| 1. ObjectiveHepatocarcinoma is the most common malignant liver tumors. Hepatocarcinoma ranked sixth in tumor incidence and third in tumor death cause sequence. The conventional therapy of hepatocarcinoma gives little hope for restoration of health because of poor diagnosis and serious side effects. Therefore, developing more effective and less toxic anticancer agents, including natural products, is necessary to prevent or retard the process of hepatocarcinogenesis. N-nitrosodiethylamine (NDEA), which exists widespreadly in nature such as in cheese, soybean, processed meats, alcoholic beverages, tobacco products, cosmetics and agricultural chemicals, is one of the most important environmental carcinogens. So the hepatocarcinoma induced by NDEA has become a major public health problem.The steam distillation method is widely used to extract and condense volatile OSCs from garlic and the final oily product is called garlic oil (GO), which contains more than30OSCs. It has been reported that diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are the three major components involved in GO. Accumulating evidence has demonstrated that the metabolic activation of NDEA and overproduction of reactive oxygen species (ROS) play key roles in the etiology of hepatocarcinoma. Because GO may regulate many metabolic enzymes and can scavenge ROS, we speculate GO could prevent NDEA-induced hepatocarcinogenesis. Therefore, the present study was designed to evaluate the effects of GO on NDEA-induced hepatocarcinogenesis and to explore the mechanisms.2. Methods2.1.60Male Wistar rats were randomly divided into4groups, i.e. normal control group, NDEA group, and two different doses of GO groups. The rats in GO group were treated with GO (20or40mg/kg body weight) by gavage for21weeks (5time/week), while other animals received equal volume of corn oil. From the second week, the rats in GO and NDEA groups were orally received NDEA (10mg/kg body weight,5time/week), while the animals in normal control group were administered volume of saline. At the end of week21, all animals were sacrificed by decapitation and blood was collected. The preventive effects of GO on NDEA induced hepatocarcinoma were evaluated by nodule incidence, number of nodules, serum biochemical indices, histopathological changes and cell proliferation capacity.2.2. The activity of CYP2E1was measured with aniline as the substrate. The activities of CYP1A1and CYP1A2were detected by measurement of the dealkylation of ethoxyresorufin and methoxyresorufin using Hitachi fluorescence spectrophotometer. The activities of cytosolic GST alpha, GST mu and GST pi were determined using cumene hydroperoxide (CuOOH),2,4-dichloro-l-nitrobenzene (DCNB) and ethacrynic acid as substrates. UDP-glucuronosyl-transferase (UGT) activity was determined with p-nitrophenol (PNP) as a substrate.2.3. Total RNA was isolated from the rat livers using Trizol reagent according to the manufacture's instructions. Complementary DNA was synthesized using the RevertAidTM First Strand cDNA Synthesis Kit. The levels of gene expression of metabolic enzymes in liver tissues were quantified by qRT-PCR.2.4. The liver tissues were homogenized in lysis buffer (50mM Tris,6.4mM magnesium chloride,0.2M sucrose, pH=7.5), then were centrifuged at12OOOg for20min. The supernatant was dissolved in strongly RIPA lysis buffer, and then was centrifuged at105,000g for60min. The supernatant and pellet were the cytosolic and microsomal samples, respectively. The protein contents of GST alpha, GST mu, GST pi in the supernatant and CYP2E1, CYP1A1, CYP1A2, UGT1A6in the pellet were measured by Western blotting.2.5. The liver tissues were homogenized in9volumes of ice-cold buffer (pH7.4) containing0.01M Tris-HCl,0.0001M EDTA-2Na,0.01M saccharose sucrose, and 0.8%saline. The homogenates were centrifuged at1000g for20min at4℃. The supernatant was collected. Lipid peroxidation (LPO) was determined by measuring the accumulation of thiobarbituric acid reactive substance and expressed as MDA content. The levels of MDA, GSH and the activities of antioxidant enzymes including SOD, CAT, GPx, GR, and GST were assayed using commercial assay kits according to the manufacturer's instructions.2.6. The liver tissues