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Toxicology Letters 193 (2010) 159–166

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

omparative evaluation of the protective potentials of humanaraoxonase 1 and 3 against CCl4-induced liver injury

ei Penga,1, Chi Zhanga,1, Haiqin Lva, Jie Zhua, Yuhui Zanga,∗, Xiaojuan Pangb,unfeng Zhanga, Junchuan Qina,∗

State Key Laboratory of Pharmaceutical Biotechnology, Life Science School, Nanjing University, Nanjing 210093, PR ChinaLife Science School, Nanjing Normal University, Nanjing 210008, PR China

r t i c l e i n f o

rticle history:eceived 2 November 2009eceived in revised form 4 January 2010ccepted 5 January 2010vailable online 15 January 2010

eywords:araoxonaseiver injury

a b s t r a c t

We previously reported that electroporation mediated hPON1 or hPON3 gene delivery could protectagainst CCl4-induced liver injury. However, substantial evidence supported that the in vivo physiologicalfunctions of hPON1 and hPON3 were distinct. To compare the protective efficacies of hPON1 and hPON3against liver injury, recombinant adenovirus AdPON1 and AdPON3, which were capable of expressinghPON1 and hPON3 respectively, were intravenously injected into mice before they were given CCl4.Adenovirus mediated expression of hPON1 and hPON3 were demonstrated by elevated serum esteraseactivity, hepatic lactonase activity, and hPON1/hPON3 mRNA expression in liver. Serum transaminaseassay, histological observation and TUNEL analysis revealed that the extent of liver injury and hepatocyte

ene deliveryxidative stress

nflammatory responsepoptosis

apoptosis in AdPON1 or AdPON3 treated mice was significantly ameliorated in comparison with control.Meanwhile, overexpression of hPON1 and hPON3 reduced the hepatic oxidative stress and strengthen thetotal antioxidant capabilities in liver through affecting the hepatic malondialdehyde (MDA), glutathione(GSH) and total antioxidant capability (T-AOC) levels, regardless of the exposure to CCl4 or corn oil.Administration of AdPON1 or AdPON3 also suppressed inflammatory response by decreasing TNF-� andIL-1� levels in CCl mice. In this study, hPON1 exhibited a slightly higher efficacy than hPON3 in alleviating

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liver injury, but the differ

. Introduction

Carbon tetrachloride (CCl4) administration can induce acute orhronic liver injury in rodents depending on the dosage and admin-stration frequency (Sipes et al., 1974). This model has been widelysed for evaluating the therapeutic potential of drugs, since it isimilar to human liver disease from the standpoint of morphologyo biochemical features of the cellular lesions (Perez, 1983). CCl4-nduced hepatic necrosis involves two sequential phases. In the firsthase, CCl4 is metabolized by cytochrome P450 system to highlyeactive trichloromethyl free radicals and reactive oxygen speciesROS), which initiate lipid peroxidation and result in hepatocellu-ar apoptosis and necrosis (McCay et al., 1984). Secondary damageccurs from inflammatory response initiated by the activated hep-

tic macrophages, mainly Kupffer cells (Edwards et al., 1993; Elsisit al., 1993), which are capable of releasing a number of inflam-atory mediators including TNF-� and IL-1�. The early-response

nflammatory cytokines may potentiate hepatic injury by stimu-

∗ Corresponding authors. Tel.: +86 25 83686672; fax: +86 25 83324605.E-mail addresses: zangyh@nju.edu.cn (Y. Zang), jcqin@nju.edu.cn (J. Qin).

1 These authors contributed equally to this work.

378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2010.01.003

between them were not significant.© 2010 Elsevier Ireland Ltd. All rights reserved.

lating other cells to express chemokines and induce hepatocytesnecrosis and apoptosis through multiple mechanisms. Therefore,anti-oxidative and anti-inflammatory therapies are thought to beeffective means of preventing and attenuating CCl4-induced liverdamage.

