- Good evening and welcome to the Pioneer Room of the Portage County Public Library.  I'm Todd Good.  I'm the Associate Dean in the College of Letters and Science at the University of Wisconsin-Stevens Point.  Welcome to the April installment of the community lecture series which is co-sponsored by the Portage County Public Library and by UWSP and the College of Letters and Science.  Tonight's speaker is Heather Molenda-Figueira, an Assistant Professor of Psychology at UWSP.  Dr. Molenda-Figueira has been at UWSP since 2011, is an alum of the University of Michigan and the University of Massachusetts at Amherst, where she received her Ph.D., and was a postdoctoral fellow at Michigan State University before joining us.  Her presentation tonight addresses Problematic Plastics: Effects on Brain and Behavior, and this talk will provide a look at how these hormone disrupting chemicals can alter brain development as well as behavior.  Please join me in welcoming Dr. Heather Molenda-Figueira.  (audience applause)  - Thank you.  To give a little bit of background on how I got interested in this topic, it started out in graduate school when I attended several seminars that talked about how wildlife biologists were noticing some changes in reproductive structure and function in animals and how they suggested that maybe this was due to endocrine-disrupting chemicals that these animals were being exposed to in their environment. 

 I also noticed that as my friends started to think about having families a lot of them were struggling with fertility issues and I was wondering if maybe some of these same chemicals that were causing difficulties in the animals were causing them troubles with reproduction as well.  So then when I decided to start my own family, I completely freaked out about all of these chemicals that we were potentially exposed to.  So I started buying organic cotton mattresses for the crib, organic cotton sheets and bedding, BPA free bottles and organic baby food, because I knew that some of these chemicals could be harmful to my child and I wanted to limit her exposure as much as possible.  However I didn't know the full extent of how these endocrine-disrupting chemicals could impact our biology, our physiology, as well as the brain and behavior.  So I decided that this would be the focus of my research at UWSP.   So what exactly is an endocrine-disrupting chemical? Well according to the CDC, an endocrine-disrupting chemical is an exogenous agent, meaning coming from outside the body, that interferes with synthesis, secretion, transport, metabolism, binding action, or elimination of natural blood-borne hormones that are responsible for homeostasis, reproduction, and developmental processes.  So as you can see, exposure to these endocrine-disrupting chemicals can cause things to go array at many different levels.   Exposure to endocrine-disrupting chemicals can disrupt things like lactation, sexual maturation, also reduce fertility, as well as potentially increase the risk for psychological disorders.   So how do these chemicals work once they get into our body?  Once these endocrine-disrupting chemicals enter our body, their next step is to enter into our endocrine cells.  So I have a very, very, basic, rudimentary diagram of how this mechanism works. 

 Here we have our lovely endocrine cell.  So these endocrine-disrupting chemicals, or EDCs for short, enter through the membrane of that cell where they interact with hormone receptors.  These can be receptors for hormones for estrogen and androgens, like testosterone as an example.   Once these EDCs bind to our hormone receptors...   Once these hormones bind to their receptors they double up with other hormone bound receptors to create what is known as a hormone dimer.  Now, many of these receptors for hormones are classified as nuclear receptors, and this is because once they bind those hormones they then move in to the nucleus of the cell where they interact with small pieces of DNA called hormone response elements.  Once this interaction takes place, they then turn on things like expression of hormone dependent genes, the production of proteins, as well as hormone dependent behaviors.   So how do we study endocrine-disrupting chemicals' actions? Well there are first are a couple of in vitro, or outside of a physiological body, couple of different tests that we can do in vitro.  So first are hormone binding assays.  These in general tell us about the strength of interactions between these endocrine-disrupting chemicals and hormone receptors. 

 Then we can compare the strength of those interactions between hormones and EDCs with natural hormones and receptors as well.   Next we can use reporter gene assays, and this gives us an idea of how easily EDCs can activate genes regulated by our endogenous hormones.  And usually this is measured by measuring fluorescence that is present in proteins in cells.   Finally, we can also look at cell proliferation studies.   This can assess whether EDCs increase cell numbers.  For example we might want to investigate whether one of these EDCs causes cancerous tumors, so cell proliferation assays would be a way that we can do this.  Next we can also do in vivo studies.  In this case we can conduct behavioral or physiological assessments in animals.  So in these studies what usually happens is that we expose animals, usually rodents like rats or mice, to endocrine-disrupting chemicals at different time points in development, or maybe even at different dosages, and see how this affects their physiology and behavior in comparison to untreated animals.  Then finally we can look at correlation studies in humans. 

