TY - JOUR
AU - Hood, Darryl B.
AB - Abstract The wild-type (WT) Cprlox/lox (cytochrome P450 oxidoreductase, Cpr) mouse is an ideal model to assess the contribution of P450 enzymes to the metabolic activation and disposition of environmental xenobiotics. In the present study, we examined the effect of in utero exposure to benzo(a)pyrene [B(a)P] aerosol on Sp4 and N-methyl-D-aspartate (NMDA)–dependent systems as well as a resulting behavioral phenotype (object discrimination) in Cpr offspring. Results from in utero exposure of WT Cprlox/lox mice were compared with in utero exposed brain-Cpr-null offspring mice. Null mice were used as they do not express brain cytochrome P4501B1–associated NADPH oxidoreductase (CYP1B1-associated NADPH oxidoreductase), thus reducing their capacity to produce neural B(a)P metabolites. Subsequent to in utero (E14–E17) exposure to B(a)P (100 μg/m3), Cprlox/lox offspring exhibited: (1) elevated B(a)P metabolite and F2-isoprostane neocortical tissue burdens, (2) elevated concentrations of cortical glutamate, (3) premature developmental expression of Sp4, (4) decreased subunit ratios of NR2B:NR2A, and (5) deficits in a novelty discrimination phenotype monitored to in utero exposed brain-Cpr-null offspring. Collectively, these findings suggest that in situ generation of metabolites by CYP1B1-associated NADPH oxidoreductase promotes negative effects on NMDA-mediated signaling processes during the period when synapses are first forming as well as effects on a subsequent behavioral phenotype. NMDA receptor, neuronal activity, neurogenesis, synaptogenesis, polycyclic aromatic hydrocarbon, benzo(a)pyrene, susceptibility-exposure paradigm, B(a)P metabolites, object discrimination task Cytochrome P450 is an important modifier of mental development at an early age. Recently, significant interactions were reported among a maternal haplotype in the cytochrome P4501B1 (CYP1B1) gene (ACCGGC), polycyclic aromatic hydrocarbon (PAH) exposure, and reductions in the mental development index in a cohort of children (Wang et al., 2010). Such studies have important implications for our society (Perera et al., 2011) and are valuable from a translational standpoint because they can inform the design of mechanistic neurotoxicology studies using animal models. Recently, Qiu et al. (2011) evaluated the effects of subchronic exposure to benzo(a)pyrene (ip injections of 6.25 mg/kg B(a)P/day for 14 weeks) versus diluent on both neurotransmitter receptor gene expression and performance in a Morris Water Maze. Microarray results revealed that 1016 genes were differentially expressed in B(a)P treated versus diluent controls. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyze those genes differentially expressed in the gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Their analysis showed that the most significantly affected category was behavior and the fourth highest was learning and memory. They ranked 22 genes involved in learning and memory and 25 genes associated with neuroactive ligand-receptor interactions. Both lists included upregulation of the ionotropic glutamate receptor N-methyl-D-asparate (NMDA) 2A (Grin2A). A conclusion was that “neuroactive ligand-receptor interactions” were among the most negatively affected by B(a)P exposure at (p < 7.7, E−6). In the Morris Water Maze test, B(a)P-treated rats had spatial learning deficits and had a decreased number of platform crossings and time spent in the target area suggesting that B(a)P caused a deleterious effect on long-term memory. Several other studies have also suggested that neurotransmitter (Xia et al., 2011) and neurotransmitter receptor gene expression (Floresco and Phillips, 2001; Iwama and Gojobori, 2002; Kemppainen et al., 2003; Riedel et al., 2003; Seamans et al., 1998) play important roles in modulating neurobehavioral effects, especially within the context of learning and memory. Our laboratory recently demonstrated a reduction in novel object discrimination following in utero exposure of Cprlox/lox (Cytochrome P450reductase, Cpr) offspring to B(a)P. Our results confirmed the notion that B(a)P can have a direct negative impact on the developmental expression of a key regulator of NMDA-mediated processes (receptor tyrosine kinase-MET) and behavioral learning and memory processes (Sheng et al., 2010). The determination of the embryonic “critical period of development” for brain structures involved in learning and memory processes in mice is based on the original work by Rodier (1976). This study described the embryonic time frames for peak neurogenesis and neuroepithelial proliferation for cerebral cortex, hippocampus, septum, amygdala, corpus striatum, thalamus, hypothalamus, cerebellum, and olfactory bulb as the period from embryonic day E14 through E17. Rodier documented almost 40 years ago that the specific time of the CNS insult is an important factor in subsequent effects on both anatomy and behavior. Therefore, this early work established what we refer to as the embryonic critical period of development. The report suggested that the behavioral effects of toxicants such as B(a)P are similar in both rats and mice and represented one of the first studies to demonstrate that mice could be used successfully in a variety of behavioral evaluations. Thus, the novel object discrimination paradigm has been used for over 20 years because it is perfectly suited to test the effects of pharmacological and genetic interventions on learning and memory processes (Dere et al., 2007). When comparisons are made among animal models of learning and memory, the object discrimination test is more closely related with those conditions under which human recognition memory is measured. This is primarily due a shortened period for training coupled with the fact that novel object discrimination does not induce high levels of arousal and stress in animals (Ennaceur and Delacour, 1988). The component of memory tested in the present study (specific to the medial prefrontal cortex [mPFC]) is termed succession, temporal order, or sometimes referred to as relative recency memory (Dere et al., 2007; Devito and Eichenbaum, 2011). In support of our argument concerning the involvement of mPFC in relative recency memory, it is known that lesions to the mPFC impair relative recency discriminations in humans, nonhuman primates, and rodents across a wide range of stimulus modalities (Fuster, 2001; Kesner and Holbrook, 1987). This is true despite the fact that in some instances, recognition of novel and familiar stimuli is preserved (Kesner et al., 1994; McAndrews and Milner, 1991). Conversely, studies have reported that after bilateral mPFC lesions, an impairment or deficit is observed with respect to the ability to differentiate between old (earlier presented) and recently presented familiar objects (Mitchell and Laiacona, 1998). Results from the present study testing in utero B(a)P aerosol–exposed Cprlox/lox offspring in an object discrimination paradigm are consistent with results from the aforementioned bilateral mPFC lesion studies and support the suggestion that the mPFC is involved (at least in part) in making a judgment regarding the sequence and order of object presentations. Additionally, the involvement of cortical glutamate receptors in performance of the novel object discrimination task in rodents is well established, as deficits and/or impairments have been reported following application of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (6-cyano-7-nitroquinoxaline-2,3-dione) or NMDA (d,l-2-amino-5-phosphono-pentanoic acid [AP5]) antagonists (Sargolini et al., 2003; Winters and Bussey, 2005). By characterizing the effects of in utero exposure to B(a)P aerosol on wild-type (WT) Cprlox/lox and brain-Cpr-null mouse offspring, we further validate novel object discrimination phenotyping as a measure of prefrontal and limbic circuit integrity by demonstrating that this behavior is negatively impacted subsequent to in utero B(a)P exposure. Our prediction was that a significant deficit in discrimination phenotype would occur following exposure and would likely be accompanied by alterations in NMDA-mediated processes (Laube et al., 1998). MATERIALS AND METHODS Cytochrome P450 reductase model Cprlox/lox. The transgenic mouse model utilized and characterized in the present study is the Cytochrome P450reductase or Cprlox/lox mouse. Nested within this study is an obligatory characterization of B(a)P disposition and metabolite bioavailability in the WT Cprlox/lox mouse against the background C57BL/6J strain. The usefulness of this model and its relevance to human POR (P450 oxidoreductase) deficiency has been widely reported and is supported by the interindividual variability in human tissue expression levels of Cpr (Wortham et al., 2007). Additionally, many mutant POR alleles have been found to be associated with congenital deficiencies in steroidogenesis/homeostasis, and/or with the Antley-Bixler Syndrome, characterized by skeletal malformation and reproductive defects (Fluck et al., 2004; Fukami et al., 2005). The brain-Cpr-null phenotype (Conroy et al., 2010; Gu et al., 2007) no longer possesses the ability to produce B(a)P metabolites from parent compound due to reduced expression of POR, thus allowing a determination of those oxidative metabolites of B(a)P contributing to any observed developmental and/or behavioral defects. Interestingly, additional phenotypes of the Cpr-low mouse have already been identified such as female infertility and an altered steroid homeostasis known to occur in human subjects (Artl et al., 2004; Fluck et al., 2004; Fukami et al., 2005, 2006; Huang et al., 2005). Other known phenotypes of the Cpr-low mouse such as reduced cholesterol levels and embryonic survival may also be found to occur in some humans. Power analysis. To determine the total number of Cpr dams (litters) as well as the number of offspring needed for developmental studies, we made the following assumptions. We