Objective To establish a fast and sensitive method for the detection of 8-hydroxy-2’-deoxyguanosine （8-OHdG） in precision-cut rat liver slices by HPLC-MS/MS and to investigate isoniazid （INH） -induced oxidative D...Objective To establish a fast and sensitive method for the detection of 8-hydroxy-2’-deoxyguanosine （8-OHdG） in precision-cut rat liver slices by HPLC-MS/MS and to investigate isoniazid （INH） -induced oxidative DNA damage. Methods Precision-cut liver slices （300 μm） were prepared from male rats, and incubated with INH （0.018 mol/L） for 2 h after 1 h preincubation. DNA in the slices was extracted and digested into free nucleosides at 37℃ . The samples were injected into HPLC-MS/MS after the proteins were removed. The level of oxidative DNA damage was estimated using the ratio of 8-OHdG to deoxyguanosine （dG）. Results The limit of detection of 8-OHdG was 1 ng/mL （S/N=3） and the intra-assay relative standard variation was 3.38% when one transition 284.3/168.4 was used as a quantifier and another two transitions 284.3/140.2, 306.1/190.2 as qualifiers. 8-OHdG and dG were well separated, as indicated by elution at 10.02 and 7.37 min, respectively. INH significantly increased the ratio of 8-OHdG to dG in rat liver slices （P〈0.05）. Conclusion 8-OHdG in precision-cut liver slices could be sensitively determined by HPLC-MS/MS. HPLC-MS/MS coupled with precision-cut tissue slices is a fast and reliable analytical technique to evaluate oxidative DNA damage of target tissues caused by procarcinogens and cytotoxins.展开更多
Precision-cut liver slice has been successfully used to study the mechanism of drug-induced hepatotoxicity, the prediction of liver toxicity, the discovery of early hepatic toxicity biomarker and the metabolism of dru...Precision-cut liver slice has been successfully used to study the mechanism of drug-induced hepatotoxicity, the prediction of liver toxicity, the discovery of early hepatic toxicity biomarker and the metabolism of drug in liver. We detected the expression of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 in precision-cut liver slice after co-cultured with monocrotaline or Tussilago farfara alkaloids to investigate the hepatotoxicity mechanism of those drugs. After co-culturing with monocrotaline or Tussilago farfara alkaloids for 6 hours, the expression of CYP3A4 in the microsome of precision-cut liver slices was detected by Western blot, and the expressions of CYP2B1 + CYP2B2 and CYP2E1 were detected by immunofluorescence. The results showed that monocrotaline induced the expression of CYP3A4 and CYP2B1 + CYP2B2, and Tussilago farfara alkaloids obviously up-regulated the expression of CYP2E1 and CYP3A4. Thus, we conclude that the up-regulation of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 may be one of the toxic mechanisms of liver injury of those drugs.展开更多
Background: Although a large number of studies have confirmed that the different levels of reactive oxygen species (ROS) in cytoplasm and nucleus have effects on cell growth, proliferation, differentiation and apoptos...Background: Although a large number of studies have confirmed that the different levels of reactive oxygen species (ROS) in cytoplasm and nucleus have effects on cell growth, proliferation, differentiation and apoptosis, the exact mechanism of ROS action is unclear. An important reason is that the production and degradation time of ROS in cells is very short, and therefore it’s difficult to understand the mechanism of action based on the traditional molecular action process through the ROS diffusion and target binding. Methods: The fresh liver tissue slices were prepared and the nuclei of hepatocytes were separated from Kunming mice according to the reported method. Liver tissue slices and hepatocyte nuclei were perfused with extracellular or intracellular fluids containing different concentrations of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), and real-time imaging monitoring of biophotonic emission was carried out using an ultra-weak biophoton imaging system. Results: The results showed that the continuous perfusion with different concentrations of H<sub>2</sub>O<sub>2</sub> (300, 400 and 500 μM, respectively) resulted in significant increase of biophotonic emissions, presenting a concentration-dependent effect in liver tissue slices and achieving the maximum effect at 400 μM, while the significant enhancement was found after 500 μM treatment on the hepatocyte nuclei. Conclusion: This study suggests that ROS generated in cells may achieve its physiological and pathological effects via biophotonic emissions, which provides a new quantum biological mechanism of ROS, while the detailed clarification requires further research.展开更多
