Opisthorchiasis viverrini (OV) infection is the major risk factor of cholangiocarcinoma (CCA) development.1, 2 OV infection and CCA development occur at high incidence in northeastern Thailand.2, 3 The antihelminthic drug praziquantel has been used for the treatment of parasite infection, including liver fluke such as OV.4 The cure rate of a single dose of praziquantel for liver fluke treatment was more than 90%.4 However, although praziquantel appears to interfere with calcium homeostasis and causes flaccid paralysis in adult flukes, its mode of action is still not clearly understood.5 It is still a matter of debate whether the therapeutic effect of praziquantel is attributed to its specific antiparasitic effect and/or antiinflammatory effect.5, 6 Accumulation of evidence verifies that DNA damage is believed to be linked between inflammation and carcinogenesis.7, 8, 9 Reactive nitrogen species can mediate the formation of 8-nitroguanine in nucleic acids.10, 11, 12 Akaike et al. have reported that 8-nitroguanine is formed in association with viral infection.13 8-Nitroguanine is a marker of nitrative DNA damage, which has been proposed to account for infection-associated carcinogenesis.14 We have demonstrated that OV infection induced DNA damage, such as the formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage. O. viverrini infection mediates iNOS-dependent DNA damage in intrahepatic bile duct epithelium and inflammatory cells of hamsters, which may play an important role in CCA development.15, 16, 17 Therefore, such DNA damage may participate in inflammation-mediated carcinogenesis.
Recently, we have reported that OV antigen induces expression of Toll-like receptor 2 (TLR2), leading to nuclear factor-κB (NF-κB)-mediated iNOS expression in macrophage cell line.18 TLRs activate homologous signal transduction pathways, leading to nuclear localization of NF-κB/Rel-type transcription factors.19 NF-κB is a key player in inflammation that regulates expression of various genes involved in controlling inflammatory response such as proinflammatory mediator and inducible nitric oxide synthase (iNOS) expression.20, 21 Relevantly, NF-κB functions as a tumor promoter in inflammation-associated cancer.22 To clarify the chemotherapeutic effect of the antihelminthic drug praziquantel, we investigated the effect of this drug on DNA damage, including the formation of 8-nitroguanine and 8-oxodG, and the expression of NF-κB and iNOS by immunohistochemistry in OV-infected hamsters. Expression of NF-κB and iNOS was also confirmed by Western blotting analysis. The amounts of nitrate plus nitrite, end products of NO, in the liver and plasma in OV-infected hamsters were measured by a rapid spectrophotometric method using Griess reaction.23
Material and methods
Male Syrian golden hamsters age ranging from 6 to 8 weeks were housed under conventional conditions and fed a stock diet and water ad libitum. We isolated metacercariae of OV from cyprinoid fish as described previously.24 Hamsters were fed with 50 metacercariae of OV by intragastric intubation. Animals were sacrificed on days 14 or 30. Praziquantel (Biltricide®, Bayer), single dose, 400 mg/kg of body weight) suspended in 2% Cremophor EI (Sigma, St Louis, MO) was given to hamsters orally 7 days before sacrifice. In addition, 2% Cremophor EI alone was given to untreated control hamsters. The protocol of this study was approved by the Animal Ethics Committee of the Khon Kaen University, Thailand.
Measurement of 8-oxodG formation in liver DNA
Each hamster liver was examined in duplicate. Approximately 200 mg of liver was scissored into small pieces, followed by homogenization in 0.25 M saccharose solution. To extract DNA, the homogenate was treated with 0.1 mg/ml RNase and 1 mg/ml proteinase K in lysis buffer (Applied Biosystems, Foster City, CA) at 60°C for 1 h under the anaerobic condition. After ethanol precipitation, DNA was dissolved in 20 mM acetate buffer (pH 5.0) and digested to deoxynucleosides by incubation with 8 units of nuclease P1 at 37°C for 30 min, and then 0.6 units of bacterial alkaline phosphatase at 37°C for 1 h in 0.1 M Tris-HCl (pH 7.5). The deoxynucleosides were analyzed using an electrochemical detector (ECD, Coulochem II 5200A, ESA Biosciences, Chelmsford, MA) coupled with HPLC equipped with a Capcell Pak C18 column (i.d. 4.6 mm × 150 mm, Shiseido, Tokyo, Japan). The mobile phase consisted of 10 mM NaH2PO4 and 6% (v/v) methanol. The analysis was carried out at a column temperature of 25°C and a flow rate of 1 ml/min. The voltage of the electrode was set at + 400 mV. The molar ratio of 8-oxodG to deoxyguanosine in each sample was measured based on the peak height of authentic 8-oxodG with ECD and the UV absorbance at 254 nm of deoxyguanosine.
