The pesticide fipronil injected into the substantia nigra of male rats decreases striatal dopamine content: A neurochemical, immunohistochemical and behavioral study
Introduction
Fipronil (FPN) belongs to the phenylpyrazole class of pesticides, which have a highly effective broad-spectrum activity as insecticides and are widely used for agricultural and non agricultural purposes, from soil injection, use on fruits, vegetables, coffee, rice and other crops and treatment of seeds to the use in poultry farms and in topical pet care products (see [1,2]). FPN acts as a GABAergic insecticide, by binding to the γ-gamma-aminobutyric acid (GABA) receptors and consequently by blocking chloride ion cellular uptake in invertebrates, leading to uncontrolled hyper-excitation of the central nervous system, convulsions, and cell death (see [3]). Due to its high lipophilic nature, FPN can become sequestered in highly lipidic tissues, including the brain, for an extended period of time [4].
Exposure to micromolar concentrations of FPN has been also reported to be able to induce cell death in vitro, either in Caco-2 cells, which are used as a model to mimic the cellular barrier of the intestinal epithelium [5,6], or in human dopaminergic neuroblastoma SH-SY5Y cells, via an apoptotic pathway mediated by reactive oxygen species (ROS) and inflammatory response [[7], [8], [9]]. The latter finding suggests that dopaminergic neurons may be a possible target of FPN neurotoxicity at central level. In agreement with this hypothesis, a progressive loss of nigrostriatal dopaminergic neurons induced by inflammatory responses to FPN has been recently reported after injection of FPN into the substantia nigra (SN) of male rats [9,10]. The inflammatory responses caused by FPN injected into the SN were revealed either by increased levels of pro-inflammatory factors such as inducible nitric oxide synthase (iNOS), ciclooxygenase 2 (COX-2), and tumor necrosis factor alpha (TNF-α) in the SN and in the striatum of FPN-treated rats, or by the up-regulation of glial fibrillary acidic protein expression and by the activation of microglia [9,10]. The increase of the above pro-inflammatory factors in FPN-treated rats was inversely correlated to the loss of nigrostriatal dopaminergic neurons, shown by the decrease in tyrosine hydroxylase (TH) immunoreactive neurons in the striatum and SN, decrease also confirmed by the decrease of TH expression by western blot analysis [9,10]. These results are in line with those obtained with other pesticides, such as rotenone and paraquat, in inducing a loss of dopaminergic neurons in the SN [[11], [12], [13], [14], [15], [16], [17]], as well as with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxy-dopamine, which are extensively used to realize animal models of Parkinson’s disease [[18], [19], [20], [21], [22], [23]].
Many studies show also that degeneration of nigrostriatal dopaminergic neurons induced by the above compounds is often correlated to deficits in the performance of the treated animals in spontaneous motor activity in the open field [[24], [25], [26], [27], [28], [29]] as well as in motor coordination in the rotarod test [[30], [31], [32], [33], [34]], in memory recognition (novel object recognition), social interaction and nociception [[35], [36], [37], [38], [39]]. In order to better characterize the central neurotoxic effects of FPN, the effect of two doses of FPN (15 μg and 25 μg) microinjected unilaterally into the SN of male rats was studied on spontaneous locomotor activity, motor coordination, the induction of rotational behavior after challenge with the dopamine agonist apomorphine, nociception, object recognition memory and social interaction, measured between 7 and 15 days after treatment. The results of these experiments show that FPN injected into the SN induces changes in spontaneous locomotor activity, motor coordination, apomorphine-induced rotational behavior, nociception, but not in novel object recognition and social interaction. These changes are related, at least in part, to the decrease of the striatal concentration of dopamine and its main metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) and of nigral TH immunoreactivity caused by the pesticide injected in the SN.
Section snippets
Animals
Adult three months old male Sprague Dawley rats, weighing 250−300 g (at the beginning of the experiments), were used in the study. Animals were housed in groups of 4 per cage, and maintained under standard conditions with 12-h light/dark cycles at room temperature (22 ± 2 °C, 60 ± 5% humidity). They were fed with standard pellet diet and tap water ad libitum along the study. The rats were handled once daily for 10 days before the injections into the SN in order to avoid the stress induced by
Body weight
As shown in Fig. 2, there was no significant difference in the body weight pattern between rats unilaterally injected into the SN either with vehicle (DMSO 1 μl, control rats) or FPN at the dose of either 15 μg or 25 μg. A slight decrease in body weight was observed in the first week after surgery in the majority of rats. The weight gain pattern, from 8 to 12 days after the microinjection, was progressive and slightly higher in control DMSO-treated rats as compared to FPN-treated rats, but the
Discussion
This study shows for the first time that FPN injected unilaterally in the SN induces changes in spontaneous locomotor activity and in motor coordination in the motility and rotarod apparatus and in rotational circling when challenged with the dopaminergic drug apomorphine. In particular, only a modest but significant time-dependent increase was seen on horizontal but not vertical activity in the first 30 min of the motility test at the dose of 25 μg of the pesticide injected into the SN (this
Author statement
RB, JB, and FS performed the behavioral and neurochemical experiments; RB, CL and FS the rotarod and nociception experiments; RB, GC and CC immunohistochemistry experiments, FS, RB and AA analyzed the experimental data; FS, PDD, AA, MRM organized and supervised the experimental design; RB, FS, AA and MRM wrote the manuscript.
Declaration of Competing Interest
The authors have nothing to declare.
Acknowledgments
This work was partially supported by grants from the University of Cagliari (CAR 2014, PRID 2015 and FIR2016, 2017, 2018) to AA, MRM and FS.
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