Fate of Pharmaceuticals in the Environment and in Water Treatment Systems - Chapter 9 docx

199 9 Antib iotic Transformation in Plants via Glutathione Conjugation Michael H. Farkas, James O. Berry, and Diana S. Aga 9.1 INTRODUCTION Pharmaceuticals have been introduced into the environment for decades, via land application of manure from antibiotic-treated livestock and via discharges from wastewater treatment plants, where only very limited removal may take place. As an increasing number of investigators report the occurrence of a wide range of phar- maceuticals in the environment, 1–3 thereisaneedtofocusmoreresearchonthe advancement of treatment technologies to remediate pharmaceutical pollutants in the environment. Efforts to enhance pharmaceutical remediation have been spurred by various concerns, ranging from the emergence of antibiotic-resistant pathogens that may infect the human population 4 to potential risks associated with the long- termexposureofconsumerstocropsthataccumulatetheantibiotics. 5 While most of these concerns have not as of yet been veried, some substantiation of these effects has come to fruition. For instance, research on human embryonic cells exposed to 13 pharmaceuticals at concentrations found in the environment has shown a signicant decrease in cell proliferation in vitro. 6 Contents 9.1 Introduction 199 9.2 Plant Uptake and Phytotoxicity of Pharmaceuticals 200 9.3 Bioavailability 201 9.4 Detoxication of Xenobiotics via Glutathione Conjugation 201 9.5 Glutathione S-Transferases: Structure, Function, and Evolution 203 9.6 InductionofGSTsin Phaseolus Vulgaris and Zea Mays by Ch lor tetr acycl ine 2 04 9.7 Mass Spectral Characterization of Antibiotic-GSH Conjugates 207 9.8 Other Environmentally Important Antibiotics 209 9.9 Hypothesis for Antibiotic-Induced Phytotoxicity 210 9.10 A reas Re quir ing Further Research 210 References 211 © 2008 by Taylor & Francis Group, LLC 200 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems Phytoremediation,theapplicationofplantsandtheirassociatedmicrobesto enhance biodegradation of contaminants in the environment, has recently been explored for the remediation of sulfadimethoxine 7 and oxytetracycline 8 antibiotics. Thephytoremediationofmetalsandorganicpollutantsinsoilisanemerging,low- cost technology, but the underlying biochemical mechanisms involved in contami - na ntuptake,detoxication,andtranslocationinplantsarelargelyunknown.One well-known detoxication pathway involves phytotransformation of contaminants via glutathione (GSH) conjugation, which is catalyzed by glutathione s-transferase (GST) enzymes. This chapter provides an overview on the role of plant GSTs in phy - to remediationoforganiccontaminantsandpresentsrecentworkonGST-mediated transformations of environmentally relevant antibiotics. 9.2 PLANT UPTAKE AND PHYTOTOXICITY OF PHARMACEUTICALS Like many heavy metals and organic contaminants, antibiotics can be taken up by plantsandcanelicitphytotoxicityinsusceptiblespecies.Infact,ithasbeenknown for quite some time that chlortetracycline, a highly used growth-promoting antibi - ot ic,isphytotoxictopintobeans(Phaseolus vulgaris). 9 More recent reports have demonstrated the phytotoxic effects of sulfadimethoxine antibiotics toward maize and barley 10 andalsothatofenrooxacinantibioticstoseveralagriculturalcrops. 11 However,someplantspeciesdonotappeartobeaffectedbyexposuretoantibiotics. For example, in the same study that reported phytotoxicity of chlortetracycline to pintobeans,itwasshownthat Zea mays was u naffected by the antibiotic exposure. 9 Pharmaceutical-induced phytotoxicity does not appear to cause plant mortality but rather leads to inhibition of plant growth and ultimately lower crop yields. For example, enrooxacin, a broad-spectrum antibiotic that is used in both human and veterinarymedicine,inhibitsrootgrowthandleafdevelopmentinavarietyofcrop plants. 