Induction of 8-hydroxydeoxyguanosine and ultrastructure alterations by silver nanoparticles attributing to placental transfer in pregnant rats and fetuses.

A quantitative assessment of the genotoxicity of silver nanoparticles (AgNPs) ascribed to its transplacental transfer and tissue distribution in pregnant rats was carried out in this study. A single intravenous (i.v.) injection of AgNPs with a size range from 4.0 to 17.0 nm was administered to pregnant rats at a dose of 2 mg/kg b.w. on the 19th day of gestation. Five groups beside control, each of the five rats were euthanized after 10 min, 1, 6, 12, or 24 h, respectively. The accumulation of nanoparticles (NPs) in mother and fetal tissues was quantified by inductively coupled plasma optical emission spectroscopy, where the highest accumulation level was recorded in maternal blood (0.523 µg/ml) after 24 h of administration. AgNPs induced accumulation in spleen tissue higher than placenta and fetal tissue homogenates. The data showed significantly detected levels of 8-hydroxydeoxyguanosine in all collected samples from administered animals compared with untreated individuals. Level of 8-OHdG in amniotic fluid exhibited the greatest values followed by maternal spleen, kidneys, and liver, respectively. Investigation by transmission electron microscope showed that the transfer of AgNPs through placental wall caused indentation of nuclei, clumped chromatin, pyknotic nuclei, and focal necrotic areas, while AgNPs appeared mainly accumulated in the macrophages of the spleen. Therefore, the data assume that the genotoxicity studies of AgNPs must be recommended during a comprehensive assessment of the safety of novel types of NPs and nanomaterials. Additionally, exposure to AgNPs must be prevented or minimized during pregnancy or prenatal periods.

Anticancer effect of metformin against 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine-induced rat mammary carcinogenesis is through AMPK pathway and modulation of oxidative stress markers.

OBJECTIVES: Metformin, the type 2 anti-diabetes medication, showed antitumor activity both in vivo and in vitro. This study was carried out to investigate the mechanisms behind the metformin anticancer effect against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female Sprague-Dawley rats. METHODS: Rats received 10 doses of PhIP (75 mg/kg, p.o., days 1-5 and 8-12). Then, rats were treated with metformin for 26 weeks at a dose of 2 mg/ml in drinking water. KEY FINDINGS: Metformin antitumor effect was mediated by increasing the adenosine monophosphate protein kinase (AMPK) activity, liver kinase B1, and decreasing the aromatase and insulin levels compared with the PhIP-administered group. Also, this treatment resulted in a significant decrease in mammary tissue oxidative stress markers and serum lipid profile. In parallel, mammary gland tumors found in PhIP+metformin group were all histologically benign included only (hyperplasia). However, most of the mammary gland tumors found in PhIP group were histologically malignant. CONCLUSIONS: These results showed that metformin antitumor effect was mediated through AMPK pathway, reducing oxidative stress and serum lipid levels. This study supports the potential benefit of using metformin as adjuvant therapy during breast cancer treatment.

Increased oxidative stress with gene alteration in urinary bladder urothelium after the Chernobyl accident.

We have previously shown that bladder urothelium of people living in the cesium-137 ((137)Cs)-contaminated areas of Ukraine demonstrates accumulation of stable p53 and p53 mutational inactivation, preferentially through G:C to A:T transition mutations at CpG dinucleotides, with a codon 245 hot spot. In the present study, we analyzed immuno-histochemically the relationship between oxidative stress markers and over-expression of p53 and H-ras in urinary bladder urothelium from 42 men with benign prostatic hyperplasia. Bladder mapping biopsies were obtained from 15 patients from a highly radiocontaminated area (group I), 14 patients from the less contaminated city of Kiev (group II) and 13 patients as a control group from "clean" (without radiocontamination) areas of Ukraine (group III). Irradiation cystitis with multiple foci of severe dysplasia and carcinoma in situ were observed in 15 of 15 (100%, group I) and 9 of 14 (64%, group II) cases, with 4 small transitional-cell carcinomas incidentally detected in groups I and II. Markedly elevated levels of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2) and 8-hydroxy-2;-deoxyguanosine (8-OHdG) were noted in these bladder urothelial lesions from groups I and II, accompanied by strong over-expression of p53 and less H-ras expression. These findings support the hypothesis that iNOS, COX-2 and 8-OHdG in bladder urothelium are induced by long-term exposure to low-dose radiation with a close relationship to p53 over-expression that could predispose to bladder carcinogenesis.

Inhibition by ginseng of 1,2-dimethylhydrazine induction of aberrant crypt foci in the rat colon.

