Normal Tissue Radiation Injury Research Articles

Biological sex differences in renin angiotensin system enzymes ACE and ACE2 regulate normal tissue response to radiation injury.

Introduction: In experimental animal models, biological sex-differences in the manifestation and severity of normal tissue radiation injury have been well-documented. Previously we demonstrated male and female rats have differential and highly reproducible responses to high-dose partial body irradiation (PBI) with male rats having greater susceptibility to both gastrointestinal acute radiation syndrome (GI-ARS) and radiation pneumonitis than female rats. Methods: In the current study, we have investigated whether differential expression of the renin-angiotensin system (RAS) enzymes angiotensin converting enzyme (ACE) and ACE2 contribute to the observed sex-related differences in radiation response. Results: During the period of symptomatic pneumonitis, the relative ratio of ACE to ACE2 (ACE/ACE2) protein in the whole lung was significantly increased by radiation in male rats alone. Systemic treatment with small molecule ACE2 agonist diminazene aceturate (DIZE) increased lung ACE2 activity and reduced morbidity during radiation pneumonitis in both sexes. Notably DIZE treatment also abrogated morbidity in male rats during GI-ARS. We then evaluated the contribution of the irradiated bone marrow (BM) compartment on lung immune cell infiltration and ACE imbalance during pneumonitis. Transplantation of bone marrow from irradiated donors increased both ACE-expressing myeloid cell infiltration and immune ACE activity in the lung during pneumonitis compared to non-irradiated donors. Discussion: Together, these data demonstrate radiation induces a sex-dependent imbalance in the renin-angiotensin system enzymes ACE and ACE2. Additionally, these data suggest a role for ACE-expressing myeloid cells in the pathogenesis of radiation pneumonitis. Finally, the observed sex-differences underscore the need for consideration of sex as a biological variable in the development of medical countermeasures for radiation exposure.

The Role of Nrf2 in the Response to Normal Tissue Radiation Injury

The transcription factor Nrf2 is an important modulator of antioxidant and drug metabolism, carbohydrate and lipid metabolism, as well as heme and iron metabolism. Regulation of Nrf2 expression occurs transcriptionally and post-transcriptionally. Post-transcriptional regulation entails ubiquitination followed by proteasome-dependent degradation. Additionally, Nrf2-mediated gene expression is subject to negative regulation by ATF3, Bach1 and cMyc. Nrf2-mediated gene expression is an important regulator of a cell's response to radiation. Although a majority of studies have shown that Nrf2 deficient cells are radiosensitized and Nrf2 over expression confers radioresistance, Nrf2's role in mediating the radiation response of crypt cells is controversial. The Nrf2 activator CDDO attenuates radiation-mediated crypt injury, whereas intestinal crypts in Nrf2 null mice are radiation resistant. Further investigation is needed in order to define the relationship between Nrf2 and radiation sensitivity in Lgr5+ and Bmi1+ cells that regulate regeneration of crypt stem cells. In hematopoietic compartments Nrf2 promotes the survival of irradiated osteoblasts that support long-term hematopoietic stem cell (LT-HSC) niches. Loss of Nrf2 in LT-HSCs increases stem cell intrinsic radiosensitivity, with the consequence of lowering the LD5030. An Nrf2 deficiency drives LT-HSCs from a quiescent to a proliferative state. This results in hematopoietic exhaustion and reduced engraftment after myoablative irradiation. The question of whether induction of Nrf2 in LT-HSC enhances hematopoietic reconstitution after bone marrow transplantation is not yet resolved. Irradiation of the lung induces pulmonary pneumonitis and fibrosis. Loss of Nrf2 promotes TGF-β/Smad signaling that induces ATF3 suppression of Nrf2-mediated target gene expression. This, in turn, results in elevated reactive oxygen species (ROS) and isolevuglandin adduction of protein that impairs collagen degradation, and may contribute to radiation-induced chronic cell injury. Loss of Nrf2 impairs ΔNp63 stem/progenitor cell mobilization after irradiation, while promoting alveolar type 2 cell epithelial-mesenchymal transitions into myofibroblasts. These studies identify Nrf2 as an important factor in the radiation response of normal tissue.

Addressing the Symptoms or Fixing the Problem? Developing Countermeasures against Normal Tissue Radiation Injury.

Addressing the Symptoms or Fixing the Problem? Developing Countermeasures against Normal Tissue Radiation Injury.

