Mitochondria are primary targets in apoptosis induced by the mixed phosphine gold species chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I) in melanoma cell lines

Based on previous evidence indicating a selective cytotoxic activity of the mixed phosphine gold complex chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I) for melanoma cells, we investigated the cellular bases of its antiproliferative effect in a panel of human melanoma cell lines (JR8, SK-Mel-5, Mel-501, 2/60, 2/21 and GRIG). The drug consistently induced a dose-dependent inhibition of cell growth, with IC50 values ranging from 0.8 to 2.3 microM and, when tested under the same experimental conditions, its cytotoxic activity was higher than (from 2- to 5-fold) or comparable to that of cisplatin as a function of cell lines. The ability of the gold complex to activate programmed cell death was assessed in JR8 and 2/60 cells, and a dose-dependent increase in cells with an apoptotic nuclear morphology was observed in both cell lines (up to 40 and 66% of the overall cell population, for JR8 and 2/60 cell lines, respectively). Such an apoptotic response was mediated by a dose-dependent loss of mitochondrial membrane potential, cytochrome c and Smac/DIABLO release from mitochondria into cytosol and enhanced caspase-9 and caspase-3 catalytic activity. A reduced or completely abrogated expression of the anti-apoptotic proteins c-IAP1, XIAP and survivin in drug-treated cells was also observed. Overall, results from the study indicate that chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I) markedly inhibits melanoma cell growth by inducing mitochondria-mediated apoptosis and suggest it as a good candidate for additional evaluation as an anticancer agent against melanoma.     

Protection against oxidant-induced apoptosis by mitochondrial thioredoxin in SH-SY5Y neuroblastoma cells

Mitochondrial oxidative stress plays important roles in aging and age-related degenerative disorders. The newly identified mitochondrial thioredoxin (mtTrx; Trx2) is a key component of the mitochondrial antioxidant system which is responsible for the clearance of reactive intermediates and repairs proteins with oxidative damage. Here, we show that in cultured SH-SY5Y human neuroblastoma 1cells, overexpression of mtTrx inhibited apoptosis and loss of mitochondrial membrane potential induced by a chemical oxidant, tert-butylhydroperoxide (tBH). The effects of calcium ionophore (Br-A23187) were not affected by mtTrx, suggesting the protection was specific against oxidative injury. The mitochondrial glutathione pool was oxidized by tBH, and this oxidation was not inhibited by increased mtTrx. Consequently, the antioxidant function of mtTrx is not redundant, but rather in addition, to that of GSH. Mutations of Cys90 and Cys93 to serines rendered mtTrx ineffective in protection against tBH-induced cytoxicity. These data indicate that mtTrx controls the mitochondrial redox status independently of GSH and is a key component of the defensive mechanism against oxidative stress in cultured neuronal cells.     

Dose- and time-dependent effects of doxorubicin on cytotoxicity,cell cycle and apoptotic cell death in human colon cancer cells

The cytostatic drug doxorubicin is a well-known chemotherapeutic agent which is used in treatment of a wide variety of cancers. A key factor in the response of cancer cells to chemotherapeutic drugs is the activation of the apoptotic pathway, a pathway that is often impaired in chemoresistant colon cancer cells. The aim of the present study was to investigate the effects of doxorubicin in Hct-116 human colon carcinoma cells in order to clarify if a time/concentration range for optimal doxorubicin-induced apoptosis exists. We compared a treatment schedule were cells were bolus incubated for 3 h with doxorubicin followed by 24 h in drug-free medium, with a continuous doxorubicin treatment schedule for 24 h. Bolus incubation was carried out to determine effects of doxorubicin accumulated during the first 3 h, whereas continuous incubation allowed further (continuous) exposure to doxorubicin. We found that bolus (3 h) treatment with doxorubicin resulted in a dose-dependent decrease of viable cells and concomitant increase of apoptosis. Additionally, bolus (3 h) doxorubicin incubation led to phosphorylation of p53 at serine 392, induction of p21, G2 arrest and increase of proapoptotic protein Bax. In contrast, continuous (24 h) treatment with doxorubicin reduced the number of living cells with no parallel raise in the amount of dead cells. Continuous (24 h) treatment with 5 μM doxorubicin resulted in cell cycle arrest in G0/G1 phase that was neither accompanied by phosphorylation and activation of p53 nor enhanced expression of p21. These results suggest that doxorubicin is able to induce cell death by apoptosis only at particular dose and treatment conditions and imply a completely different cellular response following bolus or continuous exposure to doxorubicin.     

