Modifying action of neonatal androgenization on 1,2-dimethylhydrazine-induced carcinogenesis in male CBA-strain mice

Newborn male CBA mice received a single treatment with 0.5 mg testosterone propionate. Weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) were given to 2-month-old mice. The incidence of pararenal angiosarcomas and colonic tumors in neonatally androgenized mice reached 78.5 and 71.0%, respectively by the 35th week after the DMH treatment was commenced. In DMH-treated control mice, the incidence of the above tumors amounted to 25 and 32%, respectively.     

Tumors induced by 1,2-dimethylhydrazine in hybrid mice with the pituitary gland transplanted in the kidney

1,2-Dimethylhydrazine (DMH) decreased the incidence of tumors in hypophyseal isografts in hybrid mice (CBA X C57BL/6)F1 and prevented the development of mammary tumors. In mice bearing hypophyseal isografts, there was a decrease in the incidence of DMH-induced hemangiomas of the ovary. The same mice showed no alterations in the incidence of DMH-induced uterine sarcomas, tumors of the large intestine, anal region and liver.     

Sulindac enhances cell proliferation in DMH-treated mouse colonic mucosa

In a previous study we reported that the NSAID sulindac had a marked inhibitory effect on the development of colonic tumours in mice treated with the carcinogen 1,2-dimethylhydrazine (DMH). In this study we examined the effects of sulindac in respect of cell-kinetic changes in mouse colonic mucosa as determined by flash labelling with the thymidine analogue bromodeoxyuridine (BrdUrd) at varying intervals during the process of colonic carcinogenesis. We also investigated the possibility that these changes may be modulated by misoprostol a prostaglandin E₁ analogue. Four groups of 36 mice each were treated for 18 weeks with the following drug/s respectively: (1) DMH; (2) DMH and sulindac; (3) DMH, sulindac and misoprostol; and (4) DMH and misoprostol. Three animals from each group were killed each week between the sixth week and the eighteenth week after the start of the experiment. A 1-h flash label technique was employed and paraffin sections of colonic mucosa were examined. For each animal a total of 50 perfect axially cut crypts were chosen and the following parameters determined: crypt length, labelling index and labelling index distribution: the data were analysed using the computer program GLIM. For each of the four groups, crypt lengths increased significantly with the duration of treatment with no significant difference between the groups. In sulindac-treated animals the labelling index for all positions increased with duration of treatment whereas for animals not treated with sulindac there was no significant difference in labelling index with respect to duration of treatment. The administration of misoprostol did not appear to significantly alter the effects of sulindac. It is postulated that the observed increase in cell proliferation could be a compensatory phenomenon occurring secondary to loss of crypt epithelial cells by apoptosis induced by sulindac. Also the finding of an increase in labelling index mediated by a chemopreventive agent indirectly questions the rationale behind the therapeutic manipulation of crypt cell proliferation in order to reduce the risk of colon cancer.     

Effect of neonatal androgenization on uterine sarcomagenesis induced by 1,2-dimethylhydrazine in female CBA mice

A single injection of testosterone propionate to newborn female CBA mice provoked earlier puberty and formation of permanent estrous in the majority of animals. This led to an abrupt reduction of the latent period and the increased incidence of 1,2-dimethylhydrazine-induced uterine sarcomas (90 versus 9% upon DMH injection to intact mice).     

Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models

We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQ's therapeutic potential in two different mice colon cancer models [1,2-dimethyl hydrazine (DMH) and xenografts]. We also examined TQ effects on the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion in vitro. Mice were treated with saline, TQ, DMH, or combinations once per week for 30 weeks and the multiplicity, size and distribution of aberrant crypt foci (ACF) and tumors were determined at weeks 10, 20 and 30. TQ injected intraperitoneally (i.p.) significantly reduced the numbers and sizes of ACF at week 10; ACF numbers were reduced by 86%. Tumor multiplicity was reduced at week 20 from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long-term; tumors did not re-grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly (P < 0.05) delayed the growth of the tumor cells. Using a matrigel artificial basement membrane invasion assay, we demonstrated that sub-cyto-toxic doses of TQ (40 microM) decreased C26 cell invasion by 50% and suppressed growth in three-dimensional spheroids. Apoptotic signs seen morphologically were increased significantly in TQ-treated spheroids. TUNEL staining of xenografts and immunostaining for caspase 3 cleavage in DMH tumors confirmed increased apoptosis in mouse tumors in response to TQ. These data should encourage further in vivo testing and support the potential use of TQ as a therapeutic agent in human colorectal cancer.     

