Frailty, sex, and poverty are associated with DNA damage and repair in frail, middle-aged urban adults.

Frailty is an age-related syndrome characterized by reduced recovery from stressors and increased risks of morbidity and mortality. Although frailty is usually studied in those over 65 years, our previous work showed that frailty is both present and a risk factor for premature mortality in midlife. We identified altered gene expression patterns and biological pathways associated with inflammation in frailty. Evidence suggests DNA oxidation damage related to inflammation accumulates with age, and that DNA repair capacity (DRC) declines with age and age-related conditions. We hypothesized that inter-individual differences in DNA oxidation damage and DRC are associated with frailty status and poverty level. Using the CometChip assay, we assessed baseline single-strand breaks and hydrogen peroxide (H2O2)-induced DNA oxidation damage and DRC in non-frail and frail middle-aged African American and White individuals with household incomes above and below poverty. Analysis of baseline single-strand breaks showed no associations with frailty, poverty, race, or sex. However, we identified an interaction between frailty and poverty in H2O2-induced DNA oxidation damage. We also identified interactions between sex and frailty as well as sex and poverty status with DRC. The social determinant of health, poverty, associates with DRC in men. Baseline DNA damage, H2O2-induced DNA damage as well as DRC were associated with serum cytokine levels. IL-10 levels were inversely associated with baseline DNA damage as well as H2O2-induced DNA damage, DRC was altered by IL-4 levels and sex, and by TNF-α levels in the context of sex and poverty status. This is the first evidence that DRC may be influenced by poverty status at midlife. Our data show that social determinants of health should be considered in examining biological pathways through which disparate age-related health outcomes become manifest.

Using the HepaCometChip Assay for Broad-Spectrum DNA Damage Analysis.

Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the "HepaCometChip" (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single-strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay.

The Parallel Transformations of Polycyclic Aromatic Hydrocarbons in the Body and in the Atmosphere.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) emitted from combustion sources are known to be mutagenic, with more potent species also being carcinogenic. Previous studies show that PAHs can undergo complex transformations both in the body and in the atmosphere, yet these transformation processes are generally investigated separately. OBJECTIVES: Drawing from the literature in atmospheric chemistry and toxicology, we highlight the parallel transformations of PAHs that occur in the atmosphere and the body and discuss implications for public health. We also examine key uncertainties related to the toxicity of atmospheric oxidation products of PAHs and explore critical areas for future research. DISCUSSION: We focus on a key mode of toxicity for PAHs, in which metabolic processes (driven by cytochrome P450 enzymes), leads to the formation of oxidized PAHs that can damage DNA. Such species can also be formed abiotically in the atmosphere from natural oxidation processes, potentially augmenting PAH toxicity by skipping the necessary metabolic steps that activate their mutagenicity. Despite the large body of literature related to these two general pathways, the extent to which atmospheric oxidation affects a PAH's overall toxicity remains highly uncertain. Combining knowledge and promoting collaboration across both fields can help identify key oxidation pathways and the resulting products that impact public health. CONCLUSIONS: Cross-disciplinary research, in which toxicology studies evaluate atmospheric oxidation products and their mixtures, and atmospheric measurements examine the formation of compounds that are known to be most toxic. Close collaboration between research communities can help narrow down which PAHs, and which PAH degradation products, should be targeted when assessing public health risks. https://doi.org/10.1289/EHP9984.

MalariaCometChip for high-throughput quantification of DNA damage in Plasmodium falciparum.

Comet assay is a standard approach for studying DNA damage in malaria, but high-throughput options are not available. The CometChip was previously developed using mammalian cells as a high-throughput version of the comet assay. It is based on the same principle as the comet assay but provides greater efficacy, automated data processing, and improved consistency between experiments. In this protocol, we present MalariaCometChip to quantitatively assess drug-induced DNA damage in Plasmodium falciparum. For complete details on the use and execution of this protocol, please refer to Xiong et al. (2020).

CometChip enables parallel analysis of multiple DNA repair activities.

DNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.

CometChip analysis of human primary lymphocytes enables quantification of inter-individual differences in the kinetics of repair of oxidative DNA damage.

Although DNA repair is known to impact susceptibility to cancer and other diseases, relatively few population studies have been performed to evaluate DNA repair kinetics in people due to the difficulty of assessing DNA repair in a high-throughput manner. Here we use the CometChip, a high-throughput comet assay, to explore inter-individual variation in repair of oxidative damage to DNA, a known risk factor for aging, cancer and other diseases. DNA repair capacity after H2O2-induced DNA oxidation damage was quantified in peripheral blood mononuclear cells (PBMCs). For 10 individuals, blood was drawn at several times over the course of 4-6 weeks. In addition, blood was drawn once from each of 56 individuals. DNA damage levels were quantified prior to exposure to H2O2 and at 0, 15, 30, 60, and 120-min post exposure. We found that there is significant variability in DNA repair efficiency among individuals. When subdivided into quartiles by DNA repair efficiency, we found that the average t1/2 is 81 min for the slowest group and 24 min for the fastest group. This work shows that the CometChip can be used to uncover significant differences in repair kinetics among people, pointing to its utility in future epidemiological and clinical studies.

