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Abstract

Benzo(a)pyrene (B(a)P) is an environmental pollutant which causes various lung toxicities. The present study was designed to evaluate the protective effects of carvacrol, a monoterpenic phenol against B(a)P-induced lung toxicity. In this study, Swiss albino mice were pretreated with carvacrol (25 mg/kg and 50 mg/kg) orally for 7 consecutive days before administering oral B(a)P (125 mg/kg). Preventive efficacy of carvacrol was assessed in terms of membrane oxidation, antioxidant enzyme activities, histopathological changes, and inflammatory (iNOS, NF-κB, and COX-2) markers. Carvacrol pretreatment in the two doses restored B(a)P-induced lipid peroxidation and increased the activities of antioxidant enzymes. Protein expressions of iNOS, NF-κB, and COX-2 in the lung tissue were found to be upregulated by B(a)P. Carvacrol treatment, however, downregulated their expressions by decreasing the marker of positive stained cells and restored the histopathological architecture of lung tissue. Our results suggest that carvacrol can be used as a protective agent against B(a)P-induced lung toxicity and inflammation.

Keywords

Lung toxicity inflammation benzo(a)pyrene carvacrol antioxidant

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are one of the major environmental contaminants generated by incomplete combustion of organic matter and forest fires, volcanic eruptions, fossil fuels and wood combustion, industrial processes.¹ Benzo(a)pyrene (B(a)P) is one of the PAHs which mostly produces from cigarette smoke and exhaust and plays major role in alteration in lung pathologies which may lead to cancer.² Its lipophilic nature makes it suitable for the easy absorption by biological membrane and also by different metabolic pathways.³

B(a)P is metabolized by cytochrome P450_1A1 enzyme to B[a]P-7,8-diol-9,10-epoxides (BPDE) which form mutagenic adducts with DNA (BPDE-N2-dG lesion) and act as carcinogen.⁴ It is also metabolized to O-quinones by dihydrodiol dehydrogenases, which undergoes redox cycling with their semiquinone free radicals to form large amounts of reactive oxygen species (ROS), which results in oxidative DNA damage and plays a critical role in pathogenesis of lung cancer.⁵

Genetic mutations and chromosomal damage by B(a)P have been shown by earlier in vivo and in vitro studies.^6,7 Detrimental effect of B(a)P on lung in short-term exposure made it to establish as a model for studying adverse effects on pulmonary system.^8,9

Scientists from worldwide are focusing on the use of the herb and dietary agents such as flavonoids, terpenoids, and polyphenol in preventing various kinds of environmental induced disorders including carcinogenesis.¹⁰ Cancer chemoprevention by the use of natural, dietary, or synthetic agents can reverse, suppress, or prevent carcinogenic progression and has become an appealing strategy to reduce the increasing cases of cancers worldwide.¹¹ Many natural products are known to exert their protective effects by scavenging free radicals and modulating antioxidant defense system and carcinogen detoxification.¹²

Carvacrol (5-isopropyl-2-methylphenol-CAR)¹³ is a monoterpenic phenol that is present in the essential oil of thyme, pepperwort, oregano, and wild bergamot. It belongs to the genera Origanum and Thymus.¹⁴ The concentration of carvacrol in thyme oil, Thymus vulgaris, savory, marjoram, and dittany of Crete is found to be 5–75%, 9–60%, 1–45%, 50% and 60–80%, respectively.¹⁵ Carvacrol is known to possess an extensive variety of pharmacological properties including anti-inflammatory, antioxidant,¹⁶ antimicrobial, antibacterial, and antiviral properties.¹⁷ Carvacrol has also been found to be protective against diethylnitrosamine-induced hepatocellular carcinoma in rats.¹⁸ It also has immunomodulatory effects in allergy, autoimmunity, and infectious diseases.¹⁹ Carvacrol has been found protective against oxidative stress and heart injury in cyclophosphamide-induced cardio-toxicity in rats²⁰ and also against oxidative stress induced by chronic stress in brain, liver, and kidney of rats.¹³ In the present study, we have evaluated the efficacy of carvacrol against B(a)P administered lung toxicities in male Swiss Albino mice was examined.

