Seminar: Extracellular Vesicles as Mediators of Environmental Stress in Human Disease.

BACKGROUND: Extracellular vesicles (EVs), membrane-bound particles containing a variety of RNA types, DNA, proteins, and other macromolecules, are now appreciated as an important means of communication between cells and tissues, both in normal cellular physiology and as a potential indicator of cellular stress, environmental exposures, and early disease pathogenesis. Extracellular signaling through EVs is a growing field of research for understanding fundamental mechanisms of health and disease and for the potential for biomarker discovery and therapy development. EVs are also known to play important roles in mediating the effects of exposure to environmental stress. OBJECTIVES: This seminar addresses the application of new tools and approaches for EV research, developed in part through the National Institutes of Health (NIH) Extracellular RNA Communication Program, and reflects presentations and discussions from a workshop held 27-28 September 2021 by the National Institute of Environmental Health Sciences (NIEHS) and the National Center for Advancing Translational Sciences (NCATS) on "Extracellular Vesicles, Exosomes, and Cell-Cell Signaling in Response to Environmental Stress." The panel of experts discussed current research on EVs and environmental exposures, highlighted recent advances in EV isolation and characterization, and considered research gaps and opportunities toward identifying and characterizing the roles for EVs in environmentally related diseases, as well as the current challenges and opportunities in this field. DISCUSSION: The authors discuss the application of new experimental models, particularly organ-on-chip (OOC) systems and in vitro approaches and how these have the potential to extend findings in population-based studies of EVs in exposure-related diseases. Given the complex challenges of identifying cell-specific EVs related to environmental exposures, as well as the general heterogeneity and variability in EVs in blood and other accessible biological samples, there is a critical need for rigorous reporting of experimental methods and validation studies. The authors note that these efforts, combined with cross-disciplinary approaches, would ensure that future research efforts in environmental health studies on EV biomarkers are rigorous and reproducible. https://doi.org/10.1289/EHP12980.

Health Research in the Wake of Disasters: Challenges and Opportunities for Sensor Science.

BACKGROUND: Disaster events adversely affect the health of millions of individuals each year. They create exposure to physical, chemical, biological, and psychosocial hazards while simultaneously exploiting community and individual-level vulnerabilities that allow such exposures to exert harm. Since 2013, the National Institute of Environmental Health Sciences (NIEHS) has led the development of the Disaster Research Response (DR2) program and infrastructure; however, research exploring the nature and effects of disasters on human health is lacking. One reason for this research gap is the challenge of developing and deploying cost-effective sensors for exposure assessment during disaster events. OBJECTIVES: The objective of this commentary is to synergize the consensus findings and recommendations from a panel of experts on sensor science in support of DR2. METHODS: The NIEHS convened the workshop, "Getting Smart about Sensors for Disaster Response Research" on 28 and 29 July 2021 to discuss current gaps and recommendations for moving the field forward. The workshop invited full discussion from multiple viewpoints, with the goal of identifying recommendations and opportunities for further development of this area of research. The panel of experts included leaders in engineering, epidemiology, social and physical sciences, and community engagement, many of whom had firsthand experience with DR2. DISCUSSION: The primary finding of this workshop is that exposure science in support of DR2 is severely lacking. We highlight unique barriers to DR2, such as the need for time-sensitive exposure data, the chaos and logistical challenges that ensue from a disaster event, and the lack of a robust market for sensor technologies in support of environmental health science. We highlight a need for sensor technologies that are more scalable, reliable, and versatile than those currently available to the research community. We also recommend that the environmental health community renew efforts in support of DR2 facilitation, collaboration, and preparedness. https://doi.org/10.1289/EHP12270.

Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease.

The rise of advanced technologies for characterizing human populations at the molecular level, from sequence to function, is shifting disease prevention paradigms toward personalized strategies. Because minimization of adverse outcomes is a key driver for treatment decisions for diseased populations, developing personalized therapy strategies represent an important dimension of both precision medicine and personalized prevention. In this commentary, we highlight recently developed enabling technologies in the field of DNA damage, DNA repair, and mutagenesis. We propose that omics approaches and functional assays can be integrated into population studies that fuse basic, translational and clinical research with commercial expertise in order to accelerate personalized prevention and treatment of cancer and other diseases linked to aberrant responses to DNA damage. This collaborative approach is generally applicable to efforts to develop data-driven, individualized prevention and treatment strategies for other diseases. We also recommend strategies for maximizing the use of biological samples for epidemiological studies, and for applying emerging technologies to clinical applications.

Biomonitoring in the Era of the Exposome.

BACKGROUND: The term "exposome" was coined in 2005 to underscore the importance of the environment to human health and to bring research efforts in line with those on the human genome. The ability to characterize environmental exposures through biomonitoring is key to exposome research efforts. OBJECTIVES: Our objectives were to describe why traditional and nontraditional (exposomic) biomonitoring are both critical in studies aiming to capture the exposome and to make recommendations on how to transition exposure research toward exposomic approaches. We describe the biomonitoring needs of exposome research and approaches and recommendations that will help fill the gaps in the current science. DISCUSSION: Traditional and exposomic biomonitoring approaches have key advantages and disadvantages for assessing exposure. Exposomic approaches differ from traditional biomonitoring methods in that they can include all exposures of potential health significance, whether from endogenous or exogenous sources. Issues of sample availability and quality, identification of unknown analytes, capture of nonpersistent chemicals, integration of methods, and statistical assessment of increasingly complex data sets remain challenges that must continue to be addressed. CONCLUSIONS: To understand the complexity of exposures faced throughout the lifespan, both traditional and nontraditional biomonitoring methods should be used. Through hybrid approaches and the integration of emerging techniques, biomonitoring strategies can be maximized in research to define the exposome.