The modifying effects of dietary administration of ginseng on the induction and development of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) were investigated in Fischer 344 (F-344) rats. In Experiment 1, starting at six weeks of age, 65 rats were injected with DMH or saline alone once a week for four weeks. Rats in Groups 1 and 2 were fed diets containing 1% ginseng for five weeks, starting one week before the first dose of DMH. Animals in Groups 3 and 4 received ginseng for eight weeks after DMH treatment; Group 5 served as a carcinogen control group. In Experiment 2, 60 rats were injected with DMH or saline alone four times at one-week intervals. They were also fed diets containing 1% ginseng or the control diet throughout the 30 days of the experiment. In Experiment 1, numbers of foci with at least four crypts were significantly reduced in Group 2 treated with red ginseng during the initiation phase (p < 0.005). In Experiment 2, treatment with red ginseng also resulted in a decrease in the total number of DMH-induced ACF accompanied by a reduction in 5-bromo-2'-deoxyuridine labeling indexes in colonic crypts comprising ACF (p < 0.005 and p < 0.05, respectively). These findings suggest that dietary administration of red ginseng in combination with DMH suppresses colon carcinogenesis of rats, and the inhibition may be associated, in part, with suppression of cell proliferation in the colonic mucosa.

Chemopreventive effect of 24R,25-dihydroxyvitamin D(3) in N, N'-dimethylhydrazine-induced rat colon carcinogenesis.

In this study we investigated the effects of 24R,25-dihydroxyvitamin D(3) [24R,25(OH)(2)D(3)] on N,N'-dimethylhydrazine (DMH)-induced rat colon carcinogenesis. For experiments 1 and 2, 50 F344 male, 6-week-old rats were divided into five groups in each experiment. Animals were given s.c. injections of DMH once a week for 4 weeks. Those in groups 1-5 were given 24R,25(OH)(2)D(3) in the diet (10, 5, 2.5, 1.25 or 0 p.p.m., respectively) during the post-initiation stage in experiment 1 and during the initiation stage in experiment 2. At termination, the numbers of aberrant crypt foci (ACF) in the rat colonic mucosa were decreased dose-dependently in rats treated with 24R,25(OH)(2)D(3) during the post-initiation stage, but not in the initiation stage. For experiment 3, 15 male, 9-week-old rats were divided into three groups and given 24R,25(OH)(2)D(3) in the diet (10, 5 or 0 p.p.m.). Animals were injected with 5-bromo-2'-deoxyuridine (BrdU) i.p. 1 h before death to examine DNA synthesis in the colon mucosa. BrdU labeling indices were decreased dose-dependently in colonic crypts of rats treated with 24R, 25(OH)(2)D(3). In experiment 4, using the multicarcinogenic protocol we could analyze our data with respect to not only one separate organ, but at the organism level. Sixty-eight male, 6-week-old rats were treated with DMH, N-methylnitrosourea, 2, 2'-dihydroxy-di-n-propylnitrosamine, diethylnitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine in weeks 1-4 and were then given 24R,25(OH)(2)D(3) in the diet (5, 1 or 0 p.p.m.) throughout weeks 5-30. Examination of the development of tumors and preneoplastic lesions in various organs revealed that 24R, 25(OH)(2)D(3) inhibited colonic tumor development significantly but exerted no effects on tumor induction in other organs. In conclusion, these results strongly indicate that 24R,25(OH)(2)D(3) inhibits colon carcinogenesis specifically, without any enhancement of carcinogenesis in other organs, when administered in the post-initiation phase.

Inhibitory effects of 1,3-diaminopropane, an ornithine decarboxylase inhibitor, on rat two-stage urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Overexpression of ornithine decarboxylase (ODC) has been shown to be characteristic of tumor development and progression in humans and experimental animals. Therefore, we have examined the effects of 1, 3-diaminopropane dihydrochloride (DAP), a potent inhibitor of ODC, on rat two-stage urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In experiment 1 (36 weeks), 6-week-old F344 male rats were administered 0.05% BBN in drinking water for 4 weeks and then divided into four groups. Animals of groups 1 and 2 received basal diet and drinking water supplemented with or without DAP (2 g/l). Groups 3 and 4 were given diet containing 5% sodium L-ascorbate (NaAsA), a typical urinary bladder tumor promoter, and drinking water with or without DAP. Administration of DAP to group 1 significantly reduced tumor size, multiplicity and incidence, particularly of papillomas, when compared with group 2 values. DAP together with NaAsA (group 3) also decreased tumor size relative to the group 4 case. To determine the effects of DAP on the early stages of bladder carcinogenesis and its mechanisms, a similar protocol was conducted (experiment 2) with death after 20 weeks. DAP treatment caused complete inhibition (0% incidence) of papillary and/or nodular hyperplasia in group 1 but was without influence in group 3, as compared with the respective controls. Moreover, the ODC activity, bromodeoxyuridine labeling indices and mRNA expression levels of cyclin D1 in the urinary bladder mucosa, determined by northern blotting, were markedly lower in group 1 than in group 2, but values were comparable for both groups administered NaAsA. Assessment of mRNA expression levels of the angiogenic vascular endothelial growth factor suggested no involvement in the inhibitory effects of DAP on urinary bladder carcinogenesis. The results indicate that inhibition of ODC could reduce urinary bladder carcinogenesis in rats, particularly in the early stages, through antiproliferative mechanisms.

Low-dose-dependent carcinogenic potential of 2-amino-3-methylimidazo[4,5-f]quinoline in the immunodeficient (SCID) mouse colon.

The carcinogenic potential of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), one of the most potent mutagenic heterocyclic aromatic amines in food, for the colon was assessed in mice with severe combined immunodeficiency (SCID). In Experiment I, 60 male animals, 7-8 weeks old, were administered 300, 100, or 0 ppm IQ in the diet for 20 weeks. The incidence of aberrant crypt foci (ACF), preneoplastic lesions, was 100% in the two IQ-administered groups, whereas no ACF were found in the controls. Larger lesions, at least four aberrant crypts per focus, were noted in the colons of both treated groups. Most ACF were located in the proximal colon, and the bromodeoxyuridine-labeling indexes were elevated in a dose-dependent manner, especially in this region. In Experiment II, IQ was administered in the diet at 50, 10, 2, or 0 ppm to 60 female and male 7- to 8-week-old SCID mice for 30 and 23 weeks, respectively. The incidence of ACF was dose dependent in both sexes: 100%, 100%, and 63% in the females administered 50, 10, and 2 ppm, respectively, and 100%, 83%, and 38%, respectively, in the males. Lesions of at least four aberrant crypts per focus were again evident with the 50-ppm dose. The long-term or higher dose administration of IQ in the diet might thus be applied to elucidate colon carcinogenesis in the SCID mouse.

Elevation of urinary enzyme levels in rat bladder carcinogenesis.

Urinary enzyme levels were investigated in rats administered different promoters in their diet for 32 weeks after being initiated by treatment with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks. All groups were composed of 10 rats each. Group 1: females treated with 3% uracil (100% carcinoma incidence). Group 2: control females kept on basal diet only (0% carcinoma incidence). Group 3: males treated with 5% sodium L-ascorbate (100% carcinoma incidence). Group 4: control males (0% carcinoma incidence). Urine was collected at the end of weeks 12, 24 and 36 and tested for lactate dehydrogenase (LDH), alkaline phosphatase, N-acetyl-beta-D-glucosaminase and aspartate aminotransferase activity. To facilitate comparison, data were related to the corresponding excreted creatinine levels. All measurements were made using a centrifugal automatic analyzer. The urine of rats with cancer lesions (groups 1 and 3) showed significant elevation in all enzyme activities at weeks 24 and/or 36 except for LDH in females (group 1). The M/H ratio of the LDH isozymes was reversed (1.10 +/- 0.10) in the tested rats with carcinomas at week 36. This study thus provides evidence of a correlation between high urinary enzyme levels and cancer development in the rat bladder. Measurement of the tested enzymes might thus provide a method to detect malignant changes in bladder epithelium by direct urine analysis.

Dose-dependent induction of aberrant crypt foci in the colons but no neoplastic lesions in the livers of heterozygous p53-deficient mice treated with low dose 2-amino-3-methylimidazo [4,5-f]quinoline.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a food derived heterocyclic amine which induces aberrant crypt foci (ACF) and tumors in the livers in mice. However, most previous studies of carcinogenicity were carried out with high dose treatments, so the practical risk associated with the low dose exposure is unclear. We, therefore, assessed whether low dose IQ causes ACF formation in the colons of mice constitutively hemizygous for functional p53. Simultaneously, we screened for development of preneoplastic foci in the liver. A total of 60 heterozygous p53-deficient mice as well as 60 wild-type mice were divided into five groups and administered IQ in the diet at concentrations of 50, 10, 2, 0.4 and 0 ppm until the end of the experiment. ACF were detected in the 50, 10 and 2 ppm-treated groups and the numbers of those comprising one aberrant crypt (AC) in p53-deficient mice treated with 10 or 2 ppm were significantly increased, compared to counterpart wild-type values. A dose-dependent increase of ACF was also observed in transgenic mice groups but no large ACF developed. In spite of extensive examination, no preneoplastic foci could be detected in either transgenic or wild-type mice. The results suggested that germline p53 deficiency may slightly enhance the development of ACF in colons but not in the liver. The fact that no ACF were detected in the lowest, 0.4 ppm, treated groups may imply a practical non-effective level of IQ for tumor induction.

Enhancement of urinary bladder carcinogenesis in nullizygous p53-deficient mice by N-butyl-N-(4-hydroxybutyl)nitrosamine.

We recently reported p53 mutations to be frequent in mouse invasive urinary bladder carcinomas, with and without metastasis. However, the role of p53 dysfunctions during carcinogenesis remains unclear. In the present study, heterozygous and nullizygous p53-deficient mice and their littermates were treated with the urinary bladder carcinogen, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), at a concentration of 0.01% in the drinking water throughout the experiment. This markedly accelerated urinary bladder carcinogenesis but not development of other tumors in the nullizygous p53-deficient mice. Thus the appearance of neoplastic urothelial lesions in nullizygotes (at day 60 of the experiment) was earlier than in wild-type mice and heterozygotes (at day 125). Moreover, malignant vascular tumors (hemangiosarcomas (HS)) were found in all four nullizygotes killed later than day 108. Mutational inactivation of the wild-type allele was not apparent in either the single transitional cell carcinoma observed in a wild-type mouse and a hemangiosarcoma in a heterozygote. Overall, it can be concluded that the number of normal p53 alleles is a significant determining factor in the susceptibility of urothelial cells to carcinogens. The role of the p53 defect in mouse urinary bladder carcinogenesis may thus be to diminish the threshold for occurrence of additional genetic alterations.

Promotion of NCI-Black-Reiter male rat bladder carcinogenesis by dimethylarsinic acid an organic arsenic compound.

Dimethylarsinic acid (DMAA) is a major metabolite of inorganic arsenicals in mammals. In the present study, we investigated its promoting effects on urinary bladder carcinogenesis in NCI-Black-Reiter (NBR) rats, which lack alpha2u-globulin synthesizing ability. Male 9-14-week-old NBR rats were treated sequentially with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) for 4 weeks and then given 100 ppm DMAA in their drinking water (group 1) for 32 weeks. Induction of preneoplastic lesions (papillary or nodular hyperplasia) in this DMAA-treated group was significantly increased as compared to the carcinogen alone control group (P < 0.01). The development of carcinomas was also enhanced and a significant increase in the 5-bromo-2'-deoxyuridine (BrdU) labeling index of the urinary bladder epithelial cells was observed for the DMAA treatment group. These results indicate that DMAA has promoting effects on urinary bladder carcinogenesis even in NBR rats, so its effects are not dependent on the presence of alpha2u-globulin.

Inhibition of development of N,N'-dimethylhydrazine-induced rat colonic aberrant crypt foci by pre, post and simultaneous treatments with 24R,25-dihydroxyvitamin D3.

It has recently been reported that new vitamin D3 derivatives can exert inhibitory effects on colon carcinogenesis in rats. In the present study the chemopreventive potential of 24R,25-dihydroxyvitamin D3 (24R,25(OH)2vitamin D3) was assessed in a murine model of colon carcinogenesis. In experiment 1, male 6-week-old F344 rats were administered N,N'-dimethylhydrazine (DMH) 20 mg/kg s.c. once a week 4 times. The rats were fed 24R,25(OH)2vitamin D3 at 10 ppm in the diet prior to (pre), together with (simultaneous) or after (post) DMH treatment. Modifying effects were assessed using aberrant crypt foci (ACF), putative preneoplastic lesions, as the end point markers in this model of colon carcinogenesis. After 8 weeks, pre and more markedly simultaneous administration of 24R,25(OH)2vitamin D3 was found to have reduced the total numbers of ACF and significantly inhibited the development of foci. After 16 weeks, numbers of foci with > or = 4 crypts, which are more likely to progress to tumors, were significantly reduced. The most pronounced inhibition of ACF development was noted in rats fed the 24R,25(OH)2vitamin D3 after DMH administration. The reduction was particularly marked in the proximal colon. Blood levels of calcium were not significantly increased over the control levels in groups administered DMH and the vitamin. Immunohistochemical staining showed numbers of proliferating cell nuclear antigen-positive cells to be lower in the colonic epithelia of rats fed the vitamin D3 metabolite than in the controls. In experiment 2, the effect of 24R,25(OH)2vitamin D3 on the alterations in c-fos, c-myc and c-jun oncogene expression in response to DMH administration was examined by northern blot analysis. The early increase in expression of ornithine decarboxylase (ODC) activity was not altered by 24R,25(OH)2vitamin D3. The results suggest that 24R,25(OH)2vitamin D3 is a cancer chemopreventive agent which may suppresses DMH induction of lesions and their subsequent development via an antiproliferative action.