Human complex diseases represent a phenotype (trait) that reflects the combination of: [a] genetics (e.g., DNA sequence variants); [b] epigenetics (chromosomal changes other than DNA sequence alterations); [c] environmental factors (pesticides, chemical pollutants); [d] endogenous influences (diabetes, heart disease); and [e] differences in one’s microbiome. Autism spectrum disorder (ASD) is a great example of a very complex disease trait that is certainly polygenic, plus influenced by epigenetic and environmental factors, and perhaps even by variability in the microbiome. Many genome-wide association studies (GWAS) have identified a number of low-effect genes, noncoding DNA loci, repetitive DNA sequences, and changes in DNA methylation that show correlations with ASD. Clinically, ASD is a developmental disorder that presents as severe social and communication impairment, combined with limited or focused interests and repetitive-type behaviors.
The prevalence of ASD has been exploding since the mid-1960s, rising from about one case of autism per 20,000 patients (in ~1960) to one case of ASD in 44 children today (~460 per 20,000(!!). One important caveat is that diagnosis of ASD is MUCH broader than it was, 60+ years ago. Throughout my clinical pediatrics training in the 1960s, I can remember only one ASD patient among the thousands of patients I was in contact with during those years.
The attached paper focuses on a possibly significant contribution of an environmental factor during pregnancy in ASD etiology. Proposed environmental chemicals that might cause ASD include: selective serotonin reuptake inhibitors (SSRIs), pesticides, herbicides, phthalates, polychlorinated biphenyls, solvents, air pollutants, fragrances, and heavy metals. For example, increases in the widely-used chemical, glyphosate [N-(phosphonomethyl)glycine], the active ingredient in the herbicide Roundup, were reported to be associated with increases in ASD rates over the same period — reported in the US public school system. Authors cite a population-based case-control study in California, showing the risk of ASD was associated with use of glyphosate (odds ratio = 1.16). For ASD children with intellectual disability, estimated odds ratios were higher with prenatal exposure to glyphosate (odds ratio = 1.33). These reports suggest that possible relationships between glyphosate and ASD should be explored in animal models.
Epidemiological studies implicate in utero maternal immune activation (MIA), playing a key role in the etiology of developmental disorders such as ASD; also, there are a number of positive associations between maternal infections or inflammatory biomarkers and ASD. Collectively, it is suggested that MIA during pregnancy can increase the risk of developmental disorders such as ASD in offspring. Authors [in the attached paper] show that soluble epoxide hydrolase (sEH), participating in metabolism of polyunsaturated
fatty acids (PFAs) might be involved in genesis of ASD in mouse offspring, following MIA; authors also found ASD-like behavioral abnormalities in juvenile offspring after maternal exposure to high levels of formulated glyphosate. [Authors state the dose of glyphosate is “0.098% in drinking water,” but authors should report what that computes to, in mg per kg per day]
Authors found higher levels of sEH in the prefrontal cortex (PFC), hippocampus, and striatum of juvenile offspring; in addition, they discovered decreased levels of epoxy-fatty acids [e.g., 8,9-EpETrE] in the blood, PFC, hippocampus, and striatum of juvenile offspring after maternal glyphosate exposure — which would be consistent with increased activity of sEH in the offspring. Moreover, authors found abnormal composition of gut microbiota and short-chain fatty acids in fecal samples of juvenile offspring after maternal glyphosate exposure. Interestingly, oral administration of TPPU (an sEH inhibitor) during pregnancy from embryonic day-5 (E5) to postnatal day 21 (P21) prevented ASD-like behaviors (e.g., social interaction deficits and increased grooming time) in the juvenile offspring after maternal glyphosate exposure.
These findings suggest that maternal “exposure to high levels of glyphosate” — causes ASD-like behavioral abnormalities and abnormal composition of gut microbiota in juvenile offspring mice, and that increased activity of sEH might play a role in ASD-like behaviors in offspring due to maternal glyphosate exposure. Authors suggest that “sEH might therefore be considered as a target for ASD in offspring,” following maternal stress from environmental exposure to contaminants. This study is a good example of gene-environment interactions, the recurrent theme of these GEITP blogs. 😊
COMMENTS:Hi Dan, Thanks for sending this around. There are several strong candidates which may play a role in the horrendous increase in ASD. And I agree that improved diagnosis is only a small part of the puzzle. Air pollution and other environmental pollution might play some role.
