DMH Treatment Research Articles

K‐ras exon 4A has a tumour suppressor effect on carcinogen‐induced murine colonic adenoma formation

K-ras encodes two isoforms, K-ras 4A and 4B, that are jointly affected by K-ras activating mutations, which are prevalent in colorectal cancer (CRC). CRC shows alterations in the expressed K-ras 4A : 4B isoform ratio in favour of K-ras 4B, in tumours both with and without K-ras mutations. The present study evaluated whether K-ras 4A expression can suppress colonic adenoma development in the absence of its oncogenic allele. Mice with homozygous targeted deletions of K-ras exon 4A (K-ras(tmDelta4A/tmDelta4A)) that can express the K-ras 4B isoform only, along with heterozygous K-ras(tmDelta4A/+) and wild-type mice, were given ten weekly 1,2-dimethylhydrazine (DMH) treatments to induce colonic adenomas. There was a significant increase in both the number and the size of colonic adenomas in DMH-treated K-ras(tmDelta4A/tmDelta4A) mice, with reduced survival, compared with heterozygous and wild-type mice. No K-ras mutations were found in any of the 30 tumours tested from the three groups. Lack of expression of K-ras 4A transcripts was confirmed, whereas the relative expression levels of K-ras 4B transcripts were significantly increased in the adenomas of K-ras(tmDelta4A/tmDelta4A) mice compared with K-ras(tmDelta4A/+) and wild-type mice. Immunohistochemical studies showed that adenomas of K-ras(tmDelta4A/tmDelta4A) mice had significantly increased cell proliferation and significantly decreased apoptosis with evidence of activation of MapKinase and Akt pathways, with increased phospho-Erk1/2 and both phospho-Akt-Thr308 and phospho-Akt-Ser473 immunostaining, compared with adenomas from K-ras(tmDelta4A/+) and wild-type mice. In conclusion, following DMH treatment, K-ras exon 4A deletion promoted increased number and size of colonic adenomas showing increased K-ras 4B expression, increased proliferation, decreased apoptosis, and activation of MapKinase and Akt pathways, in the absence of K-ras mutations. Therefore, K-ras 4A expression had a tumour suppressor effect on carcinogen-induced murine colonic adenoma formation, explaining the selective advantage of the altered K-ras 4A : 4B isoform ratio found in human colorectal cancer.

Oxidative Stress and Stress-signaling in Chemoprevention of Early Colon Cancer by Diclofenac

Role of oxidative stress and NF-κB signalling was evaluated in the chemopreventive effects of diclofenac, a dual cycloxygenase inhibitor in 1,2-dimethylhydrazine dihydrochloride (DMH)-induced colon carcinogenesis in rat model. After a 6 week long treatment with DMH (early stage), morphological analysis revealed marked occurrence of preneoplastic features such as multiple plaque lesions (MPLs), aberrant crypts (ACs) and aberrant crypt foci (ACF) in the colonic mucosa. Histologically well characterized dysplasia and hyperplasia were also observed. Simultaneous administration of diclofenac with DMH resulted in a significant reduction of these, proving the chemopreventive efficacy of diclofenac at the chosen dose. Diclofenac only group didn’t show any prominent carcinogenic feature. The colonic mucosa showed decreased reactive oxygen species (ROS) generation, nitric oxide (NO) levels and inducible nitric oxide synthase (iNOS) expression in the DMH group. Apoptosis was quantified in the colonic mucosa by measuring caspase-1 and 3 activities and cleavage of PARP by Western blot analysis. Immunohistochemical localization of PARP was also done in colonic paraffin sections. The activities of both the caspases and cleavage of PARP decreased after DMH treatment. Also, the number of PARP positive cells was few in the DMH group, while it featured prominently in all the other groups. The expression of nuclear factor-κB (NFκB) and IκB kinase (IKK) was elevated while that of inhibitory κB (IκB) reduced after DMH treatment, as assessed both by Western blot and immunohistochemistry. Diclofenac significantly altered the modulations induced by DMH in all the studied parameters, thus confirming the role of oxidative stress and signalling in its chemopreventive action.

