Cockayne syndrome B protects against methamphetamine-enhanced oxidative DNA damage in murine fetal brain and postnatal neurodevelopmental deficits

Abstract

Methamphetamine (METH) increases the oxidative DNA lesion 8-oxoguanine (8-oxoG) in fetal mouse brain, and causes postnatal motor coordination deficits after in utero exposure. Like oxoguanine glycosylase 1 (OGG1), the Cockayne syndrome B (CSB) protein is involved in the repair of oxidatively damaged DNA, although its function is unclear. Here we used CSB-deficient Csb(m/m) knockout mice to investigate the developmental role of DNA oxidation and CSB in METH-initiated neurodevelopmental deficits. METH (40 mg/kg intraperitoneally) administration to pregnant Csb females on gestational day 17 increased 8-oxoG levels in Csb(m/m) fetal brains (p < 0.05). CSB modulated 8-oxoG levels independent of OGG1 activity, as 8-oxoG incision activity in fetal nuclear extracts was identical in Csb(m/m) and Csb(+/+)mice. This CSB effect was evident despite 7.1-fold higher OGG1 activity in Csb(+/+) mice compared to outbred CD-1 mice. Female Csb(m/m) offspring exposed in utero to METH exhibited motor coordination deficits postnatally (p < 0.05). In utero METH exposure did not cause dopaminergic nerve terminal degeneration, in contrast to adult exposures. This is the first evidence that CSB protects the fetus from xenobiotic-enhanced DNA oxidation and postnatal functional deficits, suggesting that oxidatively damaged DNA is developmentally pathogenic, and that fetal CSB activity may modulate the risk of reactive oxygen species-mediated adverse developmental outcomes.

FIG. 1.

Increased DNA oxidation in Cockayne syndrome B (CSB)-deficient fetal brain after in utero methamphetamine (METH) exposure. METH was dissolved in 0.9% saline and administered in a single dose (40 mg/kg intraperitoneally) to gestational day (GD) 17 pregnant Csb^+/m dams mated with Csb^+/m males. Control dams were treated with the saline vehicle. The dams were sacrificed 4 h after injection. Tissue was isolated from fetal brain and analyzed for oxidative DNA damage reflected by 8-oxoguanine (8-oxoG) formation. ^ap < 0.05 indicates a difference between +/+ and m/m fetal brains exposed to METH. ^bp < 0.02 indicates a difference between 40 mg/kg and the corresponding saline control in +/m fetal brains. ^cp = 0.0002 indicates a difference between METH and the corresponding saline control in m/m fetal brains. (X,Y) indicates the number of dams (X) and fetuses (Y) analyzed.

FIG. 2.

No increase in fetal liver oxidative DNA damage after in utero METH exposure. Pregnant Csb^+/m dams were treated on GD 17 as described in fig. 1, and 8-oxoG levels were analyzed in fetal liver 4 h later. No significant differences were observed in oxidative DNA damage among saline-exposed +/+, +/m, or m/m littermates, nor among METH-exposed fetuses of different CSB genotypes.

FIG. 3.

Motor coordination deficits in CSB-deficient offspring exposed in utero to METH. Pregnant Csb^+/m dams were treated on GD 17 as described in Figure 1, and motor coordination impairment was assessed in the offspring using the rotarod test at 6, 8, 10, and 12 weeks after birth. The average latency or time at which the mice fell from the rotating rod was calculated. (x,y) indicates the number of dams (x) and fetuses (y) analyzed. (A) Females. *p < 0.05 indicates a difference between METH exposed m/m compared to the corresponding saline control. +/+[saline (14,16), METH (11,11)] +/m [saline (15,20), METH (11,13)]; m/m [saline (9,10), METH (10,10). (B) Males. +/+ [saline (11,13), METH (14,14)]; +/m [saline (16,18), METH (12,21)]; m/m [saline (10,10), METH (9,11).

FIG. 4.

Prenatal METH exposure does not cause postnatal degeneration of dopaminergic nerve terminals in the striatum of offspring. Pregnant Csb^+/m dams were treated on GD 17 as described in Figure 1, and the progeny were sacrificed at 12 weeks of age. Brain sections were stained for tyrosine hydroxylase (TH) indicative of dopaminergic nerve terminals. Immunohistochemical staining is representative of n = 4/treatment group at 400× magnification. (Inset) Positive control: reduced TH staining of adult CD-1 mice treated 1 week previously with 40 mg/kg of METH.

FIG. 5.

Endogenous 8-oxoG repair activity in fetal brain nuclear extracts from CSB mice. (A) About 2.5–20 μg of pooled fetal brain nuclear extract (lanes 1–4) from three Csb^+/+ mice was incubated with 25 nM of a biotin-labeled oligonucleotide containing 8-oxoG that was annealed to its complementary strand containing C opposite 8-oxoG. Reaction products were resolved by 15% denaturing polyacrylamide gel electrophoresis. (B) Lanes 1 and 2: 25 nM oligonucleotide incubated with 5 μg of nuclear extract from GD 17 brain from Csb^+/+ fetuses from different litters. Lanes 3 and 4: 25 nM oligonucleotide incubated with 5 μg of nuclear extract from GD 17 brain from Csb^m/m fetuses from different litters.

FIG. 6.

Densitometric analysis of endogenous fetal brain and liver 8-oxoG repair activity in nuclear extracts from Csb^+/+ and Csb^m/m mice. Extracts were incubated with a biotin-labeled oligonucleotide containing 8-oxoG that was annealed to its complementary strand containing C opposite 8-oxoG. 25 nM oligonucleotide incubated with 5 μg of nuclear extract from GD 17 brain or liver (n = 8; mean ± SD). Fetuses were selected randomly and evenly from six different litters.

FIG. 7.

Endogenous 8-oxoG repair activity in fetal brain nuclear extracts from Csb^+/+ and CD-1 mice. (A) A 10 μg aliquot of pooled fetal brain nuclear extract from Csb^+/+ mice (lanes 1 and 2) and CD-1 mice (lanes 3 and 4) was incubated with 25 nM of a biotin-labeled oligonucleotide substrate containing 8-oxoG that was annealed to its complementary strand containing C opposite 8-oxoG. Reaction products were resolved by 15% denaturing polyacrylamide gel electrophoresis. (B) Densitometric analysis of endogenous fetal brain 8-oxoG repair activity in nuclear extracts from Csb^+/+ and CD-1 mice. The oligonucleotide substrate (25 nM) was incubated with 10 μg of nuclear extract from GD 17 brain (n = 4; mean ± SD). *A difference from CD-1 mice (p < 0.05).