"The current study provides the first analysis of potential transgenerational impacts of glyphosate in mammals"

'Carefully' is a word that applies to any science in terms of 'meaning' and 'generalisation'. Don't get me wrong, I'm a big fan of science and scientific evidence when it comes to life, the universe and everything. But I also understand that science can be used/manipulated in many, many different ways, and rarely, if at all, do we see something that could be considered perfect science. Hence my use of the word 'carefully' in opening this blog post. With all that in mind, I bring the findings reported by Deepika Kubsad and colleagues [1] to the blogging table and the proposal that: "glyphosate can induce the transgenerational inheritance of disease and germline (e.g. sperm) epimutations."

Why am I talking about such a study on a blog primarily hosting autism research? Well, a few reasons (see here and see here), predominantly based around the observations that (a) herbicides and insecticides whilst very useful, are not seemingly without a risk-benefit profile, and (b) autism (and other related labels) have been 'talked about' in the context of risk and 'exposure' to such compounds. I emphasised the words 'talked about' to ensure that the current peer-reviewed science base is not contorted into something that it is currently not (i.e. cause-and-effect). I might also add that 'transgenerational inheritance' is something that has also been discussed in the peer-reviewed science literature in the context of autism too (see here).

The basics of the Kubsad paper: "There are an increasing number of conflicting reports regarding the direct exposure toxicity (risk) of glyphosate, but no rigorous investigations on the generational actions." So, using a rodent (rat) model of study, researchers exposed mummy rats (F0 'gestating female') to glyphosate - "daily intraperitoneal injections of glyphosate" - at doses that probably would be considered 'excessive', and then followed successive generations of rats (F1, F2, F3) looking for any "transgenerational pathologies observed... including prostate disease, obesity, kidney disease, ovarian disease, and parturition (birth) abnormalities." They also included a set of control animals - "F0 generation gestating females administered vehicle control dimethyl sulfoxide (DMSO) or phosphate buffered saline (PBS)" - and whilst they were at it, checked for any germline transmission issues/differences via the inspection of sperm; specifically looking for differential DNA methylation regions (DMRs). Those DMRs are important when it comes to whether specific genes are expressing or not ('switched on' or 'off').

Results: with that word 'carefully' in mind, we are told that there were: "negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology." That's important because it suggests that those directly exposed and their immediate offspring don't seemingly show adverse effects.

But... "In contrast, the F2 generation grand-offspring, derived from a direct exposure F1 generation germline, had significant increases in testis disease, kidney disease, obesity, and multiple diseases in males" and: "The F2 generation females had significant increases in ovary disease, obesity, mammary gland tumors, parturition abnormalities, and multiple disease susceptibility." Researchers also reported seeing a few things in their F3 generation too, as the words "generational toxicology" entered discussions.

As to the DMRs results, well, there were some interesting findings there too: "the glyphosate lineage sperm were found to have altered DNA methylation in direct exposure F1 and F2 generations, as well as the transgenerational F3 generation." This led Kubsad et al to conclude that glyphosate exposure could "promote germline epigenetic alterations in DNA methylation" but more needs to be done on what this actually means for gene expression and whether it tied into the disease risk(s) noted in their exposed cohort and subsequent generations.

The authors mention how their study had limitations including the use of "an environmentally relevant exposure of twice the allowed industry exposure" and the use of an animal model (bearing in mind the logical fallacies that can follow). Lots more investigation is indicated before any big headlines are generated.

But... the Kubsad results are not to brushed under the scientific carpet. They add to the range of other compounds that seemingly carry transgenerational biological correlates and imply further inspection of glyphosate in that same potential category. Whether also there may be additive or interactive effects with other compounds could also be part of the research agenda.

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[1] Kubsad D. et al. Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology. Scientific Reports. 2019; 9: 6372.

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"To examine associations between early developmental exposure to ambient pesticides and autism spectrum disorder"

The quote heading this post - "To examine associations between early developmental exposure to ambient pesticides and autism spectrum disorder" - reflects the aim of the study published by Ondine von Ehrenstein and colleagues [1]. Said aim was accomplished by examining data from "California’s main agricultural region, Central Valley, using 1998-2010 birth data from the Office of Vital Statistics" with regards to autism diagnoses (see here) and pesticide use and exposure. The results: "Findings suggest that an offspring’s risk of autism spectrum disorder increases following prenatal exposure to ambient pesticides within 2000 m of their mother’s residence during pregnancy, compared with offspring of women from the same agricultural region without such exposure." Also: "Infant exposure could further increase risks for autism spectrum disorder with comorbid intellectual disability."

The von Ehrenstein findings also come with an accompanying editorial [2] both published in the British Medical Journal (BMJ). That editorial provides a good overview of the findings and, importantly, some of their limitations. The editorial notes for example, that: "the study included only children with a diagnosis of “autistic disorder” by the California Department of Developmental Services, based on criteria described in the Diagnostic and Statistical Manual of Mental Disorders version IV-R." It cautions that the von Ehrenstein findings might not generalise to "milder forms of autism spectrum disorder." This 'lack of generalisation to milder forms' is a real reversal of what is typically seen in the peer-reviewed autism research literature (see here).

I don't want to linger too much on the von Ehrenstein study but a few points are worth noting. The question of what specific pesticides *might* influence risk of offspring autism is a complicated one. The researchers reported that "small to moderately increased risks for the disorder in offspring with prenatal exposure to the organophosphates chlorpyrifos, diazinon, and malathion, the pyrethroids permethrin and bifenthrin, as well as to glyphosate, avermectin, and methyl bromide" were observed. They (and the editorial) talk about how further research is required to "examine the joint effects of multi-exposure mixtures to more effectively protect human health." This is important in the context of other research (see here and see here) and also to avoid any big media headlines (see here) about specific preparations 'causing' autism (which has not yet been authoritatively established). Given that various different classes of pesticides have somewhat slightly different biological actions, there is a lot of work to be done on the possible biological mechanisms/targets and any synergistic effects.

Also: "children with autism spectrum disorder and co-occurring intellectual disability were examined as a separate outcome." The fact that von Ehrenstein et al observed something like a 'stronger association' between pesticide exposure and those at the "more severe end of the autism spectrum" (the description according to one media source) is another important point requiring further study. I say this in the context that 'comorbidity' might not always be the best description for symptoms and diagnoses accompanying autism (see here).