 So we can look for the relationship between exposure levels and outcomes for physiology as well as behavior and development.   All right.  So how are we exposed to these endocrine-disrupting chemicals? One of the main routes is through our foods.  Not all endocrine disruptors come from man-made chemicals.  There are natural endocrine disruptors as well.  So genistein is an estrogenic component that is present in soy products, in soy foods.   This chemical can bind to estrogen receptors and mimic some of estrogen's activity in the body.   Other foods are also large sources for exposure to EDCs, and in particular canned foods.   Acids in these foods can help to leach out some of these endocrine-disrupting chemicals that typically line the inside of cans.   And water sources. 

 So lakes and rivers and oceans all have detectable levels of endocrine-disrupting chemicals and so this again is one way in which wildlife becomes exposed to endocrine disruptors.   Okay.   Other products also contain EDCs as well, including phthalates and bisphenols, like BPA which you guys might have heard of previously.  So one of those major sources is plastics.  This comes in the form of plastic bottles, so containers that we use for water, sodas, as well as some baby bottles.  It also comes from plastics used in medical supplies, including syringes as well as medical tubing.  So of course when endocrine disruptors leach out of these plastics into the fluids that pass through these medical supplies they're going right into your blood stream, so probably not such a good thing.  Another source within plastics is in plastic toys.   Children's toys, a lot of them are made out of plastic, and they likely contain levels of bisphenol-A, although there are some toys that are now on the market that are BPA free.   Also cosmetics and personal care products. 

 So things like lotions and shampoos and soaps all have endocrine-disrupting, many of them have endocrine-disrupting chemicals in them as well.  In this case we kind of get a double whammy, because a lot of these products also are housed within plastic bottles, so that's one source, and then the products themselves often contain fragrances, and fragrances is another source of phthalates, one of those endocrine-disrupting chemicals.   So what disorders are associated with these endocrine-disrupting chemicals? Number one, it can affect pregnancy outcomes.   In humans, some but not all studies have shown that exposure to endocrine-disrupting chemicals can cause lower birth rates and others have found that exposures to EDCs like BPA and phthalates correlates with preterm birth.   It also affects various hormone-dependent physiological functions.  Some studies have shown that exposure to endocrine-disrupting chemicals can advance puberty in girls, and this is problematic because advances in puberty have been associated with reproductive cancers as well as the development of psychological disorders like depression.   It also can alter hormone release, so many endocrine disruptors can alter the release of hormones in the brain as well as the rest of the body.  It's also been associated with problems in reproductive structures in males.   Particularly when it comes to phthalates, phthalate exposure has been associated with smaller penis size, also undescended testes, lower testosterone, and also gynecomastia, or the development of breasts.  It also is associated with decreases in fertility for both men and women. 

 It also alters neural development, so one of the most harmful time points for exposure to these EDCs tends to be during in utero development.  This is a time point when there's a lot of changes in the brain going on, and one area in which some of these EDCs can affect development is the hippocampus, which some of you might already know is important for memory.   We have seen in animal studies that are problems with memory with exposure to some of these chemicals.  Also it's been suggested that some of these EDCs might play a role in development of ADHD.   EDC exposure has shown to alter dopamine function in the brain, causing hyperactivity.   And finally, EDCs are also associated with lower IQ in humans.   One last note is it also affects the risk of cancer, particularly hormone-dependent cancers like breast cancer, prostate cancer, and testicular cancer.   Okay, so why are plastics problematic? Well one of the reasons is that they are one of the main sources for the endocrine disruptor bisphenol-A, or BPA.  In the U.S. it's one of the highest volume chemicals produced and it is found in a variety of products, including plastics, where it's used to make plastics more heat resistant and clear.  So it's found, again, in water bottles and containers that are used to house food, plastic food packaging. 

  BPA is also a component of PVC, and it also is part of the epoxy resins that line food cans and beverage cans.  So again as I mentioned earlier, some of this BPA can leach out into our food products, particularly if those food products are high in acid.   It's also found in a couple other products including dental sealant and also thermal receipts.   So again, because a lot of our foods are contained in plastic as well as in cans, a major route of exposure of course is our diet.   In one study by Liao and Kannan, shown here, they looked at the content of different bisphenols in several different categories of foods.   BPA is shown in the red bars on this figure, so over 40% of foods in these categories had detectable levels of BPA, with the highest rates being in things like fats and oils, fish and seafood, meats and meat products, and vegetables.  So again, particularly those canned vegetables.   This study also looked at and estimated the daily exposure to bisphenol-A from these food products across different age groups.  What they found was the highest levels of exposure actually occurred in infants, toddlers, and children.   Although these were very minute doses, so in the case of toddlers, which had the highest rate of exposure, estimated rate of exposure, it was only about 243 nanograms, which is much, much, lower than what has been deemed safe by the CDC. 

