catfish were divided into 8 groups and treated for 3 weeks as follow: the control group, OTA-treated group (1 mg/kg b.w.), the groups treated with CSNPs, GA-CSNPs or OG-CSNPs (280 mg/kg b.w.) anole or in combination with OTA. Blood, liver and kidney samples were collected for different analyses. OTA induced a significant biochemical disturbances accompanied with oxidative stress in liver and kidney, histological changes and increase DNA fragmentation in the kidney. Co-treatment with OTA plus the different CSNPs resulted in a significant improvement in all tested parameters and histological picture of the kidney. This improvement was more pronounced in the group treated with GA-CSNPs. It could be concluded that grafting of GA or its ester improved the properties of CSNPs. Moreover, GA-CSNPs showed strong scavenging properties than OG-CSNPs due to the blocking of carboxyl groups responsible of the scavenging activity in OG.     

MAPK-ERK activation in kidney of male rats chronically fed ochratoxin A at a dose causing a significant incidence of renal carcinoma

Kidney samples of male Fischer 344 (F-344) rats fed a carcinogenic dose of OTA over 7 days, 21 days and 12 months were analysed for various cell signalling proteins known to be potentially involved in chemical carcinogenicity. OTA was found to increase the phosphorylation of atypical-PKC. This was correlated with a selective downstream activation of the MAP-kinase extracellular regulated kinases isoforms 1 and 2 (ERK1/2) and of their substrates ELK1/2 and p90RSK. Moreover, analysis of effectors acting upstream of PKC indicated a possible mobilisation of the insulin-like growth factor-1 receptor (lGFr) and phosphoinositide-dependent kinase-1 (PDK1) system. An increased histone deacetylase (HDAC) enzymatic activity associated with enhanced HDAC3 protein expression was also observed. These findings are potentially relevant with respect to the understanding of OTA nephrocarcinogenicity. HDAC-induced gene silencing has previously been shown to play a role in tumour development. Furthermore, PKC and the MEK-ERK MAP-kinase pathways are known to play important roles in cell proliferation, cell survival, anti-apoptotic activity and renal cancer development.     

Teratogenic effects of Ochratoxin A in rats with impaired renal function

The teratogenic potential of Ochratoxin A (OA), was compared in impaired renal function (IRF) and sham-operated (SO) female rats. Surgical removal of approximately 70% of the total renal tissue was accomplished utilizing unilateral ligation/electrocoagulation procedures. Control animals were sham-operated. All animals were allowed a period of 27 days to recover post surgery. IRF rats exhibited normal mating tendencies and the pregnancy rate was 100%. A single, subcutaneous teratogenic dose of OA (1.75 mg/kg) on gestation day 7 resulted in significantly increased fetal resorptions, decreased fetal body weights and increased fetal malformations in both IRF and SO animals, although the incidence of gross malformations was greater in IRF rats. A subthreshold teratogenic dose (i.e. 1 mg/kg) did not produce any significant increase in embryotoxicity or fetal malformations in IRF animals compared to SO rats.     

Investigation on the absorption kinetics of chlorogenic acid in rats by HPLC

Chlorogenic acid (ChA), a major phenolic compound in the Flos Lonicerae, is widely used in the traditional Chinese medicine practice. The purpose of this study is to report the pharmacokinetic parameters of ChA in rats after oral administration and explore its absorption profile briefly. A two-compartment model was proposed and validated through the program to explain the apparent triphasic phenomenon of ChA in rats after intragastric administration. A rapid absorption and a relatively slow distribution followed by a slower elimination phase were observed. At the administered doses of 200, 400 and 600 mg/kg, the values of absorption half-life (t 1/2 Ka) were 10.23, 18.66 and 28.13 min. The values of distribution half-life (t1/2) were 12.35, 31.04 and 39.19 min. And the values of elimination half-life (t1/2P) were 231.64, 337.23 and 420.81 min. The volume of distribution at the three doses were 55.26, 35.56, 32.22 L/kg, respectively. The AUC0-->infinity (area under the concentration-time curve) was not proportional to the administered dose. In the range of the doses examined, the absorption pharmacokinetics of ChA in rats was based on nonlinear kinetics.     

Diphenyl diselenide protects against glycerol‐induced renal damage in rats

In this study we evaluated the effect of diphenyl diselenide (PhSe)₂ on glycerol‐induced acute renal failure in rats. Rats were pre‐treated by gavage every day with (PhSe)₂ (7.14 mg kg^?1) for 7 days. On the eighth day, rats received an intramuscular injection of glycerol (8 mL kg^?1). Twenty‐four hours afterwards, rats were euthanized and the levels of urea and creatinine were measured in plasma. Catalase (CAT), glutathione peroxidase (GPx), glutathione S‐transferase (GST), δ‐aminolevulinate dehydratase (δ‐ALA‐D) and Na⁺, K⁺‐ATPase activities and ascorbic acid levels were evaluated in renal homogenates. Histopathological evaluations were also performed. The results demonstrated that (PhSe)₂ was able to protect against the increase in urea and creatinine levels and histological alterations in kidney induced by glycerol. (PhSe)₂ protected against the inhibition in δ‐ALA‐D, CAT and GPx activities and the reduction in ascorbic acid levels induced by glycerol in kidneys of rats. In conclusion, the present results indicate that (PhSe)₂ was effective in protecting against acute renal failure induced by glycerol. Copyright © 2009 John Wiley & Sons, Ltd.     

