Changes in Hepatic Phase I and Phase II Biotransformation Enzyme Expression and Glutathione Levels Following Atrazine Exposure in Female Rats

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Changes in hepatic phase I and phase II

biotransformation enzyme expression and
glutathione levels following atrazine exposure in
female rats
Arthur D. Zimmerman, Charles B. Breckenridge, Kun D. Yi, Pragati Sawhney

Coder, Desiree Wanders, Robert L. Judd & Chad D. Foradori
To cite this article: Arthur D. Zimmerman, Charles B. Breckenridge, Kun D. Yi, Pragati Sawhney
Coder, Desiree Wanders, Robert L. Judd & Chad D. Foradori (2017): Changes in hepatic phase
I and phase II biotransformation enzyme expression and glutathione levels following atrazine
exposure in female rats, Xenobiotica, DOI: 10.1080/00498254.2017.1374486
To link to this article: http://dx.doi.org/10.1080/00498254.2017.1374486
Accepted author version posted online: 08

Sep 2017.
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http://www.tandfonline.com/action/journalInformation?journalCode=ixen20
Download by: [Australian Catholic University] Date: 09 September 2017, At: 07:31
Changes in hepatic phase I and phase II biotransformation enzyme expression and
glutathione levels following atrazine exposure in female rats
Arthur D. Zimmerman1, Charles B. Breckenridge2, Kun D. Yi2, Pragati Sawhney Coder3, Desiree
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Wanders4, Robert L. Judd1 and Chad D. Foradori1
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Downloaded by [Australian Catholic University] at 07:31 09 September 2017
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1
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Department of Anatomy, Physiology and Pharmacology, Auburn University, College of
Veterinary Medicine, 210 Greene Hall, Auburn, AL 36849-5518, USA
2
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Syngenta Crop Protection LLC, 410 Swing Road, PO Box 18300, Greensboro, NC 27419-8300,
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USA
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Battelle Memorial Institute, Columbus, OH 43201
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Department of Nutrition, Georgia State University, 140 Decatur St SE, Atlanta, GA 30303-
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3995, USA
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Funding: Syngenta Crop Protection LLC.

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Running Head: Changes in liver enzymes due to ATR treatment
Summary: ATR treatment leads to an up regulation of multiple phase I and II hepatic
components by 4 days of treatment (100 mg/kg). However, by 14 days of treatment the majority
of assayed components had returned to control levels, suggesting a possible habituation or an
establishment of dominant mediators of ATR metabolism over time.

Abstract
1. To determine the effects of repeated atrazine (ATR) treatment on hepatic phase I and II
enzymes, adult female rats were treated with vehicle or 100mg/kg of ATR for 1, 2, 3 or 4
days. Glutathione-s-transferases (GST) mRNA expression, protein levels (mu, pi, alpha,
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omega), and activity (cytosolic and microsomal), along with bioavailable glutathione
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(GSH) were assayed.
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2. GST expression, concentrations, and activity were increased, along with GSH levels, in
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animals treated with ATR for 3 and 4 days.
3. A subsequent study was performed with animals treated with vehicle, 6.5, 50 or
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100mg/kg/day for 4, 8 or 14 days. Expression of hepatic Phase I CYP 450 enzymes was
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evaluated in conjugation with GST expression, protein, and activity. Nineteen of the 45
CYP enzymes assayed displayed increased mRNA levels after 8 days of treatment in
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animals treated with 50 or 100mg/kg/day. After 14 days of treatment, all CYP expression
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levels returned to control levels except for CYP2B2, CYP2B3, CYP2C7, CYP2C23,
CYP2E1, CYP3A9, CYP4A3, and CYP27A1, which remained elevated.

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4. Results indicate that there may be a habituation or adaptation of liver Phase I and Phase II
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expression following repeated ATR treatment.
Keywords: Liver enzymes, herbicides, rat, female, habituation

Introduction
Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine; ATR) belongs to the class
of chloro-s-triazine herbicides that are applied pre- and early post-emergence to control weeds in
a number of triazine-tolerant crops including corn, sorghum, and sugarcane (USDA, 2006). ATR
blocks electron transfer at the reducing site of photosystem complex II in the chloroplasts of
plants by binding to the plastoquinone-binding protein (Gysin and Knuesli, 1960; Good, 1961),
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resulting in energy depletion and oxidative damage. In mammals, ATR is rapidly absorbed,
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dealkylated in the liver by CYP P450 liver enzymes (Lang et al., 1996) and eliminated in urine
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and feces, predominantly as glutathione (GSH) conjugated chlorotriazine mercapturates. ATR
has a terminal half-life of elimination of approximately 31 hrs in humans (Campbell et al., 2016)
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and plasma clearances of 2.4, 6.9, 6.0 and 8.1 hrs for atrazine and its three chlorotriazine (DEA,
DIA and DACT) metabolites, respectively, in female rats administered ATR by gavage
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(McMullin et al., 2003).
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ATR administered to rats by gavage results in a rapid rise in corticosterone (Pruett et al.,
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2003; Fraites et al., 2009; Laws et al., 2009; Foradori et al., 2011). ATR-induced rise in plasma
corticosterone levels is partially responsible for the ATR-induced reduction in luteinizing

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hormone (LH) release in rats (Foradori et al., 2011). High peak plasma ATR levels are thought to
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be required for ATR to increase plasma corticosterone or inhibit LH release because bolus
delivery of ATR is effective in inducing a rise in corticosterone concentrations and reducing LH
release, whereas similar doses distributed throughout the day by incorporating ATR in the diet
are ineffective. We have recently demonstrated that even bolus ATR-induced corticosterone
release and LH release inhibition are attenuated after repeated treatments (7 to 21 days of
treatment) in female rats (Breckenridge et al., 2017; Foradori et al., 2017). The habituation or
tolerance to ATR over repeated treatment in female rats may be due to alterations in the hepatic
enzymes and substrates responsible for ATR metabolism leading to reduced peak plasma levels.
Previous studies have examined some components of both phase I and phase II
biotransformation after ATR exposure. In vitro studies have demonstrated that CYP1A1/2,
CYP2B1/2, CYP2C11, CYP2D1 and CYP2E1 are involved in ATR metabolism, with
CYP2B1/2 being implicated as the predominant enzyme (Hanioka et al., 1998a; Hanioka et al.,
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1998b; Hanioka et al., 1999). In vivo studies using male rats suggest that CYP1A1/2 may
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contribute more to the phase I metabolism of ATR than CYP2B1/2 (Islam et al., 2002; Pogrmic-
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Majkic et al., 2012). However, these studies were limited in scope and did not examine hepatic
CYP expression levels after repeated exposures and doses.