Much of the research has been conducted using PURE glyphosate (i.e., the active ingredient which is what is required for pesticide testing). The problem is that it may be the formulations that are part of the problem. Anyhow, lots of interesting work happening now on glyphosate — demonstrating that it is NOT innocuous to humans. LSB
COMMENT: Dan, I just did a back-of-the-envelope calculation on doses of glyphosate used in this ASD study:
They used ~1,000 ppm (million) in drinking water. The levels in tap water range from 85-330 ppt (trillion) — which means the dose they used is 3- to 12-million times higher than the average level found in tap water.
The difference with respect to food is more reasonable and that is likely the major glyphosate exposure clinically. The acceptable daily intake (ADI) for glyphosate is 0.5 ppm; therefore, the dose they used in mice (without allometric correction) is 2,000 times the human ADI.
Is this study relevant to toxicology? It irritates me that the authors use descriptors in the Abstract such as “…suggest that maternal exposure…” whereas in the title, the authors state: “Maternal glyphosate exposure CAUSES autism-like…” Association is NOT causation — even if this paper gets published in Proc Natl Acad Sci USA !! DW
COMMENT: This is GREAT, Dave. I saw the authors described in the text their treatment as “0.008% in drinking water” — where did you find all the additional details about the regimen used? DWN
COMMENT: Dan: I was going by the information in their Supplemental Materials section — “In this study, we used commercially available RoundupⓇ Maxload [48% (w/v) glyphosate (N-phosphonomethylglycine) potassium salt, having 52% other ingredients such as water and surfactant. [Lot #11946898. Nissan Chemical Corporation, Tokyo, Japan]. Previous studies used drinking water containing 0.38% (w/v) glyphosate (expressed as free base: 1% RoundupⓇ), during pregnancy and lactation, equivalent to 50 mg/kg/day of glyphosate (1,2). This corresponds to 1/20th of the glyphosate no-observed-adverse-effect level, as described previously (3). Therefore, water or formulated glyphosate [or 0.1, 0.25, 0.50, 0.75, 1.0 % RoundupⓇ] was given to the pregnant mice from E5 to P21 (weaning).”
“Measurement of glyphosate in the blood. Water or 0.098% (w/v) formulated glyphosate was given to pregnant mice from E5 to P21, as described above.”
The glyphosate dose of 0.1% would be 1000 ppm, and that is what I used, to compare with the reported levels in tap water (85-330 ppt). If mice drink 4 mL water/day at 0.1%, that would be 4 mg/day or 160 mg/kg for a 25 g mouse.
If the doses were between 0.1% and 1% of a formula containing 48% glyphosate, that would be about 2 mg/day (80 mg/kg), i.e., 20 mg/day (800 mg/kg).
The acceptable daily intake (ADI) for humans in food is 0.5 ppm or 0.5 x 10-7 g/g food. If a human eats 1,800 g food/day, that works out to 0.9 mg/day glyphosate, or 0.013 mg /kg for a 70-kg human. So, whatever the dose they actually used, it is many orders of magnitude higher than the human ADI. If my math is off, I will blame it on being quarantined in this pandemic, going on now, for more than 2 years. ☹ DW
COMMENT: I had intended to emphasize the authors’ use of the word “causes” in the title of their article (notice in the subject line of this email, it more conservatively says “possible association,” which is much more scientifically accurate thing to say). Kudos to Professor David Williams for bringing this topic up. Far too often, authors see a correlation — and immediately assume it is the cause-and-effect. “Possible association” is the much more accurate way to describe any observed correlation.
