Modulation of hypersensitivity to oxidative DNA damage in ATM defective cells induced by potassium bromate by inhibition of the Poly (ADP-ribose) polymerase (PARP).

The ataxia telangiectasia mutated (ATM) protein is a pivotal multifunctional protein kinase predominantly involved in DNA damage response, as well as in maintaining overall functional integrity of the cells. Apart from playing its major role in regulating the cellular response to DNA damage, ATM, when mutated, can additionally determine oxidative stress, metabolic syndrome, mitochondrial dysfunction and neurodegeneration. In the present paper we aim to investigate the levels of oxidative stress potentially induced by the oxidizing rodent renal carcinogen KBrO3 in ATM-defective lymphoblastoid cell lines (LCLs) established from four classical AT patients (with different ATM mutations), one AT variant with reduced hypersensitivity to X rays, obligate AT heterozygotes and wild type intrafamilial control. A possible modulatory involvement of PARP in potentially induced oxidative stress is also evaluated following its inhibition with 3-aminobenzamide (3-AB). Treatments with KBrO3 clearly showed a marked hypersensitivity of the ATM-defective LCLs, including the AT variant. A marked and statistically significant reduction of KBrO3-induced chromosomal damage following inhibition of PARP by 3-AB, was observed in all AT LCLs, but not in those from the AT variant, AT heterozygotes and wild type intrafamilial control. This result is suggestive of a modulatory involvement of PARP in the hypersensitivity of ATM-defective cells to DNA oxidative damage.

Cytogenetic evidence that DNA topoisomerase II is not involved in radiation induced chromsome-type aberrations.

ICRF-187 (Cardioxane™, Chiron) is a catalytic inhibitor of DNA topoisomerase II (Topo II), proposed to act by blocking Topo II-mediated DNA cleavage without stabilizing DNA-Topo II-"cleavable complexes". In this study ICRF-187 was used to evaluate the potential involvement of DNA topoisomerase II in the formation of the radiation-induced chromosome-type aberrations in the G0 phase of the cell cycle in human lymphocytes from three healthy male donors. This is based on many evidences that DNA topoisomerases are involved in DNA recombination, mainly of illegitimate type (non-homologous) both in vitro and in vivo. The results obtained clearly indicated that ICRF-187 did not induce per se any chromosomal damage. When challenged with the non-catalytic Topo II poison VP-16 (etoposide), which acts by stabilizing the "cleavable complex" generating "protein concealed" DSB's and thus chromosomal aberrations, it completely abolished the significant induction of chromosome-type aberrations and formation of dicentric chromosomes. This indicates that ICRF-187 acts effectively as catalytic inhibitor of Topo II. On the other hand, when X-ray treatments were challenged with ICRF-187 using experimental conditions as for VP-16 treatments, no modification of the incidence of chromosome-type aberrations and dicentric chromosomes was observed. On this basis, we conclude that Topo II is not involved in the formation of X-ray-induced chromosome-type aberrations and dicentric chromosomes in human lymphocytes in the G0 phase of the cell cycle.

Properties of mixed DOTAP-DPPC bilayer membranes as reported by differential scanning calorimetry and dynamic light scattering measurements.

We have investigated the effect of a cationic lipid [DOTAP] on both the thermotropic phase behavior and the structural organization of aqueous dispersions of dipalmitoyl-phosphatidylcholine [DPPC] by means of high-sensitivity differential scanning calorimetry and dynamic light scattering measurements. We find that the incorporation of increasing quantities of DOTAP progressively reduces the temperature and the enthalpy of the gel-to-liquid crystalline transition. We are further showing that, in mixed DOTAP-DPPC systems, the reduction of the phase transition temperature is accompanied by a reduction of the average size of the structures present in the aqueous mixtures, whatever the DOTAP concentration is. These results, which extend a previous investigation by Campbell et al. (Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M.; Biochim. Biosphys. Acta 2001, 27, 1512.) limited to a DOTAP concentration below 20 mol %, confirm that the insertion of cationic head groups in zwitterionic phosphatidylcholine bilayers facilitates the formation of stable, relatively small, unilamellar vesicles. This self-assembling restructuring from an aqueous multilamellar structure toward a liposomal phase is favored by decreasing the phospholipid phase transition temperature and by increasing the temperature of the system. This reduction of the average size and the appearance of a stable liposomal phase is also promoted by a heating and cooling thermal treatment.

