Extra-adrenal regeneration of glucocorticoids by 11beta-hydroxysteroid dehydrogenase type 1: physiological regulator and pharmacological target for energy partitioning

The major glucocorticoid in man, cortisol, plays important roles in regulating fuel metabolism, energy partitioning and body fat distribution. In addition to the control of cortisol levels in blood by the hypothalamic-pituitary-adrenal axis, intracellular cortisol levels within target tissues can be controlled by local enzymes. 11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyses the regeneration of active cortisol from inert cortisone, thereby amplifying cortisol levels and glucocorticoid receptor activation in adipose tissue, liver and other tissues. 11Beta-HSD1 is under complex tissue-specific regulation and there is evidence that it adjusts local cortisol concentrations independently of the plasma cortisol concentrations, e.g. in response to changes in diet. In obesity 11beta-HSD1 mRNA and activity in adipose tissue are increased. The mechanism of this up-regulation remains uncertain; polymorphisms in the HSD11B1 gene have been associated with metabolic complications of obesity, including hypertension and type 2 diabetes, but not with obesity per se. Extensive data have been obtained in mice with transgenic over-expression of 11beta-HSD1 in liver and adipocytes, targeted deletion of 11beta-HSD1, and using novel selective 11beta-HSD1 inhibitors; these data support the use of 11beta-HSD1 inhibitors to lower intracellular glucocorticoid levels and treat both obesity and its metabolic complications. Moreover, in human subjects the non-selective 'prototype' inhibitor carbenoxolone enhances insulin sensitivity. Results of clinical studies with novel potent selective 11beta-HSD1 inhibitors are therefore eagerly awaited. The present article focuses on the physiological role of glucocorticoids in regulating energy partitioning, and the evidence that this process is modulated by 11beta-HSD1 in human subjects.     

Expression of the glucocorticoid receptor and 11 beta-hydroxysteroid dehydrogenase 2 in pig testes cells along fetal development

The glucocorticoid (GC)-cortisol receptor (GCR)-11 beta-hydroxysteroid dehydrogenase 2 (11 beta-HSD2) system is involved in the regulation of Leydig cell function and spermatogenesis in mature animals. Herein, we describe the expression of the GCR and 11 beta-HSD2 and the occurrence of apoptosis during fetal development. Male fetuses were collected from Weeks 6, 10, 13, and 15 of pregnancy and from neonates. The testes were used for the immunocytochemical staining of GCR, 11 beta-HSD2 and for terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining of apoptosis. Apoptosis did not occur in any Leydig cells, but approximately 30% expressed GCR and 11 beta-HSD2. The number of GCR-positive cells was similar at all stages, but the number of 11 beta-HSD2-positive cells tended to be higher at Weeks 6 and 15. Steroid synthesis was also higher compared with Weeks 10 and 13. Apoptosis occurred in only a few germ cells. Nearly all germ cells were GCR positive at Weeks 10 and 13, when 11 beta-HSD2 was also increased. The total number of 11 beta-HSD2-positive germ cells was approximately 30%. Thus, elevated GCR expression coincided with the differentiation of gonocytes to spermatogonia and their migration to the basal lamina.     

Structure-based virtual screening for identification of novel 11beta-HSD1 inhibitors

Structure-based pharmacophore models were built by using LigandScout and used for virtual screening of the SPECS database to identify new potential 11beta-HSD1 inhibitors. As a refinement of the results obtained from virtual 3D pharmacophore screening, the best fitting virtual hits were subjected to docking study. The resulting compounds were tested in an enzyme assay and revealed several compounds with novel scaffolds that show sub-micromolar activity and high selectivity for 11beta-HSD1 against 11beta-HSD2.     

Steroidal lactones as inhibitors of 17beta-hydroxysteroid dehydrogenase type 5: chemical synthesis, enzyme inhibitory activity, and assessment of estrogenic and androgenic activities

