# **Oxidative Stress and Inflammation From Mechanisms to Therapeutic Approaches**

Edited by Juan Gambini Printed Edition of the Special Issue Published in *Biomedicines*

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## **Oxidative Stress and Inflammation: From Mechanisms to Therapeutic Approaches**

## **Oxidative Stress and Inflammation: From Mechanisms to Therapeutic Approaches**

Editor

**Juan Gambini**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Juan Gambini University of Valencia Spain

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## **Contents**

RAW 264.7 Mouse Macrophages Reprinted from: *Biomedicines* **2020**, *8*, 11, doi:10.3390/biomedicines8010011 ............ **103**

### *Editorial* **Oxidative Stress and Inflammation: From Mechanisms to Therapeutic Approaches**

**Juan Gambini \* and Kristine Stromsnes**

Freshage Research Group, Department of Physiology, Faculty of Medicine, Insitute of Health Research-INCLIVA, University of Valencia and CIBERFES, Avda. Blasco Ibañez, 15, 46010 Valencia, Spain<EMAIL_ADDRESS>**\*** Correspondence<EMAIL_ADDRESS>
Oxidative stress and inflammation are two phenomena that are directly involved in practically all pathologies and especially in aging. Oxidative stress, which is associated with the redox state, constitutes an important mechanism in many physiological processes, such as adaptations to physical exercise, cell signaling, and hypothalamic appetite regulation. Inflammatory mediators are known to be essential in mechanisms such as the generation of gastric mucus for the protection of the stomach and for the repair of tissues via the mobilization of stem cells. However, when these two phenomena are deregulated, their actions are harmful. In this Special Issue, we ask ourselves several questions: How and when should we allow or block oxidative stress and inflammation? What is the advisable dose of antioxidant or anti-inflammatory therapy associated with aging? Are diet, physical exercise, and decreased psychological stress the best therapies for oxidative stress and inflammation control? In this Special Issue, we have published 14 articles: 7 original research articles and 7 reviews. They describe everything from the inflammatory and oxidative processes involved in different diseases to functional foods containing molecules that counteract these phenomena.

In order to reflect upon the comments made in this Special Issue in a coherent way, the publications have been divided into two sections that we will comment on below.

Environmental pollution has become a major public health risk in recent decades, causing millions of deaths worldwide. Among pollutants, we find harmful or harmful components that can be both physical, chemical, and biological. Identifying these compounds is the starting point to combat them.

In their original research article, Heba Al Housseiny et al. [1] have identified and reflected how the exposure of a product called PM2.5, which is derived from the combustion of matter, is associated with a decrease in lung function, impaired immunity, and exacerbations in lung disease. Their results show how these particles are capable of reducing cell viability and increasing the inflammatory parameters and oxidative stress in lung epithelial cells. These data suggest that effective mitigation strategies are necessary to address not only the identification of polluting particles but also their possible neutralization and treatments that avoid their toxicity [1].

Acute respiratory distress syndrome is one of the pathologies of the respiratory system. This condition presents with the acute onset of respiratory failure that leads to bilateral pulmonary infiltrates seen on chest radiographs, hypoxemia, etc., and that can result in direct lung lesions, pneumonia, sepsis, and even death. Globally, it has an incidence of around 15 to 80/100,000 inhabitants per year, and so far, no beneficial drug therapy has been developed; however, it has been found that platelets play a fundamental role in this disease. Therefore, Chuan-Mu Chen et al. [2] describe studies that show the possible benefits of antiplatelet therapy for prevention and treatment via the control of platelet activity and describe the mechanisms of action that are involved.

Dry eye syndrome affects approximately 40% of the adult population. It manifests as hyperemia, glare, fatigue, and eye irritation. The most critical consequences of this

**Citation:** Gambini, J.; Stromsnes, K. Oxidative Stress and Inflammation: From Mechanisms to Therapeutic Approaches. *Biomedicines* **2022**, *10*, 753. https://doi.org/10.3390/ biomedicines10040753

Received: 22 February 2022 Accepted: 11 March 2022 Published: 23 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

1

desiccant stress are inflammation and damage to the ocular surface, which, in severe cases, can lead to blindness, scarring, opacification or ulceration of the cornea, and blurred vision. Treatments for this condition include lubricating drops, anti-inflammatory therapy, or immunosuppressants such as cyclosporine. However, all of these treatments can cause adverse effects that limit their use. For this reason, in their original article, Dmitry V. Chistyakov et al. [3] have tested a therapy with a new ophthalmic formulation containing a zileuton solution based on dimethylsulfoxide. Their results showed the high efficacy of the proposed therapy. On the one hand, DMSO decreased the activity of pro-inflammatory protagladins, and on the other hand, zileuton decreased the cytokine levels released by T cells. In this way, the two compounds presented synergistic activity that opens the door to more effective anti-inflammatory therapies against said pathology [3].

Gender differences in cardiovascular diseases (CVD) as well as the identification of biomarkers for disease detection are challenges that are of great importance today. In the original study by Laura Bordoni et al. [4], they recruited 547 individuals comprising both men and women with or without cardiovascular disease to identify useful biomarkers to characterize gender differences in CVD. The study evaluated blood parameters such as plasma membrane fluidity, erythrocyte lipid hydroperoxides, and trimethylamine (TMA). Lower plasmatic TMA was observed in male CVD patients compared to in the healthy male controls, while higher levels of TMA were measured in female CVD patients with respect to the female controls. Diphenyl−1-pyrenylphosphine (DPPP) was significantly lower in male CVD patients compared to in the healthy controls, while no significant changes were measured in females with or without CVD. The results obtained allowed Laura Bordoni et al. to conclude that TMA could be an effective marker for the detection and prevention of CVD and that the DPPP value could be predictive biomarker of CVD in men [4].

Breast cancer remains as one of the most common cancers today. As advances in diagnostic and treatment methods continue to become more effective, the survival rate continues to increase. However, the chemotherapy that is administered, although effective, still presents important adverse reactions, such as difficulty concentrating and remembering or alterations in processing speed. Patients may also experience symptoms of gastrointestinal (GI) issues, such as diarrhea and vomiting as well as long-term hepatotoxicity or liver damage. In their review, Taurean Brown et al. [5] describe how the series of symptoms experienced by cancer patients are interconnected and mediated by inflammatory responses. Therefore, new therapeutic approaches could help improve the quality of life in breast cancer patients after treatment [5].

Protecting the kidneys is essential in the management of diabetes-derived problems. There is good evidence to illustrate the causal link between oxidative stress and the progression of diabetes-related kidney disease (DKD). Currently, the only therapeutic options that are for DKD are limited to systemic interventions for the metabolic changes related to diabetes, such as dyslipidemia, hypertension, and hyperglycemia. Given the important role that oxidative stress plays in various aspects of kidney injury, the redox imbalance could be considered to be potential therapeutic target for renal failure in diabetic patients. This review article written by Keiichiro Matoba et al. [6] shows recent therapeutic approaches to prevent DKD by targeting the antioxidant effects of newly developed antihyperglycemic agents.

In their review, Spyridon Methenitis et al. [7] discuss the possibility of exercise-induced muscle damage being a suitable model of inflammation for the evaluation of nutraceutical anti-inflammatory properties. Low-grade, subclinical inflammation is one of the main pathophysiological mechanisms underlying most chronic and non-communicable diseases. In this sense, the use of functional foods, those with bioactive compounds and, in this case, with an anti-inflammatory capacity, could be a model to take into consideration in humans. The capacity of these compounds could be highly valued since it is known that bioavailability is a key point in their effectiveness [7].

Compounds with pharmacological activities with the possibility of having great therapeutic power have been found in nature.

Telomere shortening and oxidative stress have been linked to aging and patients with cancer and inflammatory diseases. In 2016, the *New England Journal of Medicine* published an article showing how danazol, a synthetic androgen, promotes telomere lengthening in circulating human leukocytes, comparing its effects to a hydroethanolic root extract that is known to stimulate telomerase activity. These findings open the door for treatments for associated diseases, telomeres, or aging. In this Special Issue, Isabelle Guinobert et al. [8] show how danazol, similar to extracts from the Astragalus mongholicus plant, has positive effects on telomerase-dependent telomere lengthening in human lymphocytes in their review. These findings create possibilities for new treatments for diseases that are associated with aging [8].

Diospirin is a medicinal compound derived from the Diospyros lotus, which has anticancer, antituberculous, and antileishmanial activities against Leishmania donovani. Adnan Shahidullah et al. [9] describe a modulatory role in the inflammatory response induced by lipopolysaccharides (LPS) in mouse macrophages through the inhibition of nitric oxide and several cytokines via the ER-stressed calcium-p38 MAPK/CHOP/Fas pathway. These effects have not been shown previously and could have important therapeutic uses [9].

Prolonged strenuous exercise can induce unfavorable biological changes and symptoms, including inflammatory responses that can lead to intestinal barrier dysfunction. Many athletes use non-steroidal anti-inflammatory drugs to treat inflammation-induced algesthesia. Sihui Ma et al. [10] have verified how hyperimmunized milk supplementation, obtained after immunizing cows against specific antigens, protects intestinal function, exerts anti-inflammatory effects, and promotes the development of immunity against various pathogens. This could be a viable nutritional approach for preventing intense exercise-induced symptoms.

Caloric restriction has been shown to be a powerful intervention to extend both lifespan and health span in various animal models, from yeast to primates. Furthermore, in humans, caloric restriction has been found to induce cardiometabolic adaptations that are associated with better health. Daniele La Russa et al. [11] studied the long-term effects of caloric restrictions on the inflammatory and redox balance in aged, obese rats. They found that caloric restriction not only induces weight loss but also positively modulates both the antiinflammatory and antioxidant pathways while also improving the circulating adiponectin levels in obese old rats. Along with achieving normal weight, these adaptations, which are induced by caloric restriction, suggest that the redox and inflammatory imbalance in obese aged rats appears to be caused by obesity rather than by aging [11].

Microglia play an important role in the development of neurodegenerative diseases, but the mechanisms of action have yet to be specified. When activated, they can express pro-inflammatory cytokines that act on the surrounding brain and spinal cord, something that can have a detrimental effect on nerve cells when they acquire a chronic inflammatory function and promote neuropathologies. In their bibliographic review, Tamotsu Tsukahara et al. [12] set out to clarify the mechanism of action by which the porcine liver decomposition product, which is rich in phospholipids, exerts beneficial effects on cognitive functions in healthy humans. The authors propose that this food is function due to its possibility of enhancing visual memory and delay recall as well as to improve Hasegawa's Dementia Scale-Revised scores and the Wechsler Memory Scale in healthy adults and discuss whether it would be convenient to use it in patients with neurodegenerative diseases. Additionally, they reinforce their hypothesis by outlining other recent findings showing the bidirectional interactions between lysophospholipids, microglia, and age-related neurodegenerative diseases [12].

Fatty acids (n-3 PUFA) are long chain polyunsaturated fatty acids that contain 18, 20 or 22 carbon atoms and that exert a multitude of benefits on human health. Specifically, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to

produce both cardioprotective and responses by modulating membrane phospholipids, thereby improving cardiac mitochondrial functions and energy production. Dietary supplementation of n-3 PUFAs has been found to reduce the endothelial cell apoptosis and mitochondrial dysfunction associated with oxidative stress. Additionally, it has the capacity to restore myocardial performance and vascular reactivity by counteracting the release of pro-inflammatory cytokines in vascular tissues and in the myocardium. In their review, Francesca Oppedisano et al. [13] have summarized the molecular mechanisms underlying the antioxidant and anti-inflammatory effects of n-3 PUFAs on vascular and cardiac tissues and their implication in the prevention and treatment of cardiovascular diseases.

Due to medical advances and lifestyle changes, the life expectancy of the population has increased. For this reason, it is important to achieve healthy aging by reducing the risk factors that cause age-related damage and pathologies. Through nutrition, one of the pillars of health, we are able to modify these factors by modulating the intestinal microbiota. In their bibliographic review, Elisa Sanchez-Morate et al. [14] show that diets such as the Mediterranean and Oriental diets exert healthy effects, mainly due to the high consumption of polyphenols and fibers, which interact with the intestinal bacteria, thereby generating beneficial effects on the body. Additionally, the low consumption of fats in these diets favors the state of the microbiota, thereby further contributing to the maintenance of good health [14].

In conclusion, this Special Issue addresses the roles that oxidative stress and inflammation play in different pathologies, such as ophthalmological, respiratory, and cardiovascular diseases as well as in obesity, cancer, and diabetes. In addition, without forgetting that pharmacological treatments are essential for their treatment, it has been revealed how natural products, diets, or certain foods could positively influence action against these diseases.

**Author Contributions:** Conceptualization, J.G.; software, K.S.; validation, J.G. and K.S.; resources, J.G. and K.S.; writing—original draft preparation, J.G.; writing—review and editing, K.S.; visualization, J.G. and K.S.; supervision, J.G. All authors have read and agreed to the published version of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Antioxidant**/**Anti-Inflammatory E**ff**ects of Caloric Restriction in an Aged and Obese Rat Model: The Role of Adiponectin**

#### **Daniele La Russa 1,2,\*, Alessandro Marrone 2,3, Maurizio Mandalà 3, Rachele Macirella 3 and Daniela Pellegrino 2,3,\***

Received: 31 October 2020; Accepted: 23 November 2020; Published: 25 November 2020

**Abstract:** Caloric restriction (CR) represents a powerful intervention for extending healthspan and lifespan in several animal models, from yeast to primates. Additionally, in humans, CR has been found to induce cardiometabolic adaptations associated with improved health. In this study, we evaluated in an aged and obese rat model the effect of long-term (6 months) caloric restriction (−40%) on the oxidative/inflammatory balance in order to investigate the underlining mechanisms. In plasma, we analyzed the oxidative balance by photometric tests and the adiponectin/tumor necrosis factor-α-induced gene/protein 6 (TSG-6) levels by Western blot analysis. In the white adipose tissue, we examined the protein levels of AdipoR1, pAMPK, NFκB, NRF-2, and glutathione S-tranferase P1 by Western blot analysis. Our results clearly showed that caloric restriction significantly improves the plasmatic oxidative/inflammatory balance in parallel with a major increase in circulating adiponectin levels. Additionally, at the level of adipose tissue, we found a positive modulation of both anti-inflammatory and antioxidant pathways. These adaptations, induced by caloric restriction, with the achievement of normal weight, suggest that inflammatory and redox imbalance in obese aged rats appear to be more linked to obesity than to aging.

