Sally A. Lorimore

Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a ‘bystander’ effect which may contribute to radiation-induced genomic instability and leukaemogenesis.

Radiation-induced genomic instability and bystander effects: inter-related nontargeted effects of exposure to ionizing radiation

The paradigm of genetic alterations being restricted to direct DNA damage after exposure to ionizing radiation has been challenged by observations in which cells that are not exposed to ionizing radiation exhibit responses typically associated with direct radiation exposure. These effects are demonstrated in cells that are the descendants of irradiated cells (radiation-induced genomic instability) or in cells that are in contact with irradiated cells or receive certain signals from irradiated cells (radiation-induced bystander effects). There is accumulating evidence that radiation-induced genomic instability may be a consequence of, and in some cell systems may also produce, bystander interactions involving intercellular signalling, production of cytokines and free-radical generation. These processes are also features of inflammatory responses that are known to have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. Thus, radiation-induced genomic instability and untargeted bystander effects may reflect inter-related aspects of inflammatory-type responses to radiation-induced stress and injury and contribute to the variety of pathological consequences of radiation exposures.

Indirect Macrophage Responses to Ionizing Radiation: Implications for Genotype-Dependent Bystander Signaling

In addition to the directly mutagenic effects of energy deposition in DNA, ionizing radiation is associated with a variety of untargeted and delayed effects that result in ongoing bone marrow damage. Delayed effects are genotype dependent with CBA/Ca mice, but not C57BL/6 mice, susceptible to the induction of damage and also radiation-induced acute myeloid leukemia. Because macrophages are a potential source of ongoing damaging signals, we have determined their gene expression profiles and we show that bone marrow-derived macrophages show widely different intrinsic expression patterns. The profiles classify macrophages derived from CBA/Ca mice as M1-like (pro-inflammatory) and those from C57BL/6 mice as M2-like (anti-inflammatory); measurements of NOS2 and arginase activity in normal bone marrow macrophages confirm these findings. After irradiation in vivo, but not in vitro, C57BL/6 macrophages show a reduction in NOS2 and an increase in arginase activities, indicating a further M2 response, whereas CBA/Ca macrophages retain an M1 phenotype. Activation of specific signal transducer and activator of transcription signaling pathways in irradiated hemopoietic tissues supports these observations. The data indicate that macrophage activation is not a direct effect of radiation but a tissue response, secondary to the initial radiation exposure, and have important implications for understanding genotype-dependent responses and the mechanisms of the hemotoxic and leukemogenic consequences of radiation exposure.

Role of hepatic cytochrome p450s in the pharmacokinetics and toxicity of cyclophosphamide: studies with the hepatic cytochrome p450 reductase null mouse.

Cyclophosphamide (CPA) is an anticancer prodrug that is dependent on cytochrome P450 (CYP) metabolism for its therapeutic effectiveness. In spite of the use of CPA in the clinic for over 50 years, little is known about the relationship between its toxicokinetics and therapeutic response. We have employed a powerful new model, the Hepatic Cytochrome P450 Reductase Null (HRN) mouse, which has almost no hepatic cytochrome P450 activity, to study the toxicokinetics of CPA and to establish in vivo the role of hepatic P450 metabolism in its pharmacokinetics. In HRN mice the in vitro metabolism and intrinsic clearance of CPA was over 6-fold lower than in wild-type animals. This change in CPA metabolism was also reflected in vivo, with a profound difference in the pharmacokinetics of both CPA and its metabolites. At a CPA dose of 100 mg/kg, the Cmax, plasma area under the curve (AUC) and half-life were increased by 2.6-, 6.2-, and 3.2-fold, respectively, in the HRN mice. Similar changes were also observed at a dose of 300 mg/kg. These data confirm that hepatic metabolism is the major route of CPA elimination and disposition. The primary metabolites of CPA, 4-hydroxycyclophosphamide (4-OH-CPA) and 3-dechloroethylcyclophosphamide, were still formed, but at altered rates in the HRN mice. At 100 mg/kg the t1/2 for 4-OH-CPA was increased 1.8-fold, the Cmax reduced 1.7-fold, and the AUC remained unchanged. This latter finding shows that P450-mediated oxidative metabolism is essential for the clearance of this compound. Toxicokinetic analysis of CPA-induced myelosuppression and granulocytopenia showed that at high doses (> or =100 mg/kg) there was no difference in myelotoxicity between the wild-type and HRN mice. However, at lower doses (< or =70 mg/kg) a significant difference was observed, with little toxicity seen in HRN mice but at least a 45% reduction in the bone marrow granulocyte population in wild-type mice. Meta-analysis of the toxicity experiments showed the myelotoxicity of CPA was found to be closely correlated with the Cmax of 4-OH-CPA (r2= 0.80, P = 0.002). As the therapeutic effectiveness of CPA has been linked to the AUC for 4-OH-CPA, the finding that 4-OH-CPA Cmax may determine its level of myelotoxicity indicates that the therapeutic index could be altered by changing the method of CPA administration. Furthermore, monitoring 4-OH-CPA Cmax may identify individuals at most risk of CPA side effects.

