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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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February 11, 2014

Pesticide Toxicity?

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Posted by Derek

There's been a report on the toxicity of various pesticides in the literature suggesting that they're far more toxic to human cells than had been thought. My eyebrows went up a bit when I heard this, because these sorts of assays had been done many times before. Then I realized that this was another paper from the Séralini group, and unfortunately, that alone is enough to account for the variance.

Update: commentors to this post have noted that the cell culture conditions used in the paper are rather unusual. Specifically, they're serum-free during the testing period, which puts the cells under stress to begin with. There's also the general problem, which others have brought up, about what it means to dispense these things directly onto cell cultures in diluted DMSO, since that's rather far from how they're going to be presented in the real world. Cell assays get run like that in the drug industry, to be sure, but you've got to be very careful drawing toxicological or other whole-animal conclusions from them. And we already have whole-animal studies on these formulations, don't we? I mean, juiced broccoli straight from the organic farmer's market might well have similar effects under these conditions.

Here's a story from Science with more background. Seralini is the guy who made headlines a couple of years ago with another report that genetically modified corn caused tumors in rodents, but that one was so poorly run and poorly controlled that its conclusions (which have not been seen in any other study) cannot be taken seriously. That's Séralini's problem right there: from all appearances, he's a passionate advocate for his positions, and he appears to be ready to go with whatever results line up with his beliefs. This is human nature, for sure, but science is about trying to work past those parts of human nature. The key is to keep the curious, inquisitive side, and correct for the confirmation bias I-know-I'm-right side. At this point, even if Séralini were to discover something real (and really worth taking seriously), it would have a hard time gaining acceptance, because his previous papers have been so unreliably over-the-top.

I'm not the only person who thinks that. An editor of the journal this latest Seralini paper appeared in has actually resigned because it got published:

When Ralf Reski read the latest paper from controversial French biologist Gilles-Eric Séralini, he quickly decided he wanted nothing to do with it. Séralini’s report in BioMed Research International describes how pesticides kill cultured human cells, with the hair-raising conclusion that pesticides may be vastly more toxic than assumed by regulatory authorities. Some scientists are criticizing the findings as neither surprising nor significant—but they have touched off a firestorm, with environmental groups calling for changes in how pesticides are regulated. That was too much for Reski. Within hours of reading the paper last week, the plant scientist at the University of Freiburg in Germany resigned as an editor of the journal and asked for his name to be removed from its website. "I do not want to be connected to a journal that provides [Séralini] a forum for such kind of agitation," he wrote in his resignation e-mail to the publisher, Hindawi Publishing Corporation.

Should pesticide toxicity be a subject of investigation? Absolutely. Should people be alert to assays that have not been run that should be investigated? Definitely. Are there things that we don't know about pesticide exposure that we should? I would certainly think so. But Séralini's history makes him (scientifically) one of the least effective people to be working on these questions. As a headline-grabber, though, he's pretty efficient. Which I suspect is the real point. If you're sure you're right, any weapon you can pick up is a good one.

Comments (17) + TrackBacks (0) | Category: The Scientific Literature | Toxicology


COMMENTS

1. ppedroso on February 11, 2014 9:19 AM writes...

I have just read the paper diagonally and the problem, it seems, is related to the adjuvants of the pesticides and not to the actual active molecule responsible for the killing.

This is totally regulated concerning drugs and you cannot use an excipient that has not been tested and cleared out as safe. By reading the paper it seems that it isn't so for pesticides, it even mentions that the companies responsible for the pesticides do not have to disclose the adjuvants of the final formulation. Is this true? This does not make any sense but if there is any truth to this I hope that, at least, this paper makes the regulatory agencies to give this problem some thought.

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2. Anonymous on February 11, 2014 9:21 AM writes...

Great, now I'll have to see more posts from Robert Michael Davidson on the ACS LinkedIn page from his TMZ-like blog supporting this horrid scientist.

Séralini’s paper on the pesticides actually showed a decrease in early onset deaths in males fed corn containing 33% GMO, as well as 33% GMO corn with Roundup pesticide in their drinking water. Not to mention (As Derek pointed out how badly the study was) it was about 6 studies done in one with only two controls, and only 10 mice in each experimental group.

He's nothing more than a sensationalist scientist, much like a wide array of journalists who prey on those who will believe anything they hear. Then again, there is a large fraction of academics who are sensationalist as well.

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3. SP on February 11, 2014 9:52 AM writes...