Ultrastructural Changes in Rat Colon Following 1,2-Dimethylhydrazine-Induced Colon Carcinogenesis: Protection by Zinc

The present study evaluated the modulatory effects of zinc on 1,2-dimethylhydrazine (DMH)-induced ultrastructural changes in rat colon as well as on [(3)H]thymidine uptake and [(14)C]D-glucose metabolism. The rats were segregated into four groups: normal control, DMH treated, zinc treated, DMH + zinc treated. Initiation and induction of colon carcinogenesis was achieved through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 8 and 16 weeks, respectively. Zinc was supplemented to rats at a dose level of 227 mg/L in drinking water, ad libitum for two different time durations of 8 and 16 weeks. The study revealed a significant decrease in zinc concentration in serum and colon following DMH treatment to rats, which upon zinc supplementation were recovered to near normal levels. A significant increase in in vitro [(3)H]thymidine uptake was observed following 16 weeks of DMH treatment. Further, a significant increase in the [(14)C]glucose turnover was observed following 8 and 16 weeks of DMH treatment. Simultaneous supplementation of zinc to DMH-treated rats for 16 weeks significantly decreased the uptake of [(3)H]thymidine and [(4)C]glucose when compared to DMH alone-treated rats. Changes in the ultrastructural architecture of colonic cells were evident following both treatment schedules of DMH; however, the changes were more distinguishable following 16 weeks of DMH treatment. The most obvious changes were seen in nuclear shape and disruption of cellular integrity, which upon zinc supplementation was appreciably improved. In conclusion, the study suggests positive beneficial effect of zinc against chemically induced colonic preneoplastic progression in rats.

Chemopreventive response of diclofenac, a non-steroidal anti-inflammatory drug in experimental carcinogenesis.

The chemopreventive response was evaluated of nonsteroidal anti-inflammatory drug, Diclofenac, a preferential cyclooxygenase-2 (COX-2) inhibitor in 1,2-dimethyhydrazine (DMH)-induced colon cancer in rat model. The signs of neoplasm were evident in the animals receiving 30mg of DMH per kg body weight in a weekly s.c injection for six weeks. The putative biomarker of carcinogenesis was visible in the form of multiple plaque lesions in DMH treatment and then regression seen in those animals which also received an oral dose of Diclofenac, 8 mg/kg body weight whereas no such macroscopic neoplastic lesions were seen in the animals receiving Diclofenac only or the control animals receiving the vehicle of the drug. Histopathological results showed the presence of early aberrant changes in the form of severe dysplasia and also numerous crypt fissions in the apical surface of the colonic mucosa. A very high expression of COX-2 was seen in the colonic epithelium of DMH-treated rats, as analyzed by immunohistochemistry. Also, the apoptotic events were assessed by terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) assay, where the DMH group shows few number of TUNEL positive cells which dramatically increased in the Diclofenac treatment. The results suggest that Diclofenac could be an effective chemopreventive agent in colon cancer, where perhaps apoptosis plays a very dominant end effect in cancer cell killings.

Intrinsic Mitochondrial Membrane Potential Change and Associated Events Mediate Apoptosis in Chemopreventive Effect of Diclofenac in Colon Cancer

The present study explored the role of intrinsic mitochondrial membrane potential (delta psi M) in NSAID-induced apoptosis in the early stages of colon cancer. 1,2-Dimethylhydrazine dihydrochloride (DMH) was used to induce colon cancer and its chemoprevention was studied by diclofenac in a rat model. After 6 weeks of treatment with DMH (early stage), morphological analysis revealed a marked occurrence of preneoplastic features [i.e., mucosal plaque lesions (MPLs) in the colonic tissue]. Coadministration of diclofenac with DMH resulted in a significant reduction of these lesions, thereby proving the chemopreventive efficacy of diclofenac at the chosen anti-inflammatory dose. DMH treatment also led to a significant increase in delta psi M in the isolated colonocytes as assessed by JC-1 fluorescent staining, measured both by fluorescence microscopy and spectrofluorometerically. Further, there was seen a reduction in the number of cells showing low delta psi M, and hence monomer intensity of JC-1 by DMH treatment. To study the mechanism of these alterations in delta psi M in the present work, we studied the protein expression of important proapoptotic proteins, cytochrome c and Bax, by Western blot analysis and immunohistochemistry. DMH treatment reduced the mitochondrial translocation of Bax whereas cytochrome c was found to be located prominently in the mitochondria. Protein expression of antiapoptotic Bcl-2 was also studied in the colonic mucosa, which was expectedly found to be overexpressed after DMH treatment. Diclofenac treatment ameliorated the elevated delta psi M and its associated events to exert its chemopreventive action against early stages of colon cancer.

