Novel approach to identifying the hepatitis B virus pre-S deletions associated with hepatocellular carcinoma.

AIM: To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers. METHODS: The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 µm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis. RESULTS: CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P < 0.001), large deletion (> 99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC. CONCLUSION: CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.

Rapid and sustainable detoxication of airborne pollutants by broccoli sprout beverage: results of a randomized clinical trial in China.

Broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can generate the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases (GST) and other cytoprotective enzymes. A broccoli sprout-derived beverage providing daily doses of 600 µmol glucoraphanin and 40 µmol sulforaphane was evaluated for magnitude and duration of pharmacodynamic action in a 12-week randomized clinical trial. Two hundred and ninety-one study participants were recruited from the rural He-He Township, Qidong, in the Yangtze River delta region of China, an area characterized by exposures to substantial levels of airborne pollutants. Exposure to air pollution has been associated with lung cancer and cardiopulmonary diseases. Urinary excretion of the mercapturic acids of the pollutants, benzene, acrolein, and crotonaldehyde, were measured before and during the intervention using liquid chromatography tandem mass spectrometry. Rapid and sustained, statistically significant (P ≤ 0.01) increases in the levels of excretion of the glutathione-derived conjugates of benzene (61%), acrolein (23%), but not crotonaldehyde, were found in those receiving broccoli sprout beverage compared with placebo. Excretion of the benzene-derived mercapturic acid was higher in participants who were GSTT1-positive than in the null genotype, irrespective of study arm assignment. Measures of sulforaphane metabolites in urine indicated that bioavailability did not decline over the 12-week daily dosing period. Thus, intervention with broccoli sprouts enhances the detoxication of some airborne pollutants and may provide a frugal means to attenuate their associated long-term health risks.

Reduced aflatoxin exposure presages decline in liver cancer mortality in an endemic region of China.

Primary liver cancer (PLC) is the third leading cause of cancer mortality globally. In endemic areas of sub-Saharan Africa and Asia, PLC largely arises from chronic infection with hepatitis B virus (HBV) and ingestion of aflatoxins. Although synergistic interactions between these two risk factors have been observed in cohort studies in China, here we determined the impact of agricultural reforms in the 1980s leading to diminished maize consumption and implementation of subsidized universal vaccination against HBV in the 2000s on PLC primary prevention. A population-based cancer registry was used to track PLC mortality in Qidong, China and was compared with the timeline of HBV immunization. Randomly selected serum samples from archived cohort collections from the 1980s to present were analyzed for aflatoxin biomarkers. More than 50% reductions in PLC mortality rates occurred across birth cohorts from the 1960s to the 1980s for Qidongese less than 35 years of age although all were born before universal vaccination of newborns. Median levels of the aflatoxin biomarker decreased from 19.3 pg/mg albumin in 1989 to undetectable (<0.5 pg/mg) by 2009. A population attributable benefit of 65% for reduced PLC mortality was estimated from a government-facilitated switch of dietary staple from maize to rice; 83% of this benefit was in those infected with HBV. Food policy reforms in China resulted in a dramatic decrease in aflatoxin exposure, which, independent of HBV vaccination, reduced liver cancer risk. The extensive HBV vaccine coverage now in place augurs even greater risk reductions in the future.

[Relationship between serum hepatitis B virus DNA load and hepatocellular carcinoma in Qidong, China: a cohort follow-up study of 14 years].