The authors conclude that their findings - with appropriate caveats - have implications. Namely: "Exposure of pregnant women and infants to ambient pesticides with a potential neurodevelopmental toxicity mode of action should be avoided as a preventive measure against autism spectrum disorder." I don't think too many people would disagree with the sentiments of 'avoiding pesticide exposure' particularly for pregnant women and those who might also be more vulnerable to their potential effects. The questions now turn to genetics and biology and the question of why...

Music to close: Hostiles on the Hill (apparently)...

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[1] von Ehrenstein OS. et al. Prenatal and infant exposure to ambient pesticides and autism spectrum disorder in children: population based case-control study. BMJ. 2019; 364: I962.

[2] Bakian AV. & VanDerslice JA. Pesticides and autism. BMJ. 2019; 364: I1149.

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Project TENDR and chemical exposures part 2: organophosphate pesticides aren't great for child health

So, without striking too many chords on the old 'we've been here before' piano, consider this post an extension of some other discussions a while back suggesting that exposure to some classes of 'chemicals' might not be particularly great for child health (see here).

The findings reported by Irva Hertz-Picciotto and colleagues [1] continue the Project TENDR theme with their assertion that there is "compelling evidence" that prenatal exposure to organophosphate (OP) pesticides "is putting children at risk for cognitive and behavioral deficits and for neurodevelopmental disorders." Their observations have also been picked up by the lay media with some striking headlines like 'Ban entire pesticide class to protect children's health, experts say' complete with the required stock photo of crops being sprayed from the air.

Organophosphate (OP) compounds such as OP pesticides have a very mixed history. As well as being the insecticide of choice in many countries as a result of their excellent pest control profile, the organophosphate chemistry has also been utilised for less desirable purposes as per its classification as a component of nerve agents. Remember all the quite recent chatter about a nerve agent called Novichok? Well, the chemistry behind Novichok apparently has a very distinctive OP "structural backbone" [2]. That's not to say that every OP pesticide is Novichok. But rather that the activity of OPs specifically targeting the action of acetylcholinesterase (AChE) enzymes, important enzymes that are required for proper nerve function, is also an important biological action of various nerve agents. This in itself would suggest caution in the use of OPs.

The Hertz-Picciotto paper (policy forum) is pretty data heavy in terms of how much OP pesticides are used across the globe, the slew of mostly observational research studies that have looked at pesticide exposure and various neurodevelopmental variables and the concerns voiced at both high and low levels of OP pesticide exposure. They make a few recommendations: better training for health professionals on the potential risks attached to OP pesticide exposure, greater moves to switching to "nontoxic approaches to pest control" and perhaps most controversially: "Governments phase out chlorpyrifos and other OP pesticides."

The caveats? Well I might mention a few, minus any charges of me somehow 'standing up for OPs'. First, I don't think it's unreasonable to suggest that children in particular, do need to be more strongly protected against pesticides that are (chemically-speaking) not a million miles away from nerve agents. As the authors mention, there is quite a bit of evidence in the peer-reviewed literature to suggest a possible *association* between pesticide exposure and diagnoses such as autism (see here and see here) or beyond (see here). The thing is that like many other non-genetic environmental factors (e.g. air pollution) *linked* to a heightened risk of autism or other neurodevelopmental labels, it's more about environment + genetics when it comes to risk (see here) rather than environment just working on its own. We need for example, to know more about the ways and means that OPs are metabolised in the body and whether there could be some interesting biochemistry potentially linked to labels like autism (see here).

Second, some thought needs to go into the possible replacements if OP pesticides were to be banned outright. Thinking back to a post not-so-long-ago talking about DDT exposure and offspring autism (see here) I was struck by how short some memories are in terms of why such products were developed in the first place. I don't think anyone would seriously contemplate that the development of OP pesticides was anything more than to stop pests attacking crops and to maintain yields that can feed the population. These compounds were developed with good intentions. Obviously, as the research literature has grown, we have come to realise that such products are not side-effect free and more stringent controls have been put into place regarding safety. But to ban them outright is not something that can be done overnight. What do we replace them with? Do we just accept that crop yields will be lower and less food will be produced? Do we instead start thinking about other ways to make crops more resistant to the pests that blight them? That last question has already had its own 'issues'.

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[1] Hertz-Picciotto I. et al. Organophosphate exposures during pregnancy and child neurodevelopment: Recommendations for essential policy reforms. PLoS Med 15(10): e1002671.

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Organophosphate exposure and ADHD?

"Children with higher urinary DMP [dimethylphosphate] concentrations may have a twofold to threefold increased risk of being diagnosed with ADHD [attention-deficit hyperactivity disorder]."

So said the results presented in the paper by Yu and colleagues [1] who looking at "97 doctor-diagnosed ADHD cases and 110 non-ADHD controls who were 4-15 years of age" examined urine and blood samples for various factors including "biomarkers of OP [organophosphate] pesticide exposure." They concluded that, adjusting for creatinine, urine levels of DMP but not other dialkylphosphate (DAP) metabolites were higher in the ADHD group compared with the non-ADHD group. Further: "Organophosphate pesticide exposure may have deleterious effects on children's neurodevelopment, particularly the development of ADHD." At the same time, Yu et al also reported nothing very much to see when it came to blood lead levels (BLLs) between the groups.

This is not the first time that examination of urinary metabolites of OPs have turned up something of a potential relationship with behavioural outcomes related to ADHD. The paper by Bouchard and colleagues [2] also reported a possible connection supporting a "hypothesis that organophosphate exposure, at levels common among US children, may contribute to ADHD prevalence." There too urine was the analytical medium and dialkylphosphate concentrations the target compounds. This and other research looking at this issue have led to statements [3] to the effect that: "Children's exposures to pesticides should be limited as much as possible." I don't think many people would disagree with that sentiment.

I've talked about OPs quite a bit on this blog (see here and see here) and how various conditions/labels might be 'associated' with this class of compounds either when used as insecticides or as something rather more ominous. I've tried not to be too alarmist about the possibility of a connection with health because OPs do serve an important purpose (as an insecticide) and have probably saved quite a few lives as a result. But it is getting increasingly difficult to ignore the possibility that this and other classes of pesticides either alone or in combination with other factors, seem to be implicated in various conditions/labels and more needs to be done looking at the hows and whys. This can however be done without scaremongering.

