Detection of genotoxic and non-genotoxic carcinogens in Xpc(-/-)p53(+/-) mice.

An accurate assessment of the carcinogenic potential of chemicals and pharmaceutical drugs is essential to protect humans and the environment. Therefore, substances are extensively tested before they are marketed to the public. Currently, the rodent two-year bioassay is still routinely used to assess the carcinogenic potential of substances. However, over time it has become clear that this assay yields false positive results and also has several economic and ethical drawbacks including the use of large numbers of animals, the long duration, and the high cost. The need for a suitable alternative assay is therefore high. Previously, we have proposed the Xpa*p53 mouse model as a very suitable alternative to the two-year bioassay. We now show that the Xpc*p53 mouse model preserves all the beneficial traits of the Xpa*p53 model for sub-chronic carcinogen identification and can identify both genotoxic and non-genotoxic carcinogens. Moreover, Xpc*p53 mice appear to be more responsive than Xpa*p53 mice towards several genotoxic and non-genotoxic carcinogens. Furthermore, Xpc*p53 mice are far less sensitive than Xpa*p53 mice for the toxic activity of DNA damaging agents and as such clearly respond in a similar way as wild type mice do. These advantageous traits of the Xpc*p53 model make it a better alternative for in vivo carcinogen testing than Xpa*p53. This pilot study suggests that Xpc*p53 mice are suited for routine sub-chronic testing of both genotoxic and non-genotoxic carcinogens and as such represent a suitable alternative to possibly replace the murine life time cancer bioassay.

DNA repair-deficient Xpa/p53 knockout mice are sensitive to the non-genotoxic carcinogen cyclosporine A: escape of initiated cells from immunosurveillance?

The DNA repair-deficient Xpa(-/-)p53(+/-) (Xpa/p53) mouse is a potent model for carcinogenicity testing, representing increased sensitivity toward genotoxic but surprisingly also toward true human non-genotoxic carcinogens. The mechanism of this increased sensitivity in Xpa/p53 mice toward non-genotoxic carcinogens is still unknown. Here, we investigated the mechanism of the human non-genotoxic carcinogen cyclosporine A (CsA) in the Xpa/p53 mouse model. Xpa/p53 mice exposed to CsA for 39 weeks showed a significantly increased lymphoma incidence as compared with untreated Xpa/p53 mice and CsA-treated wild-type (WT) mice. We excluded concealed genotoxicity of CsA in Xpa/p53 mice by mutant frequency analyses. As a next step, we used a genetic approach: immunodeficient DNA-PKcs mice, defective in the catalytic subunit of the DNA-dependent protein kinase, were crossed with Xpa and Xpa/p53 mice. Xpa/p53 mice had an increased lymphoma incidence with shorter latency times as compared with DNA-PKcs-deficient WT and Xpa mice. Surprisingly, also six of 15 DNA-PKcs/Xpa/p53 females had developed an adenocarcinoma of the mammary gland. Tumor responses in CsA-treated and DNA-PKcs-deficient Xpa/p53 mice were comparable as both genotypes developed mainly splenic lymphomas enriched in B lymphocytes. From our present studies, we hypothesize that levels of initiated precancerous cells are elevated in Xpa/p53 mice. These cells are insufficiently eliminated due to either suppression of the immune system by CsA or through immune-related DNA-PKcs deficiency. Based on the current studies and those conducted previously, we conclude that the Xpa/p53 model is an excellent adjunct to the current chronic rodent bioassay.

Tissue response to partially in vitro predegraded poly-L-lactide implants.

