Rapid, one-pot procedure to synthesise 103Pd:Pd@Au nanoparticles en route for radiosensitisation and radiotherapeutic applications.

The radioisotope palladium (103Pd), encapsulated in millimetre-size seed implants, is widely used in prostate cancer brachytherapy. Gold nanoparticles (Au NPs) distributed in the vicinity of 103Pd radioactive implants, strongly enhance the therapeutic dose of radioactive implants (radiosensitisation effect). A new strategy under development to replace millimetre-size implants, consist in injecting radioactive NPs in the affected tissues. The development of 103Pd@Au NPs distributed in the diseased tissue, could increase the uniformity of treatment (compared with massive seeds), while enhancing the radiotherapeutic dose to the cancer cells (through Au-mediated radiosensitisation effect). To achieve this goal, it is necessary to develop a rapid, efficient, one-pot and easy-to-automatise procedure, allowing the synthesis of core-shell Pd@Au NPs. The novel synthesis route proposed here enables the production of Pd@Au NPs in not more than 4 h, in aqueous media, with minimal manipulations, and relying on biocompatible and non-toxic molecules. This rapid multi-step process consists of the preparation of ultra-small Pd NPs by chemical reduction of an aqueous solution of H2PdCl4 supplemented with ascorbic acid (AA) as reducing agent and 2,3-meso-dimercaptosuccinic acid (DMSA) as a capping agent. Pd conversion yields close to 87% were found, indicating the efficiency of the reaction process. Then Pd NPs were used as seeds for the growth of a gold shell (Pd@Au), followed by grafting with polyethylene glycol (PEG) to ensure colloidal stability. Pd@Au-PEG (TEM: 20.2 ± 12.1 nm) formed very stable colloids in saline solution as well as in cell culture medium. The physico-chemical properties of the particles were characterised by FTIR, XPS, and UV-vis. spectroscopies. The viability of PC3 human prostate cancer cells was not affected after a 24 h incubation cycle with Pd@Au-PEG NPs to concentrations up to 4.22 mM Au. Finally, suspensions of Pd@Au-PEG NPs measured in computed tomography (CT) are found to attenuate X-rays more efficiently than commercial Au NPs CT contrast media. A proof-of-concept was performed to demonstrate the possibility synthesise radioactive 103Pd:Pd@Au-PEG NPs. This study reveals the possibility to synthesise Pd@Au NPs rapidly (including radioactive 103Pd:Pd@Au-PEG NPs), and following a methodology that respects all the strict requirements underlying the production of NPs for radiotherapeutic use (rapidity, reaction yield, colloidal stability, NPs concentration, purification).

Tailored biological retention and efficient clearance of pegylated ultra-small MnO nanoparticles as positive MRI contrast agents for molecular imaging.

A majority of MRI procedures requiring intravascular injections of contrast agents are performed with paramagnetic chelates. Such products induce vascular signal enhancement and they are rapidly excreted by the kidneys. Unfortunately, each chelate is made of only one paramagnetic ion, which, taken individually, has a limited impact on the MRI signal. In fact, the detection of molecular events in the nanomolar range using T1-weighted MRI sequences requires the design of ultra-small particles containing hundreds of paramagnetic ions per contrast agent unit. Ultra-small nanoparticles of manganese oxide (MnO, 6-8 nm diameter) have been developed and proposed as an efficient and at least 1000× more sensitive "positive" MRI contrast agent. However no evidence has been found until now that an adequate surface treatment of these particles could maintain their strong blood signal enhancement, while allowing their rapid and efficient excretion by the kidneys or by the hepatobiliairy pathway. Indeed, the sequestration of MnO particles by the reticuloendothelial system followed by strong uptake in the liver and in the spleen could potentially lead to Mn2+-induced toxicity effects. For ultra-small MnO particles to be applied in the clinics, it is necessary to develop coatings that also enable their efficient excretion within hours. This study demonstrates for the first time the possibility to use MnO particles as T1 vascular contrast agents, while enabling the excretion of >70% of all the Mn injected doses after 48 h. For this, small, biocompatible and highly hydrophilic pegylated bis-phosphonate dendrons (PDns) were grafted on MnO particles to confer colloidal stability, relaxometric performance, and fast excretion capacity. The chemical and colloidal stability of MnO@PDn particles were confirmed by XPS, FTIR and DLS. The relaxometric performance of MnO@PDns as "positive" MRI contrast agents was assessed (r1 = 4.4 mM-1 s-1, r2/r1 = 8.6; 1.41 T and 37 °C). Mice were injected with 1.21 µg Mn per kg (22 µmol Mn per kg), and scanned in MRI up to 48 h. The concentration of Mn in key organs was precisely measured by neutron activation analysis and confirmed, with MRI, the possibility to avoid RES nanoparticle sequestration through the use of phosphonate dendrons. Due to the fast kidney and hepatobiliairy clearance of MnO particles conferred by PDns, MnO nanoparticles can now be considered for promising applications in T1-weighted MRI applications requiring less toxic although highly sensitive "positive" molecular contrast agents.

