Desferrithiocin is a more potent antineoplastic agent than desferrioxamine

Desferrithiocin (DFT) is an orally effective Fe chelator, with a similar high affinity and selectivity for Fe to desferrioxamine (DFO), which has been shown clinically to possess antineoplastic activity. In this study, DFT was assessed for antineoplastic potential in hepatocellular carcinoma cell lines (HCC). This was done as there are few treatments for this aggressive neoplasm. The effects of DFT on cell proliferation, cell cycle progression, Fe uptake and toxicity were examined. To establish whether DFT was selective for cancer cells a comparison was made with normal (non-proliferating) hepatocytes and non-tumorigenic (proliferating) fibroblasts (SWISS-3T3). DFT was a potent inhibitor of HCC proliferation (IC(50) approximately 40 microM). DFO also inhibited HCC proliferation under the same conditions, but was much less active (IC(50)=110 - 210 microM). When saturated with Fe, the activity of DFT, like DFO, was greatly diminished, suggesting it may act by depriving the cells of Fe or inactivating essential Fe pool(s). Indeed DFT rapidly decreased Fe uptake from Tf-(59)Fe by hepatoma cells and also by normal hepatocytes. However, DFT (and DFO) had much less effect on cell survival in hepatocytes and fibroblasts than in hepatoma cells. DFT may, like DFO, inhibit the cell cycle in the S phase of DNA synthesis. Both chelators showed low toxicity. These results indicate that DFT has potent antineoplastic activity in HCC. Further investigation into the DFT class of Fe chelators seems warranted, particularly in view of its high activity in relation to DFO, a chelator which is already in clinical trial for neuroblastoma.     

Chloroacetonitrile-induced toxicity and oxidative stress in rat gastric epithelial cells.

Chloroacetonitrile (CAN), is a disinfectant by-product of chlorination of drinking water. Epidemiological studies indicate that exposure to CAN via drinking water might present a potential hazard to human health. The objective of the present work was to investigate the cytotoxic effects as well as the oxidative stress induced by CAN in cultured rat gastric epithelial cells (GECs). GECs were exposed in vitro to different concentrations of CAN (5-40 microm) for 60 min. Also, GECs were incubated with CAN (10 microm) for different time intervals extending to 180 min. Cytotoxicity was determined by assessing cell viability and lactate dehydrogenase (LDH) release, glutathione (GSH) level and lipid peroxidation as indicated by malondialdehyde (MDA) production. Exposure of GECs CAN (10 microm) for 60 min caused a 50% decrease in cell viability and induced an eightfold increase of LDH leakage. In the same experiment, CAN caused a decrease in cellular GSH content to approximately 50% and significantly enhanced MDA accumulation (approx. sevenfold). These toxic responses to CAN were dependent on both concentration and duration of exposure to CAN. There was a good correlation between LDH release and GSH depletion (r =0.96, P<0.05). Treatment of GECs with 5 m mN -acetyl- l -cysteine (NAC) prior to exposure to CAN afforded some degree of protection as indicated by a significant decrease in the LDH leakage (32% of total leakage) and lipid peroxidation (54%) as compared to CAN alone-treated cells. Also, pretreatment of GECs with vitamin C (1 m m) or vitamin E (10 microm) significantly inhibited LDH leakage (20 and 36% of total leakage, respectively). Preincubation with 1 m m desferroxiamine (DFO), a ferric iron chelator, or 10 microm phenanthroline (PHE), a ferrous iron chelator, diminished CAN-induced LDH leakage by 16 and 21% of total leakage, respectively and MDA production by 40 and 44%, respectively. In conclusion, our results suggest that CAN has a potential cytotoxic effect in rat GECs; and thiol group-donors, antioxidants and iron chelators can play a critical role against CAN-induced cellular damage.     

