Sulforaphane and autism continued: metabolomics wades in...

"We identified 77 urinary metabolites that were correlated with changes in symptoms, and they clustered into pathways of oxidative stress, amino acid/gut microbiome, neurotransmitters, hormones, and sphingomyelin metabolism."

So said the findings reported by Stephen Bent and colleagues [1] continuing a theme in autism research circles examining the use of a compound called sulforaphane - "a supplement with indirect antioxidant effects that are derived from broccoli sprouts and seeds" - in the context of [some] autism (see here). Once again, I'm sure that there may be people out there with brows furrowing when it comes to talk of a 'broccoli chemical' potentially impacting on the presentation of autism. But peer-reviewed science (placebo-controlled) is peer-reviewed science [2] and not just to be 'put to one side' because it doesn't follow the trends or [research] fashions of the day.

This latest work from Bent et al represents a not-so-methodologically strong attempt (i.e. not placebo-controlled) to bring the science of metabolomics into research proceedings to "examine changes in physiological markers that may underlie beneficial treatment effects from sulforaphane by analyzing changes in urinary metabolites." Metabolomics by the way, as well as being music to my research ears, is something that percolates through quite a lot of autism research these days (see here and see here for examples) as small molecules in urine, blood and other biofluids are separated, detected and elucidated all in the name of science.

A small group of children participated in the Bent study; all had a "formal diagnosis of autism", all were reasonably happy to swallow a tablet containing sulforaphane ("weight-based dosing of sulforaphane") and all were able to provide urine samples before the study started and at the conclusion of the 12 week research period. Parents of participants were also willing and able to complete a couple of behavioural schedules: "the Aberrant Behavior Checklist (ABC) and... the Social Responsiveness Scale (SRS)" at "baseline, 4 weeks, and 12 weeks using an online and secure platform" about their children too.

Results: alongside looking at pre- and post-intervention behavioural scores, the authors also "examined the number of participants who had a clinical response." This is a particularly important detail in the context of autism and the continuing discussions about how the spectrum is well and truly heterogeneous (the autisms?) and so one shouldn't expect every single person diagnosed to somehow have the same genetics and/or biochemistry; also affecting response to any particular intervention. With that in mind, authors also reported that (group) scores on one of their primary outcome measures - the SRS - were significant, indicative of some positive change noted to behaviour over the course of the intervention period. I say this bearing in mind that this was an open-trial, where everyone took sulforaphane and everyone knew that they were taking sulforaphane (including the parents who did the scoring). As for those potential 'best-responders' to sulforaphane use, we are told that: "Eight participants had a clinical response compared to seven who were classified as non-responders."

Then to those metabolomic results, and from a total of nearly 700 compounds identified in urine, approaching 80 of them seemed to show some correlation with the behavioural symptom changes noted. They were put into various categories depending on their biochemical form and/or action, and correlations with behavioural scores (and significance) were presented. Some of the best correlations that I could see were with regards to sphingomyelin metabolism. The authors did seem a bit surprised by results in this area, but added: "It is not clear how sulforaphane might alter sphingomyelin metabolism or availability and whether this is related to clinical benefits, but if this association is confirmed, it has important clinical and treatment implications." I daresay that examination of sphingomyelin metabolism in other contexts (see here for example) might be revealing, particularly in the context of other behavioural/psychiatric labels [3] that could (and do) overlap with autism.

There is a further scheme of work to be followed when it comes to sulforaphane and autism on the basis of these and other scientific results [4]. I'd also suggest that the continued incorporation of the science of metabolomics is a good thing, and adds a further tier of investigation when it comes to studying intervention more generally in the context of autism. It's also an important step in (eventually) coming up with a 'test' for who might be a best-responder to the use of sulforaphane and perhaps associated compounds...

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[1] Bent S. et al. Identification of urinary metabolites that correlate with clinical improvements in children with autism treated with sulforaphane from broccoli. Molecular Autism. 2018; 9: 35.