Oral administration of Cr(VI) induced oxidative stress, DNA damage and apoptotic cell death in mice

Potassium dichromate (Cr(VI)) was given orally to Swiss mice for 1 and 5 days with the dose of 25, 50 and 100 mg/kg body weight per day, respectively. Oxidative stress including the level of reactive oxygen species (ROS), the extent of lipid peroxidation and the activity of antioxidant enzymes in liver and kidney was determined. DNA damage in peripheral blood lymphocytes was determined by single-cell gel electrophoresis (comet assay). Apoptotic cell death in liver was detected using transmission electron microscopy and TUNEL assay. The results indicated that administration of Cr(VI) had caused a significant increase of ROS level in liver both after 1 and 5 days of exposure, accompanied with a dose-dependent decrease in superoxide dismutase (SOD) and catalase (CAT) activities. The malondialdehyde (MDA) content in liver was not changed as compared to the control animals. In contrast to the liver, no significant changes were observed in kidney on ROS, SOD, CAT and MDA as compared to the control animals. Dose- and time-dependent effects were observed on DNA damage after 1 and 5 days treatment. Significant difference was observed on the number of TUNEL positive liver cells between the control and Cr(VI) treatment groups. The apoptotic cells were also identified by characteristic ultrastructural features. The results obtained from the present study showed that Cr(VI) given orally to mice could induce dose- and time-dependent effects on DNA damage, hepatic oxidative stress and hepatocytes apoptosis. No significant oxidative stress observed in kidney in the study may suggest that the way of Cr(VI) exposure is an important factor affecting its toxicity.     

Mitochondrial thioredoxin reductase inhibition by gold(I) compounds and concurrent stimulation of permeability transition and release of cytochrome c

The effects of auranofin, chloro(triethylphosphine)gold(I) (TEPAu), and aurothiomalate on mitochondrial respiration, pyridine nucleotide redox state, membrane permeability properties, and redox enzymes activities were compared. The three gold(I) derivatives, in the submicromolar range, were extremely potent inhibitors of thioredoxin reductase and stimulators of the mitochondrial membrane permeability transition (MPT). Auranofin appeared as the most effective one. In the micromolar range, it inhibited respiratory chain and glutathione peroxidase activity only slightly if not at all. TEPAu and aurothiomalate exhibited effects similar to auranofin, although TEPAu showed a moderate inhibition on respiration. Aurothiomalate inhibited glutathione peroxidase at concentrations where auranofin and TEPAu were without effect. Under nonswelling conditions, the presence of auranofin and aurothiomalate did not alter the redox properties of the mitochondrial pyridine nucleotides indicating that membrane permeability transition occurred independently of the preliminary oxidation of pyridine nucleotides. Under the same experimental conditions, TEPAu showed a moderate stimulation of pyridine nucleotides oxidation. Mitochondrial total thiol groups, in the presence of the gold(I) derivatives, slightly decreased, indicating the occurrence of an oxidative trend. Concomitantly with MPT, gold(I) compounds determined the release of cytochrome c that, however, occurred also in the presence of cyclosporin A and, partially, of EGTA, indicating its independence of MPT. It is concluded that the specific inhibition of thioredoxin reductase by gold(I) compounds may be the determinant of MPT and the release of cytochrome c.     