Colocalization of 8-nitroguanine and 8-oxodG formation in the liver was assessed by double immunofluorescence labeling study as described previously.17 Briefly, paraffin sections (6-μm thickness) were incubated with a rabbit polyclonal anti-8-nitroguanine antibody and a mouse monoclonal anti-8-oxodG antibody (2 μg/ml, Trevigen, Gaithersburg) overnight at room temperature. Rabbit polyclonal anti-8-nitroG antibody without cross reaction was produced as described elsewhere.17 Next, the sections were incubated for 3 h with an Alexa 594-labeled goat antibody against rabbit IgG and an Alexa 488-labeled goat antibody against mouse IgG (each, 1:400, Molecular Probes, Eugene, Oregon). The stained sections were examined under an inverted Laser Scan Microscope (LSM 410, Zeiss, Gottingen, Germany). In an additional experiment, to demonstrate 8-nitroguanine formation in DNA more clearly, tissue sections were pretreated with 10 μg/ml RNase at 37°C for 1 h according to the method described by Tsuruya et al.25
In addition, colocalization of iNOS and NF-κB in the hamster liver was also assessed by double immunofluorescence labeling study as described earlier. Paraffin sections were incubated with the primary antibodies [polyclonal anti-NOS antibody (1:500, Calbiochem-Novabiochem Corporation, San Diego, CA) and mouse monoclonal anti-NF-κB p65 antibody (not specific for phosphorylated NF-κB) (1:500, Santa Cruz Biotechnology, Santa Cruz, CA)] overnight at room temperature. The same second antibodies were used as described earlier. We also performed immunohistochemistry to examine the expression of TLR2 by using anti-TLR2 rabbit polyclonal antibody (1:300, Santa Cruz Biotechnology).
The histopathological change was assessed by hematoxylin eosin staining in paraffin sections as described previously.1
Detection of iNOS and NF-κB by Western blotting
To examine iNOS expression, ∼200 mg of frozen hamster liver was scissored into small pieces, followed by homogenization in PBS containing proteinase inhibitors [10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin A and 0.1 mM phenylmethyl sulphonyl fluoride (PMSF)]. The homogenate was centrifuged at 14,000g for 10 min at 4°C, and the supernatant was used for Western blotting. To examine the nuclear translocation of NF-κB, the nuclear fraction was obtained by the method as described previously.26 Approximately 100 mg of frozen hamster liver was homogenized in 500 μl of homogenization buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF and 0.1 mM EDTA] on ice. The homogenate was incubated for 15 min on ice and centrifuged at 14,000g for 15 min. The pellet was further suspended in ice-cold buffer B [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.42 M NaCl, 25% glycerol, 0.5 mM DTT, 0.2 mM PMSF and 0.2 mM EDTA] and incubated for 30 min on ice with frequent vortex. After the suspensions were centrifuged at 14 000g for 15 min, the supernatants were collected as nuclear extracts and stored at −80°C.
