Americium biodistribution in rats after wound contamination with different physicochemical forms in the presence or absence of plutonium: analyses using STATBIODIS.

Americium (Am) biodistribution data obtained after wound contamination in rats were analysed to evaluate and quantify the influence of different physicochemical forms of Am in the presence or absence of plutonium (Pu). The biodistribution data were individual Am daily urinary excretion and tissue retention. The data were analysed with STATBIODIS, a statistical tool developed in the laboratory and based on the R language. Non-parametric methods were selected to comply with the data characteristics. Am systemic tissue retention and urinary excretion data were much greater for contamination with soluble physicochemical forms than insoluble forms. Meanwhile, Am relative biodistribution between the main retention tissues (skeleton, liver and kidney) remained the same. Hence, after absorption into blood the radionuclide behaviour was independent of the physicochemical form. The presence of Pu did not change the Am biodistribution. Comparisons of the biodistribution data from the laboratory with mean values published by other laboratories showed that soluble to moderately soluble forms of Am resulted in similar urine excretion after contamination, whether it was intravenous, intramuscular, subcutaneous injection or incision. Findings from this work will contribute to improve the understanding and interpretation of wound contamination cases with different physicochemical forms and mixtures of actinides including Am.

Analysis methodology and development of a statistical tool for biodistribution data from internal contamination with actinides.

The aim of this work was to develop a computational tool that integrates several statistical analysis features for biodistribution data from internal contamination experiments. These data represent actinide levels in biological compartments as a function of time and are derived from activity measurements in tissues and excreta. These experiments aim at assessing the influence of different contamination conditions (e.g. intake route or radioelement) on the biological behavior of the contaminant. The ever increasing number of datasets and diversity of experimental conditions make the handling and analysis of biodistribution data difficult. This work sought to facilitate the statistical analysis of a large number of datasets and the comparison of results from diverse experimental conditions. Functional modules were developed using the open-source programming language R to facilitate specific operations: descriptive statistics, visual comparison, curve fitting, and implementation of biokinetic models. In addition, the structure of the datasets was harmonized using the same table format. Analysis outputs can be written in text files and updated data can be written in the consistent table format. Hence, a data repository is built progressively, which is essential for the optimal use of animal data. Graphical representations can be automatically generated and saved as image files. The resulting computational tool was applied using data derived from wound contamination experiments conducted under different conditions. In facilitating biodistribution data handling and statistical analyses, this computational tool ensures faster analyses and a better reproducibility compared with the use of multiple office software applications. Furthermore, re-analysis of archival data and comparison of data from different sources is made much easier. Hence this tool will help to understand better the influence of contamination characteristics on actinide biokinetics. Our approach can aid the optimization of treatment protocols and therefore contribute to the improvement of the medical response after internal contamination with actinides.

Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells.

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (>10(40) cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients.

Research on the Radiotoxicology of Plutonium Using Animals: Consideration of the 3Rs-Replace, Reduce, Refine.

To characterize the health effects of incorporated plutonium, many experiments have been conducted using different animal models. These range from (1) applied (tissue uptake/retention determination, decorporation therapy efficacy), (2) fundamental (gene expression, cancer induction), and (3) dosimetry models. In recent years, the use of animals for scientific purposes has become a public concern. The application of the 3Rs - Replace (use of alternative methods or animals not considered capable of experiencing pain, suffering, and distress), Reduce (reduction in animal numbers), and Refine (better animal welfare and minimization of suffering, pain and distress) - has increased to address ethical concerns and legislative requirements. The introduction of novel non-animal technologies is also an important factor as complementary options to animal experimentation. In radiotoxicology research, it seems there is a natural tendency to Replace given the possibility of data reuse obtained from contamination cases in man and animal studies. The creation of "registries" and "repositories" for nuclear industry workers (civil and military) is now a rich legacy for radiotoxicological measurements. Similarly, Reduction in animal numbers can be achieved by good experimental planning with prior statistical analyses of animal numbers required to obtain robust data. Multiple measurements in the same animal over time (external body counting, excreta collection) with appropriate detection instruments also allow Reduction. In terms of Refinement, this has become "de rigueur" and a necessity given the societal and legal concerns for animal welfare. For research in radiotoxicology, particularly long-term studies, better housing conditions within the constraints of radiation protection issues for research workers are an important concern. These are all pertinent considerations for the 3Rs remit and future research in radiotoxicology.

The combined effects of xeroderma pigmentosum C deficiency and mutagens on mutation rates in the mouse germ line.