were homogenized in lysis buffer (50mM Tris-HCl,150mM NaCl,1%Triton X-100,1%sodium deoxychlolate,0.1%sodium dodecylsulphate,50mM beta-glycerophosphate,1mM Na3VO4,5mM NaF,1%cocktail protein inhibitors and1mM phenylmethylsulfphonylfloride), then were centrifuged at14OOOg for15min. Western blotting was used to detect the protein contents of phosphoinositide3-kinase (PI3K), AKT, NF-κB, Bcl-2, Bcl-xl, β-arrestin-2, Bax and Caspase-3.2.7. Total RNA was isolated from the rat livers using Trizol reagent. Complementary DNA was synthesized using cDNA Synthesis Kit. The levels of gene expression of apoptotic related genes (Bcl-2, Bcl-xl, β-arrestin-2, Bax and Caspase-3) in liver tissues were quantified by qRT-PCR.3. Results3.1. The preventive effect of GO on NDEA-induced hepatocarcinoma in rats3.1.1. Morphological changes of the liverThe appearance of livers in normal control group was normal and no macroscopically detectable nodules. The rats in NDEA group revealed enlarged liver. The nodule incidence of NDEA group was100%and the maximun diameter of nodules was about10mm. At the same time, there were clear necrosis regions in livers of NDEA-treated rats. Interestingly, a significant reduction in liver enlargement, nodule incidence and average nodule numbers per nodule-bearing liver was observed in GO-treated rats compared to that of NDEA group.3.1.2. Changes of body weight and relative liver weightIn the first week following NDEA treatment, the rats began to show a slow growth. Along with the NDEA exposure, the mean weight gain decreased gradually. After14weeks of exposure, the body weight in NDEA group showed negative growth. NDEA treatment increased the relative liver weight when compared with the normal control group (P<0.01). Administration of20and40mg/kg.bw GO significantly reduced the relative liver weight when compared with the NDEA group (P<0.01).3.1.3. Serum biochemical assays.NDEA administration led to the elevation of serum ALT, AST, ALP, γ-GT and AFP levels compared with those in normal control group (P<0.01), indicating liver damage and preneoplastic lesion. All these above adverse effects of NDEA were suppressed by GO.3.1.4The histological changes of the liverThe histological examinations were performed to evaluate the effects of GO and NDEA on the morphological changes and the collagen accumulation. The liver tissues of rats in normal control group revealed regular hepatic lobules without obvious fibrotic area, which were illustrated by H&E staining and sirius red staining, respectively. In contrast, the liver sections from the rats in the NDEA group showed collapse of hepatic lobules and the obvious pleomorphism such as multiple nucleoli, pyknotic nuclei, and intranuclear vacuoles. Furthermore, the sirius red positive areas were markedly increased in the NDEA-treated rats. Interestingly, the liver sections of rats in GO pretreatment groups showed fewer degenerative changes, and less sirius red positive areas.3.1.5. The changes of PCNA expression by immunohistochemistryThe positive staining of PCNA occasionally appeared in the liver sections of the normal control group, which was dramatically increased in the hepatic sections of NDEA group. However, GO pretreatment significantly decreased the number of PCNA positive nuclei when compared with the NDEA group.3.2. The changes of phase â… and phase â…¡ metabolic enzymes3.2.1. Effects of GO and NDEA on the activities, mRNA and protein levels of phase â… enzymes 3.2.1.1. Effects of GO and NDEA on the activities of phase I enzymesCompared with the normal control group, the activities of CYP2E1and CYP1A2were decreased by72%and89%(P<0.01), respectively, while the activity of CYP1A1was increased by66%in rats of NDEA group (P<0.01). The changes induced by NDEA were inhibited by GO pretreatment.3.2.1.2. Effects of GO and NDEA on the mRNA levels of phase I enzymesCompared with the normal control group, the mRNA levels of Cyp2e1and Cyp1a1were decreased by26%and51%(P<0.01), respectively, while the mRNA level of Cyp1a1was about2-fold in rats of NDEA-groups (P<0.01). GO significantly inhibited these changes induced by NDEA.3.2.1.3. Effects of GO and NDEA on the protein levels of phase I enzymesCompared with the normal control group, the