Researchers Assess the Impacts of Living in a Chemical Soup
Inside Oregon State University’s Sinnhuber Aquatic Research Laboratory are rooms lined with tanks filled with thousands of zebrafish—silver, striped, one-to-two-inch creatures often found in home aquariums. The lab is the domain of Robert Tanguay, professor of molecular toxicology at Oregon State, whose zebrafish are helping to answer one of the most pressing questions in environmental health and toxicology: what are the health effects of chemical mixtures?
Tanguay has pioneered the use of zebrafish in toxicology. His lab is on the leading edge of this research, designing new experimental techniques and equipment that are enabling scientists to assess the impacts of multiple chemical exposures. Using zebrafish, Tanguay and other scientists are zeroing in on why certain chemical components of crude oil spilled by the Exxon Valdez in 1989 continue to adversely affect fish survival in Alaska’s Prince William Sound. With zebrafish, they also are learning why pesticide runoff can impair Pacific Northwest salmon’s ability to navigate and how oil from the ruptured Deepwater Horizon well is affecting marine species’ health in the Gulf of Mexico.
But zebrafish research goes well beyond the impact of chemical exposures on marine life. Zebrafish studies are also being used to assess the potential human health impacts of the chemical stew in U.S. Superfund sites. Tanguay’s lab is conducting research for the U.S. Environmental Protection Agency (EPA), evaluating the toxicity of thousands of chemicals used in countless consumer products. Zebrafish are also proving key to advancing our understanding of how particular chemical compounds affect the expression of individual genes that maintain and influence virtually every body system.
“Some people are still skeptical about how a fish can model human health,” said Tanguay during a tour of his lab earlier this month. “But during early development we are more similar to fish than at any other time in life.” Genes are particularly susceptible to environmental chemicals in early development. What happens then can set the stage for health throughout the rest of life.
The work of Tanguay and colleagues on mixtures is particularly important because, historically, chemicals have been evaluated and regulated one at a time. In reality, however, we are exposed to multiple chemicals daily throughout our lives. Because we now know that environmental chemical exposure can profoundly influence the development of disease, understanding how mixtures may affect human health has taken on an urgency.
“We live in a chemical soup,” says Linda Birnbaum, director of the U.S. National Toxicology Program and the National Institutes of Environmental Health Science (NIEHS).
Zebrafish have been used since the 1990s for genetic modeling and screening, but it was Tanguay, while working as a post-doctoral fellow in 1995, who suggested they might be used in toxicology. Because they are vertebrates, these prolifically reproducing fish turn out to be an extremely useful model for understanding how cells in other vertebrates, including humans, respond to environmental chemicals. Zebrafish, which progress from a translucent egg to a recognizable baby fish in just 24 hours, are ideal for investigating how chemicals may affect biological processes at various stages of development.
Because zebrafish eggs develop outside the mother, they can be manipulated, and Tanguay’s lab has devised equipment and techniques for isolating the eggs to examine the effects of chemicals. In one lab, Tanguay showed me a petri dish containing translucent zebrafish eggs, no bigger than a poppy seed, and the thread-fine, glass needles used to extract genetic material. Tanguay’s researchers use that material to determine which base pair—a rung in the DNA double-helix ladder—is targeted by a particular chemical exposure.
The results of these perturbations will tell the scientists if the exposure prompts any physical or health abnormalities. The zebrafish genome has been sequenced, enabling scientists to identify genes associated with specific diseases. Because many zebrafish genes have human counterparts, what’s learned from these fish can be used to inform human health research.
By figuring out which chemical targets a specific genetic receptor, scientists can begin to connect chemical exposure to health effects. Zebrafish development is readily visible, enabling scientists to identify changes in a fish’s phenotype—its physical characteristics. The zebrafish’s rapid development and short generational cycle make it possible to pinpoint effects that could be difficult to see in other animals. After linking chemical and physical effects, scientists then work to connect the phenotype with the genetic mechanisms that prompt it—or, as Tanguay says, to “connect phenotype with genotype.”
