Wednesday, November 5, 2014

Microbiomes and GMOs

What's up everyone? This post reviews an article that I stumbled upon on Twitter. I don't think that I ever anticipated that joining Twitter would cause me to read more papers, but it's been a very pleasant surprise. The paper has a freakishly long title, way longer than 140 characters: "High-Throughput Sequence-Based Analysis of the Intestinal Microbiota of Weanling Pigs Fed Genetically Modified MON810 Maize Expressing Bacillus thuringiensis Cry1Ab (Bt Maize) for 31 Days" and is publicly available. The jargon-free title of the paper would be "DNA sequencing analysis of the bacteria in the gut of baby pigs fed an insect-resistant-GM-corn for 31 days". The study is independently funded and its authors claim no conflict of interest.

The reason I wanted to read this paper is that a) it combines two trendy topics: GMOs and microbiomes (hence the buzzword-laden title to this blog), and most importantly, b) I wanted to find out if this paper lent credibility to the Institute for Responsible Technology's hypothesis that GMOs are to be blamed for poor gut health. Here's a quote from their website, which I'll explain further below:

"A recent analysis of research suggests that Bt-toxin, glyphosate, and other components of GMOs, are linked to five conditions that may either initiate or exacerbate gluten-related disorders:

  • Intestinal permeability
  • Imbalanced gut bacteria
  • Immune activation and allergies
  • Impaired digestion
  • Damage to the intestinal wall"

  • As you may know, trillions of bacteria live in our gut. These little critters help digest our food, generate vitamins for us, and breakdown many different compounds. The collection and distribution of these bacteria is known as our "microbiome". There's a lot of research going on right now exploring different aspects of our microbiomes, but it's a fairly new field: we don't know what constitutes a "good" or "healthy" microbiome or if such a thing exists at all. We know that the composition of the bacteria in our gut fluctuates a lot and very quickly (as examples, this study suggests that microbiomes change very quickly when you eat meat; this study suggests that jet lag impacts your microbiome, possibly causing metabolic imbalances). Odds are, your microbiome is changing at this very moment to account for many different factors, particularly if you had to deal with the ridiculous daylight savings time change this weekend. 

    So what does this have to do with GMOs? One of the many arguments made by the Institute for Responsible Technology and other anti-GMO groups is that the Bt-toxin, which is a bacterial protein introduced into specific crops such as corn and cotton to make them insect resistant, negatively impact our gut. Here's an overview on how the Bt-toxin (or protein) works from UCSD: "The Bt toxin dissolve in the high pH insect gut and become active. The toxins then attack the gut cells of the insect, punching holes in the lining. The Bt spores spills out of the gut and germinate in the insect causing death within a couple days." Basically, if a bug that is sensitive to Bt eats a crop that has the Bt-toxin transgene, then it will die pretty quickly.

    The reason why this same pathway doesn't work in humans is because a) our guts don't have high pH (our stomach has low/acidic pH), and b) we don't have the receptors in our gut that recognize Bt. 

    So, that's why I was curious to read this paper. Would a single statement from the Institute for Responsible Technology finally be correct/accurate? Aren't you excited??? Let's get started!!

    The paper starts by outlining the importance of this field of research (my comments/thoughts in brackets): any change in the microbiome caused by a GM-plant could impact the host [i.e. the person], particularly if the individual is immunocompromised; the European Food Safety Authority guidelines recommend examining the impact of GMOs during animal feeding trials, but most research examining the Bt-toxin has examined the impact of the protein on bacteria of the soil [which is also important from an ecological standpoint]; in vitro [in a petri dish] tests suggest that the Bt toxin has anti-bacterial properties and that it doesn't actually get completely degraded in the intestine, so it's important to figure out what it does in vivo [in a living organism]. 

    The paper also reviews results of previous Bt-corn feeding studies that have examined the microbiome: 2 feeding studies in cows found no impact, a study in sheep also found no impact, but a feeding study in rats found a difference in the distribution/location of a bacteria after feeding Bt for 90-days (I've said it before and I'll say it again: if you read that there are no studies about GMOs and their safety, you are being misled). The authors explain that their study is important because a) the pig's digestive system is similar to that of humans and b) the DNA sequencing analysis that they perform is far superior to that used in previous studies. 

    We interrupt this post for BioChica's shameless self-promotion: please read my previous post on DNA sequencing if you need an introduction to the technology.

