From Silent Spring to Silent Night: A Tale of Toads and Men

so these introductions people always start off saying it’s a pleasure to introduce so-and-so and yeah when it’s a longtime friend former adviser and mentor I come up here they say with conviction that it really is a pleasure to introduce Tyrone today so Tyrone started off his life from Columbia South Carolina and went off to Harvard for his undergrad and then to Berkeley for PhD as well as a postdoc and then that’s where he’s a faculty member now no that’s impressive in and of itself but I’d like to give a little timeline together self-concepts so he graduated in 1985 I graduated in 1987 he started at Berkeley two years before I did and he and I were graduate school colleagues we were strapping students together largely because he was on the graduate admissions committee and I think he made sure that my application was able we’ve never met each other at that time he ended up finishing his PhD in four years Kezia applied for an NSF postdoctoral fellowship thinking that there’s no way he would get it in his third year and it would use that feedback to write a story proposal next year well he actually did get it funded so he wrapped up his PhD in four years with six months into his postdoc when his advisor at Berkeley left for a nice position and so he was essentially hired back four and a half years after starting his PhD at Berkeley to essentially replace his major professor my adviser left in my fourth year I’d like finishing in Tyrone’s lab so yeah before I was even you know I think a year or so into my first post I Tyrone was already tenure three years after starting at Berkeley I think he became a full professor just about the same time I got my first job so he’s been the full professor for like what like almost 10 years now but I’m sure I’d make a lot less than you do I’m sure he you know he started off he’s been an enderman developmental biologist studying amphibians for ages and ages he was starting some work many years ago that you’ll you’ll hear the story of today where he received some funds by this manufacturer called Novartis that makes compound called atrazine and it’s totally transformed both his research project as well as his political and social view of the world so thank you thanks Paul I can’t me give me a hug they did that last after the talk I mean not while you’re asleep we got a good story I’d like to meet this guy so – and I also want to thank all of you for coming out and and for the invitation and all that kind of stuff and as usual if you’ve ever seen me speak before I talk about science I always do my acknowledgments first that way if I run out of time the important stuff got done my first acknowledgement is always my wife Catherine Kevin my son of my daughter for their love and support and my second in line I want to thank my funding sources and and also as a matter of disclosure I’ve been funded by those companies as you just heard Novartis and Syngenta although they’ve sense as you’ll see why decided they don’t want to really hang out with me anymore they deferred to the other side throughout my talk I will give you perspectives from the industry so they have slightly different perspectives things that I call words from our former sponsor and then I want to thank all of the students that have been involved in the work I’m going to talk to you really for about 10 years worth of stuff and and we’d also like to point out here that everybody’s in blue is an undergraduate so I had a wonderful experience as an undergraduate starting with an REU NSF when I was a freshman and really I really started graduate school in 1986 when I started that program because I had a professor who treated me that way and so trying to give back to that I work as I said with a lot of undergraduates here’s my new crew and as you can also see from the photograph we enjoy quite a bit of diversity in my lab

and that I don’t not only makes for some crazy pot Luck’s which I lost one of my best cooks there’s but also makes for some really great science and finally just a shout out to my parents my family in general he comes also see we enjoy a lot of diversity there as well and finally a dedication to my grandmother who passed away in 2005 it was her level of teaching that got passed on to me and that also has really been with me throughout my entire career so dedicated to my grandmother and as I say speaking of my grandmother this is not my grandmother this is a quote that for the longest time I thought was mine actually but turns out as I’m sure we did somebody must murder understand something unless you can explain it to your grandmother and that’s not an insult but that’s sort of really because I’ve been involved in in a lot of public speaking not to academic audiences this is sort of really a philosophy that I think I’ve had adapted I’ve tried to eliminate jargon I try to rather than give a speech or give a lecture I try to tell a story as another great orator said in a language that everybody can can understand and that’s what I’m gonna try to do today is really just tell you a story honestly how I got from point A to B now I don’t have time to start I don’t have time to start with my African roots the beginning of that story I’ve never had time to start with my five-year-old or at least from when I was five years old love of em phibian biology but I’m gonna pick it up here and actually some work that Paul Barbara and I became involved in back when he was working with me in my lab we started working in East Africa actually trying to understand what I call evolutionary developmental endocrinology of African read frogs in particular this is hyper ileus artists where the males and the females are differently colored so much so that if you didn’t know any better you think there were two different species if you saw them in the wild and my interest in general and trying to understand how hormones regulate development metamorphosis and growth and sex differentiation but also in trying to understand how the environment regulates those hormones that regulate development and trying to understand the evolution of those processes and this is where Paul and I really interacted in a very unique a very neat way when I was particularly interested in the species and in this complex at the time was one how these animals end up differently colored then again evolve and why what is the ecological and evolutionary significance of this extreme sexual dimorphism opposed to paper with an undergraduate Kahneman then does where we shade that in fact for the first time we raise these animals in the laboratory and we show that they all start out green whether or not you’re male or female then we showed that the male hormone testosterone has no impact on these frogs that the female hormone estrogen in this case that’s true by all will induce a color change regardless of whether or not you’re male or female and this is significant because essentially what happens is the females at puberty at sexual maturity start to make estrogen and that estrogen then binds to a receptor in the skin and induces two colors nature so what Paul and I are still trying to do if NSF would ever listen is trying to understand not just a hormonal regulation of this developmental process but how and why it evolved why it is the way that it is turns out though that as you just heard that there was an interesting detour about twelve or thirteen years ago when my wife is in the audience that beautiful woman you saw in the beginning and heard me giving this talk and she said no you’re tired why don’t you patent that frog and I and I said even why would I ever patent a frog you can’t patent the Frog and her brother said you can’t patent the Frog heretofore to be referred to as to a sentence he was a lawyer so he had although all the words for that and we’ve been I thought it sounded kind of neat so we called it the hyper Alice Argus endocrine screen or the haze test and here’s why your patent the Frog here’s my neva patent the Frog in this case a process or the use of this frog so here’s a control they all start out green and we can raise these out of tens of thousands they’re about the size of your pinky nail when they metamorphose we can dip them in estradiol a natural estrogen that circulates in every moment in this room and we can induce a color change we can dip them in ethanol estradiol the synthetic estrogen that’s used in birth control pill and we can induce you see the spots developing a color change in a matter of days we can get them in diaster still withdrawal a non-steroidal synthetic pharmaceutical estrogen that was used a few years ago in women to treat various conditions or we can dip them in compounds like DDT which isn’t an hormone or an estrogen but we know it binds to the estrogen receptor this pesticide and can turn it on so here is the thing we tested dozens of compounds or examined dozens of compounds and what we found is that every one of these chemicals that act like an estrogen and caused color change in this frog was also known to bind to receptor in humans and promote breast cancer so we had this