The in vivo local reaction of as-polymerized poly-L-lactide composed of 96% L-lactide and 4% D-lactide (PLA96) was investigated by histology at 2, 13 and 26 weeks after subcutaneous implantation in rats. In order to simulate possible end stage reactions the PLA96 was also predegraded in vitro until approximately 50% weight loss. The local reaction of predegraded PLA (PLA96(168)) was compared to the local reaction of polyethylene (PE) and non-predegraded PLA (PLA96). For PE and PLA96 a mild local reaction was observed at all time points consisting of a minimal layer of macrophage like cells with incidentally multinucleated giant cells at the implant interface, surrounded by a mild connective tissue capsule. For PLA96 at weeks 13 and 26 some minimal alterations in terms of degradation and ingrowth of cells was noted. The in vitro incubation (90 degrees C for 168 h) of PLA96(168) resulted for the thin 0.2 mm samples in complete degradation. Predegraded 0.5, 1.0 and 2.0 mm PLA96(168) samples were implanted and evaluated. The 1.0 and 2.0 mm samples could be evaluated for all time points investigated, but some 0.5 mm PLA96(168) samples were already completely resorbed at week 2 after implantation. In general, responses found for the predegraded PLA96(168) at weeks 2, 13 and 26 were similar with a pronounced macrophage infiltrate containing birefringent material, encapsulation of polymer fragments, and the presence of a debris area consisting of polymer and cellular remnants. In lymph nodes foamy macrophages with birefringent material were only observed in lymph nodes draining sites with predegraded PLA96(168). Immunohistochemistry was performed for further characterization of the cellular infiltrate. At the implant interface of the non-degrading PE and PLA96, ED1 and OX6 (MHC class II) positive cells were identified. In the capsule macrophage like cells expressed all three macrophage markers ED1, ED2, and ED3. CD4 and CD8 positive cells, indicating T helper and T supressor/cytotoxic cells, respectively, could be observed in low numbers, CD4 more than CD8. Both CD4 and CD8 were occasionally observed within the degrading PLA96(168) implant. Polymorphonuclear neutrophilic granulocytes were mainly observed at 2 weeks after implantation. We showed that predegradation could be used as a means to study late tissue reactions to polymers. Complete degradation may be studied with relatively thin implants, but this may lead to rather optimistic interpretation of resorption periods. When materials are intended to be used for screws and/or plates for bone fixation, implants of at least 1.0-2.0 mm thickness should be used as these may show a more realistic representation of the resorption characteristics of the material under investigation.

Slow accumulation of mutations in Xpc-/- mice upon induction of oxidative stress.

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa(-/-) and Xpc(-/-) exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc(-/-) mice, in contrary to Xpa(-/-) and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc(-/-) mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.

Life spanning murine gene expression profiles in relation to chronological and pathological aging in multiple organs.

Aging and age-related pathology is a result of a still incompletely understood intricate web of molecular and cellular processes. We present a C57BL/6J female mice in vivo aging study of five organs (liver, kidney, spleen, lung, and brain), in which we compare genome-wide gene expression profiles during chronological aging with pathological changes throughout the entire murine life span (13, 26, 52, 78, 104, and 130 weeks). Relating gene expression changes to chronological aging revealed many differentially expressed genes (DEGs), and altered gene sets (AGSs) were found in most organs, indicative of intraorgan generic aging processes. However, only ≤ 1% of these DEGs are found in all organs. For each organ, at least one of 18 tested pathological parameters showed a good age-predictive value, albeit with much inter- and intraindividual (organ) variation. Relating gene expression changes to pathology-related aging revealed correlated genes and gene sets, which made it possible to characterize the difference between biological and chronological aging. In liver, kidney, and brain, a limited number of overlapping pathology-related AGSs were found. Immune responses appeared to be common, yet the changes were specific in most organs. Furthermore, changes were observed in energy homeostasis, reactive oxygen species, cell cycle, cell motility, and DNA damage. Comparison of chronological and pathology-related AGSs revealed substantial overlap and interesting differences. For example, the presence of immune processes in liver pathology-related AGSs that were not detected in chronological aging. The many cellular processes that are only found employing aging-related pathology could provide important new insights into the progress of aging.

Broad segmental progeroid changes in short-lived Ercc1(-/Δ7) mice.

Genome maintenance is considered a prime longevity assurance mechanism as apparent from many progeroid human syndromes that are caused by genome maintenance defects. The ERCC1 protein is involved in three genome maintenance systems: nucleotide excision repair, interstrand cross-link repair, and homologous recombination. Here we describe in-life and post-mortem observations for a hypomorphic Ercc1 variant, Ercc1(-/Δ7), which is hemizygous for a single truncated Ercc1 allele, encoding a protein lacking the last seven amino acids. Ercc1(-/Δ7) mice were much smaller and median life span was markedly reduced compared to wild-type siblings: 20 and 118 weeks, respectively. Multiple signs and symptoms of aging were found to occur at an accelerated rate in the Ercc1(-/Δ7) mice as compared to wild-type controls, including a decline in weight of both whole body and various organs, numerous histopathological lesions, and immune parameters. Together they define a segmental progeroid phenotype of the Ercc1(-/Δ7) mouse model.