**Keywords:** caloric restriction; inflammation; oxidative balance; adiponectin; plasma; white adipose tissue

#### **1. Introduction**

Obesity prevalence is constantly growing worldwide in all age groups and represents a serious publichealth problem, given its close correlation with several chronic diseases [1,2]. Obesity is not always the result of an excessive intake of food and/or lack of physical activity, but it is also favored by other factors (stress, drugs, or hormonal metabolic alterations) that alter the physiological mechanisms capable of regulating the supply of energy in relation to consumption, as happens, for example, with advancing age. In particular, the age-related changes in body fat distribution (increase in abdominal obesity) and metabolism (insulin resistance and metabolic syndrome) represent key factors in a vicious cycle that can accelerate the aging process itself and the progression of age-related diseases [3,4]. Lines of evidence from several studies have shown that obesity adversely affects health status and life span through cellular processes in a manner similar to aging [3,4].

Aging is a complex, multifactorial process driven by both intrinsic (genetic) and extrinsic (environmental) factors. The aging process is linked to homeostasis deterioration characterized by an alteration of both oxidative and inflammatory states. The free radical theory of aging has been proposed since 1956 and highlights how the accumulation of oxidized biomolecules during chronic oxidative stress is the basis of the age-related structural and functional alterations of all types of cells [5,6]. Over the past decades, the close link between free radicals and aging has found corroboration in several studies [7–9], although some published papers appear to contradict this theory [10,11]. The aging process is also characterized by a chronic low-grade of inflammation, defined as inflamm-aging [12], with an upregulation of proinflammatory cytokines and inflammatory compounds, all of which have been shown to be involved in the pathogenesis of age-related diseases [13]. The theory of oxidation and inflammation of aging, termed oxi-inflamm-aging, was proposed to better define what occurs during the aging process [14,15].

Additionally, obesity is characterized by a redox/inflammatory imbalance that is probably due to the endocrine activity of adipose tissue via the production of several adipokines involved in the development of inflammation, oxidative stress, abnormal lipid metabolism, increased production of insulin, and insulin resistance [16–21]. In particular, visceral obesity is associated with the development of chronic metabolic diseases through a convergence of a chronic inflammatory state and enhanced production of reactive oxygen species [21]. Lines of evidence from several studies have shown that obesity adversely affects health status and life span through cellular processes in a manner similar to aging, and the link between these processes appears to be adiponectin [3,4]. This adipokine, mainly secreted from adipocytes, modulates a number of metabolic processes and represents a key molecule in maintaining the functionality of many organs [22]; it is also involved in the development and progression of several obesity-related malignancies such as breast cancer [23,24]. As adiponectin physiologically decreases with age [25] and obesity negatively affects the decline in adiponectin levels [26], it can be assumed that obesity alters the aging process through the regulation of adiponectin.

Caloric restriction (CR) is a dietary intervention with a chronic reduction of total calorie intake without incurring malnutrition. CR activates a complex series of events and represents a powerful intervention for extending healthspan and lifespan in several animal models, from yeast to primates [27]. Although the detailed mechanisms remain to be established, several sets of experimental data have confirmed that CR induces metabolic remodeling in several organs and tissues, including white adipose tissue (WAT). In particular, CR modulates the adipokine expression profile in rodent WAT [28,29]. WAT plays a central role in the regulation of both energy storage and expenditure and is also directly involved in the regulation of lifespan. The current knowledge on the molecular and cellular events associated with CR underline how the beneficial effects of this dietary intervention can be due to an improvement in the plasma and tissue redox inflammatory profile [30–32].

Given the close relationship between obesity and aging/age-related diseases, the present studyundertakes to determine the similarities in underlying mechanisms related to both obesity and aging, focusing our attention on the key role played by adiponectin. Since the use of old animals is very important to phenocopy the systemic aging context, we used an elderly and obese rat model, a useful model that mimics the weight gain due to aging that occurs in humans.

#### **2. Materials and Methods**

#### *2.1. Animals*

Experiments were performed on young (13–15 weeks old, *n* = 6) and aged (72 weeks old, *n* = 12) male Sprague–Dawley rats. Animals were housed in the animal care facility of the University of Calabria (Italy) in a 12:12 h light–dark cycle and temperature-controlled rooms (22 ◦C) and had free access to food and water. The old animals were then divided into two subgroups: (1) Control rats were continued on an ad libitum diet of a standard laboratory chow (ssniff diet V1535, German; metabolizable energy 3.057 Kcal/Kg), while (2) food-restriction rats were fed a diet of the same chow, restricted to 60% of the intake measured by weight in pairedcontrol chow-fed rats. The food restriction diet was carried on for a total period of 6 months, and thenthe aged animals (control and treated) were

sacrificed at 24 months old. Water and food intakes were recorded every other day, while body mass was recorded monthly. Animals were euthanized with isoflurane (4%) followed by cervical transection, and several tissueswere immediately removed: abdominal fat, kidney, blood, and skeletal muscle. All experiments were carried out in accordance with the European Guidelines for the care and use of laboratory animals (Directive 26/2014/EU) and were approved by the local ethical committee of the University of Calabria and by the Italian Ministry of Health (license n.295/2016-PR).

#### *2.2. Measurement of Plasma Oxidative Status*

Plasma oxidative status was evaluated through photometric measurement kits combined with a free radical analyzer system and a spectrophotometric device reader (FREE Carpe Diem, Diacron International, Grosseto, Italy), which are routinely used in our laboratory in both human and rat models [33–38]. The diacron-reactive oxygen metabolite (dROM) test was employed for analyzing the total amount of hydroperoxides in plasma samples. The normal range of the test results was 250–300 U.CARR (Carratelli Units), where 1 U.CARR corresponds to 0.8 mg/L of hydrogen peroxide. Total plasma antioxidant capacity was measured using a biological antioxidant capacity (BAP) test. Results are expressed in μmol/L of the reduced ferric ions.

#### *2.3. Western Blot and Densitometric Analysis*

White adipose tissue samples were lysed in ice-cold RIPA buffer containing a protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy) and centrifuged for 20 min at 20,817× *g* at 4 ◦C. After removing the layer of lipids, supernatants were collected and the protein concentration was quantified using the Bradford method (Sigma, St Louis, MO, USA). For blood samples, after centrifuging for 15 min at 1500× *g* at 4 ◦C, plasma was collected and diluted (1:10 *v*/*v*) in ice-cold RIPA buffer containing a protease inhibitor cocktail (Sigma-Aldrich, Milan, Italy). The same amounts of total protein lysate were heated for 5 min in Laemmli buffer (SigmaAldrich, Milan, Italy), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) in a Bio-Rad Mini Protean III machine and then electroblotted onto a nitrocellulose membrane (NitroBind, Maine Manufacturing, Maine, USA) using a mini transblot (BioRad Laboratories, Hercules, CA, USA). The gels for immunoblot analyses were transferred to a nitrocellulose membrane and incubated overnight at 4 ◦C, with primary antibodies diluted in TBS-T directed against AdipoR1, pAMPK, NFκB, NRF-2, GSTP1, TSG6, and adiponectin, followed by species-specific peroxidase-linked secondary antibodies (1:2000; Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 1 h at room temperature. Immunodetection of protein bands was performed with an enhanced chemiluminescence kit (Western Blotting Luminol Reagent, Santa Cruz Biotechnology Inc.), and the membranes were exposed to X-ray films (Ultracruz Autoradiography Film, Santa Cruz Biotechnology Inc.). The films were then scanned, and densitometric analysis of the bands was performed using ImageJ software (1.52a version, National Institutes of Health, Bethesda, MD, USA). Beta actin and serum albumin were used as loading controls, respectively, for tissue and blood protein normalization.

#### *2.4. Statistical Analysis*

Data were analysed by one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test using GraphPad/Prism version 5.01 statistical software (SAS Institute, Abacus Concept Inc., Berkeley, CA, USA. All the results are expressed as the mean ± SE.

#### **3. Result**

#### *3.1. E*ff*ects of CR on Obesity and Plasmatic Oxidative Balance*

In laboratory rodents, obesity is defined as the achievement of a 20% increase in body mass index [39]. In our obesity model, the aged rats (2 years old) showed a significant increase in body weight, greater than 45%, compared to young rats (13–15 weeks old). Furthermore, these aged

specimens had a significant plasma oxidative imbalance, with a considerable increase in oxidative stress and a parallel and severe decrease in the antioxidant barrier. In our model, the calorie restriction (−40%) for 6 months determined a significant decrease in body weight (−34%) vs. control and, at the same time, significantly improved the oxidative balance with a remarkable decrease in oxidative stress, independent of changes in the plasmatic antioxidant barrier. The body weight data and the trend of both oxidative stress (d-ROMs) and antioxidant capacity efficiency (BAP) in plasma of young rats (Y), obese aged (OA), and aged undergoing calorie restriction (CRA) rats are reported in Figure 1.

**Figure 1.** Plasmatic values of oxidative stress (d-ROMs) (**a**) and antioxidant capacity efficiency (BAP) (**b**) tests in young rats (Y), obese aged (OA), and aged undergoing calorie restriction (CRA)rats. Data are means ± SE of five determinations for each animal (*n* = 6). Statistical differences were evaluated by one-way ANOVA, followed by Bonferroni's multiple comparison test (\* *p* < 0.05; \*\* *p* < 0.001; \*\*\* *p* < 0.0001).

#### *3.2. E*ff*ects of CR on Plasmatic Adiponectin Levels and Inflammation Markers*

To assess the possible role played by adiponectin in the antioxidant effect of CR, we evaluated its plasmatic protein levels through Western blotting analysis. In parallel, we also evaluated the plasmatic protein levels of TSG-6, a multifunctional protein associated with inflammation. Our results clearly indicate that the obesity condition associated with aging does change the circulating adiponectin levels and also induces a significant increase in TSG-6 levels, confirming the low-grade proinflammatory state found in both obesity and aging (Figure 2a). The caloric restriction determines an increase in the adiponectin plasma levels (doubled compared to both young and aged rats) and a significant reduction of the TSG-6 proinflammatory marker, which reverts to the values of the young rats (Figure 2b).

**Figure 2.** Western blotting of adiponectin (**a**) and TSG6 (**b**) in plasma samples of Y, OA, and CRA rats; (**a1**,**b1**) show the densitometric quantification of the blots. Protein loading was verified by plasmatic albumin level. Data are means ± SE of five determinations for each animal (*n* = 6). Statistical differences were evaluated by one-way ANOVA, followed by Bonferroni's multiple comparison test (\* *p* < 0.05; \*\* *p* < 0.001).

#### *3.3. E*ff*ects of CR on AdipoR1–pAMPK–NF*κ*B Pathway in Adipose Tissue*

Since adipose tissue represents a crucial junction in inflammatory processes and it is also a reserve of adipokines, we analyzed the AdipoR1–pAMPK–NFκB pathway at the level of adipose tissue. Through its receptor, adiponectin mediates AMPK activation (pAMPK), which is involved in the inhibition of NFκB activation and the suppression of inflammation. In our model, the obesity condition associated with aging determines an upregulation of AdipoR1, with a consequent increase of the AMPK active form in adipose tissue (Figure 3a,b). Despite the activation of this anti-inflammatory pathway, NFκB levels found in obese and aged subjects (control) were high compared to the young rats. CR, with the achievement of normal weight and the rise of both AdipoR1 and pAMPK, significantly inhibited NFκB expression (Figure 4) and consequently suppressed the proinflammatory state, which appears to be more linked to obesity than aging (Figure 2b).

**Figure 3.** Western blotting of AdipoR1 (**a**) and AMPK activation (pAMPK) (**b**) in adipose tissue samples of Y, OA, and CRA rats; (**a1**,**b1**) show the densitometric quantification of the blots. Protein loading was verified by using the anti-β-actin antibody. Data are means ± SE of five determinations for each animal (*n* = 6). Statistical differences were evaluated by one-way ANOVA, followed by Bonferroni's multiple comparison test (\*\* *p* < 0.001).

**Figure 4.** Western blotting of NFκB (**a**) in adipose tissue samples of Y, OA, and CRA rats; (**a1**) shows the densitometric quantification of the blots. Protein loading was verified by using the anti-β-actin antibody. Data are means ± SE of five determinations for each animal (*n* = 6). Statistical differences were evaluated by one-way ANOVA, followed by Bonferroni's multiple comparison test (\*\*\* *p* < 0.0001).

#### *3.4. E*ff*ects of CR on Antioxidant Enzymes in Adipose Tissue*

To analyze the oxidative imbalance linked to obesity detected at the plasma level, we evaluated two important cytoplasmatic antioxidant enzymes, SOD1 and GSTP1, in adipose tissue. SOD1, the most important preventive antioxidant, showed no significant changes in the three groups of rats (data not shown). Regarding the GSTP1 monomer (23 kDa), the form with antioxidant and proliferative activity, our results showed a significant increase in obese rats while it is reduced in CRArats (Figure 5a). We also evaluated NRF2, a transcription factor physiologically present in the cytosol that migrates to the nucleus only under stress conditions, where it stimulates the expression of antioxidant genes. Our results clearly showed that in obese aged rats, the cytoplasmic fraction of NRF2 is significantly increased, while in CRA rats, it is significantly decreased (Figure 5b).

