Activation of the astrocytic Nrf2/ARE system ameliorates the formation of demyelinating lesions in a multiple sclerosis animal model.

Oxidative stress critically contributes to the pathogenesis of a variety of neurodegenerative diseases such as multiple sclerosis. Astrocytes are the main regulators of oxidative homeostasis in the brain and dysregulation of these cells likely contributes to the accumulation of oxidative damage. The nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of the anti-oxidant stress defense. In this study, we elucidate the effects of astrocytic Nrf2-activation on brain-intrinsic inflammation and lesion development. Cells deficient for the Nrf2 repressor kelch-like ECH-associated protein 1 (Keap1) are characterized by hyperactivation of Nrf2-signaling. Therefore, wild type mice and mice with a GFAP-specific Keap1-deletion were fed with 0.25% cuprizone for 1 or 3 weeks. Cuprizone intoxication induced pronounced oligodendrocyte loss, demyelination and reactive gliosis in wild type animals. In contrast, astrocyte-specific Nrf2-activation was sufficient to prevent oligodendrocyte loss and demyelination, to ameliorate brain intrinsic inflammation and to counteract axonal damage. Our results highlight the potential of the Nrf2/ARE system for the treatment of neuroinflammation in general and of multiple sclerosis in particular. © GLIA 2016;64:2219-2230.

Genetic disruption of the Nrf2 compromises cell-cycle progression by impairing GSH-induced redox signaling.

Genetic disruption of Nrf2 greatly enhances susceptibility to prooxidant- and carcinogen-induced experimental models of various human disorders; but the mechanisms by which this transcription factor confers protection are unclear. Using Nrf2-proficient (Nrf2(+/+)) and Nrf2-deficient (Nrf2(-/-)) primary epithelial cultures as a model, we now show that Nrf2 deficiency leads to oxidative stress and DNA lesions, accompanied by impairment of cell-cycle progression, mainly G(2)/M-phase arrest. Both N-acetylcysteine and glutathione (GSH) supplementation ablated the DNA lesions and DNA damage-response pathways in Nrf2(-/-) cells; however only GSH could rescue the impaired colocalization of mitosis-promoting factors and the growth arrest. Akt activation was deregulated in Nrf2(-/-) cells, but GSH supplementation restored it. Inhibition of Akt signaling greatly diminished the GSH-induced Nrf2(-/-) cell proliferation and wild-type cell proliferation. GSH depletion impaired Akt signaling and mitosis-promoting factor colocalization in Nrf2(+/+) cells. Collectively, our findings uncover novel functions for Nrf2 in regulating oxidative stress-induced cell-cycle arrest, especially G(2)/M-checkpoint arrest, and proliferation, and GSH-regulated redox signaling and Akt are required for this process.

Vitamin D analogs as anti-carcinogenic agents.

Deltanoids are the class of compounds comprising all natural and synthetic vitamin D molecules. The anti-proliferative, pro-differentiation, and pro-apoptotic properties of deltanoids have garnered interest in the fields of cancer chemoprevention and chemotherapy. The naturally occurring, biologically active form of vitamin D, 1,25(OH)2D3, causes hypercalcemia at pharmacologically relevant doses which forms a major obstacle in the clinical development of this compound. Design of new deltanoids has shown promise in separating the beneficial effects from the toxic effects. The Vitamin D receptor (VDR) is a major target for deltanoid design, and the structural features of deltanoid binding have been described. Effective compounds must also exhibit beneficial pharmacokinetic properties in vivo, and the plasma vitamin D binding protein (DBP) is likely to play an important role in the success of deltanoids in the clinic. Further, dual strategies of avoiding vitamin D toxicity through altering the dosing schedule and using less toxic deltanoids are in development. The three main categories of structural modification to the vitamin D backbone include the C,D-ring, the A-ring, and the C,D-ring side chain, and the ways each area has impacted efficacy and toxicity have been described through structure-activity relationships (SARs). Lastly, there is evidence that deltanoids can enhance the activity of other chemopreventive agents. The use of a cocktail approach will be discussed as a potential avenue for deltanoids in chemoprevention and chemotherapy.

