Quaternary Ammonium Compounds: A Chemical Class of Emerging Concern.

Quaternary ammonium compounds (QACs), a large class of chemicals that includes high production volume substances, have been used for decades as antimicrobials, preservatives, and antistatic agents and for other functions in cleaning, disinfecting, personal care products, and durable consumer goods. QAC use has accelerated in response to the COVID-19 pandemic and the banning of 19 antimicrobials from several personal care products by the US Food and Drug Administration in 2016. Studies conducted before and after the onset of the pandemic indicate increased human exposure to QACs. Environmental releases of these chemicals have also increased. Emerging information on adverse environmental and human health impacts of QACs is motivating a reconsideration of the risks and benefits across the life cycle of their production, use, and disposal. This work presents a critical review of the literature and scientific perspective developed by a multidisciplinary, multi-institutional team of authors from academia, governmental, and nonprofit organizations. The review evaluates currently available information on the ecological and human health profile of QACs and identifies multiple areas of potential concern. Adverse ecological effects include acute and chronic toxicity to susceptible aquatic organisms, with concentrations of some QACs approaching levels of concern. Suspected or known adverse health outcomes include dermal and respiratory effects, developmental and reproductive toxicity, disruption of metabolic function such as lipid homeostasis, and impairment of mitochondrial function. QACs' role in antimicrobial resistance has also been demonstrated. In the US regulatory system, how a QAC is managed depends on how it is used, for example in pesticides or personal care products. This can result in the same QACs receiving different degrees of scrutiny depending on the use and the agency regulating it. Further, the US Environmental Protection Agency's current method of grouping QACs based on structure, first proposed in 1988, is insufficient to address the wide range of QAC chemistries, potential toxicities, and exposure scenarios. Consequently, exposures to common mixtures of QACs and from multiple sources remain largely unassessed. Some restrictions on the use of QACs have been implemented in the US and elsewhere, primarily focused on personal care products. Assessing the risks posed by QACs is hampered by their vast structural diversity and a lack of quantitative data on exposure and toxicity for the majority of these compounds. This review identifies important data gaps and provides research and policy recommendations for preserving the utility of QAC chemistries while also seeking to limit adverse environmental and human health effects.

Costs of abdominal aortic aneurysm care at a regional Veterans Affairs medical center with the implementation of an abdominal aortic aneurysm screening program.

BACKGROUND: Abdominal aortic aneurysm (AAA) screening has demonstrated to be cost-effective in reducing AAA-related morbidity and all-cause mortality. However, the downstream care costs of an implemented AAA screening in clinical practice have not been reported. The purpose of this study is to determine direct regional Department of Veterans Affairs (VA) costs in implementing and sustaining an AAA screening program over a 10-year period. METHODS: A cost data analysis (adjusted to 2021 U.S. dollars) of an AAA screening program was conducted from 2007 to 2016, where 19,649 veteran patients aged 65-75 with a smoking history were screened at a regional VA medical center. A decision support system tracked direct and indirect encounter costs from Medicare billing codes associated with AAA care. Costs from a patient's initial screening, follow-up imaging, to AAA repair or at the end of the analysis period, March 31, 2021, were recorded. Costs for AAA repairs outside the VA system were also tracked. RESULTS: A total of 1,183 patients screened were identified with an AAA ≥3.0 cm without history of repair. Estimated screening costs were $2.8 million or $280,000 annually ($143/screening) in the care of 19,649 screened patients. There were 221 patients who required repair (143 repairs in VA, 78 repairs outside VA). The average cost of elective endovascular repair was $43,021 and that of open repair was $49,871. The total costs for all elective repairs were $9,692,591. Screening, implementation, maintenance, and surgical repair cost involved in the management of patients with AAA disease was $13.7 million, with $10,686 per life-year lived after repair (5.8 ± 3.5 mean life-years) and $490 per life-year lived after screening (6.9 ± 3.5 mean life-years) for all patients screened. There were 13 deaths of unknown causes and one patient with a ruptured AAA that required emergency repair at a cost of $124,392. CONCLUSIONS: Despite known limitations, the implementation of an AAA ultrasound screening program is feasible, cost-effective, and a worthwhile endeavor.

PPARα-targeted mitochondrial bioenergetics mediate repair of intestinal barriers at the host-microbe intersection during SIV infection.

