Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation

The present study was carried out to examine the chemopreventive effects of carotenoids such as fucoxanthin, lycopene and lutein as well as curcumin and its derivative, tetrahydrocurcumin (THC), on development of putative preneoplastic aberrant crypt foci (ACF) in colons of mice initiated with 1,2-dimethylhydrazine dihydrochloride (DMH). Influence on proliferation of colonic crypt epithelial cells was also assessed in terms of 5-bromo-2'-deoxyuridine (BrdU) incorporation. Five-week-old B6C3F1 male mice were divided into three groups, groups 1 and 2 being given DMH (20 mg/kg body wt, s.c.) twice a week for 3 weeks. Animals of group 1 were then treated with one of the test compounds, lycopene (0.005% and 0.0025%) or fucoxanthin (0.01%) in the drinking water and lutein (0.05%), curcumin (0.5%) or THC (0.5% and 0.2%) in the diet from weeks 5-12. Group 2 served as a carcinogen alone control and group 3 mice were given test compounds alone. All animals were killed at week 12. Numbers of ACF/mouse in the group 1 treated with fucoxanthin (47.1 +/- 13.7), lutein (42.6 +/- 19.6) or 0.5% THC (46.6 +/- 17.7) were significantly decreased as compared to the control group 2 value (63.3 +/- 19.4) (P < 0.01). Numbers of aberrant crypts (ACs)/mouse were also significantly lower after treatment with lutein (79.9 +/- 34.7) or 0.5% THC (81.8 +/- 32.5) than in the control group (115.1 +/- 37.1) (P < 0.01). BrdU labeling indices (LI) in mice treated with lutein and 0.5% THC were significantly decreased in both upper and lower half compartments of colonic crypts as compared to the controls (P < 0.05 and 0.01, respectively), especially the upper half data corresponding to reduction of ACs/mouse. The results thus suggest that fucoxanthin, lutein, and THC may have potential as chemopreventive agents against colon carcinogenesis.      

Increased carcinogenic action of dimethylnitrosamine after prior administration of carbon tetrachloride

Rats were given a single dose of dimethylnitrosamine (DMN, 20 mg/kg body weight) alone or 42 or 60 hours after a non-lethal hepatotoxic dose of carbon tetrachloride (CC1₄) and killed 12 months later. DMN alone produced no tumours in the kidney and a few in the liver, but when given 42 hours after CC1₄, tumours formed in the kidneys and the number in the liver was increased. When given after 60 hours, the incidence of kidney tumours was less but that of liver tumours was further increased. A larger dose of DMN (40 mg/kg) was tolerated 42 hours after CC1₄ and enhanced the number of kidney and liver tumours, the latter apparently due to an increased proportion of cholangiomata. Numerous small focal proliferations of atypical liver cells and of bile duct epithelium were observed after treatment with DMN. The incidence of these lesions in the different experimental treatments varied in a similar manner to the liver tumours.     

Tumor-promoting activities of hydroquinone and 1,1-dimethylhydrazine after initiation of newborn mice with 1-methyl-1-nitrosourea.

To clarify the suitability of a newborn-mouse carcinogenesis assay to detect tumor-promoting activities of carcinogens, the non-genotoxic hydroquinone (HQ) and genotoxic 1,1-dimethylhydrazine (UDMH) were administered to mice during the promotion stage after treatment with 1-methyl-1-nitrosourea (MNU) (20 mg/kg body wt, single intraperitoneal injection) at day 9 after birth. Initiated males and females thus received either HQ at 0.8% in basal diet, or UDMH, at 20 mg/kg body wt once weekly by subcutaneous injection, from day 14 until the end of the experiment at 30 weeks of age. Uninitiated newborn mice, given an injection of the vehicle (0.01 M citrate buffer (pH 5.5), 20 mg/kg body wt), also received HQ or UDMH in the same way. Histopathologically, focal proliferative lesions were found in the livers of male mice and in the lungs of both male and female mice in the MNU-treated groups. HQ significantly increased the incidence and multiplicity of altered hepatocellular foci, the combined incidence of hepatocellular adenomas and carcinomas in males and the incidence and multiplicity of lung adenomas and the combined incidence of lung adenomas and carcinomas in female mice. In addition, four out of eleven MNU + HQ-treated male mice developed lung carcinomas, showing a significant elevation in multiplicity. UDMH also exhibited a tendency to increase the incidence and multiplicity of lung adenomas in female mice. Thus tumor-promoting effects of HQ or UDMH were apparently exerted in the target organs and the MNU-initiated two-stage newborn-mouse carcinogenesis assay may be useful for detection of genotoxic or non-genotoxic carcinogenicity.     

