🖨️ Print post
The human body is over 2/3 water, but it’s not the water you get from the tap. It’s also not a solid, liquid, or gas. Dr. Gerald Pollack, scientist, researcher, and author, explains the Fourth Phase of Water that is in the body. He goes over what it is exactly, how we can build more of it (through certain foods and light exposure) and how it helps us age more slowly and live more healthily.
Visit his website: pollacklab.org
Join our podcast celebration at Sally Fallon Morell’s farm
Check out our sponsors: Second Spring Foods, RaOptics, and Optimal Carnivore
—
Listen to the podcast here
Episode Transcript
Within the below transcript the bolded text is Hilda.
You may have heard that the human body is 2/3 water but that water is not the same as the water in a drinking glass. It’s a different kind of water that’s not liquid, solid, or gas. This is Episode 407. Our guest is a researcher, scientist, author, and expert in the fourth phase of water. He helped discover this fourth phase, known also as Exclusion Zone or EZ water.
Understanding what helps build this water in the body can help us strengthen our bodies, build resilience, help us feel better, and age more slowly. Our guest is Dr. Gerald Pollack. He explains exactly what EZ water is, how we can generate more of it through sunlight, infrared light, exercise, and certain foods, and why it matters.
Before we dive into the conversation, I want to invite you to a party. Join us to celebrate the 10 million downloads of this show. It’s 10 million downloads strong, thanks to you. The party is on Sunday, March 26th, 2023 at Sally Fallon Morell’s Farm in Brandywine, Maryland. We will eat hors d’oeuvres, mix and mingle, and hear from some local guests who have been on the show.
There will even be a place for you to record your testimonials of what the show and the work of the Weston A. Price Foundation have meant to you. There is only room for 130 folks to show up, so head over to Weston A. Price, go to the events tab, and save your place. Follow us on Instagram @WestonAPrice for more details on what’s in store as we celebrate the milestone. Thank you so much for sharing the show and helping us get to this place. We are beyond thankful.
Visit Ra Optics to place your order and use the discount code WISE for 10% off any order.
—
Visit his website: Pollack Lab
Join our Podcast Celebration at Sally Fallon Morell’s farm
Check out our sponsors: Second Spring Foods, Ra Optics, and Optimal Carnivore
—
Welcome to the show, Jerry.
I’m delighted to be here once again. I will try my best to answer whatever questions you might have.
I find your work fascinating. When you and I first spoke years ago, it all went over my head. I had no idea what you were talking about, EZ water and the fourth phase of water, but now having spoken to other experts, I get the idea. I want to make sure all of us, including the audience, get the idea of how this phase of water can benefit our health and how we might be able to even get in contact with it more. First, let’s take it bit by bit and help us understand. Is EZ water or this fourth phase of water the same thing as structured water?
It is and it isn’t. Structured water is a generic term. It means water with some structure but everything has structure. The term is not so useful. I’m not sure where it started. It might have started with Gilbert Ling who spent his life studying what he called structured water and what a lot of people called structured. The idea is that ordinary liquid water in a sense is not structured because it consists of individual molecules that are randomly disposed and bouncing around a furious number of times each second or each femto-second even. You would imagine that a mix like that has no structure to it.
On the other hand, what Gilbert Ling and others were talking about is the idea that water molecules are ordered in some way. That’s how it all started. Instead of using the term ordered, Gilbert and others use the term structured. Structure does convey, in general, the idea that this water is this. You might call it halfway between water and ice. Ice has a structure to it. It’s a crystal. All the molecules are organized in certain ways. This water is not ice. It’s water but it’s water that is organized or structured.
What Gilbert Ling and some others had surmised is that the structure consists of individual molecules that are stacked on one another like soldiers at attention. I spoke at length about that in my 2001 book, Cells, Gels, and the Engines of Life, whose theme was, “We should pay attention to what Gilbert Ling has been talking about because it’s important.” Once we started doing experiments immediately after that book had been published, we found that it was not quite the case. The so-called structured water, which we gave a different name, and I’ll get to that in a moment, is different.
The liquid water has undergone a transition from individual molecules to sheet-like structures that stack upon one another. The term structured is vague. Initially, we called it EZ or Exclusion Zone. It’s easy to remember unless you’re in England or some other place where it’s zed. EZ is Exclusion Zone. We gave it that name a long time ago because what we noticed is that there was a zone of water that excluded almost everything. It was pure. If you initially had some salutes or particles in the water in this form, they would get kicked out. There was a purity to it. It excluded virtually everything.
We called it Exclusion Zone water. It was suggested by an Australian colleague but that’s not a great name even though it’s easy to remember because it doesn’t tell you very much except that this is a kind of water that tends to exclude, and that’s all. Later as we kept finding that this Exclusion Zone water had properties so vastly different from ordinary liquid water, it seemed like another phase of water. We began calling it the fourth phase. The fourth phase is accurate. We know that the standard three phases are solid, liquid, and vapor. This is the fourth.
What you were alluding to at the end of your question is, “Is it important for health?” There’s a lot to say about that but for a starter, our bodies are filled with EZ water or fourth-phase water. It’s not liquid water. The way you can easily discern that is to cut yourself. Take a razor blade, and if you wish, slice. If the water inside your body were liquid water, it will come pouring out like from a breached water pipe but that’s not the case. It doesn’t come out. The water is more gel-like. It stays in and sticks to the solids inside your cells, so the water doesn’t come pouring out.
It’s a different kind of water. That’s the water that we have been talking about. That’s the fourth phase of water, which has gel-like features. It builds next to hydrophilic water-loving surfaces. Your body is filled with all of the solids inside your cells, proteins, nucleic acids, and others. The majority of their surfaces are hydrophilic and water-loving. Therefore, the water that sits next to them will be EZ or fourth-phase water, not liquid water. Your cell is densely packed with those solids that all the water inside your cell is EZ water. That’s why it doesn’t come pouring out when you slice yourself.
Let me ask you because I like to take this heady information, translate it for the readers, and make it accessible and understandable. If I’m hearing you correctly, there is a phase of water that you and your colleagues dub the fourth phase of water or EZ water. That is a different state that is more easily absorbed by our bodies. Our bodies thrive with this kind of water but when we are drinking regular liquid water, it’s not as easily absorbed by the cells as this fourth phase of water. That’s what I’m trying to figure out. How can I make this water structured so it’s better absorbed by cells so I can be living my healthiest and best life?
You have to know something about what it is that converts the water that you’ve been drinking into EZ water. We understand what converts the water. There are several things. First of all, to build any structure or organization, you need energy. That’s a given. You can surmise that by thinking about your office. You sit down at a desk that you have and throw down some papers, pencils, and your thumb drive. After a few weeks, it gets awfully messy. It requires no energy for you to make it messy but if you want to get it organized, structured, and ordered, you need to take 30 minutes out and focus on the ordering. That requires energy.
That’s a simple example of the fact that if you want to go from a random state to an ordered state, you need to put in energy. We struggled with where the energy comes from for doing this. It turned out it was light. It’s not any kind of light but it’s infrared light. Most people are not so familiar with it. They think you turn on the toaster and push it down. The bread goes down. If you look inside, you see those coils glowing bright orange and say, “It’s warm. It’s generating something. That’s infrared energy.” That’s correct. However, everything is generating infrared energy, including the screen behind you, yourself, and the hinges on the screen.
If you want to go from a random state to an ordered state, you need to put in energy.
The way you can demonstrate that is to turn off all the lights. You will see nothing. Your latest iPhone camera will see nothing despite its high sensitivity but if you have a camera with a sensor that is sensitive to infrared energy instead of visible light energy, infrared is a little bit longer in wavelength. If you whip out one of those cameras and turn it on, you get a beautiful image of everything including your necklace, your dress, the screen behind you, and the hinges on the screen. You see it all, which means that everything is generating infrared energy. The energy required for converting ordinary liquid water to EZ water is in the environment. It’s also inside your body because your metabolism is generating heat.