Iron chelation by pyridoxal isonicotinoyl hydrazone and analogues in hepatocytes in culture

Pyridoxal isonicotinoyl hydrazone (PIH) and several analogues were synthesized and assessed in the rat hepatocyte culture for their potential in iron chelation therapy. Pyridoxal isonicotinoyl hydrazone and pyridoxal benzoyl hydrazone were as effective as desferrioxamine (DFO) in reducing both net uptake of rat transferrin-59Fe and incorporation into ferritin by hepatocytes. Dialysis studies showed that this was due to a cellular action and not to the extracellular chelation of transferrin-bound 59Fe. The analogues of PIH were more effective in mobilization studies than PIH and DFO, releasing more 59Fe from ferritin as well as from the stroma-mitochondrial membranes in hepatocytes prelabelled using transferrin-59Fe. Chelator action was dependent on incubation time, concentration, temperature and lipophilicity. Pyridoxal benzoyl hydrazone, the most effective iron chelator, was also the most lipophilic, suggesting that access to cellular iron compartments as well as iron-binding affinity is important in effective iron chelation.     

Reactive oxygen species and non-peroxidative mechanisms of cocaine-induced cytotoxicity in rat hepatocyte cultures

Primary short-term cultures of hepatocytes derived from phenobarbital-induced male Sprague-Dawley rats were used to investigate the mechanisms of cocaine-induced cytotoxicity. Exposure of cells to cocaine resulted in a time and concentration-dependent release of lactate dehydrogenase (LDH) into the culture medium which became evident after 7 h of incubation. Over the course of 24 h incubation with cocaine (0.3 mM) there was no significant lipid peroxidation (measured as the formation of thiobarbituric acid-reactive substances. TBA-RS). The addition of the ferric iron chelator, deferoxamine (DFO), prevented in part cocaine-induced LDH release. Alternatively, addition of the antioxidant, alpha-tocopherol polyethylene glycol 1000 succinate (TPGS), did not protect against hepatocyte injury. Depletion of the intracellular glutathione (GSH) with diethyl maleate (DEM) to below critical levels for antioxidative protection markedly accelerated the onset and increased the extent of cocaine-induced LDH release, concomitant with massive production of lipid peroxidation. During the first four hours of incubation DFO and TPGS protected against cocaine-induced cytotoxicity in GSH-depleted cells. However, at later stages (24 h), the protective effect was lost even in the absence of lipid peroxidation. These results suggest that reactive oxygen species are involved in cocaine-mediated hepatocyte injury. However, lipid peroxidation can be dissociated from other, non-peroxidative, iron-dependent mechanisms of oxidative cell injury.     

Cytotoxic effect and role of exogenous antioxidants in carpet dust mediated toxicity in rat hepatocytes in vitro.

Carpet industries bear a great deal of economic and commercial significance in India. In order to safe guard the workers against the health hazards caused by dust in their occupational environment; it necessitates studying the biological importance of these dusts. The present study was designed to investigate the toxicity of carpet dust (knotted and tuffted) on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and cells were incubated with different concentration of carpet dust (100-5000 microg/10(6) cells) with various time (30-180 min) intervals. An exogenous antioxidant vitamin-E also used to find out the role of antioxidants and free radical production in carpet dust mediated toxicity. Cell viability by trypan blue exclusion and leakage of enzyme lactate dehydrogenase (LDH) were determined. Reduced glutathione (GSH), formation of thiobarbituric acid reactive substance (TBARS) were also measured. A significant decrease in the cell viability was observed after 60, 180 min upon incubation with tuffted carpet dust, while knotted carpet dust caused a significant decrease in the viability after 180 min. LDH leakage was parallel to the cell viability. Thiobarbituric acid reactive substance was significantly increased at 30 and 60 min with carpet dust treated hepatocytes. Dust at 1000 and 5000 microg dose level showed significantly increased formation of TBARS at 30 min incubation. However, when hepatocytes were co-incubated with carpet dust and Vit-E (10, 15 microM), a significant decrease in LDH release and TBARS production was observed while 15 microM Vit-E showed an enhanced protection than 10 microM Vit-E treated hepatocytes. The effect of carpet dust on cell viability, LDH leakage, TBARS production, GSH depletion was time and dose-dependent. Moreover, we observed that tuffted carpet dust causes greater effect than knotted one on the above mentioned parameters. Our studies also revealed that Vit-E in culture media diminishes the carpet dust mediated toxicity.     

