Mitochondrial decay is involved in BaP-induced cervical damage

Free Radical Biology & Medicine 49 (2010) 1735–1745

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Mitochondrial decay is involved in BaP-induced cervical damage Meili Gao a,⁎, Jiangang Long b, Yongfei Li c, Walayat Shah a,d, Ling Fu a, Jiankang Liu b, Yili Wang a a Institute of Cancer Research, Department of Biological Science and Engineering, Key Laboratory of Biomedical Information Engineering of the Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China b Institute of Mitochondrial Biology and Medicine, Department of Biological Science and Engineering, Key Laboratory of Biomedical Information Engineering of the Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China c School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an 710032, China d Institute of Basic Medical Sciences, Khyber Medical University, Peshawar 25000, Pakistan

a r t i c l e

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Article history: Received 8 May 2010 Revised 24 August 2010 Accepted 3 September 2010 Available online 16 September 2010 Keywords: Benzo[a]pyrene Lipid peroxidation Antioxidants ROS Mitochondria Cervix Free radicals

a b s t r a c t Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon and a potent inducer of carcinogenesis. Many studies have reported that the carcinogenic effects of BaP might be due to its intermediate metabolites and to reactive oxygen species (ROS) that cause oxidative damage to the cells. However, the mechanisms of BaPinduced oxidative damage in cervical tissue are still not clear. We studied these mechanisms in female ICR mice treated with BaP either orally or intraperitoneally by measuring (1) several general biomarkers of oxidative stress in serum, (2) mitochondrial function in the cervix, and (3) the morphology of mitochondria in cervical tissue. BaP treatment (1) significantly lowered levels of vitamins A, C, and E and of glutathione; (2) reduced activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferases; and (3) significantly increased lipid peroxidation levels. In addition, significant increases in the levels of superoxide anion, hydrogen peroxide, and hydroxyl radical were observed. These results were confirmed by morphological changes in mitochondria and by decreases in membrane potential levels and in succinate dehydrogenase and malate dehydrogenase activities. The changes in these biomarkers and mitochondrial damage were BaP-dose-dependent and eventually induced both cell apoptosis and necrosis in cervical tissue. As mitochondria are the major sites of ROS generation, these findings show that mitochondrial decay greatly contributes to BaP-induced cervical damage. © 2010 Elsevier Inc. All rights reserved.

Polycyclic aromatic hydrocarbons (PAHs) are by-products produced during pyrolysis or incomplete combustion of organic compounds [1]. As toxic and persistent organic pollutants, PAHs are widely distributed in the environment. Benzo[a]pyrene (BaP) is a representative PAH and a well-known carcinogen. To explain the carcinogenic potency of BaP, it has been proposed that its higher toxicity could be due to elevated levels of BaP metabolites and reactive oxygen species (ROS) [2]. BaP, and other PAHs in general, are subject to biotransformation by the cytochrome P450 enzymes, which play a key role in the metabolism of drugs and environmental chemicals [3,4]. The BaP metabolites so formed include several

Abbreviations: PAH, polycyclic aromatic hydrocarbon; BaP, benzo[a]pyrene; ROS, reactive oxygen species; RONS, reactive oxygen and nitrogen species; O•− 2 , superoxide anion; •OH, hydroxyl radical; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferase; GSH, reduced glutathione; LPO, lipid peroxidation; MDA, malondialdehyde; SDH, succinate dehydrogenase; MDH, malate dehydrogenase; HPLC, high-pressure liquid chromatography; TBA, 2-thiobarbituric acid; HRP, horseradish peroxidase type II; PMS, phenazine methosulfate; NBT, nitroblue tetrazolium; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method; H&E, hematoxylin and eosin. ⁎ Corresponding author. Fax: + 86 29 82663925. E-mail address: [email protected] (M. Gao). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.09.003

phenols, epoxides, dihydrodiols, dihydrodiol epoxides, and the ultimate carcinogenic metabolite anti-7,8-dihydroxy-9,10-epoxy7,8,9,10-tetrahydro-BaP [5]. Production of higher amounts of BaP metabolites results in greater ROS generation [2]. • ROS, including superoxide anion (O•− 2 ), hydroxyl radical ( OH), and hydrogen peroxide (H2O2), are generated as by-products of normal cellular metabolism. Mitochondria are the primary energy-generating organelles in the cell. The final step of the electron transport energygenerating process involves adding four electrons to oxygen to form water. Approximately 1–2% of the oxygen so consumed accepts a single electron to form reactive oxidant by-products. These oxidants attack mitochondrial membrane proteins, lipids, and nucleic acids, resulting in a lower efficiency of electron transfer. In turn, the damaged electron respiratory chain increases the production of oxidants, leading to a cycle of increasing oxidant production and mitochondrial damage [6]. Generally, ROS attack lipids and proteins and modify antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Several metabolites of BaP are subject to further transformation by conjugation with reduced glutathione (GSH), a reaction catalyzed by glutathione S-transferases (GSTs), a family of enzymes that are also involved in the prevention of lipid peroxidation (LPO) [7–9]. LPO is perhaps the most recognized biological effect of

