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

Phytoremediation, the application of plants and their associated microbes to enhance biodegradation of contaminants in the environment, has recently been The phytoremediation of metals a

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

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

these concerns have not as of yet been verified, 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 significant

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 Detoxification of Xenobiotics via Glutathione Conjugation 201

9.5 Glutathione S-Transferases: Structure, Function, and Evolution 203

9.6 Induction of GSTs in Phaseolus Vulgaris and Zea Mays by Chlortetracycline 204

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 Areas Requiring Further Research 210

References 211

Phytoremediation, the application of plants and their associated microbes to enhance biodegradation of contaminants in the environment, has recently been

The phytoremediation of metals and organic pollutants in soil is an emerging, low-cost technology, but the underlying biochemical mechanisms involved in contami-nant uptake, detoxification, and translocation in plants are largely unknown One well-known detoxification 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-toremediation of organic contaminants and presents recent work on GST-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 plants and can elicit phytotoxicity in susceptible species In fact, it has been known for quite some time that chlortetracycline, a highly used growth-promoting

demonstrated the phytotoxic effects of sulfadimethoxine antibiotics toward maize

However, some plant species do not appear to be affected by exposure to antibiotics For example, in the same study that reported phytotoxicity of chlortetracycline to

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, enrofloxacin, a broad-spectrum antibiotic that is used in both human and veterinary medicine, inhibits root growth and leaf development in a variety of crop

for human health, can inhibit 3-hydroxyl-3-methylglutaryl coenzyme A reductase

in plants, blocking isoprenoid biosynthesis, which is important for a multitude of endogenous functions.12

Direct quantification of the amount of contaminants that accumulate in plant tissues is difficult because of the analytical challenges associated with detecting low levels of analytes within the complex plant biomass Recently, however, Kumar and

accumu-lated in maize, cabbage, and green onions at the parts per billion (ppb) range using enzyme-linked immunosorbent assay (ELISA) techniques Beyond pharmaceuti-cals, other toxic organic compounds have been shown to accumulate in the fruits of

used for industrial and military purposes in the United States, has been found in the environment at levels as high as 500 ppm A controlled greenhouse study has shown that within 2 years of exposure, apple and peach trees were able to accumulate as much as 34 ppm

9.3 BIOAVAILABILITY

The bioavailability of pharmaceuticals is an important factor to consider when investigating their interactions with plants Generally, the toxicity of a contaminant

is directly related to its bioavailability.14In soil, the bioavailability of an antibiotic depends on two major factors: (1) the degree to which it adsorbs to the soil and (2) the organisms inhabiting the soil Sorption of antibiotics to soil is dependent on the soil composition and its chemical characteristics, such as pH and ionic strength Soil composition can vary with regard to its clay, sand, organic matter, and min-eral content, all of which play an important role in antibiotic sorption For instance, sulfonamide antibiotics adsorb more strongly to clay than to sand, and in general,

also be factored into antibiotic bioavailability Sulfonamides and tetracyclines are adsorbed more tightly in the presence of acidic soils, whereas the opposite is true for

char-acteristics and cannot dissolve in water without additional factors, but it appears that hydrophobicity is not directly related to the strength of sorption of these

which are regulated by the soil content and chemistry, are all important in determin-ing antibiotic bioavailability

The rhizosphere, the area surrounding a plant’s roots, is very dynamic and full of microorganisms that play a role in the fate and bioavailability of antibiotics and other contaminants in soil Root exudates in the rhizosphere may contain reactive oxygen

response to oxidative stress Oxytetracycline has been shown to induce the release

inactivation of oxytetracyline via oxidation

9.4 DETOXIFICATION OF XENOBIOTICS

VIA GLUTATHIONE CONJUGATION

Plants have an intricate defense system that is capable of combating a variety of intru-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

a physiological response that is pathogen-/xenobiotic-specific The ability of some plants to detoxify harmful compounds upon uptake via these specific 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 detoxification reactions involving a large number of xenobiotics found in living systems, including plants