OBJECTIVE: To explore the relationship between serum HBV DNA load and hepatocellular carcinogenesis in Qidong HBsAg carriers. METHODS: In 1997, 477 HBsAg carriers and 477 age, gender and residence matched HBsAg negative controls were enrolled as a prospective cohort in Qidong city. The entry serum samples were detected for the levels of HBeAg and HBV DNA. The relationship between baseline HBV DNA load and hepatocellular carcinoma (HCC) during the follow-up period from June 1997 to June 2011 were analyzed. RESULTS: The total observed person-years (PY) were 12 200. Eighty-seven patients developed HCC with an incidence of 1498/100 000 PY in the HBsAg positive group versus 6 with an incidence of 94/100 000 PY (P = 0.000) in the HBsAg negative group. The relative risk (RR) was 15.96. N o significant difference existed between the incidences of other tumors in two groups (P = 0.161). Compared with the HBsAg negative group, the RR of HCC was 11.38 (95%CI 4.87 - 26.62, P < 0.01)in the HBsAg+/HBeAg- group and 29.08 (95%CI 12.37 - 68.37, P < 0.01) in the HBsAg+/HBeAg+ group; 5.80 (95%CI 2.29 - 14.70, P < 0.01) in the HBsAg+/HBV DNA- group and 27.75 (95%CI 12.07 - 63.81, P < 0.01) in the HBsAg+/HBV DNA+ group. In HBsAg positive subjects, while the HBV DNA load was classified into 5 levels namely 250 - 10(4), 10(4)-, 10(5)-, 10(6)- and ≥ 10(7) copies/ml, the relative risks for HCC at each level were 2.84 (95%CI 1.44 - 5.61, P < 0.01), 5.75 (95%CI 2.77 - 11.95, P < 0.01), 9.05 (95%CI 4.71 - 17.41, P < 0.01), 6.39 (95%CI 2.79 - 14.64, P < 0.01) and 4.35 (95%CI 2.21 - 8.56, P < 0.01) respectively versus the < 250 copies/ml group. CONCLUSION: HBV DNA is an important risk predictor of hepatocellular carcinoma. The HBsAg carriers with the serum loads of HBV DNA between 10(5) - 10(6) copies/ml are most likely to present with HCC.

Modulation of the metabolism of airborne pollutants by glucoraphanin-rich and sulforaphane-rich broccoli sprout beverages in Qidong, China.

Epidemiological evidence has suggested that consumption of a diet rich in cruciferous vegetables reduces the risk of several types of cancers and chronic degenerative diseases. In particular, broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can release the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases. Two broccoli sprout-derived beverages, one sulforaphane-rich (SFR) and the other glucoraphanin-rich (GRR), were evaluated for pharmacodynamic action in a crossover clinical trial design. Study participants were recruited from the farming community of He Zuo Township, Qidong, China, previously documented to have a high incidence of hepatocellular carcinoma with concomitant exposures to aflatoxin and more recently characterized with exposures to substantive levels of airborne pollutants. Fifty healthy participants were randomized into two treatment arms. The study protocol was as follows: a 5 days run-in period, a 7 days administration of beverage, a 5 days washout period and a 7 days administration of the opposite beverage. Urinary excretion of the mercapturic acids of acrolein, crotonaldehyde, ethylene oxide and benzene were measured both pre- and postinterventions using liquid chromatography tandem mass spectrometry. Statistically significant increases of 20-50% in the levels of excretion of glutathione-derived conjugates of acrolein, crotonaldehyde and benzene were seen in individuals receiving SFR, GRR or both compared with their preintervention baseline values. No significant differences were seen between the effects of SFR versus GRR. Intervention with broccoli sprouts may enhance detoxication of airborne pollutants and attenuate their associated health risks.

Bioavailability of Sulforaphane from two broccoli sprout beverages: results of a short-term, cross-over clinical trial in Qidong, China.