The paraoxonase (PON) gene family contains three members,PON1, PON2, and PON3, which share 81–91% identity at nucleotidelevel and 79–90% identity at amino acid level among mammalianspecies (Primo-Parmo et al., 1996). Among the paraoxonase mem-bers, PON1 is understood by far better than PON2 and PON3.PON3 is the least studied member of the family, and its physi-ological role and natural substrates are still uncertain. However,PON3 exhibits considerable structural and functional homology toPON1. PON1 and PON3 share the lactonase activity and antioxidantproperty which are proposed to participate in preventing low den-sity lipoprotein (LDL) oxidation (Reddy et al., 2001). Nevertheless,abundant evidences have proved that the expression, protein local-ization and enzymatic activities of PON1 and PON3 are different,

suggesting that their physiological functions are far from redun-dant (Draganov et al., 2005; Ng et al., 2005). Several investigationshave been done to compare the anti-oxidative abilities of PON1 andPON3, but the results were conflicting. Liu et al. found that recom-binant human PON1 was more efficient than PON3 in retarding

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60 W. Peng et al. / Toxicolog

he oxidation of LDL in vitro (Liu et al., 2008), while the other initro study showed that rabbit serum PON3 was more efficient thanON1 in preventing copper-induced LDL oxidation (Draganov et al.,000).

Numerous studies have focused on the relationship betweenON1/PON3 and the development of oxidative stress related dis-ases, especially on atherosclerosis (Mackness et al., 2006; Shiht al., 1998, 2007). PON1 was found to be a useful index for theiagnosis of liver impairment and play multi-roles in regulatingxidative stress, fibrosis, and hepatic cell apoptosis in chronic liverisease (Ferre et al., 2006). In our previous studies, we foundhat electroporation mediated hPON1 or hPON3 gene deliveryould protect against CCl4-induced liver injury (Peng et al., 2009;hang et al., 2008). However, the efficiency of electroporationediated gene delivery was too low to compare the protective

bilities of hPON1 and hPON3 against liver injury. To intensivelynvestigate the protective mechanisms of hPON1 and hPON3 andompare their protective effects on CCl4-induced liver injury, weonstructed recombinant adenovirus AdPON1 and AdPON3, whichere capable of expressing hPON1 or hPON3. Our results showed

hat adenovirus mediated hPON1 and hPON3 gene delivery couldntroduce efficient and stable hPON1 and hPON3 expression, andhereby resulting in significant protective effects against livernjury. The protective mechanisms of hPON1 and hPON3 werelosely related to their abilities in regulating hepatic oxidativetress, hepatocyte apoptosis and inflammatory response. hPON1xhibited a slightly higher efficacy than hPON3 in alleviating livernjury, but the difference between them was not significant.

. Materials and methods

.1. Generation of recombinant adenovirus

Recombinant adenovirus was constructed using the Adenovirus Expression Vec-or Kit (Takara, Kyoto, Japan). The plasmid pBluescript-hPON1 containing hPON1DNA was kindly provided by Professor Manji Sun from Academy of Military Medicalcience, China. The plasmid pUC18-hPON3 containing hPON3 cDNA was constructedy Lv et al. (2005). The recombinant cosmids pAxCAwtit-hPON1 and pAxCAwtit-PON3 were constructed by inserting blunt-ended hPON1 or hPON3 cDNA into thenique SmiI site of pAxCAwtit. pAxCAwtit-LacZ containing LacZ gene (encoding �-alactosidase) was provided with the Kit. Recombinant cosmids were digested byspT104I to generate linear recombinant adenoviral genomics, and then transfected

nto human embryonic kidney 293 cells which provide the E1 gene product neces-ary for viral replication. Adenovirus expressing hPON1 (AdPON1), hPON3 (AdPON3)r �-galactosidase (AdlacZ) was screened and propagated in 293 cells, respectively.fter large-scale preparation, adenoviruses were concentrated by ViraBind Aden-virus Purification Kit (Cell Biolabs, San Diego, USA), dialyzed in phosphate bufferedaline (PBS) and stored in aliquots at −80 ◦C until use. The titer of each virus stockas determined by plaque assay on 293 cells.

.2. Animals and adenovirus infection

Five-week old male ICR mice, weighing 20 ± 2 g, were obtained from the Animalentre of Nanjing Medical University (Nanjing, China). Animals were maintained

n a ventilated, temperature-controlled room (23 ◦C) with a 12 h light:12 h darkycle to acclimatize for 1 week prior to experimentation. All animals had free accesso rodent chow and water. Mice were given an intravenous injection of 1 × 109

laque-forming units AdPON1 (n = 8), AdPON3 (n = 8) or AdlacZ (n = 8) via tail vein.he experiments were conducted according to the use and care guidelines of thexperimental animals of Jiangsu Province, China.