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In vitro studies of phase II metabolism of ATR in rat (Egaas et al., 1993) and human liver
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fractions (Lucas et al., 1993; Jaeger et al., 1998; Buchholz et al., 1999) indicate that GSH
conjugation is involved with glutathione-s-transferase pi (GST P) activity. GST P is increased in

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the rat (Islam et al., 2002; Pogrmic-Majkic et al., 2012) and mouse (Egaas et al., 1995a; Abel et
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al., 2004) following ATR treatment. However, these studies characterized only a few of the GST
isoforms and did not systematically evaluate the time course of enzyme induction.
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In the present study, we examined the effects of ATR exposure on the expression of
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hepatic CYP and GST enzymes, GST protein/activity levels, and GSH levels after a range of
ATR doses (0, 6.5, 50 or 100 mg/kg) and treatment durations (daily for 1, 2, 3, 4, 8 or 14 days) in
the female Sprague Dawley rat. We identified changes in Phase I and Phase II enzymes which
are dose- and duration-dependent, implicating several factors in xenobiotic metabolism which
may affect ATR metabolism.

Materials and Methods
Animals. Eight to nine-week-old female Sprague Dawley rats (Crl:CD(SD)) were received in
two shipments from Charles River Laboratories Inc. (Raleigh, NC) and acclimated and
maintained in AAALAC approved animal facilities at WIL Research Laboratories LLC.
(Ashland, Ohio). All animal procedures and experimental protocols were approved by the WIL
Research Institutional Animal Care and Use Committee. Rats were a minimum of 12 weeks old
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at the beginning of the study. Animals were individually housed in clean suspended wire-mesh
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cages in an environmentally controlled room (22 ± 3°C; 50 ± 20% humidity) on a 14-hour
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light/10-hour dark photoperiod (lights on at 0500h, off at 1900h) with water and food (Rodent
LabDiet, PMI Nutrition International LLC) available ad libitum. All animals were
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ovariectomized and implanted with a 12-14 mm long estradiol (4 mg/mL in sesame oil) silastic
capsule before additional procedures were performed. All animals received a single daily dose
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of vehicle (1% methylcellulose sodium salt in deionized water; 5 mL/kg) 7 days prior to the first
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day of treatment.
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Atrazine Formulation and Administration

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ATR was supplied by Syngenta Crop Protection, LLC, as an analytically-certified, 98.8% pure,
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white powder that was stable for use during the period of the study. ATR was prepared
approximately weekly as a suspension in 1% methylcellulose and deionized water at
concentrations of 1.3, 10, or 20 mg/mL. Suspensions were stored refrigerated (at 2 to 8°C) until
use. Sample storage stability and sample homogeneity were established by high-pressure liquid
chromatography (HPLC) prior to study conduct. The concentration of ATR, which was assessed
in every dose suspension by HPLC, ranged from of 94.7 to 101% of target.

Experiment 1: Effects of ATR treatment (100 mg/kg/day) on Phase II hepatic components
after 1, 2, 3, or 4 days of treatment
Animals were administered ATR (100 mg/kg) or vehicle by gavage once daily for 1, 2, 3 or 4
consecutive days (10-15 animals per group). After 1, 2, 3 or 4 days of treatment, blood was
collected periodically after lights-on in order to evaluate the effects of repeated ATR on the
luteinizing hormone (LH) surge; the in-life data and the LH results are provided in a separate
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publication (Breckenridge et al., 2017). Following completion of the last treatment, animals were
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euthanized by carbon dioxide inhalation and the right lobe of the liver was collected. Tissue was
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flash frozen in liquid nitrogen and stored at -70°C until analyzed for Phase II hepatic
components.
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Experiment 2: Effects of ATR treatment (6.5, 50 or 100 mg/kg/day) on Phase I and II hepatic
components after 4, 8 or 14 days of treatment

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Animals were administered ATR (6.5, 50 or 100 mg/kg) or vehicle by gavage once daily for 4, 8
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or 14 consecutive days (10-15 animals per group). After 4, 8 or 14 days of treatment, blood was
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collected periodically after lights-on in order to evaluate the effects of repeated ATR on the
luteinizing hormone (LH) surge; the in-life data and the LH results are provided in a separate
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publication (Breckenridge et al., 2017). Following completion of the last treatment, animals were
euthanized by i.p. injection of pentobarbital sodium and the right lobe of the liver was collected.
Liver tissue was flash frozen in liquid nitrogen and stored at -70°C until analyzed. Considering
the profound changes in Phase II hepatic components identified in experiment 1, experiment 2