Iron mobilization, cytoprotection, and inhibition of cell proliferation in normal and transformed rat hepatocyte cultures by the hydroxypyridinone CP411, compared to CP20: a biological and physicochemical study

The present study analyzes the iron mobilization, the cytoprotective, and the antiproliferative effects of the lipophilic hydroxypyridinone CP411, in comparison with the hydrophilic chelator CP20 or deferiprone used in the treatment of iron overload. Primary rat hepatocyte cultures and the rat hepatoma cell line Fao were used. Chelator cell uptake was evaluated by mass spectrometry in the two models. This method was also used to investigate the stability of the chelators in an acellular system as well as their scavenging and chelating effects against the hydroxyl radical generated by the Fenton reaction. The iron mobilization and the cytoprotective effects of the chelators were evaluated in primary cultures by measuring respectively 55Fe and lactate dehydrogenase release in the culture medium. The antiproliferative effect of the chelators was studied using the Fao cell line and measuring DNA synthesis by thymidine incorporation and DNA content by flow cytometry. We observed that CP411 entered the hepatocytes and the Fao cells respectively 4 and 13 times more than CP20. CP411 was 2.5 times more effective than CP20 to mobilize iron from preloaded hepatocytes. Pretreatment of the hepatocytes with CP20 or CP411 decreased the toxic effect of iron and CP411 was 1.6 times more effective than CP20. A dose-dependent decrease of DNA synthesis, correlated to an accumulation of cells in S phase, was observed in the Fao cell line in the presence of CP411, while CP20 was without effect. CP411 effect was inhibited by addition of iron simultaneously with the chelator, the addition of Zn or Cu was without effect. The inhibitory effect of CP411 was reversible since, 24hr after removal of the chelator, DNA replication reached the control level. The results show that CP411 is more efficient to protect the hepatocyte from the toxic effect of iron load and to inhibit tumor cell proliferation. Its higher efficiency may result from its better cell uptake since equimolar solutions of the two chelators in an acellular system exhibit the same ability to inhibit the Fenton reaction.     

Protection against hydrogen peroxide-mediated cytotoxicity in Friedreich's ataxia fibroblasts using novel iron chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone class

Iron-loading diseases remain an important problem because of the toxicity of iron-catalyzed redox reactions. Iron loading occurs in the mitochondria of Friedreich's ataxia (FA) patients and may play a role in its pathogenesis. This suggests that iron chelation therapy could be useful. We developed previously the lipophilic iron chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) ligands and identified 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH) as the most promising analog. Hence, this study assessed the efficacy of PCTH and other PCIH analogs compared with various chelators, including deferiprone and desferrioxamine (DFO). Age- and sex-matched control and FA fibroblasts were preincubated with iron chelators and subsequently challenged with 50 microM H2O2 for up to 24 h. The current study demonstrates an interesting structure-activity relationship among the closely related PCIH series of ligands, with only PCTH being highly effective at preventing H2O2-induced cytotoxicity. PCTH increased FA fibroblast cell viability by up to 70%, whereas DFO rescued viability by 1 to 5% only. Hence, PCTH, which was well tolerated by cells was far more effective than DFO at preventing oxidative stress. It is noteworthy that kinetic studies demonstrated PCTH to rapidly penetrate cells to induce 59Fe efflux, whereas DFO, PCIH, 2-pyridylcarboxaldehyde benzoyl hydrazone, and 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone were far slower, indicating it is the rate of chelator permeation that is crucial for protection against H2O2. In addition, PCTH was found to be as effective as or more effective than conventional radical scavengers or the antioxidant idebenone (which has undergone clinical trials) at protecting cells against H2O2-mediated cytotoxicity. These findings further indicate the potential of PCTH for treatment of iron overload.     