Some of these changes may result in physical abnormalities. Zebrafish exposed to certain dioxins, for example, had deformed vertebrae and skulls. Dioxin exposure also impaired regeneration of wounded fins. Other changes may become apparent in neurological, cognitive and behavioral testing. Still others may be exhibited as abnormalities in reproduction, altered developmental timing or changes in how metabolism and other body systems function.
Zebrafish are instrumental in the research that Nat Scholz, the ecotoxicology program manager at the National Oceanic and Atmospheric Administration’s Northwest Fisheries Science Center, is conducting to investigate the effects of what he calls “cocktails of pollution” on different fish species, including Pacific Northwest salmon and herring. Among the combinations of chemicals he’s studying are those of various fuel oil compounds, particularly polycyclic aromatic hydrocarbons (PAHs) and pesticides.
Using zebrafish, Scholz and his colleagues have discovered that certain PAHs adversely impact cardiovascular development, leading to a heart failure syndrome that can be lethal to fish larvae and reduce adult fish survival. The work with zebrafish has enabled the scientists to discover why fish that looked normal were dying. By examining how low levels of PAH exposure interfere with genes involved in heart development, they have begun to understand how oil from the Exxon Valdez and Deepwater Horizon spills has impaired the cardiovascular function of fish at a cellular level.
While the PAH exposure prompts changes early in development, these effects may not become apparent until later on, when defects—in how the heart processes potassium and calcium and in heart structure—reduce cardiac function and cause swimming problems. Experiments with zebrafish have helped deduce which enzymes and genetic receptors are affected by which hydrocarbon compounds, something that’s not been possible with fish in the wild.
Tanguay’s lab is also working with the EPA and NIEHS on an ongoing project known as Tox21, which is assessing more than 10,000 commercially produced chemicals for a wide number of health effects. Zebrafish are being used as part of what is called “high-throughput” screening—the rapid testing of lots of chemicals. The goal is to fill the many gaps in toxicity data that exist for these chemicals and identify which chemical compositions or concentrations prompt specific health effects. Results of this screening are expected later this year.
“We call this Robot Row,” said Tanguay, showing me the lab area devoted to robotic machines designed to precisely dose hundreds of zebrafish eggs and genetic material samples. Because zebrafish develop so quickly, lots of variables can be examined concurrently using these machines. Tanguay’s lab uses what look like tiny paint palettes with an array of 96 little wells, each of which gets a zebrafish sample that is then dosed by a machine with a particular chemical at different concentrations. Some experimentally treated eggs are allowed to develop, while others are halted to examine impacts during embryonic development.
Tanguay is also working with Terry Collins, professor of chemistry and director of Carnegie Mellon University’s Institute for Green Science, to assess the toxicity of molecules Collins has invented to clean up environmental contaminants. These compounds, known as TAML activators, can be used to remove dyes and other toxic chemicals from water. The TAML compounds appear to be successful in degrading these hazardous chemicals, but Collins wants to make sure their use doesn’t produce other harmful compounds or effects. Collins’ and Tanguay’s labs have been testing TAML activators on zebrafish to determine any potential toxicity, including on endocrine hormones.
The scientists are examining a complex range of possible outcomes that may result from the chemicals’ interaction with specific genetic receptors at different stages of development. If no abnormal effects are seen after the various TAML exposures, Collins will be closer to determining the safety of his chemical products. If adverse effects are seen, he will have the information he needs to begin redesigning his compounds so they are safe to use.
Tanguay’s lab has also designed equipment to test cognitive and behavioral responses in zebrafish. Similar to tests used with other lab animals, including humans, these experiments reveal how the brain, nervous system and muscles may be affected by certain chemicals, such as organophosphate pesticides, known neurotoxicants that have been linked to adverse neurological effects in children. By using zebrafish, scientists have begun to locate which neurons these chemicals may affect at particular stages of development. These experiments are also being used to test the effects on fish of pesticide-laden water running off fields and roads.