conservatively estimated that the variance between measures from litters would be approximately 10% of the mean response, so that using littermates from four to five different litters within an experimental group would be sufficient detect a significant difference. Based on these assumptions, the power analysis indicated that three replicates or cohorts from four to five different litters would suffice. Because the litter is the statistical unit, and sampling was from at least four to five individual litters within an experimental group, the litter representation would be sufficient to detect a 20% change in any of our experimental endpoints with an 80% power and a type I error rate of 5%. Pooling of samples. All experiments were approved by the Institutional Animal Care and Use Committee of Meharry Medical College and were performed according to Guidelines for Animal Experimentation. The numbers of pups within the control and exposure groups were not standardized on the day of birth due to the need to remove pups from litters for the conduct of disposition and developmental expression studies. On postnatal days (PNDs) 1, 3, 5, and 7, one pup was removed from each of at least four litters (per experimental group), and the pooled whole brains were added to 2-ml screw top vials and stored frozen at −80 until use for protein expression profiling, B(a)P metabolite bioavailability/disposition studies and/or F2-isoprostane analyses. On PNDs 9, 11, 13, and 15, one pup was removed from each of at least four litters per experimental group, left and right cortices were exposed, removed, dissected, and pooled per experimental day in 10-ml screw top vials and stored frozen at −80 until use. We assumed nine mice per litter in each of the following four experimental groups: (1) control (carbon black–exposed) Cprlox/lox, (2) B(a)P-exposed Cprlox/lox, (3) control (carbon black–exposed) brain-Cpr-null, and (4) B(a)P-exposed brain-Cpr-null mice. At least four to five litters were used per experimental group. Generation of timed-pregnant Cpr dams. Cpr mating pairs (four to five per genotype), consisting of Cprlox/lox and brain-Cpr-null mice, were bred in our Association for Assessment and Accreditation of Laboratory Animal Care accredited facility. One male stud of the Cprlox/lox genotype was cohabitated overnight with two females of the brain-Cpr-null genotype or vice versa. The morning after cohabitation was designated as embryonic day 1 (E1). Females were weighed daily and by E12, pregnant females were assigned to an exposure group based on visual observation and a consistent weight gain of 1 g/day × 3 days. At this point, all dams were moved into a preexposure protocol. During this 24-h period, dams were closely monitored and received a nutritional high fat diet supplement (Lovemash, Bioserv, Frenchtown, NJ) shown to increase litter size and improve pup survival rates (Sheng et al., 2010). During this time (E12 and E13), Cpr timed-pregnant dams were acclimated to the nose-only exposure protocol by spending 2 h/day breathing clean air in the exposure tubes. On the night of E13, timed-pregnant dams were allowed to rest in their home cages in preparation for the E14–E17, 4 h/day exposure period. Animal husbandry. Cprlox/lox and brain-Cpr-null genotypes. Dams generally gave birth on E20. Following delivery, all dams were supplemented with Supreme Mini-Treats (Bioserv), a product proven to reduce cannibalism. The numbers of pups born within both groups were not standardized on the day of birth due to the need to remove pups from litters to conduct B(a)P metabolite and F2-isoprostane bioavailability, disposition, and developmental expression profiling studies. Litter size, pup weights, and sex distribution were recorded. Generally, on PNDs 1, 3, 5, 7, 9, 11, 13, and 15, pups were removed from litters and separated into two portions (head and body) while under anesthesia. At autopsy, a 0.5 cm piece of the tail was collected for genotyping. Brains were then exposed, neocortex (left and right) removed, snap frozen in dry ice, and stored in liquid N2 until homogenates were prepared for various analyses. Each data point for metabolite disposition and expression profiling represented pooled 75–100 mg pieces of neocortex (or whole brain for early PNDs 1, 3, 5, and 7) from at least four different litters within each experimental group. Both Cprlox/lox and brain-Cpr-null litters were maintained in a controlled environment with a temperature at 21 ± 2°C and a relative humidity of 50 ± 10% with a 12/12 h light/dark cycle. All dams were fed commercial food (Rat Chow 5012, Purina Mills, St Louis, MO). Water and food was available ad libitum. Nose-only B(a)P aerosol inhalation exposure. On E14–E17 Cprlox/lox and/or brain-Cpr-null timed-pregnant dams were exposed to B(a)P in a carbon black aerosol (100 g/m3) versus carbon black only for 4 h/day (Hood et al., 2000). Briefly, a dual-component mouse aerosol generator was developed (CH Technologies, Westwood, NJ). Table 1 shows our model for the generation of B(a)P:carbon black aerosols. Air generated from a dedicated compressor was heated at a flow rate denoted by Qvap (lpm). The temperature of this air, referred to as the B(a)P stream, was monitored at the exit of a heater as well as at the exit of a B(a)P reservoir with thermocouples denoted by TCs and TCv, respectively. Hot humidified air entered with a flow rate denoted by Qhum. Convective heating of the combined airflow rate (Qvap + Qhum) passing through a premixing chamber and was monitored by a thermocouple denoted by TCp. This stream is referred to as the humidified B(a)P stream. The air containing carbon black dust from a rolling brush generator is referred to as the carbon stream with a flow rate denoted by QWright. An orifice located at the exit of the premixing chamber promotes mixing of the hot humidified B(a)P stream with the relatively cool carbon black stream to give a total flow rate denoted by Qtot. Convective heating of the humidified B(a)P stream prior to mixing with an ambient carbon black stream promotes condensation of B(a)P vapor onto carbon black particles via cooling of B(a)P vapor in close proximity to the carbon black particles. Estimation of the amount of B(a)P vapor converted to the particulate phase is based on the difference between the vapor concentration of the humidified B(a)P stream before mixing and the equilibrium vapor concentration at the temperature resulting from mixing. B(a)P vapor that is not adsorbed by carbon black is assumed to condense on the walls of the condensation chamber. All other procedures were as previously reported (Wu et al., 2003). TABLE 1 Modeling of B(a)P:Carbon Black Aerosols for Cpr Studies Qtot (slpm) Qvap (slpm) Qhum (slpm) QRBG (lpm) Tmixch (°F) Tvap (°F) ρ(Tvap) (μg/m3) Tsat (°F) TCp (°F) Tpremix1 (°F) Tparticle2 (°F) Tmix (°F) Mixture ρ(Tmix) (μg/m3) Mixture CB(a)P at Tmix (μg/m3) 10 2 7 1 132 234 3 192 276 270 79 132 5900 100 10 2 7 1 114 234 3 192 288 281 80 135 7500 100 10 2 7 1 104 234 3 192 295 288 80 137 8800 100 10 2 7 1 115 234 3 192 297 290 80 138 9100 100 Qtot (slpm) Qvap (slpm) Qhum (slpm) QRBG (lpm) Tmixch (°F) Tvap (°F) ρ(Tvap) (μg/m3) Tsat (°F) TCp (°F) Tpremix1 (°F) Tparticle2 (°F) Tmix (°F) Mixture ρ(Tmix) (μg/m3) Mixture CB(a)P at Tmix (μg/m3) 10 2 7 1 132 234 3 192 276 270 79 132 5900 100 10 2 7 1 114 234 3 192 288 281 80 135 7500 100 10 2 7 1 104 234 3 192 295 288 80 137 8800 100 10 2 7 1 115 234 3 192 297 290 80 138 9100 100 Note. TCp and Tpremix were generally high enough for topside of orifice plate > Tsat, rows 1–3 of each group of four rows. For each group, values greater than the second row are given in the fourth row for the purposes of evaluating overheating of the humidified B(a)P stream. Assume TWright = 70°F; m = 0.565; b = 37.432. RBG, rolling brush generator. Open in new tab TABLE 1 Modeling of B(a)P:Carbon Black Aerosols for Cpr Studies Qtot (slpm) Qvap (slpm) Qhum (slpm) QRBG (lpm) Tmixch (°F) Tvap (°F) ρ(Tvap) (μg/m3) Tsat (°F) TCp (°F) Tpremix1 (°F) Tparticle2 (°F) Tmix (°F) Mixture ρ(Tmix) (μg/m3) Mixture CB(a)P at Tmix (μg/m3) 10 2 7 1 132 234 3 192 276 270 79 132 5900 100 10 2 7 1 114 234 3 192 288 281 80 135 7500 100 10 2 7 1 104 234 3 192 295 288 80 137 8800 100 10 2 7 1 115 234 3 192 297 290 80 138 9100 100 Qtot (slpm) Qvap (slpm) Qhum (slpm) QRBG (lpm) Tmixch (°F) Tvap (°F) ρ(Tvap) (μg/m3) Tsat (°F) TCp (°F) Tpremix1 (°F) Tparticle2 (°F) Tmix (°F) Mixture ρ(Tmix) (μg/m3) Mixture CB(a)P at Tmix (μg/m3) 10 2 7 1 132 234 3 192 276 270 79 132 5900 100 10 2 7 1 114 234 3 192 288 281 80 135 7500 100 10 2 7 1 104 234 3 192 295 288 80 137 8800 100 10 2 7 1 115 234 3 192 297 290 80 138 9100 100 Note. TCp and Tpremix were generally high enough for topside of orifice plate > Tsat, rows 1–3 of each group of four rows. For each group, values greater than the second row are given in the fourth row for the purposes of evaluating overheating of the humidified B(a)P stream. Assume TWright = 70°F; m = 0.565; b = 37.432. RBG, rolling brush generator. Open in new tab Characterization of B(a)P:carbon black particulate aerosol. Samples for analysis of particle size distributions were obtained from the collection substrates in a seven-stage IN-TOX impactor, located downstream from the condensation chamber on one of the 12 sample ports on the cannon exposure chamber. Substrate postweights were recorded and entered into a WinCIDRS (Windows-Cascade Impactor Data Reduction Program) to generate particle size distributions for the carbon black particulate aerosol. A representative example of dM/dlogD plot is shown in Figure 1. FIG. 1. Open in new tabDownload slide Differential particle size distribution representative of B(a)P:carbon black aerosol delivered to timed-pregnant Cprlox/lox and brain-Cpr-null dams (100 μg/m3). During a typical exposure, aerosol was collected on substrates every 30 min during a 4-h exposure period from E14 to E17. Subsequent to the exposure period each day, substrate