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Oltipraz and related dithiolethiones are an important class of chemopreventive agents. Studies were undertaken to identify cancer chemopreventive dithiolethiones more active than oltipraz. Largely based upon enzyme induction activities in vitro, 17 dithiolethiones, including oltipraz, were analyzed for their ability to induce hepatic phase II enzyme activities in vivo. Of these compounds, 15 produced greater induction of NAD(P)H:quinone reductase and 11 yielded greater induction of glutathione S-transferase than oltipraz. All 17 dithiolethiones were then tested for their ability to inhibit acute hepatotoxicity by aflatoxin B1 (AFB1), which previously has been shown to be an intermediate predictor of chemopreventive activity. Rats were pretreated with dithiolethiones (0.3 mmol/kg body wt, three times a week per os) and challenged with two acutely toxic doses of AFB1 (0.5 mg/kg body wt, once daily for two successive days per os). Inhibition of hepatotoxicity was measured by changes in body weight gain during AFB1 challenge, reduction in levels of hepatic enzymes in serum and diminution of bile duct cell proliferation. Nine dithiolethiones spanning a range of responses in this toxicity screen were further tested for their ability to prevent AFB1-induced tumorigenicity, as assessed by a reduction in hepatic burden of putative preneoplastic foci. Six dithiolethiones were found to be considerably more effective than oltipraz in preventing AFB1-induced tumorigenesis. In general, dithiolethiones that were very effective in inhibition of acute hepatotoxicity were also found to be effective in prevention of hepatic tumorigenesis. [ABSTRACT FROM PUBLISHER]

Exploration of animal models leads to discoveries that can reveal candidate biomarkers for translation to human populations. Herein, a model of hepatocarcinogenesis and protection was used in which rats treated with aflatoxin (AFB 1 ) daily for 28 days (200 µg/kg BW) developed tumors compared with rats completely protected from tumors by concurrent administration of the chemoprotective agent, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Differential expression of miRNAs in tumors (AFB 1 ) and nontumor (AFB 1  + CDDO-Im) bearing livers and their levels in sera over the life-course of the animals was determined. miRNA transcriptome analysis identified 17 miRNAs significantly upregulated at greater than five-fold in the tumors. The ten most dysregulated miRNAs judged by fold-change and biological significance were selected for further study, including liver-specific miR-122-5p. Validation of sequencing results by real-time PCR confirmed the upregulation of the majority of these miRNAs in tumors, including miR-182, as well as miR-224-5p as the most dysregulated of these miRNAs (over 400-fold). The longitudinal analysis of levels of miR-182 in sera demonstrated significant and persistent increases (5.13-fold; 95% CI: 4.59-5.70). The increase in miR-182 was detected months before any clinical symptoms were present in the animals. By the terminal time point of the study, in addition to elevated levels of serum miR-182, serum miR-122-5p was also found to be increased (>1.5-fold) in animals that developed hepatocarcinomas. Thus, using the data from an unbiased discovery approach of the tissue findings, serum miR-182 was found to track across the complex, multistage process of hepatocarcinogenesis opening an opportunity for translation to human populations., (© 2019 Wiley Periodicals, Inc.)

Aflatoxin B 1 (AFB 1 ) is a potent human and animal hepatocarcinogen. To investigate the effects of aflatoxin on miRNA expression during the initiation phase of carcinogenesis, next-generation sequencing was used to analyze liver tissues from F344 rats exposed to 200 μg/kg per day AFB 1 for 4 week. A panel of miRNAs was identified that was upregulated with AFB 1 treatment compared to controls: rno-miR-434-3p, rno-miR-411-5p, rno-miR-221-3p, rno-miR-127-3p, rno-miR-205, rno-miR-429, rno-miR-34a-5p, rno-miR-181c-3p, rno-miR-200b-3p, and rno-miR-541-5p. Analysis of rat livers exposed to AFB 1 plus the chemopreventive triterpenoid CDDO-Im revealed a striking abrogation of this upregulation. These changes were validated by real-time PCR. We also explored the temporal variation in expression of the candidate miRNAs during the 4-week dosing period. Most of the candidate miRNAs were upregulated at week 1 and increased for the duration of AFB 1 dosing over the 4-week period. Treatment with CDDO-Im ameliorated these effects at all time points. All candidate miRNAs were detectable in serum from aflatoxin treated animals; however, there was no significant difference in expression for 7 of the 11 miRNAs examined. Exposure to AFB 1 upregulated miR-122-5p (fivefold), 34a-5p (13-fold), and 181c-3p (170-fold) compared with controls. The findings from this study give insight into epigenetic changes induced by aflatoxin taking place during the initial step of carcinogenesis., (© 2017 Wiley Periodicals, Inc.)

In experimental animals and humans, aflatoxin B1 (AFB1) is a potent hepatic toxin and carcinogen. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects against AFB1-induced toxicity and preneoplastic lesion formation (GST-P-positive foci). This study assessed and mechanistically characterized the chemoprotective efficacy of CDDO-Im against AFB1-induced hepatocellular carcinoma (HCC). A lifetime cancer bioassay was undertaken in F344 rats dosed with AFB1 (200 μg/kg rat/day) for four weeks and receiving either vehicle or CDDO-Im (three times weekly), one week before and throughout the exposure period. Weekly, 24-hour urine samples were collected for analysis of AFB1 metabolites. In a subset of rats, livers were analyzed for GST-P foci. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined. CDDO-Im completely protected (0/20) against AFB1-induced liver cancer compared with a 96% incidence (22/23) observed in the AFB1 group. With CDDO-Im treatment, integrated level of urinary AFB1-N(7)-guanine was significantly reduced (66%) and aflatoxin-N-acetylcysteine, a detoxication product, was consistently elevated (300%) after the first AFB1 dose. In AFB1-treated rats, the hepatic burden of GST-P-positive foci increased substantially (0%-13.8%) over the four weeks, but was largely absent with CDDO-Im intervention. The toxicogenomic RNA expression signature characteristic of AFB1 was absent in the AFB1 + CDDO-Im-treated rats. The remarkable efficacy of CDDO-Im as an anticarcinogen is established even in the face of a significant aflatoxin adduct burden. Consequently, the absence of cancer requires a concept of a threshold for DNA damage for cancer development., (©2014 American Association for Cancer Research.)

1,4-Dioxan-2-one, 1, was synthesized, and the equilibrium constant between it and the hydrolysis product 2-(2-hydroxyethoxy) acetic acid, 2, was determined as K(O) = 70 ± 4 in acidic aqueous media, 25 °C, ionic strength 1 M (KCl), and 5% by volume acetonitrile. The carboxylic acid dissociation constant of 2 was determined under the same conditions to be pK(a) = 3.31 ± 0.02. On the basis of these two determinations, the equilibrium constant between 1 and carboxylic acid anion, 3, and the proton was calculated to be K(OA) = 0.034 ± 0.002 M. The stability of 1 was determined in the range of pH between 1 and 8.5 in buffered aqueous solutions under the conditions above by UV spectrophotometric methods and exhibited simple first order kinetics of decay. On the basis of buffer dilution plots, the values of k(o), the rate constant for solvent mediated decomposition, were determined. The plot of log k(o) against pH is consistent with a three term rate law for solvolysis with a hydrogen ion catalyzed rate constant k(H+) = 1.1 (±0.1) M(-1) min(-1), a water catalyzed rate constant, k(w) = 9.9 (±0.5) × 10(-4) min(-1), and a hydroxide ion catalyzed rate constant, k(OH) = 4.1 (±0.3) × 10(4) M(-1) min(-1). The t(1/2) for decay at pH 7.0, at 25 °C, is ∼2 h. Treatment of F344 rats with aflatoxin B(1) (AFB(1)) (positive control) elicited the expected preneoplastic foci in the livers of each rat tested, while subsequent administration of 1 (a total of 1.32 g over 12 weeks) failed to statistically increase focal number or focal volume percent. In 8 rats administered 1 (1.32 g, 12 weeks) alone, no increase above background foci was detected. This study does not support compound 1 as a common carcinogen.