牛磺酸对半乳糖胺及硫酸亚铁-抗坏血酸所致大鼠肝细胞损伤的保护作用

以半乳糖胺(Galn)和硫酸亚铁-抗坏血酸(Fe-Vit C)与原代肝细胞共同培养,制成不同的肝细胞损伤模型,用不同浓度的牛磺酸预处理,以观察其对肝细胞的保护作用,结果表明:Fe—Vit C作用60min即可引发肝细胞的脂质过氧化(LPO),使培养液中的丙二醛含量明显升高,但不造成肝细胞破坏,0.6～3mmol·L~(-1)牛磺酸可明显减轻LPO程度,而浓度为30～60mmol·L~(-1)时非但不能减少LPO,反而使乳酸脱氢酶(LDH)的释放明显升高,Galn则可造成明显的肝细胞损伤,使LDH释放,0.6～3 mmol·L~(-1)牛磺酸对于这种肝细胞损伤有一定的保护作用,而30～60 mmol·L~(-1)的牛磺酸本身则可造成正常及中毒肝细胞的损伤。     

Desferrithiocin and desferrioxamine B. Cellular pharmacology and storage iron mobilization

3H-Desferrithiocin (DFT) has been synthesized from desmethyl desferrithiocin. The uptake and release of this 3H siderophore and of its iron complex have been studied in cultured rat hepatocytes and systematically compared to 14C desferrioxamine B (DFO). At 37 degrees, the uptake of both chelators is strictly proportional to the extracellular concentration and no toxicity is observed up to, at least, 1 mM. Uptake of 3H DFT is rapid and reaches a plateau after ca. 1 hr. The accumulation of 3H DFT attains a maximum three times that of 14C DFO and the plateau is reached much more rapidly. Upon reincubation in a drug-free medium of cells that had accumulated 3H DFT, most of the 3H label is rapidly released in the culture medium. These kinetic parameters suggest that the accumulation of these two chelators results from their diffusion across cellular membranes, as a function of the gradient of concentration between the cellular compartment and the extracellular medium. Differential centrifugation of homogenates from hepatocytes incubated with 3H DFT shows that the bulk of cell associated 3H-label (82%) is found in the cytosol, whereas a small proportion (14.5%) is present in the particulate fraction. Isopycnic centrifugation on sucrose gradients suggests that 3H-label associated with the particulate fraction is localized within mitochondria. In contrast, 14C DFO distributes in almost equal proportions between cytosol and the particulate fraction (MLP). At least part of the 14C-label in MLP is associated with lysosomes. Rat hepatocytes cultivated for long term in synthetic culture medium have been used to study iron mobilization by chelators from 59Fe loaded cells. DFT mobilizes iron more rapidly than DFO. This effect is also observed in vitro with ferritin, where, in addition, DFT is much more efficient than DFO to mobilize iron at acidic pH. These results strongly suggest that different iron mobilization from cultured hepatocytes results from differences in the cellular pharmacology of these two chelators and, in particular, in their rate of uptake, cellular accumulation levels and subcellular localizations. DFT could mobilize iron from cytosol and, possibly, to a small extent from mitochondria, whereas DFO would do so from cytosol and lysosomes.     

Iron mobilization from cultured hepatocytes: effect of desferrioxamine B

When cultured rat hepatocytes prelabelled for different times at 37 degrees with 59Fe are reincubated for 1 hr in a fresh medium, radiolabelled iron is released in the washout medium as a function of the prelabelling time, and behaves like low molecular weight material on isokinetic centrifugation in sucrose gradients. When apotransferrin or desferrioxamine B are present in the reincubation medium, the kinetics of iron release are similar but the absolute amounts of radiolabelled iron found in the culture medium are much greater. In the presence of apotransferrin, most of the 59Fe released from the cells distributes as transferrin whereas with desferrioxamine B, almost all the 59Fe is extracted by benzyl alcohol indicating its chelation by the drug. Cell fractionation data indicate that iron accumulated by hepatocytes is rapidly incorporated into cytosol ferritin, and this seems to be a preferred source of iron for the chelator.     

