Depletion Of Endogenous Glutathione Research Articles

Engineering 2D Multienzyme-Mimicking Pyroptosis Inducers for Ultrasound-Augmented Catalytic Tumor Nanotherapy.

Overcoming apoptosis resistance is necessary to ensure an effective cancer treatment; however, it is currently very difficult to achieve. A desirable alternative for cancer treatment is the targeted activation of pyroptosis, a unique type of programmed cell death. However, the pyroptosis inducers that are efficient for cancer therapy are limited. This work reports the engineering of 2D NiCoOx nanosheets as inducers of the production of harmful reactive oxygen species (ROS), which promote intense cell pyroptosis, and that can be applied to ultrasound (US)-augmented catalytic tumor nanotherapy. The main therapeutic task is carried out by the 2D NiCoOx nanosheets, which have four multienzyme-mimicking activities: peroxidase- (POD), oxidase- (OXD), glutathione peroxidase- (GPx), and catalase- (CAT) mimicking activities. These activities induce the reversal of the hypoxic microenvironment, endogenous glutathione depletion, and a continuous ROS output. The ROS-induced pyroptosis process is carried out via the ROS-NLRP3-GSDMD pathway, and the exogenous US activation boosts the multienzyme-mimicking activities and favors the incremental ROS generation, thus inducing mitochondrial dysfunction. The anti-cancer experimental results support the dominance of NiCoOx nanosheet-induced pyroptosis. This work expands on the biomedical applications of engineering 2D materials for US-augmented catalytic breast cancer nanotherapy and deepens the understanding of the multienzyme activities of nanomaterials.

Nanoparticles for combined photo- and chemodynamic therapy ofcancer cells involving endogenous glutathione depletion.

Background: Reactive oxygen species (ROS) are powerful weapons for various anticancer therapies. However, high glutathione (GSH) levels in cancer cells can significantly reduce the efficacy of such therapies. Methods: In this study, pH-responsive fluorescein-encapsulated zeolitic imidazolate framework-8 nanoparticleswere synthesized for ROS-mediated combination therapy. Results: Upon blue light activation, fluorescein displayed a high singlet oxygen photogeneration ability for photodynamic therapy. Concurrently, accumulated Zn2+ from degraded zeolitic imidazolate framework-8 stimulated simultaneous ROS generation and GSH depletion, thereby successfully inducing chemodynamic therapy. This triggered a cascade of photo-physical and chemical processes culminating in the localized generation of ROS, ultimately breaking the intracellular redox equilibrium. Conclusion: This nanoformulation canpotentially be used for light-activated ROS-mediated therapy for the management of superficial tumors.

Glutathione for Food and Health Applications with Emphasis on Extraction, Identification, and Quantification Methods: A Review.

Glutathione is a naturally occurring compound that plays a crucial role in the cellular response to oxidative stress through its ability to quench free radicals, thus mitigating the risk of potential damage, including cell death. While glutathione is endogenously present in different plants and animal cells, their concentration varies considerably. The alteration in glutathione homeostasis can be used as a potential marker for human diseases. In the case of the depletion of endogenous glutathione, exogenous sources can be used to replenish the pool. To this end, both natural and synthetic glutathione can be used. However, the health benefit of glutathione from natural sources derived from fruits and vegetables is still debated. There is increasingly growing evidence of the potential health benefits of glutathione in different diseases; however, the determination and in situ quantification of endogenously produced glutathione remains a major challenge. For this reason, it has been difficult to understand the bioprocessing of exogenously delivered glutathione in vivo. The development of an in situ technique will also aid in the routine monitoring of glutathione as a biomarker for different oxidative stress-mediated diseases. Furthermore, an understanding of the in vivo bioprocessing of exogenously delivered glutathione will also aid the food industry both towards improving the longevity and profile of food products and the development of glutathione delivery products for long-term societal health benefits. In this review, we surveyed the natural plant-derived sources of glutathione, the identification and quantification of extracted glutathione from these sources, and the role of glutathione in the food industry and its effect on human health.

Ultrasound-Amplified Enzyodynamic Tumor Therapy by Perovskite Nanoenzyme-Enabled Cell Pyroptosis and Cascade Catalysis.