Influence of the rhythm and route of 1,2-dimethylhydrazine administration on its carcinogenic effect in mice

Effects of route of administration (subcutaneous, intraperitoneal, by stomach tube and with drinking water) and dose fractionation on the carcinogenicity of 1,2-dimethylhydrazine (DMH) were studied in CBA and (C57B1/6j X CBA)F1 mice. Fractionation of DMH dose given subcutaneously exerted different effects on tumors at various sites: decrease of colon and anal tumor incidence, increase of vascular liver tumors and renal adenomas and no influence on hepatoma, lung adenoma and uterine sarcoma induction. When given with drinking water, DMH did not induce colon and anal tumors but produced high incidence of vascular neoplasms. No such effect was observed when DMH was administered weekly by stomach tube. Alteration of the organotropism of DMH given with drinking water is attributed by authors to the decrease of single DMH dose and not to peroral route of administration.     

Pathology of liver necrosis and regeneration after administration of dimethylnitrosamine in massive doses

Two groups of dogs were given dimethylnitrosamine (DMNA), a hepatotoxic drug, in a single lethal dose. Control dogs were allowed to die. Experimental dogs received life-saving auxiliary liver transplants which were removed after recovery of the host liver or when rejection occurred. The histopathologic findings in the host livers of the two groups are presented. Remarkable variations were noted even among livers from the same group. Centrilobular and midzonal congestion and necrosis were the main findings but appeared less severe than one would expect in view of the lethality of DMNA in control dogs. In contrast to the changes noted with long-term administration of the drug to dogs, there was nothing in this series to suggest cirrhosis.     

Organ-specific effects of 1,2-dimethylhydrazine in hamster

Uptake and metabolism of the carcinogen 1,2-dimethylhydrazine (DMH) were compared in isolated epithelial cells from the colon and the small intestine. A new method was developed to separate colonic epithelial cells into surface columnar cells and crypt cells without the use of any proteolytic enzymes. Colonic columnar cell-enriched fraction exhibited DMH metabolism two to three times higher than that of crypt cells. The carcinogen binding was much lower in the small intestine as compared to the colon. In the small intestine, the crypt cell-enriched fraction showed higher carcinogen binding as compared to villus cells. Pyrazole was found to inhibit DMH binding by isolated small intestinal and colonic epithelial cells. The extent of inhibition was maximum in cells showing the greatest ability to incorporate DMH.     

Disruption of circadian rhythms of biogenic amines in rat hypothalamus upon administration of 1,2-dimethylhydrazine]

Levels of norepinephrine (NE), dopamine (DA) and the main metabolite of serotonin 5-hydroxyindoleacetic acid (5-HIAA) have been measured in the suprachiasmatic nuclei (SCN), preoptic area (PA), and median eminence (ME) of hypothalamus of rats after sole subcutaneous injection of 1,2-dimethylhydrazine (SDMH). Circadian changes of DA in all the brain structures under study as well as of NE in PA were observed in the control group, their levels in the mornings being higher than in the evenings; a circadian change of 5-HIAA in SCN had an opposite tendency. Both the evening (11 p.m.) and morning (11 a.m.) administrations of SDMH at the dose of 21 mg/kg body weight resulted in disturbances of all the circadian rhythms observed in control. In some cases only a 12 hrs circadian rhythms phase shift was found, in the others these rhythms of neurotransmitters disappeared entirely. The evening administration of SDMH, unlike the morning one, resulted in an increase in total NE content in the hypothalamic structures under study. It is suggested that the effect of SDMH on the levels and circadian rhythms of neurotransmitters in the hypothalamic structures under study is due to affecting activities of the enzymes of biogenic amines synthesis, synaptic transmission, melatonin synthesis and secretion rhythms, as well as to its genotoxic influence upon the genes controlling circadian actions.     