Acrylonitrile-induced toxicity and oxidative stress in isolated rat colonocytes

Acrylonitrile (ACN), an environmental toxic pollutant, has been detected in drinking water, food products and occupational environment. The objective of the present work was to investigate the cytotoxic effects as well as the oxidative stress induced by ACN in cultured rat colonocytes. Colonocytes were exposed in vitro to different concentrations of ACN (0.1-2.0mM) for 60min. Also, colonocytes were incubated with ACN (1.0mM) for different time intervals extending to 180min. Cytotoxicity was determined by assessing cell viability and lactate dehydrogenase (LDH) release. Oxidative stress was assessed by determining reduced glutathione (GSH) level and lipid peroxidation as indicated by thiobarbituric acid reactive substances (TBARS) production. Exposure of colonocytes to ACN (1.0mM) for 60min caused nearly a 50% decrease in cell viability and induced a 2.5-fold increase of LDH leakage. In the same experiment, ACN caused a significant decrease in cellular GSH content as well as a significant enhancement of TBARS accumulation. These toxic responses to ACN were dependent on both concentration and duration of exposure to ACN. There was a good correlation between LDH release and TBARS formation (r(2)=0.97, p<0.05). Treatment of colonocytes with GSH, N-acetyl-l-cysteine (NAC) or dithiothreitol (DDT) prior to exposure to ACN afforded different degrees of protection as indicated by significant decrease in the LDH leakage and TBARS formation as compared to ACN alone-treated cells. Also, pretreatment of colonocytes with the antioxidant enzyme superoxide dismutase (SOD) or catalase (CAT) significantly inhibited LDH leakage and TBARS production. Preincubation with dimethyl sulfoxide (DMSO), a hydroxyl radical scavenger or desferroxiamine (DFO), an iron chelator, diminished ACN-induced LDH leakage and TBARS generation. Our results suggest that ACN has a potential cytotoxic effect in rat colonocytes; and thiol group-donors, antioxidant enzymes, hydroxyl radical scavengers and iron chelators can play an important role against ACN-induced colonotoxicity.     

儿茶素和漆黄素对柠檬酸铁诱导大鼠原代肝细胞损伤的保护作用

目的　分别从中药苞蔷薇根和叶中提取获得 (+ ) 儿茶素 (CAT )和漆黄素 (FIS)两种黄酮 ,研究其对柠檬酸铁致肝细胞铁超载的药理作用。方法　原代大鼠肝细胞加入 50μmol·L- 1柠檬酸铁诱导肝细胞铁超载。致损伤前加入CAT及FIS或损伤后 2 4h ,加入CAT及FIS ,继续培养 2 4h后 ,测定培养液中的超氧化物歧化酶 (SOD )、乳酸脱氢酶(LDH )、谷草转氨酶 (AST)、谷丙转氨酶 (ALT)活性和白蛋白 (ALB)含量。培养 48h后 ,用MTT法测肝细胞的存活率。结果　在致损伤前加入CAT或FIS ,仅见CAT 5× 10 - 5mol·L- 1和FIS 5× 10 - 5 mol·L- 1对大鼠原代肝细胞的恢复作用与模型对照组相比差异有显著性 (P <0 0 5或P <0 0 1)。在致损伤后加入CAT或FIS ,可见CAT 5× 10 - 5 、5× 10 - 6 、5× 10 - 7mol·L- 1组 ,FIS 5× 10 - 5 、5× 10 - 6 、5×10 - 7mol·L- 1组均可不同程度的提高培养液中的SOD活性 ,降低LDH水平 ,抑制ALT、AST的释放 ,提高ALB的含量 ,提高培养液中肝细胞的存活率 ,与模型对照组比 ,差异有显著性 (P <0 0 1,P <0 0 5)。FIS 5× 10 - 5 mol·L- 1组与正常对照组相比 ,对于提高SOD活性、ALB含量 ,差异无显著性 (P >0 0 5)。结论　CAT、FIS对柠檬酸铁所致大鼠原代肝细胞的损伤具有保护作用     

Prevention of lipopolysaccharide-induced injury by 3,5-dicaffeoylquinic acid in endothelial cells