Heat is not the same as infrared energy but for all intents and purposes, it’s the same. You take some water and drink it. There’s a tendency for that water to convert to EZ water because as you drink the water, much of the water will be sitting next to one or another of those hydrophilic surfaces but you also need the energy. The energy is available. It’s available not only inside your body from the metabolic heat but also outside your body because everything is generating infrared energy. The energy is there, which means EZ water is always present. If you drink ordinary liquid water, a fraction of it will turn into EZ water. The rest of it gets peed out.
I see people with special wands. They say, “I’m structuring my water.” They put the water outside in the sunlight and say, “I’m trying to structure my water,” as if our bodies couldn’t do the same thing you were describing.
Our bodies can do it but the question is, “How effective is the body? How much EZ water do you need?” When you’re six months older, we know that the body then contains 85% water. As we get wrinkly with age, we have a smaller percentage of water. Part of the reason is we don’t have the capacity to structure the water as effectively as when we’re born when everything is right usually. We need some help. We can get help from infrared energy that’s inside the body and outside the body. Many people have turned, for example, to infrared lamps. You shine the infrared lamp. What does that do? It builds EZ water.
A critical part that I neglected to mention, which is described in my Cells, Gels, and the Engines of Life book is everything the cell does, whatever kind of cell, whether it’s a muscle cell and it contracts, if it’s a secretory cell and it secretes, or if it’s a nerve cell and it conducts. Always the action of the cell involves a transition from EZ water to ordinary water and then back again. That transition is central. This is not part of standard physiology understanding or anything like that but the evidence is clear. Anybody who might be interested could read the book and make their judgment but the main point is that you need it for you to function properly. You look as though you function pretty properly.
I feel like I do.
You look like you do. What you need is a full complement of EZ water inside your cells. You want to make sure that you’ve got enough of it. Kids usually have plenty of it but as we become wrinkly with age, then we have less of it. We need all the help we can get. The second half of your question is, “Can we create EZ water? What about these wands?” There are dozens or even more than dozens of ways various companies are advertising that they sell various things that create structured water, EZ water, fourth-phase water, or whatever they call it. Probably some of them do.
It would be nice if these companies provide evidence that they do. Many of them say, “We can structure the water,” but we don’t generally see the evidence for it. It’s not trivial to do this. I’m not suggesting that there are ways for anybody to go into their kitchen and make structured water. Maybe they can. Maybe they can’t. It needs to be explored but there are several ways at least theoretically to do it. One is to have plenty of solid surfaces that build EZ water.
One of those, for example, is chia seed. Chia seed is amazing in its capacity to do that. You throw a few seeds in, add some water, and get this gel-like substance that’s filled with EZ water. That’s one way to do it but then you have to separate the chia seed from the water. Who knows what happens when you attempt to do that? That’s one way. Another way is to put an electrical charge in the water.
We found that if you stick an electrode in the water, you need to stick two electrodes in the past current. Right next to that negative electrode, EZ water builds. The water is converted from neutral ordinary water to negatively charged EZ water. EZ water typically is negatively charged. You need to put electrons in the water, and the water will transition. That’s another way that you can build it. It’s not so convenient depending on what you have available.
Another way is to expose the water to sunlight. Fifty percent roughly of the energy of the sun is in the infrared region. That’s why the sun feels warm. It’s not just light that is conferred by the sun but also heat. We feel the heat. If you put water out in the sun, the sunlight at least in theory ought to build EZ water. For example, in India, it was common. I’m not sure how common it is now to take some jugs of water, color them, and put them out in the sun.
If someone’s ill, you choose the appropriate one. That appropriate one will be good for your health. When the Ayurvedic people came to that understanding, I’m not sure if they understood what they were doing but what’s going on is that EZ water is getting built. EZ water is good for health. When you’re ill, you drink one of those jugs of water, and you should get better in theory.
I wanted to ask you about data that backs up the statement you made that EZ water is good for health. Tell us a little bit about that.
I’ll tell you in the form of a story. Maybe that’s the best place to start. The story is as is well-known for 5,000 years. I don’t know the height of the Ayurvedic culture. You know better than I do. It’s some thousands of years. The people in that culture were interested as we are in optimum health. They must be. What did they do? Typically, they would take certain herbs, for example, turmeric. Apparently, it helped with all kinds of afflictions. You take turmeric and get better.
We started thinking, “How does this work at least in terms of what we know about modern physiology and health?” One idea that we didn’t like a whole lot but it’s possible that the body has receptors all over the place for turmeric. In your heart, you have turmeric receptors. If you take turmeric, the receptors say, “Turmeric is great.” We get better. The same is true with your big toe. You’ve got pain in the big toe. There’s a turmeric receptor there. You take turmeric, and the pain in your toes disappears.
That’s one idea. Another idea is that turmeric has one single effect, and that effect impacts everything in your body. That’s a more attractive hypothesis because it’s simpler. Based on the principle of Occam’s razor, you’ve got two options to explain something. The simpler one is going to be the one that is probably correct. We were hypothesizing, “Maybe turmeric builds order and EZ water. If it builds EZ water, that EZ water is all over our body.” If we ingest turmeric, eventually it spreads throughout our body, or at least the water that it comes into contact with is spreading all around the body.
The hypothesis was maybe small amounts of turmeric build EZ water. We tested it, got a positive result, and published it. It’s not only turmeric but other agents known to be beneficial for health for thousands of years, not in conventional medicine terms but known in these ancient cultures. For example, we tested basil. The so-called holy basil is the same thing. We also tested poison to see if it did the opposite. We used glyphosate or weed killer. In theory, it doesn’t kill us but it kills the weeds. One wonders about the distinction.
Small amounts of turmeric build EZ water.
There’s a lot from Stephanie Seneff and others about glyphosate and its harmful impacts. We added a small amount of glyphosate to the water that formed EZ water. Sure enough, at every concentration we looked at, it diminished the amount of EZ water. We don’t know if the poison glyphosate acts the way other poisons act but a simple mechanism of action of poisons that remains to be tested is that they deplete EZ water.
Without EZ water, the cells can’t function. Without cell function, you die or at least get sick. We also tried ghee or clarified butter. That had a tremendous impact. There’s a tremendous growth of EZ water more than practically anything. The point is we have tried a multiplicity of agents that are known to be positive for health. They all build EZ water. It doesn’t prove that it’s EZ water that is responsible for health but it certainly is consistent with that hypothesis.
—
Coming up, Gerald gets specific about the food and healthy habits that build more EZ in the body.
—
Go to Second Spring Foods to find your favorite sprouted pantry staples and use the code WISE for 10% off.
Go to Optimal Carnivore and use the code WESTON10 to receive 10% off all of Optimal Carnivore’s products.
—
Apart from structuring the water or making it EZ water with infrared light or electrodes, are there ways in which we can build up the EZ water in our bodies?
There are. The first way is to follow what I was expressing. Take turmeric and basil. Use ghee when you’re preparing foods and such. It’s been known for thousands of years that those agents are good for health. That’s certainly one way. Another way is to go out in the sun because the sun contains infrared energy. That infrared energy builds EZ water. That will be another way. Another way to achieve much the same is to go into a sauna. It’s heat. Whether it’s moist heat or dry heat, I wouldn’t say it’s irrelevant but it’s heat. Heat is full of infrared energy.
Take your clothes off, go into the sauna, and expose yourself for 20 or 30 minutes. I’ve had the experience. I come out feeling better than when I went in. Most people feel that. Why is that? There are various theories and such. The reason is it is infrared energy. Infrared energy is building EZ water throughout your body. Without a full complement of EZ water, you’re not functioning properly.
If you’ve got muscle aches, for example, and your muscle is cramped or such, you go in and expose yourself and your muscles to the infrared energy that builds EZ water and returns the muscle to its natural state because the natural state is full of EZ water. If your muscle is cramped, it surely doesn’t have enough EZ water, at least transiently. You have to build up that EZ water, which leads to the muscle relaxation back into the high potential energy state ready to contract again.
One bit of advice you gave years ago when you were on the show was to get near natural bodies of water, waterfalls, or the beach. Does that exposure to that water somehow help also build the EZ water in our bodies?
