Molecular markers of radiation-related normal tissue toxicity

Over the past five decades, those interested in markers of radiation effect have focused primarily on tumor response. More recently, however, the view has broadened to include irradiated normal tissues-markers that predict unusual risk of side-effects, prognosticate during the prodromal and therapeutic phases, diagnose a particular toxicity as radiation-related, and, in the case of bioterror, allow for tissue-specific biodosimetry. Currently, there are few clinically useful radiation-related biomarkers. Notably, levels of some hormones such as thyroid-stimulating hormone (TSH) have been used successfully as markers of dysfunction, indicative of the need for replacement therapy, and for prevention of cancers. The most promising macromolecular markers are cytokines: TGFbeta, IL-1, IL-6, and TNFalpha being lead molecules in this class as both markers and targets for therapy. Genomics and proteomics are still in nascent stages and are actively being studied and developed.     

Sensitization to hyperthermia induced in a normal tissue by step-down heating

The effect of step-down heating was investigated in the skin of the CDF1 mouse foot. Step-down heating was induced with a 44.7 degrees C/10 min pretreatment followed by a test treatment at a lower temperature for variable time. Step-up heating, that is, a test treatment followed by a 44.7 degrees C/10 min treatment, and single heating were used as controls. The normal tissue reaction was scored at five levels of damage (from slight redness and oedema to loss of a toe or greater reaction), and the heating time to induce each level in 50% of the animals, RD50, was used as the endpoint. The effect of step-down heating was quantified by the step-down ratio, calculated as the ratio of test heating times to obtain the endpoint. A significant reduction of the RD50 was seen at all score levels when the 44.7 degrees C/10 min was given in a step-down heating schedule, and the effect increased with decreasing test treatment temperature. In contrast, the heat sensitivity was only marginally influenced by step-up heating. An analysis of the time-temperature relationship demonstrated a log-linear relationship between temperature and RD50 for single heating in the range 42.2-44.7 degrees C and for step-down heating in the range 41.7-44.7 degrees C. The curve for step-down heating showed a lesser slope indicating a decrease of the activation energy. The kinetics of the SDH effect were investigated by inserting an interval between a primary 44.7 degrees C/10 min treatment and a test treatment performed at 42.2 degrees C. The effect of step-down heating was maximal with no interval between the priming treatment and the test treatment. As the interval was increased to 1.5 hr the step-down sensitization disappeared, and with even longer intervals thermotolerance developed. From a clinical point of view, the present data indicate that step-down heating may increase the extent of both reversible and irreversible heat damage in the normal tissue.     

Genetic markers for prediction of normal tissue toxicity after radiotherapy

During the last decade, a number of studies have supported the hypothesis that there is an important genetic component to the observed interpatient variability in normal tissue toxicity after radiotherapy. This review summarizes the candidate gene association studies published so far on the risk of radiation-induced morbidity and highlights some recent successful whole-genome association studies showing feasibility in other research areas. Future genetic association studies are discussed in relation to methodological problems such as the characterization of clinical and biological phenotypes, genetic haplotypes, and handling of confounding factors. Finally, candidate gene studies elucidating the genetic component of radiation-induced morbidity and the functional consequences of single nucleotide polymorphisms by studying intermediate phenotypes will be discussed.     

Prospective prediction of radiotherapy induced normal tissue injuries

Variation of the normal tissue response to radiotherapy in animals of a given age, sex and species, and predictive analysis of the post radiotherapy normal tissue injuries for each case were reviewed. The degree of response of mouse hind legs to 25 Gy in a single dose varies from less than 20 Gy equivalent to more than 30 Gy equivalent injuries, implying that there still remains an unknown dose modifying factor whose modification factor is at large. It has been proposed to predict prospectively the post irradiation response of several normal tissues including the lung. Although, it is possible to predict the average degree of normal tissue injuries to a given dose, to predict the response of each case appears to be impossible.     

Apoptosis induced in vivo by photodynamic therapy in normal brain and intracranial tumour tissue

The apoptotic response of normal brain and intracranial VX2 tumour following photodynamic therapy (PDT) mediated by 5 different photosensitizers (Photofrin, 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX), chloroaluminium phthalocyanine (AlCIPc), Tin Ethyl Etiopurpurin (SnET(2)), and meta -tetra(hydroxyphenyl)chlorin (m THPC)) was evaluated following a previous analysis which investigated the necrotic tissue response to PDT at 24 h post treatment. Free DNA ends, produced by internucleosomal DNA cleavage in apoptotic cells, were stained using a TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling) assay. Confocal laser scanning microscopy (CLSM) was used to quantify the local incidence of apoptosis and determine its spatial distribution throughout the brain. The incidence of apoptosis was confirmed by histopathology, which demonstrated cell shrinkage, pyknosis and karyorrhexis. At 24 h post PDT, AlClPc did not cause any detectable apoptosis, while the other photosensitizers produced varying numbers of apoptotic cells near the region of coagulative necrosis. The apoptotic response did not appear to be related to photosensitizer dose. These results suggest that at this time point, a minimal and fairly localized apoptotic effect is produced in brain tissues, the extent of which depends largely on the particular photosensitizer.     