Tributyltin chloride induced testicular toxicity by JNK and p38 activation,redox imbalance and cell death in sertoli-germ cell co-culture

The widespread use of tributyltin (TBT) as biocides in antifouling paints and agricultural chemicals has led to environmental and marine pollution. Human exposure occurs mainly through TBT contaminated seafood and drinking water. It is a well known endocrine disruptor in mammals, but its molecular mechanism in testicular damage is largely unexplored. This study was therefore, designed to ascertain effects of tributyltin chloride (TBTC) on sertoli-germ cell co-culture in ex-vivo and in the testicular tissue in-vivo conditions. An initial Ca²⁺ rise followed by ROS generation and glutathione depletion resulted in oxidative damage and cell death. We observed p38 and JNK phosphorylation, stress proteins (Nrf2, MT and GST) induction and mitochondrial depolarization leading to caspase-3 activation. Prevention of TBTC reduced cell survival and cell death by Ca²⁺ inhibitors and free radical scavengers specify definitive role of Ca²⁺ and ROS. Sertoli cells were found to be more severely affected which in turn can hamper germ cells functionality. TBTC exposure in-vivo resulted in increased tin content in the testis with enhanced Evans blue leakage into the testicular tissue indicating blood–testis barrier disruption. Tesmin levels were significantly diminished and histopathological studies revealed marked tissue damage. Our data collectively indicates the toxic manifestations of TBTC on the male reproductive system and the mechanisms involved.     

Chemical behavior and in vitro activity of mixed phosphine gold(I) compounds on melanoma cell lines

(31)P nuclear magnetic resonance and electrospray ionization-mass spectrometry studies on the melanoma cytotoxic chlorotriphenylphosphine-1,3-bis(diphenylphosphino)propanegold(I), [Au(DPPP)(PPh 3)Cl], show partial decomposition that includes the novel dinuclear cation [Au 2(DPPP) 2Cl] (+); its structure was calculated by using the density functional theory (DFT). Unexpectedly, by using the diphosphine ligand 1,2-bis(diphenylphosphino)ethane (DIPHOS), [{AuCl(PPh 3)} 2(mu 2-DIPHOS)] was obtained. Its X-ray crystal structure shows a unique triangular coordination sphere in contrast to the T-shaped geometry of related Au(I)-DIPHOS compounds. Its cytotoxic activity in JR8, SK-Mel-5, and 2/60 melanoma cell lines is dose-dependent and lower than that of [Au(DPPP)(PPh 3)Cl] because of its nonchelating nature. An in vitro study of the effect of both Au compounds on the B16V melanoma cell line gives credence to this structure-activity relationship. IC 50 indicates that both Au species are 10 times more active in B16V than in JR8, SK-Mel-5, and 2/60. Oxidation of [Au(DPPP)(PPh 3)Cl] toward Au(III) compounds and phosphine-oxides is observed upon reaction with hypochlorite in water/dimethyl sulfoxide solution, mimicking endogenous hypochlorite. A related reaction involving the formation of [AuCl 4] (-) is thermodynamically feasible according to DFT calculations.     

Mitigation of 5-Fluorouracil induced renal toxicity by chrysin via targeting oxidative stress and apoptosis in wistar rats

5-Fluorouracil (5-FU) is a potent antineoplastic agent commonly used for the treatment of various malignancies. It has diverse adverse effects such as cardiotoxicity, nephrotoxicity and hepatotoxicity which restrict its wide and extensive clinical usage. It causes marked organ toxicity coupled with increased oxidative stress and apoptosis. Chrysin (CH), a natural flavonoid found in many plant extracts, propolis, blue passion flower. It has antioxidative and anti-cancerous properties. The present study was designed to investigate the protective effects of CH against 5-FU induced renal toxicity in wistar rats using biochemical, histopathological and immunohistochemical approaches.Rats were subjected to prophylactic oral treatment of CH (50 and 100 mg/kg b.wt.) for 21 days against renal toxicity induced by single intraperitoneal administration of 5-FU (150 mg/kg b.wt.). The possible mechanism of 5-FU induced renal toxicity is the induction of oxidative stress; activation of apoptotic pathway by upregulation of p53, bax, caspase-3 and down regulating Bcl-2. However prophylactic treatment of CH decreased serum toxicity markers, increased anti-oxidant armory as well as regulated apoptosis in kidney. Histopathological changes further confirmed the biochemical and immunohistochemical results. Therefore, results of the present finding suggest that CH may be a useful modulator in mitigating 5-FU induced renal toxicity.     