Unlabelled: Aldo-keto reductase-7A (AKR7A) is an enzyme important for bioactivation and biodetoxification. Previous studies suggested that Akr7a might be transcriptionally regulated by oxidative stress-responsive transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a protein highly responsive to acetaminophen (APAP) or its intermediate metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). This study was, therefore, carried out to investigate whether Akr7a is involved in the protection against APAP-induced oxidative stress and hepatotoxicity. We found that in response to APAP or NAPQI exposure, Akr7a3 mRNA and protein were significantly up-regulated in vitro in human HepG2 and LO2 cells. Similarly, strong induction was observed for Akr7a5 in mouse AML12 hepatocytes exposed to APAP. In vivo in wild-type rats, significant up-regulation of hepatic AKR7A1 protein was observed after administration of APAP. On the other hand, depletion of Nrf2 reduced the expression of Akr7a3, suggesting that Nrf2, indeed, contributes significantly to the induction of Akr7a. Moreover, loss of cell viability in Nrf2-depleted cells was significantly rescued by coexpression of AKR7A3. Furthermore, increased AKR7A3 in HepG2 cells was associated with the up-regulation of oxidative stress-related enzymes to enhance cellular antioxidant defense, which appeared to contribute significantly to protection against APAP-induced toxicity. In a line of transgenic rats overexpressing AKR7A1, increased AKR7A1 stimulated the expression of Nrf2 and other Nrf2-regulated genes, but did not better protect rats from APAP insults. In contrast, depletion of Akr7a5 in vitro in cultured AML12 cells or depletion of Akr7a1 in vivo in rat liver greatly increased APAP-induced hepatotoxicity., Conclusion: AKR7A proteins are significantly up-regulated in response to APAP/NAPQI exposure to contribute significantly to protection against APAP-induced hepatotoxicity. AKR7A mediates this protection, in part, through enhancing hepatocellular antioxidant defense., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Among these compounds, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((±)-31) is the most potent in these bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.

Since their discovery 50 years ago, the aflatoxins have become recognized as ubiquitous contaminants of the human food supply throughout the economically developing world. The adverse toxicological consequences of these compounds in populations are quite varied because of a wide range of exposures leading to acute effects, including rapid death, and chronic outcomes such as hepatocellular carcinoma. Furthermore, emerging studies describe a variety of general adverse health effects associated with aflatoxin, such as impaired growth in children. Aflatoxin exposures have also been demonstrated to multiplicatively increase the risk of liver cancer in people chronically infected with hepatitis B virus (HBV) illustrating the deleterious impact that even low toxin levels in the diet can pose for human health. The public health impact of aflatoxin exposure is pervasive. Aflatoxin biomarkers of internal and biologically effective doses have been integral to the establishment of the etiologic role of this toxin in human disease through better estimates of exposure, expanded knowledge of the mechanisms of disease pathogenesis, and as tools for implementing and evaluating preventive interventions.

In both experimental animals and humans, aflatoxin B(1) (AFB(1)) is a potent hepatic toxin and carcinogen against which a variety of antioxidants and experimental or therapeutic drugs (e.g., oltipraz, related dithiolethiones, and various triterpenoids) protect from both acute toxicity and carcinogenesis. These agents induce several hepatic glutathione S-transferases (GST) as well as aldo-keto reductases (AKR) which are thought to contribute to protection. Studies were undertaken in transgenic rats to examine the role of one inducible enzyme, AKR7A1, for protection against acute and chronic actions of AFB(1) by enhancing detoxication of a reactive metabolite, AFB(1) dialdehyde, by reduction to alcohols. The AFB(1) dialdehyde forms adducts with protein amino groups by a Schiff base mechanism and these adducts have been theorized to be at least one cause of the acute toxicity of AFB(1) and to enhance carcinogenesis. A liver-specific AKR7A1 transgenic rat was constructed in the Sprague-Dawley strain and two lines, AKR7A1(Tg2) and AKR7A1(Tg5), were found to overexpress AKR7A1 by 18- and 8-fold, respectively. Rates of formation of AFB(1) alcohols, both in hepatic cytosols and as urinary excretion products, increased in the transgenic lines with AKR7A1(Tg2) being the highest. Neither line offered protection against acute AFB(1)-induced bile duct proliferation, a functional assessment of acute hepatotoxicity by AFB(1), nor did they protect against the formation of GST-P positive putative preneoplastic foci as a result of chronic exposure to AFB(1). These results imply that the prevention of protein adducts mediated by AKR are not critical to protection against AFB(1) tumorigenicity.

3H-1,2-dithiole-3-thione (D3T) and its analogues 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (OLT) and 5-tert-butyl-3H-1,2-dithiole-3-thione (TBD) are chemopreventive agents that block or diminish early stages of carcinogenesis by inducing activities of detoxication enzymes. While OLT has been used in clinical trials, TBD has been shown to be more efficacious and possibly less toxic than OLT in animals. Here, we utilize a robust and high-resolution chemical genomics procedure to examine the pharmacological structure-activity relationships of these compounds in livers of male rats by microarray analyses. We identified 226 differentially expressed genes that were common to all treatments. Functional analysis identified the relation of these genes to glutathione metabolism and the nuclear factor, erythroid derived 2-related factor 2 pathway (Nrf2) that is known to regulate many of the protective actions of dithiolethiones. OLT and TBD were shown to have similar efficacies and both were weaker than D3T. In addition, we identified 40 genes whose responses were common to OLT and TBD, yet distinct from D3T. As inhibition of cytochrome P450 (CYP) has been associated with the effects of OLT on CYP expression, we determined the half maximal inhibitory concentration (IC(50)) values for inhibition of CYP1A2. The rank order of inhibitor potency was OLT >> TBD >> D3T, with IC(50) values estimated as 0.2, 12.8 and >100 microM, respectively. Functional analysis revealed that OLT and TBD, in addition to their effects on CYP, modulate liver lipid metabolism, especially fatty acids. Together, these findings provide new insight into the actions of clinically relevant and lead dithiolethione analogues.

A novel acetylenic tricyclic bis-(cyano enone), TBE-31, is a lead compound in a series of tricyclic compounds with enone functionalities in rings A and C. Nanomolar concentrations of this potent multifunctional molecule suppress the induction of the inflammatory protein, inducible nitric oxide synthase, activate phase 2 cytoprotective enzymes in vitro and in vivo, block cell proliferation, and induce differentiation and apoptosis of leukemia cells. Oral administration of TBE-31 also significantly reduces formation of aflatoxin-DNA adducts and decreases size and number of aflatoxin-induced preneoplastic hepatic lesions in rats by >90%. Because of the two cyano enones in rings A and C, TBE-31 may directly interact with DTT and protein targets such as Keap1 that contain reactive cysteine residues. The above findings suggest that TBE-31 should also be tested for chemoprevention and chemotherapy in relevant models of cancer and against other chronic, degenerative diseases in which inflammation and oxidative stress contribute to disease pathogenesis.