If you’re near a waterfall, there are lots of little droplets that come off the waterfall. The studies are published in a book called The Fourth Phase of Water, which has been out for some time. It’s popular. I’m flattered by its popularity. It’s high on the Amazon review list. I’m not sure why. Much of what I want to say or have said is described in that book. We described in that book droplets. What does a droplet consist of? We found through experimentation one thing about droplets. They’re always close to being spherical. When you think about a spherical droplet, why is that?
Our studies show that the droplet contains a membrane of EZ water. It’s like onion layers. There are many layers on the periphery. That’s the outer shell of a droplet. Inside the droplet are positive charges, which are repelling one another and pushing out against the membrane of the EZ. The droplet contains EZ. It turns out that there are more EZ than there are positive charges. There are more negative charges than there are positive charges. We have evidence for that. You’re going near the waterfall. You breathe in those little droplets. What you’re doing is you’re getting a negative charge. You’re getting EZ water principally. Getting EZ water means good for your health. I hope that answers that particular question.
As we start to close up, I have a couple of personal questions for you. Have your findings changed how you relate to water in general? In other words, do you seek out waterfalls? Do you speak to your water as Emoto has done? Veda Austin has done some studies on this too. Do you relate to water differently now?
I would like to answer yes. There is a reverence that I’ve developed for water but in terms of my personal habits, it hasn’t changed a whole lot. I’m hesitant to admit this but I know I should be drinking a lot more water to remain hydrated but I go for coffee every time. Maybe I do get some hydration from coffee, fresh fruits, and such but I don’t drink enough water, and I know I need to.
I have heard health experts saying, “Stop drinking water. You’re not getting hydrated by the water you drink. You need some other elements to come with it for it to be absorbed into the cells.” I’m curious about your take on that.
If we drink, for example, distilled water, it’s got none of the minerals that we need. Some people tout distilled water as being the water you should drink. Other people say, “Stay away from that water because it’s not good for your health.” I have a tendency to revert to two things natural back 5,000 years ago. What did the people drink? Spring water has a tendency to fill that bill. You can imagine people going to springs, finding the right spring, and drinking it.
It turns out that not all but many of the spring waters contain a large fraction of EZ water. There’s an easy way of testing to see if you’ve got EZ water or how much EZ water you have. It involves shining light on the water. EZ water absorbs certain wavelengths of light. They happen to be in the ultraviolet range which is 270 nanometers. If you have a sample of water and shine a light, and it absorbs a lot at 270 nanometers, you’ve got a lot of EZ water. If nothing absorbs at 270 nanometers, you don’t have any EZ water. You can test to see how much EZ water is in what you drink.
It turns out that the spring waters vary. Some of them don’t have too much EZ content and others have a huge amount of EZ content. I’m hopeful that one day, the granting agencies will understand that the word water does exist in the biological language or any language. It deserves to be studied. Water is considered in the mainstream as nothing more than a background carrier of the more important molecules of life.
Everything that we have done in the past couple of decades argues squarely against that concept. Water is not merely a background carrier. It’s essential for everything that the body does. The failure to recognize that is one of the reasons why biology or biochemistry seems as complicated as it is. If you start with the wrong foundation, everything appears to be complicated. You build one complication upon another. It becomes practically impossible to deal with.
Water is not merely a background carrier. It’s essential for everything that the body does.
I’m sorry if it doesn’t sound very humble but the evidence for me is without a doubt the direction that water is so central to everything that we do and unrecognized in principle. To figure out which water is the best for you or which water is the best under varied circumstances if you’ve got this illness or that illness, studies need to be carried out. The granting agency has never heard the word water in relation to biology. It’s almost hopeless because someone has to be incented to carry out the studies.
There’s maybe not much money, reward, or demand from the public to understand it better but I liked your advice about going more natural. The wise traditions way is going to what our ancestors were served by and how they thrived. You’re exactly right about the spring water with its trace minerals and its happy structure.
It’s important. The more water you drink, the more EZ is in the water. It should that the logic is better for health. That’s one reason why yet another expedient is what some people do, and I bet you do it yourself. You go into your backyard, pull the green leaves from the plants, crush them into a juice, and drink it. My late wife used to do that. Maybe that’s what helped her retain some modicum of health despite the gray hair and the loss of hair.
It got to be tiresome after a while but what you’re doing is you’re squeezing the water out of the plant cells. Those plant cells are freshly grown, vital, and healthy. They’re full of EZ water. We were able to confirm that. What you’re doing by crushing those leaves and squeezing out the juices is you’re drinking the water from inside the plant cells. You’re drinking EZ water. That water should be good for your health because if you drink EZ water, your body doesn’t need to convert ordinary water into EZ water. It comes directly.
The plants have gotten the light from the sun. It’s gotten all the right conditions to create the EZ water within itself. There isn’t much funding for research in this area. What do you say to the skeptic who turns his nose up at this whole water thing anyway? “You’re overrating that. I prefer my Gatorade.” What do you say to a person who’s simply not paying attention to the water inside their body or this special phase of water?
There are some things I could say that is not appropriate. It has been my experience that there are some people who are in that category that you mentioned but far more people are interested in delving more deeply into EZ water. It’s common in science that we reject any paradigm that comes up that’s new and that challenges the existing paradigm. We don’t feel comfortable because it upsets our sense of equilibrium. Scientists especially feel comfortable being in a situation where they’re in command.
They understand there are certain scientific principles. Those principles are foundational. If somebody challenges them, we feel uncomfortable because we have learned a set of paradigms. It’s built on foundations. We feel uneasy. If you look back at science and some of the major scientific discoveries, you will find that it happens uniformly when someone comes up with something that shakes the foundation. People who have walked on that foundation and felt comfortable feel most uneasy even Einstein.
In 1905, he had three findings that shook the Earth but the idea of relativity was at first rejected by the scientific community. They thought, “This is nonsense. I don’t understand it. This is crazy speaking.” This was 1905. It wasn’t until 1921 or 1922 when he won a Nobel Prize not for relativity but for one of the other discoveries that people began to take him seriously, “This guy won a Nobel Prize. Therefore, he must be a smart guy. Therefore, whatever he says is right.” Relativity took hold but this is an example that the scientific establishment has a lot of inertia to it. It doesn’t like to move.
Albert Szent-Györgyi is considered to be the Father of Modern Biochemistry. He’s a Hungarian guy. He’s full of wisdom and memorable aphorisms. He’s a creative guy. He won a Nobel Prize for discovering vitamin C. He also was into water structures. This was years ago. He’s a hero to many people although his reputation tends to fade over time. One of the things he said was relevant, “When I came forth with a fresh idea, the only time I knew it was significant was when the reactions were polarized.”
“Some people thought, ‘This is absolute nonsense.’ Other people thought, ‘This is genius.’ I knew I was onto something because there always had to be people who objected to it. If it was potentially important, they would object. If it’s not so important or it was bland, then it wasn’t so important.” I’m mentioning all of this about Einstein, Szent-Györgyi, Galileo, and the guy who talked about continental drift. They thought he was nuts. Continents drift until they found evidence that it was incontrovertible.
New findings are always challenged by the people whom they impact. Someone who has been doing computer models of water and trying to figure out the behavior of 200 molecules every femto-second or less in what they’re doing with these models based on a lot of assumptions is not happy. If what we found is correct, then what they’re doing doesn’t have a whole lot of meaning. People are human. If someone challenged you, you have a belief system that you’ve developed over the eighteen years of your life. You wouldn’t be happy about that. You might object.
On the flip side of it, at least for fourth-phase water, the number of applications that are now underway is phenomenal. There’s so much interest in it. I spend half my day responding to emails centered around the idea of EZ water because there’s so much that stems from it. If you ask me, I would say to a person like someone you mentioned, “Look at the evidence and draw your judgment.”
Even in my department, I have people who refuse to believe it, “It can’t be right. It must be nonsense,” without seriously looking at the evidence and challenging me by saying, “On page 36 of your book, you say, X, Y, and Z but the evidence for that is lacking. What’s the evidence?” If I can’t answer the question, then they have the perfect right to say, “I’m not so sure about this.” There’s a lot of reflexive reaction to it saying, “The fourth phase of water is nonsense. Everybody knows there are three phases. A fourth phase is crazy-making.”