The protein concentration in homogenated hamster liver and the nuclear extract were measured using Coomassie Protein Assay Reagent Kit (Pierce Biotechnology, Rockford). Proteins were solubilized in sample buffer [2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromphenol blue and 50 mM Tris-HCl (pH 6.8)] and boiled for 5 min. Twenty μg of the samples was separated by 4–20% SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA) as described previously.27 Standard proteins of iNOS and NF-κB were purchased from Sigma and Lab Vision (Dr. Fremont, CA), respectively. The membranes were incubated with 5% skim milk in Tris-buffered saline (TBS, pH 7.4) containing 0.1% Tween 20 (TTBS). They were then incubated with the primary antibody [mouse monoclonal ant-iNF-κB p65 antibody (1:4,000) or anti-mouse iNOS antibody (1:4,000)]. Next, they were washed in TTBS and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (1:10,000, Santa Cruz Biotechnology). After extensive washings in TBS, they were incubated with ECL plus Western blotting detection reagents (Amarsham Biosciences, Buckinghamshire, UK). We performed quantitative analysis by measuring the band intensity of iNOS or NF-κB versus that of tubulin obtained from at least 3 animals, using a laser densitometer (Personal Densitometer SI, Amarsham Biosciences). The data represent means ± standard errors.
Analysis of NO products in the liver and plasma
The amounts of nitrate and nitrite in the plasma and homogenated liver were determined by the vanadium-based simple spectrophotometric method using Griess reaction as described previously by Miranda et al.23 with a minor modification. The assay was performed in a standard flat-bottomed 96-well polystyrene microtiter plate. Nitrate concentration in biological samples was measured after reduction into nitrite, using the catalyst, vanadium (III) chloride (VCl3). The protein concentration in homogenated hamster liver was measured as described earlier. Two hundred μl of plasma or homogenated liver was deproteinized to reduce turbidity with 400 μl of cold absolute ethanol for 30 min at 4°C. The samples were centrifuged at 10,000g for 10 min. The supernatant was used for analysis of nitrate and nitrite in the plasma and homogenated liver. After 100 μl of the supernatant or standard nitrate was added to a well, 100 μl of VCl3 was added to each well, followed by immediate addition of 100 μl of the Griess reagents (premixed 50 μl of 2% sulfanilamide in 5% HCl and 50 μl of 0.1% of N-(1-naphthyl)ethylenediamine dihydrochloride). The contents were vigorously mixed and the plate was incubated for 60 min at 37°C. The absorbance at 540 nm was measured to assess the total amounts of nitrate and nitrite.
Effect of praziquantel on histopathological changes in the liver of OV-infected hamsters
Figure 1 shows histopathological changes in the liver of OV-infected hamsters and the effect of praziquantel treatment on days 14 and 30. In OV-treatment, bile duct dilation, thickness of fibrosis and predominated inflammatory cell infiltration around bile ducts were markedly observed. After drug administration, although praziquantel did not kill parasites completely, these histopathological changes were reversed (Fig. 1) on day 14. After praziquantel treatment, remaining OVs were detected in the liver of 1 and 2 hamsters out of 7 hamsters on days 14 and 30, respectively. Marked fibrosis remained on day 30 although infiltration of inflammatory cells decreased. In contrast, no histological change was observed in hamsters without OV infection (data not shown).
Inhibitory effect of praziquantel on DNA damage in the liver of OV-infected hamsters
Figure 2 shows the formation of 8-oxodG and 8-nitroguanine in the liver of hamsters infected with OV and the effect of praziquantel treatment. 8-oxodG and 8-nitroguanine immunoreactivity was not observed in the liver of normal hamsters without OV infection (Fig. 2). Both 8-oxodG and 8-nitroguanine formation was observed mainly in the same inflammatory cells and bile duct epithelial cells on days 14 and 30. The formation of these DNA lesions was clearly observed mainly in the nucleus of bile duct epithelium with RNase treatment. In the hamsters with praziquantel treatment, little or no 8-oxodG and 8-nitroguanine formation was observed in bile duct epithelial cells.
Inhibitory effect of praziquantel on 8-oxodG formation in the liver of OV-infected hamsters
8-oxodG content in the liver of OV-infected hamsters was determined by an HPLC-ECD, and the effect of praziquantel treatment is shown in Figure 3. 8-oxodG level in the liver of OV-infected hamsters was significantly increased on days 14 and 30 compared with the nontreated control (p<0.01). 8-oxodG level in the liver of OV-infected hamsters was significantly decreased by praziquantel treatment on days 14 (p< 0.01) and 30 (p<0.05).
