Paracellular and transcellular migration of metastatic cells through the cerebral endothelium.

Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases. Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood-brain barrier. By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps. We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia-like membrane protrusions towards the tumour cells. Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route. On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells. When co-culturing melanoma cells with cerebral endothelial cells, we observed N-cadherin enrichment at melanoma-melanoma and melanoma-endothelial cell borders. However, for breast cancer cells N-cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo. Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.

The anemonin content of four different Ranunculus species.

The Ranunculus species are poorly known as medicinal plants. They have potential toxicity given by the ranunculin and its enzymatic degradation compounds: protoanemonin and anemonin. This paper aims to evaluate the anemonin content of four species: R. bulbosus, R. ficaria, R. sardous and R. sceleratus. The evaluation was performed by TLC and HPLC. There were evaluated two types of extracts hydroalcoholic (HA) and glycerol-ethanol (GE). The most concentrated extract in anemonin was found to be the R. sardous aerial part HA extract: 2.66 mg/ml. The lowest anemonin content is in R. sceleratus: 0.13-0.19 mg/ml. In R. bulbosus aerial part the anemonin content is less than the used HPLC method detection limits (7.68 mg/ml). In all cases the GE extracts are less concentrated in anemonin, being more safely for human administration.

Biochemical, Histopathological and Molecular Responses in Gills of Leuciscus cephalus Exposed to Metals.

Gills are major targets for acute metal toxicity in fish, due to their permanent contact with aquatic pollutants. To assess the effects of metals on gills of the Leuciscus cephalus (chub), fish individuals were collected from two sites in the Tur River, Romania, in upstream (site 1) and downstream (site 2) of a metal pollution source. Quantitative and hyperspectral analyses showed that Zn, Sr, and Fe concentrations were significantly higher in gills from site 2 compared with site 1. Malondialdehyde and advanced oxidation protein products levels increased 17 and 28%, respectively, whereas reduced glutathione level diminished significantly in the gills of fish collected from site 2 compared to site 1. The activities of superoxide dismutase, catalase, and glutathione-S-transferase increased significantly at 41, 21, and 28%, respectively. Proliferating cell nuclear antigen (PCNA) protein levels, as well as the amount of DNA damage, were significantly increased for site 2 compared with site 1. The induced oxidative stress generated hyperplasia, hypertrophy, and inflammation in the epithelial cells and apoptosis. Hence, this could suggest that gill cells have tried to counteract the oxidative stress-induced DNA fragmentation by PCNA up-regulation, but the PCNA expression decreased on longer time due to the low level of GSH, resulting in apoptosis.

Histopatological alterations and oxidative stress in liver and kidney of Leuciscus cephalus following exposure to heavy metals in the Tur River, North Western Romania.

Pollution of the aquatic environment by heavy metals is a great concern worldwide. Freshwater fish ingests various metals through gills, skin or diet. Our aim was to investigate the oxidative stress and histopathological injuries induced by Fe, Cu, Zn, Pb, Cd in the liver and kidney of Leuciscus cephalus. Fish samples were collected from two sites in the Tur River, NW Romania, in upstream and downstream of a pollution source. Metals were differently distributed in the liver and kidney of fish. The highest concentrations of Fe, Cu and Pb were found in liver, whereas Zn and Cd concentrations were the highest in kidney in specimens collected from the downstream site. The histopathological changes were associated with metal bioaccumulation, being more severe in kidney than liver. Malondialdehyde (MDA) and advanced oxidation protein products (AOPP) increased significantly in the liver and kidney of fish from downstream site compared to upstream one, whereas reduced glutathione (GSH) decreased. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) increased significantly in livers, whereas SOD increased in kidney. Our study revealed that liver has a higher capacity and adaptability to counteract ROS compared to kidney. The more pronounced increase of hepatic SOD, CAT and GST activities is related milder structural changes observed in liver compared to kidney, where lesions were not reduced by antioxidant defense system.

Exposure to Iron Oxide Nanoparticles Coated with Phospholipid-Based Polymeric Micelles Induces Biochemical and Histopathological Pulmonary Changes in Mice.

