The role of serum uric acid as an antioxidant protecting against cancer: prospective study in more than 28 000 older Austrian women.

BACKGROUND: It has been hypothesized that serum uric acid (SUA), via its antioxidant properties may protect against carcinogenesis. However, few epidemiological investigations have addressed this association and previous findings are inconsistent. PATIENTS AND METHODS: We prospectively investigated the relation of SUA levels to subsequent cancer mortality in a large cohort of 28613 elderly Austrian women with a median follow-up of 15.2 years. Adjusted Cox proportional hazards models were calculated to evaluate SUA as an independently related factor to fatal cancer events. RESULTS: High SUA (>5.41 mg/dL) was independently associated with increased risk of total cancer mortality (p<0.0001); the adjusted hazard ratio for the highest versus lowest quartile of SUA was 1.27 (1.08-1.48). SUA levels were further positively related to deaths from malignant neoplasms of breast and female genital organs (P = 0.02) and nervous system and unspecified sites (P = 0.02). We found no evidence for an inverse relationship between SUA levels and risk of total or site-specific cancer mortality. CONCLUSION: Our results are contrary to the proposed antioxidant and protective effect of SUA against cancer and rather suggest high SUA concentrations to be associated with outcome possibly reflecting more serious prognostic indication.     

Evaluation of Azadirachta indica leaf fractions for in vitro antioxidant potential and in vivo modulation of biomarkers of chemoprevention in the hamster buccal pouch carcinogenesis model

We evaluated the chemopreventive potential of Azadirachta indica (neem) leaf fractions based on in vitro antioxidant assays, and in vivo inhibitory effects on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. In addition we also identified the major constituents in neem leaf fractions by HPLC. Analysis of the free radical scavenging activities and reducing potential of crude ethanolic extract (CEE), ethyl acetate fraction (EAF) and methanolic fraction (MF) of neem leaf revealed a concentration-dependent increase in antioxidant potential that was in the order EAF>MF>CEE. Administration of neem leaf fractions reduced the incidence of DMBA-induced HBP carcinomas at a lower concentration compared to the crude extract. Chemoprevention by neem leaf fractions was associated with modulation of phase I and phase II xenobiotic-metabolising enzymes, lipid and protein oxidation, upregulation of antioxidant defences, inhibition of cell proliferation and angiogenesis, and induction of apoptosis. However, EAF was more effective than MF in terms of antiproliferative and antiangiogenic effects, and expression of CYP isoforms. The greater efficacy of EAF may be due to higher content of constituent phytochemicals as revealed by HPLC analysis. The results of the present study suggest that the antioxidant properties of neem leaf fractions may be responsible for modulating key hallmark capabilities of cancer cells such as cell proliferation, angiogenesis and apoptosis in the HBP carcinogenesis model.     

Quantitative detection of molecular markers ProEx C (minichromosome maintenance protein 2 and topoisomerase IIa) and MIB‐1 in liquid‐based cervical squamous cell cytology

BACKGROUND.

In this study, the authors conducted a comparative quantitative evaluation of the proliferation markers ProEx C (an aberrant S‐phase induction marker, human papillomavirus E6‐E7 correlated) and MIB‐1 in squamous intraepithelial lesions (SIL) to identify a biomolecular profile informative for the diagnosis of high‐grade SIL/cervical intraepithelial neoplasia 3 or greater that was complementary to the morphologic Papanicolaou (Pap) test (“biomolecular Pap test”).

METHODS.

After the cytologic diagnosis, reflex immunocytochemistry was carried out on 76 unstained SurePath cell samples (20 routine samples that were negative for intraepithelial lesion or malignancy and 56 positive samples that were selected with matching histology). Both a morphometric analysis with a software imaging analysis system and a quantitative analysis of atypical squamous clusters were performed.

RESULTS.

The quantitative evaluation revealed an excellent, direct correlation between the 2 markers, although ProEx C was more selective and more informative for the progression of low‐ and moderate‐grade lesions, because it only revealed cells in aberrant S‐phase cell cycle. The quantitative morphometric analysis revealed the increased presence of atypical, positive clusters and the percentage of positive cells within, both paralleling the severity of the lesions. The threshold of a 3% ProEx C‐positive nuclear area was useful for splitting lesions into groups with a low risk or high risk of progression.