Expression of iNOS and NF-κB in the liver of OV-infected hamsters and inhibition by praziquantel treatment
Figure 4 shows the expression of iNOS and NF-κB in the liver of hamsters infected with OV and the effect of praziquantel treatment. Both iNOS and NF-κB expressions were observed in inflammatory cells and the epithelium of bile ducts on days 14 (Fig. 4a) and 30 (data not shown). The immunoreactivity of NF-κB was strongly observed in the nucleus of the bile duct epithelial cells (Fig. 4a). In the hamsters with praziquantel treatment, iNOS expression was reversed and weak NF-κB expression was observed in the cytoplasm of bile duct epithelium (Fig. 4a). No or weak immunoreactivities of iNOS and NF-κB were observed in the liver of normal hamsters without OV infection (data not shown). Western blotting showed that OV infection increased iNOS expression in the liver, and induced NF-κB accumulation in the nuclear fraction (Fig. 4b). The expression of these proteins was decreased by praziquantel treatment in comparison with OV infection alone (Fig. 4b). On day 14, the band intensity of iNOS versus that of tubulin was increased by OV infection to (128 ± 9.9)% (mean ± standard error) of the control (100 ± 15.1)% and decreased by praziquantel treatment (113 ± 4.6)% in comparison with OV infection alone. The band intensity of NF-κB versus that of tubulin was increased by OV infection to (165 ± 17.5)% of the control (100 ± 13.8)%, and decreased by praziquantel treatment (124 ± 13.8)% in comparison with OV infection alone.
NO production in OV-infected hamsters and inhibition by praziquantel treatment
Figure 5 shows the level of nitrate plus nitrite in the plasma (Fig. 5a) and the liver (Fig. 5b) in OV-infected hamsters. The levels of nitrate plus nitrite in the plasma (day 30, p <0.01) and the liver (days 14 and 30, p < 0.01) were significantly increased in OV-infected hamsters compared with nontreated controls (Fig. 5). On day 14, the level of nitrate plus nitrite in the liver in praziquantel treatment group was significantly reduced in comparison with OV-treatment alone (p < 0.01, Fig. 5b). On day 30, the level of nitrate plus nitrite in the plasma (p < 0.05, Fig. 5a) and the liver (p < 0.01, Fig. 5b) was significantly decreased by praziquantel treatment in comparison with OV-treatment alone. In addition, the levels of nitrate plus nitrite in the plasma and the liver on days 14 and 30 after drug treatment were not significantly different from those of normal hamster.
Expression of TLR2 in the liver of OV-infected hamsters and inhibition by praziquantel treatment
We performed immunohistochemistry to detect TLR2 expression in the liver of OV-infected hamsters and the effect of praziquantel treatment. OV infection induced strong TLR2 expression along the cell membrane in bile duct epithelial cells on day 30 (Fig. 6). TLR2 immunoreactivity was also observed in inflammatory cells around the bile ducts. After praziquantel treatment, this immunoreactivity was almost completely reduced. No or slight immunoreactivity was observed in hamsters without OV infection.