One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation, and dose schedule of the intervention agent. Therefore, a cross-over clinical trial was undertaken to compare the bioavailability and tolerability of sulforaphane from two of broccoli sprout-derived beverages: one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR). Sulforaphane was generated from glucoraphanin contained in GRR by gut microflora or formed by treatment of GRR with myrosinase from daikon (Raphanus sativus) sprouts to provide SFR. Fifty healthy, eligible participants were requested to refrain from crucifer consumption and randomized into two treatment arms. The study design was as follows: 5-day run-in period, 7-day administration of beverages, 5-day washout period, and 7-day administration of the opposite intervention. Isotope dilution mass spectrometry was used to measure levels of glucoraphanin, sulforaphane, and sulforaphane thiol conjugates in urine samples collected daily throughout the study. Bioavailability, as measured by urinary excretion of sulforaphane and its metabolites (in approximately 12-hour collections after dosing), was substantially greater with the SFR (mean = 70%) than with GRR (mean = 5%) beverages. Interindividual variability in excretion was considerably lower with SFR than with GRR beverage. Elimination rates were considerably slower with GRR, allowing for achievement of steady-state dosing as opposed to bolus dosing with SFR. Optimal dosing formulations in future studies should consider blends of sulforaphane and glucoraphanin as SFR and GRR mixtures to achieve peak concentrations for activation of some targets and prolonged inhibition of others implicated in the protective actions of sulforaphane. Cancer Prev Res; 4(3); 384-95. ©2011 AACR.

[The association of hepatitis B virus genotype and the basal core promoter mutation in Qidong, China].

OBJECTIVES: To investigate the prevalence of hepatitis B virus (HBV) genotypes and the association with hepatocellular carcinoma (HCC) or basal core promoter (BCP) mutation in Qidong, China. METHODS: The whole genome of HBV or X gene sequences were obtained from serum samples of HBV infected patients by using PCR and direct sequencing methods. Phylogenetic tree was constructed to determine the genotypes or subgenotypes of HBV. RESULTS: According to the phylogenetic tree constructed from full-length sequence of HBV, genotype C2 was predominant in Qidong area. It was prevalent in 44 out of the 48 cases (91.7%), whereas genotype B2 only existed in 4 cases (8.3%). No other genotypes or recombinant types were found in Qidong patients. The result of genotyping based on X gene sequence confirmed the above observation. In a total of 182 samples, 169 (92.9%) showed genotype C2 and 10 (5.5%) showed genotype B2. There were 3 (1.6%) patients showed a coinfection with C2 and B2. The infection rate of genotype C in Qidong was significantly higher than that in neighboring city Shanghai (chi(2) = 12.252, P less than 0.01). There was no significant difference of genotype distribution between HCC and chronic hepatitis groups (P is more than 0.05). The frequency of T1762/A1764 double mutation in genotype C2 (70.3%) was significantly higher than that in genotype B2 (30.8%, P less than 0.05). The other two types of point mutation which also occurred in BCP, i.e. T1766 and A1768, were only seen in genotype C2. CONCLUSION: (1) Genotype C2 is the predominant genotype in Qidong, China. (2) There is no association between genotype C and HCC in Qidong. (3) Genotype C has a higher prevalence of BCP mutation than genotype B.

Hepatitis B virus x gene and cyanobacterial toxins promote aflatoxin B1-induced hepatotumorigenesis in mice.

AIM: To assess the combinative role of aflatoxin B1(AFB1), cyanobacterial toxins (cyanotoxins), and hepatitis B virus (HBV) x gene in hepatotumorigenicity. METHODS: One-week-old animals carrying HBV x gene and their wild-type littermates were intraperitoneally (ip) injected with either single-dose AFB1 [6 mg/kg body weight (bw)], repeated-dose cyanotoxins (microcystin-LR or nodularin, 10 microg/kg bw once a week for 15 wk), DMSO (vehicle control) alone, or AFB1 followed by cyanotoxins a week later, and were sacrificed at 24 and 52 wk post-treatment. RESULTS: AFB1 induced liver tumors in 13 of 29 (44.8%) transgenic mice at 52 wk post-treatment, significantly more frequent than in wild-type mice (13.3%). This significant difference was not shown in the 24-wk study. Compared with AFB1 exposure alone, MC-LR and nodularin yielded approximately 3-fold and 6-fold increases in the incidence of AFB(1)-induced liver tumors in wild-type animals at 24 wk, respectively. HBV x gene did not further elevate the risk associated with co-exposure to AFB1 and cyanotoxins. With the exception of an MC-LR-dosed wild-type mouse, no liver tumor was observed in mice treated with cyanotoxins alone at 24 wk. Neither DMSO-treated transgenic mice nor their wild-type littermates had pathologic alterations relevant to hepatotumorigenesis in even up to 52 wk. CONCLUSION: HBV x gene and nodularin promote the development of AFB(1)-induced liver tumors. Co-exposure to AFB1 and MC-LR tends to elevate the risk of liver tumors at 24 wk relative to exposure to one of them. The combinative effect of AFB1, cyanotoxins and HBVx on hepatotumorigenesis is weak at 24 wk.

Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a randomized clinical trial in He Zuo township, Qidong, People's Republic of China.

Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of aflatoxin-contaminated foods, and are exposed to high levels of phenanthrene, a sentinel of hydrocarbon air toxics. Cruciferous vegetables, such as broccoli, contain anticarcinogens. Glucoraphanin, the principal glucosinolate in broccoli sprouts, can be hydrolyzed by gut microflora to sulforaphane, a potent inducer of carcinogen detoxication enzymes. In a randomized, placebo-controlled chemoprevention trial, we tested whether drinking hot water infusions of 3-day-old broccoli sprouts, containing defined concentrations of glucosinolates, could alter the disposition of aflatoxin and phenanthrene. Two hundred healthy adults drank infusions containing either 400 or < 3 micromol glucoraphanin nightly for 2 weeks. Adherence to the study protocol was outstanding; no problems with safety or tolerance were noted. Urinary levels of aflatoxin-N(7)-guanine were not different between the two intervention arms (P = 0.68). However, measurement of urinary levels of dithiocarbamates (sulforaphane metabolites) indicated striking interindividual differences in bioavailability. An inverse association was observed for excretion of dithiocarbamates and aflatoxin-DNA adducts (P = 0.002; R = 0.31) in individuals receiving broccoli sprout glucosinolates. Moreover, trans, anti-phenanthrene tetraol, a metabolite of the combustion product phenanthrene, was detected in urine of all participants and showed a robust inverse association with dithiocarbamate levels (P = 0.0001; R = 0.39), although again no overall difference between intervention arms was observed (P = 0.29). Understanding factors influencing glucosinolate hydrolysis and bioavailability will be required for optimal use of broccoli sprouts in human interventions.

Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas.

Hepatocellular carcinoma is one of the most common cancers worldwide. Infection with hepatitis B virus and exposure to aflatoxins in the diet act synergistically to amplify risk. From a public health perspective, hepatitis virus vaccination programs and efforts to both reduce aflatoxin exposures and to attenuate the toxicological consequences of unavoidable exposures should have major impacts on the global incidence of this disease. Experimentally, aflatoxin-induced hepatocarcinogenesis can be inhibited by over a score of different chemopreventive agents with multiple mechanisms of action. One agent, oltipraz, is a potent inducer of phase 2 enzymes involved in the detoxication of carcinogens including aflatoxin. A second agent, chlorophyllin, impedes the bioavailability of carcinogens by forming molecular complexes and enhances their elimination in the fecal stream. This review highlights the findings of recent randomized clinical trials with oltipraz and chlorophyllin conducted in individuals exposed to dietary aflatoxins and at high risk for development of liver cancer. Both chemopreventive agents modulated levels of aflatoxin biomarkers in the study participants in manners consonant with protection. Although pharmacological approaches establish proof of principle and help identify key molecular targets for interventions, food-based approaches that also use these molecular targets may be the most practical for widespread application in high-risk populations.

Specific mutations of hepatitis B virus in plasma predict liver cancer development.