Chronic gut inflammatory diseases are associated with disruption of intestinal epithelial barriers and impaired mucosal immunity. HIV-1 (HIV) causes depletion of mucosal CD4+ T cells early in infection and disruption of gut epithelium, resulting in chronic inflammation and immunodeficiency. Although antiretroviral therapy (ART) is effective in suppressing viral replication, it is incapable of restoring the "leaky gut," which poses an impediment for HIV cure efforts. Strategies are needed for rapid repair of the epithelium to protect intestinal microenvironments and immunity in inflamed gut. Using an in vivo nonhuman primate intestinal loop model of HIV/AIDS, we identified the pathogenic mechanism underlying sustained disruption of gut epithelium and explored rapid repair of gut epithelium at the intersection of microbial metabolism. Molecular, immunological, and metabolomic analyses revealed marked loss of peroxisomal proliferator-activated receptor-α (PPARα) signaling, predominant impairment of mitochondrial function, and epithelial disruption both in vivo and in vitro. To elucidate pathways regulating intestinal epithelial integrity, we introduced probiotic Lactobacillus plantarum into Simian immunodeficiency virus (SIV)-inflamed intestinal lumen. Rapid recovery of the epithelium occurred within 5 h of L. plantarum administration, independent of mucosal CD4+ T cell recovery, and in the absence of ART. This intestinal barrier repair was driven by L. plantarum-induced PPARα activation and restoration of mitochondrial structure and fatty acid ß-oxidation. Our data highlight the critical role of PPARα at the intersection between microbial metabolism and epithelial repair in virally inflamed gut and as a potential mitochondrial target for restoring gut barriers in other infectious or gut inflammatory diseases.

Rescue of cell death and inflammation of a mouse model of complex 1-mediated vision loss by repurposed drug molecules.

Inherited mitochondrial optic neuropathies, such as Leber's hereditary optic neuropathy (LHON) and Autosomal dominant optic atrophy (ADOA) are caused by mutant mitochondrial proteins that lead to defects in mitochondrial complex 1-driven ATP synthesis, and cause specific retinal ganglion cell (RGC) loss. Complex 1 defects also occur in patients with primary open angle glaucoma (POAG), in which there is specific RGC loss. The treatment of mitochondrial optic neuropathy in the US is only supportive. The Ndufs4 knockout (Ndufs4 KO) mouse is a mitochondrial complex 1-deficient model that leads to RGC loss and rapid vision loss and allows for streamlined testing of potential therapeutics. Preceding RGC loss in the Ndufs4 KO is the loss of starburst amacrine cells, which may be an important target in the mechanism of complex 1-deficient vision loss. Papaverine and zolpidem were recently shown to be protective of bioenergetic loss in cell models of optic neuropathy. Treatment of Ndufs4 KO mice with papaverine, zolpidem, and rapamycin-suppressed inflammation, prevented cell death, and protected from vision loss. Thus, in the Ndufs4 KO mouse model of mitochondrial optic neuropathy, papaverine and zolpidem provided significant protection from multiple pathophysiological features, and as approved drugs in wide human use could be considered for the novel indication of human optic neuropathy.

A descriptive pilot study of cytokine production following stimulation of ex-vivo whole blood with commercial therapeutic feline hydrolyzed diets in individual healthy immunotolerant cats.

BACKGROUND: Hydrolyzed diets are used in companion animals for the diagnosis and treatment of adverse food reaction. Similarly, hydrolyzed formulas are used in human infants with severe inflammatory bowel disease or milk allergy, and these must meet the standard of hypoallergenicity through rigorous testing. Unfortunately, no standards are currently applied to hydrolyzed veterinary therapeutic diets, and data for the immunogenicity of feline diets is also not available. Therefore, the main aim of this pilot study was to determine if ex-vivo whole blood stimulation assays could be used to characterize the cytokine response to hydrolyzed commercial diets in a small number of individual healthy immunotolerant cats. This approach has also been used to investigate cytokine production in response to cow milk protein in humans and currently similar studies do not exist in companion animals. Nine healthy cats previously eating the same basal diet were divided into groups and fed one of three hydrolyzed diets exclusively for 6 weeks. Heparinized whole blood was collected from each cat before and after the feeding trial. Ex-vivo whole blood stimulation assays were performed using crude extracts of the basal diet as a positive control, as this diet contained the same proteins present in the hydrolyzed diet but were intact, saline as a negative control, and each cat's respective hydrolyzed diet. Supernatants were collected and analyzed for tumor necrosis factor-alpha, interleukin-10 (IL-10), and interleukin-4 using enzyme-linked immunosorbant assay. RESULTS: Seven cats produced detectable amounts of the anti-inflammatory cytokine IL-10 upon stimulation with the basal diet. Two cats produced detectable amounts of IL-10 upon stimulation with a hydrolyzed soy-based diet and one cat produced a detectable amount of IL-10 upon stimulation with a hydrolyzed chicken-based diet (>125 pg/mL). CONCLUSIONS: Results from this pilot study suggest that in some healthy immunotolerant cats, some hydrolyzed diets may elicit a similar cytokine response compared to their basal diet, which contained the same proteins intact. Therefore, animals may be able to recognize and react to some hydrolyzed forms of tolerated proteins, and may also suggest IL-10 as a target for investigation as a potential marker for dietary tolerance in cats, however further studies would be necessary to corroborate this. Further studies are also needed to determine if this would also be the same in immunologically naïve, sensitized and clinically hypersensitized cats.

Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.

Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity.

Toxins in botanical dietary supplements: blue cohosh components disrupt cellular respiration and mitochondrial membrane potential.

Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

Mitochondria dysfunction induced by decyl-TPP mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6 sensitizes cisplatin lung anticancer therapy due to a remodeling of energy metabolism.

The pharmacological interest in mitochondria is very relevant since these crucial organelles are involved in the pathogenesis of multiple diseases, such as cancer. In order to modulate cellular redox/oxidative balance and enhance mitochondrial function, numerous polyphenolic derivatives targeting mitochondria have been developed. Still, due to the drug resistance emergence in several cancer therapies, significant efforts are being made to develop drugs that combine the induction of mitochondrial metabolic reprogramming with the ability to generate reactive oxygen species, taking into consideration the varying metabolic profiles of different cell types. We previously developed a mitochondria-targeted antioxidant (AntiOxCIN6) by linking caffeic acid to lipophilic triphenylphosphonium cation through a 10-carbon aliphatic chain. The antioxidant activity of AntiOxCIN6 has been documented but how the mitochondriotropic compound impact energy metabolism of both normal and cancer cells remains unknown. We demonstrated that AntiOxCIN6 increased antioxidant defense system in HepG2 cells, although ROS clearance was ineffective. Consequently, AntiOxCIN6 significantly decreased mitochondrial function and morphology, culminating in a decreased capacity in complex I-driven ATP production without affecting cell viability. These alterations were accompanied by an increase in glycolytic fluxes. Additionally, we demonstrate that AntiOxCIN6 sensitized A549 adenocarcinoma cells for CIS-induced apoptotic cell death, while AntiOxCIN6 appears to cause metabolic changes or a redox pre-conditioning on lung MRC-5 fibroblasts, conferring protection against cisplatin. We propose that length and hydrophobicity of the C10-TPP+ alkyl linker play a significant role in inducing mitochondrial and cellular toxicity, while the presence of the antioxidant caffeic acid appears to be responsible for activating cytoprotective pathways.

Comparative study of chromatographic medium-associated mass and potential antitumor activity loss with bioactive extracts.

Natural product drug discovery programs often rely on the use of silica (Si) gel, reversed-phase media, or size-exclusion resins (e.g., RP-C18, Sephadex LH-20) for compound purification. The synthetic polymer-based sorbent Diaion HP20SS (cross-linked polystyrene matrix) is used as an alternative to prepare purified natural product libraries. To evaluate the impact of chromatographic media on the isolation of biologically active, yet chromatographically unstable natural products, Diaion HP20SS was evaluated side-by-side with normal-phase sorbents for irreversible binding of extract constituents and their effects on bioactivity. An array of chemically diverse natural product-rich extracts was selected as a test panel, and a cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) was employed to monitor potential change(s) in bioactivity. Silica gel caused significant irreversible binding of three out of 10 extracts. Curcuma longa, Saururus cernuus, and Citrus reticulata extracts showed decreased HIF-1 inhibitory activity after elution through Si gel. An additional nonpolar column wash of HP20SS with EtOAc retained considerable bioactivities of active extracts. In general, Si gel produced the greatest loss of bioactivity. However, HP20SS elution reduced significantly HIF-1 inhibitory activity of certain extracts (e.g., Asimina triloba).

Structures and mechanisms of antitumor agents: xestoquinones uncouple cellular respiration and disrupt HIF signaling in human breast tumor cells.

The organic extract of a marine sponge, Petrosia alfiani, selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1-7) including three new compounds: 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1-7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), which possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC(50) value of 0.2 µM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.

Glycolysis inhibitor screening identifies the bis-geranylacylphloroglucinol protonophore moronone from Moronobea coccinea.

Tumor cells exhibit enhanced glucose consumption and lactate production even when supplied with adequate oxygen (a phenomenon known as the Warburg effect, or aerobic glycolysis). Pharmacological inhibition of aerobic glycolysis represents a potential tumor-selective approach that targets the metabolic differences between normal and malignant tissues. Human breast tumor MDA-MB-231 cells were used to develop an assay system to discover natural product-based glycolysis inhibitors. The assay employed was based on hypersensitivity to glycolytic inhibition in tumor cells treated with the mitochondrial electron transport inhibitor rotenone. Under such conditions, ATP supply, and hence cell viability, depends exclusively on glycolysis. This assay system was used to evaluate 10648 plant and marine organism extracts from the U.S. National Cancer Institute's Open Repository. Bioassay-guided isolation of an active Moronobea coccinea extract yielded the new bis-geranylacylphloroglucinol derivative moronone (1). Compound 1 exhibited enhanced antiproliferative/cytotoxic activity in the presence of rotenone-imposed metabolic stress on tumor cells. Surprisingly, mechanistic studies revealed that 1 did not inhibit glycolysis, but rather functions as a protonophore that dissipates the mitochondrial proton gradient. In the presence of rotenone, tumor cells may be hypersensitive to protonophores due to increased ATP utilization by the ATP synthase.

Accuracy of Point-of-Care Ultrasound in Follow Up Abdominal Aortic Aneurysm Imaging.

BACKGROUND: Point-of-care ultrasound (POCUS) has been reported as a valuable tool for bedside diagnoses of abdominal Aortic Aneurysms (AAA). However, no data exist regarding POCUS in measuring follow-up AAA diameter studies in patients with existing AAAs. The purpose of this study was to determine the variability of aortic measurements performed by a non-physician using POCUS vs standard of care (SOC) measurements by a registered vascular technologist or an abdominal/pelvic CT scan. METHODS: A prospective observational ultrasound study was performed from 1/1/2019 to 3/31/2021 on patients with a diagnosis of an AAA (≥3.0 cm). A research coordinator (non-physician) underwent a 3-hour training session in ultrasound operation and basic human anatomy to measure AAA diameter. The maximum aortic diameter was documented and compared to measurements obtained by SOC ultrasonography or CT scan. The POCUS and SOC ultrasounds were separated by no more than 90 days. Clinical risk factors including age, race, body mass index, coronary artery disease, hypertension, peripheral vascular disease, cerebrovascular disease, diabetes, and current smoking were also collected. RESULTS: Eighty-one patients (mean age: 73.6 ± 5.8 years, body mass index: 29.5 ± 6.2 kg/m2) were being followed in a vascular clinic and underwent both a POCUS and SOC ultrasounds. One indeterminant study was reported in identifying an AAA diagnosis, due to an overlying colostomy. The average follow-up time from initial screening aortic diameter to POCUS was 4.4 ± 3.7 years. Overall average aortic diameter measurements obtained were 4.1 ± .9 cm for POCUS and 4.0 ± .9 cm for SOC (P = NS). Average difference in aortic measurement for POCUS and SOC was -.1 ± .3 cm. CONCLUSIONS: POCUS is an accurate method to follow AAA diameter in patients. POCUS could improve patient follow up with AAA diameter measurements, streamline care and reduce overall burden for both patients and Radiology Departments in assessing follow up AAA diameters.

Mitochondrial respiration inhibitors suppress protein translation and hypoxic signaling via the hyperphosphorylation and inactivation of translation initiation factor eIF2α and elongation factor eEF2.

Over 20,000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods.

Repurposing FDA approved drugs inhibiting mitochondrial function for targeting glioma-stem like cells.