postweights were uploaded into a custom impactor data reduction program to generate average particle size distributions as previously described (Wu et al., 2003). FIG. 1. Open in new tabDownload slide Differential particle size distribution representative of B(a)P:carbon black aerosol delivered to timed-pregnant Cprlox/lox and brain-Cpr-null dams (100 μg/m3). During a typical exposure, aerosol was collected on substrates every 30 min during a 4-h exposure period from E14 to E17. Subsequent to the exposure period each day, substrate postweights were uploaded into a custom impactor data reduction program to generate average particle size distributions as previously described (Wu et al., 2003). Substrate extraction method and analysis (QA/QC). A modified version of U.S. EPA method 3510B was employed for the extraction of B(a)P:carbon black–impacted substrates. Subsequent to recording the substrate postweights, a 250-ml beaker was used to rinse the substrates with gas chromatography (GC) grade dichloromethane to remove particulate aerosol. A 1-ml aliquot of internal standard (4.0 mg/ml, Accu Standard, Inc., New Haven, CT) consisting of acenaphthalene, chrysene, dichlorobenzene, naphthalene, perylene, and phenanthrene was added to each rinsate. The rinses were then filtered through 5 g anhydrous sodium sulfate into individual Turbo Vap II (Zymark Corp., Hopkinton, MS) sample tubes. Two additional rinses of the beakers with dichloromethane followed by filtering assured removal of residual particulate from the beakers. Samples were then dried under nitrogen on a Turbo Vap II, reconstituted in 1 ml dichloromethane, and transferred to amber crimp-top vials for GC-mass spectroscopy (GC-MS) analysis. B(a)P quantitation was performed using a Hewlett Packard 6890/5973 gas chromatograph mass spectrometer equipped with an autosampler. Quantitative HPLC analyses of samples. Neocortical tissue extracts were resolved by a high pressure liquid chromatograph (HPLC) (model 1050, Hewlett Packard, Wilmington, DE) equipped with a fluorescence detector. Using an autosampler, 30 μl of sample was injected onto a C18 reverse-phase column (ODS Hypersil, 5 μm, 4.6 × 250 mm; Hewlett Packard). The column was eluted at 33°C at a flow rate of 1.0 ml/min with a ternary gradient of water:methanol:ethanol (%) as follows: 40:40:20 in 20 min, followed by 30:46:24 in 10 min, 100% methanol in 10 min, and 40:40:20 for 5 min. The excitation and emission wavelengths for the fluorescence detector were 244 and 410 nm, respectively. The B(a)P concentrations in neocortical tissue were calculated by comparing the retention times and peak areas of samples with those of standards using an HPLC 2D (software [DOS series]). Computer monitoring techniques—data reduction. The aerosol generation system is real time monitored for temperature by thermocouples, mass flow controllers with digital readouts with Strawberry Tree (Sunnyvale, CA) and Labtech Notebook software (Wilmington, MA). WinCIDRS (Windows-based Cascade Impactor Data Reduction System, Pacwill Environmental, Beamsville, ON, Canada) was utilized to analyze impactor data. This program provides the capabilities to reduce velocity traverse data and aids in the selection of sampling flow rates, generates files containing the hardware specifics on the impactor configurations used in sampling for later use in calculating stage D50 values, reduces the data from individual impactor runs and generates size distribution information from data at a set of standard conditions for a standardized array of particle sizes; it also combines and appropriately average the results from multiple sample runs as well as plots the particle size distributions and fractional efficiencies. Determination of prefrontal cortical glutamate concentrations: in vivo microdialysis in control and B(a)P-exposed Cprlox/lox offspring. On PND100, control and B(a)P aerosol–exposed Cprlox/lox offspring mice along with C57BL background controls (n = 4 offspring from four different litters within the group) were anesthetized with pentobarbital and mounted in a stereotaxic frame (Stoelting, Wood Dale, IL) (Figs. 4A and 4B). A guide cannula (BASi) with a dummy probe was placed and fixed by cranioplastic cement (Plastic One, Roanoke, VA) onto the skull. All mice then received an mPFC probe implantation (coordinates: A + 2.1, L + 0.2, V −2.0 mm, relative to bregma) under slight anesthesia with isoflurane, 3 h before sample collection. The probes were perfused at a flow rate of 1.5 μl/min for 165 min. Samples were collected every 15 min. Glutamate concentrations were determined by the Vanderbilt Brain Institute Neurochemistry Core. Data were analyzed using a two-factor (time × treatment) analyses of variance with repeated measures of the time factor to detect significant interactions. A Bonferroni's post hoc test was used to determine the source of the variation. B(a)P metabolite disposition and analysis from neocortical tissue. The transplacental disposition of B(a)P metabolites in neocortical tissues was determined by extraction followed by HPLC analysis as previously reported (Ramesh et al., 2001). For the early PNDs (P1–P5) whole brain tissue was used from carbon black control Cprlox/lox, carbon black control brain-Cpr-null, B(a)P-exposed Cprlox/lox, and brain-Cpr-null offspring. For the later PNDs (P7–P15) right and/or left neocortex was used from both carbon black control and B(a)P-exposed Cprlox/lox or brain-Cpr-null offspring. F2-isoprostane disposition/analysis from neocortical tissue. The ability to quantify F2-isoprostanes in Cpr offspring provided a unique opportunity to assess oxidative damage occurring primarily in neuronal membranes in utero. On PNDs 1, 3, 5, 7, 9, 11, 13, and 15, samples (75–100 mg) of dissected brain/neocortex (or whole brain for early PNDs as in PNDs 1, 3, and 5) from control and B(a)P-exposed Cprlox/lox offspring and/or brain-Cpr-null offspring were processed as previously described (Milatovic and Aschner, 2009). Briefly, brain tissue was homogenized in Folch solution, the chloroform layer evaporated, lipids chemically hydrolyzed using KOH and a stable deuterium labeled internal standard (8-iso-prostaglandin F2a-d4) added. Following extraction using C-18 and silica Sep-Pac cartridges, purification by thin layer chromatography, conversion to O-methyloxime pentafluorobenzyl ester trimethylsilyl derivative, the compound was dissolved in undecane that was dried over a bed of calcium hydride. GC was performed using a 15 m, 0.25 mm diameter, 0.25 mm film thickness, DB1701 fused silica capillary column (Fisons, Folsom, CA). The column temperature was from 190 to 300°C at 15°C/min. The methane carrier gas flow rate was 1 ml/min. Negative ion chemical ionization MS was performed using a Hewlett-Packard HP5989A instrument interfaced with monitoring ions for F2-IsoPs (m/z 569), the [2H4]15-F2t-IsoP internal standard (m/z 573), and F4-NeuroPs (m/z 593). Expression profiling via Western blot analysis. A comparison of the protein content of Sp4 transcription factor, Sp4 target gene NMDA subunit 2A, and Sp4 nontarget gene NMDA subunit 2B in neocortical tissue derived from Cpr offspring was conducted and compared with a housekeeping gene (β-actin). Pooled whole brains were used on PNDs 1, 3, and 5, and aliquots (70–100 mg) of neocortices were used on P7, 9, 11, 13, and 15 from offspring in (1) carbon black Cprlox/lox, (2) B(a)P-exposed Cprlox/lox, (3) carbon black control brain-Cpr-null, and (4) B(a)P-exposed brain-Cpr-null experimental groups. Statistical analysis of protein expression. Developmental expression profiling protein data were quantitated from Western blots using Image J software (National Institutes of Health, Bethesda, MD; open source Image J software available at http://rsb.info.nih.gov/ij/). The resulting densitometric signal band intensities ± SEM from at least three experiments were normalized to the corresponding β-actin loading levels and plotted as bar graphs. In proteins expressed as double bands, each of the bands was separately quantified and the values combined. Percentage band intensities ± SEM relative to β-actin was plotted on the y-axis and time (PND) on the x-axis for Cprlox/lox and brain-Cpr-null data sets. Immunohistochemistry. Age-matched Cprlox/lox offspring from at least four to five different litters within an experimental group of carbon black control Cprlox/lox or B(a)P-exposed Cprlox/lox was anesthetized with an ip injection of sodium pentobarbitol (100 mg/kg) and perfused first with PBS and then with 4% paraformaldehyde in PBS and postfixed for 24 h on PND100. The brains were dehydrated in 70% ethanol and left in xylene overnight before being embedded in paraffin. Paraffin blocks were coronally sectioned with a cryostat setting of 10μM. The sections were floated on a warm water bath and mounted on SuperFrost-Plus (Menzel-Glazer) glass slides. With a section interval of 10, series of sections were collected in a systematic random manner spanning 2.0 mm of the rostral-caudal extent of the hippocampus from each animal. Each series contained 20 sections. Sections were kept overnight at 37°C and then stored at room temperature. Sections were rehydrated through an ethanol series, blocked for 1 h at room temperature in PBS containing 5% normal horse serum (Vector Laboratories, Burlingame, CA) and 0.01% Triton T-100. Sections were then incubated overnight at 4°C in a 1:1000 dilution in blocking solution of monoclonal mouse anti-NeuN (Millipore #MAB377, Billerica, MA), an antibody that stains a nuclear antigen found only in neurons. After washing in PBS, the sections were stained in a 1:1000 dilution of an affinity purified biotinylated horse anti-mouse secondary antibody (Vector) for 1 h at room temperature, washed again, and incubated in Vectastain Elite ABC reagent according to the manufacturer's instructions. After washing, the color reaction was precipitated by incubation in PBS containing 0.05% diaminobenzidine and 0.01% H2O2 (Sigma-Aldrich) for 10 min. Sections were then dehydrated through an ethanol series, cleared in Citrisolv (Fisher Scientific Company), and mounted with Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI). Stereology. Sterology was performed on an Olympus BX50WI microscope equipped with Stereologer software (Chester, MD). From the series described above, four sections at equal intervals were selected for analysis. Reference spaces were drawn in the right cortex of each selected section and NeuN-stained cells counted with the Stereologer software for three age-matched (P100) male control and prenatally B(a)P-exposed animals. Cells were sampled in counting frames of 9000 μm two moved in x and y steps of 200 × 200 μm. Cells were counted using a 40× lens (NA 0.65) and were included in the measurement only when they came into focus within the dissector (dissector height of 10 μm). Total cell density was estimated using the following equation: Nv = ∑Q−/∑Volsamp, where Q− is the number of cells counted and Volsamp is the total dissector volume. The Student's t-test revealed no significant difference in neuronal density in the selected cortical area between the control and the exposed animals (p = 0.43). Object discrimination task. On P100, Cpr offspring mice (four to five offspring from four to five different litters per experimental group, where the litter represents the statistical unit) underwent one 30-min habituation session in a square activity chamber (43.2 × 43.2 cm, Med Associates, St 1). Briefly as reported in Sheng et al. (2010), an automated activity chamber monitored the horizontal and vertical movement of the mouse by quantifying the photobeam disruption. Following this habituation, the mouse was returned to its home cage for 24 h, after which each animal underwent four habituation and testing sessions, each 15 min long, separated by 15-min intervals. In the first session, the mouse was allowed to habituate to the empty activity chamber for 30 min. In session 2, the animal was allowed to familiarize itself with two objects (familiar) placed in the rear of chamber for 6 min. In session 3, the mouse was again placed in the activity chamber with the same arrangement of objects. Finally, in session 4, the session that tested novelty discrimination, one of the familiar objects from the previous session was replaced with a novel object for 6 min. Both the presentation and the position of objects were alternated as a control. Statistical analysis of responses to object discrimination in control and B(a)P aerosol–exposed Cpr offspring. Activity in the chamber was measured using open field activity software (Med Associates). This software allows for post hoc definition of zones of interest. A zone of any size can be defined by selecting grid squares of photobeam intersections. With the open field activity interface, a small zone (5 × 5 photobeam grid) and a larger zone (6 × 6 photobeam grid) were designated around each object. The software collected and documented the total activity, number of entries into each zone (object approaches) and the time spent in each zone. Simultaneously, time spent with the objects was recorded using a stopwatch by an experimenter blinded to the identity of the experimental group. Observational time included only that time during which the animal was in close proximity (within 2 cm) or actively touching or sniffing the object. The object discrimination index was calculated using the formula NI = (n − f)/(n + f), where n is the time with the novel object and f is the time with the familiar object. In this instance, the index ranged from 0 to 10, with a 0 signifying a greater preference for the familiar object and 10 signifying complete preference for the novel object. Statistical evaluations were made using ANOVA and planned comparisons. An α of 0.05 was considered significant for all statistical tests employed. Whole-cell patch clamp electrophysiology of “ex vivo” primary cortical neuronal cultures derived from control and B(a)P-exposed Cprlox/lox offspring. Cortical neuronal cultures derived from PND1 control and B(a)P-exposed Cprlox/lox offspring were prepared as previously described with the following modifications (Brown et al., 2007). PND1 control and B(a)P-exposed Cprlox/lox offspring pups were collected from respective litters within 24 h of birth. Pups were decapitated, and brains rapidly removed and placed in 35-mm Petri dishes with cold Hank's balanced salt solution (HBSS). The cortices were dissected under a dissection microscope and then placed in another dish containing HBSS to further remove blood vessels and meninges from cortical tissues. The isolated cortices were then transferred to a Petri dish containing 0.6% (wt/vol) 0.22 mm filtered trypsin in HBSS for 30 min. After two washes in HBSS, the cortical tissues were mechanically dissociated with a glass Pasteur pipette. Dissociated cortical cells were plated on poly-L-ornithine-treated glass coverslips in six-well plates, using a plating medium of glutamine-free Dulbecco's Modified Eagle Medium (DMEM)-Eagle's salts (Invitrogen, Carlsbad, CA), supplemented with Ham's F12 (Hyclone, Logan, UT), heat-inactivated fetal bovine serum (Hyclone), and penicillin/streptomycin (Sigma, St Louis, MO). The density was approximately 700,000 cells per well. After 2 days in vitro, nonneuronal cell division was halted by a 1-day exposure to 10mM cytosine arabinoside (Sigma), and cultures were shifted to Neurobasal media (Invitrogen) supplemented with B27 (Invitrogen) and penicillin/streptomycin. Cells were maintained by changing the media every 2–3 days and grown at 37°C in a humidified atmosphere of 5% CO2 in air. NMDA (Sigma) was used as a positive control for cytotoxicity at a final concentration of 100mM in conjunction with 10mM glycine. On the seventh day in culture, ex vivo primary cortical neurons derived from control and B(a)P-exposed Cprlox/lox offspring were voltage clamped using the whole-cell configuration and held at −60 mV, in a Mg2+ free external solution. The experimenter was blind to the type of treatment. RESULTS Toxicological Observations and Aerosol Characteristics In general, Cprlox/lox and brain-Cpr-null timed-pregnant dams were exposed to a B(a)P aerosol on E14–E17 that exhibited a trimodal distribution with a 93% cumulative mass less than 5.85 μm, 91% cumulative mass less than 10 μm, 57.6% cumulative mass less than 2.5 μm, and 43% less than 1 μm (Fig. 1). The characterization of the aerosol atmospheres generated in these mouse studies was comparable to those generated previously for rat studies (Hood et al., 2000). The B(a)P aerosol used in our earlier rat studies comprised a mass median aerodynamic diameter + geometric SD of 0.9 ± 0.09 μm compared with 1.0 ± 0.07 in the present Cpr mouse experiments. Analysis of live birth indices revealed no significant differences in the number of mouse pups born per litter between control Cprlox/lox dams and B(a)P-exposed Cprlox/lox dams. The analysis of the live birth index for control Cprlox/lox dams was 6.12 ± 0.42 compared with 5.9 ± 0.9 for B(a)P-exposed Cprlox/lox dams and 5.7 ± 0.31 compared with 5.99 ± 0.41 for the control and B(a)P-exposed brain-Cpr-null dams, respectively. Statistical analysis (p = 0.241) indicated no significant difference in this index between these groups. This finding is consistent with our previous reports using rat and mouse models (Brown et al., 2007; Hood et al., 2006, 2000; Sheng et al., 2010). During the prenatal exposure period as well as the subsequent preweaning period, there were no identifiable B(a)P-related effects on conventional reproductive indices of toxicity, and thus, there were no convulsions, tremors, or abnormal movements noted in any of control or B(a)P-exposed Cpr litters. In Situ Generation of “Oxidative Metabolites” in Neocortical Tissue from Cpr Offspring The quantitation of B(a)P metabolites from Cprlox/lox and brain-Cpr-null offspring is shown as Figures 2A and 2B. In neocortical tissue from in utero B(a)P aerosol–exposed Cprlox/lox offspring, we found 4,5-,7,8-,9,10-diols, 3,6- and 6,12-diones, and 3-OH and 9-OH B(a)P metabolites, indicating the presence of an active one biotransformation pathway (Fig. 2A). In contrast, in brain-Cpr-null offspring, all metabolites were essentially below the level of detection (note change in “y”-axis in Fig. 2B values). The important point in Figure 2A is the identification of the developmental period over which there is formation/accumulation of the reactive 3-OH and 9-OH metabolites. These metabolites can further oxidize to form B(a)P quinones that can undergo redox cycling and generate reactive oxygen species (Kerzee and Ramos, 2000; Kiruthiga et al., 2007). FIG. 2. Open in new tabDownload slide Metabolite distribution of cortical B(a)P metabolites during the critical postnatal period when synapses are forming for the first time in B(a)P-exposed Cpr offspring. Timed-pregnant Cprlox/lox or brain-Cpr-null dams received either carbon black only or 100 μg/m3 B(a)P via nose-only inhalation on E14–E17 for 4 h/day. Offspring were sacrificed on P1, 3, 5, 7, 9, 11, 13, and P15. Shown are the distributions of metabolites detected in B(a)P-exposed (A) Cprlox/lox offspring or (B) brain-Cpr-null offspring. FIG. 2. Open in new tabDownload slide Metabolite distribution of cortical B(a)P metabolites during the critical postnatal period when synapses are forming for the first time in B(a)P-exposed Cpr offspring. Timed-pregnant Cprlox/lox or brain-Cpr-null dams received either carbon black only or 100 μg/m3 B(a)P via nose-only inhalation on E14–E17 for 4 h/day. Offspring were sacrificed on P1, 3, 5, 7, 9, 11, 13, and P15. Shown are the distributions of metabolites detected in B(a)P-exposed (A) Cprlox/lox offspring or (B) brain-Cpr-null offspring. F2-isoprostanes measures assessed the in vivo prefrontal cortex oxidative milieu occurring primarily in neuronal membranes. Figure 3 shows the quantitation of F2-isoprostanes derived from