Met-myoglobin association in dilute solution during pressure-induced denaturation: an analysis at pH 4.5 by high-pressure small-angle X-ray scattering.

In this paper, we report on the original global fit procedure of synchrotron small-angle X-ray scattering (SAXS) data applied to a model protein, met-myoglobin, in dilute solution during temperature- and pressure-induced denaturation processes at pH 4.5. Starting from the thermodynamic description of the protein unfolding pathway developed by Hawley (Hawley, S. A. Biochemistry 1971, 10, 2436), we have developed a new method for analyzing the set of SAXS curves using a global fitting procedure, which allows us to derive the form factor of all the met-myoglobin species present in the solution, their aggregation state, and the set of thermodynamic parameters, with their p and T dependence. This method also overcomes a reasonably poor quality of the experimental data, and it is found to be very powerful in analyzing SAXS data. SAXS experiments were performed at four different temperatures from hydrostatic pressures up to about 2000 bar. As a result, the presence of an intermediate, partially unfolded, dimeric state of met-myoglobin that forms during denaturation has been evidenced. The obtained parameters were then used to derive the met-myoglobin p, T phase diagram that fully agrees with the corresponding phase diagram obtained by spectroscopic measurements.

Effect of p53 haploinsufficiency on melphalan-induced genotoxic effects in mouse bone marrow and peripheral blood.

Mice heterozygous for a p53 null mutation develop tumours induced by genotoxic carcinogens with a shorter latency than wild type mice and have been proposed as an alternate animal model for carcinogenicity testing. Some literature data suggest that p53+/- mice might also be more sensitive to the short-term effects of genotoxic agents and manifest a haploinsufficiency phenotype that could contribute to the higher tumour susceptibility. We have compared the induction of micronuclei in bone marrow and blood of p53+/- and p53+/+ isogenic mice after treatment with a single or multiple doses of melphalan (MLP), a crosslinking genotoxic carcinogen. We have also characterized the mechanism of micronucleus induction with CREST staining of kinetochore proteins to distinguish between chromosome break- and chromosome loss-induced micronuclei. Significant increases of micronucleated bone marrow polychromatic erythrocytes and blood reticulocytes were induced under all MLP exposure conditions. The frequency of micronucleated blood erythrocytes increased linearly with duration of exposure. Micronuclei were essentially a consequence of chromosome break events. After a single MLP dose, a significant reduction of the frequency of polychromatic erythrocytes in bone marrow of p53+/+ animals suggested the induction of cytotoxicity/cell cycle delay. This effect was not observed in p53+/- mice. We believe this finding to provide some evidence of a haploinsufficiency phenotype in the modulation of cell cycle/apoptotic pathways mediated by the p53 protein. In bone marrow of wild type mice, an increased effect of multiple MLP doses was detected over that of a single administration, whereas, in p53+/- mice, no differential effect was found of different exposure durations. Possibly, the probability of micronucleus formation increased under chronic exposure because of increased cell division in response to peripheral anemia and a reduction of p53 protein level had a small effect on cell cycle modulation and on such indirect mechanism of micronucleus induction. However, pairwise comparisons between the frequencies of cells with micronuclei in wild type and p53+/- mice under all exposure conditions did not show statistically significant differences, suggesting that the observed effects of p53 haploinsufficiency were weak and temporary and a higher/faster induction of irreversible chromosome damage could not account for the increased susceptibility of p53+/- mice to MLP-induced tumours.

Dielectric behavior of lysozyme and ferricytochrome-c in water/ethylene-glycol solutions.

This work deals with a dielectric study at radio frequencies of the influence at room temperature of two organic molecules, known as cryo-protectants, ethylene-glycol and glycerol, on conformational and dynamic properties of two model proteins, lysozyme (lys) from chicken egg-white and ferricytochrome-c (cyt-c) from horse heart. Cyt-c is a compact globular protein whereas lys is composed of two structural domains, separated by the active site cleft. Measurements were carried out at the fixed temperature of 20 degrees C varying the concentration of the cosolvent up to 90% w/w. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius and the electric dipole moment of the protein were calculated as a function of the cosolvent concentration. The data show that glycerol does not modify significantly the conformation of both proteins and cyt-c is also stable in the presence of ethylene-glycol. On the contrary ethylene-glycol strongly affects the dielectric response of lysozyme denoting a specific effect on its conformation and dynamics. The data are coherently interpreted hypothesizing that glycol molecule wedges between and separates the two domains of lys making them rotationally independent.