BACKGROUND: During the past years, the number of liver transplantation has increased greatly, but the number of available organs has not increased. In view of the critical shortage of organs, the indications for livin...BACKGROUND: During the past years, the number of liver transplantation has increased greatly, but the number of available organs has not increased. In view of the critical shortage of organs, the indications for living-related liver transplantation (LRLT) have broadened since experience with the procedure has been achieved. This study was undertaken to assess the value of multi-slice spiral CT (MSCT) angiography in evaluating the hepatic arterial and veinous anatomy of potential donors for LRLT. METHODS: MSCT was performed after intravenous injection of contrast material at 3 ml/s. The total dose was calculated as 2 ml/kg. Twenty LRLT donors (2 men and 18 women) were subjected to MSCT angiography of hepatic blood vessels. These were generated by volume rendering and maximum intensity projection, while curved planar reformation was added in 5 patients. RESULTS: We identified 10 important hepatic vascular variants in 9 of the 20 donors (4 arterial, 4 venous, and 2 portal venous variants). In hepatic arterial variants, two had a replaced right hepatic artery arising from the superior mesenteric artery, an accessory right hepatic artery from the superior mesenteric artery and a replaced left hepatic artery arising from the left gastric artery. In hepatic venous variants, three had an accessory inferior right hepatic vein and one had two accessory inferior right hepatic veins. In hepatic portal venous variants, two had trifurcation of the main portal vein. CONCLUSIONS: As a non-invasive and reliable method, MSCT angiography is of value in the clinical evaluation of LRLT donors. MSCT angiography should be recommended as a routine preoperative examination for potential LRLT donors.展开更多
The onset of alcoholic liver disease (ALD) is initiated by different cell types in the liver and a number of different factors including: products derived from ethanol-induced inflammation, ethanol metabolites, and th...The onset of alcoholic liver disease (ALD) is initiated by different cell types in the liver and a number of different factors including: products derived from ethanol-induced inflammation, ethanol metabolites, and the indirect reactions from those metabolites. Ethanol oxidation results in the production of metabolites that have been shown to bind and form protein adducts, and to increase inflammatory, fibrotic and cirrhotic responses. Lipopolysaccharide (LPS) has many deleterious effects and plays a significant role in a number of disease processes by increasing inflammatory cytokine release. In ALD, LPS is thought to be derived from a breakdown in the intestinal wall enabling LPS from resident gut bacterial cell walls to leak into the blood stream. The ability of adducts and LPS to independently stimulate the various cells of the liver provides for a two-hit mechanism by which various biological responses are induced and result in liver injury. Therefore, the purpose of this article is to evaluate the effects of a two-hit combination of ethanol metabolites and LPS on the cells of the liver to increase inflamma-tion and fi brosis, and play a role in the development and/or progression of ALD.展开更多
BACKGROUND Diffusion-weighted imaging(DWI)has become a useful tool in the detection,characterization,and evaluation of response to treatment of many cancers,including malignant liver lesions.DWI offers higher image co...BACKGROUND Diffusion-weighted imaging(DWI)has become a useful tool in the detection,characterization,and evaluation of response to treatment of many cancers,including malignant liver lesions.DWI offers higher image contrast between lesions and normal liver tissue than other sequences.DWI images acquired at two or more b-values can be used to derive an apparent diffusion coefficient(ADC).DWI in the body has several technical challenges.This include ghosting artifacts,mis-registration and susceptibility artifacts.New DWI sequences have been developed to overcome some of these challenges.Our goal is to evaluate 3 new DWI sequences for liver imaging.AIM To qualitatively and quantitatively compare 3 DWI sequences for liver imaging:free-breathing(FB),simultaneous multislice(SMS),and prospective acquisition correction(PACE).METHODS Magnetic resonance imaging(MRI)was performed in 20 patients in this prospective study.The MR study included 3 separate DWI sequences:FB-DWI,SMS-DWI,and PACE-DWI.The image quality,mean ADC,standard deviations(SD)of ADC,and ADC histogram were compared.Wilcoxon signed-rank tests were used to compare qualitative image quality.A linear mixed model was used to compare the mean ADC and the SDs of the ADC values.All tests were 2-sided and P values of<0.05 were considered statistically significant.RESULTS There were 56 lesions(50 malignant)evaluated in this study.The mean qualitative image quality score of PACE-DWI was 4.48.This was significantly better than that of SMS-DWI(4.22)and FB-DWI(3.15)(P<0.05).Quantitatively,the mean ADC values from the 3 different sequences did not significantly differ for each liver lesion.FB-DWI had a markedly higher variation in the SD of the ADC values than did SMS-DWI and PACE-DWI.We found statistically significant differences in the SDs of the ADC values for FB-DWI vs PACE-DWI(P<0.0001)and for FB-DWI vs SMS-DWI(P=0.03).The SD of the ADC values was not statistically significant for PACE-DWI and SMS-DWI(P=0.18).The quality of the PACE-DWI ADC histograms were considered better than the SMS-DWI and FB-DWI.CONCLUSION Compared to FB-DWI,both PACE-DWI and SMS-DWI provide better image quality and decreased quantitative variability in the measurement of ADC values of liver lesions.展开更多
基金This research was supported by the National Natural Science Foundation of China (No. 30600773).