analysis was expanded to included quantification of hepatic CYP enzymes along with Phase II
components.
RNA Extraction
RNA extraction was performed using the Qiagen (Valencia, CA) RNeasy Microarray Tissue kit
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(73304). Tissue (40-60 mg) from the right lobe of the liver was weighed and placed in 1 mL of
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QIAzol lysis reagent in a DNase/Rnase-free 1.7 mL microcentrifuge tube. Tissue was
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homogenized for 30-40 seconds using a pellet mixer (VWR, Radnor, PA; 47747-370). Following
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homogenization, the homogenate was placed on the bench top at room temperature for 5 minutes
to promote dissociation of the nucleoprotein complexes. Chloroform (200 µL) was added to the
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homogenate, mixed and allowed to sit at room temperature for 3 minutes. Homogenates were
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then centrifuged at 12,000 x g for 15 minutes at 4°C. The upper aqueous layer was transferred to
a new microcentrifuge tube and mixed with 600 μL of 70% ethanol. The mixture was transferred
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to an Rneasy Mini spin column in a 2 mL collection tube and centrifuged at 8,000 x g for 15
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seconds at room temperature. On-column DNase Digestion was performed using the Rnase-free
DNase set (Qiagen; 79254). RNA was eluted off the spin column and collected in a 1.5 mL
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collection tube. Sample concentration (ng/μL) and purity (260/280 ratio) were determined using
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the Thermo Scientific (Pittsburgh, PA) NanoDrop ND-1000 Spectrophotometer. cDNA synthesis
was performed using the Qiagen RT² First Strand Kit (330401).
Real-time PCR Analysis-GST
Real-time PCR analysis was performed using a custom Qiagen RT² Profiler PCR array. Eight
liver samples were selected at random from each control and ATR-treated groups, and mRNA
expression levels for 13 isoforms of GST were analyzed: GST alpha 3 (GSTA3), GST alpha 4
(GSTA4), GST kappa 1 (GSTK1), GST mu 1 (GSTM1), GST mu 2 (GSTM2), GST mu 3
(GSTM3), GST omega 1 (GSTO1), GST omega 2 (GSTO2), GST pi 1 (GSTP1), GST theta 1
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(GSTT1), GST theta 2 (GSTT2), microsomal GST 1 (mGST1) and microsomal GST 2 (mGST2).
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In order to find an appropriate reference gene for qPCR normalization, gene expression analysis
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was carried out on four potential genes (Gapdh, Ldha, Rpl13a and Rplp1) from liver samples
from all treatment groups. For all groups, Rplp1 was found to be the most stable and was chosen
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to be the optimal gene expression for normalization in the female rat liver after ATR treatment.
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The commercially generated plates were calibrated for optimal amplification efficiency. Internal
controls included tests for genomic DNA control (GDC), reverse-transcription control (RTC),
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and positive PCR control(PPC). The GDC specifically detects nontranscribed genomic DNA
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contamination with a high level of sensitivity. The RTC tested the efficiency of the reverse-
transcription reaction performed with the RT2 First Strand Kit by detecting template synthesized
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from the kit’s built-in external RNA control. The PPC consists of a predispensed artificial DNA
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sequence. This control tests the efficiency of the polymerase chain reaction itself. In all of the
plates run, GDC, RTC, and PPC controls, as well as, interwell and intra-plate consistency were
within the recommended limits pre-determined by Qiagen. All primer sets were verified by
Qiagen for the custom PCR array. PCR was carried out using the Bio-rad iCycler iQ real-time
detection system with RT² SYBR Green qPCR mastermix (Qiagen; 330513). Fluorescence data
collection was performed for 40 cycles at 95°C for 15 seconds and 40 cycles at 60°C for 1
minute. The fold change of each target mRNA expression relative to Rplp1 under experimental
and control conditions were calculated based on the threshold cycle (CT) as r = 2-Δ(ΔCT), where
ΔCT = CT(target)−CT(Rplp1) and Δ(ΔCT) = ΔCT(experimental)−ΔCT(control).
GSH Analysis
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Total GSH levels were determined using the Cayman Chemical GSH assay kit (Ann Arbor,
Michigan). Liver samples (100-200 mg) were homogenized using the Precellys 24 (Bertin
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Technologies, Rockville, MD) at 5000 rpm, twice for 10 seconds each time and spun at 10,000 x
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g for 15 minutes at 4 °C. Total protein content of the supernatant was measured according to the
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Bradford method (Bradford, 1976) with bovine serum albumin (BSA) as a protein standard.
Protein levels for all samples were standardized to 10 mg/mL. Samples were deproteinated with
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metaphosphoric acid followed by the reaction of the sulfhydryl groups of GSH with 5,5'-dithio-
bis-2-nitrobenzoic acid (DTNB, Ellman’s reagent) to produce a yellow colored product, 5-thio-2-
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nitrobenzoic acid. The product was measured at 412 nm using the Molecular Devices
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(Sunnyvale, CA) Spectra Max Plus384, and the absorbance was compared to a standard curve
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range of 0-8.0 μM oxidized glutathione (GSSG) using the Softmax Pro (Molecular Devices) data
software. All samples were assayed in duplicate, and the results were expressed as nmol
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GSH/mg protein.
Western Blotting. The same animals from the GST mRNA analysis were used to measure GST
protein levels. Protein extracts (75 µg) and low molecular weight standards (Bio-rad, 161-0305)
were separated by electrophoresis at 200 V for 50 minutes in a 12% Criterion TGX

polyacrylamide gel (Bio-rad; 567-1045). Protein extracts were transferred for 1 hour at 100 V to
a nitrocellulose membrane (Bio-rad; 162-0116). Blots were blocked using equal volumes of Li-
Cor (Lincoln, NE) Odyssey Blocking Buffer (927-40000) and PBS (.1M; pH 7.2), and placed on
a rotator for 1 hour. Blots were incubated overnight at 4°C on a rotator with 12 mL of primary
antibody cocktail [Primary antibody cocktails used were as follows: Mouse anti-GST alpha
(MyBioSource, San Diego, CA, MBS560696; 1:1000) with Goat anti-GST mu (Abcam,
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Cambridge, MA, Ab53942; 1:1000), Goat anti- GST pi (Abcam, Cambridge, MA, Ab53943;
1:1000) with Rabbit anti-GST omega (Abcam, Ab129106; 1:1000)]. Blots were rinsed and
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incubated in secondary antibody cocktail [secondary antibody cocktails used were as follows:
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Donkey anti-Goat (Odyssey 800 CW; 926-32214; 1:10,000) with Donkey anti-Mouse (Odyssey
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680 RD; 926-68072; 1:10,000) or Donkey anti-Rabbit (Odyssey 680 RD; 926-68073; 1:10,000)].
Blots were washed and stored in PBS until imaged. All blots were imaged using the Li-Cor
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Odyssey Infrared Imaging System, and analyzed using the Odyssey Software v2.0. Beta-actin
(Mouse anti-Actin; Millipore, Billerica, MA, MAB1501; 1:1000) was run as a loading control
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protein, and protein levels for each GST isoform were standardized to beta-actin levels.
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GST Analysis
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GST enzymatic activity was determined using the Cayman Chemical GST assay kit. Using 10
mg/mL standardized protein samples, homogenate was diluted to 1 mg/ml in potassium
phosphate buffer (100 mM potassium phosphate, 2 mM EDTA, pH 7.0), placed into 3.0 mL
sterile ultracentrifuge tubes (Beckman Coulter, 355870), heat sealed and spun in an ultra-speed
centrifuge (Beckman L8-70M, Brea, CA) at 100,000 x g for 60 minutes at 4C. Cytosolic
fraction was placed in a new sterile tube, and microsomal pellet was re-suspended in 500L of
cold homogenization buffer. Protein concentration of both fractions was determined by protein
assay (BioRad). GST activity for both fractions were determined spectrophotometrically by
measuring the rate of formation of the conjugate of GSH and 1-chloro-2,4-dinitrobenzene
(CDNB). The absorbance was read at 340 nm every 60 seconds for 6 minutes using the
Molecular Devices Spectra Max Plus384 plate reader. All samples were assayed in duplicate,
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and enzymatic activity was expressed as nmol/min/mg protein. Data were analyzed using
Softmax Pro software.
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Real-time PCR Analysis-CYP Enzymes an
Real-time PCR analysis was performed using a custom Qiagen RT² Profiler PCR array. Liver
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samples (4-5) were randomly selected from the 8 liver GST mRNA samples analyzed from
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experiment 2. mRNA expression levels for 45 isoforms of Cytochrome P450 (CYP) enzymes
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were analyzed: CYP1A1, CYP1A2, CYP1B1, CYP2A3, CYP2B2, CYP2B3, CYP2C6,