Ethnic variability in the allelic distribution of human aryl hydrocarbon receptor codon 554 and assessment of variant receptor function in vitro.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcriptional regulator of several genes including the cytochrome P4501 (CYP1) family as well as genes encoding factors involved in cell growth and differentiation. In mice, several polymorphic forms of the AHR are known, some of which have altered affinity for toxic and carcinogenic ligands. Remarkably little genetic variation has been detected in the human AHR gene. In studies on human AHR, Kawajiri et al. (Pharmacogenetics 1995; 5:151-158) reported a variation at codon 554 that results in an amino acid change from arginine to lysine; the frequency of the variant allele in a Japanese population (n = 277) was 0.43. We investigated the Lys554 allele in 386 individuals of various ethnic origins and found the frequency to be: 0.58 in Ivory Coast Africans (n = 58); 0.53 in a mixed African group (n = 20); 0.39 in Caribbean-Africans (n = 55); 0.32 in Canadian Chinese (n = 41); 0.14 in North American Indians (n = 47); 0.12 in French Canadian Caucasians (n = 20); 0.11 in a mixed ethnicity North American group (n = 45); 0.09 in Canadian Inuits (n = 22); and 0.07 in German Caucasians (n = 78). We expressed the human Lys554 allele in an in-vitro transcription-translation system and found that the receptor bearing the R554L substitution had an equivalent ability to that of the wild-type receptor to bind to a dioxin-responsive element following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Lys554 allele also was equivalent to the wild-type receptor at stimulating CYP1A1 mRNA expression when transfected into TCDD-treated receptor-deficient mouse Hepa-1 cells. It is not yet known if any of the wide variations in allele frequency at codon 554 are related to ethnic differences in susceptibility to adverse effects of environmental chemicals.

Cytochrome P450 CYP1A1 enzyme activity and DNA adducts in placenta of women environmentally exposed to organochlorines.

Organochlorine compounds bioaccumulate in fishing and hunting products included in the daily diet of many coastal populations. Prenatal and perinatal exposure to large doses of PCBs and PCDFs was shown to be deleterious on fetal and neonatal development, but information is scarce regarding possible effects of chronic low-dose exposure. This study investigates biomarkers of early effects in newborns from women exposed to organochlorines through the consumption of species from marine food chains, in two remote coastal regions of the province of Quebec (Canada). A CYP1A1-dependent enzyme activity (EROD) and DNA adducts were measured in placenta samples obtained from 30 women living on the Lower-North-Shore of the St. Lawrence River and 22 Inuit women from Nunavik (Arctic Quebec). These biomarkers were also assessed in 30 women from a Quebec urban center (Sept-Iles) as a reference group. Prenatal organochlorine exposure was determined by measuring these compounds in umbilical cord plasma. The amount of bulky polycyclic aromatic hydrocarbon (PAH)-related DNA adducts was significantly greater in the Lower-North-Shore group than in the reference group. Placental EROD activity and the amount of less bulky (OC-related) DNA adducts were significantly higher in the Nunavik group than in the reference group. For both biomarkers, smoking was found to be an important confounding factor. Organochlorine exposure was significantly associated with EROD activity and DNA adduct levels when stratifying for smoking. This study confirms that CYP1A1 enzyme induction and DNA adducts in placental tissue constitute useful biomarkers of early effects induced by environmental exposure to organochlorines.

Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions.

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are important nuclear enzymes that cooperate to minimize genomic damage caused by DNA strand interruptions. DNA strand interruptions trigger the ADP-ribosylation activity and phosphorylation activity of PARP and DNA-PK respectively. In order to understand the relationship of PARP and DNA-PK with respect to DNA binding required for their activation, we analyzed the kinetics of the reactions and determined the apparent dissociation constants (Kd app) of the enzymes for DNA strand interruptions. PARP has a high binding affinity for blunt ends of DNA (Kd app=116 pM) and 3' single-base overhangs (Kd app=332 pM) in comparison to long overhangs (Kd app=2.6-5.0 nM). Nicks are good activators of PARP although the affinity of PARP for nicks (Kd app=467 pM) is 4-fold less than that for blunt ends. The Kd app of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs is 704 pM, 1.3 nM and 1.4-2.2 nM respectively. These results demonstrate that (1) PARP, when compared to DNA-PK, has a greater preference for blunt ends and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP. The possible implications of the preferences of PARP and DNA-PK for DNA strand interruptions in vivo are discussed.

A microassay for the detection of low levels of cytochrome P450 O-deethylation activities with alkoxyresorufin substrates.