  So what is that safe dose? Well the CDC suggests that a safe dose of exposure is about 50 micrograms per kilogram of body weight per day.   So in nanograms this would be 50,000 nanograms.   And BPA clears the body in about six hours.   But what's important to note here is that a lot of animal studies that have shown adverse effects with exposure to BPA come at doses much lower than what is acceptable for human exposure.   BPA is also found throughout many different tissues and fluids in the body, including the blood, urine, so one study conducted by the CDC showed that in 93% of human participants they had detectable levels of BPA in their urine.  It's also present in amniotic fluid and placental tissue and can actually cross the placenta, so this is one way in which a developing fetus can be exposed to BPA.   It also is found in breast milk, so this is one source for infants to be exposed to BPA, and it is also found in fat tissue.   BPA is found in higher levels in infants and children as we saw in the previous slide, and another source beyond breast milk that some infants can find themselves being exposed to BPA, again, is if they have spent any time in the NICU.  So again, if children are in the NICU they'll likely be hooked up with IVs, and possibly feeding tubes, etcetera, again which are sources of bisphenol-A.  So they've noticed that in urine of children who've spent time in the NICU, they have higher levels of BPA compared to children who have not spent time in the NICU. 

 In terms of children, as we noticed on the previous slide, they have the highest estimated dietary intake of BPA.   And this is likely due to a couple factors.  Number one is they typically eat more per kilogram of body weight than adults do, and also they are more likely to consume foods that might be a source of BPA exposure.   So BPA also has estrogen or estrogen-mimicking effects within the body, and also anti-androgenic effects.  So what I mean by this term is that it blocks the effects of androgens such as testosterone and a related hormone called dihydrotestosterone.  Both of these androgens are important for sexual differentiation of the brain, as well as the body, in males.   So how does BPA exposure effect our physiology? Number one, it reduces fertility in both men and women.  So in women it increases the risk for polycystic ovarian syndrome.  It also increases the risk of endometriosis, or build up of the tissue that lines the uterus, and also increases risk for miscarriage.   And in men, it increases testosterone, and this might seem like a good thing at first, more testosterone, more manly, but in fact it's more likely an indication that there are problems in the regulation of release of hormones within the body. 

 It also causes a decrease in sperm motility in men.   So it's also associated with preterm birth and lower birth weights.  In one study they looked at maternal levels of BPA in urine in women that gave birth prematurely or those that gave birth on time, and higher levels are found in those women who gave birth preterm.   Again, as with some of the other EDCs, this associated with a higher risk of breast, prostate, and testicular cancer, and then also with other disorders such as obesity and diabetes, indicating that bisphenol-A exposure may affect metabolism and homeostasis in the body, and is also associated with cardiovascular disease.   So numerous animal studies have also demonstrated that BPA exposure can alter brain function and related behaviors.  Typically in these studies what occurs is that animals are exposed to BPA either during gestation, through administration to the mom, or shortly after birth.  These are important time points for the development and organization of brain circuits, including organization in the brain in terms of circuits that are necessary for learning, memory, and social behaviors.  It is also during this time point that their sex differences in the brain become apparent.  So next I'm gonna walk you through a couple changes that have been observed in the brain as a result of BPA exposure, and some associated behavioral changes that have been found with BPA exposure.  So first we have the overproduction of neurons in the hypothalamus, and the hypothalamus is an important area of the brain for regulating a wide variety of things, including hormone release, reproduction, social behaviors, homeostasis, etcetera. 