Methods

Chemicals

Carvacrol, B(a)P, reduced glutathione (GSH), oxidized glutathione (GSSG), nicotinamide adenine dinucleotide phosphate reduced (NADPH), flavin adenine dinucleotide (FAD), thiobarbituric acid (TBA), trichloroacetic acid (TCA), bovine serum albumin (BSA), xylene, alcohol, and ethylenediamine tetra acetic acid (EDTA) were obtained from Sigma (St Louis, Missouri, USA). All the antibodies, chemicals, and reagents used were of the highest purity and standard commercially available.

Animals

About 30 male Swiss albino mice (aged 7–8 weeks and 38–45 g) were obtained from the Central Animal House Facility of Jamia Hamdard (Hamdard University), New Delhi, India and were housed in a ventilated room in polypropylene cages at a temperature of 25°C ± 2°C under a 12 h light/dark cycle. The animals were acclimatized for 1 week before beginning the experiment. They had free access to standard laboratory feed (Hindustan Lever Ltd., Mumbai, India) and water ad libitum. The study was approved by the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) with the registration no.: 173/Go/Re/S/2000/CPCSEA.

Experimental design

Mice were divided into five groups with six animals in each group, receiving the following treatment. Carvacrol was administered orally at two doses in accordance to Nafees et al.,¹⁷ and the dose of B(a)P was selected in accordance to Sehgal et al.²¹

Group I: Served as control and was administered normal saline (10 ml/kg) for 7 days and corn oil at the dose of 10 ml/kg at 7th day only.

Group II: Served as toxicant group and received a single dose of B(a)P (125 mg/kg in corn oil, orally) on the 7th day only.

Group III: Pretreated with carvacrol at the dose of 25 mg/kg b.w. from day 1 to day 7 and B(a)P (125 mg/kg in corn oil) was given on 7th day after 2 h of the pretreatment with carvacrol.

Group IV: Pretreated with carvacrol at the dose of 50 mg/kg from day 1 to day 7 and B(a)P (125 mg/kg in corn oil) was given on 7th day after 2 h of the pretreatment with carvacrol.

Group V: Received only higher dose of carvacrol (50 mg/kg) up to 7 days.

All the animals were sacrificed by cervical dislocation 24 h after the last treatment regime and processed for subcellular fractionation. Lungs were collected for examination of various biochemical and others parameters. Before sacrifice, mice underwent mild ether anesthesia. Later, blood was withdrawn from the retro-orbital sinus, and serum was obtained by centrifugation.

Assessment of various biochemical parameters

Post mitochondrial supernatant (PMS) preparation

Lungs were removed and cleaned with ice-cold saline (0.85% sodium chloride). Lung tissues were homogenized in chilled phosphate buffer (0.1 M, pH 7.4) using a homogenizer (Remi Process Plant and Machinery Ltd., Mumbai, India) and were centrifuged at 3000 × g for 10 min at 4°C in a cooling centrifuge (Remi Process Plant and Machinery Ltd., Mumbai, India) to separate the nuclear debris. The aliquot obtained was centrifuged at 12,000 × g for 20 min at 4°C to obtain PMS, which was used as the source of enzymes. The various parameters analyzed in PMS were as follows:

Glutathione reductase (GR) activity

GR activity was measured as described by Carlberg and Mannervik.²²

Glutathione peroxidase (GPx) activity

GPx activity was estimated as described by Rashid et al.²³

Glutathione-S-transferase (GST) activity

Assay for activity was estimated by the method of Habig et al.²⁴

Assay for lipid peroxidation (LPO)

The assay for LPO was carried out as described by Nafees et al.¹⁷

Quinone reductase (QR) activity

The QR activity was carried out as described by Benson et al.²⁵

Xanthine oxidase (XO) activity

XO activity was carried out by the method described by Nafees et al.¹⁷

Assay of LDH activity

Lactate dehydrogenase (LDH) activity has been estimated in serum by the method of Kornberg.²⁶

Superoxide dismutase (SOD) activity

SOD activity was measured by the method of Marklund et al.²⁷

Catalase activity

Catalase activity was measured by the method of Claiborne et al.²⁸

Reduced glutathione (GSH) activity

GSH activity was determined by the method of Jollow et al.²⁹

Estimation of protein concentration

The protein concentration in all the samples was determined by the method described by Lowry et al.³⁰