Androgens are well known to play a predominant role in prostate cancer and other androgen-dependent diseases. To decrease the level of androgen testosterone in the prostate, we are interested in developing inhibitors of 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD5). This enzyme expressed in the prostate is one of the two enzymes able to convert 4-androstene-3,17-dione into testosterone. From a screening study, it was found that a series of steroid derivatives bearing a lactone on D-ring demonstrated potent inhibition of 17beta-HSD5 over-expressed in HEK-293 cells. The results of enzymatic assays using intact cells indicated that a C18-steroid (estradiol or 3-deoxyestradiol) backbone and a spiro-delta-lactone (six-member ring) are important for a strong inhibitory activity. Moreover, the presence of a dimethyl group at the alpha-position of the lactone carbonyl increases the selectivity of the inhibitor toward 17beta-HSD5. Compound 26, a 3-deoxyestradiol derivative with a dimethylated spiro-delta-lactone at position 17, possesses the most potent inhibitory activity for 17beta-HSD5 (IC(50)=2.9 nM). It showed no binding affinity for estrogen, androgen, progestin and glucocorticoid receptors (ER, AR, PR and GR). A weak proliferative effect was, however, observed on ZR-75-1 (ER+) cells in culture at high concentration (1 microM), but not at 0.03 microM. Interestingly, no significant proliferative effect was detected on Shionogi (AR+) cells in culture in the presence of 0.1 and 1 microM of lactone 26.     

N-Butyl-N-methyl-11-(3'-hydroxy-21', 17'-carbolactone-19'-nor-17'alpha-pregna-1',3', 5'(10')-trien-7'alpha-yl)-undecanamide: an inhibitor of type 2 17beta-hydroxysteroid dehydrogenase that does not have oestrogenic or androgenic activity

It is well known that 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the formation and inactivation, from circulating precursors, of several active androgens and oestrogens. These enzymes can thus regulate tumoural cell proliferation in androgen- and oestrogen-dependent cancers. Recently, we discovered that adding a spiro-gamma-lactone to the oestradiol nucleus results in a novel inhibitor of type 2 17beta-HSD, an enzyme that catalyses the interconversions between 4-androstene-3,17-dione and testosterone, and between oestrone and oestradiol. This finding motivated our introducing the spiro-gamma-lactone moiety onto an anti-oestrogenic nucleus. The N-butyl-N-methyl-11-(3'-hydroxy-21', 17'-carbolactone-19'-nor-17'alpha-pregna-1',3', 5'(10')-trien-7'alpha-yl)-undecanamide (4) was then efficiently synthesized and its biological activity was assessed in vitro. Despite the presence of a bulky alkylamide side chain, the spiro-gamma-lactone function conserved its ability to inhibit type 2 17beta-HSD (IC(50) = 0.35 and 0.25 microM, with and without side chain, respectively). Furthermore, the selective inhibition by lactone 4 toward type 2 17beta-HSD (microsomal fraction of human placenta) was demonstrated by the absence of inhibitory activity toward type 1 17beta-HSD (cytosolic fraction of human placenta). Cell proliferation assays indicated that compound 4 had no oestrogenic activity but did show anti-oestrogenic activity on ER(+) cell line ZR-75-1. No androgenic activity could be detected when assayed on the AR(+) cell line Shionogi either. Based on these facts, we report the synthesis of a new steroidal derivative, one that inhibits type 2 17beta-HSD while possessing anti-oestrogenic activity.     

The thymus gland is a target in malnutrition

Malnutrition, secondary to deficiency in uptake of proteins, metal elements or vitamins, consistently results in changes in the thymus gland. The organ undergoes a severe atrophy due to apoptosis-induced thymocyte depletion, particularly affecting the immature CD4(+) CD8(+) cells, as well as a decrease in cell proliferation. Such a feature is apparently linked to a hormonal imbalance, involving decrease of leptin and consequent raise of glucocorticoid hormone levels in the serum. Interestingly, this picture can be reversed after appropriate diet rehabilitation. The thymic microenvironment is also affected in malnutrition: morphological changes in thymic epithelial cells were found, together with a decrease of thymic hormone production by these cells. Additionally, intrathymic contents of extracellular proteins, such as fibronectin, laminin and collagens, are increased in the thymuses from malnourished children. Conjointly, the bulk of data discussed herein clearly points to the notion that the thymus gland is a target in malnutrition. Nevertheless, further relevant information regarding the physiology of the thymus, including the cytokine/chemokine secretion as well as the positive and negative selection events driven by TCR/MHC-peptide interactions in malnutrition, remains to be defined. These are questions that need to be answered in order to have a better understanding of the immunodeficiency seen in malnourished individuals.     