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

Residents of Qidong, People's 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-N(7)-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-N(7)-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.

GSTA1 expression in normal, preneoplastic, and neoplastic human prostate tissue.

BACKGROUND: Glutathione S-transferases (GSTs), inducible enzymes that catalyze the detoxification of reactive electrophiles and oxidants, protect against neoplastic transformation. Prostatic adenocarcinoma and high-grade prostatic intraepithelial neoplasia (HGPIN) fail to express GSTP1, a major class of GST. This failure of expression is associated with methlyation of the GSTP1 promoter, a somatic alteration proposed to be a critical step in prostatic carcinogenesis. However, simple atrophy and post-atrophic hyperplasia-proliferative lesions associated with chronic inflammation, which we have termed "proliferative inflammatory atrophy" (PIA)-express elevated levels of GSTP1. We postulated that this increase in GSTP1 expression in PIA occurs in response to increased oxidative stress. We examined the expression of another major class of GST, GSTA1, in the human prostate. METHODS: We performed immunohistochemistry against GSTA1 on formalin-fixed radical prostatectomies (n = 45). A stereological grid point counting method was used to estimate the percent of cells staining positive for GSTA1 in normal prostate, PIA, HGPIN, and adenocarcinoma. RESULTS: In contrast to GSTP1, normal peripheral zone epithelium was virtually devoid of GSTA1. Strikingly, though, epithelial cells in PIA demonstrated strong staining for GSTA1 (median of percent of cells staining positive = 44) as compared to those in normal peripheral zone (median = 3.0, P <.00001), HGPIN (median = 2.9, P <.00001), and adenocarcinoma (median = 3.8, P <.00001). Variations in GSTA1 were also detected between normal anatomic zones: the central zone showed an increase in the percentage of cells staining positive (median = 20.9) as compared to the transition (median = 0.47, P <.0002) and the peripheral (P <.0001) zones. CONCLUSIONS: Expression of GSTA1 is increased in PIA, supporting the concept that cells within these lesions are subject to localized increases in oxidative stress. Low levels of GSTA1 and GSTP1 in HGPIN and adenocarcinoma suggest a broad lack of detoxification activity in these cells, which may be associated with carcinogenesis in the prostate.

A non-calcemic sulfone version of the vitamin D(3) analogue seocalcitol (EB 1089): chemical synthesis, biological evaluation and potency enhancement of the anticancer drug adriamycin.

Novel side-chain diene sulfones 5, analogues of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (calcitriol, 1), were designed to incorporate some of the therapeutically most favorable structural features of the Leo Pharmaceutical Company's drug candidate diene EB 1089 (seocalcitol, 4) and of the Hopkins' non-calcemic side-chain sulfone analogues 2 and 3. Synthesis of diene sulfones 5 features selective Swern oxidation of a primary silyl ether in the presence of a secondary silyl ether (9-->10) and Horner-Wadsworth-Emmons aldehyde addition by a 1-phosphonyl-3-sulfonyl stabilized carbanion regiospecifically at the 1-position to form E,E-diene sulfone 11. Sulfone diene analogue 5a with natural 1alpha,3beta-diol functionality, but not its diastereomer 5b with unnatural A-ring stereochemistry, is antiproliferative in vitro toward murine keratinocytes and malignant melanoma cells, as well as toward MCF-7 human breast cancer cells. Combining diene sulfone 5a with the currently used anticancer drug adriamycin (ADR) caused a noteworthy 3-fold enhancement of ADR antiproliferative potency in MCF-7 cells. Sulfone diene analogue 5a is weakly active transcriptionally in MCF-7 and ROS 17/2.8 cells, binds poorly but measurably to the vitamin D receptor (VDR), and desirably is non-calcemic in vivo at a daily dose (7 days) of 10 microg/kg of rat body weight.