neocortical homogenates from control and B(a)P-exposed Cprlox/lox offspring (Fig. 3A) and control and B(a)P-exposed brain-Cpr-null offspring (Fig. 3B). (Note: There are no control brain-Cpr-null mouse isoprostane data for P3–P15 due to the lack of available tissue.) The data document a sustained high neocortical tissue burden of F2-isoprostanes in B(a)P-exposed Cprlox/lox, including the period from P7 to P14, the extent of which was not observed in null mice (Fig. 3B). FIG. 3. Open in new tabDownload slide In utero exposure to B(a)P aerosol (100 μg/m3) produces an approximate 10× higher concentration of F2-isoprostanes on P3 in B(a)P-exposed Cprlox/lox offspring as compared with B(a)P-exposed brain-Cpr-null offspring. Upper panel (A): F2-isoprostanes levels derived from control (white bars) and B(a)P-exposed Cprlox/lox (black bars) offspring neocortex. Lower panel (B): F2-isoprostane levels for derived from control PND9 (white bar) and B(a)P-exposed (black bars) brain-Cpr-null offspring neocortex. Due to the limited number of control offspring, only PND9 was used for time points in favor of reserving pups for behavioral determinations in later life. *p ≤ 0.05 versus control. FIG. 3. Open in new tabDownload slide In utero exposure to B(a)P aerosol (100 μg/m3) produces an approximate 10× higher concentration of F2-isoprostanes on P3 in B(a)P-exposed Cprlox/lox offspring as compared with B(a)P-exposed brain-Cpr-null offspring. Upper panel (A): F2-isoprostanes levels derived from control (white bars) and B(a)P-exposed Cprlox/lox (black bars) offspring neocortex. Lower panel (B): F2-isoprostane levels for derived from control PND9 (white bar) and B(a)P-exposed (black bars) brain-Cpr-null offspring neocortex. Due to the limited number of control offspring, only PND9 was used for time points in favor of reserving pups for behavioral determinations in later life. *p ≤ 0.05 versus control. Temporal Modulation of NMDA-mediated Developmental Processes in Cpr Offspring Evidence supporting the hypothesis that an early insult to NMDA-mediated signaling system leads to later-life phenotypical changes that manifest as elevated glutamate concentrations in mPFC is provided in Figure 4A. The top panel shows a time course plot indicating basal levels of glutamate in C57BL background controls, Cprlox/lox, B(a)P-exposed Cprlox/lox, and B(a)P-exposed brain-Cpr-null offspring obtained by microdialysis. Studies were carried out in awake behaving control and B(a)P aerosol–exposed Cpr offspring over a 120-min sampling period. Quantitation of the results in Figure 4 shows there was a statistically significant increase in the basal concentration of glutamate in the mPFC of B(a)P-exposed Cprlox/lox offspring compared with WT C57BL, WT Cprlox/lox, or B(a)P aerosol–exposed brain-Cpr-null offspring. The basal glutamate concentrations obtained in our Cpr mouse model are identical to recent reports in the literature for dorsal hippocampus in a 129/SvEv mice KATII KO model (Potter et al., 2010) and for prefrontal cortex in a Wistar rat ethanol model (Chefer et al., 2011). These findings are consistent with the idea that increases in glutamate concentration promote altered inward currents via the NMDA receptor. FIG. 4. Open in new tabDownload slide In utero exposure to B(a)P aerosol (100 μg/m3) results in a robust increase in prefrontal cortical glutamate concentrations as assessed in awake behaving Cprlox/lox offspring but not in brain-Cpr-null offspring (PND100). Top panel (A): Glutamate concentrations assessed from medial prefrontal cortex as a function of time. Bottom panel (B): Quantitation of average glutamate concentrations over this time period (M) assessed in control C57BL, control Cprlox/lox offspring versus B(a)P-exposed Cprlox/lox and brain-Cpr-null offspring. For details, see text (C57BL = blue, Cpr = red, B(a)P-Cpr = green, and B(a)P brain-Cpr-null = purple). *p ≤ 0.05 versus Cpr. FIG. 4. Open in new tabDownload slide In utero exposure to B(a)P aerosol (100 μg/m3) results in a robust increase in prefrontal cortical glutamate concentrations as assessed in awake behaving Cprlox/lox offspring but not in brain-Cpr-null offspring (PND100). Top panel (A): Glutamate concentrations assessed from medial prefrontal cortex as a function of time. Bottom panel (B): Quantitation of average glutamate concentrations over this time period (M) assessed in control C57BL, control Cprlox/lox offspring versus B(a)P-exposed Cprlox/lox and brain-Cpr-null offspring. For details, see text (C57BL = blue, Cpr = red, B(a)P-Cpr = green, and B(a)P brain-Cpr-null = purple). *p ≤ 0.05 versus Cpr. As a means of assessing the impact of in utero exposure to B(a)P aerosol on developmental NMDA-mediated processes, we evaluated developmental expression profiles for NMDA receptor subunits compared with those of Sp4 and Sp1 proteins with and without in utero B(a)P exposure (Hood et al., 2000). Inspection of the expression profile (P1 through P15) in control Cprlox/lox offspring (Fig. 5A, left panel) revealed that Sp4 expression is constitutively low on P1, reaches peak expression levels on P7, and subsides to constitutive levels by P15, consistent with earlier reports (Li and Pleasure, 2005). Conversely, the Sp4 developmental expression profile in B(a)P-exposed Cprlox/lox offspring (Fig. 5A, right panel) indicates that as early as P1, Sp4 reaches near maximal levels and by P3, it reaches peak expression levels and remains at maximal levels through P7. The interpretation of this finding is that the leftward shift in peak Sp4 expression is in response to in utero B(a)P aerosol exposure occurring on E14–E17. The consequence of this exposure is the mistiming of peak Sp4 expression in B(a)P-exposed Cprlox/lox offspring, which could significantly impact downstream signaling processes perhaps in a way similar to those reported for Sp4-null mice (Zhou et al., 2007). FIG. 5. Open in new tabDownload slide (A) In utero B(a)P aerosol exposure (100 μg/m3) induces a leftward shift in the peak temporal developmental expression of Sp4 and alters NMDA subunit ratios in B(a)P-exposed Cprlox/lox offspring (right panel) as compared with control Cprlox/lox offspring (A, left panel). Representative results from a typical experiment. The panels show developmental expression profiles from PND1 to PND15 for NR2B-Sp4 nontarget gene, NR2A-Sp4 target gene and Sp4-Sp1 proteins following SDS-PAGE of neocortical tissue from offspring gauged relative to internal β-actin controls. Lower panels represent quantitation of left and right upper panels. Post hoc analysis with the Bonferroni's test with significance shown at p < 0.05. (B) In utero B(a)P aerosol exposure (100 μg/m3) upregulates developmental expression of Sp4 in brain-Cpr-null offspring (right panel) with NMDA subunit ratios approximating those of control Cprlox/lox offspring (A, left panel). Representative results from a typical experiment. The panels show developmental expression profiles from PND1 to PND15 for NR2B-Sp4 nontarget gene, NR2A-Sp4 target gene, and Sp4-Sp1 proteins following SDS-PAGE of neocortical tissue from offspring gauged relative to internal β-actin controls. Lower panels represent quantitation of left and right upper panels. Post hoc analysis with the Bonferroni's test with significance shown at p < 0.05. FIG. 5. Open in new tabDownload slide (A) In utero B(a)P aerosol exposure (100 μg/m3) induces a leftward shift in the peak temporal developmental expression of Sp4 and alters NMDA subunit ratios in B(a)P-exposed Cprlox/lox offspring (right panel) as compared with control Cprlox/lox offspring (A, left panel). Representative results from a typical experiment. The panels show developmental expression profiles from PND1 to PND15 for NR2B-Sp4 nontarget gene, NR2A-Sp4 target gene and Sp4-Sp1 proteins following SDS-PAGE of neocortical tissue from offspring gauged relative to internal β-actin controls. Lower panels represent quantitation of left and right upper panels. Post hoc analysis with the Bonferroni's test with significance shown at p < 0.05. (B) In utero B(a)P aerosol exposure (100 μg/m3) upregulates developmental expression of Sp4 in brain-Cpr-null offspring (right panel) with NMDA subunit ratios approximating those of control Cprlox/lox offspring (A, left panel). Representative results from a typical experiment. The panels show developmental expression profiles from PND1 to PND15 for NR2B-Sp4 nontarget gene, NR2A-Sp4 target gene, and Sp4-Sp1 proteins following SDS-PAGE of neocortical tissue from offspring gauged relative to internal β-actin controls. Lower panels represent quantitation of left and right upper panels. Post hoc analysis with the Bonferroni's test with significance shown at p < 0.05. Having demonstrated that B(a)P exposure shifts the Sp4 expression profile to the left and given that NR2A is an Sp4 target gene, it is not surprising that B(a)P exposure also modulated the temporal expression of NR2A. The Sp4 nontarget NR2B developmental expression profile, on the other hand, for control Cprlox/lox offspring (Fig. 5A, left panel) revealed that expression was low from P1 to P3 and gradually increased until peak NR2B levels were present on P15. The NR2B developmental expression profile in B(a)P-exposed Cprlox/lox offspring (Fig. 5A, right panel) is strikingly similar to that of controls. What is apparent is the significant change in the NMDA subunit ratio of NR2B:NR2A on P7 in control Cprlox/lox offspring (NR2B:NR2A = 1/0.4 seen in Fig. 5A, lower left panel quantitation) as compared with B(a)P-exposed Cprlox/lox offspring (NR2B:NR2A = 1/0.78 seen in Fig. 5A, lower right panel quantitation). Reminiscent of Sp4 expression, the NR2A developmental expression profile in B(a)P-exposed Cprlox/lox offspring (right panel) is constitutively “on,” beginning from P1 to P5, and, by P13 reaches maximal expression levels compared with P15 in B(a)P-exposed offspring. The developmental expression profiles for brain-Cpr-null offspring are shown in Figure 5B. Inspection of