.3. RT-PCR and semi-quantitative RT-PCR

The total RNA was extracted from 0.01 g mouse liver using Fast RNA Iso-ation & Purification kit (Waston, Shanghai, China) and digested with DNaseto avoid DNA contamination. Total RNA was reverse-transcribed using SYBRrimeScript RT-PCR Kit (Takara, Kyoto, Japan). Aliquots of the reverse tran-cription mixture were used as templates for PCR amplification. hPON1 and

PON3 specific primers were designed to avoid amplifying mouse PON1 orON3 as follows: hPON1-forward, 5′-cggctgattgcgctcaccctct-3′ and hPON1-reverse,′-cgtacagttaggaagttctacg-3′; hPON3-forward, 5′-gccaccagagaccactattttacca-3′ andPON3-reverse, 5′-atcaccttcagttgagttaaatcc-3′ . Mouse GAPDH was amplifieds an internal control (forward, 5′-aactttggcattgtggaagg-3′ and reverse, 5′-gatgcagggatgatgttct-3′). PCR was performed as follows: initial melting at 94 ◦C for

ers 193 (2010) 159–166

5 min, followed by 30 cycles of 94 ◦C for 15 s, 60 ◦C for 30 s, 72 ◦C for 30 s, and finishedwith 7 min at 72 ◦C. The expression level of target mRNA was semi-quantitativelyevaluated by the intensity ratios of target gene to mouse GAPDH.

Mouse CYP2E1 specific primers were designed as follows, forward, 5′-ctcctcctcgtatccatctg-3′ and reverse, 5′-cagccaatcagaaaggtagg-3′ . PCR was performedas previously reported (Morris and Davila, 1996). Briefly, the PCR was initiated bymelting at 94 ◦C for 5 min, followed by 26 cycles of 94 ◦C for 30 s, 56 ◦C for 1 min,72 ◦C for 1 min, and finished with 7 min at 72 ◦C.

2.4. Esterase and lactonase activity assays

Blood samples were collected from mouse canthus vena. Serum esterase activitywas assayed using phenyl acetate as the substrate as previously described (Zhanget al., 2008). Briefly, the activity was determined by measuring the increase inabsorbance at 270 nm after 15 min incubation. One unit of esterase activity wasdefined as 1 �mol of phenyl acetate hydrolyzed/ml/min.

To determine hepatic lactonase activity, mouse liver was homogenized. After-wards, the assays were performed in a final volume of 1 ml containing 1 mMdihydrocoumarin (DHC) and 1 mM CaCl2 in 50 mM Tris/HCl (pH 8.0) buffer in thepresence of 10 �l 10% liver homogenates (Draganov et al., 2000). The hydrolysisof DHC was monitored by the increase in UV absorbance at 270 nm. One unit ofenzymatic activity is defined as 1 �mol of substrate metabolized per minute.

2.5. CCl4 exposure to induce liver injury

Mice received a single dose of 0.1 ml CCl4 (Nanjing Chem. Ltd., Nanjing, China)/kgbody weight in a 1% (v/v) corn oil solution by intraperitoneal injection on the 7th dayafter administration with adenovirus. The control group mice were administeredequal volume of corn oil. All mice were then sacrificed at 24 h after administrationof CCl4 or corn oil.

2.6. Serum aminotranferase estimation

Serum ALT and AST activities were measured using commercial kits (JianchengInstitute of Biotechnology, Nanjing, China) according to the manufacture’s instruc-tions. The results were expressed as IU/L.

2.7. Measurements of hepatic MDA, GSH and T-AOC levels

Liver tissue was homogenized rapidly in a buffer containing 9 volumes 0.15 MKCl-1.0 mM EDTA to obtain 1:10 (w/v) homogenates. Homogenates were then cen-trifuged at 12,000 rpm (4 ◦C) for 30 min to collect the supernatant for determiningMDA, GSH, T-AOC levels and protein concentrations. MDA level was evaluated bythe thiobarbituric acid reactive substances method (TBARS) (Ohkawa et al., 1979).The final concentration of MDA was expressed as nmol/mg protein. GSH and T-AOClevels were also measured using commercially available kits (Jiancheng Instituteof Biotechnology, Nanjing, China) and expressed as mg/g protein and U/mg pro-tein, respectively. Protein concentration of liver homogenate was determined bythe Bradford method, using bovine serum albumin as a standard (Bradford, 1976).