Oxidative stress induced apoptosis of human lung carcinoma (A549) cells by a novel copper nanorod formulation

This study elucidates the process of synthesis of copper (Cu) nanorods using almond skin extract as stabilizing cum capping agent. These nanorods were (about 200 nm long and 40 nm wide) characterized by transmission electron microscopy (TEM). Further, cytotoxicity potential of these nanorods was evaluated in A549 cells (Human lung carcinoma cell line) via cell viability assay and extracellular lactate dehydrogenase (LDH) release. Also, reduced glutathione (GSH), lipid peroxidation (LPO), cellular oxidative stress (Rhodamine 123 florescence) and apoptosis (Annexin V FITC/Propidium iodide staining) were also investigated in control and treated cells. Results indicated that Cu nanorods induced apoptotic death of cancer cells by induction of oxidative stress, depletion of cellular antioxidants and mitochondrial dysfunction. This study reports a novel process of synthesis of almond skin extract capped Cu nanorods and its potential as an anticancer agent against A549 lung carcinoma cells.     

Frequent biphasic cellular responses of permanent fish cell cultures to deoxynivalenol (DON)

Contamination of animal feed with mycotoxins is a major problem for fish feed mainly due to usage of contaminated ingredients for production and inappropriate storage of feed. The use of cereals for fish food production further increases the risk of a potential contamination. Potential contaminants include the mycotoxin deoxynivalenol (DON) which is synthesized by globally distributed fungi of the genus Fusarium. The toxicity of DON is well recognized in mammals. In this study, we confirm cytotoxic effects of DON in established permanent fish cell lines. We demonstrate that DON is capable of influencing the metabolic activity and cell viability in fish cells as determined by different assays to indicate possible cellular targets of this toxin. Evaluation of cell viability by measurement of membrane integrity, mitochondrial activity and lysosomal function after 24 h of exposure of fish cell lines to DON at a concentration range of 0-3000 ng ml⁻¹ shows a biphasic effect on cells although differences in sensitivity occur. The cell lines derived from rainbow trout are particularly sensitive to DON. The focus of this study lies, furthermore, on the effects of DON at different concentrations on production of reactive oxygen species (ROS) in the different fish cell lines. The results show that DON mainly reduces ROS production in all cell lines that were used. Thus, our comparative investigations reveal that the fish cell lines show distinct species-related endpoint sensitivities that also depend on the type of tissue from which the cells were derived and the severity of exposure.     

Safflor yellow B suppresses pheochromocytoma cell (PC12) injury induced by oxidative stress via antioxidant systemand Bcl-2 /Bax pathway

Our previous study demonstrates that SYB produces a neuroprotective effect in vivo. In the present study, we investigated the protective effect of safflor yellow B (SYB) on the acute oxidative injury induced by H₂O₂ and mechanisms in PC12 cells. H₂O₂ was used to mimic in vitro model of the oxidative injury and to induce apoptosis in PC12 cells. The cells were pretreated with the different concentrations of SYB. The cell viability, lactate dehydrogenase (LDH) release, malondialdehyde (MDA), and superoxide anion levels, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were measured. Caspase 3 activity, Bcl-2 and Bax expressions were also observed. The results showed that exposure of the cells to H₂O₂ significantly decreased the cell viability, SOD and GSH-Px activities and Bcl-2 expression, and increased LDH release, superoxide anion and MDA generations, caspase 3 activity and Bax expressions. Pretreatment of the cells with SYB was able to remarkably antagonize the H₂O₂-induced changes in dose-dependent way. These suggest that SYB is able to protect PC12 cells from H₂O₂-induced injury and apoptosis via antioxidant and anti-apoptotic mechanisms.     