Arabinoxylan-oligosaccharides (AXOS) reduce preneoplastic lesions in the colon of rats treated with 1,2-dimethylhydrazine (DMH)

Prebiotics are non-digestible compounds that beneficially affect the host by stimulating the growth and/or activity of one or a limited number of resident colonic bacteria in the gut. Reported beneficial effects of prebiotics include reduced gut infections, better absorption of minerals, and notably, antitumorigenic effects. Arabinoxylan (AX)-oligosaccharides (AXOS) have been suggested to exert prebiotic effects in the gut, but their effect on colon carcinogenesis has not been studied so far. To test the effect of AXOS in a rat colon carcinogenesis model. We determined the occurrence of two types of preneoplastic lesions [aberrant crypt foci (ACF) and mucin depleted foci (MDF)] in the colon of rats treated with the colon carcinogen 1,2-dimethylhydrazine (DMH) and fed either a control diet or a diet containing AXOS (4.8% w/w) (15 rats in each group). Thirteen weeks after DMH treatment, MDF counts were significantly lower in the entire colon of AXOS fed rats (MDF/colon were 7.5 +/- 0.6 and 5.5 +/- 0.6, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). Although the number of ACF in the entire colon was not significantly different between Control and AXOS fed rats, AXOS fed rats had significantly fewer ACF in the distal part of the colon than Control group rats (ACF/distal colon were 135.5 +/- 15 and 84.4 +/- 11, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). The present study shows that dietary intake of AXOS by rats reduces the occurrence of two types of preneoplastic lesions, thus suggesting a chemopreventive effect on colon carcinogenesis that should be confirmed in a long-term carcinogenesis experiment.

Efficacy of the potential chemopreventive agent, hesperetin (citrus flavanone), on 1,2-dimethylhydrazine induced colon carcinogenesis

Our current study is an effort to identify a potent chemopreventive agent against colon cancer. Here we have investigated the efficacy of hesperetin on tissue lipid peroxidation, antioxidant defense system and colonic histoarchitecture in male Wistar rats in colon carcinogenesis. Rats in groups 3, 4, 5 and 6 were treated with DMH (20 mg kg body weight s.c.) once a week for 15 weeks. Group 1 rats received modified pellet diet and served as control; group 2 received modified pellet diet along with hesperetin (20 mg/kg body weight, p.o., every day); and hesperetin was given to the rats as in-group 2 during the initiation, post-initiation and entire period stages of colon carcinogenesis. Lipid peroxidation was studied by measuring the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and conjugated dienes (CD), and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), reduced glutathione (GSH), in the liver and colonic tissues of DMH administered rats. (1) Decreased levels of lipid peroxidation in the colonic tissues; (2) decreased activities of antioxidant enzymes SOD, CAT, GPX, GR and GSH levels in the tissues on DMH treatment. Hesperetin supplementation during the initiation, post-initiation and entire period stages of carcinogenesis significantly reversed these activities. These results indicate that hesperetin may be a potential chemopreventive agent against DMH-induced colon cancer.

Na+-Stimulated Na+/H+ Exchange and an Unfavorable Ca2+ Homeostasis Initiate the Cycloxygenase-2 Inhibitors-Induced Apoptotic Signals in Colonic Epithelial Cells During the Early Stage of Colon Carcinogenesis

Evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cycloxygenase (COX) and production of the proinflammatory prostaglandin, PGE2, and thus prevent carcinogenesis in the colon. Indeed, one of the specific COX-2 inhibitors, celecoxib, had been accepted by the US FDA for the treatment of familial adenomatous polyposis. However, the molecular mechanism of such inhibition is not clear, although apoptosis appears to be the dominant antiproliferative end effect. The present study delineates the intracellular ionic milieu in the colonocytes that could generate strong apoptotic signals where DMH-induced carcinogenesis was studied in the initiation stage in rats and its regression with the COX inhibitors. While DMH treatment produced a significant elevation in the Na+/H+ exchanger activity and resultant proton efflux, this was reversed by the NSAIDs, particularly so with celecoxib and etoricoxib compared to aspirin. Similarly, the intracellular pH was changed, with more alkalosis noted in DMH, which was reversed by NSAIDs. Also, an intracellular Ca2+ build up was noted by Fura 2 AM, which was also supported by a reduced Ca2+ ATPase and an enhanced inward movement of Ca2+. Further, mitochondrial dysfunction-related cyt C release, increased DNA ladder formation, activation of caspase-3, and cleavage product of poly (ADP-ribose) polymerase (PARP) were not seen in DMH but well noted in NSAIDs. Our results indicate that NSAIDs can induce apoptosis through a change in the colonic Na+/H+ exchange, intracellular pH, and an unfavorable Ca2+ homeostasis.

