Comparison of two different cryopreservation protocols for freezing goat semen

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n = 12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300 mM Tris, 28 mM glucose, 95 mM citric acid 5% glycerol to a concentration of 200 × 10⁶ sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100 mg/mL skimmed milk powder and 27.75 mM glucose (without glycerol) to a concentration of 400 × 10⁶ sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200 × 10⁶ sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25 mL straws, held for 2 h at 4 °C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean ± SEM) were significantly lower (P < 0.05) in P1 as compared to P2 (47.50 ± 1.23% vs. 55.63 ± 1.72%; 80.04 ± 1.29% vs. 84.04 ± 1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P > 0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.     

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O₂. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506–2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3′-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.     

Induction of MHC class I-related chain B (MICB) by 5-aza-2'-deoxycytidine

5-Aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, exerts antitumor activity through induction of cell cycle arrest, apoptosis and DNA damage. In this study, we showed that MHC class I-related chain B (MICB), a ligand of the NKG2D receptor expressed by natural killer cells and activated CD8(+) T cells, was upregulated following 5-aza-dC treatment. The upregulation of MICB was accompanied by promoter DNA demethylation and DNA damage. Furthermore, the upregulation of MICB was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase. Our results suggest that promoter DNA demethylation, in combination with DNA damage, contribute to the upregulation of MICB induced by 5-aza-dC.     

Fuel smoke condensate induced DNA damage in human lymphocytes and protection by turmeric (Curcuma longa)

Summary Twigs-dry leaves smoke condensate (TDS) was investigated for its DNA damaging activity in human peripheral lymphocytes, by using a sensitive method, fluorescence analysis of DNA unwinding (FADU). An aqueous turmeric component (Aq.T) was studied as a protective agent. TDS at one to 100 folds dilution induced 55% DNA damage at 20 min, while 12-0-tetradecanoylphorbol-13-acetate (TPA) at 10 ng/ml induced only 25% damage. Aq.T at 300 ng/1 afforded 90% protection to DNA against TPA and 65% against TPA. The mechanism of Aq.T protection was investigated by using (i) inhibitors of arachidonate cascade, viz., indomethacin (28 M), NDGA (10 M), DBAP (36 M), (ii) antioxidant enzymes viz., CAT (0.2 U/l), SOD (0.6 U/1), (iii) antioxidants - BHA, curcumin (40 M), mixed gangliosides (20 nM) and protease inhibitor TLCK (100 M). These compounds offered the following extents of protection to DNA against TDS: indomethacin-40%, NDGA-83%, DBAP-70%, SOD-38%, CAT-40%, BHA-38%, curcumin-60%, mixed gangliosides-88%, TLCK-85%. Against TPA as clastogenic agent, the extents of protection were: indomethacin-73%, NDGA-32%, DBAP-72%, SOD-60%, CAT, BHA-negligible, curcumin-23%, mixed gangliosides - 60%, TLCK - 59%. These results indicate that (i) TDS and TPA induce DNA damage possibly by different mechanisms, (ii) Aq.T is a more effective protectant against TDS whereas it is on par with other inhibitors against TPA.Abbreviations FADU Fluoroscence Analysis of DNA Unwinding - Aq.T Aqueous component of turmeric - TDS Twigs-Dry leaves Smoke condensate - PBS Phosphate Buffered Saline, 20 mM, 150 mM NaCl, pH 7.4 - TPA 12-O-Tetradecanoyl Phorbol-13-Acetate - NDGA Nordihydroguaiaretic Acid - DBAP 2,4-Dibromo Acetophenone - CAT Catalase - SOD Superoxide Dismutase - BHA Butylated Hydroxyanisole - TLCK Tosyl Lysyl Chloromethyl Ketone - ROS Reactive Oxygen Species - PAH Polycyclic Aromatic Hydrocarbons - DMSO Dimethyl Sulfoxide - Buffer B 250 mM m-inositol, 10 mM sodium phosphate, 1 mM magnesium chloride, pH 7.3 - BSC Beedi Smoke Condensate - CSC Cigarette Smoke Condensate     