As with >90% (or probably >99%) of all toxicological studies, authors choose a dose WAY beyond “environmental reality,” in order to find “an effect.” This paper is a great example of this. In My Humble Opinion, this paper should never have been published — at least not in the high-visibility prestigious Proc Natl Acad Sci USA journal. ☹
What are the likely causes of ASD? Clearly, it must be a polygenic disease — with dozens if not hundreds of genes, each contributing a “small-effect” to the phenotype. In addition, to explain the incredible increase in frequency of this disease in the Western World since 1970, there must be epigenetic effects (the time for this increase to appear, i.e., <50 years, is way too short for DNA mutations to be involved). Environmental factors, lifestyle suspects, include things around us today — that were not there 50-60 years ago: e.g., [a] steady and excessive exposure of TV, electronic games and fast-moving videos to children almost from birth; [b] dietary changes (today there is so much “fast foods,” excessive sugar, and lack of a nutritious well-balanced diet); and [c] tendency of teachers, school counselors and parents diagnosing every transient behavioral defect in every school kid as “likely having ASD.” DwN
COMMENT: Wow, this discussion is very harsh on the authors. I think the authors mentioned that their results in animals are “not readily translated into human populations,” because the concentration they used here was way higher than environmental levels. The purpose of this study was to demonstrate that glyphosate (at some level of exposure) could cause behavioral disorders that are similar to an ASD-like phenotype (for example, “arsenic causes cancers”). Based on their study, I believe it is safe to say glyphosate "is associated with" ASD-like symptoms in mice — at the concentrations at which the authors chose to use. ZL
COMMENT: Findings in a mouse model, or any other nonhuman model for that matter, means nothing more than a hypothesis — unless the model has been validated in clinical studies with human patients. We at P & G lost many millions of dollars, trying to develop new drugs for humans, based on animal preclinical work. We saw many more failures, compared to successes.
Academicians who publish these preclinical studies are only interested in getting their names in lights. And, of course, their University PR departments trump up the potential value. R D'A
COMMENT: All of this is an interesting discussion — especially because the renewal assessment report of glyphosate is now on the board of the European Food Safety Authority (EFSA). Their license should be either renewed or discarded in the EU by the end of this year. Evaluation by the EFSA is a huge effort; we have thousands of pages and public opinions comprising more than 2,000 comments. ☹ OP
**[Read emails from oldest to newest]**
This email-chain just keeps going and going. Maybe there is lots to ventilate about? Here are two more of the latest comments. One from the Editor of Critical Reviews in Toxicology. And this last one is from Dr,, Jim Adams, who has a sobering message from “someone in the trenches” — a general practitioner for 40+ years. 😊 I hope everyone enjoys his frankness, honesty and humor as much as I do. 😊
COMMENT: J A
I’ve been saving the Roundup emails and now I feel that I am at a point where I can attempt to sound off — which is the point of my reply to this, and other, related trends in modern problem-solving that crosses many fields of interest. (I’ll try not to ramble too much.)
Every day in my practice, I see people who haven’t been diagnosed — even after thousands of dollars of tests, or they have had their problems solved by thousands of dollars of medications. If it’s a broken bone, no problem. If it’s fatigue or one of the myriad chronic pain or even more obvious illnesses almost always based on obesity — not so easy.
Medicine has become compartmentalized. If you take “fatigue” to a cardiologist, you get thousands of dollars of cardiac tests. If you send “fatigue” to an internist, you get another set of tests, and a CAT scan or two. If you send “fatigue” to one of the “second-tier practitioners,” you get a repeat of some of the above — plus a sleep study and more and more pills that are like throwing darts in the darkness of failed knee-jerk approaches to complex problems.
So finally, for all our deviations from normal hard work and the struggles over the millennia that kept us from becoming walking eggplants or other vegetables, the “alternate” problem-solvers get a shot: psychiatry, functional medicine, dietary guides, and a whole host of herbal incantations, etc. Then, maybe watching TV before bed — turns out to be the “real cause."
This mirrors, in a sense, the Roundup studies, wherein the millions of chemicals floating around become targets, “searches for causality,” — especially if a company like Monsanto with deep pockets is involved. Let’s consider all the food additives, chemicals in cosmetics, herbicides and pesticides. Why not add PVC pipe, residuals of all food processing, and, more realistically, every new and old drug or potion on the market? For example, take psychostimulants for children who can’t do well in school. The adverse effect studies extend at best two years…!! We're talking about habit-forming drugs that alter brain structure, and there is no feasible way to look at subtle, or not-so-subtle, mental or physical effects 20 years from now. Now, add in statins, etc. How can one account for possible drug effects and combination-drug long-term effects from something as minute as the amount of Roundup sprayed on weeds…??
Then, I have this problem with all pesticide and herbicide fears.