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CYP2C7, CYP2C8, CYP2C11, CYP2C22, CYP2C23, CYP2C37, CYP2D2, CYP2D4, CYP2E1,

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CYP2F4, CYP2R1, CYP2S1, CYP2T1, CYP2W1, CYP3A9, CYP3A18, CYP3A23/3A1,
CYP4A3, CYP4A8, CYP4B1, CYP4F1, CYP4F4, CYP4F6, CYP4F18, CYP4F40 CYP7A1,

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CYP7B1, CYP8B1, CYP11A1, CYP11B1, CYP17A1, CYP19A1, CYP24A1, CYP26A1,
CYP26B1, CYP26C1, CYP27A1, and CYP27B1. All primer sets were verified by Qiagen for the
custom PCR array. After reverse transcription, PCR was carried out using the Bio-rad iCycler iQ
real-time detection system with RT² SYBR Green qPCR mastermix (Qiagen; 330513). One cycle
was performed at 95°C for 10 minutes to activate the HotStart DNA Taq Polymerase.
Fluorescence data collection was performed for 40 cycles at 95°C for 15 seconds and 40 cycles
at 60°C for 1 minute. The fold change of each target mRNA expression relative to Rplp1 was
calculated based on the threshold cycle (CT) as r = 2-Δ(ΔCT), where ΔCT = CT(target)−CT(Rplp1)
and Δ(ΔCT) = ΔCT(experimental)−ΔCT(control).
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Statistical Analysis
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Hepatic CYP mRNA expression data were analyzed on day four of ATR treatment and compared
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to same day control animals using Student’s t-test. All other data were analyzed for all time
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points using 2-way analysis of variance (ANOVA) for treatment, days of treatment and treatment
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× days of treatment interactions with Bonferroni post-hoc tests. The level of statistical
significance was set at P ≤ 0.05. All values are reported as the mean ± SEM. Prism 5 for Mac
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(GraphPad Software, Inc.) was used for all data analysis.
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Results
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Experiment 1: Effects of ATR treatment (100 mg/kg/day) on Phase II hepatic components
after 1, 2, 3, or 4 days of treatment

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GST isoform expression
ATR-treated animals did not have a significant increase in liver mRNA expression levels after
one or two days of exposure for any of the GST enzymes measured (Table 1). After three days of
exposure, ATR-treated animals showed an increase in liver mRNA expression levels compared
to their corresponding control group for 5 GST isoforms (GSTA3, GSTM1, GSTM3, GSTP1 and
mGST2; Figure 1; Table 1). Following four days of ATR treatment, eleven GST isoforms
showed increased mRNA expression levels as compared to control (GSTA3, GSTK1, GSTM1,
GSTM2, GSTO1, GSTO2, GSTP1, GSTT1, GSTT2, mGST1, mGST2; Table 1). Four GST
isoforms, which were elevated above controls levels after 3 days of treatment, remained elevated
(GSTA3, GSTM1, GSTP1, mGST2; Figure 1). Seven additional GST isoforms were only
elevated after 4 days of treatment (GSTK1, GSTM2, GSTO1, GSTO2, GSTT1, GSTT2, mGST1;
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Table 1). GSTM3 was the only isoform which was elevated after 3 days of treatment but was not
different from control levels after 4 days of treatment.
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GST protein levels an
Western blots analysis identified no differences in protein levels for GST mu (GST M), GST pi
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(GST P), GST alpha (GST A) or GST omega (GST O) after 1, 2, 3 or 4 days of ATR treatment
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(Figure 2, Supplemental Table 1).

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Bio-reactive GSH levels and GST activity

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ATR-treated animals showed increased liver GSH levels after 3 and 4 days of treatment
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compared to control (Figure 3A). ATR treatment (100 mg/kg/day) led to increased hepatic cGST
activity after 3 and 4 days of treatment but not after 1 or 2 days (Figure 3B). There was no effect
of ATR treatment on microsomal GST activity (Figure 3C).