I'm not sure about the cell culture method they use- growing the cells to 80% then removing serum and treating with the compounds in serum-free medium 24 hours, they claim the method was valid for seeing the same effect at 24 hours that you would at 72hr in +serum media, but the validation reference is their own paper in another journal (and I'm not paying $40 to read it.) Could it be that in the absence of serum you have more sensitivity to solvents/detergents? Also, when you're using a pure compound and making a standard DMSO stock it will be ~100% DMSO plus x mM compound. When you're diluting from formulations that are themselves different starting concentrations, your stock solution is going to be x mM compounds plus some random mix of DMSO and the adjuvants depending on what the starting formulation concentration of AP was.
I also don't like his curves- he says he's fitting to a 5-point formula but then plays connect the dots so it's hard to determine an IC50, and he then uses the different points from his curves for the single point assays for apoptosis and membrane permeabilization so it's hard to compare the bar graphs.
But the biggest problem seems to be the reported numbers- the graph of Roundup seems to be a top tested concentration of 10,000 ppm, which is 10 parts per thousand, or 1%. But he says that a 100% DMSO stock is diluted 200x to 0.5% DMSO for cell treatment. I'm curious how you can dilute a stock 200x and end up with a compound being 1% final concentration. Perhaps he's reporting stock concentrations instead of final?
Anyway, his hypothesis isn't unreasonable- adjuvants are added to make things more effective- but he certainly knows how to write an abstract to get attention: "The human placental JEG3 cells appeared to be the most sensitive." Oh noes, human placentas, think of the poor fetuses!

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4. Cellbio on February 11, 2014 10:51 AM writes...

Serum free culture moves the cells closer to reading out positive in apoptosis assays, be they metabolic measures or caspase activity. Whether this is valid for predicting toxicity is an open question and likely a compound specific question, I would guess related to compound aggregation and partitioning (non-specific membrane effects) and MOA of toxicity.

Personally, I think these sorts of assays are useful only in supporting the pharmacology associated with cultured cell responses and not predictive of compound toxicity in vivo where compound exposure is not accompanied by DMSO and not under starvation culture conditions.

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5. B on February 11, 2014 11:27 AM writes...

@3, on dilutions:

That doesn't strike me as impossible, just sloppy mixing of "%" as weight/volume and volume/volume. You could have 1 mL of a solution containing (say) 2 g of compound and 0.5 g of solvent, to make up some numbers. Dilute this v/v to 0.5%, and the w/v is 1%.

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6. Anonymous on February 11, 2014 11:50 AM writes...

As was mentioned elsewhere, what is the point of introducing these actives directly onto cells. The only way they will get into our cells is to be ingested or inhaled, unless you feel like injecting them into your bloodstream.

Also, any inert ingredient that goes into an agrochemical formulation does have to be approved by the EPA and be listed on 40CFR (Part 180). They have no idea what they are talking about.

They also failed to mention all of the other surfactants and additives that are present in those formulations. Each one probably has 2-5 different emuslifiers, dispersants, rheology modifiers, etc.

I would love to see a counter to this study done with food extracts or sodas.

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7. SP on February 11, 2014 11:54 AM writes...

"2 g of compound and 0.5 g of solvent"
That sounds like a joy to dispense using a pipettor.

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8. PPedroso on February 11, 2014 11:56 AM writes...

@Anonymous 6.

"Also, any inert ingredient that goes into an agrochemical formulation does have to be approved by the EPA and be listed on 40CFR (Part 180). They have no idea what they are talking about."

Thanks for bringing some reassurance to my world.

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9. Justin Peukon on February 11, 2014 12:32 PM writes...

mmhhh... Hindawi.

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10. Anonymous on February 11, 2014 12:48 PM writes...

@8 - as Derek mentions sometimes about pharma, (paraphrasing) "its not a perfect system, but its the system we have." The same goes for the EPA (specifically, their approval process for agrochemicals and their inert ingredients). There is a system in place that is far from perfect, but at least its something. I'd be the first to admit that I don't have a ton of confidence in the people reviewing inert ingredient applications. But, 50 years ago people were spraying organo-arsenic compounds all over their crops (and our food), so we are in a better place because of some regulations.

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11. JAB on February 11, 2014 1:08 PM writes...

And guess what, surfactants are going to be active in cell culture at modest concentrations! We used to spend time avoiding saponins from plants in cell culture - many of them lyse cell membranes - but rapidly, in hours, which is how we could identify and ignore them.

I'm more worried about pesticides in bees, where the neonicotinoids don't kill at low doses, but they apparently confuse the bees sufficiently that they can't forage effectively. That's more subtle but important nonetheless! Also overall effects on soil ecology when large expanses of farmland are saturated with herbicides.

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12. Anonymous on February 11, 2014 4:30 PM writes...

@5: That would imply a solid density of 2g/ml, which seems quite high for most organic compounds. Not sure if this fits the compounds studied here?