Mitigation of normal tissue radiation injury: evidence from rat radiation nephropathy models

Normal tissue radiation injury is a common complication of radiation therapy and is also a major concern after accidental or belligerent radiation exposure, and until recently, it was deemed untreatable. Both experimental and clinical evidence now show that radiation injury can be alleviated by agents started after irradiation but before manifestation of that injury (a therapeutic approach called “mitigation”). Mitigation of normal tissue injuries would improve clinical radiation therapy, and it will be an essential medical countermeasure for accidental or belligerent radiation exposures. In rat radiation nephropathy models over 30 potential mitigators have been tested, some (e.g., angiotensin converting enzyme inhibitors and angiotensin II receptor blockers) have been found to be quite effective, but many others appear ineffective, and a few have actually made injury worse. For the most part, work with the successful agents, and the agents that made radiation injury worse, is in the peer-reviewed literature. However, we have found it difficult to publish information on the agents that were ineffective, although we suspect that others have tried some of the same agents and also found them ineffective. Here, we review all agents we know of that have been tested to date in rat radiation nephropathy models, with the goal of helping to prevent needless duplication of studies.

Mitigation of experimental radiation nephropathy by renin-equivalent doses of angiotensin converting enzyme inhibitors

Purpose: We tested five different angiotensin converting enzyme inhibitors (ACEI) as mitigators of experimental radiation nephropathy at drug doses calibrated to the plasma renin activity (PRA). This was done to determine whether all ACEI had the same efficacy as mitigators of radiation nephropathy when used at drug doses that gave equivalent suppression of the renin angiotensin system.Method: 10 Gy total body irradiation with bone marrow transplantation was used to cause radiation nephropathy in barrier-maintained rats. Equivalent ACEI doses were determined based on their effect to inhibit angiotensin converting enzyme (ACE) and raise the PRA in unirradiated animals.Results: PRA-equivalent doses were found for captopril, lisinopril, enalapril, ramipril and fosinopril. These doses overlap the human doses of these drugs on a body surface area basis. All ACE inhibitors, except fosinopril, mitigated radiation nephropathy; captopril was a somewhat better mitigator than lisinopril, enalapril or ramipril.Conclusions: Most, but not all, ACEI mitigate radiation nephropathy at doses that overlap their clinically-used doses (on a body surface area basis). Fosinopril is known to be an ineffective mitigator of radiation pneumonitis, and it also does not mitigate radiation nephropathy. These pre-clinical data are critical in planning human studies of the mitigation of normal tissue radiation injury.

Chronic oxidative stress after irradiation: An unproven hypothesis

Injury and organ failure after irradiation of late-responding tissues is a substantial problem in radiation oncology and a major threat after accidental or belligerent exposures. The mechanisms of injury may include death of clonogens, vascular injury, activation of cytokine networks, and/or chronic oxidative stress. Knowledge of mechanisms may guide optimal use of mitigators. The hypothesis of chronic oxidative stress as a mechanism for late radiation injury has received much attention. We review herein the published evidence for chronic oxidative stress in vivo, and for use of antioxidants as mitigators of normal tissue radiation injury. We conclude that there is only indirect evidence for chronic oxidative stress after irradiation, and there are only limited published reports of mitigation by antioxidants. We did not find a differentiation of persistent markers of oxidative stress from an ongoing production of oxygen radicals. It is thus unproven that chronic oxidative stress plays a major role in causing radiation injury and organ failure in late-responding tissues. Further investigation is justified, to identify chronic oxidative stress and to identify optimal mitigators of radiation injury.

Variation in Telangiectasia Predisposing Genes Is Associated With Overall Radiation Toxicity