Histopathological examination of lungs

At the end of experiment, the lungs were quickly removed and preserved in 10% buffered formalin for histopathological processing. Tissue dehydration was done with ascending grades of ethanol, followed by tissue clearing with xylene. The tissues were then transferred to molten paraffin for impregnation and embedded in paraffin blocks. After fine sectioning with microtome, staining was done with hematoxylin and eosin (H&E) stain and was examined under light microscope under 40× magnification.³¹

Immunohistochemistry

Expressions of inducible nitric oxide synthase (iNOS), transcription factor NF-κB,^32,33 and inflammatory protein (enzyme) cyclooxygenase-2 (COX-2)^34,35 were examined in the lungs of all the groups of animals.

Sections of formalin-fixed, paraffin-embedded lung tissues were placed on poly-L-Lysine coated slides. The sections were de-paraffinized with xylene. Antigen retrieval was performed by incubating slides in citrate buffer (pH: 6.0) (10 mM) at 95°C for 20 min and then rehydrated through descending grade of alcohol. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 min. The sections were then incubated with primary antibodies as desired under humid conditions and kept overnight at 4°C. Further processing was done using Lab Vision™ Ultra Vision™ Detection System: anti-polyvalent, horseradish peroxidase/diaminobenzidine staining kit (Thermo Fisher Scientific, Mumbai). The slides were then washed with tris-buffered saline and Tween 20 (TBST) five times for 2 min each. The peroxides complex was visualized with 3,3-diaminobenzidine. After immunoreactivity, the slides were dipped in distilled water, counterstained with Harris H&E. Thereafter, the slides were passed through ascending grades of ethyl alcohol to dehydrate. The slides were then cleaned with xylene in and dried and finally the sections were mounted with DPX and examined under fluorescent microscope (Olympus BX-51, U-LH-100HG, Olympus Corporation, Tokyo, Japan).

Statistical analysis

The differences between the groups were analyzed using one-way analysis of variance followed by Tukey-Kramer multiple comparisons test. All data points are presented as the treatment groups mean ± standard deviation (SD). p < 0.05 was considered as significant. Statistical program R (version 3.1.0) was used for the analysis.

Results

Effects of carvacrol pretreatment on enzymatic and non-enzymatic antioxidant activities

The effect of carvacrol pretreatment on the activity of different antioxidants was assayed. There was a significant difference (p < 0.001) in the activity of different antioxidant enzymes (GPx, GR, GST, CAT, and SOD) and non-enzymatic antioxidant (GSH) between control group I and B(a)P-treated group II. Our results showed that carvacrol (25 mg/kg) significantly increased the activities of GPx (p < 0.01), GR (p < 0.01), GST (p < 0.001) in group III animals as compared to group II ones; and at higher dose carvacrol (50 mg/kg) effects were intensified (Table 1).

Table 1.

Effect of carvacrol pretreatment on superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, reduced glutathione, and catalase on benzo(a)pyrene-induced lung toxicity (results represent mean ± SD, n = 6 animals per group).

Groups	Superoxide dismutase (U/mg protein)	Glutathione peroxidase (nmol NADPH/min/mg protein)	Glutathione reductase (nmol NADPH/oxidized/min/mg protein)	Glutathione-S- transferase (nmol dinitrochlorobenzene conjugate formed/min/mg protein)	Reduced glutathione (nmol GSH/g in tissues)	Catalase (nmol H₂O₂ consumed/min/mg protein)
I	79.97 ± 1.11	1.83 ± 0.14	0.22 ± 0.03	876.72 ± 3.62	0.94 ± 0.05	82.13 ± 2.43
II	39.86 ± 1.65^a	0.57 ± 0.13^a	0.06 ± 0.01^a	541.43 ± 6.57^a	0.52 ± 0.01^a	42.24 ± 2.73^a
III	54.99 ± 1.77^b	1.08 ± 0.14^b	0.12 ± 0.01^b	776.79 ± 5.26^c	0.69 ± 0.02^c	55.45 ± 1.98^b
IV	73.28 ± 0.86^c	1.42 ± 0.08^c	0.15 ± 0.01^c	831.71 ± 8.10^c	0.83 ± 0.02^c	67.66 ± 3.21^c
V	77.48 ± 0.54	1.67 ± 0.03	0.19 ± 0.01	864.55 ± 2.68	0.89 ± 0.03	78.47 ± 3.50

^a p < 0.001, when compared to group I.

