EWG gives many many reasons why they think you should use the guide, specifying that you (the consumer) should eat organic or at least choose the Clean 15™ over the  Dirty Dozen™:

The 12 most contaminated fruits and vegetables (the “Dirty Dozen”) are contaminated with an average of 10 different pesticides, with many tainting more than one type of produce. In contrast, the “Clean 15,” the 15 least contaminated fruits and vegetables, contain an average of less than 2. Eating organic food lowers pesticide body burdens as well. Research shows that concentrations of pesticides in children’s bodies peak during seasons that they eat the most produce, but fall to below detectable levels in just 5 days when they eat organic food.

The list of reasons has a lot of scary facts about how many pesticides detected on food, just how “polluted” our bodies are from the things we eat, and explains how our government barely regulates pesticides. Near the bottom, EWG lets us know that despite the scary facts that the need to eat fresh produce outweighs any risk from pesticide residues. They also remind consumers of the importance of eating fresh produce on their FAQ page. Unfortunately, I’m not sure if anyone gets to that part, considering that media coverage of the Shopper’s Guide rarely mentions it, instead focusing on the scary facts (as in ‘Dirty dozen’ produce carries more pesticide residue, group says on CNN Health, which dismisses the silly government for thinking that small amounts of pesticides won’t hurt us).

The truth is, pesticides are scary. As EWG’s Amy Rosenthal says, “Pesticides are designed to kill things.”

The devil, as always, is in the details.

We need the EWG

Before we get into those details, I’d like to say a few things about the Environmental Working Group in general, or really any group that does what EWG tries to do. EWG has the ability to provide a very important benefit to society. Government spending on science has decreased over the years, leaving most toxicity research to the companies that make the products being tested. Until we follow the wise leadership of India and develop a network of government certified independent testing labs, we’re all kind of left with less information than I’d prefer for many products we use every day. It’s not that I think every corporation is driven by people who choose profits over safety (on the contrary, they have to at least think their products are safe or suffer bad press or worse if people get sick) but results of corporate funded tests are often not made available to the public which leaves regulators with less info than they need to make good science-based decisions. Our system works fairly well (the grand majority of people get through life without health problems caused by things they can’t control other than their own genetics*) but it could always be better. EWG works to get information to regulators and presents a non-industry point of view, which is much needed. Unfortunately, despite their outwardly awesome intentions, some of the results are less than awesome.

Details, details

Danger, elephants. Taken by Adam Foster at Knowsley Safari Park in England. via Flickr.

In the materials accompanying the Shopper’s Guide, there are two details that are never discussed.

The first elephant in the room is dose. For any compound, from water to arsenic to ricin to organophosphates, there are amounts that are safe and amounts that are hazardous. There are amounts that will cause acute (immediate) reactions and amounts that will cause chronic problems after long term exposure. Are the amounts of pesticides found on produce enough to cause acute or chronic health problems? The EWG list does consider amount, but does not compare the amounts to EPA guidelines. The accompanying materials focus on the number of pesticides, not the dose.

The second elephant is the type of pesticides that were found on produce. There isn’t any weighting in the Shopper’s Guide of individual pesticides based on relative toxicity. This could be a problem because not all pesticides are created equal. Organophosphates, for example, are extremely dangerous because they affect cholinesterase, an enzyme that is essential for the human nervous system. Glyphosate, on the other hand, affects EPSPS, an enzyme that is only found in plants so human toxicity is low (surfactants and other ingredients in glyphosate containing herbicides may be dangerous in their own right, but EWG to my knowledge isn’t talking about those types of ingredients).

Careful consideration of dose and toxicity of pesticides on produce may mean a reordering of the list is necessary in order to truly keep consumers safe. It may also mean that many of the scary facts need some sober facts alongside to help us keep things in perspective. Let’s look at the  methods that EWG used to make the list and at the original USDA data.

EWG’s Methods

I have to tip my hat to EWG for providing their methods on their website. I don’t know how many people look at it, but I certainly did! They provide justifications for not discussing dose or type of pesticide:

The goal is to include a range of different measures of pesticide contamination to account for uncertainties in the science. All categories were treated equally; for example, a pesticide linked to cancer is counted the same as a pesticide linked to brain and nervous system toxicity, and the likelihood of eating multiple pesticides on a single food is given the same weight as the amounts of the pesticide detected or the percent of the crop on which pesticides were found.

The problem is that, as strange as it may sound, there are safe amounts of pesticides. With the incredibly low detection limits that advanced methods provide us, we can expect many positive results that aren’t biologically significant. This is why the EPA bothers to determine tolerance limits for each pesticide (see below: The Data). The EWG continues:

The EWG’s Shopper’s Guide is not built on a complex assessment of pesticide risks but instead reflects the overall pesticide loads of common fruits and vegetables. This approach best captures the uncertainties of the risks of pesticide exposure and gives shoppers confidence that when they follow the guide they are buying foods with consistently lower overall levels of pesticide contamination.