Glioblastoma Multiforme (GBM) tumors contain a small population of glioma stem-like cells (GSCs) among the various differentiated GBM cells (d-GCs). GSCs drive tumor recurrence, and resistance to Temozolomide (TMZ), the standard of care (SoC) for GBM chemotherapy. In order to investigate a potential link between GSC specific mitochondria function and SoC resistance, two patient-derived GSC lines were evaluated for differences in their mitochondrial metabolism. In both the lines, GSCs had significantly lower mitochondrial -content, and -function compared to d-GCs. In vitro, the standard mitochondrial-specific inhibitors oligomycin A, antimycin A, and rotenone selectively inhibited GSC proliferation to a greater extent than d-GCs and human primary astrocytes. These findings indicate that mitochondrial inhibition can be a potential GSC-targeted therapeutic strategy in GBM with minimal off-target toxicity. Mechanistically the standard mitochondrial inhibitors elicit their GSC-selective cytotoxic effects through the induction of apoptosis or autophagy pathways. We tested for GSC proliferation in the presence of 3 safe FDA-approved drugs--trifluoperazine, mitoxantrone, and pyrvinium pamoate, all of which are also known mitochondrial-targeting agents. The SoC GBM therapeutic TMZ did not trigger cytotoxicity in glioma stem cells, even at 100 µM concentration. By contrast, trifluoperazine, mitoxantrone, and pyrvinium pamoate exerted antiproliferative effects in GSCs about 30-50 fold more effectively than temozolomide. Thus, we hereby demonstrate that FDA-approved mitochondrial inhibitors induce GSC-selective cytotoxicity, and targeting mitochondrial function could present a potential therapeutic option for GBM treatment.

Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure.

Humans are frequently exposed to Quaternary Ammonium Compounds (QACs). QACs are ubiquitously used in medical settings, restaurants, and homes as cleaners and disinfectants. Despite their prevalence, nothing is known about the health effects associated with chronic low-level exposure. Chronic QAC toxicity, only recently identified in mice, resulted in developmental, reproductive, and immune dysfunction. Cell based studies indicate increased inflammation, decreased mitochondrial function, and disruption of cholesterol synthesis. If these findings translate to human toxicity, multiple physiological processes could be affected. This study tested whether QAC concentrations could be detected in the blood of 43 human volunteers, and whether QAC concentrations influenced markers of inflammation, mitochondrial function, and cholesterol synthesis. QAC concentrations were detected in 80 % of study participants. Blood QACs were associated with increase in inflammatory cytokines, decreased mitochondrial function, and disruption of cholesterol homeostasis in a dose dependent manner. This is the first study to measure QACs in human blood, and also the first to demonstrate statistically significant relationships between blood QAC and meaningful health related biomarkers. Additionally, the results are timely in light of the increased QAC disinfectant exposure occurring due to the SARS-CoV-2 pandemic. MAIN FINDINGS: This study found that 80 % of study participants contained QACs in their blood; and that markers of inflammation, mitochondrial function, and sterol homeostasis varied with blood QAC concentration.

Cetylpyridinium chloride is a potent AMP-activated kinase (AMPK) inducer and has therapeutic potential in cancer.

AMP-activated protein kinase (AMPK) is a eukaryotic energy sensor and protector from mitochondrial/energetic stress that is also a therapeutic target for cancer and metabolic disease. Metformin is an AMPK inducer that has been used in cancer therapeutic trials. Through screening we isolated cetylpyridinium chloride (CPC), a drug known to dose-dependently inhibit mitochondrial complex 1, as a potent and dose-dependent AMPK stimulator. Mitochondrial biogenesis and bioenergetics changes have also been implicated in glioblastoma, which is the most aggressive form of brain tumors. Cetylpyridinium chloride has been administered in humans as a safe drug-disinfectant for several decades, and we report here that under in vitro conditions, cetylpyridinium chloride kills glioblastoma cells in a dose dependent manner at a higher efficacy compared to current standard of care drug, temozolomide.

Novel mTORC1 Inhibitors Kill Glioblastoma Stem Cells.

Glioblastoma (GBM) is an aggressive tumor of the brain, with an average post-diagnosis survival of 15 months. GBM stem cells (GBMSC) resist the standard-of-care therapy, temozolomide, and are considered a major contributor to tumor resistance. Mammalian target of rapamycin Complex 1 (mTORC1) regulates cell proliferation and has been shown by others to have reduced activity in GBMSC. We recently identified a novel chemical series of human-safe piperazine-based brain-penetrant mTORC1-specific inhibitors. We assayed the piperazine-mTOR binding strength by two biophysical measurements, biolayer interferometry and field-effect biosensing, and these confirmed each other and demonstrated a structure-activity relationship. As mTORC1 is altered in human GBMSC, and as mTORC1 inhibitors have been tested in previous GBM clinical trials, we tested the killing potency of the tightest-binding piperazines and observed that these were potent GBMSC killers. GBMSCs are resistant to the standard-of-care temozolomide therapy, but temozolomide supplemented with tight-binding piperazine meclizine and flunarizine greatly enhanced GBMSC death over temozolomide alone. Lastly, we investigated IDH1-mutated GBMSC mutations that are known to affect mitochondrial and mTORC1 metabolism, and the tight-binding meclizine provoked 'synthetic lethality' in IDH1-mutant GBMSCs. In other words, IDH1-mutated GBMSC showed greater sensitivity to the coadministration of temozolomide and meclizine. These data tend to support a novel clinical strategy for GBM, i.e., the co-administration of meclizine or flunarizine as adjuvant therapy in the treatment of GBM and IDH1-mutant GBM.

