Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat

To determine whether supplemental dietary calcium and/or vitamin D deficiency are involved in modulating colon cancer induced by 1,2-dimethylhydrazine (DMH), Sprague-Dawley rats were fed diets containing either: (a) a normal content of calcium (0.87%) and phosphorus (0.60%) with 2.2 IU of vitamin D3 per g of feed (group A); (b) the same diet as group A, but with calcium and phosphorus increased to 1.80 and 0.80%, respectively (group B); or (c) a vitamin D-deficient diet with supplemental calcium (1.80%) and phosphorus (0.80%) (group C). After 6 weeks on their respective diets, one-half the animals in each group were given s.c. injections of either vehicle or DMH (20 mg/kg body weight/week) for 26 weeks. Animals were then sacrificed and the incidence of tumors as well as the number of tumors per tumor-bearing rat were determined. Colonic mucosal polyamine levels were measured after 15 weeks of exposure to vehicle or DMH, before development of histologically recognizable neoplasms. The results of these experiments demonstrated that neither calcium supplementation alone nor supplemental calcium in conjunction with vitamin D deficiency altered the incidence of colonic cancer induced by this carcinogen. Supplemental calcium, however, significantly decreased the number of rats with multiple tumors and reduced tumor size. Moreover, vitamin D deficiency abolished these protective effects of calcium on colon cancer in this experimental model. DMH treatment increased polyamine levels in the premalignant colonic mucosa in group A rats. This carcinogen-induced effect was blunted by high dietary calcium. Vitamin D-deficient, calcium-supplemented rats (group C) showed an increase in N1-acetylspermidine, but not the other polyamines, with DMH treatment.     

Chemopreventive effects of early-stage and late-stage supplementation of vitamin E and selenium on esophageal carcinogenesis in rats maintained on a low vitamin E/selenium diet

Low vitamin E and selenium (Ve/Se) nutritional status is known to be associated with increased risk of esophageal squamous cell carcinoma (ESCC). A previous human intervention trial demonstrated that Ve/Se supplementation decreased the occurrence of esophageal cancer death among younger participants but not among older ones. In this study, we intended to mimic this human nutritional status to determine the chemopreventive effects of Ve/Se supplementation at the early or late stage of esophageal carcinogenesis in rats maintained on a low Ve/Se diet. ESCC was induced in F344 rats with N-nitrosomethylbenzylamine (NMBzA) (0.35 mg/kg body wt, subcutaneously, three times per week for 5 weeks). The rats were maintained on a modified AIN-93M diet with low levels of Ve/Se or supplementation to the normal level by using the AIN-93M diet. At Week 25, the numbers of visible tumors and ESCC were significantly lower in rats on AIN-93M diet during the entire experimental period (Group D) or during the early stage (Group B) but not during the late stage (Group C). Ve/Se supplementation (switching from the low Ve/Se diet to the AIN-93M diet) also decreased cell proliferation, angiogenesis, 8-hydroxy-2'-deoxyguanosine, biosynthesis of prostaglandin E2 and leukotriene B4, expression of cyclooxygenase 2 and 5-lipoxygenase in the esophagus. Our results demonstrated that Ve/Se supplementation inhibited NMBzA-induced esophageal carcinogenesis in rats on low Ve/Se diet, and supplementation during the early stage is more effective than during the late stage of carcinogenesis.     

Enhancement of methylbenzylnitrosamine-induced esophageal carcinogenesis in zinc-deficient rats: effects on incorporation of [3H]thymidine into DNA of esophageal epithelium and liver

[³H]Thymidine incorporation was measured in esophageal epitheliumand liver DNA of rats fed a control diet ad libitum, a zinc-deficientdiet or a control diet, pair fed to the food intake of zinc-deficientrats. Additional groups of rats on each diet were dosed i.g.(2.5 mg/kg body wt) one or six times with the esophageal carcinogenmethylbenzylnitrosamine (MBN). The zinc-deficient diet significantlyincreased [3H]thymidine incorporation into esophageal epitheliumDNA at 14 days and reached a maximum at 28 days which was approximatelythree times that of the control ad libitum diet rats. Pair feedingsignificantly decreased incorporation into the esophageal epitheliumDNA relative to the control ad libitum diet, by approximately60% after 14 days. None of the diets affected [3H]thymidineincorporation in liver DNA. [³H]Thymidine incorporation wassignificantly inhibited for 48–72 h in the esophagealepithelium and liver DNA of all dietary groups after a singlei.g. dose of MBN before returning to predos-ing levels. Theperiod of inhibition of [3H]thymidine incorporation was lengthenedin all groups after six doses of MBN; the greatest increase(10 days) being noted in the esophageal epithelium DNA of thezinc-deficient rats. This inhibition was followed by a significantincrease above predosing levels in the esophageal epitheliumbut not in the liver. The maximum increase occurred 7 days afterthe completion of MBN dosing in the control ad libitum group,at 14 days in the control pair-fed group and at 36 days in thezinc-deficient group. These findings suggest that the enhancementof MBN-induced esophageal tumors by zinc deficiency is due inpart to the increased proliferation of the target cells witha concomitant greater accessibility of the cellular DNA to thecarcinogen.     