Spontaneous and induced mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germ line of xeroderma pigmentosum group C (Xpc) knockout mice defective in global genome nucleotide excision repair. Spontaneous and radiation-induced mutation rates in homozygous Xpc(-/-) males were significantly higher than those in isogenic wild-type (Xpc(+/+)) and heterozygous (Xpc(+/-)) mice. In contrast, exposure to the monofunctional alkylating agent ethylnitrosourea resulted in similar increases in ESTR mutation rates across all genotypes. ESTR mutation spectra in the germ line of Xpc(-/-), Xpc(+/-) and Xpc(+/+) did not differ. Considering these data and the results of other publications, we propose that the Xpc-deficient mice possess a mutator phenotype in their germ line and somatic tissues that may significantly enhance carcinogenesis across multiple tissues.

Long-term XPC silencing reduces DNA double-strand break repair.

To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA(KD) (knock down) and XPC(KD) cells were highly sensitive to UVC. DNA-PKcs(KD) and XRCC4(KD) cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPC(KD) cells also showed intolerance toward acute gamma-ray irradiation. We showed that XPC(KD) cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPC(KD) and DNA-PKcs(KD) cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair.

A quick and simple in vitro assay to predict bioavailability of actinides following accidental exposure.

Physicochemical properties of actinides highly influence internal intake and biodistribution. An a priori knowledge of the dissolution properties of compounds involved in accidental exposure would be of great help in early dose assessment. However, this information is rarely available, leading to difficulties in interpreting excretion data from contaminated victims. We developed an in vitro acellular assay to predict in vivo bioavailability of actinides and improve medical handling of the victims. Various actinides of different physicochemical properties were used to validate the reliability of the assay to mimic in vivo behavior of the contaminants. Our assay was designed as a dynamic muticompartmental system in which an agarose gel represents the retention compartment of actinides and a dynamic phase the transfer compartment. Relevant physiological conditions were obtained by introducing various components both in the static and dynamic phases. The proposed model may provide a good prediction of in vivo behavior and could be used as a first assessment to predict the fraction of actinides that could be potentially transferred from retention compartments, as well as the fraction available to chelating drugs.

Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein.

Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

A tandem of SH3-like domains participates in RNA binding in KIN17, a human protein activated in response to genotoxics.

The human KIN17 protein is an essential nuclear protein conserved from yeast to human and expressed ubiquitously in mammals. Suppression of Rts2, the yeast equivalent of gene KIN17, renders the cells unviable, and silencing the human KIN17 gene slows cell growth dramatically. Moreover, the human gene KIN17 is up-regulated following exposure to ionizing radiations and UV light, depending on the integrity of the human global genome repair machinery. Its ectopic over-expression blocks S-phase progression by inhibiting DNA synthesis. The C-terminal region of human KIN17 is crucial for this anti-proliferation effect. Its high-resolution structure, presented here, reveals a tandem of SH3-like subdomains. This domain binds to ribonucleotide homopolymers with the same preferences as the whole protein. Analysis of its structure complexed with tungstate shows structural variability within the domain. The interaction with tungstate is mediated by several lysine residues located within a positively charged groove at the interface between the two subdomains. This groove could be the site of interaction with RNA, since mutagenesis of two of these highly conserved lysine residue weakens RNA binding.

Actinide bioimaging in tissues: Comparison of emulsion and solid track autoradiography techniques with the iQID camera.

This work presents a comparison of three autoradiography techniques for imaging biological samples contaminated with actinides: emulsion-based, plastic-based autoradiography and a quantitative digital technique, the iQID camera, based on the numerical analysis of light from a scintillator screen. In radiation toxicology it has been important to develop means of imaging actinide distribution in tissues as these radionuclides may be heterogeneously distributed within and between tissues after internal contamination. Actinide distribution determines which cells are exposed to alpha radiation and is thus potentially critical for assessing absorbed dose. The comparison was carried out by generating autoradiographs of the same biological samples contaminated with actinides with the three autoradiography techniques. These samples were cell preparations or tissue sections collected from animals contaminated with different physico-chemical forms of actinides. The autoradiograph characteristics and the performances of the techniques were evaluated and discussed mainly in terms of acquisition process, activity distribution patterns, spatial resolution and feasibility of activity quantification. The obtained autoradiographs presented similar actinide distribution at low magnification. Out of the three techniques, emulsion autoradiography is the only one to provide a highly-resolved image of the actinide distribution inherently superimposed on the biological sample. Emulsion autoradiography is hence best interpreted at higher magnifications. However, this technique is destructive for the biological sample. Both emulsion- and plastic-based autoradiography record alpha tracks and thus enabled the differentiation between ionized forms of actinides and oxide particles. This feature can help in the evaluation of decorporation therapy efficacy. The most recent technique, the iQID camera, presents several additional features: real-time imaging, separate imaging of alpha particles and gamma rays, and alpha activity quantification. The comparison of these three autoradiography techniques showed that they are complementary and the choice of the technique depends on the purpose of the imaging experiment.

