Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis

Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys₂), T cell proliferation is dependent of Cys₂. However, the exact roles of Cys₂ in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys₂ in order to proliferate. We activated purified naïve human CD4⁺ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys₂ and increased in parallel with increasing concentrations of Cys₂. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys₂ uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys₂ to proliferate and that this is partly explained by the fact that Cys₂ is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication.     

Cell surface-directed interaction of anthracyclines leads to cytotoxicity and nuclear factor kappaB activation but not apoptosis signaling

Anthracyclines are, above all, DNA intercalators, which induce genetic damage leading to cell death. However, increasing evidence firmly suggests that the underlying mechanism for anthracycline cytotoxicity is the induction of apoptosis through intracellular-mediated signaling pathways. Whether drug/DNA interaction is necessary for such apoptosis signaling is unknown. We investigated the cellular effects of the anthracyclines daunorubicin (DNR) and doxorubicin (DOX) using the myeloid leukemia cell line U937. By comparing free drug against agarose bead-immobilized drug iDNR and iDOX (which cannot accumulate within the cell), we observed that whereas both free and immobilized anthracyclines were cytotoxic, only the former induced apoptosis; the latter induced necrosis. Indeed, we did not observe ceramide generation, neutral sphingomyelinase activation, poly (ADP-ribose) polymerase cleavage, or other apoptotic events with iDNR or iDOX. However, both free and immobilized drug were similarly capable of triggering nuclear factor kappaB activation. These observations demonstrate that whereas activation of certain cellular signaling pathways can be achieved solely through membrane interaction, apoptosis signaling requires anthracycline internalization. These results also show that the initiation of cell survival pathways (illustrated by nuclear factor kappaB activation) is independent of intracellular drug/target interaction.     

Antioxidant enzymes in oligodendroglial brain tumors: association with proliferation, apoptotic activity and survival

Summary Purpose of the study was to investigate the relationship between antioxidant enzyme expression and clinicopathological features in oligodendroglial tumors. The expression of antioxidant enzymes and related proteins (AOEs),␣manganese superoxide dismutase (MnSOD), thioredoxin (Trx), thioredoxin reductase (TrxR) and gammaglutamylcysteine synthetase catalytic and regulatory subunits (GLCL-C and GLCL-R), was studied in 85 oligodendroglial tumors. The material included 71 primary (43 grade II and 28 grade III) and 14 recurrent (6 grade II and 8 grade III) tumors. Fifty-seven cases were pure oligodendrogliomas and 28 were mixed oligoastrocytomas. Immunoreactivity for MnSOD was found in 89%, Trx in 29%, TrxR in 76%, GLCL-C in 70% and GLCL-R in 68% of cases. Increased Trx expression was associated with higher tumor grade, cell proliferation and apoptosis (P=0.006, P=0.001 and P=0.003, Mann–Whitney test). Pure oligodendrogliomas showed more intense staining than oligoastrocytomas, especially for MnSOD (P=0.002, Mann–Whitney test). In the total series Trx was associated with poor prognosis in univariate survival analysis (P=0.0343, log-rank test) and furthermore in Cox multivariate analysis (P=0.009) along with age (P=0.002). The results suggest that the expression of Trx has a correlation to patient outcome and that there may be some association between AOEs, like MnSOD and Trx, and clinicopathological features of oligodendrogliomas.     

Distinct mechanisms of site-specific oxidative DNA damage by doxorubicin in the presence of copper(II) and NADPH-cytochrome P450 reductase

The anticancer mechanism of doxorubicin (DOX), an anthracycline antibiotic, is believed to involve DNA damage through topoisomerase II inhibition and free radical generation. The free radical generation may also participate in genotoxicity, as well as cardiotoxicity, in normal human cells. The present study showed that DOX generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), an indicator of oxidative DNA damage, in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. Since DOX has both p-quinone and p-hydroquinone residues, free radical generation can be initiated by either reduction or oxidation of DOX. To clarify whether the oxidized or reduced form is more important for DOX-induced H(2)O(2) generation, we investigated the site-specific DNA damage induced by DOX in the presence of Cu(II), in comparison with that in the presence of cytochrome P450 reductase, using (32)P-labeled DNA fragments. DOX caused DNA damage in the presence of Cu(II) or cytochrome P450 reductase. The degree of Cu(II)-mediated DNA damage, including 8-oxodG formation, was much greater than that of cytochrome P450 reductase-mediated DNA damage. DOX plus Cu(II) caused DNA damage specifically at guanine, thymine and cytosine residues, particularly at 5'-GG-3', 5'-GT-3' and 5'-TG-3' sequences. Scavenger experiments suggested the involvement of reactive species generated from H(2)O(2) and Cu(I). When cytochrome P450 reductase and NADPH were used instead of Cu(II), every nucleotide was uniformly damaged, suggesting the participation of.OH. We conclude that DOX may induce carcinostatic and genotoxic effects through oxidation of its p-hydroquinone moiety by metal ion rather than through p-quinone reduction by cytochrome P450 reductase.     

DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells

Purpose

Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. The anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene.

Methods

We assessed the induction of apoptosis (Sub-G₁) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay).

Results

Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. The effects of cosmomycin D were equivalent to those obtained with doxorubicin. The broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay.

Conclusions

Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.     

Single-strand DNA breaks induced by chromophore-modified anthracyclines in P388 leukemia cells

Single-strand DNA breaks induced by chromophore-modified anthracyclines related to doxorubicin (including 11-deoxydaunorubicin, 4-demethoxydaunorubicin, 4-demethoxy-11-deoxy-4'-epi-daunorubicin, 4-demethyl-6-O-methyldoxorubicin) in cultured P388 leukemia cells were determined by the filter alkaline elution method. The tested analogues differed markedly in their cytotoxic potency. In the range of cytotoxic concentrations, 11-deoxydaunorubicin produced single-strand DNA break frequency of the same order of magnitude as that produced by doxorubicin, while other derivatives caused much more marked damage on DNA than doxorubicin. Since DNA breaks were found to be protein associated, the type of DNA damage produced by all tested derivatives presumably resulted by action of DNA topoisomerases II, as proposed for doxorubicin and other intercalating agents. Although the "potent" (with respect to DNA damage) derivatives, except 4-demethyl-6-O-methyldoxorubicin, showed an increased cellular drug accumulation as compared to doxorubicin, this did not account for the marked differences in ability to damage DNA. 4-Demethyl-6-O-methyldoxorubicin was the most efficient derivative, producing DNA breaks in a lower range of cellular drug content. A striking biphasic dose-response curve was observed for the 4-demethoxy derivatives, suggesting a complex mechanism of interaction among drug, DNA, and enzyme. A lack of correlation was noted among DNA binding affinity, induction of strand breaks, and cytotoxic activity of these chromophore-modified derivatives. From these observations, it is suggested that multiple actions of anthracyclines at the DNA level are responsible for their cytotoxic activity, which is not simply related to inhibition of a specific DNA-dependent enzyme and/or function.     

Free radical generation from heterocyclic amines by cytochrome b5 reductase in the presence of NADH.

We previously reported findings that NADPH/cytochrome P450 reductase can generate superoxide anion radical (O2*-) from heterocyclic amines (HCA) and from many anticancer agents in vitro. Here we present more evidence in which O2*- is generated when recombinant human cytochrome b5 reductase (rh-Cytb5Rd) was incubated with HCAs such as IQ and MeIQ in the presence of NADH in vitro. This indicates that free radical generation by rh-Cytb5Rd in the presence of HCA may add new insight into the damage of DNA in addition to the previously known mechanism: interaction of activated HCA-intermediates to form DNA adduct.     

Doxorubicin inhibits human DNA topoisomerase I

Summary Purified human DNA topoisomerase I was assayed quantitatively by enzyme titrations with supercoiled pHC624 DNA in the presence of 0–2.0 m doxorubicin. Supercoiled and relaxed DNAs were resolved by agarose gel electrophoresis in the presence of ethidium bromide, and the pereentage of conversion of supercoiled DNA to relaxed DNA was quantified by scanning microdensitometry. The inhibition of DNA topoisomerase I activity was measured at varying concentrations of doxorubicin. Doxorubicin inhibited enzyme activity at an IC₅₀ value (the concentration required to inhibit 50% of the total activity) of 0.8 m. Similar inhibition was observed for daunomycin, a structurally related anthracycline antitumor drug. These results indicate that anthracyclines inhibit human DNA topoisomerase I activity at concentrations that cause DNA damage and cytotoxicity in vivo.This investigation was supported by grants from the Research Council of Rutgers University and the Charles and Johanna Busch Bequest     

Non-enzymatic and enzymatic activation of mitomycin C: Identification of a unique cytosolic activity

Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH: cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COSI cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductase₁ (NQO₁ or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQO₁ because of a lack of correlation between NQO₁ (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADH:cytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreductase) and its activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH). © 1996 Wiley-Liss, Inc.     