2.8. Measurements of serum cytokines

Serum TNF-� and IL-1� concentrations were quantified using commercialmouse TNF-� and IL-1� ELISA assay kits (eBioscience, San Diego, USA).

2.9. TUNEL analysis

A modified TUNEL assay was performed using DeadEnd Colorimetric TUNEL Sys-tem (Promega, Madison, USA) according to the manufacture’s protocol. Apoptosisindex (AI) was defined as the percentage of positive cells/total cells in the samemicroscopic field (200×). Thirty randomly chosen fields under 200× magnificationwere counted and the AI values were calculated.

2.10. Histopathologic analysis

Excised livers were fixed in 10% buffered formalin for at least 24 h, embeddedin paraffin wax, cut into 5 �m thick sections, stained with hematoxylin and eosinand examined by light microscopy. The degrees of necrosis were evaluated semi-quantitatively by two pathologists in a blinded fashion as follows: 0, absence ofnecrosis; 1, minimal, spotty necrosis; 2, moderate necrosis with nonconfluent areas;

2.11. Statistical analysis

All results were expressed as mean ± S.D., which were analyzed statisticallyusing one way ANOVA followed by Student’s t-test using SPSS (version 15.0) soft-ware. P values less than 0.05 were considered to be significant difference, P < 0.01indicated very significant difference.

W. Peng et al. / Toxicology Letters 193 (2010) 159–166 161

Fig. 1. In vivo expression of hPON1 and hPON3 in mice injected with AdPON1 and AdPON3. (A) Serum esterase activity was determined every 3 days after AdPON1 injection.From day 5 to day 15, serum esterase activities in AdPON1 injected mice were significantly higher than those of control groups injected with PBS or AdlacZ (P < 0.05). Datawere shown as means ± S.D. (n = 8). (B) Hepatic lactonase activity was measured using liver homogenates on the 7th day after AdPON3 administration. Data were shown asmeans ± S.D. (n = 8). *P < 0.05 versus AdlacZ and PBS treated mice. (C) hPON1 and hPON3 mRNA expression were detected by RT-PCR analysis with total liver RNA isolatedo N1 speA se; lanw RNA et

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n the 7th day after adenovirus injection. Lanes 1 and 2 were amplified with hPOdPON1 treated mouse; lane 2, AdlacZ treated mouse; lane 3, AdPON3 treated mouas analyzed by semi-quantitative after AdPON3 injection. (E) Ratios of hPON3 m

hree independent experiments (n = 8).

. Results

.1. Adenovirus mediated expression of hPON1 and hPON3

Recombinant adenovirus AdPON1, AdPON3 and AdlacZ, whichere capable of expressing hPON1, hPON3 and �-galactosidase

espectively, were prepared and intravenously injected into mice

ig. 2. Effects of adenovirus mediated expression of hPON1 and hPON3 on mice serum ASo CCl4. Values were mean ± S.D. (n = 8). *P < 0.01 versus AdlacZ control group. **P < 0.01 v

cific primers. Lanes 3 and 4 were amplified with hPON3 specific primers. Lane 1,e 4, AdlacZ treated mouse. (D) Sequential hPON3 mRNA expression in mouse liverxpression to mGAPDH mRNA expression in Part D. Results were means ± S.D. for

as described in Section 2. After AdPON1 injection, serum esteraseactivity toward phenyl acetate reached the peak on the 7th day

(Fig. 1A), which was about 4.8-fold higher than those of controlmice injected with PBS or AdlacZ (P < 0.01). The area under thecurve (AUC) of serum esterase activity during 3 weeks in AdPON1treated mice was about 2.3-fold higher than control. However,serum esterase or lactonase activity in AdPON3 treated mice did

T and ALT levels. Serum ALT and AST activities were determined 24 h after exposureersus AdlacZ group. #P < 0.05 versus AdlacZ group.

