Chen-Yang Shi

HPLC ANALYSIS OF ISOFLAVONOIDS AND OTHER PHENOLIC AGENTS FROM FOODS AND FROM HUMAN FLUIDS

Abstract A fast, precise and selective diode array HPLC method is presented for the extraction and analysis of soy isoflavonoids from foods and from human urine, plasma, and breast milk in support of mechanistic and epidemiologic studies assessing the potential cancer protective role of soya or isoflavones. Solid phase or solvent extraction was chosen for isolation, and enzymatic or acid hydrolysis procedures were used for aglycone production depending on the matrix to be analyzed. C-18 reversed-phase HPLC was applied to selectively separate and quantitate daidzein (1), 2 glycitein (3), 2 and genistein (4), 2 including their malonyl (a) 2 and acetyl (b) 2 esters, and their mammalian metabolites equol (6) 2 and Odesmethylangolensin (7), 2 as well as formononetin (2), 2 biochanin-A (5), 2 and coumestrol (8) 2 using a gradient elution system. UV absorbance scans and authentic standards were applied for identification purposes, additional to fluorometric monitoring, electrochemical detection, and GC/MS analysis after trimethyl silylation. Detection limits of 20-μl injections were found to be 1.09, 0.53, 3.28, and 1.00 pmoles for daidzein, genistein, equol, and Odesmethylangolensin (DMA), respectively, by monitoring at the individual compounds absorption maximum. The proposed method was applied to monitor isoflavone levels in soy foods and in human plasma, urine and breast milk after challenge with roasted soybeans. Implications of the presented results on the potential activity of isoflavones to prevent cancer by exposing newborn infants to these agents are discussed.

Involvement of reactive oxygen species in aflatoxin B1-induced cell injury in cultured rat hepatocytes

The role of reactive oxygen species (ROS) in AFB1-induced cell injury was investigated using cultured rat hepatocytes. Malonaldehyde (MDA) generation and lactate dehydrogenase (LDH) release were determined as indices of lipid peroxidation and cell injury, respectively. Exposure to AFB1 for up to 72 h resulted in significantly elevated levels of LDH being released into the medium as well as the MDA generation in cultured hepatocytes. These effects were dose-dependent, indicating that AFB1 was capable of inducing oxidative damages in the cell. Further, MDA generation and LDH release were effectively inhibited by the addition of the following: (1) superoxide dismutase (500 units/ml), (2) catalase (1500 units/ml), (3) 10 mM desferrioxamine (a specific iron chelator), or (4) 260 mM dimethyl sulfoxide (a hydroxyl radical scavenger). These evidences therefore suggest that ROS, such as superoxide radicals, hydroxyl radicals and hydrogen peroxides, are involved in AFB1-induced cell injury in cultured rat hepatocytes.

Biomarkers of exposure to low concentrations of benzene: a field assessment.

OBJECTIVE: To carry out a comprehensive field investigation to evaluate various conventional and recently developed biomarkers for exposure to low concentrations of benzene. METHODS: Analyses were carried out on environmental air, unmetabolised benzene in blood and urine, urinary trans, transmuconic acid, and three major phenolic metabolites of benzene: phenol, catechol, and hydroquinone. Validations of these biomarkers were performed on 131 never smokers occupationally exposed to the time weighed average benzene concentration of 0.25 ppm (range, 0.01 to 3.5 ppm). RESULTS: Among the six biomarkers studied, unmetabolised benzene in urine correlated best with environmental benzene concentration (correlation coefficient, r = 0.76), followed by benzene in blood (r = 0.64). When urinary metabolites were compared with environmental benzene, trans, trans-muconic acid showed a close correlation (r = 0.53) followed by hydroquinone (r = 0.44), and to a lesser extent with urinary phenol (r = 0.38). No correlation was found between catechol and environmental benzene concentrations. Although unmetabolised benzene in urine correlates best with benzene exposure, owing to serious technical drawbacks, its use is limited. Among the metabolites, trans, trans-muconic acid seems to be more reliable than other phenolic compounds. Nevertheless, detailed analyses failed to show that it is specific for monitoring benzene exposures below 0.25 ppm. CONCLUSION: The overall results suggest that most of the currently available biomarkers are unable to provide sufficient specificity for monitoring of low concentrations of benzene exposure. If a lower occupational exposure limit for benzene is to be considered, the reliability of the biomarker and the technical limitations of measurements have to be carefully validated.

