Association of Vitamin D Receptor Gene Polymorphisms with Serum Vitamin D Levels in a Greek Rural Population (Velestino Study).

BACKGROUND/AIM: An alarming increase in vitamin D deficiency even in sunny regions highlights the need for a better understanding of the genetic background of the vitamin D endocrine system and the molecular mechanisms of gene polymorphisms of the vitamin D receptor (VDR). In this study, the serum levels of 25(OH)D3 were correlated with common VDR polymorphisms (ApaI, BsmI, FokI, and TaqI) in 98 subjects of a Greek homogeneous rural population. METHODS: 25(OH)D3 concentration was measured by ultra-HPLC, and the VDR gene polymorphisms were identified by quantitative real-time PCR followed by amplicon high-resolution melting analysis. RESULTS: Subjects carrying either the B BsmI (OR: 0.52, 95% CI: 0.27-0.99) or t TaqI (OR: 2.06, 95%: 1.06-3.99) allele presented twice the risk for developing vitamin D deficiency compared to the reference allele. Moreover, subjects carrying 1, 2, or all 3 of these genotypes (BB/Bb, Tt/tt, and FF) demonstrated 2-fold (OR: 2.04, 95% CI: 0.42-9.92), 3.6-fold (OR: 3.62, 95% CI: 1.07-12.2), and 7-fold (OR: 6.92, 95% CI: 1.68-28.5) increased risk for low 25(OH)D3 levels, respectively. CONCLUSIONS: Our findings reveal a cumulative effect of specific VDR gene polymorphisms that may regulate vitamin D concentrations explaining, in part, the paradox of vitamin D deficiency in sunny regions, with important implications for precision medicine.

Methionine restriction delays aging-related urogenital diseases in male Fischer 344 rats.

Dietary methionine restriction (MR) has been found to enhance longevity across many species. We hypothesized that MR might enhance longevity in part by delaying or inhibiting age-related disease processes. To this end, male Fischer 344 rats were fed control (CF, 0.86% methionine) or MR (0.17% methionine) diets throughout their life until sacrifice at approximately 30 months of age, and histopathology was performed to identify the incidence and progression of two important aging-related pathologies, namely, chronic progressive nephropathy (CPN) and testicular tumorigenesis. Although kidney pathology was observed in 87% CF rats and CPN in 62% of CF animals, no evidence of kidney disease was observed in MR rats. Consistent with the absence of renal pathology, urinary albumin levels were lower in the MR group compared to controls throughout the study, with over a six-fold difference between the groups at 30 months of age. Biomarkers associated with renal disease, namely, clusterin, cystatin C, and ß-2 microglobulin, were reduced following 18 months of MR. A reduction in testicular tumor incidence from 88% in CF to 22% in MR rats was also observed. These results suggest that MR may lead to metabolic and cellular changes providing protection against age-related diseases.

Comparison of the Serum Metabolic Fingerprint of Different Exercise Modes in Men with and without Metabolic Syndrome.

Exercise plays a beneficial role in the treatment of metabolic syndrome (MetS). Metabolomics can provide new insights and facilitate the optimization of exercise prescription. This study aimed to investigate whether the response of the human serum metabolic fingerprint to exercise depends on exercise mode or the presence of MetS. Twenty-three sedentary men (nine with MetS and fourteen healthy) completed four trials: Resting, high-intensity interval exercise (HIIE), continuous moderate-intensity exercise (CME), and resistance exercise (RE). Blood samples were collected pre-exercise, immediately after exercise, and 1 h post-exercise for targeted metabolomic analysis in serum by liquid chromatography-mass spectrometry. Time exerted the strongest differentiating effect, followed by exercise mode. The largest changes from baseline were found in the immediate post-exercise samples. RE caused the strongest responses overall, followed by HIIE, while CME had minimal effect. Unlike previous results in urine, no valid model could separate the two groups in serum. Exercise exerted a beneficial effect on prominent serum biomarkers of metabolic risks, such as branched-chain amino acids, alanine, acetylcarnitine, choline, and betaine. These findings contribute to the ongoing research efforts to map the molecular responses to exercise and to optimize exercise guidelines for individuals at cardiometabolic risk.