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oxygen radicals and results in damage to both cellular and mitochondrial membranes [10–12]. Numerous reports have shown that mitochondria are sensitive to both reactive oxygen and nitrogen species (RONS)-mediated damage and alterations to function [13–15]. For example, mitochondrial membrane potential and activities of enzymes such as succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) are affected by oxidative damage [15,16]. Oxidative damage develops if the production of free radicals is increased and/or the level of antioxidants is decreased. Our defenses against free radical damage are the enzymatic and nonenzymatic antioxidant systems, which enable the scavenging of RONS. Nonenzymatic antioxidants include vitamin A (retinol and retinyl palmitate), vitamin C (ascorbic acid), vitamin E (α-tocopherol), and other bioactive molecules that, owing to their natural molecular structure, are able to influence redox reactions [17,18]. Vitamin A is a known antioxidant that is especially responsible for healthy development. Vitamin C has been widely reported to have the capability of protecting cells from oxidative damage [18,19]. Vitamin E, the major lipid-soluble antioxidant in biological systems, can effectively counteract ROS generated by toxicant- or carcinogen-induced oxidative damage by trapping reactive oxyradicals and preserving membrane integrity. Vitamin E is essential for protection against chemical insult because the major criterion for irreversibility of cellular injury is damage to the plasma membrane [20,21]. Cigarette smoking has been established as a risk factor for the development of cervical cancer. Extensive research over the past several years has suggested that free radicals play a role in a number of diseases, including carcinogenesis [22]. Tobacco smoke contains toxic PAHs such as BaP. Based on the observation that free radicals are generated during the formation of intermediate metabolites from BaP, it has been presumed that BaP might affect cervical tissue. In fact, several studies have been carried out to investigate variations in LPO and antioxidant levels in cervical cancer [17,21,23], whereas few studies have examined the effects of BaP on antioxidant status in cervical precancerous or normal cervical tissue. The goal of this work was to investigate whether and how the target organ (the cervix) is affected by BaP-induced oxidative damage. Materials and methods Chemicals Horseradish peroxidase type II (HRP; 200 U·mg− 1 solid), phenazine methosulfate (PMS), homovanillic acid (4-hydroxy-3-methoxyphenylacetic acid), BaP, decylubiquinone, and GSH were purchased from Sigma Chemicals (St. Louis, MO, USA). A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit was obtained from Roche (Darmstadt, Germany). Other reagents of analytical grade were purchased locally (China). Animals Female ICR mice (18–22 g) were purchased from the Experimental Animal Center of Xi'an Jiaotong University (Shaanxi Province, China). The mice were kept in our departmental animal house in a crossventilated room at 22 ± 2 °C, with relative humidity of 50 to 60% and a 12-h light–dark cycle. Animals had free access to conventional laboratory feed and water and were acclimatized for a period of 1 week before the beginning of the experimental procedures. Experiments on animals were performed according to the animal ethics guidelines of the institutional animal ethics committee. Experimental design BaP was dissolved in sesame oil shortly before oral gavage or intraperitoneal injection. A total of 100 mice were randomly divided

into two groups, the intraperitoneal injection group and the oral gavage group. The two groups were further divided into five subgroups: control, vehicle, low-dose, middle-dose, and high-dose subgroups. The low-, middle-, and high-dose subgroups were treated with 2.5, 5, and 10 mg/kg BaP, respectively, twice a week. The doses of BaP administered were in the range of doses used in previous in vivo studies in mice, such as 50 mg/kg (about 1 mg per mouse) [24], 10 to 60, 125, or 200 mg/kg [25]. The vehicle subgroups received equal volumes of sesame oil twice a week and the control subgroups were left untreated. The experiment was stopped 12 weeks later, after the last administration of BaP. Some of the mice died from the BaP administration during the experimental period, so the number of mice in each of the groups was lowered to 10 to maintain equality. For studying the effects of BaP on mitochondrial ultrastructure and necrosis, 30 mice were used; for the apoptosis assay, 60 mice were used. In each subgroup 3 or 6 animals were treated as described above. Mice were sacrificed, blood was sampled, and their cervices were removed immediately.

Biochemical analysis After an overnight fast, all the animals were weighed and blood was sampled. Serum was separated by centrifugation and was used for assaying vitamin A, vitamin C, and vitamin E. All the animals were killed by decapitation, and their cervices were immediately excised, weighed, and then homogenized in 0.01 M Tris–HCl buffer (pH 7.4) for biochemical assays.

Measurement of LPO levels and antioxidative enzyme activities The homogenates were centrifuged at 10,000 g for 20 min at 4 °C to obtain postmitochondrial supernatants. The supernatants were used as the source of LPO or antioxidative enzymes [26]. LPO was assayed using a method based on that of Wright et al. [27]. The reaction mixture contained 0.58 ml Na phosphate (0.1 M, pH 7.4), 0.2 ml tissue homogenate (10%, w/v), 0.2 ml ascorbic acid (100 mM) and 0.02 ml ferric chloride (100 mM). The reaction was stopped by the addition of 1.0 ml trichloroacetic acid (10%, w/v); finally 1.0 ml of 2-thiobarbituric acid (TBA; 0.67%, w/v) was added. The optical density was measured at 532 nm with a spectrophotometer. SOD activity was estimated by monitoring the autoxidation of pyrogallol as previously described [28]. After catalysis by SOD of the dismutation of superoxide radical to hydrogen peroxide and oxygen, the absorbance of the sample was measured at 420 nm. A single unit of enzyme was defined as the quantity of SOD required to produce 50% inhibition of autoxidation. CAT activity was determined by measuring sample absorbance at 240 nm with a UV/visible spectrophotometer as described [29]. One unit of CAT was defined as the amount of enzyme required to decompose 1 μmol of H2O2 per minute, at 25 °C and pH 7.0. Results are expressed as units of CAT activity per milligram protein. GSH was estimated as total nonprotein sulfhydryl group content as described [30]. The reaction mixture, containing equal volumes of 4% sulfosalicylic acid and tissue sample, was homogenized in 4 vol of ice-cold 0.1 M phosphate buffer (pH 7.4). GPx activity was measured following the protocol described in [31]. A reaction mixture containing 0.2 ml 0.4 M phosphate buffer (pH 7.0), 0.2 ml 0.4 mM EDTA, 0.1 ml 10 mM sodium azide, and 0.2 ml cervical tissue homogenate was incubated with 0.1 ml H2O2 and 0.2 ml GSH for 10 min. The amount of H2O2 consumed was determined by estimating the remaining GSH content according to the method of Anderson [30]. GST activity was determined as described [32]. The reaction mixture contained 0.1 M Na phosphate (pH 6.5), 30 mM GSH, 30 mM 1chloro-2,6-dinitrobenzene, and cervical tissue homogenate. The optical density was measured at 340 nm with a spectrophotometer.

