Fungicide Exposure from Grapes May Be Linked to Thyroid Cancer: Another Reason to Go Organic?

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Disclaimer: While this post has a sponsor, all facts and opinions expressed are those of the research article I am presenting as well as my own, none of which have been influenced in any way, shape, or form by the sponsor.  Enjoy!

Fungicides are used throughout agriculture, and are certainly no stranger to viticulture / winegrowing.   They are utilized in the vineyard to help protect the grapes against fungal attack, which is more common in more humid environments than in drier environments.  One major concern regarding the use of fungicides, which in part has contributed to the organic viticulture movement, is the unintended environmental and human health impacts of the use of synthetic fungicides in the vineyard.  Studies have found significant health impacts related to the use of conazole fungicides, including thyroid tumors and potentially other cancers.  It is thought that these fungicides are related to higher incidences of cancerous tumors as a result of their endocrine disruption properties.  In fact, endocrine disrupting compounds have been shown in hundreds (if not thousands) of studies to cause significant health problems in both wildlife and humans, including but not limited to reproductive health issues, neurological problems, and cancer.

One particular type of conazole fungicide that is used almost exclusively in vineyards is called

Penconazole Photo By Klever (Own work) [Public domain], via Wikimedia Commons

Penconazole
Photo By Klever (Own work) [Public domain], via Wikimedia Commons

“penconazole”.  In 2008, the Commission of European Communities established a maximum amount of penconazole allowed to be used in vineyards, with the Maximum Residue Level reaching no higher than 0.2ppm for both table and wine grape vineyards.  Studies have shown that many endocrine disruptors can still cause significant health problems at these very low doses, and that these established maximums may not be appropriate for curtailing negative health impacts in humans or wildlife.

The purpose of todays’ study, which is currently in press in the journal Toxicology in Vitro, was to look at genetic changes in human cells as a result of penconazole use in grapes, specifically looking at those genes that undergo a “change in behavior” after exposure of the fungicide.  This study also aimed to determine if any of these genes could be used as indicators of penconazole exposure, which could be helpful in determining a treatment plan early and well ahead of any major carcinogenic tumor growth, as the prognosis for many cancers is almost certainly much better if the tumors are caught at a very early stage.

Methods

The cells used in this study were the human T-47D cell line, which had been originally isolated from a woman with an infiltrating ductal carcinoma (i.e. tumor) of the breast.  These cells were used for several reasons: 1) because they are a human cell line; 2) because these cells possess several of the receptors required for endocrine activity (and thus endocrine disruption when applicable), including the estrogen receptor, androgen receptor, and progesterone receptor; and 3) because this cell line has been already studied and shown to be sensitive to endocrine disruptor in laboratory tests.

The fungicide penconazole was isolated from 50g of grapes that had been treated twice with the compound during the growing season. (Note: the paper did not indicate any more details than this in terms of the conditions in the vineyard).

Sensitivity of the cells was determined using the MTT assay, which basically gives information as to the percent survival of the cells after exposure to a toxin (in this case, penconazole).  Penconazole was isolated from the grapes in concentrations between 0.1uM and 200uM.

Total RNA was isolated and analyzed to determine what (if any) genes were affected by the penconazole treatment.

The program Pathway-Express was employed to analyze the genes found to be activated to determine what the final outcome of this activation would be if complete.  In other words, if a certain gene were activated due to penconazole exposure, the Pathway-Express program takes this information and basically says “hey, I see this gene is activatedàlooks like you could end up with prostate cancer based on which gene is activated!”.

Results

  • Penconazole exposure did not inhibit cell growth at the 0.1 and 1uM concentrations.
  • Penconazole exposure at 10uM resulted in a 15% reduction in cell growth.
  • Penconazole exposure resulted in the activation/modulation (or in other words, regulation of gene expression) of 2274 genes, with 507 of those genes being modulated in a similar fashion (and thus used for further analysis).
  • Using the Pathway-Express program to analyze these 507 activated/modulated genes after penconazole exposure, the only significant pathway found was that of thyroid cancer.
    • In other words, based on the genes that were activated after penconazole exposure, the end result (if the cells were actually in a human body) would be thyroid cancer.
    • This pathway included 6 different genes: 2 down-modulated and 4 up-modulated genes.
    • Down-modulated genes: Cyclin D1 (CCND1) and nuclear receptor coactivator 4 (NCOA4, a.k.a. PTC3)
      • Cyclin D1 is involved in the regulation of CDK kinases, in particular the regulation of CDK4 and CDK6, which are critical for cellular growth processes.
      • NCOA4 interacts with the androgen receptor in such a way to increase transcriptional activity (i.e. increase the protein-making process).
        • These two genes were down-regulated, so in other words, they weren’t as active as they would be under normal conditions.
  • Up-modulated genes: retinoid X receptor alpha (RXRA), transcription factor 7-like 1 (TCF7L1), tropomyosin 3 (TPM3, a.k.a. TRK), and the translocated promoter region (TPR).
    • RXRA codes for steroid and thyroid hormone transcriptional regulators, or in other words, is responsible for producing steroid and thyroid hormones.
    • TCF7L1 functions in the regulation of cell cycle genes and cell death.
    • TPM3 and TPR encode for the proteins that provide stability to the structurally stabilizing filaments of the cells, as well as for other proteins related to actin-binding.
      • These four genes were up-regulated, so in other words, they are overexpressed and are producing a little too much of what they are responsible for.