 And there's also been shown to be an overproduction of neurons in the cortex, which is necessary for some of our higher order cognitive functions as well as things like sensation and perception.  Now you might think that having more neurons is a good thing but it's actually not, particularly when it occurs during development.   If we have an excess of neurons we might have problems when our brain is developing in utero in terms of pruning of those neurons.  So it's normal over the course of development prior to birth that the brain loses some neurons and loses some connections.  What's going on during this process is they're making more efficient and better connections.  So with an excess of neurons we might not have proper pruning, and this could potentially lead to problems in behaviors.  So because this is occurring in the hypothalamus, it could alter reproduction and it could also, overall, in terms of improper pruning, potentially lead to problems from mental health in humans.   In fact, some of the changes that take place in the brain with BPA exposure, are similar to those that we find in humans that have ADHD or schizophrenia.   We also find that there are decreases in dendritic spines, particularly in an area of the brain called the hippocampus, which is important for memory.  So what is a dendritic spine? Well to back up even further, a dendrite is a part of the structure of the neuron which receives information from neighboring neurons. 

 Dendritic spines are small projections on these dendrites that kind of offer more areas of connection for neighboring neurons, so essentially more input to those neurons.   With the loss of these dendrites in the hippocampus it can impact memory function.  Another thing that can impact memory function is a decrease in acetylcholine activity.  Acetylcholine is a neurotransmitter, or chemical, that is released in the brain that is important for memory as well.   Okay.  Also, with BPA exposure, we have a loss of sex differences in the brains of animals.   This in particular takes place in the hypothalamus.  So there's one region called the sexually dimorphic nucleus of the preoptic area.  You guys of course don't need to remember that, there will not be a quiz on this.  But this nucleus typically is larger in size in males, and there's another nucleus within the hypothalamus, called the AVPV, that is typically larger in females. 

 And with BPA exposure, the size of these nuclei decreases.   This can impact things like social behaviors, reproduction, and the regulation of hormone release, which these nuclei are responsible for.  So some of the related behavior changes that are found in animals, is that it alters social behaviors.  In both males and females it increases aggressive behaviors, and in females it's shown to increase exploratory behaviors.  So typically female rodents are a little bit more shy than their male counterparts and don't really want to explore that much, particularly if they are in an unfamiliar environment, but BPA seems to somewhat reverse this typical behavior in the females.   It also alters oxytocin receptors.  These are receptors in the brain that are important for oxytocin actions.  Oxytocin is a hormone that is also important for social behaviors as well as parental behaviors.  So with a decrease in these receptors, we see that there's an alteration in sexual behaviors in rats.  So females are more sexually receptive, and more motivated to engage in sex. 

 The opposite is true of males, so it decreases their interest in sex.  Also in terms of maternal behaviors, we see a decrease in maternal behaviors in rats.  So rat mothers are less likely to spend time in contact with their pups following BPA exposure.   BPA also alters dopamine function.  Dopamine is involved in a lot of different things in the brain, including the regulation of mood, movement, and also plays a role with reward systems in the brain.   With alterations to dopamine function with BPA exposure we see that there's an increase in movement, so hyperactivity in animal models.  And we also see that there are greater rewarding effects with drugs, including methamphetamine and morphine.   Finally, we see that there's a decrease in glutamate function.  Glutamate is an excitatory neurotransmitter, kind of speeds things up and gets things going in the brain, and what we find with BPA exposure is that there is an alteration in the levels of glutamate in the amygdala as well as the hippocampus.  These are two structures that are important for processing the emotion of fear. 

 So what we see as a result is, in BPA-treated rats, there's an increase in anxiety and depression-like behaviors in rodent models.   Okay.   So that's a lot that we've seen in rat studies, but how can BPA impact functions and behaviors in humans? There have been a few studies that have looked at prenatal exposure to BPA as measured in the mother's urine, and how this level of BPA in the urine correlates with later behaviors in young children.   So using the Child Behavior Checklist, or the Behavioral Assessment System for children, they found that with higher levels of BPA exposure there is an increase in anxiety and depression.   Also there is an increase in social problems.  So in one study they showed that BPA exposure was associated with higher levels of aggression and also emotional reactivity in males, but the opposite was found for females.   A separate study on the other hand, did find that there was an increase in aggressive behaviors and disruptive behaviors in females.  This was particularly associated with the levels of BPA in the mother's urine around 16 weeks of gestation, so kind of the beginning of the second trimester.   Also with higher levels of BPA exposure there's an association with hyperactivity and attention problems.  So this is one of those suggestions that this particular endocrine-disrupting chemical may potentially play a role in the development of ADHD. 

