Laminin 332 deposition is diminished in irradiated skin in an animal model of combined radiation and wound skin injury.

Skin exposure to ionizing radiation affects the normal wound healing process and greatly impacts the prognosis of affected individuals. We investigated the effect of ionizing radiation on wound healing in a rat model of combined radiation and wound skin injury. Using a soft X-ray beam, a single dose of ionizing radiation (10-40 Gy) was delivered to the skin without significant exposure to internal organs. At 1 h postirradiation, two skin wounds were made on the back of each rat. Control and experimental animals were euthanized at 3, 7, 14, 21 and 30 days postirradiation. The wound areas were measured, and tissue samples were evaluated for laminin 332 and matrix metalloproteinase (MMP) 2 expression. Our results clearly demonstrate that radiation exposure significantly delayed wound healing in a dose-related manner. Evaluation of irradiated and wounded skin showed decreased deposition of laminin 332 protein in the epidermal basement membrane together with an elevated expression of all three laminin 332 genes within 3 days postirradiation. The elevated laminin 332 gene expression was paralleled by an elevated gene and protein expression of MMP2, suggesting that the reduced amount of laminin 332 in irradiated skin is due to an imbalance between laminin 332 secretion and its accelerated processing by elevated tissue metalloproteinases. Western blot analysis of cultured rat keratinocytes showed decreased laminin 332 deposition by irradiated cells, and incubation of irradiated keratinocytes with MMP inhibitor significantly increased the amount of deposited laminin 332. Furthermore, irradiated keratinocytes exhibited a longer time to close an artificial wound, and this delay was partially corrected by seeding keratinocytes on laminin 332-coated plates. These data strongly suggest that laminin 332 deposition is inhibited by ionizing radiation and, in combination with slower keratinocyte migration, can contribute to the delayed wound healing of irradiated skin.

Lyman-Kutcher-Burman NTCP model parameters for radiation pneumonitis and xerostomia based on combined analysis of published clinical data.

Knowledge of accurate parameter estimates is essential for incorporating normal tissue complication probability (NTCP) models into biologically based treatment planning. The purpose of this work is to derive parameter estimates for the Lyman-Kutcher-Burman (LKB) NTCP model using a combined analysis of multi-institutional toxicity data for the lung (radiation pneumonitis) and parotid gland (xerostomia). A series of published clinical datasets describing dose response for radiation pneumonitis (RP) and xerostomia were identified for this analysis. The data support the notion of large volume effect for the lung and parotid gland with the estimates of the n parameter being close to unity. Assuming that n = 1, the m and TD(50) parameters of the LKB model were estimated by the maximum likelihood method from plots of complication rate as a function of mean organ dose. Ninety five percent confidence intervals for parameter estimates were obtained by the profile likelihood method. If daily fractions other than 2 Gy had been used in a published report, mean organ doses were converted to 2 Gy/fraction-equivalent doses using the linear-quadratic (LQ) formula with alpha/beta = 3 Gy. The following parameter estimates were obtained for the endpoint of symptomatic RP when the lung is considered a paired organ: m = 0.41 (95% CI 0.38, 0.45) and TD(50) = 29.9 Gy (95% CI 28.2, 31.8). When RP incidence was evaluated as a function of dose to the ipsilateral lung rather than total lung, estimates were m = 0.35 (95% CI 0.29, 0.43) and TD(50) = 37.6 Gy (95% CI 34.6, 41.4). For xerostomia expressed as reduction in stimulated salivary flow below 25% within six months after radiotherapy, the following values were obtained: m = 0.53 (95% CI 0.45, 0.65) and TD(50) = 31.4 Gy (95% CI 29.1, 34.0). Although a large number of parameter estimates for different NTCP models and critical structures exist and continue to appear in the literature, it is hard to justify the use of any single parameter set obtained at a selected institution for the purposes of biologically based treatment planning. Our expectation is that the proposed model parameters based on cumulative experience at various institutions are more representative of the overall practice of radiation therapy than any single-institution data, and could be more readily incorporated into clinical use.

Fast Monte Carlo simulation of DNA damage formed by electrons and light ions.

The passage of ionizing radiation through living organisms initiates physical and chemical processes that create clusters of damaged nucleotides within one or two turns of the DNA. These clusters are widely considered an important initiating event for the induction of other biological endpoints, including cell killing and neoplastic transformation. Monte Carlo simulations of the DNA damage formation process are a useful adjunct to experiments because they provide additional information about the spatial configuration of damage within a cluster. In this paper, the fast Monte Carlo damage simulation (MCDS) algorithm is re-parameterized so that yields of double-strand breaks, single-strand breaks and sites of multiple base damage can be simulated for electrons, protons and alpha particles with kinetic energies on the order of GeV. The MCDS algorithm provides a useful, quasi-phenomenological scheme to interpolate damage yields from computationally expensive, but more detailed, track-structure simulations. The predicted characteristics of various classes of damage produced by electrons, protons and alpha particles, such as average number of lesions per DNA damage cluster and cluster length in base pairs, are presented. A study examining the effects on damage complexity of an extrinsic free radical scavenger, dimethyl sulfoxide, is also presented. The reported studies provide new information that will aid efforts to characterize the relative biological effectiveness of high-energy protons and other light ions, which are sometimes used in particle therapy for the treatment of cancer.

Retrospective analysis of double-strand break rejoining data collected using warm-lysis PFGE protocols.