**Figure 5.** Western blotting of GSTP1(**a**) and NRF2(**b**) in adipose tissue samples of Y, OA, and CRA rats; (**a1**,**b1**) show the densitometric quantification of the blots. Protein loading was verified by using the anti-β-actin antibody. Data are means ± SE of five determinations for each animal (*n* = 6). Statistical differences were evaluated by one-way ANOVA, followed by Bonferroni's multiple comparison test (\* *p* < 0.05; \*\*\* *p* < 0.0001).

#### **4. Discussion**

This study was designed to evaluate the effect of long-term (6 months) caloric restriction (−40%) on the oxidative/inflammatory balance in an aged and obese rat model and the putative role of adiponectin. Our results showed notable alterations in redox/inflammatory status in our aging obesity model compared to young rats and revealed that CR treatment improves the plasmatic and cellular capacity to neutralize oxidative insults and induces a significant reduction of proinflammatory markers. This improvement of the redox/inflammatory status during CR is connected with an important increase in adiponectin plasma levels, thus suggesting an important role of this adipokine in the antioxidant/anti-inflammatory properties of the organism. Additionally, at the level of adipose tissue, we found a positive modulation of both anti-inflammatory and antioxidant pathways.

During the aging process, there is often a gradual increase in body weight and, typically, the fat is redistributed from the subcutaneous to the abdominal deposits and the liver, and this phenotypic change can affect energy metabolism and systemic insulin resistance [40]. Aged rodents develop increased fat mass, with close similarities to aged humans [41], making these animals excellent experimental models for analyzing both obesity and aging. In our experimental model, age-related physiological weight gain leads to two-year-old rats with 45% increased body weight compared to the young animals and, therefore, with overt obesity [39]. These aged and obese specimens, according to the oxidation–inflammation theory of aging [14,15], showed a considerable increase in oxidative stress, a parallel and severe decrease in the antioxidant barrier, and a significant upregulation of TSG-6, a multifunctional protein associated with inflammation [42]. Our results clearly indicate that the obesity condition associated with aging induces oxidative/inflammatory stress situations, confirming the oxidative imbalance and low-grade proinflammatory state found in both obesity and aging [4]. In our aged and obese rat model, we did not detect changes in plasma adiponectin levels with respect to the young animals. Several factors may influence adiponectin levels and activity. In obesity, as the metabolic role of adipocytes changes, the secretion of adiponectin decreases in both humans and rodents, and this is associated with chronic inflammation [4,26,43]. Data on adiponectin levels and aging have revealed conflicting results; some authors have indicated that plasmatic adiponectin levels increase with advancing age [44], while others have reported no age-related changes in its levels [4,25].

Interestingly, in our model, the long-term caloric restriction reversed obesity, determining a significant decrease in body weight (−34%) and, in parallel, increasing the adiponectin plasma levels, which were doubled compared to both young and obese aged control rats. The increased adiponectin plasma levels led to the improvement of the proinflammatory state, with a severe reduction of the TSG-6 proinflammatory marker, which reverted to the values of the young rats. The protein TSG6, a critical anti-inflammatory cytokine detected in the context of many inflammatory diseases, is produced in response to inflammatory mediators, mostly after TNF-α stimulation [42]. The anti-inflammatory and cytoprotective effects of adiponectin have long been known [45–47], and caloric restriction is also considered a possible strategy to better body metabolic and inflammatory profiles [48,49]. The role played by adiponectin and caloric restriction in maintaining/restoring the oxidative balance is less clear. A wide number of studies have reported a negative correlation of adiponectin levels with markers of oxidative stress [50] and a cause–effect relationship between the metabolic syndrome and a deteriorated oxidative status related to the altered secretion of adipokines [51]. In addition, it has been suggested that oxidative stress can inhibit adiponectin expression in obesity, but the mechanism underlying this regulation is unclear [19]. Our results show a clear improvement in the plasma redox status with a remarkable decrease in oxidative stress, independent of changes in the plasmatic antioxidant barrier.

To investigate the mechanisms triggered by caloric restriction in the improvement of oxidative/inflammatory status in our model, we analyzed anti-inflammatory and antioxidant pathways at the level of adipose tissue, focusing on the key role played by adiponectin. Adipose tissue represents a crucial junction in inflammatory/oxidant processes, and it is also a reserve of adipokines. The biological activities of adiponectin are closely related to the activation of AMP-activated protein kinase (AMPK), a key enzyme in the regulation of cellular energy homeostasis and fatty acid metabolism [52,53]. Several authors have reported that AMPK plays an important role in the modulation of inflammation, and AMPK activators exert a protective effect in animal models of inflammatory diseases [45]. In addition, AMPK can inhibit ROS generation [53,54]. Recently, it has been suggested that adiponectin may control inflammation by upregulating AMPK phosphorylation and then reducing the ROS-initiated inflammatory response [54]. Moreover, in cellular models, adiponectin treatment linearly decreased the production of ROS [54]. Through its receptor, adiponectin exerts an anti-inflammatory response by AMPK activation (pAMPK), which is involved in the inhibition of NFκB activation and consequent TNF-α and IL-1β downregulation [45].

In our model, the obesity condition associated with aging determines an upregulation of AdipoR1, with a consequent increase of the AMPK active form in adipose tissue. Despite the activation of this anti-inflammatory pathway, the NFκB levels found were high compared to young rats. CR, with the achievement of normal weight, enhances the stimulation of both AdipoR1 and pAMPK, with significant inhibition of NFκB expression and, consequently, suppression of the proinflammatory state.

To better understand the tissue redox balance, we also focused on the expression of two important cytoplasmatic antioxidant enzymes, SOD1 and GSTP1, in adipose tissue. CR has been shown to positively affect tissue redox homeostasis by the enhancement of endogenous antioxidant systems [55]. In our model, the expression of SOD1, the most important preventive antioxidant, showed no significant changes in all groups of rats used, while the GSTP1 monomer, the form with antioxidant and proliferative activity, showed a negative modulation by CR treatment. We also evaluated the expression of NRF2, a transcription factor that is physiologically present in the cytosol that migrates to the nucleus only under stress conditions, where it stimulates the expression of antioxidant genes. Our results clearly show that in obese aged rats, the cytoplasmic fraction of NRF2 is significantly increased and that CR induces a substantial decrease, probably due to a translocation effect in the nucleus to modulate the expression of antioxidant genes.

Overall, our results highlight that the long-term caloric restriction leads to the achievement of normal weight and induces a number of adaptations that improve the redox/inflammatory status at both plasma and tissue levels, suggesting that inflammatory and redox imbalances in obese aged rats appear to be more linked to obesity than to aging.

**Author Contributions:** Conceptualization, D.P. and D.L.R.; methodology, D.P., D.L.R., and A.M.; software, D.P., D.L.R., and A.M.; validation, D.P. and D.L.R.; formal analysis, D.P., D.L.R., A.M., R.M., and M.M.; investigation, D.P., D.L.R., and A.M.; resources, D.P., D.L.R., and M.M.; data curation, D.P., D.L.R., and A.M.; writing—original draft preparation, D.P. and D.L.R.; writing—review and editing, D.P., D.L.R., and A.M.; visualization, D.P., D.L.R., A.M., R.M., and M.M.; supervision, D.P. and D.L.R.; project administration, D.P. and D.L.R.; funding acquisition, D.P. and M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **Identification of Toxicity Parameters Associated with Combustion Produced Soot Surface Chemistry and Particle Structure by In Vitro Assays**

**Heba Al Housseiny 1, Madhu Singh 2, Shaneeka Emile 3, Marvin Nicoleau 4, Randy L. Vander Wal 2,5 and Patricia Silveyra 1,3,\***

Received: 3 August 2020; Accepted: 8 September 2020; Published: 11 September 2020

**Abstract:** Air pollution has become the world's single biggest environmental health risk of the past decade, causing millions of yearly deaths worldwide. One of the dominant air pollutants is fine particulate matter (PM2.5), which is a product of combustion. Exposure to PM2.5 has been associated with decreased lung function, impaired immunity, and exacerbations of lung disease. Accumulating evidence suggests that many of the adverse health effects of PM2.5 exposure are associated with lung inflammation and oxidative stress. While the physical structure and surface chemistry of PM2.5 are surrogate measures of particle oxidative potential, little is known about their contributions to negative health effects. In this study, we used functionalized carbon black particles as surrogates for atmospherically aged combustion-formed soot to assess the effects of PM2.5 surface chemistry in lung cells. We exposed the BEAS-2B lung epithelial cell line to different soot at a range of concentrations and assessed cell viability, inflammation, and oxidative stress. Our results indicate that exposure to soot with varying particle surface composition results in differential cell viability rates, the expression of pro-inflammatory and oxidative stress genes, and protein carbonylation. We conclude that particle surface chemistry, specifically oxygen content, in soot modulates lung cell inflammatory and oxidative stress responses.

**Keywords:** air pollution; soot; particulate matter; lung inflammation; functional groups

#### **1. Introduction**

Air pollution has become one of the greatest environmental health hazards to millions around the world and is primarily caused by years of industrialization and population growth, particularly in developing countries [1,2]. In the past decade, air pollution alone has been linked to 7 million annual deaths worldwide. The Global Burden of Disease Study identified air pollution as a risk factor for cardiovascular disease and respiratory infections, and it is estimated to have contributed to nearly 5 million premature deaths worldwide in 2017 alone [3,4].

Air pollutants are separated into two categories depending on the source of production. Primary pollutants such as heavy petroleum products (soot) and oxides of nitrogen (NOx) and sulfur (SOx) are emitted into the air by the combustion of fossil fuels, vehicle exhaust, natural fires, industrial practices, and natural dust [5]. Secondary pollutants are formed when primary pollutants react with

other molecules in the atmosphere, altering their toxin absorption ability. Secondary pollutants include ozone, acid rain, and particulate air pollution [5].

Particulate matter (PM) is a complex mixture of volatile organic and inorganic compounds, including solid particles and liquid droplets, with different physicochemical properties and toxicity [6,7]. Carbon black, produced by an incomplete combustion of soot, comprises a significant portion of PM. The toxicity of PM is characterized by its size, surface chemistry, solubility, and ability to form reactive oxygen species. Fine PM size ranges from 2.5 μm to 10 μm in aerodynamic diameter (PM2.5 to PM10). Notably, PM2.5 is known to exacerbate many pre-existing heart and respiratory diseases that increase the risk of death [8]. The finest particles, PM2.5, have been reported to induce inflammation and oxidative stress in the respiratory tract, and they exacerbate respiratory and cardiovascular diseases including long-term chronic diseases such as cancer and asthma. Yet, the mechanisms underlying PM2.5-induced inflammation are still unknown [8–12]. The mortality rate due to PM2.5 is more prominent in the elderly, pregnant women, infants, and low-income populations [13,14]. Exposure to fine particles affects lower-income populations in which a significant proportion have an income below the poverty threshold, leading to environmental inequality and propagating economic burden [15,16].

Exposure of the nasal and lung epithelia to PM2.5 triggers an inflammatory response within the respiratory system that, if not resolved, can contribute to chronic inflammation. This inflammatory response is characterized by the production of extracellular signaling proteins, including cytokines and chemokines secreted by epithelial and immune cells, and it plays a critical role in the lung's defense mechanism against oxidative stress [17]. Previous studies have described an increase in the expression of cellular pro-inflammatory cytokines such as interleukin 6 (IL-6) and interleukin 1 beta (IL-1β) following exposure to PM2.5 [18–20]. Protein carbonylation has also been reported as a biomarker of oxidative stress-induced protein damage and has been associated with the development of cardiovascular and respiratory diseases [21,22]. A few studies have shown that protein carbonylation is increased in PM2.5-treated human keratinocytes and rat epithelial lung cells [23]. Thus, while cytokine expression and protein carbonylation may serve as important biomarkers for numerous lung diseases, their role in PM2.5-induced lung inflammation has not been extensively investigated in human cells.

Many of the adverse health effects of PM2.5 exposure are hypothesized to be derived from oxidative stress, which is initiated by the formation of reactive oxygen species (ROS) within cells [24]. Although several studies have demonstrated the ROS potential and inflammatory action of diesel particulates from diesel engine combustion, the causative factors are less clear. Most studies use organics diluted and extracted from real exhaust particulates, and results reported may be skewed relative to cell exposure to the "complete" soot, i.e., PM2.5 along with its heteroatoms and condensed fractions. It is increasingly being recognized that extracts of diesel soot are a poor representation of the full range of toxicity and mutagenic effects [25]. Rather, the particles, and specifically their surface chemistry, are thought to have considerable direct health impacts. Yet, no studies have assessed and quantified the effect of parameters such as particle size, morphology, and surface chemistry in the cellular response to combustion-formed PM2.5 exposure. Therefore, we investigated these effects in human lung epithelial cells by using modified functionalized carbon blacks (soot) as surrogates for combustion-formed particulates. Our goal in using this approach was to understand the hierarchy of detrimental effects of soot from a combustion engine, subject to atmospheric aging, including the health effects of the primary particle itself, as compared to those of the functional groups on the carbonaceous backbone.

This work focuses on the identification of oxidative and pro-inflammatory factors induced as a response to exposure of human lung epithelial cells to synthetic soot in various surface chemistry forms. By exposing a male lung epithelial cell line (BEAS-2B) to lab-generated R-250 carbon black (nascent, nitric acid-treated, and ozone-treated carbon black), we tested the hypothesis that exposure to soot with different surface composition results in differential toxicity, inflammatory, and oxidative stress responses in human lung epithelial cells. We assessed the cell viability and expression of genes related to the inflammatory response and oxidative stress, including IL-1β, IL-6, superoxide dismutase

2 (SOD2), nuclear factor erythroid 2-related factor 2 (NFE2L2), and protein carbonylation levels at different time points and concentrations of soot. Our data indicate that the surface chemistry and concentration of soot, specifically higher oxygen content and concentration, play a critical role in the inflammatory and immune response initiated in the lung epithelium, and they are associated with a decrease in cell viability and an increase in protein carbonylation levels and expression of inflammatory cytokine genes.

