Paraquat Inhalation, a Translationally Relevant Route of Exposure: Disposition to the Brain and Male-Specific Olfactory Impairment in Mice

Abstract

Epidemiological and experimental studies have associated oral and systemic exposures to the herbicide paraquat (PQ) with Parkinson's disease. Despite recognition that airborne particles and solutes can be directly translocated to the brain via olfactory neurons, the potential for inhaled PQ to cause olfactory impairment has not been investigated. This study sought to determine if prolonged low-dose inhalation exposure to PQ would lead to disposition to the brain and olfactory impairment, a prodromal feature of Parkinson's disease. Adult male and female C57BL/6J mice were exposed to PQ aerosols in a whole-body inhalation chamber for 4 h/day, 5 days/week for 4 weeks. Subsets of mice were sacrificed during and after exposure and PQ concentrations in various brain regions (olfactory bulb, striatum, midbrain, and cerebellum) lung, and kidney were quantified via mass spectrometry. Alterations in olfaction were examined using an olfactory discrimination paradigm. PQ inhalation resulted in an appreciable burden in all examined brain regions, with the highest burden observed in the olfactory bulb, consistent with nasal olfactory uptake. PQ was also detected in the lung and kidney, yet PQ levels in all tissues returned to control values within 4 weeks post exposure. PQ inhalation caused persistent male-specific deficits in olfactory discrimination. No effects were observed in females. These data support the importance of route of exposure in determination of safety estimates for neurotoxic pesticides, such as PQ. Accurate estimation of the relationship between exposure and internal dose is critical for risk assessment and public health protection.

Keywords: Parkinson’s disease; inhalation; olfactory impairment; paraquat.

Figure 1.

Experimental timeline. Male and female C57BL/6J mice were exposed to either HEPA-filtered air (99.9% effective) or PQ aerosols (130 μg/m³) beginning on experimental day 0. Subsets of male mice were sacrificed on experimental days 10, 28, 56, and 303 (Time points Nos 1–4) and brain, lung, and kidney were collected for PQ quantitation. Bronchoalveolar lavage fluid was collected from a subset of males immediately following the end of exposure (Time point No. 2) to assess the acute effects of PQ inhalation on the lung. In order to assess the long-term effects of PQ inhalation on the lung, perfused lungs were collected from additional subset of males at Time point No. 4 for histological analysis. Olfactory discrimination training/testing began on experimental day 163, 135 days after the end of exposure. Abbreviations: PQ, paraquat; HEPA, high-efficiency particulate air.

Figure 2.

Exposure parameters. Daily (A) mean ± SD particle concentration, (B) median ± SD particle diameter, (C) mean ± SD particle mass concentration and, (D) TEM image of paraquat aerosols. Abbreviations: SD, standard deviation; TEM, transmission electron microscopy.

Figure 3.

Lung endpoints. Group means ± SE for LDH activity (A) and protein content (B) of bronchoalveolar lavage fluid collected from a subset of males (n = 3/group) immediately following the end of exposure (Time point No. 2). Group means ± SE for fibrous connective tissue area in lungs collected from a separate subset of males (n = 4–5/group) at the conclusion of the experiment (Time point No. 4), more than 300 days after the end of exposure (C). Representative images of lungs sections stained with Gomorri’s trichrome from air-control (D) and PQ-exposed (E) mice. Abbreviations: SE, standard error; LDH, lactate dehydrogenase; PQ, Paraquat.

Figure 4.

PQ quantitation in tissues. A, Group mean ± SE PQ concentration (pg/g); B, Group mean ± SE tissue burden (pg/organ) in PQ-exposed male brain regions, lung, and kidney. Samples size of n = 5/time point/region. Abbreviations: PQ, paraquat; SE, standard error.

Figure 5.

Male olfactory discrimination performance. Total number of correct and incorrect responses made by air control and PQ-exposed males (n = 8–9/group) in a 2-choice olfactory discrimination test at (A) 100:0 ratio of positive: negative scents in the correct choice (least difficult), (B) 90:10, (C) 80:20, and (D) 70:30 (most difficult). As there was a possibility that a mouse with impaired olfaction could guess correctly in a 2-option test, the residual (%) of mice within each group that never made an error was also visualized to further highlight the differences between treatment groups (E). All mice were able to correctly identify single scents during training. Olfactory discrimination training/testing began 135 days after the end of exposure. *, significant main effect of PQ at p ≤ .05 following Chi square analysis. Abbreviation: PQ, paraquat.

Figure 6.

Female olfactory discrimination performance. Total number of correct and incorrect responses made by air control and PQ-exposed females (n = 5/group) in a 2-choice olfactory discrimination test at (A) 100:0 ratio of positive: negative scents in the correct choice (least difficult), (B) 90:10, (C) 80:20, and (D)70:30 (most difficult). As there was a possibility that a mouse with impaired olfaction could guess correctly in a 2-option test, the residual (%) of mice within each group that never made an error was also visualized to further highlight the differences between treatment groups (E). All mice were able to correctly identify single scents during training. Olfactory discrimination training/testing began 135 days after the end of exposure. No significant treatment effects were noted following Chi square analysis. Abbreviation: PQ, paraquat.