No abstract

In this study, peripheral-blood mononuclear cells from patients with chronic hepatitis B and spontaneous or therapy-induced disappearance of HBsAg were examined for HBV DNA. Samples were evaluated by in situ hybridization and polymerase chain reaction both before and after clearance of HBsAg. By in situ hybridization, positive signals were observed in 2 of 13 samples collected after HBsAg loss, in 8 of 15 samples before HBsAg loss and in 0 of 4 control patients without serological markers of active or prior HBV infection. When polymerase chain reaction analyses were performed, HBV DNA was detected in 5 of 12 HBsAg-negative samples and 10 of 15 HBsAg-positive samples from the study group. Testing of mononuclear cells after disappearance of HBsAg revealed that two of eight patients were HBV DNA positive by in situ hybridization and by polymerase chain reaction, whereas two additional patients were positive by polymerase chain reaction alone. Mononuclear cell-associated HBV DNA was detected between 2 and 9 mo after the disappearance of circulating HBsAg by in situ hybridization and as long as 4 yr later by polymerase chain reaction. These data indicate that patients who have undergone HBsAg seroconversion may nonetheless harbor HBV DNA in their peripheral-blood mononuclear cells for prolonged periods.

Isolated hepatocytes from young (4–6 mo) and old (24–26 mo) F344 rats were exposed to increasing concentrations of menadione, a vitamin K derivative and redox cycling agent, to determine whether the age-related decline in Nrf2-mediated detoxification defenses resulted in heightened susceptibility to xenobiotic insult. An LC50 for each age group was established, which showed that aging resulted in a nearly 2-fold increase in susceptibility to menadione (LC50 for young: 405 μM; LC50 for old: 275 μM). 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. Lipid peroxidation was 2-fold higher, and 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). We therefore provided 400 µM N-acetyl-cysteine (NAC) to hepatocytes from old rats before menadione exposure to alleviate limits in cysteine substrate availability for GSH synthesis during challenge. NAC pretreatment 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. This data identifies cellular targets for intervention in order to limit age-related toxicological insults to menadione and potentially other redox cycling compounds.

Aging is considered a risk factor for developing many chronic diseases which includes cardiovascular diseases, cancer, and neurodegenerative diseases. Researchers are now showing that many of these diseases are associated with cellular senescence. Cellular senescence is an important process for the removal of damaged cells, but if senescent cells are not removed a chronic pro-inflammatory environment ensues, increasing the risk of many age-related diseases. These cells accumulate as a result of cancer chemotherapy, are associated with age-related diseases, and are important for development. Recent work has shown that specific targeting of senescent cells results in increased life expectancy in a progeroid Ercc1-/Δ mouse model. As such there is great interest in identifying, characterizing and targeting senescent cells. Senescent cells are identified by the release of pro-inflammatory cytokines and chemokines, increased beta galactosidase activity (b-gal), senescence associated heterochromatin foci (SAHF), and changes in morphology which are increased size and a flattened appearance. There is no single good marker for determining senescence, so these cells are identified through the collective interpretation of multiple markers. Senescence associated b-gal (SA-b-gal) is considered the “gold standard” for identifying senescent cell, and it is detected with the blue-green precipitating colorimetric x-gal substrate. However, this substrate is limited use in that it cannot be used in flow cytometry or other fluorescence based platforms. Even though C12FDG, a fluorescein based b-gal substrate, has been used since the mid 90’s, it has limited utility restricted by its lack of fix-ability. Newer b-gal substrates are demonstrated here which do not leak out of cell; enabling researchers to more easily identify and evaluate cellular senescence. In this study we use a fluorescence approach to assess the senescence phenotype based on a combination of western blots, fluorescence based imaging and ICC, flow cytometry, cell sorting, and RT-qPCR. We examined several cell-based models include replicative senescence characterized by telomere shortening in primary fibroblasts, cellular senescence characterized through DNA damage pathway by doxorubicin, oxidative stress senescence induced by confluency in atmospheric oxygen environment (SIPS), and senescence induced with palbociclib treatment (a CDK 4/6 inhibitor). Overall, we found that phenotypic characteristics of senescence vary depending on the cellular model and that a multiplex fluorescence based approach to identifying senescent cells will contribute to the understanding of cellular senescence and the age-related diseases. Citation Format: Scott T. Clarke, Taryn Jackson, Amanda Kelley, Quentin Low, Timothy Huang, Yi-Zhen Hu, Veronica Calderon, Rong Wang, Michael O'Grady. Evaluation of cellular senescence through fluorescence characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 898.

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 hepatocytes from young and old F344 rats under a redox cycling (300 μM menadione) challenge. While the rate of mGSH loss under menadione challenge was similar in both age groups, a 47% decline in initial mGSH levels with age resulted in a 50% loss of mGSH in old rat hepatoctyes versus 28% in young within 10 minutes of exposure. Examination of mitochondrial membrane potential (Δψm), which is acutely regulated by mGSH content, showed a 21% basal loss with age. Additionally, within five minutes of menadione challenge, Δψm in young was not markedly reduced but had collapsed 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 but not in young. Examination of proton leak, complex I, and complex II contributions to mitochondrial oxygen consumption rates under menadione challenge revealed no marked effect in young rat hepatocytes, while old rat hepatocytes demonstrated a significant loss in complex I activity as well as increases in proton leak and a compensatory increase in complex II activity. These data clearly demonstrate an age‐related increase in mitochondrial susceptibility to a redox‐cycling challenge, particularly in complex I, and provide a plausible mechanism that links this vulnerability to mGSH perturbations.Support or Funding InformationN.T. is supported by an NIH grant. Title: CAM RESEARCH TRAINING IN NEUROSCIENCE & STRESS; number: 2T32AT002688.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.