#### **2. Experimental Section**

#### *2.1. Synthesis and Characterization of Soot*

Commercially produced carbon black (Regal 250, Cabot Corporation, Apharetta, GA, USA) was used as the model carbon black for its chemical purity and absence of organic content [25]. Synthetic soot was produced by functionalizing R250 carbon black via controlled oxidation by the methods described below. Four soot preparations were used for experiments:

All synthetic soot preparations were further characterized for their atomic oxygen content and functional groups introduced onto the carbon black surface. Characterization was performed by X-ray photoelectron spectroscopy (XPS) and thermogravimetric analysis (TGA), as indicated below.

#### *2.2. X-Ray Photoelectron Spectroscopy (XPS)*

X-ray photoelectron spectroscopy was used to identify and quantify possible different functional groups on the carbon surface and their contribution to the total surface atomic oxygen. XPS is a surface analysis technique based on the photoelectric effect where incoming X-rays of a known wavelength (λ) are used to eject surface electrons. With the known total energy of a photon, the kinetic energy (KE) of the ejected electron is measured, and the binding energy (BE) is calculated. The BE of a core–shell electron is characteristic of the element from which it is ejected and is used to identify the elements present. XPS experiments were performed using a Physical Electronics VersaProbe II instrument equipped with a monochromatic Al kα x-ray source (hν = 1486.7 eV) and a concentric hemispherical analyzer. Charge neutralization was performed using both low-energy electrons (<5 eV) and argon ions. The binding energy axis was calibrated using sputter-cleaned Cu foil (Cu 2p3/2 = 932.7 eV, Cu 2p3/2 = 75.1 eV). Peaks were charge referenced to the C-C band in the carbon 1s spectra at 284.5 eV. Quantification of the elements was done by curve-fitting of the high-resolution scan using the CasaXPS software.

#### *2.3. Thermogravimetric Analysis (TGA)*

A TA instruments Thermogravimetric Analyzer TA 5500 coupled with a Discovery Mass Spectrometer (MS) was used to analyze mass loss and the composition of the evolved gases. The temperature was ramped up at 5 ◦C/min in an inert atmosphere. Thermal analysis of the material gives a qualitative assessment of the volumetric uniformity of functionalization of the carbon; therefore, TGA is used as a bulk material characterization tool to complement the results observed by XPS. When subject to a steady temperature ramp, functional groups on the carbon oxidize (leave) at different temperatures. The subsequent mass loss curve can be used to qualitatively identify the oxygen functional groups present on the carbon blacks.

#### *2.4. Cell Culture and Soot Exposure*

Cells from the male bronchial epithelial cell line BEAS-2B (ATCC® CRL-9609™) were thawed and cultured in RPMI-1640 medium (Gibco), supplemented with 2 mM glutamine (Gibco), 10% (*v*/*v*) heat-inactivated fetal bovine serum (VWR), and 10,000 units of Penicillin–Streptomycin (Gibco). Cells were grown in a 75 cm2 (T75) cell culture Flask (Corning) and incubated at 37 ◦C in a humidified chamber with 5% CO2.

#### 2.4.1. Cell and Soot Exposure for Cell Viability Assessment

For cell viability studies, BEAS-2B cells were plated in 24-well plates (Corning) at a density of 50,000 cells per well overnight prior to treatment with the soot preparations. The powder soot preparations were dissolved in a small amount of DMSO and then diluted in PBS. The final concentration of DMSO in the cell exposure medium was <1%. Moreover, the volume of soot solution added did not exceed 10% of the total media volume in the well. Cells were incubated with the soot preparations described above for either 6 h or 24 h at concentrations ranging from 1.56 μg/mL to 100 μg/mL at 37 ◦C in a humidified chamber with 5% CO2.

#### 2.4.2. Cell Culture and Soot Exposures for Gene Expression Assessment

To quantify the expression of inflammatory and oxidative stress genes, BEAS-2B cells were plated in 24-well plates (Corning) at a density of 50,000 cells per well overnight prior to treatment with the soot preparations. The powder soot preparations were dissolved in methanol (MeOH, negative control) and then diluted in PBS. The final concentration of MeOH in the cell exposure media was <0.5%. Moreover, the volume of soot solution added did not exceed 10% of the total media volume in the well. Cells were incubated for 6 h at 37 ◦C in a humidified chamber with 5% CO2 with all soot preparations, at concentrations of 1.56 to 12.5 μg/mL for inflammatory genes expression and of 3.125 μg/mL for oxidative stress genes expression.

#### 2.4.3. Cell Culture and Soot Exposures for Protein Carbonylation Assessment

For protein carbonylation studies, BEAS-2B cells were grown in a 75 cm2 (T75) cell culture Flask (Corning) and incubated at 37 ◦C in a humidified chamber with 5% CO2. To quantify protein carbonylation, cells were plated in 12-well plates (Corning) at a density of 100,000 cells per well overnight prior to treatment with the soot preparations. The powder soot preparations were dissolved in methanol (MeOH, negative control) and then diluted in PBS. The final concentration of MeOH in the cell exposure media was <1%. Moreover, the volume of soot solution added did not exceed 10% of the total media volume in the well. Cells were incubated for 24 h at 37 ◦C in a humidified chamber with 5% CO2 with all the soot preparations at concentrations of 25 μg/mL.

#### *2.5. Cell Viability Assay*

Cell viability was assessed using the MultiTox-Fluor Multiplex Cytotoxicity Assay Kit (Promega, Madison WI, USA), according to the manufacturer's instructions at 6 h and 24 h post-exposure with the different soot preparations. The assay was tested for cell viability and cytotoxicity using specific positive controls: digitonin (toxicity) and ionomycin (necrosis).

#### *2.6. RNA Purification and cDNA synthesis*

After exposure to soot preparations, cells were harvested in TRizol (Life Technologies, Austin, TX, USA) and RNA was extracted using the Direct-zol kit (Zymo Research, Irvine, CA, USA) in the presence of DNAse. Total RNA was quantified by nanodrop measurement and stored at −80 ◦C until further analysis. For cDNA synthesis, 500 ng of RNA were retrotranscribed using the High Capacity cDNA kit (Life Technologies), following the manufacturer's protocol. The cDNA reactions were stored at −20 ◦C until further use.

#### *2.7. Real-Time PCR*

The expression of inflammatory and oxidative stress-related genes was measured in 40 ng of cDNA by Real-Time PCR with TaqMan™ assays (Life Technologies). The following probes were used: IL1B (assay Hs01555410), IL6 (assay Hs00174131), NFE2L2 (assay Hs00232352), and SOD2 (assay Hs00167309). A housekeeping gene 18S (assay Hs03003631, Life Technologies) was used as a normalization control from 2 ng of cDNA. The reactions were conducted in triplicate using the TaqMan™ Fast Advanced Master Mix in 10 μL of final volume, following the manufacturer's protocol. Expression results (Ct values) were monitored and extracted using the QuantStudio 12K Flex Software, and data were analyzed using the relative quantification method [26].

#### *2.8. Total Protein Determination*

After exposure to soot, cells were harvested in 150 μL of 20 mM Tris-HCl lysis buffer at pH 7.5. The total protein concentration in lysates was determined by the Bicinchoninic Acid (BCA) protein assay (Pierce), following the manufacturer's protocol, using bovine serum albumin as a standard.

#### *2.9. Protein Carbonylation Assay*

The high sensitivity Protein Carbonyl ELISA Kit (Enzo Life Sciences, Farmingdale, NY, USA; cat #ALX-850-312-KI01) was used to determine the concentration of protein carbonylation starting from a sample volume containing 200 μg of protein and following the manufacturer's protocol.

#### *2.10. Statistical Analysis*

Cell proliferation, gene expression, and protein carbonylation data are presented as means ± SE. Data were plotted, and statistical analyses were performed using the GraphPad Prism software (v.8.4.3). Interactions of treatment and concentration were determined by two-way ANOVA followed by Dunnett's post hoc test, and differences among treatment groups were analyzed by one-way ANOVA followed by Tukey's post hoc analysis with the GraphPad Prism software (v.8.4.3). Values of *p* < 0.05 were considered statistically significant.

#### **3. Results**

#### *3.1. Characterization of Soot*

An illustrative transmission electron micrograph (TEM) of a carbon black aggregate and primary particle is shown in Figure 1. After wet and dry chemical treatment, and prior to conducting cell exposures, the different soot preparations were characterized to determine their atomic oxygen content and functional groups introduced onto the carbon surface by XPS and TGA, as indicated below.

XPS was performed using survey and high-resolution scans, to identify the elements present on the surface of treated soot, specifically oxygen groups. As a baseline, nascent (untreated) carbon black (S1) was also subject to the same analytical procedure. Elements in each soot preparation were quantified via curve-fitting of the high-resolution scans using the commercial software CasaXPS. Table 1

shows the measured atomic percentages of the elements present in the analyzed soot. Table 2 gives the relative percentages of oxygen functional groups present in the soot samples. As expected, the oxygen content is higher in the acid-treated soot (S2) than the ozone-treated soot (S3) and acid + heat-treated soot (S4). Wet acid reflux treatment of carbon black resulted in ≈32 atomic % oxygen compared to a ≈10 atomic % from dry gaseous treatment, the latter being a relatively mild oxidant with oxidation performed at room temperature, explaining this difference in the type and degree of functionalization. Moreover, being the most reactive group, the -COOH functional group is selectively removed with heat treatment from the carbon black, significantly reducing the oxygen content to ≈12 at (%) overall as measured by XPS. TGA-MS revealed a negligible mass loss for the ozone-treated sample (S3) between 300 and 500 ◦C despite the curve fit value of ≈2 at (%) carboxylic assignment. Various factors account for this observation. First, lactone (i.e., ester) and carboxylic anhydride groups can also register as "carboxylic" groups by the XPS curve fit, given similar C1s binding energy. (Notably, these groups do not possess the labile hydrogen nor form an anionic state.) Possessing greater thermal stability, these groups do not decompose at 300 ◦C. Moreover, the 2 at (%) carboxylic content as extracted by deconvolution is near the minimal level detectable by this fitting procedure, which is estimated as ≈1 at (%). Correspondingly, ozone treatment is known to introduce minimal carboxylic (-COOH) functionality in carbons as well.

**Figure 1.** TEM image of a carbon black (**A**) aggregate and (**B**) primary particle.

**Table 1.** Elemental content measured as atomic percent for samples S1–S4. S1: nascent soot, S2: nitric acid-treated soot, S3: ozone-treated soot, S4: nitric acid and heat-treated soot.

1 C: carbon, O: oxygen, N: nitrogen, S: sulfur.

**Table 2.** Oxygen group percentages for samples S1–S4.

#### *3.2. Cell Viability and Cytotoxicity in BEAS-2B Cells Exposed to Synthetic Soot*

BEAS-2B cell viability was measured at 6 and 24 h after exposure to soot preparations (Figures 2 and 3). A significant interaction of concentration and particle effect was found at both time points (*p* < 0.0001, two-way ANOVA). Treatment with ionomycin (positive control) resulted in a 95% reduction in cell viability after 6 h. When assessing independent effects, we found that after 6 h of treatment with S1 and S4 (soot with the lowest -COOH functional group content) (Table 2), there were no significant differences in cell viability at all the concentrations tested (Figure 2). Furthermore, the treatment of cells with soot containing the highest -COOH content (S2, S3) resulted in a reduction of viable cells in a dose-dependent manner. Treatment with S2 (9.4% -COOH content) resulted in a significant loss of cell viability at concentrations of 12.5 μg/mL and above. For S3 (2.4-COOH content), this effect was observed at a higher concentration (at least 50 μg/mL) (Figure 2).

**Figure 2.** BEAS-2B cell viability expressed as percentage of viable cells at 6 h after exposure to four different soot preparations (S1–S4). The bars summarize data from three independent experiments (*n* = 3 replicates per experiment) with results normalized to control (cells exposed to DMSO). Data are expressed as mean ± SEM. \* *p* < 0.001 (Dunnett's post hoc multiple comparisons test).  For a description of S1–S4, see Section 2.

**Figure 3.** BEAS-2B cell viability expressed as percentage of viable cells after exposure to four different soot preparations for 24 h. The graph summarizes data from three independent experiments (*n* = 3 replicates per experiment), with results normalized to control cells (exposed to DMSO). Data are expressed as mean ± SEM. \* *p* < 0.001 (Dunnett's post hoc multiple comparisons test).  For a description of S1–S4, see Section 2.

At the 24-h time point, the exposure of cells to S1 and S4 at a concentration of 50 μg/mL or above induced a significant reduction in cell viability (*p* < 0.001) (Figure 3). The effect was observed at a much lower concentration (6.25 μg/mL and above) for S2, the soot with the highest -COOH content. Finally, the effect was observed at a concentration of 25 μg/mL or higher for S3 (Figure 3).

#### *3.3. Expression of Pro-Inflammatory Genes in Cells Exposed to Synthetic Soot*

To assess the ability of the different soot to induce an inflammatory response in BEAS-2B cells, we conducted real-time PCR experiments on extracts from cells exposed to S1–S4. To avoid significant cell death, we selected concentrations below 12.5 μg/mL and the 6-h time point based on the results obtained in Figure 2. We measured the expression of two pro-inflammatory cytokines (IL-1β and IL-6) that have been previously reported to increase in response to particulate matter exposure in human bronchial epithelial cells [20,27]. We limited our focus to IL-6 and IL-1β to compare the inflammatory effects of all soot preparations on IL-6 and IL-1β expression to these previously established outcomes. A significant interaction of concentration and particle effects was observed after 6 h of exposure to S1–S4 for both genes (IL-1β, *p* = 0.022; IL6, *p* < 0.0001, two-way ANOVA) (Figures 4 and 5).