In other words, science-based risk assessment is bad because it’s complex? A less complex and unscientific method gives consumers more confidence than a science-based method? Perhaps, but this explanation of the method is a little too close to fibbing for my taste. Maybe we need to look deeper.

EWG looked at contamination in 6 different ways:

• “Percent of samples tested with detectable pesticides.” Assuming that the data was used properly, this is a good metric. It tells us how many of all the samples within a category had pesticide residues.
• “Percent of samples with two or more pesticides.” This metric might be useful if we are concerned about potential effects of consuming more than one pesticide.
• “Average number of pesticides found on a single sample.” This isn’t as useful as a median number of pesticides could be. If most of the samples contain 0 pesticides, the average would be lower than the median. If only one of the samples contains a very large number of pesticides, the average would be artificially high.
• “Average amount (level in parts per million) of all pesticides found.” Here’s where the science gets thrown out. The type of pesticide isn’t considered even though we know that some pesticides are dangerous at low doses while other pesticides are safe at much higher doses. The ppm of different pesticides should not be averaged unless they have similar toxic doses. No where on the Shopper’s Guide site  is there a discussion of how the pesticide levels found in produce match up to EPA guidelines, or how those guidelines are created (in most cases the guidelines from the EPA are at least 10 times lower than the actual dangerous dose).
• “Maximum number of pesticides found on a single sample.” This isn’t very useful either. Perhaps one sample was grown by a particularly zealous farmer who used more pesticides than she should. Perhaps the single sample was accidentally contaminated. Should the entire category of produce be condemned because of this single sample, out of hundreds of samples? Using the media number of pesticides for all of the samples make much more sense.
• “Total number of pesticides found on the commodity.” Again, this number could be based on one or a few samples which are not representative of all of the samples.

The Data

High speed capture of dye droplets by Derek Purdy. via Flickr.

Since 1991, the Agricultural Marketing Service (part of the USDA) has collected data on pesticide residues in food as part of the Pesticide Data Program (PDP) using pretty rigorous methods (pdf). In addition to this testing, the FDA tests domestic and imported food to ensure that pesticide residues are below the tolerance levels (FDA probably doesn’t test enough samples due to funding cuts but that’s another post). The results are compared to tolerance levels (maximum pesticide residue limits) that are set by the EPA (you can find the tolerance for each crop/pesticide/country combo at Maximum Residue Levels database). According to the Latest PDP Findings of Interest to Consumers (pdf), ”the vast majority of samples tested are well below the tolerance levels”. Specifically:

The USDA provides some comparisons to help us understand what 1 part per million is: 1 ounce of salt in a mountain of 62,500 pounds of sugar or 1 ounce of dye in 7,350 gallons of water.

The most recent Annual Summary of the PDP (pdf) contains data that was collected in 2008 and was released in December 2009. The Executive Summary tells us that 11,960 samples were analyzed, including fresh and processed fruit and vegetables (9,028 and 1,354 samples respectively), almonds, honey, corn, and rice (municipal drinking water is also tested). The positive pesticide residue detections were combined by food type; on average 1.6% of samples had positive residue detections. For fresh produce, positive samples ranged from 0 to 3.3% with an average of 1.9%. They go on to say:

For samples containing residues, the vast majority of the detections were well below established tolerances and/or action levels. Before allowing the use of a pesticide on food crops, EPA sets a tolerance, or maximum residue limit, which is the amount of pesticide residue allowed to remain in or on each treated food commodity. Established tolerances are listed in the Code of Federal Regulations, Title 40, Part 180. In setting the tolerance, EPA must make a safety nding that the pesticide can be used with “reasonable certainty of no harm” and that residues at (or below) the tolerance are safe. The reporting of residues present at levels below the established tolerance serves to ensure and verify the safety of the Nation’s food supply.

To restate, the methods used to detect pesticides are very sensitive, but a positive sample does not indicate a problem unless the detected level is above the established tolerance level. “A tolerance violation occurs when a residue is found that exceeds the tolerance level or when a residue is found for which there is no established tolerance.”

There were 60 samples that exceeded tolerance levels, making up 0.5% of all the samples (58 with 1 residue exceeding the tolerance and 2 with 2). There were 442 samples that had pesticide residues that don’t have established tolerance levels, making up 3.7% of all the samples (one reason why there isn’t an established tolerance level is that the pesticide in question isn’t labeled for use on the specific crop being tested). “In most cases, these residues were detected at very low levels and some residues may have resulted from spray drift or crop rotations.” Starting on page 51 of 202, the results are presented in a table the includes the number of samples tested, the number of positive samples by pesticide type, the amount of pesticide detected, and the EPA tolerance for that pesticide. I encourage you to see the report for all the details. The actual data can be downloaded from the Agriculture Marketing Service, although sadly it isn’t in any sort of convenient format (I’m wrestling with the data right now).