DNA polymerase zeta （pol ζ） in higher eukaryotes

Most current knowledge about DNA polymerase zeta （pol ζ） comes from studies of the enzyme in the budding yeast Saccharomyces cerevisiae, where pol ζ consists of a complex of the catalytic subunit Rev3 with Rev7, which associates with Revl. Most spontaneous and induced mutagenesis in yeast is dependent on these gene products, and yeast pol can mediate translesion DNA synthesis past some adducts in DNA templates. Study of the homologous gene products in higher eukaryotes is in a relatively early stage, but additional functions for the eukaryotic proteins are already apparent. Suppression of vertebrate REV3L function not only reduces induced point mutagenesis but also causes larger-scale genome instability by raising the frequency of spontaneous chromosome translocations. Disruption of Rev3L function is tolerated in Drosophila, Arabidopsis, and in vertebrate cell lines under some conditions, but is incompatible with mouse embryonic development. Functions for REV3L and REV7（MAD2B） in higher eukaryotes have been suggested not only in translesion DNA synthesis but also in some forms of homologous recombination, repair of interstrand DNA crosslinks, somatic hypermutation of immunoglobulin genes and cell-cycle control. This review discusses recent developments in these areas.     

Flavonoids suppresses the enhancing effect of beta-carotene on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A549 cells

This study investigated the individual and combined effects of beta-carotene with a common flavonoid (naringin, quercetin or rutin) on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-related carcinogen in human. A human lung cancer cell line, A549, was pre-incubated with beta-carotene, a flavonoid, or both for 1h followed by incubation with NNK for 4 h. Then, we determined DNA strand breaks and the level of 7-methylguanine (7-mGua), a product of NNK metabolism by cytochrome P450 (CYP). We showed that beta-carotene at 20 microM significantly enhanced NNK-induced DNA strand breaks and 7-mGua levels by 90% (p < 0.05) and 70% (p < 0.05), respectively, and that the effect of beta-carotene was associated with an increased metabolism of NNK by CYP because the concomitant addition of 1-aminobenzotriazole, a CYP inhibitor, with beta-carotene to cells strongly inhibited NNK-induced DNA strand breaks. In contrast to beta-carotene, incubation of cells with naringin, quercetin or rutin added at 23 microM led to significant inhibition of NNK-induced DNA strand breaks, and the effect was in the order of quercetin > naringin > rutin. However, these flavonoids did not significantly affect the level of 7-mGua induced by NNK. Co-incubation of beta-carotene with any of these flavonoids significantly inhibited the enhancing effect of beta-carotene on NNK-induced DNA strand breaks; the effects of flavonoids were dose-dependent and were also in the order of quercetin > naringin > rutin. Co-incubation of beta-carotene with any of these flavonoids also significantly inhibited the loss of beta-carotene incorporated into the cells, and the effects of the flavonoids were also in the order of quercetin > naringin > rutin. The protective effects of these flavonoids may be attributed to their antioxidant activities because they significantly decreased intracellular ROS, and the effects were also in the order of quercetin > naringin > rutin. These in vitro results suggest that a combination of beta-carotene with naringin, rutin, or quercetin may increase the safety of beta-carotene.     

Metformin (dimethyl-biguanide) induced DNA damage in mammalian cells

Metformin (dimethyl-biguanide) is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays) and in mice (micronucleus assays). Concentrations of 114.4 μg/mL and 572 μg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 μg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.     

Reduction of Cr(VI) by cysteine: Significance in human lymphocytes and formation of DNA damage in reactions with variable reduction rates