Skin absorption of actinides: influence of solvents or chelates on skin penetration ex vivo.

PURPOSE: To evaluate skin penetration and retention of americium (Am) and plutonium (Pu), in different chemical forms relevant to the nuclear industry and to treatment by chelation. MATERIALS AND METHODS: Percutaneous penetration of different Am and Pu forms were evaluated using viable pig skin with the Franz cell diffusion system. The behavior of the complex Pu-tributyl phosphate (Pu-TBP), Am or Pu complexed to the chelator Diethylene triamine pentaacetic acid (DTPA) and the effect of dimethyl sulfoxide (DMSO) was assessed. Radioactivity was measured in skin and receiver compartments. Three approaches were used to visualize activity in skin including the recent iQID technique for quantification. RESULTS: Transfer of Am was 24-fold greater than Pu and Pu-TBP complex penetration was enhanced by 500-fold. Actinide-DTPA transfer was greater than the Am or Pu alone (17-fold and 148-fold, respectively). The stratum corneum retained the majority of activity in all cases and both DMSO and TBP enhanced skin retention of Am and Pu, respectively. Histological and bioimaging data confirmed these results and the iQID camera allowed the quantification of skin activity. CONCLUSIONS: Skin penetration and fixation profiles are different depending on the chemical actinide form. Altered behavior of Pu-TBP and actinide-DTPA complexes reinforces the need to address decontamination protocols.

Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing.

We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPA(KD) and XPC(KD) (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced (HSA)KIN17. Several clones displaying undetectable protein levels of XPA, XPC, or (HSA)kin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, (HSA)KIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPA(KD) and XPC(KD) HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPC(KD) HeLa clones were more sensitive to UVC than their XPA(KD) or KIN17(KD) counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPA(KD) cells recovered a normal UVC sensitivity, XPC(KD) cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, (HSA)kin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.

Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

Several proteins involved in DNA synthesis are part of the so-called 'replication factories' that are anchored on non-chromatin nuclear structures. We report here that human kin17, a nuclear stress-activated protein, associates with both chromatin and non-chromatin nuclear structures in a cell cycle- and DNA damage-dependent manner. After L-mimosine block and withdrawal we observed that kin17 protein was recruited in the nucleus during re-entry and progression through S phase. These results are consistent with a role of kin17 protein in DNA replication. About 50% of the total amount of kin17 protein was detected on nuclear structures and could not be released by detergents. Furthermore, the amount of kin17 protein greatly increased in both G(1)/S and S phase-arrested cells in fractions containing proteins anchored to nuclear structures. The detection of kin17 protein showed for the first time its preferential assembly within non-chromatin nuclear structures in G(1)/S and S phase-arrested cells, while the association with these structures was found to be less stable in the G(2)/M phase, as judged by fractionation of human cells and immunostaining. In asynchronous growing cells, kin17 protein interacted with both chromatin DNA and non-chromatin nuclear structures, while in S phase-arrested cells it interacted mostly with non-chromatin nuclear structures, as judged by DNase I treatment and in vivo UV cross-linking. In the presence of DNA damage in S phase cells, the distribution of kin17 protein became mainly associated with chromosomal DNA, as judged by limited formaldehyde cross-linking of living cells. The physical interaction of kin17 protein with components of the nuclear matrix was confirmed and visualized by indirect immunofluorescence and immunoelectron microscopy. Our results indicate that, during S phase, a fraction of the human kin17 protein preferentially associates with the nuclear matrix, a fundamentally non-chromatin higher order nuclear structure, and to chromatin DNA in the presence of DNA damage.

Human kin17 protein directly interacts with the simian virus 40 large T antigen and inhibits DNA replication.