I hope we have skeptics reading who perhaps are going to give a deeper dive into this subject, check out some of the books you’ve mentioned and the resources, and come to their conclusions. If the evidence bears out, then they will find the help of the EZ water. Maybe it will change their worldview. If it doesn’t, then so be it. I want to ask you the question now that I pose at the end of the show. If the reader could do one thing to improve their health that could be related to this fourth phase of water or not, what would you recommend that they do?
I hesitate because I’m not sure which one is best. I mentioned several experiences along the way like going to a waterfall and such, walking on the beach barefoot, and juicing from the vegetables in your backyard but at the center of it all is to make sure you’re well-hydrated. Drink enough water or enough vegetables that contain water. You will be well off if you do that. If you have the capacity to drink water that contains a sizable fraction of EZ water, that will be even better. At the core, stay hydrated because hydration is not just a matter of filling the cell with stuff. It’s a matter of feeling the cell with the most important ingredient, and that is water or EZ water.
That’s perfect. Thank you so much for this conversation. I am so happy to talk to you. I hope we do it again before more years go by. Maybe I’ll see you at the Wise Traditions Conference next time around.
Thank you so much. I appreciate your great questions. We will see you again. Take care.
That sounds great. Thank you, Jerry. Take care. Thanks for your patience too.
—
Our guest was Dr. Gerald Pollack. Visit his website, Pollack Lab. You can find me at Holistic Hilda. For a recent review from Apple Podcasts, Nomdska had this to say, “Following the Weston A. Price Foundation advice, in 2011, I had some puzzling dental problems. I knew of but had not got around to reading Nutrition and Physical Degeneration by Weston Price, so I finally did.”
“I applied the suggestions in the book. Within one week, my dental sensitivity disappeared. I don’t even bother going every six months to the dentist anymore after the dentist repeatedly told me I don’t have any tartar on my teeth at all. I also have not been sick since 2011. I owe my health to applying what I learned from the Weston A. Price Foundation. Thank you, Sally, for your work and courage.”
Thank you so much for your review. This means a lot. Every review tells audiences that this show is worth checking out. If you would like to leave us a review, go to Apple Podcasts, click on the ratings and reviews, give us a bunch of stars, and tell the world why you like the show. Help us keep going strong at 10 million downloads plus. We appreciate your support. Thank you for reading, my friend. Keep your feet on the ground and your face to the sun.
Important Links
- Pollack Lab
- @WestonAPrice – Instagram
- Ra Optics
- Cells, Gels, and the Engines of Life
- Second Spring Foods
- Optimal Carnivore
- The Fourth Phase of Water
- Holistic Hilda
- Apple Podcasts – Wise Traditions
- Nutrition and Physical Degeneration
- Podcast Celebration
About Gerald Pollack
Gerald Pollack maintains an active laboratory at the University of Washington in Seattle. He is the Founding Editor-in-Chief of WATER: A Multidisciplinary Research Journal; Executive Director of the Institute for Venture Science; co-founder of 4th-Phase Inc.; and founder of the Annual Conference on the Physics, Chemistry, and Biology of Water. He has received numerous honors including: the Prigogine Medal for Thermodynamics; the University of Washington Annual Faculty Lecturer; the NIH Director’s Transformative Research Award; and the 1st Emoto Peace Prize. He is recognized internationally as an accomplished speaker and author.
Health Effects from Swimming Training in Chlorinated Pools and the Corresponding Metabolic Stress Pathways
Jiang-Hua Li, Zhi-Hui Wang, […], and Ya-Jun Lin
Additional article information
Associated Data
Supplementary Materials
Data Availability Statement
Abstract
Chlorination is the most popular method for disinfecting swimming pool water; however, although pathogens are being killed, many toxic compounds, called disinfection by-products (DBPs), are formed. Numerous epidemiological publications have associated the chlorination of pools with dysfunctions of the respiratory system and with some other diseases. However, the findings concerning these associations are not always consistent and have not been confirmed by toxicological studies. Therefore, the health effects from swimming in chlorinated pools and the corresponding stress reactions in organisms are unclear. In this study, we show that although the growth and behaviors of experimental rats were not affected, their health, training effects and metabolic profiles were significantly affected by a 12-week swimming training program in chlorinated water identical to that of public pools. Interestingly, the eyes and skin are the organs that are more directly affected than the lungs by the irritants in chlorinated water; instead of chlorination, training intensity, training frequency and choking on water may be the primary factors for lung damage induced by swimming. Among the five major organs (the heart, liver, spleen, lungs and kidneys), the liver is the most likely target of DBPs. Through metabolomics analysis, the corresponding metabolic stress pathways and a defensive system focusing on taurine were presented, based on which the corresponding countermeasures can be developed for swimming athletes and for others who spend a lot of time in chlorinated swimming pools.
Introduction
Chlorination is the most popular method for disinfecting swimming pool water. However, although pathogens are being killed, many toxic compounds, called disinfection by-products (DBPs), are formed. Numerous publications have indicated that DBPs exposure may be related to several diseases [1–3], and Thomas Lachocki, the head of the National Swimming Pool Foundation of USA, has emphasized that the health benefits from swimming must be weighed against the risks of chemical exposure [4]. The epidemiological evidence for adverse health effects from swimming in chlorinated water primarily originate from studies concerning respiratory function and asthma, althoughVillanueva et al. reported a significant increased risk of bladder cancer for swimmers compared with nonswimmers [5]. The chlorination of pools has been associated with an increase in lung epithelium permeability [6], a risk of developing asthma [7], and with respiratory complaints [8]. Typically, trihalomethanes and trichloramines are blamed [4]. However, the findings regarding the association of chlorination with illness are not always consistent. Font-Ribera et al. reported that swimming did not increase the risk of asthma or allergic symptoms in British children [9] but was associated with slightly less respiratory tract symptoms [10], increased lung function and with a lower risk of asthma symptoms, particularly among children with preexisting respiratory conditions [9]. A meta-analysis performed by Goodman et al. demonstrated that the association between asthma and swimming could only be confirmed among competitive swimmers and could not be confirmed among non-competitive swimmers [11]. Extremely few toxicological studies have been performed in the area of swimming exposure and health thus far. Therefore, the health effects from swimming in chlorinated pools and the corresponding stress reactions occurring in our bodies are unclear. Generally, competitive swimmers are the most possible victims of DBPs exposure, because they have to do a lot of high intensive training in swimming pools for years. To reveal the health effects of DBPs exposure from swimming training, the experimental animals were trained in chlorinated water as competitive swimmers for twelve weeks in this study (according to the lifespan of the animals, twelve weeks for rats almost equals ten years for human being, which is a nessary period for an athlete to get a best performance). Their behaviors and appearances were observed during the training program, and then histopathological and metabolomic approaches were used to analyze the health effects and corresponding metabolic stress pathways.
Materials and Methods
Animals
Animal welfare and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and were approved by the animal ethics committee of Jiangxi Normal University. Twenty-four Sprague-Dawley rats, which were three weeks old and weighed 207.1 ± 43.9 g, were commercially obtained from the Department of Laboratory Animal Science, Nanchang University, China. Throughout the study periods, all rats were housed in 590×380×200 mm plastic cages under the following conditions: 20–24°C room temperature, natural light, standard food and free water.
Treatment
After acclimatization for one week, the 24 rats were randomly distributed into a control group (CG, n = 6) and an experimental group (EG, n = 18), and then a 12-week swimming training program was performed for both groups. Unfortunately, one of the rats accidentally drowned during swimming training; therefore, the final animal number of the EG was 17. The water for the EG was purified using a water purifier and then disinfected using calcium hypochlorite, similar to public swimming pools, whereas the water for the CG was only purified, not chlorinated. Free chlorine in the swimming water was monitored using the N, N-diethyl-p-phenylenediamine (DPD) method. The level of free chlorine in the water for the EG was adjusted to 1.4–1.6 mg/L before swimming training (the ideal level recommended by the World Health Organization for public pools [12]); no free chlorine was detected in the water for the CG. The training was performed once a day, five days a week. When training, a screw nut approximately 3% of their mean body weight was tied to the top end of the tail of each rat, and all rats were kept in the special pools with water of 60 cm depth (water temperature 25–30°C, pH 6.5–7.0) until fatigued (submerged below the surface for five seconds twice). The fatigued rats ceased training immediately, were removed from the water for a short break, showered with running water and then dried with hair dryers.