**Figure 4.** Expression of human interleukin-1 mRNA (IL1B) (relative to 18S expression) in BEAS-2B cell after exposure to soot preparations for 6 h. The graph summarizes data from three independent experiments (*n* = 3 replicates per experiment), with results normalized to controls (exposed to methanol) and expressed as mean ± SEM. Two-way ANOVA interaction: *p* = 0.0109, F (12, 71) = 2.415; concentration effect: *p* < 0.0001, F (4, 71) = 30.51; soot type effect (*p* < 0.0001, F (3, 71) = 15.48). \* *p* < 0.05, different from control (Dunnett's multiple comparison's test). For a description of S1–S4, see Section 2.

The expression of IL-1β was significantly increased in a dose-dependent manner, when cells where exposed to S2, S3, or S4 at concentrations above 3.125 μg/mL (Figure 4). At the lowest concentration tested (1.56 μg/mL), only soot with a higher total oxygen content and C=O groups (S2 and S4) induced significant changes in IL-1β expression (Figure 4). A comparison of IL-1β expression in cells exposed to all soot at the highest concentration (12.5 μg/mL) revealed a significant effect of functionalized particles (S2, S3, S4) vs. nascent particles (*p* < 0.05, one-way ANOVA) (Figure 6A). This effect was also observed at concentrations as low as 3.125 μg/mL (Figure 6B).

**Figure 5.** Expression of human interleukin-6 mRNA (IL6) (relative to 18S expression) in BEAS-2B cell after exposure to soot preparations for 6 h. The graph summarizes data from three independent experiments (*n* = 3 replicates per experiment), with results normalized to controls (exposed to methanol) and expressed as mean ± SEM. Two-way ANOVA interaction: *p* < 0.0001, F (12, 61) = 5.563 concentration effect: *p* < 0.0001, F (4, 61) = 36.99; soot type effect (*p* < 0.0001, F (3, 61) = 22.84). \* *p* < 0.05, different from control (Dunnett's multiple comparison's test). For a description of S1–S4, see Section 2.

**Figure 6.** Human IL-1β mRNA (IL1B) expression (relative to 18S expression) in BEAS-2B cells treated with S1–S4 soot at 3.125 μg/mL (**A**) or 12.5 μg/mL (**B**) for 6 h. The graphs summarize data from three independent experiments (*n* = 3 replicates per experiment), with results normalized to controls (exposed to methanol) and expressed as mean ± SEM. a: different from control, b: different from S1 (*p* < 0.05). One-way ANOVA (*p* < 0.001, F (4, 20) = 20.47).

Similarly, the expression of IL-6 was significantly higher in cells exposed to functionalized soot (S2, S3, S4) at 6.25 μg/mL and 12.5 μg/mL, and it was significantly higher at 3.125 μg/mL only in cells exposed to S2 (Figure 5). When comparing the effects of different soot exposure at the highest concentration (12.5 μg/mL), we found significant differences among particles, with S2 inducing a significantly higher IL-6 mRNA expression than S3 and S4 (Figure 7A), and all three functionalized soot stimulating higher expression than the nascent (S1) particle (*p* < 0.05). In contrast, when comparing the effects of different soot at 3.125 μg/mL, the IL-6 response mimicked that of IL-1β (Figure 7B).

**Figure 7.** Human IL-6 mRNA (IL6) expression (relative to 18s) in BEAS-2B cells treated with S1–S4 soot at 3.125 μg/mL (**A**) or 12.5 μg/mL (**B**) for 6 h. The graphs summarize data from three independent experiments (*n* = 3 replicates per experiment), with results normalized to controls (exposed to methanol) and expressed as mean ± SEM. a: different from control and S1 (*p* < 0.001), b: different from S2 (*p* < 0.05), c: different from control. One-way ANOVA (*p* < 0.001, F (3, 8) = 25.7).

#### *3.4. Expression of Oxidative Stress-Related Genes in Cells Exposed to Synthetic Soot*

To assess the ability of the different soot preparations to induce the expression of oxidative stress-related genes in BEAS-2B cells, we conducted real-time PCR experiments on extracts from cells exposed to S1, S2, S3, and S4. We selected a concentration of 3.125 μg/mL at a 6-h time point considering that all soot preparations elicited changes in IL-6 and IL-1β gene expression at this concentration (Figure 4), while minimizing cell death (Figure 2). We measured the expression of two genes related to oxidative stress (SOD2 and NFE2L2), as previous studies have observed that PM2.5-induced oxidation alters the expression of these genes in respiratory tract cells [28]. Both NFE2L2 and SOD2 are critical molecules in the lung's defense mechanism against oxidative stress, and their disruption enhances susceptibility to airway inflammation [29,30].

A significant interaction of particle effects on SOD2 and NFE2L2 expression (SOD2, *p* = <0.0001; NFE2L2, *p* = 0.0001, one-way ANOVA) was observed after 6 h of exposure to all soot particles at 3.125 μg/mL. SOD2 expression was significantly downregulated in cells exposed to S2, S3, and S4, but not S1 (Figure 8A). Cell exposure to S2 and S3 resulted in the most significant downregulation in SOD2 expression, both being the highest in O-C=O content compared to S1 and S4 (Table 2). Similarly, the expression of NFE2L2 was downregulated by cell exposure to all soot preparations, including S1, with the greatest effect observed in S2 and S3 exposure (Figure 8B). Comparison of SOD2 and NFE2L2 expression in cells exposed to all soot preparations at 3.125 μg/mL revealed a significant effect of functionalized particles (S1, S2, S3, S4) vs. control particles.

#### *3.5. Protein Carbonylation in Cells Exposed to Synthetic Soot*

To assess the ability of the soot preparations to alter protein carbonylation, we measured carbonylation levels in protein extracts from BEAS-2B cells exposed to S1, S2, S3, and S4 at a concentration of 25 μg/mL at a 24-h time point. We chose this concentration because it is the minimum concentration that results in a reduction in cell viability at this time point. The exposure of cells to S2 and S4, i.e., the soot with the highest oxygen, C-O, and C=O content, resulted in the greatest increase in total of protein carbonylation compared to S1 and S3 (Figure 9). Interestingly, the increase of protein carbonylation triggered by S2 and S4 mimics the effects observed on IL-1β expression at 12.5 μg/mL at 6 h of exposure.

**Figure 8.** Expression of oxidative stress genes (relative to 18S expression) in BEAS-2B cells treated with S1–S4 soot at 3.125 ug/mL for 6 h. (**A**) Superoxide dismutase 2 (SOD2) mRNA. (**B**) Nuclear factor erythroid 2-related factor 2 (NFE2L2) mRNA. The graphs summarize data from three independent replicates, with gene expression results normalized to controls (exposed to DMSO) and expressed as mean ± SEM. a: different from control, b: different from S1, c: different from S4. One-way ANOVA (*p* < 0.05).

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**Figure 9.** Protein carbonylation levels in BEAS-2B cells treated with S1–S4 soot at 25 ug/mL for 24 h. Results are expressed as nmol/mg of total protein. a: different from S1 and S3. One-way ANOVA (*p* < 0.001, F (3, 12) = 63.07).

#### **4. Discussion**

Air pollution is a significant health hazard that is associated with the worsening of cardiopulmonary disease and reduction of life expectancy. It is estimated that over 90% of the world population is exposed to higher than recommended levels of ambient air pollution, including outdoor particle pollution such as PM2.5 [31]. Decades of epidemiological and toxicological research have demonstrated that exposure to PM2.5 is detrimental to lung health and contributes significantly to the development and exacerbation of a multitude of lung diseases [32]. Nonetheless, the cellular and molecular mechanisms driving these outcomes remain poorly understood, and the influence of physicochemical characteristics of soot particles in these mechanisms has not been yet explored. Accumulating evidence from in vivo and in vitro studies using environmental samples or purified PM2.5 mixtures have indicated that the lung epithelium responds to particle exposures in a dose- and time-dependent manner, by activating redox responses, initiating and/or exacerbating inflammation, and causing DNA damage and epigenetic alterations [14]. In this study, rather than evaluating the effects of purified particles from environmental samples, we investigated the effects of exposing lung epithelial cells to synthetic soot. We assessed the effects of particles directly on oxidative stress and inflammatory responses to understand the contributions of their surface chemistry vs. the carbonaceous soot backbone itself. Our data revealed that surface chemistry, and specifically oxygen and carboxylic acid content, contributes to cell death, the

expression of inflammatory and oxidative stress genes, and protein oxidative damage (carbonylation) in human lung epithelial cells in a concentration and time-dependent way.

Many of the adverse health effects of PM2.5 exposure are hypothesized to be derived from oxidative stress and inflammation, which is initiated by the formation of reactive oxygen species (ROS) and expression of pro-inflammatory genes within cells [33]. Although several studies have demonstrated the ROS potential and inflammatory action of diesel particulates from diesel engines [34], the causative factors are less clear. Most studies assessing these effects have used organics diluted and extracted from real exhaust particulates, and the results reported may be skewed relative to cell exposure to the "complete" soot, i.e., particulate matter along with its heteroatoms and condensed fractions. It is increasingly being recognized that extracts of diesel soot are a poor representation of the full range of toxicity [35]. Rather, the particles and, in particular, their surface chemistry have considerable direct impacts. To understand and potentially quantify the effect of parameters such as particle size, morphology, and chemistry, this study used surface-modified carbon black being a close substitute in its make and morphology. Functionalized carbon black is a surrogate for soot to understand the hierarchy of detrimental effects of soot from a combustion engine, i.e., the health effects of the primary particle itself as compared to those of the functional groups on the carbonaceous backbone. We chose carbon black to be able to control the extent of functionalization introduced and study the cell response by selectively modifying the material to exclude acidic functional groups. Exposure of a human lung epithelial cell line to model soot that closely resembles real combustion-generated soot in structure, particle size, and chemistry allowed us to identify and associate specific outcomes with particle physicochemical characteristics.

To our knowledge, this is the first study to examine the effects of surface particle chemistry and nanostructure on human lung epithelial responses. Prior studies using purified environmental samples have reported limited and often contradictory outcomes. Moreover, the majority of in vitro studies testing for toxicity use washing soot extracts that convolve condensed organics on the particles with extracted organics from the particle, ignoring the fixed particle surface chemistry. This has complicated the toxicity assessment of engine exhaust products, which is mainly due to our limited understanding of the specific contributions of different soot components. Our study provides new information on the contributions of surface chemistry to the particle's cytotoxicity. Our data indicate that both oxygen content and functional groups enhance the particle's ability to induce cell death. We demonstrate here that particles with the highest surface oxygen percentage and carboxylic acid content impair cell viability at lower concentrations than those with lower oxygen and carboxylic acid content. In addition, we show that this effect is also concentration- and time-dependent. Higher exposure times (24 h vs. 6 h) resulted in a significantly lower number of viable cells for a given concentration in particles with lower carboxylic acid content (S1, S4), but not in those with higher content (S2, S3).

It has been recently shown that exposure to PM2.5 below the current U.S. Environmental Protection Agency standards is associated with increased mortality [36]. This suggests that the inhalation of soot at very low concentrations may induce cellular damage that accumulates over time. While it is not possible to determine how closely the concentrations tested in this study reflect those of actual in vivo exposures in the human lung epithelium, we aimed to use concentrations that do not cause significant cytotoxicity when determining the contributions of particle chemistry on inflammation and oxidative stress effects. Thus, we examined the expression of two pro-inflammatory markers known to be expressed by lung epithelial cells (IL-1β and IL-6) [37] in BEAS-2B cells exposed for 6 h to soot at concentrations that were not toxic and did not have major effects on cell viability (12.5 μg/mL or lower). Our results demonstrate that the presence of functional groups in the particle's surface was sufficient to induce an inflammatory response, as indicated by the stimulation of IL-1β and IL-6 gene expression by S2, S3, and S4, but not S1. Moreover, this effect was dose-dependent, indicating a potential mechanism involving the activation of pattern recognition receptors [38]. These receptors are known for recognizing specific ligands such as pathogen- and damage-associated molecular patterns, which are endogenous ligands derived from stressed cells [39]. The activation of pattern recognition

receptors such as Toll-like-receptors in response to PM exposure results in the release of cytokines and chemokines to attract immune cells to the site of injury [40,41]. At the highest concentration tested (12.5 μg/mL), we did not observe a differential effect on IL-1β expression among different soot preparations. However, the expression of IL-6 mRNA was significantly higher in cells exposed to S2. This soot not only presents the higher oxygen atomic content, but also the higher percentage of -COOH, C=O, and C-O groups. In a previous study, 100 μg/mL ultrafine carbon black was observed to increase mRNA and protein expressions of IL-1β and IL-6 in primary rat epithelial lung cells, but IL-6 protein expression was delayed compared to IL-6 mRNA expression, and IL-1β mRNA and protein expression were not correlated [42]. Other studies have shown that IL-1β mRNA levels do not necessarily reflect IL-1β protein secretion [43]. Furthermore, it has been observed that endotoxin contamination, a component of some environmental PM preparations, may cause an intracellular accumulation of cytokines, including IL-1β [44], although previous findings indicate that endotoxin is usually found on coarse PM [45]. To further investigate the inflammatory effects of the synthetic soot, future studies should also examine the expression of IL-8 and TNF-α, as these genes have been implicated in numerous human lung diseases [46,47] and found to be upregulated in human bronchial epithelial cells exposed to PM [48,49].