[2] Singh K. et al. Sulforaphane treatment of autism spectrum disorder (ASD). Proc Natl Acad Sci U S A. 2014 Oct 28;111(43):15550-5.

[3] Castillo RI. et al. From Molecules to the Clinic: Linking Schizophrenia and Metabolic Syndrome through Sphingolipids Metabolism. Frontiers in Neuroscience. 2016;10:488.

[4] Sedlak TW. et al. Sulforaphane Augments Glutathione and Influences Brain Metabolites in Human Subjects: A Clinical Pilot Study. Mol Neuropsychiatry. 2018 May;3(4):214-222.

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On genes, environment, broccoli and autism (again)

Picture: Carl Warner: http://www.carlwarner.com/

I'm serving up two peer-reviewed papers for your reading delight today which draw attention to the ideas that (a) the 'causes' of autism are likely complex and as heterogeneous as the label itself, (b) gene x environment interactions affecting risk of autism are starting to get some good scientific research airtime and (c) don't 'dis the broccoli [chemical] autism connection just yet...

The first paper by Brandon Pearson and colleagues [1] (open-access) has already found some media interest as per the Guardian headline: 'Agricultural fungicides are 'bad news for neurons', study suggests'. Exposing mouse neurons - "cortical neuron-enriched cultures" - to several hundred chemicals (careful of that word) found in the modern environment, researchers concluded that several compounds "produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer’s disease and Huntington’s disease)." That is, several types of chemicals quite commonly found in the modern environment seemed to alter gene expression in those mouse neuron enriched cultures that weren't a million miles away from that noted previously in conditions such as autism for example.

Mouse neurons, you might be thinking? Well, obviously one has to be a little cautious about extrapolating from mouse to humans (see here) but researchers did include some comparison analysis looking at "the gene expression profile of our cultures with brain cell-type-specific expression data sets and human brain gene expression data sets." The result: "cortical cultures show strong transcriptional similarities to the human brain."

Clustering chemicals based on "concordant gene expression changes", six groups emerged. Cluster 2 chemicals, containing such pesticides as rotenone, pyridaben and fenpyroximate  and also various compounds under the heading of the strobilurins seemed show some particularly interesting results insofar as they "mimicked the transcriptional changes of two post-mortem ASD [autism spectrum disorder] brain expression data sets in a bidirectional manner." The effects of this cluster of compounds also seemed to unite various conditions with autism including Alzheimer’s disease and Huntington’s disease and the "aging brain". My interest was particularly piqued by that last association in light of other research results (see here).

When it came to the 'effects' of those chemicals in terms of genetic and biological processes, researchers put forward some not unfamiliar potential roles: "These chemicals, most of which inhibit mitochondrial complex I or III, stimulated free radical production and disrupted microtubules." Words like 'oxidative stress' start to emerge as they have done in previous autism research (see here) and yet again, inflammation or inflammatory processes seem also to be indicated. Indeed, the authors also make mention of how effects such as free radical production "can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane." Yes indeed, sulforaphane - the chemical found in broccoli - might indeed be moving back up the autism research agenda (see here for some previous background).

There is obviously lots more work to do in this area before anyone gets too carried away. The authors note: "While usage and residue levels of cluster 2 chemicals on conventionally grown foods are increasing, in the absence of causality, it is premature to draw correlations with the increased prevalence of ASD and other brain disorders." Lessons could be learned from other blanket suggestions about 'chemicals' and autism (see here) as well as an appreciation for the concept of the the plural autisms (see here). Then there are the practicalities of whether ingesting such compounds on food or in water is the same as direct exposure to cortical neuron-enriched cultures? Or indeed, whether there may be other routes of contact? I might also suggest that further studies should focus on looking for the metabolites of such agents too [2] bearing in mind the concept of statistically significant thresholds...