Role of phase 2 enzyme induction in chemoprotection by dithiolethiones.

One of the major mechanisms of protection against carcinogenesis, mutagenesis, and other forms of toxicity mediated by carcinogens is the induction of enzymes involved in their metabolism, particularly phase 2 enzymes such as glutathione S-transferases (GSTs), UDP-glucuronosyl transferases, and quinone reductases. Animal studies indicate that induction of phase 2 enzymes is a sufficient condition for obtaining chemoprevention and can be achieved by administering any of a diverse array of naturally-occurring and synthetic chemopreventive agents. Indeed, monitoring of enzyme induction has led to the recognition or isolation of novel, potent chemopreventive agents such as 1,2-dithiole-3-thiones, terpenoids and the isothiocyanate sulforaphane. For example, oltipraz, a substituted 1,2-dithiole-3-thione originally developed as an antischistosomal agent, possesses chemopreventive activity against different classes of carcinogens targeting multiple organs. Mechanistic studies in rodent models for chemoprevention of aflatoxin B(1) (AFB(1))-induced hepatocarcinogenesis by oltipraz indicates that increased expression of phase 2 genes is of central importance, although inhibition of phase 1 activation of AFB(1) can also contribute to protection. Exposure of rodents to 1,2-dithiole-3-thiones triggers nuclear accumulation of the transcription factor Nrf2 and its enhanced binding to the "antioxidant response element" (ARE), leading to transcriptional activation of a score of genes involved in carcinogen detoxication and attenuation of oxidative stress. Nrf2-deficient mice fail to induce many of these genes in response to dithiolethiones; moreover, basal expression of these genes is typically repressed. To test the hypothesis that enzyme induction is a useful strategy for chemoprevention in humans, three key elements are necessary: a candidate agent, an at-risk population and modulatable intermediate endpoints. Towards this end, a placebo-controlled, double blind clinical trial of oltipraz was conducted in residents of Qidong, PR China who are exposed to dietary aflatoxins and who are at high risk for the development of liver cancer. Oltipraz significantly enhanced excretion of a phase 2 product, aflatoxin-mercapturic acid, a derivative of the aflatoxin-glutathione conjugate, in the urine of study participants administered 125 mg oltipraz by mouth daily. Administration of 500 mg oltipraz once a week led to a significant reduction in the excretion of the primary oxidative metabolite of AFB(1), AFM(1), when measured shortly after drug administration. While this study highlighted the general feasibility of inducing phase 2 enzymes in humans, a longer term intervention is addressing whether protective alterations in aflatoxin metabolism can be sustained for extended periods of time in this high-risk population.

Kinetic constraints for the thiolysis of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) and related dithiole-3-thiones in aqueous solution.

This report summarizes an investigation of the reactions of biological and other thiols with the cancer chemopreventive oltipraz and other dithiolethiones. Analysis of the kinetics of reaction of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) 1 with monothiols and dithiols in the range of 0.75-20 mM in aqueous 15% ethanol, at pH 7.5 (0.1 M Tris buffer) and at 37 degrees C has been undertaken. A plot of k(obsd) against [thiol] shows that reactions of mono- and dithiols are first order in thiol concentration. The dependence on pH of these reactions shows that the active species is the thiolate anion. Specific second-order rate constants, k(2) (M(-1) s(-1)) for reaction of the thiolate anions with oltipraz have been determined to be cysteine, 0.040 +/- 0.001; 2-mercaptoethanol, 2.0 +/- 0.02; glutathione, 0.099 +/- 0.001; mercaptoacetic acid anion, 4.0 +/- 0.01; dithiothreitol, 1.33 +/- 0.02; 1,3-propanedithiol, 10 +/- 0.5; 1-mercaptopropane-3-ol, 6.5 +/- 0.1; 1-mercaptopropane-2,3-diol, 1.26 +/- 0.05. A plot of pK(a) against log k(2) for monothiols shows a linear dependence of k(2) on pK(a), beta(nuc) 1.1 +/- 0.07, which accounts for most of the reportedly enhanced reactivity of dithiols over monothiols. The pseudo-first-order rate constant for the solvolysis of oltipraz has been measured as 2.2 (+/-0.2) x 10(-8) s(-1). The kinetics of reaction of three other dithiole-3-thiones with glutathione has also been studied for comparison with oltipraz. The specific second-order rate constants, k(2) (M(-1) s(-1)) are 5-phenyl-1,2-dithiole-3-thione, 4.7 x 10(-)(4); 5-(4-methoxyphenyl)-1,2-dithiole-3-thione, 4.1 x 10(-4); and 1,2-dithiole-3-thione 0.08. Important implications for the mode of biological action of these compounds and the nature of the putative biological targets of the compounds are discussed.

Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dimethiole-3-thione.

BACKGROUND: The induction of phase 2 enzymes by dithiolethiones such as oltipraz is an effective means for achieving protection against environmental carcinogens in animals and humans. Transcriptional control of the expression of at least some of these protective enzymes is mediated through the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of proto-typical phase 2 enzymes such as glutathione S-transferase (GST) Ya, Yp, and NAD(P)H: quinone reductase (NQO1) in vivo. MATERIALS AND METHODS: In the present study, 3H-1,2-dithiole-3-thione (D3T) was used as a potent model inducer whose effects on gene expression and chemopreventive efficacy have been extensively characterized in the rat. Over a dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted mice by Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also affects the induction of a broader representation of phase 2 and antioxidative enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also examined in vivo by Northern blot hybridization, electromobility shift assay, and Western blot analysis. RESULTS: Specific activities of hepatic GST and NQO1 were increased by D3T in wild-type mice and were largely blunted in the nrf2-deficient mice. However, changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted mice were multidirectional, dependent upon the particular gene examined. Although elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and gamma-glutamylcysteine synthetase regulatory chain were blocked in the mutant mice, elevation of GST Yp mRNA was largely unimpeded. Increases in levels of mRNA for the heavy and light chains of ferritin were only seen in the nrf2-disrupted mice. Transcript levels of UDP-glucuronyl-transferase 1A6, heme oxygenase-1, maganese superoxide dismutase, which were inducible in the wild-type mice, actually decreased in the mutant mice, whereas levels of mRNA for GST Yc, aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in the mutant mice in the absence of any inductive effect by D3T in the wild-type mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic Nrf2 mRNA levels within several hours following dosing as assessed by Northern blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2) binding site showed increased intensity of binding with nuclear extracts prepared from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover, immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from hepatic nuclei of D3T-treated mice at the same time points. CONCLUSIONS: Nrf2 plays a central role in the regulation of constitutive and inducible expression of multiple phase 2 and antioxidative enzymes by chemoprotective dithiolethiones in vivo, although patterns of response vary among different genes. Knowledge of the factors controlling the specificity of actions of enzyme inducers will be exceedingly helpful in the design and isolation of more efficient and selective chemoprotective agents.

Liquid chromatography electrospray-mass spectrometry of urinary aflatoxin biomarkers: characterization and application to dosimetry and chemoprevention in rats.

A liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) method for the measurement of aflatoxin biomarkers in urine has been developed and validated. The two major aflatoxin-DNA adducts formed in rat tissues, aflatoxin N(7)-guanine and its imidazole ring opened derivative, 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-aflatoxin B(1), were detected and quantified in urine by the LC-ESI-MS/MS technique. Other metabolites derived from the conjugation and/or oxidation of aflatoxin B(1) measured in the urine of dosed rats included aflatoxin P(1), aflatoxin P(1)-glucuronide, aflatoxin Q(1), aflatoxin M(1), 8,9-dihydro-8,9-dihydroxy aflatoxin B(1), aflatoxin B(1)-mercapturic acid, the aflatoxin-cysteine glycine adduct derived from the aflatoxin-glutathione conjugate, aflatoxin M(1)P(1) and the aflatoxin B(1)-dialcohol. For in vivo studies to determine the dosimetry of certain aflatoxin metabolites, aflatoxin B(2) was used as an internal standard for recovery since this compound is not naturally produced in rats. In the final method using the internal standard, the coefficient of variation of six replicate analyses of in vivo rat urine samples for aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid, and aflatoxin M(1) was 12.5, 12.8, and 5.8%, respectively. Further, the LC-ESI-MS/MS method to detect aflatoxin N(7)-guanine in in vivo rat urine samples was at least 20-fold more sensitive than prior techniques. Using the LC-ESI-MS/MS technique, the dosimetry, on a weekly basis, of major urinary aflatoxin metabolites was assessed in animals chronically dosed over a 5-week period. Of particular importance was the application of this method to determine the modulation of levels of urinary aflatoxin metabolites by treatment with oltipraz, a chemopreventive agent that can completely ablate aflatoxin hepatocarcinogenesis in the rat. After 1 week, oltipraz administration diminished urinary aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) levels by 83, 92, and 82%, respectively. The magnitude of this reduction was persistent at the day 14, 21, 28, and 35-day time points with the average decrease of aflatoxin N(7)-guanine, aflatoxin B(1)-mercapturic acid and aflatoxin M(1) being 73, 92, and 90%, respectively. Importantly, even under circumstances where the oltipraz intervention was most efficient in reducing aflatoxin metabolite levels, the LC-ESI-MS/MS method was still sensitive enough to detect the reduced biomarker content. This outcome has important translational implications for the application and analysis of the efficacy of primary and secondary prevention interventions in human populations where ambient exposure levels are low, but the toxicologic hazards of these exposures remain high.

Oltipraz chemoprevention trial in Qidong, People's Republic of China: results of urine genotoxicity assays as related to smoking habits.

A Phase II chemoprevention trial was carried out in Qidong, Jiangsu Province, People's Republic of China. The recruited subjects, all of whom were positive for serum aflatoxin-albumin adducts, were divided into three treatment arms: placebo; oltipraz ([5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione]) given daily at 125 mg p.o.; and oltipraz given once per week at 500 mg p.o. Besides biomarkers related to aflatoxin B(1) exposure, the genotoxicity of blind-coded urine XAD-2 concentrates was evaluated in 201 subjects on the fifth and seventh week of intervention. Genotoxicity was assessed both in the Ames reversion test in strain YG1024 of Salmonella typhimurium, in the presence of an exogenous metabolic system (S9 mix), with or without beta-glucuronidase, and in a DNA repair test in Escherichia coli. Heating of concentrated urine samples or of cigarette smoke condensates was discovered to result in a significant enhancement of their mutagenicity. It was also found that the mutagenicity of condensates from the most extensively used brands of cigarettes in Qidong was much lower than that of Western cigarette brands. Urine mutagenicity was unrelated to treatment with oltipraz, intervention time, gender, and supplement of S9 mix with beta-glucuronidase. Mutagenicity was significantly but variably higher in cigarette smokers than in nonsmokers, which suggests that the urinary excretion of mutagens in the examined population was not exclusively attributable to smoking. Nevertheless, within smokers (28% of the recruited subjects; 67% of all males), the mutagenic potency was significantly correlated with the self-reported number of cigarettes smoked per day and, even more sharply, with the cotinine concentrations in urines. In conclusion, this study demonstrated the validity of urine mutagenicity assays as a biomarker of tobacco smoke exposure that can be investigated on a relatively large scale in chemoprevention trials and provided evidence that oltipraz treatment had no influence on this parameter in the examined population.