In patients receiving radiotherapy for breast cancer where the heart is within the radiation field, cutaneous telangiectasiae could be a marker of potential radiation-induced heart disease. We hypothesized that single nucleotide polymorphisms (SNPs) in genes known to cause heritable telangiectasia-associated disorders could predispose to such late, normal tissue vascular damage. The relationship between cutaneous telangiectasia as a late normal tissue radiation injury phenotype in 633 breast cancer patients treated with radiotherapy was examined. Patients were clinically assessed for the presence of cutaneous telangiectasia and genotyped at nine SNPs in three candidate genes. Candidate SNPs were within the endoglin (ENG) and activin A receptor, type II-like 1 (ACVRL1) genes, mutations in which cause hereditary hemorrhagic telangiectasia and the ataxia-telangiectasia mutated (ATM) gene associated with ataxia-telangiectasia. A total of 121 (19.1%) patients exhibited a degree of cutaneous telangiectasiae on clinical examination. Regression was used to examine the associations between the presence of telangiectasiae in patients who underwent breast-conserving surgery, controlling for the effects of boost and known brassiere size (n=388), and individual geno- or haplotypes. Inheritance of ACVRL1 SNPs marginally contributed to the risk of cutaneous telangiectasiae. Haplotypic analysis revealed a stronger association between inheritance of a ATM haplotype and the presence of cutaneous telangiectasiae, fibrosis and overall toxicity. No significant association was observed between telangiectasiae and the coinheritance of the candidate ENG SNPs. Genetic variation in the ATM gene influences reaction to radiotherapy through both vascular damage and increased fibrosis. The predisposing variation in the ATM gene will need to be better defined to optimize it as a predictive marker for assessing radiotherapy late effects.

Salen Mn Complexes Mitigate Radiation Injury in Normal Tissues

Salen Mn complexes, including EUK-134, EUK-189 and a newer cyclized analog EUK-207, are synthetic SOD/catalase mimetics that have beneficial effects in many models of oxidative stress. As oxidative stress is implicated in some forms of delayed radiation injury, we are investigating whether these compounds can mitigate injury to normal tissues caused by ionizing radiation. This review describes some of this research, focusing on several tissues of therapeutic interest, namely kidney, lung, skin, and oral mucosa. These studies have demonstrated suppression of delayed radiation injury in animals treated with EUK-189 and/or EUK-207. While an antioxidant mechanism of action is postulated, it is likely that the mechanisms of radiation mitigation by these compounds in vivo are complex and may differ in the various target tissues. Indicators of oxidative stress are increased in lung and skin radiation injury models, and suppressed by salen Mn complexes. The role of oxidative stress in the renal injury model is unclear, though EUK-207 does mitigate. In certain experimental models, salen Mn complexes have shown "mito-protective" properties, that is, attenuating mitochondrial injury. Consistent with this, EUK-134 suppresses effects of ionizing radiation on mitochondrial function in rat astrocyte cultures. In summary, salen Mn complexes could be useful to mitigate delayed radiation injury to normal tissues following radiation therapy, accidental exposure, or radiological terrorism. Optimization of their mode of delivery and other key pharmaceutical properties, and increasing understanding of their mechanism(s) of action as radiation mitigators, are key issues for future study.

Can cutaneous telangiectasiae as late normal-tissue injury predict cardiovascular disease in women receiving radiotherapy for breast cancer?

Background:Overall, ∼5% of patients show late normal-tissue damage after radiotherapy with a smaller number having a risk of radiation-induced heart disease. Although the data are conflicting, large studies have shown increased risks of cardiovascular disease (CVD) for irradiated patients compared with non-irradiated ones, or for those treated to the left breast or chest wall compared with those treated to the right. Cutaneous telangiectasiae as late normal-tissue injury have so far only been regarded as a cosmetic burden.Methods:The relationship between late normal-tissue radiation injury phenotypes in 149 irradiated breast cancer patients and the presence of cardiovascular disease were examined.Results:A statistically significant association between the presence of skin telangiectasiae and the long-term risk of CVD was shown in these patients (P=0.017; Fisher's exact test).Interpretation:This association may represent initial evidence that telangiectasiae can be used as a marker of future radiation-induced cardiac complications. It could also suggest a common biological pathway for the development of both telangiectasiae and CVD on the basis of a genetically predisposed endothelium. To our knowledge this is the first reported study looking at this association.

