Pentachlorophenol (PCP) is an organochloride pesticide that is ubiquitous within the
environment due to its chemical stability. It is classified as a persistent organic
pollutant, and has been predominantly used in the wood preservation industry.
Workers and populations living close to PCP usage and production are exposed to it
via inhalation and dermal absorption, and ingestion of contaminated food and water.
It is lipophilic and is able to accumulate within various bodily systems, including the
brain. Adverse effects of PCP have been reported to varying degrees in the immune,
hepatic, and endocrine systems. Although neurological symptoms have been
associated with PCP exposure, knowledge of mechanisms of neurotoxicity is limited.
Elucidation of molecular mechanisms at a cellular level within neuronal cells is
required to contribute toward the current gap in the knowledge of PCP neurotoxicity.
The aim of the study was to evaluate the effects of PCP and its active metabolites,
tetrachloro-1,4-benzoquinone (TCBQ) and tetrachlorohydroquinone (TCHQ) in
human neuroblastoma SH-SY5Y cells.
Effects on cell proliferation were assessed using the sulforhodamine B (SRB) assay.
Flow cytometric analysis was employed to investigate effects on cell cycle using
propidium iodide (PI), mode of cell death using Annexin V-FITC and PI, reactive
oxygen species (ROS) using dichlorofluorescein, and mitochondrial membrane
potential (Δᴪm) using JC-1 fluorescence. Caspase-3 activity was assessed with Ac-
DEVD-AMC, and glutathione (GSH) with monochlorobimane fluorescence. Effects on
acetylcholinesterase (AChE) were assessed in vitro using the Ellman esterase
assay, as well as in silico via molecular docking and molecular dynamics simulation.
The IC50 concentrations of PCP, TCBQ and TCHQ were 80.0, 35.4, and 63.7 μM,
respectively. Cell cycle disruptions were revealed in the form of a G1 block and a
G2/M block as a result of PCP and TCHQ exposure, respectively, while TCBQ
resulted in a prolonged S phase traverse. The predominant mode of cell death of
PCP was necrosis, while TCBQ induced apoptosis. Exposure to TCHQ resulted in
one of two fates, being either predominantly apoptotic or necrotic cell death.
Decreased Δᴪm was an early event for all compounds, however, differed in their
involvement of inducing ROS. Oxidative stress was an evident mechanism of PCP
and TCBQ toxicity, as increased ROS was accompanied by lowered GSH, while reductive stress leading to subsequent oxidative stress was indicated by increased
ROS and GSH for TCHQ. All compounds yielded increased caspase-3 activity. The
fate of TCHQ exposed cells was postulated as a switch from apoptosis to necrosis
due to overwhelming ROS insult on apoptotic machinery, surpassing a threshold for
apoptosis capability. Inhibition of AChE was observed by only TCHQ in vitro, the
Ellman IC50 of which was 79.7 μM. In silico assessment supported a hypothesis of
TCHQ inhibition of AChE, with TCHQ-acetate bound ligands binding AChE receptors
with binding energies corresponding to the Ellman IC50. Binding stability was
confirmed by molecular dynamics.
Pentachlorophenol and its active metabolites exhibited different mechanisms of
toxicity toward neuronal cells, leading to different modes of cell death. A new
hypothesis for the molecular mechanism of TCHQ AChE inhibition was developed,
and sets a platform for further investigation.