In vivo chromosomal instability and transmissible aberrations in the progeny of haemopoietic stem cells induced by high- and low-LET radiations

Purpose : To study stable and unstable chromosomal aberrations in the haemopoietic cells of CBA/H mice after exposure to both high- and low-LET radiations. Materials and methods : Chromosomal aberrations were scored in the clonal progeny of X-, α - or non-irradiated short-term repopulating stem cells using the spleen colony-forming unit (CFU-S) assay, 12 days post-transplantation and in the bone marrow reconstituted by X-, neutron- or non-irradiated exogenous (transplanted) or endogenous (X- or neutron whole-body-irradiated) long-term repopulating stem cells for up to 24 months. Results : Chromosomal instability was demonstrated in 3-6% of cells in all cases. After transplantation of X- or neutron-irradiated bone marrow ~8% of cells with stable aberrations were recorded at all times. After 3 Gy X- or 0.5Gy neutron- whole-body irradiation stable aberrations were detected in ~17 and 5% of cells respectively. Conclusions : Chromosomal instability induced in vitro can be transmitted in vivo by transplantation of haemopoietic stem cells exposed to high- or low-LET radiations. Comparable instability can be induced and shown to persist for the remaining lifetime after whole-body irradiation. There was no direct relationship between the expression of stable and unstable aberrations and significant interanimal variation in the expression of both stable and unstable aberrations.

Chromosomal Instability in Unirradiated Hemaopoietic Cells Induced by Macrophages Exposed In vivo to Ionizing Radiation

The tumorigenic potential of ionizing radiation has conventionally been attributed to DNA damage in irradiated cells induced at the time of exposure. Recently, there have been an increasing number of reports of damage in unirradiated cells that are either neighbors or descendants of irradiated cells, respectively, regarded as bystander effects and genomic instability and collectively termed nontargeted effects. In this study, we show that descendants of normal murine hemaopoietic clonogenic stem cells exposed to bone marrow-conditioned medium derived from gamma-irradiated mice exhibit chromosomal instability unlike the descendants of directly gamma-irradiated cells. The instability is expressed in bone marrow cells of the radiation-induced acute myeloid leukemia (r-AML) susceptible strain (CBA/Ca) but not in mice resistant to r-AML (C57BL/6). Furthermore, crossgenetic experiments show the induction of the instability phenotype requires both the producer and responder cells to be of the susceptible CBA/Ca genotype. Macrophages are the source of the bystander signals, and the signaling mechanism involves tumor necrosis factor-alpha, nitric oxide, and superoxide. The findings show a genotype-dependent chromosomal instability phenotype induced by radiation-induced macrophage-mediated bystander signaling. As the majority of accidental, occupational, and therapeutic exposures to ionizing radiation are partial body exposures, the findings have implications for understanding the consequences of such exposure.

Tumour induction by methyl-nitroso-urea following preconceptional paternal contamination with plutonium-239

We have investigated the possibility that transgenerational effects from preconceptional paternal irradiation (PPI) may render offspring more vulnerable to secondary exposure to an unrelated carcinogen. 239Pu (0, 128 or 256 Bq g(-1)) was administered by intravenous injection to male mice, 12 weeks before mating with normal females. Two strains of mouse were used -- CBA/H and BDF1. Haemopoietic spleen colony-forming units (CFU-S) and fibroblastoid colony-forming units (CFU-F), a component of their regulatory microenvironment, were assayed independently in individual offspring at 6, 12 and 19 weeks of age. Bone marrow and spleen from each of these mice were grown in suspension culture for 2 or 7 days for assessment of chromosomal aberrations. Female BDF1 were injected with methyl-nitroso-urea (MNU) as a secondary carcinogen at 10 weeks of age and monitored for onset of leukaemia/lymphoma. Mean values of CFU-S and CFU-F were unaffected by preconceptional paternal plutonium-239 (PP-239Pu), although for CFU-F in particular there was an apparent increase in variation between individual animals. There was significant evidence of an increase in chromosomal aberrations with dose in bone marrow but not in spleen. By 250 days, 68% of MNU-treated control animals (no PPI) had developed thymic lymphoma (62%) or leukaemia (38%). The first case arose 89 days after MNU administration. In the groups with PPI, leukaemia/lymphoma developed from 28 days earlier, rising to 90% by 250 days. Leukaemia (65%) now predominated over lymphoma (35%). This second generation excess of leukaemia appears to be the result of PPI and may be related to inherited changes that affect the development of haemopoietic stem cells.

