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Protein carbonyl content the toxin in corn oil (CPO) or corn oil alone. For
Protein carbonyl content was determined by the these studies, three experimental paradigms were
OxiSelect Protein Carbonyl Kit (Cell Biolabs, Inc.) employed [Figure 1]. (1) Pregnant APP female mice
and ELISA. Briefly, bovine serum albumin (BSA) were fed CPO daily for 3-week from the beginning of
standards or protein samples (10 μg/mL) were gestation; (2) female APP mice were fed CPO daily for
adsorbed onto a 96-well plate for 2 h at 37 °C. The 3-week from the beginning of lactation; and (3) weaned
protein carbonyls present in the sample or standard APP offspring were fed CPO daily for up to 6 months
were derivatized to dinitrophenyl (DNP) hydrazone of age. CPO has been shown to cross the placental
and probed with an anti-DNP antibody, followed and is secreted into the milk following treatment of
by an HRP conjugated secondary antibody. The rodents. [34,35]
protein carbonyl content in an unknown sample
was determined by comparing with a standard curve Administration of chlorpyrifos oxon exacerbates memory
that was prepared from predetermined reduced and deficits in the amyloid precursor protein mice
oxidized BSA standards. The effect of the CPO (1 mg/kg) on the various
paradigms was assessed for nonspatial pretraining for
Statistical evaluation four consecutive days to learn the location of the hidden
Experiments consisted of 10 mice in each group. platform. Analyzes by the Morris water maze test on
Each biochemical analysis consisted of two or three each day of the training period showed that the mice
replicates. Statistical analyzes and data display were do learn, indicated by the reduced latency time for the
conducted utilizing computer software designed for mice to reach the hidden platform during the training
scientific data analysis (Prism 4 GraphPad, Prism, La period [Figure 2]. By the 4th day of training, the
Jolla, CA). Quantitative data are displayed as the mean control APP in all groups showed the shortest latency
and standard error of the mean. Differences between period, representing enhanced learning, compared
groups were determined by ANOVA analysis and to the CPO APP mice. Mice administered CPO under
Dunnett’s multiple comparison tests used to determine each paradigm showed a worsening of the latency time
differences between transgenic control mice and treated suggesting that the treatment with CPO perturbs the
animals. learning process.

RESULTS Two days following training, mice were subjected
to the final behavioral Morris water maze test to
Experimental protocol determine the memory deficits. CPO-treated mice
To investigate the influence of environmental toxins exhibited substantial worsening of memory deficits,
on AD, we used the APP transgenic mice expressing assessed by the latency period and distance traveled,
the mutant form of human presenilin-1 (DeltaE9) and which is the time and distance, respectively,
the mutant chimeric mouse/human APP695 residue that it took the animal to swim to the submerged
form (51). The mouse prion protein promoter directs platform [Figure 3]. The shorter time and distance
the expression of both transgenes. APP mice start traveled indicates better memory. The CPO treatment
developing amyloid plaques around 3-4 months of age.
This will allowed us to study the process in a relatively
short period and to allow for the generation of sufficient
animals for the studies. APP mice were exposed to
either 1 mg/kg CPO via ingestion by suspending

Figure 2: Treatment of amyloid precursor protein (APP) mice with chlorpyrifos
Figure 1: Experimental protocol for treatment of mice with chlorpyrifos oxon. oxon (CPO) results in diminished memory acquisition. APP mice (control, corn
This figure illustrates the protocol for the treatment of transgenic Alzheimer’s oil alone) and APP mice treated with CPO (CPO) from the different groups at 6
disease mouse model utilized in this study. (a) Treatment of pregnant female months of age were trained in the Morris water maze test on each of 4 consecutive
amyloid precursor protein (APP) mice for 3‑week during gestation; (b) treatment days to learn the location of a submerged, invisible platform in a pool of water.
of female APP mice after birth and for 3‑week during lactation; (c) treatment The time that it took the mice to swim to the platform was recorded each day,
of APP mice following weaning until termination of the study at 6 months. All measured as the latency period (in seconds, s), with shorter latency times
animals were assessed behaviorally prior to sacrifice. -3w, indicates time of plug indicating better memory acquisition. Latency (s) is shown as mean ± standard
following when male and female APP mice were placed together; 0, indicates error of the mean (statistical significance, *P < 0.05, **P < 0.01, compared to
time at birth; +3w, indicates time of weaning of APP mice APP control group; n = 12 per group)

34 Neuroimmunol Neuroinflammation | Volume 2 | Issue 1 | January 15, 2015