The Yu results whilst interesting are not however without some cautions. DAP metabolites as markers for OP exposure still requires further investigations [4], not least from which specific OP they are derived from. That other factors such as exposure to second-hand tobacco smoke might also link into the presentation of specific metabolites such as DMP [5] is another consideration. Continuing the theme that combinatorial exposures might also exert an effect [6] other research illustrates how difficult it might be to pin one specific type of exposure to specific behavioural outcomes. And then also we have the added layer of complexity that is the genetics of xenobiotic metabolism with specific focus on OPs. Relationships are likely to be pretty complicated as a result.

Having said all that does not however mean that results like the ones from Yu et al can be just brushed under the carpet...

Music to close, and having watched Guardians of the Galaxy for the Nth time last evening, all I can say is the film soundtrack is kinda cool...

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[1] Yu CJ. et al. Increased risk of attention-deficit/hyperactivity disorder associated with exposure to organophosphate pesticide in Taiwanese children. Andrology. 2016 Apr 12.

[2] Bouchard MF. et al. Attention-deficit/hyperactivity disorder and urinary metabolites of organophosphate pesticides. Pediatrics. 2010 Jun;125(6):e1270-7.

[3] Roberts JR. et al. Pesticide exposure in children. Pediatrics. 2012 Dec;130(6):e1765-88.

[4] Sudakin DL. & Stone DL. Dialkyl phosphates as biomarkers of organophosphates: the current divide between epidemiology and clinical toxicology. Clin Toxicol (Phila). 2011 Nov;49(9):771-81.

[5] Jain RB. Levels of dialkylphosphate metabolites in urine among general U.S. population. Environ Toxicol Pharmacol. 2016 Feb 26;43:74-82.

[6] Osaka A. et al. Exposure characterization of three major insecticide lines in urine of young children in Japan-neonicotinoids, organophosphates, and pyrethroids. Environ Res. 2016 May;147:89-96.

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Yu CJ, Du JC, Chiou HC, Chung MY, Yang W, Chen YS, Fuh MR, Chien LC, Hwang B, & Chen ML (2016). Increased risk of attention-deficit/hyperactivity disorder associated with exposure to organophosphate pesticide in Taiwanese children. Andrology PMID: 27070915

On glyphosate and autism (without scaremongering)

Glyphosate use and autism rates - should I blog about it?

Well, after some deliberation I decided it was a topic worthy of an entry. There is some peer-reviewed science discussions behind it and, as per other areas of controversy on the autism research landscape, the idea that 'science is about debate' (why else does everyone keep going on about open-access and transparency) should always prevail.

I'm assuming most people have heard of the organophosphonate (that's phosphonate not phosphate) herbicide glyphosate - N-phosphonomethylglycine - over the course of its discovery and use under the trade name Roundup. First patented as a chelating agent (another area of 'discussion' with autism in mind) glyphosate has been successfully killing weeds for quite a few years by interfering with some of the chemistry of the aromatic amino acids tryptophan, tyrosine and phenylalanine via its actions on the shikimate pathway. As per many other herbicides, such formulations always require a degree of care in their use given the possibility of side-effects following accidental over-exposure. Indeed, government agencies are cottoning on to this fact (see here).

The debate on glyphosate and its potential effects on human and animal health has been a hot one in recent times. Partly overlapping with the introduction of genetically modified (GM) crops that are glyphosate-tolerant (Roundup Ready) by the company who initially brought glyphosate to the market, the safety angle of glyphosate has been scrutinised and debated. Only this year (2015), an arm of the World Health Organisation (WHO) described glyphosate (and several organophosphate pesticides I might add) as "probably carcinogenic to humans". Following such an announcement, quite a few media column inches were produced as one might expect.

Set within that context, discussions have turned to whether there may be other 'effects' following the fairly widespread use of glyphosate. Perhaps inevitably, autism, and particularly the quite phenomenal increase in cases of autism, has been suggested to be 'linked' to the use of glyphosate. One might say that in this respect, glyphosate is a victim of its own success insofar as how widely it has been and is currently used.

The paper that seems to have started the ball rolling suggesting a link between glyphosate and autism is that from Anthony Samsel and Stephanie Seneff [1]. A review paper focussing specifically on the proposed inhibitory actions of glyphosate on CYP enzymes, mention of autism in the text and particularly that "glyphosate may be the most significant environmental toxin contributing to autism" was bound to stir up debate. With added soundbites about how half of children will be born with autism in the United States by 2025 and graphs correlating autism prevalence and glyphosate use, such observations were ripe for speculation and discussion.

Further papers from Samsel & Seneff [2] have continued the theme that autism may be one of many conditions/labels where glyphosate might be implicated. Drawing on animal and plant studies implicating glyphosate use with specific depletion of manganese (Mn), a whirlwind of research studies and references have been used to link such depletions to autism (and associated aspects such as anxiety) among various other conditions. To quote: "Many diseases and conditions are currently on the rise in step with glyphosate usage in agriculture, particularly on GM crops of corn and soy. These include autism, AD [Alzheimer's disease], PD [Parkinson's disease], anxiety disorder, osteoporosis, inflammatory bowel disease, renal lithiasis, osteomalacia, cholestasis, thyroid dysfunction, and infertility. All of these conditions can be substantially explained by the dysregulation of Mn utilization in the body due to glyphosate." Just in case you thought that Samsel / Seneff are the sole research team talking about glyphosate use and autism rates, I'll also refer you to the paper by Cynthia Nevison [3] that has been previously discussed on this blog (see here).

As other commentators have noted, there are a few considerations to mention about the discussions so far on any relationship between glyphosate use and autism. That 'correlation is not the same as causation' is perhaps the most important element to the data as they currently stand given that, to my knowledge, no-one has actually published any peer-reviewed results on levels of glyphosate and/or metabolites in people with autism and/or other family members compared to asymptomatic controls for example. It's not as if such a feat is too complicated given the various means and methods already published on this topic [4].

'Correlation is not the same as causation' is one of the themes also discussed in the review paper by Miguel Faria [5] who provides an important discussion to some of the points raised by Samsel & Seneff. I don't want to head too much into this quite long commentary on the Samsel / Seneff paper (complete with author replies) but it does strike me as providing something of a counter-balance to the very hypothesis-based writings of the original authors. That glyphosate is one of a multitude of herbicides and insecticides in use is an important point made in terms of the difficulties in disentangling it from the 'chemical soup' (not to demonise) that we live our lives in these days. The only other thing that I might mention is my much-used notion about the continued pluralisation of autism (see here) and its varied comorbidity as being something to bear in mind when it comes to any discussions about aetiology.

