Chronic stress decreases ornithine decarboxylase expression and protects against 1,2-dimethylhydrazine-induced colon carcinogenesis.

Biological response to stress depends on the type, timing, and severity of the stressor. Acute stressful environments may positively activate molecular and cellular mechanisms to favor adaptation; however, chronic stress is often associated with detrimental health effects. Colon cancer (CC) is one of the leading causes of death associated with cancer and has been mentioned as a stress-related disease. In the present work, the effect of chronic stress on the initial phase of CC was evaluated, and special emphasis was placed on ornithine decarboxylase (ODC) expression and polyamines for their role in hyperproliferative diseases. BALB/c mice (n = 5/group) were administered the pro-carcinogen 1,2-dimethylhydrazine (DMH) for 8 weeks (20 mg/kg body weight/week) to induce colon carcinogenesis, and then exposed for 4 weeks to two physical stressors: restraint and forced-swimming. Distal colon inflammatory lesions and histomorphological changes were evaluated by hematoxylin-eosin staining; plasma corticosterone levels, colon ODC expression, and urinary polyamines were determined by competitive ELISA, RT-qPCR, Western Blot, and HPLC, respectively. The short-term exposure to DMH triggered colon inflammation, initiated colon carcinogenesis and increased ODC expression; meanwhile, the exposure to chronic stress activated the hypothalamic-pituitary-adrenal (HPA) axis, elicited the production of plasmatic corticosterone, and decreased ODC expression. The exposure of DMH-treated mice to chronic stress counteracted the inflammatory effect of DMH and maintained ODC homeostasis. In early phase of carcinogenesis, the exposure of DMH-treated mice to chronic stress had a positive effect against colon inflammation and maintained ODC homeostasis. The cross-talk between corticosterone, ODC expression, and inflammation in a tumor environment is discussed.

Comparing metabolite profiles of habitual diet in serum and urine.

BACKGROUND: Diet plays an important role in chronic disease etiology, but some diet-disease associations remain inconclusive because of methodologic limitations in dietary assessment. Metabolomics is a novel method for identifying objective dietary biomarkers, although it is unclear what dietary information is captured from metabolites found in serum compared with urine. OBJECTIVE: We compared metabolite profiles of habitual diet measured from serum with those measured from urine. DESIGN: We first estimated correlations between consumption of 56 foods, beverages, and supplements assessed by a food-frequency questionnaire, with 676 serum and 848 urine metabolites identified by untargeted liquid chromatography mass spectrometry, ultra-high performance liquid chromatography tandem mass spectrometry, and gas chromatography mass spectrometry in a colon adenoma case-control study (n = 125 cases and 128 controls) while adjusting for age, sex, smoking, fasting, case-control status, body mass index, physical activity, education, and caloric intake. We controlled for multiple comparisons with the use of a false discovery rate of <0.1. Next, we created serum and urine multiple-metabolite models to predict food intake with the use of 10-fold crossvalidation least absolute shrinkage and selection operator regression for 80% of the data; predicted values were created in the remaining 20%. Finally, we compared predicted values with estimates obtained from self-reported intake for metabolites measured in serum and urine. RESULTS: We identified metabolites associated with 46 of 56 dietary items; 417 urine and 105 serum metabolites were correlated with ≥1 food, beverage, or supplement. More metabolites in urine (n = 154) than in serum (n = 39) were associated uniquely with one food. We found previously unreported metabolite associations with leafy green vegetables, sugar-sweetened beverages, citrus, added sugar, red meat, shellfish, desserts, and wine. Prediction of dietary intake from multiple-metabolite profiles was similar between biofluids. CONCLUSIONS: Candidate metabolite biomarkers of habitual diet are identifiable in both serum and urine. Urine samples offer a valid alternative or complement to serum for metabolite biomarkers of diet in large-scale clinical or epidemiologic studies.

Genetic polymorphisms of dihydropyrimidinase in a Japanese patient with capecitabine-induced toxicity.