the expression profiles from these null mice (P1 through P15) in control brain-Cpr-null offspring (Fig. 5B, left panel) revealed that Sp4 expression is constitutively low on P1 and reaches peak expression levels on P13. Conversely, the Sp4 developmental expression profile for B(a)P-exposed brain-Cpr-null offspring (Fig. 5B, right panel) indicates that as early as P1, Sp4 expression is evident and reaches maximal levels by P3 then drops to constitutive expression levels by P7. What is apparent in the brain-Cpr-null offspring data is that the NMDA subunit ratio of NR2B:NR2A on early PNDs (P1, P3, P5, and P7) in B(a)P-exposed brain-Cpr-null offspring (NR2B:NR2A = 1/0.5 seen in Fig. 5B, lower right panel quantitation) is approximately the same as compared with P7 in control Cprlox/lox offspring (NR2B:NR2A = 1/0.5 seen in Fig. 5A, lower left panel quantitation). Based on the results from a recent NR2A subunit knockout study in mice (Brigman et al., 2008), it would be reasonable to predict that in our null model, the absence of modulation in NMDA subunit ratios would mean that these mice would not exhibit a behavioral deficit phenotype. Such a finding would be in support of our hypothesis that a significant deficit in behavioral learning results from the loss of proper temporal functioning of the NR2A subunit. Rescue of Discrimination Deficit Phenotype in the Brain-Cpr-Null Mouse Novel object discrimination testing was used to measure B(a)P-induced behavioral effects as described in our recent report (Sheng et al., 2010). Data in Figure 6 show that control Cprlox/lox offspring mice were better able to better discriminate between novel and familiar objects (8.1 ± 0.31) as compared with B(a)P-exposed Cprlox/lox offspring (2.0 ± 0.17). An ANOVA revealed significant differences between the control Cprlox/lox offspring (observational, small zone, and large zone) with regard to entries into the novel zone compared with the B(a)P-exposed Cprlox/lox offspring. The exposed group exhibited a diminished ability to discriminate novel from familiar objects as indicated by a significantly reduced time as compared with controls (p < 0.05 for the 100 g/m3 group). Post hoc analysis using the Bonferroni's test revealed the significance at p < 0.01. Testing of control brain-Cpr-null offspring versus B(a)P-exposed brain-Cpr-null offspring in the 100 g/m3 group revealed no significant differences in object discrimination at 9.1 ± 1.4 versus 9.0 ± 0.8. Given that brain-Cpr-null offspring are incapable of producing significant levels B(a)P metabolites via CYP1B1, these data strongly implicate a sustained-oxidative neocortical tissue metabolite burden during the critical period from P0–P5 as contributing to the discrimination deficit phenotype observed in B(a)P aerosol–exposed Cprlox/lox offspring. FIG. 6. Open in new tabDownload slide In utero B(a)P aerosol exposure induces a significant deficit in object discrimination in B(a)P-exposed Cprlox/lox offspring as compared with control Cprlox/lox, WT brain-Cpr-null, or B(a)P-exposed brain-Cpr-null offspring. Object discrimination index for control Cprlox/lox offspring mice was 8.1 ± 0.31 and 2.0 ± 0.17 for B(a)P-exposed Cprlox/lox offspring. An ANOVA revealed a significant difference (p < 0.05 for the 100 μg/m3 group). Testing of control brain-Cpr-null offspring revealed an index of 9.1 ± 1.4 versus 9.0 ± 0.8 for the B(a)P-exposed brain-Cpr-null offspring. Post hoc analysis with the Bonferroni's test failed to reveal a statistically significance difference at p < 0.05. FIG. 6. Open in new tabDownload slide In utero B(a)P aerosol exposure induces a significant deficit in object discrimination in B(a)P-exposed Cprlox/lox offspring as compared with control Cprlox/lox, WT brain-Cpr-null, or B(a)P-exposed brain-Cpr-null offspring. Object discrimination index for control Cprlox/lox offspring mice was 8.1 ± 0.31 and 2.0 ± 0.17 for B(a)P-exposed Cprlox/lox offspring. An ANOVA revealed a significant difference (p < 0.05 for the 100 μg/m3 group). Testing of control brain-Cpr-null offspring revealed an index of 9.1 ± 1.4 versus 9.0 ± 0.8 for the B(a)P-exposed brain-Cpr-null offspring. Post hoc analysis with the Bonferroni's test failed to reveal a statistically significance difference at p < 0.05. Figure 7 shows coronal sections of adult neocortical sections from Cprlox/lox offspring that were analyzed for cytoarchitecture by immunohistochemical staining for NeuN. Neocortical neurons were counted in Cprlox/lox offspring that had not been exposed to B(a)P in utero (panel A, low magnification, panel B, higher magnification) and compared with Cprlox/lox offspring that were exposed to the B(a)P in utero (panel C, low magnification, panel D, higher magnification). Panel E in Figure 7 shows the plot resulting from stereological analysis of neocortical neurons subsequent to application of a two-tailed t-test. As can be seen, no significant differences were seen in the density of neocortical neurons as a result of in utero exposure to B(a)P aerosol. FIG. 7. Open in new tabDownload slide Postbehavior characterization of neocortical cytoarchitecture. Coronal sections of adult neocortical sections from Cprlox/lox offspring were analyzed for cytoarchitecture by immunohistochemical staining for NeuN. Neocortical neurons were counted in Cprlox/lox offspring that had not been exposed to B(a)P in utero (panel A, low magnification; panel B, higher magnification) and compared with Cprlox/lox offspring that were exposed to the B(a)P in utero (C, 4× low magnification; D, 20× high magnification). Panel (E) shows the plot resulting from stereological analysis of neocortical neurons subsequent to application of a two-tailed t-test. As can be seen, no significant differences in the density of neocortical neurons are observed as a result of in utero exposure to B(a)P aerosol. FIG. 7. Open in new tabDownload slide Postbehavior characterization of neocortical cytoarchitecture. Coronal sections of adult neocortical sections from Cprlox/lox offspring were analyzed for cytoarchitecture by immunohistochemical staining for NeuN. Neocortical neurons were counted in Cprlox/lox offspring that had not been exposed to B(a)P in utero (panel A, low magnification; panel B, higher magnification) and compared with Cprlox/lox offspring that were exposed to the B(a)P in utero (C, 4× low magnification; D, 20× high magnification). Panel (E) shows the plot resulting from stereological analysis of neocortical neurons subsequent to application of a two-tailed t-test. As can be seen, no significant differences in the density of neocortical neurons are observed as a result of in utero exposure to B(a)P aerosol. Negative Modulation of Cortical Inward Currents in Neurons Derived from B(a)P-Exposed Cprlox/lox Offspring Lastly, electrophysiology experiments were performed on ex vivo primary cortical neurons as a means of ascertaining potential B(a)P exposure–induced effects on cortical currents. On the seventh day in culture, ex vivo primary cortical neurons derived from control and B(a)P-exposed Cprlox/lox offspring were voltage clamped using the whole-cell configuration and held at −60 mV, in an Mg2+ free external solution. The current-voltage (I-V) relationship of cortical neurons derived from control and B(a)P-exposed (100 μg/m3) Cprlox/lox offspring was nearly linear between −100 and −20 mV (Fig. 8A). Figure 8B shows a representative current trace obtained from carbon black control and cortical neurons derived from 100 μg/m3 B(a)P aerosol–exposed offspring at −80 mV using the same experimental configuration as in Figure 8A. Although there were no apparent differences between the current recorded in control and B(a)P-exposed cortical neurons at positive membrane potentials there was a statistically significant voltage-dependent decrease in the inward currents recorded at negative membrane potentials as shown in Figure 8C (t = −2.92789, p < 0.0429). FIG. 8. Open in new tabDownload slide B(a)P aerosol exposure (100 μg/m3) induces a voltage-dependent decrease in the inward currents of cortical neurons derived from B(a)P-exposed Cprlox/lox offspring. Cortical neurons were voltage clamped using whole-cell configuration in a Mg2+ free external solution. Current-voltage relations were generated using a voltage step (1 s) protocol ranging from −80 to 20 mV separated by 20 mV from a holding potential of −60 mV. The experimenter was blind to the type of treatment. (A) Representative I-V currents for cortical neurons derived from carbon black and 100 g/m3 B(a)P aerosol–exposed Cprlox/lox offspring. There is a voltage-dependent decrease in the magnitude of inward currents at negative membrane potentials in the cortical neurons derived from 100 μg/m3 B(a)P-exposed Cprlox/lox offspring. (B) Representative current traces obtained from carbon black control and cortical neurons derived from 100 μg/m3 B(a)P aerosol–exposed offspring at −80 mV using the same experimental configuration as in panel (A). (C) Bar graph shows pA inward current recorded at −100 mV in control and B(a)P-exposed cortical neurons (t = −2.92789, p < 0.0429, n = 3–5). FIG. 8. Open in new tabDownload slide B(a)P aerosol exposure (100 μg/m3) induces a voltage-dependent decrease in the inward currents of cortical neurons derived from B(a)P-exposed Cprlox/lox offspring. Cortical neurons were voltage clamped using whole-cell configuration in a Mg2+ free external solution. Current-voltage relations were generated using a voltage step (1 s) protocol ranging from −80 to 20 mV separated by 20 mV from a holding potential of −60 mV. The experimenter was blind to the type of treatment. (A) Representative I-V currents for cortical neurons derived from carbon black and 100 g/m3 B(a)P aerosol–exposed Cprlox/lox offspring. There is a voltage-dependent decrease in the magnitude of inward currents at negative membrane potentials in the cortical neurons derived from 100 μg/m3 B(a)P-exposed Cprlox/lox offspring. (B) Representative current traces obtained