Effect of epidermal chalones on induction and growth of tumors of the cervicovaginal epithelium of mice

Tumors of the cervicovaginal epithelium were induced in BALB/c female mice with local application of dimethylbenzanthra cene. In the course of inducing tumors or after their appearance the mice were treated intravaginally with 55-81% ethanolic extract of the rat skin or liver (control) containing 1% chalones. The skin extract rather than liver one delays slightly the growth and incidence of cervical and vaginal tumours, and increases the survival of animals.     

Effects of 2.45-GHz microwave radiation and phorbol ester 12-O-tetradecanoylphorbol-13-acetate on dimethylhydrazine-induced colon cancer in mice

The purpose of this study was to investigate the effects of 2.45 GHz microwave (MW) radiation on dimethylhydrazine (DMH)-induced colon cancer in mice. The subjects were 115 Balb/c mice 4 weeks of age. The animals were divided into group A (control), group B (DMH), group C (DMH + MW), and group D [DMH + 12-O-tetradecanoylphorbol-13-acetate (TPA)]. Radiation (10 mW/cm²) was delivered dorsally with the E field parallel to the mouse's long body axis in an anechoic chamber. Radiations were administered 3 hr daily, 6 days per week, over a period of 5 months. The average SAR was estimated to be 10–12 W/kg. During the course of radiation treatments, DMH was injected once per week. The tumor promoter TPA was administered once per week for 10 weeks, from the third week on, after the initial treatment. The incidence of tumors did not significantly differ between the three test groups (groups B, C, and D; P > 0.25). However, the number of tumors, the size of the tumors, and the incidence of protuberant and infiltrative types in tumor-bearing animals were higher in group D compared to groups B and C (P < 0.05). No difference was found between groups B and C (P > 0.25). The study indicates that 2.45 GHz microwave radiation at 10 mW/cm² power density did not promote DMH-induced colon cancers in young mice. The study also showed that TPA could accelerate colon tumor production if a tumor was initiated. © 1994 Wiley-Liss, Inc.     

Effect of quality and quantity of dietary fat and dimethylhydrazine in colon carcinogenesis in rats.

The effect, quality, and quantity of dietary fat on colon tumor induction by DMH were studied in rats exposed to a given regimen for two generations prior to treatment with DMH. Animals fed a 20% corn oil or 20% lard and treated with DMH had a higher incidence of colonic tumors than did rats fed a 5% corn oil, 5% lard or Purina lab chow and treated similarly. The quality of fat had no major difference on the incidence of colonic tumors.     

Fenugreek seeds modulate 1,2-dimethylhydrazine-induced hepatic oxidative stress during colon carcinogenesis

1,2-dimethylhydrazine (DMH) is a colon carcinogen which undergoes oxidative metabolism in the liver. We have investigated the modulatory effect of fenugreek seeds (a spice) on colon tumor incidence as well as hepatic lipid peroxidation (LPO) and antioxidant status during DMH-induced colon carcinogenesis in male Wistar rats. In DMH treated rats, 100% colon tumor incidence was accompanied by enhanced LPO and a decrease in reduced glutathione (GSH) content as well as a fall in glutathione peroxidase (GPx), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) activities. Inclusion of fenugreek seed powder in the diet of DMH treated rats reduced the colon tumor incidence to 16.6%, decreased the LPO and increased the activities of GPx, GST, SOD and CAT in the liver. We report that fenugreek modulates DMH-induced hepatic oxidative stressduring colon cancer     

Chemopreventive efficacy of green tea drinking against 1,2‐dimethyl hydrazine‐induced rat colon carcinogenesis

Colorectal cancer is one of the leading causes of tumour‐related deaths. In the present study, the chemopreventive effect of green tea on 1,2‐dimethylhydrazine (DMH)‐induced colon carcinogenesis was studied in male Wistar rats. The DMH group received subcutaneous injections of DMH (30 mg kg^?1 body weight) once a week for 30 weeks, the normal group received the vehicle of DMH, and the DMH + green tea group received DMH simultaneously with 1% green tea as their sole source of drinking fluid throughout the experimental period. In the DMH group treated with green tea, significant reductions in gene overexpressions of colonic nuclear factor κB (NF‐κB), tumour necrosis factor α, inducible nitric oxide synthase and cyclooxygenase 2, and NF‐κB immunostaining indicates the anti‐inflammatory effect of green tea in attenuating colon cancer. Moreover, the anti‐angiogenic and anti‐invasiveness effects of green tea were revealed as reductions of both vascular endothelial growth factor and matrix metalloproteinase‐7 mRNA expression levels. These effects were confirmed by the significant reduction of serum tumour necrosis factor α, C‐reactive protein levels, inhibition of tumour incidence, and nearly normal survival rate and colonic architecture. It can be concluded that green tea exerts a potent chemopreventive effect on colon carcinogenesis possibly due to the inhibition of NF‐κB. Copyright © 2012 John Wiley & Sons, Ltd.     