Genetic variation of 3β‐hydroxysteroid dehydrogenase type II in three racial/ethnic groups: Implications for prostate cancer risk

Elevated prostatic dihydrotestosterone (DHT) has been suggested to increase the risk of prostate cancer. The HSD3B2 gene encodes the type II 3β‐hydroxysteroid dehydrogenase: one of two enzymes that initiate the inactivation of DHT. Thus, the HSD3B2 gene is a candidate gene for predisposition to prostate cancer.

BENZENE METABOLITES ENHANCE REACTIVE OXYGEN SPECIES GENERATION IN HL60 HUMAN LEUKEMIA CELLS

Benzene is myelotoxic and leukemogenic in humans. The mechanisms leading to these effects, however have not been fully elucidated. One of the underlying mechanisms is believed to be the oxidative damage caused by its metabolites. A comparative study was undertaken to examine the relationships between reactive oxygen species (ROS) production, lipid peroxidation and subse quent cytotoxicity induced by five major benzene meta bolites. The generation of ROS by benzene metabolites was demonstrated by the significant and dose-dependent increase of intracellular ROS formation in HL60 human promyelocytic leukemia cells in vitro. 1,4-Benzoquinone (BQ) was found to be the most potent metabolite in induction of ROS formation, followed by 1,2,4-benzene triol (BT) and to a lesser extent, phenol (PH) and trans,trans-muconaldehyde (MD). No significant effect was observed when the cells were treated with trans, trans-muconic acid (MA). The enhancement of ROS production by BQ was effectively inhibited by the addition of catalase, deferoxamine (DFO) and dimethyl sulfoxide (DMSO), but unchanged by superoxide dismutase (SOD), suggest that hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) are the two major forms of ROS involved. The results also demonstrate that the ability of benzene metabolites in enhancing ROS generation is closely correlated to their capacity in causing lipid peroxidation and subsequent cytotoxicity. These findings together with earlier parallel observations on DNA damage suggest that ROS play an important role in the mechanism of carcinogenesis induced by benzene metabolites.

Simultaneous determination of hydroquinone, catechol and phenol in urine using high-performance liquid chromatography with fluorimetric detection

A method was developed for simultaneous determination of urinary hydroquinone, catechol and phenol using high-performance liquid chromatography (HPLC) with variable-wavelength fluorimetric detection. Urine samples, after acid hydrolysis, were saturated with sodium sulphate and extracted by diethyl ether. The two buffers used for gradient elution were (A) 10 mM sodium acetate containing 0.5% (v/v) acetic acid and (B) the same as buffer A but containing an additional 20% (v/v) acetonitrile. Hydroquinone, catechol and phenol were separated in a C18 column and detected at 2.9, 6.8 and 13.6 min, respectively. The recovery and reproducibility were generally over 90%. Over 300 extracted samples were analysed and no change in column efficiency was noted. Comparisons were also made with HPLC using ultraviolet (UV) detection and with gas chromatography (GC). The proposed method appears to be more sensitive and reliable than other existing methods. This new method was also validated with urine samples collected from cigarette smokers and from refinery workers exposed to low concentrations of benzene.

Evaluation of biomarkers for occupational exposure to benzene.