Where next, or is there even 'a next' for looking at any possible relationship between glyphosate use and autism rates? Well, I'd like to think that autism research can learn a lesson or two about not throwing out baby and bathwater when it comes to theories about [some] autism as per what seems to have happened to something like methylmalonic acid (MMA) and autism (see here) for example. It's not beyond the realms of possibility that certain chemicals or mixtures might be linked to autism risk and/or onset as per preliminary research talk about dioxin exposure being potentially linked to [some] autism or autistic traits (see here) albeit with a lot more follow-up research required.

But such research needs to be done with care and assiduity; mindful that this is a topic that will inevitably garner significant attention (as per the editorial sentence on the original Samsel / Seneff paper: "Note added by the Publisher: This paper attracts great attention.") and knowing what can happen sometimes when science and media mix (see here).

Perhaps a little less focus on soundbites and more on cold, objective science is the best way forward in this area?

Music: Fleetwood Mac- Dreams.

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[1] Samsel A. & Seneff S. Glyphosate’s Suppression of Cytochrome P450 Enzymes and Amino Acid Biosynthesis by the Gut Microbiome: Pathways to Modern Diseases. Entropy 2013. 15; 1416-1463.

[2] Samsel A. & Seneff S. Glyphosate, pathways to modern diseases III: Manganese, neurological diseases, and associated pathologies. Surgical Neurology International. 2015;6:45.

[3] Nevison CD. A comparison of temporal trends in United States autism prevalence to trends in suspected environmental factors. Environ Health. 2014 Sep 5;13:73.

[4] Yoshioka N. et al. Rapid determination of glyphosate, glufosinate, bialaphos, and their major metabolites in serum by liquid chromatography-tandem mass spectrometry using hydrophilic interaction chromatography. J Chromatogr A. 2011 Jun 10;1218(23):3675-80.

[5] Faria MA. Glyphosate, neurological diseases – and the scientific method. Surgical Neurology International. 2015;6:132. doi:10.4103/2152-7806.162550.

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Faria MA (2015). Glyphosate, neurological diseases - and the scientific method. Surgical neurology international, 6 PMID: 26322242

Effects of prenatal organophosphate pesticide exposure on a mouse model of autism

Pesticides and autism is a topic previously covered on this blog (see here for example). The idea that some of the various preparations that we use to control the pests which have an ability to blight our crops or cause serious health issues could be linked to an increased risk of autism is an area likely to invoke some divisions within elements of the autism and wider scientific community.


Part of the problem with the available evidence suggesting a link between autism and pesticide exposure is that it is, to a large extent, observational and correlational. As per the paper from Janie Shelton and colleagues [1], results are based on variables such as looking at what types and amounts of pesticides are used in a specific area and correlating proximity to that area as a function of a diagnosis of autism or not. Similar to the work looking at other environmental factors linked (or not) to autism such as air pollution (see here), it's all about where and when you were, followed by calculations on your probable exposure being tied into risk.

The paper from Alessia De Felice and colleagues [2] (open-access) approaches the research area of pesticide exposure and autism (a model of autism) from a slightly different angle. Their findings were based on the use of a mouse model of autism and what happened to mouse offspring when mother mice were exposed to a particular pesticide "at doses devoid of maternal or systemic toxicity."

The mouse model in question was a favourite in autism research circles, the BTBR T+tf/J strain (see here). The pesticide used was "the organophosphate insecticide chlorpyrifos (CPF)." Organophosphate (OP) by the way, refers to the chemical make-up of the compound and its particular effects on the enzyme acetylcholinesterase working in a similar fashion to certain warfare agents.

The De Felice paper is open-access but as ever, a few details are provided:

• Recognising that pesticide exposure already has quite a long history in terms of adverse health effects (pesticide applicators of the world take note), the authors set about looking to "evaluate in the offspring of both sexes the effect of the gestational CPF exposure on spontaneous locomotion, ultrasonic vocalization and neurodevelopment during the first two weeks of postnatal life, to evidence early changes in behavioral profile." Further they decided to "evaluate the long-term effects of CPF on selected markers of the peculiar behavioral repertoire of this mouse strain." You'll note that the word autism does not appear in either of those aims.
• Male and female BTBR mice were allowed to 'get it on' in breeding cages. Females mice were regularly inspected for evidence of carrying a baby mouse and 14 days into the pregnancy were either given peanut oil (vehicle) or CPF dissolved in peanut oil by oral gavage for 4 days. This was given as a single dose.
• Baby mice were born ("Twenty-four litters (13 Vehicle-treated and 11 CPF-treated"). Various behavioural assessments were conducted including an analysis of ultrasonic vocalisations and spontaneous movements. Various other behavioural measures were also made when the baby mice grew into adult mice.
• Results: "prenatal CPF exposure significantly modifies spontaneous motor activity in BTBR pups, delaying their motor development and further enhancing the abnormally high vocalization rates." The authors also reported reduced weight gain in CPF exposed offspring during the early days. 
• Looking into adulthood, CPF exposure seemed to show more pronounced effects for males over females with an "altered pattern of investigation of a sexual partner." This finding has however been downplayed by the authors to a certain extent. They conclude with the need for further research in this area "to evaluate the role of environmental chemicals in the etiology of neurodevelopment disorders."

Taking a few steps back, one has to remember that this was a study of mouse exposure to pesticides not humans. Indeed, other similar work from some of the authors has also recently seen the light of day [3]. As per other discussions, mice might be good 'models' of something like autism but they are never ever going to reflect autism (and its important comorbidities) in their entirety. Given also that the BTBR mouse "exhibit a 100% absence of the corpus callosum and a severely reduced hippocampal commissure" according to suppliers, one has to be careful about any sweeping generalisations to all autism. The timing and route of CPF exposure described by De Felice et al might also be seen as potentially important variables insofar as their focus some time into pregnancy and sole reliance on the oral route of exposure. 

That all being said, these are potentially important results not least because of their focus on how both genes and environment might be implicated in [models of] cases of autism. As I've mentioned, this was a study of offspring BTBR mice thus implying that there may be some familial predisposition to mimicking the effects of autism anyway. The idea that in predisposed individuals there may be additional effects from an environmental factor is an important one and perhaps complements the whole 'air pollution effects modified by genotype' suggestion (see here) with autism in mind. Certainly, I think there is quite a bit more research to see and do in this area.