The Cyclooxygenase-2 Inhibitor Etoricoxib Is a Potent Chemopreventive Agent of Colon Carcinogenesis in the Rat Model

Cyclooxygenase-2 (COX-2), an inducible prostaglandin G/H synthase, is overexpressed in several human cancers, including colon cancer, and therefore the potential ability of a selective COX-2 inhibitor, etoricoxib, is considered in the prevention of the 1,2-dimethyl hydrazine (DMH)-induced colon carcinogenesis in the rat model. DMH was injected s.c. for 6 weeks, whereas etoricoxib was fed orally to the rats on a daily basis. The results showed that DMH produced a very high number of multiple plaque lesions (MPLs), putative neoplastic biomarkers, localized throughout the colon, whereas considerable regression was observed with etoricoxib treatment. In addition, the etoricoxib group was the only group that exhibited very few of these lesions. Histopathological analysis revealed extreme dysplasia, a few adenomas, and other carcinogenic changes in the DMH group, which are distinctly absent in the etoricoxib-treated group. COX-2 was also seen to be highly expressed following DMH treatment. The DMH treatment caused very few apoptotic cells, as determined by the TUNEL assay of the colonic mucosa in paraffin sections whose number greatly increased following etoricoxib treatment. Because all these changes were clearly reversed by etoricoxib in DMH-treated animals, and the use of etoricoxib alone did not produce a neoplastic effect per se, it appears that etoricoxib, a selective COX-2 inhibitor, might be a safe and potentially chemopreventive agent in colon cancer.

Selenium as a chemopreventive agent in experimentally induced colon carcinogenesis

To elucidate the chemopreventive efficacy of selenium during experimentally induced colon carcinogenesis. Thirty-two male wistar rats were divided into four groups: group I (normal control); group II [1,2-dimethylhydrazine (DMH) treated]; group III (selenium treated); and group IV (DMH + selenium treated). Groups II and IV were given subcutaneous injections of DMH (30 mg/kg body weight) every week for 20 wk. Selenium, in the form of sodium selenite, was given to groups III and IV at 1 ppm in drinking water ad libitum for 20 wk. At the end of the study, rats were sacrificed and their colons were analyzed for the development of tumors, antioxidant enzyme levels and histological changes. 100% of the DMH treated rats developed tumors, which was reduced to 60% upon simultaneous selenium supplementation. Similarly, tumor multiplicity decreased to 1.1 following selenium supplementation to DMH treated rats. Levels of lipid peroxidation, glutathione-S-transferase, superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) decreased following DMH treatment, whereas levels of glutathione (GSH) and glutathione reductase (GR) significantly increased in DMH treated rats. Selenium administration to DMH treated rats led to an increase in the levels of lipid peroxidation, SOD, catalase, glutathione-S-transferase and GPx, but decreased the levels of GSH and GR. Histopathological studies on DMH treated rats revealed dysplasia of the colonic histoarchitecture, which showed signs of improvement following selenium treatment. The study suggests the antioxidative potential of selenium is a major factor in providing protection from development of experimentally induced colon carcinogenesis.