Dihydropyrimidinase (DHP) is the second enzyme in the catabolic pathway of uracil, thymine, and chemotherapeutic fluoropyrimidine agents such as 5-fluorouracil (5-FU). Thus, DHP deficiency might be associated with 5-FU toxicity during fluoropyrimidine chemotherapy. We performed genetic analyses of the family of a patient with advanced colon cancer who underwent radical colectomy followed by treatment with 5-FU prodrug capecitabine and developed severe toxicity attributable to a lack of DHP. We measured urinary uracil and dihydrouracil, and genotyped DPYS in the patient and her family. We also measured the allele frequency of DPYS polymorphisms in 391 unrelated Japanese subjects. The patient had compound heterozygous missense and nonsense polymorphisms comprising c.1001A>G (p.Gln334Arg) in exon 6 and c.1393C>T (p.Arg465Ter) in exon 8, which are known to result in a DHP enzyme with little or no activity. The urinary dihydrouracil/uracil ratio in the patient was 17.08, while the mean ± SD urinary dihydrouracil/uracil ratio in family members who were heterozygous or homozygous for wild-type DPYS was 0.25 ± 0.06. In unrelated subjects, 8 of 391 individuals were heterozygous for the c.1001A>G mutation, while the c.1393C>T mutation was not identified. This is the first report of a DHP-deficient patient with DPYS compound heterozygous polymorphisms who was treated with a fluoropyrimidine, and our findings suggest that polymorphisms in the DPYS gene are pharmacogenomic markers associated with severe 5-FU toxicity in Japanese patients.

Sources of variability in metabolite measurements from urinary samples.

BACKGROUND: The application of metabolomics in epidemiological studies would potentially allow researchers to identify biomarkers associated with exposures and diseases. However, within-individual variability of metabolite levels caused by temporal variation of metabolites, together with technical variability introduced by laboratory procedures, may reduce the study power to detect such associations. We assessed the sources of variability of metabolites from urine samples and the implications for designing epidemiologic studies. METHODS: We measured 539 metabolites in urine samples from the Navy Colon Adenoma Study using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectroscopy (GC-MS). The study collected 2-3 samples per person from 17 male subjects (age 38-70) over 2-10 days. We estimated between-individual, within-individual, and technical variability and calculated expected study power with a specific focus on large case-control and nested case-control studies. RESULTS: Overall technical reliability was high (median intraclass correlation = 0.92), and for 72% of the metabolites, the majority of total variance can be attributed to between-individual variability. Age, gender and body mass index explained only a small proportion of the total metabolite variability. For a relative risk (comparing upper and lower quartiles of "usual" levels) of 1.5, we estimated that a study with 500, 1,000, and 5,000 individuals could detect 1.0%, 4.5% and 75% of the metabolite associations. CONCLUSIONS: The use of metabolomics in urine samples from epidemiological studies would require large sample sizes to detect associations with moderate effect sizes.

Urinary nucleosides as biomarkers of breast, colon, lung, and gastric cancer in Taiwanese.

Urinary nucleosides are associated with many types of cancer. In this study, six targeted urinary nucleosides, namely adenosine, cytidine, 3-methylcytidine, 1-methyladenosine, inosine, and 2-deoxyguanosine, were chosen to evaluate their role as biomarkers of four different types of cancer: lung cancer, gastric cancer, colon cancer, and breast cancer. Urine samples were purified using solid-phase extraction (SPE) and then analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The Mann-Whitney U test and Principal Component Analysis (PCA) were used to compare differences in urinary nucleosides between patients with one of four types of cancer and healthy controls. The diagnostic sensitivity of single nucleosides for different types of cancer ranged from 14% to 69%. In contrast, the diagnostic sensitivity of a set of six nucleosides ranged from 37% to 69%. The false-positive identification rate associated with the set of six nucleosides in urine was less than 2% compared with that of less than 5% for a single nucleoside. Furthermore, combining the set of six urinary nucleosides with carcinoembryonic antigen improved the diagnostic sensitivity for colon cancer. In summary, the study show that a set of six targeted nucleosides is a good diagnostic marker for breast and colon cancers but not for lung and gastric cancers.

Proteinuria predicts 10-year cancer-related mortality in patients with type 2 diabetes.