Conclusions

Unless you’ve recently had a class in developmental biology or genetics of some sort, a lot of what I just presented to you might sound a little confusing.  Completely understandable!  This isn’t an easy topic to grasp, but once you are able to associate the more confusing terms with a more simplified approach, it doesn’t look as foreboding.

Let’s take a step back and review the main finding of this research, and then break it down from there.

Daniels, Gene, photographer, Photographer (NARA record: 8463941).  This media is available in the holdings of the National Archives and Records Administration, cataloged under the ARC Identifier (National Archives Identifier) 542503.

Daniels, Gene, photographer, Photographer (NARA record: 8463941). This media is available in the holdings of the National Archives and Records Administration, cataloged under the ARC Identifier (National Archives Identifier) 542503.

The primary result from this research was that a human cell line exposed to the fungicide penconazole, which was isolated from grapes that had been exposed to the compound during the growing season, had notably altered gene expression such that thyroid cancer would be the most significant outcome.  Simply put: exposure to the endocrine-disrupting fungicide penconazole increases the chances of getting thyroid cancer.  This result is in agreement with other studied examining the effects of fungicides and pesticides on human health.

“OK, pretend I don’t have a clue about this sort of thing: what the hell do you mean by all of this?”

From the detailed genetic information found in this research, how is it that thyroid cancer is a possibility after exposure of human cells to penconazole?

Well, 6 genes were found to be related to this pathway, and that they were all significantly altered in the cells.  Two of the genes were down-regulated, while 4 of the genes were up-regulated.  Down-regulated in very basic terms means that the cells aren’t producing as much as they would be under normal conditions, and up-regulated in very basic terms means that the cells are producing too much of what they would be under normal conditions.

So, in this case, two genes were malfunctioning after fungicide exposure to result in a poor showing in the cells, while four genes were malfunctioning after fungicide exposure to result in them going crazy with producing way too much “stuff”.

In this situation, the two genes that are down-regulated are ones that help regular cell division and proliferation.  After exposure to penconazole, these genes get very “confused” and they are no longer able to effectively tell the cell where it is in the cell division and proliferation process, which can result in the process spinning out of control.  Additionally, when you throw in the 4 up-regulated genes after penconazole exposure, you get even more cellular division and proliferation mayhem, as the cell no longer has the ability to stop producing steroid and thyroid hormones, AND it basically tells the cell to keep growing MORE.  This sort of information (plus a lot more details) is what the Pathway-Express program was using to determine the ultimate outcome from this sort of down- and up-regulation, and with confidence it determined that these genes, when “messed up” by the exposure to the fungicide penconazole, could result in thyroid cancer in whoever was initially exposed to the toxic compound.

What’s next?

According to the authors of this research, any of the genes that were shown to be affected by penconazole exposure, even at relatively low doses, could be possible “biomarkers of penconazole exposure”.  This could be an important tool, as it would identify possible at-risk individuals for thyroid cancer prior to the formation of any tumor, and perhaps preventative measures could be taken to attempt to reduce this risk, and at the very least monitor more frequently so that if a tumor were to appear later on after penconazole exposure, it could be found very early and effectively treated.

It is important to note who might be at-risk for exposure to penconazole, as well as other possible avenues

Photo By Stefan Kühn (Own work) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC-BY-SA-2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons

Photo By Stefan Kühn (Own work) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC-BY-SA-2.5-2.0-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons

that weren’t tested in this study but would be important to study in the future.  The penconazole exposure in this study came from the grapes after they had been treated a couple of times with the fungicide during the growing season.  I think the most obvious people at risk for this type of situation are the vineyard workers, particular those with direct and frequent contact with a lot of grapes.  I doubt there is enough penconazole on one grape that an inquisitive visitor would sneak in order to elicit this pathway to thyroid cancer, but certainly those exposed to the compound on a more regular and long-term basis may be at the greatest risk.

What about drinking the wine made from these grapes?  Could penconazole fungicide exposure to the grapes result in thyroid cancer in the person that drank the wine made from those grapes?  Now, that’s a loaded question, and one I can’t answer with certainty from this particular study.  It is highly likely that during the winemaking process, the penconazole is cleaned off and diluted so much that levels are too low to show this type of negative health impact, BUT then again, a lot of endocrine disruptors have been shown to be effective even at teeny tiny doses, so I don’t want to just out and say “it’s fine” without seeing the evidence.  Also, there is also the possibility of interactions between the penconazole and the other chemicals/compounds used in the winemaking process, which could result in either negative effects, or a negated effect.  Perhaps the synergistic interaction between penconazole and a particular winemaking component makes the fungicide more harmful to human health.  Or on the other hand, perhaps the synergy results in the “shutdown” of penconazole and it is no longer a threat.