Bis(tri-n-butyltin)oxide induces programmed cell death (apoptosis) in immature rat thymocytes

In order to characterise the mechanism of cytotoxicity of the immunotoxic organotin compound bis(tri-n-butyltin)oxide (TBTO) to lymphoid cells, isolated thymocytes from immature rats were exposed to TBTO (0.1–5 M) for up to 6 h. At lower TBTO concentrations (0.1 and 1 M) vital staining showed that only marginal loss of viability occured, although morphological studies demonstrated increased numbers of cells with abnormal features indicative of programmed cell death (apoptosis). These changes included nuclear chromatin condensation (which was associated with increased DNA fragmentation), cytoplasmic contraction and formation of membrane bound apoptotic bodies. When visualised by agarose gel electrophoresis, genomic DNA appeared as a series of fragments with a repeat multiple of 180–200 base pairs. Comparable morphological changes and cleavage of DNA into oligonucleosomal fragments were evident in thymocytes incubated with 10 M methyl prednisolone hemisuccinate (MPS); a glucocorticoid hormone known to induce programmed cell death in thymocytes. Marked cytotoxicity associated with degenerative changes indicative of necrosis was observed in thymocytes incubated with 5 M TBTO. These findings indicate that, at levels which are not overtly cytotoxic, TBTO is capable of inducing programmed cell death in rat thymocytes. This suggest a possible mechanism for the T-cell immunodeficiency previously reported for TBTO in vivo.     

Toxic effects of bis(thiosemicarbazone) compounds and its palladium(II) complexes on herpes simplex virus growth

Here, we present data on the activity of benzyl bis(thiosemicarbazone); 3,5-diacyl-1,2,4-triazole bis(4-methylthiosemicarbazone) and their Pd(II) complexes against the replication of wild type and of acyclovir (ACV)-resistant, herpes simplex virus type 1 (HSV 1) and type 2 (HSV 2) strains. The data were compared to those under the action of acyclovir. The testing of cytotoxic activity suggests that these compounds may be endowed with important antiviral properties. It is interesting to note that the Pd(II)-benzyl bis(thiosemicarbazone) complex, 2, exhibits a significant activity against acyclovir-resistant viruses R-100 (HSV 1) and PU (HSV 2) with an in vitro selectivity index (SI) of 8.0 vs. 0.01 for acyclovir. This complex also negatively influenced the expression of key structural HSV 1 proteins (VP23, gH and gG/gD), thus suppressing simultaneously virus entry, transactivation of virus genome, capsid assembly, and cell-to-cell spread of infectious HSV progeny.     

Aldo–keto reductase 7A5 (AKR7A5) attenuates oxidative stress and reactive aldehyde toxicity in V79-4 cells

Aldo–keto reductase (AKR) enzymes are critical in the detoxification of endogenous and exogenous aldehydes. In previous studies, we have shown that AKR7A5 enzyme is catalytically active towards aldehydes arising from lipid peroxidation (LPO) and that it can significantly protect against 4-hydroxynonenal-induced apoptosis, suggesting a protective role against the consequences of oxidative stress. The aim of this study was to elucidate the cytoprotective effect of AKR7A5 against oxidative stress using a transgenic mammalian cell line expressing AKR7A5. Results show that expression of AKR7A5 in V79-4 cells provides significant protection against the cytotoxicity of H₂O₂ and menadione, with its expression altering the IC₅₀ of H₂O₂ from 1.1 to 2.3 mM and the IC₅₀ of menadione from 8.6 to 9.6 μM, thus providing direct evidence for its anti-oxidant activity. Cells expressing AKR7A5 were also found to be more resistant to several LPO-derived aldehydes – trans-2-nonenal, hexanal and methylglyoxal. In addition the ability of AKR7A5 to enable the cells to cope with ROS accumulation and glutathione depletion was assessed. V79-4 cells overexpressing AKR7A5 were able to lower cellular ROS levels following treatment with H₂O₂ and menadione. AKR7A5 was also able to maintain cellular glutathione homeostasis in the presence of H₂O₂ and menadione. These findings indicate the importance of AKR7A5 in protecting cells from the damaging effects of oxidative stress, and that this cytoprotective function is carried out through multiple pathways.     