Ursolic Acid and Oleanolic Acid Suppress Preneoplastic Lesions Induced by 1,2-Dimethylhydrazine in Rat Colon

Ursolic acid (UA) and oleanolic acid (OA) are pentacyclic triterpenoid compounds found in plants used in the human diet and in medicinal herbs, in the form of aglycones or as the free acid. These compounds are known for their hepatoprotective, anti-inflammatory, antimicrobial, hypoglycemic, antimutagenic, antioxidant, and antifertility activities. In the present study, we evaluated the effects of UA and OA on the formation of 1,2-dimethyl-hydrazine (DMH)-induced aberrant crypt foci (ACF) in the colon of the male Wistar rat. The animals received subcutaneous (sc) injections of DMH (40 mg/kg body weight) twice a week for two weeks to induce ACF. UA, OA and a mixture of UA and OA were administered to the rats five times a week for four weeks by gavage at doses of 25 mg/kg body weight/day each, during and after DMH treatment. All animals were sacrificed in week 5 for the evaluation of ACF. The results showed a significant reduction in the frequency of ACF in the group treated with the triterpenoid compounds plus DMH when compared to those treated with DMH alone, suggesting that UA and OA suppress the formation of ACF and have a protective effect against colon carcinogenesis.

Chemopreventive potential of zinc in experimentally induced colon carcinogenesis

The present study was performed to evaluate the efficacy of zinc treatment on colonic antioxidant defense system and histoarchitecture in 1,2-dimethylhydrazine- (DMH) induced colon carcinogenesis in male Sprague–Dawley rats. The rats were segregated into four groups viz., normal control, DMH treated, zinc treated, DMH + zinc treated. Colon carcinogenesis was induced through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 16 weeks. Zinc (in the form of zinc sulphate) was supplemented to rats at a dose level of 227 mg/L in drinking water, ad libitum for the entire duration of the study. Increased tumor incidence, tumor size and number of aberrant crypt foci (ACF) were accompanied by a decrease in lipid peroxidation, glutathione- S-transferase, superoxide dismutase (SOD) and catalase. On the contrary, significantly increased levels of reduced glutathione (GSH) and glutathione reductase (GR) were observed in DMH treated rats. Administration of zinc to DMH treated rats significantly decreased the tumor incidence, tumor size and aberrant crypt foci number with simultaneous enhancement of lipid peroxidation, SOD, catalase and glutathione- S-transferase. Further, the levels of GSH and GR were also decreased following zinc supplementation to DMH treated rats. Well-differentiated signs of dysplasia were evident in colonic tissue sections by DMH administration alone. However, zinc treatment to DMH treated rats greatly restored normalcy in the colonic histoarchitecture, with no apparent signs of neoplasia. EDXRF studies revealed a significant decrease in tissue concentrations of zinc in the colon following DMH treatment, which upon zinc supplementation were recovered to near normal levels. In conclusion, the results of this study suggest that zinc has a positive beneficial effect against chemically induced colonic preneoplastic progression in rats induced by DMH.

Inhibitory Effect of Natural Coumarin Compounds, Esculetin and Esculin, on Oxidative DNA Damage and Formation of Aberrant Crypt Foci and Tumors Induced by 1,2-Dimethylhydrazine in Rat Colons

The effects of esculetin (6,7-dihydroxycoumarin) and its 6-glycoside, esculin, on 8-oxo-2'-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, 1,2-dimethylhydrazine (DMH), were examined in the colons of male Fischer 344 rats. Animals were given water containing esculetin or esculin for 7 d before subcutaneous injection of DMH (20 mg/kg body wt), killed 24 h after DMH treatment, and the levels of thiobarbituric acid reactive substances (TBARS) and 8-oxodG in the colons were determined. Both esculetin and esculin suppressed significantly the DMH-induced increases in 8-oxodG and TBARS in rat colon mucosa. We further investigated the modifying effect of esculin intake on the development of DMH-induced colonic aberrant crypt foci (ACF). Animals were given DMH once a week for 4 weeks to induce ACF. They then received water containing esculin ad libitum for 5 weeks (initiation phase) or 11 weeks after DMH treatment (post-initiation phase). Animals in the positive control group received tap water throughout the experiment. At the end of the experiment (16 weeks), the ingestion of esculin during the initiation phase significantly reduced the incidence of gross tumors, the number of ACF per rat and the mean number of AC per focus, while the esculin treatment during the post-initiation phase significantly decreased only the number of ACF per rat. These results suggest that esculin intake has an inhibitory effect on DMH-induced oxidative DNA damage and carcinogenesis in rat colons.

