Antimutagenic activity of wheat β-purothionin Tk-AMP-BP

Antimutagenic activity on human RD cells was studied for β-purothionin Tk-AMP-BP isolated from seeds of wheat Triticum kiharae, which has high stress resistance. Cadmium chloride at 5 × 10⁻⁶ M was used as a mutagen. The numbers of DNA breaks in mutagen-treated and intact cells were inferred from the single-stranded to double-stranded DNA ratio and expressed as protection coefficients. The protective effect was simultaneously assayed for aqueous plant extracts known to possess antioxidant properties. Wheat thionin was the most active among all of the antimutagens examined; its protection coefficient reached 85–88% at micromolar peptide concentrations (8–32 μg/ml). Thus, wheat β-purothionin was for the first time demonstrated to be highly efficient in protecting human cell DNA from the damaging effect of cadmium chloride.     

The amino-acid sequence of a purothionin from Triticum monococcum, a diploid wheat

Purothionins were extracted and purified from the diploid wheat Triticum monococcum. Two proteins were obtained, one of which was present in only very small amounts. The major purothionin of T. monococcum was sequenced and it had an amino acid sequence identical with that of the beta-purothionin of Triticum aestivum (hexaploid bread wheat). It is known that T. monococcum contains the wheat A genome, so the structural gene coding for the beta-purothionin must comprise a part of the A genome. There have been no observable (as amino acid replacements) changes in the DNA comprising either the beta-purothionin gene of T. aestivum or the purothionin gene of T. monococcum, since T. monococcum (or its wild equivalent, Triticum boeoticum) hybridized with the diploid wheat B genome progenitor and started the evolution from diploid to allohexaploid wheat. All of the investigated characteristics of the purothionin-like protein isolated in small amounts suggested that it was essentially identical in amino acid sequence with the T. monococcum purothionin. It may be a dimerized form of beta-purothionin.     

Interaction between beta-Purothionin and dimyristoylphosphatidylglycerol: a (31)P-NMR and infrared spectroscopic study

The interaction of beta-purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of dimyristoylphosphatidylglycerol (DMPG) was investigated by (31)P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of beta-purothionin. The results indicate that beta-purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that beta-purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by beta-purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of beta-purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24 degrees C to ~33 degrees C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20 degrees C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of beta-purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the alpha-helices and the beta-sheet of beta-purothionin have tilt angles of ~60 degrees and 30 degrees, respectively, relative to the normal of the ATR crystal.     

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.     

Is there a difference in metal ion-based inhibition between members of thionin family: Molecular dynamics simulation study

Thionins have a considerable potential as antimicrobial compounds although their application may be restricted by metal ion-based inhibition of membrane permeabilizing activity. We previously reported the properties associated with the proposed mechanism of metal ion-based inhibition of beta-purothionin. In this study, we investigated the effects of metal ions on alpha-hordothionin which differs from beta-purothionin by eight out of 45 residues. Three of the differing residues are thought to be involved in the mechanism of metal ion-based inhibition in beta-purothionin. The structure and dynamics of alpha-hordothionin were explored using unconstrained molecular dynamics (MD) simulations in explicit water as a function of metal ions. Although the global fold is almost identical to that of beta-purothionin, alpha-hordothionin displays reduced fluctuating motions. Moreover, alpha-hordothionin is more resistant to the presence of metal ions than beta-purothionin. Mg(+2) ions do not affect alpha-hordothionin, whereas K(+) ions induce perturbations in the alpha2 helix, modify dynamics and electrostatic properties. Nevertheless, these changes are considerably smaller than those in beta-purothionin. The proposed mechanism of metal ion-based inhibition involves the hydrogen bonding network of Arg5-Arg30-Gly27, which regulates dynamic unfolding of the alpha2 C-end which is similar to beta-purothionin response. The key residues responsible for the increased resistance for alpha-hordothionin are Gly27 and Gly42 which replace Asn27 and Asp42 involved into the mechanism of metal ion-based inhibition in beta-purothionin. Comparison of MD simulations of alpha-hordothionin with beta-purothionin reveals dynamic properties which we believe are intrinsic properties of thionins with four disulphide bonds.     

Assessment of the genotoxic effects of methyl chloride in human lymphoblasts

The activity of methyl chloride was measured in 4 genotoxicity assays. In an established human lymphoblast line, a 3-h treatment with 0-5% methyl chloride resulted in a dose-related increase in mutant fraction at the thymidine kinase locus and induction of sister-chromatid exchange. No increase in DNA damage, as measured by alkaline elution, was detected in the lymphoblasts at concentrations of methyl chloride shown to be mutagenic. Also, a concentration-related increase in 8-azaguanine-resistant fraction in Salmonella typhimurium was observed following a 3-h treatment with atmospheres containing 0-20% methyl chloride. Thus, methyl chloride is a weak, direct-acting mutagen for bacteria and human cells in culture.     