The reduction of the aflatoxin B 1 (AFB 1) dialdehyde metabolite to its corresponding mono and dialcohols, catalyzed by aflatoxin B 1-aldehyde reductase (AFAR, rat AKR7A1, and human AKR7A3), is greatly increased in livers of rats treated with numerous chemoprotective agents. Recombinant human AKR7A3 has been shown to reduce the AFB 1-dialdehyde at rates greater than those of the rat AKR7A1. The activity of AKR7A1 or AKR7A3 may detoxify the AFB 1-dialdehyde, which reacts with proteins, and thereby inhibits AFB 1-induced toxicity; however, direct experimental evidence of this hypothesis was lacking. Two human B lymphoblastoid cell lines, designated pMF6/1A2/AKR7A1 and pMF6/1A2, were genetically engineered to stably express AKR7A1 and/or cytochrome P4501A2 (1A2). The pMF6/1A2/AKR7A1 cells were refractory to the cytotoxic effects of 3 ng/mL AFB 1, in comparison to pM6/1A2 cells, which were more sensitive. Diminished protection occurred at higher concentrations of AFB 1 in pMF6/1A2/AKR7A1 cells, suggesting that additional factors were influencing cell survival. COS-7 cells were transfected with either vector control, rat AKR7A1, or human AKR7A3, and the cells were treated with AFB 1-dialdehyde. There was a 6-fold increase in the dialdehyde LC 50, from 66 microM in vector-transfected cells to 400 microM in AKR7A1-transfected cells, and an 8.5-fold increase from 35 microM in vector-transfected cells to 300 microM in AKR7A3-transfected cells. In both cases, this protective effect of the AFAR enzyme was accompanied by a marked decrease in protein adducts. Fractionation of the cellular protein showed that the mitochondria/nuclei and microsomal fractions contained the highest concentration of protein adducts. The levels of human AKR7A3 and AKR7A2 were measured in 12 human liver samples. The expression of AKR7A3 was detectable in all livers and lower than those of AKR7A2 in 11 of the 12 samples. Overall, these results provide the first direct evidence of a role for rat AKR7A1 and human AKR7A3 in protection against AFB 1-induced cytotoxicity and protein adduct formation.

The aflatoxin B 1 aldehyde reductases (AFARs), inducible members of the aldo-keto reductase superfamily, convert aflatoxin B 1 dialdehyde derived from the exo- and endo-8,9-epoxides into a number of reduced alcohol products that might be less capable of forming covalent adducts with proteins. An isotope dilution tandem mass spectrometry method for quantification of the metabolites, C-8 monoalcohol, dialcohol, and C-6a monoalcohol, was developed to ascertain their possible role as urinary biomarkers for application to chemoprevention investigations. This method uses a novel (13)C 17-aflatoxin B 1 dialcohol internal standard, synthesized from (13)C 17-aflatoxin B 1 biologically produced by Aspergillus flavus. Chromatographic standards of the alcohols were generated through sodium borohydride reduction of the aflatoxin B 1 dialdehyde. This method was then explored for sensitivity and specificity in urine samples of aflatoxin B 1-dosed rats that were pretreated with 3 H-1,2-dithiole-3-thione to induce the expression of AKR7A1, a rat isoform of AFAR. One of the two known monoalcohols and the dialcohol metabolite were detected in all urine samples. The concentrations were 203.5 +/- 39.0 ng of monoalcohol C-6a/mg of urinary creatinine and 10.0 +/- 1.0 ng of dialcohol/mg of creatinine (mean +/- standard error). These levels represented about 8.0 and 0.4% of the administered aflatoxin B 1 dose that was found in the urine at 24 h, respectively. Thus, this highly sensitive and specific isotope dilution method is applicable to in vivo quantification of urinary alcohol products produced by AFAR. Heretofore, the metabolic fate of the 8,9-epoxides that are critical for aflatoxin toxicities has been measured by biomarkers of lysine-albumin adducts, hepatic and urinary DNA adducts, and urinary mercapturic acids. This urinary detection of the alcohol products directly contributes to the goal of mass balancing the fate of the bioreactive 8,9-epoxides of AFB 1 in vivo.

Chemoprevention by chlorophyll (Chl) was investigated in a rat multi-organ carcinogenesis model. Twenty-one male F344 rats in three gavage groups (N = 7 rats each) received five daily doses of 250 microg/kg [(3)H]-aflatoxin B(1) ([(3)H]-AFB(1)) alone, or with 250 mg/kg chlorophyllin (CHL), or an equimolar amount (300 mg/kg) of Chl. CHL and Chl reduced hepatic DNA adduction by 42% (P = 0.031) and 55% (P = 0.008), respectively, AFB(1)-albumin adducts by 65% (P < 0.001) and 71% (P < 0.001), respectively, and the major AFB-N(7)-guanine urinary adduct by 90% (P = 0.0047) and 92% (P = 0.0029), respectively. To explore mechanisms, fluorescence quenching experiments established formation of a non-covalent complex in vitro between AFB(1) and Chl (K(d) = 1.22 +/- 0.05 microM, stoichiometry = 1Chl:1AFB(1)) as well as CHL (K(d) = 3.05 +/- 0.04 microM; stoichiometry = 1CHL:1AFB(1)). The feces of CHL and Chl co-gavaged rats contained 137% (P = 0.0003) and 412% (P = 0.0048) more AFB(1) equivalents, respectively, than control feces, indicating CHL and Chl inhibited AFB(1) uptake. However, CHL or Chl treatment in vivo did not induce hepatic quinone reductase (NAD(P)H:quinone oxidoreductase) or glutathione S-transferase (GST) above control levels. These results are consistent with a mechanism involving complex-mediated reduction of carcinogen uptake, and do not support a role for phase II enzyme induction in vivo under these conditions. In a second study, 30 rats in three experimental groups were dosed as in study 1, but for 10 days. At 18 weeks, CHL and Chl had reduced the volume percent of liver occupied by GST placental form-positive foci by 74% (P < 0.001) and 77% (P < 0.001), respectively compared with control livers. CHL and Chl reduced the mean number of aberrant crypt foci per colon by 63% (P = 0.0026) and 75% (P = 0.0004), respectively. These results show Chl and CHL provide potent chemoprotection against early biochemical and late pathophysiological biomarkers of AFB(1) carcinogenesis in the rat liver and colon.

Background: Cells from patients with t(15;17) acute promyelocytic leukemia (APL) express the fusion protein between the promyelocytic leukemia protein and retinoic acid receptor alpha (PML/RAR alpha). Patients with APL respond to differentiation therapy with all-trans-retinoic acid, which induces PML/RAR alpha degradation. When resistance to all-trans-retinoic acid develops, an effective treatment is arsenic trioxide (arsenite), which also induces this degradation. We investigated the mechanism of arsenite-induced PML/RAR alpha degradation., Methods: NB4-S1 APL cells were treated with clinically relevant concentrations of arsenite. Lysosomes were visualized with a lysosome-specific dye. Lysosomal protein esterase was measured by immunoblot analysis. Lysosomal cathepsin L was detected by immunogold labeling and transmission electron microscopy, and its activity was measured in cytosolic cellular fractions. In vitro degradation assays of PML/RAR alpha in cell lysates were performed with and without protease inhibitors and assessed by immunoblot analysis. Only nonparametric two-sided statistical analyses were used. The nonparametric Wilcoxon test was used for group comparison, and the nonlinear regression technique was used for analysis of dose-response relationship as a function of arsenite concentration., Results: Arsenite treatment destabilized lysosomes in APL cells. Lysosomal proteases, including cathepsin L, were released from lysosomes 5 minutes to 6 hours after arsenite treatment. PML/RAR alpha was degraded by lysate from arsenite-treated APL cells, and the degradation was inhibited by protease inhibitors. At both 6 and 24 hours, substantially fewer arsenite-treated APL cells, than untreated cells, contained cathepsin L clusters, a reflection of cathepsin L delocalization. Cells with cathepsin L clusters decreased as a function of arsenite concentration at rates of -2.03% (95% confidence interval [CI] = -4.01 to -.045; P = .045) and -2.39% (95% CI = -4.54 to -.024; P = .029) in 6- and 24-hour treatment groups, respectively, per 1.0 microM increase in arsenite concentration. Statistically significantly higher cytosolic cathepsin L activity was detected in lysates of arsenite-treated APL cells than in control lysates. For example, the mean increase in cathepsin activity at 6 hours and 1.0 microM arsenite was 26.3% (95% CI = 3.3% to 33%; P < .001), compared with untreated cells., Conclusions: In APL cells, arsenite may cause rapid destabilization of lysosomes.

