Altered glutathione homeostasis in animals prenatally exposed to lipopolysaccharide

Abstract

We previously reported that injection of bacterial lipopolysaccharide (LPS) into gravid female rats at embryonic day 10.5 resulted in a birth of offspring with fewer than normal dopamine (DA) neurons along with innate immunity dysfunction and many characteristics seen in Parkinson's disease (PD) patients. The LPS-exposed animals were also more susceptible to secondary toxin exposure as indicated by an accelerated DA neuron loss. Glutathione (GSH) is an important antioxidant in the brain. A disturbance in glutathione homeostasis has been proposed for the pathogenesis of PD. In this study, animals prenatally exposed to LPS were studied along with an acute intranigral LPS injection model for the status of glutathione homeostasis, lipid peroxidation, and related enzyme activities. Both prenatal LPS exposure and acute LPS injection produced a significant GSH reduction and increase in oxidized GSH (GSSG) and lipid peroxide (LPO) production. Activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, was up-regulated in acute supranigral LPS model but was reduced in the prenatal LPS model. The GCS light subunit protein expression was also down-regulated in prenatal LPS model. GSH redox recycling enzyme activities (glutathione peroxidase, GPx and glutathione reducdase, GR) and glutathione-S-transferase (GST), gamma-glutamyl transpeptidase (gamma-GT) activities were all increased in prenatal LPS model. Prenatal LPS exposure and aging synergized in GSH level and GSH-related enzyme activities except for those (GR, GST, and gamma-GT) with significant regional variations. Additionally, prenatal LPS exposure produced a reduction of DA neuron count in the substantia nigra (SN). These results suggest that prenatal LPS exposure may cause glutathione homeostasis disturbance in offspring brain and render DA neurons susceptible to the secondary neurotoxin insult.

Fig. 1

γ-Glutamylcysteine synthetase (GCS) subunit protein in the midbrain. Heavy subunit (GCS-HS) and light subunit (GCS-LS) of GCS were detected in the midbrain following supranigral saline (Sal) or lipopolysaccharide (LPS) injection. β-actin was used as a control of protein loading. (A) Representative Western blot images. (B) Relative optical density of immunoblot band. Data are the means ± SEM from 5 animals each group. **P < 0.01, compared with saline control.

Fig.2

Reduced glutathione (GSH), glutathione disulfide (GSSG) levels and GSH/GSSG ratio in the brain. Glutathione concentration was measured in cortex, striatum, midbrain and cerebellum in prenatally LPS or saline (Sal)-exposed rats at 4 and 17 months of age. (A): GSH; (B): GSSG; (C): Ratio of GSH/GSSG. Each bar from left to right in different regions represents Sal/4 months, LPS/4 months, Sal/17 months and LPS/17 months, respectively. Data are the means ± SEM from 5 animals each group. *P < 0.05, **P < 0.01, compared with the corresponding group at 4 months of age. ^#P < 0.05, ^##P < 0.01, compared with the corresponding saline group.

Fig. 3

Lipid peroxide (LPO) level in the brain. LPO was measured in cortex, striatum, midbrain and cerebellum in prenatally LPS or saline (Sal)-exposed rats at 4 and 17 months of age. Each bar from left to right in different regions represents Sal/4 months, LPS/4 months, Sal/17 months and LPS/17 months, respectively. Data are the means ± SEM from 5 animals each group. *P < 0.05, **P < 0.01, compared with the corresponding group at 4 months of age. ^##P < 0.01, compared with the corresponding saline group.

Fig. 4

γ-Glutamylcysteine synthetase (GCS) activity in the brain. GCS activity was measured in cortex, striatum, midbrain and cerebellum in prenatally LPS or saline (Sal)- exposed rats at 4 and 17 months of age. Each bar from left to right in different regions represents Sal/4 months, LPS/4 months, Sal/17 months and LPS/17 months, respectively. Data are the means ± SEM from 5 animals each group. *P < 0.05, **P < 0.01, compared with the corresponding group at 4 months of age. ^#P < 0.05, ^##P < 0.01, compared with the corresponding saline group.

Fig. 5

γ-Glutamylcysteine synthetase (GCS) subunit protein in the brain. Heavy subunit (GCS-HS) and light subunit (GCS-LS) of GCS were detected in cortex (COR), striatum (STR), midbrain (MID) and cerebellum (CER) in prenatally LPS or saline (Sal)-exposed rats at 4 and 17 months (m)of age. β-actin was used as a control of protein loading. (A, B) Representative Western blot images from four groups of animals; (C, D) Relative optical density of immunoblot band. Each bar from left to right in different regions represents Sal/4 months, LPS/4 months, Sal/17 months and LPS/17 months, respectively. Data are the means ± SEM from 5 animals each group. No significant changes of GCS-HS protein were observed in all groups. **P < 0.01, compared with the corresponding group at 4 months of age. ^#P < 0.05, ^##P < 0.01, compared with the corresponding saline group.

Fig.6

Glutathione peroxidase (GPx) and glutathione reductase (GR) activities in the brain. (A) GPx and (B) GR activities were measured in cortex, striatum, midbrain and cerebellum in prenatally LPS or saline (Sal)-exposed rats at 4 and 17 months of age. Each bar from left to right in different regions represents Sal/4 months, LPS/4 months, Sal/17 months and LPS/17 months, respectively. Data are the means ± SEM from 5 animals each group. *P < 0.05, **P < 0.01, compared with the corresponding group at 4 months of age. ^#P < 0.05, ^##P < 0.01, compared with the corresponding saline group.

Fig. 7

γ-Glutamyl transpeptidase (γ-GT) and glutathione Stransferase (GST) activities in the brain. (A) γ-GT and (B) GST activiies were measured in cortex, striatum, midbrain and cerebellum in prenatally LPS or saline (Sal)-exposed rats at 4 and 17 months of age. Each bar from left to right in different regions represents Sal/4 months, LPS/4 months, Sal/17 months and LPS/17 months, respectively. Data are the means ± SEM from 5 animals each group. *P < 0.05, **P < 0.01, compared with the corresponding group at 4 months of age. ^#P < 0.05, ^##P < 0.01, compared with the corresponding saline group.

Fig. 8

Upper panel: Representative photomicrographs of tyrosine hydroxylase immunoreactivity (TH-ir) in mesencephalon of the animals exposed prenatally to saline (Sal) or lipopolysaccharide (LPS) at 4 and 17 months old. A: Sal/4 months; B: LPS/4 months; C: Sal/17 months; D: LPS/17 months. Magnification bar = 0.25 mm. Lower panel: Figure shows TH-ir cell counts in the substantia nigra (SN). Data are the means ± SEM from 5 animals each group. *P < 0.05, compared with the corresponding group at 4 months of age. ##P < 0.01, compared with the respective saline group.