162 W. Peng et al. / Toxicology Letters 193 (2010) 159–166

Fig. 3. Adenovirus mediated expression of hPON1 and hPON3 alleviated CCl4-induced liver injury. Mice from different group were killed 24 h after injection with CCl4 orcorn oil. Paraffin-embedded liver sections were stained with hematoxylin and eosin. Representative sections are shown for each group. (A) AdlacZ control group; (B) AdlacZg ar inds rkedlyv

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roup; (C) AdPON1 group; (D) AdPON3 group. Original magnification: 200×. Scale bcore. The results showed that necrosis areas in AdPON1 or AdPON3 group were maersus AdlacZ group.

ot exhibit detectable difference from those of control during 3eeks (data not shown). So, we measured the hepatic lactonase

ctivity with homogenized liver tissue. The hepatic lactonase activ-ty toward DHC on the 7th day after AdPON3 administration wasbout 2 times higher than those of control groups injected with PBSr AdlacZ (Fig. 1B).

To confirm the hepatic expression of hPON1 and hPON3, RT-CR was performed with hPON1 or hPON3 specific primers 7 daysfter adenovirus inoculation. As shown in Fig. 1C, hPON1 andPON3 mRNA expression were detected in the liver of AdPON1 and

dPON3 treated mice respectively, but not in that of AdlacZ treatedontrol. Semi-quantitative RT-PCR analysis demonstrated that hep-tic hPON3 mRNA expression peaked at the 7th day (Fig. 1D and E),nd the higher expression level maintained at least for 14 days afterdPON3 administration.

icated 100 �m. (E) The extents of necrosis were assessed with a semi-quantitativereduced in comparison with AdlacZ group. **P < 0.01 versus AdlacZ group. #P < 0.05

3.2. Protective effects of hPON1 and hPON3 gene delivery onCCl4-induced hepatocyte damage

To evaluate the protective effects of hPON1 and hPON3 onliver injury, three group of mice were intravenously injected withAdPON1 (AdPON1 group), AdPON3 (AdPON3 group) and AdlacZ(AdlacZ group) respectively, and treated by a single intraperitonealinjection of CCl4 7 days after adenovirus inoculation. Meanwhile,three control group mice, titled as AdlacZ control, AdPON1 con-trol and AdPON3 control, were administrated with the same dose

of AdlacZ, AdPON1 and AdPON3 respectively and intraperitoneallyinjected with corn oil 7 days later. Subsequently, serum ALT andAST activities, which were used as sensitive markers of hepaticdamage, were determined 24 h after CCl4 or corn oil administra-tion. There was no difference in serum ALT and AST levels among

W. Peng et al. / Toxicology Letters 193 (2010) 159–166 163

Table 1Effects of hPON1 and hPON3 gene delivery on hepatic oxidative stress.

AdLAcZ control group AdPON1 control group AdPON3 control group AdlacZ group AdPON1 group AdPON3 group

MDA (nmol/mg protein) 0.31 ± 0.11 0.19 ± 0.03# 0.21 ± 0.05# 1.37 ± 0.33* 0.64 ± 0.12** 0.72 ± 0.41**

GSH (mg/g protein) 0.066 ± 0.014 0.082 ± 0.007# 0.077 ± 0.011# 0.021 ± 0.002* 0.062 ± 0.018** 0.049 ± 0.006**

T-AOC (U/mg protein) 2.10 ± 0.41 2.57 ± 0.31# 2.58 ± 0.59# 1.20 ± 0.26* 2.36 ± 0.69** 2.10 ± 0.50**

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** Denotes a very significant difference (P < 0.01) versus AdlacZ group.# Denotes a significant difference (P < 0.05) versus AdlacZ control group.

dPON1, AdPON3 and AdlacZ control mice. The mean activities oferum ALT and AST in AdlacZ control group were 24.11 ± 10.59 IU/Lnd 26.02 ± 4.47 IU/L respectively, while serum ALT and AST valuesn AdlacZ group dramatically increased to 9657.14 ± 731.68 IU/Lnd 3900.85 ± 1114.3 IU/L (P < 0.01). However, serum ALT and ASTctivities in AdPON1 and AdPON3 group were significantly lowerhan those of AdlacZ group (P < 0.05) (Fig. 2), indicating that over-xpression of hPON1 and hPON3 could ameliorate hepatocyteamage.