From gene to disease; 'apparent mineralocorticoid excess' syndrome, a syndrome with an apparent excess of mineral corticoids

Apparent mineralocorticoid excess (AME) is an autosomal recessive disease caused by deficiency of the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). 11beta-HSD2 converts cortisol into inactive cortisone and prevents the stimulation of the mineralocorticoid receptor by cortisol. In patients with AME, an enhanced stimulation of mineralocorticoid receptors by cortisol in the distal nephron causes an elevated sodium reabsorption and increased potassium excretion. Sodium retention leads to severe low renin hypertension. The diagnosis of AME is based on the detection of an increased concentration of cortisol metabolites and a low or undetectable concentration of cortisone metabolites in urine. Molecular analysis of the HSD11B2 gene confirms the diagnosis. AME is successfully treated by potassium-sparing diuretics, sometimes in combination with loop diuretics (furosemide). Mild forms of AME might occur more frequently than is currently known and should be suspected in patients with hypertension, hypokalemia and decreased plasma renin concentration. Since liquorice can induce the clinical symptoms of AME due to reversible inhibition of the 11beta-HSD2 enzyme by glycyrrhetinic acid, the active ingredient of liquorice, patients suspected of having AME should not consume liquorice.     

Discovery of selective CYP11B2 (aldosterone synthase) inhibitors for the therapy of congestive heart failure and myocardial fibrosis

An increased aldosterone concentration due to congestive heart failure leads to a further progression of the disease as well as to myocardial fibrosis. To interfere with these fatal processes selective inhibition of aldosterone synthase (CYP11B2) is required. CYP11B1, a key enzyme in glucocorticoid biosynthesis showing a high homology to the target enzyme (>93%), must not be inhibited. Screening of our P450 inhibitor library for inhibition of bovine aldosterone synthase resulted in a high number of compounds showing reasonable inhibition. In the next step substances were tested for oral absorption using two artificial membrane assays. The inhibition of human CYP11B2 was evaluated using assays in fission yeast and V79MZ cells stably expressing the active human target enzyme. For selectivity, inhibition of CYP11B1, CYP11A1, CYP17, CYP19 and CYP5 was determined. Rather potent and selective compounds obtained in this way were structurally further optimised, finally leading to inhibitors showing IC(50) values within the low nanomolar range.     

Aromatase and 11beta-hydroxysteroid dehydrogenase 2 localisation in the testes of pigs from birth to puberty linked to changes of hormone pattern and testicular morphology

Oestrogens and glucocorticoids are important for spermatogenesis and are regulated via aromatase for oestradiol synthesis and 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD 2) as an inactivator of cortisol. In the present study postnatal changes of these two enzymes were monitored together with testicular development and hormone concentrations. Pigs were assigned to three periods: Weeks 0-5, Weeks 5-11 or Weeks 11-17. In Period 1, groups of four piglets were killed after each week. Blood plasma and testes were sampled immediately post mortem. For Periods 2 and 3, groups of six pigs were fitted with vein catheters for daily blood collection. Testes from all pigs were obtained after killing. Levels of testosterone, oestradiol, LH, FSH and cortisol were determined radioimmunologically. The 11beta-HSD 2- and aromatase-expressing cells were stained immunocytochemically. All hormones were maximal 2 weeks after birth. A rise of LH, testosterone and oestradiol occurred again at Week 17. FSH and cortisol remained basal. Parallel to the first postnatal rise, the presence of aromatase and 11beta-HSD 2 in Leydig cells increased, together with germ and Sertoli cell numbers. Expression was low from 3 to 5 weeks, was resumed after Week 5 and was maximal at Week 17. The amount of 11beta-HSD 2 in germ cells was greatest at birth, decreased thereafter and was absent after Week 3.     