Picosecond internal dynamics of lysozyme as affected by thermal unfolding in nonaqueous environment.

A neutron-scattering investigation of the internal picosecond dynamics of lysozyme solvated in glycerol as a function of temperature in the range 200-410 K has been undertaken. The inelastic contribution to the measured intensity is characterized by the presence of a bump generally known as "boson peak", clearly distinguishable at low temperature. When the temperature is increased the quasielastic component of the spectrum becomes more and more intrusive and progressively overwhelms the vibrational bump. This happens especially for T > 345 K when the protein goes through an unfolding process, which leads to the complete denaturation. The quasielastic term is the superposition of two components whose intensities and linewidths have been studied as a function of temperature. The slower component describes motions with characteristic times of approximately 4 ps corresponding to reorientations of polypeptide side chains. Both the intensity and linewidth of this kind of relaxations show two distinct regimes with a crossover in the temperature range where the melting process occurs, thus suggesting the presence of a dynamical transition correlated to the protein unfolding. Conversely the faster component might be ascribed to the local dynamics of hydrogen atoms caged by the nearest neighbors with characteristic time of approximately 0.3 ps.

Structural characterization of the pH-denatured states of ferricytochrome-c by synchrotron small angle X-ray scattering.

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.

pH-dependent local structure of ferricytochrome c studied by x-ray absorption spectroscopy.

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.

Solvent isotope effects on the phase-transition properties of lipid bilayers.

Highly sensitive differential scanning microcalorimetry (DSC) has been used to investigate the phase transition properties of lipid vesicles prepared from 1,2-distearoyl-L-3-glyceryl-phosphatidylcholine (DSPC) in H(2)O and D(2)O. The data show that the response of pre-transition properties to D(2)O-->H(2)O substitution is stronger than the main transition properties. We find that there is a small increase in the phase transition temperature (DeltaT approximately 0.5 K) and in the co-operative unit in the main transition. The increase in enthalpy (DeltaH congruent with1 kJ(.)mol(-1)) and in transition temperature (DeltaT congruent with2 K) observed in the pre-transition is comparable with that observed in quite different processes and systems, i.e. melting of nucleic acids and proteins and gel formation. It is suggested that D(2)O-->H(2)O substitution affects the thermal transition in these systems in such a way that the contributions of enthalpy and entropy to structural reorganization of water in these processes is modified.

Instability of three-dimensional structures in ribosomal cores evidenced by microcalorimetric studies.

In this paper we show a microcalorimetric investigation carried out on the so-called cores, i.e. ribosomes deprived of select proteins by LiCl treatment. Thermal degradation of native ribosomes gives rise to two thermal transitions occurring at different temperatures. In the cores the high temperature peak persists even after treatment at very high ion strength (2 M LiCl). This strongly suggests the existence of a very stable structure that was previously observed also in particles treated with agents that hydrolyze the RNA moiety. The low temperature peak gradually but dramatically decreases even though it never disappears completely. This indicates that the treatment to obtain ribosomal cores does not cause complete unfolding of the particle but only the destabilization of a structural three-dimensional domain present in native ribosomes. These data are discussed in the light of previous results obtained by dielectric spectroscopy and microcalorimetric studies on ribosomal particles.

Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry.

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.

Differential scanning calorimetry of chromatin at different levels of condensation.

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 degrees C, while the other two occur at 63 degrees C and 74 degrees C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 degrees C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.

Evaluation of 10 aliphatic halogenated hydrocarbons in the mouse bone marrow micronucleus test.

Ten halogenated aliphatic hydrocarbons (carbon tetrachloride, 1-chlorohexane, 2,3-dichlorobutane, 1,2-dichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, hexachloroethane, 1,1,2-trichloroethane, 1,2,3-trichloropropane and 1,1,3-trichloropropene), previously assayed in genetic assays in fungi, were evaluated in the mouse bone marrow micronucleus test in order to assess their genotoxicity in vivo. All chemicals were administered once i.p. at 40 and 70-80% of their respective LD50 to male and female CD-1 mice, 24 and 48 h before killing. All treatments produced evident clinical symptoms, but no marked depression of bone marrow proliferation. No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes over the control values were observed at any sampling time with any of the 10 halogenated hydrocarbons assayed. The comparison of the results obtained in this study with the findings provided by in vitro micronucleus assays on the same chemicals, reported by other authors, indicate that mouse bone marrow is weakly sensitive to the genotoxic effects induced by halogenated hydrocarbons in other test systems. This suggests that the role of such an assay in carcinogen screening may be questionable for this chemical class. An examination of mouse bone marrow micronucleus test results with the halogenated aliphatic hydrocarbons classified as carcinogens by IARC supports this conclusion.