Septicemia is a major cause of death associated with noncoronary intensive care. Systemic production of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) is a major cause of hypotension and poor organ perfusion seen in septic shock. Here, we show that pretreatment of F344 rats with the cancer chemoprotective agent 3H-1,2-dithiole-3-thione (D3T) blocks lipopolysaccharide (LPS)-mediated induction of hepatic iNOS and significantly reduces the associated serum levels of NO metabolites and enzyme markers of toxicity provoked by treatment with LPS. Immunohistochemical analysis shows that this protective effect is largely due to suppression of iNOS expression in hepatocytes. Importantly, pretreatment of animals with D3T blunts LPS-mediated hypotension and dramatically increases their survival. Inasmuch as iNOS expression can be regulated by nuclear factor (NF) kappaB, mechanistic studies show that D3T blocks NFkappaB nuclear translocation and DNA binding and that these effects are accompanied by changes in the levels of phospho-inhibitor of NFkappaB. In conclusion, this study identifies new drug classes and targets that may improve the prevention and treatment of septic shock, as well as chronic diseases associated with the NFkappaB and iNOS pathways.

Synthetic triterpenoid analogues of oleanolic acid are potent inducers of the phase 2 response as well as inhibitors of inflammation. We show that the triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), is a highly potent chemopreventive agent that inhibits aflatoxin-induced tumorigenesis in rat liver. The chemopreventive potency of CDDO-Im was evaluated by measuring inhibition of formation of putative preneoplastic lesions (glutathione S-transferase P positive foci) in the liver of rats exposed to aflatoxin B1. CDDO-Im produces an 85% reduction in the hepatic focal burden of preneoplastic lesions at 1 micromol/kg body weight and a >99% reduction at 100 micromol/kg body weight. CDDO-Im treatment reduces levels of aflatoxin-DNA adducts by approximately 40% to 90% over the range of 1 to 100 micromol/kg body weight. Additionally, changes in mRNA levels of genes involved in aflatoxin metabolism were measured in rat liver following a single dose of CDDO-Im. GSTA2, GSTA5, AFAR, and EPHX1 transcripts are elevated 6 hours following a 1 micromol/kg body weight dose of CDDO-Im. Microarray analysis using wild-type and Nrf2 knockout mice confirms that many phase 2 and antioxidant genes are induced in an Nrf2-dependent manner in mouse liver following treatment with CDDO-Im. Thus, low-micromole doses of CDDO-Im induce cytoprotective genes, inhibit DNA adduct formation, and dramatically block hepatic tumorigenesis. As a point of reference, oltipraz, an established modulator of aflatoxin metabolism in humans, is 100-fold weaker than CDDO-Im in this rat antitumorigenesis model. The unparalleled potency of CDDO-Im in vivo highlights the chemopreventive promise of targeting Nrf2 pathways with triterpenoids.

Chlorophyllin (CHL) is a promising chemopreventive agent believed to block cancer primarily by inhibiting carcinogen uptake through the formation of molecular complexes with the carcinogens. However, recent studies suggest that CHL may have additional biological effects particularly when given after the period of carcinogen treatment. This study examines the post-initiation effects of CHL towards aflatoxin B1 (AFB1)-induced preneoplastic foci of the liver and colon. The single concentration of CHL tested in this study (0.1% in the drinking water) had no significant effects on AFB1-induced foci of the liver and colons of rats.

Generalized cellular hyperplasia has long been associated as a factor in the causation of liver cancer. Parenchymal cell hyperplasia resulting from hepatotoxins, viruses, parasites, or malnutrition is exceedingly variable as to when it occurs, its extent, and its duration. Partial hepatectomy has been used as an experimental tool precisely because the timing and extent of hyperplasia can be known and controlled. With regards to aflatoxin B1 (AFB1) carcinogenesis, partial hepatectomy has produced variable results. An explanation appears to reside in the hepatotoxic properties of AFB1 that enhance the early stages of carcinogenesis., (Copyright 2003 Wiley-Liss, Inc.)

Oltipraz and related dithiolethiones constitute an important class of chemopreventive agents that enhance the expression of carcinogen detoxication and antioxidant genes. Dose-response studies were undertaken to characterize the cancer chemopreventive activities of several dithiolethiones that are at least as active as oltipraz as inducers. Inhibition of formation of pre-neoplastic lesions and formation of DNA adducts in livers of rats exposed to aflatoxin B1 (AFB1) was monitored. In the tumorigenesis experiment, the dithiolethiones were orally gavaged 3 days/week for 3 successive weeks and at four doses ranging from 0.03 to 0.3 mmol/kg body wt. AFB1 was gavaged beginning 1 week after the start of the dithiolethiones and for two successive weeks. The burden of AFB1-induced putative pre-neoplastic lesions (glutathione S-transferase-placental isoform positive foci) was quantified by light microscopy. Reduction in AFB-DNA adduct burden was assessed 24 h following the first dose of AFB1. Both the parent 1,2-dithiole-3-thione (D3T) and its 5-tert-butyl derivative were more potent inhibitors than oltipraz against these endpoints, while two of the seven tested analogs were slightly less inhibitory. D3T, the most potent dithiolethione of this series, was examined by microarray analysis for induction of hepatic genes at an intermediate chemopreventive dose (0.1 mmol/kg). Transcript levels of eight genes, including two known to detoxify aflatoxin, namely, glutathione S-transferase A5 (GSTA5) and AFB1 aldehyde reductase (AFAR) were elevated. Western analysis indicated that induction of hepatic GSTA5 and AFAR were directly related to the dose of D3T. At the highest dose of D3T (0.3 mmol/kg), protein levels of GSTA5 and AFAR were induced by 7- and 27-fold, respectively. While efficacy in humans has yet to be tested, D3T is clearly more potent than oltipraz and serves as a useful molecular probe for determining the key events associated with protection by this class of agents.

Epidemiological studies have shown that exposure to aflatoxin B(1) (AFB(1)) and concurrent infection with hepatitis B lead to a multiplicative risk of developing liver cancer. This chemical-viral interaction can be recapitulated in the tree shrew (Tupia belangeri chinensis). As an initial characterization of this model, the metabolism of AFB(1) in tree shrews has been examined and compared to a sensitive bioassay species, the rat. Utilizing LC/MS/MS, an unreported product, aflatoxin M(1)-N(7)-guanine (AFM(1)-N(7)-guanine), was detected in urine and hepatic DNA samples 24 h after administration of 400 microg/kg AFB(1). In hepatic DNA isolated from tree shrews, AFM(1)-N(7)-guanine was the predominant adduct, 0.74 +/- 0.14 pmol/mg DNA, as compared to 0.37 +/- 0.07 pmol/mg DNA of AFB(1)-N(7)-guanine. Conversely, in rat liver, 6.56 +/- 2.41 pmol/mg DNA of AFB(1)-N(7)-guanine and 0.42 +/- 0.13 pmol/mg DNA of AFM(1)-N(7)-guanine were detected. Rats excreted 1.00 +/- 0.21 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.29 +/- 0.10 pmol AFM(1)-N(7)-guanine/mg creatinine as compared to 0.60 +/- 0.12 pmol AFB(1)-N(7)-guanine/mg creatinine and 0.69 +/- 0.16 pmol AFM(1)-N(7)-guanine/mg creatinine excreted by the tree shrew. Furthermore, tree shrew urine contained 40 times more of the hydroxylated metabolite, AFM(1), than was excreted by rats. In vitro experiments confirmed this difference in oxidative metabolism. Hepatic microsomes isolated from tree shrews failed to produce aflatoxin Q(1) or aflatoxin P(1) but formed a significantly greater amount of AFM(1) than rat microsomes. Bioassays indicated that the tree shrew was considerably more resistant than the rat to AFB(1) hepatocarcinogenesis, which may reflect the significant differences in metabolic profiles of the two species.