Cancer chemoprevention using natural vitamin D and synthetic analogs.

Substantial epidemiologic data support a role for vitamin D in cancer prevention. However, dose-limiting hypercalcemic effects have proved a major obstacle to the development of natural vitamin D as a cancer chemopreventive. Structure-activity studies have sought to disassociate the toxicities and chemopreventive activities of vitamin D, and a number of synthetic deltanoids (vitamin D analogs) have shown considerable promise in this regard. Several such compounds have chemopreventive efficacy in preclinical studies, as does natural vitamin D. Data supporting further development of agents of this class include in vitro and in vivo evidence of antiproliferative, proapoptotic, prodifferentiating and antiangiogenic activities. Ongoing studies are aimed at further defining the molecular mechanisms through which vitamin D and synthetic deltanoids affect gene expression and cellular fate. Additional efforts are focused on establishing the chemopreventive index (efficacy vs toxicity) of each synthetic deltanoid.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50-80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Protection against 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine cytotoxicity and DNA adduct formation in human prostate by glutathione S-transferase P1.

The prostate has been identified as a target for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced carcinogenesis. Humans are exposed to PhIP through ingestion of well-done cooked meats, and there is evidence from epidemiological studies that implicates red meat consumption in prostate carcinogenesis. The alpha and pi class isoforms of glutathione S-transferases (GSTs) have been shown to inhibit adduction of activated PhIP metabolites to DNA in cell-free systems. In humans, silencing of GST pi(GSTP1) through CpG island hypermethylation is found in nearly all prostate carcinomas and is believed to be an early event in prostate carcinogenesis. We hypothesized that suppressed GSTP1 expression in prostate cells would increase their vulnerability to cytotoxicity and DNA adduct formation mediated by activated PhIP metabolites. To test this hypothesis, the human prostate adenocarcinoma cell line, LNCaP, which contains a silenced GSTP1 gene, was genetically modified to constitutively express high levels of GSTP1. Both LNCaP and LNCaP-GSTP1 cells exposed to N-OH-PhIP, but not parent PhIP, for 24 h showed a dose-dependent decrease in cell viability. GSTP1-overexpressing cells had LC50s 30-40% higher than cells transfected with the vector alone. PhIP-DNA adducts isolated from LNCaP-derived cells and primary human prostate tissue cultures exposed to N-OH-PhIP were analyzed by liquid chromatography/electrospray ionization mass spectrometry. Primary cultures of human prostate tissue and LNCaP-GSTP1 cells had approximately 50% lower adduct levels than parental LNCaP and vector control cells. Bioactivation assays using LNCaP cytosols showed that enzymatic activation of N-OH-PhIP to a DNA binding species was dependent on ATP and could be inhibited by recombinant human GSTP1 in the presence of glutathione. This evidence confirms that N-OH-PhIP can be bioactivated to a DNA binding species in human prostate and human prostate-derived cells. These observations provide the basis for using LNCaP and LNCaP-GSTP1 cells as a model system for studying the role of this enzyme in protection against N-OH-PhIP induced DNA damage in prostate carcinogenesis. Loss of GSTP1 expression in human prostate may, therefore, enhance its susceptibility to carcinogenic insult by compounds such as N-OH-PhIP. Conversely, induction of GSTs in early-stage prostate carcinogenesis may be a useful protective strategy.

Specific p53 mutations detected in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry.

Hepatocellular carcinoma (HCC), a common cause of cancer deaths worldwide, has several major etiological risk factors, including infection with the hepatitis viruses and exposure to aflatoxin B1. A specific missense mutation resulting from a guanine to thymine transversion at the third position of codon 249 in the p53 tumor suppressor gene has been reported in 10-70% of HCCs from areas of high dietary exposure to aflatoxin B1. Short oligonucleotide mass analysis was compared with DNA sequencing in 25 HCC samples for specific p53 mutations. Mutations were detected in 10 samples by short oligonucleotide mass analysis in agreement with DNA sequencing. Analysis of another 20 plasma and tumor pairs showed 11 tumors containing the specific mutation, and this change was detected in six of the paired plasma samples. Four of the plasma samples had detectable levels of the mutation; however, the tumors were negative, suggesting possible multiple independent HCCs. Ten plasma samples from healthy individuals were all negative. This molecular diagnostic technique has implications for prevention trials and for the early diagnosis of HCC.

