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Background: Hemorrhagic cystitis from chemical or radiation bladder injury can cause life-threatening bleeding and/or urothelial cancer. Fibroblast growth factor 7 (FGF7) blocks urothelial apoptosis induced by cyclophosphamide (CPP) or radiation, but there are limitations to its use in patients, including an inability to apply it by direct bladder infusion and the high cost due to the large size of the protein (18.8 kDa). Purpose: We previously identified a small peptide that equaled or exceeded the mitigation effect of full length FGF2 on acute radiation syndrome. Benefits of developing a similar FGF7 biomimetic peptide include lower likelihood of inflammation, longer shelf life, higher purity, and lower cost than the full-length protein. Methods: Based on the 3-dimensional structure of FGF2 peptide, a corresponding FGF7 peptide (FGF7p) consisting of 19 amino acids was identified and synthesized. FGF7p or vehicle was given subcutaneously (SQ) to female mice subjected to sham injury, intraperitoneal (IP) CPP or external beam radiation over the bladder. One day after injury, bladders were harvested. Slides with paraffin embedded tissues underwent H&E staining, TUNEL and immunofluorescence (IF) assays. Results: In uninjured control mice, a 20 mg/kg threshold dose of FGF7p induced expression of phosphorylated (activated) FRS2a (pFRS2a) and pAKT in urothelium (consistent with cytoprotective effects of FGF7, albeit at a 4x higher dose than full length FGF7). Unexpectedly, FGF7p activation of urothelial FRS2a and AKT was delayed 24 hours compared to FGF7. FGF7p (20 mg/kg) or vehicle was given at 72 and 48 hours prior to CPP (150 mg/kg). One day after CPP, TUNEL staining revealed an increase in apoptotic and sloughing urothelial cells in vehicle-treated mice compared to in FGF7p-treated mice. IF for pAKT and its targets, pS6K and pBAD, revealed minimal staining in vehicle-treated mice, but strong urothelial staining for all protective markers in FGF7p-treated mice. Using the same dosing strategy, we subjected anesthetized mice to 10 Gy radiation over the bladder. One day after injury, TUNEL staining revealed many more apoptotic urothelial nuclei in control mice, than in FGF7p-treated mice. Conclusions: FGF7p appears to block bladder urothelial apoptosis via AKT and its targets, in a similar manner to FGF7. FGF7p is 200x less expensive to make vs. FGF7 and is likely to work via direct bladder infusion (avoiding systemic side effects) due to its small size. Our future studies will clarify long term benefits of FGF7p and assess effectiveness of direct bladder infusion. Citation Format: Sridhar Narla, Lori P. Rice, David Ostrov, Steven G. Swarts, Dietmar W. Siemann, Carlton M. Bates. FGF7 peptide (FGF7p) mimetic mitigates urothelial injury from cyclophosphamide or radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5431.

Molecular products resulting from the reaction of radiation-produced dry and aqueous electrons with DNA hydrated between 2.5 and 367 waters per nucleotide (Γ) are quantified in this work. Specifically, the yields of the 5,6-dihydropyrimidines (formed from the reaction of electrons with thymine and cytosine) and molecular hydrogen (H2) (formed in the reaction of intra-spur electrons) have been studied. We find the yields for the 5,6-dihydropyrimidines are highest (41–108 nmol/J) and the yields of hydrogen lowest (14–18 nmol/J) at low hydrations (Γ Γ = 32 . This result would suggest that the H2 produced in the irradiated hydrated DNA is derived mainly from intra-spur reactions in bulk water. However, an analysis of the H2 G-values calculated based on the dry mass of DNA (i.e., an analysis of the contribution of the water alone to the H2 yields) suggests that H2 produced in irradiated hydrated DNA is derived not only from intra-spur reactions in bulk water (44–45 nmol/J) but also from DNA (61 nmol/J). This is an interesting finding that suggests fully hydrated DNA (Γ>33) forms H2 in competition with dihydropyrimidines.

ESR spectroscopy at low temperatures is employed to investigate electron transfer within DNA doped with randomly spaced electron traps. The traps were introduced by careful bromination of DNA in ice-cooled aqueous solution. The procedure is shown by NMR and GC/MS techniques to modify thymine, cytosine, and guanine 2‘-deoxyribosides, transforming them into 5-bromo-6-hydroxy-5,6-dihydrothymine, T(OH)Br, 5-bromocytosine, CBr, and 8-bromoguanine, GBr, derivatives. The bromination products formed in molar ratio close to T(OH)Br/CBr/GBr = 0.2:1:0.23 and serve as internal electron scavengers on γ-irradiation. Paramagnetic products that result from electron scavenging in DNA by T(OH)Br and CBr units at 77 K have been identified by ESR as the 6-hydroxy-5,6-dihydrothymin-5-yl (TOH•) radical and the 5-bromocytosine σ* radical anion, CBr•-. Our quantitative estimates show that electron scavenging by T(OH)Br in bromine-doped DNA is over an order of magnitude more efficient than the more abundant CBr traps. This indica...

