Veterinary Pharmaceuticals in the Environment - American Chemical

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Chapter 11

Characterization of Chlortetracycline-induced Glutathione S-Transferase to Conjugate Chloroacetanilide and Chlorotriazine Herbicides Michael H. Farkas1, James O. Berry1, and Diana S. Aga2 1

Department of Biological Sciences, University at Buffalo 2 Department of Chemistry, University at Buffalo

Glutathione S-transferases (GST) induced in maize by chlortetracycline (CTC) appear to inhibit the ability to detoxify chloroacetanilide herbicides, as determined by liquid chromatography/mass spectrometry. Furthermore, the same CTC-induced GSTs were able to conjugate chlorotriazine herbicides at a rate undistinguishable from GSTs isolated from untreated maize plants. This data suggests that CTC, while not toxic to maize, may have indirect effects on herbicide dtoxification in a class-specific manner. Chlortetracycline (CTC) is a commonly used antibiotic in animal husbandry. A majority of the antibiotic passes through the animal nonmetabolized and the manure is applied to crop fields as fertilizer where it has been shown to have phytotoxic tendencies in certain crops, as well as accumulate in others. Soil amended with CTC has been shown to induce expression of glutathione S-transferases (GST) in maize and subsequent analysis by liquid chromatography/ion-trap mass spectrometry (LC/IT-MS) identified stable CTC products conjugated with glutathione (GSH). Purified GSTs isolated from maize treated with CTC were able to produce nearly twice as much conjugated product relative to the GSTs isolated from nontreated control plants. Due to the prevalence of antibiotics in the environment, this work has raised concerns with regards to inducing herbicide resistance among target weeds or susceptibility among non-target crop plants. Herbicide © 2009 American Chemical Society In Veterinary Pharmaceuticals in the Environment; Henderson, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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conjugation by GSTs has been well characterized and is one of the main determinants of plant susceptibility. When GSTs isolated from maize control and CTC-treated plants were added to separate in vitro reactions containing three chloroacetanilide herbicides (metolachlor, propachlor, and alachlor) and one chlorotriazine herbicide (atrazine), the GSTs from the CTC-treated maize showed a reduced ability to conjugate the herbicides. In addition, analysis via LC/IT-MS has made it possible, for the first time, to detect chloroacetanilide herbicides that have been conjugated with two GSH molecules, in addition to the single GSH conjugate.

Introduction Pharmaceutical Usage in Agriculture In the United States, veterinary pharmaceuticals are used to both treat illnesses and promote growth in every type of livestock. There is very little data as to the quantity of antibiotics used for animal husbandry purposes. The estimates that do exist are conflicting and most likely inaccurate. The Animal Health Institute has estimated that antibiotic use in agriculture is 10% of the total antibiotic consumption, of which 87% is used for therapeutic purposes (1). On the other hand, in a book sponsored by the Union of Concerned Scientists estimates that antibiotics used for growth promotion is 8 times higher than what is used in all of human medicine (2). Regardless, of the disparity in usage estimates, it is known with great certainty that in both humans and animals a majority of the pharmaceuticals pass through the organism unaltered, in their active form. In some cases, 70% of the pharmaceutical is not metabolized and it is excreted in both urine and feces (3). While humans are a source of antibiotics in the environment, the focus this book is with regards to agricultural sources. Excrement from animals, largely in confined animal feeding operations (CAFOs), is collected and spread as a slurry onto crop fields where the pharmaceuticals are able to leach into the soil and into surrounding waters. Tetracyclines are the most commonly used antibiotics for animal husbandry. For example, surveyed dairy farmers in Pennsylvania reported using tetracyclines 70% relative to other antibiotics (4). In Denmark, where actual usage numbers do exist, tetracyclines again accounted for a minimum of 25% of the total usage of antibiotics in all food animals (5). Tetracyclines raise greater concerns because they are also used in human therapeutics and multiple forms of resistant bacteria have been identified. Tetracyclines in the environment are probably the most studied of all the antibiotics, largely for the reasons stated.

In Veterinary Pharmaceuticals in the Environment; Henderson, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Pharmaceuticals in the Environment An important factor to consider when studying antibiotics in the environment is their interactions in soil. In other words, will they be tightly adsorbed to the soil or will they move freely through the soil and into surrounding waters? In either case, it is necessary to consider both the physical properties of the particular antibiotic, as well as the properties of the soil. Sulfonamides and tetracyclines are two examples that exhibit different distribution coefficients (Kd) in soil. Sulfonamides have a relatively low coefficients ranging from 0.6-4.9, whereas tetracyclines are more adsorbed to soil with coefficients varying from 290-1620(6,7). Therefore, it would be expected that sulfonamides are more likely to enter groundwater, and ultimately be more problematic. However, there are other factors to consider such as soil characteristics, i.e. soil composition, pH, soil minerals, and soluble organic matter (SOM)(6). For example, the relative composition of clay in the soil will increase the adsorption of tetracyclines. In addition, tetracyclines are wellknown chelators of divalent cations. Soil composed of higher concentrations of Ca2+ will lead to increased adsorption. Kulshrestha, et al, determined that oxytetracycline adsorption to montmorillonite, in fact, decreases as the pH of the soil increases(8). This is likely due to a number of factors that account for both the antibiotic properties (it is positively charged at low pH and repulsed by soil cations) and soil properties (at higher soil pH, hydrophobic interactions are predominant). As the understanding of antibiotics in the environment increases, there are a growing number of concerns that are being raised. The most prevalent and likely the most controversial concern is that sub-therapeutic levels of antibiotics in the environment will lead to an increase in drug-resistant pathogens. This is an area that requires careful consideration and investigation since many antibiotics are used for both veterinary purposes as well as for human therapeutics. Chee-Sanford and colleagues (2001) found that hog lagoons contained tetracycline resistance genes and these same genes could be found in groundwater up to 250 m downstream(9). More specifically, a similar study found that Tet M (a ribosome protection protein)(10) is the most prevalent resistance gene in hog lagoons and its prevalence is season-dependent(11). Typically, when the effects of pharmaceuticals in the environment are studied, a single compound is used for the study. A more recent investigation looked at the effects of a mixture of pharmaceuticals at sub-therapeutic levels (ng/L) on human embryonic cells, in vivo(12). The investigators found that cell proliferation was significantly decreased and morphological changes were observed. This suggests potential adverse human health effects can occur from low levels of pharmaceuticals that can leach into groundwater and a potential adverse effect on aquatic organisms. An area of concern that is beginning to be investigated more fully is that of plant uptake and accumulation of antibiotics. Uptake by plants leads to two different concerns: 1) accumulation of antibiotics in edible portions of plants will expose consumers to the antibiotic and 2) uptake will cause phytotoxicity by the antibiotic, thus resulting in lower crop yields or death of the crop. Many modeling studies have shown that antibiotics at environmentally-relevant