BACKGROUND: We conducted a cohort study to determine if proteinuria predicts cancer-related mortality in type 2 diabetic subjects. METHODS: Between July 1996 and June 2003, we enrolled 646 type 2 diabetic subjects. Participants were followed-up until December 31, 2008. The vital status was ascertained by linking records with computerized death certificates in Taiwan. RESULTS: During a median follow-up of 10.4 years, 158 subjects had died, including 59 from cancers. Subjects with proteinuria had a hazard ratio (HR) of 2.77 (95% CI 1.82-4.21) for all-cause mortality and 1.99 (95% CI 1.00-3.94) for cancer-related mortality after adjustment for demographic factors and medical conditions. Specifically, proteinuria showed a trend of increased colon cancer death. The presence of proteinuria significantly improved the predictive ability of cancer-related mortality (increase in concordance statistics or area under the ROC curve=0.03). Patients with both proteinuria and estimated glomerular filtration rate <60 ml/min per 1.73 m² showed higher HR for all-cause mortality than patients with proteinuria only (adjusted HRs (95% CI), 4.01 (2.42-6.67) vs. 2.69 (1.51-4.79), both p<0.01). CONCLUSIONS: Proteinuria can predict 10-year all-cause and cancer-related mortalities independently in type 2 diabetic subjects, over and above the established risk factors associated with type 2 diabetes.

Oxidative stress and 8-oxoguanine repair are enhanced in colon adenoma and carcinoma patients.

Oxidative stress is involved in the pathogenesis of colon cancer. We wanted to elucidate at which stage of the disease this phenomenon occurs. In the examined groups of patients with colorectal cancer (CRC, n = 89), benign adenoma (AD, n = 77) and healthy volunteers (controls, n = 99), we measured: vitamins A, C and E in blood plasma, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in leukocytes and urine, leukocyte 8-oxoGua excision activity, mRNA levels of APE1, OGG1, 8-oxo-7,8-dihydrodeoxyguanosine 5'-triphosphate pyrophosphohydrolase (MTH1) and OGG1 polymorphism. The vitamin levels decreased gradually in AD and CRC patients. 8-OxodG increased in leukocytes and urine of CRC and AD patients. 8-OxoGua was higher only in the urine of CRC patients. 8-OxoGua excision was higher in CRC patients than in controls, in spite of higher frequency of the OGG1 Cys326Cys genotype, encoding a glycosylase with decreased activity. mRNA levels of OGG1 and APE1 increased in CRC and AD patients, which could explain increased 8-oxoGua excision rate in CRC patients. MTH1 mRNA was also higher in CRC patients. The results suggest that oxidative stress occurs in CRC and AD individuals. This is accompanied by increased transcription of DNA repair genes, and increased 8-oxoGua excision rate in CRC patients, which is, however, insufficient to counteract the increased DNA damage.

ELISA measurement for urinary 3-hydroxyproline-containing peptides and its preliminary application to healthy persons and cancer patients.

BACKGROUND/AIM: We reported that endogenous urinary 3-hydroxyproline (3-Hyp) is useful for cancer screening because cancer invasion involves the destruction of basement membrane. A simple and sensitive assay is desired. PATIENTS AND METHODS: An ELISA method using a specific antibody against a synthetic peptide of 10 amino acids including 3-Hyp corresponding to the amino acid sequences of collagen type IV alpha chain was applied to urine samples from 180 healthy controls and 22 cancer patients. RESULTS: The values in controls were 2.44+/-1.90 (SD) mg peptide/g creatinine for 52 men and 2.87+/-2.01 for 128 women, while the values in 22 cancer patients were very low at 0.110+/-0.137 (p<0.001). DISCUSSION: The discrepancy in the data between our previous and present studies is based on the difference of targets measured. 3-Hyp-containing peptides in cancer patients might be destroyed by the elevated peptidase levels found in these patients. CONCLUSION: This ELISA assay may be useful for cancer screening.

No evidence of an association of JC virus and colon neoplasia.

JC virus (JCV) is an ubiquitous human polyomavirus that frequently resides in the kidneys of healthy individuals and is excreted in the urine of a large proportion of the adult population. Polyomaviruses are associated with disease largely in immunocompromised individuals (progressive multifocal leukoencephalopathy). Colorectal cancers can show chromosome instability and it was hypothesized that JCV may account for some of this instability. We screened urine from 45 healthy donors and 233 colorectal cancer/normal tissue pairs for the presence of JCV sequences using a Taqman assay. This assay could detect 1 virus genome in 10 human genomes. In the urine samples, we found an infection rate of approximately 70%. The JCV isolates in these samples could be categorized into four JCV types (2B, 4, 7, and 8), none of which had a rearranged regulatory region. Among the colon tissues, one normal tissue (<0.5%) and none of the matched tumors tested positive for JCV. There is no evidence in these data to indicate that JCV is the cause of genetic instability in colorectal cancer.