Apparent synergism between radiation and the carcinogen 1,2-dimethylhydrazine in the induction of colonic tumors in rats

We have evaluated the interaction of radiation and 1,2-dimethylhydrazine (DMH) with respect to colon carcinogenesis in the Fischer 344 rat and have demonstrated the utility of this model for future more detailed mechanistic studies. In initial experiments, single doses of abdomen-only radiation (9 Gy) or DMH (150 mg/kg) were employed alone or in combination. Radiation was administered 3.5 days prior to the DMH. At 8 months post-treatment, the incidence of DMH-induced colon tumors was doubled by prior radiation exposure. When the protocol was repeated employing a DMH dose of 135 mg/kg with a 6-month observation period, the incidence of tumors induced by DMH alone was reduced, but the combination of radiation plus DMH still resulted in an augmentation of tumor incidence. When the protocol of radiation plus DMH was repeated three times at monthly intervals, a 15-fold increase in tumor incidence (from 5 to 74%) was observed at 6 months post-treatment. This finding demonstrates an apparent synergy between the radiation and the chemical carcinogen. Throughout these studies, the appearance of carcinomas was associated with preexisting colonic lymphoid nodules. The reproducibility of tumor induction as well as range of tumor incidence generated by variations in this system may be adequately sensitive to examine the combination of much lower doses of radiation and/or chemical carcinogen. The relationship between existing lymphoid aggregates which alter local epithelial cell kinetics and which are associated with fenestrations in the basement membrane, and the development of colon cancer in congruent sites may assist in defining dose-response curves for combined agents as well as providing a system for evaluating the mechanisms underlying their interactions.     

Superoxide Dismutase Activity During Dimethylhydrazine Colon Carcinogenesis and the Effects of Cholic Acid and Indole

Copper, zinc superoxide dismutase (Cu, ZnSOD) and manganese superoxide dismutase (MnSOD) activities were measured in mouse large intestinal mucosa during dimethylhydrazine (DMH) carcinogenesis. Mice were divided into five groups. Group A was subcutaneously injected with DMH (20mg/kg) weekly and fed with a diet containing 0.2% cholic acid (C) and 0.8% indole (I). Group B was injected with DMH and given indole feeding. Group C was treated with DMH injection and cholic acid feeding. Group D was given DMH injection alone. Group E was an age-matched control group given 0.9% NaCl injection. The experiment last 21 weeks. The Cu, ZnSOD activity of intestinal mucosa in group A animals began to increase significantly at the 7th week of the experiment. In groups B, C and D, however, this enzyme was not elevated statistically until the 16th week, and then each of these groups kept an increased Cu, ZnSOD level the rest of the experimental period. MnSOD activity was elevated statistically in group C animals at the 7th week. The enzyme activity in group A and D animals increased at the 9th week, but the enzyme activity did not increase statistically until the 11th week in group B. After the 16th week of the experiment the increased activity of MnSOD in all experimental groups returned to the level of the control group. Large intestinal cancer tissues had increased Cu, ZnSOD activity and decreased MnSOD activity.     

Superoxide Dismutase Activity During Dimethylhydrazine Colon Carcinogenesis and the Effects of Cholic Acid and Indole

Copper, zinc superoxide dismutase (Cu, ZnSOD) and manganese superoxide dismutase (MnSOD) activities were measured in mouse large intestinal mucosa during dimethylhydrazine (DMH) carcinogenesis. Mice were divided into five groups. Group A was subcutaneously injected with DMH (20mg/kg) weekly and fed with a diet containing 0.2% cholic acid (C) and 0.8% indole (I). Group B was injected with DMH and given indole feeding. Group C was treated with DMH injection and cholic acid feeding. Group D was given DMH injection alone. Group E was an age-matched control group given 0.9% NaCl injection. The experiment last 21 weeks. The Cu, ZnSOD activity of intestinal mucosa in group A animals began to increase significantly at the 7th week of the experiment. In groups B, C and D, however, this enzyme was not elevated statistically until the 16th week, and then each of these groups kept an increased Cu, ZnSOD level the rest of the experimental period. MnSOD activity was elevated statistically in group C animals at the 7th week. The enzyme activity in group A and D animals increased at the 9th week, but the enzyme activity did not increase statistically until the 11th week in group B. After the 16th week of the experiment the increased activity of MnSOD in all experimental groups returned to the level of the control group. Large intestinal cancer tissues had increased Cu, ZnSOD activity and decreased MnSOD activity.     