156 concentrations will not kill plants, however others have shown decreased growth and crop production (13,14). A factor that has not been considered, and is the basis of this chapter investigates the indirect effects that may be caused by uptake of antibiotics into plants. All of these areas of plant uptake will be discussed in more detail in the subsequent sections.


Antibiotic Uptake into Plants Relatively few studies have investigated uptake of pharmaceuticals into plants. A few studies have reported that some crop plants do take up commonly used veterinary pharmaceuticals into the edible portions of plants. The most recent studies have shown that chlortetracycline (CTC) is taken up by maize, green onion, and cabbage at ppb (ng g-1) levels (15). However, this same study reported that tylosin, an antibiotic in the macrolide family used mainly for swine production, was not detected in any of the three plants. On the other hand, a study conducted by Boxall and colleagues (2006) observed uptake of a wider range of veterinary pharmaceuticals including: oxytetracycline, enrofloxacin, and tylosin to name a few into lettuce and carrots(14). Interestingly, uptake of compounds that are of xenobiotic nature into a plant is commonly believed to occur via absorption of the compound into the root of the plant. So, the octanol-water partition coefficient of the compound is a key factor in uptake (16). In other words, lipophilic compounds are expected to be taken up, and accumulated to a greater extent than would be more polar compounds. However, oxytetracycline uptake into alfalfa was shown to be facilitated in an energy dependent manner (17). Although the authors of this work were unable to deduce a specific channel responsible for uptake of oxytetracycline, they were able to show that uptake did follow Michaelis-Menten kinetics. The first study to report antibiotic phytotoxicity was that of Batchelder in 1982 (13). The author showed that pinto bean development and nutrient uptake were stunted in the presence of CTC and oxytetracycline. However, the concentration of the antibiotics to observe a significant decrease in development and nutrient uptake was at a concentration of 160 mg kg-1, a concentration 8 times higher than observed in the environment (18). Even though the concentration was so much greater than environmental concentrations, the study also showed that radish, wheat, and maize were not affected by the antibiotics in the soil (13). Since this first demonstration of antibiotic phytotoxicity, others have been published using environmentally relevant concentrations. For example, sulfadimethoxine is toxic to maize at a concentration of 1 mg kg-1 (19). Enrofloxacin, is toxic to radish, lettuce, pinto beans, and cucumber (20). In fact, enrofloxacin is efficiently accumulated in these plants in the range of μg g-1 and approximately 25% of this is converted to another active antibiotic, ciprofloxacin. Antibiotics are capable of leaching into surrounding waters, but in other cases such as flumequine (a quinolone derivative) it is directly introduced into waters used for aquaculture. This antibiotic is accumulated and phytotoxic to aquatic weeds, which are consumed by various organisms and possibly promoting dissipation throughout the food chain (21).



157 Plants are not totally defenseless against contaminants in the environment, including antibiotics. An aquatic fern, Azolla filiculoides Lam., efficiently accumulates sulfadimethoxine and it is believed to be degraded by symbiotic microorganisms (22). Degradation by microorganisms reduced uptake of active sulfadimethoxine into the plant, although metabolites were not identified. Hairy root cultures of sunflowers also showed rhizosphere degradation of antibiotics, however degradation was caused by recognition of a stressor and a defense response by the plant (23). In this study, oxytetracycline was applied to the hairy root cultures of sunflowers and a general defense response was activated where the roots produce Reactive Oxygen Species (ROS). The ROS consists of free radical oxygen species and are produced by enzymes such as NADPH oxidase and peroxidases, which in this case are exuded from the roots and into the media. This leads to oxidation of oxytetracycline, likely producing quinone derivatives (24). Glutathione S-Transferase-mediated Antibiotic Detoxification in Plants Our early research has focused on the observations of Batchelder (1982) where pinto bean development and nutrient uptake are directly inhibited in the presence of CTC, while maize plants are unaffected (13). We exposed maize and pinto bean seedlings to soil treated with 20 mg kg-1 of CTC and sampled the roots, shoots, and leaves of the plants for total protein content at 1, 2, and 3 days (specific methods can be found in (25)). Using SDS-PAGE as an initial screening tool, in maize we were able to determine that a difference in banding pattern existed between the control (untreated) and CTC-treated roots in the range of 20-30 kDa. Although this increased band could be any number of proteins, under the experimental conditions it was suggestive of an increase in glutathione s-transferases (GSTs). No significant changes were noticed in the shoots or leaves, but all were tested for GST activity, as were the pinto bean samples. The shoots and stems did not show any significant increase in GST activity, but the maize roots showed a significant (p

Veterinary Pharmaceuticals in the Environment - American Chemical

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