protein levels of CYP2E1and. CYP1A2were decreased by97%and96%(P<0.01). GO pretreatment can inhibit these decreases induced by NDEA. The protein level of CYP1A1in rats of NDEA group was almost50-fold greater than the corresponding control value (P<0.01). Compared with the NDEA group, the protein content of CYP1A1was decreased by40%and81%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively.3.2.2. Effects of GO and NDEA on the activities, mRNA and protein levels of GST alpha, GST mu and GST pi3.2.2.1. Effects of GO and NDEA on the activities of GST alpha, GST mu and GST piCompared with the control value, the activities of GST alpha and GST mu were slightly but significantly decreased, however, the activity of GST pi was sharply increased (about3.1fold) in NDEA group (P<0.01). Compared with the NDEA group, GO pretreatment significantly inhibited the activation of GST pi, and the inactivation of GST alpha and GST mu (P<0.01). Interestingly, all these above changes induced by NDEA were simultaneously ameliorated by GO pretreatment.3.2.2.2. Effects of GO and NDEA on the mRNA levels of Gst alpha, Gst mu and Gst piThe mRNA levels of Gst alpha and Gst mu in rats of NDEA group were decreased by37%and24%(P<0.01) compared with those in normal control group, respectively, while the mRNA levels of Gst pi was increased by67%(P<0.01). The changes of the mRNA levels induced by NDEA were also significantly attenuated by GO pretreatment.3.2.2.3. Effects of GO and NDEA on the protein levels of GST alpha, GST mu and GST piGST pi was not detected in the normal control group. NDEA significantly induced GST pi protein expression, but reduced the protein levels of GST alpha and GST mu. Again, GO obviously suppressed the decreases of the GST alpha and GST mu protein levels and the increase of the GST pi protein level.3.2.3. Effects of GO and NDEA on the activities, mRNA and protein levels of UGTs3.2.3.1. Effect of GO and NDEA on the activity of UGTsThe activity of UGTs in rats of NDEA group was decreased by47%compared with the control value (P<0.01), which was significantly inhibited by GO pretreatment.3.2.3.2. Effects of GO and NDEA on the mRNA levels of UgtsCompared with the normal control group, the mRNA level of Ugt1a6was significantly decreased by35%(P<0.01) in NDEA group. GO suppressed NDEA-induced decrease of the mRNA level of Ugt1a6. However, no significant alteration of the mRNA level of Ugt1a1was observed in all groups.3.2.3.3. Effect of GO and NDEA on the protein level of UGT1A6The protein level of UGT1A6in NDEA group was significantly decreased by50%(P<0.01), when compared to normal control group. GO pretreatment significantly suppressed NDEA-induced decrease of the protein level of UGT1A6.3.3. Effects of GO and NDEA on LPO and antioxidant systems in NDEA-induced hepatocarcinoma3.3.1. Effect of GO and NDEA on MDA level in rat liverNDEA significantly increased the MDA level (about1.6-fold), when compared to the normal control group. Compared with that of NDEA group, the MDA level was decreased by34%and44%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively. No significant difference of MDA level was observed between GO treatment groups and normal control group (P>0.05).3.3.2. Effect of GO and NDEA on GSH level in rat liverCompared with the normal control group, the GSH level was significantly decreased by94%in NDEA group. Compared with the NDEA group, GO pretreatment significantly increased the GSH level. The GSH level was about2.4-fold and7.4-fold in20and40mg/kg.bw GO treatment groups(P<0.01), respectively, when compared to the NDEA group.3.3.3. Effects of GO and NDEA on the activities of antioxidant enzymes in rat liverNDEA caused significant decreases in the activities of SOD, CAT, GPx, GR, and GST (P<0.01). Compared with NDEA group, GO pretreatment significantly increased the activities of SOD, CAT, GPx, GR and GST by29%,27%,75%,47%,122%(P<0.05or P<0.01) and24%,34%,94%,98%,187%(P<0.01) in20and40mg/kg.bw GO treatment groups, respectively.3.3.4. GO inhibited the formation of8-OHdG in liver DNA induced by NDEAAfter20weeks of NDEA treatment, the content of8-OHdG was augmented to2.4-fold (P<0.01) compared with that in normal control group. However, this elevation of8-OHdG induced