The biochemical and histopathological changes induced by the exposure to iron oxide nanoparticles coated with phospholipid-based polymeric micelles (IONPs-PM) in CD-1 mice lungs were analyzed. After 2, 3, 7 and 14 days following the intravenous injection of IONPs-PM (5 and 15 mg Fe/kg bw), lactate dehydrogenase (LDH) activity, oxidative stress parameters and the expression of Bax, Bcl-2, caspase-3 and TNF-α were evaluated in lung tissue. An increase of catalase (CAT) and glutathione reductase (GR) activities on the second day followed by a decrease on the seventh day, as well as a decline of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity on the third and seventh day were observed in treated groups vs. controls. However, all these enzymatic activities almost fully recovered on the 14th day. The reduced glutathione (GSH) and protein thiols levels decreased significantly in nanoparticles-treated groups and remained diminished during the entire experimental period; by contrast malondialdehyde (MDA) and protein carbonyls increased between the 3rd and 14th day of treatment vs. control. Relevant histopathological modifications were highlighted using Hematoxylin and Eosin (H&E) staining. In addition, major changes in the expression of apoptosis markers were observed in the first week, more pronounced for the higher dose. The injected IONPs-PM generated a dose-dependent decrease of the mouse lung capacity, which counteracted oxidative stress, thus creating circumstances for morphopathological lesions and oxidation processes.

Silica Nanoparticles Induce Oxidative Stress and Autophagy but Not Apoptosis in the MRC-5 Cell Line.

This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO NPs) on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The nanoparticles' morphology, composition, and structure were investigated using high resolution transmission electron microscopy, selected area electron diffraction and X-ray diffraction. Our study showed a decreased cell viability and the induction of cellular oxidative stress as evidenced by an increased level of reactive oxygen species (ROS), carbonyl groups, and advanced oxidation protein products after 24, 48, and 72 h, as well as a decreased concentration of glutathione (GSH) and protein sulfhydryl groups. The protein expression of Hsp27, Hsp60, and Hsp90 decreased at all time intervals, while the level of protein Hsp70 remained unchanged during the exposure. Similarly, the expression of p53, MDM2 and Bcl-2 was significantly decreased for all time intervals, while the expression of Bax, a marker for apoptosis, was insignificantly downregulated. These results correlated with the increase of pro-caspase 3 expression. The role of autophagy in cellular response to SiO2NPs was demonstrated by a fluorescence-labeled method and by an increased level of LC3-II/LC3-I ratio. Taken together, our data suggested that SiO2 NPs induced ROS-mediated autophagy in MRC-5 cells as a possible mechanism of cell survival.

Hepatoprotective effects of Berberis vulgaris L. extract/ß cyclodextrin on carbon tetrachloride-induced acute toxicity in mice.

The present study investigated the capacity of formulated Berberis vulgaris extract/ß-cyclodextrin to protect liver against CCl(4)-induced hepatotoxicity in mice. Formulated and non-formulated extracts were given orally (50 mg/kg/day) to mice for 7 days and were then intra-peritoneally injected with 1.0 mL/kg CCl(4) on the 8th day. After 24 h of CCl(4) administration, an increase in the levels of apartate-amino-transferase (AST), alanine-amino-transferase (ALT) and malondialdehyde (MDA) was found and a significant decrease in superoxide-dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione-peroxidase (GPx) levels could be detected. This was accompanied by extended centrilobular necrosis, steatosis, fibrosis and an altered ultrastructure of hepatocytes. Pre-treatment with formulated or non-formulated extract suppressed the increase in ALT, AST and MDA levels and restored the level of antioxidant enzymes at normal values. Histopathological and electron-microscopic examination showed milder liver damage in both pre-treated groups and the protective effect was more pronounced after the formulated extract was administered. Internucleosomal DNA fragmentation induced by CCl(4) was reduced in the group which received non-formulated extract and absent in the group which received formulated extract. Taken together, our results suggest that Berberis vulgaris/ß-cyclodextrin treatment prevents hepatic injury induced by CCl(4) and can be considered for further nutraceutical studies.

Flucytosine and Amphotericin B Coadministration Induces Dose-Related Renal Injury.