Peaches

There do seem to be some discrepancies between what EWG says the USDA data says and what the USDA data says.

The EWG says “more than 96 percent of peaches tested positive for pesticides”, and “peaches had been treated with more pesticides than any other produce, registering combinations of up to 67 different chemicals.” That sounds pretty bad.

Table 3 of the 2008 USDA report lists the “Number of Samples Analyzed and Summary of Results per Commodity” (page 34). According to this table, 616 peach samples were analyzed, with an average number of 130 different analyses conducted on each individual sample, resulting in a total of 80,184 tests done on the 616 peach samples. Of these tests, 2,155 were positive for pesticide residues, and 52 different pesticides were detected. While the number of positive detections out of all the tests isn’t the same as the number of positive samples out of all the samples, it is still interesting to know that only 2.7% of all the tests conducted on peaches were positive.

52 isn’t 67. 2.7% isn’t 96%. What’s happening here?

EWG didn’t use the most recent data. Instead, they seem to have combined data from 2000 to 2008. That seems very strange to me, considering that EPA regulations for allowed pesticide use and allowed pesticide tolerances have been changing over the years, becoming more strict. At least they didn’t include pre-2000 data, but still this isn’t the best way to find the information that consumers want. We need to know how many fruits and vegetables today are positive for pesticides, not all the fruits and vegetables in the past decade.

Even when we consider the fact that the EWG isn’t working with the best dataset, that still doesn’t answer how they decided that more than 96% of peaches were positive for pesticides. Hopefully the answer will be clear once I’ve looked at the USDA data myself.

If not scary “facts”, then what?

I am definitely an advocate of using science-based approaches to farming that reduce input use overall, and of careful Integrated Pest Management strategies that use the safest possible solutions to any pest problem, only using inputs if other options have been unsuccessful, and using the safest possible pesticide whether that pesticide is natural or synthetic.

How do we encourage government to introduce regulation that will make this happen and how do we encourage consumers to care about this enough to talk to their elected officials?

The best course of action would be to present the information in a less agenda driven way. Provide the data along with the EPA guidelines, which would show that the great majority of produce is well within guidelines. There are ways to advocate for reduced pesticide use without alarming people unnecessarily.

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* In the developed world, health problems caused by our own choices (bad nutrition, lack of exercise, smoking, and so on) dwarfs any problems that might be caused by normal use of household chemicals, plastics, foods, etc.

Note: A group called Alliance for Food and Farming, called an “industry front group” by EWG has challenged the Shopper’s Guide, saying that it unnecessarily alarms consumers. I have not read any materials from AFF on this subject prior to writing this post to be sure that my comments were not based even subconsciously on their comments. I heard about the AFF response through the Iowa State Sustainable Agriculture Listserv, which led me to write a few responses about the Shopper’s Guide to the original poster which then were turned into this post. This year’s Shopper’s Guide came out in June 2010.

An Arabidopsis stomate showing two guard cells exhibiting green fluorescent protein and native chloroplast (red) fluorescence. via Wikipedia.

This image is an extreme closeup of a stomate (singular, the plural form is stomata). These two cells, called guard cells, control the plant’s respiration: how much carbon dioxide gets in and how much oxygen and water vapor gets out. The control isn’t very good, though. Most plants just have their stomata open all day every day so they can pull in lots of CO2 to use during photosynthesis to make sugar. And that means a lot of water, painstakingly pulled up from the soil, through the roots, gets lost. If stomata could be more selective, only opening when more CO2 was needed for photosynthesis, then water could be conserved.

An enzyme called carbonic anhydrase raises the levels of CO2 in chloroplasts so the plant can make plenty of sugar. It does this by converting CO2 from its storage form carbonic acid back to it’s useable form: CO2 + H2O ⇌ H2CO3.

Carbonic anhydrase also appears in the guard cells, where it controls the opening and closing of stomata.

Julian Schroeder, Professor of Biology at UC, San Diego hypothesized that more carbonic anhydrase in the guard cells would place tighter control over opening and closing. His group tried shutting off the carbonic anhydrase gene in the stomata of a little plant called Arabidopsis. Those plants were unable to respond to increased CO2 concentrations in the air, remaining open all day. They also tried expressing additional copies of the carbonic anhydrase gene in the stomata. Those plants closed their stomata when water was scarce. This makes sense – carbonic anhdrase needs water to function, so it can’t function when water’s not around.