11 Statins, which are cholesterol-reducing agents that are highly prescribed for human health, can inhibit 3-hydroxyl-3-methylglutaryl coenzyme A reductase inplants,blockingisoprenoidbiosynthesis,whichisimportantforamultitudeof endogenousfuncti ons. 12 Direct quantication of the amount of contaminants that accumulate in plant tissuesisdifcultbecauseoftheanalyticalchallengesassociatedwithdetectinglow levels of analytes within the complex plant biomass. Recently, however, Kumar and coworkers 5 were able to measure concentrations of chlortetracycline that accumu- latedinmaize,cabbage,andgreenonionsatthepartsperbillion(ppb)rangeusing enzyme-linked immunosorbent assay (ELISA) techniques. Beyond pharmaceuti - cal s,othertoxicorganiccompoundshavebeenshowntoaccumulateinthefruitsof apple and peach trees. 13 Trichloroethylene, a cleaning and degreasing agent widely usedforindustrialandmilitarypurposesintheUnitedStates,hasbeenfoundinthe environmentatlevelsashighas500ppm.Acontrolledgreenhousestudyhasshown thatwithin2yearsofexposure,appleandpeachtreeswereabletoaccumulateas much as 34 ppm. © 2008 by Taylor & Francis Group, LLC Antibiotic Transformation in Plants via Glutathione Conjugation 201 9.3 BIOAVAILABILITY The bioavailability of pharmaceuticals is an important factor to consider when investigatingtheirinteractionswithplants.Generally,thetoxicityofacontaminant is directly related to its bioavailability. 14 In soil, the bioavailability of an antibiotic dependsontwomajorfactors:(1)thedegreetowhichitadsorbstothesoiland(2) theorganismsinhabitingthesoil.Sorptionofantibioticstosoilisdependentonthe soilcompositionanditschemicalcharacteristics,suchaspHandionicstrength. Soil composition can vary with regard to its clay, sand, organic matter, and min - er alcontent,allofwhichplayanimportantroleinantibioticsorption.Forinstance, sulfonamide antibiotics adsorb more strongly to clay than to sand, and in general, antibiotics adsorb less tightly to soil minerals than to organic matter. 14 However, the soil’smineralcompositionplaysaroleintetracyclinebinding.ItisknownthatCa 2+ sorption to clay increases oxytetracycline adsorption relative to Na + .SoilpHmust also be factored into antibiotic bioavailability. Sulfonamides and tetracyclines are adsorbedmoretightlyinthepresenceofacidicsoils,whereastheoppositeistruefor tylosin (macrolide family of antibiotics). 15 Some antibiotics have hydrophobic char- acteristicsandcannotdissolveinwaterwithoutadditionalfactors,butitappearsthat hydrophobicity is not directly related to the strength of sorption of these antibiot- ic stosoil. 16,17 In general, electrostatic forces, complexation, and hydrogen bonding, whichareregulatedbythesoilcontentandchemistry,areallimportantindetermin- in g antibiotic bioavailability. The rhizosphere, the area surrounding a plant’s roots, is very dynamic and full of microorganismsthatplayaroleinthefateandbioavailabilityofantibioticsandother contaminants in soil. Root exudates in the rhizosphere may contain reactive oxygen species (ROS) such as H 2 O 2 ,whichareproducedbyplantsasageneraldefense response to oxidative stress. Oxytetracycline has been shown to induce the release of H 2 O 2 inhairyrootculturesofsunowers(Helianthus annuus), 18 resultinginthe inactivation of oxytetracyline via oxidation. 