model where we could raise these animals by tens of thousands and test individual chemicals with a very simple cheap visual assay test that would allow us to tell you oh yeah this chemical is a potential promoter of breast cancer well as more as we showed that if we use the estrogen blocker tamoxifen we could block estrogens ability induce color change in these animals so we could not only screen potential breast cancer promoters but we could screen potential estrogen blockers that can be used potentially by a pharmaceutical company and the treatment and of course should do to human self screening but this again we could do by the tens of thousands the other thing we propose is because people were just now starting to talk about endocrine disruptors and contaminants in the water that might act like hormones that you could send me a sample of water and I could put some tadpoles in envelope they change color I’d be concerned about your water supply that was the idea the idea was a little mom-and-pop shop on the side my wife at the NBA my brother-in-law with a law degree mean when in the science and and boy things sure took a turn from there we haven’t gone back to solve the basic problems of hyper aliens yet because we actually filed a patent and we actually we actually had a customer so in the virus they were the Flies that the time came along and said well we’d like for you to use frogs to test anthracene and this is literally and literally just telling you to store almost an exact chronological order I had never heard of atrazine and it is now in case you’re chemically inclined we’re sort of joined at the carbon bond now so to speak but if you google tyrone after xing comes up with google apps you see Wikipedia links is everything after Xena tiger and it is an herbicide or weed killer when I say pesticide if I do I mean anything that kills a pest I know some people in equate insecticide and pesticide but to me anything that kills a pest is a pesticide so an herbicide is a pesticide in this case a weed killer of course and it’s mostly used on corn in this country it’s been used since 1958 so it’s we have a long legacy here and we use 80 million pounds annually United States and I say it that way cuz I can’t fathom 80 million new pounds of atrazine every year being put in the environment here it’s the second biggest selling pesticide in the world it’s used in more than 80 countries so we’re talking about a global issue the things that I’m going to talk to you about but it’s now outlawed in Europe where’s the lawyers like for me to say it has been denied regulatory approval the point being that the hand of the farthest Mouse ingenta is in Europe so we’re using 80 million pounds of a chemical that’s been outlawed on home turf no pun intended so let me now secondly in case you haven’t met him yet introduce you to the African clawed frog Xenopus face-to-face and I introduced this frog this way for a couple reasons I believe in whole animal so before we break it apart and look at cells and how I want to introduce you to the main character the only reason I love the introduces frog is it is the lab rat of the amphibian world I never thought I’d be working on such a non frog it can’t jump it has no tongue it’s completely aquatic it has a lateral line system it’s a weird frog but the reason everybody works on and the reason of people sequenced actually a related species because everybody in the world knows Xenopus and who knows the reason why and I know you know it’s an interesting story this was the very first pregnancy test developed in 1920 it turns out that the human pregnancy hormone HCG is so similar to what’s called a luteinizing hormone in this frog that it’ll make this frog lay eggs so in nineteen forty or so if you thought you were pregnant you’d go to the doctor and they would inject your pee into the Frog and if it lays eggs they say okay fix up the you know baby room now I love this story because we’re just talking about MSF what I don’t know is who’s the first argument what’ll happen if I inject P into a frog and receptive like that better less having different meals back then now the other reason they pointed out and I know I probably don’t have to do it for this audience is that in the same way that the testosterone and estrogen that worked in hyper areas artists are exactly the same hormones in human synthesized the same way functioning the same way and here’s another example of where the hormone of pregnancy is so similar to the hormone regulating reproduction and miss frog that the costs react the idea being that as I talk to you now about endocrine disrupting compounds and frogs keep in mind that we’re probably not just talking about frogs and when we get to the end of the top a tail of toads and Men I’ll show you direct evidence that but that’s the case so maybe long story short we showed some effects and the virus didn’t like it I said I’m leaving they said goodbye and then we published a paper after we repeated the work which technically belonged to them at the time that paper was published in Proceedings of match Academy of Sciences hermaphroditic D masculinized frogs at

the exposure to the herbicide atrazine at low ecologically relevant doses you have seen how why they didn’t like me I don’t pull any words back here’s what we found just briefly this is the old stuff so this is back in 2002 we found that after seeing inhibited growth of the Voicebox atelerix and males which is of concern to the manufacturer because male larynx or voice box is testosterone dependent the same reason that the men in this room have deeper voices in general and the women because we have got more testosterone at puberty same things regulating these frogs suggesting that they’re chemically castrate it would I like to use they’d like for me to say damask utilized but I like chemical castration better and here’s why if you look at the goatman cities Hannah most a proportion depending on the dose look like this so here’s the kidneys either the gonads and this is from our original PNAS paper those testes then it has two ovaries that it has a large testa’s then it has more overs we published another paper in Environmental Health Perspectives that show that there are number of abnormalities that are defined by atrazine exposure or to other hormones one is we call single sex polygon it is omits just a fancy way of saying mini testes which which I love the story cuz this one actually mated on Jay Leno he made some comment about what guy wouldn’t want a few extra my comment of course is to get you in enough trouble and in the case we shall see no matter how much they have they don’t work we had lateral hermaphrodites test us on one side over Eon the other test is in the front ovaries in the back and then these mixed ones which regardless how you look at it none of this is normal and I point that out one because the manufacturer likes to go around saying although haze is making a big deal out enough in its natural variation I’ve looked at tens of thousands of frogs does not normally a naturally occur the other reason is and I won’t ask you to do it well who knows what people think frogs are hermaphrodites naturally somebody always knows somebody always has it anybody Jurassic Park for 10 years that have people asked me wasn’t that natural because apparently and I didn’t realize was either apparently in Jurassic Park it was frog DNA that made the dinosaurs change sex and that came originally from that graffiti a per who actually somebody we were talking about who misidentified male and female vanity fathers hyper always the