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

Clinical usefulness of doxorubicin (DOX) is limited by the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1) responsible for the active efflux of drugs out of resistant cells. Doxorubicin is a well-known bioreductive antitumour drug. Its ability to undergo a one-electron reduction by cellular oxidoreductases is related to the formation of an unstable semiquionone radical and followed by the production of reactive oxygen species. There is an increasing body of evidence that the activation of bioreductive drugs could result in the alkylation or crosslinking binding of DNA and lead to the significant increase in the cytotoxic activity against tumour cells. The aim of this study was to examine the role of reductive activation of DOX by the human liver NADPH cytochrome P450 reductase (CPR) in increasing its cytotoxic activity especially in regard to MDR tumour cells. It has been evidenced that, upon CPR catalysis, DOX underwent only the redox cycling (at low NADPH concentration) or a multistage chemical transformation (at high NADPH concentration). It was also found, using superoxide dismutase (SOD), that the first stage undergoing reductive activation according to the mechanism of the redox cycling had the key importance for the metabolic conversion of DOX. In the second part of this work, the ability of DOX to inhibit the growth of human promyelocytic-sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. Our assays showed that the presence of CPR catalysing only the redox cycling of DOX had no effect in increasing its cytotoxicity against sensitive and MDR tumour cells. In contrast, an important increase in cytotoxic activity of DOX after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells.     

A unique cytosolic activity related but distinct from NQO1 catalyses metabolic activation of mitomycin C

Mitomycin C (MMC) is a prototype bioreductive drug employed to treat a variety of cancers including head and neck cancer. Among the various enzymes, dicoumarol inhibitable cytosolic NAD(P)H:quinone oxidoreductase1 (NQO1) was shown to catalyse bioreductive activation of MMC leading to cross-linking of the DNA and cytotoxicity. However, the role of NQO1 in metabolic activation of MMC has been disputed. In this report, we present cellular and animal models to demonstrate that NQO1 may play only a minor role in metabolic activation of MMC. We further demonstrate that bioreductive activation of MMC is catalysed by a unique cytosolic activity which is related but distinct from NQO1. Chinese hamster ovary (CHO) cells were developed that permanently express higher levels of cDNA-derived NQO1. These cells showed significantly increased protection against menadione toxicity. However, they failed to demonstrate higher cytotoxicity due to exposure to MMC under oxygen (normal air) or hypoxia, as compared to the wild-type control CHO cells. Disruption of the NQO1 gene by homologous recombination generated NQO1-/- mice that do not express the NQO1 gene resulting in the loss of NQO1 protein and activity. The cytosolic fractions from liver and colon tissues of NQO1-/- mice showed similar amounts of DNA cross-linking upon exposure to MMC, as observed in NQO1+/+ mice. The unique cytosolic activity that activated MMC in cytosolic fractions of liver and colon tissues of NQO1-/- mice was designated as cytosolic MMC reductase. This activity, like NQO1, was inhibited by dicoumarol and immunologically related to NQO1.     

Maternal carriage of extended-spectrum beta-lactamase-producing Escherichia coli isolates in Argentina

Abstract

The aim of this study was to determine the prevalence of vaginal Escherichia coli colonization and perianal carriage of Enterobacteriaceae resistant to third generation cephalosporins in pregnant women. Vaginal and perianal samples from 259 pregnant women were studied. Vaginal swabs were inoculated onto MacConkey agar plates and perianal swabs were inoculated onto CHROMagar extended-spectrum beta-lactamase (ESBL) plates. The minimal inhibitory concentration of the isolates was determined using the Epsilometer test method. The phenotypic detection of ESBLs was performed by the combined disc method using cefotaxime versus cefotaxime plus clavulanate. The prevalence of vaginal E. coli colonization during pregnancy was 14·3%. The resistance rate to ampicillin, gentamicin, and cefotaxime was 48·6, 10·8, and 0·8%, respectively. Enterobacteriaceae resistant to third generation cephalosporins were recovered in 7·3% of all perianal specimens. Among them, 5·4% of pregnant women were colonized with E. coli ESBL-producer strains. The present study revealed that colonization with Enterobacteriaceae resistant to third generation cephalosporins is significant in pregnancy. ESBL-producing E. coli were the most prevalent organisms. Screening strategies designed to monitor for ESBL-producing E. coli could be useful in endemic areas to prevent perinatal transmission and the introduction of multiresistant strains to the maternity ward.     