Invasive fungal infections remain an important clinical problem, and despite recent approaches, they bring high morbidity and mortality. Combination therapies are the most effective; however, adverse effects need to be considered. In this study, we aimed to evaluate the nephrotoxicity induced by combined therapy of flucytosine (FL) and amphotericin B (AMF) at 3 different doses administered to mice for 14 days: 300 µg/kg AMF+50 mg/kg FL; 600 µg/kg AMF+100 mg/kg FL; 900 µg/kg AMF+150 mg/kg FL. Antifungal coadministration triggered nuclear translocation of NF-κB and upregulated nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65 (NF-κB p65) messenger RNA mRNA level in dose-dependent manner. The immunopositivity of tumor necrosis factor-α and interleukin-6 (IL-6), together with IL-6 gene expression, increased both in tubular and glomerular cells. Amphotericin B-flucytosine cotreatment increased significantly the number of terminal deoxy-nucleotidyl transferase (TdT)-mediated dUTP nick end-labeling positive nuclei. Apoptotic cells in renal tubuli were confirmed by electron microscopy. Histopathological analysis revealed collagen accumulation at the glomerular level. Collagen was also evidenced in the glomeruli at the dose of 900 µg/kg AMF+150mg/kg FL by Masson-Goldner trichrome staining and electron microscopy. Moreover, antifungal cotherapy induced upregulation of transforming growth factor beta 1 (TGF-ß1) gene expression in a dose-dependent manner. Inflammation and epithelial tubular apoptosis are associated with TGF-ß1 activation and initiation of the early stage of glomerular fibrosis at higher doses, leading to tubule-interstitial fibrosis.

Berberis vulgaris extract/ß-cyclodextrin complex increases protection of hepatic cells via suppression of apoptosis and lipogenesis pathways.

Berberis vulgaris (Bv) is well known worldwide for its healing properties. However, limited information is available concerning its mechanism of action and the increased hepatoprotective activity of formulated extracts. This study evaluated the protective effect of Bv bark extract against CCl4-induced cytotoxicity in Huh7 cells, as well whether ß-cyclodextrin complexation of the extract resulted in increased hepatoprotective effects. Huh7 cells were incubated for 48 h with 5, 7.5 and 10 µg/ml of unformulated or formulated Bv extract alone and in co-treatment with CCl4. The effects on Huh7 cell growth and apoptosis were evaluated by MTT assay, caspase-3/7 activity and caspase-3 expression, whereas fatty acid changes were investigated by Oil red O staining and the detection of peroxisome proliferator-activated receptor-γ (PPARγ) expression using immunofluorescence. Ultrastructural alterations were observed by electron microscopy. The MTT assay showed that co-exposure to CCl4 and 7.5 µg/ml formulated extract led to a 1.25-fold increase in cell viability compared with the non-formulated extract. Caspase-3/7 activity decreased by 50% and 70% following co-treatment with unformulated or formulated extract, compared with that in cells treated with CCl4 alone. Furthermore, hepatocyte ultrastructure was protected from CCl4-induced injury in the two co-treated groups, intracytoplasmic lipid accumulation decreased significantly and PPARγ expression was restored, in comparison with CCl4-treated cells alone. Formulated and unformulated extracts were efficient against the anti-proliferative and pro-apoptotic actions of CCl4 through suppression of CCl4-induced caspase-3 activation and lipid accumulation. The protective effect of the formulated extract was more pronounced than that of the unformulated one, which may be due to its increased solubility.

Protective effects of silymarin against bisphenol A-induced hepatotoxicity in mouse liver.

Bisphenol A (BPA) is an endocrine-disrupting chemical released into the environment, with severe consequences for human health, including metabolic syndrome and associated pathological conditions. Due to limited information on BPA-induced hepatotoxicity, the present study focused on investigating the association between BPA-induced toxicity and inflammatory markers in the liver, and how these injuries may be alleviated using the natural agent silymarin, a flavonoid with antioxidant properties obtained from Silybum marianum. Administration of BPA to male CD-1 mice for 10 days caused a significant increase in the number of cells immunopositive for interleukin 6 and tumor necrosis factor-α, pro-inflammatory cytokines that mediate the hepatic inflammatory response. Treatment with 200 mg/kg of silymarin concurrently with BPA for 10 days resulted in a diminished level of pro-inflammatory cytokines and in significantly reduced ultrastructural injuries. Additionally, silymarin was able to restore the significantly decreased glycogen deposits observed following BPA exposure to normal levels, thus favoring hepatic glycogenesis. This study represents the first report of silymarin ability to reduce hepatic lesions and to counteract inflammation caused by BPA in mice. A dose of 200 mg/kg silymarin was sufficient to induce a protective effect against structural and ultrastructural injuries induced by BPA and to lower the levels of pro-inflammatory cytokines observed in murine liver tissue following exposure to BPA.