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Estimation of ROS levels were measured using the method described by Levels of O•− 2 Nishikimi et al. [33]. The reaction mixture contained 15 μM PMS, 78 μM NADH, 30 μM nitroblue tetrazolium (NBT), and postmitochondrial fraction in 0.01 M Tris–HCl buffer (pH 7.4). After the sample was incubated at ambient temperature for 5 min, the color was read at 560 nm against a blank. The content of H2O2 in the postmitochondrial fraction was determined according to the method of Ruch et al. [34]. The reaction mixture contained 10 μl of the fraction, 90 μl of phosphate buffer, and 100 μl of a solution of HRP (1 U·ml− 1) and homovanillic acid; the mixture was incubated for 60 min at 37 °C. Afterward, each sample was mixed with 300 μl of phosphate buffer and 125 μl of 0.1 M glycine–NaOH buffer (pH 12) and 25 mM EDTA; the resulting mixture was determined spectrofluorimetrically. Slit widths were set at 10 nm for both emission and excitation, the integration time was 0.1 s, excitation was at 312 nm, and emission was measured at 420 nm. Hydroxyl radical levels in cervix were measured by the degradation of deoxyribose as described by Gutteridge [35]. Briefly, reaction tubes contained 0.73 M H2O2, 0.8 mM deoxyribose in 10 mM phosphate buffer (pH 7.4) and postmitochondrial fraction. The final volume was 1.0 ml. The reaction was initiated by adding 0.42 mmol of FeSO4, and the mixture was incubated for 10 min at 37 °C. Deoxyribose degradation by the hydroxyl radicals so generated was measured by the TBA-reactive substances assay. This assay yields a stable colored substance with a maximum absorbance at 532 nm, which was measured against a blank. Determination of nonenzymatic antioxidant levels Nonenzymatic antioxidants, including vitamin A, vitamin C, and vitamin E, were assayed in the serum of experimental animals. Vitamin A concentrations were determined by high-pressure liquid chromatography (HPLC), as described by Bortolotti et al. [36].Vitamin C was measured photometrically as described in [37]; when dehydroascorbic acid is coupled with dinitrophenylhydrazone and treated with sulfuric acid, it forms an orange-red compound, which was measured at 520 nm. Vitamin E was assayed using the method described by Baker et al. [38]. In brief, the method involves the reduction of ferric ions to ferrous ions by α-tocopherol and the subsequent formation of a colored complex with 2,2′-dipyridyl. Absorbance of the chromophore was measured at 520 nm.

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The reaction mixture contained 1 mM EDTA, freshly neutralized 0.2 mM oxaloacetate, 0.5 mM NADH, and 50 mM potassium phosphate buffer (final pH 6.9) and the mitochondrial fraction. At the dilution of the extract used in the assay NADH oxidase activity was negligible. Detection of mitochondrial ultrastructure Mitochondrial ultrastructure was examined by transmission electron microscope. Cervical tissue was fixed, embedded, sectioned, and stained with uranyl acetate and lead citrate. The sections were viewed and photographs taken using a Hitachi H-600 transmission electron microscope (Japan, JEM-100SX) at 80 kV accelerating voltage. Apoptosis assay Apoptosis was assessed by the TUNEL method using the In Situ AllDeath Detection Kit (Roche). Formalin-fixed tissues were embedded in paraffin and were then sectioned at 5 μm thickness. The tissue sections were mounted on slides and deparaffinized, diluted proteinase K was added, and the sections were blocked with 3% H2O2 in phosphate-buffered saline. The sections were then treated with the TUNEL reaction mixture composed of terminal deoxynucleotidyl transferase, rinsed, and visualized using Converter-POD with 0.03% 3,3′-diaminobenzidine and then mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount. Apoptotic cells within each epithelial region were counted using a light microscope and the epithelial cell apoptotic rate was quantified by calculating the percentage of apoptotic cells. Examination of necrotic cell death Necrosis was observed with hematoxylin and eosin (H&E) as performed in routine histological examination. The tissue specimens were obtained from mice treated with various doses of BaP. The cervical tissues were fixed with 10% phosphate-buffered neutral formalin, dehydrated in graded (50–100%) alcohol, and embedded in paraffin. Serial sections were cut into 5-μm-thick slices and stained with H&E for cell necrosis examination. The examination was cursory and qualitative, for the purpose of determining whether BaP caused necrotic cell death in mouse cervical tissue.