Association between lifestyle and anthropometric parameters and thyroid nodule features.

PURPOSE: Thyroid nodularity has been associated with obesity, but data regarding associations of body composition parameters with specific ultrasound features of thyroid nodules are lacking. The aim of the present study was to assess associations between thyroid nodule ultrasound characteristics, lifestyle, and anthropometric parameters. SUBJECTS AND METHODS: This was a cross-sectional study in the general apparently healthy population of Northern Greece. Thyroid ultrasound data together with medical history, demographic, and anthropometric characteristics were individually recorded. Body composition was evaluated using bioelectrical impedance. RESULTS: Three hundred and six subjects [215 females (70.3%), aged 20-83 years] were included. Ultrasound revealed one or more thyroid nodules in 168 subjects (54.9%). Subjects with thyroid nodules were more frequently females (p = 0.033), older (p < 0.001) and had higher fat mass (p = 0.011), total body fat percentage (p < 0.001) and waist circumference (p = 0.045) than subjects without nodules. In logistic regression analyses, age and female gender were the only independent predictors of presence of thyroid nodules, as well as specific sonographic features. Additionally, total body fat percentage was positively correlated with nodule size (rho = 0.210, p = 0.006) and was the only independent predictor of hypoechoic thyroid nodule(s) and peripheral vascularity, while lack of exercise was predictive of internal vascularity. CONCLUSIONS: Body fat accumulation and lack of exercise, used as surrogate markers of sedentary lifestyle, influence thyroid nodule size and could predict some ultrasonographic characteristics, like hypoechoicity and internal vascularity. Therefore, routine thyroid examination of obese patients and promotion of active lifestyle may be warranted to prevent thyroid nodule formation and possibly progression to malignancy.

Effects of Different Exercise Modes on the Urinary Metabolic Fingerprint of Men with and without Metabolic Syndrome.

Exercise is important in the prevention and treatment of the metabolic syndrome (MetS), a cluster of risk factors that raises morbidity. Metabolomics can facilitate the optimization of exercise prescription. This study aimed to investigate whether the response of the human urinary metabolic fingerprint to exercise depends on the presence of MetS or exercise mode. Twenty-three sedentary men (MetS, n = 9, and Healthy, n = 14) completed four trials: resting, high-intensity interval exercise (HIIE), continuous moderate-intensity exercise (CME), and resistance exercise (RE). Urine samples were collected pre-exercise and at 2, 4, and 24 h for targeted analysis by liquid chromatography-mass spectrometry. Time exerted the strongest differentiating effect, followed by exercise mode and health status. The greatest changes were observed in the first post-exercise samples, with a gradual return to baseline at 24 h. RE caused the greatest responses overall, followed by HIIE, while CME had minimal effect. The metabolic fingerprints of the two groups were separated at 2 h, after HIIE and RE; and at 4 h, after HIIE, with evidence of blunted response to exercise in MetS. Our findings show diverse responses of the urinary metabolic fingerprint to different exercise modes in men with and without metabolic syndrome.

Changes in Thyroid Hormone Levels Within the Normal and/or Subclinical Hyper- or Hypothyroid Range Do Not Affect Circulating Irisin Levels in Humans.