Overcoming apoptosis resistance to achieve efficient breast cancer treatment remains a challenge. The precise induction of another form of programmed cell death, pyroptosis, is an excellent alternative for treating cancer. Ultrasound (US)-enhanced enzyme dynamic (enzyodynamic) therapy is developed by employing LaFeO3 (LFO) perovskite nanocrystals as a substrate to increase the rate of deleterious reactive oxygen species (ROS) generation for intensive cell pyroptosis. LFO nanocrystals possess quadruple enzyme-mimicking activities, including oxidase-, peroxidase-, glutathione peroxidase-, and catalase-mimicking activities, which undertake the dominant therapeutic task through cascade catalytic reactions, including the reversal of hypoxic microenvironment, depletion of endogenous glutathione, and continuous output of ROS. US exogenous stimulation increases the transition rate of the intermediate complex to Fe (II) and favors incremental ROS production, by which the ROS burst-induced pyroptosis process is accomplished through the ROS-TXNIP-NLRP3-GSDMD pathway. Both in vitro and in vivo antineoplastic outcomes affirm the ascendancy of LFO nanozyme-induced pyroptosis. This work highlights the critical role of US coupled with nanocatalytic reactors in pyroptosis-dominant breast cancer treatment with the apoptosis resistance circumvention feature.

Nano-assembly of ruthenium(II) photosensitizers for endogenous glutathione depletion and enhanced two-photon photodynamic therapy.

Photodynamic therapy (PDT) is a promising noninvasive cancer treatment. PDT in the clinic faces several hurdles due to the unique tumor environment, a feature of which is high levels of glutathione (GSH). An excess amount of GSH consumes reactive oxygen species (ROS) generated by photosensitizers (PSs), reducing PDT efficiency. Herein, nano-photosensitizers (RuS1 NPs and RuS2 NPs) are reported. These consist of ruthenium complexes joined by disulfide bonds forming GSH sensitive polymer nanoparticles. The NPs achieve enhanced uptake compared to their constituent monomers. Inside cancer cells, high levels of GSH break the S-S bonds releasing PS molecules in the cell. The level of GSH is also then reduced leading to excellent PDT activity. Furthermore, RuS2 NPs functionalized with tumor targeting hyaluronic acid (HA@RuS2 NPs) assessed in vivo were highly effective with minimal side effects. To the best of our knowledge, RuS NPs are the first metal complex-based nano-assembled photosensitizers which exhibit enhanced specificity and consume endogenous GSH simultaneously, thus achieving excellent two-photon PDT efficiency in vitro and in vivo.

UVA Radiation Enhances Lomefloxacin-Mediated Cytotoxic, Growth-Inhibitory and Pro-Apoptotic Effect in Human Melanoma Cells through Excessive Reactive Oxygen Species Generation.

Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths. Thus, searching for new melanoma treatment options is an important field of study. The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation. C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system. The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells. The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells’ viability in a concentration-dependent manner. This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug. For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells’ viability by 50% (EC50) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively. Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells’ cell cycle distribution (including G2/M, S as well as Sub-G1-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation. This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy. The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.

Nabag (Zizyphus spina-christi) Extract Prevents Aberrant Crypt Foci Development in Colons of Azoxymethane-Treated Rats by Abrogating Oxidative Stress and inducing Apoptosis

Zizyphus spina-christi (ZSC) fruit is a rich source of bioactive compounds but any medicinal properties in chemoprevention of colon cancer have hitherto not been studied. The aim of the present study was to examine in vivo protective effects of ZSC water extract on colon carcinogenesis in azoxymethane (AOM)-treated rats. Our results showed that ZSC significantly reduced AOM-induced colonic aberrant crypt foci development and AOM-induced oxidative stress as indicated by restoration of endogenous glutathione depletion and abrogating the impairment of total antioxidant capacity. Caspase-3 cleavage, which has been considered as an apoptotic index, was almost undetectable in AOM-treated rats and ZSC exhibited pro-apoptotic effects evidenced by increased levels of cleaved caspase-3. In the studied model, our findings provide the first in vivo evidence that ZSC extract could inhibit the early stage of colon carcinogenesis by preventing oxidative stress and inducing apoptosis.