Assay of mitochondrial enzymes and membrane potential

Statistical analysis

A 10% homogenate was prepared in 0.25 M sucrose and centrifuged at 600 g for 10 min at 4 °C. The supernatant fraction was decanted and then again centrifuged at 15,000 g for 10 min at 4 °C to obtain the mitochondrial fraction. Mitochondrial membrane potential was monitored as described in [39]. Briefly, the mitochondrial fraction (1 mg/ml of protein) was incubated for 1.5 min at 20 °C with rhodamine 123 in basic reaction medium containing 150 mM sucrose, 5 mM MgCl2, 10 mM disodium succinate, 2.5 mM rotenone, 2 mM K buffer, 20 mM potassium Hepes buffer (pH 7.4). After centrifugation for 2 min at 10,000 g, free dye in the supernatant was determined using a fluorescence spectrometer at an excitation wavelength of 495 nm and an emission wavelength of 535 nm. Mitochondrial SDH was assayed as described by the method of Rustin et al. [40]. The reaction mixture contained 50 mM potassium phosphate, pH 7.8, 2 mM EDTA, 0.1% bovine serum albumin, 3 μM rotenone, 80 μM 2,6dichlorophenol, 50 μM decylubiquinone, 1 μM antimycin A, 0.2 mM ATP, 0.3 mM KCN, and the mitochondrial fraction. The mixture was preincubated for 10 min at 37 °C; the reaction was started by addition of 10 mM succinate and followed for 6 min at 600 nm. Mitochondrial MDH was measured by the method of Turner and Manchester [41].

All values are expressed as means ± SD. Data analyses were performed using the SPSS 13.0 software package. Analysis of variance was applied for the comparison of the means of the various treatment groups. Post hoc testing was performed for intergroup comparisons applying the least-significant difference. Values were considered statistically significant at P b 0.05. Results Effects of vehicle on antioxidant status and mitochondrial ultrastructure Normally corn oil [10] or sesame oil [24] has been used to dissolve BaP for treatment of rats or mice; sesame oil was used as the vehicle in this study. Vehicle groups received the same volume of sesame oil as used in the BaP-treated groups. To assess whether the mice were affected by sesame oil, we compared the biomarkers of vehicle groups with control groups. Biomarkers of oxidative stress were not affected by sesame oil compared with concurrent control groups. Likewise, mitochondrial ultrastructure, membrane potentials, and activities of the mitochondrial enzymes SDH and MDH were not affected by

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vehicle. Additionally, neither apoptosis nor necrosis was induced in either vehicle groups or controls. These findings imply that sesame oil as vehicle does not affect cervical tissue.

Effects of BaP on LPO and antioxidants To examine the role of oxidative damage induced by BaP, LPO levels and antioxidants were assayed in postmitochondrial fractions of cervical tissue. ICR mice were treated with BaP at low, middle, and high doses by either oral gavage or intraperitoneal injection, and the effects of these treatments on LPO and antioxidants in the cervix were determined (Fig. 1). The levels of GSH and the activities of the antioxidant enzymes SOD, CAT, GPx, and GST were significantly

B MDA content (nmol/mg protein)

GPx activity (U/mg protein)

A

decreased, whereas MDA levels were significantly increased in BaPtreated groups compared to the concurrent control groups. We simultaneously determined whether nonenzymatic antioxidants such as vitamins A, C, and E were affected by BaP. As shown in Table 1, the levels of vitamin A, vitamin C, and vitamin E were significantly decreased in BaP-treated groups compared with the concurrent control groups. In addition, the activities of SOD, CAT, GST, and GPx as well as the levels of GSH and vitamins A, C, and E were lower, and MDA levels were higher, if the dose of BaP was higher. Moreover, no significant differences were observed between the orally and the intraperitoneally treated mice in all the biomarkers studied here (Fig. 1, Table 1). These data show that oxidative damage may be induced by BaP in cervical tissue and may cause decreases in antioxidant levels.

1200 1000

***

***

*** 800 *** 600 ***

***

400 200 0 Oral gavage

6

*** ***

5 4

*** *** *** ***

3 2 1 0 Oral gavage

Intraperitoneal injection

Treatments

Treatments

D

C *

**

**

***

300

*** ***

200

100

SOD activity (U/mg protein)

400

CAT activity (U/mg protein)

Intraperitoneal injection

300 250

* *

** **

200

***

100 50 0

0 Oral gavage

Intraperitoneal injection

F 60

2.5 *** ***

***

***

***

1.5

***

1.0 0.5

GST activity (U/mg protein)

GSH content (mg/mg protein)

Treatments

3.0

2.0

Intraperitoneal injection

Oral gavage

Treatments

E

***

150

50 * 40

** ***

*** ***

30

***

20 10 0

0.0 Oral gavage

Intraperitoneal injection

Treatments

Oral gavage

Intraperitoneal injection

Treatments

Fig. 1. Effects of BaP on LPO and antioxidative enzymes in the cervix of mice. (A) GPx activity. (B) MDA levels. (C) CAT activity. (D) SOD activity. (E) GSH levels. (F) GST activity. Postmitochondrial fractions were obtained and were assayed for LPO levels and antioxidants as described under Materials and methods. Values are means ± SD, n = 7. *P b 0.05 vs concurrent control, **P b 0.01 vs concurrent control, ***P b 0.01 vs concurrent control.