The ultimate goal is to learn which chemical combinations, concentrations and structures alter the normal function of genes and hormones. There is strong evidence, for example, that interference with endocrine system hormones and the genes that regulate them can lead to metabolic disorders such as diabetes and obesity. Research has also shown that endocrine-disrupting chemicals can upset reproductive, developmental, immune system and neurological health. In addition, scientists have discovered that altered gene expression—distinctly different from genetic mutation—can be passed from one generation to another in what are called epigenetic effects. Pinpointing these precursors to disease is key to protecting and improving human health.
The ability to connect specific chemicals in the environment with mechanisms of disease is essential to finding and “eliminating those that are nasty,” explains Tanguay. In a world where manufactured chemicals are ubiquitous, the tiny, fecund zebrafish is playing a big role in discovering how this chemical stew is affecting our health.
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By Ana Maldonado-Contreras
- Your gut is home to trillions of bacteria that are vital for keeping you healthy.
- Some of these microbes help to regulate the immune system.
- New research, which has not yet been peer-reviewed, shows the presence of certain bacteria in the gut may reveal which people are more vulnerable to a more severe case of COVID-19.
You may not know it, but you have an army of microbes living inside of you that are essential for fighting off threats, including the virus that causes COVID-19.
How Do Resident Bacteria Keep You Healthy?<p>Our immune defense is part of a complex biological response against harmful pathogens, such as viruses or bacteria. However, because our bodies are inhabited by trillions of mostly beneficial bacteria, virus and fungi, activation of our immune response is tightly regulated to distinguish between harmful and helpful microbes.</p><p>Our bacteria are spectacular companions diligently helping prime our immune system defenses to combat infections. A seminal study found that mice treated with antibiotics that eliminate bacteria in the gut exhibited an impaired immune response. These animals had low counts of virus-fighting white blood cells, weak antibody responses and poor production of a protein that is vital for <a href="https://doi.org/10.1073/pnas.1019378108" target="_blank">combating viral infection and modulating the immune response</a>.</p><p><a href="https://doi.org/10.1371/journal.pone.0184976" target="_blank" rel="noopener noreferrer">In another study</a>, mice were fed <em>Lactobacillus</em> bacteria, commonly used as probiotic in fermented food. These microbes reduced the severity of influenza infection. The <em>Lactobacillus</em>-treated mice did not lose weight and had only mild lung damage compared with untreated mice. Similarly, others have found that treatment of mice with <em>Lactobacillus</em> protects against different <a href="https://doi.org/10.1038/srep04638" target="_blank" rel="noopener noreferrer">subtypes of</a> <a href="https://doi.org/10.1038/s41598-017-17487-8" target="_blank" rel="noopener noreferrer">influenza</a> <a href="https://doi.org/10.1371/journal.ppat.1008072" target="_blank" rel="noopener noreferrer">virus</a> and human respiratory syncytial virus – the <a href="https://doi.org/10.1038/s41598-019-39602-7" target="_blank" rel="noopener noreferrer">major cause of viral bronchiolitis and pneumonia in children</a>.</p>
Chronic Disease and Microbes<p>Patients with chronic illnesses including Type 2 diabetes, obesity and cardiovascular disease exhibit a hyperactive immune system that fails to recognize a harmless stimulus and is linked to an altered gut microbiome.</p><p>In these chronic diseases, the gut microbiome lacks bacteria that activate <a href="https://doi.org/10.1126/science.1198469" target="_blank" rel="noopener noreferrer">immune cells</a> that block the response against harmless bacteria in our guts. Such alteration of the gut microbiome is also observed in <a href="https://doi.org/10.1073/pnas.1002601107" target="_blank" rel="noopener noreferrer">babies delivered by cesarean section</a>, individuals consuming a poor <a href="https://doi.org/10.1038/nature12820" target="_blank" rel="noopener noreferrer">diet</a> and the <a href="https://doi.org/10.1038/nature11053" target="_blank" rel="noopener noreferrer">elderly</a>.