Honghong Hu, a postdoctoral research working on the project, said in the press release Newly Identified Enzymes Help Plants Sense and Respond to Elevated Carbon Dioxide and Could Lead to Water-wise Crops: “The guard cells respond to CO2 more vigorously. For every molecule of CO2 they take in, they lose 44 percent less water.”

This research, Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells, published in January 2010, indicates that increasing the number of carbonic anhydrase genes in the stomata could potentially decrease the water lost through stomata in crops. The implications for drought prone regions are obvious. Plants could need less water and could hold on to the water they have longer. It won’t be plug and play, though. As stated in the press release, water that evaporates from stomata cools the plants just like water evaporating from our pores cools us. Increased expression of carbonic anhydrase will have to be tested to determine its effects on plants in high temperature environments.

Hu H, Boisson-Dernier A, Israelsson-Nordström M, Böhmer M, Xue S, Ries A, Godoski J, Kuhn JM, & Schroeder JI (2010). Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells. Nature cell biology, 12 (1) PMID: 20010812

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Thanks to @RivenCactus for bringing this research to my attention by Tweeting a link to the TreeHugger article Newly Discovered Enzyme Could Create Crops That Thrive in Dry, High CO2 Conditions.

If a gene like this was used to make crops more drought tolerant, could it spread to weeds and make weeds weedier?

Yes and no.

If there was a sexually compatible wild relative or weed species growing nearby the drought tolerant crop, it is possible that weed/crop hybrids could include the gene. Sexual compatibility means that the weed not only has to be a fairly close relative to the crop but also means that they have to be pollinated by the same method, have pollen shed at the same time, not have any incompatibility genes, etc. In the United States, there are few weed species that are sexually compatible with crop species, but there are some. In these cases, farmers can use the same sort of strategies to reduce gene flow that they would use to avoid spread of a conventionally bred trait.

If gene flow does happen, the gene will only be present in the weed population at low levels, unless the gene makes the weeds that have it able to outcompete weeds that don’t have it. See Escape! Crop-Specific Gene Flow to Wild Relatives and Those naughty plants! on Biofortified for more discussion of gene flow.

In Proposed US law to mandate GMOs?, I posted the actual text of the The Global Food Security Act of 2009, S.384, introduced by Senators Richard Lugar (R-IN) and Robert Casey (D-PA), in response to authors of blog posts and petitions that didn’t quite seem to have read it before getting all excited about it.

The next big GMO scandal involves recommended changes to the Codex Alimentarius Commission of the Joint FAO/WHO Food Standards Programme. All relevant documents have been posted by the Codex Committee on Food Labeling (CCFL), apparently unknown to those who would have us up in alarm.

The Institute for Responsible Technology (founded by Jeffery Smith) wants us to pay attention to their Action Alert – Codex Conference (emphasis original):

Please send this URGENT message to US Government leaders to protect your right to know which foods are made from genetically modified organisms (GMOs)…

They must stop US negotiators at an international (Codex) conference from May 3-7, from pushing an agenda that could make it difficult for anyone, anywhere in the world to label foods as genetically modified (GM) food—or even make non-GMO claims on their product’s label.

A petition on CREDO Action Network is even more alarming (emphasis mine):

…the current U.S. draft position paper declares that mandatory labeling laws such as they have in Europe are “false, misleading or deceptive.” If the U.S. succeeds in writing the proposed Codex regulations, any attempts here in the U.S. to label foods as genetically engineered, whether voluntary or by law, would become far more difficult.

What’s actually “misleading or deceptive” is the way the US recommendations are presented by these two groups. Ironically, the US actually seems to be recommending most recently that nothing be changed at all, in Government Comments at Step 3 (pdf). The US recommendations, presented 3-7 May 2010, as “Proposed draft recommendations for the labelling of foods and food ingredients obtained through certain techniques of genetic modification/genetic engineering”, are as follows:

We strongly encourage CCFL to discontinue further discussion of the provisions in Appendix VII of ALINORM 09/32/22 so that the Committee may focus its resources on the agenda items dealing with the implementation of the WHO Global Strategy on Diet, Physical Activity, and Health, an agenda item of immense public health significance and directly related to the mandate of Codex—to protect the health of consumers.

Why would the US want the Codex Committee to stop working on plans to make rules for mandatory labeling of GMOs? In short, member countries have such different regulatory frameworks and such different ideas that it’s unlikely that any consensus will be reached. In fact, no consensus has been reached in over a decade of discussion on this topic, and by Codex rules, where there is no basis for consensus, discussion should end. This is laid out in full in the above referenced document. Other countries and some other groups put out Comments at Step 3, which you can find at the Codex Committee on Food Labeling website. Perhaps it is inappropriate for the US to propose that discussion be stopped, but stopping the discussion is far from being the same as banning labeling of GMOs.

