Jin Bing Wang

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.

Chlorophyllin intervention reduces aflatoxin–DNA adducts in individuals at high risk for liver cancer

Residents of Qidong, Peoples Republic of China, are at high risk for development of hepatocellular carcinoma, in part from consumption of foods contaminated with aflatoxins. Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, we tested whether chlorophyllin could alter the disposition of aflatoxin. One hundred and eighty healthy adults from Qidong were randomly assigned to ingest 100 mg of chlorophyllin or a placebo three times a day for 4 months. The primary endpoint was modulation of levels of aflatoxin-N7-guanine adducts in urine samples collected 3 months into the intervention measured by using sequential immunoaffinity chromatography and liquid chromatography–electrospray mass spectrometry. This aflatoxin–DNA adduct excretion product serves as a biomarker of the biologically effective dose of aflatoxin, and elevated levels are associated with increased risk of liver cancer. Adherence to the study protocol was outstanding, and no adverse events were reported. Aflatoxin-N7-guanine could be detected in 105 of 169 available samples. Chlorophyllin consumption at each meal led to an overall 55% reduction (P = 0.036) in median urinary levels of this aflatoxin biomarker compared with those taking placebo. Thus, prophylactic interventions with chlorophyllin or supplementation of diets with foods rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers.

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.

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.

Oltipraz chemoprevention trial in Qidong, Jiangsu Province, People's Republic of China

Oltipraz has been used clinically in many regions of the world as an antischistosomal agent and is an effective inhibitor of aflatoxin hepatocarcinogenesis in rats. This chemopreventive action of oltipraz results primarily from an altered balance in aflatoxin metabolic activation and detoxication. In 1995, a randomized, placebo‐controlled, double‐blind intervention was conducted in residents of Qidong, Peoples Republic of China, who are at high risk for exposure to aflatoxin and development of hepatocellular carcinoma. The major study objectives were to define a dose and schedule for oltipraz that would reduce levels of aflatoxin biomarkers in biofluids of the participants, and to further characterize dose‐limiting side effects. Two hundred thirty‐four healthy eligible individuals, including those infected with HBV, were randomized to receive either 125 mg oltipraz daily, 500 mg oltipraz weekly, or placebo. Blood and urine specimens were collected to monitor potential toxicities and evaluate biomarkers over the 8‐week intervention and subsequent 8‐week follow‐up periods. Overall, compliance in the intervention was excellent; approximately 85% of the participants completed the study. Objective evaluation of adverse events was greatly facilitated by inclusion of a placebo arm in the study design. A syndrome involving numbness, tingling, and pain in the fingertips was the only event that occurred more frequently among the active groups (18 and 14% of the daily 125 mg and weekly 500 mg arms, respectively) compared to placebo (3%). These symptoms were reversible and could be relieved with non‐steroidal antiinflammatory agents. A more complete understanding of the chemopreventive utility of oltipraz awaits completion of an assessment of the efficacy of oltipraz in modulating levels of aflatoxin biomarkers. J. Cell. Biochem. Suppls. 28/29:166–173.

Genetic Variants in Five Novel Loci Including CFB and CD40 Predispose to Chronic Hepatitis B

Hepatitis B virus affects more than 2 billion people worldwide, 350 million of which have developed chronic hepatitis B (CHB). The genetic factors that confer CHB risk are still largely unknown. We sought to identify genetic variants for CHB susceptibility in the Chinese population. We undertook a genome‐wide association study (GWAS) in 2,514 CHB cases and 1,130 normal controls from eastern China. We replicated 33 of the most promising signals and eight previously reported CHB risk loci through a two‐stage validation totaling 6,600 CHB cases and 8,127 controls in four independent populations, of which two populations were recruited from eastern China, one from northern China and one from southern China. The joint analyses of 9,114 CHB cases and 9,257 controls revealed significant association of CHB risk with five novel loci. Four loci are located in the human leukocyte antigen (HLA) region at 6p21.3, including two nonsynonymous variants (rs12614 [R32W] in complement factor B [CFB], Pmeta=1.28 × 10−34; and rs422951 [T320A] in NOTCH4, Pmeta = 5.33 × 10−16); one synonymous variant (rs378352 in HLA‐DOA corresponding to HLA‐DOA*010101, Pmeta = 1.04 × 10−23); and one noncoding variant (rs2853953 near HLA‐C, Pmeta = 5.06 × 10−20). Another locus is located at 20q13.1 (rs1883832 in the Kozak sequence of CD40, Pmeta = 2.95 × 10−15). Additionally, we validated seven of eight previously reported CHB susceptibility loci (rs3130542 at HLA‐C, rs1419881 at TCF19, rs652888 at EHMT2, rs2856718 at HLA‐DQB1, rs7453920 at HLA‐DQB2, rs3077 at HLA‐DPA1, and rs9277535 at HLA‐DPA2, which are all located in the HLA region, 9.84 × 10−71 ≤ Pmeta ≤ 9.92 × 10−7). Conclusion: Our GWAS identified five novel susceptibility loci for CHB. These findings improve the understanding of CHB etiology and may provide new targets for prevention and treatment of this disease. (Hepatology 2015;62:118‐128)

Acceleration to Death from Liver Cancer in People with Hepatitis B Viral Mutations Detected in Plasma by Mass Spectrometry