Experiment 2: Effects of ATR treatment (6.5, 50 or 100 mg/kg/day) on Phase I and II hepatic
components after 4, 8 or 14 days of treatment
CYP enzyme expression
All CYP isoform expression data are presented in Table 2 with a select number depicted in
Figure 4. Animals treated once daily for 4, 8 or 14 days with 6.5, 50, or 100 mg/kg of ATR had
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significantly increased liver mRNA expression for CYP1A2, CYP26A1, CYP27A1, CYP2B2,
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CYP2B3, CYP2C22, CYP2C23, CYP2C37, CYP2C7, CYP2D2, CYP2E1, CYP2F4, CYP2R1,
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CYP2T1, CYP3A18, CYP3A23/3A1, CYP3A9, CYP4A3, CYP4F1, CYP4F6 and CYP7A1
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(Table 2, Figure 4). There was no treatment effect on expression levels of CYP17A1, CYP4B1 ,
CYP8B1, CYP2C6, CYP2D4, and CYP4F4. CYP7B1 was the only isoform found to be
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decreased in expression (6.5 and 100 mg/kg after 4 days), and although CYP8B1 expression was
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also decreased, it was not statistically significant.
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Of the CYP isoforms found to have increased expression after ATR treatment, 3 were
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elevated above control after 4, 8 and 14 days of treatment (CYP2B3, CYP2C23, CYP3A9;
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Figure 4B). Of these isoforms, CYP3A9 was the only isoform increased in 6.5 mg/kg treated
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animals. All other increases were only identified in 50 or 100 mg/kg ATR-treated groups.
CYP1A2 was only elevated after 4 days of ATR but was no different from control levels after 8
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and 14 days of treatment. CYP3A23/3A1, CYP2C22, and CYP2R1 were elevated compared to
control animals after 4 and 8 days of treatment but not 14 days (Table 2, Figure 4D). CYP26A1,
CYP2C37, CYP2D2, CYP2F4, CYP2T1, CYP3A18, CYP4F1, CYP4F6 and CYP7A1 were
increased only after 8 days of treatment (Figure 4E). CYP27A1, CYP2C7, CYP2E1 and
CYP4A3 were increased after 8 days of treatment and remained high after 14 days (Figure 4C
and 4F). Although CYP2B2 was only statistically significantly elevated after 14 days of
treatment at 100 mg/kg, it had the largest increase in expression compared to control levels
(Table 2, Figure 4A). Expression of CYP2C6, CYP2D4, CYP11A1, CYP11B1, CYP19A1,
CYP1A1, CYP1B1, CYP24A1, CYP26B1, CYP27B1, CYP2A3, CYP2C11, CYP2C80,
CYP2S1, CYP2W1, CYP4A8, CYP4F18 and CYP4F40 were below detection.
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GST isoform expression
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All GST isoform expression data from experiment 2 are presented in Table 3 with a select
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number depicted in Figure 5. Animals treated with ATR once daily for 4 days with 50 and 100
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mg/kg showed a significant increase in expression levels in GSTA3, GSTO1 and mGST2
compared to control (Figure 5). GSTA4 and GSTK1 were elevated compared to controls after 4
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days of treatment but only at the highest dose of 100 mg/kg/day (Figure 5; Table 3). After 8 days
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of treatment, all GST isoforms, with the exception of GSTO2, mGST1 and mGST2, showed an
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increase in expression at 100 mg/kg/day compared to controls (Table 3). Furthermore, animals
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treated for 8 days with ATR also showed an increased expression for GSTt1 at 6.5, 50 and 100
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mg/kg/day compared to control. GSTP1, mGST1, GSTA3, GSTA4, GSTK1, GSTM2, GSTM3,
GSTO1 and GSTT1 all showed an increase in expression after 8 days at 50 mg/kg/day compared
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to control (Figure 5; Table 3). Conversely, after 14 days of treatment, only mGST2 at 100
mg/kg/day was significantly elevated above control. None of the other GST expression levels
were significantly elevated above control levels after 14 days of treatment. Three GST isoforms,
GSTA4, GSTK1, and mGST1, were below control levels in the 50 mg/kg/day ATR animals.
While not statistically significant in all cases, multiple GST isoform expression levels (GSTA3,
GSTA4, GSTK1, GSTM3, GSTT1 and mGST1) were trending lower after 14 days of 50 and/or
100 mg/kg of atrazine treatment (Table 3).
GST protein levels
Using western blots, we found no significant difference in protein levels for GST M, GST P or
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GST A after 4, 8 or 14 days of ATR treatment (Figure 6A-C). GST O was elevated in the
animals treated with 100 mg/kg/day when compared to controls but only on day 4 of treatment
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(Figure 6D, Supplemental Table 2)
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Bio-Reactive GSH levels and GST activity
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There was a significant treatment effect of ATR on GSH levels. ATR-treated animals showed
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increased liver GSH levels at 6.5, 50 and 100 mg/kg/day after 4 days of treatment compared to
e
control and 100 mg/kg/day after 14 days of treatment (Figure 7A). ATR treatment did not have a
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significant effect on liver GSH levels after 8 days of treatment at any dose. There was no
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increase in cGST or mGST activity after 4 or 8 days of treatment, but ATR treated animals
showed an increase in cGST activity after 14 days of treatment in the 100 mg/kg/day group
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compared to control (Figure 7B). ATR treatment had no effect on mGST activity at any dose or
duration of treatment (Figure 7C).

Discussion
In the present study, we characterized the changes in hepatic GST mRNA expression,
GST protein levels, GST activity, and GSH levels after 1, 2, 3 and 4 daily doses of 100 mg/kg of
ATR. Considering the changes in phase II components identified in experiment 1, in a follow up
study, the hepatic expression 45 isoforms of CYP enzymes were analyzed in conjugation with
phase II mediators GST expression, protein concentration and activity, along with GSH
t
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concentration after 4, 8 and 14 days of ATR at daily doses of 6.5, 50 or 100 mg/kg. Our results
cr
are consistent with previous studies that showed that both hepatic phase I CYP P450 and phase II
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GST enzymes play a role in the metabolism of ATR. In addition, by examining multiple time
points and different doses, we demonstrated the presence of dynamic changes in the expression
an
of hepatic enzymes linked to xenobiotic metabolism.
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Of the 45 CYP enzyme isoforms evaluated for hepatic mRNA expression, levels were
consistently below the limits of detection for 16 isoforms (CYP1A1, CYP1B1, CYP2A3,
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CYP2C11, CYP2C80, CYP2S1, CYP2W1, CYP4A8, CYP4F18, CYP4F40, CYP11A1,

e
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CYP11B1, CYP19A1, CYP24A1, CYP26B1, and CYP27B1,). Expression of mRNA was not
significantly altered in an additional 6 isoforms (CYP2C6, CYP2D4, CYP4B1, CYP4F4,