9.4 DETOXIFICATION OF XENOBIOTICS VIA GLUTATHIONE CONJUGATION Plantshaveanintricatedefensesystemthatiscapableofcombatingavarietyofintru- sions ranging from pathogens to exogenous chemicals. In fact, the plant’s defense system is controlled by numerous biochemical pathways and is capable of producing aphysiologicalresponsethatispathogen-/xenobiotic-specic.Theabilityofsome plants to detoxify harmful compounds upon uptake via these specic pathways has promoted interest in the area of phytoremediation, which is still in its early stages. As mentioned earlier, GST enzymes are responsible for many of these detoxication reactionsinvolvingalargenumberofxenobioticsfoundinlivingsystems,including plants. The GSTenzymesareprimarilyfoundinthecytosolofplants,mammals,bac- te ria,fungi,andinsects.TheGSTsarepartofathree-phasedetoxicationsystem involved in detoxifying xenobiotics in living organisms. Phase I includes enzymes © 2008 by Taylor & Francis Group, LLC 202 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems such as cytochrome P450 monooxygenases. The purpose of the Phase I enzymes is to introduce reactive functional groups via hydroxylation and epoxidation reactions to xenobiotic compounds. 19 The introduction of functional groups prepares a xenobiotic to be acted upon by Phase II enzymes, of which GSTs are a major component. GSTs detoxify xenobiotics that are typically electrophilic, and they do so by substituting glutathione (GSH) at an electrophilic site, which renders the xenobiotic more polar and more readily translocated. 20 GSHisatripeptide(H-glutamyl-cysteinyl-glycine) that is conjugated to many endogenous and xenobiotic compounds ( Figure 9.1). GST- m ediated conjugations occur very rapidly, and the general mechanism takes place via a nucleophilic attack of the thiol group of GSH on an electrophilic atom in the xenobiotic. The rst documented cases of plant GST-mediated detoxication were in the metabolism of herbicides. There are a few cellular mechanisms that render a herbicide selective toward some unwanted species of weeds, but most commonly GSTs are involved in detoxifying herbicides in nontarget species. Both GSTs and GSH must be already present in abundance (or be induced) for a plant to be able to detoxify a xenobiotic via the glutathione pathway. For example, maize is tolerant to chloracetanilide and chlorotriazine herbicides by using a Type IGST 21,22 to detoxify these chlorinated compounds after plant uptake. Type I GSTs are constitutively expressed in maize; hence, maize plants are inherently able to detoxify atrazine. 21 Type II GSTs, however, are only induced by exogenous chemi- cals. Safeners are chemicals that are applied to plants that do not have the inherent abilitytodetoxifyachemical,thusinducingGSTexpression.Forexample,maize has a slight tolerance for thiocarbamate herbicides (EPTC), but pretreatment with a safenersuchasdichlormidorbenoxacorgreatlyincreasesinductionofTypeIIGSTs that specically protect the maize from EPTC exposure. 23 While plants are capable of metabolizing xenobiotics using different mechanisms, they may be susceptible if the metabolic pathway they use for detoxication is not efcient. For instance, H 2 N H N N H COOH COOH CH 2 O O S NO 2 NO 2 H 2 N H N N H COOH COOH CH 2 O O SH Cl NO 2 NO 2 –HCl FIGURE 9.1 The reaction between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) as catalyzed by GST enzyme proceeds via the electrophilic substitution of chlorine atom in CDNBbythesulfuratomofglutathione,producingadechlorinatedconjugate. © 2008 by Taylor & Francis Group, LLC Antibiotic Transformation in Plants via Glutathione Conjugation 203 maize detoxies atrazine via GST conjugation, which is an efcient mechanism. In contrast, a pea plant metabolizes atrazine slowly and inefciently via N-dealkyl- ation. 24 Theslowrateofatrazinemetabolismbypeaplantsleavesthemsusceptible to atrazine toxicity. GST-mediated transformations of xenobiotics is not the only mechanism of detoxication in the Phase II pathway. Glucosyl- and malonyltransferases are Phase IIenzymesthataddglucoseandmalonicacidtoxenobiotics,respectively. 19 These enzymes serve similar functions as GST with respect to “tagging” a xenobiotic and making it more polar. In Duckweed ( Lemna gibba), several chlorinated pesticides aretransformedusingtheselatterPhaseIIenzymes. 