Assessment of the potential irritancy of oleic acid on human skin: Evaluation in vitro and in vivo

As skin barrier modulating compounds, fatty acids are frequently used in formulations for transdermal or topical delivery. In this study the effects of oleic acid on keratinocytes in vitro was compared with its in vivo skin irritancy in humans. Dose- and time-dependent effects of oleic acid were examined in submerged human keratinocyte cultures, in reconstructed human epidermis (RE-DED), and in excised human skin, using alterations in morphology and changes in interleukin-1 mRNA levels as endpoints. In vitro results were compared with responses of living human skin after topical application of oleic acid, using non-invasive bioengineering methods. Direct interaction of oleic acid and submerged keratinocyte cultures resulted in cell toxicity at very low concentrations of the fatty acid. By contrast, when oleic acid was applied topically on RE-DED or on excised skin, no alterations in morphology were observed. Modulation of stratum corneum thickness indicated a key role of the stratum corneum barrier in the control of oleic acid-induced toxicity. In agreement with these findings, no epidermal tissue damage was seen in vivo, whereas oleic acid induced a mild but clearly visible skin irritation and inflammatory cells were present in the upper dermal blood vessels. Small amounts of oleic acid induced IL-1 mRNA expression in submerged keratinocyte cultures, whereas in RE-DED and in excised skin, IL-1 mRNA levels were increased only when the concentration applied topically was at least two orders of magnitude higher. It is concluded that minute amounts of oleic acid are sufficient to cause local (i.e. inside the viable epidermis) modulation of cytokine production. These concentrations do not affect morphology but induce skin irritation in vivo. To achieve comparable effects in the skin, much higher topical doses are needed than expected according to the locally required levels, owing to the rate-limiting transport of the fatty acid across the stratum corneum barrier.     

Cytotoxicities of the l and d isomers of phenylalanine mustard in L1210 cells

The cytotoxicity of d-phenylalanine mustard (medphalan) to murine L1210 leukemia cells in culture was reduced by both the d and l isomers of leucine. l-Leucine only partially protected L1210 cells from medphalan cytotoxicity at drug concentration's above 10 μM, suggesting that medphalan uptake occured via both an amino acid carrier and an as yet undetermined agency, possibly passive diffusion. At equitoxic concentrations of l-phenylalanine mustard (melphalan) and medphalan, l-leucine reduced medphalan cytotoxicity by only one-sixth that obtained with melphalan. Analysis of melphalan and medphalan inhibition of the initial rate of l-leucine transport indicated a melphalan K_i of 0.085 mM, a value one-seventh that of melphalan (K_i, 0.635 mM).     

Indirect immunoperoxidase localization of aflatoxin B1 in rat liver

Aflatoxin B2a (AFB2a) antibody was used as a histochemical probe in the indirect immunoperoxidase localization of aflatoxin B1 (AFB1) bound to rat liver. The efficacy of the indirect method was initially demonstrated by detecting AFB1 covalently bound to DNA in an enzyme-linked immunosorbent assay. AFB1-modified DNA was attached to a polystyrene microtissue culture plate (solid phase) and then subjected to sequential incubation with AFB2a antiserum followed by goat anti-rabbit peroxidase conjugate. Assays for bound peroxidase revealed that the AFB2a antiserum could be diluted 200,000-fold and still yield a signal-to-noise ratio of 10 when compared to an unmodified DNA control. When the same indirect immunoperoxidase protocol was applied to the light-microscopic localization of AFB1 in liver sections of rats treated in vivo with the mycotoxin, bound toxin could be identified in excellent detail in tissues fixed with periodate-lysine-paraformaldehyde and embedded in glycol methacrylate, but was detectable with only poor resolution in unfixed cryostat sections. Peroxidase-positive reactions in hepatocytes typically exhibited strong nuclear and relatively lighter cytoplasmic staining. Greater concentrations of peroxidase-positive hepatocytes were detected in the periportal area than in the area of the central vein, suggesting a circulatory pattern for AFB1 binding in the liver.     

Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells

In this study, we investigated the cell death process induced by imidazoacridone C-1311 (Symadex) in HT-29 human colon carcinoma cells which have been shown to be preferentially sensitive to this compound in experimental tumour models both in vitro and in nude mice. Compound C-1311 at the EC(99) dose delayed progression of cells through the S phase which was followed by G2 arrest. At 48-96 h after drug exposure, an increasing fraction of cells rounded up and detached from the substratum which suggested the induction of cell death. This was confirmed by the induction of DNA fragmentation as revealed by pulse field electrophoresis and DNA strand breaks by the TUNEL assay. The dying cells had also mitotic features which were evidenced by various biochemical and morphological criteria such as activation of Cdk1 kinase, presence of the mitotic epitope MPM-2 and condensation of chromatin into mitotic chromosomes in drug-treated cells. These results show that C-1311 does not induce rapid apoptosis in HT-29 cells, instead drug exposure leads to prolonged G2 arrest followed by G2 to M transit and cell death during mitosis in the process of mitotic catastrophe.     

Fumonisins as a possible contributory risk factor for primary liver cancer: A 3-year study of corn harvested in Haimen, China, by HPLC and ELISA

Employing HPLC fluorometry, gas-liquid chromatography (GLC) and a novel enzymelinked immunosorbent assay (ELISA) based on a monoclonal antibody, 40 corn samples, each collected in 1993 from agricultural stocks for human consumption in Haimen (Jiangsu County) and Penlai (Shandong Province), high- and low-risk areas for primary liver cancer (PLC) in China, respectively, were analysed for fumonisins (FBs), aflatoxins (AFs) and trichothecenes. Levels and positive rates of FBs and deoxynivalenol (DON) were significantly higher in Haimen than in Penlai. ELISA of the 40 corn samples harvested in the two areas in 1994 revealed that FB contamination levels and rates in these areas were comparable to those observed in 1993 in Haimen. ELISA analysis of 1993 and 1994 products revealed a wide occurrence of AFB₁ but the positive rates as well as levels were not significantly different between these areas. ELISA of the same sample number of corn harvested in 1995 revealed that FB contamination in Haimen was significantly higher than in Penlai. These 3-yearly surveys of corn samples (240 in total) demonstrated that corn harvested in Haimen was highly contaminated with FBs and that the contamination level, as well as positive rate in 1993 and 1995, were 10–50-fold higher than those in Penlai, suggesting FBs as a risk factor for promotion of PLC in endemic areas, along with the trichothecene DON. Co-contamination with AFs, potent hepatocarcinogens, was assumed to play an important role in the initiation of hepatocarcinogenesis.     

Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3 h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.     

Production and characterization of monoclonal antibodies against ochratoxin B.

Monoclonal antibodies against ochratoxin B (OTB) were generated by immunizing Balb/c mice with OTB conjugated to keyhole limpet hemocyanin (KLH) via carbodiimide reactions with CHMC and EDAC. A stable hybridoma cell line 2F1.E10 was produced by fusion of murine splenocytes and myeloma cells. The obtained antibodies were characterized using an indirect competitive ELISA. The detection limit was calculated (27+/-2 nM OTB) and 50% binding inhibition was reached at 500 nM free OTB. A low cross-reactivity to ochratoxin A (OTA) of 3.3% and no cross-reactivities to either coumarin or DL-phenylalanine were observed, suggesting a highly specific OTB antibody. The antibody type was identified as IgG class 1 with the light chain being of the kappa configuration. These antibodies can be used in an indirect competitive ELISA to detect OTB in the nanomolar to micromolar concentration range and may be useful for the analysis of contaminated food items.     

Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, a precursor of cholesterol that is also required for cell proliferation. Mevalonate depletion results in a G1 phase cell cycle arrest that is mediated in part by impaired activity of cyclin-dependent kinase (CDK) 2, and decreased expression of positive regulators of G1 to S phase progression. Inhibition of mevalonate synthesis may, therefore, be a useful strategy to impair the growth of malignant cells. Plant isoprenoids, including beta-ionone and geraniol, have previously been shown to inhibit rodent mammary tumor development, and rodent and avian hepatic HMG-CoA reductase activity. We hypothesized that the putative anti-proliferative and cell cycle inhibitory effects of beta-ionone and geraniol on MCF-7 human breast cancer cells in culture are mediated by mevalonate depletion resulting from inhibition of HMG-CoA reductase activity. Flow cytometric analysis showed a G1 arrest in isoprenoid-treated MCF-7 cells, and also a G2/M arrest at higher concentrations of isoprenoids. These compounds minimally affected the growth of MCF-10F normal breast epithelial cells. Both beta-ionone and geraniol inhibited CDK 2 activity and dose-dependently decreased the expression of cyclins D1, E, and A, and CDK 2 and 4, without changing the expression of p21cip1 or p27kip1. Although both beta-ionone and geraniol also inhibited MCF-7 proliferation, only geraniol inhibited HMG-CoA reductase activity. While these effects were significantly correlated (r2=0.89, P <0.01), they were not causally related, since exogenous mevalonate did not restore growth in geraniol-inhibited cells. These findings indicate that mechanisms other than impaired mevalonate synthesis mediate the anti-proliferative and cell cycle regulatory effects of beta-ionone and geraniol in human breast cancer cells.     