Music: Buddy Holly - Rave on!

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[1] Shelton JF. et al. Neurodevelopmental disorders and prenatal residential proximity to agricultural pesticides: The CHARGE study. Environ Health Perspect. 2014: June 23.

[2] De Felice A. et al. Prenatal Exposure to a Common Organophosphate Insecticide Delays Motor Development in a Mouse Model of Idiopathic Autism. PLoS One. 2015 Mar 24;10(3):e0121663.

[3] Venerosi A. et al. Effects of maternal chlorpyrifos diet on social investigation and brain neuroendocrine markers in the offspring – a mouse study. Environmental Health 2015, 14:32.

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De Felice, A., Scattoni, M., Ricceri, L., & Calamandrei, G. (2015). Prenatal Exposure to a Common Organophosphate Insecticide Delays Motor Development in a Mouse Model of Idiopathic Autism PLOS ONE, 10 (3) DOI: 10.1371/journal.pone.0121663

Pesticides and autism: chapter II

I've labelled this entry a chapter II post reflecting some continued interest in how agricultural pesticide exposure might fit into autism research (see here for the chapter I post). In that previous post, I talked about various issues such as the old correlation-is-not-necessarily-causation mantra and indeed, how use of something like galantamine for cases of autism spectrum disorder (ASD) [1] might present something of a paradox for certain types of pesticides being involved in the condition, as a function of its similar acetylcholinesterase inhibitor activity (albeit reversible).

Altogether Now? @ Wikipedia 

Continuing the pesticide theme, I'm talking today about the paper by Janie Shelton and colleagues [2] (open-access) and their results strengthening "the evidence linking neurodevelopmental disorders with gestational pesticide exposures, and particularly, organophosphates". Some of the press on this study be seen here.

Based on data derived from the CHARGE study (beincharge!) authors reported that maternal residence during pregnancy close to locations undertaking "agricultural pesticide application" might elevate the risk of offspring autism; something previously discussed by some of the authors [3]. Results from CHARGE by the way, have already talked about other environmental associations with autism risk such as air pollution (see here). This initiative also recently confirmed what many people already knew in saying the gastrointestinal (GI) symptoms seem to be over-represented in cases of autism (see here).

The Shelton paper is open-access but here are a few pointers:

• With an authorship list of the great and the good associated with CHARGE, commercial pesticide use data was linked to the addresses of mothers when pregnant for groups diagnosed with autism (n=486) or developmental delay (DD) (n=168) compared with asymptomatic controls (n=316) living in California, USA. As alluded to in a post on that most undesirable of jobs - the commercial pesticide applicator - commercial pesticide use tends to be quite tightly regulated as a result of the potential for health effects when mis-used.
• Various models and algorithms built up a statistical picture of different classes of pesticides, their use and when and where they sprayed. The authors aimed to ascertain whether gestational exposure was linked to autism risk and whether there were "specific windows of vulnerability during gestation". 
• Results: bearing in mind there were quite a few estimates built into this study, a few points are worth mentioning. "Proximity to organophosphates at some time during gestation was associated with a 60% increased risk for ASD". Organophosphate (OP) pesticides were also "the most commonly applied agricultural pesticide near the home during pregnancy" and chlorpyrifos exposure in particular, during the 2nd trimester, seemed to show some association with offspring ASD risk.
• "Children of mothers residing near pyrethroid insecticide applications, just prior to conception or during 3rd trimester were at greater risk for both ASD and DD". Pyrethroids were the "second most commonly applied class of pesticides".
• A few additional points: males were slightly more likely than females to be exposed to pesticides during gestation, and the effects of multiple exposures (various different classes of pesticides) was generally "not found to be higher that the observations of the individual classes of pesticides".

Reiterating that this was a study based on estimation rather than looking at actual individual pesticide exposure during pregnancy or any biological testing for said exposure, this is an interesting study. I say that not to further condemn pesticides, which actually do quite a good job at helping to maintain our food supply and reduce our exposure to various pests. But rather that further study is indicated in this area as a result. The authors note the various strengths of their study based to a large extent on the fairly extensive data held on CHARGE participants. Likewise they note that their study did not for example, take into account "external non-agricultural sources" of pesticides such as those which many of us see sprayed around our homes, gardens and other areas of residence/work which could have affected their data. An 'underestimate' in actual exposure according to some external commentary on the study.

The question of pesticide exposure being potentially linked to autism risk carries quite a bit of the same baggage as the air pollution correlation. Yes, to some degree, we're all pretty unfit for consumption (see here) as a function of our 'chemical load' - bearing in mind the mis-representation of that word. Some people use this generality as a stick to beat such hypotheses on environment being potentially linked to conditions like autism (yes, we know the autisms are a complicated set of conditions). But as we've seen with the air pollution work, it may be the sum of the environmental risks combined with some genetic fragility which eventually provides the more important answers (see here); something which Shelton et al conclude: "Further research on gene-by-environment interactions may reveal vulnerable sub-populations". I might at this point also throw in a related post on Reelin and OPs as one area where we might begin searching.

In terms of the mechanism of effect, well if other autism research is anything to go by, it's gonna be complicated and probably not just confined to old the grey-pink matter. The obvious place to start looking would be the biological mechanisms which we rely on to metabolise things like OPs. PON1 is a good example, and as I've mentioned in other posts (see here) how PON1 has already seen some autism research action [4]. Indeed the paper by Gaita and colleagues [5] adds to the interest here and their findings of decreased serum arylesterase activity in case of autism. Paşca and colleagues [6] further suggested that a correlation (that word again) between high levels of homocysteine and low serum paraoxonase 1 arylesterase activity in cases of autism might be important. Certainly, when it comes to the 'big H' there is quite a bit of data with autism in mind (see here) but I'm not going to get ahead of myself here. I'll also direct your attention to other research talking about in-utero OP exposure being linked to shortened gestational duration [7] as an example of how effects may be peripheral as well as central. Oh and how PON1 enzyme levels might also tie into birth measurements [8].

If there is a take-home point to this post it is that the Shelton results suggestive of a link between gestational pesticide exposure and autism risk invite quite a bit more further scientific inspection of this area. I don't give clincial or medical advice on this blog but certainly the idea that pregnant women should perhaps "take special care to avoid contact with agricultural chemicals whenever possible" seems like a sensible statement to make (see here).