BACKGROUND: Both thyroid hormones and irisin increase energy expenditure and induce browning of adipose tissue. However, irisin physiology and regulation remain largely unknown, and existing data are mainly derived from observational studies. In this study, we aimed to elucidate whether changes in thyroid-axis hormones alter circulating irisin levels in humans, thereby exerting a direct downstream effect on serum irisin. SUBJECTS AND METHODS: Samples from a cross-sectional evaluation and two interventions were utilized, including patients who had previously undergone thyroidectomy. In the cross-sectional study, 96 consecutively enrolled subjects were divided into a euthyroid group and a subclinical hyperthyroid group, according to their serum thyrotropin (TSH) levels (TSH cutoff 0.3 mIU/L). In interventional study A, 34 patients who had undergone thyroidectomy due to thyroid cancer were withdrawn from their thyroxine replacement treatment for five weeks. In interventional study B, 13 patients underwent a recombinant human TSH stimulation protocol, and blood samples were drawn at baseline, day 3 (i.e., at least 24 hours after the second intramuscular injection), day 5, and day 10. RESULTS: Irisin concentrations were not associated with thyroid-axis hormones (i.e., TSH, free thyroxine, and free triiodiothyronine) cross-sectionally in either the overall cohort or in the euthyroid and/or subclinical hyperthyroid subgroups (p > 0.05). There was no significant difference between euthyroid and subclinical hyperthyroid subjects (p = 0.60). Levothyroxine withdrawal did not result in any changes in irisin concentrations (p = 0.33). Recombinant human TSH stimulation did not induce any significant changes in circulating irisin (p = 0.60). CONCLUSIONS: Changes in thyroid-axis hormone levels within the physiological or supraphysiological range do not affect circulating irisin levels in humans. Therefore, their metabolic effects are most likely independent of each other. Other regulators of irisin levels should be identified in the future.

The novel agent phospho-glycerol-ibuprofen-amide (MDC-330) inhibits glioblastoma growth in mice: an effect mediated by cyclin D1.

Given that glioblastoma multiforme (GBM) is associated with poor prognosis, new agents are urgently needed. We developed phospho-glycerol-ibuprofen-amide (PGIA), a novel ibuprofen derivative, and evaluated its safety and efficacy in preclinical models of GBM, and its mechanism of action using human GBM cells and animal tumor models. Furthermore, we explored whether formulating PGIA in polymeric nanoparticles could enhance its levels in the brain. PGIA was 3.7- to 5.1-fold more potent than ibuprofen in suppressing the growth of human GBM cell lines. PGIA 0.75× IC50 inhibited cell proliferation by 91 and 87% in human LN-229 and U87-MG GBM cells, respectively, and induced strong G1/S arrest.In vivo, compared with control, PGIA reduced U118-MG and U87-MG xenograft growth by 77 and 56%, respectively (P< 0.05), and was >2-fold more efficacious than ibuprofen. Normal human astrocytes were resistant to PGIA, indicating selectivity. Mechanistically, PGIA reduced cyclin D1 levels in a time- and concentration-dependent manner in GBM cells and in xenografts. PGIA induced cyclin D1 degradation via the proteasome pathway and induced dephosphorylation of GSK3ß, which was required for cyclin D1 turnover. Furthermore, cyclin D1 overexpression rescued GBM cells from the cell growth inhibition by PGIA. Moreover, the formulation of PGIA in poly-(L)-lactic acid poly(ethylene glycol) polymeric nanoparticles improved its pharmacokinetics in mice, delivering PGIA to the brain. PGIA displays strong efficacy against GBM, crosses the blood-brain barrier when properly formulated, reaching the target tissue, and establishes cyclin D1 as an important molecular target. Thus, PGIA merits further evaluation as a potential therapeutic option for GBM.

Phospho-ibuprofen (MDC-917) suppresses breast cancer growth: an effect controlled by the thioredoxin system.

INTRODUCTION: We have recently synthesized phospho-ibuprofen (P-I; MDC-917), a safer derivative of ibuprofen, which has shown anti-cancer activity. We investigated its efficacy and mechanism of action in the treatment of breast cancer in preclinical models. METHODS: We evaluated the anti-breast-cancer efficacy of P-I alone or incorporated into liposomes (Lipo-P-I) in human estrogen receptor-positive (MCF-7) and triple-negative, i.e., estrogen receptor-negative, progesterone receptor-negative and HER2-negative (MDA-MB231) breast cancer cell lines - as they represent the most frequent (estrogen receptor-positive) and the most difficult-to-treat (triple-negative) subtypes of breast cancer - and their xenografts in nude mice. We assessed the effect of P-I on the levels of reactive oxygen nitrogen species in response to P-I using molecular probes, on the thioredoxin system (expression and redox status of thioredoxin-1 (Trx-1) and thioredoxin reductase activity), on cyclooxygenase 2, NF-κB and mitogen-activated protein kinase cell signaling; and on the growth of xenografts with stably knocked-down Trx-1. RESULTS: Compared with controls, P-I 400 mg/kg/day inhibited the growth of MDA-MB231 xenografts by 266%, while the growth of MCF-7 xenografts was inhibited 51% byP-I 300 mg/kg/day and 181% by Lipo-P-I 300 mg/kg/day. In both cell lines, P-I induced oxidative stress and suppressed the thioredoxin system (oxidized Trx-1 and decreased its expression; inhibited thioredoxin reductase activity). These changes triggered downstream redox signaling: the activity of NF-κB was suppressed and the Trx-1-ASK1 complex was dissociated, activating the p38 and JNK mitogen-activated protein kinase cascades. Trx-1 knockdown abrogated the anti-cancer effect of P-I in vitro and in vivo. CONCLUSION: P-I is safe and effective against breast cancer. Liposomal formulation enhances its efficacy; the effect is heavily dependent on the induction of oxidative stress and the suppression of the thioredoxin system. P-I merits further evaluation as an agent for the treatment of breast cancer.

Chemotherapeutic properties of phospho-nonsteroidal anti-inflammatory drugs, a new class of anticancer compounds.

Nonsteroidal anti-inflammatory drugs (NSAID) exhibit antineoplastic properties, but conventional NSAIDs do not fully meet safety and efficacy criteria for use as anticancer agents. In this study, we evaluated the chemotherapeutic efficacy of 5 novel phospho-NSAIDs, each of which includes in addition to the NSAID moiety a diethylphosphate linked through a butane moiety. All 5 compounds inhibited the growth of human breast, colon, and pancreatic cancer cell lines with micromolar potency. In vivo investigations confirmed the antitumor activity of phospho-aspirin (PA) and phospho-sulindac (PS) in inhibiting tumor growth in established human xenograft models, in which cell proliferation was suppressed and apoptosis enhanced in the absence of detectable animal toxicity. Notably, all of the phospho-NSAIDs tested induced reactive oxygen and nitrogen species in cultured cells, with PA and PS inducing detectable levels of oxidative stress in vivo that were associated positively with apoptosis and negatively with proliferation. Potentially explaining these effects, all of the phospho-NSAIDs tested also inhibited the thioredoxin system and the redox sensitive transcription factor NF-κB. Taken together, our findings show the strong anticancer efficacy and promising safety of phospho-NSAIDs in preclinical models of breast, colon, and pancreatic cancer, suggesting further evaluation as anticancer agents.

Phospho-sulindac (OXT-328) combined with difluoromethylornithine prevents colon cancer in mice.