Genotoxicity and cell death induced by tinidazole (TNZ)

Tinidazole (TNZ), a second-generation 5-nitroimidazole compound chemically related to metronidazole (MTZ), has been widely used throughout Europe and developing countries for the treatment of amoebic and parasitic infections. Despite TNZ's increasing use in therapeutics, scarce experimental reports are available in literature on its potential genotoxicity in human cells. Therefore, the aim of the present study was to achieve a precise characterization of the cytotoxic and genotoxic activities of this nitroimidazole in cultured human lymphocytes at therapeutic concentrations (0.1, 1, 10 and 50 microg/ml of culture) and evaluate the possible cell death mechanism associated with it. The endpoints analyzed included: mitotic index (MI), replication index (RI), sister chromatid exchange (SCE) and chromosomal aberrations (CA). A significant decrease (p<0.0001) in MI as well as an increase in SCE (p<0.0001) and CA (p<0.0001) frequencies were observed. No modifications in RI were found. The results suggest a genotoxic and cytotoxic effect of TNZ related with cell death process. Therefore, we evaluated this mechanism by DNA fragmentation (laddering), fluorescence microscopy using acridine orange/ethidium bromide (AO/EB) staining and flow cytometry propidium iodide (PI). DNA extracts of TNZ-treated cells resulted in nucleosomal DNA ladder pattern after 48 h of cell treatment; meanwhile no differences were detected in untreated cells. This pattern correlated with the observed decrease in cellular viability (p<0.05), morphological evidence of apoptosis and increase in the percentage of nuclei with hypodiploid DNA content of TNZ exposed cultures compared with control (p<0.05). We concluded that TNZ is genotoxic, cytotoxic and is able to modulate cell death through apoptotic mechanisms in the experimental design employed.     

A protective study of curcumin associated with Cr6+ induced oxidative stress,genetic damage,transcription of genes related to apoptosis and histopathology of fish,Channa punctatus (Bloch, 1793)

Ameliorative potential of curcumin against Cr⁶⁺-induced eco-toxicological manifestations was assessed in liver of exposed Channa punctatus (Actinopterygii) in six groups for 45 d; Group I as control. Group II with 3 mg/L of curcumin; group III with 7.89 mg/L of Cr⁶⁺. Groups IV, V and VI were simultaneously co-exposed with 7.89 mg/L of Cr⁶⁺ and three different curcumin concentrations, 1, 2, and 3 mg/L, respectively. In group III, SOD-CAT, GR significantly (p < 0.05) increased; decreased GSH level; elevated MN and AC frequencies; and a significant (p < 0.05) up-regulation of cat (2.72-fold), p53 (1.73-fold), bax (1.33-fold) and apaf-1 (2.13-fold) together with a significant (p < 0.05) down-regulation of bcl-2 (0.51-fold). Co-exposure significantly (p < 0.05) brought down activities of SOD-CAT, GR, raised GSH, decreased micronuclei and apoptotic frequencies along with recovery of histopathological anomalies in liver. This study establishes the protective role of curcumin against Cr⁶⁺-induced hepatotoxicity in fish.     

Alterations in human red blood cell properties induced by 3-(dimethylamino)phenol (in vitro)

3-(Dimethylamino)phenol (3-DMAP) exists in the environment as a transformation product of ureic herbicides and may also be considered as a derivative of phenoxyherbicides.

In this study, the activity of glutathione peroxidase, catalase and superoxide dismutase, as well as the level of free radicals and changes in cell morphology were measured in human erythrocytes exposed (in vitro) to 3-(dimethylamino)phenol. Human erythrocytes were incubated for 1 h in 3-DMAP at concentrations of 10–500 μg per 1 ml erythrocytes of 5% haematocrit.

The results show that 3-(dimethylamino)phenol increased the level of free radicals and changed the activity of glutathione peroxidase, catalase, superoxide dismutase and acetylcholinesterase. It also changed cell morphology.

All these results corroborated the thesis that 3-DMAP induces oxidative stress in cells. 3-DMAP changed the properties of the cell membrane, caused strong oxidation of haemoglobin, inhibited the levels of enzymatic and non-enzymatic antioxidants, which, in result, lead to generation of free radicals (ROS and semiquinones) that occurred in the exposed cells, predisposing them to oxidative damage.     