Differential stability of E. coli ribosomal particles and free RNA towards thermal degradation studied by microcalorimetry.

We investigated the thermal degradation of E. coli ribosomes by differential scanning microcalorimetry. The 70S particles show two distinctive and irreversible peaks upon thermal degradation. Free rRNA in solution produces, on the contrary, an unstructured denaturation profile. The thermal analysis of 50S particles shows a profile substantially identical to that observed in 70S, while 30S particles produce an unstructured denaturation pattern. Therefore the thermal behavior of the 70S particle is essentially attributable to the denaturation of the 50S subunit. Our data validate previous observations that the 50S has a more rigid structure as compared to 30S, which behaves as a 'floppy' particle. In addition our data suggest that protein/RNA interactions play a significant role to stabilize three-dimensional structures of the ribosome.

Light-dependent activation of 7,12-dimethylbenz[a]anthracene to a potent genotoxicant.

7,12-Dimethylbenz[a]anthracene (DMBA), which is widely used in mutagenesis and experimental carcinogenesis, is activated to a mutagen by white fluorescent light. A 40 min exposure to white fluorescent light of Salmonella typhimurium TA98 plates treated with DMBA, in the absence of exogenous metabolism, resulted in an approximately 30-fold increase in the number of histidine revertants. This phenomenon also occurs, with lesser intensity, with other promutagens, such as benzo[a]pyrene or 2-acetylaminofluorene, and in other Salmonella tester strains. Moreover, white fluorescent light is able to activate DMBA to a toxicant for Chinese hamster V79 cells in culture, resulting in very low cell survival. Under these conditions, white fluorescent light-activated DMBA was shown to cause chromosomal aberrations, but not gene mutations, as determined by resistance to thioguanine. This white fluorescent light-dependent activation of DMBA seems to be related to the formation of reactive species, as the addition of vitamin E results in a reduction in the number of histidine revertants induced by white fluorescent light in S. typhimurium TA98.

Clastogenic effects of the dithiocarbamate fungicides thiram and ziram in Chinese hamster cell lines cultured in vitro.

We report here the results obtained using the dithiocarbamate fungicides thiram and ziram to investigate the induction of chromosomal aberrations (CAs) in Chinese hamster ovary (CHO) cells both in the absence and presence of S9 metabolism, and in a Chinese hamster epithelial liver (CHEL) cells which retain metabolic competence to activate different classes of promutagens/procarcinogens. Both thiram and ziram proved to be strong chromosome breaking agents in the CHEL cells and CHO cells in the presence of S9 metabolism. These findings suggest that thiram and ziram require metabolic conversion to become genetically active, and corroborate the evidence that CHEL cells are suitable to activate and detect a broad spectrum of chemical procarcinogens including these two pesticides.

Mutagenicity testing of imidazole and related compounds.

Ames tests have been performed with imidazole and its principal metabolites, hydantoin and hydantoic acid. N-Acetyl-imidazole, a potential metabolite resulting from the action of intestinal bacteria, and histamine, a structurally related compound which is widely distributed in mammalian tissues, have also been tested. Imidazole and histamine were also tested in the UDS assay in primary rat hepatocytes, while imidazole alone was tested in the M2-C3H mouse fibroblast malignant transformation assay. Imidazole gave consistently negative results in the Ames test, the UDS assay and the transformation assay. The three metabolites of imidazole, namely hydantoin, hydantoic acid and N-acetyl-imidazole, all gave negative results in the Ames test. Histamine gave no evidence of mutagenic activity in the Ames test or of genotoxicity in the UDS assay. These results indicate that imidazole and its metabolites are unlikely to present a mutagenic or carcinogenic hazard.

Alternative methods in toxicology tests: in vitro toxicity.

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develop alternative methods to reduce the required number of animals. The new in vitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by the in vitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity with in vitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems for in vitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mitochondrial activity, intracellular content and leakage of enzymes into the medium.

Alternative methods in toxicology tests: In vitro toxicity.

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develope alternative methods to reduce the required number of animals. The newin vitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by thein vitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity within vitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems forin vitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mytochondrial activity, intracellular content and leakage of enzymes into the medium.