The nonsteroidal anti-inflammatory drug (NSAID) sulindac and the ornithine decarboxylase (ODC) antagonist difluoromethylornithine (DFMO), individually and together, are effective inhibitors of colon carcinogenesis. However, chronic use of sulindac is associated with significant side effects. We evaluated the chemopreventive efficacy of phospho-sulindac (P-S, OXT-328), an apparently safe derivative of sulindac, together with DFMO, in HT-29 human colon cancer xenografts. Nude mice were divided into four groups as follows: group 1 received vehicle (corn oil); group 2 received P-S (100 mg/kg/d) by oral gavage; group 3 received DFMO (2% in drinking water); and group 4 received P-S (100 mg/kg/d) by gavage plus DFMO (2% in drinking water; P-S/DFMO). Eighteen days after implantation, compared with controls, tumor volume was inhibited 65.9% by P-S, 52.9% by DFMO, and 70.9% by P-S/DFMO (P < 0.01 for all). P-S/DFMO reduced cell proliferation 27.1% and increased apoptosis 38.9% compared with controls (P < 0.05 for both). Compared with controls, P-S reduced the levels of thioredoxin-1 (Trx-1) and thioredoxin reductase (TrxR), whereas DFMO reduced polyamine content (putrescine and spermidine) and TrxR levels. Importantly, P-S/DFMO decreased putrescine and spermidine levels and the expression of Trx-1, TrxR, and cyclooxygenase (COX) 2. Of these molecular targets, TrxR most consistently correlated with tumor growth. Study results show that P-S/DFMO is an efficacious drug combination for colon cancer prevention and also show the safety of P-S, which may overcome the limiting side effects of conventional sulindac. P-S/DFMO has an intricate mechanism of action extending beyond polyamines and including the thioredoxin system, an emerging regulator of chemoprevention. P-S/DFMO merits further evaluation.

Phospho-ibuprofen (MDC-917) is a novel agent against colon cancer: efficacy, metabolism, and pharmacokinetics in mouse models.

We have developed a novel chemical modification of conventional nonsteroidal anti-inflammatory drugs to reduce their toxicity and enhance their efficacy. Phospho-ibuprofen [(PI) 2-(4-isobutyl-phenyl)-propionic acid-4-(diethoxy-phosphoryloxy)-butyl ester (MDC-917)], a novel derivative of ibuprofen, strongly inhibited the growth of human colon cancer cells in vitro and SW480 human colon cancer xenografts in nude mice. PI was metabolized minimally by cultured cells, but extensively by liver microsomes and mice, undergoing regioselective oxidation to produce 1-OH-PI and carboxyl-PI, which can be hydrolyzed to 1-OH-ibuprofen and carboxyl-ibuprofen, respectively. PI also can be hydrolyzed to release ibuprofen, which can generate 2-OH-ibuprofen, carboxyl-ibuprofen, and ibuprofen glucuronide. After a single oral administration (400 mg/kg) of PI, ibuprofen and ibuprofen glucuronide are the main plasma metabolites of PI; they have, respectively, C(max) of 530 and 215 µM, T(max) of 1 and 2 h, elimination t(1/2) of 7.7 and 5.3 h, and area under the concentration-time curve (0-24 h) of 1816 and 832 µM × h. Intact PI was detected in several tissues but not in plasma; at a higher PI dose (1200 mg/kg), PI plasma levels were 12.4 µM. PI generated the same metabolites in mouse plasma as conventional ibuprofen, but with much lower levels, perhaps accounting for the enhanced safety of PI. The antitumor effect of PI was significantly associated with plasma ibuprofen levels (p = 0.016) but not with xenograft ibuprofen levels (p = 0.08), suggesting a complex anticancer effect. These results provide a pharmacological basis to explain, at least in part, the anticancer efficacy and safety of this promising compound and indicate that PI merits further evaluation as an anticancer agent.

Phospho-sulindac (OXT-328), a novel sulindac derivative, is safe and effective in colon cancer prevention in mice.

BACKGROUND & AIMS: Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective cancer chemopreventive agents. However, chronic administration of NSAIDs is associated with significant side effects, mainly of the gastrointestinal tract. Given these limitations, we synthesized phospho-sulindac (P-S; OXT-328), a novel sulindac derivative. METHODS: Here, we evaluated the safety and efficacy of P-S in preclinical models, including its mechanism of action with human colon cancer cell (HCCC) lines and animal tumor models. RESULTS: (1) Compared with sulindac, P-S is much more potent in inhibiting the growth of cultured HCCCs and more efficacious in preventing the growth of HT-29 xenografts in nude mice. P-S also prevents the growth of intestinal tumors in Apc/Min mice. (2) In combination with difluoromethylornithine (DFMO), P-S reduced tumor multiplicity in Apc/Min mice by 90%. (3) P-S is much safer than sulindac as evidenced by its in vitro toxicologic evaluation and animal toxicity studies. Mechanistically, P-S increases the intracellular levels of reactive oxygen and nitrogen species, which are key early mediators of its chemopreventive effect. Moreover, P-S induces spermidine/spermine N(1)-acetyltransferase enzymatic activity, and together with DFMO it reduces polyamine levels in vitro and in vivo. CONCLUSIONS: P-S displays considerable safety and efficacy, two pharmacologic properties that are essential for a potential cancer chemopreventive agent, and thus merits further evaluation.

Phospho-sulindac (OXT-922) inhibits the growth of human colon cancer cell lines: a redox/polyamine-dependent effect.

Non-steroidal anti-inflammatory drugs such as sulindac are promising chemoprevention agents against colon cancer, but their weak potency and side effects limit their use for both chemoprevention and chemotherapy. Here, we evaluated the effect of a new sulindac derivative, phospho-sulindac or OXT-922, on the growth of human cancer cell lines and its mechanism of action. OXT-922 inhibited the growth of human cancer cell lines originating from colon, pancreas and breast ~11- to 30-fold more potently than sulindac. This effect was mediated by a strong cytokinetic effect. Compared with control, OXT-922 inhibited cell proliferation by up to 67%, induced apoptosis 4.1-fold over control and blocked the G(1) to S cell cycle phase transition. OXT-922 suppressed the levels of cell cycle regulating proteins, including cyclins D(1) and D(3) and Cyclin-dependent kinases (CDK) 4 and 6. The levels of intracellular reactive oxygen species (ROS), especially those of mitochondrial O2ⁱ⁻, were markedly elevated (5.5-fold) in response to OXT-922. ROS collapsed the mitochondrial membrane potential and triggered apoptosis, which was largely abrogated by antioxidants. OXT-922 suppressed nuclear factor-kappaB activation and downregulated thioredoxin-1 expression. It also suppressed the production of prostaglandin E(2) and decreased cyclooxygenase-1 expression. Similar to sulindac, OXT-922 enhanced spermidine/spermine N(1)-acetyltransferase activity, reduced the cellular polyamine content and synergized with difluoromethylornithine to inhibit cancer cell proliferation and induce apoptosis. Our results suggest that OXT-922 possesses promising anticancer properties and deserves further evaluation.

Induction of colon tumorigenesis by glutathione depletion in p53-knock-out mice.

In order to examine the role of glutathione (GSH), a key cellular antioxidant, on spontaneous tumor development, we tested the effects of buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, and 1,2-oxothiazolidine-4-carboxylic acid (OTCA), a cysteine and GSH precursor, on tumor incidence and spectrum in p53 nullizygous (p53-/-) transgenic mice. Mice were randomly assigned to three groups: control (no treatment), BSO (20 mM in drinking water) or OTCA (6 g/kg in the diet) (n=30 per group). After 10 weeks, GSH levels were decreased 29-88% in all tissues except liver and brain in BSO-treated mice, while no changes were observed in most tissues from OTCA-treated animals. Mice in all groups showed similar survival patterns as well as incidence of the most commonly observed tumors: i.e., lymphomas (80%) and other tumors (38%). However, a 5-fold increase in incidence of colonic tumors (from 4-20%) was observed in the BSO-treated group, suggesting that GSH deficiency and loss of p53 function play contributory roles in colon carcinogenesis.

Enhanced levels of glutathione and protein glutathiolation in rat tongue epithelium during 4-NQO-induced carcinogenesis.

High glutathione (GSH) levels are commonly found in oral tumors and are thought to play an important role in tumorigenesis. While posttranslational binding of GSH to cellular proteins (protein glutathiolation) has recently been recognized as an important redox-sensitive regulatory mechanism, no data currently exist on this process during carcinogenesis. Our goal was to determine the effects of 4-nitroquinoline-N-oxide (4-NQO)-induced carcinogenesis on tongue levels of protein-bound and free GSH and related thiols in the rat. Male F-344 rats (6 weeks of age) were administered either 4-NQO (20 ppm) in drinking water or tap water alone (controls) for 8 weeks. Twenty-four weeks after cessation of 4-NQO, squamous cell carcinomas of the tongue were observed in all rats. The levels of both free and bound GSH in tumors, as well as in adjacent tissues, were 2- to 3-fold greater than in tongue epithelium from control rats (p < 0.05). Prior to tumor formation, at 8 weeks after cessation of 4-NQO, hyperplasia, dysplasia and carcinoma in situ were observed in 100%, 25% and 12.5% of 4-NQO-treated rats, respectively. At this early stage of carcinogenesis, levels of free and bound GSH were increased 50% compared with tongue tissues from control rats (p<0.05). Glutathione disulfide (GSSG) levels were also 2-fold greater in tongue tissues from 4-NQO treated vs. control rats (p<0.05). Altogether, these results suggest that protein glutathiolation, together with GSH and GSSG levels, are induced during oral carcinogenesis in the rat possibly as a result of enhanced levels of oxidative stress.

Methionine restriction inhibits colon carcinogenesis.

Previously, we demonstrated that life-long methionine restriction (MR) in rats increases life span and inhibits aging-related disease processes. The present study examines the effects of MR on the formation of preneoplastic aberrant crypt foci (ACF) in the colon of azoxymethane (AOM)-treated rats. Six-week-old male F344 rats were placed on essential amino acid-defined diets containing either 0.86% Met (control diet) or 0.17% Met (MR diet) and 1 wk later were given AOM (15 mg/kg/wk, s.c.) for 2 consecutive wk. Ten weeks after the final AOM treatment, ACF formation was markedly reduced in rats fed the MR diet with ACF containing > or = 4 crypts/focus being reduced by over 80% compared to controls (P < 0.001). A similar 83% reduction in ACF containing > or = 4 crypts/focus was observed in rats fed the MR diet only during the post-initiation period (after the final dose of AOM; P < 0.001). Five weeks after AOM administration, a 12% reduction in colonic cell proliferation was observed in MR rats compared to controls (P < 0.05). These results show that MR inhibits colonic tumor development in the rat, an effect that occurs primarily during post-initiation phases of carcinogenesis and may be due, in part, to an inhibition of colonic cell proliferation.

Glutathione depletion enhances the formation of endogenous cyclic DNA adducts derived from t-4-hydroxy-2-nonenal in rat liver.

Earlier, we detected the cyclic adducts of deoxyguanosine (dG) derived from t-4-hydroxy-2-nonenal (HNE), a long chain alpha,beta-unsaturated aldehyde (enal) product from oxidation of omega-6 polyunsaturated fatty acids, in tissue DNA of rats and humans as endogenous DNA damage. Recent evidence implicates the cyclic HNE adducts in human liver carcinogenesis. Because glutathione (GSH) protects against oxidative stress, we undertook a study to examine the effect of GSH depletion on the HNE-derived cyclic adducts in vivo. Four F344 rats were administered L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at 10 mM in drinking water for 2 weeks. Rats in the control group were given water only. Livers were harvested, and each liver was divided into portions for GSH and DNA adduct analyses. The BSO treatment depleted hepatic GSH by 77%; the GSH levels were reduced from 6.3 +/- 0.3 in the control rats to 1.5 +/- 0.1 micromol/g tissues in the treated group. The formation of HNE-dG adducts, analyzed by an HPLC-based 32P-postlabeling assay, was increased by 4-fold, from 6.2 +/- 2.2 nmol/mol dG in liver DNA of control rats to 28.5 +/- 16.1 nmol/mol dG in the rats treated with BSO (p <0.05). The formation of 8-oxodG in liver DNA was also increased as a result of BSO treatment, although the increase was not statistically significant. These results further support the endogenous origin of HNE-dG adducts and, more importantly, indicate a critical role that GSH plays in protecting against in vivo formation of the promutagenic cyclic DNA adducts derived from HNE.