Hepatocyte initiation during continuous administration of diethylnitrosamine and 1,2-sym-dimethylhydrazine

Hepatocyte initiation, as indexed by growth-selected gamma-glutamyl transferase-positive foci, was measured during continuous exposure to diethylnitrosamine (DEN) at concentrations used in previous DEN bioassay, DNA adduct, and cell replication studies. Hepatocyte initiation increased in a dose- and lobe-specific manner. The efficiency of DEN as an initiating agent was not affected by DEN dose rate over concentrations ranging from 4 to 40 ppm. In a similar experiment the initiating abilities of 1,2-sym-dimethylhydrazine and DEN were compared under continuous exposure regimens. Foci induction increased in a lobe- and time-dependent manner. When left and median lobes were compared, the initiating ability of the two compounds correlated with their carcinogenicity. However, when corresponding anterior lobes were compared, no such correlation was observed.     

Degenerative behavior of epithelial cells in the colonic crypt of the mouse following administration of colonic carcinogen, 1,2-dimethylhydrazine

The degenerative behavior of cells following administration of 1,2-dimethylhydrazine was analyzed in its target organ, the distal colon of the mouse. Within 3–6 h after carcinogen treatment, an increasing number of epithelial cells in the proliferative compartment of the crypt degenerated. Degenerating cells were present most frequently as phagosomes in the neighboring epithelial cells, and infrequently as pyknotic nuclei being extruded from the epithelial lining in the crypt. Epithelial cells prelabeled with [³H] thymidine degenerated first, followed by those not prelabeled, indicating that the carcinogen-induced degeneration of cells occurred after passage of cells through the DNA synthesis phase.     

Colonic cancer induced by 1,2-dimethylhydrazine (DMH) in rats after partial colectomy

Using colonic cancer induced by DMH as experimental model, carcinogenic rate in the rats with or without partial colectomy was compared in order to study the etiology of the local recurrence of large bowel cancer after radical resection and observe the influence of operative injury on carcinogenesis. Sixty five male Wistar rats were divided into two groups: 48 with a partial colectomy (group 1) and 17 controls (group 2). All were given subcutaneous injection of DMH 20 mg/kg weekly for twenty weeks. Then, some rats were killed on scheduled time, the others were sacrificed in the 29th week. The results showed that carcinogenic rate was 87.5% and 58.8% in groups 1 and 2 (P less than 0.05). The tumor number in anastomotic site in group 1 (57.1%) was much higher than that at corresponding site in group 2 (28.6%) (P less than 0.05). It is suggested that the trauma itself be one of the promoting factors for cancer recurrence in addition to implantation during operation, residual tumor, etc. Large bowel cancer induced by DMH in rats may be used as an experimental model in studying the same cancer in the human being. Furthermore, after having given DMH, large bowel cancer incidence of the rats in different intervals is described.     

Tumours of the anal region induced in mice by 1,2-dimethylhydrazine

CBA female mice treated with 1,2-dimethylhydrazine developed a high incidence of benign and malignant tumours in the anal region. Many of these tumours originated from the perianal glands rather than the epidermis.     

Lack of initiation activity in rat liver of low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

It has been generally accepted that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged for cancer risk assessment in humans. Here we examined low dose carcinogenicity of a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), using an in vivo medium-term bioassay to detect initiating activity for rat hepatocarcinogenesis. With MeIQx initiation at various doses followed by administration of phenobarbital, a well known hepatopromoter, no induction of glutathione S-transferase placental form-positive foci, assessed as preneoplastic lesions, was noted at doses of 0.001-1 ppm. The results imply a no-observed effect level for hepatocarcinogenicity with this genotoxic agent.     

Suppression of diethylnitrosamine-initiated preneoplastic foci development in the rat liver by combined administration of four antioxidants at low doses.

Potential synergism between 4 antioxidants acting at low doses on development of glutathione S-transferase placental form (GST-P)-positive liver cell foci was examined in male rats initially given diethylnitrosamine (200 mg/kg, i.p.). Beginning 2 weeks after the initiation, rats received the antioxidants, individually or in combination, in the diet for 6 weeks. All rats were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. The numbers and areas of GST-P-positive foci were significantly decreased by single treatment with butylated hydroxyanisole (BHA, 1%), tert-butylhydroquinone (TBHQ, 1%) and catechol (0.8%), but not with sesamol (0.5%). Combined treatments (BHA + TBHQ, catechol + sesamol, or all 4 chemicals) at a quarter of the above dose levels resulted in decrease in numbers and areas of foci to levels less than the sums of individual inhibition data obtained with the one-quarter levels. Although these combined effects were not statistically significant in the additive model, the results indicate possible synergistic suppression of carcinogenesis by low-dose combined treatment with anti-cancer agents and the usefulness of the present protocol for this type of analysis.