Mediators and Mechanisms of Radiation Nephropathy

Abstract. Normal tissue radiation injury occurs after sufficient irradiation, thus limiting the curative potential of x-ray therapy. In the kidney, radiation injury results in fibrosis and, ultimately, renal failure. The mediators of fibrosis in radiation nephropathy have received scant attention. Therefore, we evaluated the sequential presence of alpha smooth muscle actin (αsma), fibrin, collagen, and TGFβ1 in a porcine model of radiation nephropathy using 9.8 Gy single-dose local kidney irradiation. During the 24-week study, there was progressive and significant collagen accumulation in glomeruli and in interstitium. In glomeruli, this was associated with significant mesangial αsma expression by 2 weeks after irradiation, a further rise at 4 weeks, and then a gradual fall to baseline. Glomerular fibrin deposition was significant by 4 weeks after irradiation, and remained elevated thereafter. There was little or no glomerular TGFβ1 expression at any time point. Tubular fibrin deposition was significant at 4 weeks after irradiation but declined thereafter. There was little or no tubulo-interstitial αsma expression at any time after irradiation. At 6 weeks after irradiation, there was a significant peak of tubular epithelial TGFβ1 expression that declined thereafter. The early glomerular injury is evident as mesangial αsma expression but is not proceeded by TGFβ1 expression. There is sustained glomerular fibrin deposition with deposition of fibrin in tubular lumens, suggesting that tubular fibrin derives and flows out from injured glomerular tufts. We conclude that i) αsma expression is an early marker of glomerular radiation injury, presaging scarring; ii) fibrin deposition is involved in glomerular and tubular radiation injury; and iii) TGFβ1 is not an early event in radiation nephropathy, and not apparent in glomeruli in this model, but may correlate with later tubulo-interstitial fibrosis. Thus, the mediators of scarring in this model differ according to time after injury and also according to the affected tissue compartment.

Inactivation of thrombomodulin by ionizing radiation in a cell-free system: possible implications for radiation responses in vascular endothelium.

Normal tissue radiation injury is associated with loss of vascular thromboresistance, notably because of deficient levels of endothelial thrombomodulin (TM). TM is located on the luminal surface of most endothelial cells and has critical anticoagulant and anti-inflammatory functions. Chemical oxidation of a specific methionine residue (Met388) at the thrombin-binding site in TM reduces its main functional activity, i.e., the ability to activate protein C. We examined whether exposure to ionizing radiation affects TM in a similar manner. Full-length recombinant human TM, a construct of epidermal growth factor-like domains 4-6, which are involved in protein C activation, and a synthetic peptide containing the methionine of interest were exposed to gamma radiation in a cell-free system, i.e., a system not confounded by TM turnover or ectodomain shedding. The influence of radiation on functional activity was assessed with the protein C activation assay; formation of a TM-thrombin complex was assessed with surface plasmon resonance (Biacore), and oxidation of Met388 was assessed by HPLC and confirmed by mass spectroscopy. Exposure to radiation caused a dose-dependent reduction in protein C activation, impaired TM-thrombin complex formation, and oxidation of Met388. These results demonstrate that ionizing radiation adversely affects the TM molecule. Our findings may have relevance to normal tissue toxicity in clinical radiation therapy as well as to the development of radiation syndromes in the non-therapeutic radiation exposure setting.

Differential Radiation Effect in Tumor and Normal Tissue after Treatment with Ramipril, an Angiotensin-Converting Enzyme Inhibitor

The angiotensin-converting enzyme inhibitor, ramipril, has been shown to mitigate radiation injury in normal tissues. Using A549 cell xenografts grown in athymic mice, we measured the effect of ramipril on radiation damage to tumors. Ramipril did not alter tumor response to radiation despite different times of drug administration with respect to radiation delivery (drug started 2 weeks before or immediately after irradiation). In contrast, using the same dose, ramipril reduced normal tissue radiation injury (30 Gy x 2 or 6 Gy x 10) as assessed by a semi-quantitative scale of skin damage and relative leg contraction. The results indicate that ramipril could offer therapeutic gain due to its different effect on normal tissues and tumors.

The late radiotherapy normal tissue injury phenotypes of telangiectasia, fibrosis and atrophy in breast cancer patients have distinct genotype-dependent causes

The relationship between late normal tissue radiation injury phenotypes in 167 breast cancer patients treated with radiotherapy and: (i) radiotherapy dose (boost); (ii) an early acute radiation reaction and (iii) genetic background was examined. Patients were genotyped at single nucleotide polymorphisms (SNPs) in eight candidate genes. An early acute reaction to radiation and/or the inheritance of the transforming growth factor-β1 (TGFβ1 −509T) SNP contributed to the risk of fibrosis. In contrast, an additional 15 Gy electron boost and/or the inheritance of X-ray repair cross-complementing 1 (XRCC1) (R399Q) SNP contributed to the risk of telangiectasia. Although fibrosis, telangiectasia and atrophy, all contribute to late radiation injury, the data suggest that they have distinct underlying genetic and radiobiological causes. Fibrosis risk is associated with an inflammatory response (an acute reaction and/or TGFβ1), whereas telangiectasia is associated with vascular endothelial cell damage (boost and/or XRCC1). Atrophy is associated with an acute response, but the genetic predisposing factors that determine the risk of an acute response or atrophy have yet to be identified. A combined analysis of two UK breast cancer patient studies shows that 8% of patients are homozygous (TT) for the TGFβ1 (C-509T) variant allele and have a 15-fold increased risk of fibrosis following radiotherapy (95% confidence interval: 3.76–60.3; P=0.000003) compared with (CC) homozygotes.

