Low-dose inhalation exposure to trichloroethylene induces dopaminergic neurodegeneration in rodents

Abstract

Trichloroethylene (TCE) is one of the most pervasive environmental contaminants in the world and is associated with Parkinson disease (PD) risk. Experimental models in rodents show that TCE is selectively toxic to dopaminergic neurons at high doses of ingestion, however, TCE is a highly volatile toxicant, and the primary pathway of human exposure is inhalation. As TCE is a highly lipophilic, volatile organic compound (VOC), inhalation exposure results in rapid diffusion throughout the brain, avoiding first-pass hepatic metabolism that necessitated high doses to recapitulate exposure conditions observed in human populations. We hypothesized that inhalation of TCE would induce significantly more potent neurodegeneration than ingestion and better recapitulate environmental conditions of vapor intrusion or off gassing from liquid TCE. To this end, we developed a novel, whole-body passive exposure inhalation chamber in which we exposed 10-month-old male and female Lewis rats to 50 ppm TCE (time weighted average, TWA) or filtered room air (control) over 8 weeks. In addition, we exposed 12-month-old male and female C57Bl/6 mice to 100 ppm TCE (TWA) or control over 12 weeks. Both rats and mice exposed to chronic TCE inhalation showed significant degeneration of nigrostriatal dopaminergic neurons as well as motor and gait impairments. TCE exposure also induced accumulation of pSer129-αSyn in dopaminergic neurons as well as microglial activation within the substantia nigra of rats. Collectively, these data indicate that TCE inhalation causes highly potent dopaminergic neurodegeneration and recapitulates some of the observed neuropathology associated with PD, providing a future platform for insight into the mechanisms and environmental conditions that influence PD risk from TCE exposure.

Trichloroethylene (TCE) is a chlorinated organic solvent used as a degreasing product and as chemical feedstock for developing refrigerants (Doherty, 2000; IARC, 1995). Due to its widespread use over much of the 20th century, TCE is now a pervasive environmental contaminant in the United States and throughout the world (Zogorski et al., 2006). TCE has been measured in 9%–34% of drinking water within the United States (Agency for Toxic Substances and Disease Registry, 1997), and nonaqueous plumes of TCE within groundwater aquifers are resistant to degradation (Morrison et al., 1964), presenting a challenge for both tracking and mitigating TCE exposures (Basu et al., 2006; Wu et al., 2019). Due to its highly volatile nature, the most common route of exposure in humans is through inhalation (Todd et al., 2019; Wu and Schaum, 2000), which can readily occur from contaminated air or volatilization from groundwater and soil (Archer et al., 2015; Forand et al., 2012). As the vaporization point for TCE is 70°F (21°C), vapor intrusion into indoor air of businesses and homes represents an environmental risk for TCE exposure (Archer et al., 2015; Todd et al., 2019), which can be impacted by indoor and outdoor temperature, humidity, and a number of other dynamic physical properties (Paciência et al., 2016; Shirazi et al., 2020; Xie and Suuberg, 2021). Consumer products are also primary sources for indoor air contamination of chlorinated volatile organic compounds (VOCs), and TCE is within the top 15 highest estimated emissions of Prop 65-listed chemicals in consumer products (Knox et al., 2023). As TCE is a known carcinogen, several agencies regulate limits of exposure in air and water (summarized in Table 1).

TCE is linked to elevated risk for Parkinson’s disease (PD) (Bove et al., 2014; Dorsey et al., 2023; Gash et al., 2008; Guehl et al., 1999; Kochen et al., 2003), with growing evidence including a large case-control study of veterans in Camp Lejeune, North Carolina (odds ratio, 1.70; 95% CI, 1.39–2.07; p < .001) (Goldman et al., 2023). We and others have shown that systemic exposure to TCE in rodents induces the selective neurodegeneration of dopamine neurons in the substantia nigra (SN) and their terminal projections in the striatum (ST), accumulation of endogenous α-synuclein (αSyn), and microglial activation (De Miranda et al., 2021; Gash et al., 2008; Guehl et al., 1999; Keane et al., 2019; Liu et al., 2010; Sauerbeck et al., 2012). However, previous studies investigating neurodegeneration caused by TCE have used oral gavage or intraperitoneal injection of high doses of TCE in rodents (Guehl et al., 1999; Liu et al., 2010; Sauerbeck et al., 2012), which limits the translational relevance to most environmental exposure conditions. Inhalation exposure may particularly influence neurodegeneration as inhaled toxicants have a direct route into the brain through olfactory pathways (Nolwen et al., 2018), and from perfused blood from the lungs (Lucchini et al., 2012).

TCE is a small, highly lipophilic molecule that readily crosses membranes, including the blood-brain barrier, and causes acute neurological effects following inhalation such as dizziness, sleepiness, and reduced reaction time (Cichocki et al., 2016; Riederer et al., 2002; Todd et al., 2019). We predicted that inhalation exposure would be especially toxic to dopaminergic neurons and produce parkinsonian pathology at doses lower than previously used in other routes of exposure. To assess this, we developed an inhalation exposure platform using a whole-body, passive exposure inhalation chamber, coupled with environmentally relevant extrapolated doses, and assessed PD-related neurodegeneration through the most common route of TCE exposure in humans. We found that exposure to a time weighted average (TWA) of 50 ppm TCE for rats and 100 ppm TCE for mice, over 8 and 12 weeks, respectively, resulted in significant loss of nigrostriatal dopaminergic neurons that corresponded with motor behavior deficits. We also observed accumulation of phosphorylated αSyn (pSer129-αSyn) within dopaminergic neurons as well as activated microglia within the ventral midbrain. Collectively, we found that TCE inhalation resulted in pathologic outcomes consistent with idiopathic PD pathology in human brain tissue, providing further support for epidemiological data that TCE is a risk factor for PD.

Materials and methods

Chemical reagents and supplies

TCE (CAS 79-01-6) and other chemicals were purchased from Sigma-Aldrich (St Louis, Missouri) unless otherwise noted. Antibody information for immunohistochemistry (IHC) is listed in Table 2.

Animals

Adult (10-month-old) male and female Lewis rats were obtained through a retired breeding program from Envigo (Indianapolis, Indiana) and adult (12-month-old) male and female C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, Maine). Upon arrival, rats and mice were acclimated to the UAB small animal facility for 1 week prior to study onset. Rodents were provided with standard rodent chow and filtered water ad libitum throughout the study period. Rats were double housed with platforms and wooden blocks for enrichment, mice were housed 3–4 to a cage with enrichment nests, and all rodents maintained at 72–74°C with 50%–60% humidity. At the study termination, rats and mice were humanely euthanized using a lethal dose of pentobarbital euthanasia and underwent transcardial perfusion with PBS, followed by organ collection. All animal experiments were conducted with approval by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC).

TCE administration

TCE and control (HEPA filtered room air) groups were randomly divided. TCE exposed rodents were placed in a whole-body passive exposure chamber inside a chemical fume hood within the lab. Control animals were placed in an adjacent fume hood with passive flow of filtered room air. Rats were exposed for 7 h a day for 8 weeks, and mice were exposed 7 h a day for 12 weeks (to compensate for metabolism rate differences). TCE concentrations were monitored using Honeywell MiniRAE3000 photoionization detector (PID monitor) placed at the level of the rat/mouse, equipped with an 11.7 UV lamp using a correction factor of 0.43 for TCE. TCE handling and disposal were carried out following UAB Environmental Health and Safety procedures for hazardous chemicals.

Motor behavior

Motor behavior was assessed using Noldus Catwalk XT 10.6 gait analysis system in the UAB Behavioral Assessment Core. Rats and mice were trained to walk across the Catwalk stage 1 day prior to testing. The camera distance was set at 70 cm for rats and 35 cm for mice. Each subject required 3 successful runs for analysis. A successful run was defined as a duration from 0.5 to 5.0 s with a maximum variation of 60%. Detection settings were automatically generated, and gain was varied from 220 to 225 to optimize signal produced from pawprints. During testing, animals were allowed to walk across the stage, and pawprints were recorded. Animals were excluded after 30 failed run attempts. Prints were analyzed in Catwalk XT software (Version 10.6). Compliant runs were automatically classified, manually validated, and extraneous signal prints were excluded manually while blinded.

Striatal terminal intensity

Serial brain sections (35 μm) spanning the volume of the rat and mouse striatum (one-sixth sampling fraction, approximately 10 sections per animal) were stained for tyrosine hydroxylase (TH) and detected using an infrared secondary antibody (IRDye 680, Licor Biosciences). Striatal tissue sections were analyzed using near-infrared imaging for density of dopamine neuron terminals (LiCor Odyssey) and analyzed using LiCor Odyssey software (V3.0; Licor Biosciences, Lincoln, Nebraska). Results are reported as striatal TH intensity in arbitrary fluorescence units.

Stereology

Stereological analysis of dopamine neuron number in the SN was adapted from Cao et al. (2010) employing an unbiased, automated system (Cao et al., 2010). Briefly, serial nigral tissue sections (one-sixth sampling fraction, 10 sections per animal) spanning the volume of the SN were stained for TH and quantified using a MBF Biosciences Widefield microscope and Stereo Investigator software (Center for Neurodegeneration and Experimental Therapeutics Core, UAB). Representative fluorescent images were processed using Nikon NIS-Elements Advanced Research software (Version 4.5, Nikon, Melville, New York), and quantitative analysis was performed on brightfield images. Results are reported as the number of TH-positive cell bodies (whole neurons) within the SN.

Immunohistochemistry

Brain sections were maintained at −20°C in cryoprotectant, stained while free-floating, and mounted to glass slides for imaging. Fluorescent immunohistochemical images were collected using a Nikon AX confocal microscope. Quantitative fluorescence measurements and all imaging parameters were kept consistent across specimens. Confocal images were analyzed using Nikon NIS-Elements Advanced Research software (Version 4.5, Nikon, Melville, New York). A minimum of 4 images per tissue slice were analyzed per animal, averaging 9–15 neurons per 60× image. Results are reported as a measure of puncta within TH-positive cells, either number of objects (no. of objects) or area (μm²).

Statistical analysis

All data were expressed as mean values±SEM. Outcome measures were evaluated for normally distributed means using a normality test for homoscedasticity, followed by appropriate parametric or nonparametric tests; Student’s unpaired 2-tailed t test to compare control and TCE groups. Statistical significance between male and female rats was evaluated for normally distributed means by 2-way analysis of variance (2-way ANOVA) with a Sidak multiple comparisons test to correct for mean comparison between multiple groups, where source of variation was defined as “sex” or “TCE” groups. Prior to study onset, an a priori power analyses were conducted for both t test and 2-way ANOVAs using G*power software to determine the sample size required for a 20%–40% difference between mean, with a 95% power at α=.05. Statistical significance between treatment groups or sex is represented in each figure as *p < .05, **p < .01, ***p < .001, ****p < .0001, unless otherwise specified on graph. Statistical outliers from each data set were determined using the extreme studentized deviate (Grubbs’ test, α = .05). Statistical analyses were carried out using GraphPad Prism software (V. 5.01). Data are visualized with violin plots with a single line representing the median, with 2 additional lines showing the interquartile range. The violin shape shows the density curve representing the estimated distribution of data points. The Kernel Density Estimation used to plot the density curves occasionally extends past zero as an artifact of the algorithm in projecting probability of data density at a given Y value. Animal treatment groups obscured and renumbered by a third party. Animals were unblinded at the conclusion of the analysis.

Results

Establishing human equivalent dose in adult rodents

To model a low human equivalent dose (HED) of TCE in rodents, we used allometric scaling to normalize TCE dose to body surface area between humans and rats or mice. Based off the equation below by Nair and Jacob (2016), HED for 50 ppm in rats and 100 ppm in mice was determined to be approximately equivalent to 8 ppm in a human.

Rat and mouse correction factors (6.2 and 12.3, respectively) were obtained from Nair and Jacob (2016) to scale body surface area for a 60 kg human. As allometric scaling does not encompass differences in the metabolism of TCE, the exposure window was increased for mice to 12 weeks to accommodate their increased metabolic rate (Cichocki et al., 2016). Allometrically scaled TCE exposure of approximately 8 ppm is below most TCE exposure limits currently maintained by the U.S. regulatory agencies (Table 1). For example, TWA limits are set at 100 ppm over an over an 8-h workday by OSHA and 10 ppm by the American Conference of Government Industrial Hygienists. TCE concentration was monitored over 7 h at a TWA of 50 ppm for rats and 100 ppm for mice and fluctuations were continuously recorded on a PID monitor (Figure 1).

Although PD is a disease of aging, toxicant exposures can occur throughout at any stage of life, and the window of exposure across the lifespan may vary depending on the source of exposure—eg, environmental or occupational. For example, occupational exposure to TCE might occur in young adulthood to middle age when working with the solvent, but environmental exposures can occur over any age range. To accommodate this, we exposed 10-month-old rats and 12-month-old mice to represent a young adulthood to middle age range (Wang et al., 2020).

TCE inhalation exposure induced nigrostriatal dopaminergic neurodegeneration and motor deficits in rats

Immunohistochemistry of striatal brain sections from TCE-exposed 10-month-old male and female Lewis rats revealed over a 50% reduction in TH intensity compared with control (p < .0001; Figs. 2A and C). In addition, TCE exposure caused a loss of approximately 50% of dopaminergic neurons assessed by unbiased stereology in the SN compared with control (p = .0003; Figs. 2B and D). The loss of neurons was confirmed by staining all cells with NeuroTrace, a fluorescent Nissl dye, which showed significant cell loss following TCE exposure (Supplementary Figure 1). No sex differences were observed in either striatal TH intensity or in dopaminergic neuron degeneration within the SN (Figs. 5A and B). Following 8 weeks of 50 ppm TCE inhalation exposure, male rats were assayed for motor behavior deficits utilizing Noldus Catwalk XT gait analysis system, which revealed a number of affected gait parameters. Left swing speed (p = .0012), front swing speed (p = .0350), and hind limb swing speed (p = .0040) and hind print area (p = .0041) were increased following TCE exposure when compared with vehicles. Conversely, front stand time (p = .0437), left (p = .0479) and front step cycle (p = .0400), and hind base of support (p = .0453) were decreased in TCE exposed animals compared with controls (Figs. 2E–N; only male rats are reported as no sex differences in neuropathological markers were identified). In all collated limb analyses, the deficits appear to be driven by left side limbs, indicating a possible asymmetric gait impairment caused by exposure to TCE inhalation.

TCE inhalation increases pSer129-αSyn accumulation in dopaminergic neurons and induces microglial activation within the SN of exposed rats

Endogenous pSer129-αSyn was assayed within dopaminergic (TH+) neurons using IHC and quantified by accumulated puncta. TCE inhalation caused significant accumulation of endogenous pSer129-αSyn compared with control (p = .0049; Fig. 3), which was predominantly located within the soma and proximal axons of dopaminergic neurons of rats exposed to TCE. In addition, rats exposed to 50 ppm TCE via inhalation displayed increased CD68 expression, a lysosomal protein and macrophage phagocytic activity marker, within IBA1 positive cells compared with control (p = .0117; Fig. 4). Microglia also displayed an activated morphological phenotype within TCE exposed rat brain tissue, appearing more hypertrophic with less ramified processes and amoeboid shape. No sex differences were observed in either pSer129-αSyn accumulation or in microglial activation (Figs. 5C and D).