文摘Objective To establish a fast and sensitive method for the detection of 8-hydroxy-2’-deoxyguanosine （8-OHdG） in precision-cut rat liver slices by HPLC-MS/MS and to investigate isoniazid （INH） -induced oxidative DNA damage. Methods Precision-cut liver slices （300 μm） were prepared from male rats, and incubated with INH （0.018 mol/L） for 2 h after 1 h preincubation. DNA in the slices was extracted and digested into free nucleosides at 37℃ . The samples were injected into HPLC-MS/MS after the proteins were removed. The level of oxidative DNA damage was estimated using the ratio of 8-OHdG to deoxyguanosine （dG）. Results The limit of detection of 8-OHdG was 1 ng/mL （S/N=3） and the intra-assay relative standard variation was 3.38% when one transition 284.3/168.4 was used as a quantifier and another two transitions 284.3/140.2, 306.1/190.2 as qualifiers. 8-OHdG and dG were well separated, as indicated by elution at 10.02 and 7.37 min, respectively. INH significantly increased the ratio of 8-OHdG to dG in rat liver slices （P〈0.05）. Conclusion 8-OHdG in precision-cut liver slices could be sensitively determined by HPLC-MS/MS. HPLC-MS/MS coupled with precision-cut tissue slices is a fast and reliable analytical technique to evaluate oxidative DNA damage of target tissues caused by procarcinogens and cytotoxins.
文摘Precision-cut liver slice has been successfully used to study the mechanism of drug-induced hepatotoxicity, the prediction of liver toxicity, the discovery of early hepatic toxicity biomarker and the metabolism of drug in liver. We detected the expression of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 in precision-cut liver slice after co-cultured with monocrotaline or Tussilago farfara alkaloids to investigate the hepatotoxicity mechanism of those drugs. After co-culturing with monocrotaline or Tussilago farfara alkaloids for 6 hours, the expression of CYP3A4 in the microsome of precision-cut liver slices was detected by Western blot, and the expressions of CYP2B1 + CYP2B2 and CYP2E1 were detected by immunofluorescence. The results showed that monocrotaline induced the expression of CYP3A4 and CYP2B1 + CYP2B2, and Tussilago farfara alkaloids obviously up-regulated the expression of CYP2E1 and CYP3A4. Thus, we conclude that the up-regulation of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 may be one of the toxic mechanisms of liver injury of those drugs.
文摘Background: Although a large number of studies have confirmed that the different levels of reactive oxygen species (ROS) in cytoplasm and nucleus have effects on cell growth, proliferation, differentiation and apoptosis, the exact mechanism of ROS action is unclear. An important reason is that the production and degradation time of ROS in cells is very short, and therefore it’s difficult to understand the mechanism of action based on the traditional molecular action process through the ROS diffusion and target binding. Methods: The fresh liver tissue slices were prepared and the nuclei of hepatocytes were separated from Kunming mice according to the reported method. Liver tissue slices and hepatocyte nuclei were perfused with extracellular or intracellular fluids containing different concentrations of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), and real-time imaging monitoring of biophotonic emission was carried out using an ultra-weak biophoton imaging system. Results: The results showed that the continuous perfusion with different concentrations of H<sub>2</sub>O<sub>2</sub> (300, 400 and 500 μM, respectively) resulted in significant increase of biophotonic emissions, presenting a concentration-dependent effect in liver tissue slices and achieving the maximum effect at 400 μM, while the significant enhancement was found after 500 μM treatment on the hepatocyte nuclei. Conclusion: This study suggests that ROS generated in cells may achieve its physiological and pathological effects via biophotonic emissions, which provides a new quantum biological mechanism of ROS, while the detailed clarification requires further research.