OBJECTIVE--To evaluate the relations between environmental benzene concentrations and various biomarkers of exposure to benzene. METHODS--Analyses were carried out on environmental air, unmetabolised benzene in urine, trans, trans-muconic acid (ttMA), and three major phenolic metabolites of benzene; catechol, hydroquinone, and phenol, in two field studies on 64 workers exposed to benzene concentrations from 0.12 to 68 ppm, the time weighted average (TWA). Forty nonexposed subjects were also investigated. RESULTS--Among the five urinary biomarkers studied, ttMA correlated best with environmental benzene concentration (correlation coefficient, r = 0.87). When urinary phenolic metabolites were compared with environmental benzene, hydroquinone correlated best with benzene in air. No correlation was found between unmetabolised benzene in urine and environmental benzene concentrations. The correlation coefficients for environmental benzene and end of shift catechol, hydroquinone, and phenol were 0.30, 0.70, and 0.66, respectively. Detailed analysis, however, suggests that urinary phenol was not a specific biomarker for exposure below 5 ppm. In contrast, ttMA and hydroquinone seemed to be specific and sensitive even at concentrations of below 1 ppm. Although unmetabolised benzene in urine showed good correlation with atmospheric benzene (r = 0.50, P < 0.05), data were insufficient to suggest that it is a useful biomarker for exposure to low concentrations of benzene. The results from the present study also showed that both ttMA and hydroquinone were able to differentiate the background level found in subjects not occupationally exposed and those exposed to less than 1 ppm of benzene. This suggests that these two biomarkers are useful indices for monitoring low concentrations of benzene. Furthermore, these two metabolites are known to be involved in bone marrow leukaemogenesis, their applications in biological monitoring could thus be important in risk assessment. CONCLUSION--The good correlations between ttMA, hydroquinone, and atmospheric benzene, even at concentrations of less than 1 ppm, suggest that they are sensitive and specific biomarkers for benzene exposure.

Colorectal carcinoma among ethnic Chinese in Singapore

Recent epidemiologic studies have suggested that the anatomic distribution of colorectal carcinoma may have undergone a distal to proximal shift over several decades, which has been attributed variously to environmental and genetic factors as well as preventive intervention.

Inhibition of aflatoxin B1-DNA binding and adduct formation by selenium in rats☆

The effect of selenium on aflatoxin B1-DNA binding and adduct formation was studied. Male Fischer 344 rats, fed with up to 8 ppm of sodium selenite in drinking water for 8 weeks, were given a single i.p. dose of aflatoxin B1. The rats were killed 24 h later and the amount of AFB1 bound to hepatic DNA and the amount of DNA adducts formed were determined. Selenium pretreatment resulted in a dose-dependent inhibition of AFB1-DNA binding as well as adduct formation. This was accompanied by an increase of reduced glutathione (GSH) in the liver of selenium-treated animals. These results suggest that selenium could effectively inhibit AFB1-induced DNA damage, which may be partially responsible for its anticarcinogenic effect against AFB1.

Cytotoxicity and DNA Strand Breaks Induced by Benzene and its Metabolites in Chinese Hamster Ovary Cells

The cytotoxicity of benzene (BZ) and its major metabolites phenol (PHE), hydroquinone (HQ), catechol (CAT), 1,4‐benzoquinone (BQ), 1,2,4‐benzenetriol (BT), trans,trans‐muconic acid (ttMA) and S‐phenylmercapturic acid (S‐PMA) was assessed by exposing Chinese Hamster Ovary (CHO) cells to these compounds. Benzene was the least toxic (LD50 = 20 mM), while BQ showed the highest potency (LD50 = 10 μM), followed by HQ (LD50 = 40 μM). It was found that the trend of cytotoxicity was: BQ > HQ >> CAT > ttMA > BT > S‐PMA >> PHE > BZ. 1,4‐Benzoquinone and HQ also demonstrated considerable ability to induce DNA strand breaks in CHO cells, which was assayed using the fluorimetric analysis of DNA unwinding. The other metabolites were unable to cause DNA strand breaks. When HQ was administered in combination with other metabolites, no synergism was observed in the induction of DNA strand breaks. From these results, it can be seen that BQ and HQ are the most bioreactive species among the benzene metabolites when tested on CHO cells. Differences between the results obtained in our study and other studies were discussed.