from carbon black control and cortical neurons derived from 100 μg/m3 B(a)P aerosol–exposed offspring at −80 mV using the same experimental configuration as in panel (A). (C) Bar graph shows pA inward current recorded at −100 mV in control and B(a)P-exposed cortical neurons (t = −2.92789, p < 0.0429, n = 3–5). DISCUSSION We have used complementary biophysical, molecular, neurochemical, and neurobehavioral approaches to examine in utero B(a)P exposure–induced effects in a Cpr mouse model. Our central findings are that in utero exposure to B(a)P aerosol (Fig. 1) in the Cpr mouse results in a substantial neocortical oxidative tissue burden of B(a)P metabolites (Fig. 2) and F2-isoprostanes (Fig. 3) during a period when synapses are developing for the first time. This sustained neocortical tissue burden would be expected to contribute to an increased cortical oxidative load. The presence of three distinct categories of metabolites (diols, diones, and hydroxy derivatives) indicate an active phase I biotransformation pathway in our control Cpr mouse model. Present in the B(a)P-exposed Cprlox/lox offspring were the 4,5-,7,8-,9,10-diols, the 3,6- and 6,12-diones, and the 3-OH and 9-OH B(a)P metabolites. The important point in interpretation of this data is the identification of the developmental period over which there is formation and accumulation of 3-OH and 9-OH metabolites. These metabolites can further oxidize to form B(a)P quinines that can undergo redox cycling and generate reactive oxygen species (Kerzee and Ramos, 2000; Kiruthiga et al., 2007). Although the data from the brain-Cpr-null support the suggestion that accumulation of hydroxy metabolites and their conversion into reactive intermediates in the Cprlox/lox mouse likely contributes to the observed neurotoxicity, the levels may be below the threshold required to cause gross alterations in neuropathology (Fig. 7). Additional experiments will be needed to gain a better mechanistic understanding on why there were no observable pathologies in the neocortical cytoarchitecture in B(a)P-exposed animals. Although speculative, one possibility may be that the plastic state of these young brains allows for certain compensations, e.g., these mice may have an improved mitochondrial capacity. Such an effect already has been noted in response to severe hyperglycemia and hyperinsulinemia (Lenaers et al., 2010). Also, no studies are available on the effect of B(a)P on NRF2 transcription factor, a master switch that regulates an antioxidant pathway. We may find that the susceptibility-exposure paradigm inherent to the present study causes upregulation of several or all antioxidant genes in response to Nrf2. Thus, despite dysregulation of glutamate homeostasis and ROS production, gross morphological alterations may be absent. Furthermore, it should be considered that the lack of change in total cell numbers would not take into consideration a more subtle change in cellular migration patterns and synaptogenesis in Cprlox/lox offspring exposed in utero to B(a)P aerosol. The present study demonstrates that in utero exposure to B(a)P aerosol in a Cpr model results in a temporal modification of upstream Sp4 transcription factor protein expression (Fig. 5) during a time when synapses are first forming. This response likely negatively impacts downstream: (1) Sp4 target gene subunit ratio protein expression (NR2A, Fig. 5), (2) homeostatic glutamate neurotransmitter concentrations (Fig. 4), (3) novel object discrimination phenotype (Fig. 6), and (4) the magnitude of inward currents in cortical neurons (Fig. 8). The latter finding presents the potential for pharmacological augmentation of NR2A-mediated currents at cortical synapses as a means of modulating evoked activity in a structure specific manner. The basal glutamate concentrations obtained in our Cpr mouse model are identical to recent reports in the literature for dorsal hippocampus in a 129/SvEv mice KATII KO model (Potter et al., 2010) and for prefrontal cortex in a Wistar rat ethanol model (Chefer et al., 2011). These findings are consistent with the idea that increases in glutamate concentration promote altered inward currents via the NMDA receptor. Whether or not these elevated concentrations of glutamate result from modulation in the activities of vesicular, glial, or astrocyte transporters (VGluT, GLT, or GLAST) remains to be seen. That the mPFC integrity is, in part, important for the expression of object discrimination memory is supported by several studies where hippocampal lesions per se were found not to impair object discrimination (i.e., the ability to recognize a familiar object and discriminate it from a novel object). These studies would suggest that the hippocampus is not totally required for the type of discrimination utilized in the present study (Forwood et al., 2005). We do agree that there are examples in the literature demonstrating that hippocampal lesions can impair object discrimination when the memory for the sample object includes spatial information. This is to say that, when an object is presented in a “complex” environment with many visual and tactile cues, it appears less salient to the animal and can therefore be encoded by the hippocampus as part of this ‘complex’ environment. Conversely, when an object is placed in an “‘impoverished” environment, it might appear highly salient and can therefore be encoded by say, the perirhinal cortex, separately from the environment. The data from these studies may facilitate a discussion regarding the currently accepted hypotheses as to why rodents with hippocampal lesions may or may not be impaired in the object discrimination paradigm under complex but not impoverished environment conditions (Winters et al., 2004). Also in support of our findings, other pharmacological studies have investigated the role of the parahippocampal glutamate receptor within the context of performance on the object discrimination task. The parahippocampal region is located dorsally to the hippocampal formation, and the evidence argues against a role of hippocampal NMDA receptors in object discrimination. In one study, intraseptal infusions of a low 0.4 mg dose of the NMDA receptor antagonist AP5, given prior to or after the sample trial or given prior to the test trial, improved object discrimination at a delay of 24 h but not 45 min in rats (Puma and Bizot, 1998). Another study reported that a presample trial sc injection of kynurenic or 5,7-dichlorokynurenic acid (antagonists at the glycine site of the NMDA receptor) in doses of 0.6 or 30 mg/kg also improved one-trial object recognition in rats at a 1-h delay (Hlinak and Krejci, 1995). These studies argue against a crucial role for the hippocampal NMDA receptor and thereby diminish the necessity for performance of in vivo microdialysis toward delineating a role for glutamate in hippocampal formations within the context of performance on the object discrimination task. It has been previously demonstrated by Grova et al. (2007) that subacute exposure to B(a)P (0–200 mg/kg) (one ip injection per day for 10 days) in adult mice modulated gene expression of NMDA-NR1 subunit in brain regions highly involved in cognitive function. Subacute exposure to B(a)P seemed to differentially affect NMDA-R1 expression in different parts of the brain. Cerebral regions, including the temporal cortex, showed no change in expression irrespective of the B(a)P dose administered. In the hippocampus, exposure to B(a)P led to increased up to 17-fold in a dose-dependent manner. In the frontal cortex, mRNA expression decreased 4–35 times with increasing doses of B(a)P. The results from these subacute studies in adult mice at relatively high doses in comparison to the present study suggest a link between B(a)P exposure dose, expression of functional obligatory NMDA-R1 mRNA, and impairments in short-term and spatial memory. We know that postnatal brain development requires experience-dependent input that can induce the release of glutamate and thereby promote critical aspects of synaptic maturation. It is during this process of postnatal synaptogenesis that the effects of in utero B(a)P exposure on neural activity are most likely to alter the expression of genes, each with its unique temporal expression profile. In neurons of the neonatal brain, NR2A mRNA progressively increases during development and is dependent upon synaptic activity (Cull-Candy et al., 2001; Kohr, 2006). In sensory pathways, Philpot et al. (2001) showed that the developmental shift from NR2B to NR2A can be postponed by sensory deprivation. Studies such as these have predicted whether an alteration in the biophysical or molecular properties of NMDARs (from insertion of NR2A and/or loss of the NR2B) places upper limit constraints on the length of the “critical period” with respect to neural activity and experience-dependent fine-tuning of certain circuits (Liu et al., 2004). A recent study by Zhou et al. (2007) reported on postnatal development of the hippocampus in the complete absence of the Sp4 gene. Notable observations were that the dentate granule cell precursors appeared to divide less during postnatal development in Sp4-null mice. Dentate granule cells from Sp4-null mice displayed less dendritic growth and arborization than those from WT mice placed into primary neuronal cultures. Additionally, Sp4-null mutant adult mice displayed both decreased neuronal cell density in the dentate granule layer and presented with a smaller dentate gyrus. The dentate gyrus is the primary gateway for hippocampal trisynaptic circuits to process the information received from the entorhinal cortex. The overall conclusion from this Sp4-null report is that abnormalities in circuitry exist in the null mouse that are characterized by a reduced hippocampal volume and that represent a significant risk factor for some neurobehavioral deficit disorders. Collectively, results from genome-wide analyses predict an overlap of the Sp protein