Genetic variants and normal tissue toxicity after radiotherapy: A systematic review

During the last decade, nearly 60 studies have addressed possible associations between various genetic sequence alterations and risk of adverse reactions after radiotherapy. We report here an overview of these studies with information on the genetic variants, tumour type, number of patients included, the endpoint studied, the mechanism(s) by which the candidate genes are involved in the pathogenesis of normal tissue toxicity, and odds ratios (ORs) for candidate variants. Though many positive results have been reported, inconsistent findings and non-replication of previous results have frequently occurred. This can presumably be attributed to certain methodological shortcomings including lack of statistical power to detect small effect sizes. Based on theoretical considerations and experiences from other scientific fields, we discuss how future studies should be designed in order to successfully unravel the genetics of normal tissue radiosensitivity. We propose a model of the allelic architecture that may underlie differences in normal tissue radiosensitivity. Genome wide association studies have proven a powerful tool to identify novel loci that affect various phenotypes. Nonetheless, genome wide association studies are extremely demanding in terms of sample size. Furthermore, certain limitations still relate to this kind of studies, emphasizing the need for international consortia such as the ESTRO GENEPI.     

Selective toxicity induced by picolinic acid in simian virus 40-transformed cells in tissue culture.

When cultured normal and SV40-transformed normal rat kidney and BALB/3T3 cells were exposed to picolinic acid, cell proliferation ceases. Most of the normal cells remained in a quiescent G1 (G0) state and viable for prolonged periods of time. In contrast, SV40-transformed cells progressed to the S and G2 phases of the cell cycle and remained viable only up to 90 to 120 hr. Then, most of the cells began to die. However, a very small fraction of the cell population (approximately 0.01 percent) developed into variants resistant to picolinic acid. Prevention of development of variants, and therefore destruction of all transformed cells, was obtained by addition of glycerol to picolinic acid-treated cells. Untransformed cells were unaffected by the same treatment. These results suggest that differential tumor toxicity should be feasible.     

Evaluation of pulmonary toxicity induced by pepleomycin

Spirometric parameters and transfer lung factor for carbon monoxide (TLCO) were determined in 14 cancer patients treated with pepleomycin at the dose of 10 mg twice weekly up to the cumulative dose of 200 mg. Mean values recorded after completion of therapy did not significantly differ from those recorded before treatment. Three patients pretreated with bleomycin had a fall in TLCO of more than 20% of pretreatment values. Three patients (2 of them pretreated with bleomycin) showed radiologic signs of pulmonary toxicity without instrumental signs of lung toxicity. In patients with no risk factors, no significant modifications in the tested parameters were observed after completion of therapy. These preliminary results suggest that, in the absence of risk factors, pepleomycin, may have no significant pulmonary toxicity at least up to the cumulative dose of 200 mg.     

Effect of surgery on normal tissue toxicity in patients treated with accelerated radiotherapy

The aim of this study was to assess the effect of surgery on normal tissue toxicity in head and neck cancer patients treated with accelerated radiotherapy. Toxicity data from two trials of accelerated radiotherapy were compared. The first group was taken from a phase III trial of definitive radiotherapy and the second group from a phase II trial of postoperative radiotherapy. The general eligibility criteria (apart from surgery), data collection and radiotherapy details for both trials were similar. The definitive group included 172 eligible patients and the postoperative group 52 eligible patients. At 3 weeks into treatment, by which time the dose and rate of dose accumulation were identical, there was no difference in acute toxicity. Analysis of late toxicity showed greater subcutaneous fibrosis in the postoperative group.     

Effect of hyperthermia on normal tissue toxicity and on adriamycin pharmacokinetics in dogs.

This study examined the effects of Adriamycin (ADR) (30 mg/m2), whole-body hyperthermia (WBH) (42 degrees C for 1 h), and the combination of the two (ADR plus WBH) on gastrointestinal and hematopoietic toxicity and the effects of WBH on ADR pharmacokinetics in the normal dog (n = 5/treatment group). Duodenal biopsies were collected from animals in each group via endoscopy and were incubated in the presence of [3H]thymidine as an index of cell turnover. Additional duodenal biopsies were assayed for the enzymes gamma-glutamyltranspeptidase, N-acetyl-beta-D-glucosaminidase, and succinate dehydrogenase. Complete blood chemistry profiles and differential blood cell counts were done prior to and following treatment. Cell turnover was most depressed 3 days after ADR or ADR plus WBH; WBH alone had little effect on cell turnover. Neither gamma-glutamyltranspeptidase, N-acetyl-beta-D-glucosaminidase, nor succinate dehydrogenase activities were significantly altered by any of the treatment protocols. High performance liquid chromatography was used to quantify Adriamycin and adriamycinol in samples collected up to 6 h after drug administration. Duodenal biopsies were collected immediately and 1 h after drug administration for measurement of tissue concentrations of Adriamycin. A significant increase in the apparent volume of distribution and whole-body clearance and decrease in area under the plasma Adriamycin concentration versus time curve occurred when drug was administered concurrently with WBH. This differs from results reported in some other mammalian species.     

Skin and perivascular toxicity induced experimentally by doxorubicin

Extravasation of antitumor drugs and particularly doxorubicin (DXR) can be followed by skin ulceration and slowly evolving perivascular necrosis. DXR lesions have some characteristics in common with those induced by ionizing radiation and, with respect to gross morphology, are reminiscent of skin lesions induced by necrotizing agents. Time course and histopathology of toxic phenomena induced by intradermal or perivascular injection of various doses of either DXR or caustic chemicals have been studied in hairy outbred and hairless inbred (MF1 hr/hr) mice. The latter strain has been found to be intrinsically more sensitive to DXR induced toxic effects, particularly as far as perivascular administration is concerned. Long lasting lesions and, in a few cases, systemic involvement have been observed. On the contrary, necrotic foci induced by caustic chemicals rapidly regressed in both strains. The perivascular administration model, which has not been previously investigated, appears to be representative of what happens in clinical conditions and can be of use for assessing either skin toxicity of antitumor compound or the protective effect of candidate antidotes.