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[1] Ghaleiha A. et al. Galantamine efficacy and tolerability as an augmentative therapy in autistic children: A randomized, double-blind, placebo-controlled trial. J Psychopharmacol. 2013 Oct 15;28(7):677-685.

[2] Shelton JF. et al. Neurodevelopmental disorders and prenatal residential proximity to agricultural pesticides: The CHARGE study. Environ Health Perspect. 2014: June 23.

[3] Shelton JF. et al. Tipping the balance of autism risk: potential mechanisms linking pesticides and autism. Environ Health Perspect. 2012 Jul;120(7):944-51.

[4] D'Amelio M. et al. Paraoxonase gene variants are associated with autism in North America, but not in Italy: possible regional specificity in gene-environment interactions. Mol Psychiatry. 2005 Nov;10(11):1006-16.

[5] Gaita L. et al. Decreased serum arylesterase activity in autism spectrum disorders. Psychiatry Res. 2010 Dec 30;180(2-3):105-13.

[6] Paşca SP. et al. High levels of homocysteine and low serum paraoxonase 1 arylesterase activity in children with autism. Life Sci. 2006 Apr 4;78(19):2244-8.

[7] Eskenazi B. et al. Association of in Utero Organophosphate Pesticide Exposure and Fetal Growth and Length of Gestation in an Agricultural Population. Environ Health Perspect. 2004; 112(10): 1116–1124.

[8] Harley KG. et al. Association of organophosphate pesticide exposure and paraoxonase with birth outcome in Mexican-American women. PLoS One. 2011;6(8):e23923.

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Janie F. Shelton, Estella M. Geraghty, Daniel J. Tancredi, Lora D. Delwiche, Rebecca J. Schmidt, Beate Ritz, Robin L. Hansen, & Irva Hertz-Picciotto (2014). Neurodevelopmental disorders and prenatal residential proximity to agricultural pesticides: The CHARGE study Environmental Health Perspectives : 10.1289/ehp.1307044

Reelin' in OPs in autism

Get that pinch of salt ready again, as hopefully I'm not straying too far outside my area of competence with this post.

A recent entry on the SFARI blog talked about the potential pitfalls associated with too heavy reliance on animal models of autism spectrum disorders (ASDs) from the point of view of replication and reproducibility. Without wishing to strike up the 'I've talked about that before' orchestra, I've talked about similar things before(!) on this blog with specific reference to one particularly interesting mouse model of autism - the BTBR dangermouse.

Bearing in mind that we really shouldn't be putting all our research eggs into the mouse model basket for autism or any other condition under investigation, I was drawn to the paper by Brian Mullen and colleagues* (open-access) discussing their findings based on a Reeler mutant mouse model - mice not producing as much reelin as they should be - exposed to chlorpyrifos oxon (CPO), a metabolite of the organophosphate (OP) pesticide, chlorpyrifos. Quite a while back I had started to talk about OPs and autism (see here) and some curious findings in relation to the use of galantamine. Keep that compound in mind for now.

OK, perhaps it would be best if I provided a few descriptions first before venturing in the nitty-gritty of the Reeler OP exposed mouse.

Wikipedia provides quite a good overview of the Reelin gene and reelin protein findings so far in autism. Suffice to say that it is a complicated picture but basically boils down to (a) the neuronal tasks undertaken by reelin potentially overlapping with some of the ideas of how autism - some autism - might come about and (b) the positioning of the Reelin gene - human chromosome 7q22 - thought to be an autism gene 'hotspot' (something which is coming back into vogue recently as per the paper by Michaelson and colleagues** on regional hypermutation hotspots in autism). This paper from Persico and colleagues*** (open-access) from a few years back is as good an introduction as any to the hows and whys of reelin and autism.

Chlorpyrifos? Well, quite a good description about this OP can be found here. As with many pesticides - OPs in particular - chlorpyrifos works by inhibiting the break down of the neurotransmitter acetylcholine (ACh) by binding to the active site of the acetylcholinesterase (AChE) enzyme. In animals (and humans) this eventually leads to a build up of ACh which leads to things like neurotoxicity and death. It is with this mechanism in mind that quite unfortunately a few nerve agents also work in a similar fashion.

So then the Mullen paper is open-access, but here are a few highlights:

• Reeler mice (heterozygous for the Reeler gene - RI +/-) became romantically involved with wild-type males. A few drinks, some flirting, some Isaac Hayes music... no, not really, but Reeler females conceived and at a specific time during pregnancy, a special pump was fitted delivering a set concentration of CPO to some mouse mothers-to-be at a critical time of cortical activity and development.
• Various combinations of offspring mice were created which were either heterozygous (+/-) or homozygous (+/+) for the Reeler gene and either received CPO or were "vehicle-treated" (placebo).
• Several lines of investigation were carried out based on offspring survival, AChE activity, mouse behaviour and various anatomical studies.
• Results: Prenatal CPO did not affect embryo survival rates. That being said, more boy mice were present in the CPO treated litters (bearing in mind the relatively small litter numbers). CPO administration affected AChE activity as one might expect, reducing it by about "85% of the starting value". In contrast, CPO administration seemed to have a "protective effect on reelin protein" which brought reelin levels up in the +/- group.
• Mouse behaviour was interesting and not exactly in line with what the authors expect (they predicted that the reduced reelin expression combined with CPO exposure would increase autistic-like behaviours). Yes they saw some changes in the +/- CPO combination but more often than not it was rather more mixed and indeed suggested some potential mitigating effect. Also "combined CPO exposure and loss of reelin expression seemed to have more of an effect in female mice rather than male mice". Mmm...
• Same goes for brain anatomy... indeed the authors again actually suggest that each variable (reelin expression or use of CPO) seemed to mitigate one and another rather than increase any issue in a cumulative fashion.

I'm intrigued by the Mullen findings, accepting again the mouse model issue and how, as much as science tries, mouse models of autism are not person models of autism. I talked earlier for example about how galantamine was being touted as a possible intervention option for some people with autism (see this open trial by Nicholson and colleagues**** and this trial by Novotny which I'm still searching around for peer-reviewed results).

The primary mode of action of galantamine is with reference to its AChE-inhibiting properties, which place it not a million miles away from the actions of compounds like OPs and indeed chlorpyrifos. The difference predominantly lies in galantamine being a reversible AChE inhibitor and OPs exerting a more final irreversible effect on AChE. If one therefore assumes that the mitigating effects of CPO exposure on reelin deficient mice are potentially as a consequence of those AChE inhibiting effects and not just as the authors suggest CPO "increasing the pool of full-length reelin" by affecting reelin degrading enzymes, where does that leave us?

