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Previous research has identified variation in cancer cell line response to high levels of extracellular H2O2 (eH2O2) exposure. This directly contributes to our understanding cellular efficacy of pharmacological ascorbate (P-AscH-) therapy. Here we investigate the factors contributing to latency of peroxisomal catalase of a cell and the importance of latency in evaluating cell exposure to eH2O2. First, we develop a mathematical framework for the latency of catalase in terms of an effectiveness factor, ηeff, to describe the catalase activity in the presence of high levels of eH2O2. A simplified relationship emerges, [Formula: see text] when mprp/Dij≪1, where mp,rp, and [Formula: see text] are the experimentally determined peroxisome permeability, average peroxisome radius, and the pseudo first-order reaction rate constant, respectively. [Formula: see text] is the catalase concentration in the peroxisome and k2=1.7x107M-1s-1. Next, previously published parameters are used to determine the latency effect of the cell lines: normal pancreatic cells (H6c7), pancreatic cancer cells (MIA PaCa-2), and glioblastoma cells (LN-229, T98G, and U-87), all which vary in their susceptibility to exposure to high eH2O2. The results show that effectiveness is not significantly different except for the most susceptible, MIA PaCa-2 cell line, which is higher when compared to all other cell lines. This result is counterintuitive and further implies that latency, as a single parameter, is ineffective in forecasting cell line susceptibility to P-AscH- therapy equivalent eH2O. Thus, further research remains necessary to identify why cancer cells vary in susceptibility to P-AscH- therapy.

For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1-0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors.

Response to Ling et al. regarding “An integrated physico-chemical approach for explaining the differential impact of FLASH versus conventional dose rate irradiation on cancer and normal tissue responses”

The high extracellular hydrogen peroxide (H2O2) concentrations generated during pharmacological ascorbate (P-AscH-) therapy has been shown to exhibit a high flux into susceptible cancer cells leading to a decrease in clonogenic survival. It is hypothesized that the intracellular H2O2 concentration for susceptibility is independent of cell type and that the variation observed in dosing is associated with differences in the cell-specific overall steady-state intracellular H2O2 concentration values. The steady-state variation in intracellular H2O2 concentration is coupled to a number of cellular specific transport and reaction factors including catalase activity and membrane permeability. Here a lumped-parameter mathematical modeling approach, assuming a catalase-dominant peroxide removal mechanism, is used to calculate intracellular H2O2 concentration for several cell lines. Experimental measurements of critical parameters pertaining to the model are obtained. The cell lines investigated are normal pancreatic cells, H6c7, the pancreatic cancer cell line, MIA PaCa-2 and the glioblastoma cell lines, LN-229, T98G, and U-87; all which vary in susceptibility. The intracellular H2O2 concentration estimates are correlated with the clonogenic surviving fraction for each cell line, in-vitro. The results showed that, despite the fact that the experimental parameters including catalase concentration and plasma membrane permeability demonstrated significant variability across cell lines, the calculated steady-state intracellular to extracellular H2O2 concentration ratio did not vary significantly across cell lines. Thus, the calculated intracellular H2O2 concentration is not unique in characterizing susceptibility. These results imply that, although intracellular H2O2 concentration plays a key role in cellular susceptibility to P-AscH- adjuvant therapy, its overall contribution in a unifying mechanism across cell types is complex.

Ferroptosis is a form of cell death characterized by a pro‐oxidative cellular milieu and iron‐dependent lipid peroxidation. Ferroptosis has been implicated in various forms of liver injury, in keeping with the major role of the liver in iron metabolism. Limited research has addressed potential differences in ferroptosis mediators with age and sex, especially in an in vivo model. The goal of this investigation was to evaluate hepatic labile iron and mediators of ferroptosis with ageing in both sexes. Because female animals generally display greater antioxidant defences than males, we hypothesized that females would display a phenotype resistant to ferroptosis. Here, we determined iron contents, protein expression of ferroptosis mediators and measures of oxidative injury in liver samples from 12‐ and 24‐month‐old male and female Fischer 344 rats. In comparison to males, the livers of female rats at both ages contained more non‐haem iron, which was associated with greater ferritin heavy chain expression and attenuated expression of transferrin receptor‐1. In female rats, the 24‐month‐old group had higher contents of thiobarbituric acid reactive substances compared with their 12‐month‐old counterparts, yet similar contents of labile iron. These results suggest a disconnect between labile iron contents and oxidative injury with age. Female animals also displayed greater expression of acyl‐CoA synthetase long‐chain family member 4 (ACSL4), a modulator of ferroptosis, and greater abundance of high molecular weight 4‐hydroxnonenal‐modified proteins. These results demonstrate clear differences in iron and ferroptosis mediators between sexes and suggest that female rats of this strain might be more susceptible to ferroptosis. What is the central question of this study?Do iron stores and ferroptosis mediators in the liver differ with age or sex?What is the main finding and its importance?We observed greater non‐haem iron, 4‐hydroxynonenal‐modified proteins and ACSL4 protein expression in female livers, suggesting greater susceptibility to ferroptosis. [ABSTRACT FROM AUTHOR]

Cancer cell toxicity to therapeutic H2O2 varies widely depending on cell type. Interestingly, it has been observed that different cancer cell types have varying peroxiporin expression. We hypothesize that variation in peroxiporin expression can alter cell susceptibility to therapeutic H2O2 concentrations. Here, we silence peroxiporin aquaporin-3 (AQP3) on the pancreatic cancer cell line MIA PaCa-2 and compare clonogenic survival response to the wild-type. The results showed a significantly higher surviving fraction in the clonogenic response for siAQP3 MIA PaCa-2 cells at therapeutic H2O2 doses (P < 0.05). These results suggest that peroxiporin expression is significant in modulating the susceptibility of cancer cells to ascorbate therapy.

Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial. There have been a number of studies suggesting that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen et al. provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of 3-day old Caenorhabditis elegans correlated inversely with lifespan. The interpretation of ‘mitoflashes’ as ‘bursts of superoxide’ radicals assumes that circularly permuted yellow fluorescent protein (cpYFP) is a reliable indicator of mitochondrial superoxide. This interpretation has been criticised because experiments and theoretical considerations both show that changes in cpYFP fluorescence are due to alterations in pH, not superoxide-. We now provide direct evidence that purified cpYFP is completely unresponsive to superoxide. Therefore ‘mitoflashes’ do not reflect superoxide generation and are not evidence for a link between mitochondrial free radical dynamics and lifespan.

Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H2O2) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H2O2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H2O2 by GPx was undertaken to determine how the reaction species (H2O2, GSH, and GPx-1) influence the rate of removal of H2O2. Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H2O2 increases with initial GPx(r), as expected, but is a function of both GPx(r) and GSH when the initial GPx(r) is less than H2O2. This simulation is supported by the biological observations of Li et al. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H2O2 was a direct function of GPx activity x GSH (effective GPx activity). The predicted cellular average GPx(r) and H2O2 for their study are approximately GPx(r) < or =1 microm and H2O2 approximately 5 microm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance.