Differential up-regulation of striatal dopamine transporter and α-synuclein by the pyrethroid insecticide permethrin

Abstract

The effects of permethrin on striatal dopaminergic biomarkers were assessed in this study. Retired breeder male C57 B1/6 mice were given an ip dose of permethrin (0.1–200 mg/kg) at 7-day intervals, over a 2-week period (Days 0, 7, and 14). Animals were then sacrificed 1 day (t = 1), 14 days (t = 14), or 28 days after the last treatment (t = 28). Dopamine transporter (DAT) protein as assayed by Western blotting was increased to 115% in the 0.8 mg/kg group over that of control mice at t = 1 (P < 0.05). At t = 14, this value increased to 140% of control, and declined slightly to 133% of control at t = 28. The mice given the 1.5 mg/kg dose displayed a significant increase in DAT protein only at t = 28, to 145% of controls. Thus, upregulation of the DAT at low doses of PM is variable 24 h after treatment, and seems to stabilize by t = 28. The threshold dose for increasing DAT expression in Western blots by t = 28 was 0.2 mg/kg permethrin. [³H]GBR 12935, used to assay DAT binding, followed the same trend as that for the Western blotting data for 0.8 and 1.5 mg/kg doses of permethrin over the 4 weeks posttreatment. At 200 mg/kg permethrin, DAT protein was unchanged vs controls (t = 1), but had significantly increased by t = 14 and continued to increase at t = 28, suggesting that the reduced dopamine transport at this dose was due to nerve terminal stress and that recovery had occurred. The protein α-synuclein was also significantly induced at the 1.5 mg/kg dose at t = 1; however, unlike DAT up-regulation, this effect had declined to control values by t = 14. Maximal induction of α-synuclein protein occurred at a dose of 50 mg/kg permethrin. These data provide evidence that the pyrethroid class of insecticides can modulate the dopaminergic system at low doses, in a persistent manner, which may render neurons more vulnerable to toxicant injury.

Introduction

Parkinson's disease (PD), a chronic neurodegenerative disease of unknown etiology, is characterized by a loss of dopaminergic neurons in the substantia nigra and depletion of striatal dopamine (DA) (Fearnley and Lees, 1991). In humans, an 80% striatal DA loss is required before overt clinical symptoms appear, after which significant correlation exists between severity of PD symptoms and subsequent DA loss (Hornykiewicz and Kish, 1986).

The etiology of idiopathic PD is thought to be multifactorial Paganini-Hill, 2001, Siderowf, 2001, Zhang et al., 2000, and it has long been known that there is an epidemiological link between PD and persons who are associated with rural living and agricultural work. In particular, persons exposed to various herbicides and insecticides in an agricultural setting show an increased risk of developing PD Semchuk et al., 1992, Semchuk et al., 1993 Gorell et al., 1998). Other work linked exposure to organochlorine insecticides such as dieldrin to PD (Fleming et al., 1994). The discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is able to produce parkinsonism in animals and humans via its oxidative metabolite MPP⁺ (Tipton and Singer, 1993) provided the original evidence for a putative environmental component in the etiology of PD (Jenner et al., 1992).

The so-called Gulf War Syndrome, which is manifest as various neurological maladies reported by veterans of the Persian Gulf War, may be linked to pesticide exposure. These compounds include pyridostigmine bromide (PB), an anticholinesterase, the insect repellent DEET, and the pyrethroid insecticide permethrin (PM), which was impregnated into the uniforms of soldiers who served in the war (Hoy et al., 2000). Personnel who served in the first Gulf War report a variety of symptoms of neurological dysfunction, with no mortality increases, which have been confirmed by epidemiological studies (Steele, 2001). Further, these symptoms may be related to a spectrum of generalized neurologic injury to the central, peripheral, and autonomic nervous systems Haley et al., 1997a, Haley et al., 1997b.

The dopamine transporter (DAT) is a membrane-bound carrier molecule that mediates the action of DA in the nerve synapse via the reuptake of DA into the dopaminergic neuron, and the DAT is also capable of neuronal DA release (Horn, 1990). In addition, MPP⁺ is transported into the neuron via the DAT, where it blocks mitochondrial complex 1 respiration and subsequently causes cell death (Tipton and Singer, 1993). Thus, the DAT is a putative molecular gateway for exogenous and endogenous dopaminergic toxicants.

Using three ip doses over a 2-week period (Days 0, 7, and 14), our laboratory found that the organochlorine insecticide heptachlor has significant effects on DA transport and causes DAT up-regulation in C57B1/6 mice at relatively low doses Miller et al., 1999, Kirby et al., 2000. We also showed that the pyrethroid insecticides deltamethrin and PM significantly increase DA uptake in treated mice Kirby et al., 1999, Karen et al., 2001. For PM, DA uptake was increased 33% above controls (P < 0.05) at a dose of 1.5 mg/kg, but reduced 56% below control values (P < 0.05) at 200 mg/kg (Karen et al., 2001). The objective of this research was to further characterize the time and dose dependence of PM's effects on transporter ligand binding and DAT protein expression. Moreover, because mutations in the α-synuclein gene are associated with some forms of familial PD Langston et al., 1998, Mizuno et al., 2001, and the herbicide paraquat causes up-regulation and increased aggregation of α-synuclein in C57B1/6 mice (Manning-Bog et al., 2001), we have expanded our studies to include Western blot analysis of α-synuclein expression in PM-treated mice. In this way, we hope to gain insight into the mechanisms by which compounds alter the dopaminergic system and render the neuron more susceptible to toxicant injury.