CONCLUSIONS.

Both ProEx C and MIB‐1 were valid proliferation markers in cytologic preparations, and nuclear positivity was quantified successfully by using computer‐assisted analysis. The analysis of atypical clusters may be a valuable tool in the diagnosis of SIL. The presence of atypical clusters and their positivity for proliferation markers are good first‐glance indicators of lesion grade. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Effect of Ethanol on Uterotropic Action of Estrogens

Female rats aging 3 months at the beginning of experiments received 5 or 15% ethanol and then were subjected to bilateral ovariectomy 2 weeks before end of the experiment. During the last 11 days they were daily injected intramuscularly with 2 g estradiol. Drinking of 5% ethanol combined with injections of estrogens induced DNA damage in the uterus detected by comet assay and abolished induction of progesterone receptors, changes in peroxidase activity, proliferation index, endometrium thickness, and other indices reflecting the hormonal effect of estradiol on the uterus. Drinking of 15% ethanol was accompanied by an increase in DNA-damaging effects of estrogens and a decrease in their hormonal uterotropic effects. It is concluded that unlike tobacco smoking, drinking of moderate ethanol concentrations modifies primarily genotoxic, but not the hormonal effect of estrogens.     

Induction of Extrahepatic Biliary Carcinoma by N-Nitrosobis(2-oxopropyl)amine in Hamsters Given Cholecystoduodenostomy with Dissection of the Common Duct

The methods we used to produce a carcinoma in the extrahepatic bile duct and gallbladder in hamsters are described along with the characteristics of the induced tumors. Female Syrian golden hamsters were first subjected to Cholecystoduodenostomy with dissection of the extrahepatic bile duct on the distal end of the common duct (CDDB) and were, 4 weeks later, treated with weekly subcutaneous injections of N-nitrosobis(2-oxopropyl)amine (BOP) at a dose of 10 mg/kg body weight for 9 weeks. The animals were killed at the 12th, 16th and 20th week after the initiation of BOP treatment. Extrahepatic bile duct carcinoma developed in 16%, 24% and 41% and gallbladder carcinoma occurred in 58%, 81% and 82% of the hamsters, respectively, at the corresponding times of killing. The incidences were significantly higher than those in sham-operated controls ( P <0.01). The induced extrahepatic bile duct carcinomas were predominantly of the polypoid type and gallbladder carcinomas were of the papillary type in growth form, being morphologically similar to early stage biliary carcinoma in humans. Immunohistochemical staining using bromodeoxynridine and anti-bromo-deoxyuridine monoclonal antibody demonstrated that the CDDB procedure greatly accelerated the cell kinetic activity of the biliary epithelium, and this was considered to be a major factor promoting the development of biliary carcinomas in this hamster model. In conclusion, this new model provides a high incidence of tumor development at the extrahepatic biliary tract and is expected to be useful for clarifying the characteristics of this highly malignant tumor.     

Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene–induced mammary tumors in BALB/c and p53 hemizygous mice

We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12-dimethylbenz[a]anthracene (DMBA)-treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53-overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild-type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild-type p53 was related to in vitro culture conditions. The effect of DMBA on mammary-tumor development was also tested in mice rendered hemizygous for p53. These mice and wild-type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridizations detected no gross alterations in the wild-type p53 allele in mammary tumors from the p53-deficient mice. Point mutation of the wild-type p53 allele was also infrequent in the DMBA-induced mammary tumors from hemizygous p53 mice; it occured in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA-induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha-ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA-induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild-type p53 was retained in both DMBA-induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild-type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration. © 1994 Wiley-Liss, Inc.     

