Genotoxicity of chloroquine in rat liver cells: Protective role of free radical scavengers

The genotoxic effect of chloroquine (CQ), a 4-aminoquinoline antimalarial drug was investigated in rat liver cells using the alkaline comet assay. Chloroquine (0–1000 μmol/L) significantly increased DNA strand breaks of rat liver cells dose-dependently. Rat liver cells exposed to CQ (100–500 μmol/L) and treated with endonuclease III and formamidopyrimidine-DNA glycosylase, the bacterial DNA repair enzymes that recognize oxidized pyrimidine and purine, respectively, showed greater DNA damage than those not treated with the enzymes, providing evidence that CQ induced oxidation of purines and pyrimidines. Treatment of cells with 5 mmol/L N-acetylcysteine, an intracellular reactive oxygen species (ROS) scavenger, and 100 μmol/L and 250 μmol/L deferoxamine, an established iron chelator, significantly decreased the CQ-induced strand breaks and base oxidation, respectively. Similarly, the formation of DNA strand breaks and oxidized bases was prevented by vitamin C (10 μmol/L) (a water-soluble antioxidant), quercetin (50 μmol/L) (an antioxidant flavonoid), and kolaviron (30 μmol/L and 90 μmol/L) (an antioxidant and a liver hepatoprotective phytochemical). The results indicate that the genotoxicity of CQ in rat liver cells might involve ROS and that free radical scavengers may elicit protective effects in these cells.     

GANRA-5 protects both cultured cells and mice from various radiation types by functioning as a free radical scavenger

Abstract

The radio-protective effects of the oxazolone derivative chemical compound 4-(4-methoxy-3-methoxyphenyl-methyl)-2-phenyl- 5(4H)-oxazolone (GANRA-5) against different types of radiation including X-rays, carbon ion beams, microwaves and ultraviolet light (UV) were studied. Cell proliferation/cytotoxicity assay and colony-forming assay were conducted to evaluate the toxicity of GANRA-5. To test its influence on the induction of double-stranded break (DSB) formation and genomic instability, γH2AX focus-forming assay as well as cytokinesis-block micronucleus assay was utilized. Our results indicate that GANRA-5 exhibits low toxicity, while providing high radio-protective effects for MRC-5 cells against different types of radiation. We also found that GANRA-5 acts as a free radical scavenger. Our animal studies provided evidence that GANRA-5 significantly increases the survival rate of mice after X-ray irradiation. Analyses of hemogram, visceral index and detection of superoxide dismutase (SOD) and malondialdehyde (MDA) in the viscera indicate both low toxicity of GANRA-5, combined with its ability to shield radiation risk. In conclusion, our results suggest that GANRA-5 has the potential to be used as a safe and efficient radio-protectant.     

Genotoxicity of idarubicin and its modulation by vitamins C and E and amifostine

Idarubicin is an anthracycline anticancer drug used in haematological malignancies. The main side effect of idarubicin is free-radicals based cardiotoxicity. Using the comet assay we showed that the drug at concentrations from the range 0.001 to 10 microM induced DNA damage in normal human lymphocytes, measured as the increase in percentage of DNA in the tail (% tail DNA). The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Recognised cell protector, amifostine at 14 mM decreased the mean % tail DNA of the cells exposed to idarubicin at all tested concentrations of the drug. So did vitamin C at 10 microM, but vitamin E (alpha-tocopherol) at 50 microM increased the % tail DNA. Lymphocytes exposed to idarubicin and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Pretreatment of lymphocytes with nitrone spin traps, N-tert-butyl-alpha-phenylnitrone and alpha-(4-pyridil-1-oxide)-N-tert-butylnitrone decreased the extent of DNA damage evoked by idarubicin. To discuss the influence of vitamins and amifostine in cancer cells we used also murine pro-B lymphoid BaF3 transformed with BCR/ABL oncogene. These cells can be treated as model cells of human acute myelogenous leukemia. The response of these cells to vitamin E was quantitatively the same as human lymphocytes. However, vitamin C did not exert any effect on DNA damage and amifostine, in spite to normal lymphocytes, potentiated this effect. The results obtained suggest that reactive oxygen species, including free radicals, may be involved in the formation of DNA lesions induced by idarubicin. The drug can also methylate DNA bases. Our results indicate that not only cardiotoxicity but also genotoxicity and in consequence induction of secondary malignancies should be taken into account as diverse side effects of idarubicin. Amifostine may potentate DNA-damage effect of idarubicin in cancer cells and decrease this effect in normal cells. Vitamin C can be considered as protective agents against DNA damage in normal cells in persons receiving idarubicin-based chemotherapy, but the use of vitamin E cannot be recommended and at least needs further research.     