A microfluorometric method for the detection of low levels of cytochrome P450 was developed to increase the sensitivity of the assay, since a low level of CYP450 associated enzymatic activities was expected in human placenta tissues and small samples of placenta (approximately 10 g) could be easily collected, stored and processed. The dual fluorescence assay of Kennedy et al. [1], which was developed to simultaneously quantitate microsomal proteins and ethoxyresorufin-O-deethylase (EROD) activity was adapted for 96 wells microtiter plates. Placental microsomes samples were analyzed. For samples obtained from non-smoking mothers from the general southern Quebec population, results ranged from less than 1-3.3 pmol/mg protein.min. Samples collected from smoking mothers showed activity levels ranging from 30-69 pmol/mg protein.min. These results showed the suitability of the microassay for measuring low level of CYP450 activity in tissues such as placenta.

Determination of genotoxicity of the metabolites of the pesticides Guthion, Sencor, Lorox, Reglone, Daconil and Admire by 32P-postlabeling.

Commercial formulations of the pesticides: Guthion (azinphos methyl), Sencor (metribuzin), Lorox (linuron), Reglone (diquat), Daconil (chlorothalonil) and Admire (imidacloprid) were studied for their genotoxicity by 32P-postlabeling. Metabolites of the pesticides were obtained enzymatically using arochlor induced rat liver S9 fraction, in an NADPH generating system. The resulting metabolites were reacted with calf thymus DNA and the DNA was analyzed for presence of adducts by either the nuclease P1 or butanol enrichment. Nuclease P1 enrichment resulted in adducts for all the pesticides. Compared to the level of adducts in control DNA, the levels in pesticide-treated DNA were higher for all the pesticides, except Daconil. The increase in adduct numbers for pesticide-treated DNAs ranged from 4.9-12.4 times the control-DNA indicating pesticide genotoxicity in this in vitro system. Enrichment using butanol extraction gave three adducts unique to Sencor-DNA. These adducts were different from those obtained with nuclease P1 enrichment of the same. B(alpha)P was the positive control for the in vitro metabolism, and two adduct enrichment procedures: nuclease P1 digestion and butanol extraction.

Equilibrium model in an in vitro poly(ADP-ribose) turnover system.

Poly(ADP-ribose) metabolism plays an important role in numerous DNA-related functions. This homopolymer is synthesized by poly(ADP-ribose) polymerase and is degraded mainly by the poly(ADP-ribose) glycohydrolase. The activities of these two enzymes in the nucleus are closely coordinated. To better understand the interactions between these enzymes, we designed an in vitro system in which both enzymes are present at the same time. In this work, we report a model describing the synthesis and degradation of the poly(ADP-ribose) in turnover conditions. Because the half-life of the polymer in the cell is close to 1 min, we studied the very early kinetic interactions of these two enzymes.

Comparative study of DNA adducts levels in white sucker fish (Catostomus commersoni) from the basin of the St. Lawrence River (Canada).

The levels of DNA adducts in the hepatic tissue of the white sucker fish species Catostomus commersoni were determined by 32P-postlabelling. The fish were caught at four sites: two sites near the city of Windsor (Québec, Canada) on the St. François River, a downstream tributary of the St. Lawrence River, and two sites in the St. Lawrence River itself, near the city of Montréal (Québec, Canada). The latter sites are known to be contaminated by many pollutants including polycyclic aromatic hydrocarbons. Total adduct levels in all fish ranged from 25.1-178.0 adducts per 10(9) nucleotides. White sucker from the selected sites of the St. Lawrence River had a significantly higher mean level of DNA adducts than those of the St. François River (129.4 vs 56.8, respectively). These results suggest that the effluents of many heavy industries (e.g. from a Soderberg aluminium plant) flowing in the St. Lawrence River are more likely to produce genotoxic damage to fish than those released in one of its tributary, and mainly associated to the activities of a small town and a nearby pulp and paper mill.

Autoantibodies reacting with poly(ADP-ribose) and with a zinc-finger functional domain of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA.

Poly(ADP-Ribose) polymerase (PARP) is a chromatin-associated enzyme that specifically binds to DNA strand breaks in a zinc-dependent manner. We describe here the presence of IgG antibodies reacting with recombinant human PARP in the serum of patients with systemic lupus erythematosus (SLE) and primary and secondary Sjögren's syndrome (pSS and sSS). The reactivity of patients' sera was further studied in ELISA with a synthetic peptide of 44 residues corresponding to the second zinc finger (F2) present in the DNA-binding domain of PARP and which was shown to effectively bind 65Zn. Thirty-five percent of SLE sera (n = 97), 42% of pSS sera (n = 67), and 56% of sSS sera (n = 16) were found to contain raised levels of IgG antibodies reacting with peptide F2 which corresponds to the domain in PARP that is directly involved in the specific recognition of single and double strand breaks in DNA. Antibodies reacting with the whole enzyme and/or peptide F2 occurred independently from antibodies reacting with poly(ADP-ribose) which is rapidly synthesized in vivo by PARP from NAD and then degraded in response to DNA strand breaks.