Did the US ever propose that voluntary labels be prohibited? Nope. They did advocate that labels ”indicate that foods derived from GM/GE were not in any way different or less safe due to their method of production provided that they had undergone safety assessments consistent with relevant Codex guidelines.” This is consistent with other labeling requirements in the CODEX and other labeling sources. A voluntary label that meets this requirement would be similar to rBST labels in the US that state that milk from cows that have not been treated with rBST is no different than milk from cows that have been treated with rBST.

The Spring 2010 entries have been narrowed down to eight finalists – one of which was submitted by yours truly. GMOs could render important antibiotics worthless is consistently one of Biofortified’s most read posts since it went up on March 15.

I hope you’ll read the entries and consider voting for any of these contributions from graduate students. Voting is open from April 1 to April 20. Each person can vote one time every day, using the button to the right of the post’s title. Click the banner below to see a list of all of the finalists.

Rat by Big Fat Rat via Flickr.

There were quite a few articles debunking the problematic study. A great source is David Tribe’s post The curious incident of silence about mistreated animals that showed how animals died in this study (in all groups, including the control) at far higher rates than is typical in rodent feeding studies. The Austrian researchers may not have had proper living conditions for their charges.

They also seem to have had some problems with simple mathematical calculations, as shown in Review of Zentek paper by James Lamb, an independent reviewer who was asked to look at the paper by Monsanto.

The German government site GMO Safety questioned the study in Does GM maize cause impotence? EFSA experts voice doubts and “GM maize causes impotence” Or: How a scientific study is used for political motives.

“Prof. Zentek himself declares his study as a preliminary draft which needs to be scrutinized and only partially delivers conclusive results.” according to the article Did the Monsanto Hybrid Transgenic Maize lower the fertility of mice in a multi generation feeding experiment? by the Public Research and Regulation Initiative (among other articles).

Not deterred by the critical analysis of scientists, Greenpeace and other activist groups led the charge with headlines like Mice! Forget about birth control – try GE maize instead! and “news” sites like Grist posted headlines like GM-OH, NO! Long-term study: GMOs lower fertility in mice. It’s a shame that they didn’t bother to investigate further before fearmongering.

The Austrian government was happy to ignore the critical reviews as well, but other officials in the EU were more cautious, calling for a review of this science-by-press-release. That review is complete, and Austria has withdrawn the study from consideration as to the safety of genetically modified crops.

Details of Austria’s withdrawl of the study as well as some background can be found in Austria withdraws study on the long-term consequences of GM maize on GMO Compass.

Margaret Clarke researches the soil amoeba Dictyostelium discoideum using biotechnology. Dr. Clarke is officially retired, but as a dedicated scientist, she’s continuing her work. She visited Iowa State yesterday and today.

Phagocytosis of a bacterium by an immune cell (on a tee shirt!) by Zazzle.

In particular, Dr. Clarke studies phagocytosis – literally “cell eating”. These amoeba are single celled organisms that eat bacteria (and just about any bacteria-sized particle that might be nutritious). Phagocytosis is the process of forming a cup that engulfs the prey, drawing the prey into the phagocyte, and digesting the prey.

Her work has important applications in human medicine, as the phagocytosis process takes place in special phagocytic cells that are part of the immune system of humans and other animals. Learning how phagocytosis works in amoeba can help us to understand how it works in the immune system.

Before biotechnology came around, Dr. Clarke used biochemical analysis to determine what sorts of compounds were at work in actively phagocytising cells. She was able to find out that both actin and myosin were present. While this was important information (people used to think myosin was only present in muscle tissue), she wasn’t able to get any information about exactly where, when, and how these proteins were produced in the phagocytosis process – especially since it happens so quickly!

Finally, about 15 years ago, people started using biotechnology to label proteins in living cells with fluorescent proteins. Dr. Clarke saw that she could use these fluorescent labels to actually see where, when, and how actin and other compounds accumulated and dissapated in amoeba that were phagocytising. Dr. Clarke labeled different proteins with either red or green fluorescent proteins, learning much about the phagocytosis process. Even better – she caught them on film!

The images above are from Dr. Clarke’s 2006 paper Phagocyte meets prey: Uptake, internalization, and killing of bacteria by Dictyostelium amoebae. This transgenic amoeba are expressing RFP-LimEΔ, which is the gene for red fluorescent protein joined to a gene for a protein that binds to actin. Where ever actin is expressed, the amoeba will be a brighter red. It is eating GFP labeled E. coli.