The induction of genotoxicity by Cr (VI) is dependent on its reductive activation inside the cell. Our recent studies have found that reduction of Cr (VI) by cysteine resulted in the formation of mutagenic Cr (III)-DNA adducts in the absence of oxidative DNA damage. In this work, we examined the formation of oxidative and Cr (III)-dependent types of DNA damage under a broader range of Cr (VI) and cysteine concentrations and investigated a potential role of this reducer in intracellular metabolism of Cr (VI). Peripheral lymphocytes from unexposed humans had 7.8-fold excess of glutathione over cysteine, whereas lymphocytes from stainless steel welders contained only 3 times higher amount of glutathione (p = 0.0009) which was entirely caused by the decrease in the concentration of glutathione. A strong correlation (r = 0.72) between the levels of both thiols was found in lymphocytes from controls. The number of DNA-protein crosslinks in lymphocytes from welders was 4.1 times higher than among controls, indicating the presence of Cr (VI)-dependent DNA damage. The average rate of Cr (VI) reduction by cysteine was approximately 5 times faster than that by glutathione. Higher reduction rate combined with the decrease in the intracellular concentration of glutathione should make cysteine a predominant Cr (VI)-reducing thiol in lymphocytes of welders. Analysis of the initial rates of Cr (VI) reduction by different concentrations of cysteine suggested the presence of one- and two-electron pathways, with one-electron mechanism dominating in the physiological range of concentrations. There was no detectable formation of DNA breaks or abasic sites under a broad range of Cr (VI) and cysteine concentrations, resulting in up to 68-fold differences in the rates of reduction and the production of as many as 3 Cr (III)-DNA adducts per 10 bp. The reactions with slow reduction rates (low concentrations of cysteine) led to the most extensive formation of Cr (III)DNA adducts. In summary, these results further establish Cr (III)-DNA adducts as the major form of DNA damage resulting from Cr (VI) metabolism by cysteine. The role of cysteine in reduction of Cr (VI) becomes more significant under conditions of occupational exposure to Cr (VI)-containing welding fumes.     

DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: synergistic effect of a second metal ion

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).     

Lipid peroxide induced DNA damage: protection by turmeric (Curcuma longa)

Summary Liposomal lipid peroxidation and peroxide induced DNA damage were investigated. Inhibition of lipid peroxidation was studied using 400 µM uric acid, -carotene, -tocopherol, curcumin and butylated hydroxyanisole (BHA). Curcumin, the active principle of turmeric (Curcuma longa), was as effective an antioxidant as BHA. An aqueous extract of turmeric was also found to be an effective inhibitor. The inhibition obtained using this aqueous extract, incorporated into the liposome itself, was 70% at 300 ng/µ1 This indicates the presence of yet another antioxidant in turmeric besides the lipophilic curcumin. The aqueous antioxidant extended 80% protection to DNA against peroxidative injury at 100 ng/µl. This component of turmeric is being characterised and investigated as an antioxidant/anticlastogen and as an antipromoter.Abbreviations GT₁b Trisialoganglioside - TBS Tris Buffered Saline - PBS Phosphate Buffered Saline - TBA Thio Barbituric acid - BHA Butylated Hydroxy Anisole - EDTA Ethylene Diamine Tetra Acetic Acid     

用彗星实验技术分析MTX对小鼠细胞DNA的损伤作用

MTX是一种抗叶酸药物 ,作用于增殖细胞 ,为了解其作用机制和探测其遗传毒性靶器官 ,以小鼠为研究对象 ,用彗星实验技术检测了MTX腹腔注射染毒后对脾、骨髓、胸腺、和外周血淋巴细胞的DNA损伤作用及其与MTX剂量间的相关。 1.2 5～ 5mg/kgMTX可诱发小鼠体内 4种细胞的DNA单链断裂 ,核DNA损伤程度与用药剂量呈正相关。不同种类细胞对MTX的易感性不同 ,脾、骨髓、胸腺、外周血淋巴细胞可能是MTX的遗传毒性靶细胞。外周血淋巴细胞在SCGE分析中的拖尾现象可作为用药后组织器官对药物敏感性反映的生物标志     

Short- and long-term freezing effects in a coastal (Lobaria virens) versus a widespread lichen (L. pulmonaria)