earlier species were talking about hey this is not normal that’s the point we propose that here’s what happened so if you imagine that this is rooted that this is your testicle you should make testosterone and I always point out the people who’s literally means testicular hormone it’s the male hormone that controls most masculine features of feel what we propose because it is evidence for this at the time is that after seen induced aroma taste and this is the only sort of technical term I want you to remember it’s gonna come up over and over again aroma taste is the enzyme in a machine that converts testosterone into estrogen the generator of estrus is where that word comes from and so when these animals were exposed they were deemed asked analyzed and I’m showing you a simple version I’ll show you a longer version later they would deem asked you lies because using up the testosterone so to speak and social can be feminized because now they’re making the female hormone which is fine if you’re a female that a little complicated if you’re a male insert of that same paper the PNAS paper again but here are control levels of testosterone measured at night his addressing treated males and his control females so we have good evidence that they were chemically castrated and feminized low testosterone small lyrics and the growing ovaries in their testes and so that was the first paper then and I thought we were done but then I sort of realized there were through some unanswered questions one we didn’t know if the hermaphrodites were males with ovaries or females but testa’s at the time even the frogs have genetic sex determination the chromosome is identical and there were no sex specific markers so we couldn’t genotype them we met a good idea because if we had 20% of Americans in a population we would usually have that 30% males and 50% females so we were missing males for every hermaphrodite that we counted so we had some idea and there are other reasons that I won’t go into here but we also didn’t know what happened when they became adults because we only looked at the metamorph exposés larvae so with these hermaphrodites remain hermaphrodites for life with a shift over and become males what they shift over and become females a couple unanswered questions and we didn’t know how to answer them other than to grow some frogs out for three years but that’s a hard thing to do because that means you have to go to students I have a project for you and maybe in three years you might have some data it’s a hard thing to get people to commit to but we did it expose these animals we raised them up for three years and and here’s what we got see these two guys I showed we should have put some ratings on the talk II didn’t know it’s going to be x-rayed hey that guy looks like if you use your imagination it looks like he’s kind of smiling

that’s his brother and we know that now because one we had an all-male population we only looked at males there’s now a genetic marker called BMW that females have but males don’t so there’s a unique chromosome with this marker on it and when we genotype that guy he’s a male and he’s a male genetically he not only performs that the female though he lays viable eggs we have three generations now in this population that hasn’t had a genetic female in it for three generations we can expose these males to addressing in a proportion of them turned into females and we can maintain the population that way so the other thing that we looked at and then factually another former student was involved in this part of the project is we again can identify true genetic females for example in our controls and we could show that in their gonads are expressing enrollment ace which is the enzyme that makes estrogen which is makes that make some function as females at the same time in our app isn’t treated animals again we know that our females are really males genetically but they express or limit ace and make estrogen assist their females that’s what turns them into females and that’s what keeps them producing eggs that’s what keeps them reproductive so we have a connection now between this molecular factors internal aromatase a biochemical event testosterone being turned into estrogen a development of morphological and reproductive behavior event which was which was pretty interesting so we sort of have this spectrum of where we have genetic males and 10% as I just told you this experiment turning the females that make estrogen have essentially an ovipositor when I call chloroquine labia and absence of male Breeden glands a female type larynx four legs and they can reproduce fully functional as a female and lay viable eggs but I thought in case of a Latino what to do because now this is only 10% of our animals ninety percent of them look like males and so the question was so we were getting ready to write this out perfect I think we may have even submit it just this part and it didn’t make it so I said to gee why are we gonna do with these other males and it seemed hard to believe that 10 percent of the animals were completely vulnerable and that the other 90% were normal functioning males but I’m not a behavioral biologist I didn’t know what to look at but I came up with some ideas I call this the pool party experiment the idea is you could fail control where females for genetic females in a pool at 6 p.m. you put in full control males and if it in for a dressing treated males and then you ask can these guys compete with these guys for a limited number of females so and I’m going to behavioral biologist I’m just sort of making this up or I guess pool party weather analogy might be the nightclub right so you you you start off in the club the lights go out the next morning the lights come on and then you just see who made the hookup and so here’s how we do that here’s what that looks like see there’s a guy who could stitch us on them so you can see who’s who so here’s a blacks th black stitch there’s a guy who lost out there’s a guy who won and there’s guy who lost out their eggs on the bottom so it’s been a full cloudy and you look at the pool party data you find that the controls are pretty good so we did this four different times everybody was a virgin so no male is tested twice no females tested twice this is the first time they’ve ever seen the opposite sex experience wasn’t an issue only to after seeing treated mils ever they’re successful got the hookup as my students say now imagine you at the club and one set out line for prints ugly lights everybody’s inspected the lights come on now imagine that some guy comes up sticks a needle in your heart and takes a blood sample inside but something want to sign up for it necessarily and so that’s what we did so we measured the blood and everywhere to control males whether or not they wanted loss here’s their average testosterone level then we measured every fantasy treated male over these four experiments so there’s sixteen animals being measured there’s their average then if you look at the individual data here’s everybody you make the love connection it is the addressee treated animals here’s the two that made it at pas argue with me if I said this is to cough I said if you can’t get your testosterone above five nanograms per mil you don’t cut it because that’s the way it looks to me only two animals were ever able to do it and they’re above that cutoff so the problem is these males whether or not their interest or not we don’t know but they don’t get it estas your own serves when their females around either they’re getting beat up better controls that a female’s don’t like them for whatever reason they don’t have enough to Stas your own to attract and successfully mate with a female so then I stay didn’t know what to do then we did what I call the motel 6 experiment the idea is again virgins they’ve never seen females before instead of making them competed the clock we just got in the room right away so you had two male and a female in here and after Xena treated male and female there’s styrofoam in between so they can’t peep and see what the other ones are doing and the idea here is to ask even if you don’t have to compete can you be a