As you can see, Dictyostelium are greedy little guys! They’ll try to eat just about anything, including things that are too large for them, like this amoeba trying to eat a yeast cell to the right (for the video of this frustrated little guy, see Honorable Mention #5 at the Olympus Bioscapes 2009 competition).

Development of Dictyostelium by M. Grimson and R. Blanton of Texas Tech University.

In addition to its awesome phagocytosis techniques, Dictyostelium has an amazing life cycle. When food becomes scarce, the amoeba will put out cAMP,a chemical that causes them to aggregate. About 100,000 individual amoeba group together and start to form what’s called a “migrating slug.” The “slug” will move through the soil toward the surface where it will develop into a fruiting body and eventually put out spores that will become single celled amoeba again.

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Clarke M, & Maddera L (2006). Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae. European journal of cell biology, 85 (9-10), 1001-10 PMID: 16782228

Of course, patents and other forms of intellectual property protection (IPP) do have their limitations. Right now, IPP may be at once too strict and too lax.

IPP is too strict in that patent holders must pursue violators of their patents, or risk loosing the patent. In other words, to follow the law, patent holders must go after even the smallest violators. This may be a waste of time for the patent holder and penalizes small patent violators who can’t afford a court case. The requirement is necessary, though, an attempt to prevent a company from sitting on a patent but not doing anything with it. One possible solution would be to make IPP more lax by creating a bottom threshold limit of the income a violator is making from the protected IP. In the case of agriculture, small farmers who willingly or accidentally violate biotech patents or plant variety protection would only be subject to a suit if their income or benefit from the IP is above a certain amount.

IPP is too lax in that there are situations where patents can be violated but the patent holder has little recompense. In agriculture, this could happen if one company used another company’s germplasm in their breeding program, although this has become more and more rare as markers have started being used to identify members of a plant’s lineage. Another example from agriculture is protected methods that a rival company might be able to use without anyone knowing.

This idea of too lax IPP came up in conversation about how to help African countries develop their own seed production facilities. Companies like Pioneer have donated germplasm and traits royalty free for use in breeding programs in Africa. Their goals here are partially altruistic – they can help people while having only a tiny effect on their own bottom line – and partially self-interested – farmers who can start making money today may be customers tomorrow.

The donor companies have legitimate concerns that existing or startup companies could take those traits and germplasm and develop new products that would eventually compete with the patent holders. As I said, this is less of a concern than it used to be because markers can increasingly be used to determine the varieties that were used to develop a new variety. It might not be so easy to track a patent protected method that was used to develop new varieties.

Marc Albertsen of Pioneer Hi-Bred International gave an exciting talk today about a new way to develop male sterile plants for hybrid production that doesn’t involve cytoplasmic male sterility (look forward to a post about it soon!). Pioneer might be interested in donating this technology to researchers developing improved crops for Africa, but with no way to track the method, they might fear that another company could access and their hard work without paying for it. I’m not sure what would be potential solutions to this problem, but potentially a more strict IPP for companies that willfully use another company’s work could be an answer.

Too lax, too strict – what do you think?

Antibiotics by AJC1 via Flickr.

We’ve seen such claims made in popular media such as the March 2010 Fury as EU approves GM potato: Critics claim plant could spread antibiotic-resistant diseases to humans in the Independent: “Opponents fear bacteria inside the guts of animals fed the GM potato – which can cause human diseases – may develop resistance to antibiotics.” Groups that actively work against deregulation of genetically engineered crops have been making such claims for years.

We’ve also seen these claims in peer-reviewed journals (although, far less frequently than in non-peer reviewed media and reports). For example, in the February 2009 issue of Critical Reviews in Food Science and Nutrition, the review Health Risks of Genetically Modified Foods: “An area of concern focuses on the possibility that antibiotic resistance genes used as markers in transgenic crops may be horizontally transferred to pathogenic gut bacteria, thereby reducing the effectiveness of antimicrobial therapy.”

Are antibiotic marker genes in genetically engineered crops really a risk to human health? Many people have raised this question and there seems to be a lot of confusion about the issue. It’s time to look into the risks and reasons more deeply.

What are antibiotic resistance genes for?

Growing plants up from cells. By Mirkov via Michigan State University.

In order to understand why these genes are used, we have to look a little at the process of genetic engineering. For some plant types, including corn and rice, immature seeds are dissected to expose the developing embryo. Pieces of carrot roots can be transformed, as can the leaves of tobacco. The desired genes are transferred into the plant cells with either a gene gun or Agrobacterium. The plant cells are then grown up into whole plants in petri dishes, with the help of plant hormones. The process is similar to other asexual plant propagation techniques, but much smaller!