The role of estrogen receptor-beta, in the early age-related bone gain and later age-related bone loss in female mice

The molecular and cellular mechanism of estrogen action in skeletal tissue remains unclear. The purpose of this study was to understand the role of estrogen receptor-beta, (ERbeta) on cortical and cancellous bone during growth and aging by comparing the bone phenotype of 6- and 13-month-old female mice with or without ERbeta. Groups of 11-14 wild-type (WT) controls and ERbeta knockout (BERKO) female mice were necropsied at 6 and 13 months of age. At both ages, BERKO mice did not differ significantly from WT controls in uterine weight and uterine epithelial thickness, indicating that ERbeta does not regulate the growth of uterine tissue. Femoral length increased significantly by 5.5% at 6 months of age in BERKO mice compared with WT controls. At 6 months of age, peripheral quantitative computerized tomography (pQCT) analysis of the distal femoral metaphysis (DFM) and femoral shafts showed that BERKO mice had significantly higher cortical bone content and periosteal circumference as compared with WT controls at both sites. In contrast to the findings in cortical bone, at 6 months of age, there was no difference between BERKO and WT mice in trabecular density, trabecular bone volume (TBV), or formation and resorption indices at the DFM. In 13-month-old WT mice, TBV (-41%), trabecular density (-27%) and cortical thickness decreased significantly. while marrow cavity and endocortical circumference increased significantly compared with 6-month-old WT mice. These age-related decreases in cancellous and endocortical bone did not occur in BERKO mice. At 13 months of age, BERKO mice had significantly higher total, trabecular and cortical bone, while having significantly lower bone resorption, bone formation and bone turnover in DFM compared with WT mice. These results indicate that deleting ERbeta protected against age-related bone loss in both the cancellous and endocortical compartments by decreasing bone resorption and bone turnover in aged female mice. These data demonstrate that in female mice, ERbeta plays a role in inhibiting periosteal bone formation, longitudinal and radial bone growth during the growth period, while it plays a role in stimulating bone resorption, bone turnover and bone loss on cancellous and endocortical bone surfaces during the aging process.     

Effect of testosterone propionate on the induction by 1,2-dimethylhydrazine of angiosarcomas of the renal capsule in castrated mice

Administration of 1,2-dimethylhydrazine (DMH) produced 83% (15/18) of renal capsule angiosarcomas in CBA mice. Castration that preceded the DMH-treatment reduced tumor incidence to 7% (2/29). Simultaneous administration of DMH and testosterone propionate (TP) to castrated males restored the tumor frequency (100%, 24/24). Castrated males that received TP after the cessation of the DMH treatment developed tumors in 10% (3/31). Combined treatment of castrated females with DMH and TP resulted in the development of angiosarcomas in 92% animals (22/24). It is concluded that TP enhances the stage of sarcomogenesis initiation induced by DMH.     

Activity of 1,2-dimethylhydrazine in the mouse bone marrow micronucleus assay using a triple- and a single-dosing protocol

In the present study the ability of 1,2-dimethylhydrazine (DMH) to induce micronuclei in mouse bone marrow erythrocytes was investigated using 2 different dosing regimens. DMH caused an increase in micronuclei in both male and female mice following single administration and sampling after 24 h. The effect was more pronounced in female than in male animals. Triple administration of DMH at concentrations corresponding to 80, 40 and 20% of the median lethal dose (MLD) did not increase the incidence of micronuclei in either sex. Small increases in micronucleus incidence were observed after triple dosing at 100% of the MLD value. These results suggest that a future micronucleus protocol should include animals of both sexes and single and repeated administration of the test substance.     