25 However, unlike GST, these enzymesappeartoconjugateatthecarboxylicacidandamineR-groupsofthepes- ti cide, instead of at the more electrophilic chlorine atoms. 9.5 GLUTATHIONE S-TRANSFERASES: STRUCTURE, FUNCTION, AND EVOLUTION The GST enzymes are hetero- and homodimeric, with an average molecular weight of around 50 kDa, and serve a variety of functions. GSTs are encoded by a large and diverse gene family. In plants, this family is divided into ve classes based on sequence identity. These classes include: phi, tau, theta, zeta, and lambda, in which theta and zeta have homology to the mammalian classes. Genomic analysis of Ara- bidopsis thaliana has located at least 48 GST genes, with the tau and phi classes being most abundant. Each monomer of the GST dimer is composed of two binding sites. The more internal binding site (G site) is responsible for binding glutathione (GSH). 24 TheGsiteisaconservedgroupofaminoacidslocatedintheNterminal domain of the polypeptide. The C terminal domain contains the binding site for the hydrophobic substrate (H site). This region is much more variable in terms of amino acid sequence relative to other GSTs, which is not unexpected when keeping in mind thelargenumberofcompoundsGSTscanbind. Electrophilic xenobiotics are particularly deleterious to living organisms, becausetheycanbecytotoxicorgenotoxic. 19 There exist two types of these electro- philes:softandhard.Anexampleofeachtypeofelectrophilethatistypicallyfound in a xenobiotic includes alkene groups (carbon-carbon double bonds) and halogens, respectively.GSTscanmediatetheconjugationofbothtypesofelectrophiles.GSH conjugation “tags” a xenobiotic for further processing. Processing of the “tagged” xenobioticisconsideredasPhaseIIIofthedetoxicationpathway,andtheendresult for the xenobiotic differs depending on the organism. In plants, the GSH-conjugated xenobiotic is either stored in the cell’s vacuole or else it is sent to the apoplast (area outsideofthecell). 26 Some evidence does exist, which suggests that GSH-labeled xenobiotics are exuded back into the soil after processing of GSH. 27 To tra nsport the xenobiotic conjugate requires Phase III proteins, which are ATP-dependent trans- po rterswiththeabilitytorecognizeandbindtoGSH. GSH plays a major role both in a plant’s endogenous cellular processes as well as in the plant’s defense responses. GSH is ubiquitous and abundant with roles spanning © 2008 by Taylor & Francis Group, LLC 204 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems protein and nucleic acid synthesis, including modulation of enzyme activity and adaptation to environmental stress. 28 Environmental stressors are quite variable, rangingfromtemperatureextremestoxenobioticstress.GSHisfoundintwoforms withinacell:anoxidizedform,inwhichadisuldebondisformedbetweentwo glutathionemolecules(GSSG),andthereducedform(GSH).Theratioofthetwo formsiscrucialtohowaplantadaptstoitsstressor.AlackoffreeGSHcandimin- ish a plant’s ability to mount an appropriate response to a stressor. Understanding theGSHbiosyntheticpathwayandthemechanismsbywhichitisutilizedbyvarious enzymeswillprovideinsightintoxenobioticdetoxicationbyplants.Activationof GSH biosynthesis is based on the ability of proteins involved in photosynthesis to actasanintricatesensorysystemtorespondtovariationsinredoxpotentialcaused by environmental stress. Environmentalstressalsoinducesotherenzymesthatarepartofthedetoxi- cation pathway. Glutathione reductase is an enzyme that reduces GSSG to GSH, thus increasing the concentration of free GSH. Glutathione peroxidases (GPX) are antioxidantenzymes.Aplant,asareactiontoenvironmentalstress,producesROS to contain the stressor within the site where the stressor is introduced. GPXs are used topreventoxidativedamagebyoxidizingtwoGSHstoformGSSG.GSTsalsohave peroxidaseactivity(althoughtheyareencodedbyadifferentgenefamily)andtheir mode of action is conjugation of GSH to the oxidant. 