Elsayed I. Salim

Chemopreventive potential of volatile oil from black cumin (Nigella sativa L.) seeds against rat colon carcinogenesis.

Chemopreventive effects of orally administered Nigella sativa oil on the induction and development of 1,2-dimethylhydrazine-induced aberrant crypt foci (ACF), putative preneoplastic lesions for colon cancer, were investigated in Fischer 344 rats. Starting at 6 wk of age, 45 male rats (groups 1-3) were subcutaneously injected with DMH once a week for 3 wk. Group 1 (15 rats) served as a carcinogen control group without N. sativa administration. Group 2 or 3 (15 rats each) were given the oil in the postinitiation stage or in the initiation stage, respectively. Animals of group 4 (11 rats) were injected with 0.9% saline and received N. sativa oil from the beginning until the termination. At sacrifice, 14 wk after the start, the total numbers of ACF as well as those with at least four crypts were significantly reduced in group 2 (P < 0.01). However, treatment with N. sativa oil in the initiation stage (group 3) did not exhibit significant inhibitory effects except on foci with only one aberrant crypt. Immunohistochemical analysis of 5-bromo-2′-deoxyuridine labeling in colonic crypts revealed the N. sativa oil to have significant antiproliferative activity in both initiation and postinitiation stages and especially in the latter. Histological examination revealed no pathological changes in the liver, kidneys, spleen, or other organs of rats treated with N. sativa. In addition, biochemical parameters of blood and urine as well as body weight gain were not affected. These findings demonstrate that the volatile oil of N. sativa has the ability to inhibit colon carcinogenesis of rats in the postinitiation stage, with no evident adverse side effects, and that the inhibition may be associated, in part, with suppression of cell proliferation in the colonic mucosa.

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 (137Cs)–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. Int. J. Cancer 86:790–798, 2000.

Lack of large intestinal carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine at low doses in rats initiated with azoxymethane

2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), an abundant food‐derived heterocyclic amine (HCA), has attracted particular attention as a human colon carcinogen. Humans are in fact exposed to continuous low doses of HCAs during lifetime. Therefore, we focused on rat large intestinal carcinogenicity of PhIP at levels that mimic practical human exposure. A total of 192 6‐week‐old male F344 rats were subcutaneously injected twice with 15 mg/kg body weight azoxymethane (AOM), then continuously fed various doses (0, 0.001, 0.01, 0.1, 1, 10, 50 and 200 ppm) of PhIP in the diet. At week 16, aberrant crypt foci (ACF) were quantitatively analyzed. At week 36, tumor occurrence was pathologically analyzed. Then immunohistochemical examinations were performed. PhIP was found to enhance strongly AOM‐initiated rat large intestinal tumorigenesis at high doses (50 and 200 ppm), while lower doses (0.001–10 ppm) had no apparent effects. High doses also caused variation in tumor histologic types and their distribution throughout the large intestinal segments. Frequencies of ACF/cm2 did not meaningfully vary between the groups. Cellular proliferation activity in normal‐appearing colonic mucosa was significantly increased at high doses. These novel findings may provide evidence of a low‐dose potential for PhIP, with a no‐observed effect level speculated to be 10 ppm in the present initiation‐promotion experimental model.

Induction of glutathione S-transferase placental form positive foci in liver and epithelial hyperplasia in urinary bladder, but no tumor development in male Fischer 344 rats treated with monomethylarsonic acid for 104 weeks.

The carcinogenicity of monomethylarsonic acid (MMA(V)), a major metabolite of inorganic arsenics in human and experimental animals, was investigated in male Fischer 344 rats. A total of 129 rats at 10 weeks of age were randomly divided into three groups and received drinking water containing MMA(V) at doses of 0 (Control), 50, and 200 ppm ad libitum for 104 weeks. No significant differences were found between the control and the MMA(V)-treated groups regarding clinical signs, mortality, hematological, and serum biochemistry findings. Quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci in liver revealed a significant increase of numbers and areas in the 200 ppm MMA(V)-treated group. In the urinary bladder MMA(V) induced simple hyperplasia and significantly elevated the proliferating cell nuclear antigen (PCNA)-positive index in the urothelium. A variety of tumors developed in rats of all groups, including the controls, but all were histologically similar to those known to occur spontaneously in F344 rats and there were no significant differences among the groups. Thus, it could be concluded that, under the present experimental conditions, MMA(V) induced lesions in the liver and urinary bladder, but did not cause tumor development in male F344 rats even after 2 years exposure.