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CYP8B1, and CYP17A1) in animals administered ATR at doses of 6.5, 50 or 100 mg/kg/day for
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4, 8 or 14 days. Of the 17 CYP isoforms where mRNA expression was significantly altered in
the high-dose (100 mg/kg/day) group, 7 isoforms displayed increased expression on Day 4
(CYP2B3, CYP2C22, CYP2C23, CYP2R1, CYP3A23/3A1 and CYP39A) and mRNA
expression was decreased in 1 isoform (CYP7B1). On day 8, mRNA expression was
significantly increased in 13 isoforms (CYP2B3, CYP2C23, CYP2C7, CYP2D2, CYP2E1,
CYP2F4, CYP3A18, CYP3A23/3A1, CYP4A3, CYP4F1, CYP4F6, CYP26C1, and CYP27A1),

but no longer significantly increased in CYP2C22, CYP2R1 or CYP3A9. Also on day 8,
CYP7B1 was no longer significantly decreased. On day 14, mRNA expression was increased in
5 isoforms that were previously increased on day 8 or day 4 (CYP2B3, CYP2C23, CYP2C7,
CYP2E1 and CYP3A9) and was not significantly decreased in any isoform. One isoform was
elevated in the 100 mg/kg group for the first time after 14 days (CYP2B2).
Based on these results it can be concluded that in the higher dose groups (50-100
t
ip
mg/kg/day), CYP isoforms were generally not induced by day 4, reached a peak by day 8 and
cr
declined thereafter. Expression of CYP2B2 displayed the largest mean dose- and time-dependent
us
increase after ATR treatment (Figure 4A, Table 2). The expression of CYP7B1 and to a lesser
extent CYP26C1, CYP2C6, CYP4A3, CYP4B1, and CYP8B1 were trending downward or
an
significantly reduced in all groups on day 4 but not thereafter, indicating that ATZ may inhibit
the expression of some CYP enzymes after initial exposure but the reduced expression is not
M
sustained after prolonged treatment.
d
Previous studies have shown that CYP1A1/2, CYP2E1, CYP2B1/2, CYP2C6/11,

e
pt
CYP3A1/2 and CYP4A3 are involved in ATR metabolism or that their expression is modified by
ATR treatment. However, previous studies were limited by the variety of CYP enzymes
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expressed in particular cell lines or microsomal preparations and by antibody and substrate
Ac
specificity. For example, Hanioka et al (1998), using liver microsomes from both male and
female SD rats, found that CYP1A2 was not involved in the metabolism of ATR and CYP1A2
protein levels were not affected by ATR exposure (Hanioka et al., 1998a). However, the same
group examined CYP1A2 and its inducibility after ATR treatment using male SD rat liver
microsomes, and found that CYP1A2 plays a minor role in the N-monodealkylation of ATR
(Hanioka et al., 1998b). Another study using immunoblots in male Fischer rats found that ATR
exposure significantly increased CYP1A2 protein levels (Islam et al., 2002). Peripubertal male
Wistar rats gavaged with 200 mg/kg/day of ATR from postnatal day 23 to day 50 displayed
increased CYP1A1/2 activity (Pogrmic-Majkic et al., 2012), again suggesting that CYP1A1/2 is
involved in the phase I biotransformation of ATR or, at the very least, is responsive to ATR
treatment. In our study, CYP1A2 was significantly elevated only after 4 days of 50 mg/kg/day of
ATR and there were no changes in CYP1A2 at other treatment duration or dose. The limited
t
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induction of CYP1A2 in the current study mirrors the inconsistent findings from previous reports
involving ATR metabolism.
cr
us
CYP2E1 has also been implicated in the biotransformation of ATR in both male and
female SD rat liver microsomes (Jaeger et al., 1998). Our results show an over 3-fold increase in
an
mRNA expression after treatment with 50 and 100 mg/kg/day of ATR after 8 days which was
sustained after 14 days, indicating ATR treatment might regulate CYP2E1, supporting its role in
M
ATR metabolism. Likewise, CYP2B2 and CYP4A3 showed a dose-dependent increase in
d
expression throughout the study. This is consistent with previous studies which have shown that
e
CYP2B1/2 and CYP2B2 play a role in the metabolism of ATR (Hanioka et al., 1998a; Hanioka
pt
et al., 1999; Islam et al., 2002; Pogrmic-Majkic et al., 2012). CYP2C6, which has been shown to
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be involved in the metabolism of ATR in male rat liver microsomes (Hanioka et al., 1998a;
Hanioka et al., 1998b), did not show a statistically significant increase in expression compared to
Ac
control groups in the present study (Table 2). Nevertheless, mRNA expression levels were
elevated, but not significantly, on day 8 in the 50 and 100 mg/kg/day dose groups and in all
groups on day 14.
ATR treatment also led to an increase in the expression of several CYP isoforms
associated with oxidative stress and inflammation. CYP4F1, CYP4F4 and CYP4F6, which have
all been shown to be involved in the omega-hydroxylation of leukotriene B4, a potent mediator of
inflammation. It is believed that the omega-hydroxylation of leukotriene B4 helps to reduce the
number of inflammatory signals and resolve the inflammation process. In our study, a transient
but significant increase in CYP4F1 and CYP4F6 expression was noted in the mid- and high-dose
groups on day 8 but not on day 14. In addition, the expression of mGST2 was increased above
control after 14 days of treatment. mGSTs are also involved in the biosynthesis of leukotrienes
t
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which help to protect against oxidative stress (Jakobsson et al., 1999). Collectively these results
suggest that the potential oxidative stress following high doses of ATR likely results in increased
cr
expression of both mGST and CYP4F enzymes. Indeed, previous studies have indicated that
us
ATR can cause oxidative stress in rats if given at high enough doses and for a long enough
an
duration of time (Abarikwu et al., 2010; Singh et al., 2010). Similarly, CYP26A1 and CYP26C1
are trans-retinoic acid hydrolyses that regulate the cellular concentrations of ATR via oxidative
M
metabolism (Topletz et al., 2012). We found a statistically significant, 2-fold increase of
CYP26A1 mRNA expression on day 8 in the 50 mg/kg dose group and non-significant decreased
e d
expression of CYP26C1 in all groups after 4 days and increased expression after 8 or 14 days.
pt
The current study identified a large number of CYP enzymes which demonstrated altered
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mRNA expression after ATR treatment but have not previously been linked to ATR treatment.
CYP2D2, CYP2C7, CYP2R1, CYP3A18 and CYP3A23/3A1 all showed increased expression
Ac
after 4 and 8 days of ATR treatment compared to control, but by 14 days of ATR treatment,
expression levels were back to control levels. These CYP isoforms are regulated following short-
term ATR exposure and might be involved in the initial metabolism of or represent an adaptive
response to ATR exposure. The expression of CYP17A1, an enzyme with 17-alpha-hydroxylase
and 17,20-lyase activities that catalyzes the conversion of pregnenolone to

