Oxidative Medicine and Cellular Longevity
Volume 2019 (2019), Article ID 1607304, 28 pages
https://doi.org/10.1155/2019/1607304

Review Article

Triclosan: An Update on Biochemical and Molecular Mechanisms

Mohammad A. Alfhili1,2 and Myon-Hee Lee¹

¹Department of Medicine (Division of Hematology/Oncology), Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA
²Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia

Correspondence should be addressed to Myon-Hee Lee; leemy@ecu.edu

Received 30 November 2018; Revised 28 February 2019; Accepted 1 April 2019; Published 2 May 2019

Academic Editor: Demetrios Kouretas

Copyright © 2019 Mohammad A. Alfhili and Myon-Hee Lee. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Triclosan (TCS) is a synthetic, chlorinated phenolic antimicrobial agent commonly used in commercial and healthcare products. Items made with TCS include soaps, deodorants, shampoos, cosmetics, textiles, plastics, surgical sutures, and prosthetics. A wealth of information obtained from in vitro and in vivo studies has demonstrated the therapeutic effects of TCS, particularly against inflammatory skin conditions. Nevertheless, extensive investigations on the molecular aspects of TCS action have identified numerous adversaries associated with the disinfectant including oxidative injury and influence of physiological lifespan and longevity. This review presents a summary of the biochemical alterations pertaining to TCS exposure, with special emphasis on the diverse molecular pathways responsive to TCS that have been elucidated during the present decade.

1. Introduction

Triclosan (TCS), or 5-chloro-2-(2,4-dichlorophenoxy)phenol, is a synthetic broad-spectrum antimicrobial developed in the 1960s. As a polychlorinated bisphenolic compound, TCS has a perceptible aromatic odor and is weakly soluble in water. It dissolves well in organic solvents including ethanol, dimethylsulfoxide (DMSO), and methanol [1], and the type of solvent and detergent availability seem to influence TCS activity [2–4]. For example, TCS dissolved in oils (e.g., olive oil) and alkali (e.g., sodium carbonate) exhibits markedly reduced efficacy when compared to other solvents such as glycerol and polyethylene glycol (PEG) [3, 5]. In fact, using propylene glycol (PG) as a solvent renders TCS more effective than using PEG, which is probably due to micellar solubilization of TCS in the larger PEG molecules [3]. Recently, we have shown that the presence of nonionic detergents (e.g., Tween 20) inhibits TCS activity in vivo, most likely due to micelle formation [6]. In contrast, sodium dodecyl sulfate (SDS) has been reported to potentiate the antibacterial effect of TCS in vitro [7].

TCS has gained enormous popularity in commerce and in healthcare owing to its antibacterial, antiviral, and antifungal properties [8–10]. This efficacy has led to the widespread use of TCS as a preservative in a variety of consumer products, including cosmetics, soaps, mouthwashes, antiperspirants, kitchen utensils, clothing textiles, bedclothes, electronics, plastics, and toys (Triclosan White Paper prepared by the Alliance for the Prudent Use of Antibiotics (APUA)). In clinical practice, TCS is used as a disinfectant and an antiseptic in surgical sutures, scrubs, implants, and medical devices [11, 12]. Annual global production of TCS was estimated at 1500 tons [13], and a total of 132 million liters of TCS-containing products was consumed in a single year in the United States (Safety and Effectiveness of Consumer Antiseptics; Topical Antimicrobial Drug Products for Over-the-Counter Human Use; Proposed Amendment of the Tentative Final Monograph. 2013 https://www.fda.gov/downloads/AboutFDA/ReportsManualsForms/Reports/EconomicAnalyses/UCM379555.pdf).

The high demand for TCS has consequently led to substantial buildup in drinking and wastewater sources and, more alarmingly, accumulation in body fluids [14–20], establishing the antimicrobial as an environmental pollutant. Pharmacokinetic studies in man show that TCS reaches the systemic circulation by rapid absorption through the skin and mucous membranes of the oral cavity and gastrointestinal tract, and variations in the bioavailability of TCS unsurprisingly affect the rate of urinary excretion [21, 22]. TCS content in commercial products may reach as high as 17 mM and comprise up to 1% of ingredients [12, 19, 23]. Moreover, absorption of up to 25% of applied TCS has been recorded [24], and metabolic studies in rats and mice revealed sulfation, glucuronidation, and hydroxylation products in tissues and excreta [25, 26].

Since the advent of TCS, early studies on the antiseptic have shown evidence of symptomatic relief from acne [27, 28] and contact dermatitis [29, 30] with fewer, or at least comparable, side effects to other therapeutic alternatives [31]. Later, TCS was found to be effective against crural ulcer [32] and chemically induced dermatitis and desquamation [33, 34], which could be attributed to its anti-inflammatory [35], hypoallergenic [36], and analgesic [37] properties. Moreover, a battery of studies collectively indicate that TCS is not a skin or oral mucosal irritant, has a very low sensitization potential (0.1-0.3% of 14,000 subjects), and is unlikely to be phototoxic to human skin (http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_166.pdf). This is in contrast to the reversible skin and eye irritation caused by up to 10% TCS reported in animals (http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_166.pdf). Also, in initial studies by Lyman and Furia, it was suggested that TCS is carcinogenic when orally administered to rats [38, 39]. Subsequent investigations in rats and mice disclosed that TCS perturbs microsomal detoxification [40], causes nephrotoxicity and hepatotoxicity [41], reduces prenatal and postnatal survival [42], and leads to central nervous system suppression [43] and hypothermia [44]. In humans, the earliest description of an adverse TCS reaction probably comes from a case report of two patients who developed contact dermatitis following application of deodorants containing 0.12% and 0.2% TCS [45]. Since then, several case reports of the same ailment have thus far been in congruence [46–49]. It is important to mention that, as is the case with healthy subjects, in patients diagnosed with, or suspected to have, contact dermatitis, TCS was similarly found to have a very low sensitization potential (0.6-0.8% of 11,887 patients) (http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_166.pdf).

In light of the dichotomous debate surrounding TCS, the US Food and Drug Administration (FDA), following extensive examination of available data, has effectively banned antiseptic products containing TCS since September 2016 [19]. In Europe, TCS was approved for use in cosmetics by the European Community Cosmetic Directive in 1986 (http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_166.pdf). However, the European Commission disapproved the use of TCS for hygienic purposes in 2017, but maintained its legality as a preservative in select cosmetics and mouthwashes in concentrations up to 0.3% and 0.2%, respectively (http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_054.pdf; http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=OJ:L:2014:107:FULL&from=EN). Furthermore, the Scientific Committee on Consumer Safety (SCCS) expressed its concern over the continued use of TCS in cosmetics, but not in antiseptics, mainly due to the cumulative pattern of exposure (http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_054.pdf). Importantly, the European Chemicals Agency (ECHA) classifies TCS, under the classification, labeling, and packaging (CLP) regulation, as an eye irritant 2 (causes serious eye irritation), skin irritant 2 (causes skin irritation), aquatic acute 1 (very toxic to aquatic life), and aquatic chronic 1 (very toxic to aquatic life with long-lasting effects) (https://echa.europa.eu/documents/10162/21680461/bpc_opinion_triclosan_pt1_en.pdf/efc985e4-8802-4ebb-8245-29708747a358). Because of the previously mentioned ecotoxic properties, TCS is currently a candidate for substitution under the Biocides European Union regulation (Reg 528/2012/EC) (https://echa.europa.eu/potential-candidates-for-substitution-previous-consultations/-/substance-rev/12/term?_viewsubstances_WAR_echarevsubstanceportlet_SEARCH_CRITERIA_EC_NUMBER=222-182-2&_viewsubstances_WAR_echarevsubstanceportlet_DISS=true).

Our aim in this review is to provide an update on current knowledge regarding TCS therapeutic and toxic potential. Emphasis is placed on the biochemical and molecular alterations, either brought about by, or in response to, TCS exposure. Data from both in vitro and in vivo studies, obtained from humans and other organisms, are incorporated into the analysis, with special attention being given to reports published during the present decade.

2. Membrane and Cytoskeletal Damage

Perhaps the earliest report describing the antimicrobial activity of TCS was by Vischer and Regös [50] which was shown through topical application. In a follow-up study, TCS was found to be more effective with the broadest spectrum against bacteria and fungi when compared to other antimicrobials such as gentamicin and clotrimazole [10]. Subsequent efforts, which continue to this day, have focused on dissecting the diverse action mechanisms and cellular targets of TCS. Initially, it was thought that TCS interacts with the prokaryotic cell membrane nonspecifically [9]. This was corroborated by the resistance of Gram-negative bacteria to TCS, which was ascribed to their outer membrane [51, 52]. Investigating the genetic response of Mycobacterium tuberculosis to TCS, Betts et al. [53] identified perturbations in a wide assortment of genes involved in cell wall, transport, detoxification, and DNA replication and transcription. Also, Klebsiella pneumoniae with inactive efflux pump KpnGH exhibit pronounced susceptibility to multiple antibiotics including TCS [54]. Several genes in the membrane stress response pathway were also studied in Escherichia coli and Rhodospirillum rubrum S1H [55–57]. During the electro-Fenton transformation of TCS, significant changes in expression patterns of genes involved in cell wall and membrane structure, cell envelope, flagella, and multidrug efflux were observed (Table 1). These findings complement an earlier report describing enhanced resistance to TCS due to overexpressed acrAB multidrug efflux pump [58]. It was recently suggested that TCS binds to the transcriptional repressor AcrR, causing conformational changes and preventing its binding to the efflux pump AcrA promoter in Agrobacterium tumefaciens [59].

Table 1: Summary of membrane and cytoskeletal targets of TCS.

Model
Target
Response

Gene/protein
Molecular identity

K. pneumoniae
KpnGH
Efflux pump
Sensitive to TCS

E. coli
AcrAB
Efflux pumps
Upregulated by TCS

acrE
Upregulated by TCS

mdtE
Upregulated by TCS

acrF
Upregulated by TCS

mdtB
Upregulated by TCS

mdtC
Upregulated by TCS

yddA
Upregulated by TCS

emrA
Upregulated by TCS

emrE
Upregulated by TCS

sanA
Cell wall/membrane structure
Upregulated by TCS

dacB
Upregulated by TCS

amiC
Cell envelope
Upregulated by TCS

clsA
Upregulated by TCS

ompX
Membrane porin
Downregulated by TCS

motA
Flagellar
Upregulated by TCS

flgM
Upregulated by TCS

R. rubrum S1H
sugE
Small multidrug resistance protein
Upregulated by TCS

mexF
RND efflux system, inner membrane transporter
Upregulated by TCS

mexB
Upregulated by TCS

mexE
RND efflux system, membrane fusion proteins
Sensitive to TCS

mexA
Upregulated by TCS

mexM
Upregulated by TCS

oprM
RND efflux system, outer membrane transporter
Upregulated by TCS

glmM
Cell envelope; phosphoglucosamine mutase
Upregulated by TCS

exoD
Cell envelope; exopolysaccharide synthesis protein D
Upregulated by TCS

wbpM
Cell envelope; polysaccharide biosynthesis protein M
Upregulated by TCS

A. tumefaciens C58
AcrA
RND efflux system, periplasmic adaptor protein
Upregulated by TCS

Human erythrocytes
Na⁺,K⁺,Mg²⁺-ATPase
Membrane ion transporter
Sensitive to TCS

C. elegans
Pmp-3
Membrane ABC transporter
Downregulated by TCS

D. rerio
Actin, cytoplasmic 2
Cytoskeleton
Downregulated by TCS

Actin α1, skeletal muscle
Downregulated by TCS

Light polypeptide 3
Downregulated by TCS

Desmin
Cytoskeleton; muscular filament structure
Upregulated by TCS

Fast skeletal muscle myosin
Sensitive to TCS

Keratin, type I cytoskeletal 18
Upregulated by TCS

Tropomyosin α-1 chain
Downregulated by TCS

Type II cytokeratin
Upregulated by TCS

Lamin B1
Cytoskeleton; nuclear lamina
Downregulated by TCS

D. polymorpha
Tubulin β-2/α-4 chain
Cytoskeleton
Upregulated by TCS

Tubulin β-4 chain
Upregulated by TCS

Myosin light chain
Cytoskeleton; muscular filament structure
Upregulated by TCS

Abbreviation: RND: resistance-nodulation-division; ABC: ATP-binding cassette.

The interaction of TCS with the cell membrane was also studied in human red blood cells (RBCs; erythrocytes). TCS exposure led to K⁺ leakage and overt hemolysis, indicating membrane damage, while antagonizing hypotonic lysis, which may be due to membrane expansion [60]. TCS also inhibited membrane-bound Na⁺,K⁺,Mg²⁺-ATPase enzymatic activity [61]. These observations suggest that TCS causes membrane destabilization, perturbs monovalent ion transport, and modulates the overall osmoregulation of erythrocytes. Evidence for membrane damage is further confirmed in numerous studies by means of compromised stability and permeability [62]. To directly observe how TCS interacts with the cell membrane, Guillén and coworkers utilized nuclear magnetic resonance (NMR) spectroscopy to demonstrate that TCS intercalates within hydrophobic pockets in the lipid bilayer, perpendicularly to phospholipid molecules [63]. Furthermore, using neutral red to evaluate membrane integrity, diminished uptake of the dye in hemocytes of the clam Ruditapes philippinarum and mussel Mytilus galloprovincialis was related to TCS-induced suppression of pinocytosis and disturbed phagocytosis [64, 65].

Along those lines, our recent findings indicate that TCS blunts the expression of the pmp3 membrane transporter in Caenorhabditis elegans nematodes and that pmp3(ok1087) mutants exhibit increased sensitivity to the disinfectant [66]. Finally, a proteomic analysis of zebrafish (Danio rerio) larvae and gills of freshwater mussel Dreissena polymorpha revealed alterations in cytoskeletal protein levels following TCS exposure (summarized in Table 1) [67, 68].

There is a consensus in the literature regarding the membranotropic nature of TCS in different membrane models across various species. The cell membrane is a primary target for TCS and among the first cellular obstacles that must be overcome by the antiseptic to exert its effects. Although evidence implicating membrane-associated efflux pumps as part of the cellular response to TCS is strong, there is paucity in reports describing TCS modulation of structural or functional membrane components in human-based systems. Similarly lacking is an understanding of the role of membrane receptors not only in pumping out TCS molecules but also in transducing both inter- and intracellular signals as a consequence to TCS presence.

3. Cellular Longevity

The interest in TCS and ultimate cell fate has originally stemmed from its use in oral hygiene products, which is reflected in two seminal studies on human gingival cells [69, 70]. TCS was shown to be cytotoxic to gingival fibroblasts and epithelial cells, identifying it as a novel stimulator of apoptosis in the latter.

Investigations have thus far followed a more comprehensive approach, relating cell death induced by TCS to other cellular adversaries, utilizing both human and non-human model systems. When TCS was treated to human choriocarcinoma placental cells (JEG-3), multiple dose- and time-dependent responses were observed [71]. While there was a proportional increase in estradiol and progesterone secretion, β-human chorionic gonadotropin (β-hCG) release was nevertheless inhibited with increasing TCS concentrations [71]. In addition to blunted proliferation, significant cell death was recognized as apoptotic in nature evidenced by activated caspase-3 and Hoechst 33342-stained fragmented DNA [71]. Similarly, using anoikis-resistant H460 human lung cancer cells, Winitthana et al. demonstrated that 24-hour exposure to 10 μM TCS causes cell death and apoptosis. Nontoxic levels (≤7.5 μM), however, enhanced cell growth (increased colony number and reduced size) without altering proliferation. TCS also promoted epithelial-to-mesenchymal transition (EMT), along with the migratory and invasive abilities of the cells [72].

A research group performed a series of in vivo and in vitro studies on the effect of TCS on growth and proliferation of human BG-1 ovarian cancer cells. Results from these studies indicate that TCS increases cellular proliferation and both gene expression and protein levels of cyclin D1 and decreases p21 and Bax gene expression and protein levels [73]. These effects were significantly antagonized by the estrogen receptor (ER) antagonist ICI 182,780, implicating ER in TCS-induced cell cycle progression and in its antiapoptotic role. Investigators from the same group also reported a similar response to TCS by MCF-7 breast cancer cells and LNCaP prostate cancer cells. In MCF-7 cells, 1 μM TCS enhanced growth and proliferation during a six-day period, which was associated with increased cyclin D1 and reduced p21 expression levels. When mice were treated with TCS for 8 weeks, brdU-positive breast tumor cells were significantly increased compared to the control group treated with corn oil [74]. Similar to BG-1 cells, TCS-promoted proliferation of MCF-7 cells was mediated through ERα signaling, demonstrated as antagonism by kaempferol and 3,3-diindolylmethane (DIM), two phytoestrogens [75]. In addition to cyclin D1 and p21, TCS caused an increase in cyclin E and a decrease in Bax and induced metastasis through elevated cathepsin D protein expression. These observations were paralleled in vivo using xenografted mouse models. Researchers from this report expanded their findings to VM7Luc4E2 cells, a variant of the MCF-7 model, to show that TCS (0.1-10 μM) is pro-proliferative and antiapoptotic by inhibiting oxidative stress, with both effects being antagonized by kaempferol [76]. In LNCaP cells exposed to concentrations of TCS ranging from 0.01 to 10 μM for up to 5 days showed enhanced proliferation and migration and reduced p21 protein expression [77]. In primary human syncytiotrophoblasts, TCS at 0.001 to 10 μM induced apoptosis as seen by condensed nuclei and fragmented DNA [78]. TCS also reduced 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) via a caspase-dependent mechanism. Other targets included both Bax and Bcl-2 proteins.

Similar to human cells, both pro- and antiapoptotic properties were observed in rodent cells treated with TCS. Beside its cytotoxicity, TCS caused caspase-dependent apoptosis in rat neural stem cells along with elevated Bax and reduced Bcl-2 [79]. In a series of studies, [80–82] mouse neurons were used to show that TCS is apoptotic through the Fas receptor (FasR), aryl hydrocarbon receptor (AhR), and caspase activation involving N-methyl-D-aspartate receptors (NMDARs). In agreement with the cytotoxicity data, TCS-treated mouse lung epithelial cells were deformed with reduced viability [83]. Conversely, TCS stimulated the proliferation of mouse epidermis-derived JB6 Cl 41-5a cells, by increasing cyclins D1 and A and reducing p27(Kip1) protein levels [84]. Examining these effects in vivo, B6C3F1 mice exhibited epidermal hyperplasia and focal necrosis following topical administration of TCS. Moreover, the pluripotency markers of mouse embryonic stem cells were analyzed following TCS exposure [85]. Alkaline phosphatase (Alp), Sox2, Oct4, and Nanog were all reduced, while miRNA-134 was elevated.

Unlike human and rodent cells, in vivo and in vitro studies on aquatic organisms uniformly agree that TCS is solely proapoptotic in these animals. Pyknotic apoptosis in the central nervous system of zebrafish D. rerio was observed following treatment with either TCS alone or TCS combined with derivatives 2,4,6-trichlorophenol (2,4,6-TCP) and 2,4-dichlorophenol (2,4-DCP) [86, 87]. The TCS-derivative mixture caused pronounced deformities and behavioral abnormalities and perturbed the expression of a panel of neurodevelopmental and apoptotic genes (Table 2). Also, TCS, following both in vivo and in vitro exposure, induced a dose- and time-dependent increase in apoptotic hemocytes of D. polymorpha [88, 89]. Likewise, when the saltwater clam Ruditapes philippinarum was treated with TCS, hemocytes exhibited significant cell death, blunted proliferation, reduced size, and promiment apoptotic DNA fragmentation [65]. TCS-induced apoptosis, or apoptosis-like cell death, was also detected in unicellular organisms, such as the green alga Chlamydomonas reinhardtii and the pathogenic fungus Cryptococcus neoformans [90, 91].

Table 2: Summary of cell survival molecules modulated by TCS.

Model
Target
Response

Gene/protein
Molecular identity

JEG-3 cells
Estradiol
Major female sex hormones
Upregulated by TCS

Progesterone
Upregulated by TCS

β-hCG
Maintenance of pregnancy
Downregulated by TCS

Caspase-3
Apoptosis regulator; proapoptotic
Upregulated by TCS

BG-1 cells
Cyclin D1
Cell cycle regulators
Upregulated by TCS

p21
Downregulated by TCS

Bax
Apoptosis regulator; proapoptotic
Downregulated by TCS

MCF-7 cells
Cyclin D1
Cell cycle regulators
Upregulated by TCS

Cyclin E
Upregulated by TCS

p21
Downregulated by TCS

Bax
Apoptosis regulator; proapoptotic
Downregulated by TCS

Cathepsin B
Metastasis markers
Upregulated by TCS

Cathepsin D
Upregulated by TCS

MMP-9
Upregulated by TCS

MMP-2
Upregulated by TCS

CXCR4
Upregulated by TCS

Snail
Mesenchymal markers
Upregulated by TCS

Slug
Upregulated by TCS

LNCaP
p21
Cell cycle regulator
Downregulated by TCS

Primary human syncytiotrophoblasts
11β-HSD2
Fetal development; anticortisol
Downregulated by TCS

Caspase-3
Apoptosis regulators; proapoptotic
Upregulated by TCS

Bax
Upregulated by TCS

Bcl-2
Apoptosis regulator; antiapoptotic
Downregulated by TCS

Rat neural stem cells
Caspase-3
Apoptosis regulators; proapoptotic
Upregulated by TCS

Bax
Upregulated by TCS

Bcl-2
Apoptosis regulator; antiapoptotic
Downregulated by TCS

Mouse neocortical neurons
GluN1
Ionotropic glutamate receptors; neurotransmission
Downregulated by TCS

GluN1
Downregulated by TCS

GluN2A
Downregulated by TCS

GluN2A
Downregulated by TCS

GluN2B
Upregulated by TCS

GluN2B
Downregulated by TCS

FasR
Apoptosis regulators; proapoptotic
Upregulated by TCS

Caspase-8
Upregulated by TCS

Caspase-9
Upregulated by TCS

Caspase-3
Upregulated by TCS

AhR
Ligand-activated receptor; detoxification
Upregulated by TCS

JB6 Cl 41-5a cells
Cyclin D1
Cell cycle regulators
Upregulated by TCS

Cyclin A
Upregulated by TCS

p27
Downregulated by TCS

B6C3F1 mice
Alp
Pluripotency markers; stem cell self-renewal and differentiation regulators
Downregulated by TCS

Oct4
Downregulated by TCS

Nanog
Downregulated by TCS

ALP
Downregulated by TCS

Oct 4
Downregulated by TCS

Nanog
Downregulated by TCS

Sox 2
Downregulated by TCS

miRNA-134
Transcriptional regulator of pluripotency markers
Upregulated by TCS

D. rerio
Oct4
Pluripotency markers
Downregulated by TCS

Nanog
Downregulated by TCS

Sox2
Upregulated by TCS

p53
Cell cycle regulator; tumor suppressor
Upregulated by TCS

Casp3
Apoptosis regulators; proapoptotic
Upregulated by TCS

Casp8
Upregulated by TCS

Shha
Early neurogenesis
Sensitive to TCS sensitive to TCS

Ngn1
Upregulated by TCS

Nrd
Upregulated by TCS

Elavl3
Upregulated by TCS

α1-tubulin
Neural maturation
Upregulated by TCS

Gap43
Upregulated by TCS

Gfap
Downregulated by TCS

Mbp
Downregulated by TCS

Abbreviation: Shha: sonic hedgehog a; Ngn1: neurogenin 1; Nrd: NeuroD; Elavl3: ELAV-like, neuron-specific RNA-binding protein 3; Gap43: growth-associated protein 43; Gfap: glial fibrillary acidic protein; Mbp: myelin basic protein.

Collectively, studies on TCS influence on cell fate indicate estrogenic, proliferative, and apoptotic activities. Genes and proteins governing the regulation of cell cycle and apoptosis are particularly sensitive to TCS modulation. The disparity in ultimate cell fate seems to point at an interspecies variation and a dose-specific response, among other experimental details such as cell type and duration of exposure. Elucidating the existence and the identity of a specific molecular “switch” that may tip the scales in favor of either cell death or survival could be an important inquiry for future investigations.

4. Oxidative Stress

Overwhelming evidence has recently accumulated in support of the prooxidative action of TCS. It is prudent to provide an overview of human-based studies first before summarizing notable findings obtained from other model organisms.

In Puerto Rican pregnant women, a correlation between exposure to TCS during pregnancy and oxidative damage, as measured by urinary 8-hydroxyguanosine (8-OHdG), and inflammation was suggested [92]. Similar observations were also mirrored in Chinese and Brazilian children [93, 94]. Conversely, in a global effort comprising nine countries from Asia, Europe, and North America, no relation between urinary TCS and 8-OHdG was established [95].

In vitro studies on human cells have also shed some light on the oxidative potential of TCS. In peripheral blood mononuclear cells (PBMC), 2,4-dichlorophenol (2,4-DCP)—a product of TCS transformation—promoted reactive oxygen species (ROS) generation, with subsequent lipid peroxidation and protein carbonylation [96]. Similarly, TCS caused elevated ROS in Nthy-ori 3-1 human follicular thyroid cells [97] and lipid peroxidation in retinoblastoma (Y79 RB) cells [98]. Our recent investigations on mesenchymal stem cells also showed TCS interference with the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the “master regulator” of detoxification, and its downstream targets, heme oxygenase 1 (HO-1) and NAD(P)H dehydrogenase [quinone 1] (NQO-1) [66]. Consistently, TCS incorporated in mouthrinse did not exhibit antioxidant activity on fibroblasts [99]. In contrast, TCS reduced ROS levels in VM7Luc4E2 cells, which contributed to its antiapoptotic activity in these malignant breast cells [76].

Mitochondrial damage was also evident in multiple mammalian cells including human PBMC and keratinocytes, exposed to 3.5-350 μM TCS [100]. At concentrations up to 100 μM, TCS caused depolarization of mitochondrial membrane, reduced oxidative phosphorylation, and suppressed ATP synthesis. Weatherly et al. [101] utilized human HMC-1.2 mast cells and primary keratinocytes to show that TCS is a proton ionophore uncoupler and interferes with ATP production.

Animal studies conducted on mice and rats have revealed a profound response in the cellular antioxidant machinery upon TCS treatment. In rat thymocytes, superoxide anions were found to be elevated following TCS treatment [102] which, as Yueh et al. [103] showed, was met with increased expression of key antioxidant enzymes including HO-1, NQO-1, and glutathione S-transferase (GST) in mouse liver. Evidence for testicular DNA damage, elevated malondialdehyde (MDA), and superoxide dismutase (SOD), in addition to diminished catalase (CAT), was related to TCS treatment in weanling rats [104]. Similarly, in lung homogenates of female albino rats, TCS was found to induce lipid peroxidation and severely deplete the levels of other crucial antioxidants: SOD, CAT, and glutathione (GSH) [105]. Increased expression of glutathione peroxidase 1 (Gpx1) and aldehyde oxidase 1 (Aox1) was also observed as a consequence to TCS exposure in C57BL/6 mice [106]. Most recently, Zhang et al. [97] showed downregulation of antioxidant enzymes, Gpx3, Cat, and Sod2, along with elevated MDA, in the hypothalamus of Sprague-Dawley rats. Moreover, it was found that TCS treatment leads to increased ROS and reduced GSH activity in rat neural stem cells [79]. TCS also increased ROS levels in mouse neocortical neurons, along with perturbed regulation of cytochrome P450 family 1, subfamily a, member 1 (CYP1a1) and CYP1b1 [81, 82]. Effects of TCS on cytochromes and hepatic detoxification were also demonstrated in Sprague-Dawley rats, showing increased levels of UDP-glucuronosyltransferase 1-1 (Ugt1a), Ugt2b1, CYP1a1, CYP1a2, CYP2b1, CYP3a1, and sulfotransferase family 1E member 1 (Sult1e1) [97].

Several terrestrial organisms have been employed in the study of TCS toxicology. Caenorhabditis elegans is among the best-studied animal models due to its ease of maintenance and high genetic homology to humans. We have recently shown that TCS leads to overproduction of ROS, inhibition of nuclear translocation of protein skinhead-1 (SKN-1) antioxidant transcription factor, and downregulation of gamma-glutamyl cysteine synthetase (Gcs1) [66]. In a subsequent report, Skn1 expression was found to be upregulated by TCS along with Sod1, Sod4, heat shock proteins (Hsp)-3, -4, -16.2, and -70; and cytochromes Cyp29A2 and Cyp34A9 (https://app.dimensions.ai/details/publication/pub.1103154992#readcube-epdf). TCS also enhanced nuclear translocation of stress-related factor DAF-16, suggesting the occurrence of oxidative stress [107]. In the Earthworm Eisenia fetida, oxidative damage by TCS was manifested as a transient elevation in CAT and GST enzymes, increased MDA, and DNA damage [108]. In a follow-up study by the same group, SOD was also increased and decreased by TCS depending on the concentration used [109], a response mirrored by CAT in the snail Achatina fulica [110]. In that study, TCS caused diminished levels of SOD and peroxidase (POD), along with elevated MDA, among other morphological anomalies.

The ubiquity of TCS in aquatic environments has made animal models from that habitat the subject of extensive investigations on TCS toxicity. Perhaps the most relevant aquatic organism is the zebrafish D. rerio, owing to a strong structural and molecular resemblance to humans. Elucidating the interaction between TCS and the antioxidant system in ZFL liver cells, Zhou et al. [111] showed evidence of induced CYP1A activity along with a general trend of suppression in phase I and II detoxification enzymes. Elevated MDA, along with perturbed homeostasis of GSH, peroxiredoxin-2 (PRD-2), and HSPs, were observed in zebrafish larvae grown in the presence of TCS ([67, 87]).

TCS has been shown to induce MDA and cause oscillations in CAT, ethoxyresorufin-O-deethylase (EROD), erythromycin N-demethylase (ERND), and aminopyrine N-demethylase (APND) in Daphnia magna [112]. Moreover, elevated amino acids, including glutamine, glutamate, and proline, have been attributed to a general oxidative stress state in daphnids [113]. Also, stress-related proteins, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hsp-70, were modulated by TCS in D. polymorpha, in addition to lipid peroxidation [68]. TCS exposure demonstrated reduced oxyradicals and lipofuscin and elevated oxidized glutathione (GSSG) in the digestive gland of swollen river mussels Unio tumidus [114]. In Tigriopus japonicus copepods treated with TCS, increased ROS, SOD, GST, GPx, and GSH content was noted [115]. TCS also caused perturbations in expressional profiles of Cyps, Sod, Gst, and Cat proteins (Table 3) [115].

Table 3: Oxidative stress patterns elicited by TCS.

Model
Target
Response

Biomarker
Molecular identity

Humans (pregnant women; children)
Urinary 8-OHdG
Oxidized deoxyguanosine; DNA damage
Upregulated by TCS

Nthy-ori 3-1 cells
ROS
Metabolic oxygen by-products
Upregulated by TCS

PBMC
ROS
Upregulated by TCS

Lipid peroxidation
Oxidized lipids
Upregulated by TCS

Protein carbonylation
Oxidized proteins
Upregulated by TCS

Y79 RB cells
Lipid peroxidation
Oxidized lipids
Upregulated by TCS

Human bone marrow-derived mesenchymal stem cells
Nrf2
Antioxidant regulator
Downregulated by TCS

Ho-1
Antioxidant enzymes
Downregulated by TCS

Nqo-1
Downregulated by TCS

VM7Luc4E2 cells
ROS
Metabolic oxygen by-products
Upregulated by TCS

Mouse liver
O₂–
Antioxidant enzymes
Upregulated by TCS

HO-1
Upregulated by TCS

NQO-1
Upregulated by TCS

GST
Upregulated by TCS

Weanling rats
MDA
Oxidized lipid marker
Upregulated by TCS

SOD
Antioxidant enzymes
Upregulated by TCS

CAT
Downregulated by TCS

Female albino rat lung homogenates
Lipid peroxidation
Oxidized lipids
Upregulated by TCS

SOD
Antioxidants
Downregulated by TCS

CAT
Downregulated by TCS

GSH
Downregulated by TCS

C57BL/6 mice liver
Gpx1
Antioxidant enzyme; glutathione homeostasis
Upregulated by TCS

Aox1
Superoxide and hydrogen peroxide formation
Upregulated by TCS

Sprague-Dawley rat hypothalamus
MDA
Oxidized lipid marker
Upregulated by TCS

Gpx3
Antioxidant enzyme; glutathione homeostasis
Downregulated by TCS

Cat
Antioxidant enzymes
Downregulated by TCS

Sod2
Downregulated by TCS

Rat neural stem cells
ROS
Metabolic oxygen by-products
Upregulated by TCS

GSH
Antioxidant
Downregulated by TCS

Mouse neocortical neurons
ROS
Metabolic oxygen by-products
Upregulated by TCS

Cyp1a1
Cytochrome family enzymes; detoxification
Downregulated by TCS

CYP1a1
Downregulated by TCS

Cyp1b1
Downregulated by TCS

Cyp1b1
Upregulated by TCS

Sprague-Dawley rat liver
Cyp1a1
Upregulated by TCS

Cyp1a2
Upregulated by TCS

Cyp2b1
Upregulated by TCS

CYP2b1
Upregulated by TCS

Cyp3a1
Upregulated by TCS

Ugt2b1
Glucuronidation enzymes; detoxification
Upregulated by TCS

Ugt2b1
Upregulated by TCS

Sult1e1
Sulfation enzyme; detoxification
Upregulated by TCS

Sult1e1
Upregulated by TCS

C. elegans
ROS
Metabolic oxygen by-products
Upregulated by TCS

Skn1
Stress response regulator
Upregulated by TCS

SKN-1
Downregulated by TCS

Gcs1
Antioxidant enzymes
Downregulated by TCS

Sod1
Upregulated by TCS

Sod4
Upregulated by TCS

Hsp-3
Stress response; protein stabilization
Upregulated by TCS

Hsp-4
Upregulated by TCS

Hsp-16.2
Upregulated by TCS

Hsp-70
Upregulated by TCS

Cyp29A2
Cytochrome family enzymes; detoxification
Upregulated by TCS

Cyp34A9
Upregulated by TCS

DAF-16
Stress response
Upregulated by TCS

E. fetida
MDA
Oxidized lipid marker
Upregulated by TCS

CAT
Antioxidant enzymes
Upregulated by TCS

GST
Upregulated by TCS

SOD
Sensitive to TCS to TCS

A. fulica
MDA
Oxidized lipid marker
Upregulated by TCS

CAT
Antioxidant enzymes
Sensitive to TCS to TCS

SOD
Downregulated by TCS

POD
Downregulated by TCS

ZFL liver cells
CYP1A
Cytochrome family enzyme; detoxification
Upregulated by TCS

D. rerio larvae
GPx
Antioxidant enzymes; glutathione homeostasis
Upregulated by TCS

GR
Downregulated by TCS

PRD-2
Antioxidant enzyme
Downregulated by TCS

Hsp-5
Stress response; protein stabilization
Upregulated by TCS

Hsp-90 β
Upregulated by TCS

D. magna
MDA
Oxidized lipid marker
Upregulated by TCS

CAT
Antioxidant enzymes
Sensitive to TCS to TCS

EROD
Detoxification enzymes
Sensitive to TCS to TCS

ERND
Sensitive to TCS to TCS

APND
Sensitive to TCS to TCS

Glutamine
Amino acids; markers of protein oxidation/breakdown
Upregulated by TCS

Glutamate
Upregulated by TCS

Proline
Upregulated by TCS

D. polymorpha gills
Hsp-70
Stress response; protein stabilization
Sensitive to TCS to TCS

U. tumidus digestive gland
GAPDH
Oxidoreductase; glucose metabolism
Sensitive to TCS to TCS

GSSG
Oxidized glutathione; antioxidant
Upregulated by TCS

Oxyradicals
Oxygen-containing radicals; prooxidants
Downregulated by TCS

Lipofuscin
Lysosomal pigment granules; toxicity marker
Downregulated by TCS

T. japonicus
ROS
Metabolic oxygen by-products
Upregulated by TCS

Sod
Antioxidant enzymes
Sensitive to TCS to TCS

SOD
Upregulated by TCS

Cat
Sensitive to TCS to TCS

Gst variants
Antioxidants; glutathione homeostasis
Sensitive to TCS to TCS

GST
Upregulated by TCS

GPx
Upregulated by TCS

GSH
Upregulated by TCS

Cyp3026a3
Cytochrome family enzymes; detoxification
Upregulated by TCS

Cyp3037a1
Upregulated by TCS

P. fulvidraco
MDA
Oxidized lipid marker
Sensitive to TCS to TCS

CAT
Antioxidant enzyme
Upregulated by TCS

Gst
Antioxidant enzyme; glutathione homeostasis
Sensitive to TCS to TCS

EROD
Detoxification enzymes
Upregulated by TCS

ERND
Upregulated by TCS

APND
Upregulated by TCS

Cyp1a
Cytochrome family enzymes; detoxification
Sensitive to TCS to TCS

Cyp3a
Sensitive to TCS to TCS

H. fossilis
CAT
Antioxidant enzymes
Upregulated by TCS

SOD
Upregulated by TCS

GSH
Antioxidants; glutathione homeostasis
Downregulated by TCS

GST
Downregulated by TCS

GPx
Downregulated by TCS

C. auratus
MDA
Oxidized lipid marker
Upregulated by TCS

CAT
Antioxidant enzymes
Upregulated by TCS

SOD
Downregulated by TCS

GSH
Antioxidant; glutathione homeostasis
Upregulated by TCS

Brachionus koreanus
ROS
Metabolic oxygen by-products
Upregulated by TCS

Gst variants
Antioxidant enzyme; glutathione homeostasis
Sensitive to TCS to TCS

Gpx
Sensitive to TCS to TCS

GST
Upregulated by TCS

Sod
Antioxidant enzymes
Sensitive to TCS to TCS

Cat
Sensitive to TCS to TCS

Cyp3042a1
Cytochrome family enzymes; detoxification
Sensitive to TCS to TCS

Cyp43a1
Sensitive to TCS to TCS

Hsp10
Stress response; protein stabilization
Sensitive to TCS to TCS

Hsp21
Upregulated by TCS

Hsp27
Upregulated by TCS

Hsp30
Sensitive to TCS to TCS

Hsp40
Sensitive to TCS to TCS

Hsp40h
Sensitive to TCS to TCS

Hsp60
Sensitive to TCS to TCS

Hsp70
Upregulated by TCS

Hsc70
Upregulated by TCS

Hsp90α1
Sensitive to TCS to TCS

Hsp90α2
Sensitive to TCS to TCS

Hsp90β
Sensitive to TCS to TCS

B. gargarizans liver
Sod
Antioxidant enzyme
Downregulated by TCS

Phgpx
Antioxidant enzyme; glutathione homeostasis
Downregulated by TCS

P. perezi larvae
GST
Upregulated by TCS

R. philippinarum digestive gland
MDA
Oxidized lipid marker
Upregulated by TCS

CAT
Antioxidant enzymes
Sensitive to TCS to TCS

SOD
Sensitive to TCS to TCS

GPx variants
Antioxidant enzymes; glutathione homeostasis
Sensitive to TCS to TCS

GST
Sensitive to TCS to TCS

GR
Sensitive to TCS to TCS

EROD
Detoxification enzyme
Sensitive to TCS to TCS

O. mykiss liver and kidney
Cat
Antioxidant enzymes
Downregulated by TCS

Sod
Upregulated by TCS

Gpx variants
Antioxidant enzymes; glutathione homeostasis
Upregulated by TCS

Gsta
Upregulated by TCS

Hsp90bb
Stress response; protein stabilization
Upregulated by TCS

Hsp90ba
Upregulated by TCS

Hsc70a
Upregulated by TCS

C. reinhardtii
ROS
Metabolic oxygen by-products
Upregulated by TCS

MDA
Oxidized lipid marker
Upregulated by TCS

Sod
Antioxidant enzyme
Upregulated by TCS

Gpx
Antioxidant enzyme; glutathione homeostasis
Upregulated by TCS

R. rubrum S1H
Gpx
Upregulated by TCS

GrxC
Antioxidant enzymes; glutathione homeostasis
Upregulated by TCS

TrxB
Upregulated by TCS

OsmC
Antioxidant enzyme
Upregulated by TCS

DnaJ
Heat shock protein; general stress marker
Upregulated by TCS

RpoN
RNA polymerase factor sigma-54; general stress marker
Downregulated by TCS

TerA
Tellurite resistance protein A; general stress marker
Upregulated by TCS

Psp variants
Phage shock proteins; general stress markers
Sensitive to TCS to TCS

ClpP
ATP-dependent protease, proteolytic subunit; general stress marker
Upregulated by TCS

HrcA
Heat-inducible transcription suppressor; general stress marker
Upregulated by TCS

E. coli K12, MG1655
OxyR
ROS sensor proteins
Upregulated by TCS

Grx
Antioxidant enzymes; glutathione homeostasis
Upregulated by TCS

Sod variants
Antioxidant enzymes
Upregulated by TCS

Cat variants
Upregulated by TCS

Ahp variants
Antioxidant enzymes
Upregulated by TCS

E. coli
ROS
Metabolic oxygen by-products
Upregulated by TCS

YgiW
Antioxidant proteins
Downregulated by TCS

SoxS
Downregulated by TCS

YhcN
Downregulated by TCS

Abbreviation: TrxB: thioredoxin; OsmC: peroxiredoxin osmotically inducible protein C-like. Effects of 2,4-DCP, a by-product of TCS degradation.

TCS treatment in the yellow catfish Pelteobagrus fulvidraco revealed induced CAT, EROD, ERND, and APND [116]. Expressional profiling of Cyp1a, Cyp3a, and Gst showed both up- and downregulation depending on TCS concentration and length of exposure, a pattern that was also seen with MDA formation. When another catfish, Heteropneustes fossilis, was treated with a cosmetic effluent rich in TCS, increased SOD and CAT activities and reduced GSH, GST, and GPx were noted [117].

Oxidative damage by TCS was also evident in the goldfish Carassius auratus, as MDA, CAT, and GSH were elevated in addition to a reduced total antioxidant capacity [118]. Variable responses by antioxidant enzymes and in MDA levels were recorded in the goldfish’s liver after TCS treatment under a pH range of 6 to 9 [119]. The oxidative potential of TCS was also evident in the rotifer Brachionus koreanus, detected as ROS overproduction and enhanced GST activity, in addition to transcriptional modulation of cytochromes, antioxidant genes Gst, Gpx, Sod, and Cat and chaperons (Table 3) [120]. Moreover, TCS inhibited Sod and phospholipid hydroperoxide glutathione peroxidase (Phgpx) expression in the liver of Bufo gargarizans tadpoles [121] and induced GST in Pelophylax perezi frog larvae [122].

Sendra et al. [123] studied the combined effect of titanium dioxide (TiO₂) and a heterogeneous mixture of organic compounds including TCS using the clam Ruditapes philippinarum. Modulations in EROD, SOD, CAT, GPx, GST, and GR enzyme activities were noted in the clam’s digestive gland, in parallel with increased lipid peroxidation. TCS exposure caused alterations in Cat, Sod, Gpx1, Gpx2, Gsta, Hsp90bb, Hsp90ba, and Hsc70a genes in rainbow trout Oncorhynchus mykiss [124]. Although in one report TCS failed to elicit oxidative stress in the green algae Chlamydomonas reinhardtii [125], another report detected ROS formation following TCS exposure [90], which was also most recently confirmed by significantly increased MDA, downregulated Gpx, and upregulated Sod expression [126].

The antimicrobial nature of TCS makes bacteria an appropriate target for mechanistic studies. Using Rhodospirillum rubrum S1H, Pycke et al. [57] detected upregulation in a host of TCS-induced oxidative response genes, most notably Gpx. In E. coli K12, MG1655, the electro-Fenton transformation of TCS caused activation of genes related to ROS sensing, along with reduced glutaredoxin (Grx), Sod, Cat, and alkyl hydroperoxide reductase (Ahpr) [55]. Very recently, ROS formation by TCS was associated with diminished expression of antioxidants in E. coli (Table 3), an event that preceded mutagenesis and enhanced drug resistance in that species [56]. TCS was also recently used to validate novel self-luminescent bioreporter strains of Nostoc sp. PCC 7120 using Sod promoters [127].

Collectively, monumental evidence demonstrates the prooxidant properties of TCS evident as both overproduction of ROS and interference with the cellular antioxidant defensome. TCS is toxic in part by inducing oxidative damage in a wide range of organisms and by targeting a defined cluster of proteins in a fashion that is conserved among diverse species. Nonetheless, the vast majority of data are collected from non-human models, and, as is the case with other toxicological reports of TCS, studies conducted on man or human-derived tissues are severely lacking.

5. Immunity and Inflammation

TCS has, for a long time, been recognized as an effective therapy for infectious dermatitis [29–31], and the observed curative capacity of the compound was solely attributed to its antimicrobial activity. It was not until the end of last century that associations between TCS exposure and remission of noninfectious inflammation were made [33, 35, 36], and the use of antibacterials as anti-inflammatory agents has gained deserved attention during the past two decades. For example, an appreciable number of antibiotics, including macrolides and quinolones, have been shown to possess anti-inflammatory activity [128–132]. Follow-up efforts have successfully provided solid evidence for the direct interaction of TCS with inflammatory pathways.

Gaffar et al. [133] reported that TCS inhibits cyclooxygenase-1 (COX-1) and COX-2, 5-lipoxygenase and (LPO), 15-LPO, and interleukin- (IL-) 1β-induced prostaglandin E2 (PGE2) in gingival cells. TCS was also shown to suppress a wider range of inflammatory mediators including IL-1β-induced prostaglandin I2 (PGI2) and arachidonic acid, tumor necrosis factor (TNF)α-induced PGE2, phospholipase A2 (PLA2), and COX [134]. Moreover, in a double-blind crossover study, participants who used a mouthrinse with added 0.15% TCS developed significantly less oral erythematous lesions than those who used a TCS-free mouthrinse [135]. By then, the anti-inflammatory properties of TCS were established and were widely accepted within the scientific and medical communities.

TCS in prosthetic devices was found to have no influence on the acute phase response [136], and only modest differences were seen between TCS and stannous fluoride dentrifice [137]. Nevertheless, TCS, when applied intracrevicularly, improved clinical parameters of gingivitis [138]. In a recent double-blind, randomized, crossover study, it was concluded that TCS-containing toothpaste inhibits inflammation in peri-implant tissue [139].

To date, elaborations on the anti-inflammatory nature of TCS have been the focus of subsequent studies. Mustafa et al. [140–142] identified IL-1β, interferon (IFN)γ, major histocompatibility complex (MHC) class II, and PGE synthase-1, as targets of TCS in human gingival fibroblasts. Of note, studies to discern the subcellular localization of TCS show preference for nuclear, as opposed to cytosolic, accumulation. Although initial uptake was considerably higher in the cytoplasm, a great proportion of cytosolic TCS was eliminated after repeated washing, while nuclear retention was observed [143]. This may explain the perturbed inflammatory signaling associated with TCS. Moreover, in primary human oral epithelial cells, TCS attenuated LPS-induced cytokine response including IL-8, IL-1α, and TNFα and aggravated the antimicrobial response, which was mediated through microRNA (miRNA) regulation of the toll-like receptor (TLR) pathway [144]. The findings were also reciprocated in cells derived from diabetic patients, with an exaggerated TLR response [145]. It was revealed that TCS, nevertheless, abrogated LPS-induced TLR response, again, through regulating miRNAs (stimulating miR146a and inhibiting miR155s).

In skin and leukocytes of mice topically treated with TCS, alterations in inflammatory responses were mediated through TLR4 [146]. Likewise, TCS downregulated parathyroid hormone- (PTH-) or PGE2-stimulated matrix metalloproteinase-13 (MMP-13) expression in rat osteoblastic osteosarcoma cells [147]. Since hyperactive MMP-13 is implicated in periodontal disease, it was suggested that TCS might have a protective role against oral inflammatory conditions through its action on that enzyme, among others [148].

Interestingly, favorable results have been observed for TCS against other inflammatory conditions including cardiovascular disease and hidradenitis suppurativa [149, 150]. Moreover, the use of TCS-impregnated ureteral stents seems to be a promising approach to combat urinary tract infections (UTI) and associated inflammation [151, 152]. Along those lines, an increased urinary TCS was related to increased serum IL-6 in pregnant women [92], pointing at a possible pro- or anti-inflammatory role.

In a unique effort by Barros et al. [153], TCS modulation of the inflammatory response in an ex vivo whole blood stimulation assay was investigated. In that study, TCS inhibited multiple inflammatory mediators induced by LPS, including interleukins, most notably IL-1 & IL-6, IFNs, and colony-stimulating factor (CSF) 2. Activation of type 1 T helper lymphocytes was interrupted through the action of TCS on CD70. In a related report, TCS also reduced the capacity of natural killer (NK) lymphocytes to lyse chronic myelogenous leukemia K562 cells [154]. Recently, chitosan-TCS particles reduced the expression of IL-1β-induced Cox2 and Il6, among other immune molecules in gingival fibroblasts (Table 4) [155], showcasing the vast amenability of this antimicrobial to nanoparticle manipulation.

Table 4: Inflammatory and immune mediators responsive to TCS.

Model
Target
Response

Biomarker
Molecular identity

Human gingival fibroblasts
COX-1/2
Inflammatory mediators
Downregulated by TCS

5/15-LPO
Downregulated by TCS

PGE2
Downregulated by TCS

PGI2
Downregulated by TCS

Arachidonic acid
Downregulated by TCS

PLA2
Downregulated by TCS

PGE synthase-1
Downregulated by TCS

IFNγ
Immune/inflammatory cytokines
Downregulated by TCS

IL-1β
Downregulated by TCS

MHC II
Cell surface proteins; adaptive immunity regulators
Downregulated by TCS

Cox2
Inflammatory mediator
Downregulated by TCS

Il6
Immune/inflammatory cytokines
Downregulated by TCS

Il1b
Downregulated by TCS

Tlr6
Innate immunity receptor
Upregulated by TCS

Human primary oral epithelial cells
IL-8
Immune/inflammatory cytokines
Downregulated by TCS

IL-1α
Downregulated by TCS

TNFα
Downregulated by TCS

miR146a
Transcriptional regulators of TLR response
Upregulated by TCS

miR155s
Downregulated by TCS

Mouse skin and leukocytes
S100A8/A9
Inflammatory modulator; Ca²⁺-binding protein
Upregulated by TCS

Tlr4
Innate immunity receptors
Upregulated by TCS

TLR4
Upregulated by TCS

Tlr1
Upregulated by TCS

Tlr2
Upregulated by TCS

Tlr6
Upregulated by TCS

Rat osteoblastic osteosarcoma cells
MMP-13
Endopeptidase; collagen degradation
Downregulated by TCS

Human oral fluids
IL-1α
Immune/inflammatory cytokines
Downregulated by TCS

IL-1β
Sensitive to TCS

IL-8
Sensitive to TCS

MCP-1
Sensitive to TCS

TIMP-2
MMP regulator proteins
Sensitive to TCS

TIMP-1
Downregulated by TCS

MMP-8/9
Endopeptidases; extracellular matrix degradation
Downregulated by TCS

Human urine
IL-6
Immune/inflammatory cytokines
Upregulated by TCS

Sprague-Dawley rats
TNFα
Upregulated by TCS

IL-6
Upregulated by TCS

Human whole blood leukocytes
Csf2
Hematopoietic stem cell growth and maintenance
Downregulated by TCS

Ifna1
Immune/inflammatory cytokines
Downregulated by TCS

Ifna2
Downregulated by TCS

Ifna4
Downregulated by TCS

Ifna8
Downregulated by TCS

Il-1f10
Downregulated by TCS

Il-1f5
Downregulated by TCS

Il-1f7
Downregulated by TCS

Il-1f8
Downregulated by TCS

Il-1f9
Downregulated by TCS

Il-6
Downregulated by TCS

Il-11
Downregulated by TCS

Il-13
Downregulated by TCS

Il-25
Downregulated by TCS

Il-19
Downregulated by TCS

Il-21
Downregulated by TCS

Il-9
Downregulated by TCS

Cd70
Cell surface receptor/ligand; activated lymphocytes
Downregulated by TCS

Bmp2
Growth factors; bone and cartilage development
Upregulated by TCS

Bmp6
Upregulated by TCS

Tnfrsf11b
TNFSF11 receptor
Downregulated by TCS

Gdf3
Growth/differentiation factors
Downregulated by TCS

Gdf2
Downregulated by TCS

Gdf5
Downregulated by TCS

Gdf9
Downregulated by TCS

Inhba
Hypothalamus-pituitary axis regulator
Downregulated by TCS

Lefty2
Left-right determination factor 2; left-right asymmetry of organs
Downregulated by TCS

Sprague-Dawley rats
TNFα
Immune/inflammatory cytokine
Upregulated by TCS

IL-6
Upregulated by TCS

Abbreviation: MCP: monocyte chemoattractant protein; TIMP: tissue inhibitor of metalloproteinase; Bmp: bone morphogenetic protein; Gdf: growth differentiation factor; Inhba: inhibin beta A chain.

Table 5: TCS genotoxicity and carcinogenicity.

Model
Effect
Classification

HepG2 cells
Global DNA hypomethylation
Limited evidence of carcinogenicity

V79 cells
Chromosomal aberrations

Mouse
Somatic mutation (positive spot test)
Increased incidence of liver tumors
Aggravated hepatocellular carcinoma
Exacerbated colon tumorigenesis

D. polymorpha
DNA damage (positive comet assay)
N/A

U. tumidus
DNA strand breaks (Hoescht 33342 fluorescence)

E. Fetida
DNA damage (positive Comet assay)

D. magna
DNA damage (positive Comet assay)

A. salina
DNA damage (positive Comet assay)

H. fossilis
DNA damage (positive Comet assay)

C. auratus
DNA damage (positive Comet assay)

O. mykiss
DNA damage (positive Comet assay)

T. thermophila
DNA damage (positive Comet assay)

A. cepa
Chromosomal stickiness, reduced mitotic activity, and ana-telophase bridges (positive Feulgen reaction)

N/A = data from non-mammalian animals are not considered for ECHA mutagenicity/carcinogenicity classification.

Other in vivo studies on rodents and marine organisms clarified further the immunomodulatory properties of TCS. For instance, in mice subjected to an acute, systemic E. coli infection, Sharma et al. [156] demonstrated that cotreatment with TCS significantly reversed the damage caused by the bacteria. Specifically, TCS prolonged survival; lessened hepatic congestion, hemorrhage, and fatty changes; and reduced blood liver enzymes, serum TNFα, and the severity of bacteremia. In accordance with published data, TCS was similarly immunosuppressive in aquatic mussels (M. galloprovincialis) and clams (R. philippinarum) [64, 65].

Contrary to the overwhelming evidence of the anti-inflammatory function of TCS, a number of studies have nonetheless identified a proinflammatory role by the antiseptic. For example, upon intratracheal instillation of TCS in Sprague-Dawley rats, elevated total cell (TC) count, polymorphonuclear leukocytes (PMNs), total protein (TP), LDH, TNFα, and IL-6 were observed in bronchoalveolar lavage (BAL) fluid [83], which, except for TP, returned to baseline levels 14 days after exposure. Consonantly, it has also been demonstrated that TCS exacerbates diethylnitrosamine-induced hepatocellular carcinoma in C57BL/6 mice [103]. Likewise, TCS was very recently found to increase Tlr4 expression to promote colitis and aggravate colitis-related cancer in C57BL/6 mice [157].

It is evident from the wealth of information present that TCS is a modulator of immune and inflammatory reactions. The sum of data from in vitro and in vivo studies indicates that TCS, on its own, is immunosuppressive. Nevertheless, increasing evidence seems to suggest that in the presence of an existing adverse condition, such as inflammation or tumor, TCS further potentiates and worsens the eventual outcome. Investigations into the molecular basis behind this unique behavior are particularly warranted.

6. Genotoxicity and Carcinogenicity

Among the most important aspects of toxicological profiling of compounds is their interaction with the molecule of life—the DNA. Early efforts [42, 158] point at a possible role for TCS in somatic mutations observed in mice. TCS also caused a significant reduction in global DNA methylation in human hepatocellular carcinoma HepG2 cells, a finding associated with liver tumor [159]. Similarly, TCS caused a dose-responsive increase in chromosomal aberrations in lung fibroblast V79 cells, but not in ovary CHO cells, of the Chinese hamster Cricetulus griseus [12]. In a comparative study on Drosophila melanogaster using three mouthwashes, namely, Cepacol® (0.05% cetylpyridinium chloride), Periogard® (0.12% chlorhexidine digluconate), and Plax® (0.03% TCS), it was concluded that only the ethanol content in Cepacol®, but not other active ingredients, caused mitotic recombination between homologous chromosomes [160]. On the other hand, TCS induced dose-responsive DNA damage in hemocytes of the zebra mussel D. polymorpha [88], and strand breaks in the digestive gland of U. tumidus mussels [114]. A similar dose-dependent DNA damage was also observed in the earthworm E. fetida [108, 109], but not in E. andrei [161].

Comparing TCS to other toxicants in the larvae of freshwater insect Chironomus riparius, Martinez-Paz et al. [162] found TCS, along with nonylphenol, to be the most potent in causing DNA breakage. It was also noted that TCS, either alone or in combination with carbendazim, induced DNA damage in D. magna [163]. Using the brine shrimp Artemia salina, a time-dependent pattern of TCS-induced genotoxicity was identified [164]. Moreover, TCS was genotoxic in the catfish Heteropneustes fossilis, goldfish C. auratus, and rainbow trout O. mykiss [117, 118, 124]. Importantly, when TCS at an environmentally relevant concentration (3 nM) was treated to the freshwater protozoan Tetrahymena thermophila, notable DNA damage, without significant perturbation in growth or cell viability, was evident [165]. In a more detailed study on E. coli, Gou et al. [55] revealed that the electro-Fenton transformation of TCS caused upregulation of a host of genes involved in the DNA repair machinery, indicative of DNA stress. These genes belong to base excision repair (mutT and nfo), nucleotide excision repair (uvrA and uvrD), mismatch repair (uvrD and ssb), and double-strand break repair (ssb and recN). Chromosomal stickiness, reduced mitotic activity, and ana-telophase bridges were also noticeable in the bulb onion Allium cepa following TCS treatment [166].

In a recent proof-of-concept study, the promising potential of a toxicogenomic approach as a follow-up to positive in vitro genotoxicity data was evaluated. Using TCS as a testing compound, it was shown that the antimicrobial is non-DNA reactive and that it is genotoxic solely in vitro as opposed to in vivo [167].

Ambiguity surrounding the carcinogenicity of TCS still remains today. Investigators have generally been able to provide evidence for carcinogenic effects in animal models but not in humans. Of the earliest studies in this regard was a report by Lyman and Furia [38] identifying TCS as a carcinogen in mice. Other studies on mice have been in agreement with that conclusion. For example, it was noted that chronic TCS exposure increased the incidence of liver neoplasms [12] and aggravated hepatocellular carcinoma [103]. Furthermore, TCS caused colonic inflammation and worsened colitis or tumorigenesis induced by dextran sodium sulfate [168]. These findings, were, however, not paralleled in rats, hamsters, or baboons [12, 169]. More importantly, in vivo human studies of TCS are scarce, and aspects related to TCS-induced oncogenesis are lacking. Consequently, whether TCS poses a carcinogenic hazard to humans is unknown and requires further investigation. Nonetheless, the interaction of TCS with human-derived cancer cells in vitro has recently gained considerable attention (reviewed under Therapeutic Proposals).

In light of available data (Table 5), TCS demonstrates carcinogenicity solely in mice and within a narrow range of tissues (the liver and colon), which constitutes limited evidence of carcinogenicity according to ECHA (https://echa.europa.eu/documents/10162/23036412/clp_en.pdf/58b5dc6d-ac2a-4910-9702-e9e1f5051cc5). Hence, TCS is not classifiable as a carcinogen (http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_054.pdf). It must be noted that in case future assessment conclusively rules out TCS as a human carcinogen, caution with its use must still be exercised given the established carcinogenicity of its transformation products—dioxins, chloroform, and anilines [170].

7. Cellular Signaling

Adaptations to the ever-changing intracellular and surrounding environments are achieved, in large part, by effective communication. Transmission of information that carries specific instructions is executed by messengers that function in tandem within a defined pathway. Tasks, however, are usually accomplished through the sequential transduction of multiple messages along a complex, intertwining network that involves a wide assortment of mediators [171]. Hence, the participation of cell signaling cascades in the response to xenobiotics cannot be overlooked.

The use of human cell lines has provided a wealth of information particularly regarding the study of signaling molecules responsive to stressors and xenobiotics, including TCS. In H460 lung cancer cells, TCS promoted migration and invasion through focal adhesion kinase/ATP-dependent tyrosine kinase (FAK/Akt) and Ras-related C3 botulinum toxin substrate 1 (Rac1) [72]. Evidence similarly exists for the classical mitogen-activated protein kinases (MAPK) as targets of TCS. For example, proliferation of JB6 Cl 41-5a cells as induced by TCS was accompanied by activation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 MAPKs, in addition to Akt [84]. Importantly, blocking either MEK1/2 or phosphoinositide 3-kinase (PI3K) significantly attenuated TCS-induced proliferation. In another study on rat neural stem cells, TCS-induced cytotoxicity and apoptosis were accompanied by activation of p38 and JNK and suppression of ERK, Akt, and PI3K [79]. This points at the involvement of these proteins in both cellular survival and death as brought about by TCS. Recently, TCS was shown to activate p38 and JNK in vivo as detected in the hypothalamus of Sprague-Dawley rats and in vitro utilizing human Nthy-ori 3-1 thyroid follicular cells [97]. In that study, TCS stimulated the thyrotropin-releasing hormone receptor through p38 MAPK, which, in turn, influenced the thyroid peroxidase (TPO) level.

In suppressing TLR signaling in whole blood leukocytes, TCS downregulated the expression of several signaling mediators, most notably, NF-κB-inducing kinase (Nik) and C-jun, which accounted for the overall blunted inflammatory response to LPS in these cells [153]. Furthermore, suppression of Mmp-13 expression in mouse osteoblastic osteocarcinoma cells by TCS was possibly related to its inhibition of Fos/Jun and AP-1 sequence binding in both the Mmp-13 and C-fos promoters [147].

The endocrine-disrupting activity of TCS, specifically its estrogenicity, has been of great interest to researchers. Kim et al. [73] utilized BG-1 ovarian cancer cells to show that the proliferative effects of TCS were mediated through ERα. Confirming the ER’s role, the use of ICI 182,780 reversed the proliferative properties of TCS along with associated perturbations in cyclin D1, p21, and Bax expression and protein levels. Likewise, the ER is implicated in TCS-induced proliferation of MCF-7 cells and increased breast tumor mass in mice [74, 75, 172]. This was similarly indicated by TCS inhibition with ICI 182,780 or kaempferol and the stimulation of insulin-like growth factor (IGF) signaling, namely, phosphorylated insulin receptor substrate (pIRS-1), pAkt, pMEK1/2, and pERK1/2 [75]. Notably, kaempferol also inhibited TCS-induced VM7Luc4E2 cell growth [76]. These observations are in congruence with an earlier report by Huang et al. [173] describing the estrogenic activities of nanomolar concentrations of TCS in the same cells. Investigating ER-responsive genes on the transcriptional and translational levels, it was shown that TCS induced pS2 but blunted ERα mRNA and protein levels, the latter of which was related to elevated miR-22, miR-206, and miR-193b miRNAs.

Recent studies have also argued for the dual effect of TCS on ER signaling. For example, Henry and Fair [174] demonstrated that, when administered alone to MCF7 cells, TCS at 7 nM to 700 μM exhibits estrogenic activity but becomes antiestrogenic in the presence of E2. Along those lines, it was shown that TCS, on its own, lacked any effect on rat uterine growth, but could still potentiate the effect of ethinylestradiol (EE) [175]. In a follow-up investigation, it was reported that TCS promotes EE-induced inhibition of ERα and ERβ expression and when given alone does not activate ER at concentrations from 30 nM to 100 μM [176]. Furthermore, TCS diminished E2 and estrogen sulfotransferase in sheep placenta [177]. This is in contrast to the increased activity of ERβ but not ERα caused by a TCS-derivative mixture, which led to neurological and behavioral abnormalities in zebrafish [87]. Also, Sprague-Dawley rats given TCS showed increased uterine weight and Calbindin-d(9k) (CaBP-9k) expression, which was also reciprocated in pituitary GH3 cells [178]. Reversal of both anomalies by ICI 182,780 and RU 486 points at a possible estrogenic role of the antimicrobial.

Very recently, Serra et al. [179] challenged accumulating evidence of TCS estrogenicity by showing the lack of agonistic or antagonistic effect in vivo and in vitro. While up to 0.3 μM TCS did not modulate ER-dependent brain aromatase in zebrafish embryos, interference with the enzyme’s activity, and with E2 activation of the enzyme observed at 1 μM, was not attributed to TCS-ER interaction. Moreover, up to 10 μM TCS lacked estrogenic effects in ER-expressing zebrafish liver cells as well as in MCF-7 cells [179]. Additionally, in a screening study of the estrogenicity of a group of endocrine-disrupting chemicals on fish species, TCS failed to significantly elicit a response in an in vitro ERα reporter gene assay [180].

In light of available evidence, the general consensus seems to indicate that the estrogenicity of TCS is contingent upon multiple factors, including concentration, species, duration of exposure, and whether TCS is administered alone or in combination with other molecules.

With regard to the androgenic properties of TCS, it was revealed that TCS interferes with testosterone- (TSN-) related transcription but promotes that dependent on androgen [181, 182]. In a recent in vivo study on weanling male rats, Riad et al. [104] reported that TCS, either alone or combined with butylparaben, reduced TSN, leutinizing hormone (LH), and follicle-stimulating hormone (FSH), while increased E2 was observed upon single TCS administration Also, TCS-induced proliferation and migration of LNCaP cells were significantly reduced in presence of bicalutamide, an androgen receptor (AR) antagonist [77]. These findings support a previous report by Ahn et al. [183] in which 1 μM TCS reduced E2-induced ER activation by 50% and AR in human BG1Luc4E2 ovarian adenocarcinoma cells and T47D-ARE breast cancer cells, respectively. Evidence for TCS estrogenicity was detected in MCF7 cells when [(3)H]estradiol was successfully displaced from the ER by the antimicrobial [184]. Furthermore, 10 μM TCS attenuated E2-dependent ERE-CAT reporter gene induction, while 0.1 and 1 μM TCS inhibited TSN-stimulated LTR-CAT reporter gene in both T47D cells and S115 mouse mammary tumor cells [184]. TCS was also determined to have a weak effect on AhR in recombinant rat hepatoma (H4L1.1c4) cells. Finally, Forgacs et al. [185] showed that TCS interferes with recombinant hCG stimulation of TSN in a novel BLTK1 murine Leydig cell model. Most recently, however, no significant influence on androgen synthesis or activity by TCS was observed in Wistar rats [186].

Controversy surrounding the interaction between TCS and members of the peroxisome proliferator-activated receptors (PPARs) has gained considerable attention as of late. This has essentially stemmed from the apparent discrepancy between data obtained from humans and those from rodents. In comparing the differential modulation of TCS on PPARα in HepG2 cells and mouse hepatoma Hepa1c1c7 cells, distinct responses were observed by Wu et al. [187]. Protein levels of PPARα downstream target, acyl-coenzyme A oxidase, were decreased in HepG2 cells but were increased in Hepa1c1c7, which also showed higher DNA synthesis and blunted apoptosis through transforming growth factor (TGF-β). PPAR signaling was similarly identified as a target of TCS through genome-wide CRISPR-Cas9 screening in HepG2 cells [188], zebrafish [189], and Gallus gallus chicken embryos [190]. In the latter model, PPAR signaling members Cyp7a1, fatty acid-binding protein 1 (Fabp1), acyl-CoA synthetase long-chain family member 5 (Acsl5), acyl-CoA oxidase 2 (Acox2), and perilipin 1 (Plin1) were upregulated, whereas angiopoietin-like 4 (Angptl) was downregulated.

TCS administered to pregnant mice caused insulin resistance, hypothyroidism, diminished glucose transporter 4 (GLUT4) expression, and inhibition of Akt and mTOR phosphorylation [191, 192]. While thyroxine corrected these adversaries, PPARγ activator, rosiglitazone, solely reversed the decrease in Akt phosphorylation in adipose tissue and in muscle [192]. PPARγ is known to ameliorate mTOR suppression-induced glucose intolerance in rats [193], further underlining the far-reaching effects of TCS action.

Although TCS has been reported to promote hepatocyte proliferation in mice through PPAR [12], Yueh et al. [103] found no appreciable induction of PPARα following TCS treatment. Importantly, the authors also identified constitutive androstane receptor (CAR) as a possible aggravator of TCS-induced tumorigenesis, given the halved tumor number in Car–/− mice compared to their Car+/− counterparts. TCS, as is the case with PPARs, is reported to exhibit varying affinities for CAR and pregnane X receptor (PXR) in humans and rodents. A weak agonist for human CAR, TCS was found to be a reverse agonist for rodent CAR, an agonist for human PXR, and had no effect on rodent PXR [194].

Calcium concentration within cells influences protein conformation and dynamics. Protein binding of Ca²⁺, on the other hand, maintains the ion’s content within a physiological range and sets forth diverse cellular activities related to gene expression, motility, secretion, and survival [195]. Beside proteins, intracellular Ca²⁺ levels are modulated by a variety of stimuli, including xenobiotic exposure. Through the Ca²⁺ channel ryanodine (Ry) receptor type 1 (RyR1), TCS increased cytosolic Ca²⁺ dose-dependently in primary skeletal myotubes irrespective of extracellular Ca²⁺ [183]. Accordingly, muscle contractility was compromised upon TCS exposure in vitro and in vivo [196]. Results from this study indicate that TCS impaired excitation-contraction coupling (ECC) in cardiac and skeletal muscles and enhanced electrically induced Ca²⁺ transients in myotubes without depleting intracellular Ca²⁺ and notwithstanding RyR1 blockage. TCS also efficiently blocked excitation-coupled Ca²⁺ entry and interfered with the bidirectional signaling between RyR1 channels and Ca²⁺ ions. Likewise, TCS compromised ECC in larval fathead minnows Pimephales promelas, as evidenced by altered RyR and dihydropyridine receptor (DHPR) mRNA and protein levels and weakened ligand binding to both receptors in adult muscle homogenates [197].

In rat thymocytes, TCS elevated intracellular Ca²⁺ levels and opened Ca²⁺-responsive K⁺ channels, eventually leading to membrane hyperpolarization [198]. Also, TCS prevented Ca²⁺-induced mitochondrial swelling in rat liver [199]. A more in-depth analysis of TCS modulation of Ca²⁺ homeostasis was conducted on rat basophilic leukemia (RBL) mast cells [24]. In this cell type, TCS caused mitochondrial fission and diminished membrane potential and translocation, with compromised ATP production and elevated ROS. These changes were associated with perturbed mitochondrial and endoplasmic reticulum Ca²⁺ and depleted cytosolic Ca²⁺ levels following antigen stimulation. Accordingly, TCS-induced degranulation of mast cell may at least in part be attributed to Ca²⁺ mobilization.

Calcium modulation by TCS has also been investigated in other organisms. In C. reinhardtii exposed to 14 μM TCS, increased Ca²⁺ levels with oxidative stress, cell and mitochondrial membrane depolarization, compromised photosynthesis, and caspase activation were noted [90]. Importantly, chelation of intracellular Ca²⁺ ions by BAPTA-AM protected the algae from TCS-induced Ca²⁺ dysregulation. These observations strongly implicate Ca²⁺ as a mediator of a wide array of toxic anomalies attributed to TCS.

Literature concerning the xenobiotic response to TCS has revealed important signaling pathways activated or suppressed by TCS (Table 6). Distinct outcomes exist among species and even within the same species based on experimental conditions and model under investigation. Although important milestones in TCS signaling have been achieved so far, there remains a lot to be discovered, especially in human-based systems, about the modulatory effects of TCS on cellular physiology. In particular, the response of many human cell types and tissues to TCS treatment is unknown, and identification of signaling pathways and their roles in cellular growth, metabolism, and overall function is therefore advised.

Table 6: TCS modulation of signaling pathways.

Model
Target
Response

Pathways
TCS role

H460 cells
FAK/Akt
Cellular migration and invasion
Upregulated by TCS

Rac1
Upregulated by TCS

JB6 Cl 41-5a cells
ERK1/2
Cell proliferation
Upregulated by TCS

JNK
Upregulated by TCS

p38
Upregulated by TCS

Akt
Upregulated by TCS

PI3K
Upregulated by TCS

Rat neural stem cells
JNK
Cytotoxicity and apoptosis
Upregulated by TCS

p38

Upregulated by TCS

ERK

Akt
Downregulated by TCS

PI3K
Downregulated by TCS

Downregulated by TCS

Sprague-Dawley rats hypothalamus and Nthy-ori 3-1 cells
JNK
Reduced TPO; hypothyroidism
Upregulated by TCS

p38
Upregulated by TCS

Whole blood leukocytes
Nik
Anti-inflammatory response
Downregulated by TCS

Cjun
Downregulated by TCS

Mouse osteoblastic osteocarcinoma
Fos
Downregulated by TCS

Jun
Downregulated by TCS

Ap1
Downregulated by TCS

BG-1
ERα
Cell proliferation
Upregulated by TCS

MCF-7 cells
ERα
Sensitive to TCS

pIRS-1
Upregulated by TCS

pAKT
Upregulated by TCS

pMEK1/2
Upregulated by TCS

pERK1/2
Upregulated by TCS

VM7Luc4E2 cells
Erα
Downregulated by TCS

Ps2
Upregulated by TCS

ERα
Downregulated by TCS

pS2
Upregulated by TCS

miR-22
Upregulated by TCS

miR-206
Upregulated by TCS

miR-193b
Upregulated by TCS

Sheep placenta
E2
Anti-estrogenicity
Downregulated by TCS

Estrogen sulfotransferase
Downregulated by TCS

BG1Luc4E2 cells
ER
Downregulated by TCS

Sprague-Dawley rats and GH3 cells
CaBP-9 k
Estrogenicity
Upregulated by TCS

LNCaP
AR
Androgenicity; cell proliferation, and migration
Upregulated by TCS

T47D-ARE cells
AR
Anti-androgenicity
Downregulated by TCS

H4L1.1c4 cells
AR
Pro(anti)-androgenicity
Sensitive to TCS

HepG2 cells
Acyl-coenzyme A oxidase
Blunted lipid metabolism
Downregulated by TCS

Hepa1c1c7 cells
Acyl-coenzyme A oxidase
Enhanced lipid metabolism and DNA synthesis
Upregulated by TCS

TGF-β
Antiapoptosis
Downregulated by TCS

D. rerio
PPARα
Enhanced lipid metabolism
Upregulated by TCS

PPARγ
Upregulated by TCS

G. gallus embryo livers
PPARα
Upregulated by TCS

ICR mice
Akt
Impaired glucose metabolism
Downregulated by TCS

mTOR
Downregulated by TCS

C57BL/6 mice
CAR
Tumorigenesis
Upregulated by TCS

HepG2 cells
CAR
Enhanced hepatic catabolism
Upregulated by TCS

PXR
Upregulated by TCS

Rodent FAO hepatoma cells
CAR
Reduced hepatic catabolism
Downregulated by TCS

Primary skeletal myotubes
Ca²⁺
Diminished muscle contractility
Upregulated by TCS

RyR1
Upregulated by TCS

P. promelas muscle homogenates
Ryr2
Sensitive to TCS

Ryr3
Downregulated by TCS

RyR
Downregulated by TCS

Rat thymocytes
Ca²⁺
Cell membrane hyperpolarization
Upregulated by TCS

RBL cells
Ca²⁺
Mast cell degranulation
Downregulated by TCS

C. reinhardtii
Ca²⁺
Dampened photosynthesis
Upregulated by TCS

TCS is anti-estrogenic in the presence of E2.

8. Therapeutic Proposals

The first specific action mechanism of TCS in prokaryotes was only demonstrated 20 years ago, when inhibition of fatty acid synthesis in Escherichia coli was noted following exposure to TCS [200, 201]. TCS irreversibly inhibited the fatty acid biosynthesis enzyme, enoyl–acyl carrier protein reductase (ACP), by mimicking its natural substrate in vivo. Further, a mutated or overexpressed ACP, encoded by fabI, was shown to confer TCS resistance in the bacterium. These findings established ACP as a specific, subcellular TCS target. Efforts have thus far revealed the susceptibility of a host of other pathogens to inhibition of fatty acid synthesis by TCS. These include Staphylococcus aureus, M. tuberculosis, Helicobacter pylori, Haemophilus influenzae, Plasmodium falciparum, Toxoplasma gondii, Leishmania spp., and Trypanosoma spp. [52, 202–208]. In humans, fatty acid synthase (FAS) is the only multienzyme complex that is responsible for the endogenous synthesis of saturated fatty acids from acetyl-CoA and malonyl-CoA [209, 210]. Although a BLAST analysis of E. coli FabI protein and FAS showed no homology, appreciable sequence similarities were nevertheless found with polyketide synthase and type I FAS of M. tuberculosis [211].

The success of cerulenin, a mycotoxin with fatty acid inhibitory action, in suppressing tumor progression in vivo has spawned several reports in support of fatty acid synthesis inhibition as an emerging target for chemotherapy [212]. The earliest study in this regard investigated the cytotoxicity of TCS in MCF-7 and SKBr-3 breast cancer cells [211]. It was revealed that TCS at 10-50 μM is cytotoxic and antiproliferative, induces morphological alterations, and inhibits FAS. These findings corroborate an earlier observation linking FAS inhibition with apoptotic death of breast cancer cells [211, 213, 214]. TCS was similarly found to inhibit the development of methylnitrosourea-induced breast cancer in Sprague-Dawley rats [209]. In human A-375 melanoma cells, TCS inhibited growth at 40 μM [215]. TCS was similarly found to be dose-dependently proapoptotic in prostate cancer cells, with IC₅₀ values as low as 4.5-7.8 μM [216]. Whereas no cytotoxicity was observed in NIH3T3 fibroblasts at concentrations up to 60 μM, values of IC₅₀ ranging from 0.74 to 62 μM were nonetheless observed in nonmalignant prostate cells. This suggests two things; first, that prostate cells are relatively more sensitive to TCS toxicity than fibroblasts and presumably other nonmalignant cell types, and second, that malignant prostate cells exhibit higher chemosensitivity compared to their nonmalignant counterparts. This differential susceptibility could be due to overexpressed FAS in malignant cells. However, in contrast to these reports, at concentrations up to 345 μM, TCS was found to be preferentially cytotoxic to Y79 RB cells over mouse 3T3 fibroblasts and human MIO-M1 Müller glial cells as indicated by IC₅₀ values, creating a large therapeutic index of 7.1 and 5.3, respectively [217]. FAS suppression, depleted fatty acid content, lipid peroxidation, and apoptotic death were noted in Y79 RB cells at the same TCS concentration range [98]. Recently, TCS at 40 μM was also shown to be effective against MiaPaCa-2 and AsPC-1 pancreatic cancer cells suppressing proliferation and eliciting apoptotic death [218]. Of note, in a related study, TCS impeded mouse preadipocyte differentiation [219]. Given the regulation of food intake by FAS, and the susceptibility of adipocyte development to TCS inhibition, it was suggested that TCS may possess anti-obesogenic properties.

The differential expression and activity of FAS in healthy and malignant tissues, where it is upregulated in the latter [220, 221], indicate a possibly high therapeutic index. The long history of human use, and the ubiquity of TCS in consumer products, coupled with encouraging in vivo results, cements the antimicrobial as a promising candidate for chemotherapy. As noted earlier, it must be stressed that variations in the final outcome of TCS treatment largely depend on experimental setup. Moreover, limited data from animal studies suggest that in the presence of a preexisting tumor, TCS administration seems to exacerbate the condition. This observation is concerning and indeed warrants further investigation before TCS can be invested in for clinical trials.

9. Conclusion

TCS is a synthetic antimicrobial with a long history of human use. At concentrations well below those present in commercial products, data from in vitro and in vivo studies have provided evidence of adverse effects on diverse molecular pathways. Most alarmingly is TCS enhancement of malignant cell proliferation in vitro and tumor growth in vivo. On the other hand, TCS has also been shown to be protective against malignant cell growth and proliferation, possibly opening the door for its use in chemotherapy. Clearly, dose and time dependence is an important factor in determining the eventual denouement of the chemical. In spite of the numerous publications dissecting the signaling pathways responsive to TCS, it is evident that a severe paucity surrounding human-based in vivo and in vitro studies still remains today. Future studies, thus, should focus on identifying signaling molecules differentially regulated by TCS and characterize their roles in toxic or protective effects in different cell types. Insights gained from such revelations will be invaluable to possibly validate targets for drug development or devise possible TCS adjuvants or inhibitors.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

We thank the members of the Lee Laboratory for helpful advice and discussion during this work. This work was supported in part by the Brody Brothers Grant (BBE216102), NIH (1R15AG060373-01) to M-H.L., and the Saudi Government Graduate Scholarship (through King Saud University) to M.A.A.

References

R. Montville and D. W. Schaffner, “A meta-analysis of the published literature on the effectiveness of antimicrobial soaps,” Journal of Food Protection, vol. 74, no. 11, pp. 1875–1882, 2011.

V. Kjaerheim, A. Skaare, P. Barkvoll, and G. Rolla, “Antiplaque, antibacterial, and anti-inflammatory properties of triclosan mouthrinses in combination with zinc citrate or polyvinylmethylether maleic acid (PVM-MA) copolymer,” European Journal of Oral Sciences, vol. 104, no. 5-6, pp. 529–534, 1996.

V. Kjaerheim, S. M. Waaler, and G. Rolla, “Organic solvents and oils as vehicles for triclosan in mouthrinses: a clinical study,” Scandinavian Journal of Dental Research, vol. 102, no. 5, pp. 306–308, 1994.

A. B. Skaare, V. Kjaerheim, P. Barkvoll, and G. Rolla, “Does the nature of the solvent affect the anti-inflammatory capacity of triclosan? An experimental study,” Journal of Clinical Periodontology, vol. 24, no. 2, pp. 124–128, 1997.

V. Kjaerheim, S. M. Waaler, and G. Rolla, “Significance of choice of solvents for the clinical effect of triclosan-containing mouthrinses,” Scandinavian Journal of Dental Research, vol. 102, no. 4, pp. 202–205, 1994.

M. A. Alfhili, D. S. Yoon, T. A. Faten et al., “Non-ionic surfactants antagonize toxicity of potential phenolic endocrine-disrupting chemicals, including Triclosan in Caenorhabditis elegans,” Molecules and Cells, vol. 41, no. 12, pp. 1052–1060, 2018.

S. M. Waaler, G. Rölla, K. K. Skjörland, and B. Ögaard, “Effects of oral rinsing with triclosan and sodium lauryl sulfate on dental plaque formation: a pilot study,” Eropean Journal of Oral Sciences, vol. 101, no. 4, pp. 192–195, 1993.

K. Bellamy, R. Alcock, J. R. Babb, J. G. Davies, and G. A. J. Ayliffe, “A test for the assessment of ‘hygienic’ hand disinfection using rotavirus,” The Journal of Hospital Infection, vol. 24, no. 3, pp. 201–210, 1993.

J. Regos and H. R. Hitz, “Investigations on the mode of action of Triclosan, a broad spectrum antimicrobial agent,” Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskramkheiten und hygiene, Erste Abteilung Originale, Reihe A: Medizinische Mikrobiologie und Parasitologie, vol. 226, no. 3, pp. 390–401, 1974.

J. Regös, O. Zak, R. Solf, W. A. Vischer, and E. G. Weirich, “Antimicrobial spectrum of triclosan, a broad-spectrum antimicrobial agent for topical application. II. Comparison with some other antimicrobial agents,” Dermatologica, vol. 158, no. 1, pp. 72–79, 1979.

R. C. Petersen, “Triclosan antimicrobial polymers,” AIMS Molecular Science, vol. 3, no. 1, pp. 88–103, 2016.

J. V. Rodricks, J. A. Swenberg, J. F. Borzelleca, R. R. Maronpot, and A. M. Shipp, “Triclosan: a critical review of the experimental data and development of margins of safety for consumer products,” Critical Reviews in Toxicology, vol. 40, no. 5, pp. 422–484, 2010.

X. Chen, J. L. Nielsen, K. Furgal, Y. Liu, I. B. Lolas, and K. Bester, “Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions,” Chemosphere, vol. 84, no. 4, pp. 452–456, 2011.

M. Adolfsson-Erici, M. Pettersson, J. Parkkonen, and J. Sturve, “Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden,” Chemosphere, vol. 46, no. 9-10, pp. 1485–1489, 2002.

T. Geens, H. Neels, and A. Covaci, “Distribution of bisphenol-A, triclosan and n-nonylphenol in human adipose tissue, liver and brain,” Chemosphere, vol. 87, no. 7, pp. 796–802, 2012.

L. Hovander, T. Malmberg, M. Athanasiadou et al., “Identification of hydroxylated PCB metabolites and other phenolic halogenated pollutants in human blood plasma,” Archives of Environmental Contamination and Toxicology, vol. 42, no. 1, pp. 105–117, 2002.

L. W. B. Olaniyan, N. Mkwetshana, and A. I. Okoh, “Triclosan in water, implications for human and environmental health,” Springerplus, vol. 5, no. 1, p. 1639, 2016.

J. Schulze, F. H. Marquardt, F. Lyman, and C. Spitzer, “Determination of free and conjugated triclosan-1 in blood by electron capture gas liquid chromatography-2,” Journal of the American Oil Chemists' Society, vol. 52, no. 7, pp. 215–218, 1975.

L. M. Weatherly and J. A. Gosse, “Triclosan exposure, transformation, and human health effects,” Journal of Toxicology and Environmental Health Part B, Critical Reviews, vol. 20, no. 8, pp. 447–469, 2017.

Q. Wu, H. Shi, C. D. Adams, T. Timmons, and Y. Ma, “Oxidative removal of selected endocrine-disruptors and pharmaceuticals in drinking water treatment systems, and identification of degradation products of triclosan,” Science of The Total Environment, vol. 439, pp. 18–25, 2012.

C. Queckenberg, J. Meins, B. Wachall et al., “Absorption, pharmacokinetics, and safety of triclosan after dermal administration,” Antimicrobial Agents and Chemotherapy, vol. 54, no. 1, pp. 570–572, 2010.

G. Sandborgh-Englund, M. Adolfsson-Erici, G. Odham, and J. Ekstrand, “Pharmacokinetics of triclosan following oral ingestion in humans,” Journal of Toxicology and Environmental Health Part A, vol. 69, no. 20, pp. 1861–1873, 2006.

S. B. Levy, “Antibacterial household products: cause for concern,” Emerging Infectious Diseases, vol. 7, Supplement 3, pp. 512–515, 2001.

L. M. Weatherly, A. J. Nelson, J. Shim et al., “Antimicrobial agent triclosan disrupts mitochondrial structure, revealed by super-resolution microscopy, and inhibits mast cell signaling via calcium modulation,” Toxicology and Applied Pharmacology, vol. 349, pp. 39–54, 2018.

J. L. Fang, M. Vanlandingham, G. G. da Costa, and F. A. Beland, “Absorption and metabolism of triclosan after application to the skin of B6C3F1 mice,” Environmental Toxicology, vol. 31, no. 5, pp. 609–623, 2016.

T. Moss, D. Howes, and F. M. Williams, “Percutaneous penetration and dermal metabolism of triclosan (2,4, 4-trichloro-2-hydroxydiphenyl ether),” Food and Chemical Toxicology, vol. 38, no. 4, pp. 361–370, 2000.

E. Franz and S. Weidner-Strahl, “The effectiveness of topical antibacterials in acne: a double-blind clinical study,” The Journal of International Medical Research, vol. 6, no. 1, pp. 72–77, 1978.

T. W. Lee, J. C. Kim, and S. J. Hwang, “Hydrogel patches containing triclosan for acne treatment,” European Journal of Pharmaceutics and Biopharmaceutics, vol. 56, no. 3, pp. 407–412, 2003.

P. Kalliomaki and K. Kuokkanen, “Comparative study on the efficacy and tolerance of the ointments CGP433 and GP41353 in the treatment of infectious dermatitis,” Zeitschrift für Hautkrankheiten, vol. 54, no. 14, pp. 668–670, 1979.

H. Weitgasser, C. Schindléry, and V. Macarol, “A comparative multicentre trial of halometasone/triclosan cream and betamethasone dipropionate/gentamicin sulphate cream in the treatment of infected acute eczematous dermatitis,” The Journal of International Medical Research, vol. 11, Supplment 1, pp. 43–47, 1983.

A. Aliaga, A. Castells, D. Kriznik et al., “An overview of two comparative multicentre trials with halometasone/triclosan cream in acute superficial bacterial skin infections,” The Journal of International Medical Research, vol. 11, Supplement 1, pp. 53–57, 1983.

L. Huber, “Role of Klion ointment in the treatment of crural ulcer,” Therapia Hungarica, vol. 39, no. 3, pp. 148–150, 1991.

P. Barkvoll and G. Rolla, “Triclosan protects the skin against dermatitis caused by sodium lauryl sulphate exposure,” Journal of Clinical Periodontology, vol. 21, no. 10, pp. 717–719, 1994.

A. Skaare, G. Eide, B. Herlofson, and P. Barkvoll, “The effect of toothpaste containing triclosan on oral mucosal desquamation. A model study,” Journal of Clinical Periodontology, vol. 23, no. 12, pp. 1100–1103, 1996.

V. Kjaerheim, P. Barkvoll, S. M. Waaler, and G. Rolla, “Triclosan inhibits histamine-induced inflammation in human skin,” Journal of Clinical Periodontology, vol. 22, no. 6, pp. 423–426, 1995.

P. Barkvoll and G. Rolla, “Triclosan reduces the clinical symptoms of the allergic patch test reaction (APR) elicited with 1% nickel sulphate in sensitised patients,” Journal of Clinical Periodontology, vol. 22, no. 6, pp. 485–487, 1995.

V. Kjaerheim, A. Roed, P. Brodin, and G. Rolla, “Effects of triclosan on the rat phrenic nerve-diaphragm preparation,” Journal of Clinical Periodontology, vol. 22, no. 6, pp. 488–493, 1995.

F. L. Lyman and T. Furia, “Toxicology of 2, 4, 4′-trichloro-2′-hydroxy-diphenyl ether,” IMS, Industrial Medicine and Surgery, vol. 38, no. 2, pp. 64–71, 1969.

F. L. Lyman and T. E. Furia, “Toxicology of 2,4,4-trichloro-2-hydroxyphenyl ether,” IMS, Industrial Medicine and Surgery, vol. 37, no. 7, p. 546, 1968.

E. Arrhenius, L. Renberg, L. Johansson, and M. A. Zetterqvist, “Disturbance of microsomal detoxication mechanisms in liver by chlorophenol pesticides,” Chemico-Biological Interactions, vol. 18, no. 1, pp. 35–46, 1977.

A. Y. K. Chow, G. H. Hirsch, and H. S. Buttar, “Nephrotoxic and hepatotoxic effects of triclosan and chlorhexidine in rats,” Toxicology and Applied Pharmacology, vol. 42, no. 1, pp. 1–10, 1977.

L. B. Russell and C. S. Montgomery, “Use of the mouse spot test to investigate the mutagenic potential of triclosan (Irgasan DP300),” Mutation Research, vol. 79, no. 1, pp. 7–12, 1980.

T. L. Miller, D. J. Lorusso, and M. L. Deinzer, “The acute toxicity of nonachloropredioxin and 3- and 4-hydroxynonachlorodiphenyl ether in mice,” Journal of Toxicology and Environmental Health, vol. 10, no. 4-5, pp. 699–707, 1982.

T. L. Miller, D. J. Lorusso, M. L. Walsh, and M. L. Deinzer, “The acute toxicity of penta-, hexa-, and heptachlorohydroxydiphenyl ethers in mice,” Journal of Toxicology and Environmental Health, vol. 12, no. 2-3, pp. 245–253, 1983.

J. Roed-Petersen, G. Auken, and N. Hjorth, “Contact sensitivity to Irgasan DP 300,” Contact Dermatitis, vol. 1, no. 5, pp. 293-294, 1975.

B. Steinkjer and L. R. Braathen, “Contact dermatitis from triclosan (Irgasan DP 300),” Contact Dermatitis, vol. 18, no. 4, pp. 243-244, 1988.

E. Storer, K. J. Koh, and L. Warren, “Severe contact dermatitis as a result of an antiseptic bath oil,” The Australasian Journal of Dermatology, vol. 45, no. 1, pp. 73–75, 2004.

S. Veronesi, S. M. P. de Padova, D. Vanni, and M. Melino, “Contact dermatitis to triclosan,” Contact Dermatitis, vol. 15, no. 4, pp. 257-258, 1986.

C. S. M. Wong and M. H. Beck, “Allergic contact dermatitis from triclosan in antibacterial handwashes,” Contact Dermatitis, vol. 45, no. 5, p. 307, 2001.

W. A. Vischer and J. Regös, “Antimicrobial spectrum of Triclosan, a broad-spectrum antimicrobial agent for topical application,” Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskramkheiten und hygiene, Erste Abteilung Originale, Reihe A: Medizinische Mikrobiologie und Parasitologie, vol. 226, no. 3, pp. 376–389, 1974.

P. Gilbert and A. J. McBain, “Literature-based evaluation of the potential risks associated with impregnation of medical devices and implants with triclosan,” Surgical Infections, vol. 3, Supplement 1, pp. S55–S63, 2002.

R. J. Heath, J. Li, G. E. Roland, and C. O. Rock, “Inhibition of the Staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene,” The Journal of Biological Chemistry, vol. 275, no. 7, pp. 4654–4659, 2000.

J. C. Betts, A. McLaren, M. G. Lennon et al., “Signature gene expression profiles discriminate between isoniazid-, thiolactomycin-, and triclosan-treated Mycobacterium tuberculosis,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 9, pp. 2903–2913, 2003.

V. B. Srinivasan, B. B. Singh, N. Priyadarshi, N. K. Chauhan, and G. Rajamohan, “Role of novel multidrug efflux pump involved in drug resistance in Klebsiella pneumoniae,” PLoS One, vol. 9, no. 5, article e96288, 2014.

N. Gou, S. Yuan, J. Lan, C. Gao, A. N. Alshawabkeh, and A. Z. Gu, “A quantitative toxicogenomics assay reveals the evolution and nature of toxicity during the transformation of environmental pollutants,” Environmental Science & Technology, vol. 48, no. 15, pp. 8855–8863, 2014.

J. Lu, M. Jin, S. H. Nguyen et al., “Non-antibiotic antimicrobial triclosan induces multiple antibiotic resistance through genetic mutation,” Environment International, vol. 118, pp. 257–265, 2018.

B. F. G. Pycke, G. Vanermen, P. Monsieurs et al., “Toxicogenomic response of Rhodospirillum rubrum S1H to the micropollutant triclosan,” Applied and Environmental Microbiology, vol. 76, no. 11, pp. 3503–3513, 2010.

L. M. McMurry, M. Oethinger, and S. B. Levy, “Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli,” FEMS Microbiology Letters, vol. 166, no. 2, pp. 305–309, 1998.

P. Nuonming, S. Khemthong, T. Dokpikul, R. Sukchawalit, and S. Mongkolsuk, “Characterization and regulation of AcrABR, a RND-type multidrug efflux system, in Agrobacterium tumefaciens C58,” Microbiological Research, vol. 214, pp. 146–155, 2018.

T. L. Miller and M. L. Deinzer, “Effects of nonachloropredioxin and other hydroxychlorodiphenyl ethers on biological membranes,” Journal of Toxicology and Environmental Health, vol. 6, no. 1, pp. 11–25, 1980.

D. J. Lorusso, T. L. Miller, and M. L. Deinzer, “Effect of hydroxychlorodiphenyl ethers (chlorinated pre-and isopredioxins) on erythrocyte membrane adenosinetriphosphatase activity,” Journal of Toxicology and Environmental Health, vol. 8, no. 1-2, pp. 215–223, 1981.

J. Villalain, C. R. Mateo, F. J. Aranda, S. Shapiro, and V. Micol, “Membranotropic effects of the antibacterial agent Triclosan,” Archives of Biochemistry and Biophysics, vol. 390, no. 1, pp. 128–136, 2001.

J. Guillén, A. Bernabeu, S. Shapiro, and J. Villalaín, “Location and orientation of Triclosan in phospholipid model membranes,” European Biophysics Journal, vol. 33, no. 5, pp. 448–453, 2004.

L. Canesi, C. Ciacci, L. C. Lorusso et al., “Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms,” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, vol. 145, no. 3, pp. 464–472, 2007.

V. Matozzo, A. Costa Devoti, and M. G. Marin, “Immunotoxic effects of triclosan in the clam Ruditapes philippinarum,” Ecotoxicology, vol. 21, no. 1, pp. 66–74, 2012.

D. S. Yoon, Y. Choi, D. S. Cha et al., “Triclosan disrupts SKN-1/Nrf2-mediated oxidative stress response in C. elegans and human mesenchymal stem cells,” Scientific Reports, vol. 7, no. 1, article 12592, 2017.

E. Falisse, A. S. Voisin, and F. Silvestre, “Impacts of triclosan exposure on zebrafish early-life stage: toxicity and acclimation mechanisms,” Aquatic Toxicology, vol. 189, pp. 97–107, 2017.

C. Riva, S. Cristoni, and A. Binelli, “Effects of triclosan in the freshwater mussel Dreissena polymorpha: a proteomic investigation,” Aquatic Toxicology, vol. 118-119, pp. 62–71, 2012.

H. Babich and J. P. Babich, “Sodium lauryl sulfate and triclosan: in vitro cytotoxicity studies with gingival cells,” Toxicology Letters, vol. 91, no. 3, pp. 189–196, 1997.

H. L. Zuckerbraun, H. Babich, R. May, and M. C. Sinensky, “Triclosan: cytotoxicity, mode of action, and induction of apoptosis in human gingival cells in vitro,” European Journal of Oral Sciences, vol. 106, Part 1, no. 2, pp. 628–636, 1998.

E. Honkisz, D. Zieba-Przybylska, and A. K. Wojtowicz, “The effect of triclosan on hormone secretion and viability of human choriocarcinoma JEG-3 cells,” Reproductive Toxicology, vol. 34, no. 3, pp. 385–392, 2012.

T. Winitthana, S. Lawanprasert, and P. Chanvorachote, “Triclosan potentiates epithelial-to-mesenchymal transition in anoikis-resistant human lung cancer cells,” PLoS One, vol. 9, no. 10, article e110851, 2014.

Y. S. Kim, H. W. Seo, M. H. Lee, D. K. Kim, H. Jeon, and D. S. Cha, “Protocatechuic acid extends lifespan and increases stress resistance in Caenorhabditis elegans,” Archives of Pharmacal Research, vol. 37, no. 2, pp. 245–252, 2014.

H. R. Lee, K. A. Hwang, K. H. Nam, H. C. Kim, and K. C. Choi, “Progression of breast cancer cells was enhanced by endocrine-disrupting chemicals, triclosan and octylphenol, via an estrogen receptor-dependent signaling pathway in cellular and mouse xenograft models,” Chemical Research in Toxicology, vol. 27, no. 5, pp. 834–842, 2014.

S. H. Kim, K. A. Hwang, and K. C. Choi, “Treatment with kaempferol suppresses breast cancer cell growth caused by estrogen and triclosan in cellular and xenograft breast cancer models,” The Journal of Nutritional Biochemistry, vol. 28, pp. 70–82, 2016.

G. A. Lee, K. C. Choi, and K. A. Hwang, “Treatment with phytoestrogens reversed triclosan and bisphenol A-induced anti-apoptosis in breast cancer cells,” Biomolecules & Therapeutics, vol. 26, no. 5, pp. 503–511, 2018.

S. H. Kim, K. A. Hwang, S. M. Shim, and K. C. Choi, “Growth and migration of LNCaP prostate cancer cells are promoted by triclosan and benzophenone-1 via an androgen receptor signaling pathway,” Environmental Toxicology and Pharmacology, vol. 39, no. 2, pp. 568–576, 2015.

N. Zhang, W. Wang, W. Li et al., “Inhibition of 11β-HSD2 expression by triclosan via induction of apoptosis in human placental syncytiotrophoblasts,” The Journal of Clinical Endocrinology and Metabolism, vol. 100, no. 4, pp. E542–E549, 2015.

B. K. Park, E. L. T. Gonzales, S. M. Yang, M. Bang, C. S. Choi, and C. Y. Shin, “Effects of triclosan on neural stem cell viability and survival,” Biomolecules & Therapeutics, vol. 24, no. 1, pp. 99–107, 2016.

K. A. Szychowski, A. M. Sitarz, and A. K. Wojtowicz, “Triclosan induces Fas receptor-dependent apoptosis in mouse neocortical neurons in vitro,” Neuroscience, vol. 284, pp. 192–201, 2015.

K. A. Szychowski, A. Wnuk, M. Kajta, and A. K. Wojtowicz, “Triclosan activates aryl hydrocarbon receptor (AhR)-dependent apoptosis and affects Cyp1a1 and Cyp1b1 expression in mouse neocortical neurons,” Environmental Research, vol. 151, pp. 106–114, 2016.

K. A. Szychowski, A. Wnuk, J. Rzemieniec, M. Kajta, T. Leszczyńska, and A. K. Wójtowicz, “Triclosan-evoked neurotoxicity involves NMDAR subunits with the specific role of GluN2A in caspase-3-dependent apoptosis,” Molecular Neurobiology, vol. 56, no. 1, pp. 1–12, 2019.

J. T. Kwon, Y. S. Yang, M. S. Kang et al., “Pulmonary toxicity screening of triclosan in rats after intratracheal instillation,” The Journal of Toxicological Sciences, vol. 38, no. 3, pp. 471–475, 2013.

Y. Wu, F. A. Beland, S. Chen, and J. L. Fang, “Extracellular signal-regulated kinases 1/2 and Akt contribute to triclosan-stimulated proliferation of JB6 Cl 41-5a cells,” Archives of Toxicology, vol. 89, no. 8, pp. 1297–1311, 2015.

X. Chen, B. Xu, X. Han et al., “The effects of triclosan on pluripotency factors and development of mouse embryonic stem cells and zebrafish,” Archives of Toxicology, vol. 89, no. 4, pp. 635–646, 2015.

J. Kim, H. Oh, B. Ryu et al., “Triclosan affects axon formation in the neural development stages of zebrafish embryos (Danio rerio),” Environmental Pollution, vol. 236, pp. 304–312, 2018.

J. Liu, L. Sun, H. Zhang et al., “Response mechanisms to joint exposure of triclosan and its chlorinated derivatives on zebrafish (Danio rerio) behavior,” Chemosphere, vol. 193, pp. 820–832, 2018.

A. Binelli, D. Cogni, M. Parolini, C. Riva, and A. Provini, “Cytotoxic and genotoxic effects of in vitro exposure to triclosan and trimethoprim on zebra mussel (Dreissena polymorpha) hemocytes,” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, vol. 150, no. 1, pp. 50–56, 2009.

A. Binelli, D. Cogni, M. Parolini, C. Riva, and A. Provini, “In vivo experiments for the evaluation of genotoxic and cytotoxic effects of Triclosan in Zebra mussel hemocytes,” Aquatic Toxicology, vol. 91, no. 3, pp. 238–244, 2009.

M. Gonzalez-Pleiter, C. Rioboo, M. Reguera et al., “Calcium mediates the cellular response of Chlamydomonas reinhardtii to the emerging aquatic pollutant Triclosan,” Aquatic Toxicology, vol. 186, pp. 50–66, 2017.

E. Movahed, G. M. Y. Tan, K. Munusamy et al., “Triclosan demonstrates synergic effect with amphotericin B and fluconazole and induces apoptosis-like cell death in Cryptococcus neoformans,” Frontiers in Microbiology, vol. 7, p. 360, 2016.

D. J. Watkins, K. K. Ferguson, L. V. Anzalota Del Toro, A. N. Alshawabkeh, J. F. Cordero, and J. D. Meeker, “Associations between urinary phenol and paraben concentrations and markers of oxidative stress and inflammation among pregnant women in Puerto Rico,” International Journal of Hygiene and Environmental Health, vol. 218, no. 2, pp. 212–219, 2015.

Y. Lv, C. Rui, Y. Dai et al., “Exposure of children to BPA through dust and the association of urinary BPA and triclosan with oxidative stress in Guangzhou, China,” Environmental Science: Processes & Impacts, vol. 18, no. 12, pp. 1492–1499, 2016.

B. A. Rocha, A. G. Asimakopoulos, M. Honda et al., “Advanced data mining approaches in the assessment of urinary concentrations of bisphenols, chlorophenols, parabens and benzophenones in Brazilian children and their association to DNA damage,” Environment International, vol. 116, pp. 269–277, 2018.

A. P. Iyer, J. Xue, M. Honda et al., “Urinary levels of triclosan and triclocarban in several Asian countries, Greece and the USA: association with oxidative stress,” Environmental Research, vol. 160, pp. 91–96, 2018.

B. Bukowska, P. Wieteska, M. Kwiatkowska, P. Sicinska, and J. Michalowicz, “Evaluation of the effect of 2,4-dichlorophenol on oxidative parameters and viability of human blood mononuclear cells (in vitro),” Human & Experimental Toxicology, vol. 35, no. 7, pp. 775–784, 2016.

P. Zhang, M. Yang, L. Zeng, and C. Liu, “P38/TRHr-dependent regulation of TPO in thyroid cells contributes to the hypothyroidism of triclosan-treated rats,” Cellular Physiology and Biochemistry, vol. 45, no. 4, pp. 1303–1315, 2018.

S. Vandhana, K. Coral, U. Jayanthi, P. R. Deepa, and S. Krishnakumar, “Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors,” Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, vol. 1831, no. 9, pp. 1458–1466, 2013.

M. Battino, M. S. Ferreiro, D. Fattorini, and P. Bullon, “In vitro antioxidant activities of mouthrinses and their components,” Journal of Clinical Periodontology, vol. 29, no. 5, pp. 462–467, 2002.

C. Ajao, M. A. Andersson, V. V. Teplova et al., “Mitochondrial toxicity of triclosan on mammalian cells,” Toxicology Reports, vol. 2, pp. 624–637, 2015.

L. M. Weatherly, J. Shim, H. N. Hashmi, R. H. Kennedy, S. T. Hess, and J. A. Gosse, “Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes,” Journal of Applied Toxicology, vol. 36, no. 6, pp. 777–789, 2016.

I. Tamura, Y. Kanbara, M. Saito et al., “Triclosan, an antibacterial agent, increases intracellular Zn²⁺ concentration in rat thymocytes: its relation to oxidative stress,” Chemosphere, vol. 86, no. 1, pp. 70–75, 2012.

M. F. Yueh, K. Taniguchi, S. Chen et al., “The commonly used antimicrobial additive triclosan is a liver tumor promoter,” Proceedings of the National Academy of Sciences of the United States of America, vol. 111, no. 48, pp. 17200–17205, 2014.

M. A. Riad, M. M. Abd-Rabo, S. A. Abd El Aziz, A. M. El Behairy, and M. M. Badawy, “Reproductive toxic impact of subchronic treatment with combined butylparaben and triclosan in weanling male rats,” Journal of Biochemical and Molecular Toxicology, vol. 32, no. 3, article e22037, 2018.

A. T. Mohammed, A. A.-R. Mohamed, and H. Ali, “Pulmonary apoptotic and oxidative damaging effects of Triclosan alone or in combination with fluoride in Sprague Dawley rats,” Acta Histochemica, vol. 119, no. 4, pp. 357–363, 2017.

Z. Wang, X. Li, and J. E. Klaunig, “Investigation of the mechanism of triclosan induced mouse liver tumors,” Regulatory Toxicology and Pharmacology, vol. 86, pp. 137–147, 2017.

K. A. Lenz, C. Pattison, and H. Ma, “Triclosan (TCS) and triclocarban (TCC) induce systemic toxic effects in a model organism the nematode Caenorhabditis elegans,” Environmental Pollution, vol. 231, Part 1, pp. 462–470, 2017.

D. Lin, Q. Zhou, X. Xie, and Y. Liu, “Potential biochemical and genetic toxicity of triclosan as an emerging pollutant on earthworms (Eisenia fetida),” Chemosphere, vol. 81, no. 10, pp. 1328–1333, 2010.

D. Lin, X. Xie, Q. Zhou, and Y. Liu, “Biochemical and genotoxic effect of triclosan on earthworms (Eisenia fetida) using contact and soil tests,” Environmental Toxicology, vol. 27, no. 7, pp. 385–392, 2012.

X. Wang, Z. Liu, W. Wang et al., “Assessment of toxic effects of triclosan on the terrestrial snail (Achatina fulica),” Chemosphere, vol. 108, pp. 225–230, 2014.

Z. Zhou, J. Yang, and K. M. Chan, “Toxic effects of triclosan on a zebrafish (Danio rerio) liver cell line, ZFL,” Aquatic Toxicology, vol. 191, pp. 175–188, 2017.

Y. Peng, Y. Luo, X. P. Nie, W. Liao, Y. F. Yang, and G. G. Ying, “Toxic effects of triclosan on the detoxification system and breeding of Daphnia magna,” Ecotoxicology, vol. 22, no. 9, pp. 1384–1394, 2013.

V. Kovacevic, A. J. Simpson, and M. J. Simpson, “(1)H NMR-based metabolomics of Daphnia magna responses after sub-lethal exposure to triclosan, carbamazepine and ibuprofen,” Comparative Biochemistry and Physiology Part D, Genomics & Proteomics, vol. 19, pp. 199–210, 2016.

H. I. Falfushynska, L. L. Gnatyshyna, O. Y. Osadchuk et al., “Diversity of the molecular responses to separate wastewater effluents in freshwater mussels,” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, vol. 164, pp. 51–58, 2014.

J. C. Park, J. Han, M. C. Lee, J. S. Seo, and J. S. Lee, “Effects of triclosan (TCS) on fecundity, the antioxidant system, and oxidative stress-mediated gene expression in the copepod Tigriopus japonicus,” Aquatic Toxicology, vol. 189, pp. 16–24, 2017.

P. Ku, X. Wu, X. Nie et al., “Effects of triclosan on the detoxification system in the yellow catfish (Pelteobagrus fulvidraco): expressions of CYP and GST genes and corresponding enzyme activity in phase I, II and antioxidant system,” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, vol. 166, pp. 105–114, 2014.

P. Banerjee, T. K. Dey, S. Sarkar, S. Swarnakar, A. Mukhopadhyay, and S. Ghosh, “Treatment of cosmetic effluent in different configurations of ceramic UF membrane based bioreactor: toxicity evaluation of the untreated and treated wastewater using catfish (Heteropneustes fossilis),” Chemosphere, vol. 146, pp. 133–144, 2016.

F. Wang, R. Xu, F. Zheng, and H. Liu, “Effects of triclosan on acute toxicity, genetic toxicity and oxidative stress in goldfish (Carassius auratus),” Experimental Animals, vol. 67, no. 2, pp. 219–227, 2018.

C. Li, R. Qu, J. Chen et al., “The pH-dependent toxicity of triclosan to five aquatic organisms (Daphnia magna, Photobacterium phosphoreum, Danio rerio, Limnodrilus hoffmeisteri, and Carassius auratus),” Environmental Science and Pollution Research International, vol. 25, no. 10, pp. 9636–9646, 2018.

J. Han, E. J. Won, U. K. Hwang, I. C. Kim, J. H. Yim, and J. S. Lee, “Triclosan (TCS) and Triclocarban (TCC) cause lifespan reduction and reproductive impairment through oxidative stress-mediated expression of the defensome in the monogonont rotifer (Brachionus koreanus),” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, vol. 185-186, pp. 131–137, 2016.

L. Chai, A. Chen, P. Luo, H. Zhao, and H. Wang, “Histopathological changes and lipid metabolism in the liver of Bufo gargarizans tadpoles exposed to Triclosan,” Chemosphere, vol. 182, pp. 255–266, 2017.

D. Martins, M. S. Monteiro, A. M. V. M. Soares, and C. Quintaneiro, “Effects of 4-MBC and triclosan in embryos of the frog Pelophylax perezi,” Chemosphere, vol. 178, pp. 325–332, 2017.

M. Sendra, M. G. Pintado-Herrera, G. V. Aguirre-Martinez et al., “Are the TiO2 NPs a “Trojan horse” for personal care products (PCPs) in the clam Ruditapes philippinarum?” Chemosphere, vol. 185, pp. 192–204, 2017.

E. Capkin, T. Ozcelep, S. Kayis, and I. Altinok, “Antimicrobial agents, triclosan, chloroxylenol, methylisothiazolinone and borax, used in cleaning had genotoxic and histopathologic effects on rainbow trout,” Chemosphere, vol. 182, pp. 720–729, 2017.

A. C. Almeida, T. Gomes, K. Langford, K. V. Thomas, and K. E. Tollefsen, “Oxidative stress in the algae Chlamydomonas reinhardtii exposed to biocides,” Aquatic Toxicology, vol. 189, pp. 50–59, 2017.

C. G. Pan, F. J. Peng, W. J. Shi, L. X. Hu, X. D. Wei, and G. G. Ying, “Triclosan-induced transcriptional and biochemical alterations in the freshwater green algae Chlamydomonas reinhardtii,” Ecotoxicology and Environmental Safety, vol. 148, pp. 393–401, 2018.

J. Hurtado-Gallego, K. Martin-Betancor, I. Rodea-Palomares, F. Leganes, R. Rosal, and F. Fernandez-Pinas, “Two novel cyanobacterial bioluminescent whole-cell bioreporters based on superoxide dismutases MnSod and FeSod to detect superoxide anion,” Chemosphere, vol. 201, pp. 772–779, 2018.

O. Culic, V. Erakovic, and M. J. Parnham, “Anti-inflammatory effects of macrolide antibiotics,” European Journal of Pharmacology, vol. 429, no. 1-3, pp. 209–229, 2001.

Y. Iino, M. Toriyama, Y. Natori, K. Kudo, and A. Yuo, “Erythromycin inhibition of lipopolysaccharide-stimulated tumor necrosis factor alpha production by human monocytes in vitro,” The Annals of Otology, Rhinology & Laryngology. Supplement, vol. 101, 10 Supplement, pp. 16–20, 1992.

O. M. Korzeniowski, “Effects of antibiotics on the mammalian immune system,” Infectious Disease Clinics of North America, vol. 3, no. 3, pp. 469–478, 1989.

S. M. Uriarte, R. E. Molestina, R. D. Miller et al., “Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 7, pp. 2538–2543, 2004.

B. Van Vlem, R. Vanholder, P. De Paepe, S. Ringoir, and D. Vogelaers, “Immunomodulating effects of antibiotics: literature review,” Infection, vol. 24, no. 4, pp. 275–291, 1996.

A. Gaffar, D. Scherl, J. Afflitto, and E. J. Coleman, “The effect of triclosan on mediators of gingival inflammation,” Journal of Clinical Periodontology, vol. 22, no. 6, pp. 480–484, 1995.

T. Modéer, A. Bengisson, and G. Rölla, “Triclosan reduces prostaglandin biosynthesis in human gingival fibroblasts challenged with interleukin-1 in vitro,” Journal of Clinical Periodontology, vol. 23, no. 10, pp. 927–933, 1996.

A. B. Skaare, G. Rolla, and P. Barkvoll, “The influence of triclosan, zinc or propylene glycol on oral mucosa exposed to sodium lauryl sulphate,” European Journal of Oral Sciences, vol. 105, Part 2, no. 5, pp. 527–533, 1997.

T. M. Hernandez-Richter, M. W. Wichmann, W. Schrodl, M. K. Angele, K. Heinritzi, and F. W. Schildberg, “The acute phase response following implantation of triclosan-bonded vascular prostheses,” Clinical and Experimental Medicine, vol. 1, no. 1, pp. 35–41, 2001.

S. Kumar, “Little difference between triclosan and stannous fluoride dentifrices on gingival inflammation,” Evidence-Based Dentistry, vol. 16, no. 1, pp. 13-14, 2015.

D. K. Suresh, K. L. Vandana, and D. S. Mehta, “Intracrevicular application of 0.3% Flurbiprofen gel and 0.3% Triclosan gel as anti inflammatory agent. A comparative clinical study,” Indian Journal of Dental Research, vol. 12, no. 2, pp. 105–112, 2001.

F. V. Ribeiro, M. Z. Casati, R. C. Casarin et al., “Impact of a triclosan-containing toothpaste during the progression of experimental peri-implant mucositis: clinical parameters and local pattern of osteo-immunoinflammatory mediators in peri-implant fluid,” Journal of Periodontology, vol. 89, no. 2, pp. 203–212, 2018.

M. Mustafa, M. Bakhiet, B. Wondimu, and T. Modeer, “Effect of triclosan on interferon-gamma production and major histocompatibility complex class II expression in human gingival fibroblasts,” Journal of Clinical Periodontology, vol. 27, no. 10, pp. 733–737, 2000.

M. Mustafa, B. Wondimu, M. Ibrahim, and T. Modeer, “Effect of triclosan on interleukin-1beta production in human gingival fibroblasts challenged with tumor necrosis factor alpha,” European Journal of Oral Sciences, vol. 106, Part 1, no. 2, pp. 637–643, 1998.

M. Mustafa, B. Wondimu, T. Yucel-Lindberg, A. T. Kats-Hallstrom, A. S. Jonsson, and T. Modeer, “Triclosan reduces microsomal prostaglandin E synthase-1 expression in human gingival fibroblasts,” Journal of Clinical Periodontology, vol. 32, no. 1, pp. 6–11, 2005.

M. Mustafa, B. Wondimu, K. Hultenby, T. Yucel-Lindberg, and T. Modeer, “Uptake, distribution and release of 14C-triclosan in human gingival fibroblasts,” Journal of Pharmaceutical Sciences, vol. 92, no. 8, pp. 1648–1653, 2003.

M. A. Wallet, N. L. Calderon, T. R. Alonso et al., “Triclosan alters antimicrobial and inflammatory responses of epithelial cells,” Oral Diseases, vol. 19, no. 3, pp. 296–302, 2013.

K. G. Neiva, N. L. Calderon, T. R. Alonso, F. Panagakos, and S. M. Wallet, “Type 1 diabetes-associated TLR responsiveness of oral epithelial cells,” Journal of Dental Research, vol. 93, no. 2, pp. 169–174, 2014.

N. B. Marshall, E. Lukomska, A. P. Nayak, C. M. Long, J. M. Hettick, and S. E. Anderson, “Topical application of the anti-microbial chemical triclosan induces immunomodulatory responses through the S100A8/A9-TLR4 pathway,” Journal of Immunotoxicology, vol. 14, no. 1, pp. 50–59, 2017.

V. M. Barnes, T. Xu, E. Shimizu et al., “Triclosan blocks MMP-13 expression in hormone-stimulated osteoblasts,” Journal of Periodontology, vol. 84, no. 11, pp. 1–9, 2013.

B. A. Pancer, D. Kott, J. V. Sugai et al., “Effects of triclosan on host response and microbial biomarkers during experimental gingivitis,” Journal of Clinical Periodontology, vol. 43, no. 5, pp. 435–444, 2016.

M. P. Cullinan, J. E. Palmer, M. J. Faddy et al., “The influence of triclosan on biomarkers of cardiovascular risk in patients in the Cardiovascular and Periodontal Study (CAPS): a randomized controlled trial,” Journal of Periodontology, vol. 86, no. 7, pp. 847–855, 2015.

S. Hessam, M. Sand, N. M. Meier, T. Gambichler, L. Scholl, and F. G. Bechara, “Combination of oral zinc gluconate and topical triclosan: an anti-inflammatory treatment modality for initial hidradenitis suppurativa,” Journal of Dermatological Science, vol. 84, no. 2, pp. 197–202, 2016.

P. A. Cadieux, B. H. Chew, B. E. Knudsen et al., “Triclosan loaded ureteral stents decrease proteus mirabilis 296 infection in a rabbit urinary tract infection model,” The Journal of Urology, vol. 175, no. 6, pp. 2331–2335, 2006.

C. N. Elwood, B. H. Chew, S. Seney, J. Jass, J. D. Denstedt, and P. A. Cadieux, “Triclosan inhibits uropathogenic Escherichia coli-stimulated tumor necrosis factor-alpha secretion in T24 bladder cells in vitro,” Journal of Endourology, vol. 21, no. 10, pp. 1217–1222, 2007.

S. P. Barros, S. Wirojchanasak, D. A. Barrow, F. S. Panagakos, W. Devizio, and S. Offenbacher, “Triclosan inhibition of acute and chronic inflammatory gene pathways,” Journal of Clinical Periodontology, vol. 37, no. 5, pp. 412–418, 2010.

F. Udoji, T. Martin, R. Etherton, and M. M. Whalen, “Immunosuppressive effects of triclosan, nonylphenol, and DDT on human natural killer cells in vitro,” Journal of Immunotoxicology, vol. 7, no. 3, pp. 205–212, 2010.

L. Pavez, N. Tobar, C. Chacon et al., “Chitosan-triclosan particles modulate inflammatory signaling in gingival fibroblasts,” Journal of Periodontal Research, vol. 53, no. 2, pp. 232–239, 2018.

S. Sharma, T. N. C. Ramya, A. Surolia, and N. Surolia, “Triclosan as a systemic antibacterial agent in a mouse model of acute bacterial challenge,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 12, pp. 3859–3866, 2003.

H. Yang, W. Wang, K. A. Romano et al., “A common antimicrobial additive increases colonic inflammation and colitis-associated colon tumorigenesis in mice,” Science Translational Medicine, vol. 10, no. 443, article eaan4116, 2018.

A. Kanetoshi, E. Katsura, H. Ogawa, T. Ohyama, H. Kaneshima, and T. Miura, “Acute toxicity, percutaneous absorption and effects on hepatic mixed function oxidase activities of 2,4,4-trichloro-2-hydroxydiphenyl ether (Irgasan DP300) and its chlorinated derivatives,” Archives of Environmental Contamination and Toxicology, vol. 23, no. 1, pp. 91–98, 1992.

H. Ma, L. Zheng, Y. Li et al., “Triclosan reduces the levels of global DNA methylation in HepG2 cells,” Chemosphere, vol. 90, no. 3, pp. 1023–1029, 2013.

F. Rodrigues, M. Lehmann, V. S. do Amaral, M. L. Reguly, and H. H. R. de Andrade, “Genotoxicity of three mouthwash products, Cepacol®, Periogard®, and Plax®, in the Drosophila wing-spot test,” Environmental and Molecular Mutagenesis, vol. 48, no. 8, pp. 644–649, 2007.

F. Chevillot, M. Guyot, M. Desrosiers et al., “Accumulation and sublethal effects of triclosan and its transformation product methyl-triclosan in the earthworm Eisenia andrei exposed to environmental concentrations in an artificial soil,” Environmental Toxicology and Chemistry, vol. 37, no. 7, pp. 1940–1948, 2018.

P. Martinez-Paz, M. Morales, J. L. Martinez-Guitarte, and G. Morcillo, “Genotoxic effects of environmental endocrine disruptors on the aquatic insect Chironomus riparius evaluated using the comet assay,” Mutation Research, vol. 758, no. 1-2, pp. 41–47, 2013.

A. R. R. Silva, D. N. Cardoso, A. Cruz et al., “Ecotoxicity and genotoxicity of a binary combination of triclosan and carbendazim to Daphnia magna,” Ecotoxicology and Environmental Safety, vol. 115, pp. 279–290, 2015.

X. Xu, Y. Lu, D. Zhang et al., “Toxic assessment of triclosan and triclocarban on Artemia salina,” Bulletin of Environmental Contamination and Toxicology, vol. 95, no. 6, pp. 728–733, 2015.

L. Gao, T. Yuan, P. Cheng et al., “Effects of triclosan and triclocarban on the growth inhibition, cell viability, genotoxicity and multixenobiotic resistance responses of Tetrahymena thermophila,” Chemosphere, vol. 139, pp. 434–440, 2015.

O. Herrero, J. M. Perez Martin, P. Fernandez Freire, L. Carvajal Lopez, A. Peropadre, and M. J. Hazen, “Toxicological evaluation of three contaminants of emerging concern by use of the Allium cepa test,” Mutation Research, vol. 743, no. 1-2, pp. 20–24, 2012.

T. Y. Doktorova, G. Ates, M. Vinken, T. Vanhaecke, and V. Rogiers, “Way forward in case of a false positive in vitro genotoxicity result for a cosmetic substance?” Toxicology In Vitro, vol. 28, no. 1, pp. 54–59, 2014.

K. Z. Sanidad, H. Xiao, and G. Zhang, “Triclosan, a common antimicrobial ingredient, on gut microbiota and gut health,” Gut Microbes, pp. 1–4, 2018.

H. N. Bhargava and P. A. Leonard, “Triclosan: applications and safety,” American Journal of Infection Control, vol. 24, no. 3, pp. 209–218, 1996.

R. U. Halden, A. E. Lindeman, A. E. Aiello et al., “The Florence statement on triclosan and triclocarban,” Environmental Health Perspectives, vol. 125, no. 6, article 064501, 2017.

I. J. Uings and S. N. Farrow, “Cell receptors and cell signalling,” Molecular Pathology, vol. 53, no. 6, pp. 295–299, 2000.

G. A. Lee, K. A. Hwang, and K. C. Choi, “Inhibitory effects of 3,3′-diindolylmethane on epithelial-mesenchymal transition induced by endocrine disrupting chemicals in cellular and xenograft mouse models of breast cancer,” Food and Chemical Toxicology, vol. 109, Part 1, pp. 284–295, 2017.

H. Huang, G. Du, W. Zhang et al., “The in vitro estrogenic activities of triclosan and triclocarban,” Journal of Applied Toxicology, vol. 34, no. 9, pp. 1060–1067, 2014.

N. D. Henry and P. A. Fair, “Comparison of in vitro cytotoxicity, estrogenicity and anti-estrogenicity of triclosan, perfluorooctane sulfonate and perfluorooctanoic acid,” Journal of Applied Toxicology, vol. 33, no. 4, pp. 265–272, 2013.

T. E. Stoker, E. K. Gibson, and L. M. Zorrilla, “Triclosan exposure modulates estrogen-dependent responses in the female Wistar rat,” Toxicological Sciences, vol. 117, no. 1, pp. 45–53, 2010.

G. W. Louis, D. R. Hallinger, and T. E. Stoker, “The effect of triclosan on the uterotrophic response to extended doses of ethinyl estradiol in the weanling rat,” Reproductive Toxicology, vol. 36, pp. 71–77, 2013.

M. O. James, W. Li, D. P. Summerlot, L. Rowland-Faux, and C. E. Wood, “Triclosan is a potent inhibitor of estradiol and estrone sulfonation in sheep placenta,” Environment International, vol. 36, no. 8, pp. 942–949, 2010.

E. M. Jung, B. S. An, K. C. Choi, and E. B. Jeung, “Potential estrogenic activity of triclosan in the uterus of immature rats and rat pituitary GH3 cells,” Toxicology Letters, vol. 208, no. 2, pp. 142–148, 2012.

H. Serra, F. Brion, J. M. Porcher, H. Budzinski, and S. Ait-Aissa, “Triclosan lacks anti-estrogenic effects in zebrafish cells but modulates estrogen response in zebrafish embryos,” International Journal of Molecular Sciences, vol. 19, no. 4, p. 1175, 2018.

S. Miyagawa, A. Lange, I. Hirakawa et al., “Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor,” Environmental Science & Technology, vol. 48, no. 9, pp. 5254–5263, 2014.

J. Chen, K. C. Ahn, N. A. Gee, S. J. Gee, B. D. Hammock, and B. L. Lasley, “Antiandrogenic properties of parabens and other phenolic containing small molecules in personal care products,” Toxicology and Applied Pharmacology, vol. 221, no. 3, pp. 278–284, 2007.

V. Christen, P. Crettaz, A. Oberli-Schrammli, and K. Fent, “Some flame retardants and the antimicrobials triclosan and triclocarban enhance the androgenic activity in vitro,” Chemosphere, vol. 81, no. 10, pp. 1245–1252, 2010.

K. C. Ahn, B. Zhao, J. Chen et al., “In vitro biologic activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens,” Environmental Health Perspectives, vol. 116, no. 9, pp. 1203–1210, 2008.

R. H. Gee, A. Charles, N. Taylor, and P. D. Darbre, “Oestrogenic and androgenic activity of triclosan in breast cancer cells,” Journal of Applied Toxicology, vol. 28, no. 1, pp. 78–91, 2008.

A. L. Forgacs, Q. Ding, R. G. Jaremba, I. T. Huhtaniemi, N. A. Rahman, and T. R. Zacharewski, “BLTK1 murine Leydig cells: a novel steroidogenic model for evaluating the effects of reproductive and developmental toxicants,” Toxicological Sciences, vol. 127, no. 2, pp. 391–402, 2012.

W. T. Farmer, G. W. Louis, A. R. Buckalew, D. R. Hallinger, and T. E. Stoker, “Evaluation of triclosan in the Hershberger and H295R steroidogenesis assays,” Toxicology Letters, vol. 291, pp. 194–199, 2018.

Y. Wu, Q. Wu, F. A. Beland, P. Ge, M. G. Manjanatha, and J. L. Fang, “Differential effects of triclosan on the activation of mouse and human peroxisome proliferator-activated receptor alpha,” Toxicology Letters, vol. 231, no. 1, pp. 17–28, 2014.

P. Xia, X. Zhang, Y. Xie, M. Guan, D. L. Villeneuve, and H. Yu, “Functional toxicogenomic assessment of triclosan in human HepG2 cells using genome-wide CRISPR-Cas9 screening,” Environmental Science & Technology, vol. 50, no. 19, pp. 10682–10692, 2016.

D. E. Haggard, P. D. Noyes, K. M. Waters, and R. L. Tanguay, “Phenotypically anchored transcriptome profiling of developmental exposure to the antimicrobial agent, triclosan, reveals hepatotoxicity in embryonic zebrafish,” Toxicology and Applied Pharmacology, vol. 308, pp. 32–45, 2016.

J. Guo, S. Ito, H. T. Nguyen et al., “Effects of prenatal exposure to triclosan on the liver transcriptome in chicken embryos,” Toxicology and Applied Pharmacology, vol. 347, pp. 23–32, 2018.

X. Cao, X. Hua, X. Wang, and L. Chen, “Exposure of pregnant mice to triclosan impairs placental development and nutrient transport,” Scientific Reports, vol. 7, no. 1, article 44803, 2017.

X. Hua, X. Y. Cao, X. L. Wang, P. Sun, and L. Chen, “Exposure of pregnant mice to triclosan causes insulin resistance via thyroxine reduction,” Toxicological Sciences, vol. 160, no. 1, pp. 150–160, 2017.

W. T. Festuccia, P. G. Blanchard, T. Belchior et al., “PPARγ activation attenuates glucose intolerance induced by mTOR inhibition with rapamycin in rats,” American Journal of Physiology Endocrinology and Metabolism, vol. 306, no. 9, pp. E1046–E1054, 2014.

K. B. Paul, J. T. Thompson, S. O. Simmons, J. P. Vanden Heuvel, and K. M. Crofton, “Evidence for triclosan-induced activation of human and rodent xenobiotic nuclear receptors,” Toxicology In Vitro, vol. 27, no. 7, pp. 2049–2060, 2013.

D. E. Clapham, “Calcium signaling,” Cell, vol. 131, no. 6, pp. 1047–1058, 2007.

G. Cherednichenko, R. Zhang, R. A. Bannister et al., “Triclosan impairs excitation-contraction coupling and Ca2+ dynamics in striated muscle,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 35, pp. 14158–14163, 2012.

E. B. Fritsch, R. E. Connon, I. Werner et al., “Triclosan impairs swimming behavior and alters expression of excitation-contraction coupling proteins in fathead minnow (Pimephales promelas),” Environmental Science & Technology, vol. 47, no. 4, pp. 2008–2017, 2013.

T. Kawanai, “Triclosan, an environmental pollutant from health care products, evokes charybdotoxin-sensitive hyperpolarization in rat thymocytes,” Environmental Toxicology and Pharmacology, vol. 32, no. 3, pp. 417–422, 2011.

V. V. Teplova, K. N. Belosludtsev, and A. G. Kruglov, “Mechanism of triclosan toxicity: mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation,” Toxicology Letters, vol. 275, pp. 108–117, 2017.

G. McDonnell and A. D. Russell, “Antiseptics and disinfectants: activity, action, and resistance,” Clinical Microbiology Reviews, vol. 12, no. 1, pp. 147–179, 1999.

L. M. McMurry, M. Oethinger, and S. B. Levy, “Triclosan targets lipid synthesis,” Nature, vol. 394, no. 6693, pp. 531-532, 1998.

J. G. Beeson, P. A. Winstanley, G. I. McFadden, and G. V. Brown, “New agents to combat malaria,” Nature Medicine, vol. 7, no. 2, pp. 149-150, 2001.

H. H. Lee, J. Yun, J. Moon et al., “Crystallization and preliminary X-ray crystallographic analysis of enoyl-acyl carrier protein reductase from Helicobacter pylori,” Acta Crystallographica Section D, Biological Crystallography, vol. 58, no. 6, pp. 1071–1073, 2002.

J. Marcinkeviciene, W. Jiang, L. M. Kopcho, G. Locke, Y. Luo, and R. A. Copeland, “Enoyl-ACP reductase (FabI) of Haemophilus influenzae: steady-state kinetic mechanism and inhibition by triclosan and hexachlorophene,” Archives of Biochemistry and Biophysics, vol. 390, no. 1, pp. 101–108, 2001.

R. McLeod, S. P. Muench, J. B. Rafferty et al., “Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I,” International Journal for Parasitology, vol. 31, no. 2, pp. 109–113, 2001.

S. L. Parikh, G. Xiao, and P. J. Tonge, “Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid,” Biochemistry, vol. 39, no. 26, pp. 7645–7650, 2000.

C. W. Roberts, R. McLeod, D. W. Rice, M. Ginger, M. L. Chance, and L. J. Goad, “Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa,” Molecular and Biochemical Parasitology, vol. 126, no. 2, pp. 129–142, 2003.

N. Surolia and A. Surolia, “Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum,” Nature Medicine, vol. 7, no. 2, pp. 167–173, 2001.

S. Lu and M. C. Archer, “Fatty acid synthase is a potential molecular target for the chemoprevention of breast cancer,” Carcinogenesis, vol. 26, no. 1, pp. 153–157, 2005.

R. Lupu and J. Menendez, “Pharmacological inhibitors of fatty acid synthase (FASN)--catalyzed endogenous fatty acid biogenesis: a new family of anti-cancer agents?” Current Pharmaceutical Biotechnology, vol. 7, no. 6, pp. 483–494, 2006.

B. Liu, Y. Wang, K. Fillgrove, and V. Anderson, “Triclosan inhibits enoyl-reductase of type I fatty acid synthase in vitro and is cytotoxic to MCF-7 and SKBr-3 breast cancer cells,” Cancer Chemotherapy and Pharmacology, vol. 49, no. 3, pp. 187–193, 2002.

E. S. Pizer, C. Jackisch, F. D. Wood, G. R. Pasternack, N. E. Davidson, and F. P. Kuhajda, “Inhibition of fatty acid synthesis induces programmed cell death in human breast cancer cells,” Cancer Research, vol. 56, no. 12, pp. 2745–2747, 1996.

F. P. Kuhajda, K. Jenner, F. D. Wood et al., “Fatty acid synthesis: a potential selective target for antineoplastic therapy,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 14, pp. 6379–6383, 1994.

F. P. Kuhajda, E. S. Pizer, J. N. Li, N. S. Mani, G. L. Frehywot, and C. A. Townsend, “Synthesis and antitumor activity of an inhibitor of fatty acid synthase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 7, pp. 3450–3454, 2000.

T. S. Ho, Y. P. Ho, W. Y. Wong, L. Chi-Ming Chiu, Y. S. Wong, and V. Eng-Choon Ooi, “Fatty acid synthase inhibitors cerulenin and C75 retard growth and induce caspase-dependent apoptosis in human melanoma A-375 cells,” Biomedicine & Pharmacotherapy, vol. 61, no. 9, pp. 578–587, 2007.

M. C. Sadowski, R. H. Pouwer, J. H. Gunter, A. A. Lubik, R. J. Quinn, and C. C. Nelson, “The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer,” Oncotarget, vol. 5, no. 19, pp. 9362–9381, 2014.

P. R. Deepa, S. Vandhana, U. Jayanthi, and S. Krishnakumar, “Therapeutic and toxicologic evaluation of anti-lipogenic agents in cancer cells compared with non-neoplastic cells,” Basic & Clinical Pharmacology & Toxicology, vol. 110, no. 6, pp. 494–503, 2012.

K. Nishi, K. Suzuki, J. Sawamoto et al., “Inhibition of fatty acid synthesis induces apoptosis of human pancreatic cancer cells,” Anticancer Research, vol. 36, no. 9, pp. 4655–4660, 2016.

B. Schmid, J. F. Rippmann, M. Tadayyon, and B. S. Hamilton, “Inhibition of fatty acid synthase prevents preadipocyte differentiation,” Biochemical and Biophysical Research Communications, vol. 328, no. 4, pp. 1073–1082, 2005.

E. S. Pizer, R. J. Kurman, G. R. Pasternack, and F. P. Kuhajda, “Expression of fatty acid synthase is closely linked to proliferation and stromal decidualization in cycling endometrium,” International Journal of Gynecological Pathology, vol. 16, no. 1, pp. 45–51, 1997.

R. E. Wilentz, L. A. Witters, and E. S. Pizer, “Lipogenic enzymes fatty acid synthase and acetyl-coenzyme A carboxylase are coexpressed with sterol regulatory element binding protein and Ki-67 in fetal tissues,” Pediatric and Developmental Pathology, vol. 3, no. 6, pp. 525–531, 2000.