Clinical significance of serum and urinary c-erbB-2 levels in colorectal cancer.

In this study we measured serum and urinary c-erbB-2 levels in 63 patients with colorectal cancer and 29 healthy controls, assessing their role in cancer-specific survival and the effects of resectional surgery. Serum and urinary c-erbB-2 levels were measured by an enzyme-linked immunosorbent assay, preoperatively and 7 days following tumor resection. Preoperative serum c-erbB-2 concentrations were significantly higher in the cancer patients and correlated with disease stage and the presence of liver metastases. Urinary c-erbB-2 was detected more often in cancer patients, although levels did not differ from controls and there was no association with any clinicopathological variable. Serum c-erbB-2 levels decreased significantly in those patients resected for cure and were an independent prognostic factor for cancer-specific survival with higher preoperative concentrations correlating with worse overall survival. These findings suggest that serum assessment of c-erbB-2 concentration may be valuable in defining colorectal cancer prognosis.

Phenotype of cytochrome P450 CYP2D6 in patients with familial adenomatous polyposis.

This paper describes an attempt to establish the distribution of the oxidative phenotype of sparteine in patients with familial adenomatous polyposis (FAP). The oxidative polymorphism of sparteine was determined in 30 patients with FAP. One hundred and twenty-six normal subjects were examined as a control group. Subjects with urinary metabolic ratios (MR) greater than 20 (the metabolic ratio of sparteine/dehydrosparteines excreted in urine) were defined as poor metabolizers of sparteine. None of the patients were classified as poor metabolizers of sparteine, although 5 control subjects were. No significant differences were found in the distribution of frequencies between patients and control subjects. However, there was a higher metabolic ratio (mean 1.58 +/- 1.13) in 5 patients with malignant changes in large bowel adenomas compared with other FAP patients without malignant changes (mean MR 0.89 +/- 0.66).

Detection of mutant k-ras sequences in the urine of cancer patients.

DNA fragments from apoptotic cells crossing the renal barrier retain their matrix functions, which allows PCR identification of mutant sequences in excreted DNA. We investigated the possibility of detecting k-ras mutations in urinary DNA of tumor patients (colon cancer). In some patients with k-ras codon 12 mutations in tumor cell DNA the same changes were detected in the urinary DNA. The possibility of using this approach for early diagnosis and monitoring of tumors is discussed.

The effect of soy isoflavones on the development of intestinal neoplasia in ApcMin mouse.

Data from epidemiological studies suggest that isoflavones in soy may have a protective effect on the development of colon cancer in humans. Therefore, we have investigated whether soy isoflavones will inhibit intestinal tumour development in Apc(Min) mice. The mice were fed a Western-type high risk diet (high fat, low fibre and calcium) containing two different isolates of soy protein as a protein source. For the control and test groups this resulted in the administration of about 16 and 475 mg of total isoflavones per kg diet, respectively. As a positive control. a third group of mice was administered a low isoflavone diet supplemented with 300 ppm sulindac. No significant differences in the incidence, multiplicity, size and distribution of intestinal tumours were observed between Min mice fed low and high isoflavone-containing diets. However, a clear reduction in the number of small intestinal tumours was observed for the sulindac diet. Thus, in contrast to epidemiological studies, our results demonstrate that high amounts of soy isoflavones present in a Western-type high risk diet do not protect against intestinal tumour development in a relevant animal model such as the Min mice.

A diet containing chickpeas and wheat offers less protection against colon tumors than a casein and wheat diet in dimethylhydrazine-treated rats.