The GST enzymes are primarily found in the cytosol of plants, mammals, bac-teria, fungi, and insects The GSTs are part of a three-phase detoxification system involved in detoxifying xenobiotics in living organisms Phase I includes enzymes

such as cytochrome P450 monooxygenases The purpose of the Phase I enzymes is to introduce reactive functional groups via hydroxylation and epoxidation reactions to

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

GST-mediated 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 first documented cases of plant GST-mediated detoxification 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

are constitutively expressed in maize; hence, maize plants are inherently able to

chemi-cals Safeners are chemicals that are applied to plants that do not have the inherent ability to detoxify a chemical, thus inducing GST expression For example, maize has a slight tolerance for thiocarbamate herbicides (EPTC), but pretreatment with a safener such as dichlormid or benoxacor greatly increases induction of Type II GSTs

of metabolizing xenobiotics using different mechanisms, they may be susceptible

if the metabolic pathway they use for detoxification is not efficient For instance,

H2N

H

H COOH COOH

CH2 O O

S

NO2

NO2

H2N

H

H COOH COOH

CH2 O

O

SH

Cl

NO2

NO2

–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 CDNB by the sulfur atom of glutathione, producing a dechlorinated conjugate.

maize detoxifies atrazine via GST conjugation, which is an efficient mechanism.

In contrast, a pea plant metabolizes atrazine slowly and inefficiently via

to atrazine toxicity

GST-mediated transformations of xenobiotics is not the only mechanism of detoxification in the Phase II pathway Glucosyl- and malonyltransferases are Phase

enzymes serve similar functions as GST with respect to “tagging” a xenobiotic and

enzymes appear to conjugate at the carboxylic acid and amine R-groups of the pes-ticide, 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 five classes based on sequence identity These classes include: phi, tau, theta, zeta, and lambda, in which

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

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 the large number of compounds GSTs can bind

Electrophilic xenobiotics are particularly deleterious to living organisms,

electro-philes: soft and hard An example of each type of electrophile that is typically found

in a xenobiotic includes alkene groups (carbon-carbon double bonds) and halogens, respectively GSTs can mediate the conjugation of both types of electrophiles GSH conjugation “tags” a xenobiotic for further processing Processing of the “tagged” xenobiotic is considered as Phase III of the detoxification pathway, and the end result 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

xenobiotic conjugate requires Phase III proteins, which are ATP-dependent trans-porters with the ability to recognize and bind to GSH

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

protein and nucleic acid synthesis, including modulation of enzyme activity and

ranging from temperature extremes to xenobiotic stress GSH is found in two forms within a cell: an oxidized form, in which a disulfide bond is formed between two glutathione molecules (GSSG), and the reduced form (GSH) The ratio of the two forms is crucial to how a plant adapts to its stressor A lack of free GSH can dimin-ish a plant’s ability to mount an appropriate response to a stressor Understanding the GSH biosynthetic pathway and the mechanisms by which it is utilized by various enzymes will provide insight into xenobiotic detoxification by plants Activation of GSH biosynthesis is based on the ability of proteins involved in photosynthesis to act as an intricate sensory system to respond to variations in redox potential caused

by environmental stress

Environmental stress also induces other enzymes that are part of the detoxi-fication pathway Glutathione reductase is an enzyme that reduces GSSG to GSH, thus increasing the concentration of free GSH Glutathione peroxidases (GPX) are antioxidant enzymes A plant, as a reaction to environmental stress, produces ROS

to contain the stressor within the site where the stressor is introduced GPXs are used

to prevent oxidative damage by oxidizing two GSHs to form GSSG GSTs also have peroxidase activity (although they are encoded by a different gene family) and their

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 MAYS BY CHLORTETRACYCLINE

The authors of this chapter performed similar experiments to those reported

in response between maize and pinto beans grown in antibiotic-treated soil Ten-day-old maize and pinto beans were transplanted into soil pretreated with 20 mg