Mode of action of poly(ADP-ribose) glycohydrolase.

The turnover of the homopolymer of ADP-ribose, which is known to be involved in many DNA-related functions, is controlled by 2 principal enzymes. Poly(ADP-ribose) polymerase (EC 2.4.2.30) synthesizes the polymer from NAD, and poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme responsible for its catabolism (Thomassin et al. (1992) Biochim. Biophys. Acta 1137, 171-181). In vivo, poly(ADP-ribose) polymers constitute a heterogeneous population of branched polymers attaining sizes of 200-400 residues. They are rapidly degraded by PARG, displaying variable kinetic parameters as a function of polymer size. Several studies have suggested that PARG acts exoglycosidically on its substrate but others observed that it could act endo/exo-glycosidically. We analysed the mode of action of PARG under conditions most suitable for expression of all the activities of PARG, using HPLC purified long free polymer and very pure PARG. We conclusively show that on large free polymers, PARG exhibits endoglycosidic activity along with exoglycosidic activity. This endoglycosidic activity could have a significant role during cellular response to DNA damage.

Poly(ADP-ribose) catabolism in mammalian cells.

Poly(ADP-ribose) catabolism is a complex situation involving many proteins and DNA. We have developed an in vitro turnover system where poly(ADP-ribose) metabolism is monitored in presence of different relative amounts of two principal enzymes poly(ADP-ribose) transferase and poly(ADP-ribose) glycohydrolase along with other proteins and DNA. Our current results reviewed here show that the quality of polymer, i.e. chain length and complexity, as well as preference for the nuclear substrate varies depending upon the availability of poly(ADP-ribose) glycohydrolase. These results are interpreted in the light of the recent data implicating poly(ADP-ribose) metabolism in DNA-repair.

Molecular and biochemical features of poly (ADP-ribose) metabolism.

In the past five years, poly(ADP-ribosyl)ation has developed greatly with the help of molecular biology and the improvement of biochemical techniques. In this article, we describe the physico-chemical properties of the enzymes responsible for the synthesis and degradation of poly(ADP-ribose), respectively poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase. We then discuss the possible roles of this polymer in DNA repair and replication as well as in cellular differentiation and transformation. Finally, we put forward various hypotheses in order to better define the function of this polymer found only in eucaryotes.

Rearrangements of the nucleosome structure in chromatin by poly(ADP-ribose).

In order to approach and clarify the effect of poly(ADP-ribose) on the nucleosomal structure, polynucleosomes from calf thymus were incubated with long poly(ADP-ribose) chains prepared in vitro and examined by ELISA with antibodies directed against the five individual histones H1, H2A, H2B, H3 and H4 as well as against two synthetic peptides in residues 1-25 of H2B and 130-135 of H3. The results showed that: (i) free ADP-ribose polymers did indeed interact with the nucleosomes; (ii) the accessibility of epitopes recognized by any of the different antibodies was altered, the binding of antibodies being increased or decreased depending on the quantity of poly(ADP-ribose) added thereby suggesting a modulation in nucleosome structure; (iii) for any ADP-ribose polymer concentration, core histones as well as histone H1 were always recognized by their respective antibodies, thus suggesting that poly(ADP-ribose) does not seem to cause complete stripping of histones from nucleosomal DNA.

Analysis of the activation of poly(ADP-ribose) polymerase by various types of DNA.

In this work, we examined the activation of poly(ADP-ribose) polymerase by various types of DNA. We have found that the best activator for poly(ADP-ribose) polymerase is an octanucleotide. However, the use of a defined activated DNA is also suitable.

Enzymological properties of poly(ADP-ribose)polymerase: characterization of automodification sites and NADase activity.

We have characterized the effect of poly(ADP-ribose) polymerase automodification on the enzyme's activities, which include poly(ADP-ribose) synthesis and NADase activity. The apparent Km of the enzyme for NAD+ during polymer synthesis is higher than the one measured for alternate NADase activity. Furthermore, we have found that there are 28 automodification sites, in contrast to the 15 sites (postulated to be on the 15 glutamic acids) reported to be present in the automodification domain. For the first time, we show that some of these acceptor sites are outside the reported automodification domain (15 kDa); we demonstrate automodification in the NAD+ binding domain (55.2 kDa) and the DNA binding domain (42.5 kDa). We have analyzed the relationship between the number of sites modified on poly(ADP-ribose) polymerase and its effect on the polymerization activity and its alternate NADase activity. Automodification greatly altered both enzyme activities, decreasing both polymer synthesis and alternate NADase activity.