Chromosomal instability in unirradiated hemopoietic cells resulting from a delayed in vivo bystander effect of γ radiation

Untargeted effects of ionizing radiation (de novo effects in the unirradiated descendants or neighbors of irradiated cells) challenge widely held views about the mechanisms of radiation-induced DNA damage with implications for the health consequences of radiation exposures particularly in the context of the induction of malignancy. To investigate in vivo untargeted effects of sparsely ionizing (low linear energy transfer) radiation, a congenic sex-mismatch bone marrow transplantation protocol has been used to repopulate the hemopoietic system from a mixture of gamma-irradiated and nonirradiated hemopoietic stem cells such that host-, irradiated donor- and unirradiated donor-derived cells can be distinguished. Chromosomal instability in the progeny of irradiated hemopoietic stem cells accompanied by a reduction in their contribution to the repopulated hemopoietic system is consistent with a delayed genomic instability phenotype being expressed in vivo. However, chromosomal instability was also shown in the progeny of the nonirradiated hemopoietic stem cells implicating a bystander mechanism. Studies of the influence of irradiated recipient stromal microenvironment and experiments replacing irradiated cells with irradiated cell-conditioned medium reveal the source of the in vivo bystander effect to be the descendants of irradiated cells, rather than irradiated cell themselves. Thus, it is possible that a radiation-induced genomic instability phenotype in vivo need not necessarily be a reflection of intrinsically unstable cells but the responses to ongoing production of inflammatory-type damaging signals as a long-term unexpected consequence of the initial single radiation exposure.

Tissue-specific p53 responses to ionizing radiation and their genetic modification: the key to tissue-specific tumour susceptibility?

Although little is understood of the underlying mechanisms, there are tissue‐specific responses to tumourigenic and therapeutic agents and these responses are influenced by genetic factors. Ionizing radiation is an important tumourigenic and therapeutic agent for which there is substantial evidence for such tissue‐dependent and genotype‐dependent responses. Because the p53 tumour suppressor protein is a major determinant of cellular responses to radiation, the present study has investigated whether modification of the p53 pathway contributes to tissue‐dependent and genotype‐dependent responses using inbred strains of mice. Comparison of responses in haemopoietic and epithelial cells in irradiated C57BL/6 and DBA/2 mice revealed significant differences in p53 and apoptotic responses in different cell types and in different cells of the same type, reflecting the complexity of damage responses operating in the whole organism. The data suggest that p53‐mediated up‐regulation of Bax is a major determinant of apoptosis in the spleen, but not in the intestine, whereas p53‐mediated induction of p21waf1 plays an anti‐apoptotic role in the spleen, but not in the intestine. It is also shown that p53 stabilization and differential transactivational activities towards Bax or p21waf1 are influenced by genetic factors that act in a tissue‐specific manner. Analysis of ATM, a potential mediator of differential p53 activation, indicates that this key regulator of radiation responses is preferentially induced in epithelial cells, but is unlikely to account for genetic modification of p53 or apoptotic responses in the mouse strains studied. Polymorphisms in the p53 or DNA‐PKcs genes are also unlikely to account for the genetic modifications that are reported here. There are numerous further potential modifiers of the p53 pathway, but analysis of backcross and inter‐cross mice demonstrates that genes responsible for the complex modification of these in vivo responses can be identified by linkage analysis. This approach has the potential to reveal new or unexpected interactions involving the p53 pathway that determine both short‐term and long‐term effects of radiation exposure and the basis of tissue‐specific responses and tumour susceptibility. Copyright

Genetic Factors Influencing Bystander Signaling in Murine Bladder Epithelium after Low-Dose Irradiation In Vivo

Abstract Mothersill, C., Lyng, F., Seymour, C., Maguire, P., Lorimore, S. and Wright, E. Genetic Factors Influencing Bystander Signaling in Murine Bladder Epithelium after Low-Dose Irradiation In Vivo. Radiat. Res. 163, 391–399 (2005). Radiation-induced bystander effects occur in cells that are not directly hit by radiation tracks but that receive signals from hit cells. They are well-documented in vitro consequences of low-dose exposure, but their relevance to in vivo radiobiology is not established. To investigate the in vivo production of bystander signals, bladder explants were established from two strains of mice known to differ significantly in both short-term and long-term radiation responses. These were investigated for the ability of 0.5 Gy total-body irradiation in vivo to induce production of bystander signals in bladder epithelium. The studies demonstrate that irradiated C57BL/6 mice, but not CBA/Ca mice, produce bystander signals that induce apoptosis and reduce clonogenic survival in reporter HPV-G-transfected keratinocytes. Transfer of medium from explants established from irradiated animals to explants established from unirradiated animals confirmed these differences in bladder epithelium. The responses to the in vivo-generated bystander signal exhibit genotypic differences in calcium signaling and also in signaling pathways indicative of a major role for the balance of pro-apoptosis and anti-apoptosis proteins in determining the overall response. The results clearly demonstrate the in vivo induction of bystander signals that are strongly influenced by genetic factors and have implications for radiation protection, medical imaging, and radiotherapy.