Sex differences from TCE inhalation were not observed in outcome measures

Age-matched adult male and female Lewis rats (10 months old) were obtained from the same commercial source (Envigo) and housed under identical conditions prior to the onset of TCE exposure; 50 ppm of TCE over 8 weeks. Histopathological analysis of the nigrostriatal tract showed no significant differences between sexes with loss of dopaminergic neuron terminals or somas of TCE exposed rats (p = .8959; p = .0868; Figs. 5A and B). Additionally, no differences were found between sexes in TCE exposed groups in the expression of pSer129-αSyn in the SN (p = .9180; Fig. 5C). Finally, in TCE animals, no sex differences were apparent in the expression of CD68 within IBA1 positive cells (p = .5115; Fig. 5D).

TCE inhalation induced nigrostriatal dopaminergic neurodegeneration and motor deficits in mice

Adult (12-month-old) C57Bl/6 male and female mice were exposed to 100 ppm TCE inhalation or control (HEPA filtered room air). Immunohistochemistry of striatal brain sections from TCE-exposed mice had approximately a 30% reduction in TH intensity when compared with vehicles (p < .0071; Figs. 6A and C). In addition, TCE exposure caused a loss of approximately 50% of dopaminergic neurons in the SN when compared with vehicles (p = .0391; Figs. 6B and D). The loss of neurons was confirmed by staining all cells with NeuroTrace which showed significant cell loss following TCE exposure (Supplementary Figure 1). Following 12 weeks of TCE exposure, motor behavior was assayed using Noldus CatwalkXT gait analysis system. Mice exposed to TCE had a significantly increased left side swing speed (p = .0220) and hind swing speed (p = .0252) with reduced right side step cycle (p = .0221) and front step cycle (p = .0217) when compared with controls (Figs. 6E–L). As observed in rats, these findings indicate an asymmetric gait impairment, similar to observations reported in human PD (Lewek et al., 2010). These results indicate that this exposure method can be applied in both rat and mouse models of disease.

Discussion

Growing evidence suggests that TCE exposure elevates risk for PD (Goldman et al., 2023), however, considerably less is known about how this pervasive environmental contaminant causes the specific neurotoxicity that drives parkinsonian neurodegeneration. The most common route of human exposure to TCE is from inhalation of its vapor, which typically occurs through occupational use of the solvent or from vapor intrusion into building airspace from contaminated soil and/or groundwater (Archer et al., 2015; Forand et al., 2012; Todd et al., 2019; Wu and Schaum, 2000). Previously reported experimental data shows that TCE exposure in rodents induces the selective degeneration of dopaminergic neurons from the nigrostriatal tract, as well as other PD-related pathologies such as motor deficits, neuroinflammation, and pSer129-αSyn accumulation (De Miranda et al., 2021; Gash et al., 2008; Guehl et al., 1999; Keane et al., 2019; Liu et al., 2010; Sauerbeck et al., 2012). However, the majority of this evidence was generated from exposure through oral gavage administration, which represents a less common TCE exposure route (ingestion) and requires higher doses of TCE in rats and mice than what is typically recorded in human populations.

For example, in both Gash et al. (2008) and Liu et al., (2010), 1000 mg/kg TCE was administered via oral gavage over 6 weeks in Fisher 344 rats to induce significant dopaminergic neurodegeneration (Gash et al., 2008; Liu et al., 2010). In C57Bl/6 mice, 400 mg/kg TCE over 8 months induced a similar severity of nigral dopaminergic cell loss as reported in rats, and also caused motor impairments (Liu et al., 2018). With interest in examining moderate experimental doses, our group reported in 2021 that daily oral gavage with 200 mg/kg TCE over 6 weeks in aged Lewis rats (10–15 months old) caused significant loss of nigrostriatal dopaminergic neurons, induced endolysosomal dysfunction and αSyn aggregation, caused neuroinflammation, and influenced aberrant kinase activation of the PD-associated protein LRRK2 (De Miranda et al., 2021).