Kin17 is an evolutionarily conserved DNA-binding protein, which forms intranuclear foci in proliferating cells. Recent data have suggested that human kin17 protein is associated with cell proliferation and unrepaired DNA lesions. Herein, we show that human fibroblasts (MRC5-V2 and CHSV4) immortalized with SV40 overexpress endogenous kin17 protein, as compared with normal diploid human fibroblasts. We observed that certain carcinoma cell lines also up-regulated kin17 protein, suggesting that increased kin17 protein levels may be a consequence of the immortalized phenotype. We report here that the endogenous kin17 protein is located in nucleoplasmic foci and colocalizes with SV40 large T antigen. Purification of human kin17 protein allowed analysis of the physical interaction with T antigen by several in vitro and in vivo assays. Large T antigen and human kin17 protein are part of the same high molecular weight multiprotein complex in human cells. Furthermore, human kin17 protein interacts with T antigen bound to the SV40 DNA origin of replication. Strikingly, the overexpression of human kin17 protein in vivo and the introduction of increased amounts of human kin17 protein in an in vitro assay reduced T-antigen-dependent DNA replication, suggesting that kin17 protein may be involved in the DNA replication process in human cells.

In vitro assessment of plutonium uptake and release using the human macrophage-like THP-1 cells.

Plutonium (Pu) intake by inhalation is one of the major potential consequences following an accident in the nuclear industry or after improvised nuclear device explosion. Macrophages are essential players in retention and clearance of inhaled compounds. However, the extent to which these phagocytic cells are involved in these processes highly depends on the solubility properties of the Pu deposited in the lungs. Our objectives were to develop an in vitro model representative of the human pulmonary macrophage capacity to internalize and release Pu compounds in presence or not of the chelating drug diethylenetriaminepentaacetate (DTPA). The monocyte cell line THP-1 was used after differentiation into macrophage-like cells. We assessed the cellular uptake of various forms of Pu which differ in their solubility, as well as the release of the internalized Pu. Results obtained with differentiated THP-1 cells are in good agreement with data from rat alveolar macrophages and fit well with in vivo data. In both cell types, Pu uptake and release depend upon Pu solubility and in all cases DTPA increases Pu release. The proposed model may provide a good complement to in vivo animal experiments and could be used in a first assessment to predict the fraction of Pu that could be potentially trapped, as well as the fraction available to chelating drugs.

Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.

Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes.

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of (HSA)kin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.

Germline mutation rates at tandem repeat loci in DNA-repair deficient mice.

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of non-exposed and irradiated severe combined immunodeficient (scid) and poly(ADP-ribose) polymerase (PARP-1-/-) deficient male mice. Non-exposed scid and PARP-/- male mice showed considerably elevated ESTR mutation rates, far higher than those in wild-type isogenic mice and other inbred strains. The irradiated scid and PARP-1-/- male mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated wild-type isogenic males. ESTR mutation spectra in the scid and PARP-1-/- strains did not differ from those in the isogenic wild-type strains. Considering these data and the results of previous studies, we propose that a delay in repair of DNA damage in scid and PARP-1-/- mice could result in replication fork pausing which, in turn, may affect ESTR mutation rate in the non-irradiated males. The lack of mutation induction in irradiated scid and PARP-1-/- can be explained by the high cell killing effects of irradiation on the germline of deficient mice.

Combined drug and surgery treatment of plutonium-contaminated wounds: indications obtained using a rodent model.

There is an important requirement following accidental actinide contamination of wounds to limit the dissemination and retention of such alpha-emitting radionuclides. To reduce wound and systemic contamination, treatment approaches include chelation therapy with or without wound excision. However, it has been hypothesized that wound excision could lead to increased contaminant release and systemic organ retention. This study in the rat addresses this question. Anesthetized rats were contaminated with plutonium nitrate following wounding by deep incision of hind leg muscle. Excision of tissue at the contaminated site was performed 7 d later with or without Diethylene Triamine Pentaacetic Acid (DTPA) treatment (30 µmol kg⁻¹ i.v.). Pu urinary excretion was then measured for a further 3 d, and animals were euthanized at 14 d after contamination. Tissue samples were evaluated for Pu activity and histology. At 7 d after contamination, around 50% of the initial activity remained at the wound site. An average of 16% of this activity was then removed by surgery. Surgery alone resulted in increased urinary excretion, suggesting release from the wound site, but no subsequent increases in organ retention (bone, liver) were observed at 14 d. Indeed, organ Pu activity was slightly reduced. The combination of surgery and DTPA or DTPA treatment alone was much more effective than excision alone as shown by the markedly increased urinary Pu excretion and decreased tissue levels. This is the first report in an experimental rodent model of resection of Pu-contaminated wound. Urinary excretion data provide evidence for the release of activity as a result of surgery, but this does not appear to lead to further Pu organ retention. However, a combination of prior DTPA treatment with wound excision is particularly effective.