Following 1 week of application of CPF, mean body weights of the mice receiving dermal applications of CPF were reduced compared to the control group. The weight loss observed in this study could be attributed to the effect of CPF causing cholinergic overstimulation, leading to increased gastric motility and a reduction in absorption . Furthermore, cholinergic overstimulation of nicotinic receptors can cause increased muscular activity (fasciculation and tremors) and thus increases energy consumption. Daily applications of CPF for seven days at the lower dose (1/10th dermal LD50) could reduce plasma cholinesterase activity compared to control group (without stress, 30.5% reduction and with stress, 49.8% reduction) but the reduction in CPF only group was not statistically significant. The reduction in stressed group was statistically significant compared to the control. Similar to the findings in this study, a previous study found that subsequent to daily low dose (12% LD50) injection of the OP soman to mice for three days, plasma cholinesterase activities were inhibited by 32% . In a separate study, 14 days after dermal applications of OP diisopropylfluorophosphate (DFP) to monkeys at low doses of 0.01 mg/kg BW, cholinesterase activity was reduced by 76% . A single dermal application of CPF in humans for four hours absorbed CPF very little (4.3%), and CPF was not completely eliminated from the body even after 120 hours, suggesting accumulation of CPF in the body . The CPF applied in this study was dissolved in the xylene, which is an organic solvent. As organic solvents dissolve fat, xylene can be easily absorbed by the layer of fat in the skin.
Compared to the previous study by this author , where application of swim stress and CPF (1/5th LD50) for 21 days facilitated the reduction in serum cholinesterase by 19.7% (compared to only application of CPF), this study showed that application of stress for lower duration (7 days) with lower dose of CPF (1/10th LD50) can bring down the serum cholinesterase levels by similar level (19.3%). The shorter duration of stress might not have potentiated the neurotoxic effects of CPF enough in all the mice. Hence the changes observed with stress remained statistically insignificant compared to the CPF only groups but became significant compared to the control.
Qualitative observations of the hippocampal neurons in this study showed that following seven days of low dose CPF application (1/10th dermal LD50), no apparent damage to the neurons was visible. However at the higher dose (1/5th dermal LD50), seven days of application resulted in visible damage in the form of pyknosis. Dendritic morphology was assessed in the prefrontal cortex, CA1 area of the hippocampus and the nucleus accumbens following repeated (14 days) low dose intraperitoneal application of OP malathion (40 mg/kg BW) in mice. Dendritic length in the hippocampus and prefrontal cortex, and density of dendritic spines in all the three areas assessed were reduced . As part of the trisynaptic circuit, afferent inputs to the hippocampus are first sent to the dentate gyrus, which then projects to the CA3 area. The CA3 neurons then send projections to CA1. Dendrites of CA1 neurons project to the subiculum and then back to the entorhinal cortex. CA3 being an early structure in this circuit, it is the first part of the hippocampus to be affected by cholinergic overactivity. This could explain the neuronal reduction observed only in CA3 after application of low dose CPF (1/5th dermal LD50) for seven days. Agricultural workers chronically exposed to low-levels of CPF and other pesticides were found performing poorly on neurobehavioral tests . Following occupational exposure to CPF, functional deficits in cognitive tests of abstraction, concentration and memory have also been reported [26, 27]. These functional deficits can be extrapolated to be caused by prolonged exposure to low dose CPF.
Quantitative examination of the hippocampal neurons showed that consequent application of stress and CPF (1/10th and 1/5th dermal LD50), even for seven days, showed marked reduction in neuronal density in all areas of the hippocampus. Neuronal density in the CA3 area of the hippocampus was also shown to be significantly reduced in rats after prolonged pain stress in the form of 13 min electric shocks for 15 days . It has been proposed that alterations in the cholinergic neurotransmitter systems due to stress are the initial events contributing to CNS impairment and that exacerbation of injury could occur with the concurrent exposure of stress and cholinesterase inhibitors . Previous study by the authors showed that toxicity on hippocampal neurons following three-weeks-long applications of CPF at high doses (1/2 dermal LD50) could be exacerbated by exposure to swim stress . It was reported that compared to just CPF application (1/2 dermal LD50), CPF with stress increased the reduction in neuronal density by 30%, 12% and 26.7% in the CA1, CA2 and CA3 areas of the hippocampus respectively. This study showed that the application of 1/10th dermal LD50 CPF with stress for 7 days only showed many pyknosed neurons surrounded by vacuolation of neuropil in the CA1and CA3 sub-fields of the hippocampus and the neuronal count was significantly reduced (p < 0.05) compared to the control. These changes were less apparent after application of CPF (1/10th dermal LD50) only. The current study has shown that stress with dermal application of CPF can cause hippocampal damage only after seven days of application at a much lower dose (1/10 dermal LD50). Stress has been demonstrated to increase permeability of the BBB to foreign chemicals . Thus the increased permeability could have caused the increased toxicity of CPF on the hippocampal neurons observed in this study.
Following one week of CPF application at both doses (1/10th and 1/5th dermal LD50), GFAP expression as measured by astrocytic density was significantly increased compared to the control group. GFAP expression has been found to be increased following toxic insult to the CNS in many studies. A single subcutaneous injection (50 μg/kg bw, 1/2 LD50) of the cholinesterase inhibitor Sarin was found to significantly increase GFAP levels in the cerebral cortex by 269% after one hour, and to 318% after two . Extended studies in rats on the effects of gestational exposure to cholinotoxicants nicotine and CPF, alone and in combination, showed increased GFAP expression in offspring in the CA1 sub-field of the hippocampus, and white matter and granular cell layer of the cerebellum [17, 18]. In the present study, GFAP expression was increased in the groups receiving combined treatments of stress and CPF 0.2 dosage as compared to those just receiving CPF 0.2 dosage, but the increase was not significant. The application of swim stress with CPF 0.1 dosage did not increase the GFAP expression compared to that in CPF 0.1 dosage only. The findings suggest that toxicity resulting from stress leads to increase in GFAP expression in response to greater injury to the hippocampus with higher sub-toxic dose of CPF. Qualitative examination showed that following seven days of CPF application, GFAP expression in the astrocytes was more prominent compared to the control groups. The astrocytic processes of the groups receiving CPF were longer, and greater in number. This may be attributed to the neuroprotective effect of astrocytes limiting neuronal damage. It has been suggested that the metabolites of CPF, trichloropyridinol (TCP), exert strong toxic effects on astrocytes, compromising their neuroprotective effects and thus increasing the neurotoxicity of CPF . The neuroprotective effects of astrocytes have been suggested in many studies. To assess the influence of glial cells on the neurotoxicity of OPs, aggregate brain cell cultures of foetal rat telencephalon were treated with CPF and parathion for 10 days. This in vitro study found that the neurotoxicity of CPF and parathion was increased in aggregate cultures deficient in glial cells . When an acute dose of the OP diisopropylfluorophosphate (DFP) was injected subcutaneously into hens, the authors discovered that GFAP expression studied in total RNAs extracted from non-susceptible parts of cerebrum was upregulated from first 2 days, indicating a neuroprotective effect from anticipated imminent neurotoxicity .