As a comparison for TCE dose in animals, the most well-known drinking water contamination event recorded in the U.S. stems from Camp Lejeune, North Carolina, where reconstruction data estimates an average of 783 µg/l TCE, with an actual measured maximum value of 1400 µg/l (Maslia et al., 2016). However, exposure to contaminated water at Camp Lejeune occurred for over 30 years, varied by time, season, and location, and included other toxicants such as tetrachloroethylene and benzene (Maslia et al., 2016), all of which are conditions that cannot be fully recapitulated in laboratory animals. Likewise, exposure to TCE at Camp Lejeune, as in other contaminated sites, included inhalation of TCE from vaporization of contaminated groundwater and soil, ingestion, and dermal exposure from TCE-contaminated drinking water.

In addition to differences in exposure conditions, interspecies variability in the oxidative metabolism of TCE indicates that rodents metabolize TCE more rapidly than humans (Lash et al., 2000). Mice even exhibit strain-dependent variability of metabolism in Bradford et al. (2011), Chiu et al. (2014), Chiu et al. (2009), and Valdiviezo et al. (2022), suggesting that greater doses beyond simple allometric scaling would be necessary in laboratory animals to accurately replicate exposure. Thus, dose conversions of TCE exposure from animal to human and vice versa, are complex, rely on imperfect conversion assumptions, and should be considered in the temporal context of a human lifespan. Toward this end, inhalation provides an environmentally relevant route of exposure with the potential for chronic timepoints that more closely match conditions observed in TCE-exposed populations. Here, we selected an inhalation exposure of 50 ppm TCE in rats and 100 ppm TCE in mice (TWA)—doses that are at or below regulated occupational exposure limits in humans (Table 1), and though nonexact, allometrically scale to a HED of approximately 8 ppm (Nair and Jacob, 2016).

Although this was not the first study to examine TCE inhalation as a primary route of exposure in rodents, to our knowledge, this is the first data on parkinsonian neurodegeneration or associated pathology from passive, chronic TCE inhalation. Previous studies investigating the effects of TCE inhalation on renal toxicity, carcinogenicity, neuronal plasticity in hippocampal and visual cortical slices, and the organization of motor behavior have been reported; however, doses used in these studies were often high, in some cases up to 1500 ppm, and were predominantly acute exposures (Altmann et al., 2002; Henschler et al., 1980; Kulig, 1987; Mensing et al., 2002). Given the selective sensitivity of dopaminergic neurons to neurotoxicants (De Miranda et al., 2016), we hypothesized that TCE inhalation would induce more severe neurodegeneration than ingestion, forgoing first-pass hepatic metabolism within the liver (Mortuza et al., 2018) and resulting in neurotoxicity at lower doses than previously used. In addition, exposure to inhaled toxicants presents more direct routes to the brain through the lungs as well as the olfactory bulb, from which some efferent pathways synapse directly in the striatum (Hillary et al., 2020; Nolwen et al., 2018). Indeed, despite a lower dose, 50 ppm TCE inhalation caused more dopaminergic neurodegeneration than 200 mg/kg TCE via oral ingestion: approximately 50% reduction in the number of dopaminergic neurons from TCE inhalation versus approximately 35% from oral gavage (De Miranda et al., 2021). This may have partially been impacted by duration of exposure (8 weeks of 50 ppm TCE exposure vs 6 weeks of 200 mg/kg exposure); however, given the vast difference in TCE concentration between the 2 exposure methods, route of exposure rather than duration likely provided the most significant impact on neurodegeneration caused by TCE. Additionally, these data suggest that previous methods using oral gavage with higher bolus doses of TCE produce similar toxic effects on the dopaminergic system as lower dose inhalation (De Miranda et al., 2021; Liu et al., 2010, 2018). This supports the premise that experimental modeling of TCE neurotoxicity is not a 1:1 equivalent of reported TCE levels in contaminated water that would be ingested by humans, rather, it reflects the complex combined routes of exposure that occur under environmental conditions.

