Dietary glucoraphanin-rich broccoli sprout extracts protect against UV radiation-induced skin carcinogenesis in SKH-1 hairless mice.

Feeding broccoli sprout extracts providing daily doses of 10 micromol of glucoraphanin to SKH-1 hairless mice with prior chronic exposure to UV radiation (30 mJ cm(-2) of UVB, twice a week, for 17 weeks) inhibited the development of skin tumors during the subsequent 13 weeks; compared to the controls, tumor incidence, multiplicity, and volume were reduced by 25, 47, and 70%, respectively, in the animals that received the protective agent.

Utility of mycobacterial interspersed repetitive unit typing for differentiating Mycobacterium tuberculosis isolates in Wuhan, China.

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis, and may represent a feasible approach to study M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating M. tuberculosis strains, isolates from 105 patients in Wuhan City, China, were genotyped by this method as compared to spoligotyping. MIRU typing identified 55 types that defined 21 clusters and 34 unique isolates. The discriminatory power was high [Hunter-Gaston discriminatory index (HGDI), 0.97]. Spoligotyping showed that 86 (81.9 %) of 105 isolates belonged to the Beijing family genotype. For Beijing family and non-Beijing strains, the discriminatory power of MIRU was high (HGDI, 0.95 and 0.98, respectively). Among the alleles of the MIRU loci for the Beijing family, only locus 26 was highly discriminative, but for non-Beijing strains, loci 10, 16 and 26 were highly discriminative. MIRU typing is a simple and fast method which may be used for preliminary screening of M. tuberculosis isolates in China.

Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast.

Consumers of higher levels of Brassica vegetables, particularly those of the genus Brassica (broccoli, Brussels sprouts and cabbage), reduce their susceptibility to cancer at a variety of organ sites. Brassica vegetables contain high concentrations of glucosinolates that can be hydrolyzed by the plant enzyme, myrosinase, or intestinal microflora to isothiocyanates, potent inducers of cytoprotective enzymes and inhibitors of carcinogenesis. Oral administration of either the isothiocyanate, sulforaphane, or its glucosinolate precursor, glucoraphanin, inhibits mammary carcinogenesis in rats treated with 7,12-dimethylbenz[a]anthracene. In this study, we sought to determine whether sulforaphane exerts a direct chemopreventive action on animal and human mammary tissue. The pharmacokinetics and pharmacodynamics of a single 150 mumol oral dose of sulforaphane were evaluated in the rat mammary gland. We detected sulforaphane metabolites at concentrations known to alter gene expression in cell culture. Elevated cytoprotective NAD(P)H:quinone oxidoreductase (NQO1) and heme oxygenase-1 (HO-1) gene transcripts were measured using quantitative real-time polymerase chain reaction. An observed 3-fold increase in NQO1 enzymatic activity, as well as 4-fold elevated immunostaining of HO-1 in rat mammary epithelium, provides strong evidence of a pronounced pharmacodynamic action of sulforaphane. In a subsequent pilot study, eight healthy women undergoing reduction mammoplasty were given a single dose of a broccoli sprout preparation containing 200 mumol of sulforaphane. Following oral dosing, sulforaphane metabolites were readily measurable in human breast tissue enriched for epithelial cells. These findings provide a strong rationale for evaluating the protective effects of a broccoli sprout preparation in clinical trials of women at risk for breast cancer.

Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study.

Broccoli sprouts are widely consumed in many parts of the world. There have been no reported concerns with respect to their tolerance and safety in humans. A formal phase I study of safety, tolerance, and pharmacokinetics appeared justified because these sprouts are being used as vehicles for the delivery of the glucosinolate glucoraphanin and its cognate isothiocyanate sulforaphane [1-isothiocyanato-(4R)-(methylsulfinyl)butane] in clinical trials. Such trials have been designed to evaluate protective efficacy against development of neoplastic and other diseases. A placebo-controlled, double-blind, randomized clinical study of sprout extracts containing either glucosinolates (principally glucoraphanin, the precursor of sulforaphane) or isothiocyanates (principally sulforaphane) was conducted on healthy volunteers who were in-patients on our clinical research unit. The subjects were studied in three cohorts, each comprising three treated individuals and one placebo recipient. Following a 5-day acclimatization period on a crucifer-free diet, the broccoli sprout extracts were administered orally at 8-h intervals for 7 days (21 doses), and the subjects were monitored during this period and for 3 days after the last treatment. Doses were 25 micromol of glucosinolate (cohort A), 100 micromol of glucosinolate (cohort B), or 25 micromol of isothiocyanate (cohort C). The mean cumulative excretion of dithiocarbamates as a fraction of dose was very similar in cohorts A and B (17.8 +/- 8.6% and 19.6 +/- 11.7% of dose, respectively) and very much higher and more consistent in cohort C (70.6 +/- 2.0% of dose). Thirty-two types of hematology or chemistry tests were done before, during, and after the treatment period. Indicators of liver (transaminases) and thyroid [thyroid-stimulating hormone, total triiodothyronine (T3), and free thyroxine (T4)] function were examined in detail. No significant or consistent subjective or objective abnormal events (toxicities) associated with any of the sprout extract ingestions were observed.