Aim： To investigate the effect of 3,5-dicaffeoylquinic acid （3,5-diCQA） on lipopolysaccharide （LPS）-induced injury in human dermal microvascular endothelial cells （HMEC-1）. Methods： The anti-oxidant effect was detected using the malondialdehyde （MDA） assay in a rat liver microsome model of lipid peroxidation. Cell viability was analyzed using the 3-（4,5-dimethylthiazol-2-yl）-2,5 diphenyltetrazolium bromide assay. Cell lipid peroxide injury was measured by lactate dehydrogenase （LDH） release. Apoptotic cells were detected by flow cytometry, and confirmed by DNA fragmentation analysis. Caspase-3 activity was measured using a specific assay kit. The level of intracellular reactive oxygen species （ROS） was determined by flow cytometry with a 2,7-dichlorodihydro-fluorescein diacetate fluorescence probe. Results： The exposure of microsomes to ascorbate-Fe^2＋ resulted in lipoperoxidation according to an increase in the level of MDA. MDA formation decreased in a dose-dependent manner on treatment with 5, 10, or 50 μmol/L 3,5-diCQA. Treatment with LPS for 16 h resulted in a 60% decrease in cell viability and an increase in LDH release from 47.6% to 61.5%. DNA laddering was observed by agarose gel electrophoresis. The level of apoptotic cells peaked at 27% after treatment with LPS for 12 h. Following treatment with LPS for 12 h, intracellular ROS and caspase-3 activity increased. Pretreatment with 3,5-diCQA at 5, 10, or 50 μmol/L for 1 h attenuated LPS-mediated endothelial cell injury. The anti-apoptotic action of 3,5-diCQA was partially dependent on its capacity for anti-oxidation and the suppression of caspase-3 activity. Conclusion： 3,5-diCQA displays anti-oxidative and anti-apoptotic activity in HMEC-1 due to scavenging of intracellular ROS induced by LPS, and the suppression of caspase-3 activity.     

Isolated hepatocytes as a model for aging effects on hepatotoxicity: studies with allyl alcohol

The influence of aging on the toxicity of allyl alcohol was studied in hepatocytes isolated from male Fischer 344 rats. Initial values for trypan blue uptake, lactate dehydrogenase (LDH) release, alanine aminotransferase release, and glutathione content were similar in cells isolated from rats aged 5, 15, or 26 months. Incubation with 0.1 to 0.8 mM allyl alcohol resulted in a dose- and time-dependent loss of viability. Dose-effect and time-effect curves were displaced to the left with hepatocytes isolated from old rats compared with those from young rats. Intermediate values were found for hepatocytes of middle-aged rats. Inhibition by pyrazole of allyl alcohol-induced LDH release from hepatocytes also was affected by age. Total protection was observed for cells from young rats whereas no protection was found for those of old rats. Cells from middle-aged rats were between the extremes. The results demonstrate that isolated hepatocytes can be used to study age-related differences in hepatotoxicity.     

Mobilization of iron from cells by hydroxyquinoline-based chelators

With the aim of identifying an iron (Fe) chelator which is effective at mobilizing intracellular Fe, two novel ligands were synthesized and tested. Hydroxyquinoline is known to possess a high affinity for Fe and was thus chosen as the Fe binding motif for the hexadentate chelators, C1 (2,2'-[ethane-1,2-diylbis(iminomethylene)]diquinolin-8-ol) and C2 (2,2'-[cyclohexane-1,2-diylbis(iminomethylene)]diquinolin-8-ol). Both chelators are lipophilic, with Fe3+ complexes slightly more hydrophilic than the free ligands. C1 and C2 were equally toxic to K562 cells, and partial protection was afforded by supplementing the culture medium with human holotransferrin, suggesting that some of the toxicity of the ligands is due to cellular Fe depletion. Micromolar concentrations of both ligands effectively mobilized 59Fe from reticulocytes and K562 cells. In reticulocytes, 50 microM C1 caused the release of 60% of the cells' initial 59Fe uptake after a 4h incubation. Under the same conditions, C2 revealed a release of 50% of the 59Fe. Overall, both ligands merit in vivo study for oral activity. Their effectiveness at low concentrations makes them candidates for therapeutic use.     