中国居民膳食有机锡污染水平和摄入量

目的 获得我国居民膳食有机锡污染的基础数据,为开展有机锡风险分析提供初步的暴露评估结果。方法 采用建立的气相色谱-脉冲火焰光度检测器方法测定了2000年中国总膳食研究的4个大区12类48份混合样品,根据样品中有机锡总量和丁基锡含量以及食物消费量,计算获得我国居民膳食有机锡的暴露量,并对部分典型的阳性样品进行污染溯源分析。结果 12类48份混合样品中,水果、糖、酒类未检出有机锡,其他类食物样品仅是个别检出。南方一区多个样品中检出二甲基锡,含量为1．5～4．1μg/kg,南方一区水产品检出丁基锡,其中三丁基锡0．9μg/kg,二丁基锡1．1μg/kg,一丁基锡1．4μg/kgg。我国居民膳食三丁基锡的暴露下限和暴露上限分别为0．003μg·kg^-1·d^-1与0.006μg·kg^-1·d^-1,分别占WHO推荐三丁基锡每日允许摄入量（ADI）的2．5％和5．0％;丁基锡暴露下限和暴露上限分别0．004μg·kg^-1·d^-1与0．019μg·kg^-1·d^-1,占WHO推荐三丁基锡ADI的3．5％和15．8％。南方一区水产样品的溯源分析显示,福建省和上海市水产样品为有机锡污染的主要来源,带鱼和小黄鱼为有机锡污染较为严重的样品,上海市水产样品检出含量较高的二甲基锡。结论 我国居民有机锡膳食暴露水平较低,有机锡污染来源有待于进一步研究。     

Fetal origins of insulin resistance and obesity

A number of epidemiological studies worldwide have demonstrated a relationship between poor early growth and an increased susceptibility to insulin resistance, visceral obesity, type 2 diabetes and other features of the metabolic syndrome in adulthood. However, the mechanistic basis of this relationship and the relative roles of genes and the environment remain a subject of debate. The 'thrifty phenotype' hypothesis proposes that poor fetal nutrition leads to programming of metabolism and an adult phenotype that is adapted to poor but not plentiful nutrition. The maternal reduced-protein rat model has been used to examine the importance of the maternal environment in determining susceptibility to adult disease. Pregnant and lactating rat dams are fed a diet containing 80 g protein/kg as compared with 200 g protein/kg, which leads to growth restriction in utero. Offspring of low-protein dams have increased susceptibility to diabetes, insulin resistance and hypertension when fed a palatable high-fat diet that promotes obesity. Administration of leptin during pregnancy and lactation to these protein-restricted dams produces offspring that have increased metabolic rate and do not become obese or insulin resistant when fed on a high-fat diet. Increased glucocorticoid exposure, particularly during late gestation, has been linked with insulin resistance in adulthood. High levels of fetal glucocorticoids may result from a decreased activity of placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2, which normally protects the fetus from high maternal glucocorticoid levels. Leptin administration to protein-restricted dams inhibits the suppression of 11beta-HSD-2 and may be one mechanism by which the metabolic syndrome is prevented.     

Recent progress in the study of analytical methods, toxicity, metabolism and health effects of organotin compounds

Over the years, a variety of uses has been found of organic tin compounds as fungicides, as stabilizers in plastics and for other industrial uses. The purpose of this article is to summarize and review the results so far obtained as to the analytical method for organotins in biological samples, the toxicity, metabolism, and biochemical and health effects of organotin compounds. 1) Many methods have been developed for analysis of organotin compounds by spectrophotometry, polarography, gas- or liquid-chromatography, etc. These methods, however, are mainly for analysis of organotins in standard solutions or in water, and are not suitable for organotin compounds in biological samples. Recently, we have developed several methods for analysis of various kinds of organotin compounds in biological samples. These methods are able simultaneously to separate and determine trace amounts (at nanogram order) of organotin compounds and their metabolites in the same biological samples. 2) Acute toxicity of organotin compounds which appeared on the literature are summarized. Trialkyl and triaryl compounds seem to be more toxic than the tetra-, di-, or mono-compounds of the same chain length. With an increase in the number of C atoms the toxicity of alkyl compounds decreases. Aryltin compounds are less toxic than alkyltin compounds. 3) Intestinal absorption sites for tetra-alkyltins are jejunum and duodenum, and those for trialkyltins are ileum and jejunum. A considerable amount of orally administered tetra- and trialkyltins of low molecular weights are absorbed, but only very little of the other organotin compounds seems to be absorbed from the gastrointestinal tract. Absorbed organotin compounds rapidly undergo dealkylation by the microsomal mono-oxygenase system dependent on cytochrome P-450 in the liver, brain or other organs, and the compounds and their metabolites distribute to the whole body, ultimately being excreted into urine, bile and faeces. The biological half life of organotin compounds in mammals is usually short, a half of the amount of tributyl- and triphenyl-tins deposited in the body disappearing in several days. A part of organotin compounds excreted into bile is demonstrated to have been absorbed from the intestine and to circulate in the body via enterohepatic circulation. 4) Specific effects of organotin compounds on the biological systems and health include disturbance of the structure and function of the central nervous system (interstitial edema of white matter), inhibited oxidative phosphorylation in mitochondria of cells, atrophy of the thymus and thymus dependent lymphoid tissues resulting in the dysfunction of T cells for immunity, inhibited enzyme activity, lesions in the liver and bile ducts etc., although some specificity is observed among species of animals and organotin compounds. Recently we found that a single oral administration of triphenyltin fluoride to rabbits induces transient diabetes and diabetic lipemia by inhibiting insulin secretion from morphologically normal pancreatic B-cells...     