M. Gao et al. / Free Radical Biology & Medicine 49 (2010) 1735–1745

n

VE (g/mg protein)

60

VC (g/mg protein)

VA (g/mg protein)

0.42 ± 0.04 0.41 ± 0.05 0.31 ± 0.03*** 0.27 ± 0.02*** 0.22 ± 0.01***

2.72 ± 0.40 2.70 ± 0.41 2.03 ± 0.38*** 1.54 ± 0.36*** 1.03 ± 0.25***

0.40 ± 0.05 0.39 ± 0.04 0.29 ± 0.03*** 0.26 ± 0.04*** 0.19 ± 0.02***

2.70 ± 0.37 2.68 ± 0.41 2.05 ± 0.36*** 1.43 ± 0.31*** 0.89 ± 0.19***

50

VE, vitamin E; VC, vitamin C; VA, vitamin A. Values indicate means ± SD. **P b 0.01 vs concurrent control. ***P b 0.001 vs concurrent control.

Effects of BaP on ROS levels As mentioned above, ROS are produced as by-products during BaP catabolism in organisms [2]. To investigate whether excessive ROS • were induced by BaP, levels of ROS such as O•− 2 , H2O2, and OH were assessed in postmitochondrial fractions from cervical tissue of control and experimental animals. As shown in Table 2, ROS levels were significantly higher in BaP-treated groups compared to the control groups, and the increases were dose-dependent. Again no significant differences in ROS levels were observed between the orally and the intraperitoneally treated mice. This finding suggests that excessive ROS are induced by BaP in cervical tissue. Effects of BaP on membrane potential levels and SDH and MDH activities in cervical mitochondria To study the effect of BaP on mitochondrial function in cervical tissue, membrane potential levels and SDH and MDH activities in cervical mitochondria were assayed in controls and in BaP-treated mice. Figs. 2 and 3 present the effects of BaP on the levels of the membrane potential and the activities of the mitochondrial enzymes SDH and MDH. Highly significant decreases were observed in the membrane potentials of middle-dose and high-dose BaP-treated groups compared to the concurrent control groups. However, the corresponding decrease was not significant in the low-dose BaP-treated group (Fig. 2). Activities of SDH and MDH significantly decreased in all three BaP-treated groups compared to their concurrent control groups. These results demonstrate that mitochondrial function in the cervix of experimental animals may be affected by BaP.

*

40

FI units

Group

Oral gavage

Intraperitoneal injection

Table 1 Effects of BaP on vitamin A, C, and E levels in serum

Oral gavage Control 10 0.52 ± 0.08 Vehicle 10 0.50 ± 0.05 Low dose 9 0.41 ± 0.04** Middle dose 9 0.36 ± 0.07*** High dose 9 0.29 ± 0.03*** Intraperitoneal injection Control 10 0.51 ± 0.09 Vehicle 10 0.50 ± 0.07 Low dose 8 0.39 ± 0.06** Middle dose 7 0.33 ± 0.08*** High dose 7 0.27 ± 0.05***

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***

30

*** ***

20 10 0

Control

Vehicle

Low dose Middle dose High dose

Fig. 2. Effects of BaP on membrane potential levels in cervical mitochondria of control and treated mice. Mitochondrial fractions were obtained and mitochondrial membrane potential levels were measured as described under Materials and methods. Each value is expressed as the mean ± SD for seven mice in each group. *P b 0.05 vs concurrent control, ***P b 0.01 vs concurrent control.

Effects of BaP on mitochondrial ultrastructure The effects of BaP on mitochondrial ultrastructure were examined using a transmission electron microscope. As shown in Figs. 4 and 5, the mitochondria appeared clearly round-shaped or band-shaped in both intraperitoneally and orally treated control groups, i.e., mitochondrial ultrastructure was normal in the control and vehicle groups. Mitochondrial membranes were intact and cristae were arranged in the form of a concentric ring or vertical line, congested and clear (Figs. 4A and 5A). In low-dose BaP-treated groups and in middle-dose orally treated groups, the mitochondrial structure was basically normal except for a slight swelling of the mitochondria and a slight decrease in clearing of cristae (Figs. 4B and 5B). However, marked detrimental effects were observed between the middle-dose intraperitoneally treated and the high-dose BaP-treated groups when mitochondrial ultrastructure was compared. Mitochondria appeared indistinctly round-shaped or band-shaped. Most mitochondrial outer and inner membranes were partially lost or fused. Cristae morphology and membranes were barely visible. In addition, swelling of the mitochondria was associated with reduced electron density of the matrix (Figs. 4C, 5C, and D). These observations showed that mitochondrial ultrastructure was more perturbed in the intraperitoneal groups than in the oral gavage groups. The results suggest that mitochondrial ultrastructure in cervical tissue may be seriously affected by BaP.

Table 2 Effects of BaP on ROS levels in cervical tissue Group

Superoxide radical (mol NBT/10 min/1012 cells)

Oral gavage Control 54.22 ± 3.28 Vehicle 55.10 ± 3.73 Low dose 75.36 ± 6.39** Middle dose 84.90 ± 6.77*** High dose 124.91 ± 12.68*** a Intraperitoneal injection Control 55.15 ± 4.11 Vehicle 55.50 ± 4.07 Low dose 80.93 ± 7.64** Middle dose 93.33 ± 9.87*** High dose 136.73 ± 12.55*** Values indicate means ± SD, n = 7. **P b 0.01 vs concurrent control. ***P b 0.001 vs concurrent control.