Liver cancer is the leading cause of cancer death in many regions of the world. With the goal to discover biomarkers that reflect subsets of high-risk individuals and their prognosis, we nested our study in a male cohort of 5,581 hepatitis B surface antigen carriers in Qidong, Peoples Republic of China, who were recruited starting in 1989. By December 2003, 667 liver cancer cases were diagnosed in this group and plasma samples collected at the initial screening at enrollment were available in 515 cases who had succumbed to liver cancer. Hepatitis B virus (HBV) DNA could be isolated in 355 (69%) of these samples. In 14%, 15%, 19%, 31%, and 22%, screening took place at ≤1.5, 1.51 to 3, 3.01 to 5, 5.01 to 9, and >9 years before death, respectively; and 39% died at age below 45 years. The relation between mutations in HBV and time to death were determined by logistic regression for the odds of mutation and by survival analyses methods with age as the time scale. In 279 (79%) of these individuals, the samples contained a two-nucleotide 1762T/1764A HBV mutation. Sixteen samples lacking the 1762T/1764A mutation had novel mutations elsewhere in the 1761 to 1767 region of the HBV genome. There was a statistically significant difference (P = 0.012) for the high prevalence of the HBV mutations in the men who died from hepatocellular carcinoma under the age of 45 years relative to those who died after 55 years of age and HBV mutations accelerated death (relative hazard, 1.40; 95% confidence interval, 1.06-1.85) and that the effect was attenuated by age from 2.04 for age 35 years to 1.0 for age 65 years with the 90% confidence band being above 1 for ages <50 years. These findings provide a conceptual framework to explain the acceleration of mortality in individuals infected with HBV. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1213–8)

Abstract LB-405: Modulation of the metabolism of air-borne pollutants by broccoli sprout beverages: Results of a short term, cross-over clinical trial in Qidong, People's Republic of China

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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 (GR) which can release the chemopreventive agent, sulforaphane (SF), an inducer of glutathione transferases. Two broccoli sprout-derived beverages, one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR), were evaluated for SF bioavailability, tolerability and pharmacodynamic action. The GRR beverage relied on the conversion of GR to SF in the gut microflora whereas the SFR beverage was prepared by conversion of GR to SF with myrosinase from daikon sprouts prior to consumption. Study participants were recruited from the farming community of He Zuo Township, Qidong, Peoples Republic of China, previously documented to have a high incidence of hepatocellular carcinoma and more recently, exposures to substantive levels of air-borne pollutants including phenanthrene, acrolein, crotonaldehyde, benzene and ethylene oxide. Two hundred five individuals were screened at village clinics and 50 willing, healthy participants were randomized into two treatment arms. The study protocol was as follows: a 5-day run-in period, a 7-day administration of beverages, a 5-day washout period, and a 7-day administration of the opposite beverage. Bioavailability of SF, as indicated by excretion of GR, SF and SF-thiol conjugates in urine collected daily 12 hours after dosing, was measured by isotope dilution mass spectrometry. The urinary excretions of SF and its thiols were substantially greater in individuals receiving a SFR beverage compared to a GRR beverage. Determination of possible pharmacodynamic action of the SFR and GRR beverages has been assessed through measures of the urinary excretion of the mercapturic acids of acrolein, crotonaldehyde, ethylene oxide, benzene and 1,3-butadiene using liquid and gas chromatography coupled to mass spectrometry. Statistically significant increases in the levels of excretion of glutathione-derived conjugates of acrolein, crotonaldehyde, and benzene were seen in individuals receiving SFR, GRR or both compared to their pre-intervention baseline values. Increases provoked by SFR were greater than for GRR, but the differences were not significantly different. Thus, chemopreventive intervention may attenuate risks to health associated with exposures to air-borne pollutants in addition to possible effects on aflatoxin exposures in this region. Supported by NIEHS grant P01 ES006052. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-405. doi:10.1158/1538-7445.AM2011-LB-405

Breast Cancer Subtypes in Northern Chinese Women.

Introduction: Breast cancer is currently regarded as a heterogeneous disease. Gene expression analysis has identified several subtypes that differ in prognosis and therapy response. Little is known about the distribution of these subtypes among native Chinese women.Patientsand Method: From December 2006 to November 2008, 1,133 women were diagnosed with invasive breast cancer in Shanxi province cancer hospital and 818 had assessable information for ER, PR, and Her-2/neu status. Based on immunohistochemical surrogates for ER, PR, and Her-2/neu status obtained from the department of pathology, the molecular subtypes were defined as: luminal A (ER+ and/or PR+, HER2−), luminal B (ER+ and/or PR+, HER2+), basal-like (ER−, PR−, HER2−), and Her-2/neu (ER−, PR−, and HER2+).Results: Luminal A was the most prevalent breast cancer subtype (70.6%), compared with the luminal B breast cancer subtype (15%), basal-like breast cancer subtype (9.6%), and Her-2/neu breast cancer subtype (4.8%). The frequency of Her2 positive breast cancer was 19.88% in the cohort studied. The luminal B breast cancer subtype was largely presented in patients aged less than 50 years old (58.8%) compared with the luminal A subtype (48.5%), basal-like subtype (52.6%) and Her-2/neu breast cancer subtypes (47.4%). The largest tumor size was basal-like breast cancer (28.1mm), followed by luminal B (25.8mm), Her-2/neu (25.7mm) and luminal A (23mm) (P = 0.003). The lymph node status did not vary among the four groups, however, the mean number of lymph nodes metastasis was significantly different with 3.2, 2.9, 5.7 and 4.9 for luminal A, luminal B, Her-2/neu and basal-like subtypes respectively (P = 0.005).Conclusions: The distribution of breast cancer subtypes in Northern Chinese women was similar to other ethnicities. The basal-like and Her-2/neu breast cancer subtypes were associated with unfavorable clinical features. Further studies will identify the difference in treatment response and prognosis among the four subtypes in this cohort. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3168.