Short-term oral toxicity of butyl ether, ethyl hexyl ether, methyl heptyl ether and 1,6-dimethoxyhexane in male rats and the role of 2-methoxyacetic acid

A 4-week oral study was conducted in male rats to characterize and compare the toxicity of four aliphatic ethers (butyl ether, BE; ethyl hexyl ether, EHxE; methyl heptyl ether, MHpE; and 1,6-dimethoxyhexane, DMH) which have been proposed as high-cetane diesel additives. Male Sprague-Dawley rats (280 ± 20 g) were divided into groups of seven animals each and were administered by gavage low (2 mg/kg body weight), medium (20 mg/kg) or high (200 mg/kg) doses of BE, EHxE, or MHpE, 5 days per week for 4 weeks. Another group of animals was administered DMH at 200 mg/kg while the control group received the vehicle (corn oil at 1 ml/100 g bw) only. At the end of the treatment period, relative testis weights and thymus weights were significantly decreased in the DMH group but not in animals receiving BE, EHxE, or MHpE. Microscopic examination revealed degeneration of the seminiferous tubules and reduction of sperm density in the epididymides in the DMH treatment group. Urinary creatine/creatinine ratio, a sensitive indicator of testicular damage, was markedly elevated in the DMH treated animals but not in those treated with BE, EHxE, or MHpE. In the bone marrow, DMH caused mild dyserythropoiesis and dysthrombopoiesis, while BE, EHxE, and MHpE produced mild increases in granulocytes and myelocyte/erythrocyte ratio. All four ethers at 200 mg/kg caused mild histological changes in the thyroid but no significant modulation in the circulating thyroxin (T4) or triiodothyronine (T3) levels. All four ethers produced hepatic effects at 200 mg/kg consisting of mild, adaptive histological changes, increased urinary ascorbic acid output, and elevation in the activities of one or more xenobiotic metabolizing enzymes (benzyloxyresorufin- O-dealkylase, UDP-glucuronosyltransferase, glutathione- S-transferases). The level of 2-methoxyacetic acid (MAA), a known testicular and developmental toxin, was significantly increased in the urine and plasma of animals treated with DMH but not in those administered the high dose BE, EHxE, or MHpE. Amomg the individual rats treated with DMH, the MAA level appeared to correlate with the severity of toxicity such as testicular and thymic weights, and urinary creatine/creatinine ratio. It is concluded that BE, EHxE, and MHpE differed from DMH in that they did not produce testicular or thymic toxicity. All four ethers at high dose caused changes to the thyroid, liver and bone marrows that were mild and adaptive in nature. MAA appeared to be the proximal toxicant in DMH treated animals but the route by which DMH is metabolized to MAA remains to be elucidated.

Modifying effect of propolis on dimethylhydrazine-induced DNA damage but not colonic aberrant crypt foci in rats

Propolis is a honeybee product with several biological and therapeutic properties, including antimutagenic and anticarcinogenic activities. The effects of an aqueous extract of propolis (AEP) were evaluated on the formation of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and DNA damage in the colon of male Wistar rats by the ACF and Comet assays, respectively. AEP was administered orally at 0.01%, 0.03%, 0.1%, and 0.3% in the drinking water, which resulted in doses of approximately 12, 34, 108, and 336 mg/kg body weight/day. Animals were also given a single subcutaneous injection of 40 mg/kg DMH and sacrificed 4 hr later for evaluating DNA damage, or 4 doses of 40 mg/kg DMH, administered 2 doses/week for 2 weeks, and sacrificed 12 weeks after the last injection for evaluating ACF development in the distal colon. Administration of AEP either simultaneously with or after the DMH treatment resulted in no statistically significant reduction of ACF. In contrast, 0.01%, 0.03%, and 0.3% AEP, given simultaneously with DMH, reduced DNA damage induction in the mid and distal colon. However, 0.3% AEP alone increased DNA damage in the colon. In conclusion, AEP had no effect on the formation of DMH-induced ACF in rat colon, but it modulated DMH-induced DNA damage in colon cells. Further investigations are recommended in order to establish the conditions under which propolis produces either protective or deleterious effects.