Structure of beta-purothionin in membranes: a two-dimensional infrared correlation spectroscopy study

Two-dimensional infrared correlation spectroscopy has been used to investigate the structure of beta-purothionin, a small basic protein found in the endosperm of wheat seeds, in the absence and presence of dimyristoylphosphatidylglycerol (DMPG) membranes. To generate the two-dimensional synchronous and asynchronous maps, hydrogen-deuterium exchange of the protein amide protons has been used as an external perturbation. This method has allowed us to separate the different secondary structure elements and side chain contributions in the regions of amide I, II, and II' bands to determine that the relative order of deuteration of the beta-purothionin protons is as follows: turns, asparagines, and lysines > unordered structure and tyrosine > beta-sheet > alpha-helices and arginines. The results also indicate that the protein undergoes significant changes both in secondary structure and in deuteration in the presence of DMPG bilayers. The helical content of beta-purothionin is higher in the presence of the lipid, and the relative order of deuteration is as follows: lysines and arginines > asparagines and beta-sheet > unordered structure and alpha-helices. The inversion in the deuteration order of the arginine residues is assigned to a change of the degree of association of the protein in the membrane. In addition, the results reveal that the part of the protein containing the tyrosine residue interacts with the lipid membrane. Our results combined with those previously published suggest that the toxicity of beta-purothionin is more associated with the formation of functional channels in cell membranes rather than with a lytic phenomenon.     

Molecular-genetic effects of cadmium chloride

We studied the effect of cadmium chloride on: (1) the DNA of human cells; (2) the mutagenic effect of reproducing Kilham virus; (3) the synthesis of virus-induced interferon, and (4) the reproduction of oncogenic (mammalian leucosis) virus. Cadmium chloride caused degradation of DNA in human- and rat-embryo cells. Culture infected by the virus in the presence of cadmium sulphate had the highest yield of cells with chromosomal aberrations. Cadmium chloride caused marked inhibition of the virus-induced synthesis of interferon. The introduction of cadmium chloride into diploid cells infected by the leucosis virus caused a 3-4 fold increase in the yield of virus-induced transformation foci.     

Use of liposomes, viral capsids, and nanoparticles as DNA carriers

We tested a variety of liposomes for parameters such as DNA binding capacity and DNase I protection of incorporated and attached DNA to elucidate their use as vehicles for DNA transfer into cells and animals. The results were compared to other potential DNA vehicles, empty viral capsids, and nanoparticles. Maximal binding capacity was achieved for positively charged nanoparticles, DNase I protection was observed for most preparations with neosome preparations being least efficient. The uptake of radiolabeled DNA by cells in culture was determined for cationic and nonionic surfactant vesicles, viral capsids, and nanoparticles. Cellular DNA uptake was best for dioleoyl-derived positively charged liposomes (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTMA) and the DNA could be shown to be physiologically active. The recombination rate for DNA fragments transfected in polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DOTMA and polyoma-mediated DNA transfer.     

The protection role of heat shock protein 70 (HSP-70) in the testes of cadmium-exposed rats

Cadmium (Cd) is an environmental carcinogenic pollutant known to inactivate several proteins involved in DNA repair systems while at the same time creating an oxidative stress that can result in additional DNA lesions. The testis and the lung are the target organs for cadmium carcinogenesis. Increased production of oxidants in vivo can cause damage to intracellular macromolecules such as DNA, proteins and lipids, which in turn lead to oxidative injury. So, this investigation aimed to evaluate the protective role of L-Carnitine through up regulation of HSPs against DNA damage induced by cadmium chloride. The current study was carried out on forty adult male rats, each with average weight 220-250g., were divided into 4 equal groups. 1(st) group was received saline solution (0.5 ml/100 g body weight) and kept as control. 2(nd) group was received 500mg / kg body weight L-Carnitine intraperitoneally (IP). 3(rd) group was administered 1.2 mg cadmium chloride IP. 4(th) group was received both cadmium chloride and L-Carnitine simultaneously. The comet assay parameters showed significantly increased HSP70 and DNA damage in testis cells after 10 and 56 days in the third group. Meanwhile, HSP70 showed significantly decreased levels after 10 days and 56 days in the fourth group after L-Carnitine treatment simultaneously with cadmium chloride. The results of the present study demonstrate a damaging effect of cadmium chloride on DNA of the testis cells (with low stress response). This damaging effect increases the synthesis of HSP70 that upregulated by L-Carnitine treatment and showed ameliorative effect of the cells for recovery.     

Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as alpha 1-, alpha 2-, and beta-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes nu 8a and nu 8b. The nu 8b frequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for alpha 1- (or alpha 2-) and beta-purothionin, respectively. The intensity of the 1176-cm-1 nu 9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong approximately 1000-cm-1 nu 12 band of phenylalanine in aqueous solution is about half the value observed in most proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytotoxic plant protein, beta-purothionin, forms ion channels in lipid membranes

Thionins are small cysteine-containing, amphipathic plant proteins found in seeds and vegetative tissues of a number of plant genera. Many of them have been shown to be toxic to microorganisms such as fungi, yeast, and bacteria and also to mammalian cells. It has been suggested that thionins are present in seeds to protect them, and the germinating seedling, from attack by phytopathogenic microorganisms, but the mechanism by which they kill cells remains unclear. Using electrophysiological measurements, we have shown that beta-purothionin from wheat flour can form cation-selective ion channels in artificial lipid bilayer membranes and in the plasmalemma of rat hippocampal neurons. We suggest that the generalized toxicity of thionins is due to their ability to generate ion channels in cell membranes, resulting in the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.     

Modification of repair DNA synthesis in mutagen-treated human fibroblasts during adaptive response and the antimutagenic effect of garlic extract

Repair DNA synthesis (RDS) in human fibroblasts during the adaptive responses (ARs) induced by cadmium chloride (CdCl2), gamma-radiation, and 4-nitroquinoline-1-oxide (4NQO) was compared in cells pretreated and not pretreated with garlic extract. The RDS was increased during the ARs induced CdCl2 and gamma-irradiation. Garlic extract stimulated RDS in cells treated by the same mutagens. 3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, decreased the RDS rate in cells treated with CdCl2 and gamma-irradiation but had no significant effect on cells treated with 4NQO. It was demonstrated that DNA repair was involved into cell protection in different ways in the cases of antimutagen treatment and AR.     

The effect of a bromide leaving group on the properties of nitro analogs of the duocarmycins as hypoxia-activated prodrugs and phosphate pre-prodrugs for antitumor therapy

Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkylating agents. A series of four nitroCBI alcohol prodrugs containing a bromide rather than chloride or sulfonate leaving group was synthesized. In assays for in vitro hypoxia-selective cytotoxicity against human tumor cell lines the two bromides with DNA minor groove binding basic side chains displayed hypoxic cytotoxicity ratios (HCRs) of 52-286 in HT29 cells and 41-43 in SiHa cells. These values compare well with a related previously reported chloride analog. The corresponding more water soluble phosphate pre-prodrugs of the bromides were synthesized and evaluated for in vivo antitumor activity against SiHa human tumor xenografts. All four phosphates, with both neutral and basic side chains, demonstrated activity providing statistically significant hypoxic log(10) cell kills of 0.87-2.80 at non-toxic doses, matching or proving superior to those of their chloride analogs.     

Cytogenetic effect of the radiosensitizing action of 5-bromo-2'-deoxyuridine on eukaryotic cells at various periods of the DNA synthesis phase

The effect of 5-bromo-2'-deoxyuridine (BUDR) radiosensitizing action on meristematic cells of wheat seedlings and human peripheral blood lymphocytes synchronized with 5-fluoro-2'-deoxyuridine at the G1-S boundary was revealed. This effect was only found in the beginning of the S phase. We show that it was conditioned by incorporation of BUDR into special early replicating DNA sequences which are specific for chromosomal mutagenesis. Using the molecular biology methods non-random distribution of 3H-BUDR in the early replicating wheat DNA sequences with different degree of repeating was found.     

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.     

Effect of vinblastine sulfate on gamma-radiation-induced DNA single-strand breaks in murine tissues

The effect of vinblastine sulfate on gamma-radiation-induced DNA strand breaks in different tissues of tumour bearing mice, was studied by single-cell gel electrophoresis. Intraperitonial administration of different doses (0.25-2.0mg/kg body weight) of vinblastine sulfate 30 min prior to 4 Gy gamma-radiation exposure showed a dose-dependent decrease in the yield of DNA strand breaks in murine fibrosarcoma, blood leukocytes and bone marrow cells. The dose-dependent protection of cellular DNA against radiation-induced strand breaks as evidenced from comet tail length, tail moment and percent DNA in the tail, was more pronounced in bone marrow cells than in the cells of the tumor fibrosarcoma. In fibrosarcoma cells, the decrease in comet tail length, tail moment and percent DNA in the tail was detected at lower doses of vinblastine sulfate administration and these parameters were not significantly altered at higher doses, from that of the control irradiated. From this study, it appears that in addition to anticancer activity, vinblastine sulfate could offer protection to the normal tissues against gamma-radiation-induced DNA strand breaks.     

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.