I live in the Mississippi Delta — where airplanes have sprayed every imaginable chemical directly in the air, since I was 10 years old. You smell them all spring, summer and fall while riding down the highways. When I was age 10, I stood in my T-shirt and marked the rows for airplanes flying over my head, smearing my glasses with 2-4-D (remember dioxin?) and every chemical, except Paraquat, a chemical we all knew “was toxic.” (My brother uses Paraquat in spray rigs every year to spray the stuff on the ground, as if that’s safer.)
What’s my obvious point?
If chemicals for bugs and weeds are truly toxic, it would seem to me that those of us in the Mississippi Delta with 60 years of intense, yearly exposure, second only to the crop-dusters and spray-rig-tractor drivers, should be more valid study subjects than rats. My friends and I rode our bikes in the DDT fog sprayers as children. But then, this returns to paragraph one. We know nothing. We have science groomed by ulterior motives…money mostly…and hysteria. None of us is as brilliant as a Cray Computer, and we are simply throwing darts in darkness to invent answers — using clever tools and tests — all depending on our particular fields of interest. Yet, from another viewpoint, are we all like the doctors, including myself years ago, who are more like college kids smoking pot…”I’ve done it, and it’s hasn’t killed me yet?” We assume safety if there aren’t visible or palpable immediate adverse effects. There is a pill for everything.
And by the way, despite the use of an incredible array of pesticides, some sprayed over the entire city overhead by airplanes at night during summer, when our mosquitoes are horrid. Right now, at 6 am in Cleveland, Mississippi, it sounds like a songbird sanctuary, and by sunrise, the air will be literally filled with insects flying or crawling over every square inch of territory in my back yard. I don’t know if the Joni Mitchell lyric “Give me spots on my apples, but leave the birds and the bees” is an intelligent assessment of reality or not. I, by the way, keep bees in my back yard, and they’re buzzing around and surviving just fine.
In summary, I’m 72 years old, and I have been breathing high doses of every evil chemical imaginable, and it’s done no harm that I can see.
(This is an old photo below…well, I did have to have a few plastic surgeries over the years.)
Subject: Fw: California regulators changing language on glyphosate and cancer risk
I have followed the recent exchanges on your blog. I have stayed on the sidelines — because I received a lot of flak — related to articles by Willliams et al. on “glyphosate's potential carcinogenicity,” which I chose to accept for publication and it got published in Critical Reviews in Toxicology; and I refused to retract them in response to requests from IARC and others.
You will find the attached article of interest. I am sure you recognize Carey Gillam has a vested position.
Keep up the great work that you do — in communicating and discussing these high-profile issues(!!)
Best regards, RM
COMMENT: Let me add to the firestorm…As a proud member of the National Academy of Schmoozers, I could not agree more with Fred. I’m continually dismayed by the number of so-called toxicology studies that draw ‘disconcerting’ conclusions for human health — based on in vitro (and sometines in vivo) studies that use doses/concentrations that are 1,000 times to, sometimes, 10s-of-thousands of times, higher than could ever conceivably be achieved from ‘environmental’ exposures.
Imagine if drug safety assessment were based on observed effects on in vitro studies that required doses thousands of times higher than physiological-based pharmacokinetic modeling and simulation (PBPK) therapeutic concentrations. We would have no new drugs….the Glyphosate ‘controversy’ is just the tip of this toxicology iceberg…. ☹
COMMENT: [Read emails from oldest to newest]**
Lastly, to bring this email-chain into perspective and full-circle, the short video clip [attached] of a small toddler on a farm shows an example of a child who is completely aware of his surroundings and acts appropriately when challenged by a cow. No doubt the child’s parents have been using glyphosate on their farm, and their entire family has been exposed. Even during her pregnancy with this child, the toddler was likely exposed to this herbicide in utero. But, as you can see here — he does NOT show any evidence of ASD. 😊😉😉
Nebert, Daniel (nebertdw)
Sent: Wednesday, April 13, 2022 5:22 PM
The raging firestorm of GLYPHOSATE — continueth, including claims of an association and possible causation not only to ASD but also to cancer. For many years, I’ve been aware of this political battle and fraudulent (glyphosate-cancer) “scientific” studies and claims. Sound science (following The Scientific Method *) continues to be replaced by government opinion and policy, and this trend seems to be accelerating during these past 2-3 decades. Why? Perhaps this has to do with the internet? or social media? or incompetent teaching of the latest generation of students? I just don’t know.