Disruption of mitochondrial function as mechanism for anti-cancer activity of a novel mitochondriotropic menadione derivative.

Menadione, also known as vitamin K3, is a 2-methyl-1,4 naphthoquinone with a potent cytotoxic activity mainly resulting from its quinone redox-cycling with production of reactive oxygen species (ROS). Although increased ROS generation is considered a relevant mechanism in cancer cell death, it may not be sufficiently effective to kill cancer cells due to phenotypic adaptations. Therefore, combining ROS-generating agents with other molecules targeting important cancer cell phenotypes can be an effective therapeutic strategy. As mitochondrial dysfunction has been implicated in many human diseases, including cancer, we describe here the discovery of a mitochondrial-directed agent (MitoK3), which was developed by conjugating a TPP cation to the C3 position of the menadione's naphthoquinone ring, increasing its selective accumulation in mitochondria, as well as led to alterations of its redox properties and consequent biological outcome. MitoK3 disturbed the mitochondrial bioenergetic apparatus, with subsequent loss of mitochondrial ATP production. The combinatory strategy of MitoK3 with anticancer agent doxorubicin (DOX) resulted in a degree of cytotoxicity higher than those of the individual molecules, as the combination triggered tumour apoptotic cell death evident by caspase 3/9 activities, probably through mitochondrial destabilization or by interference with mitochondrial redox processes. The results of this investigation support the importance of drug discovery process in developing molecules that can be use as adjuvant therapy in patients with specific cancer subtypes.

The Eye Drop Preservative Benzalkonium Chloride Potently Induces Mitochondrial Dysfunction and Preferentially Affects LHON Mutant Cells.

Purpose: Benzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as "dry eye" and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects. Method: Mitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3). Results: Benzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 µM) and O2 consumption (IC50, 10.9 µM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107-667 µM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 µM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 µM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect. Conclusions: Benzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients.

In Vitro Evaluation of Mitochondrial Function and Estrogen Signaling in Cell Lines Exposed to the Antiseptic Cetylpyridinium Chloride.

BACKGROUND: Quaternary ammonium salts (QUATS), such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BAK), are frequently used in antiseptic formulations, including toothpastes, mouthwashes, lozenges, throat and nasal sprays, and as biocides. Although in a recent ruling, the U.S. Food and Drug Administration (FDA) banned CPC from certain products and requested more data on BAK's efficacy and safety profile, QUATS, in general, and CPC and BAK, in particular, continue to be used in personal health care, food, and pharmaceutical and cleaning industries. OBJECTIVES: We aimed to assess CPC's effects on mitochondrial toxicity and endocrine disruption in vitro. METHOD: Mitochondrial O2 consumption and adenosine triphosphate (ATP) synthesis rates of osteosarcoma cybrid cells were measured before and after CPC and BAK treatment. Antiestrogenic effects of the compounds were measured by a luciferase-based assay using recombinant human breast carcinoma cells (VM7Luc4E2, ERalpha-positive). RESULTS: CPC inhibited both mitochondrial O2 consumption [half maximal inhibitory concentration (IC50): 3.8µM] and ATP synthesis (IC50: 0.9µM), and additional findings supported inhibition of mitochondrial complex 1 as the underlying mechanism for these effects. In addition, CPC showed concentration-dependent antiestrogenic activity half maximal effective concentration [(EC50): 4.5µM)]. BAK, another antimicrobial QUATS that is structurally similar to CPC, and the pesticide rotenone, a known complex 1 inhibitor, also showed mitochondrial inhibitory and antiestrogenic effects. In all three cases, there was overlap of the antiestrogenic activity with the mitochondrial inhibitory activity. CONCLUSIONS: Mitochondrial inhibition in vitro occurred at a CPC concentration that may be relevant to human exposures. The antiestrogenic activity of CPC, BAK, rotenone, and triclosan may be related to their mitochondrial inhibitory activity. Our findings support the need for additional research on the mitochondrial inhibitory and antiestrogenic effects of QUATS, including CPC and BAK. https://doi.org/10.1289/EHP1404.