Repeated, intermittent exposures to diisopropylfluorophosphate in rats: protracted effects on cholinergic markers, nerve growth factor-related proteins, and cognitive function

Abstract

Organophosphates (OPs) pose a constant threat to human health due to their widespread use as pesticides and their potential employment in military and terrorist attacks. The acute toxicity of OPs has been extensively studied; however, the consequences of prolonged or repeated exposure to levels of OPs that produce no overt signs of acute toxicity (i.e. subthreshold levels) are poorly understood. Further, there is clinical evidence that such repeated exposures to OPs lead to prolonged deficits in cognition, although the mechanism for this effect is unknown. In this study, the behavioral and neurochemical effects of repeated, intermittent, and subthreshold exposures to the alkyl OP, diisopropylfluorophosphate (DFP) were investigated. Rats were injected with DFP s.c. (dose range, 0.25-1.0 mg/kg) every other day over the course of 30 days, and then given a 2 week, DFP-free washout period. In behavioral experiments conducted at various times during the washout period, dose dependent decrements in a water maze hidden platform task and a spontaneous novel object recognition (NOR) procedure were observed, while prepulse inhibition of the acoustic startle response was unaffected. There were modest decreases in open field locomotor activity and grip strength (particularly during the DFP exposure period); however, rotarod performance and water maze swim speeds were not affected. After washout, DFP concentrations were minimal in plasma and brain, however, cholinesterase inhibition was still detectable in the brain. Moreover, the 1.0 mg/kg dose of DFP was associated with (brain region-dependent) alterations in nerve growth factor-related proteins and cholinergic markers. The results of this prospective animal study thus provide evidence to support two novel hypotheses: (1) that intermittent, subthreshold exposures to alkyl OPs can lead to protracted deficits in specific domains of cognition and (2) that such cognitive deficits may be related to persistent functional changes in brain neurotrophin and cholinergic pathways.

Fig 1

Effect of repeated exposure (subcutaneous injections every other day) to subthreshold doses of DFP on weight gain over a 30 day period. Each symbol represents the mean ± S.E.M. N=11-26.

Fig 2

Open Field Activity on day 8 (A) and day 22 (B) of DFP exposure and on day 7 of a DFP-free washout period (C). Left to right: horizontal activity measured as the number of ambulatory counts (photobeam breaks) at each time point over a 30 min evaluation period; vertical activity (photobeam breaks activated by rearing activity); stereotypical movements (repetitive photobeam breaks); fear/anxiety related behavior measured as the time spent in the central versus the peripheral zone of the activity monitor. N=11-26. * = significant difference (p<0.05) in time spent in the center versus peripheral zone.

Fig 3

(A) Forelimb grip strength measured in kg of force. Day 7, day 14, day 21, and day 28 of DFP exposure are depicted as well as day 6 and day 13 of a DFP-free washout period (WO). (B) Accelerating rotarod performance expressed as time maintained on a rotating bar that accelerated from 4 to 40 rpm over a 5-min period. Day 16, and day 30 of CPF exposure are depicted as well as day 4 and day 14 of a DFP-free washout period (WO). The bars in each figure represent the mean ± S.E.M. N=11-26.

Fig 4

Effects of prior chronic exposure to DFP (i.e., testing beginning day 7 of a DFP-free washout period) on a water maze spatial learning procedure. (A) Hidden platform test (mean ± S.E.M), 2 trials/day over 6 consecutive days of testing. (B) Daily swim speeds (mean ± S.E.M. cm/sec) during water maze hidden platform trials. (C) Water maze probe trials (mean platform area crossings ± S.E.M.) conducted on day 14 after the last day of a 30 day exposure period to DFP or vehicle. * = significantly different from vehicle controls (p<0.05). N=11-26.

Fig 5

Effects of prior chronic exposure to DFP (i.e., testing on days 2-5 of a DFP-free washout) on the performance of a spontaneous novel object recognition task. The illustrations at the left indicate the preference for the novel object compared with the familiar object (*= p<0.05) at each of the 3 delays. Discrimination (d2) indices (i.e., averaged across delay) are presented at the right. d2 was calculated on each A/B trial and was defined as the difference in time spent exploring the novel and familiar objects divided by the total exploration time for both objects: d2 index = (novel-familiar)/(novel + familiar). Data are expressed as the mean ± S.E.M. N=11-26. * = significantly different from vehicle controls (p<0.05); † = p<0.07.