</p><p>In the U.S., 117 million individuals – about half the adult population – <a href="https://health.gov/our-work/food-nutrition/2015-2020-dietary-guidelines/guidelines/" target="_blank" rel="noopener noreferrer">suffer from Type 2 diabetes, obesity, cardiovascular disease or a combination of them</a>. That suggests that half of American adults carry a faulty microbiome army.</p><p>Research in my laboratory focuses on identifying gut bacteria that are critical for creating a balanced immune system, which fights life-threatening bacterial and viral infections, while tolerating the beneficial bacteria in and on us.</p><p>Given that diet affects the diversity of bacteria in the gut, <a href="https://www.umassmed.edu/nutrition/melody-trial-info/" target="_blank" rel="noopener noreferrer">my lab studies show how diet can be used</a> as a therapy for chronic diseases. Using different foods, people can shift their gut microbiome to one that boosts a healthy immune response.</p><p>A fraction of patients infected with SARS-CoV-2, the virus that causes COVID-19 disease, develop severe complications that require hospitalization in intensive care units. What do many of those patients have in common? <a href="https://www.cdc.gov/mmwr/volumes/69/wr/mm6912e2.htm" target="_blank" rel="noopener noreferrer">Old age</a> and chronic diet-related diseases like obesity, Type 2 diabetes and cardiovascular disease.</p><p><a href="http://doi.org/10.1016/j.jada.2008.12.019" target="_blank" rel="noopener noreferrer">Black and Latinx people are disproportionately affected by obesity, Type 2 diabetes and cardiovascular disease</a>, all of which are linked to poor nutrition. Thus, it is not a coincidence that <a href="https://www.cdc.gov/mmwr/volumes/69/wr/mm6933e1.htm" target="_blank" rel="noopener noreferrer">these groups have suffered more deaths from COVID-19</a> compared with whites. This is the case not only in the U.S. but also <a href="https://www.washingtonpost.com/world/europe/blacks-in-britain-are-four-times-as-likely-to-die-of-coronavirus-as-whites-data-show/2020/05/07/2dc76710-9067-11ea-9322-a29e75effc93_story.html" target="_blank" rel="noopener noreferrer">in Britain</a>.</p>
Discovering Microbes That Predict COVID-19 Severity<p>The COVID-19 pandemic has inspired me to shift my research and explore the role of the gut microbiome in the overly aggressive immune response against SARS-CoV-2 infection.</p><p>My colleagues and I have hypothesized that critically ill SARS-CoV-2 patients with conditions like obesity, Type 2 diabetes and cardiovascular disease exhibit an altered gut microbiome that aggravates <a href="https://theconversation.com/exercise-may-help-reduce-risk-of-deadly-covid-19-complication-ards-136922" target="_blank" rel="noopener noreferrer">acute respiratory distress syndrome</a>.</p><p>Acute respiratory distress syndrome, a life-threatening lung injury, in SARS-CoV-2 patients is thought to develop from a <a href="http://doi.org/10.1016/j.cytogfr.2020.05.003" target="_blank" rel="noopener noreferrer">fatal overreaction of the immune response</a> called a <a href="https://theconversation.com/blocking-the-deadly-cytokine-storm-is-a-vital-weapon-for-treating-covid-19-137690" target="_blank" rel="noopener noreferrer">cytokine storm</a> <a href="http://doi.org/10.1016/S2213-2600(20)30216-2" target="_blank" rel="noopener noreferrer">that causes an uncontrolled flood</a> <a href="http://doi.org/10.1016/S2213-2600(20)30216-2" target="_blank" rel="noopener noreferrer">of immune cells into the lungs</a>. In these patients, their own uncontrolled inflammatory immune response, rather than the virus itself, causes the <a href="http://doi.org/10.1007/s00134-020-05991-x" target="_blank" rel="noopener noreferrer">severe lung injury and multiorgan failures</a> that lead to death.</p><p>Several studies <a href="https://doi.org/10.1016/j.trsl.2020.08.004" target="_blank" rel="noopener noreferrer">described in one recent review</a> have identified an altered gut microbiome in patients with COVID-19. However, identification of specific bacteria within the microbiome that could predict COVID-19 severity is lacking.