29 The functional difference between the two types of peroxidases is based on the substrate acted upon. GPXs reduce ROS, while GST peroxidases conjugate electrophiles such as lipid peroxides that are the result of ROS. 9.6 INDUCTION OF GSTS IN PHASEOLUS VULGARIS AND ZEA MA YS BY CHLORTETRACYCLINE The authors of this chapter performed similar experiments to those reported ear- lier by Batchelder 9 to examine the physiological basis of the observed differences in response between maize and pinto beans grown in antibiotic-treated soil. Ten- day-oldmaizeandpintobeansweretransplantedintosoilpretreatedwith20mg kg –1 ofchlortetracycline(CTC),withconcentrationssimilartothosefoundinthe OH OH NH 2 OH N H 3 C CH 3 O O O OH H 3 C OH Cl FIGURE 9.2 Thechemicalstructureofchlortetracycline.Thearrowsdepictpotentialsites of glutathione conjugation. © 2008 by Taylor & Francis Group, LLC Antibiotic Transformation in Plants via Glutathione Conjugation 205 environment. 30 CTCisagoodcandidateforinitialinvestigationsintoaplant’s responsefortworeasons:itiswidelyusedwithhighapplicationratesinagriculture, anditcontainsbothhardandsoftnucleophiles (Figure 9 . 2). The plants were then harvesteddailyfor3daysandextractedforanalysisoftotalproteins.Togainagen- er al perspective of the response of the plants to the antibiotic, the total proteins were subjectedtoSDS-PolyacrylamideGelElectrophoresis(SDS-PAGE).Interestingly, a distinct increase in bands at the range of 20 to 30 kDa from the maize samples growninCTC-treatedsoilwasobserved,relativetothemaizecontrol(untreated) samples. This was not observed in CTC-treated pinto beans. The increase in the proteins banding at this size range was indicative of GST induction in the treated maize plants. Days Posttreatment 123 Days Posttreatment 123 GST Activity (µmol/mL/min/µg protein) 0.00 0.02 0.04 0.06 0.08 MCR MTR GST Activity (mmol/mL/min/mg protein) 0.00 0.02 0.04 0.06 0.08 PCR PTR FIGURE 9.3 GSTactivitymeasuredfromtotalproteinextractsat1,2,and3daysafter plants were treated with CTC. (A) Maize control (MCR) and CTC-treated (MTR) plants. (B)Pintobeancontrol(PCR)andCTC-treated(PTR)plants.Valuesrepresentthemeanand standard deviation of six replicates. Asterisks denote statistically signicant data (p < 0.05). A B © 2008 by Taylor & Francis Group, LLC 206 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems To verify that these induced proteins were in fact GSTs, enzyme activity assays wereperformedusingthecrudeextractsfromtheplants.Theassayusedwasbased on the standard GST-catalyzed conjugation reaction of GSH to 1-chloro-2,4-dini- tr obenzene (CDNB). 31 Indeed,GSTactivityintheCTC-treatedmaizesampleswas signicantly higher relative to the control (untreated plants) on the rst and third days of exposure (Figure 9.3A).T heGSTactivitiesintheproteinextractsfrompinto beansshowednosignicantdifferencesbetweentreatedandcontrolplantsinanyof thedayssampled(Figure9. 3B), consistent with the SDS-PAGE results. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (min) (A) 0 20 40 60 80 100 10.42 9.81 9.16 Relative Abundance Relative Abundance OH OH NH 2 OH N H 3 C CH 3 O OO OH H 3 C OH N H H N NH 2 O O CH 2 COOH S HOOC Time (min) (B) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 0 20 40 60 80 100 2.20 1.93 1.54 HOOC N H H N NH 2 O OCH 2 COOH SH Cl OH OH NH 2 OH N H 3 C CH 3 O OO OH H 3 C OH Glutathione S-Transferase + FIGURE 9.4 (Continued) © 2008 by Taylor & Francis Group, LLC Antibiotic Transformation in Plants via Glutathione Conjugation 207 9.7 MASS SPECTRAL CHARACTERIZATION OF ANTIBIOTIC-GSH CONJUGATES TodemonstratetheinvolvementofGSTsinCTCtransformation,in vitro conjugation reactionswereperformed usingafnity-puried GSTs from CTC-treated maize. 