Inhibition of MDR1 expression by retinol treatment increases sensitivity to etoposide (VP16) in human neoplasic cell line

Multidrug resistance (MDR) is the major obstacle to cancer chemotherapy. MDR phenotype is mainly related to the over-expression of MDR1 gene, being responsible for tumor resistance to several chemotherapeutic drugs. It has been suggested that MDR1 expression is redox-regulated and we have recently described a pro-oxidative effect of retinol. Here we tested the therapeutic use of retinol as a modulator of MDR1 gene expression in tumor cell lines, and verified in situ the enhancement of anticancer drug efficacy. Two human colorectal adenocarcinoma cell lines (HT29, SW620) with different degrees of MDR1 expression were used. Cells were pre-treated with a sublethal dose of retinol and then challenged with the etoposide (VP16) drug. The drug GI50 was assessed by SRB method and levels of MDR1 expression were determined by semi-quantitative rtPCR. Retinol treatment caused a 40% decrease in MDR1 expression and increased VP16 toxicity. MDR1 expression and drug sensitivity were restored to control values when mannitol (a hydroxyl radical scavenger) was co-administrated. Our data point a role to the use of retinol as an adjuvant in the treatment of tumors with MDR phenotype.     

Propofol, an anesthetic possessing neuroprotective action against oxidative stress, promotes the process of cell death induced by H2O2 in rat thymocytes

Propofol (2,6-diisopropylphenol) is a general anesthetic possessing a neuroprotective action against oxidative stress produced by H₂O₂. H₂O₂ induces an exposure of phosphatidylserine on outer surface of cell membranes, resulting in change in membrane phospholipid arrangement, in rat thymocytes. Since propofol is highly lipophilic, the agent is presumed to interact with membrane lipids and hence to modify the cell vulnerability to H₂O₂. Therefore, to test the possibility, we have examined the effect of propofol on rat thymocytes simultaneously incubated with H₂O₂. Although propofol (up to 30 μM) alone did not significantly affect the cell viability, the agent at 10 μM started to increase the population of dead cells in the presence of 3 mM H₂O₂ and the significant increase was observed at 30 μM. Propofol at clinically relevant concentrations (10–30 μM) facilitated the process of cell death induced by H₂O₂ in rat thymocytes. However, propofol protected rat brain neurons against the oxidative stress induced by H₂O₂ under same experimental condition. Therefore, the action of propofol may be dependent on the type of cells.     

Detection of key enzymes,free radical reaction products and activated signaling molecules as biomarkers of cell damage induced by benzo[a]pyrene in human keratinocytes

Benzo[a]pyrene (BaP) is a known carcinogenic and cell damaging agent. The underlying cell damaging pathomechanisms have not been totally revealed. Especially BaP-related induction of oxidative and nitrosative stress has not been previously investigated in detail. The presented study investigated these effects in order to elucidate the pathomechanism and as well to identify potential biological markers that may indicate a BaP exposure. Human immortalized keratinocytes (HaCaT cells) were exposed to BaP (1 μM) for either 5 min or 6 h, respectively. BaP-induced cellular damage was evaluated by immunocytochemistry analysis of multiple signaling cascades (e.g. apoptosis, Akt, MAPK, NOS, nitrotyrosine and 8-isoprostane formation), detection of nitrosative stress using diaminofluorescein (DAF-FM) and oxidative stress using 3′ -(p-aminophenyl)fluorescein (APF).Our results show that BaP exposure significantly enhanced NO and ROS productions in HaCaT cells. BaP led to eNOS-phosphorylation at Ser¹¹⁷⁷, Thr⁴⁹⁵ and Ser¹¹⁶ residues. Using specific inhibitors, we found that the Erk1/2 pathways seemed to have strong impact on eNOS phosphorylation. In addition, BaP-induced apoptosis was observed by caspase-3 activation and PARP cleavage.Our results suggest that BaP mediates its toxic effect in keratinocytes through oxidative and nitrosative stress which is accompanied by complex changes of eNOS phosphorylation and changes of Akt and MAPK pathways.