Sequence and Nuclease Requirements for Breakage and Healing of a Structure-Forming (AT)n Sequence within Fragile Site FRA16D.

Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication. Fragility at Flex1 is initiated by structure-specific endonuclease Mus81-Mms4 acting together with the Slx1-4/Rad1-10 complex, whereas Yen1 protects Flex1 against breakage. Sae2 is required for healing of Flex1 after breakage. Our study shows that breakage within a CFS can be initiated by nuclease cleavage at forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also are impaired in their ability to heal, and this deleterious combination accounts for their fragility.

Mrc1 and Tof1 prevent fragility and instability at long CAG repeats by their fork stabilizing function.

Fork stabilization at DNA impediments is key to maintaining replication fork integrity and preventing chromosome breaks. Mrc1 and Tof1 are two known stabilizers that travel with the replication fork. In addition to a structural role, Mrc1 has a DNA damage checkpoint function. Using a yeast model system, we analyzed the role of Mrc1 and Tof1 at expanded CAG repeats of medium and long lengths, which are known to stall replication forks and cause trinucleotide expansion diseases such as Huntington's disease and myotonic dystrophy. We demonstrate that the fork stabilizer but not the checkpoint activation function of Mrc1 is key for preventing DNA breakage and death of cells containing expanded CAG tracts. In contrast, both Mrc1 functions are important in preventing repeat length instability. Mrc1 has a general fork protector role that is evident at forks traversing both repetitive and non-repetitive DNA, though it becomes crucial at long CAG repeat lengths. In contrast, the role of Tof1 in preventing fork breakage is specific to long CAG tracts of 85 or more repeats. Our results indicate that long CAG repeats have a particular need for Tof1 and highlight the importance of fork stabilizers in maintaining fork integrity during replication of structure-forming repeats.

The role of fork stalling and DNA structures in causing chromosome fragility.

Alternative non-B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single-stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT-rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance.

Evidence for grow-through penetration of 0.2-µm-pore-size filters by Serratia marcescens and Brevundimonas diminuta.

We find that both Brevundimonas diminuta and Serratia marcescens can grow through sterilizing grade filter membranes of different membrane polymer compositions. Although this passage does not occur on a consistent basis, generation of "grow-through positive" results indicate that grow-through can occur stochastically at basal levels. This observation argues that the following risk mitigation strategies during pharmaceutical aseptic processing are warranted: minimization of processing times, and monitoring, minimizing and characterizing pre-filter bioburden.

A survey of quality attributes of virus spike preparations used in clearance studies.

Demonstration of effective and consistent viral clearance by small scale models of downstream processing, typically cited as logarithmic reduction value (LRV), is an important safety requirement for biotech products. LRVs have anecdotally been reported to be inconsistent in these small-scale studies, even under controlled conditions when all process parameters are held constant. It was postulated that the quality of virus spike preparations used can in some cases adversely affect performance of these studies, which, from a regulatory standpoint, would be undesirable. This, along with topics discussed in PDA's Technical Report 47 (TR47), "Virus Preparations Used in Viral Clearance Studies," suggests that improving the quality and consistency of virus spike quality and utilizing testing procedures as described within should make these studies more reliable. However, an extensive survey to assess overall quality attributes to date has not been performed. To scout the landscape of spike preparation quality, we systematically characterized 18 commercially available virus preparations, focusing on key attributes identified in TR47: (1) infectious/total- and infectious/particle-associated copy numbers, (2) exogenous DNA/protein content and banding patterns, and (3) presence of aggregates. We found substantial variation across many of the preparations tested, often in more than one category. By modeling small-virus retentive filtration and low-pH inactivation unit operations, we show that virus preparation quality can potentially affect unit operation performance and viral clearance outcome. Our data supports the notion that during early-phase development, characterization of virus stock quality may provide an added level of control. LAY ABSTRACT: Demonstration of effective and consistent viral clearance is an important safety requirement for biotech products. However, accumulating evidence suggests that the quality of virus preparations used in clearance studies often vary, and thus potentially affect their performance. To scout the landscape of virus preparation quality, we systematically characterized 18 commercially available virus preparations, focusing on key attributes identified in PDA's Technical Report 47 (TR47). Virus Preparations Used in Viral Clearance Studies. We found substantial variation across many of the preparations tested, often in more than one attribute category. By performing small-virus retentive filtration and low-pH inactivation unit operations on a small scale, we also show that virus preparation quality can affect unit operation performance and viral clearance outcome. Our data supports the notion that during early-phase development, characterization of virus stock quality may provide an added level of control.