Histological and immunohistochemical observations of mucin‐depleted foci (MDF) stained with Alcian blue, in rat colon carcinogenesis induced with 1,2‐dimethylhydrazine dihydrochloride

The usefulness of mucin-depleted foci (MDF), which have recently been proposed as a new preneoplastic biomarker in rat colon carcinogenesis, was histologically investigated in rat colonic tissues treated with 1,2-dimethylhydrazine dihydrochloride (DMH). The relationship among aberrant crypt foci (ACF), MDF and beta-catenin accumulated crypts (BCAC) was examined by comparing the corresponding computer-captured images. Twelve male F344 rats were given DMH s.c. at a dose of 40 mg/kg body weight, once a week for 2 weeks, and randomly divided into two groups. Rats in group 1 were given normal drinking water, while those in group 2 were given drinking water containing indomethacin (IND) at 16 ppm for 6 weeks. All animals were sacrificed 8 weeks after the first DMH treatment. The resected colons were fixed in 10% formalin, and stained with Alcian blue for observation of ACF and MDF. Histological and immunohistochemical analysis revealed that the numbers of ACF, MDF and overlapping lesions in group 2 (treated with IND) were significantly decreased, compared with those in group 1. The number of BCAC in group 2 was also significantly lower than that in group 1. The reduction (61.5%) of MDF by IND was much greater than that (29.3%) of ACF. Analyses of the computer-captured images indicated that MDF had more frequent dysplastic changes and overexpression of beta-catenin than did ACF. MDF having over 4 crypts or MDF with the appearance of ACF corresponded well to BCAC. These results suggest that MDF may be useful as an early biomarker in colon carcinogenesis.

Nutritional-pharmacological combinations--a novel approach to reducing colon cancer incidence.

Recent studies have suggested that n-9 fatty acids in olive oil prevent colon carcinogenesis while n-6 PUFA seems to activate this process. To evaluate the effects of nutritional-pharmacological combinations made up of olive or soy oil-based diets and the drug sulindac, on colon cancer incidence in a chemically induced (1,2-dimethylhydrazine, DMH) rat cancer model. Male rats were assigned to two different dietary regimes based on a standard murine defined diet (AIN-76A) containing either a low (4%) or high (15 %) concentration of olive or soy oil. Some groups also received sulindac in their food (80 mg/kg food) starting from the ninth week following the first DMH or vehicle administration. Oleic and linoleic acid reached higher levels in plasma and liver lipids when rats were fed high concentrations of olive or soy oil, respectively. Rats fed a low or high soy oil-based diet showed no significant difference in the number of aberrant crypt foci (ACF) in proximal or distal colon specimens. In contrast, rats fed a higher olive oil-based diet developed a significantly lower number of ACF than rats fed a low concentration of olive oil. Addition of sulindac reduced the number of ACF in rats fed the 4%, but not the 15%, soy oil diet. In contrast, the effect of sulindac was significant when combined with both the low and high concentrations of olive oil. High soy oil-based diet or DMH treatment upregulated colon expression of Bcl-2, but not that of cyclooxygenase-2 (COX-2). In contrast, olive oil dose-dependently downregulated the expression of both Bcl-2 and COX-2 in colonic mucosa and also abrogated the upregulation of Bcl-2 by DMH. Olive oil/sulindac combinations were effective in downregulating colonic mucosa Bcl-2 expression (with the 4% oil diet) and COX-2 expression (with the 15% oil diet). These effects were not observed in rats fed the soy oil/sulindac combinations. Caspase-3 activity in colonic mucosa was unaffected by soy oil or soy oil/sulindac combinations. The addition of olive oil, on the other hand, significantly enhanced colonic caspase-3 activity. Diets containing high levels of olive oil exert a significant protective effect from tumor development that is additive with the inhibitory effect of sulindac. These inhibitory effects are mediated by regulating the expression and activity of key proteins involved in prostaglandin-biosynthesis and apoptosis-induction pathways. It may be concluded that appropriate dietary-pharmacological combination can improve anti-tumor efficacy over either dietary or pharmacological intervention alone.

Effect of vanadium on colonic aberrant crypt foci induced in rats by 1,2 dimethyl hydrazine.