.3. Histological analysis

To analyze the extent of liver injury, liver sections were stainedith hematoxylin and eosin. No apparent damage was found in the

iver sections from three control group mice. In contrast, exten-ive damage was detected in the sections from AdlacZ group mice.epatocyte necrosis was the predominant histopathologic lesionnd the affected livers displayed hemorrhage, vacuolar change,ydropic degeneration of hepatocytes and infiltration of inflam-atory cells in AdlacZ group (Fig. 3B). However, in AdPON1 anddPON3 group mice, typical histological changes were significantlylleviated and necrosis areas were remarkably reduced (Fig. 3C, D).ccording to semi-quantitative analysis results, the extents of liver

njury were significantly reduced in AdPON1 and AdPON3 group inomparison with that of AdlacZ group (Fig. 3E).

.4. Effects of hPON1 and hPON3 gene delivery on hepaticxidative stress

The generation of reactive oxygen species and increase of hep-tic lipid peroxidation are important features of chronic liveriseases. To examine the effects of hPON1 and hPON3 on hepaticxidative stress, liver tissue was homogenized to determine hepaticDA, GSH and T-AOC levels 24 h after CCl4 administration (Table 1).

ig. 4. Effects of adenovirus mediated expression of hPON1 and hPON3 on mice serumetermined 24 h after administration of CCl4 using ELISA kits. Values are presented as mea*P < 0.01 versus AdlacZ group.

s using commercially available kits. Values are presented as mean ± S.D. (n = 8).

MDA is the end product of lipid peroxidation, whose levels indi-rectly reflect the degrees of oxidative stress. Of three control groupsinjected with corn oil, hepatic MDA concentration in AdlacZ controlgroup was 0.31 ± 0.11 nmol/mg protein, significantly higher thanthose of AdPON1 and AdPON3 control group. Of CCl4 treated groups,the hepatic MDA level in AdlacZ group was 1.37 ± 0.33 nmol/mgprotein, which was about 2 times higher as those of AdPON1 andAdPON3 group.

GSH is the first line of defense against free radicals and playsa key role in detoxifying the reactive toxic metabolites of CCl4(Park et al., 2008). T-AOC is an indicator of enzymatic and non-enzymatic antioxidants. Of three control groups, the hepatic GSHand T-AOC levels in AdPON1 and AdPON3 control group were sig-nificantly higher than those of AdlacZ control group. Meanwhile, ofCCl4 treated groups, hepatic GSH and T-AOC levels in AdPON1 andAdPON3 group mice were also significantly higher than those ofAdlacZ group. Collectively, our results supported that overexpres-sion of hPON1 and hPON3 could reduce mouse hepatic oxidativestress and strengthen the total antioxidant capabilities, regardlessof the exposure to CCl4 or corn oil.

3.5. Effects of hPON1 and hPON3 gene delivery on serumpro-inflammatory cytokines level after CCl4 administration

Inflammatory cytokines are early mediators of tissue damageand repair. In the present study, mouse serum TNF-� and IL-1� concentrations were measured to evaluate the influence ofhPON1 and hPON3 on CCl4-induced inflammatory responses. Therewas no significant difference in serum TNF-� and IL-1� levels

among three control groups. CCl4 exposure markedly stimulatedTNF-� and IL-1� releasing in comparison with those of controlgroups. Serum TNF-� and IL-1� level in AdlacZ group increasedto 114.43 ± 51.08 pg/ml and 35.84 ± 22.32 pg/ml respectively afterCCl4 administration, which were about 8.7- and 6.8-fold higher

pro-inflammatory cytokines levels. Serum TNF-� and IL-1� concentrations weren ± S.D. (n = 8). *P < 0.01 versus AdlacZ control group. #P < 0.05 versus AdlacZ group.

164 W. Peng et al. / Toxicology Letters 193 (2010) 159–166

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ig. 5. Representative photomicrographs of serial sections of mice liver biopsies staontrol group; (B) AdlacZ group; (C) AdPON1 group; (D) AdPON3 group. TUNEL-po00 �m. (E) Apoptosis index values are presented as mean ± S.D. (n = 30). *P < 0.01 v

han those of AdlacZ control group mice. However, the serum TNF-level in AdPON1 and AdPON3 group were 43.48 ± 37.23 pg/ml

nd 56.07 ± 39.68 pg/ml respectively, and the serum IL-1� levelsere 10.80 ± 7.25 pg/ml in AdPON1 group and 8.17 ± 8.14 pg/ml

n AdPON3 group (Fig. 4). These results suggested that AdPON1r AdPON3 pretreatment suppressed CCl4-induced inflammatoryesponse to a great extent.