Sample preparation procedures for the pulsed-field gel electrophoresis (PFGE) assay usually involve a lysis step at temperatures as high as 50 degrees C. During this warm-lysis procedure, multiply damaged sites containing heat-labile sites (HLS) can be converted into double-strand breaks (DSB). Once formed, these DSB cannot be distinguished from the DSB formed directly by ionizing radiation. This paper develops a method to correct DSB estimates for the effects of HLS in warm-lysis protocols. A first-order repair model is used to predict the number of HLS available for conversion into DSB as a function of the time available for repair before initiating warm-lysis. A mathematical expression is derived to separate prompt DSB from those formed through the artefactual conversion of HLS into DSB. The proposed formalism only requires the specification of two adjustable parameters, both of which can be estimated from measured data. Estimates of prompt DSB yields obtained by correcting warm-lysis data are in good agreement with estimates obtained using cold-lysis protocols, which do not include the effect of HLS. The retrospective analyses of two published datasets suggest that corrections for HLS have a substantial impact on DSB yields within the first 20-30 min after irradiation. Bi-exponential fits to the DSB data for Chinese hamster ovary cells suggest that corrections for HLS reduce the half-time for fast DSB rejoining by about 15%, whereas the half-time for the slow DSB rejoining only decreases by 4%. The total DSB yield and the fraction of fast-rejoining DSB decrease by 24 and 38%, respectively, when the correction is applied. The proposed formalism can be used to characterize trends and uncertainties in DSB rejoining kinetics associated with the artefactual conversion of HLS into DSB. The retrospective application of the methodology to warm-lysis data enhances their relevance and usefulness for studies of DSB rejoining kinetics.

Monte Carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. I. Model properties and predicted trends.

DNA is constantly damaged through endogenous processes and by exogenous agents, such as ionizing radiation. Base excision repair (BER) and nucleotide excision repair (NER) help maintain the stability of the genome by removing many different types of DNA damage. We present a Monte Carlo excision repair (MCER) model that simulates key steps in the short-patch and long-patch BER pathways and the NER pathway. The repair of both single and clustered damages, except double-strand breaks (DSBs), is simulated in the MCER model. Output from the model includes estimates of the probability that a cluster is repaired correctly, the fraction of the clusters converted into DSBs through the action of excision repair enzymes, the fraction of the clusters repaired with mutations, and the expected number of repair cycles needed to completely remove a clustered damage site. The quantitative implications of alternative hypotheses regarding the postulated repair mechanisms are investigated through a series of parameter sensitivity studies. These sensitivity studies are also used to help define the putative repair characteristics of clustered damage sites other than DSBs.

Monte carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. II. Comparisons of model predictions to measured data.

Clustered damage sites other than double-strand breaks (DSBs) have the potential to contribute to deleterious effects of ionizing radiation, such as cell killing and mutagenesis. In the companion article (Semenenko et al., Radiat. Res. 164, 180-193, 2005), a general Monte Carlo framework to simulate key steps in the base and nucleotide excision repair of DNA damage other than DSBs is proposed. In this article, model predictions are compared to measured data for selected low-and high-LET radiations. The Monte Carlo model reproduces experimental observations for the formation of enzymatic DSBs in Escherichia coli and cells of two Chinese hamster cell lines (V79 and xrs5). Comparisons of model predictions with experimental values for low-LET radiation suggest that an inhibition of DNA backbone incision at the sites of base damage by opposing strand breaks is active over longer distances between the damaged base and the strand break in hamster cells (8 bp) compared to E. coli (3 bp). Model estimates for the induction of point mutations in the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene by ionizing radiation are of the same order of magnitude as the measured mutation frequencies. Trends in the mutation frequency for low- and high-LET radiation are predicted correctly by the model. The agreement between selected experimental data sets and simulation results provides some confidence in postulated mechanisms for excision repair of DNA damage other than DSBs and suggests that the proposed Monte Carlo scheme is useful for predicting repair outcomes.

A fast Monte Carlo algorithm to simulate the spectrum of DNA damages formed by ionizing radiation.

Ionizing radiation produces both singly and multiply damaged DNA sites. Multiply damaged sites (MDS) have been implicated in radiation-induced cell killing and mutagenesis. The spatial distribution of elementary damages (strand breaks and base damages) that constitute MDS is of special interest, since the complexity of MDS has an impact on damage repair. A fast and easy-to-implement algorithm to simulate the local clustering of elementary damages produced by ionizing radiation is proposed. This algorithm captures the major trends in the DNA damage spectrum predicted using detailed track- structure simulations. An attractive feature of the proposed algorithm is that only four adjustable parameters need to be identified to simulate the formation of DNA damage. A convenient recipe to determine the parameters used in the fast Monte Carlo damage simulation algorithm is provided for selected low- and high-LET radiations. The good agreement among the damage yields predicted by the fast and detailed damage formation algorithms suggests that the small-scale spatial distribution of damage sites is determined primarily by independent and purely stochastic events and processes.

NOREC, a Monte Carlo code for simulating electron tracks in liquid water.

The work reported here was originally motivated by a discussion of Monte Carlo computer codes for electron transport in water given in Report No. 130 by the National Council on Radiation Protection and Measurements (NCRP). It was pointed out (correctly) that a published depth-dose distribution calculated by the Oak Ridge electron transport code, OREC, for 800 keV electrons normally incident on a water slab was apparently in error, possibly due to inadequate treatment of elastic scattering. In this paper we describe the replacement of the original OREC elastic cross sections by current ones from the National Institute of Standards and Technology (NIST). This investigation led also to the critical examination and revision of some other parts of the program, as described here. The revised code, which we have renamed NOREC, represents the first substantial review and modification of the Oak Ridge code in a number of years. We also present some comparisons of results calculated with the old and new versions and discuss their implications with respect to earlier studies. We have also written a version of NOREC in C++ language, which is available to other investigators. This paper provides a record of a response to the NCRP published statement and documentation for the revised code, NOREC.