Finally, thinking about the post we discussed not too long ago about resveratrol and it being a seemingly healthy compound that happens to have endocrine disrupting-like characteristics:  could resveratrol compete with the penconazole and come out “on top”, resulting in the shutdown of the fungicide in the system?  Does wine inherently protect itself again this sort of disruptor simply by the nature of its own natural antioxidative, anti-carcinogenic, and other important health-benefiting qualities?

Certainly, I think these results show cause for concern for those people actually handling the grapes themselves after they have been treated with the fungicide penconazole, at least concern enough to do a long-term clinical observational study of those workers exposed to the compound on a somewhat regular basis and how many of them end up with some form of thyroid or other cancer after a certain number of years.

As far as exposure from wine is concerned, I think the jury is still out, but certainly these results should prompt other researchers to take this next step, as we really won’t know the true answer until someone actually does the study.

Even though I covered a lot, I know I didn’t cover all of it.  Please feel free to leave your comments either on what I’ve presented here, or perhaps questions, comments, or concerns that popped up in your mind based on the results that you read.  Let’s have a healthy discussion about this!  All are welcome!

Source: Perdichizzi, S., Mascolo, M.G., Silingardi, P., Morandi, E., Rotondo F., Guerrini, A., Prete, L, Vaccari, M., and Colacci, A. 2013. Cancer-related genes transcriptionally induced by the fungicide penconazole. Toxicology in Vitro, http://dx.doi.org/10.1016/j.tiv.2013.06.006.

12 comments for “Fungicide Exposure from Grapes May Be Linked to Thyroid Cancer: Another Reason to Go Organic?

  1. October 14, 2013 at 11:26 am

    And just think of how few winemakers even wash their grapes.

    I’m also interested in the difference between the effects from drinking grape juice versus wine, both made from the same grapes. I wonder what influence alcohol and fermentation have on the compounds found in the grapes you discussed above?

    Great detailed article and solid research!

    • Becca
      October 14, 2013 at 12:58 pm

      Thanks for your comments, Diana! I’m glad you enjoyed the post! You raise a really interesting question regarding the difference between the effects from drinking grape juice versus wine. I hadn’t thought of that, and am not really sure of the answer, but seeing as how the two are processed differently, I would not be surprise if there was such an effect! Good thinking!

  2. Hal Hinkle
    October 14, 2013 at 11:29 am

    Becca – good post on a complicated and important topic: estrogen disruptors (ERs). Reading the article at face value, doesn’t make clear if the level found in the treated grapes is a real problem for consumers (or more importantly for vineyard workers and applicators), because the key issue with ERs is that they are mostly fat soluble and therefore accumulate in the consumer’s body and bioaccumulate up the food chain. One older study demonstrated that levels of ERs that accumulate in the food chain are more potent carcinogens that direct sources consumed, in their case from tainted water (Water Sci Technol. 2003;47(9):71-6.).

    In the particular here for Penconzole, since I believe it has an aliphatic side chain (http://www.efsa.europa.eu/en/efsajournal/doc/175r.pdf), it would be fat soluble and therefore subject to bioaccumulation potentially.

    Even without bioaccumulation, repeated exposure can result in cancer for certain toxic compounds (http://www.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC-113961_10-May-06_a.pdf).

    Not great stuff.

    • Becca
      October 14, 2013 at 1:01 pm

      Thanks for your fantastic comments, Hal! Those links you recommended are definitely helpful in terms of addressing this question/problem. Makes me wonder—how much of the penconzole leaches into the soil and into the water supply? Perhaps it could then accumulate that way (after long-term consumption anyway).

  3. pricklypear
    October 14, 2013 at 12:00 pm

    Does this explain the high cancer rates in the Napa Valley? shhhhhh…. wouldn’t want to hurt the ever so perfect image here.

  4. Mary Macdonald
    October 17, 2013 at 5:24 pm

    This is a very compelling topic and your presence in the field of wine making most appreciated.
    We do not have Penconzole listed as one of the fungicide treatments under the Guide to Fruit Production 2012 -2013 (Publication 360)published by the Ontario Ministry of Rural and Agricultural Affairs (OMAFRA)
    The compound closest (judging by the word-root) is difenoconazole Trade name : Inspire which is available for use in Ontario but we ourselves do not use in our vineyard in Prince Edward County Ontario.

  5. James TED KEARNS
    December 13, 2013 at 11:46 am

    what are the trade names of products that contain penconazole? Is this fungicide approved for use on grapes in the united states?

  6. February 8, 2014 at 4:24 pm

    In relation to the possible danger to vineyard workers (of which I am one at times), how is the exposure deemed dangerous, I.e. ingestion through breathing, or through skin contact?

    Thanks
    Nick

    • Becca
      February 10, 2014 at 6:40 pm

      Hi Nick,

      Thanks for reading and commenting! I don’t think they really mentioned this issue in this particular paper, however, in general, pesticides can enter one’s body through breathing/inhalation, eating/ingesting, or through the skin/touching.

      In general, it’s probably wise to wear gloves (which I’m sure you already do), and maybe some sort of mask to try and avoid breathing it in. Oh, and don’t eat any of it ;)

      I hope this was somewhat helpful! Cheers!

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