Lichens are considered freezing tolerant, although few species have been tested. Growth, a robust measure of fitness integrating processes in all partners of a lichen thallus, has not yet been used as a viability measure after freezing. We compared relative growth rates (RGR) after freezing with short-term viability measures of photo- and mycobiont functions in the coastal Lobaria virens and the widespread L. pulmonaria to test the hypothesis that low temperature shapes the coastal distribution of L. virens. Hydrated thalli from sympatric populations were subjected to freezing at −10, −20 and −40 °C for 5 h. The rate of cooling and subsequent warming was 5 °C h⁻¹. Short-term viability measures of photobiont (maximal photosystem II efficiency, effective PSII yield) and mycobiont viability (conductivity index), as well as subsequent RGR, were assessed. The exotherms showed that L. virens froze at −3 °C; L. pulmonaria, at −4 °C. Freezing significantly impaired short-term viability measures of both photo- and mycobiont, particularly in the coastal species. Lobaria pulmonaria grew 2.1 times faster than L. virens, but the short-term damage after one freezing event did not affect the long-term RGR in any species. Thereby, short-term responses were impaired by freezing, long-term responses were not. While the lacking RGR-responses to freezing suggest that freezing tolerance does not shape the coastal distribution of L. virens, the significant reported adverse short-term effects in L. virens may be aggravated by repeated freezing-thawing cycles in cold winters. In such a perspective, repeated freezing may eventually lead to reduced long-term fitness in L. virens.     

Cytotoxic and genotoxic effects of (R)- and (S)-ricinoleic acid derivatives

(R)-ricinoleic acid is the main component of castor oil from Ricinus communis L. Due to the presence of the hydroxyl group in homoallylic position and asymmetrically substituted carbon atom, it may undergo a number of chemical and biochemical transformations resulting in the products with some specific bioactivities. Conversion of (R)-ricinoleic acid into its (S)-enantiomer enables synthesis of both (R)- and (S)-ricinoleic acid derivatives and comparison of their biological activities. In the present research, (R)- and (S)-ricinoleic acid amides synthesized from methyl ricinoleates and ethanolamine or pyrrolidine as well as acetate derivatives of ethanolamine amides were studied to demonstrate their biological activities using HT29 cancer cells. Double staining of cells with fluorochromes (Hoechst 33258/propidium iodide) as well as 2,′7′-dichlorodihydrofluorescein (DCF) and comet assays were performed. Both the tested amides and acetates caused DNA damage and induced apoptotic and necrotic cell death. In the case of (R)- and (S)-enantiomers of one of the tested acetates, significant difference in the ability to induce DNA damage was observed, which showed the impact of the stereogenic center on the activities of these compounds.     

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.     

More pronounced salt dependence and higher reactivity for platination of the hairpin r(CGCGUUGUUCGCG) compared with d(CGCGTTGTTCGCG)

The DNA interference pathways exhibited by cisplatin and related anticancer active metal complexes have been extensively studied. Much less is known to what extent RNA interaction pathways may operate in parallel, and perhaps contribute to both antineoplastic activity and toxicity. The present study was designed with the aim of comparing the reactivity of two model systems comprising RNA and DNA hairpins, r(CGCGUUGUUCGCG) and d(CGCGTTGTTCGCG), towards a series of platinum(II) complexes. Three platinum complexes were used as metallation reagents; cis-[PtCl(NH₃)₂(OH₂)]⁺ (1), cis-[PtCl(NH₃)(c-C₆H₁₁NH₂)(OH₂)]⁺ (2), and trans-[PtCl(NH₃)(quinoline)(OH₂)]⁺ (3). The reaction kinetics were studied at pH 6.0, 25 °C, and 1.0 mM ≤ I ≤ 500 mM. For both types of nucleic acid targets, compound 3 was found to react about 1 order of magnitude more rapidly than compounds 1 and 2. Further, all platinum compounds exhibited a more pronounced salt dependence for the interaction with r(CGCGUUGUUCGCG). Chemical and enzymatic cleavage studies revealed similar interaction patterns with r(CGCGUUGUUCGCG) after long exposure times to 1 and 2. A substantial decrease of cleavage intensity was found at residues G4 and G7, indicative of bifunctional adduct formation. Circular dichroism studies showed that platinum adduct formation leads to a structural change of the ribonucleic acid. Thermal denaturation studies revealed platination to cause a decrease of the RNA melting temperatures by 5–10 °C. Our observations therefore suggest that RNA is a kinetically competitive target to DNA. Furthermore, platination causes destabilization of RNA structural elements, which may lead to deleterious intracellular effects on biologically relevant RNA targets.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review)