successful male motel 6 and here’s the answer the way we answer that question is we put them in those tanks the next morning we collected the eggs we’ll let the edge develop for three days and some undergraduate counted thousands and thousands of eggs and figured out how many hatched and how many didn’t how many turned into tadpoles about 80% of the controls 80% of the eggs and a control tank are fertilized whereas we’re down to about 15% and atrazine treated males so even if they get a female with no competition they’re incapable of successfully fertilizing most of the eggs and here’s the reason why so now now that they’ve done all the behavioral stuff you know we can sacrifice um and look at what’s going on and it’s lucky if we did that in direction in that order because it’s kind of hard to get dead animals reproduce anyway so these are the test is the accepted cross-section you can probably guess which one is which I will make you vote and here’s why these guys these guys can’t fertilize eggs if I blow up a section see that’s what testicular tubular these are all premature sperm developing all the dark stuff in the middle there’s a lot of millions of sperm heads all lined up in that to be ready to go throughout this test is this testicular tube you there’s the outline of it this has a bunch of like sort of debris and junk in it and most of the tubers are like that there’s a few with sperm but that’s it these guys don’t have enough testosterone to attract a successful mate with a female even if they’re given the opportunity to mate with a female and even if they show you behavior which we don’t matter they do they don’t make enough sperm because they don’t have enough testosterone to support sperm production so they are truly chemically castrated this is just lettuce stuff straight out of the paper by the way the paper just came out a month ago in PNAS this is stuff straight out of the paper here’s the breeding glands which is testosterone dependent the large and controls faint and small and anthracene treatment animals statistically significant further indication of a lack of testosterone here’s the lyrics of the Voicebox the muscle goes all the way around the fall halo and a control in the anthracene treated animal it stops at the power higher just like a dozen of female so there’s not a size difference but the arrangement of the muscle is much more feminine like and atrazine treated animals so they are low testosterone they don’t show reproductive behavior in that they can’t compete with control males even when they do they don’t have enough sperm the fertilized eggs they’re missing their androgen dependent breeding glands they’re larynxes or voice boxes feminized they’re truly chemically castrated and another paper then working on with former graduate student dan buckles we’re looking at the more thoroughly it turns out that in frogs GnRH of the natural permanece of hormone controls luteinizing hormone and followed the stimulating hormone from the pituitary just like a dozen humans the Elat stimulates testosterone from the testes but in the presence of atrazine testosterone is converted into estrogen and 10% of the animals is estrogen makes them completely females and the other 80% the damask UI’s ones that I just told you about enough estrogen feeds back and shuts off the reproductive axis this is exactly how birth control pill works you make enough estrogen and guys if we took birth control pill same thing would happen we progressed and then we would lose our fertility so these animals was like they’re on a little bit of estrogen on a birth control pill that’s shutting down their fertility because these hormones are required for testosterone and sperm production and that’s what’s happening these animals and we’re putting this together in a separate paper now so again all this stuff just came out after zillion deuces complete feminization and chemical castration and their African clawed frogs just came out a month ago again in PNAS now what have I gotten myself into I thought I was done is that okay my curiosity is now I’m settled that about three years ago or two years ago when we first started putting together the most recent pns paper we also did a series of replicants other families our lineages to ask how repeatable this finding was and that there is variation and a susceptibility and I can tell you it’s not something is 45% turn into females but any mates where these tanks are all males they’re all brothers and my student Maness knock my win came up she would come up every day and she’d say you know I was cleaning the tanks and there a couple tanks I have to pull these guys apart all the time I simply talking about their all-male she was oh no I have to pull them apart every morning and so I went down and then they saw us there’s two guys two brothers actually sometimes you see this look at that it’s just a free-for-all this three there’s another pair coming up the set that Wow in in in 20 years in working with Xenopus and I’m not gonna exaggerate hundreds of thousands of frogs I’ve never seen this before but I didn’t know what to do about it I’m not a behavioral biologist so I said well okay we need some data then we need more than photographs we need some data so I said go down each day and just count the

number of same-sex parents cuz I know females we’ve removed that with females from those tanks it’s all males first day she comes back and she counts so these are brothers by the way this is 40 brothers of a tub this is 40 brothers count them again third day she counts now sometimes that happens in controls there’s a background level so there’s a background level and controls but it’s highly inducible or enhanced by an after scene exposure so didn’t know what to do with it okay so I’m thinking now sounds like a nature paper you got a plan so I’m thinking right now what do we do so I said well and I apologize just like it did last night I don’t know if I’m being politically correct or not so I apologize I may use some wrong language or say something wrong but I said now I said now my we got to figure out who’s gay the guy on the top would have died in the bottom right and so it turns out if you look at the number of pairs same set of Tanks hears that we did that on day one she goes down when she came out so again there’s a background level but you can see it’s enhanced more than three-fold and the after seeing tanks side-by-side there we did is we took the guy off the bottom the guys off the bottom and we put him in the control tank and then you double the number of same-sex pairs in the control tank and now there’s only one pair in the atrazine then in the next day we took the guy off the bottom out of controls we put it back in the actors entry to tank and then look what happens then we take the ones off the bottom out and they’re marked we know which ones are which we clearly the control tank and and now there’s no more there’s no more party in the atrazine tank then we put them back in the afternoon tank and now there’s party again then you take them out and now the party goes away again and and and so on so the ones on the bottom are determining the number of same-sex pairs and and here I think I’ve got summarized here with the receptive males are the ones at the bottom of the tank you have much higher same-sex payers than it controls and when you put the receptive ones in the control tank again it doesn’t go as high as the at the the atrazine treated ones but you do get an increase and you get an absence with the section of this one paired not only the ones in the bottom control the number of pairs so either they’re soliciting or just not resisting that they’re fixed and by fixed I mean if you look at these pairs the ones on the top that’s kind of different ones every time the ones in the bottom it’s the same twelve males every time they’re fixed in that behavior and in the controls this is just sort of random there’s different ones in the bottom and top all the time these guys are increasing the number of pairs because they’re fixed and this female type behavior their gender identity they think as I’ll show you they think that their females so I definitely did that again I’m not a behavioral biologist I’m just to figure out what we do so here’s the next thing we did we looked at the number of pairs and twelve observations in the different subgroups so here’s one that’s the one I’ve been showing you here’s another one and they’re actually cousins and here’s another one and here’s another one so now you see the trouble I’m