Enhancement of methylene blue-induced cytotoxicity in human brain tumor cells by an iron chelator,deferoxamine

Previously, we have reported that methylene blue (MB) induces cytotoxicity in human brain tumor cells through the generation of free radicals. In this study the effect of deferoxamine (DFO), an iron chelator, on MB-induced cytotoxicity was investigated using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells as model cellular systems. The cytotoxic effect of MB was potentiated by DFO. The potentiation effect of DFO was significantly blocked by either stoichiometric amounts of ferric ion, various antioxidants, hydroxyl radical scavengers or intracellular Ca²⁺ release blockers. These results suggest that hydroxyl radical DFO. These results further suggest that the combined treatment with MB and DFO may be useful for the therapeutical applications of human brain tumors.     

Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine.

The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG:GSH ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2',7'-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release, and trypan blue uptake were determined from 0 to 12 h. After glutathione depletion at 3 h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12 h. At 1.5 h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9 h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6 h, with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1 h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2 h after AAP exposure prevented further deterioration of XTT reduction at 3 h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.     

o-hydroxyphenylacetaldehyde is a hepatotoxic metabolite of coumarin.

o-Hydroxyphenylacetaldehyde (o-HPA), the product of coumarin 3, 4-epoxide, was synthesized and its contribution to the hepatotoxic effects of coumarin in the rat was determined. The relative toxicity of coumarin and o-HPA were initially assessed in Chinese hamster ovary K1 (CHO K1) cells, a cell line that does not contain cytochrome P450. In CHO K1 cells, o-HPA-mediated toxicity greatly exceeded that of coumarin. CHO K1 cell viability, determined via the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), was decreased by 95 and 6% in cultures containing o-HPA and coumarin (4 mM), respectively. Coumarin and o-HPA were then incubated in metabolically competent primary rat hepatocyte cultures. Cell viability was determined via the reduction of MTT, and lactic dehydrogenase (LDH) release was used as a measure of cytotoxicity. Concentration-dependent decreases in cell viability and increased LDH release were observed using 0.2 to 0.8 mM o-HPA and coumarin, with coumarin being consistently less toxic than o-HPA. Cell viability was decreased by 11 and 50% at 0.5 mM coumarin or o-HPA, respectively. Hepatocyte LDH release increased 5-fold after a 6-h exposure to 0.8 mM o-HPA, corresponding to a greater than 90% loss of cell viability in these cultures. In contrast, 0.8 mM coumarin decreased cell viability by 60%, an effect likely due to the conversion of coumarin to coumarin epoxide and o-HPA. Furthermore, 3-hydroxycoumarin (0.8 mM), which is not a product of coumarin epoxidation, had no effect on cell viability or hepatocellular LDH release. These studies demonstrate that metabolically active rat hepatocytes convert coumarin into toxic metabolites, and strongly suggest that o-HPA and coumarin 3, 4-epoxide mediate the toxicity of coumarin in rodents in vivo.     

Protective effect of deferoxamine on chromium(VI)-induced DNA single-strand breaks, cytotoxicity, and lipid peroxidation in primary cultures of rat hepatocytes

Incubation of primary cultures of rat hepatocytes with K₂Cr₂O₇ and deferoxamine (DFO), an iron chelator, resulted in a marked decrease in cellular levels of DNA single-strand breaks caused by K₂Cr₂O₇. Cellular treatment with DFO also suppressed both dichromate-induced cytotoxicity – evaluated by the leakage of lactate dehydrogenase, and lipid peroxidation – as monitored by malondialdehyde formation. In addition, treatment with DFO attenuated the suppression of the levels of vitamin E and C as well as the inhibition of alkaline phosphatase and glutathione peroxidase activity attributed to K₂Cr₂O₇. However, DFO had no influence on the cellular level of glutathione or the activity of glutathione reductase and superoxide dismutase suppressed by dichromate. Under the same experimental conditions, cellular uptake and distribution of chromium were not affected by DFO. These results indicate that DFO protects cells from chromium(VI)-induced DNA strand breaks, cytotoxicity, lipid peroxidation, vitamin E and C depression, and glutathione peroxidase inhibition. The role of antioxidants in chromium(VI)-induced cytotoxicity, DNA breaks, and lipid peroxidation is discussed. Received: 17 September 1996 / Accepted: 25 November 1996