by NDEA was significantly reduced in GO treatment groups and decreased by29%(P<0.05) and44%(P<0.01) in20and40mg/kg.bw treatment groups, respectively.3.4. Effects of GO and NDEA on PI3K-AKT-NFκB and apoptosis3.4.1. Eeffect of GO and NDEA on PI3KNDEA caused significant increases in the protein contents of p85and p110. Compared with the normal control group, the protein contents of p85and p110were about1.8-fold (P<0.01) and2.5-fold (P<0.01) in NDEA group. GO pretreatment can significantly inhibit the increases induced by NDEA. Compared with those of NDEA group, the protein content of p85was decreased by41%and44%(P<0.01) in20and40mg/kg.bw GO pretreatment groups; while the protein content of p110was decreased by24%and61%in20and40mg/kg.bw GO pretreatment groups (P<0.01), respectively. 3.4.2. Effects of GO and NDEA on AKT and AKT phosphorylationNDEA significantly increased the protein content of total AKT (about1.7-fold), when compared with the normal control group. Compared with that of NDEA group, the protein content of total AKT was decreased by41%and47%in20and40mg/kg.bw GO pretreatment groups (P<0.01), respectively.Phosphorylated AKT is the active form of AKT. NDEA administration led to the elevation of the protein contents of p-AKT (Thr308) and p-AKT (Ser473). The protein contents of p-AKT (Thr308) and p-AKT (Ser473) in NDEA group was about3.0-fold and12.6-fold (P<0.01), when compared to the normal control group.Compared with the NDEA group, the protein content of p-AKT (Thr308) was decreased by13%and35%in20and40mg/kg.bw GO pretreatment groups (P<0.05), while the protein content of p-AKT (Ser473) was decreased by36%(P<0.05) and64%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively.3.4.3. Effects of GO and NDEA on IκB and NF-κBCompared with the normal control group, the protein level of p-IκB was decreased by77%(P<0.01) in NDEA group. GO pretreatment can enhance the phosphorylation of IκB. Compared with the NDEA group, the protein content of p-IκB was about1.5-fold and2.7-fold (P<0.05) in20and40mg/kg.bw GO pretreatment groups, respectively. NDEA significantly increased the protein content of NF-κB p65(about1.9-fold), when compared with the normal control group. Compared with that of NDEA group, the protein content of NF-kB p65was decreased by33%and39%(P<0.01) in20and40mg/kg.bw GO pretreatment groups, respectively.3.4.4. Effects of GO and NDEA on Bcl-2and Bcl-xl3.4.4.1. Effects of GO and NDEA on the mRNA levels of Bcl-2and Bcl-xlQuantitative real-time PCR results showed significant increases in mRNA expression levels of anti-apoptotic Bcl-2and Bcl-xl in NDEA group. Compared with the normal control group, the mRNA expression levels of Bcl-2and Bcl-xl in NDEA group were increased to3.8-fold and1.5-fold (P<0.01), respectively. Compared with the NDEA group, the mRNA level of Bcl-2in20and40mg/kg.bw GO pretreatment groups was decreased by69%and80%(P<0.01), respectively, while the mRNA expression level of Bcl-xl was decreased by51%and41%(P<0.01), respectively.3.4.4.2. Effects of GO and NDEA on the protein levels of Bcl-2and Bcl-xlCompared with the normal control group, the protein contents of Bcl-2and Bcl-xl in NDEA group were increased to1.6-fold and4.0-fold (P<0.01), respectively. GO pretreatment inhibited the increases of Bcl-2and Bcl-xl induced by NDEA treatment. Compared with the NDEA group, the protein content of Bcl-2in20and40mg/kg.bw GO pretreatment groups was decreased by41%and60%(P<0.01), respectively, while the protein content of Bcl-xl was decreased by33%and80%(P<0.01), respectively.3.4.5. Effect of GO and NDEA on Bax3.4.5.1. Effect of GO and NDEA on the mRNA level of BaxCompared with the normal control group, the mRNA expression level of Bax in NDEA group was decreased by47%(P<0.01). Compared with the NDEA group, the mRNA level of Bax in20and40mg/kg.bw GO pretreatment groups was increased to1.7-fold and2.7-fold (P<0.01), respectively. Furthermore, the mRNA level of Bax in40mg/kg.bw GO pretreatment group was higher than that of the normal control group (P<0.05).3.4.5.2. Effect of GO and NDEA on the protein level of BaxCompared with the normal control group, the protein content of Bax in NDEA group was decreased by42%(P<0.01). Compared with the NDEA group, the protein