  And also, higher levels of BPA exposure are associated with problems in reading, writing, and math, in both males and females.  Now there are some discrepancies in some of these studies, as some produce different sex-dependent results, and in many cases girls were more negatively affected by BPA exposure prenatally than boys, so this might suggest that potentially female fetuses are more sensitive to these potentially negative effects of BPA.   Because of all of these concerns about health and development with BPA, it's been removed from a lot of products, but it has been replaced with a structurally similar chemical BPS.   BPS has mainly replaced BPA in a lot of plastic products, it's more heat stable than BPA is, and that's thus maybe a little bit less likely to leach out of plastics and into your food supply for example.   But another negative of BPS is that it's less biodegradable, so it might have more negative effects on the environment in general.   So it's been found in the urine in different studies in anywhere between 81-97% of participants, and again, as with BPA, it's found in higher levels in children and also adolescents.   Like BPA it's found in bottles, but also pesticides, leather tanning agents, cleaning products, personal care products, and thermal paper as well.   It is found in lower levels in foods compared to BPA, so it's only in about 10% of foods from those previous slides.  However, like BPA, some of the highest levels of BPS were found in meats, fish, and seafood.   Okay. 

 So how does BPS affect our physiology?  We know that it has estrogenic effects at two of those nuclear estrogen receptors, estrogen receptor alpha and beta.  It also has anti-androgenic effects, so again, blocking the effects of androgens like testosterone and DHT.   It may also alter cell survival, so some studies have shown that BPS exposure increases enzymes that promote cell death.   And it can affect physiology in vivo as well.  So many of the studies on effects of BPS in vivo have come from studies of Zebrafish in which their water has been treated with BPS.  And we found, in terms of compromising their reproduction, is that number one we see an increase in estrogen in males and females, and a decrease in testosterone in male fish, and this indicates that, again, there might be problems with regulation of hormone release.  We also find that there is a decrease in gonad weight in these fish that have been exposed to BPS.  They also produce fewer viable offspring and more of those offspring are malformed.  Also, in terms of brain development, we see that BPS exposure in these fish resulted in a 240% increase in neurogenesis in the hypothalamus.  So again, with more neurons we have more chances for miswiring and then later problems with physiological function and behavior. 

  In terms of rodents, exposure to BPS alters reproduction.  It decreases testosterone production and also impairs spermatogenesis, or the production of new sperm.  This is also occurring at doses lower than what has been deemed safe for BPS exposure, which like BPA is around 50 micrograms per kilogram per day.   Finally, BPS exposure has shown to increase uterine growth in rats.  So like BPA, it's potentially a chemical that could promote a disorder like endometriosis.  Okay, so what do we know about the effects of BPS on behavior? The answer is pretty much nothing.  So I had, after reading these studies on BPA and how it affects anxiety, I was wondering if potentially exposure to BPS could impact levels of anxiety as well.   Why I chose to look at anxiety is, is because it's one of the most prevelant psychiatric illnesses in the U.S. population with about 16 million people suffering from the disorder.   So in our study we used a rat model.  So pregnant rats were administered BPS-treated water that resulted in daily exposures of about 15 micrograms, and then we had a group of control rats that received untreated water. 

  This treatment began at 12 days of gestation, so equivalent to kind of the end of the second trimester in humans, and it continued until 45 days of life in the pups.  So they were exposed to BPS in the water consumed by their mothers, transferred through the milk, and then also from their water bottles once they were old enough to consume water and actually reach the spouts on their own.  So it continued until 45 days of life, and this is just past puberty.  Yes, rats do go through puberty, in case you were all wondering.  So this is just past puberty, because puberty is another important time point where hormones organize and change circuits in the brain.  We wanted to make sure we covered that time point as well.  So we had four experimental groups.  We were also interested in looking at whether there are potentially sex differences in levels of anxiety, so we had males and females that were treated with BPS or controlled water.   Following treatment with this, or also I guess, during some of the treatment with this water, we assessed anxiety using the three most common tests used to quantify anxious behavior in rodent models.   Number one was the elevated plus maze, also the open field test, and lastly the light-dark box. 

 And I'll explain what these tests are in a moment.   So testing of these three different assessments took place on three consecutive days and tests were run at three different developmental time points.  So first it was starting at 21 days of age, so this was a juvenile testing point, 37 days, which is around the time of puberty in rats, and at 56 days or older for their adult testing point.   Okay, so what do these tests look like? First of all we have the elevated plus maze, and as the name suggests it has a plus confirmation, and it's elevated off of the floor about a foot and a half.   It has two open arms and two closed arms.  So if rats are more anxious, what they do is they will spend more time in those closed arms, 'cause rodents tend to feel more secure in dark, cozy, little places.   The next test is the open field test, which simply is a square box that is divided into nine different squares.  Here in order to assess anxiety, we see what duration of time they spent in the very center of the chamber versus the corners and sides.  So the more anxious the animals are, the more time they will spend in those corners and sides versus the center of the chamber.  Lastly, we have the light-dark box. 