getting into same-sex behavior homosexuality what everyone call it there’s a genetic component some families have zero backgrounds some have some background there’s an environmental factor that’s inducible by atrazine which is all sugar causes a hormone imbalance and it’s context dependent if you take the ones from the top and put them in a pool party they will choose females over these receptive males now you might be asking yourself is is it just that you have some big males that are overpowering little males right and the answer is no here’s the body weight the ones on the top here’s the ones in the bottom the ones on the bottom are always bigger which is interesting because normally the females are bigger than the males if you think it estas your own levels however the ones at the top with one exception have higher testosterone levels and the ones in the bottom if you look at estrogen the ones on the top of zero the ones in the bottom have estrogen levels as if they’re females and even if you inject these guys with gnrh the testosterone levels go up but so do their estrogen levels they behave like females they’re always on the bottom and their hormone profile is like a female so we propose them that here’s what we have we have the spectrum of males that don’t respond to atrazine they have high testosterone reading glands and their tight legs test us for the sperm and they’re always on top we have males that are damasc utilized that have a male type cloaca and no breeding glams female typically eggs no sperm and they’re uninvolved they’re asexual sort of then you have these animals that I don’t damask utilized or slightly feminized where they have a slightly feminine cloaca no breeding glands and even though they’re still males as I’ll show you in a minute they’re always on the bottom as if through a female and then you have these completely feminized animals that I just told you about so now that we’ve done all the behavior of stuff we constructed dissect out some of these receptive males and we find stuff like this his

kidneys there’s a nice-looking test dissection this is all over the duct so they’re still male but this is the equipment of a man with a uterus there’s still mail they still have male gonads but they have some female parts and they behave as if they’re females they think that they’re females their gender identity as female now this shouldn’t be surprising I could show you kind of the same thing with estrogen but the analogy I often use is it’s like smoking if everybody in this room smokes cigars some of us would get lung cancer some of us would get asthma some of us will be George Burns and never show any effect at all so there’s a range of effects even within a tub when you’re exposed to something like atrazine we should probably expect it and not be surprised so there briefly we’ve shown this is a paper published in Nature that similar effects occur in other species these are leopard frogs or this is a leopard frog this which I usually call the junk in the trunk our eggs that have yoked up into this males test us and are bursting through this males test us so it’s not hermaphrodite it is a male but it’s growing eggs instead of sperm we’ve shown that the basis that we’re using or ecologically relevant in other words this isn’t some wild crazy dose that nobody would ever see the package of atrazine recommends application at levels that are up to 290 million times our effective dose which is point one parts per billion okay which is one one thousandth of a grain of salt and two liters as our ace to give you a visual these are minimum and maximum levels of agricultural runoff temporary pools permanent water and precipitation again here’s what it takes to make hermaphrodites and here the habitats at risk there’s enough after Zenon rainwater to chemically castrate and make hermaphroditic frogs a half million pounds of after zine go up on dust and come down and rainfall in the US every year and they can travel over 600 miles they can measured in the rain water in Minnesota from when it’s applied in Kansas same thing in Europe that’s why it was banned instead of country by country but as all European Union is the level allowed in our drinking water three parts per billion which is 30 times what it takes to chemically castrate and make hermaphroditic from so do effects occur in the while again you publish a paper in nature here’s an animal I’m going to show you a cross-section through its testes blow it up blow it up to stick your tubules that are missing sperm but include eggs throughout in that particular study we showed the areas of highest atrazine user in red this is the range historically of leopard frogs and everywhere in red now are the areas where we found hermaphrodites and 100% of the time anthracene contamination I used to tell people we were controlling for latitude in this study but this is i-80 and we were driving to a hurt meeting in Indiana and and we collected along the way and got a nature paper process now what we’re doing that’s that we’re finishing up the summer we finish up all the analysis is instead of following the rules we follow up the North Platte and the Missouri and the Mississippi River now we have a sample size that’s like five hundred thousand over ten years and the idea is to not just look for a correlation between atrazine and and hermaphrodites but also to determine if there gradients whether these rivers start off relatively clean all three and asked if their gradients of increasing frequency or severity of hermaphrodites with the increase in anthracene the other thing they’re looking at is whether or not their temporal gradients as well as spatial because because we did this over because because because because we did this over ten years and some places at the North Platte where there is antecedent contamination and 92% of Maronites years later there’s no act racine and no hermaphrodites which doesn’t mean that these guys got well and it doesn’t mean at the atrazine degrade it it just washed on down to the Gulf of Mexico but we’re looking at the young of the year so what it means is once the anthracene is gone the new animals aren’t affected an important finding because it says that this was not normal because if it were there’d be hermaphrodites every year not just in years when there was at regime so not only are there only hermaphrodites where this after seen their own Manfred is in our studies winners anthracene all I’m showing here is I wanted a few academics who brags about what I haven’t done a number of papers since hours have come out supporting after zine effects on gonads and amphibians so we’re unlike the industry might tell you they’ll tell you a few things this being one of them and let the industry might tell you we’re not the only ones who are finding these types of effects words for modern former sponsor they speak for themselves so the reason I call this from Silent Spring the silent night is that I think that much the same way that Rachel Carson talked about the death of birds in the role of pesticides as tell her that something that can output pending Silent Spring was telling us something

