Age-related decrease in resilience against acute redox cycling agents: Critical role of declining GSH-dependent detoxification capacity Public Deposited


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  • Over the past century, life expectancy in the United States has dramatically increased leading to an increasingly aging population with people reaching, and spending more years in ‘old age’. While this unprecedented shift in population demographics represents great strides for humanity, it is not without cost. One consequence of longer life is the increased accrual of age-associated diseases and chronic pathophysiological conditions. This is evident in the fact that over 80% of Americans over the age of 65 have at least one chronic medical condition [275]. Thus, lifespan has outpaced ‘healthspan’, or the time of one’s life spent free from disease and disuse syndromes. The work in this dissertation is defined by the investigation of “health assurance” biochemical pathways, the failure of which lead to heightened risk for age-related diseases. In particular, I have focused on why resiliency to oxidative stresses decline significantly with age. This focus has led to a research project that ultimately pinpoints a loss in glutathione-dependent defenses as an underlying aging factor, which could enhance risk for a variety of age-related diseases. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a major transcriptional regulator of numerous anti-oxidant, anti-inflammatory, and metabolic genes. We observed that, paradoxically, Nrf2 protein levels decline in the livers of aged rats despite the inflammatory environment evident in that organ. To examine the cause(s) of this loss, we investigated the age-related changes in Nrf2 protein homeostasis and activation in cultured hepatocytes from young (4–6 months) and old (24–28 months) Fischer 344 rats. While no significant age-dependent change in Nrf2 mRNA levels was observed, Nrf2 protein content was attenuated by ~40% with age (p<0.05 , N=4). Treatment with anethole trithione (A3T), along with bortezomib to inhibit degradation of existing protein, caused Nrf2 to accumulate significantly in cells from young animals (p < 0.05), but not old, indicating a lack of new Nrf2 synthesis. We hypothesized that the loss of Nrf2 protein synthesis with age may partly stem from an age-related increase in microRNA inhibition of Nrf2 translation. miRNA-146a, increases by > 2-fold with age and is predicted to bind Nrf2 mRNA. Transfection of hepatocytes from young rats with a miRNA-146a mimic caused a 55% attenuation of Nrf2 translation that paralleled the age-related loss of Nrf2. Overall, these results provide novel insights for the age-related decline in Nrf2 and identify new targets to maintain Nrf2-dependent detoxification with age. Having established that this major transcriptional regulator is attenuated with age, we next examined what effects this would have on toxicological resilience.Isolated hepatocytes from young and old rats were exposed to increasing concentrations of menadione, a vitamin K derivative and redox cycling agent, an LC50 for each age group was established, and results showed a nearly 2-fold increase in susceptibility to menadione (LC50 for young: 405 μM; LC50 for old: 275 μM) with age. Examination of the known Nrf2-regulated pathways associated with menadione detoxification revealed, surprisingly, that NAD(P)H:quinone oxido-reductase 1 (NQO1) protein levels and activity were induced 9-fold and 4-fold with age, respectively (p=0.0019 and p=0.018; N=3), but glutathione peroxidase 4 (GPX4) declined by 70% (p=0.0043; N=3). These results indicate toxicity may stem from vulnerability to lipid peroxidation instead of inadequate reduction of menadione semi-quinone. GSH declined by a 3-fold greater margin in old versus young rat cells given 300 µM menadione (p<0.05 and p≤0.01 respectively; N=3), and providing GSH synthesis substrates (400 µM N-acetyl-cysteine ) to hepatocytes from old before menadione resulted in a >2-fold reduction in cell death, suggesting that the age-related increase in menadione susceptibility likely stems from attenuated GSH-dependent defenses. Additionally, young rat hepatocytes maintained ~30% of their GSH content which suggested the possibility that mitochondrial GSH preservation may be critical for cell survival. In order to determine the role of mitochondrial GSH (mGSH) loss in increased susceptibility to xenobiotic insult with age, markers of mitochondrial function were measured in intact and digitonin permeabilized isolated hepatocytes from young and old rats under a redox cycling challenge (300 μM menadione; ~LC50 for old). Preliminary results show that, while the rate of mGSH loss under menadione challenge was similar in both age groups, the difference in basal mGSH with age (68 vs 36 nmol GSH mg protein, N=1) ultimately resulted in a 50% loss of mGSH in old rat hepatoctyes versus only 28% in young within 10 minutes of exposure. Examination of mitochondrial membrane potential (Δψm), which is acutely regulated by mGSH content, showed a distinct loss in basal mitochondrial membrane potential (Δψm) (~21%). Additionally, within five minutes of menadione treatment, Δψm was not markedly reduced in young but had collapsed and passed the threshold for mitochondrial permeability transition pore opening (~100 mV) in old rat hepatocytes. Further characterization demonstrated that basal respiration and respiratory reserve capacity, indicators of cellular bioenergetic capacity, were both significantly reduced upon menadione treatment in old rat hepatocytes (34% and 72% respectively, N=4, p<0.05) but not in young. These results suggested that the age-related difference in mitochondrial function under menadione challenge might stem from mGSH regulated electron transport chain (ETC) components. We therefore examined proton leak, complex I, and complex II contributions to mitochondrial oxygen consumption rates under menadione challenge in both age groups. Results showed no marked effect in young rat hepatocytes, while old rat hepatocytes demonstrated significant increases in proton leak and complex II contributions to oxygen consumption rate (4.2 and 3.6-fold respectively, N=4, p<0.05), along with a significant decline in complex I contribution (4.8-fold, N=4, p<0.05). This data clearly demonstrates an age-related increase in mitochondrial susceptibility to menadione challenge, particularly in complex I, and provide a plausible mechanism that links this vulnerability to age-related mGSH perturbations.
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