The pesticide fipronil injected into the substantia nigra of male rats decreases striatal dopamine content: A neurochemical, immunohistochemical and behavioral study

https://doi.org/10.1016/j.bbr.2020.112562Get rights and content

Highlights

  • Fipronil injected in the substantia nigra of male rats induces changes in several behaviors.

  • Among these are changes in spontaneous motor activity and motor coordination.

  • The pesticide also induces changes in nociception and object recognition performance.

  • These changes are parallel to decreases in striatal dopamine and nigral tyrosine hydroxylase.

Abstract

Experimental evidence shows that the phenylpyrazole pesticide fipronil exerts neurotoxic effects at central level in rodents, and in particular on nigrostriatal dopaminergic neurons, whose degeneration is well known to cause motor and non-motor deficits in animals and in humans. In order to characterize better the central neurotoxic effect of fipronil, we injected fipronil (15 and 25 μg) dissolved in dimethyl sulfoxide (DMSO) unilaterally into the substantia nigra of male rats. Male rats injected with DMSO unilaterally into the substantia nigra were used as controls. Control and fipronil-treated rats were then tested in different motor (i.e., open field arena, rotarod, tail flick) and non motor tests (novel object recognition, social interaction) 15 days after injection. A systemic challenge dose of the dopamine-agonist apomorphine was also used to study the presence of a rotational behavior. Sixteen days after fipronil or DMSO injection into the substantia nigra, rats were sacrificed, and either striatal dopamine content or substantia nigra tyrosine hydroxylase (TH) immunoreactivity were measured. The results confirm that the unilateral injection of fipronil into the substantia nigra caused the degeneration of nigrostriatal dopaminergic neurons, which leads to a decrease around 50 % in striatal dopamine content and substantia nigra TH imunoreactivity. This occurred together with changes in motor activity and coordination, and in nociception but not in recognition memory and in social interaction, as revealed by the results of the behavioral experiments performed in fipronil-treated rats compared to vehicle-treated rats 15 days after treatment, as found with other compounds that destroy nigrostriatal dopaminergic neurons.

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.

References (72)

  • S. Walsh et al.

    Time-course of nigrostriatal neurodegeneration and neuroinflammation in the 6-hydroxydopamine-induced axonal and terminal lesion models of Parkinson’s disease in the rat

    Neuroscience

    (2011)
  • E.Y. Lee et al.

    Rosiglitazone, a PPAR-(agonist, protects against striatal dopaminergic neurodegeneration induced by 6-OHDA lesions in the substantia nigra of rats

    Toxicol. Lett.

    (2012)
  • R. Deumens et al.

    Modeling Parkinson’s disease in rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway

    Exp. Neurol.

    (2002)
  • E.A. Jackson et al.

    Behavioral activity of some novel aporphines in rats with 6-hydroxydopamine lesions of caudate or nucleus accumbens

    Eur. J. Pharmacol.

    (1983)
  • M.M. Ferro et al.

    Comparison of bilaterally 6-OHDA- and MPTP-lesioned rats as models of the early phase of Parkinson’s disease: histological, neurochemical, motor and memory alterations

    J. Neurosci. Methods

    (2005)
  • M. Geed et al.

    Silibinin pretreatment attenuates biochemical and behavioral changes induced by intrastriatal MPP+ injection in rats

    Pharmacol. Biochem. Behav.

    (2014)
  • J. Bové et al.

    Neurotoxin-based models of Parkinson’s disease

    Neuroscience

    (2012)
  • C.S. Lee et al.

    Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by instrastriatal 6-hydroxydopamine in the rat

    Neuroscience

    (1996)
  • A. Rahimmi et al.

    N-acetylcysteine prevents rotenone-induced Parkinson’s disease in rat: an investigation into the interaction of parkin and Drp1 proteins

    Brain Res. Bull.

    (2015)
  • S. Loiodice et al.

    Implication of nigral dopaminergic lesion and repeated L-dopa exposure in neuropsychiatric symptoms of Parkinson’s disease

    Behav. Brain Res.

    (2019)
  • L. Angioni et al.

    Involvement of nigral oxytocin in locomotor activity: a behavioral, immunohistochemical and lesion study in male rats

    Horm. Behav.

    (2016)
  • C. Lobina et al.

    Different sensitivity to the motor incoordinating effects of gamma-hydroxybutyric acid (GHB) and baclofen in GHB-sensitive and GHB-resistant rats

    Brain Res.

    (2005)
  • K. Vedovelli et al.