about human health I think in the same way over 70% of all infants are in decline to some extent after zena’s but one pesticide and pesticides were that one cause of that decline but I think that after zine and pesticides are a significant one and in that’s the same way that our Silent Spring should have been telling us something I think our Silent Night should be telling us something and you can probably see where I’m going with it and a tale of toads and Men because the hormones we’re talking about our synthesize had functioned exactly the same a colleague of mine wrote an echo epidemiology the occurrence of an association in more than one species and species population is very strong evidence for causation I’ve shown you evidence from more than one species of amphibian more than one generally more than one faintly even there’s also evidence and fish that I’ll tell you a little bit about not from my lab there’s evidence in reptiles and birds that I’m going to talk to a little bit about of toads and men the evidence in mammals including humans via the same types of mechanisms so doses testosterone decrease in the presence of atrazine leading to a decrease in sperm production and other vertebrate classes the first example is in fish where normally salmon when exposed to females urine testosterone levels go up and the presence of atrazine testosterone levels decline and don’t go up seems like a story you’ve heard before if you look at milk or sperm convection in the same fish it goes down as well very similar to what we’ve shown in our frogs this is work that was done in England on salmon so we see a decline in testosterone and fish a decline in testosterone in frogs as is our work and their studies and rats that I’ll show you in more detail that show a decline in testosterone again you have to be convinced that across vertebrate classes that this was just a coincidence rather than a cause-effect relationship in humans Shanna Swan showed the fall if you look at controlled men in Columbia Missouri compared to men subfertile men she called him who had no sperm count and couldn’t get their wives pregnant there’s significantly more atrazine in their urine and i don’t want to overstate her data but if you look at this there’s enough atrazine in the urine to chemically castrate and produce the effects that we’ve produced in frogs about point one parts per billion if you look at that i’m going to change the axis now if you look at levels from a study in california in 1993 here the levels in few workers and that will also give access again because here the levels of men who apply anthracene then reapply after that you have up to 2,500 parts per billion atrazine in their urine that’s 24,000 times what’s associated with low fertility and men in Columbia Missouri that’s 25,000 times what we use in a laboratory to chemically castrate and make hermaphroditic frogs or as I tell my so-called public audiences one of these guys could pee in a bucket I got diluted 24 thousand times and used the anthracene in their urine to chemically castrate and make hermaphrodites out of 24,000 buckets of 30 tadpoles each now one issue had become involved in as the environmental justice issue because we know nothing about the health of these men because they’re primarily Mexican Mexican American workers with life expectancies of 50 that in addition to atrazine are exposed to chemicals like Corp ikran which was originally developed as a nerve gas in California and as I said the vast majority are Mexican Mexican American workers with little access to health care but whereas in terms of environmental justice issue if you look at California as you may know California at least until recently was the fifth largest economy in the world that economy was based on agriculture one in ten jobs in agriculture thirty percent of the land as an agriculture we produced 350 agricultural products 50% of the u.s. is food fifty percent of the United States is food comes from California we use more pesticides than any other state and ninety percent of the workers are Hispanic if you now plot onto here the top ten counties for agriculture the top ten pesticide users using counties and now dot the thirty poorest towns in California look at the overlap so the poorest people living in the most contaminated areas doing the jobs that expose them to the most pesticides are the ones that make us the fifth largest economy in the world or whether wherever we stand now so this becomes a significant environmental justice issue because there is a targeted group that we need to worry about so the other side equation is aroma taste turned on by atrazine it won’t produce egg yolk and egg yolk production in humans but we know the aroma taste and estrogen are associated with mammary tumors and prostate cancer so is there evidence that we should be concerned

about for prostate cancer I’m going to read to you from a paper published and the International Journal of occupational and environmental health the paper was the study was conducted in a plant that produces a cuisine in San Gabriel Louisiana and a community that does 80% black or african-american an immediate to you because I later say that I’m misrepresenting their findings so I’m going to read to you exactly what they wrote out of their own factory they they take the increase in all cancers combined seem and the overall study group was concentrated in the company employee group sounds like a nice place to work they wrote quote the increase in prostate cancer yes subjects was concentrated and company employees they were quote the prostate cancer increase was further concentrated in actively working company employees so if you actually go to work and more likely to get prostate cancer they work late all that one of these cases occurred in men with ten or more years since hire so the more loyal you are to the company the more likely you are to get prostate cancer and finally and I think most significantly analyses restricted to company employees also found that the prostate cancer increase was limited to men under sixty years of age so a disease that most men get when are over 65 every one of these men with 50 years old asset one and the increase was 8.4 for the reason I point out as an african-american community I visited some Gento they wouldn’t let me come in but they’re waste or whatever comes about a pipe dumps right into the Mississippi most of which looks like that I mentioned that 80% african-american community because here the top 13 cancers in the US what’s in red now are those 13 we’re asking how many are african-americans more likely to get 11 out of the 13 is that because there’s a biological difference or is that because African Americans like Latin Americans are more likely for Latino Americans are more likely to live and work in areas where they’re exposed to pesticides and other chemicals associated with these kinds of health hazards what’s more is if you look at mortality relative duck asian-americans african-americans are more likely to die corrected for healthcare access of 13 out of 13 with prostate cancer being 2.5 just by being a black man they’re two and a half times more likely to die from prostate cancer and the response of the industry and in Xiamen toxicology chemistry the society that’s tried to ban me from speaking there was that they say that this is a defamatory remark and their response was we don’t put our factory in places just because they’re black people there they put out factoring the places with the cheapest real estate and that just happens to be where most bi people cliff they actually said that to me so with regards to breast cancer as I told you max testosterone goes down with after zine and there’s a concomitant increase in estrogen just like we showed in frogs just like they’ve shown in fish just like they’ve shown in Turtles just like they’ve shown in alligators coils chickens now rats this is a study done by the EPA and there’s same less there’s an increase in the incidence of mammary tumors that are estrogen dependent in humans if you take a normal human adrenal cell it doesn’t express aroma taste or make estrogen but you explains that the after scene and it does there’s now not only this lab but my lab and several others have repeated this effect so we shown aroma taste induction just by coincidence and fish frogs alligators turtles chickens quails rats now human cell line which is quite a coincidence at least one study showed with a p-value point zero zero zero one that women whose world was contaminated with atrazine are more likely to develop breast cancer relative to women who live in the same community study that in Kentucky but don’t use their world water so that alone didn’t tell us that when we have experimental evidence showing that after zien can do and this aquatic organism that sort of backs up what a person can do in this aquatic organism at the same time the human cell line studies are helping us understand on the molecular level how the after zine regulates aromatisse they think we pretty much have that figured out and that significant is and what they’re most concerned about is this is from one of my graduate students who’s now saw an induction of aroma taste in human breast cancer cells this is significant because of the following cancer happens when a cell those bands he has red that means Pam the estrogen receptors important and breast cancer because breast tissue is estrogen dependent now how much sense does that make have you ever thought about this women most women get breast cancer after menopause you’re getting an estrogen dependent cancer at a time when your estrogen levels are the lowest they’ve ever been in your life the is because one breast cancers depended a lifetime exposure to estrogen or