Cisplatin-evoked DNA fragmentation in normal and cancer cells and its modulation by free radical scavengers and the tyrosine kinase inhibitor STI571

Cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP) is well studied anticancer drug, whose activity can be attributed to its ability to form adducts with DNA, but this drug can also form DNA-damaging free radicals, however this mechanism of cisplatin action is far less explored. Using the comet assay we studied cisplatin-induced DNA damage in the presence of spin traps: DMPO and PBN, Vitamins A, C and E as well as the tyrosine kinases inhibitor STI571 in normal human lymphocytes and leukemic K562 cells. The latter cells express the BCR/ABL fusion protein, which can be a target of the tyrosine kinase inhibitor STI571. A 20 h incubation with cisplatin at 1-10 microM induced DNA cross-links and DNA fragmentation in normal and cancer cells. Cisplatin could induce intra- and interstrand DNA-DNA cross-links as well as DNA-protein cross-links. DNA damage in K562 cells was more pronounced than in normal lymphocytes. In the presence of spin traps and vitamins we noticed a decrease in the DNA fragmentation in both cell types. Co-treatment of the lymphocytes with cisplatin at 10 microM and STI571 at 0.25 microg/ml caused an increase of DNA fragmentation in comparison with DNA fragmentation induced by cisplatin alone. In the case of K562 cells, an increase of DNA fragmentation was observed after treatment with cisplatin at 1 microM. Our results indicate that the free radicals scavengers could decrease DNA fragmentation induced by cisplatin in the normal and cancer cells, but probably they have no effect on DNA cross-linking induced by the drug. The results obtained with the BCR/ABL inhibitor suggest that K562 cells could be more sensitive towards co-treatment of cisplatin and STI571. Our results suggest also that aside from the BCR/ABL other factors such as p53 level, signal transduction pathways and DNA repair processes can be responsible for the increased sensitivity of K562 cells to cisplatin compared with normal lymphocytes.     

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 microM) and NBQX (10 microM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase--oligomycin, an uncoupler of oxidative phosphorylation--CCCP, a chelator of intracellular Ca2+--BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport--ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+](i). Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.     

In vivo reduction of chromium (VI) and its related free radical generation

Chromium (VI) compounds are widely recognized as human carcinogens. Extensive studies in vitro and in model systems indicate that the reactive intermediate, Cr (V), generated by cellular reduction of Cr (VI), is likely the candidate for the ultimate carcinogenic form of chromium compounds. Here we review our current understanding of the in vivo reduction of Cr (VI) and its related free radical generation. Our results demonstrate that Cr (V) is indeed generated from the reduction of Cr (VI) in vivo, and that Cr (V) thus formed can mediate the generation of free radicals. Cr (V) and its related free radicals are very likely to be involved in the mechanism of Cr (VI)induced toxicity and carcinogenesis. These studies also illustrate that in vivo EPR spectroscopy and magnetic resonance imaging can be very useful and powerful tools for studying paramagnetic metal ions in chemical and biochemical reactions occurring in intact animals.     

Lipid peroxidation in hepatocyte cell cultures: Modulation by free radical scavengers and iron

Summary Rat hepatocytes were isolated and then maintained in serum-free cell culture medium for 24 h. The amount of malondialdehyde (MDA) accumulated in the medium was assayed and used as a measure of lipid peroxidation. The acivity of lactate dehydrogenase (LDH) and urea were measured in the medium and used as indicators of hepatocellular viability and function. The effects of iron; desferrioxamine mesylate (Desferal), an iron chelator; and mannitol, a hydroxyl free radical scavenger were investigated. The addition of iron, Fe² resulted in a three-fold increase in the levels of MDA. Desferal inhibited the production of MDA and blocked the effect of Fe²⁺. Neither iron nor Desferal had any effect on LDH or urea levels. Mannitol had no effect on MDA or urea production, but caused a 4 to 8-fold increase in the LDH levels in the medium. The results show that iron is involved in the mechanism of lipid peroxidation in hepatocyte cultures but suggest that as a pathologic event lipid peroxidation is not expressed in terms of viability during the first 24 h of hepatocyte culture.     