    Effects of increased opportunity for physical exercise and learning experiences on recognition memory and brain-derived neurotrophic factor levels in brain and serum of rats

    Neuroscience

    (2011)
  • M.N. de Lima et al.

    Reversal of age-related deficits in object recognition memory in rats with l-deprenyl

    Exp. Gerontol.

    (2005)
  • M.N. de Lima et al.

    Reversion of age-related recognition memory impairment by iron chelation in rats

    Neurobiol. Aging

    (2008)
  • A. Cavoy et al.

    Spatial but not object recognition is impaired by aging in rats

    Physiol. Behav.

    (1993)
  • M. Palkovits

    Punch sampling biopsy technique

    Methods Enzymol.

    (1983)
  • J. Blesa et al.

    Compensatory mechanisms in Parkinson’s disease: circuits adaptations and role in disease modification

    Exp. Neurol.

    (2017)
  • R.K. Schwarting et al.

    The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments

    Prog. Neurobiol.

    (1996)
  • A. Fil et al.

    Pain in Parkinson disease: a review of the literature, Parkinsonism Relat

    Disord.

    (2013)
  • K.M. Sindhu et al.

    Rats with unilateral median forebrain bundle, but not striatal or nigral, lesions by the neurotoxins MPP+ or rotenone display differential sensitivity to amphetamine and apomorphine

    Pharmacol. Biochem. Behav.

    (2006)
  • L. Truong et al.

    Developing a preclinical model of Parkinson’s disease: a study of behaviour in rats with graded 6-OHDA lesions

    Behav. Brain Res.

    (2006)
  • R. Kumar et al.

    Bioefficacy and fate of fipronil and its metabolites in basmati rice under sub-tropical climatic conditions

    Crop Protection

    (2013)
  • C.C. Tingle et al.

    Fipronil: environmental fate, ecotoxicology, and human health concerns

    Rev. Environ. Contam. Toxicol.

    (2003)
  • X. Wang et al.

    Fipronil insecticide toxicology: oxidative stress and metabolism

    Crit. Rev. Toxicol.

    (2016)
  • E.G. Stafford et al.

    Consequences of fipronil exposure in egg-laying hens

    J. Am. Vet. Med. Assoc.

    (2018)
  • Cited by (17)

    • Oxytocin-conjugated saporin injected into the substantia nigra of male rats alters the activity of the nigrostriatal dopaminergic system: A behavioral and neurochemical study

      2021, Brain Research
      Citation Excerpt :

      In fact, it is difficult sustaining that this effect is secondary to the decreased number of nigrostriatal dopaminergic neurons expressing oxytocinergic receptors of OXY-SAP-treated rats. This because a decrease in the nigrostriatal dopaminergic activity as that occurring in OXY-SAP-treated rats, usually reduces rather than increase locomotor activity (see Bharatiya et al., 2020; Jáidar et al., 2019; Nuckolls et al., 2012; Robinson et al., 1994; Rohlfs et al., 1997; Schwarting and Huston, 1996). An increase in locomotor activity is usually observed when marked dopamine receptor hypersensitivity takes place in the lesioned striatum, and this does not seem the case of this study.

    • Mitochondrial and transcriptome responses in rat dopaminergic neuronal cells following exposure to the insecticide fipronil

      2021, NeuroToxicology
      Citation Excerpt :

      In addition to cytotoxicity and apoptosis, in vitro investigations demonstrate fipronil is likely a potent uncoupler of oxidative phosphorylation (Vidau et al., 2011). This observation may in part explain the rapid death of dopamine neurons in the substantia nigra of rats injected with fipronil (Bharatiya et al., 2020), as altered oxidative phosphorylation (OXPHOS) can result in redox imbalances and an increase in reactive oxygen species. Taken together, both in vitro and in vivo data reveal that fipronil can induce neurotoxicity to neuronal cells including dopamine neurons.

    • The effects of fipronil on emotional and cognitive behaviors in mammals

      2021, Pesticide Biochemistry and Physiology
      Citation Excerpt :

      However, sufficient toxicity studies have not been conducted on the effects of fipronil on the emotional and cognitive behaviors of mammals. Interestingly, neurotransmitters dopamine and serotonin or 5-hydroxytryptamine (5-HT) in each part of the brain are associated with the results of behavioral tests in response to other chemicals (Ait Bali et al., 2017; Bharatiya et al., 2020a, 2020b). Fipronil is primarily metabolized by cytochrome P450 (CYP), and fipronil sulfone is a by-product.

    View all citing articles on Scopus
    View full text