Cadmium is an important toxic environmental heavy metal. Occupational and environmental pollution with cadmium results mainly from mining, metallurgy industry and manufactures of nickel-cadmium batteries, pigments and plastic stabilizers. Important sources of human intoxication are cigarette smoke as well as food, water and air contaminations. In humans, cadmium exposures have been associated with cancers of the prostate, lungs and testes. Acute exposures are responsible for damage to these organs. Chronic intoxication is associated with obstructive airway disease, emphysema, irreversible renal failure, bone disorders and immuno-suppression. At the cellular level, cadmium affects proliferation, differentiation and causes apoptosis. It has been classified as a carcinogen by the International Agency for Research on Cancer (IARC). However, it is weakly genotoxic. Indirect effects of cadmium provoke generation of reactive oxygen species (ROS) and DNA damage. Cadmium modulates also gene expression and signal transduction, reduces activities of proteins involved in antioxidant defenses. Several studies have shown that it interferes with DNA repair. The present review focuses on the effects of cadmium in mammalian cells with special emphasis on the induction of damage to DNA, membranes and proteins, the inhibition of different types of DNA repair and the induction of apoptosis. Current data and hypotheses on the mechanisms involved in cadmium genotoxicity and carcinogenesis are outlined.     

Suppression of 8-oxo-2'-deoxyguanosine formation and carcinogenesis induced by N-nitrosobis (2-oxopropyl)amine in hamsters by esculetin and esculin

Effects of esculetin (6,7-dihydroxycoumarin) and its glycoside, esculin, on 8-oxo-2'-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were administered esculetin by gastric intubation into the stomach 30 min before BOP administration or ingestion of a diet containing esculin for 7 days before BOP administration, and killed 1 or 4 h after BOP treatment, and the contents of thiobarbituric acid-reacting substrates (TBARS) and 8-oxodG in the pancreas were determined. Both compounds suppressed significantly the BOP-induced increases in 8-oxodG and TBARS contents in hamster pancreas. We further investigated the effect of esculin on pancreatic carcinogenesis by the rapid production model induced by augmentation pressure with a choline-deficient diet, ethionine, methionine and BOP. Esculin was given ad libitum as a 0.05% aqueous solution in either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly smaller than in the control group, while esculin given during the promotion phase showed no apparent effects. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.     

Modulation of oxidative DNA damage and DNA-crosslink formation induced by cis-diammine-tetrachloro-platinum(IV) in the presence of endogenous reductants

Platinum(IV) [Pt(IV)] complex, satraplatin, is currently in clinical trials for the treatment of various cancers. As a key step of the anti-cancer effect exertion, satraplatin is supposed to be reduced by endogenous reductants to platinum(II) [Pt(II)] complex. In this study, we investigated the interaction of DNA, Pt(IV), and the endogenous reductants such as ascorbic acid (AsA) and glutathione (GSH). As a model Pt(IV) compound, cis-diammine-tetrachloro-Pt(IV) [cis-Pt(IV)], which is a prodrug of cisplatin [cis-diammine-dichloro-Pt(II), cis-Pt(II)], was incubated with calf thymus DNA in the presence of AsA or GSH. In the presence of AsA, cis-Pt(IV) induced oxidative DNA damage. Hydroxyl radical scavengers suppressed the AsA-associated oxidative damage, thereby suggesting that hydroxyl radicals are involved in the DNA oxidation. cis-Pt(II)-like CD spectral change and crosslink formation in calf thymus DNA were also observed during this DNA oxidation, suggesting cis-Pt(IV) reduction by AsA and DNA conformational change induced by the newly formed cis-Pt(II) binding to DNA. GSH did not induce oxidative DNA damage likely due to its own hydroxyl radical scavenging ability. Further, GSH suppressed the Pt(II)-mediated DNA conformational change and crosslink formation, suggesting that GSH sequesters the cis-Pt(II) away from DNA by GSH-cis-Pt(II) complex formation.