Protection against UV-light-induced skin carcinogenesis in SKH-1 high-risk mice by sulforaphane-containing broccoli sprout extracts.

Aerobic life, UV solar radiation, genetic susceptibility, and immune status contribute collectively to the development of human skin cancers. In addition to direct DNA damage, UV radiation promotes the generation of reactive oxygen intermediates that can cause oxidative damage and inflammation, and ultimately lead to tumor formation. Treatment of murine and human keratinocytes with the isothiocyanate sulforaphane elevated phase 2 enzymes and glutathione and protected against oxidant toxicity. Topical application of sulforaphane-containing broccoli sprouts extracts induced the phase 2 response in mouse skin in vivo. Sulforaphane inhibited cytokine-dependent (gamma-interferon or lipopolysaccharide) induction of iNOS in RAW 264.7 macrophages. The UV-radiation-induced skin carcinogenesis in "initiated high-risk mice" was substantially inhibited by broccoli sprout extracts containing sulforaphane. After completion of the UV irradiation schedule (30 mJ/cm(2)/session twice a week for 20 weeks), groups of approximately 30 mice were treated topically on their backs (5 days a week for 11 weeks) with broccoli sprout extract containing either the equivalent to 0.3 micromol (low dose) or 1.0 micromol (high dose) sulforaphane, respectively. At this time point, the tumor incidence had reached 100% in the control mice. Tumor burden, incidence, and multiplicity were reduced by 50% in the animals that received the high dose of protector. Tumor incidence and multiplicity did not differ between the low dose-treated and the control groups, but the low dose treatment resulted in a substantial reduction of the overall tumor burden. Thus, topical application of sulforaphane-containing broccoli sprout extracts is a promising strategy for protecting against skin tumor formation after exposure to UV radiation.

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.

Regulation of heart insulin receptor tyrosine kinase activity by magnesium and spermine.

Insulin action and aspects of the insulin-signaling pathway have been studied in the heart although the direct regulation of the heart's insulin receptor has not been explored. This study describes the first purification and characterization of the mammalian (rabbit, rat and bovine) heart insulin receptor. The rabbit heart IR showed maximum insulin binding of 18 microg/mg (approximately 1 mole insulin/mole (alpha2beta2) receptor) and a curvilinear Scatchard plot with a high affinity KD for insulin binding of approximately 4 nM at optimal pH (7.8) and NaCl concentration (150 mM). The insulin receptor tyrosine kinase activity was stimulated by insulin, Mg2+ (half-maximum response at approximately 5.6-10.6 nM and approximately 8.5 mM, respectively) and by the physiological polyamines, spermine and spermidine. The stimulation by Mg2+ and the polyamines occurred with and without insulin. These characteristics of the heart insulin receptor provide a mechanism for regulating the activity of the receptor's tyrosine kinase activity by the intracellular free Mg2+ concentration and the polyamines in the absence and presence of insulin.

Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans.

BACKGROUND: Humans are exposed to substantial quantities of isothiocyanates and glucosinolates from vegetables. Since dietary isothiocyanates are widely regarded as potentially important chemoprotectors against cancer, reliable methods for measuring the plasma and tissue pharmacokinetics of isothiocyanates and their dithiocarbamate metabolites are essential for defining dosing regimens. METHODS: Isothiocyanates (ITC) and dithiocarbamates (DTC) react quantitatively with 1,2-benzenedithiol to produce 1,3-benzodithiole-2-thione that can be quantified spectroscopically. Although this cyclocondensation reaction has been highly useful for analyzing plant material and urine samples, the determination of DTC/ITC (the total quantity of DTC and ITC components in a sample that react in the cyclocondensation reaction) in blood and tissues has been hampered by their low levels and the high concentrations of proteins that interfere with the cyclocondensation reaction. The protein content of blood and tissues was reduced by the precipitation with polyethylene glycol (PEG) or ultrafiltration, and the sensitivity of the method was increased substantially by the solid phase extraction of the cyclocondensation product. RESULTS: Pharmacokinetic measurements were made in four human volunteers who received single doses of about 200 micromol of broccoli sprout isothiocyanates (largely sulforaphane, with lesser amounts of iberin and erucin). Isothiocyanates were absorbed rapidly, reached peak concentrations of 0.943-2.27 micromol/l in plasma, serum and erythrocytes at 1 h after feeding and declined with first-order kinetics (half-life of 1.77+/-0.13 h). The cumulative excretion at 8 h was 58.3+/-2.8% of the dose. Clearance was 369+/-53 ml/min, indicating active renal tubular secretion. CONCLUSION: A sensitive and specific method for quantifying DTC levels in human plasma, serum, and erythrocytes has been devised. Determinations of ITC/DTC levels are important because: (i) dietary isothiocyanates are of potential value in reducing the risk of cancer, and (ii) humans are extensively exposed to DTC as fungicides, insecticides, pesticides and rubber vulcanization accelerators.