Mitochondria, energetics, epigenetics, and cellular responses to stress.

BACKGROUND: Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria-nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation. OBJECTIVES: We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function. METHODS: The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25-26 March 2013. Here, we summarize key points and ideas emerging from this meeting. DISCUSSION: A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways. CONCLUSIONS: Understanding mitochondria-cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to environmental stressors.

Harnessing genomics to identify environmental determinants of heritable disease.

Next-generation sequencing technologies can now be used to directly measure heritable de novo DNA sequence mutations in humans. However, these techniques have not been used to examine environmental factors that induce such mutations and their associated diseases. To address this issue, a working group on environmentally induced germline mutation analysis (ENIGMA) met in October 2011 to propose the necessary foundational studies, which include sequencing of parent-offspring trios from highly exposed human populations, and controlled dose-response experiments in animals. These studies will establish background levels of variability in germline mutation rates and identify environmental agents that influence these rates and heritable disease. Guidance for the types of exposures to examine come from rodent studies that have identified agents such as cancer chemotherapeutic drugs, ionizing radiation, cigarette smoke, and air pollution as germ-cell mutagens. Research is urgently needed to establish the health consequences of parental exposures on subsequent generations.

Inhibition of fried meat-induced colorectal DNA damage and altered systemic genotoxicity in humans by crucifera, chlorophyllin, and yogurt.

UNLABELLED: Dietary exposures implicated as reducing or causing risk for colorectal cancer may reduce or cause DNA damage in colon tissue; however, no one has assessed this hypothesis directly in humans. Thus, we enrolled 16 healthy volunteers in a 4-week controlled feeding study where 8 subjects were randomly assigned to dietary regimens containing meat cooked at either low (100°C) or high temperature (250°C), each for 2 weeks in a crossover design. The other 8 subjects were randomly assigned to dietary regimens containing the high-temperature meat diet alone or in combination with 3 putative mutagen inhibitors: cruciferous vegetables, yogurt, and chlorophyllin tablets, also in a crossover design. Subjects were nonsmokers, at least 18 years old, and not currently taking prescription drugs or antibiotics. We used the Salmonella assay to analyze the meat, urine, and feces for mutagenicity, and the comet assay to analyze rectal biopsies and peripheral blood lymphocytes for DNA damage. Low-temperature meat had undetectable levels of heterocyclic amines (HCAs) and was not mutagenic, whereas high-temperature meat had high HCA levels and was highly mutagenic. The high-temperature meat diet increased the mutagenicity of hydrolyzed urine and feces compared to the low-temperature meat diet. The mutagenicity of hydrolyzed urine was increased nearly twofold by the inhibitor diet, indicating that the inhibitors enhanced conjugation. Inhibitors decreased significantly the mutagenicity of un-hydrolyzed and hydrolyzed feces. The diets did not alter the levels of DNA damage in non-target white blood cells, but the inhibitor diet decreased nearly twofold the DNA damage in target colorectal cells. To our knowledge, this is the first demonstration that dietary factors can reduce DNA damage in the target tissue of fried-meat associated carcinogenesis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00340743.

XRCC1 and DNA polymerase beta in cellular protection against cytotoxic DNA single-strand breaks.

Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1(-/-) mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase beta (pol beta) is specific to this pathway, whereas pol beta is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1(-/-), and to a lesser extent in pol beta(-/-) cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol beta(-/-) cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1(-/-) cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair.

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1(-)) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3 weeks) treatment of HCT116 cells with VAN at minimally toxic concentrations (0.5-2.5mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/10(6) survivors) in a concentration-related manner by 19-73%. A similar treatment with CIN at 2.5-7.5microM yielded a 13-56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116+chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations.

Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair.

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, DeltauvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage-resulting in an antimutagenic effect on spontaneous mutation.

Comparative mutagenicity of halomethanes and halonitromethanes in Salmonella TA100: structure-activity analysis and mutation spectra.

Halonitromethanes (HNMs) are a recently identified class of disinfection by-products (DPBs) in drinking water that are mutagenic in Salmonella and potent inducers of DNA strand breaks in mammalian cells. Here we compared the mutagenic potencies of the HNMs to those of their halomethane (HM) homologues by testing all nine HNMs and seven of the nine HMs (minus bromomethane and chloromethane) under the same conditions (the pre-incubation assay) in Salmonella TA100 +/- S9. We also determined the mutation spectra for several DBPs. In the presence of S9, all nine HNMs, but only three HMs, dibromomethane (DBM), dichloromethane (DCM), and bromochloromethane (BCM), were mutagenic. Only two DBPs of each class were mutagenic in the absence of S9. The HNMs were generally more potent mutagens than their HM homologues, and the brominated forms of both classes of DBPs were more mutagenic and cytotoxic than their chlorinated homologues. The HNMs were at least 10 times more cytotoxic than the HMs, and the cytotoxicity rankings in the presence of S9 were similar for the HNMs and the HMs. The addition of a nitro-group to BCM did not change the mutation spectra significantly, with both homologues inducing primarily (55-58%) GC --> AT transitions. The greater cytotoxic and mutagenic activities of the HNMs relative to the HMs are likely due to the greater intrinsic reactivity conferred by the nitro-group. Energy calculations predicted increased reactivity with increasing bromination and greater reactivity of the HNMs versus the HMs (Elumo values were approximately 20 kcal/mol lower for the HNMs compared to their HM homologues). Given that the HNMs also are potent genotoxins in mammalian cells [Environ. Sci. Technol. 38 (2004) 62] and are more mutagenic and 10x more cytotoxic in Salmonella than the HMs, whose levels are regulated in drinking water, further study of their occurrence and potential health effects is warranted.