Chlorination DBP measurements
Two typical classes of chlorination DBPs, chloroform and chloramines, were measured at the 20th minute of every training session. Chloroform was measured using a gas chromatograph (SHIMADZU GC-2010) with a HP-5 chromatographic column (30 m×0.32 mm×0.25 μm) in accordance with the Chinese standard test methods for organic substances in drinking water (GB/T 5750.8–2006). Chloramines were measured using a colorimeter (HKM II, Guangdong Huankai Microbial Sci. & Tech. Co. Ltd.) based on the DPD method.
Behaviors and appearances
Behaviors and appearances of the rats were observed throughout the study. Body weights were measured using an electronic balance.
Swimming capacity test
After the 12-week swimming training and one day of rest, a swimming capacity test was performed. The duration from entry into water until the exhaustion of each rat was recorded. The pool water used in the test was running water supplied by the municipal water company. To increase the intensity and to shorten the time, a 13.7 g screw nut was tied to the top end of the tail of each rat in the test. The exhaustion criterion was set such that rats remained submerged below the surface for ten seconds [13].
Urine samples
The day before the swimming capacity test, 24-hour urine samples were collected with metabolic cages (NaN3 preservation) and then centrifuged at 3000 r/min at 4°C for 10 min. Approximately 4 ml supernatant aliquots were transferred into 5 ml Eppendorf tubes and stored at −80°C for nuclear magnetic resonance (NMR) testing.
Histopathology
The day after the swimming capacity test, animals were sacrificed with an overdose administration of pentobarbiturate (120 mg/kg). First, a gross anatomy dissection was performed on the animals, and tissues of their five major organs, i.e., heart, liver, spleen, lungs and kidneys, were collected and fixed with 4% formaldehydum polymerisatum. Twelve hours later, the fixed tissues were embedded in paraffin for sectioning, and then a regular histopathological analysis was performed using hematoxylin and eosin staining and optical microscopes.
NMR measurements
Urine samples were thawed at room temperature, and then 400 μL urine was mixed with 200 μL phosphate buffer (pH 7.4,0.2 M NaH2PO4/Na2HPO4), with 10% D2O as a field lock and with 0.05% sodium 3-trimethylsilyl-(2,2,3,3-2H4)-1-propionate (TSP) as a chemical shift reference. After centrifugation at 13000 g for 10 min, the supernatants were transferred into 5 mm NMR tubes and measured using a standard one dimensional 1H pulse sequence with water suppression (Noesypresat) on a Bruker DRX400 spectrometer operating at 400.13 MHz 1H resonance frequency and at 298 K.
Data processing
All 1H NMR Spectra were manually corrected for phase and baseline distortions and referenced to the TSP signal at 0 ppm using Top Spin software version 3.0 (Bruker Biospin, Germany). Integration was performed over a 10.00–0.02 ppm region, with a bucket width of 0.02 ppm. Regions corresponding to the spectrum signals of water and of urea (6.20–4.20 ppm) were excluded, and the integration of each region was normalized to the sum of the total spectrum to obtain the urine metabolite data (S1 Table).
Statistical analysis
Principal component analysis (PCA) was conducted on the urine metabolite data for pattern recognition using SIMCA-P+ software version 10.0 (Umetrics, Umea, Sweden). Before PCA, data were subjected to orthogonal signal correction (OSC) and unit variance scaling. Other statistical analyses were performed using IBM SPSS Statistics software version 20.0 (SPSS Inc., Chicago, IL, USA). The durations of the animals in swimming capacity test were expressed as the mean value ± standard deviation (SD) and compared using independent t-tests. The positive rates in histopathological analysis were evaluated by Chi-square (χ2) test. The significance level was set at 0.05.
Results and Discussion
Concentrations of the typical chlorination DBPs
Concentrations of the two typical classes of chlorination DBPs (chloroform and chloramines), which were measured at the 20th minute of the swimming training, were 0.7±0.05 μg/L and 1.05±0.12 mg/L in the water for the EG, and none of these chemicals were detected in the water for the CG. The 20th minute was approximately the middle point of the training session (the duration of each training session was approximately 40 minutes); thus, the concentrations of the DBPs at this point were selected to represent the exposure doses. The exposure doses of these two typical classes of DBPs for the EG rats were significantly higher than those for the CG rats, and this experiment was a typical chronic low-dose exposure experiment.
Behaviors and Appearances
The final body weights of the rats measured before euthanasia were extremely close between the EG and the CG (344.34 ± 34.95 g vs. 337.07 ± 46.00 g, p>0.05). No significant behavior differences were observed between the two groups during the entire experimental period; however, some unusual appearance changes appeared in the EG rats. First, the skin around their eyes became increasingly red with the development of the experiment, and in the ending period of the experiment, bloodstains could be observed in the rims of most rats’ eyes. Second, from the third experimental week on, an increasing number of rats had bloodstains appearing at the tips of their noses; however, approximately two weeks later, this symptom gradually disappeared. Third, their fur became increasingly dry and lackluster, and significant signs of hair loss were observed during the last month.
These results indicated that the fur, respiratory tracts and eyes of the EG rats were severely affected by chlorinated water, although their growth was essentially unaffected. According to our observations, the daily behaviors and sizes of the EG rats were normal, and their final body weights were even slightly heavier than those weights of the control group. Nevertheless, dried and lackluster fur, hair removal, bloody noses and eyes did occur in the EG rats and not to the CG rats. In fact, similar symptoms, red and swollen eyes, dried skin and nasal mucosal congestion, always appear after humans swim in a chlorinated pool; however, the long-term (12 weeks) and high-frequency (5 days a week) of the experimental swimming training caused even worse symptoms in these experimental rats.
Additionally, an interesting phenomenon was observed by comparing the development of the bloody noses and bloody eyes. The bloody noses commonly appeared in the third and in the fourth week; however, approximately two weeks later, this symptom gradually disappeared. The significantly bloody eyes commonly appeared in the ending period of the experiment; however, this problem was becoming worse during the study, and no signs of improvement appeared. The bloody noses appeared first, suggesting that respiratory tracts may be more vulnerable to the irritants from the chlorinated water than eyes; the gradually disappearing symptom suggests that respiratory tracts may have some adaptability to chlorinated water possibly because of the protection from nasal mucous. In contrast, without the mucosal protection, the bloody eyes were becoming increasingly significant during the entire experiment, although this symptom appeared later than the bloody noses.
Therefore, the eyes and skin may be the organs that must be the focus of concern regarding permanent damage induced by irritants from chlorinated water, rather than respiratory tracts, although respiratory symptoms were the most emphasized toxic risk of swimming exposure in recent decades.
Swimming capacity test
The duration period from the entry into water until the rats reached exhaustion was significantly shorter for the EG rats compared with the CG rats (29.74±11.50 vs. 39.15±9.85 minutes, p<0.05), indicating that the training effects were significantly impaired by the chlorinated water.
Histopathological analysis
The results of histopathological analysis is shown in Table 1. The color and size of the five major organs, the heart, liver, spleen, lungs and kidneys, of all rats were normal, and no significant exudation, hyperplasia, edema, atrophy and other disease symptoms were observed during the gross anatomy dissection. Under light microscopy, no disease symptoms were found in the heart, spleen and kidney sections; however, some disease symptoms were observed in the liver and lung sections.
Table 1
Table 1
The results of histopathological analysis.