Gene expression arrays as a tool to unravel mechanisms of normal tissue radiation injury and prediction of response

Over the past 5 years there has been a rapid increase in the use of microarray technology in the field of cancer research. The majority of studies use microarray analysis of tumor biopsies for profiling of molecular characteristics in an attempt to produce robust classifiers for prognosis. There are now several published gene sets that have been shown to predict for aggressive forms of breast cancer, where patients are most likely to benefit from adjuvant chemotherapy and tumors most likely to develop distant metastases, or be resistant to treatment. The number of publications relating to the use of microarrays for analysis of normal tissue damage, after cancer treatment or genotoxic exposure, is much more limited. A PubMed literature search was conducted using the following keywords and combination of terms: radiation, normal tissue, microarray, gene expression profiling, prediction. With respect to normal tissue radiation injury, microarrays have been used in three ways: (1) to generate gene signatures to identify sensitive and resistant populations (prognosis); (2) to identify sets of biomarker genes for estimating radiation exposure, either accidental or as a result of terrorist attack (diagnosis); (3) to identify genes and pathways involved in tissue response to injury (mechanistic). In this article we will review all (relevant) papers that covered our literature search criteria on microarray technology as it has been applied to normal tissue radiation biology and discuss how successful this has been in defining predisposition markers for radiation sensitivity or how it has helped us to unravel molecular mechanisms leading to acute and late tissue toxicity. We also discuss some of the problems and limitations in application and interpretation of such data.

Radiation injury and the protein C pathway.

To summarize current knowledge regarding the role of the thrombomodulin (TM)-protein C system in acute and chronic radiation responses in normal tissues. Studies published in the biomedical literature during the past three decades and cited in PubMed and unpublished clinical and laboratory data from our own research program. STUDY SUMMARY: The risk of injury to normal tissues limits the cancer cure rates that can be achieved with radiation therapy. Microvascular injury is a prominent feature of normal tissue radiation injury and plays a critical role in both acute (inflammatory) and chronic (fibrotic) radiation responses. Evidence from our own and other laboratories strongly suggests that dysfunction of the TM-protein C system plays a key role in the pathogenesis of radiation-induced injury to normal tissue. Exposure of normal tissues to ionizing radiation causes a pronounced, sustained deficiency of endothelial TM. This is likely to be due to a combination of initial inactivation of TM by reactive oxygen species, reduced transcription of TM, and release of TM into the circulation. Deficient levels of endothelial TM cause loss of local vascular thrombo-resistance, excessive activation of protease-activated receptor-1 by thrombin, and insufficient activation of protein C. These changes are presumed to be critically involved in many aspects of acute radiation toxicity and in sustaining the fibroproliferative processes that lead to chronic radiation-induced organ dysfunction and clinical complications. Injury of vascular endothelium may be key to the acute responses of normal tissues to ionizing radiation and to the progressive nature of chronic radiation fibrosis. Restitution of the TM-protein C pathway is an appealing strategy by which to prevent or treat normal tissue toxicity associated with radiation treatment of cancer.

Radiation toxicity and proteinase‐activated receptors

AbstractDespite recent technological advances, the cure rates that can be achieved with radiation therapy are limited by the risk of injury to the normal tissues that surround the tumor. This review summarizes current knowledge regarding proteinase‐activated receptors (PARs) and their possible role in the pathogenesis of normal tissue radiation injury. In particular, data are presented indicating that activation of PAR1 by thrombin, and PAR2 by trypsin and mast cell proteinases, may be involved both in acute radiation responses, as well as in the chronic fibroproliferative processes that lead to the development of radiation‐induced tissue fibrosis and clinical complications. If effective pharmacologic modulators of PAR1 or PAR2 were available, these compounds would be potentially useful as preventive or therapeutic agents in patients receiving radiation therapy, especially if blockade could be targeted to specific tissues or cellular compartments. Drug Dev. Res. 60:1–8, 2003. © 2003 Wiley‐Liss, Inc.

Prophylactic hyperbaric oxygen treatment and rat spinal cord re-irradiation

Normal tissue injury may lead to severe, life threatening, late side effects after therapeutic use of irradiation. Neurological complications caused by radiation of the spinal cord are ascribed to progressive, irreversible damage to the vasculature. Hyperbaric oxygen (HBO) is known to induce angiogenesis in irradiated tissue and has been proven to reduce late radiation injury in several normal tissues when applied during the latent period before complications become manifest. In the present study: (1) the prophylactic potential of HBO; (2) optimal timing of HBO therapy after spinal cord irradiation, i.e. during the latent period; and (3) effect of HBO on the re-irradiation tolerance of the spinal cord were investigated. The rat cervical spinal cord was locally X-ray irradiated with ten fractions of 6.5 Gy in 11 days. Five treatment groups ( n=10) included: irradiation alone and irradiation followed by 30 HBO treatments (100% oxygen at 240 kPa for 90 min) during latency, with HBO starting either immediately, 5, 10 or 15 weeks after the primary irradiation course. One year after the primary treatment, the same spinal cord volume was re-irradiated with 20 Gy single dose. During life span, the animals were observed on the incidence of myelitis and the duration of the latent period. The actuarial analysis revealed no significant difference in neurological complications free survival between the irradiation alone and the irradiation+HBO treatment groups. A tendency towards radiosensitization was found in the group in which the primary irradiation course was immediately followed by the HBO treatment course. The data show that HBO applied during the latent period of progressively developing irradiation damage to the spinal cord does not increase the re-irradiation tolerance of this tissue.

Mediators and mechanisms of radiation nephropathy.

Normal tissue radiation injury occurs after sufficient irradiation, thus limiting the curative potential of x-ray therapy. In the kidney, radiation injury results in fibrosis and, ultimately, renal failure. The mediators of fibrosis in radiation nephropathy have received scant attention. Therefore, we evaluated the sequential presence of alpha smooth muscle actin (alphasma), fibrin, collagen, and TGFbeta1 in a porcine model of radiation nephropathy using 9.8 Gy single-dose local kidney irradiation. During the 24-week study, there was progressive and significant collagen accumulation in glomeruli and in interstitium. In glomeruli, this was associated with significant mesangial alphasma expression by 2 weeks after irradiation, a further rise at 4 weeks, and then a gradual fall to baseline. Glomerular fibrin deposition was significant by 4 weeks after irradiation, and remained elevated thereafter. There was little or no glomerular TGFbeta1 expression at any time point. Tubular fibrin deposition was significant at 4 weeks after irradiation but declined thereafter. There was little or no tubulo-interstitial alphasma expression at any time after irradiation. At 6 weeks after irradiation, there was a significant peak of tubular epithelial TGFbeta1 expression that declined thereafter. The early glomerular injury is evident as mesangial alphasma expression but is not proceeded by TGFbeta1 expression. There is sustained glomerular fibrin deposition with deposition of fibrin in tubular lumens, suggesting that tubular fibrin derives and flows out from injured glomerular tufts. We conclude that i) alphasma expression is an early marker of glomerular radiation injury, presaging scarring; ii) fibrin deposition is involved in glomerular and tubular radiation injury; and iii) TGFbeta1 is not an early event in radiation nephropathy, and not apparent in glomeruli in this model, but may correlate with later tubulo-interstitial fibrosis. Thus, the mediators of scarring in this model differ according to time after injury and also according to the affected tissue compartment.

