Geng Sun Qian

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, Peoples 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 μmol glucoraphanin nightly for 2 weeks. Adherence to the study protocol was outstanding; no problems with safety or tolerance were noted. Urinary levels of aflatoxin-N7-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.

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.

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.

Sequential accumulation of the mutations in core promoter of hepatitis B virus is associated with the development of hepatocellular carcinoma in Qidong, China

BACKGROUND/AIMS To investigate the mutations in hepatitis B virus (HBV) that might be related to hepatocellular carcinoma (HCC) in the high-risk area Qidong, China. METHODS DNA sequences of HBV basal core promoter (BCP) and the overlapping X gene were determined in 58 HCC and 71 chronic hepatitis (CH) patients. In addition, a consecutive series of plasma samples from 15 HCC cases were employed to compare the CP/X sequences before and after the occurrence of HCC. RESULTS T1762/A1764 double mutation was frequently found in Qidong patients, regardless of clinical status (65.5% in HCC and 73.2% in CH, P>0.05). Unexpectedly, the adjacent T1766/A1768 mutation significantly increased the risk of HCC (P<0.05). Moreover, the prevalence of triple mutations in BCP was significantly higher in patients with HCC than those with CH (P<0.05). The longitudinal study demonstrated that the mutations in BCP were gradually accumulated during the development of HCC. Colony formation assay showed while A1764 mutation alone did not alter the colony-inhibitory activity of HBx, double or triple mutations largely abrogated this effect. CONCLUSIONS The complex mutation involving T1766/A1768 was closely related to HCC. The enhanced risk of HCC caused by BCP variants could be attributable partially to the aberrant activity of HBx.

Genetic alterations in hepatocellular carcinomas: association between loss of chromosome 4q and p53 gene mutations.

The major risk factors for hepatocellular carcinomas (HCC) in high incidence areas include infection with hepatitis B and C viruses (HBV, HCV) and exposure to aflatoxin. Genetic alterations in 24 liver resection specimens from Shanghai and Qidong were studied. Hepatitis B virus was integrated in all patient samples, and a null phenotype for the GSTM1 enzyme was present in 63% of patients. Alteration of p53 was present in 95% (23/24) of cases: mutations of the p53 gene in 12 HCC, p53 overexpression in 13 and loss of heterozygosity (LOH) of chromosome 17p in 17. All seven HCCs with a p53 mutation from Qidong and three of five from Shanghai had the aflatoxin-associated point mutation with a G to T transversion at codon 249, position 3. No HCC had microsatellite instability. LOH of chromosome 4q, 1p, 16q and 13q was present in 50%, 46%, 42% and 38%, respectively, and 4q was preferentially lost in HCCs containing a p53 mutation: LOH of 4q was present in 75% (9/12) of HCC with, but only 25% (3/12) of HCC without, a p53 gene mutation (P = 0.01). These data indicate a possible interaction between p53 gene mutation and 4q loss in the pathogenesis of 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. Cancer Prev Res; 7(8); 813–23. ©2014 AACR.

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.

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. Cancer Prev Res; 6(10); 1038–45. ©2013 AACR.

Comparison Study on the Complete Sequence of Hepatitis B Virus Identifies New Mutations in Core Gene Associated with Hepatocellular Carcinoma

Background: Mutations in the hepatitis B virus (HBV) genome may influence the activity of liver disease. The aim of this study was to identify new viral variations associated with hepatocellular carcinoma (HCC). Methods: We carried out a comparison study on the complete sequence of HBV isolated from 20 HCC and 35 non-HCC patients in Qidong, China, an area with a high incidence of HCC. We compared the HBV sequences in a consecutive series of plasma samples from four HCC cases before and after the occurrence of HCC. In addition, we selected four mutations in the HBV core (C) gene to verify their relationships to HCC in an independent set of 103 HCC cases and 103 sex- and age-matched non-HCC controls. Results: The pre-S deletion and 12 point mutations, namely, the pre-S2 start codon mutation, T53C in the pre-S2 gene, T766A in the S gene, G1613A, C1653T, A1762T, G1764A in the X gene, and G1899A, C2002T, A2159G, A2189C, and G2203W (A or T) in the pre-C/C gene, showed close associations with HCC. In the validation study, A2159G, A2189C, and G2203W showed consistent associations with HCC by univariate analysis. Multivariate analysis showed that A2189C and G2203W were independent risk factors for HCC. The odds ratios (95% confidence interval) were 3.99 (1.61-9.92) and 9.70 (1.17-80.58), respectively, for A2189C and G2203W. Conclusions: These results implicate A2189C and G2203W as new predictive markers for HCC. Impact: The complete genome analysis of HBV provided pilot data for the identification of novel mutations that could serve as markers for HCC. Cancer Epidemiol Biomarkers Prev; 19(10); 2623–30. ©2010 AACR.

Reevaluation of glypican-3 as a serological marker for hepatocellular carcinoma

BACKGROUND Glypican-3 (GPC3) is a novel histochemical marker of hepatocellular carcinoma (HCC). However, its utility as a serologic marker for HCC is not conclusive. METHODS A total of 1037 subjects, including 155 patients with HCC, 180 with chronic hepatitis, 124 with liver cirrhosis, 442 with non-HCC cancer and 136 healthy controls, were analyzed for serum GPC3 (sGPC3) by an ELISA constructed with 2 monoclonal antibodies. RESULTS The average level of sGPC3 in HCC patients was 99.94±267.2ng/ml, which was significantly higher than in patients with chronic hepatitis (10.45±46.02ng/ml, P<0.0001), liver cirrhosis (19.44±50.88ng/ml, P=0.0013), non-HCC cancer (20.50±98.33ng/ml, P<0.0001) and healthy controls (4.14±31.65ng/ml, P<0.0001). The sensitivity of sGPC3 in HCC diagnosis was 40.0%, whereas the specificity was 98.5%, 94.4% and 87.1% in healthy controls, chronic hepatitis patients and liver cirrhosis patients, respectively. In addition, 13.5% (28/207) of lung cancer patients and 13.2% (9/68) of thyroid cancer patients had positive results with sGPC3. CONCLUSION Serum GPC3 is a potential marker for HCC. However, the presence of sGPC3 in patients with lung cancer and thyroid cancer might limit its application as a single marker in the diagnosis of HCC.