A major risk factor for hepatocellular carcinoma (HCC) is hepatitis B virus (HBV), whose pathogenesis is exacerbated by the acquisition of mutations that accelerate carcinogenesis. We examined, with mass spectrometry, the temporality of an HBV 1762(T)/1764(A) double mutation in plasma and tumors. Initial studies found that 52 of 70 (74.3%) tumors from patients residing in Qidong, People's Republic of China, contained this HBV mutation. Paired plasma samples were available for six of the tumor specimens; four tumors had the HBV 1762(T)/1764(A) mutation, whereas three of the paired plasma samples were also positive. The potential predictive value of this biomarker was explored by using stored plasma samples from a study of 120 residents of Qidong who had been monitored for aflatoxin exposure and HBV infection. After 10 years of passive follow-up, there were six cases of major liver disease including HCC (four cases), hepatitis (one case), and cirrhosis (one case). All six cases had detectable levels of the HBV 1762(T)/1764(A) mutation up to 8 years before diagnosis. Finally, 15 liver cancers were selected from a prospective cohort of 1,638 high-risk individuals in Qidong on the basis of available plasma samples spanning the years before and after diagnosis. The HBV 1762(T)/1764(A) mutation was detected in 8 of the 15 cases (53.3%) before cancer. The persistence of detection of this mutation was statistically significant (P = 0.022, two-tailed). We therefore found that a prediagnosis biomarker of specific HBV mutations can be measured in plasma and suggest this marker for use as an intermediate endpoint in prevention and intervention trials.

[Studies of hereditary conditionality index on some morphological characters of Tupaia belangeri chinensis].

Tree shrew is a kind of excellent experimental animal resource in medical science and biology. In this paper, 35 Tupaia blangeri chinensises (TBCs) captured from Kunming,Yunnan province were investigated. We analyzed hereditary conditionality index about some morphological characters. According to Rife-Buranamanas law, we analyzed the appearance characteristics with hereditary conditionality including color of fur, orbit etc. The results showed the following: wild fur with seasonal red spot, white fur of abdomen, non-white orbit, flesh-color palm, non-cocked ear, round tip tail, and the line between the breasts of both sides in a vertical position with axis line.

Prospective detection of codon 249 mutations in plasma of hepatocellular carcinoma patients.

A specific missense mutation in the p53 tumor gene at codon 249 has been reported in over 50% of hepatocellular carcinoma (HCC) tumors and in paired blood samples from areas of high dietary exposure to aflatoxin B1, including Qidong, People's Republic of China. Using a combination of pre-digestion with HaeIII, PCR and mass spectrometry, the temporality of this mutation in plasma before and after the clinical diagnosis of HCC was examined. Sixteen liver cancer cases, diagnosed between 1997 and 2001, were selected from a prospective cohort of 1638 high-risk individuals in Qidong on the basis of available annual plasma samples spanning the years before and after diagnosis. The codon 249 mutation was detected in plasma samples obtained after diagnosis in seven of the 15 cases (46.7%) with PCR amplifiable DNA, which is in accord with the reported prevalence of this mutation in HCC. The persistent detection of this mutation in plasma collected annually following diagnosis was statistically significant (P = 0.024, two-tailed) in repetitive samples following diagnosis. Moreover, the mutation was detected in the plasma of four of eight cases positive at the time of diagnosis at least 1 year and in one case 5 years prior to diagnosis. Tracking of the marker in pre-diagnostic samples was borderline statistically significant (P = 0.066). None of the 18 healthy US control plasma samples had any detectable mutations. We have therefore found that pre-diagnosis biomarkers of specific p53 mutations can be measured in plasma and this suggests a paradigm for developing these markers for use in prevention and intervention trials.

Translational strategies for cancer prevention in liver.

Unlike many other types of human cancer, the aetiology of liver cancer is well understood. Infection with hepatitis viruses, coupled with dietary exposure to the fungal toxin aflatoxin, increases the risk of the disease. Although primary prevention, based on vaccination and avoiding exposure to these agents, is an appealing option, such strategies will require considerable investment of time and resources to be successful. In the developing world--where the burden of liver cancer is highest--immediate, practical and economical approaches are essential. So, targeted chemoprevention might be most appropriate for the present generation of individuals at risk.