If you're still here after all that, the second paper I want to talk about is that from Sarah Wong and colleagues [3] that has also received a bit of media attention. The focus this time was on a gene called p53 (see here for some background) and how issues with this gene might be 'over-represented' when it comes to autism following on from other work by some of the same authors [3]. First of all, please don't get too fixated by mention of the words 'cancer gene' when it comes to p53 given it's [protein] tumour suppressing capabilities. As I've discussed before, the risk of cancer does not seem to be elevated any more than the general population risk when it comes to autism (see here). Perhaps of greater relevance to the Wong findings is the idea that p53 has other 'activities' such as that related to oxidative stress (yes, that again) and "DNA repair, bioenergetics and mitochondrial DNA (mtDNA) copy number maintenance."

Based on data from CHARGE (beincharge!), researchers garnered blood samples from 66 children diagnosed with an autism spectrum disorder (ASD) and "race-, gender-, and age-matched typically neurodeveloping children (n = 46)" (authors words not mine). They analysed for mtDNA copy number and deletions and p53 gene copy ratios and found them to be "more common in children with AU [autism] and their fathers." The authors translate their findings as pointing to "a role for deficient DNA repair capacity not driven by paternal age." They also suggest that environment might intersect with genetics in relation to 'severity' scores of autism obtained for their cohort: "gene x environment interaction seems to play a greater role in children with autism with less severe symptoms."

Taken together the Pearson and Wong findings point to some interesting 'associations' potentially relevant to [some] autism. The idea that certain components of the modern-day environment might increase the risk of autism is nothing new but the way that Pearson et al went about studying the possible relationship is. The results from Wong et al suggesting that there might be issues with the gene 'whose role is to suppress cellular damage from environmental stressors' suggests that exposure patterns might not necessarily be where it's all at when looking at compound/chemical X or Y in relation to autism risk. I'm also inclined to direct you to some previous discussion about the caspases and autism (see here) in light of the involvement of p53 with the process of apoptosis (programmed cell death) in mind. As I've mentioned before, the biological mechanisms for how people deal with various xenobiotics needs a lot more investigation in autism research circles (see here); something that might similarly extend to genetic mechanisms too.

Oh, and just in case you think that I'm pushing the either/or of genetic and environment when it comes to autism, I'm not, as words like epigenetics spring to mind and the idea that genomic instability might, for example, have quite a few different dimensions (see here)...

To close, I'm thinking of branching out... football (soccer) pundit perhaps?

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[1] Pearson BL. et al. Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration. Nat Commun. 2016 Mar 31;7:11173.

[2] Domingues VF. et al. Pyrethroid Pesticide Metabolite in Urine and Microelements in Hair of Children Affected by Autism Spectrum Disorders: A Preliminary Investigation. Int. J. Environ. Res. Public Health 2016; 13: 388.

[3] Wong S. et al. Role of p53, Mitochondrial DNA Deletions, and Paternal Age in Autism: A Case-Control Study. Pediatrics. 2016. March 31.

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Pearson, B., Simon, J., McCoy, E., Salazar, G., Fragola, G., & Zylka, M. (2016). Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration Nature Communications, 7 DOI: 10.1038/ncomms11173




Wong, S., Napoli, E., Krakowiak, P., Tassone, F., Hertz-Picciotto, I., & Giulivi, C. (2016). Role of p53, Mitochondrial DNA Deletions, and Paternal Age in Autism: A Case-Control Study PEDIATRICS, 137 (4) DOI: 10.1542/peds.2015-1888

Yes folks... broccoli chemical impacts on autism presentation

Please do not adjust your set. Broccoli, or least a chemical found in broccoli called sulforaphane has, under placebo-controlled, double-blind experimental conditions, been reported to impact on the presentation of autism according to the paper by Kanwaljit Singh and colleagues [1] (open-access).

Eat your greens @ Fir0002/Flagstaffotos

I had to do a bit of a double-take myself when I first read about these results (see here). Indeed, even the authors themselves seemed to be a little taken aback by their own findings if other media on this study is to be believed (see here). Still peer-reviewed science is peer-reviewed science and that goes just as much for this study as any other.