Tissue glutathione and cysteine levels in methionine-restricted rats.

OBJECTIVE: Previously, we demonstrated that lifelong methionine (Met) restriction (MR) increases lifespan, decreases the incidence of aging-related diseases, increases blood glutathione (GSH) levels, and prevents loss of GSH during aging in rats. Our present objective was to elucidate the effects of MR on GSH metabolism and transport by determining the time course and nature of GSH and cysteine changes in blood and other tissues in young and mature rats. METHODS: Male F-344 rats were placed on control (0.86% Met) or MR (0.17% Met) defined amino acid diets at age 7 wk and killed at different times thereafter. MR was also initiated in adult (12-mo-old) rats. RESULTS: Throughout the first 2 mo of MR, blood GSH levels increased 84% and liver GSH decreased 66% in relation to controls. After this period, liver GSH levels remained constant through at least 6 mo. GSH levels also decreased in the pancreas (80%) and kidney (22%) but remained unchanged in other tissues examined after 11 wk of MR. The increase in blood GSH was evident as soon as 1 wk after initiating MR and reached a plateau by 6 wk. A similar increase in erythrocyte GSH levels was observed when MR was administered to mature adult rats. Fasting decreased liver GSH in controls but had no further effect in MR animals. By 1 mo, cysteine levels had decreased in all tissues except brain. CONCLUSION: These results suggest that adaptive changes occur in the metabolism of Met, cysteine, and/or GSH as a result of MR in young and adult rats. These early metabolic changes lead to conservation of GSH levels in most extrahepatic tissues and increased GSH in erythrocytes by depleting liver GSH to a critical level.

Insulin resistance and its contribution to colon carcinogenesis.

The insulin resistance-colon cancer hypothesis, stating that insulin resistance may be associated with the development of colorectal cancer, represents a significant advance in colon cancer, as it emphasizes the potential for this cancer to become a modifiable disease. The fact that the incidence of insulin resistance has been increasing in the United States and much of the rest of the Western world where colon cancer remains the second leading cause of cancer death makes the exploration of the interrelationship of these conditions a subject of high priority. Here, we review the salient features of insulin resistance, defined as impaired biological response to the action of insulin. Recent epidemiological studies, evaluating potential associations between colon cancer risk and diabetes mellitus, dietary intake and metabolic factors, and IGF levels in several clinical settings, provide strong support of the insulin resistance-colon cancer hypothesis (without establishing causality). Mechanistically, insulin resistance has been associated with hyperinsulinemia, increased levels of growth factors including IGF-1, and alterations in NF-kappaB and peroxisome proliferator-activated receptor signaling, which may promote colon cancer through their effects on colonocyte kinetics. It is a reasonable expectation that in the not too distant future, critical interventions to the already mapped molecular sequence of events, which link two apparently disparate entities, combined with lifestyle changes could abrogate the development of colon cancer.

Protein glutathiolation in human blood.

Glutathione (GSH) exists in both free and protein-bound (glutathiolated) forms (GSSP). Protein glutathiolation may represent an important post-translational regulatory mechanism for proteins. However, there are little data regarding the regulation of glutathiolation in blood. Our objectives were to examine GSSP levels of human blood by determining the distribution and variability of blood GSSP, as well as its relationship to free GSH and hemoglobin in healthy adults. To this end, we used a newly modified method allowing for rapid analysis of both GSH and GSSP in blood. GSSP was found in red cells with levels ranging from 4 to 27% of total (free+bound) GSH (mean+/-SD: 12.1+/-4.5%) with a concentration of 0.13+/-0.05 microEq GSH/mL (mean+/-SD). No correlations were observed between GSSP and either GSH (r=-0.085) or hemoglobin (r=0.086). Together these results suggest that the extent of protein glutathiolation in blood is substantial ( approximately 0.1 mmol/L). While the interindividual variation in GSSP is large (34%), its levels are apparently not regulated by GSH content.