Mistletoe and Garlic Extracts as Polyurethane Carriers - A Possible Remedy for Choroidal Melanoma.

BACKGROUND: Melanoma is known as the most dangerous form of skin cancer; whereas the malignant choroidal melanoma is an orphan disease known as the most common primary intraocular malignancy in adults. Literature suggests that the consumption of garlic and mistletoe leads to a reduced risk of developing cancer. OBJECTIVE: The aim of this study was the obtaining and the characterization of polymer structures containing mistletoe or garlic extract. METHODS: The structures were obtained in a polyaddition process combined with a spontaneous emulsification; they were characterized by pH, size, Zeta potential and DSC measurements, evaluation of encapsulation efficacy, penetrability through membranes and in vitro cytotoxicity tests. RESULTS: The microstructures present sizes between 1.05 and 2.60 µm and Zeta potentials between -7 and +36 mV. A good encapsulation was observed on different evaluations (88-92%). It was determined that approx. 30% of polymer microstructures containing vegetal extracts pass through an artificial membrane in 4 days. An in vitro cytotoxicity test revealed that these products are safe for administration. The analysis of antitumor efficacy indicates that garlic extracts have important effects after 48 and 72 hours on A375 cells; however, polymer microstructures with herbal extracts did not reveal antiproliferative activities on A375 cells because these polymer structures present a slow degradation. CONCLUSION: Sterile eye drops solutions based on polymer microstructures containing garlic or mistletoe extracts were obtained; the sample based on garlic extracts may be used in the pharmaceutical field as drug carrier with an antiproliferative effect which occurs after a prolong period.

Plant-Derived Biomolecules and Drug Delivery Systems in the Treatment of Liver and Kidney Diseases.

Chronic liver and kidney diseases are among the most fearful pathologies affecting an increasing number of people, having severe consequences on life quality. Although much progress has been made in recent years, availability of cost-effective and innovative therapies is still an unmet medical need. One of the major challenges in the therapy of liver and kidney diseases is to selectively deliver drugs to achieve therapeutically relevant concentration in the target organ, in order to decrease the amount of drug needed and to reduce unwanted side effects. In this review we summarize recent advances in selective drug targeting to liver or to kidney including cell-selective therapeutic approaches as well. We pay special attention to plant-derived bioactive molecules which constitute promising tools for the treatment of chronic liver and kidney diseases. We discuss cellular, subcellular and molecular mechanisms underlying the observed pharmacological effects of plant-derived drugs and give an overview of formulations, which can increase therapeutic effectiveness of these biomolecules in the treatment of chronic liver and kidney disorders.

Evaluation of Hepatotoxicity with Treatment Doses of Flucytosine and Amphotericin B for Invasive Fungal Infections.

Invasive fungal infection is a well-known cause of morbidity and mortality in immunocompromised patients. In this study we aimed to evaluate the hepatotoxicity induced by combined therapy of flucytosine and amphotericin B, at three different doses administered to mice for 14 days: 50 mg/kg flucytosine and 300 µg/kg amphotericin B; 100 mg/kg flucytosine and 600 µg/kg amphotericin B; 150 mg/kg flucytosine and 900 µg/kg amphotericin B. Liver injuries were evaluated by analysis of optic and electron microscopy samples, changes in TNF-α, IL-6, and NF-κB inflammation markers levels of expression, and evaluation of mRNA profiles. Histological and ultrastructural analysis revealed an increase in parenchymal and portal inflammation in mice and Kupffer cells activation. Combined antifungal treatment stimulated activation of an inflammatory pathway, demonstrated by a significant dose-dependent increase of TNF-α and IL-6 immunoreactivity, together with mRNA upregulation. Also, NF-κB was activated, as suggested by the high levels found in hepatic tissue and upregulation of target genes. Our results suggest that antifungal combined therapy exerts a synergistic inflammatory activation in a dose-dependent manner, through NF-κB pathway, which promotes an inflammatory cascade during inflammation. The use of combined antifungal therapy needs to be dose limiting due to the associated risk of liver injury, especially for those patients with hepatic dysfunction.