Significant signs of cytoplasm rarefaction and ballooning degeneration were found in the liver sections of 3 rats from the EG (Fig. 1A), whereas the CG rats had primarily intact nuclei and normal cell shapes (Fig. 1B). Three rats from the EG were found liver damage, indicating that the positive rate reached 18 percent. Although this ratio did not show statistical significance when compared with the CG, it still suggests that the liver is most likely an important target organ of DBPs. Because of its unique metabolism and relation to the gastrointestinal tract, the liver is an important target of the toxicity of drugs, xenobiotics, and of oxidative stress [14]. Numerous reports have demonstrated the hepatoxicity of chloroform, which is the most common toxic DBP in chlorinated swimming pools. For instance, Tumasonis et al. reported that a significantly increased incidence of hepatic neoplastic nodules in female rats and a significantly increased incidence of hepatic adenofibrosis in both male and female rats were induced by the chronic ingestion of chloroform [15]. Additionally, Reuber claimed to have found a statistically significant number of hepatic cholangiocarcinomas in female rats with chronic chloroform administration [16].
Fig 1
Fig 1
Representative hematoxylin and eosin staining (H&E, 400×) of liver sections.
Dilatation and congestion of the capillary vessels around the alveoli, edema fluid in the alveoli, and lymphadenosis under the bronchial mucosae were commonly observed in the lung sections from both groups (Fig. 2). No significant differences were found between the EG and the CG.
Fig 2
Fig 2
Representative hematoxylin and eosin staining (H&E, 100×) of lung sections.
Compared with liver damage, lung damage was much more commonly detected under microscopes. However, notably, both the rats from the EG and from the CG had almost identical symptoms in their lungs. This phenomenon, combined with the disappearing bloody noses discussed above, suggests that chlorination disinfection may not be the primary factor for lung damage during swimming because the alveoli and bronchi are also protected by attached mucosal-like respiratory tracts. In contrast, we believe that the training intensity, training frequency and water choking might be the primary factors for the observed lung damage. In this study, both groups of rats were trained similar to competitive swimmers, with high frequency and high intensity, which made them inevitably choke much water and become more vulnerable to lung damage. Similarly, studies of elite swimmers in the United States, Canada, Great Britain, Australia, Finland, and in Ireland confirmed that asthma is more common only among competitive swimmers and was not common among non-competitive swimmers11. We suppose that the primary differences between the competitive swimmers and non-competitive swimmers are training intensity and frequency, not the pool water.
Metabolomics
Principal component analysis (PCA) The first three principal components (PCs) explain 57.3% of the total variance of the raw data and are sufficient to present the differences between the EG and the CG. All samples are clearly and correctly clustered into two different groups in the PCA score plot (Fig. 3), which suggests that the 12-week swimming training in chlorinated water induced a significant change in the metabolic profiles of the EG rats. To identify the primary metabolites that changed under the conditions of this experiment, the chemical shifts in the peaks with large loadings in the first three PCs are marked in the PCA loading plot (Fig. 3). In Fig. 3, we can see that the majority of the differences between the EG and the CG are explained by PC 1 and PC 3. The T tests further confirm that the differences between the groups are significant on PC 1 and PC 3 (p0.05). Thereby, the interpretation of PCs in the following parts will focus on PC 1 and PC 3.
Fig 3
Fig 3
Score plot (left) and loading plot (right) of the principal component analysis.
Peak assignment and changing trends of the assigned metabolites The peaks with large loadings in the first three PCs (Fig. 3) are assigned in the 1H NMR spectra (Fig. 4), and the corresponding metabolites and their changing trends in the experimental group are shown in Table 2.
Fig 4
Fig 4
Assignment and intensity comparison of peaks with large loadings.
Table 2
Table 2
Metabolites with large loadings and their changing trends in the experimental group.
Meanings of PC 1 As shown in Table 2, the first representative metabolite of PC 1 is taurine, followed by citrate and by creatinine. Citrate is also the first representative metabolite of PC 2, which will be discussed later. The changes in taurine and in creatinine primarily reflect the metabolic stress reactions in organism due to chlorinated water.
Taurine, which is a derivative of cysteine, is called a conditionally essential amino acid and plays many protective roles in the body. Taurine acts as a substrate in the conjugation of bile acids; exerts osmoregulatory, membrane stabilizing, and cytoprotective effects; possesses antioxidative properties; modulates intracellular Ca2+ concentration; regulates neurotransmitters and ion movement; and reduces pro-inflammatory cytokines in various organs [17]. As an antioxidant, taurine can directly scavenge hypochlorous acid (HClO) and prevents changes in membrane permeability due to oxidative impairment [18]; as a protector of organs against toxicity, taurine reduces nitrosative stress and oxidative stress by increasing the activities of antioxidant enzymes and intracellular GSH and by normalizing the activities of Na+-K+-ATPase, Ca2+, and Mg2+-ATPase during various toxin-induced pathophysiological conditions [19–21].
Creatinine, which is a metabolic byproduct of skeletal muscle creatine and phosphocreatine, is generally eliminated from the kidneys by glomerular filtration with partial tubular excretion. Generally, urinary creatinine excretion is constant in healthy animals, and changes in urine excretion are usually connected with changes in muscle mass or in muscle energy metabolism. Considering that the body weights of the rats are so close between the two groups, the differences in muscle mass should be also slight. Therefore, the elevated level of urine creatinine detected in the EG rats might primarily be related to an unusual muscle energy metabolism.
Meanings of PC 3 Apart from taurine, which was discussed above, the second representative metabolites of PC 3 is hippurate (Table 1). Thus, PC 3 primarily reflects the changes in acid-base balance.
Hippurate belongs to the uremic toxin family and participates in various physiological processes of energy metabolism and of acid-base balance [22]. Hippurate is an inhibitor of glucose utilization in the muscle and in the kidneys, an inhibitor of glucose utilization in the kidneys and in the liver, a modulator of fatty acid metabolism, and a stimulator of ammoniagenesis. Metabolic acidosis stimulates hippurate synthesis in the liver and in the kidneys and increases urine excretion by the kidneys, whereas alkalinization decreases its synthesis and excretion [23]. The following possible mechanism of hippurate action in the correction of metabolic acidosis was proposed by Dzúrik et al. [22]: Metabolic acidosis stimulates hippurate synthesis and its tubular secretion; hippurate synthesized in the liver is released into the blood, from which hippurate is filtered in the glomeruli and taken up from the interstitium by the organic anion transport system; hippurate synthesized in the kidneys is secreted directly into primary urine; the increased hippurate promotes ammonia production by activating P-independent glutaminase (PIG) at the proximal luminal membrane, which initiates the metabolism of glutamine and of glutamate with ammonia formation, which is a dominant elimination product of H+.
Meanings of PC 2 As shown in Table 2, the first representative metabolite of PC 2 is citrate, followed by taurine and hippurate, which indicates that the elevated urine excretion of citrate might be related to the changes in taurine and hippurate.
Citrate is a dominant product of the tricarboxylic acid cycle (TCA) and occupies a critical crossroad step in the intermediary metabolism of most mammalian cells [24]. Citrate is synthesized in mitochondria and then becomes the entry substrate into the TCA. Its oxidation provides the major source of cellular ATP production; hence, elevated urinary excretion of citrate is an obvious sign of perturbed energy metabolism. Abnormal urinary excretion of citrate indicates mitochondrial inefficiencies in energy production and explains the biochemical basis of excessive fatigue and of weakness [25]. In addition to these roles, citrate also possesses a modulatory function in the correction of acid-base balance, as well as hippurate, because of its anion properties [25].
Thus, PC 2 primarily reflects the roles of citrate in energy metabolism and in acid-base balance. Considering the differences between the two groups are not significant on PC 2 (p>0.05), the modulatory functions of citrate in energy metabolism and in acid-base balance may be not enough to make significant differences.
Metabolism of chlorination DBPs
Chlorine is a necessary element for our bodies, and nontoxic. HClO is the active ingredient of chlorination disinfectants, removing a variety of parasites, bacteria and viruses. However, although attacking microbes and viruses, HClO also reacts with many pool water substances and produces various DBPs. DBPs can be inhaled and ingested during swimming or absorbed dermally, and two classes of DBPs, chloroform and chloramines, have been the focus of most swimming pool studies thus far [26]. To better understand the corresponding metabolic stress pathways, the metabolism of these two classes of DBPs is illustrated in Fig. 5.