Hepatocellular carcinoma and aflatoxin exposure in Zhuqing Village, Fusui County, People's Republic of China.

Hepatocellular carcinoma (HCC) is a common cause of cancer morbidity and mortality in Asia and Africa. Epidemiological studies have found that dietary exposure to aflatoxin B1 (AFB1) and chronic infection with hepatitis B virus are two major risk factors for HCC. We have collated the incidence and mortality data of malignant tumors from 1973 to 1999 in Zhuqing Village, Fusui County, an area with very high HCC rates, and found that this cancer accounted for 64% of the total cancer incidence. Dietary intake of AFB1 was monitored for 1 week in a study group consisting of 15 males and 14 females from different households in this village. Four of 29 participants (13.8%) and 3 of 15 (20%) male participants were hepatitis B virus surface antigen positive. AFB1 was detectable in 76.7% (23 of 30) of ground corn samples (range, 0.4-128.1 ppb), 66.7% (20 of 30) of cooking peanut oil samples (range, 0.1-52.5 ppb), and 23.3% (7 of 30) of rice samples (range, 0.3-2.0 ppb) collected from each household. Mean levels of serum AFB1-albumin adducts in this group were 1.24 +/- 0.31 pmol/mg of albumin at the beginning of the study and 1.21 +/- 0.19 pmol/mg of albumin at the end of the period. Urinary AFB1 metabolites were detectable in 88.9% (24 of 27) samples (range, 0.9-3569.7 ng/24-h urine). These data provide the exposure and disease risk information for establishing intervention studies to diminish the impact of aflatoxin exposure in this high-risk population.

Biomarkers and surrogacy: relevance to chemoprevention.

Clinical cancer prevention trials that use disease as the end-point are of necessity large, lengthy and costly. While such trials will always remain the 'gold standard' for establishing efficacy, they are unwieldy and inefficient for the rapid translation of our accelerating understanding of the molecular basis of cancer into preventive strategies. The inclusion of biomarkers in the process of chemopreventive agent development is crucial for the advancement of the field. This overview highlights the types of approach that are being used in the development and application of biomarkers in chemoprevention studies. Biomarkers, which measure exposure, susceptibility or risk factors, can be used in selecting study cohorts, assessing participant compliance and/or determining agent efficacy. Key features of biomarkers include reliability, precision, accuracy and validity. Not all biomarkers are suitable for all purposes and are likely to be imperfect in any single setting. Judicious selection and matching of biomarkers with agents and study cohorts is required for their effective utilization. A critical but non-dichotomous element of risk biomarkers is their degree of surrogacy. A classification scheme is provided that relates the degree of surrogacy of risk biomarkers to their utility in preventive interventions.

Preneoplastic prostate lesions: an opportunity for prostate cancer prevention.