Gas chromatography/mass spectrometry (GC/MS-SIM) is an excellent technique for performing both qualitative and quantitative analysis of DNA base damage products that are formed by exposure to ionizing radiation or by the interaction of intracellular DNA with activated oxygen species. This technique commonly uses a hot formic acid hydrolysis step to degrade the DNA to individual free bases. However, due to the harsh nature of this degradation procedure, the quantitation of DNA base damage products may be adversely affected. Consequently, we examined the effects of various formic acid hydrolysis procedures on the quantitation of a number of DNA base damage products and identified several factors that can influence this quantitation. These factors included (1) the inherent acid stabilities of both the lesions and the internal standards; (2) the hydrolysis temperature; (3) the source and grade of the formic acid; and (4) the sample mass during hydrolysis. Our data also suggested that the N,O-bis (trimethylsilyl)trifluoroacetamide (BSTFA) derivatization efficiency can be adversely affected, presumably by trace contaminants either in the formic acid or from the acid activated surface of the glass derivatization vials. Where adverse effects were noted, modifications were explored in an attempt to improve the quantitation of these DNA lesions. Although experimental steps could be taken to minimize the influence of these factors on the quantitation of some base damage products, no single procedure solved the quantitation problem for all base lesions. However, a significant improvement in the quantitation was achieved if the relative molecular response factor (RMRF) values for these lesions were generated with authentic DNA base damage products that had been treated exactly like the experimental samples.

Quercetin, a plant-derived aglycone form of flavonoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of quercetin. The reduction potential of quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monocytic leukemia cells was determined by flow cytometry using CM-H2DCFDA dye. The results were compared with curcumin, a natural product exhibiting a similar range of reported health benefits.1) Quercetin has a higher reduction potential compared with curcumin at three different pH settings and is comparable to Trolox at pH 7-9.5; 2) its TAC is 3.5 fold higher than curcumin; 3) it reduced LPS-induced ROS to near normal levels; 4) it reduced LPS-induced NO production. These data provide a physico-chemical basis for comparing antioxidants, with potential benefits individually or in combination.

Fifty-eight semicarbazone and pyrazole derivatives of curcumin have been developed as potential mitigation agents to treat acute radiation syndrome (ARS). Pyridyl (D12, D13), furyl (D56), and phenyl (D68) derivatives of curcumin semi-carbazones were found to provide the highest dose modifying factors (DMF) with respect to survival in sub-TBI (bone marrow sparing) exposures in mouse models. To investigate the basis for the mitigating effects of these agents on ARS, we examined their oxidation potentials and radical scavenging properties in comparison to other semicarbazone and pyrazole curcumin derivatives with less effective DMFs. Comparisons between D12, D13, D56, and D68 and other semicarbazone and pyrazole derivatives of curcumin did not show a sufficient difference in reducing properties and hydrogen atom donating properties for these properties to be the basis of the dose modifying activities of these compounds. Therefore, their DMFs likely reflect structure-activity relationship(s),wherein interaction with key receptors or alteration of enzyme expression result in modifications of cellular or tissue responses to radiation, rather than on the derivatives' ability to modify radiation-induced flux of free radicals through direct interaction with these radicals.

The radiosensitivities and the kinetics for removal of radiation-induced DNA damage were compared for proliferative (P) and quiescent (Q) cells of the lines 66 and 67 derived from a mouse mammary adenocarcinoma. As determined from cell survival assays, the 66 and 67 Q cells were more radiosensitive than their 66 and 67 P counterparts. The rank order of their radiosensitivity was: 67 Q greater than 66 Q greater than or equal to 67 P greater than 66 P. Induction of radiation damage in the DNA of these cells, as measured by the alkaline elution technique, was identical for 66 and 67 P and Q cells. The repair of this DNA damage was biphasic for 66 and 67 P and Q cells. The half-times for the fast and slow repair phases in 66 Q cells were identical to those previously measured in 67 Q cells. The half-times of the fast and slow repair phases in 66 P cells were also identical to those previously measured in 67 P cells. However, the half-times for the fast and slow repair phases in 66 and 67 Q cells were longer than those measured in their 66 and 67 P counterparts. The 66 cell data are consistent with our previously published hypothesis that Q cells are more radiosensitive than their corresponding P cells because they repair their radiation-induced DNA damage slower. However, our results are not consistent with hypotheses that attempt to explain the radiosensitivity differences between lines 66 and 67 solely on the basis of measurable induction and repair of DNA damage.