estrogen like compounds but also when you develop the breast cancer turns out that breast cancers the fiberglass Express aromatase locally so even though your blood levels are low you make estrogen locally which stimulate those damaged cells to turn into tumors this local expression is so important that the top treatment for breast cancer right now is a chemical called letrozole which knocks out all imitates how much sense does that make when knocking out aroma taste a message in the treat breast cancer when we’re using 80 million pounds of a chemical that’s the most common contaminant of drinking water that does exactly the opposite what’s more is Novartis oncology offers treatment for cancers that range from breast cancer the same company that gave us 80 million pounds of addressing an aroma taste inducer associated with breast cancer now gives us lectures all which blocks aroma taste to treat breast cancer so if you’re living in a community I’m taking letrozole to treat your breast cancer how does that impact that by the atrazine in your drinking water now I get into a little bit of trouble the lawyers wrote to my Dean because I read a paper called a one-stop shop chemical causes and cures for cancer that went to Obama’s straight to Obama’s office and the factors of press release coming out this week they wrote to my Dean they said while Syngenta does share some corporate history with Novartis and Astra Zeneca had has been completely undependable since November 2000 some gender simply does not manufacture self pharmaceutical products November 2000 now it turns out in January 2000 they published a paper not me where they wrote the observed induction of aroma taste of rate-limiting enzyme and the conversion of androgens estrogens may be an underlying explanation for some of the reported hormonal disrupting and tumor promoting properties of these herbicides in vivo in January 2000 and just by coincidence in July 2000 a marketing application for first-line letrozole treatment of postmenopausal women was applied for by the FDA so I’m not saying is any kind of conspiracy going on I’m just saying there’s an interesting set of chronological coincidences when in January thousand they discovered adapters and induces aroma taste and promotes breast cancer by July of 2000 they start selling aroma taste blocker to treat breast cancer then by November 2000 they stop selling a cuisine and spin-offs in Ghent that’s just an interesting set of coincidence so I’ve talked about prostate and mammary cancer anthracene also causes immune fillion rat near damaging rats that are exposed in utero an EPA laboratory showed that after seen because of the hormone imbalances it creates causes abortion and four strains of rats those that don’t abort their sons are born with prostate disease when their mothers exposed to atrazine the dollars are born with impaired mammary development which looks like that he’s a three different EPA laboratories and when those rats grow up their offspring shall growth and development such that this rat suffers from effects of atrazine that his grandmother was exposed to so when I look at I have to read this one outloud some Gentile Sue’s no obligation to update forward-looking statements to reflect actual results that’s a word for my response to folks when I think about the fact that after seeing even after use in France is still an environment after 20 years when I think about that issue and the impact on public health I’m moved to become you heard politically active because this is that this issue is not about us we’ve been exposed our children and our grandchildren will be exposed and if we take these rat studies to tell us anything about us which is that’s why we do them in the first place right our grandchildren’s grandchildren will be potentially affected by after seeing than other chemicals that we’re putting into the environment today when I think about that and I think about statements like this from the old EPA I have faith in the new one the ultimate decision is much bigger than science and raised in public opinion it raises me to a whole new level of awareness and sense of responsibility I have a website that I don’t maintain as well as I could but from there you can if you’re inclined there’s a button you push called a canal and you can write directly to Congress you can write directly to sin center you can write directly to the EPA with your concerns if you are moved to be concerned keith Ellison has just written a bill and US Congress had worked with him when he was in Minnesota to ban a cuisine you can write directly to keep that he dropped a bill on Earth Day and in support of this after reading the data the the the papers yourself if you like if you had Facebook I group a college students that I speak to started to paint global citizens against a cuisine and from there there’s a petition on the breast cancer site that is looking for twenty thousand signatures they have about ten if you care more about frauds there’s another petition Melanie Zaremba me save the frogs they’re looking for a hundred thousand signatures I think they’re about five thousand right now and

obviously folks as I close up today I think I was told I have to do it I’ve crossed a line and park is a woman once told in Minnesota she said dr Hayes that was a great talk but it was half a talk she said you told us what problem was you didn’t tell us what to do about it and now I’m telling you there are things that that we can do about it some disagree including my adviser who I love and admire and respect but if the other side’s put information out there I think we have an obligation to become politically active and in fact another great orator in person that I respect said just the opposite default he said those who have the privilege to know have the duty to act and I’ve taken that as my responsibility as well so thank you for your time questions or comments yes California’s away because we grow food if you live in the Midwest you’ve got corn soy corn soy and actually now mostly corn corn corn corn so you’re using a lot of atrazine that’s all 90% of crops I’m making that number up but it’s a big number California we have 350 agricultural products and atrazine is only used on corn the biggest use of atrazine actually and it’s not louis it’s still significant the biggest use is actually in forestry in california because i use it after they cut the log so that we’s don’t grow back for replanting not actually on corn so even those there’s colors you saw you notice that they don’t match over with the agricultural counties I drew in their matching over whether the lumber same thing as you go up through Oregon and Washington there’s a lot of use on the trees tree farms like some question it’s read an endocrinology we call organizational versus activational effect if you’re exposed developmentally no Darnell who we call him will be a female for the rest of his life if we remove the atrazine and the estrogen levels go down he doesn’t produce eggs as much but he’s still a female if you’re exposed as an adult to atrazine and I believe we published this or either that is coming out the next one if you’re the dull your testosterone levels go down but you remove the after scene presumably you turn to normal as far as we can tell but if your brain is fixed on that gender identity or if you completely turn into a female that’s a permanent effect I don’t know no and again that’s that’s all court of work and and that we talked about this yesterday with the undergraduates that’s difficult data to get because you’re talking about a population especially in Arizona right who’s not really interested in going in and complaining about things and trusting officials and all that kind of stuff so those would be really difficult data to get with the human participation and and you know kinds of you know things that you would need to get the information yes that’s a hard question to answer because to be honest we don’t look at females anymore and the reason is that females already have so much estrogen that a little boost in estrogen that they’re getting from a cuisine probably doesn’t have an impact on their physiology at least that