Reactive oxygen species induce signals that lead to apoptotic DNA degradation in primary CD4+ T cells

Reactive oxygen species are toxic to cells but they may also have active roles in transducing apoptotic events. To study the role of reactive oxygen species in growth factor depletion induced apoptosis of human primary CD4+ T cells, we used a synthetic manganese porphyrin superoxide dismutase mimetic to detoxify superoxide anions formed during apoptosis. Apoptosis of primary CD4+ T cells was characterized by generation of superoxide anions, plasma membrane phosphatidyl-serine translocation, loss of mitochondrial membrane potential, activation of caspase 3, condensation of chromatin, as well as DNA degradation. The detoxification of superoxide anions did not influence plasma membrane phosphatidyl-serine translocation, or chromatin condensation, and only marginally inhibited the loss of mitochondrial membrane potential and the formation of DNA strand breaks. In contrast, the detoxification of superoxide anions significantly reduced caspase 3 activity and almost completely inhibited the apoptotic decrease in total cellular DNA content as measured by propidium iodide staining. Our results indicate that reactive oxygen anions induce signals leading to efficient DNA degradation after the initial formation of DNA strand breaks. Thus, reactive oxygen anions have active roles in signaling that lead to the apoptotic events.     

RAS/MAPK signaling functions in oxidative stress,DNA damage response and cancer progression

Mitogen-activated protein kinase (MAPK) signaling pathways organize a great constitution network that regulates several physiological processes, like cell growth, differentiation, and apoptotic cell death. Due to the crucial importance of this signaling pathway, dysregulation of the MAPK signaling cascades is involved in the pathogenesis of various human cancer types. Oxidative stress and DNA damage are two important factors which in common lead to carcinogenesis through dysregulation of this signaling pathway. Reactive oxygen species (ROS) are a common subproduct of oxidative energy metabolism and are considered to be a significant physiological modulator of several intracellular signaling pathways including the MAPK pathway. Studies demonstrated that the MAP kinases extracellular signal-regulated kinase (ERK) 1/2 and p38 were activated in response to oxidative stress. In addition, DNA damage is a partly common circumstance in cell life and may result in mutation, cancer, and even cell death. Recently, accumulating evidence illustrated that the MEK/ERK pathway is associated with the suitable performance of cellular DNA damage response (DDR), the main pathway of tumor suppression. During DDR, the MEK/ERK pathway is regularly activated, which contributes to the appropriate activation of DDR checkpoints to inhibit cell division. Therefore, the aim of this review is to comprehensively discuss the critical function of MAPK signaling in oxidative stress, DNA damage, and cancer progression.     

Acute effects of blood flow restriction on exercise-induced free radical production in young and healthy subjects

The main purpose of this study was to investigate the acute local and systemic effects of low-load resistance exercise (30% 1RM) with partial vascular occlusion on exercise-induced free radical production and to compare these effects with other established training methods.

Fifteen young and healthy males (25?±?3 years) performed the following four sessions in a counterbalanced order on separate days: low-load resistance exercise (LI: 30% 1RM), low-load resistance exercise with blood flow restriction (LIBR: 30% 1RM), high-load resistance exercise (HI: 80% 1RM) and an additional session without exercise but blood flow restriction only (BR). Blood samples were obtained 15?min prior to and immediately after exercise sessions from the right index finger and first toe. To analyze concentrations of reactive oxygen species (ROS), electron paramagnetic resonance (EPR) spectroscopy was used. Additionally, mitochondrial ROS production was measured by adding inhibitors of electron transport chain complex III. There was an increased systemic ROS generation after the LIBR session from 0.837?±?0.093 to 0.911?±?0.099?µmol/l/min. However, no local or systemic time?×?condition interaction was detected for ROS production. Regarding mitochondrial ROS production, results were not different between the conditions. Although the low-load resistance exercise session with partial vascular occlusion elicited systemic increases of ROS production, no significant changes were seen on a local level. We assume that this ROS concentration might not be high enough to induce cellular damage but is rather involved in muscle remodulation. However, this needs to be confirmed by future research.     