Interactions of selenium deficiency, vitamin E, polyunsaturated fat, and saturated fat on azoxymethane-induced colon carcinogenesis in male F344 rats

The effect of the interaction of selenium deficiency, excess vitamin E, and type of fat on colon carcinogenesis induced by azoxymethane (AOM) was studied in male F344 rats. The experimental diets, based on a Torula yeast diet and containing 20% stripped corn oil or 20% stripped lard, were as follows: 1) selenium deficient with adequate (50 mg/kg diet) vitamin E, 2) selenium deficient with excess (750 mg/kg diet) vitamin E, 3) selenium adequate with adequate vitamin E, and 4) selenium adequate with excess vitamin E. Starting at about 3 weeks of age, animals were fed the experimental diets, and at 7 weeks of age all animals except the vehicle-treated controls were given sc injections of AOM (15 mg/kg body wt) once weekly for 2 weeks. Animals were fed the experimental diets until termination of the experiment. Selenium deficiency significantly inhibited the incidence (percentage of animals with tumors) and multiplicity (tumors per animal) of colon adenocarcinomas and adenomas, whereas excess vitamin E had no effect on colon carcinogenesis. There was no interaction between the selenium status and vitamin E; the selenium status and type of fat; vitamin E and type of fat; and among selenium status, vitamin E, and type of fat.     

Time-selective chemoprevention of vitamin E and selenium on esophageal carcinogenesis in rats: the possible role of nuclear factor kappaB signaling pathway

Previous human intervention trial demonstrated that vitamin E (Ve) and selenium (Se) supplementation decreased esophageal cancer deaths among younger participants, but may have no effect or produce an opposite effect among older ones. In our study, we intended to mimic this human nutritional trial to determine the chemopreventive effects of Ve/Se supplementation at the early or late stage of esophageal carcinogenesis in rats. Esophageal squamous cell carcinoma (ESCC) was induced in Fischer 344 rats with N-nitrosomethylbenzylamine (NMBzA, 0.35 mg/kg BW, s.c., three times per week for 5 weeks). The rats were maintained on a modified AIN-93M diet with low levels of Ve/Se or supplemented with high levels of Ve/Se at different stages. At Week 25, the number and volume of visible tumors, the numbers of dysplasia and ESCC were significantly lower in rats of supplementation during the early stage (Group C) or during the entire experimental period (Group E), but not during the late stage (Group D). Ve/Se supplementation at the early stage also significantly decreased cell proliferation, nuclear factor kappaB (NFκB) activation, protein and mRNA expression of cyclooxygenase 2 and 5-lipoxygenase and biosynthesis of prostaglandin E2 and leukotriene B4 during the carcinogenesis of rat esophagus. Our results demonstrated that the chemopreventive efficacy of Ve/Se supplementation on NMBzA-induced esophageal cancer is time selective and that supplementation during the early stage is clearly effective but probably ineffective during the late stage of carcinogenesis. NFκB signaling pathway activation and aberrant arachidonic acid metabolism might be the underlying mechanism.     

Dietary zinc deficiency increases the methylbenzylnitrosamine- induced formation of O6-methylguanine in the esophageal DNA of the rat

Dietary zinc deficiency in combination with environmental exposureto methylbenzylnitrosamine (MBN) is associated with an increasedincidence of esophageal carcinoma in man. The proposed mechanismof MBN-induced esophageal carcinoma is through metabolic activationof MBN to form benzaldehyde, and a carbonium ion which methylatesDNA. MBN is known to methylate DNA forming O⁶-methylguanine(O⁶ MeG) adducts. These adducts can induce guanine to adeninepoint mutations and such mutations are responsible for certaincarcinogen-induced tumors. Rats maintained on a zinc-deficientdiet exhibit an increased incidence of MBN-induced esophagealcarcinoma when compared with ad libitum and pair-fed controls.The caloric restriction of the pair-fed controls was associatedwith a lower incidence of MBN-induced esophageal carcinoma thanwas observed in the ad libilum controls. These differences intumor incidence were associated with alterations in the formationand clearance of MBN-induced esophageal O⁶-MeG. Weanling maleSprague-Dawley rats were raised on egg protein diets containing2.3 p.p.m. zinc (low zinc) or 50 p.p.m. zinc (control). Onegroup of control animals was fed the control diet ad libitumand a second group pair-fed the control diet to match the intakeof the zinc-deficient group. After 3 weeks on the diets theanimals were injected with a single dose of MBN (2.0 mg/kg b.w.)and levels of esophageal O⁶-MeG were determined after 1, 3,6 and 24 h. O⁶-MeG was significantly higher in the zinc-deficientanimals than in controls, with the pair-fed controls demonstratingO⁶-MeG levels lower than the ad libitum controls. Thus, dietaryzinc deficiency results in significantly increased levels ofMBN-induced esophageal O⁶-MeG. and caloric restriction resultsin decreased levels of MBN-induced esophageal O⁶-MeG. Thesechanges in esophageal O⁶-MeG may in part explain the increasedincidence of MBN-induced esophageal carcinoma observed withdietary zinc deficiency.     