I'm not for one minute suggesting that OPs are, in any way, shape or form 'protective' against autism or anything else given their potentially important effects on neurodevelopment*****. But perhaps that the cholinergic system might require a little more investigation with autism in mind as per studies like this one from Ray and colleagues****** and talk of some blanket effect from pesticide exposure on the rising numbers of cases of autism is a little premature.

Finally, it also make me wonder if that graph of autism prevalence and organic food sales (shown above) which seems to be doing the rounds might actually provide something more than just a chuckle about correlation and autism, particularly if reelin is involved?

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* Mullen BR. et al. Decreased Reelin expression and Organophosphate pesticide exposure alters mouse behaviour and brain morphology. ASN Neuro. January 2013.

** Michaelson JJ. et al. Whole-genome sequencing in autism identifies hot spots for de novo germline mutation. Cell. 2012; 151: 1431-1442.

*** Persico AM. et al. Reelin gene alleles and haplotypes as a factor predisposing to autistic disorder. Mol Psychiatry. 2001; 6: 150-159.

**** Nicolson R. et al. A prospective, open-label trial of galantamine in autistic disorder. J Child Adolesc Psychopharmacol. 2006; 16: 621-629.

***** Potera C. Newly discovered mechanism for chlorpyrifos effects on neurodevelopment. Environ Health Perspect. 2012; 120: a270–a271.

****** Ray MA. et al. Neuronal nicotinic acetylcholine receptor subunits in autism: an immunohistochemical investigation in the thalamus. Neurobiol Dis. 2005; 19: 366-377.

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Mullen BR, Khialeeva E, Hoffman DB, Ghiani CA, & Carpenter EM (2013). Decreased Reelin Expression and Organophosphate Pesticide Exposure Alters Mouse Behavior and Brain Morphology. ASN neuro PMID: 23298182

Top 10 environmental pollutants and autism

I did promise in a recent post on rich/poor kids, older mums/dads and autism, that I would pass comment on the editorial by Landrigan and colleagues* (full-text) published in the journal Environmental Health Perspectives. I couldn't let this paper go by without some reference given that it provides quite a bit of background as to where autism research should be looking if we are to assume that certain aspects of the environment might play a role in symptom onset in at least some cases of autism and perhaps other related conditions.

Indeed there are several other papers in the same journal which might also be relevant to this post, including:

• This one by Sheldon and colleagues** (open-access) looking at pesticide exposure and autism,
• This paper by Kalkbrenner and colleagues*** (open-access) looking at maternal smoking during pregnancy,
• This paper by Wayman and colleagues**** (open-access) looking at non-dioxin like polychlorinated biphenyls (PCBs) and dendritic growth,
• and another paper by Wayman and colleagues***** (open-access) on ryanodine receptor dysregulation following PCB exposure.

I have to say that I don't have the time, energy or expertise to go through each and every one of these papers but would instead refer to quite a nice summary here on two of the papers and invite any comments from those that did read them. Please also consider this entry as a sort of continuation of my interest in pesticide exposure and health as per this and this post.

There are a few things to note about the Landrigan commentary including:

• Acceptance that autism is a family of conditions of which the authors suggest that about 30-40% of cases might be attributable to genetic inheritance.
• An estimated 3% of all neurobehavioural conditions are "caused directly by toxic environmental exposures" and some 25% caused by interactions between environmental factors and inherited susceptibilities.
• There is accumulating 'proof of principle' evidence for some role for environmental factors in cases of autism. 
• Added to what is already known about the neurological and developmental effects of some of these environmental agents combined with the growth and growth in areas such as epigenetics, there are some pieces of the research puzzle which seem to overlap with evidence from autism research.
• A workshop titled 'Exploring the Environmental Causes of Autism and Learning Disabilities' generated a list of 10 chemicals widely persistent in the environment which might cause developmental neurotoxicity. These were: Lead, Methylmercury, Polychlorinated biphenyls (PCBs), Organophosphate (OP) pesticides, Organochlorine (OC) pesticides, Endocrine disruptors, Automotive exhaust, Polycyclic aromatic hydrocarbons (PAHs), Brominated flame retardants, Perfluorinated compounds. The chemicals with links will take you to previous posts on this blog which have covered the compound in question; some with autism in mind, others with a more peripheral connection.

I'm sure you will agree that there are quite a few very bold statements included in this editorial not least the top 10 chemical targets for further research. I imagine that there will be some discussions on the rights and wrongs of naming and shaming the potential chemical offenders as time goes on.

The good news about these chemicals / compounds is that with the technology available these days, all are perfectly testable, and can be analysed in various biological fluids. One would therefore imagine that designing a large enough study covering people with autism, their parents, even siblings and appropriate comparators, controlling for comorbidity, even incorporating some degree of 'endophenotype' research and genetic / epigenetic elements is a perfectly feasible way of testing one or more of the '10 chemical hypotheses'.

Assuming some connection is confirmed and bearing in mind the complexity of cause-and-effect, the next question might be 'what can we do about it'? Minimising contact with these agents, particularly during conception and pregnancy and into early childhood, utilising the various ways and means available to remove these chemicals from the body - yes, even using that dreaded biomedical 'mumbo-jumbo' word detox - and assessing outcome both behavioural and biochemical (see here). Who knows perhaps even looking at the various detoxification mechanisms such as glutathione and its relations which have ascended up the evidence ranks quite recently to note any possible association.

Stop already with the speculation, I hear you cry. And I will.

To finish, do you feel compelled to walk like an Egyptian?

* Landrigan P. et al. A research strategy to discover the environmental causes of autism and neurodevelopmental disabilities. Environmental Health Perspectives. April 2012
DOI: 10.1289/ehp.1104285

** Sheldon JF. et al. Tipping the balance of autism risk: potential mechanisms linking pesticides and autism. Environmental Health Perspectives. April 2012
DOI: 10.1289/ehp.1104553

*** Kalkbrenner AE. et al. Maternal smoking during pregnancy and the prevalence of autism spectrum disorders using data from the Autism and Developmental Disabilities Monitoring Network. Environmental Health Perspectives. April 2012
DOI: 10.1289/ehp.1104556

**** Wayman GA. et al. PCB 95 promotes dendritic growth via ryanodine receptor-dependent mechanisms. Environmental Health Perspectives. April 2012
DOI: 10.1289/ehp.1104832

***** Wayman GA. et al. PCB 95 modulates calcium-dependent signaling pathway responsible for activity-dependent dendritic growth. Environmental Health Perspectives. April 2012
DOI: 10.1289/ehp.1104833

Bitty and autism

'I want bitty' says Harvey, the archetypal Peter Pan 'mummy's boy'.