The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger

Free radicals are by-products of metabolism and exist in a homeostasis between generation and scavenging in vivo. Excessive free radicals cause various diseases, including nervous system diseases. Neuroglobin (Ngb), a nervous system-specific oxygen-binding protein, has been suggested to be a potential free radical scavenger in the nervous system in vivo; however, its underlying mechanism remains unclear. In this study, we investigated the antioxidant potential and free radical scavenging properties of recombinant human Ngb (rhNgb) in vitro. Interestingly, we found that the rhNgb protein itself has a direct and distinct antioxidant capacity and can efficiently scavenge a variety of free radicals, including the [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid)] (ABTS) cation, superoxide anion, hydrogen peroxide, and hydroxyl radical. The capacity of rhNgb to scavenge the superoxide anion and hydrogen peroxide was even comparable to that of vitamin C. In addition, rhNgb had Fe(2+) chelating activity but hemoglobin did not. In conclusion, our results indicated that the rhNgb protein itself has antioxidant and free radical scavenging activities, providing fundamental evidence for the neuroprotective function of Ngb. These data provide key information for the origin of the neuroprotective and physiological role of Ngb and will promote the treatment of reactive oxygen species (ROS)-related diseases using this novel oxygen-binding globin.     

Natural and newly synthesized hydroxy-1-aryl-isochromans: A class of potential antioxidants and radical scavengers

We investigated the antioxidant and radical scavenging activity of polyphenolic isochromans. To assess the relation between structure and scavenging properties the natural occurring 1-(3′-methoxy-4′-hydroxy)phenyl-6,7-dihydroxy-isochroman (ISO-3, three OH groups) was compared with three newly synthesized derivatives that differ in their degree of hydroxylation by substitution with methoxy-groups (ISO-4: four OH groups; ISO-2: two OH groups and ISO-0: fully methoxylated). We found that ISO-4 is a 2-fold better scavenger for the artificial radical 1,1-diphenyl-2-picrylhydrazyl (DPPH, 100?μM) with an EC₅₀=10.3?μM compared to the natural ISO-3 (EC₅₀=22.4?μM) and to ISO-2 (EC₅₀=25.1?μM), while ISO-0 did not react with DPPH. The scavenging capacity for superoxide enzymatically generated in a hypoxanthin-xanthinoxidase reaction was the highest for ISO-4 (EC₅₀=34.3?μM) compared to those of ISO-3 (EC₅₀=84.0?μM) and ISO-2 (EC₅₀=91.8?μM), while ISO-0 was inactive. In analogy, ISO-4 scavenged peroxynitrite (ONOO^?, EC₂₅=23.0?μM) more effective than ISO-3, ISO-2 and ISO-0.

When C6 rat glioma cells loaded with the reactive oxygen/nitrogen (ROS/RNS)-sensitive fluorochrome 2,7-dichlorodihydrofluorescein, were exposed to hydrogen peroxide, the lowest stress level as indicated by the fluorescence signal was detected when the cells were pretreated with ISO-4 or ISO-2 but to a much lesser extent with ISO-3, while ISO-0 did not show any effect. All tested hydroxyisochromans superceded the scavenging effect of trolox.

The excellent radical and ROS/RNS scavenging features of the hydroxy-1-aryl isochromans and their simple synthesis let these compounds appear to be interesting candidates for pharmaceutical interventions that protect against the deleterious action of ROS/RNS.     

Oxygen free radical induced damage during intestinal ischemia/reperfusion in normal and xanthine oxidase deficient rats

This study looks at the role of xanthine oxidase (XO) in ischemia/reperfusion (I/R) induced intestinal mucosal damage using normal and xanthine oxidase deficient rats. Tungstate feeding for 3 days depleted the intestinal mucosal XO by 80%. A ligated loop of the rat small intestine (both normal and XO-deficient) was subjected to 1 h of total ischemia followed by 5 min revascularisation. The ensuing mucosal damage was assessed by biochemical and histological studies. Ischemia or I/R increased the XO levels in normal rats without any change in XO-deficient rats. Myeloperoxidase (a neutrophil marker) level was increased in both group of rats but it was comparatively higher in the XO-deficient rats. Accumulation of peroxidation products such as malondialdehyde, conjugated diene and increased production of hydroxyl radicals by microsomes were seen after ischemia and I/R and were similar in normal and XO-deficient rats. Studies on other parameters of peroxidation showed a decrease in polyunsaturated fatty acids and alpha-tocopherol, an increase in cysteine and cystine levels after I/R and were similar in both normal and XO-deficient rats. Histological results indicated gross morphological changes in the intestinal mucosa due to ischemia and I/R, and the damage was more severe in XO-deficient rats. These observations suggest that oxygen-derived free radicals are involved in the intestinal mucosal damage during I/R and infiltrated neutrophils rather than XO may be the primary source of free radicals under these conditions.     

Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks

The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis.     

The GO system prevents ROS-induced mutagenesis and killing in Pseudomonas aeruginosa

Inactivation of the Pseudomonas aeruginosa mutM, mutY , or mutT gene conferred a 2.4-, 17.2-, or 38.1-fold increase in spontaneous mutation frequency, respectively. Importantly, the mutY and mutT strains each displayed a robust H₂O₂-induced mutation frequency. In addition, the mutM, mutY , and mutT mutations severely sensitized P. aeruginosa to killing by H₂O₂, suggesting that these gene products act to repair one or more cytotoxic lesions in P. aeruginosa . Nucleotide sequence analysis of a fragment of the rpoB gene from rifampicin resistant mutM -, mutY -, and, mutT -deficient strains was consistent with this conclusion. These findings are discussed in terms of possible roles for mutM, mutY , and mutT in contributing to survival and mutagenesis of P. aeruginosa colonizing the airways of cystic fibrosis patients.     

Retinol-induced elevation of ornithine decarboxylase activity in cultured rat Sertoli cells is attenuated by free radical scavenger and by iron chelator

We investigated retinol effects in ornithine decarboxylase activity in Sertoli cells. We also tested the hypothesis that free radical scavengers and iron chelators may attenuate the effect of retinol. Sertoli cells isolated from 15-day-old Wistar rats were previously cultured for 48 h and then treated with retinol by 24 h with or without mannitol (1 mM) or 1,10 phenanthroline (100 M). We measured ornithine decarboxylase and catalase activities and malondialdehyde concentrations in response to retinol treatment. In response to 7 M retinol treatment ornithine decarboxylase activity increased 30%. Retinol-induced ornithine decarboxylase activity was significantly decreased by addition of free radical scavenger (mannitol) or iron chelator (1,10 phenanthroline). In addition the same effect was observed in catalase increased activity and in malondialdehyde concentrations. These results suggest that retinol treatment induced ornithine decarboxylase and catalase activity and increased malondialdehyde concentration. These effects appear to be mediate by ROS.     

Nox4-dependent ROS modulation by amino endoperoxides to induce apoptosis in cancer cells

Tumor metastasis is the main cause of death in cancer patients. Anoikis resistance is one critical malefactor of metastatic cancer cells to resist current clinical chemotherapeutic treatments. Although endoperoxide-containing compounds have long been suggested as anticancer drugs, few have been clinically employed due to their instability, complex synthesis procedure or low tumor cell selectivity. Herein, we describe a one-pot strategy to synthesize novel amino endoperoxides and their derivatives with good yields and stabilities. In vitro cell-based assays revealed that 4 out of the 14 amino endoperoxides selectively induce metastatic breast carcinoma cells but not normal breast cells to undergo apoptosis, in a dose-dependent manner. Mechanistic studies showed that the most potent amino endoperoxide, 4-Me, is selective for cancer cells expressing a high level of Nox4. The anticancer effects are further shown to be associated with reduced O₂⁻:H₂O₂ ratio and increased ·OH level in the cancerous cells. Animal study showed that 4-Me impairs orthotopic breast tumor growth as well as tumor cell metastasis to lymph nodes. Altogether, our study suggests that anticancer strategies that focus on redox-based apoptosis induction in tumors are clinically viable.     

Time-related alterations of superoxide radical levels in diverse organs of bile duct-ligated rats

The time-related alterations of superoxide radical measured in vivo by employing an ultrasensitive fluorescent assay in the liver, intestine, kidney and brain of rats with experimentally induced obstructive jaundice was investigated. Eighteen rats were randomly divided into Group A, rats subjected to sham operation, and Group B, rats subjected to bile duct ligation (BDL). Three rats from each group were subsequently killed at different time points post-operatively (1, 5 and 10 days). As compared to sham-operated, BDL rats showed a gradual increase with time of superoxide radical in the intestine, liver, kidney and brain: for animals sacrificed on the 1^st, 5^th and 10^th day the increase was 45%, 50% and 96% in the liver, 76%, 81% and 118% in the intestine, 64%, 71% and 110% in the kidney and 76%, 95% and 142% in the brain, respectively. This study provides direct evidence of an early appearance of oxidative stress in diverse organs, implying a uniform systemic response to biliary obstruction and emphasizing the need of early bile flow restoration.