dehydroepiandrosterone (DHEA) (Payne and Hales, 2004), was not statistically significantly
elevated in our study. CYP27A1, which is known to perform the 27-hydroxylation of cholesterol,
displayed an approximate 2-fold increased expression on day 8 and 14 in the mid- and high-dose
(50 and 100 mg/kg/day) groups.
In the assessment of the effect of ATR on hepatic GST expression (Experiment 1),
significant elevations in mRNA levels were observed in five GST isoforms of GST on day 3 and
t
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in 11 GST isoforms on day 4. Hepatic GSTM2, GSTK1, GSTO1, GSTO2, GSTT1, GSTT2 and
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two microsomal GST isoforms, mGST1 and mGST2, which have not been previously examined
us
in ATR-treated animals, all showed increased expression. GSTP1 and GSTM1 showed more
than a two-fold increase in expression. This increase in GSTP1 expression is consistent with
an
previous studies, which indicated that GSTP is responsible for the majority of the GSH-
dependent Phase II metabolism of ATR in the liver of mice (Egaas et al., 1993; Abel et al.,
M
2004). However, to our knowledge, we are the first to report increased hepatic GSTM1 after
d
ATR treatment.
e
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After 8 days of ATR treatment at 100 mg/kg/day, mRNA expression levels of all GST
isoforms with the exception of GSTO2, mGST1 and mGST2 were elevated above controls.
ce
Animals administered 8 daily ATR doses of 50 mg/kg also showed significantly increased
Ac
expression of the majority of GST isoforms examined. GSTT1 expression was also elevated
above control levels in all dose groups on day 8 but not on day 4 or 14. Islam et al. (2002)
reported increased GSTP expression following treatment with 10 mg/kg of ATR after 3 days of
treatment, suggesting that the regulation of phase II metabolism components is responsive to
relatively low doses of ATR.

By day 14 of treatment, all GST isoform expression was at control levels except for
elevated mGST2 expression and a reduction in GSTA4 and GSTT2 expression. Considering the
reduction in expression of several GST isoforms in the liver after 14 consecutive days with high
doses of ATR, the possibility that prolonged dosing resulted in liver necrosis was considered.
However, as reported by Campos-Pereira et al., high doses of ATR (400 mg/kg) administered for
14 days failed to produce necrosis in the liver, so it is unlikely the lower doses used in the
t
ip
present study resulted in liver damage (Campos-Pereira et al., 2012). Furthermore, there was no
evidence of hepatotoxicity in either male or female rats administered ATR in the diet for 90 days
cr
at average daily doses as high as 34.5 mg/kg (Breckenridge et al., 2010).
us
To determine if increased mRNA expression of GST enzymes corresponded with
an
increased GST protein level, we evaluated the effect of ATR on protein levels of four GST
isoforms (GST M, GST O, GST P and GST A). In Experiment 1, protein levels of these isoforms
M
in liver were not increased in rats administered ATR at a dose of 100 mg/kg/day for 1 to 4 days.
d
In Experiment 2, GST O was increased after 4 days of treatment with 100 mg/kg of ATR, but
e
there was no other significant difference between ATR-treated animals at any dose or time point.
pt
However, the mean GST O protein level in the control group on day 4 was significantly less than
ce
the control group mean on day 8 and 14. This difference may account for the significant
difference in mean GST O protein level in the 100 mg/kg ATR group on day 4 in Experiment 2.
Ac
Overall, the results are consistent with those of Campos-Pereira et al. and Islam et al., who
reported that there was no significant difference in the protein level of GST protein levels,
following ATR treatment (Islam et al., 2002; Campos-Pereira et al., 2012). ATR-induced
increase in mRNA expression of GST isoforms, without a corresponding rise in protein levels in
the same liver samples, could suggest that repeated ATR treatment upregulates transcription of
GST isoforms without driving significant translation and protein production. Alternatively, high-
doses of ATR might increase protein levels of specific GST isoforms without altering the level
of all detectable GST biotransformation enzymes. Increased protein levels and/or activity in a
specific isoform is difficult to discern due to limitations in current antibody specificity.
Considering the limitation in GST isoform protein quantification, we evaluated cytosolic
and microsomal GST activity and quantified the amount of the liver GST substrate, GSH. In
t
ip
Experiment 1, cytosolic GST activity and GSH levels were both significantly increased after
cr
three or four daily doses of 100 mg/kg ATR (Figure 2), whereas in Experiment 2, GSH levels
us
were increased after 4 daily doses of 6.5, 50 or 100 mg/kg ATR but cytosolic GST activity was
unaffected at days 4 and 8 but increased in the high-dose group on day 14. It is interesting to note
an
that on day 8, the majority of GST isoforms were at their highest expression levels, yet overall
cytosolic GST levels were not increased. This suggests that the increased expression of GST
M
levels on day 8 may have been triggered by higher level of GSH utilization earlier on day 4 and
d
perhaps between day 4 and day 8. These results suggest that repeated doses of ATR upregulates
e
the expression and availability of GSH, thereby facilitating GST-mediated phase II elimination
pt
of ATR and the other chlorotriazine metabolites as suggested by results from other laboratories
ce
(Lucas et al., 1993; Egaas et al., 1995a; Egaas et al., 1995b; Jaeger et al., 1998; Buchholz et al.,
1999).
Ac
The tolerance of maize to chlorotriazines has been linked to higher levels of expression
and activity of GST (Timmerman, 1989) and that when weeds develop resistance to ATR, it is
often attributed to increased expression/activity of GST in the resistant weed (Anderson and
Gronwald, 1991; Evans et al., 2017). In a pharmacokinetic model of ATR (Campbell et al.,
2016) it has been demonstrated that the clearance of chlorotriazines from plasma was most
sensitive to the rate of elimination of the ATR metabolite, DACT (Breckenridge et al., 2016).
Thus, the rate of the clearance of ATR and its metabolites from plasma is directly dependent on
GSH levels and GST expression and activity in the liver and kidney. The present study has
shown that hepatic levels of GSH and GST isoform expression reaches a maximum around day
8. In the animals that displayed time-dependent changes in GSH levels and GST expression,
these same animals also displayed a time-dependent change in their biological response to ATR
t
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(Breckenridge et al., 2017). Thus, a dose-dependent maximum attenuation of the LH surge was
observed in animals administered ATR for 4 days but decreases with longer ATR treatment.
cr
Similarly, the ATR-induced rise in plasma corticosterone levels is present at 1 to 4 days of
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treatment but absent by 7 days of treatment (Breckenridge et al., 2017). The altered hormone
an
responses to repeated ATR treatment align with our present findings that expression of
components of both phase I and II hepatic metabolisms of ATR are elevated at similar time
M
points after repeated ATR treatment.
d
In the current study, we examined the hepatic expression, protein levels and activity
e
levels of the primary component enzymes and substrates for GST-mediated phase II
pt
biotransformation as well as the expression of 45 CYP isoforms involved in phase I xenobiotic