NH2 OH N

H3C CH3

OH

H3C

OH Cl

FIGURE 9.2 The chemical structure of chlortetracycline The arrows depict potential sites

of glutathione conjugation.

environment.30 CTC is a good candidate for initial investigations into a plant’s response for two reasons: it is widely used with high application rates in agriculture,

harvested daily for 3 days and extracted for analysis of total proteins To gain a gen-eral perspective of the response of the plants to the antibiotic, the total proteins were subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Interestingly,

a distinct increase in bands at the range of 20 to 30 kDa from the maize samples grown in CTC-treated soil was observed, relative to the maize control (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

Days Posttreatment

0.00 0.02 0.04 0.06

0.08

MCR MTR

0.00 0.02 0.04 0.06

0.08

PCR PTR

FIGURE 9.3 GST activity measured from total protein extracts at 1, 2, and 3 days after

plants were treated with CTC (A) Maize control (MCR) and CTC-treated (MTR) plants (B) Pinto bean control (PCR) and CTC-treated (PTR) plants Values represent the mean and standard deviation of six replicates Asterisks denote statistically significant data (p < 0.05).

A

B

To verify that these induced proteins were in fact GSTs, enzyme activity assays were performed using the crude extracts from the plants The assay used was based

on the standard GST-catalyzed conjugation reaction of GSH to

significantly higher relative to the control (untreated plants) on the first and third

beans showed no significant differences between treated and control plants in any of the days sampled (Figure 9.3B), 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

9.81

9.16

NH2 OH N H3C CH3

O OHO O H3C OH

N H

NH2 O

O CH2

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

1.93

1.54 HOOC

N H

NH2 O

O CH2

COOH

SH

Cl

OH OH

NH2 OH N H3C CH3

O OHO O H3C OH

Glutathione S-Transferase

+

FIGURE 9.4 (Continued)

9.7 MASS SPECTRAL CHARACTERIZATION

OF ANTIBIOTIC-GSH CONJUGATES

To demonstrate the involvement of GSTs in CTC transformation, in vitro conjugation

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 were conducted containing all reactants,

excluding the GST enzyme (no plant extract added) Results from the LC/MS/MS

CTC-GSH conjugate (Figure 9.4A) These peaks were characterized by the absence

of a chlorine signature in the mass spectra and very short chromatographic retention time, indicating increased polarity relative to the unconjugated CTC (Figure 9.4B) MS/MS analysis revealed an ion with a m/z of 677, which corresponds to the GSH-CTC conjugate with the loss of glycine (MW 75 Da) (Figure 9.4C) 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

m/z (C) 0

20

40

60

80

100

548

659

530 506 404

OH OH

NH2

OH N H3C CH3

O O O

OH H3C OH

H

NH2 O

O CH2

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

to the observed conjugate and the similarities in isomeric peaks (C) chemical structure of the fragment ion m/z 677 and its MS/MS fragmentation spectrum.

CTC standard Furthermore, the mass spectra of these nonenzymatically formed

This suggests that GSH was able to conjugate to CTC nonenzymatically but at sites other than the chlorine atom

Enzymatic conjugation of GSH to CTC occurred when either maize or pinto bean 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)

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+H2O)] +

654

OH OH

NH2 OH N H3C CH3

O OHO O

H3C OH HN

NH2

O

O H2 C HOOC

S

Cl

H3C

0

20

40

60

80

100

[M695–(Glu+H2O)] +

548

566

602 531

402

m/z 695

D C B A

695 [M695–H2O]+

[M695–Glu]+

[M695–(Glu+H2O+NH3)] +

OH OH

NH2 OH N H3C CH3

O O O

OH

OH

NH H2N

O

O H2 C COOH

S

Cl

FIGURE 9.5 LC/ESI-MS/MS spectra of m/z 654 (A) and m/z 695 (B) formed in

nonenzy-matic in vitro reactions Insets represent hypothesized position of GSH conjugation.