Chemopreventive effect of JTE-522, a selective cyclooxygenase-2 inhibitor, on 1, 2-dimethylhydrazine-induced rat colon carcinogenesis

Selective COX-2 inhibitors have been suggested to be an effective strategy in the prevention of colon cancer without the adverse side effects of non-selective, nonsteroid anti-inflammatory drugs. The present experiment was designed to assess the potential chemopreventive properties of JTE-522, a new selective cyclooxygenase-2 inhibitor, on the induction of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), a marker of rat colon carcinogenesis. A total of 80 male F344 rats were treated with 3 or 10 mg/kg of body weight JTE-522 or vehicle by oral gavage five times weekly from the start of the experiment. One week later, rats received s.c. injections of saline or 20 mg/kg of body weight DMH once weekly for four successive weeks. At the end of 12 weeks after the start of experiment, all rats were sacrificed and colons were evaluated for ACF. 10 mg/kg JTE522 significantly suppressed the total ACF/colon. No inhibitory effect was observed in the 3 mg/kg JTE-522 treatment group. This result suggests that JTE-522 possesses chemopreventive activity against colon carcinogenesis.

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.

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 (24ff,25(OH)2vitainin D3) was assessed in a murine model of colon carcinogenesis. In experiment 1, male 6‐week‐old F344 rats were administered N2N‐dimethylhydrazme (DMH) 20 mg/kg s.c. once a week 4 times. The rats were fed 24R,25(OH)2vitaniin 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 ≥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)2vitaniin 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.

Persistence of Liver Cirrhosis in Association with Proliferation of Nonparenchymal Cells and Altered Location of α-Smooth Muscle Actin-Positive Cells

This study was carried out to achieve pathological understanding for the persistence of cirrhosis induced by thioacetamide (TAA). Forty-five, male, 21-day-old, F344 rats were randomly allocated to group 1 and received drinking water as a control, and groups 2 and 3 given 0.015% or 0.03% TAA, respectively for 12 weeks. Two-third of animals per group were sacrificed, and remainder were maintained for a further 4 weeks without TAA treatment. Liver cirrhosis was induced in all animals in group 3 at week 12, with obvious increase of collagen content, and this persisted after cessation of TAA. Proliferating cell nuclear antigen (PCNA) positive labeling indices of nonparenchymal cells were increased significantly after cessation in groups 2 and 3 (p < 0.01). RT-RCR analysis of α-smooth muscle actin (α-SMA) showed significant increase in group 3 compared to that of control at both time points (p < 0.05). Immunohistochemical staining of it demonstrated positive cells to mainly be located around regenerating hepatic nodules at week 12, however, they were focused into enlarged portal areas consisting of fibrous tissues and pseudo-bile ductular cells after the cessation. Taken together, we conclude persistence of liver cirrhosis could be associated with the proliferation of nonparenchymal cells and altered location of α-SMA positive cells.

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.

High susceptibility of p53 knockout mice to esophageal and urinary bladder carcinogenesis induced by N, N-dibutylnitrosamine

In human cancer, alterations in the p53 tumor suppressor gene are the most common genetic alterations. The aim of the present study was to detect sensitivity of the p53 (+/-) mice and their littermates p53 (+/+) mice to N, N-dibutylnitrosamine (DBN) carcinogenicity. In experiment 1, 6-7-week-old p53 (+/-) and p53 (+/+) mice were treated with 0, 0.025 and 0.05% DBN, respectively, in drinking water for 20 weeks. Esophageal squamous cell and urinary bladder transitional cell carcinomas (TCCs) and fibrosarcomas were found to be significantly increased in p53 (+/-) mice treated with doses of DBN compared to p53 (+/+) mice administered similar doses. In experiment 2, 6-7-week-old p53 (+/-) and p53 (+/+) mice were administered 0 or 0.05 % DBN in drinking water for 8 weeks. Immunohistochemical staining revealed a significant increase in numbers of p53 and bromodeoxyuridine (BrdU) positive cells in the esophageal and urinary bladder epithelia of DBN-treated p53 (+/-) mice compared to p53 (+/+) mice administered DBN. Molecular analysis revealed point mutations in the residual p53 allele in four of eight (50%) esophageal mucosa of DBN-treated p53 (+/-) mice, and in three of eight (38%) of treated p53 (+/+) mice. The results show that p53 (+/-) mice were sensitive to DBN treatment with respect to esophageal and bladder tumor development, with a mechanism that could be confined to early mutations of the residual p53 allele and increased cellular proliferation in the target organs.