ce
metabolism in ATR-treated animals. Our research reports novel findings that ATR exposure
differentially regulates the expression of a number of CYP and GST isoforms as well as GST-
Ac
mediated metabolism components. ATR treatment over a longer period of time leads to
variations in the expression of hepatic phase I CYP and phase II GST enzymes compared to
shorter-term ATR treatment, which may be due to liver adaptation. We have validated previous
findings, but have also identified novel players in the hepatic effects of high dose ATR
treatment. From a human safety perspective, it is likely that human exposure to ATR and the
chlorotriazines is too low to trigger either adverse effects on reproduction (DeSesso et al., 2014;
Foradori et al., 2014; Breckenridge et al., 2016) or the apparent adaptive changes in CYP and
GST enzyme expression observed in the present study after repeated doses of ATR.
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Changes in Hepatic Phase I and Phase II Biotransformation Enzyme Expression and Glutathione Levels Following Atrazine Exposure in Female Rats

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Xenobiotica

the fate of foreign compounds in biological systems

ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20

Changes in hepatic phase I and phase II

Arthur D. Zimmerman, Charles B. Breckenridge, Kun D. Yi, Pragati Sawhney

To link to this article: http://dx.doi.org/10.1080/00498254.2017.1374486

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glutathione levels following atrazine exposure in female rats

Veterinary Medicine, 210 Greene Hall, Auburn, AL 36849-5518, USA

Funding: Syngenta Crop Protection LLC.

Running Head: Changes in liver enzymes due to ATR treatment

Summary: ATR treatment leads to an up regulation of multiple phase I and II hepatic

of assayed components had returned to control levels, suggesting a possible habituation or an

establishment of dominant mediators of ATR metabolism over time.

(GSH) were assayed.

CYP2E1, CYP3A9, CYP4A3, and CYP27A1, which remained elevated.

expression following repeated ATR treatment.

Keywords: Liver enzymes, herbicides, rat, female, habituation

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine; ATR) belongs to the class

corticosterone levels is partially responsible for the ATR-induced reduction in luteinizing

delivery of ATR is effective in inducing a rise in corticosterone concentrations and reducing LH

CYP expression levels after repeated exposures and doses.

conjugation is involved with glutathione-s-transferase pi (GST P) activity. GST P is increased in

may affect ATR metabolism.

maintained in AAALAC approved animal facilities at WIL Research Laboratories LLC.

Atrazine Formulation and Administration

approximately weekly as a suspension in 1% methylcellulose and deionized water at

in every dose suspension by HPLC, ranged from of 94.7 to 101% of target.

after 1, 2, 3, or 4 days of treatment

components after 4, 8 or 14 days of treatment

the profound changes in Phase II hepatic components identified in experiment 1, experiment 2

QIAzol lysis reagent in a DNase/Rnase-free 1.7 mL microcentrifuge tube. Tissue was

(GSTA4), GST kappa 1 (GSTK1), GST mu 1 (GSTM1), GST mu 2 (GSTM2), GST mu 3

ΔCT = CT(target)−CT(Rplp1) and Δ(ΔCT) = ΔCT(experimental)−ΔCT(control).

were separated by electrophoresis at 200 V for 50 minutes in a 12% Criterion TGX

mg/mL standardized protein samples, homogenate was diluted to 1 mg/ml in potassium

measuring the rate of formation of the conjugate of GSH and 1-chloro-2,4-dinitrobenzene

Softmax Pro software.

were analyzed: CYP1A1, CYP1A2, CYP1B1, CYP2A3, CYP2B2, CYP2B3, CYP2C6,

CYP2C7, CYP2C8, CYP2C11, CYP2C22, CYP2C23, CYP2C37, CYP2D2, CYP2D4, CYP2E1,

CYP2F4, CYP2R1, CYP2S1, CYP2T1, CYP2W1, CYP3A9, CYP3A18, CYP3A23/3A1,

CYP4A3, CYP4A8, CYP4B1, CYP4F1, CYP4F4, CYP4F6, CYP4F18, CYP4F40 CYP7A1,

CYP7B1, CYP8B1, CYP11A1, CYP11B1, CYP17A1, CYP19A1, CYP24A1, CYP26A1,

and Δ(ΔCT) = ΔCT(experimental)−ΔCT(control).

Experiment 1: Effects of ATR treatment (100 mg/kg/day) on Phase II hepatic components

after 1, 2, 3, or 4 days of treatment

GST isoform expression

different from control levels after 4 days of treatment.

(Figure 2, Supplemental Table 1).

Bio-reactive GSH levels and GST activity

of ATR treatment on microsomal GST activity (Figure 3C).

components after 4, 8 or 14 days of treatment

CYP enzyme expression

CYP2B3, CYP2C22, CYP2C23, CYP2C37, CYP2C7, CYP2D2, CYP2E1, CYP2F4, CYP2R1,

(Table 2, Figure 4A). Expression of CYP2C6, CYP2D4, CYP11A1, CYP11B1, CYP19A1,