Circumvention of confluence-dependent resistance in a human multi-drug-resistant colon-cancer cell line

Colorectal adenocarcinomas are inherently resistant to anthracyclines and other topoisomerase-II inhibitors. Resistance to doxorubicin of colon cancer cells (Caco2) depends on 2 main mechanisms. The first is typical multi-drug resistance, characterized by the mdr1 gene and its product the P170 membrane glycoprotein. P170 effluxes anthracyclines out of cancer cells and is antagonized in vitro by verapamil. The second mechanism, which develops when cell-culture density increases, we have designated confluence-dependent resistance. Confluence-dependent resistance depends on the reduced topoisomerase II content of the G₀/G₁,-phase cells which accumulate in the confluent population. We show here that short treatments of confluent Caco2 cells with slightly toxic concentrations of DNA-damaging agents (cisplatin, melphalan or mitomycin C) produced a transient accumulation of cells in S- and G₂/M-phases of the cell cycle. Concomitantly with the increase in the S-phase population, the topoisomerase II cellular level and the sensitivity of cells to doxorubicin were greatly enhanced. Over-coming confluence-dependent resistance through S-phase accumulation and inhibition of multi-drug resistance by verapamil were fully additive, and a nearly complete reversal of confluent Caco2 cells' resistance to doxorubicin was obtained when both strategies were combined. © 1995 Wiley-Liss, Inc.     

Homologous recombination repair is essential for repair of vosaroxin-induced DNA double-strand breaks

Vosaroxin (formerly voreloxin) is a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II, inducing site-selective double-strand breaks (DSB), G2 arrest and apoptosis. Objective responses and complete remissions were observed in phase 2 studies of vosaroxin in patients with solid and hematologic malignancies, and responses were seen in patients whose cancers were resistant to anthracyclines. The quinolone-based scaffold differentiates vosaroxin from the anthracyclines and anthracenediones, broadly used DNA intercalating topoisomerase II poisons. Here we report that vosaroxin induces a cell cycle specific pattern of DNA damage and repair that is distinct from the anthracycline, doxorubicin. Both drugs stall replication and preferentially induce DNA damage in replicating cells, with damage in G2 / M > S > G1. However, detectable replication fork collapse, as evidenced by DNA fragmentation and long tract recombination during S phase, is induced only by doxorubicin. Furthermore, vosaroxin induces less overall DNA fragmentation. Homologous recombination repair (HRR) is critical for recovery from DNA damage induced by both agents, identifying the potential to clinically exploit synthetic lethality.     

Deoxynivalenol induced mouse skin tumor initiation: Elucidation of molecular mechanisms in human HaCaT keratinocytes

Among food contaminants, mycotoxins are toxic to both human and animal health. Our prior studies suggest that Deoxynivalenol (DON), a mycotoxin, behaves as a tumor promoter by inducing edema, hyperplasia, ODC activity and activation of MAPK's in mouse skin. In this study, topical application of DON, 336 and 672 nmol significantly enhanced ROS levels, DNA damage and apoptosis with concomitant downregulation of Ki‐67, cyclin D, cyclin E, cyclin A and cyclin‐dependent kinases (CDK4 and CDK2) thereby resulting in tumor initiation in mouse skin. Further, the elucidation of molecular mechanisms of tumor initiation by DON (0.42–3.37 nmol/ml) in HaCaT keratinocytes, revealed (i) enhanced ROS generation with cell cycle phase arrest in G0/G1 phase, (ii) increase in levels of 8‐OxoG (6–24 hr) and γH2AX protein, (iii) significant enhancement in oxidative stress marker enzymes LPO, GSH, GR with concomitant decrease in antioxidant enzymes catalase, GPx, GST, SOD and mitochondrial membrane potential after DON (1.68 nmol) treatment, (iv) suppression of Nrf2 translocation to nucleus, enhanced phosphorylation with subsequent activation ERK1/2, p38 and JNK MAPK's following DON (1.68 nmol) treatment, (v) overexpression of c‐jun, c‐fos proteins, upregulation of Bax along with downregulation of Bcl‐2 proteins, (vi) increase in cytochrome‐c, caspase‐9, caspase‐3 and poly ADP ribose polymerase levels leads to apoptosis. Pretreatment of superoxide dismutase, mannitol and ethanol to HaCaT cells resulted in significant reduction in ROS levels and apoptosis indicating the role of superoxide and hydroxyl radicals in DON induced apoptosis as an early event and skin tumor initiation as a late event.     