Altered expression of DJ-1 in the hippocampal cells following in vivo and in vitro neuronal damage induced by trimethyltin

Trimethyltin chloride (TMT) is known to produce neuronal damage in the dentate gyrus at least in part via oxidative stress. DJ-1, an oncogene product, is known to act as an anti-oxidant to prevent neuronal damage in dopaminergic neurons. The aim of this study was to determine the alterations in DJ-1 expression in the hippocampal cells of mice after in vivo and in vitro treatment with TMT. In naïve animals, DJ-1 was ubiquitously expressed in the hippocampus, in which the CA1 pyramidal cell layer and dentate granule cell layer had lower and higher levels of it, respectively. An intraperitoneal injection of TMT at the dose of 2.8 mg/kg produced DJ-1 up-regulation in the CA1 pyramidal cell layer, CA3 stratum lucidum, dentate molecular layer, and dentate hilus, but not in the dentate granule cell layer, on day 3-5 post-treatment. Temporary depletion of endogenous glutathione by the prior subcutaneous injection of 2-cyclohexen-1-one was effective in facilitating neuronal damage and DJ-1 up-regulation in the dentate gyrus induced by an intraperitoneal injection of TMT at the dose of 2.0 mg/kg. In primary cultures of mouse hippocampal cells, DJ-1 was present in neurons, but not in astrocytes. TMT treatment produced a dramatic expression of DJ-1 in the astrocytes in the cultures. Taken together, our data suggest that the DJ-1 protein is positively regulated in response to oxidative stress induced by TMT.

Suppression of phosphoenolpyruvate carboxykinase gene expression by reduced endogenous glutathione level

Glutathione is a small tripeptide to maintain overall reducing environment in vivo. Reduced endogenous glutathione level has been associated with aging, obesity and diabetes. In this study, the direct impact of low endogenous glutathione level on energy homeostasis is investigated at molecular level. Depletion of endogenous glutathione in rat primary hepatocytes by BSO, an inhibitor of γ-glutamylcysteine synthase, leads to reduced mRNA levels of several key enzymes in energy homeostasis, including phosphoenolpyruvate carboxylkinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. Supplementation of various reducing reagents, including N-acetylcysteine, DTT and glutathione, reverses the inhibitory effect of BSO on PEPCK mRNA level. The suppressive effect of BSO on PEPCK mRNA level is also reversed through co-treatment with either SB210290, a specific p38 kinase inhibitor, or wortmannin and LY294002, the well-established PI-3 kinase inhibitors, suggesting the involvement of these kinases in this process. These observations correlate well with the observations that reduced endogenous glutathione level and reduced gluconeogenesis coincide with aging process, implying a causal relationship between these changes in aged population. More importantly, this study suggests that endogenous glutathione level tightly associates with energy homeostasis at molecular level, identifying reduced endogenous glutathione level as a potential contributing factor to dysregulated metabolic processes in aging, obese and diabetic populations. In addition, the different responses of PEPCK expression to the alteration of endogenous glutathione level in rat hepatoma cells from primary hepatocytes raises caution against using established cell lines in examining the dysregulated metabolic process related to altered endogenous glutathione level.

In vivo depletion of endogenous glutathione facilitates trimethyltin-induced neuronal damage in the dentate gyrus of mice by enhancing oxidative stress

Acute treatment with trimethyltin chloride (TMT) produces neuronal damage in the hippocampal dentate gyrus of mice. We investigated the in vivo role of glutathione in mechanisms associated with TMT-induced neural cell damage in the hippocampus by examining mice depleted of endogenous glutathione by prior treatment with 2-cyclohexen-1-one (CHO). In the hippocampus of animals treated with CHO 1 h beforehand, a significant increase was seen in the number of single-stranded DNA-positive cells in the dentate gyrus when determined on day 2 after the injection of TMT at a dose of 2.0 mg/kg. Immunoblot analysis revealed that CHO treatment induced a significant increase in the phosphorylation of c-Jun N-terminal kinase in the cytosolic and nuclear fractions obtained from the dentate gyrus at 16 h after the TMT injection. There was also a concomitant increase in the level of phospho-c-Jun in the cytosol at 16 h after the injection. Expectedly, lipid peroxidation was increased by TMT in the hippocampus, and was enhanced by the CHO treatment. Moreover, CHO treatment facilitated behavioral changes induced by TMT. Taken together, our data indicate that TMT-induced neuronal damage is caused by activation of cell death signals induced at least in part by oxidative stress. We conclude that endogenous glutathione protectively regulates neuronal damage induced by TMT by attenuating oxidative stress.