The paper is open-access but a few details might be useful:

• The study was previously registered in the US ClinicalTrials.gov database (see here). It involved comparing "capsules of sulforaphane-rich broccoli sprout extracts" with "indistinguishable placebo capsules" containing microcrystalline cellulose (wood pulp) in a small group of young men (aged 13-27 years) diagnosed with an autism spectrum disorder (ASD) administered daily over 18 weeks. As I mentioned, the study was also double-blind.
• Various measures - behavioural and physiological - were recorded at baseline (prior to study start) and at choice points during the 18 week study period. The results of the behavioural measures including the Aberrant Behaviour Checklist (ABC) and Social Responsiveness Scale (SRS) completed by parents/caregivers and the Clinical Global Impression Severity (CGI-S) and the Clinical Global Impression Improvement (CGI-I) scales completed by "study physicians" are included in the main paper results and conclusions.
• Results: well, first and foremost there were a few adverse events reported during the trial. The authors note that: "Sulforaphane treatment effectively improved core aberrant behaviors of ASD, and was safe and well-tolerated". But... "the sulforaphane group gained significantly more weight over the 18-wk period, compared with placebo" and there was mention of "single unprovoked seizures" occurring in two participants taking the active treatment. These seizures may well be unconnected to the sulforaphane capsules but one cannot rule out the possibility that they were connected.
• Forty participants completed the trial, or at least "part of the outcome measure evaluations" boiling down to "14 placebo and 26 sulforaphane". The statistical evaluation undertaken involved looking at "the differences between scores of individuals at 4, 10, 18, and 22 wk from their respective average pretreatment values". But the authors also undertook a separate intention-to-treat analysis that "included all 44 participants".
• The headlines: "many of the participants who were treated with sulforaphane in this study had statistically significant and clinically meaningful improvements during treatment with sulforaphane". With all due respect to parent/caregiver reports, I was particularly drawn to the fact that study physicians although blinded to who was on active treatment and who was taking a placebo were able to rate "13 of the 40 participants" as showing noticeable improvements in behaviour and sociability and "all were receiving sulforaphane". That's quite a feat by any study's standard.
• The authors conclude: "The substantial improvements of individual ASD patients’ trajectories were conspicuous and suggest that further investigation of sulforaphane in ASD is promising".

These are interesting results crying out for further independent [longer term] replication. The fact also that this was a trial of adolescents and adults with autism also fills a gap in the autism research market alluded to in previous posts on this blog (see here). 

Mechanism of effect? Well, there does seem to be quite a bit more to do in this area. The authors note that sulforaphane "was selected because it upregulates genes that protect aerobic cells against oxidative stress, inflammation, and DNA-damage, all of which are prominent and possibly mechanistic characteristics of ASD". Oxidative stress does indeed appear on the research radar when it comes to autism, at least some autism (see here) and sulforaphane fits the bill in terms of its potential 'protective' effects [2]. I've also talked about such mechanisms with another source of sulforaphane in mind (see here). That all being said, I don't doubt that there may be other biological processes at work.

So, in conclusion 'eat your greens' might very well be an important phrase for some on the autism spectrum. Whether eating the source material carries the same effect or will be equally well received as taking a daily pill is another matter...

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[1] Singh K. et al. Sulforaphane treatment of autism spectrum disorder (ASD). PNAS. 2014. October 13.

[2] Guerrero-Beltrán CE. et al. Protective effect of sulforaphane against oxidative stress: recent advances. Exp Toxicol Pathol. 2012 Jul;64(5):503-8.

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Kanwaljit Singh, Susan L. Connors, Eric A. Macklin, Kirby D. Smith, Jed W. Fahey, Paul Talalay, & Andrew W. Zimmerman (2014). Sulforaphane treatment of autism spectrum disorder (ASD) PNAS : 10.1073/pnas.1416940111