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13. SP on February 11, 2014 7:28 PM writes...

We can speculate on mostly-solid stock solutions, but let's be honest- most people in screening (except fragments) start with 10 mM stocks, which is 1.69 mg of glyphosate per mL of DMSO solution. Let's be generous and assume he can make a 1M stock, or 169 mg per mL. That's still roughly 13-16% depending if you use w/w or w/v. Basically there's no way he made a DMSO stock that was then diluted 200x to 10,000 ppm. The numbers on the graph are clearly a mistake and minimally require a correction.

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14. Anonymous CP on February 12, 2014 10:29 AM writes...

I read the article and there are a number of flaws to the study. The most important being that there is no mention that it is not the "pure" formulations that are sprayed by the farmer. Take imidacloprid for example as as a 200SC (200g/L) formulation. The label guidelines are, that for most applications 25ml of this formulation would be diluted with 100L of water before spraying......quite a big oversight?!

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15. Lane Simonian on February 13, 2014 11:47 AM writes...

This is from an earlier Seralini (2008) paper and he may have gotten this one right in regards to Roundup.

Thus, POEA (polyethyloxylated tallow amine--a surfactant) could be considered as the active ingredient on human cell death and more damaging than G (glyphosate).

And a very recent study leading further credence to this finding.

Int J Toxicol. 2014 Jan 16. [Epub ahead of print]
Glyphosate Commercial Formulation Causes Cytotoxicity, Oxidative Effects, and Apoptosis on Human Cells: Differences With its Active Ingredient.
Chaufan G, Coalova I, Molina MD.
Author information

Abstract
In the present study, the effects on oxidative balance and cellular end points of glyphosate, aminomethylphosphonic acid (AMPA), and a glyphosate formulation (G formulation) were examined in HepG2 cell line, at dilution levels far below agricultural recommendations. Our results show that G formulation had toxic effects while no effects were found with acid glyphosate and AMPA treatments. Glyphosate formulation exposure produced an increase in reactive oxygen species, nitrotyrosine formation, superoxide dismutase activity, and glutathione (GSH) levels, while no effects were observed for catalase and GSH-S-transferase activities. Also, G formulation triggered caspase 3/7 activation and hence induced apoptosis pathway in this cell line. Aminomethylphosphonic acid exposure produced an increase in GSH levels while no differences were observed in other antioxidant parameters. No effects were observed when the cells were exposed to acid glyphosate. These results confirm that G formulations have adjuvants working together with the active ingredient and causing toxic effects that are not seen with acid glyphosate.

Or from a clearly written summary of this study:

To further investigate the mechanism of cytotoxicity the group sought to measure reactive oxygen species (ROS) production. They found that G formulation increased ROS production in HepG2 cells, but that glyphosate and AMPA did not. To identify whether nitrosative stress was involved the researchers measured peroxynitrite formation and found that G formulation resulted in a significant increase in nitrated proteins. Neither glyphosate or AMPA resulted in an increase in peroxynitrite formation. Finally the group showed that G formulation treatment lead to an increase in caspase 6/7 activity, while glyphosate and AMPA treatment did not.

So it may be glyphosate working in concert with POEA that explains the potential toxicity of Roundup.

The acute effects of heavy exposures to certain pesticides and herbicides is well-known, but the chronic effects of lesser long-term exposures are only slowly coming to light (see recent study on DDT and Alzheimer's disease).

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16. Davet on February 13, 2014 5:00 PM writes...

The use of "pesticide" plays upon people's ignorance about the term - that they are evil chemicals. Virtually anything can be a pesticide: garlic is a registered pesticide. Caffeine is as well. Disinfectants also. Water purification and pool chemicals are pesticides. Simple cloth matress covers for beg bugs are also registered pesticides.

The most ironic was a wash introduced to remove pesticides from fruit. It also claimed to remove bacteria, which is a pesticidal claim, so the company had to register it as a pesticide. So they were selling a pesticide wash off pesticides.

JAB-Environmental assessments are a significant part of pesticide approvals. As noted, in the old days, arsenicals used as insecticides and herbicides did build up in the soil (pre-DDT lead arsenate was the top insecticide on crops). However, modern herbicides don't have this residence time.

The only modern equivalent to the above I know is that organic farmers in California used heavy metal seed treatments such as lead chromate, until EPA banned them in the 1990s. There are naturally occuring sources of lead chromate, so organic farmers naively thought "natural"= safe.

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17. eugene on February 15, 2014 6:22 AM writes...

Derek, there is a very interesting article on pesticides and suppression of research in the New Yorker. I was wondering if you could do a take on it?

www.newyorker.com/reporting/2014/02/10/140210fa_fact_aviv?currentPage=all

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