The influence of depleted glutathione levels on the photodynamic action of zinc phthalocyanine in CHO K1 cells.

The current study focuses on any influence that depletion of endogenous glutathione in CHO K1 cells may have on the photodynamic action of zinc phthalocyanine (ZnPc). Two lasers--a HeNe laser, 632.5 nm, maximum power output 3.5 mW, and a Toshiba semiconducting laser, 670 nm, maximum power of 7 mW--were used. Chinese Hamster Ovary cells (CHO K1) were exposed to light, 2-10 J. Cellular reduced glutathione levels [GSH] were depressed prior to exposure to ZnPc and laser light, using buthionine sulphoximine, a potent inhibitor of gamma-glutamylcysteine synthetase. The influence of hypoxic intracellular conditions was studied by reduction of oxygen content of cells by 80% following purging of cell cultures with nitrogen. In well-aerated cells, doubling times are reduced by the photodynamic action of ZnPc by 29 +/- 6%, fig 2 (p = 0.01). Cells with lowered [GSH] do not show this effect (p = 0.1). When hypoxic cells are studied at normal [GSH], no photodynamic effect is observed (p = 0.1). When cell viability is studied, using the 670-nm laser, a photodynamic effect is observed, (80% fall from controls, p < 0.001), irrespective of the cellular [GSH] level for a single dose of 6 J. This effect is observed in cells with normal [GSH], for varied doses of 2 J and higher (63% fall at 2 J, p < 0.001). Lowered [GSH] was observed to depress the photodynamic effect of ZnPc when cell-doubling times were the endpoint. The photostimulating effect of ZnPc was similarly suppressed by hypoxic conditions. When cell viability was the endpoint, then a photodynamic effect of ZnPc was observed irrespective of the endogenous [GSH] values.

Gastroprotective effect of Neem ( Azadirachta indica) bark extract: Possible involvement of H +-K +-ATPase inhibition and scavenging of hydroxyl radical

The antisecretory and antiulcer effects of aqueous extract of Neem ( Azadirachta indica) bark have been studied along with its mechanism of action, standardisation and safety evaluation. The extract can dose dependently inhibit pylorus-ligation and drug (mercaptomethylimidazole)-induced acid secretion with ED 50 value of 2.7 and 2 mg Kg −1 b.w. respectively. It is highly potent in dose-dependently blocking gastric ulcer induced by restraint-cold stress and indomethacin with ED 50 value of 1.5 and 1.25 mg Kg −1 b.w. respectively. When compared, bark extract is equipotent to ranitidine but more potent than omeprazole in inhibiting pylorus-ligation induced acid secretion. In a stress ulcer model, it is more effective than ranitidine but almost equipotent to omeprazole. Bark extract inhibits H +-K +-ATPase activity in vitro in a concentration dependent manner similar to omeprazole. It offers gastroprotection against stress ulcer by significantly preventing adhered mucus and endogenous glutathione depletion. It prevents oxidative damage of the gastric mucosa by significantly blocking lipid peroxidation and by scavenging the endogenous hydroxyl radical ( OH)-the major causative factor for ulcer. The OH-mediated oxidative damage of human gastric mucosal DNA is also protected by the extract in vitro. Bark extract is more effective than melatonin, vitamin E, desferrioxamine and α-phenyl N-tert butylnitrone, the known antioxidants having antiulcer effect. Standardisation of the bioactive extract by high pressure liquid chromatography indicates that peak 1 of the chromatogram coincides with the major bioactive compound, a phenolic glycoside, isolated from the extract. The pharmacological effects of the bark extract are attributed to a phenolic glycoside which is apparently homogeneous by HPLC and which represents 10% of the raw bark extract. A single dose of 1g of raw extract per kg b.w. (mice) given in one day and application of 0.6g raw extract per kg b.w. per day by oral route over 15 days to a cumulative dose of 9g per kg was well tolerated and was below the LD 50. It is also well tolerated by rats with no significant adverse effect. It is concluded that Neem bark extract has therapeutic potential for the control of gastric hyperacidity and ulcer.