The risk of liver cancer is greatest in people both infected with hepatitis B virus (HBV) and highly exposed to aflatoxin B(1) (AFB(1)). The tree shrew (Tupaia belangeri chinensis) is a unique species that can be infected with human HBV, is susceptible to AFB(1)-induced liver cancer, and shows a synergistic interaction between HBV and AFB(1) for liver cancer. In this regard, the tree shrew may be useful for evaluating experimental chemoprevention strategies relevant to high-risk human populations as it mirrors the human epidemiology of liver cancer. To begin developing the model for chemoprevention study, two groups of tree shrews were fed 400 microg AFB(1)/kg b.wt. in milk daily for 4 weeks. One week prior to AFB(1) administration, one group also received oltipraz (0.5 mmol/kg, p.o.) daily for 5 weeks. At weekly intervals, 1 ml of blood and a 24-h urine sample were obtained from each animal. Aflatoxin-albumin adducts in serum were determined by a radioimmunological assay and aflatoxin-N(7)-guanine adducts in urine were measured by HPLC. Aflatoxin-albumin adducts increased rapidly in 2 weeks to plateau at 20 pmol/mg protein, and they diminished after cessation of AFB(1) exposure. Oltipraz significantly attenuated the overall burden of aflatoxin-albumin adducts throughout the exposure period with a median reduction of 80%. In a single cross-sectional analysis at the end of AFB(1) dosing, oltipraz treatment decreased urinary aflatoxin-N(7)-guanine by 93%. Collectively, these results indicate that oltipraz reduces AFB(1) risk biomarkers in the tree shrew in a manner similar to that observed in rodents and humans, and establishes a rationale to evaluate cancer chemoprevention by oltipraz in human HBV-infected, AFB(1) exposed tree shrews.

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, 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. Alliaceous and cruciferous plants are rich in organosulfur compounds with inducer activity. Indeed, monitoring of enzyme induction has led to the recognition or isolation of novel, potent chemopreventive agents such as 1,2-dithiole-3-thiones, dithiins 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 B1 (AFB1)-induced hepatocarcinogenesis by oltipraz indicates that increased expression of phase 2 genes is of central importance, although inhibition of phase 1 activation of aflatoxin B1 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, leading to transcriptional activation of a score of genes involved in carcinogen detoxification and attenuation of oxidative stress. Nrf2-deficient mice fail to induce many of these genes in response to oltipraz and the impact of this genotype on the chemopreventive efficacy of dithiolethiones is currently under investigation. 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, P.R. 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 AFB1, aflatoxin M1, 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. Food-based approaches to chemoprotection, targeted both to the general population and high-risk individuals, offer many practical advantages compared to the use of pharmaceutical agents. Thus, identification and utilization of naturally-occurring organosulfur chemoprotectors including dithiins should be a high priority.

Intake of dairy products and major dairy constituents (e.g., calcium) has been proposed to reduce the risk of colorectal cancer, although epidemiological studies have yielded inconclusive results. We conducted a randomized cross-over trial to test the effects of high- and low-dairy consumption diets on rectal mucosal proliferation, a possible intermediary marker for large bowel cancer. From a gastroenterology clinic at an academic medical center, we recruited 40 patients, ages 25-79 years, who had either a history of a large bowel adenoma or a first-degree relative with large bowel cancer. Participants completed a baseline questionnaire covering demographic characteristics, health history, and habits and a food frequency questionnaire. They were randomized to a 12-week diet of either high dairy intake (six dairy servings/day) or low dairy intake (<0.5 serving of dairy products/day), with an intervening 12-week washout period in which they were asked to resume their usual diet before crossing over to the alternate study diet for the last 12-week period of the study. Adherence to the study diets was monitored by a daily dairy intake checklist and periodic, unscheduled 24-h dietary recalls. Biopsies of the rectal mucosa were obtained at the beginning and end of each intervention phase. Two assays of rectal mucosal cell proliferation were performed: immunohistochemical determination of proliferating cell nuclear antigen and whole crypt mitotic count. We found no statistically significant changes in either of these proliferation measures as a result of high or low dairy intake. There was no correlation between the labeling index for proliferating cell nuclear antigen and whole crypt mitotic count; however, measures of the location and intensity of cell proliferation within the rectal crypt were highly correlated between the two assays. Thus, our study indicates that greater consumption of dairy products over a 12-week period does not change rectal mucosal cell proliferation.

Clinical cancer prevention studies that use disease as an endpoint are of necessity, large, lengthy, and extremely costly. Development of the field of cancer chemoprevention is being accelerated by the application of intermediate markers to preclinical and clinical studies. Sensitive and specific analytic methods have been developed for detecting and quantifying levels of covalent adducts of aflatoxins with cellular DNA and blood proteins at ambient levels of exposure. Such biomarkers can be applied to the preselection of exposed individuals for study cohorts, thereby reducing study size requirements. Levels of these aflatoxin-DNA and albumin adducts can be modulated by chemopreventive agents such as oltipraz and chlorophyllin in experimental models. Overall, a good concordance is seen between diminution of biomarkers and reductions in tumor incidence and/or multiplicity in these settings. Thus, these markers can also be used to rapidly assess the efficacy of preventive interventions. However, the successful application of these biomarkers to clinical prevention trials will be dependent upon prior determination of the associative or causal role of the marker to the carcinogenic process, establishment of the relationship between dose and response, and appreciation of the kinetics of adduct formation and removal. The general approach that has been utilized for the development, validation and application of aflatoxin-DNA and protein adduct biomarkers to cancer chemoprevention trials is summarized., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Conclusion: The morphological and quantitative findings of the present study suggest that atypical acinar cell foci are not neoplastic in nature., Background: Atypical acinar cell foci (AACF) are rare and unusual lesions in the human pancreas. The biological nature of AACF is poorly understood, and is not clear whether they represent neoplastic or degenerative changes in the acinar cells., Methods: To further characterize and understand the significance of AACF in relation to acinar cell tumor development, we have examined these lesions by light and electron microscopy and evaluated the growth pattern by measuring cell proliferation and the size of the foci in the pancreas of a 16-yr-old male., Results: The pancreas was grossly unremarkable. AACF were randomly distributed throughout the pancreas, well delineated, and showed minimal variation in sizes. The constituent cells contained uniform nuclei, pale vacuolated cytoplasm, and exhibited low nuclear-cytoplasmic ratio. Electron microscopic examination showed a few zymogen granules and markedly dilated rough endoplasmic reticulum. Proliferative index in AACF (13%) was less than in adjacent uninvolved acinar tissue (19%). Quantitative stereological analysis showed the pancreas to contain approximately 1800 AACF/cm3 with a mean focal diameter of 360 microns.

Studies in animals and humans have established serum aflatoxin-albumin adducts as biomarkers of exposure to aflatoxin B1 (AFB1), a food-borne hepatocarcinogen. To assess the utility of measurements of aflatoxin-albumin adducts to predict risk of hepatocellular carcinoma (HCC), 123 male F344 rats were dosed with 20 microg of AFB1 daily for 5 weeks after randomization into three groups: no intervention; delayed-transient (500 ppm of oltipraz, weeks 2 and 3 relative to AFB1); or persistent (500 ppm oltipraz, weeks -1 to 5). Serial blood samples were collected from each animal at weekly intervals throughout aflatoxin B1 exposure and assayed for levels of aflatoxin-albumin by radioimmune assay. Area under the curve (AUC) values for aflatoxin-albumin adducts decreased 20 and 39% in the delayed-transient and persistent oltipraz intervention groups, respectively, as compared to no intervention. Similarly, the total incidence of HCC dropped from 83 to 60% (P = 0.03) and 48% (P < 0.01) in these groups. Tumor multiplicity was also reduced in the two oltipraz intervention groups, whereas time to HCC was increased. Mononuclear cell leukemia, a common neoplasm in F344 rats, was seen in 39% of the control animals, whereas the two oltipraz interventions reduced incidence to 18% (P = 0.05) and 13% (P = 0.01), respectively. Overall, a significant association was seen between biomarker AUC and risk of HCC (P = 0.01). However, when the predictive value of aflatoxin-albumin adducts was assessed within treatment groups, there was no association between AUC and risk of HCC (P = 0.56). Thus, aflatoxin-albumin adducts can be useful for monitoring population-based changes induced by interventions, such as in chemoprevention trials, but have limited utility in identifying individuals destined to develop HCC. As a consequence, the use of this biomarker in quantitative risk assessment should be pursued cautiously.