.6. Effects of hPON1 and hPON3 gene delivery on hepaticpoptosis

Although necrosis in centrilobular area is a major cause of CCl4-nduced liver injury, hepatocyte apoptosis also contributes to itsathogenesis (Shi et al., 1998). In this study, apoptotic hepatocytes

ere detected by the in situ TUNEL assay (Fig. 5). No TUNEL-positive

ells were detected in the liver of three control group mice, whereaspproximately 18% hepatocytes were TUNEL positive in AdlacZroup after CCl4 administration. The percentage of TUNEL-positiveells in the liver of AdPON1 and AdPON3 group were about 8.5%

or TUNEL. Mice were sacrificed 24 h after injection with CCl4 or corn oil. (A) AdlacZcells were indicated by arrows. Original magnification: 200×. Scale bar indicatedAdlacZ group.

and 8%, respectively. These results suggested that hPON1 or hPON3could significantly reduce the extent of apoptosis caused by CCl4exposure.

4. Discussion

Recombinant adenovirus can efficiently mediate foreign genesexpression and easily accumulate in liver, where it is mainly takenup by hepatocytes and Kupffer cells (Shayakhmetov et al., 2004).Therefore, adenovirus is an ideal tool for gene therapy in liverdisease. In the present study, AdPON1 injection resulted in about4.8-fold increase in serum esterase activity on the 7th day afterinoculation. Hepatic lactonase activity toward DHC in AdPON3group was about 2 times higher than those of control on the 7th

day after AdPON3 injection. The absence of serum hPON3 activityafter AdPON3 administration was possibly due to the translocationand secretion efficiency of hPON3. More specifically, the N-terminalsequence of hPON3 might be less effective than that of hPON1 inguiding hPON3 releasing into circulation (Draganov, 2007).

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W. Peng et al. / Toxicolog

CCl4 administration destroyed functional integrity of cell mem-rane in liver and damaged hepatocytes released their contents

ncluding ALT and AST into the extracellular space (Drotman andawhorn, 1978). Of CCl4 treated groups, serum ALT and AST activ-ties in AdPON1 and AdPON3 group mice were significantly lowerhan those of AdlacZ group, suggesting that overexpression ofPON1 or hPON3 could help to maintain liver function. Subsequentistopathologic observations also confirmed that administration ofdPON1 or AdPON3 significantly reduced the extent of liver injury.

Reactive oxygen metabolites (ROM) have been implicated inhe pathogenesis of many kinds of liver diseases including CCl4-nduced liver injury. When liver cells are exposed to excess ROM,xidative stress occurs and affects many cellular functions. In theresent study, we monitored hepatic MDA, GSH and T-AOC levelso evaluate oxidative stress. MDA level indirectly reflects the extentf cellular damage attacked by free radicals and is widely used asn index of free radical mediated lipid peroxidation (Ohkawa et al.,979). GSH constitutes the first line of defense against free radicalsOokhtens and Kaplowitz, 1998) and depletion of mitochondrialSH will lead to necrosis as a result of the inability to defendgainst normally produced ROM in mitochondria. T-AOC reflectshe total antioxidant capabilities. According to our results, admin-stration of AdPON1 or AdPON3 could reduce the hepatic oxidativetress not only in mice treated by CCl4, but also in the control micereated by corn oil. Lower hepatic GSH and T-AOC levels in AdlacZroup mice might be due to deficiency of antioxidants exhaustedy increased oxidative stress. So, the protective effects of hPON1nd hPON3 should be closely related to their antioxidant potentialshich balanced the increased oxidative stress. Although there were

lready substantial evidences supporting that hPON1 and hPON3xerted their antioxidant effects in different manner, administra-ion of AdPON1 and AdPON3 did not exhibited significant differencen reducing hepatic oxidative stress in this study.