Sensitivity of electrospray ionization mass spectrometry detection of codon 249 mutations in the p53 gene compared with RFLP.

Hepatocellular carcinoma (HCC) has several major etiological risk factors, including infection with hepatitis viruses and exposure to aflatoxin B(1). A specific missense mutation resulting from a guanine to thymine transversion at the third position of codon 249 in the p53 tumor suppressor gene has been reported in 10-70% of HCCs from areas of high dietary exposure to aflatoxin B(1.) This mutation has not only been detected in tumor samples but has also been measured in DNA isolated from the blood of patients with HCC in two separate studies by two independent methods: RFLP and short oligonucleotide mass analysis (SOMA), an electrospray ionization mass spectrometry technique. To compare the relative sensitivities of these methodologies, a set of serially diluted samples was analyzed by both techniques. The detection limits of RFLP and SOMA were 6% and 2.4% mutant alleles in the presence of wild-type alleles, respectively. When the DNA samples were predigested with HaeIII before SOMA, the detection limit was improved to 0.4% mutant allele in the presence of wild-type alleles. We have therefore found that SOMA is about 2.5-15-fold more sensitive than RFLP for detection of specific p53 mutations. A set of 26 DNA samples from HCC and normal liver was analyzed by RFLP and SOMA, and 5 samples were positive for the p53 mutation. An additional 4 samples were found to be positive for the mutation when SOMA was repeated after HaeIII predigestion.

Strategies for chemoprevention of liver cancer.

Liver cancer is one of the most common cancers worldwide. Infection with hepatitis B virus and exposure to aflatoxins in the diet act synergistically to amplify risk. From a public health perspective, hepatitis virus vaccination programs and efforts to both reduce aflatoxin exposures and to attenuate the toxicological consequences of unavoidable exposures should have major impacts on the incidence of this disease. Experimentally, aflatoxin-induced hepatocarcinogenesis can be inhibited by over a score of different chemopreventive agents with multiple mechanisms of action. One agent, oltipraz, is a potent inducer of phase 2 enzymes involved in the detoxication of carcinogens including aflatoxin. A second agent, chlorophyllin, impedes the bioavailability of carcinogens by forming molecular complexes and enhances their elimination in the fecal stream. This review highlights the findings of recent randomized clinical trials with oltipraz and chlorophyllin conducted in individuals exposed to dietary aflatoxins and at high risk for development of liver cancer. Both chemopreventive agents modulated levels of aflatoxin biomarkers in the study participants in manners consonant with protection.

Preliminary study on the production of transgenic mice harboring hepatitis B virus X gene.

AIM:To establish transgenic mice lineage harboring hepatitis B virus X gene and to provide an efficient animal model for studying the exact role of the HBx gene in the process of hepatocarcinogenesis.METHODS:The HBx transgenic mice were produced by microinjecting the construct with X gene of HBV (subtype adr) DNA fragment into fertilzed eggs derived from inbred C57BL/6 strain; transgenic mice were identified by using Nested PCR; expression and phenotype of HBx gene were analyzed in liver from transgenic mic at the age of 8 weeks by RT-PCR, pathologic examination and periodic acid-schiff staining (PAS), respectively.RESULTS:Five hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant retroviral DNA fragment, and 368 survival eggs injected were transferred to the oviducts of 18 pseudopregnant recipient mice, 8 of them became pregnant and gave birth to 20 F1 offspring. Of 20 offsprings, four males and two females carried the hybrid gene (HBx gene). Four male mice were determined as founder, named X 1, X 5, X 9 and X 15. These founders were back crossed to set up F1 generations with other ibred C57BL/6 mice or transgenic littermates, respectively.Transmission of HBx gene in F1 offspring of X 1, X 5 and X 9 except in X 15 followed Mendelian rules. The expression of HBx mRNA was detected in liver of F1 offspring from the founder mice (X 1 and X 9), which showed vacuolation lesion and glycogen positive foci.CONCLUSION:Transgenic mice harboring HBx gene were preliminarily established.