Chrysin attenuates liver fibrosis and hepatic stellate cell activation through TGF-ß/Smad signaling pathway.

We investigated the protective effect of chrysin on chronic liver fibrosis in mice and the potential mechanism underlying TGF-ß1-mediated hepatic stellate cells (HSCs) activation on fibrogenesis. Experimental fibrosis was established by intraperitoneal injection of mice with 20% v/v, 2 ml/kg CCl4 twice a week, for 7 weeks. Mice were orally treated with 3 doses of chrysin (50, 100 and 200 mg/kg) or with vehicle as control. For the assessment of the spontaneous reversion of fibrosis, CCl4 treated animals were investigated after two weeks of recovery time. Silymarin was used as standard hepatoprotective flavonoid. Histopathological investigations showed that hepatic fibrosis grade was markedly reduced in the chrysin groups compared to the fibrotic one. Moreover, CCl4 activated HSCs induced an upregulation of smooth muscle actin (α-SMA), an increased number of TGF-ß1 immunopositive cells and marked up-regulation of TGF-ß1. α-SMA and TGF-ß1 levels were significantly reduced in all chrysin treated groups in a dose-dependent manner, whereas the level of spontaneous reversal of fibrosis was lower compared to all flavonoid treated groups. Liver mRNA levels of Smad 2 in the 50, 100 and 200 mg/kg chrysin treated groups were significantly reduced by about 88.54%, 92.15% and 95.56% of the corresponding levels in the fibrosis mice group. The results were similar for mRNA levels of Smad 3. The protective response to silymarin was almost similar to that seen with the highest doses of chrysin. In this study, we have shown that chrysin has the efficacy to reverse CCl4-stimulated liver fibrosis by inhibition of HSCs activation and proliferation through TGF-ß1/Smad pathway. These results suggest that chrysin may be useful in stopping or reversing the progression of liver fibrosis and might offer the possibility to develop a new therapeutic drug, useful in treatment of chronic liver diseases.

Magnetite nanoparticles induced adaptive mechanisms counteract cell death in human pulmonary fibroblasts.

Magnetite nanoparticles (MNP) have attracted great interest for biomedical applications due to their unique chemical and physical properties, but the MNP impact on human health is not fully known. Consequently, our study proposes to highlight the biochemical mechanisms that underline the toxic effects of MNP on a human lung fibroblast cell line (MRC-5). The cytotoxicity generated by MNP in MRC-5 cells was dose and time-dependent. MNP-treated MRC-5 cells accumulated large amount of iron and reactive oxygen species (ROS) and exhibited elevated antioxidant scavenger enzymes. Reduced glutathione (GSH) depletion and enhanced lipid peroxidation (LPO) processes were also observed. The cellular capacity to counteract the oxidative damage was sustained by high levels of heat shock protein 60 (Hsp60), a protein that confers resistance against ROS attack and inhibition of cell death. While significant augmentations in nitric oxide (NO) and prostaglandine E2 (PGE2) levels were detected after 72 h of MNP-exposure only, caspase-1 was activated earlier starting with 24h post-treatment. Taken together, our results suggest that MRC-5 cells have the capacity to develop cell protection mechanisms against MNP. Detailed knowledge of the mechanisms induced by MNP in cell culture could be essential for their prospective use in various in vivo biochemical applications.

Fenugreek powder exerts protective effects on alcoholised rats' kidney, highlighted using ultrastructural studies.