CCl4 toxicity results from the bioactivation of the CCl4 moleculeo the trichloromethyl free radical by cytochrome P450 isozymesP450s) of the endoplasmic reticulum (Koch et al., 1974; Slater,984). Of the multiple forms of P450s present in liver endoplas-ic reticulum, cytochrome P450 2E1 (CYP2E1) has been implicated

s a key metabolizing enzyme for CCl4 bioactivation and CCl4ediated liver injury in animal studies (Shibayama, 1988). More-

ver, previous study using cyp2e1−/− mice demonstrated thatice which lacked CYP2E1 expression were resistant to liver dam-

ge after exposure to CCl4 (Wong et al., 1998). To investigatehether CYP2E1 involved in the protective effects of PON1 and

ON3 against CCl4-induced liver injury, we designed mouse CYP2E1pecific primers to detect CYP2E1 mRNA expression using semi-uantitative RT-PCR according to previously reported methodsMorris and Davila, 1996). However, our results showed that theYP2E1 mRNA expression level did not exhibited detectable changefter AdPON1 or AdPON3 inoculation (data not shown).

In addition to mediate lipid peroxidation, CCl4 administrationlso induced severe inflammatory response and hepatocytes apop-osis. The inflammatory mediators releasing from activated hepatic

acrophages augmented CCl4-induced hepatic injury (Badger etl., 1996). Therefore, neutralizing and suppressing the release ofnflammatory mediators were suggested to alleviate the extent ofepatic injury (DeCicco et al., 1998). TNF-� is a multifunctionalytokine, which is rapidly secreted by macrophages in response toissue damage (Morio et al., 2001). IL-1�, which exhibits remark-ble similar properties to those of TNF-� (Dinarello, 1996), cantimulate inflammatory cells to release many other cytokines

ncluding TNF-�, IL-6 and IL8. The present study confirmedhat serum TNF-� and IL-1� levels in AdlacZ group significantlyncreased after CCl4 administration. However, TNF-� and IL-1� lev-ls in AdPON1 and AdPON3 group were less than half of thosen AdlacZ group, suggesting that hPON1 and hPON3 might exert

ers 193 (2010) 159–166 165

their protective effects against liver injury through suppressing theinflammatory responses. Except for TNF-� and IL-1�, serum Il-6levels were also measured in this study. Although serum IL-6 lev-els in AdlacZ group significantly increased after exposure to CCl4,AdPON1 and AdPON3 inoculation did not exhibit significant effectson IL-6 levels in AdPON1 and AdPON3 group (data not shown).

Apoptosis is an essential feature of liver injury ranging fromacute to chronic liver diseases. Targeting toward the apoptoticpathways therefore may constitute a future therapeutic possibilityin alleviating liver damage. Hepatocyte apoptosis can be initiatedeither by death ligands on the cell surface, such as TNF-� and Fas lig-ands, or by intracellular stresses, such as toxic metabolites of drugsand reactive oxygen metabolites (ROM) which profoundly disruptmitochondrial function. In CCl4-induced hepatic apoptosis, thesetwo pathways are not mutually exclusive, but closely interlinked.CCl4 administration caused significant increase in serum TNF-�level and hepatic oxidative stress, which were two principle factorsresponsible for hepatic apoptosis (Ding and Yin, 2004; Simon et al.,2000). Although the main apoptotic effect of TNF-� is mediated byTNF-R1 receptor, TNF-� stimulates the generation of reactive oxy-gen species (ROS), which may in turn promote hepatic apoptosis.Therefore, the efficacies of hPON1 and hPON3 in ameliorating apop-tosis were closely related to their abilities in reducing oxidativestress and inflammatory response. In this study, hPON1 and hPON3exhibited nearly same efficacies in alleviating hepatic apoptosis.

Taken together, the data presented in this study demonstratedthat adenovirus mediated expression of hPON1 and hPON3 wereassociated with lower hepatic oxidative stress, reduced inflamma-tory response and fewer TUNEL-positive cells in CCl4 administratedmice in comparison with control. These results will help us fullyunderstand the mechanism of PON1 and PON3 in protecting againstliver injury, and ultimately enable us to develop more effectivetherapies for liver diseases in the future.

Conflict of interest statement

There are no conflicts of interest.

Acknowledgements

The study was supported by the National Natural Science Foun-dation of China (Grant No. 30670858) to Junchuan Qin and KeyProject of National Natural Science Foundation of China (Grant No.90713015) to Ju Huangxian.

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