In addition to studying the inflammatory response triggered by soot exposure, we examined the expression of SOD2 and NFE2L2, which play a critical role in antioxidant defense mechanisms against ROS. We used a concentration that did not significantly reduce cell viability (3.125 μg/mL) but was enough to elicit an inflammatory response by altering IL-6 and IL-1β gene expression. We found that the presence of functional groups in the particles' surface was sufficient to alter the expression of both SOD2 and NFE2L2, as indicated by the gene expression changes in cells exposed to S1, S2, S3, and S4. Interestingly, SOD2 mRNA expression was significantly lower in cells exposed to all soot variants, including S1, but NFE2L2 gene expression was not significantly affected in cells treated with S1. The expression of SOD2 and NFE2L2 was the lowest in cells exposed to S2 and S3, the highest total oxygen carboxylic acid-containing soot, indicating a potential role of these groups in the inhibition of these gene's expression.

Combined, these findings suggest that PM2.5 toxicity induces a decrease in the expression of antioxidant response genes SOD2 and NFE2L2. Under physiologic conditions, ROS generation is minimized by antioxidant proteins, but an overproduction of ROS disturbs the antioxidant defense system, leading to oxidative stress [30]. ROS are produced by the reduction of molecular oxygen and the formation of superoxide anions. Superoxides are precursors to most ROS but are metabolized by SOD2 to hydrogen peroxide (H2O2). Catalase (CAT) serves as a protective enzyme that breaks down H2O2 and eliminates ROS formation. Some studies have shown that exposure to PM2.5 induces a loss of SOD2 and CAT activity, leading to an accumulation of ROS [24]. PM2.5 has also been observed to downregulate protein kinase B (Akt) signaling, which is a known modulator of SOD2 and NFE2L2, decreasing the expression of SOD2 and NFE2L2 [49,50]. Another study observed that NFE2L2 expression in human BEAS-2B bronchial epithelial cells is downregulated when cells are subjected to high concentrations of PM2.5 or repeated exposure protocols [51].

An impairment in the antioxidant defense mechanism by the downregulation of SOD2 and NFE2L2 increases the expression of IL-1β and IL-6, inducing an inflammatory response, as observed in our findings [52]. Based on our data, a decrease in SOD2 and NFE2L2 gene expression is associated with a significant increase in IL-1β and IL-6, particularly in cells treated with S2 and S4, potentiating an inflammatory response as expected. The high oxygen content of S2 and S4 makes them potential good oxidants that can cause changes in oxygen saturation and can react with molecular oxygen to form H2O2 and the most potent form of ROS, hydroxyl radicals. Exposure to PM2.5 can downregulate CAT activity, causing an overaccumulation of these ROS, resulting in production and oxidative stress, increasing the inflammatory response [53]. While S3 (O3 treated PM2.5) did not cause the most significant increase in IL-1 β and IL-6 gene expression, S3 along with S2 did cause the most significant decrease in SOD2 and NFE2L2 gene expression. O3 generates ROS, including H2O2, thus exerting similar effects to soot with

higher oxygen content [54]. Overall, more research is needed to elucidate the mechanisms by which the expression of antioxidant enzymes and transcription factors relates to oxidative stress responses induced by soot with varying surface chemistry.

Carbonylation is an irreversible protein modification induced by oxidative stress and is associated with chronic inflammation. Thus, we examined protein carbonylation as a potential biomarker of oxidative stress induced by exposure to PM2.5 in our studies. Protein carbonylation is a well-used biomarker for studying numerous human diseases, including Alzheimer's disease, diabetes, and chronic lung disease. Despite this, very little is known about PM2.5-induced protein carbonylation and its role in oxidative stress. We exposed BEAS-2B cells for 24 h to soot preparations at a concentration that elicited changes in cell viability at 24 h (25 μg/mL) but no cell death. Protein carbonylation has been previously observed to increase in human keratinocytes exposed to PM2.5 at 50 μg/mL at a 24-h time point [23], indicating that changes in protein carbonylation require higher concentrations of PM than those required to elicit changes in gene expression. We found that that protein carbonylation was significantly higher in cells exposed to S2 and S4 compared to cells exposed to S1 and S3, thus following a similar trend observed in IL-1β and IL-6 gene expression. Targets of carbonylation are dependent on the oxidative environment of the cell and abundance of ROS. In the NFE2L2/Kelch-like ECH-associated protein 1 (KEAP1) mechanism, cytoplasmic KEAP1 binds NFE2L2, targeting it for proteasomal degradation. When carbonylated, KEAP1 releases NFE2L2, which translocates to the nucleus and, along with other transcription factors, binds to antioxidant response elements to produce cytoprotective proteins [22].

Our study has several limitations. First, while our characterization of lung epithelial cellular responses to synthetically modified soot revealed important contributions of surface chemistry, we used a submerged monolayer culture model wherein cells have been removed from their physiological conditions, including interactions with neighboring cells in the airway epithelium. Therefore, our studies will need to be replicated and investigated by alternative methods such as exposures in air–liquid interface cell cultures, using cell lines and primary cells of the lung and nasal epithelia, and using in vivo exposure models. Second, the BEAS-2B cell line is derived from an adult male individual; therefore, our results do not reflect the potential effects of sex/gender and/or age in the observed responses. Considering the known differential effects of air pollution exposure in men and women [55], and in individuals of different age groups and disease status [56], it will be important to expand these investigations to models that are representative of such populations. Third, while according to the literature, a particle concentration of 100 μg/mL corresponds to approximately 16μg/cm2 if all the suspended particles are deposited on the cells on the surface of the plates [57], we have not directly measured the proportion of particles that were in contact with cultured cells in our experiments. Finally, we have reported a differential reduction on the percent of viable cells as well as changes in mRNA expression of selected inflammatory and oxidative stress-related genes upon exposures, although in the current study, we have not evaluated changes in cytokine protein expression, the activity of oxidative stress enzymes, nor mechanisms previously associated with PM2.5 responses, including mitochondrial damage, apoptosis, autophagy, DNA damage, and epigenetic changes [52,58–60].

Our study has potential implications for environmental health. Vehicular traffic and consequent engine exhaust and industrial emissions are among the top contributors to outdoor pollution that results in millions of annual causalities worldwide. In light of the recent COVID-19 outbreak, one pilot (unpublished) study highlighted that an increase of only 1 μg/m3 in PM2.5 is associated with an 8% increase in the COVID-19 death rate [61]. While particle air pollution regulations are currently based on mass, the discussion is ongoing as to whether to include particle number concentration as a pseudo-surrogate for particle surface area. The results from our study reflect the impact of particle surface chemistry and physical structure as direct operative factors in health impacts, suggesting that such factors should be considered when developing environmental policies. These physicochemical properties are relatable to combustion conditions and fuel, indicating that precautions for exposure to

small particles from various sources may be improved, and corresponding improvements in mitigation strategies may be more rapidly engineered.

In summary, we report that particle surface chemistry contributes to the cellular and molecular responses exerted in lung epithelial cells. We show that higher oxygen content and carboxylic acid functional groups in soot result in greater cell damage and death and cause the cells to react by activating inflammatory responses. These effects are exacerbated by soot concentration and exposure time. Together, our results demonstrate the role of particle surface chemistry as a direct operative factor impacting health. This physicochemical property is relatable to combustion conditions and fuel type. Thus, precautions for exposure to other soot types with similar surface (oxygen) chemistry may be improved, enabling better assessment from other anthropogenic sources and thereby formulating effective mitigation strategies to tackle this problem worldwide.

#### **5. Conclusions**

We conclude that particle surface chemistry, and specifically oxygen content and the presence of carboxylic acid groups in soot, differentially affect inflammatory and oxidative stress responses in human lung epithelial cells.

**Author Contributions:** Conceptualization, R.L.V.W., P.S.; methodology, H.A.H., S.E., M.S., M.N., R.L.V.W., P.S.; formal analysis, H.A.H., S.E., M.S., M.N., R.L.V.W., P.S.; investigation, H.A.H., S.E., M.S., M.N., R.L.V.W., P.S.; writing—original draft preparation, H.A.H., M.S., R.L.V.W., P.S.; writing—review and editing, H.A.H., S.E., M.S., R.L.V.W., P.S.; supervision, R.L.V.W., P.S.; validation, H.A.H., R.L.V.W., P.S.; visualization, H.A.H., R.L.V.W., P.S.; project administration, R.L.V.W., P.S.; funding acquisition, R.L.V.W., P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Pennsylvania State University Human Health & the Environment Seed Grant (R.L.V., P.S.), and the National Heart Lung and Blood Institute of the National Institutes of Health under award number HL133520 (PS).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Inflammation in Dry Eye Syndrome: Identification and Targeting of Oxylipin-Mediated Mechanisms**

**Dmitry V. Chistyakov 1,\*, Olga S. Gancharova 1, Viktoriia E. Baksheeva 1, Veronika V. Tiulina 1,2, Sergei V. Goriainov 3, Nadezhda V. Azbukina 4, Marina S. Tsarkova 2, Andrey A. Zamyatnin, Jr. 1,5, Pavel P. Philippov 1, Marina G. Sergeeva 1, Ivan I. Senin 1 and Evgeni Yu. Zernii 1,5,\***

Received: 21 July 2020; Accepted: 8 September 2020; Published: 11 September 2020

**Abstract:** Dry eye syndrome (DES) is characterized by decreased tear production and stability, leading to desiccating stress, inflammation and corneal damage. DES treatment may involve targeting the contributing inflammatory pathways mediated by polyunsaturated fatty acids and their derivatives, oxylipins. Here, using an animal model of general anesthesia-induced DES, we addressed these pathways by characterizing inflammatory changes in tear lipidome, in correlation with pathophysiological and biochemical signs of the disease. The decline in tear production was associated with the infiltration of inflammatory cells in the corneal stroma, which manifested one to three days after anesthesia, accompanied by changes in tear antioxidants and cytokines, resulting in persistent damage to the corneal epithelium. The inflammatory response manifested in the tear fluid as a short-term increase in linoleic and alpha-linolenic acid-derived oxylipins, followed by elevation in arachidonic acid and its derivatives, leukotriene B4 (5-lipoxigenase product), 12-hydroxyeicosatetraenoic acid (12-lipoxigeanse product) and prostaglandins, D2, E2 and F2α (cyclooxygenase products) that was observed for up to 7 days. Given these data, DES was treated by a novel ophthalmic formulation containing a dimethyl sulfoxide-based solution of zileuton, an inhibitor of 5-lipoxigenase and arachidonic acid release. The therapy markedly improved the corneal state in DES by attenuating cytokine- and oxylipin-mediated inflammatory responses, without affecting tear production rates. Interestingly, the high efficacy of the proposed therapy resulted from the synergetic action of its components, namely, the general healing activity of dimethyl sulfoxide, suppressing prostaglandins and the more specific effect of zileuton, downregulating leukotriene B4 (inhibition of T-cell recruitment), as well as upregulating docosahexaenoic acid (activation of resolution pathways).

**Keywords:** dry eye syndrome; inflammation; oxidative stress; corneal damage; tear lipidome; 5-lipoxigenase; leukotriene B4; prostaglandins; dimethyl sulfoxide; zileuton

#### **1. Introduction**

Dry eye syndrome (DES; also known as dry eye, dry eye disease, or keratoconjunctivitis sicca) is a common multifactorial ocular surface disease, characterized by decreased production/increased evaporation of the tear, resulting in its hyperosmolarity and instability of the tear film [1]. DES affects up to 40% of the adult population and manifests as eye irritation, hyperemia, glare, eye fatigue, and blurred vision [2,3]. Etiologically, alterations in tear homeostasis are associated with dysfunction of the lachrymal and/or Meibomian glands (such as in Sjögren syndrome and Meibomian Gland Dysfunction (MGD)) as well as a number of other factors, including contact lens wearing, adverse effects of various medications, complications of ocular surgery and general anesthesia [1,4–7].

In DES pathogenesis, the reduced tear production and/or alterations in the tear composition results in the loss of lubricating, nourishing and protective qualities of tears against the ocular surface tissues [8]. The most critical consequences of this desiccating stress are ocular surface inflammation and damage, which in severe cases can cause blindness, due to corneal scarring, opacification or ulceration [3]. Consistently, the generally accepted treatment of DES involves not only using lubricating eye drops and ointments, but also topical anti-inflammatory therapy [9,10]. Unfortunately, the administration of common ocular anti-inflammatory medications, such as corticosteroid eye drops or cyclosporine, is associated with a number of adverse effects in patients with dry eye, which generally limits their employment [11]. Thus, there is a great demand for a new safe and effective anti-inflammatory therapy for DES.

The solution to this problem requires an understanding of the mechanisms underlying inflammation in DES, which can be selectively targeted. Currently, it is recognized that an inflammatory reaction in DES is triggered by cytokines (such as interleukin (IL) 1 beta and tumor necrosis factor alpha (TNF-α)) secreted in tear fluid (TF) during the early stages of the disease. Indeed, hyperosmolarity of the tear film stimulates an inflammatory cascade, resulting in the release of these cytokines by limbal epithelial cells [12]. In addition, the desiccating stress leads to compensatory reflex stimulation of the lachrymal gland, which may activate a neurogenic inflammatory cytokine response [3,8]. These events induce maturation and an increase in the density of antigen-presenting dendritic cells in the cornea, subsequent activation of T-cells and their recruitment to the ocular surface (including conjunctiva and lacrimal glands) and release of additional effector cytokines in the TF, thereby causing further damage to the corneal epithelium [3,13]. These processes are accompanied by infiltration of the cornea by leucocytes and other immune cells [3,14]. Consistently, DES can be treated by using drugs blocking the activation of T cells (i.e., lifitegrast, LFA-1 antagonist) or inhibiting cytokine production by these cells (i.e., cyclosporine, suppressor of calcineurin regulated transcription of cytokine genes), as well as using cytokine receptor inhibitors [13,15,16]. Inflammatory changes in DES are also associated with oxidative stress of the ocular surface tissues, stemming from TF deficiency. Indeed, TF contains low molecular weight antioxidants (glutathione, ascorbic acid, and others) and reactive oxygen species (ROS) scavenging enzymes (glutathione reductase, glutathione peroxidase, superoxide dismutase, etc.) thereby providing local antioxidant defense [17,18]. Accordingly, the inflammatory component of DES can be attenuated by the topical administration of antioxidants [6,19].