Apoptotic differences in experimentally induced colorectal rat tumours

The presence of apoptotic bodies and of intraepithelial lymphocytes (IELs) were assessed in colorectal adenomas and adenocarcinomas induced in 158 rats by two different carcinogens: 1,2 dimethylhydrazine (DMH) and glutamic acid pyrolysate (GLU-P-1 and 2). Apoptotic granules were present in 97.5% ( n=40) of the 41 GLU-induced adenomas and adenocarcinomas, but only in 20.5% ( n=24) of the 117 DMH-induced tumours. IELs were found in 95.1% ( n=39) of the 41 GLU-induced tumours but only in 21.4% (n=25) of the 117 DMH-induced neoplasias. The differences were significant ( p< 0.001). The presence of IELs and apoptotic granules in GLU tumours (and their absence in the majority of the DMH tumours) is new evidence that IELs are the cells from which many of the apoptotic granules — seen in colorectal neoplasias — derive. GLU neoplasias were induced following daily treatment, for 24 months (about half the life span of the animals) and DMH neoplasias by weekly doses, for a period of only 2.8-6 months. It would appear that 'slowly growing' colorectal GLU neoplasias often attract IELs and trigger lymphocytic apoptosis whereas 'quickly growing' DMH tumours seldom evoke those reactions.     

Folic acid and sodium butyrate prevent tumorigenesis in a mouse model of colorectal cancer

Colorectal cancer is a leading cause of morbidity and mortality worldwide, and its incidence has been increasing in recent years. The role of epigenetic modifications, including DNA methylation and histone modifications, has only recently been investigated. In this study, the effects of epigenetic agents such as folic acid (FA) and sodium butyrate (NaBu) on the development of colorectal cancer induced by 1,2-dimethylhydrazine (DMH) using ICR mice was examined. Of the mice treated in a chemopreventive manner with epigenetic agents, FA and NaBu, 15–50% developed colorectal cancer at 24 weeks compared with a 95% incidence of colorectal cancer in DMH-treated control mice. Folate deficiency can alter cytosine methylation in DNA leading to inappropriate activation of the proto-oncogene c-myc. We detected lower levels of p21WAF1 gene expression in colorectal cancer samples, as well as significantly lower levels of acetylated histone H3, compared with samples from corresponding normal colorectal mucosa. In contrast, administration of NaBu increased levels of p21WAF1 mRNA and p21WAF1 protein, and was associated with an accumulation of histone acetylation. In summary, our results show that FA and NaBu reduce the incidence of colorectal cancer induced by DMH-induced in ICR mice, and therefore we hypothesize that targeting epigenetic targets should be further investigated for the prevention of colorectal cancer in humans.     

Protective role of luteolin in 1,2-dimethylhydrazine induced experimental colon carcinogenesis

The aim of the present study was to unravel the chemopreventive effect of luteolin on bacterial enzymes such as beta-glucuronidase and mucinase in a colon carcinogenesis model induced by 1, 2-dimethyl hydrazine (DMH). Twenty mg/kg body weight of DMH were administered subcutaneously once a week for the first 15 weeks and then discontinued. Luteolin (0.1, 0.2, or 0.3 mg/kg body weight/everyday (p.o.) was administered in a dose dependent manner at the initiation and also at the post-initiation stages of carcinogenesis to DMH treated rats. The animals were sacrificed at the end of 30 weeks. Colon cancer incidence and the activities of bacterial enzymes beta-glucuronidase (in the proximal colon, distal colon, intestines, liver and colon contents) and mucinase (colon and fecal contents) were significantly increased in DMH -treated rats compared to the control rats. On luteolin administration, colon cancer incidence, number of tumors per rat and the activities of beta-glucuronidase and mucinase, were significantly decreased both in the initiation and post-initiation stages of colon carcinogenesis dependent on the three different doses given. The increase in beta-glucuronidase activity may augment the hydrolysis of glucuronide conjugates, liberating toxins, while the increase in the mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Thus our results demonstrate for the first time that luteolin, a dietary flavonoid, exerts chemopreventive and anticarcinogenic effects against DMH induced colon cancer.     

Reinfection of virus free mice with mouse mammary tumour virus

BR6/Icrf mice carrying a milk-transmitted mammary tumour virus (MMTV) develop tumours after several pregnancies. If the mice are freed from MMTV, no tumours develop. In the experiments described in this paper, MMTV was reintroduced into MMTV-free mice by foster nursing, which was least effective if the pups were exposed to the virus only during the first week of life. Exposure for even a short time after that age led to a tumour incidence similar to that found in normally infected mice. Reinfection was also achieved by injection of MMTV-containing milk into weanling or pregnant mice, and was then transmitted naturally to the next generation.