Dipyridamole protects cultured rat embryonic cortical neurons from neurotoxic insult

The effects of a clinically useful cardiovascular agent, dipyridamole, were examined in a rodent tissue culture model of neural protection. Dipyridamole effectively protected rat embryonic day 18 (E18) cortical neurons from either trophic deprivation or endogenous glutathione depletion by l‐buthionine (R,S) sulfoximine (BSO). Trophic deprivation was associated with an increase in intracellular oxidative stress, as determined by the increased fluorescence of dichloro, dihydrofluorescein (H2DCFDA). Dipyridamole's neural protection was time and concentration‐dependent (EC50 = 342 nm), and its continuous presence in the culture medium was required. Dipyridamole or exogenously added glutathione markedly decreased trophic deprivation induced H2DCFDA fluorescence, indicating a reduction in neuronal oxidative stress. These results demonstrate that dipyridamole protects primary neuronal cultures against either trophic or chemically mediated insults, and suggest that dipyridamole has a potent antioxidant ability that compensates for glutathione depletion in primary neuronal cells.

Sensitization by prolonged glutathione depletion of kainic acid to potentiate DNA binding of the nuclear transcription factor activator protein-1 in murine hippocampus

In four of four mice intracerebroventricularly injected with the inhibitor of glutathione synthesis l-buthionine-[S,R]-sulfoximine (BSO) 2 days before, an intraperitoneal injection of kainic acid (KA) invariably led to marked potentiation of DNA binding activity of the nuclear transcription factor activator protein-l (AP1) in the hippocampus at a dose which was ineffective in animals previously injected with vehicle alone. However, KA failed to potentiate binding in animals injected with BSO 1 day before. The intracerebroventricular injection of BSO induced marked and prolonged depletion of a total glutathione content in murine hippocampus for 1–2 days after administration. These results suggest that prolonged depletion of endogenous glutathione for a period longer than 1 day may lead to sensitization of KA signals to potentiate AP1 DNA binding in cell nuclei and thereby modulate de novo synthesis of particular proteins at the level of gene transcription in murine hippocampus.

Influence of intense sound exposure on glutathione synthesis in the cochlea

Previous studies have shown that depletion of endogenous glutathione (GSH) potentiates noise-induced hearing loss (NIHL), whereas replenishment of GSH attenuates NIHL (Yamasoba et al., Brain Res. 784 (1998) 82–90). Since these findings indicate an important role of GSH in protection from NIHL, we assessed the influence of intense sound exposure (broadband noise, 105 dB SPL, 5 h) on GSH and cysteine levels in the guinea pig cochlea using high performance liquid chromatography. GSH levels were significantly increased in the lateral wall 2 and 4 h post-exposure and returned to normal 6 h post-exposure. GSH levels in the sensory epithelium and modiolus did not show significant changes following noise. Cysteine levels were unchanged in any of the cochlear segments. For the cochlea as a whole, intense sound exposure did not significantly change GSH or cysteine levels throughout the 6-h measurement period post-exposure. These results indicate that GSH synthesis is markedly upregulated selectively in the lateral wall by noise exposure, presumably in response to the robust consumption of GSH, as it is utilized in scavenging reactive oxygen species.

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization.

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.