Novel approaches using alkaline or acid/guanidine treatment to eliminate therapeutic antibody interference in the measurement of total target ligand

Measurement of the total target ligand can help to provide pharmacokinetic (PK) and pharmacodynamic (PD) informations. However, the presence of monocloncal antibody therapeutics (ThAs) interferes with ELISA determinations of the total target proteins. The interferences can cause over- or under-estimation of the target protein analysis. The nature of interferences was dependent upon the ThA, target protein, antibody reagents and assay conditions of the ELISA. We have developed novel alkaline and acid/guanidine treatment approaches to dissociate the protein binding and preferentially denature the ThA. The neutralized target proteins can be determined by ELISA. These methods provide reproducible measurements of total target protein without ThA interference. Serum samples, standards and QCs containing target protein and ThA were treated with alkaline buffer (pH > 13) containing casein or acid/guanidine buffer (pH < 1). Total target proteins for two different ThA systems were successfully measured and interferences were completely eliminated by the treatments. These methods were successfully applied to analysis in pre-clinical serum samples.     

Influence of carboxylic acid functionalization on the cytotoxic effects induced by single wall carbon nanotubes on human endothelial cells (HUVEC)

A vast variety of nanomaterials have been developed in the recent years, being carbon nanotubes (CNTs) the ones that have attracted more attention, due to its unique properties which make them suitable for numerous applications. Consequently, it is predicted that tons of CNTs will be produced worldwide every year, being its exposure of toxicological concern. Nanomaterials, once into the body, can translocate from the uptake sites to the blood circulation or the lymphatic system, resulting in distribution throughout the body. Thus, the vascular endothelium can be in contact with them and can suffer from their toxic effects. In this regard, the aim of this work was to investigate the cytotoxicity of single-walled carbon nanotubes (SWCNTs) on human endothelial cells evaluating the influence of acid carboxylic functionalization and also the exposure time (24 and 48 h). Biomarkers assessed were neutral red uptake, protein content, a tetrazolium salt metabolization and cell viability by means of the Trypan blue exclusion test. Cells were exposed to concentrations between 0 and 800 μg/mL SWCNTs for 24 and 48 h. Results have shown that both SWCNTs and carboxylic acid functionalized single-walled carbon nanotubes (COOH-SWCNTs) induce toxic effects in HUVEC cells in a concentration- and time-dependent way. Moreover, the carboxylic acid functionalization results in a higher toxicity compared to the SWCNTs.     

Suppression of mast cell-mediated allergic reaction by Amomum xanthiodes.

The mast cell-mediated immediate-type allergic reaction is involved in many allergic diseases such as asthma, allergic rhinitis and sinusitis. Stimulation of mast cells starts the process of degranulation resulting in release of mediators such as histamine and an array of inflammatory cytokines. In this report, we investigated the effect of aqueous extract of Amomum xanthiodes (Zingiberaceae) (AXE) on the mast cell-mediated allergy model and studied its possible mechanisms of action. AXE inhibited compound 48/80-induced systemic reactions and serum histamine release in mice. AXE decreased immunoglobulin E (IgE)-mediated passive cutaneous anaphylaxis reaction. AXE reduced histamine release and intracellular calcium from rat peritoneal mast cells activated by compound 48/80. Furthermore, AXE decreased the activation of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase and c-jun N-terminal kinase, and downstream tumor necrosis factor (TNF)-alpha production in phorbol 12-myristate 13-acetate and calcium ionophore A23187-stimulated human mast cells. Our findings provide evidence that AXE inhibits mast cell-derived allergic reactions, and that intracellular calcium, TNF-alpha, and p38 MAPK are involved in these effects.     