Not every cell receives the gene of interest, however, so researchers need a way to find the cells that have it. Enter antibiotic resistance genes. If the cells are transformed with the gene of interest and an antibiotic resistance gene, the appropriate antibiotic can be added to the media in the petri dish so that any cells that didn’t get the genes will die. The antibiotic resistance gene is being used as a selectable marker, since it allows the researcher to select only the desired cells.

Of course, just because these genes are useful doesn’t mean that they are safe or that they should be used. What does the research tell us?

Risk: Gene transfer from plants to bacteria

Soil bacteria by Michigan State University.

Fear of antibiotic resistance markers is mainly due to fear of gene transfer from genetically modified plants to bacteria in the soil or bacteria in human or animal guts. There are at least two reasons why this fear is unwarranted. First, soil and gut bacteria naturally contain a variety of antibiotic resistance genes without any human intervention. Second, transfer of genes from a plant to a bacterium is extremely unlikely.

Natural antibiotic resistance

Life for a bacterium isn’t easy. They have to compete fiercely for resources, so it’s not surprising that some bacteria have evolved to produce poison that kills their competitors: antibiotics. The producers of these antibiotics also evolved antibiotic resistance mechanisms so they could survive their own weapons. Additionally, bacteria develop resistance to antibacterial compounds in the environment.

Often, antibiotic resistance is conferred by a single gene. Any bacteria that can find that resistance gene and use it have an advantage. Consequently, antibiotic resistance genes are widespread in natural environments. When humans intervene, using antibiotics in ways that encourage development of resistance in bacteria, that resistance is passed around even faster (no GMOs needed). For some information on how humans, check out the CDC’s pages on antibiotic resistance.

Gene swapping

Genetically modified crops: methodology, benefits, regulation and public concerns, a 2000 review in the British Medical Bulletin has a summary of the risks of horizontal gene transfer from genetically modified crops:

…horizontal transfer of a gene from ingested plant material to bacteria has never been demonstrated, and there is no indication that it has ever occurred during evolution. The probability that it could occur is, therefore, considered to be so low that it is not relevant when compared with the natural occurrence of antibiotic resistance genes.

They sound awfully confident, don’t they?

Bacteria (prokaryotes) are fairly promiscuous when it comes to genes. Many types of bacteria (not all) have the ability to take up DNA from the environment and from other bacteria and integrate it into their own genome during parts or all of their life cycle. This process is called horizontal gene transfer, and the bacteria that have this ability are called competent. Since they have this ability, it makes sense to worry about bacteria picking up antibiotic resistance genes (and other genes as well) from other organisms, including genetically modified crops.

Interestingly, eukaryotes (multicellular organisms like plants and people) also have the ability to take up DNA into their genomes. For example, the August 2007 Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes shows transfer of the entire genome from endosymbiotic bacteria into their hosts’ genomes. A more recent example appeared in January 2010 in the New York Times: Hunting Fossil Viruses in Human DNA. An entire virus genome resides in the human genome, and has been passed down from our simian ancestors.

So we know that bacteria can swap DNA and that eukaryotes can take up DNA from bacteria and viruses. Can prokaryotes take up DNA from eukaryotes?

It doesn’t look like it.

The European Food Safety Authority has the following to say about the subject in their excellent 2009 Statement of EFSA on the consolidated presentation of opinions on the use of antibiotic resistance genes as marker genes in genetically modified plants (section 2.1.1.2., edited slightly for clarity):

While many studies support the evolutionary significance of horizontal gene transfer between bacteria, eukaryotic genes in prokaryotic genomes are a rarity. There is no definitive report of DNA transfer from eukaryotes to bacteria.

As of 24 September 2008 the public genome databases included more than 750 completed prokaryotic genomes. In the first annotation of the putative genes there are frequent cases where closest matches are found with eukaryotic genes, but these preliminary results have not manifested into demonstrations of horizontal gene transfer from eukaryotes to prokaryotes, as judged by the scientific publications interpreting the genomic sequencing data. For one functional gene (phosphoglucose isomerase), phylogenetic analyses indicated that the gene might have been transferred from a eukaryote to bacteria. The transfer was estimated to have happened approximately 500 million years ago.

Homologous recombination from Duke University.

Multiple studies have found that bacteria can take up eukaryote DNA, but only in certain conditions, where the researchers used a sort of genetic trick called homologous recombination. In short, homologous recombination can occur when the ends of the donor DNA have sequences similar to part of the acceptor DNA. The homologous sequences can bind together, and in the next round of replication, the donor DNA can be integrated. In studies that aimed to find evidence of transfer of DNA from eukaryotes to prokaryotes without genetic tricks, none was found.

Table 1 of the EFSA Statement lists all studies prior to its publishing that examined horizontal gene transfer in bacteria (25 studies in all). Of the 18 studies that looked for gene transfer with homologous recombination, 15 found gene transfer and 3 did not. Of the 16 studies that looked for gene transfer without homologous recombination, no evidence of gene transfer was found.