 This has two adjoining chambers with a door in the middle so they can pass through from side to side.  The light side of the compartment has white walls that's illuminated by a bright, white, light, and the dark side has black walls and a lid, so not too much illumination penetrates in to that side.   So for each of these assessments the duration of the test was five minutes, we recorded all of these behaviors, and then myself as well as my team of five or six students analyzed the tapes with a computer aided program to determine duration spent in the various areas within these testing chambers.  So what we found was, first of all, with the elevated plus maze that we didn't see an effect of BPS treatment or any sex differences, but as they aged they did tend to spend less time in those open arms, so essentially becoming a little bit more anxious as they got older.  We found similar effects for the open field test, so no treatment effects and no sex differences, but in this case they seemed to be a little more anxious around the time of puberty, so around that pubertal time point they spent less time in the middle of that chamber.  Then lastly we found completely nothing with the light-dark box.  And the reason for this is that the light side is very, very, aversive.  So usually within 10 seconds they flew into the dark side and they did not exit for the remainder of the test.   So, as a scientist, finding negative results kind of is a disappointing thing, but for the rest of humanity, it might not be such a bad thing.  There are some reasons why we might not have found an association between BPS treatments and levels of anxiety. 

 The good news could be that there simply is no association between BPS and levels of anxiety as has been observed in previous studies with BPA.  Second, we might have used too low of a dose of BPS treatment to elicit any changes in levels of anxiety.  So currently what we are doing in the lab is we are going to be increasing our dose and also using a different administration paradigm where we are more directly feeding the animals BPS in corn oil.  Apparently rats really like to drink corn oil.  So this will also help us to get a better idea of direct dosing rather than having to estimate it based on water consumption.   Okay.   My second to last question for everyone is is bisphenol exposure safe? In my mind this still is a little bit up for debate.  While exposure to one of these endocrine-disrupting chemicals such as BPA at doses deemed to be safe as determined by the CDC might be okay.  This is certainly not the only endocrine-disrupting chemical we're exposed to on a daily basis.   So there have been studies that have shown that these mixtures of EDCs that we take in through environmental exposures, through our diet, etcetera, can have detrimental effects on health and animal models. 

 Also, maybe because we are exposed to so many of these different chemicals, we might need to reevaluate what is considered a safe dose for individual chemical exposure.   This is something that has occurred recently in Europe.  They have lowered their daily exposure dose for BPA from 50 micrograms to four micrograms.   So quite a bit different.  So according to the National Toxicology Program at the National Institutes of Health, they do suggest that there is some concern for effects on brain behavior and the prostate gland, for exposures to fetus, infants, and young children.  They also suggest that there is minimal concern for adverse effects for mammary gland and early puberty in females, as well as reproductive toxicity in workers.  So for example, those individuals that work as cashiers and are exposed to BPA through those cash register receipts.   Finally, negligible concern for reproductive toxicity in adult men and women, and malformation in newborns.  But again, this is based on fairly limited studies that have looked at the associations between exposure levels and health outcomes in humans.  I also think that it's important to further investigate exposure and whether there are particular times in development where exposure to endocrine-disrupting chemicals in general might be more harmful to development and later behaviors and physiology. 

  So, what can we do to reduce our exposure?  Number one, we can avoid plastics that have their recycling labels numbered three, six, and seven.  Safer bets are numbers one, two, three, and four.  We can also look for glass or metal containers for our food and drink.   Next we can avoid microwaving our food in plastic containers or washing plastic containers in dishwashers with harsh detergents, so again, heating up these plastic components or plastic products is one way to promote the leaching of these chemicals into your food sources.   Next we can reduce or eliminate the consumption of canned foods and try to eat more fresh foods or frozen foods.  And finally, with concerns for exposure to children, you can look for BPA and BPS free baby bottles, as well as toys.   There are quite a few BPA free bottles on the market, as well as some toys.  Looking for BPS free items currently is a little bit more challenging but hopefully as more research comes about and suggests that there might be some harm with exposure to this chemical as well, we'll start removing BPS from products as well.   All right.  Thank you all for being such good listeners. 

 (audience applause)