Fig 5
Fig 5
Metabolism of the representative chlorination disinfection by-products (DBPs) in swimming pools.
Chloroform is primarily metabolized in the liver, which explains its hepatoxicity; however, chloroform metabolism also occurs in other tissues, such as the kidneys. Chloroform metabolism may occur via two pathways, oxidative and reductive, but primarily via the oxidative pathway (Fig. 5A), except under special conditions of high chloroform doses in preinduced animals [27]. Extensive rodent studies have demonstrated that chloroform may be metabolized oxidatively to trichloromethanol and spontaneously decomposed to the electrophilic phosgene (COCl2) [28–29]. COCl2 is highly reactive, and binds covalently to cell components containing nucleophilic groups, including proteins, reduced glutathione, and phospholipid polar heads [30–31], and may be hydrolyzed by reacting with water, yielding carbon dioxide and hydrochloric acid (HCl). Chloramines, including monochloramine (NH2Cl), dichloramine (NHCl2) and trichloramine (NCl3), are not persistent and can change into one another easily while producing HClO and NH3 (Fig. 5B).
Overall, DBP-induced toxicity primarily originates from 4 reactive compounds, HCLO, COCL2, HCl and NH3 (Fig. 5). These compounds are all highly toxic to cells. These compounds can attack cells directly or indirectly by reacting with amino acids, destroying membranes, changing the construction and function of proteins and lipids, unbalancing the acid-base balance, blocking metabolism and by inducing respiratory burst.
Corresponding metabolic stress pathways
To eliminate the health hazards of these 4 compounds, the corresponding metabolic stress reactions occur in organisms. As shown in Table 1, taurine is the No. 1 representative metabolite of PC 1, the No. 2 representative metabolite of PC 2 and the No. 1 representative metabolite of PC3, which indicates that taurine plays the key role in the stress reactions induced by DBPs. As shown in Fig. 6A, first, taurine can react directly with HClO to form taurine chloramines (TaurineCL) [32–33]. Second, by increasing the activities of intracellular GSH, taurine also plays an important role in eliminating COCl2. To convert TaurineCL to sulfoacetaldehyde and COCl2 to CO(GS)2, NH3 and HCl are formed in these procedures (Fig. 6A) [34], thus explaining why taurine is also involved in the acid-base balance and is related to the urinary excretion of hippurate and of citrate (this function will be discussed in the next section). Additionally, because taurine reacts directly with HClO and indirectly with other oxidative radicals, the urinary excretion of the EG rats showed a declining trend.
Fig 6
Fig 6
Corresponding metabolic stress pathways to the chlorination disinfection by-products (DBPs).
As above-mentioned, NH3 and HCl are also highly toxic to cells. To eliminate ammonia toxicity and acidosis induced by these two compounds, NH3 and H+ must be excreted as soon as possible. In these procedures, hippurate and citrate play important roles. First, the increased H+ stimulates hippurate synthesis in the liver and in the kidneys, thus increasing the hippurate plasma concentration. Then, the increased hippurate stimulates PIG desamidating glutamine with the formation of ammonia, and finally, ammonia and H+ are secreted into urine as NH4 + (Fig. 6B) [22]. To balance increased NH4 + excretion, the excretion of hippurate and citrate are also increased because of their anion properties.
However, to eliminate the ammonia toxicity and acidosis, energy metabolism in cells is seriously impaired because two substrates of the TCA, citrate and α-ketoglutarate, are involved in the excretion of NH3 and of H+ (Fig. 6B). On the one hand, elevated ammonia stimulates glutamate dehydrogenase to catalyze the reductive amination of α-ketoglutarate, resulting in the depletion of α-ketoglutarate and of NADH. On the other hand, to balance the increased NH4 + excretion, citrate, which is another substrate of the TCA, is secreted more than normal. Thus, the TCA and oxidative phosphorylation are inhibited [25]. To meet the increased demand of energy for recovery from the fatigue caused by swimming training and by stress reactions against the toxic compounds, the skeletal muscle must catalyze more phosphocreatine and creatine, thus resulting in elevated urine creatinine. Meanwhile, the inhibited TCA and the inhibited oxidative phosphorylation also explain the inferior performance of the EG rats in the swimming capacity test.
Conclusions
In summary, the 12-week swimming training in chlorinated pool water did induce some disease symptoms, an impairment of training effects and a significant change in the metabolic profiles, although the growth and behaviors of the experimental animals were not affected. The bloody noses, bloody eyes, lackluster fur and hair removal reflected the direct irritation of the DBPs to the respiratory tract, eyes and skin because these organs were in direct contacted with the DBPs. However, possibly because of the protection from nasal mucous, the respiratory tract showed some adaptability to the DBPs. Thus, the eyes and skin might be the organs that require greater attention for permanent damage. Symptoms found in the liver and lung sections indicate that the liver is most likely the most possible target organ of DBPs, and training intensity, training frequency and water choking may be the primary factors for lung damage induced by swimming, instead of chlorination. The result of the swimming capacity test showed that training effects were significant affected by chlorinated water through perturbing the TCA cycle and oxidative phosphorylation. Through analyzing the changes in metabolic profiles using a multivariate analysis, corresponding metabolic stress pathways were proposed, as shown in Fig. 6, in which a defense system centering on taurine and related metabolites is presented. Based on these pathways, the underlying toxicological mechanisms of DBPs are illustrated, and corresponding countermeasures can be developed.
Supporting Information
S1 ARRIVE Guidelines Checklist
Completed “The ARRIVE Guidelines Checklist” for reporting animal data in this manuscript.
(PDF)
Click here for additional data file.(481K, pdf)
S1 Table
The normalized urine metabolite data of the individual animal (data < 0.000001 excluded).
(XLS)
Click here for additional data file.(144K, xls)
Acknowledgments
We would like to thank the Physical and Chemical Testing Center of Jiangxi Normal University for help in the NMR experiments.
Funding Statement
This research was supported by the National Science Foundation of China (NSFC 21365013) and by the science project from the Jiangxi Provincial Education Department (GJJ 13240). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Data Availability
All relevant data are within the paper and its Supporting Information files.
Article information
PLoS One. 2015; 10(3): e0119241.
Published online 2015 Mar 5. doi: 10.1371/journal.pone.0119241
PMCID: PMC4351252
PMID: 25742134
Jiang-Hua Li, 1 , 2 ,* Zhi-Hui Wang, 3 Xiao-Juan Zhu, 1 Zhao-Hui Deng, 1 Can-Xin Cai, 1 Li-Qiang Qiu, 1 Wei Chen, 1 and Ya-Jun Lin 1
Hans-Joachim Lehmler, Academic Editor
1 Key Laboratory of Training Monitoring and Intervention on the Sports in Water, State Sports General Administration, Institute of Physical Education, Jiangxi Normal University, Nanchang, China
2 Jiangxi Provincial Key Laboratory of Chemical Biology, Jiangxi Normal University, Nanchang, China
3 Beijing Economic Management School, Beijing, China
The University of Iowa, UNITED STATES
Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: JHL. Performed the experiments: ZHD CXC LQQ WC YJL. Analyzed the data: XJZ. Contributed reagents/materials/analysis tools: ZHW. Wrote the paper: JHL ZHW.
* E-mail: moc.anis@8auhgnaijil
Received 2014 May 7; Accepted 2015 Jan 27.