Molecular screening of the 11beta-HSD1 gene in men characterized by the metabolic syndrome

Adipose tissue type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), which generates hormonally active cortisol from inactive cortisone, has been shown to play a central role in adipocyte differentiation and abdominal obesity-related metabolic complications. The objective was to investigate whether genetic variations in the human 11beta-HSD1 gene are associated with the metabolic syndrome among French-Canadian men. We sequenced all exons, the exon-intron splicing boundaries, and 5' and 3' regions of the human 11beta-HSD1 gene in 36 men with the metabolic syndrome, as defined by the National Cholesterol Education Program-Adult Treatment Panel III, and two controls. Three intronic sequence variants were identified: two single-nucleotide polymorphisms in intron 3 (g.4478T>G) and intron 4 (g.10733G>C) and one insertion in intron 3 (g.4437-4438insA). The relative allele frequency was 19.6%, 22.1%, and 19.6% for the g.4478G, g.10733C, and g.4438insA alleles, respectively. One single-nucleotide polymorphism was identified in exon 6 (c.744G>C or G248G). The frequency of the c.744C allele was only 0.46% in a sample of 217 men. Variants were not associated with components of the metabolic syndrome except for plasma apolipoprotein B levels. In conclusion, molecular screening of the 11beta-HSD1 gene did not reveal any sequence variations that can significantly contribute to the etiology of the metabolic syndrome among French-Canadians.     

Tissue glutathione as a cyst(e)ine reservoir during cystine depletion in growing rats

Previous data suggest that liver glutathione (GSH) serves as a cyst(e)ine reservoir in rats starved or fed cyst(e)ine-deficient diets. In the present study we investigated whether extrahepatic tissue GSH concentrations also decreased during cystine (Cys-Cys) depletion and whether excess dietary cystine increased tissue GSH and cysteine (Cys) concentrations. Five groups of growing rats (80-100 g) were fed diets containing crystalline L-amino acids differing in methionine (Met) and cystine (Cys-Cys) content for 15 days. All diets were isonitrogenous (1.3 g/100 g diet) and provided the minimum Met (0.17%) required by growing rats. Diet 1 provided 0.17% Met and 0% Cys-Cys, diet 2 (the recommended diet) provided 0.17% Met and 0.26% Cys-Cys, diet 3 provided 0.50% Met and 0% Cys-Cys, diet 4 provided 0.17% Met and 0.39% Cys-Cys and diet 5 provided 0.17% Met and 0.52% Cys-Cys. Diets 2 and 3 were isosulfurous at 3.3 mmol/100 g diet. Diets 4 and 5 provided 50 and 100%, respectively, more Cys-Cys than required when 0.17% Met was present. Animals fed diet 1 (Cys-Cys depletion) had significantly decreased (P less than 0.05) liver, muscle, spleen, heart and thymus GSH concentrations, whereas brain, small intestine and erythrocyte GSH concentrations remained unchanged. Although brain and small intestine Cys concentrations were not affected by Cys-Cys depletion, spleen, heart and liver Cys concentrations decreased significantly (P less than 0.05). Feeding Cys-Cys above the requirement level did not increase GSH and Cys concentrations of any tissue except liver where Cys levels were elevated. The data indicate that liver, muscle, spleen, heart and thymus GSH serve as Cys reservoirs during Cys-Cys depletion.     