Effect of verapamil on hepatic ischemia/reperfusion injury

Alterations in cellular calcium homeostasis are a critical factor in the pathogenesis of hepatic ischemic damage and may mediate oxygen free radical injury during the reperfusion period. We investigated the effect of the calcium channel blocker verapamil on hepatic ischemia/reperfusion injury in normal rats and rats sensitized to oxidative injury by chemical depletion of the endogenous antioxidant glutathione. Forty-five minutes of complete hepatic ischemia followed by reperfusion caused an increase in the serum glutamic pyruvic transaminase (SGPT) level and a decline in the endogenous hepatic glutathione level but no increase in hepatic lipid peroxidation products. Chemical depletion of hepatic glutathione with diethylmaleate did not induce hepatocellular injury but augmented hepatic ischemia/reperfusion-induced SGPT release and promoted lipid peroxidation. Pretreatment with the calcium entry blocker verapamil protected against the ischemia/reperfusion-induced drop in hepatic glutathione but did not reduce SGPT release in normal rats. In rats sensitized to oxidative injury by chemical depletion of endogenous glutathione, the calcium channel blocker verapamil protected against ischemia/reperfusion-induced lipid peroxidation and reduced the release of SGPT. These findings indicate that the rat liver is protected against oxidative injury after short periods of total ischemia by its rich supply of endogenous glutathione. A beneficial effect of verapamil occurs only in rats sensitized to oxidative injury, suggesting that the calcium channel blocker protects against oxygen radical attack.

UVA damages depend on depletion of endogenous glutathione induced by UVA irradiation in cultured human fibroblasts(HF)

UVA damages depend on depletion of endogenous glutathione induced by UVA irradiation in cultured human fibroblasts(HF)

Influence of N-acetylcysteine on the antitumor activity of doxorubicin.

C ONSIDERABLE ATTENTION has been given to doxorubicin-induced cardiomyopathy since its recognition nearly 10 yr ago. Although the mechanism by which doxorubicin (DOX) produces cardiotoxicity is unknown, evidence suggests that it is linked to the effect of free radicals formed during the drug’s metabolism’ (Fig. 1). Handa and Sate* initially showed that DOX metabolism in a microsomal NADPH oxidase system yielded superoxide anion (0;;). Goodman and Hochstein3 demonstrated that, in the reduction-oxidation cycle of DOX 1 molecule in the quinone form could cycle to a semiquinone then back to the quinone form many times to generate molecules of superoxide anion and initiate lipid peroxidation. When Bachur et aL4 demonstrated that heart microsomes from rats were capable of supporting the generation of free radicals from DOX, it was suggested that a free radical oxidative mechanism might mediate the cardiotoxicity of this drug. This concept was supported by data from Myers et al.’ and Olson et al.6 demonstrating that the antioxidant vitamin E and the free radical scavenger cysteamine attenuated the acute cardiotoxicity of DOX in mice. The glutathione peroxidase pathway (Fig. 2), which detoxifies potentially damaging peroxides by reducing them to alcohols, also was shown to be an important modulator of DOX-induced cardiotoxicity.‘.’ Depletion of endogenous glutathione by diethyl maleate’.’ and inhibition of glutathione peroxidase by reduction of endogenous selenium stores (selenium is a required cofactor of glutathione peroxidase activity)‘dramatically potentiated DOX-induced cardiotoxicity in laboratory animals. Furthermore, pretreatment of rabbits with diethyl maleate to lower endogenous cardiac glutathione levels to less than 30% of basal values significantly potentiated the negative inotropic activity of DOX in isolated papillary muscles obtained from pretreated rabbits.’ Additionally, studies with Nacetylcysteine (NAC) have shown this agent to provide significant protection against DOXinduced cardiotoxicity.“,”

Conjugation and bioactivation of chlorotrifluoroethylene

Halogenated vinyl cysteine conjugates, which are potent, specific nephrotoxins, have been chemically synthesized from both chlorotrifluoroethylene (CTFE) and trichloroethylene (TCE) but have not been shown to be formed in a biological system. The vinyl cysteine conjugates are stable, but rapidly activated and degraded enzymatically with the release of pyruvate. Increased concentrations of pyruvate after incubation with CTFE may be an indirect index of the formation and bioactivation of a vinyl cysteine conjugate of CTFE. Incubation of CTFE with 10,000 x g rat liver and kidney supernatants resulted in 60 and 100 nmole/mg/hr increases in pyruvate concentration with concomitant depletion of endogenous glutathione. A renal cortex brush border preparation high in C-S lyase activity but deficient in cytosolic transferases did not generate pyruvate during incubation with CTFE. The evidence presented suggests that halogenated vinyl cysteine conjugates are formed in vitro by conjugation of the halogenated ethylene with glutathione which subsequently degrades to form the cysteine conjugate.