Absence of p53 mutations and various frequencies of ki-ras exon 1 mutations in rat hepatic tumors induced by different carcinogens

Mutations of p53 and Ki-ras exon 1 were investigated in rat hepatic lesions induced by four kinds of hepatocarcinogenic protocols: continuous feeding of 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB), daily intraperitoneal injection of aflatoxin B, (AFB,), and the Soft and Farber regimen (Nature 236:701–703, 1976), in which diethylnitrosamine (DEN) or nitrosomethylurea (NMU) was used as initiating agents. DNA from microdissected tissue sections was amplified by the polymerase chain reaction (PCR) directly using primers for p53 exons 5–8 and Ki-ras exon 1 and analyzed for mutations by denaturing gradient gel electrophoresis (DGGE) or constant denaturant gel electrophoresis (CDGE). One or both of the p53 PCR primers were located within introns to prevent amplifying the p53 processed pseudogenes. In a total of 59 hepatocellular carcinomas (HCCs), no p53 aberrations were detected, indicating that p53 mutations are not very important in rat hepatic carcinogenesis. On the other hand, Ki-ras codon 12 mutations were found at low frequency in HCCs, hyperplastic foci, and cholangiof ibroses induced by 3′-Me-DAB and by AFB! but not in the lesions induced by the Solt and Farber regimen. Although Ki-ras codon 12 mutations are generally infrequent in rat hepatic tumors, their incidence thus appears to vary depending on the carcinogen used for their induction. © 1994 Wiley-Liss, Inc.     

Absence of ras Mutations and Low Incidence of p53 Mutations in Renal Cell Carcinomas Induced by Ferric Nitrilotriacetate

Renal cell carcinomas induced in male Wistar rats by iron chelate of nitrilotriacetate (Fe-NTA) were examined for mutations in ras oncogenes and p53 tumor suppressor gene. Fourteen primary tumors and two metastatic tumors from 11 animals were evaluated. Exons 1 and 2 of the H-, K-, and N- ras genes were amplified by polymerase chain reaction (PCR), and the presence of mutations was examined by direct sequencing. Exon 5 through exon 7 of p53 gene, including the 3'half of the conserved region II and the entire conserved region III through V, were surveyed for point mutations by PCR-single stranded conformation polymorphism (SSCP) analysis. Direct sequencing of the ras genes showed no mutations in codon 12, 13, or 61 among the tumors evaluated. SSCP analysis of p53 gene exon 6 indicated conformational changes in two primary tumors. One tumor had a CCG-to-CTG transition at codon 199, and the other had an ATC-to-ATT transition at codon 229 and two nonsense C-to-T transitions. These results suggest that neither ras genes nor p53 gene play a major role in the development of renal cell carcinomas induced by Fe-NTA.     

Hepatocyte Growth Factor Enhancement of Preneoplastic Hepatic Foci Development in Rats Treated with Diethylnitrosamine and N-Ethyl-N-hydroxyethylnitrosamine

Effects of hepatocyte growth factor were investigated in a two-stage rat liver carcinogenesis protocol. Male F344 rats were first treated with diethylnitrosamine (200 mg/kg, i.p.) and then, starting two weeks later, with N-ethyl-N-hydroxyethylnitrosamine (EHEN) for 6 weeks at a dose of 0.01% in drinking water. Hepatocyte growth factor, which was injected i.v. at a dose of 200 μg/kg body weight one (at week 3) or two times (at weeks 3 and 4) during EHEN administration, significantly increased the development of preneoplastic glutathione S-transferase placental form-positive foci. Although the observed effects of hepatocyte growth factor were weaker than that of the two-thirds partial hepatectomy (PH) performed at week 3, the present results suggest that the enhancing effects of PH performed during the promotion stage may be largely mediated through induction of hepatocyte growth factor.     

A p53 mutation in exon 5 associated with adenovirus transformation

The adenovirus type 5 (Ad5) 55-kDa E1B oncoprotein has been shown to form complexes with the p53 tumor suppressor protein. These complexes are thought to interfere with normal p53 activity and may be responsible for the paucity of p53 mutations in cells transformed by these viruses. This report describes an example of a p53 mutation in exon 5 in an Ad5-transformed cell line that exhibited less expression of E1B 55-kDa protein and a longer tumor-latency phenotype than another Ad5-transformed cell line expressing wild-type p53. The finding of a p53 mutation in an Ad5-transformed cell line is unusual, especially considering the current theory that p53-E1B interactions play an important role in adenovirus transformation. This mutation could represent an alternative method of inactivating p53 function in the absence of sufficient levels of E1B 55-kDa oncoprotein. © 1995 Wiley-Liss Inc.