Fig 6

(A) Effects of prior chronic exposure to DFP (i.e., testing on day 13 of a drug free washout) on the percentage of prepulse inhibition (PPI) in rats for three prepulse intensities (5, 10, and 15 dB above background). (B) DFP effects on the mean startle amplitude to 120-dB, 20-ms noise burst. (C) DFP effects on the percentage of prepulse inhibition averaged across the three prepulse intensities. Bars represent mean ± S.E.M. for each treatment. N=11-26.

Fig 7

(A) Plasma concentrations of DFP during a treatment regimen in which DFP was administered s.c. on alternate days over a 30 day period followed by a 14 day wash-out period. The arrows indicate days during which DFP was administered prior to removing the blood sample for analysis. Each value represents the mean ± S.E.M. derived from 6 rats. (B). Plasma cholinesterase activity as a percent of control during a treatment regimen in which DFP was administered s.c. on alternate days over a 30 day period followed by a 14 day wash-out period. The arrows indicate days during which DFP was administered prior to removing the blood sample for analysis. Each value represents the mean ± S.E.M. derived from 5-6 rats. (C) Brain acetylcholinesterase activity as a percent of control measured in 5 brain regions on the last day of a 30-day alternate injection regimen as well as in separate groups of animals at 7 and 14 days after the last DFP administration. Each value represents the mean ± S.E.M. derived from 5-6 rats. *p<0.05 with respect to vehicle control mean.

Fig 8

α₇ nicotinic acetylcholine receptor (α₇ nAChR) levels measured by Western Blot in brain lysates derived from rats that had completed the 1.00 mg/kg alternate day regimen of DFP plus the two-week washout period. A. Representative blot illustrating a molecular weight marker and α₇ nAChR protein (~56 kDa). B. Blots illustrating α₇ nAChR protein and β-actin (~38-40 kDa) in the same samples from basal forebrain (BF), hippocampus (HIPP), and prefrontal cortex (PFC). C. Data presented in the bar graphs were obtained from densitometry measurements of the bands for α₇ nAChR and β-actin and the represent the mean ± SEM for the ratio. *** indicates significant difference compared to vehicle-treated control rats, p<0.001, two-tailed Student’s t-tests. N = 5-6.

Fig 9

Pro-NGF and mature NGF (mNGF) levels measured by Western Blot in brain lysates derived from rats that had completed the 1.00 mg/kg alternate day regimen of DFP plus the two-week washout period. A. Representative blot illustrating a molecular weight marker, proNGF (~32 kDa), and NGF (~14 kDa). B. Blots illustrating proNGF, NGF, and β-actin (~38-40 kDa) in the same samples from basal forebrain (BF), hippocampus (HIPP), and prefrontal cortex (PFC). C. Data presented in the bar graphs were obtained from densitometry measurements of the bands for proNGF, NGF, and β-actin and the represent the mean ± SEM for the ratio. * and ** indicates significant difference compared to vehicle-treated control rats, p<0.05, p<0.01 (respectively), two-tailed Student’s t-tests. N = 5-6.

Fig 10

(A) TrkA levels measured by Western Blot in brain lysates derived from rats that had completed the 1.00 mg/kg alternate day regimen of DFP plus the two-week washout period. A. Representative blot illustrating a molecular weight marker and TrkA (~140 kDa). B. Blots illustrating TrkA and β-actin (~38-40 kDa) in the same samples from basal forebrain (BF), hippocampus (HIPP), and prefrontal cortex (PFC). C. Data presented in the bar graphs were obtained from densitometry measurements of the bands for TrkA and β-actin and the represent the mean ± SEM for the ratio. * and ** indicates significant difference compared to vehicle-treated control rats, p<0.05, p<0.01 (respectively), two-tailed Student’s t-tests. N = 5-6.

Fig 11

p75^NTR levels measured by Western Blot in brain lysates derived from rats that had completed the 1.00 mg/kg alternate day regimen of DFP plus the two-week washout period. A. Representative blot illustrating a molecular weight marker and p75^NTR (~75 kDa). B. Blots illustrating p75^NTR protein and β-actin (~38-40 kDa) in the same samples from basal forebrain (BF), hippocampus (HIPP), and prefrontal cortex (PFC). C. Data presented in the bar graphs were obtained from densitometry measurements of the bands for p75^NTR protein and β-actin and the represent the mean ± SEM for the ratio. ** indicates significant difference compared to vehicle-treated control rats, p<0.01, two-tailed Student’s t-tests. N = 5-6.