Zinc deficiency and methylbenzylnitrosamine-induced esophageal cancer in rats.

Dietary zinc deficiency increases the incidence of and shortens the lag time for induction of esophageal tumors in rats by methylbenzylnitrosamine (MBN). Groups of control and zinc-deficient outbred Charles River CD rats were given 24, 17, 8, or 4 doses of MBN, administered twice weekly by intragastric intubation at doses of 2 mg/kg body weight. Between 58 and 93 days after the beginning of treatment, all rats were killed and examined. The frequency of esophageal tumors in the zinc-deficient groups was significantly higher than in the corresponding control groups. Following ip injection of [methyl-14C]MBN, DNA and RNA of esophagus and liver were more noticeably labeled than kidney, lung, and small intestine, which exhibited only modest labeling. In vitro incubation with [14C]MBN of tissue slices from esophagus, liver, kidney, and small intestine produced similar results. This pattern correlates well with the results of our studies of MBN-induced esophageal tumorigenesis.     

Differential induction of DNA strand breaks by nitrosamines in the rat liver and esophagus

Hepatic and esophageal nuclei were isolated from Sprague-Dawley rats treated with 5 mg/kg dimethylnitrosamine (DMN), 100 mg/kg diethylnitrosamine (DEN) and 2.5 mg and 10 mg/kg methylbenzylnitrosamine (MBN) and subjected to alkaline elution to determine DNA strand breaks and their subsequent repair. Results obtained showed that hepatic nuclei isolated from rats 4 h after treatment by either DMN or DEN had about 60% of the DNA eluting through the filter. However, at 12 h post treatment, while about 50% of the single-strand breaks in dimethylnitrosamine treated rats were repaired, only about 10-15% of such breaks induced by DEN were repaired in the liver. That DEN is a more effective inducer of hepatic preneoplastic lesions could thus be attributed to this slow repair of the DEN induced lesions. Strand breaks were neither induced by DMN in the esophagus nor by MBN in the liver, the nontarget tissues. More surprising, however, was the finding that MBN induced little or no single-strand breaks in its target tissue, the esophagus. Furthermore, there was no evidence for DNA-protein cross-linking or alkali labile sites in the esophageal DNA. The results indicate that DNA damage induced by the initiating carcinogen in the target tissue may not necessarily involve strand breaks.     

Effect of deficiencies of selenium and vitamin E alone or in combination on the induction of mammary carcinogenesis by 1-methyl-1-nitrosourea

Experiments were conducted to determine the dietary levels ofselenium and vitamin E that could be fed chronically to inducea deficiency of one or both nutrients. It was observed thatdiets containing <0.02 mg Se/kg and 3 IU vitamin E/kg induceda deficiency without drastically altering growth performanceor survival. Once established, these dietary conditions wereused to investigate the effect of single or combined deficienciesof selenium and vitamin E on the promotion phase of mammarycarcinogenesis induced by 1-methyl-1-nitrosourea (MNU). In thecarcinogenesis experiment, 21 day old female Sprague-Dawleyrats were fed a torula yeast formulated diet deficient in seleniumand vitamin E until they were 50 days of age. At that time,each rat was injected i.p. with 12.5 mg MNU/kg body weight.Seven days thereafter, rats were randomly assigned to one offour treatment groups that were deficient or adequate in seleniumand/or vitamin E. The experiment was terminated 30 weeks aftercarcinogen treatment. In comparison to rats fed the adequatediet, final cancer incidence and number were higher and cancerlatency shortened in rats consuming the diet deficient in bothselenium and vitamin E. Single deficiencies of either nutrientfailed to significantly alter the tumorigenic response.     