Hydrogen peroxide (mmol/1012 cells)

Hydroxyl radical (nmol/1012 cells/h)

68.24 ± 6.46 70.13 ± 6.55 98.58 ± 8.32*** 127.74 ± 11.69*** 150.19 ± 10.28***

15.26 ± 0.97 16.09 ± 0.81 34.10 ± 3.21*** 41.47 ± 3.02*** 48.54 ± 4.16***

68.44 ± 6.15 68.39 ± 6.04 109.94 ± 9.72*** 138.37 ± 12.09*** 168.15 ± 11.21***

15.73 ± 0.86 16.55 ± 0.81 38.17 ± 3.13*** 45.39 ± 3.52*** 57.89 ± 4.15***

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µ

A

B

involving mitochondria can lead to mitochondrial DNA damage, destabilization of mitochondrial function, and induction of cell death [42]. Here, we investigated the effects of BaP on cervical cell apoptosis using TUNEL staining to visualize DNA fragmentation, a marker of apoptotic cell death [43]. As shown in Fig. 6, BaP induced apoptosis of cervical epithelial cells. Apoptotic cells were labeled mainly in the lower layers of the epithelium (Figs. 6A–D). There was a significant (P b 0.001) increase in the rate of epithelial cell apoptosis in BaP-treated animals compared with the control or vehicle group (Fig. 6E). Likewise, the rate of apoptosis increased with increasing BaP dose, but there was no significant difference between the intraperitoneally and the orally treated groups. Moreover, necrotic cells and inflammatory cells were observed in cervical precancerous lesions (such as cervical epithelial hyperplasia and atypical hyperplasia) in tissue from BaP-treated groups (Figs. 7B–D) compared with the control group (Fig. 7A). These results suggest that BaP can induce apoptosis or necrosis in cervical tissue and also support the conclusion that the oxidative stress induced by BaP may be involved in cell death.

Discussion

Fig. 3. Effects of BaP on mitochondrial enzymes in cervix from control and treated mice. (A) Effect of BaP on mitochondrial SDH activities. (B) Effect of BaP on mitochondrial MDH activities. Mitochondrial fractions were obtained and SDH and MDH activities were measured as described under Materials and methods. Each value is expressed as the mean ± SD for seven mice in each group. *P b 0.05 vs concurrent control, **P b 0.01 vs concurrent control, ***P b 0.001 vs concurrent control.

Effects of BaP on cervical cell apoptosis and necrosis The results described above suggest that BaP can induce oxidative stress in cervical tissue. It has been shown that oxidative stress

Recent work has shown that excessive ROS are involved in a number of diseases, including precancer and cancer, aging, neurodegenerative diseases, malaria, and arteriosclerosis [24]. Because the potential toxicity of BaP that is important in the etiology of precancerous and cancerous lesions is generated by the interaction of ROS and BaP metabolites, evaluation of oxidative stress markers would reveal the molecular mechanisms underlying the ability of BaP to mediate carcinogenesis. In this study, we report an assessment of the levels of LPO and antioxidants, the pathological changes in mitochondria, and apoptosis or necrosis, all these being consequences of the oxidative damage induced by BaP in mouse cervix. LPO is a well-established mechanism of oxidative damage caused by excessive ROS, which are cytotoxic and act as tumor promoters or cocarcinogenic agents. The measurement of MDA provides a convenient index of LPO [27,44]. In this study MDA levels were higher in BaPtreated groups than in controls and the differences were statistically significant. The results indicate that treatment with a range of doses of BaP results in oxidative damage because the LPO product MDA is an indicator of ROS generation in tissue [1,45]. It is well known that free radicals participate in BaP epoxidation and probably cause the increased MDA excretion observed after BaP treatment [46]. MDA

Fig. 4. Electron micrographs of mitochondria from cervical endometrium of mice treated by oral gavage. (A) Representative image of mitochondria from control, vehicle, and lowdose groups. (B) Mitochondria from middle-dose BaP-treated group. (C) Mitochondria from high-dose BaP-treated group. Experimental conditions were as described under Materials and methods. Bar, 0.5 μm.

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Fig. 5. Electron micrographs of mitochondria from cervical endometrium of mice treated by intraperitoneal injection. (A) Representative image of mitochondria from control and vehicle groups. (B) Mitochondria from low-dose BaP-treated group. (C) Mitochondria from middle-dose BaP-treated group. (D) Mitochondria from high-dose BaP-treated group. Experimental conditions were as described under Materials and methods. Bar, 0.5 μm.

content in the BaP-treated groups was higher than in the concurrent controls, indicating that the cervices of these animals suffered more serious oxidative damage. Distinct increases in the products of LPO were accompanied by marked decreases in the activities of SOD, CAT, GPx, and GST and in GSH levels, in the BaP-treated groups compared with the control groups. SOD is important in preventing LPO by catalyzing the dismutation of the LPO initiator and the transformation of superoxide radicals to H2O2 and O2; CAT and GPx catalyze H2O2 to molecular water [1]. The significant decreases in antioxidant enzymes such as SOD, CAT, and GPx suggest their increased utilization to scavenge LPO products by the excessive ROS generated in BaP-treated mice [11]. In addition, GST plays a crucial role in detoxification processes because it catalyzes the conjugation of both endogenous substances and xenobiotics with GSH. GSH constitutes the first line of defense against free radicals and plays an important role in the detoxification of electrophilic substances and prevention of cellular oxidative stress [11,47]. Inhibition of GST activity and depletion of GSH below its basal level would promote the generation of ROS, with a cascade of effects