Copyright © 2015 Li et al
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Articles from PLOS ONE are provided here courtesy of PLOS
References
1. Bernard A, Voisin C Voisin C, Sardella A. Respiratory risks associated with chlorinated swimming pools: a complex pattern of exposure and effects. Am J Respir Crit Care Med. 2011; 183: 570–572. 10.1164/rccm.201008-1306ED [PubMed] [CrossRef] [Google Scholar]
2. Bougault V, Loubaki L, Joubert P, Turmel J, Couture C, Laviolette M, et al. Airway remodeling and inflammation in competitive swimmers training in indoor chlorinated swimming pools. J Allergy Clin Immunol. 2012; 129: 351–358. 10.1016/j.jaci.2011.11.010 [PubMed] [CrossRef] [Google Scholar]
3. Florentin A, Hautemanière A, Hartemann P. Health effects of disinfection by-products in chlorinated swimming pools. Int J Hyg Environ Health. 2011; 214: 461–469. 10.1016/j.ijheh.2011.07.012 [PubMed] [CrossRef] [Google Scholar]
4. Lubick N. Swimming in chlorine byproducts. Environ Sci Technol. 2007; 41: 6634 [PubMed] [Google Scholar]
5. Villanueva CM, Cantor KP, Cordier S, Jaakkola JJ, King WD, Lynch CF, et al. Disinfection byproducts and bladder cancer. Apooled analysis. Epidemiology. 2004; 15: 357–367. [PubMed] [Google Scholar]
6. Bernard A, Carbonnelle S, Michel O, Higuet S, De Burbure C, Buchet JP, et al. Lung hyperpermeability and asthma prevalence in schoolchildren: unexpected associations with the attendance at indoor chlorinated swimming pools. Occup Environ Med. 2003; 60: 385–394. [PMC free article] [PubMed] [Google Scholar]
7. Nickmilder M, Bernard AM. Ecological association between childhood asthma and availability of indoor chlorinated swimming pools in Europe. Occup Environ Med. 2007; 64: 37–46. [PMC free article] [PubMed] [Google Scholar]
8. Levesque B, Duchesne JF, Gingras S, Lavoie R, Prud'Homme D, Bernard E, et al. The determinants of prevalence of health complaints among young competitive swimmers. Int. Arch Occup Environ Health. 2006; 80: 32–39. [PubMed] [Google Scholar]
9. Font-Ribera L, Villanueva CM, Nieuwenhuijsen MJ, Zock JP, Kogevinas M, Henderson J, et al. Swimming pool attendance, asthma, allergies, and lung function in the Avon Longitudinal Study of Parents and Children cohort. Am J Respir Crit Care Med. 2001; 183: 582–588. [PMC free article] [PubMed] [Google Scholar]
10. Font-Ribera L, Kogevinas M, Zock JP, Nieuwenhuijsen MJ, Heederik D, Villanueva CM, et al. Swimming pool attendance and risk of asthma and allergic symptoms in children. Eur Respir J. 2009; 34: 1304–1310. 10.1183/09031936.00180608 [PubMed] [CrossRef] [Google Scholar]
11. Goodman M, Hays S. Asthma and swimming: a meta-analysis. J Asthma. 2008; 45: 639–647. 10.1080/02770900802165980 [PubMed] [CrossRef] [Google Scholar]
12. WHO. Guidelines for safe recreational water environments: swimming pools and similar environments (VOL. 2) World Health Organization; 2006. [Google Scholar]
13. Dawson CA, Horvath SW. Swimming in small laboratory animals. Med Sci Sports. 1970; 2: 51–78. [PubMed] [Google Scholar]
14. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ, et al. Mechanisms of hepatotoxicity. Toxicol Sci. 2002; 65: 166–176. [PubMed] [Google Scholar]
15. Tumasonis CF, McMartin DN, Bush B. Toxicity of chloroform and bromodichloromethane when administered over a lifetime in rats. Ecotoxicol Environ Saf 1985; 9: 233–240. [PubMed] [Google Scholar]
16. Reuber MD. Carcinogenicity of chloroform. Environ Health Perspect. 1979; 31: 171–182. [PMC free article] [PubMed] [Google Scholar]
17. Das J, Roy A, Sil PC. Mechanism of the protective action of taurine in toxin and drug induced organ pathophysiology and diabetic complications: a review. Food Funct. 2012; 3: 1251–1264. 10.1039/c2fo30117b [PubMed] [CrossRef] [Google Scholar]
18. Timbrell JA, Seabra V, Waterfield CJ. The in vivo and in vitro protective properties of taurine. Gen Pharmacol. 1995; 26:453–462. [PubMed] [Google Scholar]
19. Das J, Ghosh J, Manna P, Sinha M, Sil PC. Arsenic-induced oxidative cerebral disorders: protection by taurine. Drug Chem Toxicol. 2009; 32: 93–102. 10.1080/01480540802564171 [PubMed] [CrossRef] [Google Scholar]
20. Manna P, Sinha M, Sil PC. 2009. Taurine plays a beneficial role against cadmium-induced oxidative renal dysfunction. Amino Acids. 2009; 36: 417–428. 10.1007/s00726-008-0094-x [PubMed] [CrossRef] [Google Scholar]
21. Roy A, Manna P, Sil PC. Prophylactic role of taurine on arsenic mediated oxidative renal dysfunction via MAPKs/ NF-kappaB and mitochondria dependent pathways. Free Radic Res. 2009; 43: 995–1007. 10.1080/10715760903164998 [PubMed] [CrossRef] [Google Scholar]
22. Dzúrik R, Spustová V, Krivosíková Z, Gazdíková K. Hippurate participates in the correction of metabolic acidosis. Kidney Int Suppl. 2001; 78: S278–281. [PubMed] [Google Scholar]
23. Spustová V, Gajdos M, Opatrný K Jr, Stefíková K, Dzúrik R. Serum hippurate and its excretion in conservatively treated and dialysed patients with chronic renal failure. Physiol Res. 1991; 40: 599–606. [PubMed] [Google Scholar]
24. Tian JS, Shi BY, Xiang H, Gao S, Qin XM, Du GH. 1H-NMR-based metabonomic studies on the anti-depressant effect of genipin in the chronic unpredictable mild stress rat model. PLoS One. 2013; 8: e75721 10.1371/journal.pone.0075721 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
25. Lord R, Bralley J (2008) Laboratory Evaluations for Integrative and Functional Medicine. 2nd ed. Georgia: Metametrix Institute; 2008. [Google Scholar]
26. LaKind JS, Richardson SD, Blount BC. The good, the bad, and the volatile: can we have both healthy pools and healthy people? Environ Sci Technol. 2010; 44: 3205–3210. 10.1021/es903241k [PubMed] [CrossRef] [Google Scholar]
27. Gemma S, Vittozzi L, Testai E. Metabolism of chloroform in the human liver and identification of the competent P450s. Drug Metab Dispos. 2003; 31: 266–274. [PubMed] [Google Scholar]
28. Mansuy D, Beaune P, Cresteil TH, Lange M, Leroux JP. Evidence for phosgene formation during liver microsomal oxidation of chloroform. Biochem Biophys Res Commun. 1997; 79: 513–517. [PubMed] [Google Scholar]
29. Pohl LR, Bhooshan B, Whittaker NF, Krishna G. Phosgene: a metabolite of chloroform. Biochem Biophys Res Commun. 1977; 79: 684–691. [PubMed] [Google Scholar]
30. Gemma S, Ade P, Sbraccia M, Testai E, Vittozzi L. In vitro quantitative determination of phospholipid adducts of chloroform intermediates in hepatic and renal microsomes from different rodent strains. Environ Toxicol Pharmacol. 1996; 2: 233–242. 10.1016/S1382-6689(96)00060-9 [PubMed] [CrossRef] [Google Scholar]
31. Vittozzi L, Gemma S, Sbraccia M, Testai E. Comparative characterization of chloroform metabolism and toxicokinetics in rodent strains differently susceptible to chloroform carcinogenicity. Environ Toxicol Pharmacol. 2000; 8: 103–110. [PubMed] [Google Scholar]
32. Jacobsen JG, Collins LL, Smith LH Jr. Urinary excretion of isethionic acid in man. Nature. 1967; 214: 1247–1248. [PubMed] [Google Scholar]
33. Ripps H, Shen W. Taurine: a “very essential” amino acid. Mol Vis. 2012; 18: 2673–2686. [PMC free article] [PubMed] [Google Scholar]
34. Cunningham C, Tipton KF, Dixon HB. Conversion of taurine into N-chlorotaurine (taurine chloramine) and sulphoacetaldehyde in response to oxidative stress. Biochem J. 1998; 330: 939–945. [PMC free article] [PubMed] [Google Scholar