The separate classes of molecules that contribute to inflammatory responses in DES and can be targeted in its prospective therapy are polyunsaturated fatty acids (PUFAs) and their derivative oxylipins, representing multipotent lipid mediators of inflammation. Oxylipins are biosynthesized from PUFAs via multiple oxidative reactions catalyzed by specific enzymes (cyclooxygenase (COX), lipoxygenase (LOX) or cytochrome P450 monooxygenase (CYP)) or proceeding in an enzyme-independent manner in the presence of ROS [20]. Previously, it was suggested that chronic inflammation in DES is associated with an imbalance between omega-3 (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) and omega-6 (arachidonic acid (AA)) PUFAs, which lead to the hyperproduction of proinflammatory lipid mediators (omega-6 derivative oxylipins, such as prostaglandins) and underproduction of proresolving (omega-3 derivative oxylipis) molecules [21]. Consistently, DES patients were characterized by upregulation of prostaglandin (PG) E2 in TF, the levels

of which correlated with their symptom grades [21–23]. Interestingly, selective inhibition of COX-2, responsible for PGE2 biosynthesis, reduced the levels of pro-inflammatory cytokines in DES apparently via the attenuating effect of PGE2 on antigen-presenting dendritic cells [24], indicating the existence of interrelation between inflammatory pathways in DES. Although COX suppression seems to be a promising route in DES therapy, the well-acknowledged inhibitors of cyclooxygenases, non-steroidal anti-inflammatory drugs (NSAIDs), are not recommended in patients with DES, due to a number of severe side effects [25,26]. Thus, it is important to develop an alternative approach to selective targeting of the oxylipin-dependent inflammatory pathways, based on an understanding of the dynamic changes of the full spectrum of these lipid mediators in DES.

Previously, we developed a rabbit model of DES, closely reproducing the pathogenesis of the human disease [27]. In this model, DES is induced by exposure of the animals to general anesthesia, which diminishes the production of basal and reflex tears and decreases the stability of the tear film thereby inducing common signs of the disease [4,28]. In a more recent study, we adopted a quantitative UPLC-MS/MS-based approach to identify and characterize baseline patterns of lipid mediators in the TF of healthy rabbits and demonstrated that they did not differ significantly from that of human TF [29]. Here, we employed these approaches to characterize TF lipidomic changes in DES, focusing on PUFAs, oxylipins and phospholipid derivatives contributing to the regulation of inflammation, and to find correlations between these changes and the pathophysiological and biochemical signs of the disease. Based on the data obtained, we proposed a novel complex anti-inflammatory therapy, targeting the specific oxylipin-mediated mechanisms revealed in DES. Using subsequent morphological, biochemical and lipidomic studies, this therapy was found to markedly improve the corneal state in DES by selective suppression of its inflammatory component, without affecting tear production rates.

#### **2. Experimental Section**

#### *2.1. Materials*

Anesthetic preparation containing 50 mg/mL tiletamine and 50 mg/mL zolazepam was from Virbac (Carros, France). Xylazine hydrochloride was from Nita-Farm (Saratov region, Russia). Ultragrade Tris was from Amresco (Solon, OH, USA). Phosphate-buffered saline (PBS) was from Thermo Fisher Scientific (Waltham, MA, USA). Zileuton, DMSO, hemoglobin, luminol, hydrogen peroxide solution, and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were from Sigma-Aldrich (St. Louis, MO, USA). Reagents for histological examination were from Biovitrum (Moscow, Russia). Bicinchoninic acid (BCA) assay kit was from Sigma-Aldrich. Glutathione peroxidase and superoxide dismutase assay kits were from Randox (Crumlin, UK). Tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) assay kits were from Immunotex (Stavropol, Russia). The Schirmer's test tear strips were from Contacare Ophthalmics and Diagnostics (Vadodara, Gujarat, India.). The deuterated oxylipins standards 6-keto PGF1α-d4, TXB2-d4, PGF2α-d4, PGE2-d4, PGD2-d4, LTC4-d5, LTB4-d4, 5(S)-HETE-d8, 12(S)-HETE-d8, 15(S)-HETE-d8, Oleoyl Ethanolamide-d4, EPA-d5, DHA-d5, and AA-d8 were from Cayman Chemical (Ann Arbor, MI, USA). Solid-phase lipid extraction cartridge Oasis® PRIME HLB was obtained from Waters, Eschborn, Germany. Other reagents and suppliers used in this study were from Sigma-Aldrich, Amresco, or Serva (Heidelberg, Germany) and were at least of reagent grade. All buffers and other solutions were prepared using ultrapure water.

#### *2.2. Experimental Animals and Ethics Statement*

The study involved a total of 102 healthy New Zealand white rabbits (6 months old, weight of 2.3 to 3 kg) purchased from a certified farm (Krolinfo, Moscow region, Russia). The rabbits were housed in individual cages (795 × 745 × 1776 mm3) under normal conditions (12 h light/12 h dark, 22–25 ◦C, 55–60% humidity) with free access to food and water. The daily monitoring of health status of all animals involved ophthalmological exam, inspection of habitus, derma and mucous membranes as well as monitoring of heart/respiratory rates and body temperature. No adverse events were observed

during the course of the study. The humane euthanasia of the animals intended for histological analysis of the anterior segment of the eye was performed by an overdose of the anesthetic (for general anesthesia conditions, see next section). The eyeballs of the animals were enucleated postmortem. Otherwise, the animals were rehabilitated for three days and returned to the farm. The animals were treated according to the 8th edition "Guide for the Care and Use of Laboratory Animals" of the National Research Council and "Statement for the Use of Animals in Ophthalmic and Visual Research" of The Association for Research in Vision and Ophthalmology (ARVO). The study was approved by the Belozersky Institute of Physico-chemical Biology Animal Care and Use Committee (Protocol number 1/2016, 12 January 2016).

#### *2.3. Experimental Model*

The experiments were performed using a single-blind method. The rabbits were divided into 17 groups, 6 animals in each group (Table 1), and treated as described in Section 3. DES was induced by introducing rabbits into prolonged general anesthesia as described in previous studies [6,30]. Particularly, the animals were placed in prone position in a restraining device and subjected to repeating intramuscular injection of anesthetic preparation containing 1:2 mixture of 50 mg/mL tiletamine/zolazepam and 20 mg/mL xylazine hydrochloride to achieve continuous narcotic sleep for 6 h. For the anti-inflammation therapy, the animals received conjunctival instillations of the eye drops, consisting of either 50% dimethyl sulfoxide (DMSO) in normal saline (0.90% *w*/*v* of NaCl), or the same solution containing 0.5 mg/mL (0.05% (*w*/*v*)) zileuton. The eye drops were prepared under sterile conditions. Each of the formulations was administrated 1 drop 3 times daily in each eye for up to 7 days, starting from the day of DES induction.

1 Measurement of total protein concentration, total antioxidant activity, antioxidant enzyme activity, and cytokine concentration in TF; 2 UPLC-MS/MS of lipid mediators (PUFAs, oxylipins and phospholipid derivatives) in TF; d: day.

#### *2.4. Histological Analysis*

Histological analysis of the cornea was performed essentially as described earlier [6,31,32]. Briefly, the eyeball was enucleated immediately postmortem and fixed in 10% neutral buffered formalin in phosphate buffer (pH 7.4) for 24 h at room temperature. The cornea and iris were trimmed out, dehydrated (absolute isopropanol, 7 portions × 5 h) and embedded in Histomix paraffin medium. Eight 3-micron thick serial nasotemporal cross-sections of each cornea were obtained and mounted on glass slides. The sections were then deparaffinized (xylene, 5 min, two times), hydrated, and stained with Carazzi's hematoxylin and 0.5% eosin Y. Stained sections were dehydrated by 96% ethanol and xylene, cleared in BioClear tissue clearing agent, mounted into BioMount synthetic medium, and examined using Leica DM4000 microscope with Leica DFC Camera (Leica, Wetzlar, Germany) or Zeiss AxioVert.A1 microscope with Axiocam IC C5 camera (Carl Zeiss, Oberkochen, Germany). Processing of the microphotographs was performed using the AxioVision 8.0 and ZEN 2 lite ZEISS Microscope Software (Carl Zeiss, Oberkochen, Germany) and Adobe Photoshop CS6 Extended (Adobe Systems, San Jose, CA, USA) software.

#### *2.5. Schirmer's Test*

Tear secretion in animals was measured without topical or general anesthesia according to the following procedure [6,30]. Schirmer's test tear strips were placed under the lower eyelid the restrained animal for 5 min, and the length of the moistened paper (in mm) was recorded. The procedure was repeated at least three times, and the average values were calculated.

#### *2.6. TF Collection*

The collection of TF for analytical purposes was performed in separate groups of animals (Table 1). For biochemical analysis, TF was obtained according to the Schirmer's test procedure, except for the strip was allowed to get moistened for exactly 20 mm. Fifteen millimeters-long fragments of the strip were then cut off, extracted with 150 μL of PBS for 30 min and the resulting solution was collected and stored until the biochemical analysis (see Sections 2.7–2.9) at −80 ◦C [6]. To collect TF samples for lipidomic analysis [29] (see Sections 2.10 and 2.11), the strip was allowed to get moistened reaching exactly 15 mm, the procedure was repeated with 3 strips and 15 mm fragments of each strip were cut off, transferred to a plastic tube with 1 mL of anhydrous methanol containing 0.1% *v*/*v* BHT and stored at −80 ◦C.

#### *2.7. Total Protein Concentration Measurement*

The concentration of total protein in TF samples was measured using BCA assay. Colorimetric reaction was monitored with Synergy H4 Hybrid plate reader (Biotek, Winooski, VT, USA). The calibration curve was plotted using standard solutions of bovine serum albumin in PBS.

#### *2.8. Total Antioxidant Activity Analysis*

The total antioxidant activity in TF was analyzed using hemoglobin/H2O2/luminol system [6,33,34]. Briefly, the TF samples were diluted 1:4 by PBS and added to the reaction mixture, containing 0.01 mM luminol and 0.5 mM hemoglobin. The reaction was initiated in the presence of 6 μM H2O2 and chemiluminescence of the samples was monitored every one second for 10 min using Glomax-Multi Detection System luminometer (Promega, Madison, WI, USA). The results were calculated in the Trolox equivalent after measuring chemiluminescence of standard solutions, containing 1–8 μM Trolox in PBS.

#### *2.9. Antioxidant Enzymes Activity and Cytokine Concentration Analysis*

The activities of superoxide dismutase and glutathione peroxidase and the contents of TNF-α and IL-10 in TF samples were measured without their additional dilution using the respective commercially available kits. The colorimetric reactions were monitored using Synergy H4 Hybrid Reader (Biotek, Winooski, VT, USA).

#### *2.10. Lipid Extraction*

The extraction of lipids for UPLC-MS/MS analysis was performed exactly as described in our resent study [29]. Briefly, TF samples were mixed with 2 ng of deuterated internal standard solutions and centrifuged (12,000× *g*, 3 min). The supernatants were mixed with 0.1% acetic acid (6 mL) and loaded onto solid-phase lipid extraction cartridge, which was washed with 15% methanol, 0.1% formic acid (2 mL), and the lipids were eluted with 500 μL of anhydrous methanol and subsequently with 500 μL of acetonitrile. The obtained TF extracts were concentrated by evaporation, reconstituted in 50 μL of 90% methanol and stored at −80 ◦C.

#### *2.11. UPLC-MS*/*MS Analysis*

TF lipids were analyzed in the obtained extracts using 8040 series UPLC-MS/MS mass spectrometer (Shimadzu, Japan) in multiple-reaction monitoring mode at a unit mass resolution for both the precursor and product ions [29,35]. The lipids separation was performed by reverse-phase UPLC on Phenomenex C8 column (0.4 mL/min, sample temperature of 5 ◦C). The compounds were eluted with 10–95% acetonitrile gradient in 0.1% (*v*/*v*) formic acid and subjected to MS analysis using electrospray ionization (in both positive and negative ion modes). The molecular ions were fragmentized by collision-induced dissociation in the gas phase and analyzed by tandem (MS/MS) mass spectrometry. The selected lipids were identified and quantified by comparing their mass-spectrometric and chromatographic data with those obtained for the corresponding oxylipins standards (see Section 2.1.) using Lipid Mediator Version 2 software (Shimadzu, Japan).

#### *2.12. Statistical Analysis*

The line charts in the figures represent mean ± SEM. Statistical significance was assessed using the repeated measure ANOVA (for analysis within the groups) and one way ANOVA (for analysis between the groups) with post-hoc pairwise comparisons using Tukey's test. The probability of less than 0.05 was considered significant.

#### **3. Results**

#### *3.1. Dynamics of Inflammation in DES: Tear Production and Morphological Changes in the Cornea*

To characterize the inflammatory responses associated with DES, a control group and four experimental groups of animals were created (groups 1–5, Table 1). To induce DES, the animals within all the experimental groups were exposed to six hours of general anesthesia in accordance with the approach developed in our previous study [6]. Using this setup, we assessed the dynamics of the pathological process in DES, focusing on its inflammatory component. To this end, we performed a histopathological examination of corneas obtained from animals euthanized immediately after anesthesia (at the sixth hour), as well as on days one, three and seven. To complete DES characterization, the intensity of tear production was analyzed in a separate group of animals (group 6, Table 1) using a standardized Schirmer's test.