on the functional and structural integrity of cells and organelle membranes [48], reducing the capacity to detoxify other chemicals and increasing the vulnerability to oxidative stress [26]. These findings are in conformity with the observations that in cervical cancer patients increased oxidative damage leads to consumption and depletion of endogenous antioxidant enzymes [49,50]. Moreover, levels of nonenzymatic antioxidants such as vitamins A, C, and E were significantly lower in BaP-treated groups compared to the control groups. The primary antioxidant action of vitamin A is to scavenge singlet oxygen and to prevent lipid peroxidation [18]. Vitamin A deprivation causes mitochondrial depolarization and increases mitochondria-derived ROS formation [51]. Vitamin C is an effective biologic antioxidant that prevents the oxidative damage to cell membranes induced by aqueous radicals; it also exists in interconvertible forms and participates in neutralizing free radicals [52,53]. Vitamin E is a donor antioxidant that reacts with and reduces peroxyl radicals and thus can limit LPO by terminating chain reactions initiated in membrane lipids. Vitamin E-deficient animals have a stronger inflammatory response and develop a more powerful oxidative stress

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Apoptotic intraepithelial cells (%)

E

Oral gavage

Intraperitoneal injection *** *** *** ***

30 25 20 15 10

***

***

5 0

Control

Vehicle

Low dose Middle dose High dose Groups

Fig. 6. Effects of BaP on cervical cell apoptosis. Apoptotic cells in the lower layers of the cervical epithelium were labeled by TUNEL (original magnification: × 100). (A) Representative image of cervical tissue from control and vehicle groups. (B) Cervical tissue from low-dose BaP-treated groups. (C) Cervical tissue from middle-dose BaP-treated groups. (D) Cervical tissue from high-dose BaP-treated groups. (E) Quantitative and statistical analysis of the effects of BaP on apoptosis of cells from control and treated mice. The rate of epithelial cell apoptosis was quantified by counting the percentage of apoptotic cells as described under Materials and methods. ***P b 0.001 compared to the concurrent control group.

response [54–56]. Hence, it is suggested that the cytotoxic effects of BaP significantly decreased the levels of vitamin A, C, and E as reported in other studies [49,54]. Low levels of vitamins A, C, and E in BaP-treated mice may be due to their increased utilization for scavenging LPO products. Organisms with antioxidant status this low are more prone to malignancy, as reported in cervical cancer patients [22,57]. These results indicate that the abnormally high concentrations of free radicals such as O•− 2 , H2O2, and •OH induced by BaP could disrupt the balance between oxidants and antioxidants and promote oxidative damage.

Mitochondria are important organelles that perform a variety of fundamental functions ranging from the synthesis of ATP to programmed cell death [58,59]. Toxic effects of BaP lead to damage of mitochondrial ultrastructure, including impairment of the outer or inner membranes and cristae, swelling of the mitochondria, and reduction of electron density. These phenomena were similar to responses of mitochondria to BaP in skeletal muscle [60], lung tissue [61], human granulosa cells [62], and RL95-2 human endometrial cancer cells [63]. This also directly correlates with the depletion of the mitochondrial enzymes SDH and MDH and of the mitochondrial

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Fig. 7. Effects of BaP on cervical cell necrosis. Necrotic cell death induced by BaP in the upper cervical epithelium was visualized using H&E staining as per routine histological examination (original magnification: × 400). (A) Representative image of cervical tissue from control and vehicle groups. (B) Cervical tissue from low-dose BaP-treated groups. (C) Cervical tissue from middle-dose BaP-treated groups. (D) Cervical tissue from high-dose BaP-treated groups.

membrane potential, as observed in BaP administration. The decreases in mitochondrial enzymes might be due to a marked deficiency in one or more electron transport chain components [16,61]. Although the exact mechanism explaining the effects of BaP on mitochondria is yet to be discovered, a few previous studies suggest that ROS generation may be associated with alterations in mitochondrial ultrastructure [59–63]. Additionally, more recent studies indicate that the carcinogenic properties of BaP could be due to increased levels of carcinogenic BaP metabolites and ROS [1,2,10]. Therefore, changes in ROS—including O•− 2 , H2O2, and •OH—may increase oxidative damage and engender the aberrant ultrastructural changes in mitochondria. The variations in LPO and antioxidants are consistent with mitochondrial impairment [64] and with reductions in the activities of the mitochondrial enzymes SDH and MDH and the mitochondrial membrane potential. The findings indicate that BaP treatment in cervical tissue increases excessive ROS and mitochondrial damage. There is evidence indicating that ROS produced by mitochondria are involved in mediating apoptosis [65]. Moreover, mitochondrial damage and dysfunction have been identified as key elements underlying both necrotic cell death and apoptosis [66,67]. Our results show that the rate of apoptotic, TUNEL-positive cells in the base of the epithelium was significantly higher after BaP induction. This increased apoptotic rate, in conjunction with mitochondrial changes, including decreased membrane potential and disruption of ultrastructural morphology, may support the suggestion that the mitochondrial (intrinsic) pathway is intimately involved in BaP-induced apoptosis, as reported elsewhere [63,68]. Our findings also support the hypothesis that