^b p < 0.01, when compared to group II.

^c p < 0.001, when compared to group II.

^d p < 0.05, when compared to group II.

Effect of carvacrol pretreatment on XO activity

B(a)P administration significantly increased the activity of XO in group II as compared to the control group I (p < 0.001). However, it was found that pretreatment of carvacrol in groups III and IV (p < 0.05) and (p < 0.01), respectively, restored the activity of XO significantly when compared to group II. There was no significant difference observed in group V as compared to group I (Figure 1(a)).

Figure 1.

Effect of carvacrol on various parameters (mean ± SD, n = 6 animals per group). (a) Effect on XO activity in lungs. B(a)P leads the significant elevated level (***p < 0.001) of XO in group II as compared to group I. Carvacrol-treated groups III and IV significantly (#p < 0.05, ##p < 0.01) decreased the level of enzyme activity as compared to group II. (b) Effect on LPO (MDA level) in lung injury induced by B(a)P. MDA level was significantly (***p < 0.001) increased in B(a)P-treated group II as compared to control group I. Carvacrol pretreatment significantly (#p < 0.05, ##p < 0.01) decreases the level of MDA content in groups III and IV as compared to group II. (c) Effect on LDH. B(a)P treatment increases the activity of LDH in group II significantly (***p < 0.001) as compared to vehicle-treated group I. Carvacrol pretreatment attenuates the value of LDH (###p < 0.001 and ###p < 0.001) in groups III and IV. There was no significant difference in the level of LDH in groups I and V. (d): Effect on QR activity in Swiss albino mice. B(a)P treatment reduces the activity in group II significantly (***p < 0.001) as compared to group I. Carvacrol pretreatment restored the activity significantly (###p < 0.001, ###p < 0.001) in group III and IV, respectively. There is no significant difference in group I and group V. B(a)P: benzo[a]pyrene; DCPIP: dichlorophenolindophenol; LDH: lactate dehydrogenase; LPO: lipid peroxidation; MDA: malondialdehyde; NADH: nicotinamide adenine dinucleotide; QR: quinone reductase; XO: xanthine oxidase.

Effect of carvacrol pretreatment on LPO

Carvacrol inhibits lipid peroxidation generated by B(a)P. Malondialdehyde (MDA) is a well-known biomarker of oxidative stress–induced membrane damage. Oral administration of B(a)P leads to significant elevation in the concentration of MDA in group II as compared to the control group I (p < 0.001). In group III, administration of carvacrol (25 mg/kg) reduced MDA formation significantly (p < 0.05) compared to group II. Also at higher dose, carvacrol (50 mg/kg) attenuated MDA formation significantly (p < 0.001) compared to group II. There was no significant change in the concentration of MDA between control and group 5 animals (Figure 1(b)).

Effect of carvacrol pretreatment on LDH activity

B(a)P-treated group showed significantly increase in serum LDH (***p < 0.001) when compared to control group. Carvacrol pretreatment significantly decreased toward the normal in group III and group IV (###p < 0.001, ###p < 0.001) as compared to group II. No significant change observed in the group V as compared to group I (Figure 1(c)).

Effect of carvacrol on QR activity

The concentration of QR was significantly (p < 0.001) decreased in the B(a)P-treated group II as compared to group I. QR concentration significantly increased in carvacrol pretreatment groups III (p < 0.01) and IV (p < 0.001) when compared to group II. No significant difference was observed between groups I and V (Figure 1(d)).

Histopathological findings of lungs

Oral administration of B(a)P caused disruptions of lung epithelium (Figure 2(b)) when compared to the control group (Figure 2(a)). It also caused severe destruction of alveolar architecture and necrosis of the alveolar epithelium (Figure 2(b)). Infiltration of inflammatory cells was also seen. Carvacrol at both the doses (25 mg/kg and 50 mg/kg) showed partial protection against B(a)P as evident by lung histology (Figures 2(c) and (d)). About 50 mg/kg carvacrol (Figure 2(e)) did not show any lung pathology.