Molecular investigation of the effects of lindane in rat hepatocytes: Microarray and mechanistic studies

Although many studies of lindane toxicity have been carried out, we still know little about the underlying molecular mechanisms. We used a microarray specifically designed for studies of the hepatotoxic effects of xenobiotics to evaluate the effects of lindane on specific gene expression in primary cultured rat hepatocytes. These genes were assigned to detoxication processes (CYP3A4, Gsta2, CYP4A1), cell signalling pathways and apoptosis (Eif2b3, Eif2b4, PKC). In this study, we demonstrate that lindane up-regulates PKC by increasing oxidative stress. TEMPO (a well known free radical scavenger) and Ro 31-8220 (an inhibitor of classical PKCs) prevented the inhibition of spontaneous and intrinsic apoptosis pathway (characterised by Bcl-xL induction, Bax down-regulation, caspases inhibition) and the induction of necrosis by lindane in rat hepatocytes. Thus, these findings indicate that several dependent key signalling pathways, including detoxification, apoptosis, PKC activity and redox status maintenance, contribute to lindane-induced toxicity in primary cultured rat hepatocytes. This may account more clearly for the acute and chronic effects of lindane in vivo, with the induction of cell death and tumour promotion, respectively.     

Impact of lactic acid bacteria on oxidative DNA damage in human derived colon cells.

It is assumed that reactive oxygen species (ROS) play a key role in inflammatory bowel diseases and colon cancer and a number of studies indicate that lactic acid bacteria (LAB) possess antioxidant properties and may prevent these diseases. In the present study, we developed a model which allowed us to investigate the prevention of oxidative DNA damage in human derived colon (HT29) cells by LAB. Furthermore, we investigated if these effects correlate with superoxide (O2(-)) resistance of the strains. The protective properties of 55 strains were monitored in single cell gel electrophoresis (SCGE) assays. After preincubation of the cells with LAB (60 min), oxidative damage was induced by exposure to plumbagin (5.0 microM, 120 min) which releases O2(-) or by hydrogen peroxide (50 microM, 10 min); O2(-) resistance was monitored in plate growth inhibition assays. 25 strains (45%) reduced plumbagin induced DNA migration while only few of them (20%) were protective towards hydrogen peroxide induced damage. The strongest effects (up to 60% reduction of O2(-) induced DNA migration) were observed with representatives of the species Streptococcus thermophilus. The prevention of DNA damage in the colon cells by the bacteria did not correlate with their O2(-) resistance. Additional experiments indicate that the reduction of oxidative damage is only seen with viable bacteria but not with heat inactivated cells and that it takes also place when the colon cells are separated from the LAB by permeable filter membranes indicating that the bacteria release ROS protective factors into the medium. Dose response experiments showed that the protection depends on the concentration of the bacteria; significant effects were observed with titers 3 x 10(6-7)cells/ml. Unexpectedly, we found that a substantial fraction of the strains (13%) induced DNA damage in untreated cells, some of them increased also the effects of the ROS generating chemicals. Preliminary experiments with tetramethylbenzidine (TMB) agar indicate that this phenomenon may be due to release of hydrogen peroxide by the bacteria. Overall, our study shows that the impact of LAB on DNA damage in human derived colon cells is ambivalent; while the majority of strains was protective against oxidative damage some of them induced per se pronounced DNA migration. Since the effects were seen with bacterial concentrations which may be reached in the intestinal tract after consumption of fermented milk products, it is likely that the effects we observed in the present study are relevant for humans.     

Effects of vitamin D on aortic smooth muscle cells in culture

Earlier investigations on vitamin-induced experimental atherosclerosis in rats suggested that smooth muscle cells (SMCs) play a pivotal role in development of these vascular abnormalities. This study demonstrates the effects of vitamin D (ergocalciferol) on SMCs of rat aorta in tissue culture. SMCs were obtained from aortas of newborn rats by enzymatic digestion and maintained for 6 wk in primary culture with vitamin D (1.2 n ) in the culture medium. The effects of vitamin D on SMCs, as compared with control SMCs cultures, were evaluated by light and electron microscopy. Growth of SMCs was characterized by cell counting, measurement of DNA and protein content, and by analysis of the nucleolar organizing regions. Vitamin D had no effect on proliferation of SMCs but stimulated synthesis and intercellular deposition of elastic fibres and had a stabilizing effect on the musculo-elastic multilayer formed by the cultured cells. In addition, it prevented degeneration of SMCs, with long-term preservation of the typical phenotype in primary culture.     

Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells

Introduction

The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA.

Methods

We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes.

Results

We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC₅₀ values of 16, 27, and 15 nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC₅₀ of 62 nM, and was sensitive to ketoconazole inhibition.

Discussion

CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.     

Role of CYP2E1 in ketone-stimulated insulin release in pancreatic B-cells

The role of CYP2E1 in ketone-stimulated insulin release was investigated using isolated pancreatic islets of Langerhans and two mammalian insulin secreting pancreatic beta-cell lines engineered to stably express human CYP2E1 (designated BRIN BD11h2E1 and INS-1h2E1). Isolated rat pancreatic islets were shown to express the CYP2E1 isoform which was inducible by pretreatment of animals with acetone. The cDNA encoded CYP2E1 was expressed and inducible in the engineered cells as shown by Western blotting. The transfected protein was enzymatically active in the heterologous cells as determined by p-nitrophenol hydroxylation rates (0.176 +/- 0.08 vs. 0.341 +/- 0.08 nmol/min/mg microsomal protein in BRIN BD11 control cells and BRIN BD11h2E1 cells respectively, P < 0.001; 0.204 +/- 0.03 vs. 0.633 +/- 0.102 nmol/min/mg microsomal protein in INS-1 and INS-1h2E1, respectively, P < 0.001). Cultivation of CYP2E1 expressing BRIN BD11h2E1 and INS-1h2E1 cells in 40 mM ethanol increased the rate of p-nitrophenol hydroxylation (0.968 +/- 0.09 nmol/min/mg microsomal protein, P < 0.001 and 0.846 +/- 0.103 nmol/min/mg microsomal protein, P < 0.001, respectively) providing further evidence that the heterologous protein is inducible. Cultivation of control cells with ethanol had no observable effect (0.186 +/- 0.05 and 0.195 +/- 0.03 in BRIN BD11 and INS-1, respectively). These cell lines also express NADPH-cytochrome P450 reductase protein which was enzymatically active (0.632 +/- 0.023 in parental BRIN BD11 vs. 0.657 +/- 0.066 without ethanol and 0.824 +/- 0.014 nmol/min/mg microsomal protein with ethanol in BRIN BD11h2E1, P < 0.05; and 1.568 +/- 0.118 in parental INS-1 vs. 1.607 +/- 0.093 without ethanol and 1.805 +/- 0.066 nmol/min/mg microsomal protein with ethanol in INS-1h2E1, P < 0.05) thereby providing a functional cytochrome P450 system. The insulin secretory response of control cell lines and islets was similar to cell lines and islets which had been chemically pretreated, to induce CYP2E1 expression, in response to known nutrient secretagogues. However, insulin output was significantly higher in pretreated islets (1.3-fold, P < 0.05) and CYP2E1 expressing cell lines (BRIN BD11h2E1 2.3-fold, P < 0.001; INS1-1h2E1 1.6-fold, P < 0.001) when stimulated with the ketone 3-hydroxybutyrate than control islets and parental cell lines respectively. Similar acute exposure to acetoacetate enhanced insulin secretion by 1.3-fold (P < 0.05) in pretreated islets, 2.6-fold (P < 0.001) in ethanol pretreated BRIN BD11h2E1 and 1.4-fold (P < 0.001) in ethanol pretreated INS-1h2E1 cells compared to the respective control islets or ethanol pretreated control parental cells. Therefore, these studies highlight a possible role for CYP2E1 in pancreatic cell dysfunction.     

Cadmium-induced apoptosis is mediated by the translocation of AIF to the nucleus in rat testes

Cadmium (Cd) is a highly toxic metal that affects a variety of cellular events, such as cell proliferation, differentiation and survival. Cd generates reactive oxygen species (ROS) that induce apoptosis. We previously demonstrated that Cd induces apoptosis in testicular germ cells and that apoptosis was prevented by the administration of ascorbic acid (AA), an ROS scavenger. However, little is known about the signaling pathways underlying Cd-induced apoptosis in rat testes. Here, we report that Cd-induced apoptosis in rat testes was associated with the translocation of apoptosis inducing factor (AIF) from mitochondria to the nucleus, and that this was prevented by treatment with AA. Cd-induced cleavage of poly ADP-ribose polymerase-1 (PARP-1), and this was also inhibited by treatment with AA. Taken together, these results suggest that Cd-induced ROS was responsible for the upregulation of PARP-1, the translocation of AIF to the nucleus, and apoptosis of testicular cells in rat testes.     