*The six steps of The Scientific Method include:
1) ask a question;
2) learn what is already known about the topic;
3) construct a hypothesis;
4) experiment to test the hypothesis (including repeat experiments to confirm the same result);
5) analyze data from the experiments and draw conclusions;
6) communicate the results to others.
[Repeat Steps 1 through 6 again and again.]
Dear Dr. Nebert,
Jim Enstrom included me on the correspondence involving you, David Williams, and others regarding the PNAS paper linking glyphosate exposure and ASD. Since there was no direct mention of cancer in the email exchange, I wanted to make you aware – if you are not already aware – of the unfortunate controversy that has been raging for seven years regarding the claim of carcinogenicity of glyphosate.
The controversy stems from IARC’s 2015 determination that glyphosate is a “probable carcinogen.” (This initial publication was followed several years later by IARC Monograph 112, which reached the same conclusion.) IARC based its conclusion of probable carcinogenicity solely on animal (rodent) studies, which it judged to constitute sufficient evidence (whereas the epidemiologic evidence was judged to be insufficient). However, the Agency’s evaluation of the rodent studies has been criticized on the grounds that the wrong statistical test was used to assess dose-response trends, erroneously declaring certain positive trends statistically significant. In addition, where comparable inverse trends occurred, these were ignored. Other irregularities have been documented by Reuters reporter, Kate Kelland, who found that non-carcinogenic findings were edited out in successive drafts. There is also the appearance of a serious conflict of interest on the part of Christopher Portier, who served as an invited guest on the Working Group but, who was also influential in IARC’s 2014 decision to evaluate glyphosate. Within days of the publication of IARC's conclusion in March, 2015, Portier signed a lucrative contract to act as a litigation consultant for two law firms that were preparing to sue Monsanto on behalf of glyphosate cancer victims.
Regarding the epidemiology, most studies have been case-control studies of occupational groups exposed to glyphosate. Many of these were conducted when glyphosate was less widely used and exposure would have been low. An association of glyphosate with non-Hodgkin’s lymphoma (NHL) was found in some of these studies. It is noteworthy that IARC ignored the results of the large prospective cohort study, the Agricultural Health Study [AHS], conducted by NCI among 54,000 pesticide applicators. Although the latest results regarding glyphosate were not published until 2018, the results of the analyses involving glyphosate had circulated far earlier, and, furthermore, the head of the Working Group that evaluated glyphosate, Aaron Blair, was one of the lead researchers on the AHS, who would have been intimately aware of the results. The overall conclusion from the AHS analysis was as follows: “In this large, prospective cohort study, no association was apparent between glyphosate and any solid tumors or lymphoid malignancies overall, including NHL and its subtypes.”
Recently, together with two colleagues, I published an updated meta-analysis of epidemiologic studies of glyphosate exposure and NHL. Using updated information from the case-control studies, where available, and examining the five different latency conditions considered in the AHS analyses, we found no association of glyphosate exposure with risk of NHL.
Given your interest in carcinogenesis and your comments on the toxicology of glyphosate, I wanted to draw your attention to the situation regarding glyphosate and cancer. This question has serious implications for agriculture, where glyphosate has been enormously useful to farmers, providing an environmentally benign means of controlling weeds and increasing yields, enabling no-till cultivation. Activists, litigators, and politicians have taken IARC’s glyphosate determination as gospel, and this has provided the basis for four (by my count) successful lawsuits in California against Monsanto and its successor company, Bayer. In addition, local jurisdictions and states (such as the state of Maine) are considering banning glyphosate, based on IARC’s verdict.
In this juggernaut, the judgment of 17 other national and international health/regulatory agencies, which have found glyphosate to be non-carcinogenic and to not pose a risk to the general population, is blithely ignored. In other words, carefully-conducted reviews, including those of the U.S. EPA, Health Canada, and the European Food Safety Authority, among others, are simply deemed to be of no weight — when counterpoised to IARC’s seriously flawed (some would say, fraudulent) conclusion.