In the present study, we demonstrated that praziquantel successfully reduced nitrative and oxidative DNA damage, the expression of iNOS and the activation of NF-κB. The antihelminthic drug praziquantel has been used for the treatment of parasite infection, including liver fluke caused by OV infection.4 Although praziquantel did not kill parasites completely, this drug dramatically reduced histopathological changes, such as infiltration of inflammatory cells around bile ducts. This result may not be simply explained by the ability of praziquantel to kill parasites. Its antiinflammatory property independent of killing parasites may be involved in reduction in the histopathological changes. OV infection induced DNA damage, such as the formation of 8-nitroguanine and 8-oxodG, in bile duct epithelial cells in consistent with our previous reports.15, 16, 17 The formation of 8-nitroguanine was clearly observed mainly in the nucleus of bile duct epithelium with RNase treatment, suggesting that 8-nitroguanine is formed in genomic DNA. The formation of 8-nitroguanine and 8-oxodG was almost completely inhibited by drug treatment. This finding is confirmed by the result of quantitative analysis of 8-oxodG by HPLC-ECD. The level of nitrate plus nitrite in the liver and plasma was significantly decreased by praziquantel treatment. These results suggest that praziquantel may be capable of inhibiting inflammatory response mediated by OV antigen, regardless of whether the parasites are dead or alive. This hypothesis is supported by our recent study showing that OV antigen causes expression of inflammation-related gene products in cultured macrophage cells.18 Increased cell proliferation in OV-treated hamsters is likely to be a secondary phenomenon due to DNA damage inflicted on epithelial bile duct. Our findings suggest that praziquantel may reduce the risk of cholangiocarcinogenesis triggered by OV infection through iNOS-dependent DNA damage.
We have previously reported that OV infection strongly induces iNOS in bile duct epithelial cells and inflammatory cells.16, 17 Activated NF-κB regulates the expression of iNOS.20, 21 We have firstly demonstrated that OV infection induces NF-κB accumulation in the nucleus, suggesting that its nuclear translocation and activation occurred and then mediates iNOS expression, resulting in NO production. Immunohistochemical analysis revealed an apparent increase in the expression of iNOS and the accumulation of nuclear NF-κB in the epithelium of bile ducts and neighboring inflammatory cells. On the other hand, Western blotting showed that OV infection slightly increased the amounts of iNOS and nuclear NF-κB in the whole liver. This difference could be explained by assuming that iNOS expression and NF-κB activation in bile ducts contributes to only a small part of their amounts in whole liver tissues. NF-κB p65 is reported to be phosphorylated and then translocated to the nucleus in the process of its activation.28 This process may be involved in OV-induced iNOS expression, which can be inhibited by praziquantel treatment. Recently, we have reported that OV antigen induces expression of TLR2, leading to NF-κB-mediated iNOS expression in macrophage cell line.18 In this study, we demonstrated that OV infection induced strong TLR2 expression in bile duct epithelial cells as well as inflammatory cells and that its expression was reversed by praziquantel treatment. TLR2 activates homologous signal transduction pathways, leading to nuclear localization of NF-κB/Rel-type transcription factors.19 TLR2 activates NF-κB dependent transcription and ROS production.29 Relevantly, NF-κB was expressed through TLR and related molecules in cultured biliary epithelial cells treated with LPS.30 The treatment with praziquantel dramatically reduced iNOS expression and the level of NO products in the liver. Thus, praziquantel-induced decrease in OV-induced DNA damage can be reasonably explained by suppression of TLR-mediated pathway involving NF-κB-mediated iNOS expression and NO generation.
Recently, we have reported that chronic inflammation triggered by repeated OV infection mediates strong iNOS expression in intrahepatic bile duct epithelium of hamster.16 We also reported that 8-nitroguanine was formed in the sites of carcinogenesis in patients with Helicobacter pylori infection31 and chronic hepatitis C.32 8-Nitroguanine formation was found in the nucleus of colonic epithelial cells of mouse model of inflammatory bowel diseases after RNase treatment, suggesting that 8-nitroguanine is formed in genomic DNA.33 NO reacts with superoxide (O2•−) to form peroxynitrite (ONOO−) and mediates nitrative and oxidative DNA damage. 8-Nitroguanine formed in DNA is spontaneously released, followed by the formation of apurinic sites,10 leading to G → T transversions.34 Therefore, 8-nitroguanine is a mutagenic lesion, leading to carcinogenesis in addition to 8-oxodG, which also causes G → T transversions.35, 36 Thus, 8-nitroguanine could not only be a promising biomarker for inflammation but also a useful indicator of the risk of carcinogenesis. We conclude that praziquantel can exhibit a preventive effect against OV-induced cholangiocarcinoma by inhibiting iNOS-dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect.
The authors also thank Mr. T. Saraboon for technical supports in the present study.