Most people in the UK who have watched the comedy show Little Britain will know about Harvey and his constant desire for 'bitty'. For those who are perhaps unfamiliar with our quite eccentric humour here in the UK (we did of course bring the world the Ministry of Silly Walks), bitty in this case, is slang for breastfeeding or breast milk. Harvey is a character who despite being a grown-up, well-to-do man with a fiancee, still quite enjoys being breastfed by his mum, having never quite moved out of his infancy on this issue. The character is a parody for all those people out there - mostly men - who despite being quite capable, refuse to grow up and break from 'mummy's apron strings'. For the mums also who want to keep their precious at that age when the bond was so close.

Anyway. A while back I posted an 'other musings' entry on early infant feeding practices in light of the disparity of views of when to start weaning children from milk to food. One of the debates included as part of that story was the issue of breastfeeding. On purpose I did not get into the nitty-gritty debate of 'breast vs. bottle' being as I am the wrong gender to comment and respectful of the fact that such a decision is a matter of personal, informed choice or in some cases no choice. I am however interested in the various research into breastfeeding with regards to autism and lots of other childhood and developmental issues and areas.

Like many people have said before me, there is no doubt that breast milk has quite a lot going for it. If you need convincing, have a look at this document from the WHO detailing the beneficial effects of breastfeeding on areas such as health and intelligence (schooling). There is, as always, an important caveat attached to this document and the research in general, in that correlation does not necessarily imply causation and hence whilst an association might be strong statistically, there could be (and are) lots of other factors which influence such effects. Still breast milk, in its various stages and forms, carries quite a lot of nourishment and important compounds which can potentially help get a developing child off to a good start as well as being good for mum also. Suffice to say that before we got so good at making the modern infant formula feed, breast milk was the only (safe) way of nourishing a young infant for many, many generations of women.

The research base regarding breastfeeding and autism covers quite a bit of ground. Kanner, in his early descriptions talked about early feeding problems in his cohort but did not specify whether due to breast or formula. I don't really want to get too stuck on looking at any general association between breastfeeding rates and autism simply because (a) I think this is perhaps too simplistic an association to make (see correlation/causation argument above), and (b) there is always that risk of stigmatising mums who did or didn't breastfeed when all too often there are some complex processes in any decision made. The brief mention I will give to the analysis of breastfeeding rates in mothers with a child/children with autism suggests that there is no cut-and-dried answer of association.  The study that my colleagues and I have published touching upon this area, suggested that breastfeeding rates were fairly high (60-69%) according to sub-diagnostic groups we looked at compared with the UK average of about 55% exclusive breastfeeding for the first 4 weeks of infancy. Other studies have shared such notions. Other parental surveys have reported slightly different results. Final answer: not cut-and-dried.

When it comes to any direct effects of breastfeeding in relation to autism, there are a couple of themes which emerge related to the composition of breast milk: a possible role of environmental pollutants and the structural composition of breast milk in relation to things like fatty acids and those pesky casomorphins which perhaps need addressing.

I am going to try and be quite careful when talking about contamination from environmental pollutants and breast milk. Careful because 'environmental pollutants' covers such a wide range of chemicals and compounds, some naturally occurring and some more man-made. Careful also because some of the studies indicating increasing environmental exposures found in breast milk have to be balanced against those indicating decreasing exposure in breast milk. Geography and other factors such as smoking seem to play their role, as does the influence of maternal diet. There is some small suggestion that higher levels of specific environmental contaminants passed through mothers milk are associated with specific outcomes for the child at later ages. Again with my balancing act in full.. er, balance, there is however quite a lot of evidence pointing in the other direction, suggesting that such contamination of milk are not associated with any specific negative outcomes, including results from some rather large studies here and here.

Where do the studies specifically with autism in mind stand on this issue? Well, they don't stand anywhere because aside from some discussion on what happens to rats who are given doses of selected pollutants equivalent to that derived from breast milk showing some neural disorganisation, there is very little direct evidence on pollutant composition of breast milk in relation to autism. Indeed, the recent CHARGE study on PBDEs in plasma of children with autism (not specifically looking at breast milk!) suggested that levels of this one class of pollutants showed no difference from controls, although all groups presented with high levels. Going back to that very complicated issue of parental age and autism, another study touched upon another possible contributing variable in that the longer we leave it to have children, the more scope there may be for parental (mother) accumulation of environmental pollutants to be potentially passed on through breast milk. Whether this could have an effect specifically on autism, I don't know.

The other issue about breast milk potentially linked to autism is its composition; with specific focus on fatty acids and casomorphins. There has been quite a bit of speculation about specific types of fatty acids in breast milk. This review provides quite a good overview of what types of fatty acids might be most important, their relative quantity in breast milk and associations with outcome. Readers of this blog might remember that fatty acids have cropped up quite a few times with regards to autism (and ADHD). There is some common sense in perhaps thinking that the fatty acids in breast milk might show some involvement with autism given the various studies indicating potential fatty acid deficiencies in some cases. I would perhaps temper this statement by saying that formula milk manufacturers have also caught on to this (and GOS and FOS), and generally include them in their formulations also.

The other side of this issue is in relation to breast milk and casomorphins. I know there has been a bit of a debate in relation to casomorphins (and gluteomorphins) with respect to autism and this is perhaps fodder for another post. What is widely accepted is that mammalian milk protein (casein) is eventually broken down to its constituent peptides and amino acids via hydrolysis. Mammalian milk covers human milk as well as milk from cows, goats, sheep, etc. Given the continued speculation on the involvement of casomorphins in some conditions including autism, there is perhaps some grounds for further research into any possible relationship. Is there any variability in the level of casomorphins according to the different stages of milk and is this consistent across all lactating mothers? This and other questions on things like lactose intolerance and breast milk might be interesting to examine in light of the recent Harvard findings on autism.

Given the length of this post and to save anymore people from switching off, I will stop there. I am not going to post any links to videos of Harvey and 'bitty' so as not to offend. Instead I offer a short excerpt from a movie that some of you may have seen which offers a new perspective on breastfeeding.