Trigonella foenum-graecum (TFG) seeds exert a protective antioxidant effect and membrane protector through their rich content in polyphenolic flavonoids. The previous research focused on the hypoglycemic action of the seeds, with scarce studies on the preventive effects in the pathology of the kidney. Our work was conducted on an experimental in vivo model; the animals were given two different concentrations of TFG seeds, consequently to alcohol intoxication. Transmission electron microscopy (TEM) analysis showed vacuolation in cytoplasm, edemas at the apical pole of the nephrocytes, diffusion of the cytoplasmic and mitochondrial matrix and the increase in number of the lysozymes and especially peroxisomes, as well as the congestion of blood capillaries. In the case of the groups T5R and T10R, which received Trigonella powder together with ethanol, the structural and ultrastructural changes produced by the ethylic intoxication were more reduced, being somewhat improved in the T5R group. Therefore, the majority of the cells nuclei have retained their spherical shape, being at the same time predominantly euchromatic, with little heterochromatin and evenly dispersed. Our results suggest the use of Trigonella seeds as a food supplement to prevent cellular deterioration and improve renal morphology and function.

Antioxidant and hepatoprotective effects of naringenin and its ß-cyclodextrin formulation in mice intoxicated with carbon tetrachloride: a comparative study.

The present study evaluated the antioxidant and hepatoprotective effects of the flavonoid naringenin (NGN) and its ß-cyclodextrin formulation at a dose of 50 mg/kg b.w. The assessment was done by the investigation of serum-enzymatic and liver antioxidant activity, histopathological and ultrastructural changes in male Swiss mice, which were subjected to acute experimental intoxication with CCl4. Formulated and free flavonoid were orally given to mice for 7 days and then were intraperitoneally injected with 1.0 mL/kg CCl4 on the 8th day. After 24 h of CCl4 administration, an increase in the levels of transaminases aspartate aminotransferase and alanine aminotransferase activities and malondialdehyde concentration occurred and a significant decrease in superoxide dismutase, catalase glutathione-peroxidase activities, and glutathione levels was detected as well. These were accompanied by extended centrilobular necrosis, steatosis, fibrosis, and an altered ultrastructure of hepatocytes. Pretreatment with formulated or free flavonoid retained the biochemical markers to control values. Histopathological and electron-microscopic examination confirmed the biochemical results. In conclusion, both NGN and NGN/ß-cyclodextrin complex showed antioxidant and hepatoprotective effects against injuries induced by CCl4.

In vitro cytocompatibility evaluation of chitosan/graphene oxide 3D scaffold composites designed for bone tissue engineering.

Extensively studied nowadays, graphene oxide (GO) has a benefic effect on cell proliferation and differentiation, thus holding promise for bone tissue engineering (BTE) approaches. The aim of this study was not only to design a chitosan 3D scaffold improved with GO for optimal BTE, but also to analyze its physicochemical properties and to evaluate its cytocompatibility and ability to support cell metabolic activity and proliferation. Overall results show that the addition of GO in the scaffold's composition improved mechanical properties and pore formation and enhanced the bioactivity of the scaffold material for tissue engineering. The new developed CHT/GO 3 wt% scaffold could be a potential candidate for further in vitro and in vivo osteogenesis studies and BTE approaches.

Protective effects of naringenin on carbon tetrachloride-induced acute nephrotoxicity in mouse kidney.

The ability of naringenin (NGN) to protect the kidney against CCl4-induced renal toxicity in male Swiss mice was investigated. The flavonoid was given orally to mice for 7days and then on the 8th day, these were intraperitoneally injected with 10mmol/kg CCl4. When the toxicant was administrated alone, an increase of malondialdehyde (MDA) concentration was observed and a significant decrease in superoxide dismutase (SOD), catalase (CAT) glutathione-peroxidase (GPx) specific activities as well as glutathione (GSH) levels was detected after 24h. These were accompanied by glomerular and tubular degenerations, vascular congestion, necrosis and fatty changes. Marked collagen deposition and strong TGF-ß1 expression were observed mainly in the mesangial cells of the glomeruli and tubulointerstitial areas. Ultrastructural investigations showed proximal and distal tubular epithelial cells alterations including numerous lysosomes and dense granular bodies, altered mitochondria, appearance of "myeloid bodies" and basal enfolding dilatation. Pre-treatment with NGN resulted in the return of biochemical markers to control values. Histopathological and electron-microscopic examinations confirmed the biochemical results. In conclusion, NGN showed antioxidant and renal protective effects against injuries induced by CCl4.