Past work using the human-hamster hybrid line GM10115 has demonstrated that exposure to a variety of DNA damaging agents can lead to the persistent destabilization of chromosomes. To gain insight into the potential biochemical mechanisms involved in perpetuating the unstable phenotype, groups of clones characterized as stable or unstable were analyzed for indications of oxidative stress. All of the clones were derived from single progenitor cells surviving exposure to ionizing radiation or chemicals. Compared with their stable counterparts, unstable clones possessed elevated levels of reactive oxygen species (ROS) as measured by their enhanced ability to oxidize fluorogenic dyes. Fluorescence automated cell sorting analysis indicated that unstable clones had significantly higher mean fluorescence signals of approximately 2-fold and approximately 1.25-fold, respectively, as derived from the dyes 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, respectively. To determine whether mitochondria might constitute a potential source of ROS, stable and unstable clones of cells were analyzed for mitochondrial content using nonyl acridine orange and function using rhodamine 123. Fluorescence automated cell sorting data indicated that compared with stable clones, unstable clones possessed an elevated number (15% increase in mean nonyl acridine orange fluorescence) of dysfunctional mitochondria (27% decrease in mean rhodamine 123 fluorescence). Interestingly, the consequences of elevated ROS did not translate to an increase in oxidative base damage in nuclear DNA. Analysis of nine different base damage adducts by gas chromatography/mass spectrometry did not reveal significant differences between stable and unstable clones. The data suggest that the perpetuation of many of the abnormal phenotypes associated with genomic instability may be linked to a state of chronic oxidative stress derived in part from dysfunctional mitochondria.

Little computational or experimental information is available on site-specific hydroxyl attack probabilities to DNA. In this study, an atomistic stochastic model of OH radical reactions with DNA was developed to compute relative OH attack probabilities at individual deoxyribose hydrogen atoms. A model of the self-complementary decamer duplex d(CCAACGTTGG) was created including Na(+) counter ions and the water molecules of the first hydration layer. Additionally, a method for accounting for steric hindrance from nonreacting atoms was implemented. The model was then used to calculate OH attack probabilities at the various C-H sites of the sugar moiety. Results from this computational model show that OH radicals exhibit preferential attack at different deoxyribose hydrogens, as suggested by their corresponding percentage solvent-accessible surface areas. The percentage OH attack probabilities for the deoxyribose hydrogens [1H(5')+2H(5'), H(4'), H(3'), 1H(2')+2H(2'), H(1')] were calculated as approximately 54.6%, 20.6%, 15.0%, 8.5% and 1.3%, respectively, averaged across the sequence. These results are in good agreement with the latest experimental site-specific DNA strand break data of Balasubramanian et al. [Proc. Natl. Acad. Sci. USA 95, 9738-9742 (1998)]. The data from this stochastic model suggest that steric hindrance from nonreacting atoms significantly influences site-specific hydroxyl radical attack probabilities in DNA. A number of previous DNA damage models have been based on the assumption that C(4') is the preferred site, or perhaps the only site, for OH-mediated DNA damage. However, the results of the present study are in good agreement the experimental results of Balasubramanian et al. in which OH radicals exhibit preferential initial attack at sugar hydrogen atoms in the order 1H(5')+2H(5')H(4')H(3')1H(2')+2H(2')H(1').

Purpose: One of the most confounding issues in the treatment of cancer is that two patients with the same diagnosis respond differently to the same treatment. The ultimate goal of this research effort is to better understand why this occurs. As a first step, the study presented here is performed to develop a system of computer codes to model the complex reactions between ionizing radiation and DNA. Method and Materials: A mathematical atomistic model of a 167 base-pair B-DNA molecule was constructed using commercially available software packages. The probability of each possible fate of an •OH approaching to this 167 base-pair molecule was determined by implementing the “near-approach” DNA-radiation chemistry computational model described by Aydogan et al. (Radiat Res 157(1), 38–44, 2002) and compiled into an outcomes database. The secondary electron spectrum generated at depth in tissue by a Co-60 beam was modeled using the code MCNP 4.B. Two microdosimetry codes, OREC and RADLYS, originally developed at Oak Ridge National Laboratory, were adapted for this research. Attack sites were logged and single strand break (ssb) and double strand break (dsb) rates were calculated. Results: At a dose of 100 Gy, the concentration scaled dsb per molecule was only 0.4 which is in good agreement with the dsb data which is 0.43 dsb/ molecule from the computational effort described in this report. Conclusion: A Monte Carlo calculation model has been shown to simulate single and double strand breaks using a novel computational approach presented authors previously that reduces the computation time and eliminates unnecessary repetition of the computationally expensive near chemistry simulation in DNA damage. Experimental comparison of the single and double strand break yields calculated in this study have shown the accuracy of the DNA damage simulation model presented.

The induction of base damage products in γ-irradiated DNA, hydrated between 2.5 and 32.8 moles of water per mole of nucleotide (Γ), was investigated using the gas chromatography/mass spectrometry-selected ion monitoring technique. In general, the yields of the measured base damage products were found to be dependent on the extent of the hydration when the DNA was irradiated under nitrogen. At low hydrations (Γ ≤ 13), the highest yields of the measured products were found for 7,8-dihydro-8-oxo-guanine, 5,6-dihydrothymine and, to a lesser extent, 2,6-diamino-4-oxo-5-formamidopyrimidine, products which are consistent with the base radicals found in low-temperature ESR studies. At higher hydrations (Γ ≤ 13), changes in DNA conformation and an increase in the attack of bulk water radicals on DNA play a significant role in the formation of radiation-induced DNA base damage products. Additional findings in our study include: (1) the sum of the yields of the products formed from electron-loss centers is greater t...

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (Γ), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with Γ = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of Γ was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with Γ between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with Γ > 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried D...