文摘BACKGROUND: During the past years, the number of liver transplantation has increased greatly, but the number of available organs has not increased. In view of the critical shortage of organs, the indications for living-related liver transplantation (LRLT) have broadened since experience with the procedure has been achieved. This study was undertaken to assess the value of multi-slice spiral CT (MSCT) angiography in evaluating the hepatic arterial and veinous anatomy of potential donors for LRLT. METHODS: MSCT was performed after intravenous injection of contrast material at 3 ml/s. The total dose was calculated as 2 ml/kg. Twenty LRLT donors (2 men and 18 women) were subjected to MSCT angiography of hepatic blood vessels. These were generated by volume rendering and maximum intensity projection, while curved planar reformation was added in 5 patients. RESULTS: We identified 10 important hepatic vascular variants in 9 of the 20 donors (4 arterial, 4 venous, and 2 portal venous variants). In hepatic arterial variants, two had a replaced right hepatic artery arising from the superior mesenteric artery, an accessory right hepatic artery from the superior mesenteric artery and a replaced left hepatic artery arising from the left gastric artery. In hepatic venous variants, three had an accessory inferior right hepatic vein and one had two accessory inferior right hepatic veins. In hepatic portal venous variants, two had trifurcation of the main portal vein. CONCLUSIONS: As a non-invasive and reliable method, MSCT angiography is of value in the clinical evaluation of LRLT donors. MSCT angiography should be recommended as a routine preoperative examination for potential LRLT donors.
文摘The onset of alcoholic liver disease (ALD) is initiated by different cell types in the liver and a number of different factors including: products derived from ethanol-induced inflammation, ethanol metabolites, and the indirect reactions from those metabolites. Ethanol oxidation results in the production of metabolites that have been shown to bind and form protein adducts, and to increase inflammatory, fibrotic and cirrhotic responses. Lipopolysaccharide (LPS) has many deleterious effects and plays a significant role in a number of disease processes by increasing inflammatory cytokine release. In ALD, LPS is thought to be derived from a breakdown in the intestinal wall enabling LPS from resident gut bacterial cell walls to leak into the blood stream. The ability of adducts and LPS to independently stimulate the various cells of the liver provides for a two-hit mechanism by which various biological responses are induced and result in liver injury. Therefore, the purpose of this article is to evaluate the effects of a two-hit combination of ethanol metabolites and LPS on the cells of the liver to increase inflamma-tion and fi brosis, and play a role in the development and/or progression of ALD.
文摘BACKGROUND Diffusion-weighted imaging(DWI)has become a useful tool in the detection,characterization,and evaluation of response to treatment of many cancers,including malignant liver lesions.DWI offers higher image contrast between lesions and normal liver tissue than other sequences.DWI images acquired at two or more b-values can be used to derive an apparent diffusion coefficient(ADC).DWI in the body has several technical challenges.This include ghosting artifacts,mis-registration and susceptibility artifacts.New DWI sequences have been developed to overcome some of these challenges.Our goal is to evaluate 3 new DWI sequences for liver imaging.AIM To qualitatively and quantitatively compare 3 DWI sequences for liver imaging:free-breathing(FB),simultaneous multislice(SMS),and prospective acquisition correction(PACE).METHODS Magnetic resonance imaging(MRI)was performed in 20 patients in this prospective study.The MR study included 3 separate DWI sequences:FB-DWI,SMS-DWI,and PACE-DWI.The image quality,mean ADC,standard deviations(SD)of ADC,and ADC histogram were compared.Wilcoxon signed-rank tests were used to compare qualitative image quality.A linear mixed model was used to compare the mean ADC and the SDs of the ADC values.All tests were 2-sided and P values of<0.05 were considered statistically significant.RESULTS There were 56 lesions(50 malignant)evaluated in this study.The mean qualitative image quality score of PACE-DWI was 4.48.This was significantly better than that of SMS-DWI(4.22)and FB-DWI(3.15)(P<0.05).Quantitatively,the mean ADC values from the 3 different sequences did not significantly differ for each liver lesion.FB-DWI had a markedly higher variation in the SD of the ADC values than did SMS-DWI and PACE-DWI.We found statistically significant differences in the SDs of the ADC values for FB-DWI vs PACE-DWI(P<0.0001)and for FB-DWI vs SMS-DWI(P=0.03).The SD of the ADC values was not statistically significant for PACE-DWI and SMS-DWI(P=0.18).The quality of the PACE-DWI ADC histograms were considered better than the SMS-DWI and FB-DWI.CONCLUSION Compared to FB-DWI,both PACE-DWI and SMS-DWI provide better image quality and decreased quantitative variability in the measurement of ADC values of liver lesions.