necrotic cell death is initiated by mitochondrial damage and is associated with decreased membrane potential, mitochondrial swelling, and progressive increases in ATP depletion [67,69]. Even inflammatory reactions elicited by necrotic cell death were observed in BaP-treated groups. Further work is required to examine the detailed mechanism of BaP-induced necrotic cell death with respect to other biomarkers of necrosis such as ATP and Ca2+ levels and especially the mitochondrial permeability transition. Interestingly, both cellular apoptosis and necrosis were observed in the cervical epithelial region, suggesting that cervical epithelia are more susceptible and are recognized as major targets for BaP [70]. In our study, the indices assessed were not affected by sesame oil; it is therefore suitable for use as a vehicle as previously described [24]. Similarly, there were no significant differences in results between the two routes of administration—intraperitoneal injection and oral gavage—except in the case of mitochondrial ultrastructure; here, administration by intraperitoneal injection effected greater changes than oral gavage. This may be due to the greater degree of injury produced by intraperitoneal injection. Further, BaP-induced damage to the organism was dose-dependent and positively correlated, as reported elsewhere [1,26,71]. The markers assessed in this study, especially LPO and antioxidants, closely correlate with cervical cancer. BaP administration increased LPO products and decreased antioxidant levels. These findings are consistent with variations in antioxidant status in cervical cancer as reported elsewhere [17,22,23,49,50,57]. These studies have shown that cervical cancer patients, compared to healthy controls,

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have higher lipid peroxidation levels and lower indices of both antioxidant enzymes, such as SOD, GPx, and GST, and nonenzymatic antioxidants, including vitamins A, C, and E. Free radicals and ROS • such as O•− 2 , H2O2, and OH play important roles in the initiation and progression of cancer. Although cervical cancer was not observed within our experimental period, cervical precancerous lesions (Fig. 7) were observed in BaP-treated animals. Hence, the findings of this study imply that the changes in LPO and antioxidant levels may have prognostic significance in cervical precancer [22,50,57]. BaP-induced oxidative stress, or oxidative damage in various species [10,24,26,72], may be postulated to be a possible mechanism of BaP-induced toxicity. Cervical cancer is the third most prevalent type of female cancer and ranks second as a cause of cancer-related deaths in women worldwide [73]. A number of risk factors, including cigarette smoking, have been associated with cervical precancer and cancer. BaP, a common environmental and occupational PAH, is present in tobacco smoke, in smoke from various types of organic combustion or exhaust, and in various occupational settings [71,74,75]. This suggests that most humans are probably more or less constantly exposed to this compound, although to a much lower extent than are active smokers. The data obtained from this study indicate that BaP can elicit oxidative damage in mouse cervix. All biomarkers were assessed in postmitochondrial or mitochondrial fractions of cervical tissue, except that nonenzymatic antioxidants were measured in serum; we can say that mitochondrial decay is involved in BaP-induced damage in the cervix. In conclusion, the results of our studies demonstrate that BaP produces excessive free radicals—especially ROS—as well as pathological changes in mitochondria from mouse cervix. This unique association between oxidative damage and BaP may help us to understand the variations in antioxidant status in cervical precancerous lesions. Our results partially support epidemiological studies, which closely link exposure to cigarette smoke as a risk factor for the development of cervical cancer. Understanding variations in the antioxidant status of other species and changes of other biomarkers is important for designing appropriate mechanistic studies of BaPinduced toxicity in the cervix and warrants further investigation. Acknowledgments The authors thank Ying Sun and Baochang Lai for their help in the experiments and Dr. Edward Sharman (University of California at Irvine, USA) for critical reading and editing of the manuscript. This study was supported by a Principal grant from Xi'an Jiaotong University of China (7114003, 01380005). References [1] Pan, L. Q.; Ren, J.; Liu, J. Responses of antioxidant systems and LPO level to benzo (a)pyrene and benzo(k)fluoranthene in the haemolymph of the scallop Chlamys ferrari. Environ. Pollut. 141:443–451; 2006. [2] Garry, S.; Nesslany, F.; Aliouat, E.; Haguenoer, J. M.; Marzin, D. Hematite (Fe2O3) acts by oxidative stress and potentiates benzo[a]pyrene genotoxicity. Mutat. Res. 563:117–129; 2004. [3] Bauer, E.; Guo, Z.; Ueng, Y. F.; Bell, L. C.; Zeldin, D.; Guengerich, F. P. Oxidation of benzo[a]pyrene by recombinant human cytochrome P450 enzymes. Chem. Res. 8: 136–142; 1995. [4] Yun, C. H.; Shimada, T.; Guengerich, F. P. Role of human liver cytochrome P4502C and 3A enzymes in 3-hydroxylation of benzo[a]pyrene. Cancer Res. 52: 1868–1874; 1992. [5] Melikian, A. A.; Sun, P.; Prokopczyk, B.; El-Bayoumy, K.; Hoffmann, D.; Wang, X.; Waggoner, S. Identification of benzo[a]pyrene metabolites in cervical mucus and DNA adducts in cervical tissues in humans by gas chromatography mass spectrometry. Cancer Lett. 146:127–134; 1999. [6] Judge, S.; Jang, Y. M.; Smith, A.; Hagen, T.; Leeuwenburgh, C. Age-associated increases in oxidative stress and antioxidant enzyme activities in cardiac interfibrillar mitochondria: implications for the mitochondrial theory of aging. FASEB J. 19:419–421; 2005. [7] Korashy, H. M.; El-Kadi, A. O. The role of aryl hydrocarbon receptor and the reactive oxygen species in the modulation of glutathione transferase by heavy metals in murine hepatoma cell lines. Chem. Biol. Interact. 162:237–248; 2006.

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