Figure 2.

Effect of carvacrol pretreatment against B(a)P-induced histological alteration (representative tissue, n = 6 animals per group). (a): Normal lung of control mice lung. (b) B(a)P (125 mg/kg)-treated group showed severe destruction of alveolar architecture and necrosis of the alveolar epithelium of the lungs. (c and d) Carvacrol at both the doses (25 mg/kg and 50 mg/kg) showed protection against B(a)P-induced lung damage. (e) Carvacrol (50 mg/kg)-treated group showed normal lung morphology (H&E stain, 40× magnification).

Immunohistochemistry

There were higher expressions of COX-2 (Figure 3(a)), NF-κB (Figure 3(b)), and iNOS (Figure 3(c)) in group II animals treated with B(a)P than the animals treated with both carvacrol and B(a)P (groups III and IV), as well as the control group I.

Figure 3.

Photomicrographs depicting immunohistochemical staining of COX-2 (a), NF-κB (b), and iNOS (c) (representative tissue, n = 6 animals per group). There were higher expressions of COX-2, NF-κB, and iNOS in group II animals treated with B(a)P than the animals treated with both carvacrol and B(a)P (groups III and IV), as well as the control group I (40× magnification).

Discussion

ROS plays an important role in the etiology of various cancers while the antioxidants combat the free radicals.^36,37 B(a)P is one of the PAHs classified human carcinogen.³⁸ The metabolic activation of B(a)P resulted in the formation of diol epoxide as ultimate carcinogen. It can bind to the nucleophilic center of DNA bases especially N⁷ position of guanine base.³⁹ This adduct formation initiates the cascade of reactions and induces initiation of the cancer process.⁴⁰ Metabolism of B(a)P leads to the formation of ROS in the form of $O_{2}^{-}$ , OH⁻, and H₂O₂.⁴¹ This free radical causes lipid peroxidation and formation of stable oxidative product viz MDA and HNE adducts. This MDA-DNA adducts may undergo different mutations leading to cancer.^4,42 B(a)P also causes severe toxicities to the system as well. Prenatal exposure to rats of B(a)P is also known to affect learning and memory disorders.⁴³

Previous studies have shown that capsaicin,⁴⁴ piperine,^45,46 and naringenin,²³ a naturally occurring flavanone, can be protective against B(a)P-induced lung cancer in experimental models. These compounds decrease the extent of oxidative stress with concomitant increase in the activities of enzymatic antioxidants.⁴⁶ Carvacrol is derived from Origanum and Thymus plants and is a monoterpene¹⁴ having antifungal, antimicrobial, antitumor, and antioxidant properties.^{16,23,45,47
–49} It also possesses anticarcinogenic, antiproliferative, and antiplatelet properties.⁵⁰ In this study, the antioxidant and anti-inflammatory property of carvacrol against BaP-induced toxicity has been investigated.

Oxidative stress is one of the major causes of carcinogenesis due to induction of gene mutation resulting from cell injury and adverse effects on signal transduction and transcription factors.⁵¹ ROS initiates LPO directly by reacting with lipids of membranes or by acting as secondary messengers for the primary free radicals.⁵² LPO plays an important role in cellular damage and is a marker of oxidative stress which in turn may ultimately lead to disease development.⁵³ It has been found that administration of B(a)P significantly increases the level of MDA formation,⁵⁴ showing its toxic effect in lung tissue. This was significantly attenuated by carvacrol treatment in our study. The modulatory effect of carvacrol on LPO level may be due to its antioxidant and free radical scavenging property.⁵⁵ Accumulation of H₂O₂ in excessive amounts causes cell toxicities.⁵⁶ Enzymatic and non-enzymatic antioxidants play an important role in the defense system against free radicals.⁵⁷ SOD is the enzyme that disrupts superoxide free radicals and protects the cells against superoxide by converting superoxide radicals into hydrogen peroxide.⁵⁶ Again GSH is a tripeptide antioxidant molecule which acts either by direct interaction of –SH group with ROS or by incriminating the enzymatic detoxification reaction for ROS.^58,59 Various GSH dependent enzymes like GST, GPx, GR, and so on, work by scavenging free radicals. With the help of GSH, GPx protects tissue from damage due to oxidative stress by converting hyperoxides into water and disulfide glutathione (GSSG); GSSG thus formed will be reconverted into GSH with the help of GR.^60
–62 We have demonstrated that B(a)P treatment reduces the level of these antioxidant enzymes by generating free radicals.⁶³ Treatment with carvacrol in both the doses (25 mg/kg and 50 mg/kg) effectively restores the activities of these enzymes. It was also observed that B(a)P administration enhances the activity of XO⁶⁴ which reduces O₂ to superoxide anion radical $O_{2}^{-}$ and consequently produces oxidative stress.⁶⁵ Treatment with carvacrol significantly attenuates the activity of XO. These results show that carvacrol protects the lungs from oxidative injury by raising the level of enzymatic antioxidants.