It was found that tear production decreased two-fold immediately after anesthesia and was completely restored only on day three (Figure 1a). These changes were associated with pronounced morphological changes in the cornea, involving denudation of the corneal stroma, which had already appeared during the period of narcotic sleep, as a result of a large number of deaths of the corneal epithelial cells (Figure 1b, C0). The denudation was preserved focally on day one (Figure 1b, C1). On the remainder of the surface, the epithelium was non-homogenous, consisting of different numbers of layers. Notably, at this stage, the epithelium was abundantly infiltrated by inflammatory cells, mainly granulocytes. Such infiltrates were even found in the areas, where its structure was not significantly altered. The corneal stroma also contains a granulocyte infiltrate. Moderate areas of denudation and multiple epithelial lesions, together with the signs of inflammatory infiltration in the stroma but without intraepithelial infiltration, remained on day three (Figure 1b, C3). In addition, at this

time-point, the stroma contained activated keratocytes and exhibited remodeling processes. The signs of inflammation were not entirely resolved until the seventh day after DES induction (Figure 1b, C7). Meanwhile, sporadic areas of denudation remained until day seven, when they were surrounded by reepithelialization rolls along the edges, indicating a developed healing process.

**Figure 1.** Dynamic changes in tear secretion and corneal morphology in general anesthesia-induced DES. (**a**) The results of standardized Schirmer's tests performed in animals at 6th hour, 1st day, 3rd day or 7th day after their exposure to 6-h general anesthesia. ∗ *p* < 0.05 compared with the values measured in the control group. (**b**) Representative microscopic images of hematoxylin and eosin-stained cross-sections of the corneas obtained at 6th hour (C0), 1st day (C1), 3rd day (C3) and 7th day (C7) after exposure of the animals to general anesthesia. The image of the normal cornea is also presented (N). Stromal denudation (complete absence of the corneal epithelium) areas (red arrowheads), loci of desquamation/destruction of the epithelial layer (orange arrowheads), and sites of reepithelialization (green arrows; the detachment of the new epithelium from the stroma represents an artifact stemmed from histological processing) are indicated. The inflammatory changes include granulocytic infiltration of the corneal epithelium (intraepithelial granulocytes; black arrowheads) and stroma (black arrows), and postinflammatory signs of regeneration (activation of stromal keratocytes) at the ex-sites of the infiltration (blue arrows). Ep: epithelium; Str: stroma; DM: Descemet's membrane and endothelium. Hematoxylin and eosin staining, magnification 200×. The central areas of the cornea are presented. Gaps, cracks and folds of preparations are of an artificial origin.

We concluded that the pathological process in our DES model involved ocular surface inflammation, reaching a maximum on day one after anesthesia, which was accompanied by a decline in tear production and prolonged corneal damage.

#### *3.2. Dynamics of Inflammation in DES: Biochemistry and Lipidomics of the Tear Fluid*

To understand the mechanisms underlying the inflammatory reactions observed on the ocular surface, we next examined biochemical markers of the associated inflammatory pathways, focusing on lipid mediators (Figure 2). The analysis was performed on TF, which is known to be responsible for nourishing the cornea and maintaining the immune responses of the ocular surface [29]. TF was collected using Schirmer's strips in experimental group 8 (Table 1), following the aforementioned timeline and analyzed by a high-performance, quantitative mass-spectrometric approach, developed in our recent studies [29]. In parallel, TF samples (group 7, Table 1) were analyzed for total protein content, characteristic proinflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines and antioxidant molecules (total antioxidant activity (low molecular weight antioxidants), superoxide dismutase and glutathione peroxidase), which were previously characterized as markers of anesthesia-induced DES [6].

**Figure 2.** Mechanisms of biosynthesis of lipid mediators. Metabolites of arachidonic acid (AA), linoleic acid (LA) and alpha-linolenic acid (ALA) are divided into the groups synthesized via lipoxygenase (LOX), cyclooxygenas (COX), cytochrome P450 monooxygenase (CYP) or reactive oxygen species (ROS) dependent pathways. The other abbreviations are as follows: AEA, N-arachidonoylethanolamine; DHA, docosahexaenoic acids; 9,10-DiHOME, 9,10-dihydroxyoctadecamonoenoic acid; 12,13-DiHOME, 12,13-dihydroxyoctadecamonoenoic acid; EPA, eicosapentaenoic acid; 9,10-EpOME, 9,10-epoxyoctadecamonoenoic acid; 12,13-EpOME, 12,13-epoxyoctadecamonoenoic acid; 9-HODE, 9-hydroxyoctadecadienoic acid; 13-HODE, 13-hydroxyoctadecadienoic acid; 9-KODE, 9-oxo-octadecadienoic acid; 13-KODE, 13-oxo-octadecadienoic acid; LTB4, leukotriene B4; lyso-PAF, lyso-platelet-activating factor; OEA, oleoylethanolamine; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2α, prostaglandin F2α. The mediators altered in DES and affected by the proposed anti-inflammatory therapy (see below) are indicated in red and gray colors, respectively.

It was noted that the pathological process reflected in TF as a rapid decline of the total antioxidant activity and moderate upregulation of glutathione peroxidase (day one) as well as a more delayed suppression of superoxide dismutase, manifested on day three (Figure 3). The observed alterations were accompanied by inflammatory changes, such as an increase in total protein concentration and a decrease in IL-10 content, without having a significant effect on TNF-α. Importantly, these changes were most pronounced on day one, which concurred with histological findings (see Figure 1).

**Figure 3.** Dynamic changes in biochemical properties of TF in DES. The animals were exposed to general anesthesia for 6 h (shown as gray box). TF samples were collected before (norm) and immediately after (6 h) the general anesthesia as well as on 1, 3, and 7 day post-exposure. The samples were analyzed for total protein, TNF-α, IL-10, as well as total antioxidant activity (in Trolox equivalent) and activity of glutathione peroxidase and superoxide dismutase using the respective assays. \* *p* < 0.05 as compared to parameters of TF of the intact (control) animals.

A UPLC-MS/MS analysis of TF samples revealed a total of 23 lipid mediators, including three PUFAs (AA, DHA and EPA), 18 oxylipins and three phospholipid derivatives (OEA, AEA, Lyso-PAF) (Figure 4; full names and biosynthesis pathways for all lipids detected in the study are presented in Figure 2). The identified oxylipins were assigned to five subgroups, namely cyclooxygenase (COX)-dependent derivatives of AA (PGD2, PGE2, PGF2α, TXB2), cytochrome p450 (CYP)-dependent derivatives of LA (9,10-DiHOME/9,10-EpOME and 12,13-DiHOME/12,13-EpOME), as well as lipoxygenase (LOX)-dependent derivatives of AA (5-HETE, 12-HETE, and LTB4), LA (9-HODE/9-KODE and 13-HODE/13-KODE) and ALA (9-HOTrE, 13-HOTrE). The detected lipid mediators exhibited different behavior in DES. Thus, the most prolonged change in TF content was observed for AA and its COX-dependent (PGD2, PGE2, PGF2α) and LOX-dependent (LTB4, 5-HETE, 12-HETE) products, most of which exhibited sustained growth until the seventh day after the anesthesia. The significant increase in LTB4 was accompanied by an increase and a subsequent decrease in the TF content of its precursor 5-HETE. In turn, the LOX/oxidative stress-dependent derivatives of LA (9-HODE/9-KODE and 13-HODE/13-KODE), LOX-dependent derivatives of ALA (9-HOTrE, 13-HOTrE), CYP derivatives of LA (9,10-EpOME and 12,13-EpOME) as well as the phospholipid derivative AEA demonstrated a short-term increase on day one indicating the critical significance of this time-point as the peak of the oxidative and inflammatory response. Finally, no significant DES-dependent changes were observed in the case of DHA and EPA, as well as OEA and Lyso-PAF.

**Figure 4.** Dynamic changes in TF content of lipid mediators in DES. The animals were exposed to general anesthesia for 6 h (shown as gray box). TF samples were collected before (norm) and immediately after (6 h) the general anesthesia as well as on 1, 3, and 7 day post-exposure. The concentrations of the lipid mediators (phospholipid derivatives, PUFAs and oxylipins) in TF were measured using quantitative UPLC-MS/MS analysis. \* *p* < 0.05 as compared to the parameters of TF of the intact (control) animals. The identified oxylipins are divided into subgroups according to their precursors (AA, ALA, LA, phospholipids) and major biosynthetic pathways, involving COX, LOX, or CYP.

Considered together, these data demonstrate that anesthesia-induced DES is associated with oxidative stress and inflammatory response, with the latter being governed mainly by AA and its derivative oxylipins, namely 5-HETE/LTB4 (5-LOX products) and 12-HETE (12-LOX product) as well as COX-dependent prostaglandins (PGD2, PGE2, PGF2α).

#### *3.3. Selective Targeting of Inflammation in DES: Rationale for the Drug Formulation*

The obtained lipidomic data indicated that ocular inflammation in DES was governed mainly by two pathways, which depend on AA release and involve its processing by 5/12-LOX and COX. Thus, its targeting may include compounds dampening these pathways. The common COX inhibitors, NSAIDs, are efficacious in relieving different forms of ocular inflammation, but they can exacerbate DES by causing severe side effects [25,26]. Therefore, we focused on alternative methods of treatment, such as targeting LOX-dependent pathways. For this purpose, we chose zileuton (N-(1-Benzo(b)thien-2-ylethyl)-N-hydroxyurea), an anti-asthmatic drug, selectively inhibiting 5-LOX and thereby suppressing the generation of leukotrienes, including LTB4 [36]. An important benefit of zileuton is that it also inhibits AA release and, consequently, downregulates prostaglandins [37].

Given that zileuton (a low water-soluble drug) had never been employed in ophthalmology, a new formulation was developed for its administration in the form of eye drops, containing dimethyl sulfoxide (DMSO). DMSO was selected as it is low toxic [38,39], high tissue permeable, and facilitates the penetration of other compounds through biological membranes [40,41]. The use of up to 50% DMSO was recognized as not causing any adverse reactions upon topical administration in the eye [38,42]. Since maximum solubility of zileuton in 50% solution of DMSO in PBS was reported to be 0.5 mg/mL [43], we settled on the formulation of complete eye drops, consisting of 0.5 mg/mL zileuton in normal saline, containing 50% DMSO. According to the results of clinical examinations performed by an experienced veterinarian (co-author of this study), this dosage was well tolerated by both healthy rabbits and the animals with DES. Thus, no signs of local (i.e., in the cornea, conjunctiva, iris or eyelids) or systemic reactions were registered. It should be emphasized that the formulation was not regarded as final, but it was approved for primary verification of the proposed anti-inflammatory approach.

#### *3.4. Selective Targeting of Inflammation in DES: A Morphological Study*

To trial the suggested anti-inflammatory therapy, DES was induced in six experimental groups of animals (groups 6–11, Table 1), which subsequently received instillations of the complete zileuton/DMSO eye drops (zileuton groups) or 50% DMSO in normal saline (DMSO groups). One drop of each of these forms was administrated three times daily in each eye for up to seven days and the animals were euthanized on days one, three and seven following the anesthesia for histological analysis.

Schirmer's tests performed on similar groups of animals receiving the same instillations of zileuton/DMSO or DMSO (groups 12 and 15, Table 1), revealed no effect of the therapy on tear production (Figure 5a). Yet, already on the first day after the anesthesia, the corneas of zileuton groups exhibited pronounced histological signs of epithelial recovery, such as reepithelization rolls (Figure 5b, Z1). The area of denudation was minimal, as single-layer epithelium covered a significant part of the surface, which was drastically in contrast to the untreated samples. Importantly, at this time-point, the cornea exhibited almost no inflammatory infiltrate both in epithelium and stroma. On day three, the epithelium was restored across most of the cornea: the stromal denudation areas were rare and their size did not exceed single cells (Figure 5b, Z3). The newly formed epithelium had a thickness of several layers, although it did not reach the full-size characteristic of normal tissue. These samples exhibited no edema and acute inflammation, but there were signs of the completed inflammatory process and subsequent regeneration, such as an increased number of activated keratocytes at the corresponding sites. Finally, on day seven, the cornea of the rabbits which received zileuton (Figure 5b, Z7) was indistinguishable from the tissue of the healthy animals (Figure 5b, N).

**Figure 5.** Dynamic changes in tear secretion and corneal morphology in DES in the course of anti-inflammatory therapy. The animals were exposed to general anesthesia for 6 h (shown as gray box) and subsequently received instillations of the eye drops containing 50% DMSO (D1, D3, D7) or 50% DMSO with 0.5% zileuton (Z1, Z3, Z7) for 7 days. (**a**) The results of standardized Schirmer's tests performed at 6th hour, 1st day, 3rd day or 7th day after the exposure. (**b**) Representative microscopic images of hematoxylin and eosin-stained cross-sections of the corneas at 1st (D1, Z1), 3rd (D3, Z3) and 7th (D7, Z7) day after exposure. The image of the normal cornea is also presented (N). Stromal denudation (complete absence of the corneal epithelium) areas (red arrowheads), loci of desquamation/destruction of the epithelial layer (orange arrowheads), and sites of reepithelialization (green arrows) are indicated. The inflammatory changes include granulocytic infiltration in the corneal epithelium (intraepithelial granulocytes; black arrowheads) and stroma (black arrows), neovascularization (yellow arrows), and postinflammatory signs of regeneration (activation of stromal keratocytes) at the ex-sites of the infiltration (blue arrows). Ep: epithelium; Str: stroma; DM: Descemet's membrane and endothelium. Hematoxylin and eosin staining, magnification 200×. The central areas of the cornea are presented. Gaps, cracks and folds of preparations are of an artificial origin.