</p><p>To address this question, my colleagues and I recruited COVID-19 hospitalized patients with severe and moderate symptoms. We collected stool and saliva samples to determine whether bacteria within the gut and oral microbiome could predict COVID-19 severity. The identification of microbiome markers that can predict the clinical outcomes of COVID-19 disease is key to help prioritize patients needing urgent treatment.</p><p><a href="https://doi.org/10.1101/2021.01.05.20249061" target="_blank" rel="noopener noreferrer">We demonstrated</a>, in a paper which has not yet been peer reviewed, that the composition of the gut microbiome is the strongest predictor of COVID-19 severity compared to patient's clinical characteristics commonly used to do so. Specifically, we identified that the presence of a bacterium in the stool – called <em>Enterococcus faecalis</em>– was a robust predictor of COVID-19 severity. Not surprisingly, <em>Enterococcus faecalis</em> has been associated with <a href="https://doi.org/10.1053/j.gastro.2011.05.035" target="_blank" rel="noopener noreferrer">chronic</a> <a href="https://doi.org/10.1016/S0002-9440(10)61172-8" target="_blank" rel="noopener noreferrer">inflammation</a>.</p><p><em>Enterococcus faecalis</em> collected from feces can be grown outside of the body in clinical laboratories. Thus, an <em>E. faecalis</em> test might be a cost-effective, rapid and relatively easy way to identify patients who are likely to require more supportive care and therapeutic interventions to improve their chances of survival.</p><p>But it is not yet clear from our research what is the contribution of the altered microbiome in the immune response to SARS-CoV-2 infection. A recent study has shown that <a href="https://doi.org/10.1101/2020.12.11.416180" target="_blank" rel="noopener noreferrer">SARS-CoV-2 infection triggers an imbalance in immune cells</a> called <a href="https://doi.org/10.1111/imr.12170" target="_blank" rel="noopener noreferrer">T regulatory cells that are critical to immune balance</a>.</p><p>Bacteria from the gut microbiome are responsible for the <a href="https://doi.org/10.7554/eLife.30916.001" target="_blank" rel="noopener noreferrer">proper activation</a> <a href="https://doi.org/10.1126/science.1198469" target="_blank" rel="noopener noreferrer">of those T-regulatory</a> <a href="https://doi.org/10.1038/nri.2016.36" target="_blank" rel="noopener noreferrer">cells</a>. Thus, researchers like me need to take repeated patient stool, saliva and blood samples over a longer time frame to learn how the altered microbiome observed in COVID-19 patients can modulate COVID-19 disease severity, perhaps by altering the development of the T-regulatory cells.</p><p>As a Latina scientist investigating interactions between diet, microbiome and immunity, I must stress the importance of better policies to improve access to healthy foods, which lead to a healthier microbiome. It is also important to design culturally sensitive dietary interventions for Black and Latinx communities. While a good-quality diet might not prevent SARS-CoV-2 infection, it can treat the underlying conditions related to its severity.</p><p><em><a href="https://theconversation.com/profiles/ana-maldonado-contreras-1152969" target="_blank">Ana Maldonado-Contreras</a> is an assistant professor of Microbiology and Physiological Systems at the University of Massachusetts Medical School.</em></p><p><em>Disclosure statement: Ana Maldonado-Contreras receives funding from The Helmsley Charitable Trust and her work has been supported by the American Gastroenterological Association. She received The Charles A. King Trust Postdoctoral Research Fellowship. She is also member of the Diversity Committee of the American Gastroenterological Association.</em></p><p><em style="">Reposted with permission from <a href="https://theconversation.com/a-healthy-microbiome-builds-a-strong-immune-system-that-could-help-defeat-covid-19-145668" target="_blank" rel="noopener noreferrer" style="">The Conversation</a>. </em></p>
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