Mitochondrial complex I dysfunction induced by cocaine and cocaine plus morphine in brain and liver mitochondria

Mitochondrial function and energy metabolism are affected in brains of human cocaine abusers. Cocaine is known to induce mitochondrial dysfunction in cardiac and hepatic tissues, but its effects on brain bioenergetics are less documented. Furthermore, the combination of cocaine and opioids (speedball) was also shown to induce mitochondrial dysfunction. In this work, we compared the effects of cocaine and/or morphine on the bioenergetics of isolated brain and liver mitochondria, to understand their specific effects in each tissue. Upon energization with complex I substrates, cocaine decreased state-3 respiration in brain (but not in liver) mitochondria and decreased uncoupled respiration and mitochondrial potential in both tissues, through a direct effect on complex I. Morphine presented only slight effects on brain and liver mitochondria, and the combination cocaine+morphine had similar effects to cocaine alone, except for a greater decrease in state-3 respiration. Brain and liver mitochondrial respirations were differentially affected, and liver mitochondria were more prone to proton leak caused by the drugs or their combination. This was possibly related with a different dependence on complex I in mitochondrial populations from these tissues. In summary, cocaine and cocaine+morphine induce mitochondrial complex I dysfunction in isolated brain and liver mitochondria, with specific effects in each tissue.     

Solanum torvum Swartz. fruit containing phenolic compounds shows antidiabetic and antioxidant effects in streptozotocin induced diabetic rats

In this study, quantification of phenolic compounds and the investigation of antidiabetic and antioxidant activities of the fruit of Solanum torvum Swartz. are described. S. torvum fruit methanol extract (STMe) was administered orally at a dose of 200 and 400 mg/kg/day to streptozotocin induced diabetic rats for 30 days. The levels of glucose, insulin, total protein, hemoglobin, glycated hemoglobin, liver glycogen and marker enzymes of carbohydrate metabolism, hepatic function and antioxidants were analyzed. High-performance liquid chromatography (HPLC) analysis revealed that STMe contained high levels of phenolic compounds, mainly rutin (1.36% w/w), caffeic acid (12.03% w/w), gallic acid (4.78% w/w) and catechin (0.46% w/w). STMe at 200 and 400 mg/kg reduced blood glucose level by 17.04% and 42.10%, respectively in diabetic rats. The levels and/or activities of other biochemical parameters were restored significantly compared to diabetic control rats due to treatment with fruit extract. Histology of liver and pancreas in STMe treated groups substantiated the cytoprotective action of the drug. Immunohistochemical observation of islets in extract treated diabetic rats showed apparent β-cells regeneration. These findings suggest that S. torvum fruit containing phenolic compounds has great potential as a natural source of antidiabetic and antioxidant drug.     

Protective effects of Passiflora alata extract pretreatment on carbon tetrachloride induced oxidative damage in rats.

The leaf extract of Passiflora alata Dryander (P. alata) has been demonstrated to possess antioxidant activity in vitro. The aim of this study was to investigate the effects of P. alata leaf extract pretreatment on carbon tetrachloride-treated rats. Male Wistar rats were randomly allocated into four groups: group 1 (control - vehicle), group 2 and 3 (P. alata extract - 1 and 5mg/kg, respectively) and group 4 (trolox - 0.18mg/kg). Rats received daily pretreatment by oral gavage for 30 days followed by a single dose of CCl(4) (3ml/kg i.p. in vegetable oil) on the 30th day and were killed after 6h. The pretreatment with the P. alata extract provided significant protection to liver, evidenced by lower degree of necrosis, decreased lipid peroxidation (TBARS) and higher catalase and superoxide dismutase activities. Additionally, pretreated-rats with P. alata (5mg/kg) showed significantly decreased cardiac TBARS levels. Our results indicate that a low oral dose of P. alata leaf extract has both hepato and cardioprotective effects on rats treated with CCl(4).