Oxidative stress results in inflammation which activates NF-κB.⁶⁶ NF-κB activation causes transcriptional upregulation of COX-2 and pro-inflammatory cytokines, such as IL-6 and TNF-α.⁶⁷ NF-κB and COX-2 are the important enzymes of inflammatory signaling pathways.⁶⁸ Our study shows that carvacrol exerted its anti-inflammatory property by attenuating the NF-κB over expression in B(a)P-treated group.

Higher concentrations of NO can cause DNA strand breakage and base alterations.⁶⁹ It forms nitrogen dioxide and peroxynitrite anion when reacts with oxygen and superoxide anion, which are cytotoxic oxygen radicals interfering with a variety of lung functional parameters. The expression of iNOS is involved in many inflammatory and neoplastic conditions.⁷⁰ The modulation of COX-2 and iNOS-mediating signaling pathway has been considered a new approach for preventing carcinogenesis.⁶⁰ In this study, carvacrol was found to be effectively modulating the over expression of iNOS by BaP which shows its protective effect against cell injury. COX is a pro-inflammatory enzyme that plays key role in inflammation. In the present study, expression of COX-2 was measured. Here, we have demonstrated that carvacrol treatment effectively suppresses the expression of activated COX-2 enzyme activity in B(a)P-treated mice lung tissues. This signifying the strong anti-inflammatory effect, and hence, it shows antitumor promoting potential of carvacrol as well.

So, finally we could conclude that carvacrol (25 mg/kg and 50 mg/kg) demonstrated significant protective (antiapoptotic, antiproliferative, and anti-inflammatory) activities against B(a)P-induced lung toxicity in experimental mice. Further studies are needed to explain the detailed mechanism of action to aid the discovery of new therapeutic agents for the treatment of B(a)P and other environmental carcinogen-induced lung toxicity.

Footnotes

Acknowledgement

We acknowledge the University Grant Commission (UGC), New Delhi, India for giving fellowship to the first author Preeti Barnwal.

Declaration of Conflicting Interests

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

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Benzo(a)pyrene induces lung toxicity and inflammation in mice: prevention by carvacrol

Abstract

Keywords

Introduction

Methods

Chemicals

Animals

Experimental design

Assessment of various biochemical parameters

Post mitochondrial supernatant (PMS) preparation

Glutathione reductase (GR) activity

Glutathione peroxidase (GPx) activity

Glutathione-S-transferase (GST) activity

Assay for lipid peroxidation (LPO)

Quinone reductase (QR) activity

Xanthine oxidase (XO) activity

Assay of LDH activity

Superoxide dismutase (SOD) activity

Catalase activity

Reduced glutathione (GSH) activity

Estimation of protein concentration

Histopathological examination of lungs

Immunohistochemistry

Statistical analysis

Results

Effects of carvacrol pretreatment on enzymatic and non-enzymatic antioxidant activities

Effect of carvacrol pretreatment on XO activity

Effect of carvacrol pretreatment on LPO

Effect of carvacrol pretreatment on LDH activity

Effect of carvacrol on QR activity

Histopathological findings of lungs

Immunohistochemistry

Discussion

Footnotes

Acknowledgement

Declaration of Conflicting Interests

Funding

References