The usefulness of urinary taurine as a non-invasive measure of hepatotoxicity of aflatoxin B1 (AFB1) was evaluated: changes in urinary taurine were characterized in a dose-response, acute toxicity experiment and in two sub-chronic, low dose exposure experiments. Urine of young, male, F344 rats was collected for 4 days prior to, and for 3 days after, the treatment with AFB1. Rats received a single p.o. dose of 0, 0.25, 0.5, 1, 2 or 3 mg AFB1/kg body wt. A transient increase in urinary taurine was noted with doses of 1, 2 or 3 mg AFB1/kg. In two sub-chronic exposure experiments, rats were gavaged with 25 microg AFB1/day for 5 successive days per week for 1 or 2 weeks (approximately 0.25 mg/kg/day). In the first experiment, only a transient increase in urinary taurine during 5 successive doses of AFB1 was observed, while in the second experiment, urinary taurine rose continuously during the 2 weeks of the AFB1 treatment. An explanation for these differing results is not obvious. Urinary taurine appeared to be a useful, non-invasive marker when hepatotoxicity was extensive. Unfortunately, at the low doses of AFB1 (0.25-0.5 mg/kg) as used in carcinogenesis experiments (10 doses of 25 microg/rat), urinary taurine appeared to be an insensitive measure of hepatic damage.

Dithiolethiones are an important class of cancer chemopreventive agents. More than 50 new dithiolethione analogs were synthesized for structure-activity studies. Using selected dithiolethiones, studies were designed to measure protection against the hepatotoxicity of aflatoxin B1 (AFB1) and relate it to the protection against carcinogenicity. Young male F344 rats were pretreated with 0.1 or 0.3 mmol dithiolethiones/kg body wt and challenged with toxic doses of AFB1 (50 micrograms/100 g rat/day) on 2 successive days. One day later, the protection from hepatotoxicity was assessed by measuring serum hepatic enzymes, hepatic necrosis, and degree of bile duct cell proliferation. The ability of these dithiolethiones to prevent AFB1-induced tumorigenicity was assessed by quantifying the hepatic burden of putative preneoplastic lesions [placental glutathione S-transferase (GST-P)-positive foci]. Significant correlations (p < 0.01) were observed between these toxicological indices and GST-P focal burden (alanine aminotransferase, r = 0.943; sorbitol dehydrogenase, r = 0.897; histological index, r = 0.893; bile duct cell proliferation, r = 0.933). These results imply that inhibition of hepatotoxicity affords protection against hepatocarcinogenicity. The extent of protection from acute hepatotoxicity offers a simple, short-term biological endpoint to screen dithiolethiones and related compounds for their chemopreventive properties.

Hepatic levels of GSH and Phase II detoxication enzymes were compared to biochemical and histological indices of hepatic damage in 4- to 76-week-old nontransgenic mice and their transgenic littermates that overexpress the hepatitis B virus large envelope protein. The mice were fed a low-sucrose AIN-76A diet ad libitum. Hepatic-specific activities of quinone reductase (QR) and glutathione S-transferase (GST) were increased 2- to 10-fold beginning at 12 weeks of age in transgenic mice and correlated with increases in serum alanine aminotransferase (ALT) (r = 0.84 and 0.59, respectively). Quantitative histological analysis demonstrated that apoptosis was the predominant feature in 4- to 12-week-old transgenic mice, whereas necrosis and inflammation predominated at later time points. Surprisingly, 3-fold elevations in ALT were observed beginning at 52 weeks of age in nontransgenic mice, and hepatic-specific activities of QR and GST were also modestly increased in elderly nontransgenic animals. In contrast to transgenic mice, apoptosis was not a prominent feature. The strongest histological correlates to ALT in 4- to 76-week-old nontransgenic mice were necrosis and inflammation (r > 0.96), which in turn may have been evoked by hepatic fat accumulation. Profiles of specific GST isoforms were quantitated chromatographically and identified by sequencing tryptic digests. The Ya1 subunit of alpha-class GST was markedly increased from undetectable levels in transgenic mice, while more modest increases were observed in nontransgenic mice more than 1 year old. Fivefold elevations of the Yb1 subunit, a constitutively expressed mu-class GST, were found in transgenic mice older than 4 weeks of age, while 2-fold increases were observed in nontransgenic animals that were more than 1 year old. These studies demonstrate that selected increases in Phase II detoxication enzymes are a stereotyped response to chronic hepatitis that is strikingly reminiscent of the treatment of mice with anticarcinogenic enzyme inducers.

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] protects against chemical carcinogenesis in several animal models and is currently under evaluation as a possible chemopreventive agent in humans. Ideally, clinical chemopreventive interventions use dosing regimens that maximize efficacy while minimizing toxicity. Toward this end, the chemopreventive efficacy achieved by administration of intermittent doses of oltipraz was evaluated in rats. F344 rats were treated with oltipraz (0.5 mmol/kg, p.o.) once weekly, twice weekly, or daily over a 5-week period. After the first week, all rats were gavaged with 20 micrograms/kg of aflatoxin B1 for 28 consecutive days. Livers were analyzed 2 months after the last aflatoxin B1 dose, and the volume of liver occupied by glutathione S-transferase (GST)-P positive foci, a presumptive marker of neoplasia, was observed to be decreased > 95%, > 97%, or > 99% in livers of rats receiving once-, twice-weekly or daily oltipraz treatments, respectively. The chemopreventive actions of oltipraz have been associated with increases in the levels of phase 2 detoxifying enzymes, such as the glutathione S-transferase isozymes. Accordingly, GST conjugation activity measured with 1-chloro-2,4-dinitrobenzene as substrate increased 1.5-, 1.8-, or 2.4-fold for the once-weekly, twice-weekly or daily treatments, respectively, throughout a 7-day period. Quantitative HPLC analyses of GST subunits 24 h after 2 or 7 daily administrations of oltipraz showed that the levels of subunits Yb1, Yp, Yc2, and Ya2 were increased with maximum elevations of 5.6-, 11.1-, 6.4-, and 10.4-fold, respectively. In comparison, levels of subunits Yb2 and Yc1 were modestly elevated 1.8- to 2.6-fold, respectively, whereas subunit Ya1 was not induced. Remarkably, the levels of subunit Yp and Ya2 remained elevated approximately 2.3-fold 7 days after a single dose of oltipraz. In contrast, the levels of subunits Yb1 and Yc2 diminished to approximate control levels within 7 days after a single dose of oltipraz. GST mRNA levels for Ya, Yb, and Yp were measured by Northern blot analysis and were found to be elevated maximally to 13.7-, 13.5-, and 3.9-fold, respectively, after two daily oltipraz doses. Interestingly, GST Ya and Yb mRNA diminished to constitutive levels after 7 daily doses of oltipraz, with no corresponding decreases in GST subunit or activity levels. The levels of GST Ya and Yb mRNA decreased to constitutive levels within 4 days after a single oltipraz administration, whereas GST Yp mRNA levels remained elevated throughout the 7-day follow-up period.(ABSTRACT TRUNCATED AT 400 WORDS)

Poly(ADP-ribose) is a homopolymer of ADP-ribose units synthesized from NAD+ on nuclear acceptor proteins and is known to be involved in DNA repair. It is not known whether large oral doses of the clinically utilized NAD precursors nicotinic acid or nicotinamide affect poly(ADP-ribose) metabolism or the cellular response to DNA damage. In our first study, using Fischer-344 rats, 2 wk of dietary nicotinic acid supplementation (500 and 1000 mg/kg diet) caused elevated levels of NAD+ in the blood, liver, heart and kidney, while nicotinamide caused elevated levels only in the blood and liver, compared with controls fed a diet containing 30 mg/kg nicotinic acid. Both nicotinic acid and nicotinamide, at 1000 mg/kg diet, caused elevations in liver NAD+, by 44 and 43%, respectively. Only nicotinamide, however, elevated liver poly(ADP-ribose) (63% higher than control group). Following treatment with the hepatocarcinogen diethylnitrosamine, higher levels of hepatic NAD+ were observed in rats fed both nicotinic acid and nicotinamide at 1000 mg/kg diet, but only nicotinic acid supplementation caused a greater accumulation of hepatic poly(ADP-ribose) (61% higher than control group). Neither of the dietary treatments significantly affected the proportion of the liver occupied by placental glutathione-S-transferase positive foci. These results show that poly(ADP-ribose) synthesis is not directly responsive to hepatic NAD+ levels during niacin supplementation, and that the mechanisms of action of nicotinic acid and nicotinamide are different. The observed changes in poly(ADP-ribose) metabolism do not appear to cause any change in susceptibility to chemically induced carcinogenesis in this organ.

