Lysosomes and trivalent arsenic treatment in acute promyelocytic leukemia.

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.

Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole.

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.

Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1.

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.

Evaluation of the cancer chemopreventive potency of dithiolethione analogs of oltipraz.

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.

Reduction of aflatoxin B(1) adduct biomarkers by oltipraz in the tree shrew (Tupaia belangeri chinensis).

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.

Chemoprotection by organosulfur inducers of phase 2 enzymes: dithiolethiones and dithiins.

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.

Identification of dithiolethiones with better chemopreventive properties than oltipraz.

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.

Effects of milk and milk products on rectal mucosal cell proliferation in humans.

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.

Use of aflatoxin adducts as intermediate endpoints to assess the efficacy of chemopreventive interventions in animals and man.

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.

Atypical acinar cell foci in human pancreas. Morphological and morphometric analysis.

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.

Predictive value of molecular dosimetry: individual versus group effects of oltipraz on aflatoxin-albumin adducts and risk of liver cancer.

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.

Urinary taurine as a non-invasive marker of aflatoxin B1-induced hepatotoxicity: success and failure.

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.

Protection against aflatoxin B1-induced hepatic toxicity as short-term screen of cancer chemopreventive dithiolethiones.

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.

Elevations of hepatic quinone reductase, glutathione, and alpha- and mu-class glutathione S-transferase isoforms in mice with chronic hepatitis: a compensatory response to injury.

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.

Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione S-transferases.

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)

Large supplements of nicotinic acid and nicotinamide increase tissue NAD+ and poly(ADP-ribose) levels but do not affect diethylnitrosamine-induced altered hepatic foci in Fischer-344 rats.

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.

The effect of niacin deficiency on diethylnitrosamine-induced hepatic poly(ADP-ribose) levels and altered hepatic foci in the Fischer-344 rat.

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.

Marginal zinc status does not exacerbate pancreatic carcinogenesis associated with dietary soybean trypsin inhibitor concentrate in rats.

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.