Mechanism and pharmacological specificity of dUTPase-mediated protection from DNA damage and cytotoxicity in human tumor cells

Purpose: We have reported previously that the expression of E. coli dUTPase (dutE) can protect HT29 cells from 5-fluorodeoxyuridine (FdUrd)-induced DNA fragmentation and cytotoxicity. In the study reported here, we further characterized the ability of dutE expression in one HT29 clone, dutE7, to alter the effects of treatment with FdUrd and other thymidylate synthase (TS) inhibitors. In addition, we developed two HuTu80 dutE-expressing clones using a pLNCX-dutE retroviral construct and tested their sensitivity to FdUrd-induced DNA fragmentation and cytotoxicity. Methods: Both a dutE retroviral expression system and a dutE antibody were developed to facilitate the generation and screening of dutE-expressing clones. HT29 and HuTu80 clones expressing dutE were tested for drug-induced DNA damage with either alkaline elution or pulsed field gel electrophoresis and drug-induced loss of clonogenicity. Results: Following a 24-h treatment with 100 μM CB3717 or 500 nM methotrexate (MTX), dutE7 cells were significantly less sensitive to drug-induced loss of clonogenicity than con3 cells. DutE7 cells were also resistant to CB3717-induced DNA fragmentation at 24 h. However, following a 48-h treatment with CB3717 or MTX there was no difference in survival between con3 and dutE7 cells, even though DNA damage was still greatly attenuated in the dutE7 cell line. In addition, expression of dutE in two HuTu80 clones, 80  C and 80  K, did not protect these cells from FdUrd-induced DNA damage or cytotoxicity. Conclusions: We conclude that the role of uracil misincorporation and subsequent DNA damage in cytotoxicity induced by TS inhibitors, in HT29 cells, is time dependent, and that cytotoxicity caused by long-term exposure to these drugs is largely independent of resultant DNA damage, in this cell line. The inability of dutE to protect HuTu80 cells from FdUrd further suggests that the significance of uracil misincorporation resulting from TS inhibition varies among cell lines. Received: 19 August / Accepted: 16 December 1997     

DNA base damage induced by ionizing radiation recognized by Escherichia coli UvrABC nuclease but not Nth or Fpg proteins

Ionizing radiation and other free radical-generating systems induce a great variety of oxidative damage to DNA bases. The major known lesions are repaired by two well-characterized DNA glycosylases of Escherichia coli, endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), which have associated AP lyase activities. To detect and characterize potentially harmful oxidative base DNA lesions that may be repaired by alternative means, we exposed plasmid DNA to low doses γ-rays and removed the major base lesions by treatment with Nth and Fpg proteins. The closed circular DNA remaining after these treatments was used as a substrate of the UvrABC endonuclease complex from E. coli and as a template in a DNA polymerase arrest assay in vitro. The circular DNA contained lesions that were recognized and incised by the UvrABC nuclease and also lesions that blocked DNA polymerization in vitro. The blocking lesions were more abundant in DNA irradiated under nitrogen than under air and occurred mainly at tandem guanines; however, they were also frequent at tandem adenines and tandem cytosines. © 1996 Wiley-Liss, Inc.     

Lunasin peptide purified from Solanum nigrum L. protects DNA from oxidative damage by suppressing the generation of hydroxyl radical via blocking fenton reaction

Oxidative DNA damage is the most critical factor implicated in carcinogenesis and other disorders. However, the protective effects of lunasin against oxidative DNA damage have not yet reported. In this study, we report here the protective effect of lunasin purified from Solanum nigrum L. against oxidative DNA. Lunasin protected DNA from the oxidative damage induced by Fe²⁺ ion and hydroxyl radical. To better understand the mechanism for the protective effect of lunasin against DNA damage, the abilities to chelate Fe²⁺, scavenge the generated hydroxyl radical and block the generation of hydroxyl radical were evaluated. Although it did not scavenge generated hydroxyl radical, lunasin blocked the generation of hydroxyl radical by chelating Fe²⁺ ion.     

Modulation of the antioxidant activities in dox-sensitive and -resistant Friend leukemia cells. Effect of doxorubicin

Tumor cell resistance to anthracyclines has been associated with increased activity against free radicals. Here, we have investigated the direct effect of doxorubicin (DOX) in the modulation of glutathione level and antioxidant activities in DOX-sensitive and-resistant cells (288 fold). The glutathione level in untreated cells was 88% greater in resistant than in sensitive cells. The activities of the superoxide dismutase, glutathione -S-transferase and glutathione reductase were respectively 24, 15 and 38% higher in resistant cells than in their sensitive counterparts. In contrast, catalase and total glutathione peroxidase were reduced in resistant cells by 18 and 21% respectively. Moreover, the activity of selenium-dependent glutathione peroxidase was lowered by 47% in the resistant as compared to the sensitive cells. Exposure of sensitive or resistant cells to low doses of DOX did not affect these levels in either cell variant. It is concluded therefore that resistance to anthracyclines may not always be associated with an elevated level of intracellular antioxidant activity enzymes.