Poly(ADP-ribose) is synthesized on nuclear proteins in response to DNA damage and plays an important role in DNA repair. Niacin and tryptophan are dietary precursors to NAD+, which is the substrate for poly(ADP-ribose) synthesis. This study examined the influence of niacin status on poly(ADP-ribose) metabolism and carcinogenesis. Diets devoid of added niacin, with different levels of tryptophan, were used to produce moderate and severe niacin deficiencies in male Fischer-344 rats. Control rats were pair fed niacin-replete diets. After a 21-day feeding period, rats were injected with diethylnitrosamine (DEN) (Expt 1, 200 mg/kg ip; Expt 2, 100 mg/kg ip). In Experiment 1, blood and liver NAD+ and liver poly(ADP-ribose) were measured over the next 15 hours. Whereas blood and liver NAD+ were decreased by niacin deficiency, blood NAD+ was not affected by DEN. Liver NAD+ decreased significantly in response to DEN treatment in the pair-fed groups, but it did not change in the niacin-deficient groups. Unexpectedly, at 10 hours postinjection, liver poly(ADP-ribose) accumulation was greater (p < 0.05) in the niacin-deficient than in the pair-fed rats (n = 9), despite lower initial NAD+ levels and a lack of NAD+ disappearance in niacin-deficient livers. In Experiment 2, livers were examined for the presence of altered hepatic foci three months after DEN exposure. There were no significant differences in the percentage of liver occupied by foci between the niacin-deficient and pair-fed groups (n = 8). These results indicate that niacin-deficient rats were able to accumulate higher concentrations of hepatic poly(ADP-ribose) in response to DEN and did not show elevated susceptibility to initiation of altered hepatic foci.

Although the etiology of pancreatic cancer is largely unknown, diet-associated factors may play a role. Male Sprague-Dawley rats (14 d of age) were given a single injection of either saline or azaserine and were weaned (21 d) to diets with either adequate (30 micrograms/g) or low (9 micrograms/g) zinc, with or without 1.0 g/100 g active trypsin inhibitor in the form of soybean trypsin inhibitor concentrate. Experimental diets were fed for 14 wk. Regardless of dietary zinc status, diets with soybean trypsin inhibitor concentrate caused hyperplasia and/or hypertrophy of the pancreas. Pancreatic zinc content was not different among groups. Low dietary zinc levels did not affect total body growth rate or serum zinc concentration. Tibia zinc was also used as an indicator of zinc status. Tibia zinc concentration was lower in rats fed diets low in zinc relative to adequate zinc diets. Azaserine-induced acidophilic foci were larger and more numerous when soybean trypsin inhibitor concentrate was present in the diet regardless of dietary zinc level. Thus, low zinc does not exacerbate the soybean trypsin inhibitor concentrate effects that promote pancreatic cancer.

The rapidly expanding understanding of the progressive processes of carcinogenesis provides opportunities for the identification of molecular biological markers reflecting events from exposure through clinical disease. These molecular biological markers can be classified into categories of markers of exposure reflecting the dose of toxic agents, markers of effect indicating a biological response to exposure, and markers of susceptibility providing information about the inherent sensitivity of individuals to the toxic agents. By definition some of these markers are chemical agent specific, such as a carcinogen-DNA or -protein adduct, while others are biological process specific, such as the altered expression of a gene. This article reviews the development and validation of molecular biomarkers of aflatoxins using experimental and human population studies. The development of molecular biomarkers for aflatoxins is based upon the extensive research database available about their metabolism, macromolecular adduct formation, and general mechanisms of action. The long-term goal of the research described in this paper is the application of aflatoxin biomarkers to the development of preventive interventions in human populations at high risk for liver cancer.

Physical activity (exercise) is a lifestyle factor that has received little attention with regard to its role in the etiology and/or prevention of cancer. These studies examined the effects of treadmill exercise on the early stages of pancreatic carcinogenesis initiated by azaserine in rats. Male Lewis rats were treated with azaserine at 2 weeks of age and weaned to experimental protocols at 3 weeks of age. Two experiments were undertaken; treadmill exercise began at 6 weeks of age (Experiment 1) or at 13 weeks of age (Experiment 2). Rats were exercised for 15-20 min/day and for 3-5 days/week. Treadmill speed and angle of incline were adjusted to afford a range of exercise intensities. The development of putative preneoplastic lesions of the pancreatic acinar cells (henceforth termed foci) was evaluated by quantitative stereological analysis using light microscopy. In Experiment 1, exercise resulted in a known paradoxical reduction in food intake by about 15% of the intake of the sedentary group fed ad libitum. The burden of azaserine-induced foci was decreased by approximately 37%, and this was attributed to the well known effects of reduced caloric intake in these young, rapidly growing rats. In Experiment 2, the higher intensity treadmill exercise group had an increased focal burden, compared to their sedentary pair-fed controls. Importantly, this enhancement occurred despite a reduction in food intake and body fat stores in this treadmill exercise group. These experiments demonstrate that exercise may suppress or promote carcinogenesis, depending upon the stage in the life cycle of the animal.

Previous studies have demonstrated that ingestion of 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) during the aflatoxin B1 (AFB1) treatment phase completely prevented hepatic cancer. In this study we evaluated the effect of feeding oltipraz during the post-AFB1 treatment phase. Fifty-five male F344 rats were divided into five groups. All rats were gavaged with 25 micrograms AFB1/rat, five times a week for two successive weeks. The rats were fed the oltipraz-supplemented diet according to three different feeding regimes: during the AFB1 treatment phase (1 week prior to, during and 1 week after the last gavage with AFB1); during the post-treatment phase; or throughout the entire time of the experiment. Phenobarbital-supplemented diet was fed during post-treatment phase to one group and this was used as a positive control for the promotion of AFB1-induced focal growth. The burden of putative, preneoplastic, hepatic glutathione S-transferase P-positive foci was evaluated at 13 weeks after the AFB1 treatment phase. As seen previously, oltipraz fed during the AFB1 treatment phase significantly inhibited focal development, i.e. the volume percent of the liver occupied with foci was reduced by 87%. Oltipraz when fed during the post-treatment phase neither inhibited nor enhanced focal development.

Cholecystokinin (CCK) has been shown to stimulate the growth of both normal pancreas and azaserine-induced putative preneoplastic pancreatic lesions in the rat. The present study was performed to determine whether these effects are mediated by way of CCK-A receptors, CCK-B receptors, or both. Sixteen-day-old male Lewis rats were given i.p. injections of azaserine at 30 mg/kg body weight. Starting on day 21, rats were given s.c. injections, 5 days/week for 16 consecutive weeks, of either (a) CCK octapeptide (nonselective CCK agonist) (2.50 micrograms/kg body weight, n = 17), (b) tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2-methylphenylaminocarbonyl++ +)-Asp- (N-methyl)-Phe-NH2 (highly selective CCK-A agonist) (1.84 micrograms/kg body weight, n = 18), (c) [(2R,3S)-beta-MePhe28,N-MeNle31]CCK26-33 (highly selective CCK-B agonist) (2.40 micrograms/kg body weight, n = 18), or (d) normal saline solution (control, n = 17). Rats were subsequently sacrificed, pancreatic weights were determined, and quantitative morphometric analysis of atypical acinar cell foci and nodules was performed. Both CCK octapeptide and the selective CCK-A agonist tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2- methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH2 stimulated pancreatic growth and the development of acidophilic atypical acinar cell foci and nodules. Furthermore, the effect produced by the selective CCK-A agonist tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2- methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH2 was greater than that produced by CCK octapeptide. In contrast, the selective CCK-B agonist [(2R,3S)-beta-MePhe28,N-MeNle31]CCK26-33 had no effect. These findings suggest that the growth of putative preneoplastic lesions (acidophilic atypical acinar cell foci and nodules) in the rat pancreas during the early stages of azaserine-induced pancreatic carcinogenesis is mediated specifically by way of CCK-A receptors.