Finding our sensitive patients

The practice of radiation oncology requires balancing tumor control probability against the risk of normal tissue injury. Using radiation to eradicate disease is acceptable only if life-threatening or severe normal tissue injury is avoided. Despite a number of advances in the field of radiation oncology, the understanding of the genetic and epigenetic phenomenon that determine an individual’s risk for normal tissue radiation injury remains rudimentary. Patients display a diverse spectrum of toxicity for any given treatment schedule. While toxicity risks for populations of patients are known, the determination of an individual’s normal tissue radiosensitivity is rarely possible prior to treatment. Therefore, current practice standards commonly prescribe radiation dose schedules according to clinical scenarios, without regard to the genotype or phenotype of the individual being irradiated. Moving away from considering normal tissue risk of radiation injury as a population phenomenon towards determining the risk for the individual being irradiated is a worthy task. For patients with a radiosensitive genotype or phenotype, the risk-benefit ratio of radiation could be more clearly defined, and lower dosages or alternatives to radiation could be explored. Equally important, the ability to identify the most radiosensitive patients has the potential to improve the tumor control probability in the remaining population. In general, practice standards limit radiation dosages to those predicted to cause serious late complications in 10% or less of the treated population. Accordingly, this dose is below the threshold for radiation injury in the remaining 90%. A normal tissue predictive assay that successfully identified the 10% most radiosensitive patients would allow radiation dosages to be escalated in the remaining 90%. Tucker et al. have mathematically modeled this concept using a set of human fibroblast radiosensitivity data (1). Their results estimated that dose escalation of 10% would be possible if the identified radiosensitive patients were excluded from treatment. For clinical situations in which dose escalation would occur over the steep portion of the tumor control probability curve, this dose increase could achieve clinically meaningful results. Despite the theoretical appeal, normal tissue predictive assays have not proven clinically useful. The major limitation of laboratory-based normal tissue predictive assays is that most radiation complications result from a number of genetic and epigenetic phenomenon. Target cells have different intrinsic radiosensitivity and cellular environments dramatically affect radiation response. In addition, host factors that impair microvasculature (diabetes, atherosclerosis) or alter immune response (scleroderma, lupus) are known to predispose to radiation injury. The contributions of epigenetic factors and the cellular environment to radiation response are difficult to reproduce in laboratory settings. Correspondingly, most in vitro predictive assays have focused on studying the genetic predisposition of an individual to radiation injury. The excitement for this strategy arose after individuals with ataxia telangiectasia (AT) were found to have a radiosensitive phenotype. In vitro studies of fibroblasts and lymphocytes from these individuals revealed an exquisite normal tissue radiosensitivity that was directly attributable to a specific genetic defect. Based on this paradigm, a number of functional normal tissue predictive assays have been developed. These assays commonly use such cells as fibroblasts and lymphocytes to predict clinical injuries that develop independently of the cell type used in the assay. The theory underlying these assays is that germline factors affecting radiosensitivity will be present in any cell, and therefore fibroblasts and lymphocytes are appropriate surrogates for the target cell of the normal tissue injury. In this issue of the IJROBP, Crompton et al. report a pilot study using a new predictive assay based, in part, on this paradigm. Their study examines the value of a lymphocyte apoptosis assay in predicting radiation injury risk. The methodology of the study compares the degree of in vitro radiation-induced apoptosis of peripheral lymphocytes in a 12-patient radiosensitive cohort with age-matched controls and with a cohort of 9 patients with AT. Their results indicated a decreased rate of radiation-induced apoptosis in the AT and hypersensitive patients compared with controls. This result seems paradoxical, in that unlike fibroblast clo-

Modulation of radiation-induced apoptosis by lazaroids

Lazaroids (21-aminosteroids) are a novel class of compounds specifically designed to inhibit the iron-dependent lipid peroxidation associated with reperfusion injury. In the present study, we compared the effect of several lazaroids on radiation-induced apoptosis in explanted rat thymocytes and cultured murine lymphoma cells irradiated in vitro. An assay for the DNA fragmentation specific for apoptosis indicated that the lazaroids U-74500A, U-75412E, and U-74389F when dissolved in ethanol protected rat thymocytes and that U-75412E when dissolved in 10% cyclodextrin protected lymphoma cells. Additional experiments were conducted to ascertain which of the mechanisms associated with lazaroids were responsible for the inhibition of apoptosis. Other, nonsteroid inhibitors of lipid peroxidation—cysteamine, Desferal, and α-tocopherol—displayed no ability to inhibit apoptosis, suggesting that the steroid moiety of the lazaroid molecule was the active component. This was confirmed by showing that the nonglucocorticoid steroid testosterone inhibited radiation-induced apoptosis in these cell systems. Thus, lazaroids may inhibit apoptosis by interfering with specific functions of the cell membrane that are required for the manifestation of apoptosis and not by the inhibition of lipid peroxidation. We conclude that lazaroids may be useful agents for blocking radiation injury in normal tissues, but this will require confirmation in animal systems. Radiat Oncol Invest 1996;4:66–73. © 1996 Wiley-Liss, Inc.