Combination of chlorogenic acid and salvianolic acid B protects against polychlorinated biphenyls-induced oxidative stress through Nrf2

Caffeic acid derivatives (CADs) are well-known phytochemicals with multiple physiological and pharmacological activities. This study aimed to investigate the combined protective effects of CADs on PCB126-induced liver damages and oxidative stress in mice. Here, we used chemiluminescence and chose chlorogenic acid (CGA), salvianolic acid B (Sal B) as the best antioxidants. Then, mice were intragastrically administered with 60 mg/kg/d CGA, Sal B, and CGA plus Sal B (1:1) for 3 weeks before exposing to 0.05 mg/kg/d PCB126 for 2 weeks. We found that pretreatment with CGA, Sal B, and CGA plus Sal B effectively attenuated liver injury and cytotoxicity caused by PCB126, but improved the expressions of superoxide dismutase (SOD), glutathione reduced (GSH), heme oxygenase-1 (HO-1) and nuclear factor E2-related factor 2 (Nrf2), CGA plus Sal B especially, was found to have the best effects that indicated a synergetic protective effect. Taken together, as the Nrf2 regulates the cyto-protective response by up-regulating the expression of antioxidant genes, we suggested that CGA plus Sal B had a combined protection on PCB126-induced tissue damages and that the Nrf2 signaling might be involved.     

102例冠心苏合丸相关性肾损害不良反应分析

目的:分析冠心苏合丸引起肾损害的不良反应临床特点。方法:回顾研究102例因服用冠心苏合丸引起相关性肾损害不良反应患者的临床资料,分析冠心苏合丸致肾损害的临床特点、ADR表现、服药情况、治疗方法等。结果:102例患者中,女性76例,占74.51%;平均年龄为(66.42±10.31)岁;平均服用药物时间为(8.45±7.55)年;服用1~9年出现ADR的患者占61.76%;102例ADR程度分级均为严重,临床表现均不典型,病情呈隐匿性进展,均伴有贫血,贫血程度与肾功能减退程度不平行;67例肾图检查均出现双侧肾功能中重度受损;98例B超双肾体积缩小,严重受损,且大小不对称,结构不清呈弥漫性改变;25例肾脏病理活检以中重度慢性小管间质性肾炎为主。结论:患者肾功能受损情况与服用冠心苏合丸的时间长短、服用剂量不平行,个体差异较大;肾功能呈进行性损害,对出现贫血、肾功能损害及肾脏大小改变的患者应追问其服药史,以求尽快确诊救治。     

Effect of ochratoxin A on cell survival and collagen homeostasis in human mesangial cells in primary culture

Ochratoxin A (OTA) is a mycotoxin produced by several fungi growing on food source material. The main target of OTA is the kidney. So far, mainly cell lines of different origin have been used to study OTA toxicity. Yet all of them derived from tubule segments and therefore only limited information is available on glomerular effects of OTA. We exposed human mesangial cells in primary culture to OTA in nanomolar concentrations for up to 14 days. Necrotic and apoptotic cell death as well as fibrotic changes were studied. Protein content decreased only when unusual high OTA concentrations were used (1 μM). By contrast, an increase of caspase-3 activity or LDH release was observed after five days already at 10 nM OTA. A decrease of collagen I secretion was accompanied by a virtually unchanged collagen III and fibronectin secretion. Collagen IV secretion was slightly increased at low OTA concentrations (0.3–10 nM). We conclude that OTA has only a minor effect on human mesangial cells in primary culture. OTA did not influence collagen homeostasis substantially. Based on the data presented here, a risk of mesangial damage by OTA exposure is unlikely.     

Influence of lipoic acid on testicular toxicity induced by bi-n-butyl phthalate in rats

Bi-n-butyl phthalate (BNBP) is an environmental pollutant. The aim of this study was to evaluate the protective effect of lipoic acid (LA) against testicular dysfunction associated with the intake of to BNBP- intoxicated rats. Adult male Wistar rats were divided into 4 groups of 6 animals each, and received medication orally for 14 days. Group I rats received 0.5 ml corn oil. Group II rats received LA (20 mg/kg B.W./day). Group III rats received BNBP (250 mg/kg B.W./day). Group IV rats received LA 24 h prior to BNBP intake. Testes weight, cauda sperm count and sperm motility were decreased significantly by 18.15%, 13.83% and 13.5%, respectively, after BNBP treatment. Significant increase by 12.1%, 10.20% and 11.51%, respectively, was observed in LA–BNBP rats. Significant increase by 1.53%, 1.5% and 1.8%, for serum follicle stimulating hormone, testosterone and total antioxidant status, respectively, were observed in LA–BNBP rats. Testicular lipid peroxides and lactate dehydrogenase enzyme were significantly decreased by 1.5 and 1.6 folds, respectively, in LA–BNBP rats were decreased after BNBP treatment. Testicular superoxide dismutase, catalase and glutathione reductase enzymes were significantly increased in LA–BNBP rats. LA–BNBP rats, decreased the damage to seminiferous tubules produced by BNBP intake. In conclusion, LA mitigated BNBP-induced testicular toxicity through antioxidant mechanism and by direct free radical scavenging activity.