we can measure so for example is easy to measure the sperm reduction that a female lays 2,000 plus eggs and to find an effect that we just haven’t seen one so it may in female frogs but not that we’ve ever detected and to be honest we aren’t even really looking anymore because we’ve never found anything so the answer is not that we’ve seen doesn’t mean no them and an estrogen in general estrogens much worse for a male ester that eventually will kill a male if you expose a male to estrogen for more than a month I think what happens is a liver has completely taken over from making video jenner egg yolk and then they eventually end up with some kind of weird kidney damage evacuated kidneys that causes anosmia a regulatory problem and they fill it with water and die females have a better way of I guess regulating and metabolizing the estrogen and that’s what and I think has to do with one in a female frog there’s more consistent estrogen levels whereas in rats knowing the estrogen cycles and when you’re exposed to atrazine the studies have shown you get an elevated level of estrogen which actually has detrimental effects on the on the rats and and it probably is a matter of measuring the right thing I once gave a talk called frogs don’t have press if they did they probably would get affected frogs but it was not a mammary gland conference so I thought was an appropriate title people people look at me like why are you giving a talk at a man yes there’s good evidence there’s good data

for that it’s and that’s what it’s correlated with there’s a lot of things because it’s correlated with the increased pesticide use and so-called endocrine disrupter use and I don’t know that any studies sorted out which chemicals but that’s been the proposal that that that’s part of the problem and a lot of this data come out of Europe where supposedly they’ve been banking and measuring sperm count the same way for some phenomenal number of years and so it’s a really good believable piece of data and it’s mostly quote in the in the developed world if I’m not mistaken where you see that in relationship northern Europe and and the US I think there’s been a 50% decline don’t quote me on that I don’t want to give out numbers that will prove inaccurate but it’s significant we’re still being ok though it’s there’s no population problem all these videos through water it’s highly water soluble is not in your corn and your exposure is going to be through drinking water unless you are a well to two things somebody told me to remind you can get it through a shower and those occupational levels are from inhalation and skin not from drinking water and in Australia they put in swimming pools to control the algae so little kids swim in it in Australia so I don’t want to say yes or no my guess is No yeah well some of it is tap water apparently somebody let’s turn the avión spelled backwards is naive and then they realize it but then I guess I’d arrived at the plastics leach them out in the father which others other questions or comments yes science question we’re going to get away from the applications a lot of the patterns that you described were fairly typical of the naturally occurring clonal species of both fishes and even there are even some things and birds that are rather similar now these things mate back in some cases hundreds of thousands of five billions of years do you think there is some natural equivalence to atrazine that are produced these similar dramatically there are there are some estrogens estrogenic phytoestrogens a perfect example and there’s some that even there’s great stories of where there’s even coevolution so there’s a type of clover I think that makes an estrogenic compound that can act like a birth control pill and the end the story is that the the latest that eat plants and bad years will switch over to eating this clover and then they get the phytoestrogens and then that decrease it’s a reproduction and then the plant material grows back and they switch back to their normal day you know and so there’s a meet stories like that and certainly even for birth control pill the first birth control pills that were made was by extracting similar compounds from the AMS in Mexico of course because it wasn’t allowed in the u.s. from yams there are natural compounds that can act as endocrine disruptors again some which may even have an evolutionary story behind them and the I mean the other evolutionary story is that the estrogen receptor which is squired for female reproduction was the first receptor to evolve and according to the phylogeny and so the idea is that receptor didn’t have to be so specific because there were no other hormones around so to speak and that that’s why a sturgeon so much more we see some into so many more endocrine disruptors because that receptors just not as specific there was no selection for it to be until now where I hear a story as we produce a thousand new chemicals a day so you’re bound to find some that interact serenity that’s an interesting word for it yeah I think so I think we have a whole new EPA one of the first things Lisa Jackson when she took over EPA said was we’re gonna throw out the Bush stuff and bring science back in to the EPA process she opened up for the reviews for atrazine under and again I’m not going to take credit for it but under the suggestion or prodding of Barbara Boxer who I’ve written to and people had written to from my website and and they’re starting to new reviews now and I think I could say this now in an interesting turn the EPA legal branch is not pursuing people who sell and use atrazine which I’m not sure how they do that when in fact they okayed it for the market but that’s what’s happening now so and I’ve had a little bit of involvement in it yes I what I think will happen a couple of things I want to say and I don’t know how long you guys build alright I got nowhere to be till 10 so I’ll stay here on what the way things happen and whatever I don’t want to take a lot of credit for it but I think awareness has been risen and on a number of different fronts there’s a class-action lawsuit right now where six states are suing Syngenta to take atrazine out of the water which will be so expensive that they wouldn’t sell it anymore

the Center for Biological Diversity is suing Syngenta for after seing contamination where there’s endangered species of fish and amphibians so there’s litigation there’s a ton of publications coming out on on humans on birth defects on low birth weight more fog stuff so they’re facing publication the EPA has opened up for new reviews so they’re facing regulation there’s bills being entertained in Minnesota and and in Illinois and on the on the federal level so what I what I what I hope is that they’re spending a lot of money fighting on all of these fronts they’re getting a lot of bad publicity and what I hope that will happen is that the EPA realized whether or not they regulate after seeing that the main point is we want regulatory to look at these things in a different way they’re 80,000 chemicals that went on the market before there was ever an EPA in existence there was no regulation there was no tests that had to be passed when things like DDT and after zine and Mottola core and and TBT and all these things went on the market so I hope there’s going to be a major reform and a difference in how we weigh out public health and environmental health relative to how we weigh out economic concerns which that’s what this is corn my corn is money after Xena increases corn yell by 1.2 percent that’s a hundred million dollars a year yes ten years ago there was a state of course everything’s in Minnesota yes or no so the most direct cause and limb deformities is at rheumatoid that burrows into the limb which uses snails as a intermediate host and they only link with atrazine is that after seen causes a change in the after seen and nitrates a change in the amount of algae that are plant material that’s available and it causes an increase in the snails which increases to host for the trematode it also lowers immune function and snails so that they get more infections and it lowers immune function in frogs so that they get more infections when the parasite leaves the snail and goes to the Frog so there’s an indirect relationship and that that’s all published by multiple laboratories none of them mine