We examined the influence of extruded chickpeas and wheat relative to casein and wheat in a dimethylhydrazine (DMH)-induced colon tumor study in male Sprague-Dawley rats. The three diets, based on a modified AIN76 rodent diet with fat present at 10 g/100 g dry matter (DM), were as follows: casein with wheat starch (Cas/S) as control, casein with wheat (Cas/W) and chickpeas with wheat (CP/W). All diets were fed from 5 wk of age throughout the 28-wk study. At 28 wk, there was a significantly lower incidence of large intestinal tumors in rats fed Cas/W relative to those fed CP/W ( 11 vs. 56%, chi-square test, P = 0.018). The colonic tumor burden (tumors/tumor-bearing animal) was not different in Cas/W-fed and CP/W-fed rats (1 vs. 1.7), but the tumor mass index was significantly lower in the former group (0.22 vs. 1.21, P = 0.026). Rats fed the CP/W diet had significantly lower plasma cholesterol concentration (P < 0.01) than rats fed the other two diets. The cecal contents of rats fed the CP/W diet had significantly greater relative weights (46%, P < 0.05) than those of the Cas/W-fed rats; this was associated with higher concentrations of all short-chain fatty acids. Fecal analyses showed significantly (P < 0.05) higher concentrations of total fat (54%), total steroids (83%) and secondary bile acids (179%) in the CP/W-fed rats relative those fed Cas/W. There were higher concentrations of nitrogen in the feces of CP/W rats relative to the Cas/W-fed rats (84%, P < 0.05), associated with greater fecal weights (67%, P < 0.05). Although wheat and its fibers have been shown to be protective against DMH-induced cancers in rats, this was not the case in this study in which chickpeas (45 g/100 g diet) provided the protein and were an important source of soluble fiber. Elevated fat, secondary bile acid concentrations and/or nitrogenous compounds could be responsible for the increased colon tumorigenesis seen and may reflect a legume effect.

Urinary excretion of modified nucleosides as biological marker of RNA turnover in patients with cancer and AIDS.

Using boronate gel affinity chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), a method for the simultaneous determination of 12 urinary modified nucleosides has been developed. The RP-HPLC fractions were identified by gas chromatography/mass spectrometry analysis. The HPLC quantitation of urinary nucleoside levels before and after surgery of cancer patients suggested that urinary 5'-deoxy-5'-methylthioadenosine and N-[(9-beta-D-ribofuranosyl-9H-purine-6-yl) carbamoyl]-L-threonine (t6A) levels were helpful in monitoring therapeutic effects in cancer patients. From the fact that molar ratios of urinary N2,N2-dimethylguanosine (m2 2G)pseudouridine (psi) and t6A/psi in cancer patients were lower than those of normal or post-surgical cancer patients, the increase of rRNA content in cancer tissues growing rapidly was estimated using the stoichiometric relationship between the ratio of the number of residues of their modified nucleoside in RNAs and the proportion of rRNA to total RNAs in average tissues of whole body. Furthermore, from the estimation of RNA turnover using urinary nucleoside levels, it was found that the half-lives of rRNA rather than tRNA of patients with cancer and those of both RNAs in the case of acquired immunodeficiency syndrome (AIDS) were extremely short compared with those of the normal. Thus, we discovered that the selected urinary modified nucleosides were very useful as a biological marker of whole-body RNA turnover in patients with cancer and AIDS.

Serum cathepsin B levels and urinary excretion of cathepsin B in the cancer patients with remote metastasis.

Serum cathepsin B levels and urinary excretion of cathepsin B in the cancer patients without remote metastasis were significantly higher than those in the control non-cancer patients. Moreover, these parameters in the cancer patients with remote (liver or lung) metastasis were significantly higher than those in the cancer patients without remote metastasis. After radical curative operations, these parameters were restored to the control values. These results suggest a possible role of lysosomal enzyme, cathepsin B in the pathogenesis of tumor metastasis, and also suggest that these parameters might be possible indicators for tumor malignancy such as remote metastasis.

Compartmental analysis of the pharmacokinetics of radioiodinated monoclonal antibody B72.3 in colon cancer patients.

Sixteen patients with colorectal cancer were administered 37-74 MBq (1 mg) of radioiodinated B72.3 monoclonal antibody. Pharmacokinetic analysis was carried out on plasma and urine samples. Elimination from the plasma was biexponential with a mean T1/2 alpha of 3.7 h and T1/2 beta of 62.4 h. The plasma clearance was fit to a two-compartmental model. This was combined with a previously reported model for radioiodine to construct a composite model. There was a good correlation (r = 0.952) between the model-predicted and observed excretion of radioiodine suggesting that the composite model is compatible with the pharmacokinetics of the radiolabelled antibody.

Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine.

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 micrograms/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.