To investigate the chemo preventive effects of vanadium on rat colorectal carcinogenesis induced by 1,2-dimethylhydrazine (DMH). Male Sprague-Dawley Rats were randomly divided into four groups. Rats in Group A received saline vehicle alone for 16 weeks. Rats in Group B were given DMH injection once a week intraperitoneally for 16 weeks; rats in Group C, with the same DMH treatment as in the Group B, but received 0.5-ppm vanadium in the form ammonium monovanadate ad libitum in drinking water. Rats in the Group D received vanadium alone as in the Group C without DMH injection. Aberrant crypt foci (ACF) were formed in animals in DMH-treated groups at the end of week 16. Compared to DMH group, vanadium treated group had less ACF (P<0.001). At the end of week 32, all rats in DMH group developed large intestinal tumors. Rats treated with vanadium contained significantly few colonic adenomas and carcinomas (P<0.05) compared to rats administered DMH only. In addition, a significant reduction (P<0.05) in colon tumor burden (sum of tumor sizes per animal) was also evident in animals of Group C when compared to those in rats of carcinogen control Group B. The results also showed that vanadium significantly lowered PCNA index in ACF (P<0.005). Furthermore, vanadium supplementation also elevated liver GST and Cyt P-450 activities (P<0.001 and P<0.02, respectively). Vanadium in the form of ammonium monovanadate supplemented in drinking water ad libitum has been found to be highly effective in reducing tumor incidence and preneoplastic foci on DMH-induced colorectal carcinogenesis. These findings suggest that vanadium administration can suppress colon carcinogenesis in rats.

Chemoprevention of 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine-induced colon carcinogenesis by 1-O-hexyl-2,3,5-trimethylhydroquinone after initiation with 1,2-dimethylhydrazine in F344 rats.

The aim of this study is to investigate the chemopreventive effects of the synthetic phenolic antioxidant 1-O-hexyl-2,3, 5-trimethylhydroquinone (HTHQ) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colon carcinogenesis in rats after initiation with 1,2-dimethylhydrazine (DMH) in male F344 rats. Groups of 20-22, 6-week-old male F344 rats were given four subcutaneous injections of 40 mg/kg body wt of DMH during the initial 4 weeks. They were then maintained on powdered basal diet containing 0.03% PhIP alone, PhIP together with 0.5 or 0.125% HTHQ, 0.5 or 0.125% HTHQ alone or basal diet for 32 weeks. A small number (1.1 +/- 1.1/rat) of colon tumors were induced by DMH treatment alone. After initiation with DMH, the number of colon tumors was greatly increased to 8.3 +/- 5.6 by the administration of PhIP. Additional treatment with HTHQ dose-dependently decreased the multiplicity of colon adenocarcinomas to 4.9 +/- 2.8 and 2.6 +/- 1.4 with 0.125 and 0.5%, respectively. This treatment similarly reduced atypical hyperplasias of the ventral prostate. Furthermore, HTHQ significantly reduced the multiplicity of duodenal adenocarcinomas induced by DMH + PhIP or DMH alone. Immunohistochemically, HTHQ was revealed to suppress PhIP-DNA adduct formation in the epithelial cells of the colon and prostate in a separate 2 weeks experiment. The present results clearly showed that HTHQ has chemopreventive potential for PhIP-associated colon and prostate carcinogenesis. The observed inhibition may largely be due to interference with PhIP-DNA adduct formation. In addition, HTHQ has been demonstrated to inhibit duodenal carcinogenesis in the post-initiation stage.

Protective action of propolis on the rat colon carcinogenesis.

Propolis is a honeybee product with several biological and therapeutical properties. Its effect on the process of colon carcinogenesis and DNA damage were evaluated in the male Wistar rats using the aberrant crypt foci (ACF) assay and the comet assay, respectively. For both tests, animals were treated with the colon carcinogen 1,2 dimethylhydrazine (DMH, 40 mg/kg, s.c.) for 2 weeks (two injections/week) in order to induce both DNA damage and ACF. The animals were divided into groups that received propolis (ethanolic extract) at three different doses (10, 30, and 90 mg/kg b.w., by gavage), either simultaneously or after DMH treatment. For the comet assay, peripheral blood samples were collected 4 h after the last DMH treatment. All animals were sacrificed at the 5th week for evaluation of ACF. The results show that only the intermediate dose (30 mg/kg) of propolis, administered after DMH initiation, is significantly associated to a smaller number of aberrant crypts in the distal colon. No effect on DNA damage in peripheral blood cells, however, was verified by the comet assay. These data suggest that propolis has a protective influence on the process of colon carcinogenesis, suppressing the development of preneoplastic lesions, and probably exerts no protection against the initiation of carcinogenesis.