Route of exposure to environmental PD risk factors may also be a key variable in disease etiology. Prodromal symptoms of PD, such as anosmia and decreased gastrointestinal (GI) motility, can occur decades prior to the onset of motor dysfunction (Berg et al., 2021; Chen and Ritz, 2018; Heintz-Buschart et al., 2018; Mahlknecht et al., 2015), and both olfactory and GI pathways are physiological entry points for environmental contaminants. Environmental influence on the Braak and dual-hit hypotheses, as predicated by Chen et al. (2022), suggests that PD may start in olfactory pathways, within the gut, or both, influenced by endogenous features like αSyn accumulation and exogenous factors such as toxicant exposures (Chen et al., 2022). TCE represents an environmental risk factor for PD that could influence disease pathogenesis through both inhalation (olfactory) and ingestion (gut) as it contaminates air and water. In line with this, we previously showed that ingestion of TCE caused gut microbiome changes that mirrored gut microbiome dysbiosis in idiopathic PD (Ilieva et al., 2022). Additionally, we observed the accumulation of pSer129-αSyn within dopaminergic neurons of TCE inhalation exposed rats, as well as microglial activation in the ventral midbrain, suggesting that αSyn dysfunction or inflammatory pathways induced by TCE could be potential triggers or facilitators of PD pathogenesis caused by exposure, which may be route dependent (Johnson et al., 2019).

As previously discussed, species differences in TCE metabolism may also impact translational relevance to human exposure and disease. Published data in wild-type (C57Bl/6) mice show that both greater doses and longer exposure times were required to produce comparable dopaminergic neurodegeneration to rats (Liu et al., 2018). Although rats may share more brain homology with humans than mice (Ellenbroek and Youn, 2016), mouse models encompass a broad scale of genetic manipulation that is not widely available in rat models, particularly for PD and other neurodegenerative disorders (Dawson et al., 2010). To address this, we evaluated whether inhalation exposure to TCE in C57Bl/6 mice would produce similar dopaminergic pathology as observed in TCE exposed rats. To account for body surface area and metabolism differences, we adjusted the dose to 100 ppm TCE over 12 weeks, which induced comparable dopaminergic neurodegeneration (approximately 50% TH+ neuron loss) as well as asymmetric gait disturbances similar to those measured in rats. Overall, fewer parameters of motor deficits were significantly changed in mice exposed to TCE than observed in rats, which is consistent with data suggesting mice display less accurate fine motor coordination than rats (Do Carmo and Cuello, 2013). Thus, this model of TCE inhalation allows both the tissue homology of rats and genetic tools of mice to be leveraged in order to evaluate mechanisms of PD pathogenesis driven by TCE exposure, several of which are under current investigation in our lab.

As with all experimental models of exposure, there are limitations of TCE inhalation in rodents. For example, though inhalation may provide more direct brain access than ingestion, lung metabolism and elimination of TCE occurs rapidly (Mortuza et al., 2018), possibly resulting in different toxic metabolites based on species. In addition, though we did not observe sex differences at the level of outcome measures reported—mostly cellular pathological changes—this does not preclude sex-specific effects that could be apparent with more sensitive analyses, such as transcriptomic or epigenetic influences as found in other PD-related toxicant exposures (Adamson et al., 2022; Gezer et al., 2020; Kochmanski et al., 2019). Finally, while nearly infinite combinations of exposure conditions could be investigated using this method, we chose to first report this proof-of-concept data in adult rodents with exposure occurring over 5 days/week, replicating a workplace or occupational exposure. Future studies to assess TCE exposure over the course of the lifespan, extremely low doses as observed in common environmental conditions, and the interaction with other PD risk factors will provide more information on the mechanisms of TCE-induced neurodegeneration.

Conclusions

TCE has been implicated in PD risk for decades. However, although animal models of TCE exposure showed important proof of principle data of dopaminergic neurodegeneration and other PD related pathology, confusion over high doses required to recapitulate key features of the disease limited some translational relevance. We predicted this was predominantly because TCE exposure in humans mostly occurs via inhalation, providing a more direct path to the brain. Our data here suggest that TCE inhalation causes potent dopaminergic neurotoxicity at much lower doses than previously examined, providing a missing mechanistic link between TCE exposure and PD risk.

Supplementary data

Supplementary data are available at Toxicological Sciences online.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by research grants from the National Institutes of Environmental Health Sciences (grant number R00ES029986, B.R.D.).

References