Changes in hepatic phase I and phase II biotransformation enzyme expression and glutathione levels following atrazine exposure in female rats

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

Changes in Sensitivity to the Effects of Atrazine on the Luteinizing Hormone Surge in Female Sprague‐Dawley Rats after Repeated Daily Doses: Correlation with Liver Enzyme Expression

“…Among the CYP450 enzymes evaluated by Zimmerman et al (2017), mRNA expression of a number of CYP enzymes (CYP1A2, CYP3A18, CYP2E1 CYP2B2, CYP3A23/31, CYP4A3, CYP3A9, and CYP2D4; Supplementary Figs. S1 and S2) displayed large, ATZ-dose-related, increased levels relative to controls.…”

“…In experiment 1, liver samples collected from 5 rats/group killed on day 1, 2, 3, or 4 were analyzed using real-time polymerase chain reaction analyses for GST mRNA expression. Total GSH levels were evaluated using the Cayman Chemical GSH assay kit (Ann Arbor, MI) as described by Zimmerman et al (2017). In experiment 2, GST, GSH, and P450 analyses were conducted on liver samples collected from 5 rats/sex/group on day 4, 8, or 14.…”

“…The samples were refrigerated overnight at 4°C and then transferred to a freezer at −70°C, until shipment on dry ice to Auburn University for analyses of messenger RNA (real-time polymerase chain reaction), enzyme expression, and total protein levels (Western blots) for the following thirteen GST liver enzymes: GSTA3, GSTA4, GSTK1, GSTM1, GSTM2, GSTM3, GSTO1, GSTO2, GSTP1, GSTT1, GSTT2, MGST1, and MGST2. Overall GST activity was determined in cytosolic (GSTC) and microsomal (GSTM) fractions (Zimmerman et al, 2017).…”

“…The right lobe of the liver was collected, weighed, and flash-frozen in liquid nitrogen, and were then shipped on dry ice to Auburn University for analyses of mRNA levels for the following 29 P450 liver enzymes: CYP17A1, CYP1A2, CYP26A1, CYP26C1, CYP27A1, CYP2B2, CYP2B3, CYP2C22, CYP2C23, CYP2C37, CYP2C6, CYP2C7, CYP2D2, CYP2D4, CYP2E1, CYP2F4, CYP2R1, CYP2T1, CYP3A18, CYP3A23/ 3A1, CYP3A9, CYP4A3, CYP4B1, CYP4F1, CYP4F4, CYP4F6, CYP7A1, CYP7B1, and CYP8B1B. In addition, total GSH levels (nmol GSH/mg protein) were measured and standardized to total protein levels in the supernatant of homogenized, centrifuged liver samples (Zimmerman et al, 2017).…”

“…The statistical significance of correlation between these parameters (R 2 ) was assessed using a two-sided t test. A similar analysis was conducted for the isoforms of GST mRNA expression after 4, 8, or 14 days of ATZ treatment (Zimmerman et al, 2017). However, since GST mRNA expression increased in a dose-dependent manner after 4, 8, or 14 days of ATZ treatment for all GST isoforms, the average fold-change in GST mRNA expression was calculated for each individual animal, relative to the control group mean, and correlated with its LH AUC by calculating the R 2 .…”