transcription factor family with the AhR network and the Grin family of ionotropic glutamate receptor subunits (ex. NR2A). Earlier studies have reported that knockdown of the transcription factor Sp4 in mice leads to increased numbers of highly branched dendrites during the maturation of cortical neurons in primary neuronal cultures (Ramos et al., 2007). The results from this report suggest that Sp4 transcription factor likely controls dendritic patterning during development by limiting branch formation and by promoting activity-dependent pruning during a time when synapses are forming for the first time. The signaling events that regulate deactivation kinetics for the establishment of fast synapses are preceded by changes in the subunit composition of the NMDA receptor at the synapse (Cull-Candy and Leszkiewicz, 2004). This occurs during the “postnatal critical switch period” and is represented by the preweaning postnatal period from P1 to P14. Results from a recent study by Brigman et al. (2008) illustrate the principle that NMDA receptor subunit proteins mediate certain forms of synaptic plasticity and learning. A touch screen system was used to assess spatial discrimination learning in an NR2A subunit protein knockout mouse (KO). The study found that NR2A KO mice exhibited a significantly retarded discrimination learning pattern (Silvers et al., 2007) supporting the currently accepted hypothesis that relative increases in the NR2A subunit protein during development ultimately serve to stabilize memories by constraining excessive synaptic plasticity (Cull-Candy and Leszkiewicz, 2004). On the other hand, NR2B-containing NMDARs appear to be the dominant form found during development, and their activity can initiate anatomical and functional plasticity, including long-term potentiation (Feldman and Knudsen, 1998; Wormley et al., 2004a,b; McCallister et al., 2008). Paradoxically, NR2B does not contain GC-box elements within the 5′ promoter region, thus suggesting a potential mechanism for regulation of NR2A by Sp4 transcription factor during the time when synapses are developing (see schematic Fig. 9). The rationale for the Sp4 transcription factor and its target genes as viable targets for B(a)P exposure–induced modulation during critical periods of development is based on the identification of canonical xenobiotic responsive element consensus sequences and GC-box elements within the 5′ promoter region (Supplementary Data), which is thought to render this gene and its target genes susceptible to modulation by B(a)P during critical phases of development. Supplementary Data shows the 5′ promoter region of the Grin2b (NR2B) gene, which contains a single Sp4 binding site (GC box) and no XRE sequence in its 5′ promoter. The NR2B gene does, however, have seven XRE sequences in its 5′UTR (Supplementary Data). Due to the absence of canonical XRE sequences and multiple GC-box elements in the 5′ promoter region, NR2B would not be classified as an Sp4 target gene. Conversely, the Grin2a gene (NR2A) contains two Sp4 binding sites (GC-boxes) and six XRE sequences in its 5′ promoter (three in a forward orientation and three in a reverse orientation) (Supplementary Data). There are two additional XRE sequences in the ORF of Grin2a, one forward and one reverse, as is shown in Supplementary Data. The NR2A receptor subunit thus qualifies as an Sp4 target gene and has been reported as such (Liu et al., 2003). FIG. 9. Open in new tabDownload slide Top panel, normal glutamate and NMDA-NR2A homeostasis: normal temporal activation of Sp4 expression and of its target genes during embryonic development in timed-pregnant control Cprlox/lox dams is depicted from E10–E20 (birth). During the early postnatal period, the Sp4 target gene NR2A facilitates in establishing constitutive NMDA-NR2A–driven cortical currents. This occurs during the “critical period of synapse formation” (P7–P15) and contributes to a normal object recognition/discrimination phenotype in Cprlox/lox offspring. Bottom panel, dysregulated (elevated) cortical glutamate and NMDA-NR2A homeostasis: premature activation of Sp4 expression and of its target gene, NR2A occurs subsequent to in utero exposure of timed-pregnant Cprlox/lox dams to B(a)P in aerosol. During the early postnatal period in exposed Cprlox/lox offspring, dysregulated Sp4 and target gene expression results in upregulated NMDA NR2A subunit expression to negatively impact NR2A-driven cortical currents. This occurs during the critical period of synapse formation when NR2A-NR2B subunit ratios are altered (P7–P15) thus contributing to an impaired object recognition/discrimination phenotype in exposed Cprlox/lox offspring. FIG. 9. Open in new tabDownload slide Top panel, normal glutamate and NMDA-NR2A homeostasis: normal temporal activation of Sp4 expression and of its target genes during embryonic development in timed-pregnant control Cprlox/lox dams is depicted from E10–E20 (birth). During the early postnatal period, the Sp4 target gene NR2A facilitates in establishing constitutive NMDA-NR2A–driven cortical currents. This occurs during the “critical period of synapse formation” (P7–P15) and contributes to a normal object recognition/discrimination phenotype in Cprlox/lox offspring. Bottom panel, dysregulated (elevated) cortical glutamate and NMDA-NR2A homeostasis: premature activation of Sp4 expression and of its target gene, NR2A occurs subsequent to in utero exposure of timed-pregnant Cprlox/lox dams to B(a)P in aerosol. During the early postnatal period in exposed Cprlox/lox offspring, dysregulated Sp4 and target gene expression results in upregulated NMDA NR2A subunit expression to negatively impact NR2A-driven cortical currents. This occurs during the critical period of synapse formation when NR2A-NR2B subunit ratios are altered (P7–P15) thus contributing to an impaired object recognition/discrimination phenotype in exposed Cprlox/lox offspring. Clearly, the novel object discrimination task is sensitive enough to detect deficits in response to object discrimination that are reflective of learning and memory impairments. The fact that B(a)P aerosol–exposed Cprlox/lox offspring mice demonstrate negative modulation in the temporal developmental expression of Sp4 demonstrates a particular sensitivity to environmental exposure during a critical period of development. Studies in the immediate future will seek to elucidate the functional changes and mechanisms undergirding alterations in Sp4 target gene-driven neural activity (NMDA-NR2A mediated) and plasticity using our Cpr-null model. Translating these new concepts into animal studies offers the promise of advancing our ability to establish the presently absent, mechanistic connections between exposure-induced diseased phenotypes due to disturbances in temporal expression patterns during critical windows of development. In order for significant advances in the field to come to fruition, molecular-level hypotheses of in utero air pollution exposure effects on later-life phenotypes must continue to be interrogated by multidisciplinary teams. FUNDING This work was supported, in part, by National Institutes of Health (S11ES014156–05, U54NS041071, and 1R56ES017448-01A1 to D.B.H, 1R01CA142845-01A1 to A.R., 1R01NS071122-01A1 and 1R01DA026947-01A1 to H.K). Also critical to the conduct of these studies were grants from the Simons Foundation Autism Research Initiative, Institutional Grant (G12RRO3032), Nuclear Regulatory Commission Grant (NRC-27-10-515) as well as Meharry Medical College-Vanderbilt University Alliance for Research Training in Neuroscience Grant (T32MH065782). We thank Xinxin Ding, PhD, of the Wadsworth Center, New York State Department of Health for providing us with the Cpr mouse model. Special thanks to our colleague Diana Marver, PhD, for a critical review of the manuscript. Author contributions: D.B.H. conceived and designed the entire study, performed the animal husbandry-inhalation exposures of timed-pregnant Cpr dams (with M.M.), analyzed-interpreted data, and wrote the manuscript; Z.L. performed the surgery and executed the microdialysis experiments and significantly contributed to writing portions of the paper; G.C. performed protein developmental expression profiles in the laboratory of S.S.; A.R designed and performed the bioavailability and disposition studies; S.S. provided intellectual input and experimental expertise toward ascertaining the role of Sp proteins in XRE activation via developmental expression profiling that was performed in his laboratory. S.S. also provided substantial drafting and revising of manuscript to ensure incorporation of important intellectual content; R.R. and R.C. performed discrimination phenotyping on Cpr offspring under the direction of M.M.; M.M. performed immunohistochemistry and with G.J. and R.C. performed timed-pregnant matings of Cpr dams; H.K. performed the whole-cell patch clamp electrophysiology analysis of cortical neurons derived from Cpr offspring; M.A. provided the expertise for performance of F2-isoprostane analysis on Cpr offspring. M.A. also provided substantial input toward revision of manuscript to ensure incorporation of important intellectual content. All authors reviewed the manuscript and made comments prior to submission. This research was supported in part by an appointment to the U.S. Nuclear Regulatory Commission HBCU Research Participation Program administered by the Oak Ridge Institute for Science and Education. 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TI - PAH Particles Perturb Prenatal Processes and Phenotypes: Protection from Deficits in Object Discrimination Afforded by Dampening of Brain Oxidoreductase Following In Utero Exposure to Inhaled Benzo(a)pyrene
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfr261
DA - 2012-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/pah-particles-perturb-prenatal-processes-and-phenotypes-protection-OiEAq9C5iv
SP - 233
EP - 247
VL - 125
IS - 1
DP - DeepDyve
ER -