In short, there are many DNA sequences that look like eukaryote genes in prokaryote genomes, but so far only one has been found that might be an actual functional gene. All evidence to date shows that gene transfer from eukaryotes to prokaryotes can only occur when homologous DNA sequences are present in donor and acceptor genomes. The lack of evidence for horizontal gene transfer in the wild suggests that there are some sort of barriers to gene transfer from eukaryotes to prokaryotes.

Barriers to gene swapping

Prokaryotic DNA and eukaryotic DNA are sort of like different computer languages. In fact, each species has slightly different ways of “personalizing” its own DNA with things like methylation and other DNA modifications, different codon preference, post translational modification of RNA, and whether or not introns are present. Eukaryotic DNA is so different from prokaryotic DNA that the bacteria just can’t take it up and use it as they would other bacterial DNA. Additionally, even if all of the barriers to gene uptake occur and all the barriers to gene expression are overcome, the likelihood that the gene will confer a positive trait for the bacterium is low. Most eukaryotic genes aren’t going to be helpful for a prokaryote, such that the few useful genes are few and far between. Even if a bacterium was able to uptake and express an antibiotic resistance gene from a genetically engineered plant, there would have to be selective pressure (i.e. an environment that included the antibiotic that the gene conferred resistance to) in order for the gene to be maintained in a bacterial colony. For more information about barriers to gene swapping, check out the EFSA Statement.

Avoiding the unlikely

Despite the fact that horizontal gene transfer from eukaryotes to prokaryotes is so unlikely (the only known example was estimated to have happened 500 million years ago), there are still precautions that can be taken to make it even more unlikely. For example, if antibiotic resistance genes are used as selectable markers in genetically modified organisms, researchers can avoid using sequences with homology to known bacterial genomes, they can be sure to only use antibiotic resistance genes that include features that make the gene unusable in bacteria, and they can avoid using promoters that are active in bacteria, just to name a few.

Alternative markers

Transformed rice cells expressing GFP via Scuola Superiore Sant'Anna.

Another option is to find alternative marker genes and alternative strategies. Herbicide resistance genes are an alternative selectable marker. Visible markers like GFP or GUS are screenable markers. Marker genes can be bred out, meaning that the final plant line will not contain the marker gene. Finally, it is possible to use no marker genes at all, but that does require far more screening of adult plants which can add expense and time to any project.

Peer-reviewed works cited

Dona A, & Arvanitoyannis I (2009). Health Risks of Genetically Modified Foods Critical Reviews in Food Science and Nutrition, 49 (2), 164-175 DOI: 10.1080/10408390701855993

Halford NG & Shewry PR (2000). Genetically modified crops: methodology, benefits, regulation and public concerns. British medical bulletin, 56 (1), 62-73 PMID: 10885105

Hotopp JC, et al. (2007). Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. Science (New York, N.Y.), 317 (5845), 1753-6 PMID: 17761848

Other works cited

Collins JD, et al. (2009). Statement of EFSA on the consolidated presentation of opinions on the use of antibiotic resistance genes as marker genes in genetically modified plants. European Food Safety Authority.

Hickman M & Roberts G (2010). Fury as EU approves GM potato: Critics claim plant could spread antibiotic-resistant diseases to humans. The Independent.

Zimmer, C (2010). Hunting Fossil Viruses in Human DNA. New York Times.

Note: This work was originally posted at Scientific Blogging as an entry in their 2010 Scientific Blogging Contest.

On the right is “tassel ear”, where silks and kernels (female, seed producing plant parts) appear on the tassel (male, pollen producing plant parts), where they are most certainly NOT supposed to be – it’s ok for sorghum and other grasses, but not for corn! On the left, there are at least 2 ears where there should be one, and those leaves poking out between the two might be more ears. Neither of these plants are transgenic or carry heritable mutations that cause these strange phenotypes. Both transgenic and non-transgenic fields are treated with a herbicide before we plant but after that the plants are grown with no additives, chemical or otherwise.

So, what the heck is going on?

I’ve always meant to look it up, but pollination season is so busy, and then it’s harvest season which is so busy, and then we’re analyzing the seeds… you get the idea.

While looking for pictures of corn borer damage, I found an awesome site by Peter Thomison and Allen Geyer of the Horticulture and Crop Science Department of Ohio State University: Troubleshooting Abnormal Corn Ears and Related Disorders.

They say that tassel ear is due to a variety of causes, including mechanical injury due to hail, which we did have pretty badly last year. No one really knows what causes “bouquet ear” with multiple ears appearing where there should be one, but it might be due to temperature stress due to cold.

There are many other common but strange corn phenotypes explained on their site. Check it out!