Glucocorticoid blockade does not abrogate tumor-induced cachexia

Cancer-induced cachexia is a common manifestation observed in patients with malignancies. Elevated levels of circulating glucocorticoids and interleukin-6 (IL-6) have been observed in cancer patients with cachexia and are implicated as major mediators in this process. The purpose of this study was to investigate the role of circulating glucocorticoid levels as primary mediators in cancer-induced cachexia. We evaluated whether inhibition of glucocorticoids with the receptor antagonist RU-486 could abrogate the detrimental wasting of muscle and adipose tissues seen in a well-characterized murine tumor-induced cachexia model. Mice (12/group) were randomized to control, tumor-bearing, control + vehicle, or tumor-bearing + glucocorticoid receptor antagonist groups. Circulating serum glucocorticoid and IL-6 levels were measured in addition to multiple body composition parameters, such as total body weight, lean body mass, and adipose content. The results of this study indicate a significant physiological alteration in the tumor-bearing host that causes severe and detrimental changes in body composition parameters. Regression analysis demonstrated a significant correlation between increased circulating glucocorticoid levels and alterations in body composition parameters. These observed defects were not abrogated with the administration of a glucocorticoid receptor antagonist. We therefore conclude that the untoward effects of tumor-induced cachexia are not mediated primarily by the peripheral effects of high circulating glucocorticoid levels but may involve a complex interaction with IL-6.     

有机锡的人体暴露分析

人类活动导致环境中广泛存在各种有机锡化合物,因此,人体普遍暴露于这些污染物中。除皮肤吸收和肺部吸收外,人体主要通过膳食途径暴露于有机锡化合物。该文在总结人体对有机锡暴露途径(包括空气、饮用水、食物和生活用品)的基础上,概述了人体对有机锡的暴露水平以及有机锡对人类健康的危害,最后对有机锡的人体健康风险评价方法进行了简要介绍,并提出了评价中存在的主要问题。     

The thymus is a common target in malnutrition and infection

Malnutrition, secondary to deficiency in intake of proteins, minerals or vitamins, consistently results in changes in the thymus. This organ undergoes a severe atrophy due to apoptosis-induced thymocyte depletion, particularly affecting the immature CD4+CD8+ cells, as well as a decrease in cell proliferation. This feature is apparently linked to a hormonal imbalance, involving a decrease in leptin and consequent increase in glucocorticoid hormone levels in the serum. The thymic microenvironment is also affected in malnutrition: morphological changes in thymic epithelial cells have been found, together with a decrease of thymic hormone production by these cells. Additionally, intrathymic contents of extracellular proteins, such as fibronectin, laminin and collagens, are increased in thymuses from malnourished children. Taken together, these data clearly point to the notion that the thymus is significantly affected in malnutrition. Similar patterns of thymic changes occur in acute infectious diseases, including a severe atrophy of the organ, mainly due to the apoptosis-related depletion of immature CD4+CD8+ thymocytes. Additionally, thymocyte proliferation is compromised in acutely-infected subjects. The microenvironmental compartment of the thymus is also affected in acute infections, with an increased density of the epithelial network and an increase in the deposition of extracellular matrix. In conclusion, it seems clear that the thymus is targeted in malnutrition as well as in acute infections. These changes are related to the impaired peripheral immune response seen in malnourished and infected individuals. Thus, strategies inducing thymus replenishment should be considered in therapeutic approaches, in both malnutrition and acute infectious diseases.     

Proteomic analysis of covalent modifications of tubulins by isothiocyanates

Although isothiocyanates (ITC), which are found in cruciferous vegetables, have been shown to inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in tumor cells, the biochemical mechanisms of cell growth inhibition by these compounds are not fully understood. Studies have reported that ITC binding to intracellular proteins may be an important event for initiating apoptosis. Specific protein target(s) and molecular mechanisms for ITC have been investigated in human lung cancer A549 cells using proteomic tools. Cells were treated with various amounts (1-100 μmol/L) of radiolabeled phenethyl-ITC (PEITC) and sulforaphane (SFN) and the extracted proteins resolved using 2-dimensional gel electrophoresis. The results of mass spectrometric analyses suggested that tubulin may be an in vivo binding target for ITC. The binding of ITC to tubulin was associated with growth arrest. The proliferation of A549 cells was significantly reduced by ITC, with benzyl-ITC (BITC) having a greater relative activity than PEITC or SFN. Mitotic arrest and apoptosis as well as disruption of microtubule polymerization were induced in the order: BITC > PEITC > SFN. An analysis of tubulins isolated from BITC-treated A549 cells showed that Cys(347), a conserved cysteine in all α-tubulin isoforms, was covalently modified by BITC. Taken together, these results suggest that tubulin is a binding target of ITC and that this interaction can lead to growth inhibition and apoptosis.