Environmental factors, especially the diet, play a prominent role in the epidemic of prostate cancer (PCA), in the United States. Many candidate dietary components have been proposed to influence human prostatic carcinogenesis, including fat, calories, fruits and vegetables, anti-oxidants, and various micronutrients, but the specific roles dietary agents play in promoting or preventing PCA remain controversial. We have collected evidence to suggest that GSTP1, the gene encoding the pi-class glutathione S-transferase (GST), may serve a "caretaker" function for prostatic cells. Although GSTP1 can be detected in normal prostatic epithelium, in almost all PCA cases, PCA cells fail to express GSTP1 polypeptides, and lack of GSTP1 expression most often appears to be the result of somatic "CpG island" DNA methylation changes. Loss of GSTP1 function also appears to be characteristic of prostatic epithelial neoplasia (PIN) lesions, thought to represent PCA precursors. We have recently learned that a new candidate early PCA precursor lesion, proliferative inflammatory atrophy (PIA), characterized by proliferating prostatic cells juxtaposed to inflammatory cells, contains epithelial cells that express high levels of GSTP1. These findings have formed the basis for a new model of prostatic carcinogenesis, in which prostatic cells in PIA lesions, subjected to a barrage of inflammatory oxidants, induce GSTP1 expression as a defense against oxidative genome damage. When cells with defective GSTP1 genes appear amongst the PIA cells, such cells become vulnerable to oxidants and electrophiles that inflict genome damage that tends to promote neoplastic transformation to PIN and PCA cells. Subsequently, PIN and PCA cells with defective GSTPI genes remain vulnerable to similar stresses tending to promote malignant progression. This new model for prostatic carcinogenesis has implications for the design of new prostate cancer prevention strategies. Rational prevention approaches might include: (i) restoration of GSTPI expression via treatment with inhibitors of CpG methylation, (ii) compensation for inadequate GSTPI activity via treatment with inducers of general GST activity, and (iii) abrogation of genome-damaging stresses via avoidance of exogenous carcinogens and/or reduction of endogenous carcinogenic (particularly oxidant) stresses.

Conceptually new 20-epi-22-oxa sulfone analogues of the hormone 1alpha,25-dihydroxyvitamin D(3): synthesis and biological evaluation.

New C,D-ring side-chain-modified sulfone 4a, with natural 1alpha, 3beta-hydroxyl groups but lacking the 25-hydroxyl group characteristic of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1), has been prepared and characterized. Novel synthetic features include: (1) chemoselective oxidation of only a primary silyl ether in a primary-secondary bis-silyl ether intermediate and (2) smooth reductive etherification without interference by a neighboring sulfonyl group. Sulfone 4a, but not its 1beta, 3alpha-diastereomer 4b, is powerfully antiproliferative and transcriptionally active in vitro but desirably noncalcemic in vivo. Although sulfone 4a, designed to resemble Leo Pharmaceutical Co.'s KH-1060 (3), is recognized by catabolic enzymes, the selective biological profile of sulfone 4a is likely not due to its metabolites that are formed in only minor amounts.

Identification and characterization of chlorin e(4) ethyl ester in sera of individuals participating in the chlorophyllin chemoprevention trial.

Chlorophyllin (CHL), a mixture of water soluble derivatives of chlorophyll, has been shown to be an effective inhibitor of aflatoxin B(1) (AFB(1)) carcinogenesis and AFB(1)-DNA adduct formation in rainbow trout and rats [Breinholt, V., Hendricks, J., Pereira, C., Arbogast, D., and Bailey, G. (1995) Cancer Res. 55, 57-62; Kensler, T. W., Groopman, J. D., and Roebuck, B. D. (1998) Mutat. Res. 402, 165-172]. The chemopreventive action of CHL has been previously attributed to molecular complexing. In 1997, a randomized, double-blind clinical trial of CHL was conducted in Qidong, Jiangsu Province, People's Republic of China. At the completion of the study, when serum samples were regrouped by subject identification number, it was noted that many of the participant samples were green in color. Using HPLC, ESI/MS, and MS/MS techniques, serum samples from individuals receiving CHL were found to contain previously unreported copper chlorin e(4) ethyl ester (CuCle(4) ethyl ester) as well as copper chlorin e(4) (CuCle(4)). Both chlorins originated in the study tablet, were absorbed into the bloodstream, and conferred a green color to the sera. This initial finding of in vivo absorption and bioavailability of two chlorin derivatives suggests that the mechanism of CHL chemoprevention may lie in the actions of these two components in vivo in addition to preventing carcinogen absorption from the gut.