    The experiment seems pretty well designed: once the pigs were weaned, they were randomly split into two groups of nine pigs each (i.e. n=9/treatment). One group was given feed that was made from Bt-corn and the second group was given feed that was made from the non-genetically modified strain of the corn (i.e. the non-GM isogenic parent line). The pigs weren't given antibiotics at any point and were allowed to eat and drink freely. It would have been good to have the numbers of how much they ate/drank to determine if there was any difference.
    The Bt-corn and the control corn were grown in neighboring plots to ensure that they had the same environmental conditions (this is an important aspect to the study since previous papers that I've reviewed suggest that the environment plays a bigger role in differences between GM and non-GM crops than whether the crop is a GMO). The Bt-corn and its corn were tested to determine if there were any differences in their nutritional composition (i.e. does the GM-corn have more/less sugars, carbs, amino acids, etc. than the non-GM corn?) and there were no alarming differences. The authors made feed for the pigs out of the Bt-corn, making sure that the ONLY GM ingredient was the Bt-corn. For the control pigs, they made the exact same feed using the control corn. The pigs were fed the corn for 31 days and then euthanized. Fecal samples (i.e. pig poop) were collected on day 1 and day 31. Once the pigs were euthanized, samples were collected from different points along the intestine. Then, DNA was collected from all the stool samples.

    Apparently, this cute little mammal's
    digestive system is quite similar to our own
    Once they had the DNA, they performed a specific analysis which allows you to determine what species were in the sample (including bacteria), as well as their distribution (for the science-y people, they amplified the v4 portion of the 16S rRNA using universal primers, sequenced on a 454 and then BLASTed against a 16s-specific database). They included a negative control throughout the DNA analysis. They compared the results from the two groups of pigs and did the necessary stats.

    So... after all that work, what did the data say?

    The authors go through the different families of bacteria found in each area of the intestine and any differences seen between the two treatments. There were no differences in the abundance of the most prevalent types of bacteria between the two treatment groups. There were differences in the abundance of some of the minor families of bacteria, most of which the authors attribute to sampling (for example, the bacteria was found in 5 pigs in one treatment and only 2 pigs in the other treatment). Other differences in the abundance of the less prevalent bacteria were primarily attributed to differences in the amount of fiber between the corn leading to differences in the amount of food ingested (which points again to the fact that the authors really should have measured the amount of food and water that the pigs were taking in). It's important to note that the differences observed were within the "normal" amount for corn.

    The authors conclude by stating that the biological importance of these small differences remains to be seen, but that, in any case, they didn't cause any issues in the intestines of the pigs examined. The authors point out that they didn't observe any anti-bacterial effects caused by the Bt-toxin, possibly due to the fact that the amount of Bt-toxin in the feed was approximately 4000x lower than the amount used in the in vitro studies where the anti-bacterial effect had been observed/measured.

    I'd like to take a moment to say "Holy Crap!! 4000x lower?? No wonder the real world relevance of in vitro studies is always an important question!!"

    The final paragraph of the study reads (my explanations in [brackets]): "In conclusion, 31 days of Bt maize consumption had only minimal impact on microbial community structure in the ceca [gut] of pigs, resulting in statistically significant differences in abundance of only 2 of 39 bacterial families and 2 of 54 genera [subfamilies] detected. However, the low abundance and frequency of detection of some taxa [types of bacteria], as well as the lack of information on their role within the intestine, make interpretation of some of the data difficult. Nonetheless, results from the present study indicate that dietary Bt maize [corn] is well tolerated at the level of the intestinal microbiota following 31 days of exposure, as the differences observed are not believed to be of major biological importance and were not associated with any adverse health effects."

    So there you have it. The paper suggests that Bt corn doesn't cause a leaky gut, doesn't kill the bacteria in your gut, and doesn't have a negative impact on your intestine. Next time you're bloated and gassy, you won't be able to blame the GMOs :P

    I have no doubt that some will read this and point to the small differences observed and yell "Aha!! There ARE differences!" The paper clearly questions the biological relevance of these differences, and as I mentioned earlier in this post, the paper adds more evidence to an existing body of data suggesting that Bt causes no harm in the gut. Some will say "31 days isn't enough!!", but again, as I mentioned at the beginning of this post, evidence suggests that changes in our microbiome take place quickly and that it's always in flux. If I were an academic scientist, I wouldn't want to investigate the question of Bt impact on microbiomes any further because of the high likelihood that I wouldn't find anything new. We hear a lot about the shortage of grant money and funding in science. Can you honestly tell me that it's the best use of your tax dollars to do yet another study on this topic with all the data that already exists? Anyhoo, that's my rant for this evening...

    Till next time!

    Update 11/6/2014: I got the following comment about the article that is worth noting "They also didn't use very strict p-values or do multiple test corrections - pretty amazing how few significant hits they found without it!"