32 The enzyme reaction products were characterized using liquid chromatography/ion-trap mass spectrometry (LC/MS/MS). Since GSH may also conjugate with xenobiotics nonenzymatically, in vitro control reactions w ere conducted containing all reactants, excluding the GST enzyme (no plant extract added). Results from the LC/MS/MS analysis of the reactionproductsr evealedpeakscorrespondingtodechlorinated CTC-GSHconjugate(Figure 9.4A).Thesepeakswerecharacterizedbytheabsence ofachlorinesignatureinthemassspectraandveryshortchromatographicretention time,indicatingincreasedpolarityrelativetotheunconjugatedCTC(Figure9.4B). MS/MSanalysisrevealedanionwitham/zof677,whichcorrespondstotheGSH- CTCconjugatewiththelossofglycine(MW75Da)(Figure9.4C) . Losses of m/z 18 (a water molecule) and m/z129 (glutamic acid) are characteristic fragmentation patterns for GSH. Another important feature of the chromatogram of the CTC-GSH conjugate was the existence of three isomeric peaks characteristic of CTC, which were maintained after conjugation. This is of interest because the products formed during nonenzymatic conjugation were two different conjugates (m/z 654 and m/z 695), each of which eluted as single peaks, with retention times very close to the 200 250 300 350 400 450 500 550 600 650 700 m/z (C) 0 20 40 60 80 100 Relative Abundance 548 659 530 506 404 OH OH NH 2 OH N H 3 C CH 3 O OO OH H 3 C OH H N NH 2 O O CH 2 COOH S m/z 677 D C B A 677 –129 –18 FIGURE 9.4 In vitro LC/MS/MS data for GST-mediated CTC-GSH conjugation. (A) Chro- matogram of m/z 677 conjugate and hypothesized product of a GST-mediated CTC-GSH reaction (inset). (B) Chromatogram of CTC demonstrating the difference in polarity relative totheobservedconjugateandthesimilaritiesinisomericpeaks.(C)chemicalstructureofthe fragment ion m/z 677 and its MS/MS fragmentation spectrum. © 2008 by Taylor & Francis Group, LLC 208 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems CTC standard. Furthermore, the mass spectra of these nonenzymatically formed conjugates indicated that the chlorine atom was retained (Figures 9.5A and 9 .5B). This suggests that GSH was able to conjugate to CTC nonenzymatically but at sites other than the chlorine atom. EnzymaticconjugationofGSHtoCTCoccurredwheneithermaizeorpintobean GSTs were used to catalyze the reaction. However, while both control samples and CTC-treated samples produced CTC-GSH conjugates, treated maize GST samples 250 300 350 400 450 500 550 600 650 700 m/z (B) 250 300 350 400 450 500 550 600 650 700 m/z (A) 0 20 40 60 80 100 507 D C B A [M+H–Glu–Cys] + 422 [M654–Glu] + 525 m/z 654 [M654–(Glu+H 2 O)] + 654 OH OH NH 2 OH N H 3 C CH 3 O OO OH H 3 C OH HN NH 2 O O H 2 C HOOC S Cl H 3 C Relative Abundance Relative Abundance 0 20 40 60 80 100 [M695–(Glu+H 2 O)] + 548 566 602 531 402 620 677 m/z 695 DA B C 695 [M695–H 2 O] + [M695–Glu] + [M695–(Glu+H 2 O+NH 3 )] + OH OH NH 2 OH N H 3 C CH 3 O OO OH OH NH H 2 N O O H 2 C COOH S Cl FIGURE 9.5 LC/ESI-MS/MSspectraofm/z654(A)andm/z695(B)formedinnonenzy- matic in vitro reactions. Insets represent hypothesized position of GSH conjugation. © 2008 by Taylor & Francis Group, LLC [...]... affecting the concentrations of pharmaceuticals released to the aquatic environment, Journal of Contemporary Water Research and Education, 1, 56–65, 2001 © 2008 by Taylor & Francis Group, LLC 212 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems 4 Khachatourians, G.G., Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria, CMAJ, 1 59, 11 29 1136,... harvested and total proteins were extracted daily for 3 days after treatment GST activity was determined using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate © 2008 by Taylor & Francis Group, LLC 210 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems sulfadimethoxine-treated plants showed the highest GST activity, which was unexpected considering the known phytotoxicity of sulfadimethoxine... the maize-treated GST samples, and these reactions incubated six times longer Therefore, these data suggest that CTC-induced toxicity in pinto beans is likely caused by the inability of GSTs to efficiently detoxify the antibiotic, allowing it to negatively affect growth These findings also indicate that nonenzymatic conjugation is much too slow to provide any support for the GSTs 9. 