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] protects against aflatoxin B1-induced hepatocarcinogenesis in rats when fed before and during carcinogen exposure; however, such an exposure-chemoprotection intervention paradigm is not directly relevant to most human populations. To model and assess the possible efficacy of short term interventions targeted at individuals at risk for sustained exposure to aflatoxins, 175-g male F344 rats were treated daily with 25 micrograms of aflatoxin B1, p.o., for 28 days. One week after the start of aflatoxin B1 exposure, half of the animals were fed a diet supplemented with 0.075% oltipraz for 10 days; these rats were then restored to the unsupplemented AIN-76A diet for the remainder of the experimental period. Livers were analyzed 2 or 3 months after the last aflatoxin B1 dose for burden of glutathione S-transferase P (GST-P)-positive foci, as an index of presumptive preneoplastic tumors. The transient intervention with oltipraz reduced the volume percent of hepatic GST-P-positive foci by 54% (P = 0.047) and 72% (P = 0.004) at 2 and 3 months, respectively. A strong positive correlation was also observed between the extent of fibrosis in the livers of these animals and the hepatic burden of GST-P-positive foci, implying that cytotoxicity is associated with the tumorigenic process. This protection may reflect alterations in the metabolism and disposition of aflatoxin B1 induced by oltipraz. Glutathione S-transferase catalyze the detoxication of aflatoxin-8,9-oxide and were found to be rapidly induced in the livers of animals after the beginning of the oltipraz intervention. Glutathione S-transferase activity remained significantly (P < 0.05) higher until 9 days after the end of the oltipraz intervention. In contrast, levels of hepatic aflatoxin-DNA adducts were not significantly reduced until 4 days after the beginning of the intervention but remained significantly (P < 0.05) lower up to 11 days after the end of the intervention. The cumulative reduction in levels of hepatic aflatoxin-DNA adducts (approximately 25%) by the oltipraz intervention underestimated the reduction in the hepatic burden of GST-P-positive foci. The significant protection against presumptive preneoplastic tumors, despite the delay of intervention, suggests that oltipraz may exert substantial activity against the cytotoxic and autopromoting action of repeated exposures to aflatoxin B1 and supports the utility of intervention trials with oltipraz in individuals chronically consuming aflatoxin B1-contaminated foods, particularly in regions with high incidences of liver cancer.

In two experiments, the effects of caloric restriction during the postinitiation phase of pancreatic carcinogenesis were evaluated. Male Lewis rats were given injections of azaserine at 14 days of age and weaned to the postinitiation test protocols at 21 days of age. In the first experiment, the caloric content of the diets was restricted by 10, 15, 20, and 30% of the intakes of the ad libitum-fed rats. A sixth group was fed diet ad libitum for only 5-6 h/day; i.e., they were "meal-fed". The development of putative preneoplastic lesions (henceforth termed foci) was evaluated by quantitative stereological (morphometric) analysis of the pancreas. Caloric restriction during the 4-month postinitiation phase resulted in a significant reduction in focal development beginning at 10% caloric restriction and increasing with more severe restriction. The caloric intake of the meal-fed group closely matched the caloric intake of the 10 or 15% caloric restriction groups and the focal response of the meal-fed rats was similar to the groups restricted in calories by 15 to 20%. In the second experiment, rats were initiated with azaserine and weaned to one of four groups: ad libitum; meal-fed; meal-fed for 2 months and ad libitum thereafter; or ad libitum for 2 months and meal-fed thereafter. Foci were evaluated at 2 and 4 months; neoplasm incidence and multiplicity were determined at 14 months postinitiation. Compared to the ad libitum group, the meal-fed group had significantly fewer foci at all times of evaluation and significantly fewer neoplasms. When rats were meal fed for 2 months and then switched to ad libitum feeding for the remainder of the experiment, the focal outcome at 4 months was similar to the group meal fed for all 4 months; and at 14 months the neoplastic outcome was intermediate between the ad libitum and the meal-fed group. Intervention in the ad libitum feeding regimen at 2 months by meal feeding for the remainder of the experiment resulted in a significant decrease in the focal and neoplastic development, as compared to the group fed ad libitum continuously. These two intervention groups were intermediate in response between the meal-fed and ad libitum-fed groups. These results indicate that the postinitiation phase of pancreatic carcinogenesis can be modulated by relatively simple dietary interventions such as moderate caloric restriction.

The cause of the yearly death of an estimated 1,000 to 2,000 migrating dabbling ducks (Anas spp.) and 10 to 50 swans (Cygnus buccinator and C. columbianus) has remained a mystery for the last ten years in Eagle River Flats (ERF), a 1,000 ha estuarine salt marsh near Anchorage, Alaska, used for artillery training by the U.S. Army. We have gathered evidence that the cause of this mortality is the highly toxic, incendiary munition white phosphorus (P4). The symptoms of poisoning we observed in wild ducks included lethargy, repeated drinking, and head shaking and rolling. Death was preceded by convulsions. Farm-reared mallards dosed with white phosphorus showed nearly identical behavioral symptoms to those of wild ducks that became sick in ERF. White phosphorus does not occur in nature but was found in both the sediments where dabbling ducks and swans feed and in the gizzards of all carcasses collected in ERF. We hypothesize that feeding waterfowl are ingesting small particles of the highly toxic, incendiary munition P4 stored in the bottom anoxic sediments of shallow salt marsh ponds.

Pancreatic cancer is the fifth most common cause of death due to cancer. Except for an association with cigarette smoking, its etiology is poorly understood. Because of the dearth of epidemiological clues as to causation, studies with experimental animal models assume greater importance. Rodent models of pancreatic cancer indicate that while dietary fat per se does not cause pancreatic cancer, it does enhance or promote tumor development. Subsequent to treatment with a pancreatic carcinogen, high intakes of dietary unsaturated fats of the n-6 series, but not saturated fats, enhance or promote tumor development. A requisite level of linoleic acid is needed for this promotion. Fats of the n-3 series (e.g., certain fish oils) are inhibitory to tumor growth. Promotion by dietary fats appears only partly related to the high caloric content of fat. Mechanistically, certain dietary unsaturated fats appear to selectively enhance the growth rate of carcinogen-induced, pre-cancerous lesions. Irrespective of precise understanding of mechanisms of promotion, it appears possible to intervene in the process of cancer development and reduce the burden of cancer. Experimentally, this may be accomplished by decreasing total fat intake, decreasing caloric intake, increasing exercise or increasing the intake of n-3 fatty acids.

Studies were undertaken to evaluate the effects of caloric restriction on voluntary exercise in a rat model of pancreatic cancer. Suckling male Lewis rats were initiated with 3 doses (30 mg/kg body weight) of the pancreatic carcinogen azaserine. At 28 days of age they were weaned to one of four experimental protocols; namely, sedentary/ad libitum fed, voluntary exercise/ad libitum fed, sedentary/food restricted, and voluntary exercise/food restricted. Voluntary exercise was provided by free access to running wheels fitted with odometers. Food restriction was intended to be mild, at less than 10% reduction of ad libitum intake. Putative pre-neoplastic pancreatic foci were identified microscopically at 4 months post-azaserine treatment. As previously shown, food restriction led to increased wheel activity, but the increased activity could not be maintained beyond the first half of the post-initiation treatment phase. Additionally, the extensive wheel running occurred only when available food was restricted by greater than 10%. Exercise per se had a lesser effect compared to food restriction on pancreatic tumorigenesis.