content of Bax in20and40mg/kg.bw GO pretreatment groups was increased to2.5-fold and4.4-fold (P<0.01), respectively. Furthermore, the protein content of Bax in40mg/kg.bw GO pretreatment group was higher than that in the normal control group (P<0.01).3.4.6. Effect of GO and NDEA on Bcl-2/Bax ratioCompared with the normal control group, the mRNA and protein expression levels of Bcl-2were increased in NDEA group, while the mRNA and protein expression levels of Bax were decreased. So the ratio of Bcl-2/Bax in NDEA group was significantly increased. Compared with the normal control group, the Bcl-2/Bax ratio of mRNA level was increased to7.1-fold (P<0.01), while the Bcl-2/Bax ratio of protein content was increased to2.7-fold (P<0.01) in NDEA group. GO pretreatment inhibited the increases of Bcl-2/Bax ratio induced by NDEA treatment. Compared with the NDEA group, the Bcl-2/Bax ratio of mRNA level in20and40mg/kg.bw GO pretreatment groups was decreased by82%and93%(P<0.01), respectively, while the Bcl-2/Bax ratio of protein content was decreased by76%and91%(P<0.01), respectively. Furthermore, the Bcl-2/Bax ratio in40mg/kg.bw GO pretreatment group was lower than that in the normal control group.3.4.7. Effect of GO and NDEA on Caspase-33.4.7.1. Effect of GO and NDEA on the mRNA level of Caspase-3Compared with the normal control group, the mRNA expression level of Caspase-3in NDEA group was decreased by23%(P<0.05). Compared with the NDEA group, the mRNA level of Bax in20and40mg/kg.bw GOpretreatment groups was increased to1.5-fold and2.3-fold (P<0.01), respectively. Furthermore, the mRNA level of Caspase-3in40mg/kg.bw GOpretreatment group was higher than that in the normal control group (P<0.01).3.4.7.2. Effect of GO and NDEA on the protein level of Caspase-3Compared with the normal control group, the protein content of Caspase-3in NDEA group was decreased by38%(P<0.01). Compared with the NDEA group, the protein content of Bax in20and40mg/kg.bw GO pretreatment groups was increased to about3.0-fold (P<0.01). Furthermore, the protein content of Caspase-3in20and40mg/kg.bw GO pretreatment groups was higher than that in the normal control group (P<0.01).3.4.8. Effect of GO and NDEA on β-arrestin-2Recently, β-arrestin-2was shown to mediate anti-apoptotic signaling, so we detected the expression levels of β-arrestin-2in liver tissues. Compared with the normal control group, the mRNA and protein expression levels of β-arrestin-2in NDEA group were increased to1.9-fold and3.4-fold (P<0.01), respectively. Pretreatment with GO caused marked decreases at both the mRNA and protein levels. Compared with the NDEA group, the mRNA level of β-arrestin-2in20and40 mg/kg.bw GO pretreatment groups was decreased by36%and43%(P<0.01), respectively, while the protein expression level of β-arrestin-2was decreased by52%and69%(P<0.01), respectively.4. Conclusion4.1. GO obviously prevented NDEA-induced hepatocarcinogenesis in terms of nodule incidence, number of nodules, serum biochemical parameters, histopathological changes and cell proliferation capacity.4.2. GO regulated the activities and expression levels of phase I metabolizing enzyme (CYP2E1, CYP1A2, CYP1A1) and phase â…¡ metabolizing enzymes (GST alpha, GST mu, GST pi, UGT1A1, UGT1A6), which maybe contribute to its prevention against NDEA-induced hepatocarcinogenesis.4.3. Chronic exposure of rats to NDEA (lOmg/kg.bw,5times/week,20weeks) significantly increased lipid peroxidation; GO pretreatment alleviated lipid peroxidation induced by NDEA.4.4. GO significantly inhibited NDEA-induced reduction of GSH content and decrease of SOD, CAT, GPx, GR, GST activities, and notably relieved DNA oxidative damage induced by NDEA, which might be associated with its prevention against NDEA-induced hepatocarcinogenesis.4.5. Chronic exposure of rats to NDEA activated the PI3K-AKT signaling pathway and downstream NF-κB signaling molecule, thus inhibiting apoptosis. GO pretreatment significantly inhibited the activation of PI3K-AKT-NF-KB-anti-apoptosis signaling pathway, which suggested the downregulation of PI3K-AKT-NF-κB-anti-apoptosis by GO pretreatment was involved in its prevention against NDEA-induced hepatocarcinogenesis.
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