8 OTHER ENVIRONMENTALLY... in sickness and in health., Trends in Plant Science, 5, 193 – 198 , 2000 29 May, M., Vernoux, T., Leaver, C., Van Montagu, M., and Inze, D., Glutathione homeostasis in plants: implications for environmental sensing and plant development, Journal of Experimental Botany 49, 6 49 667, 199 8 30 Aga, D.S., O’Connor, S., Ensley, S., Payero, J.O., Snow, D., and Tarkalson, D., Determination of the persistence of. .. detoxify the antibiotics via GSH conjugation, as in the case of sulfadimethoxine in maize The data suggest that there are two different mechanisms for antibioticinduced phytotoxicity First, in the case of CTC toxicity in pinto beans, it appears that the plant GSTs do have an innate ability to bind and conjugate CTC to GSH; however, pinto beans do not appear to have the ability to induce expression of GSTs... cannot bind the compound to effect its transformation These two hypothetical mechanisms need further investigation and better understanding of the GST biochemical pathways, as well as clarification of mechanisms that trigger a response under antibiotic stress 9. 10 AREAS REQUIRING FURTHER RESEARCH Residues of veterinary antibiotics that are unintentionally applied to soil through the land application of. .. Giese, R.F., and Aga, D.S., Investigating the molecular interactions of oxytetracycline in clay and organic matter: Insights on factors affecting its mobility in soil, Environ Sci Technol 38, 4 097 –4105, 2004 17 Tolls, J., Sorption of veterinary pharmaceuticals in soils: a review, Environ Sci Technol., 35, 3 397 –3406, 2001 18 Gujarathi, N.P., Haney, B.J., Park, H.J., Wickramasinghe, S.R., and Linden, J.C.,... agriculture and needs to be studied more thoroughly On the other hand, antibiotic-induced herbicide resistance could be beneficial in phytoremediation For instance, if chlortetracycline can induce GST expression in maize, then perhaps the isozymes induced can enhance the plant’s ability to detoxify the herbicide Using in vitro assays similar to those described earlier, preliminary results comparing CTC-treated... prevent the accumulation of persistent antibiotics in soil Phytoremediation is an expanding area of research where field studies have shown to be effective in remediating sites contaminated with pollutants such as heavy metals, pesticides, and explosives.33 The findings that GST detoxification is involved in the biotransformation of various antibiotics is encouraging; it suggests they may be removed by the. .. sulfadimethoxine to maize In vitro conjugation reactions provided insight as to the results of the GST activity assays It was found that tylosin was the only antibiotic that formed a GSH conjugate when catalyzed by maize GSTs; no conjugate was formed in the absence of GSTs The inability of GSTs to catalyze the transformation of sulfadimethoxine via GSH conjugation may explain the reported toxicity of this . Group, LLC 204 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems protein and nucleic acid synthesis, including modulation of enzyme activity and adaptation to environmental. LLC 208 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems CTC standard. Furthermore, the mass spectra of these nonenzymatically formed conjugates indicated that the chlorine. Group, LLC 206 Fate of Pharmaceuticals in the Environment and in Water Treatment Systems To verify that these induced proteins were in fact GSTs, enzyme activity assays wereperformedusingthecrudeextractsfromtheplants.Theassayusedwasbased on