Prevention by aspirin and its combination with alpha-difluoromethylornithine of azoxymethane-induced tumors, aberrant crypt foci and prostaglandin E2 levels in rat colon

The dose-response relationship in male F344 rats was determined for the ability of aspirin administered in the diet to prevent azoxymethane (AOM)-induced colon cancer and aberrant crypt foci (ACF) and to reduce prostaglandin E2 (PGE2) levels. Starting at either 7 or 22 weeks of age, the rats received aspirin. All rats received two doses of AOM (15 mg/kg each on days 7 and 14) and were killed on day 36. The lowest concentrations of aspirin to prevent ACF or reduce PGE2 levels were 600 and 400 mg/kg, respectively. To evaluate the prevention of tumors, rats received either 0 or 400 mg/kg aspirin for a total of 39 weeks with AOM (30 mg/kg) administered 7 days after the start of treatment. Aspirin had no effect on the yield of colon tumors. In a second experiment, rats started to receive 0, 200, 600 or 1800 mg/kg aspirin or 1000 mg/kg alpha-difluoromethylornithine (DFMO) +/- aspirin. Eight and 15 days later, all the rats received 15 mg/kg AOM. Eleven weeks later, animals that were receiving the control diet started to receive 0, 200, 600 or 1800 mg/kg aspirin; 1000 or 3000 mg/kg DFMO; or 1000 mg/kg DFMO + 200 or 600 mg/kg aspirin. The animals were killed 32 weeks later. DFMO effectively reduced the yield of colon tumors when administered starting either before or after AOM while aspirin was much weaker. The combination of aspirin + DFMO administered after AOM was synergistic. Both aspirin and DFMO decreased the Mitotic Index, while apoptosis was increased only by DFMO. Our results demonstrated that aspirin and DFMO could prevent colon cancer when administered after AOM. Furthermore, aspirin reduced ACF, PGE2 levels and mitosis at concentrations that did not prevent cancer. In contrast, the ability to enhance apoptosis did correlate with the prevention of cancer.     

Effects of vitamin E and selenium supplementation on esophageal adenocarcinogenesis in a surgical model with rats

Two well-known antioxidative nutrients, vitamin E and selenium, were used in this study to investigate possible inhibitory action against the formation of esophageal adenocarcinoma (EAC) in rats. In this model, carcinogenesis is believed to be driven by oxidative stress. Male Sprague-Dawley rats (8 weeks old) were divided into four groups and received esophagoduodenal anastomosis (EDA) surgery plus iron supplementation (12 mg/kg/week). Vitamin E and selenium were supplemented in the diet in the forms of alpha-tocopheryl acetate (750 IU/kg) and sodium selenate (1.7 mg Se/kg), which were 10 times the regular amounts in the basic AIN93M diet. At 40 weeks after surgery, all the EDA groups had lower body weights than the non-operated control group. Iron nutrition (hemoglobin, total serum iron and transferrin saturation) was normal as a result of iron supplementation after EDA. Vitamin E supplementation maintained the normal plasma level of alpha-tocopherol in EDA rats, but not those of gamma-tocopherol and retinol. Selenium supplementation increased the serum and liver selenium contents of the EDA rats. Histopathological analysis showed that selenium supplementation increased the incidence of EAC and the tumor volume. The selenium level in the tumor is higher than that in the duodenum of the same animal.Vitamin E supplementation, however, inhibited carcinogenesis, especially in the selenium-supplemented group. We believe that vitamin E exerts its effect through its antioxidative properties, and a high dose of inorganic selenium may promote carcinogenesis by enhancing oxidative stress.     

Efficacy of Targretin on methylnitrosourea-induced mammary cancers: prevention and therapy dose-response curves and effects on proliferation and apoptosis

Various aspects of the chemopreventive and chemotherapeutic properties of the RXR receptor agonist Targretin (LGD 1069) were examined in the methylnitrosourea (MNU)-induced model of mammary cancer. The administration of Targretin at dose levels of 60, 20 or 6.7 mg/kg body wt/day by gavage decreased the number of mammary tumors by 96, 85 and 78%, respectively. When Targretin was administered in the diet at 92 and 275 mg/kg diet cancer multiplicities were reduced by 78 and 92%, respectively. A wider range of dietary doses of Targretin at 15, 50 and 150 mg/kg diet reduced the number of mammary tumors by 38, 55 and 70%, respectively. Treatment of rats with different regimens of Targretin (250 mg/kg diet) yielded cancer multiplicities of 4.3 for non-treated rats, 0.5 for rats treated continuously with Targretin, 2.1 for rats treated with Targretin for 8 weeks followed by 10 weeks of the control diet and 1.6 for rats treated with Targretin alternating 3 days on and 4 days off. Targretin was also examined as a therapeutic agent by treating rats with at least one palpable mammary tumor for 5 weeks. A high dose of Targretin (272 mg/kg diet) caused partial or complete regression of approximately 65% of the cancers over this time period. In contrast, in animals treated with 15 mg Targretin/kg diet only 1 of 12 cancers showed significant regression. Finally, the effect of a limited exposure to Targretin (7 days) on cell proliferation and apoptosis in small mammary tumors was determined. Targretin at 150 mg/kg diet strongly decreased proliferation (75%) and increased apoptosis (300%), while a lower dose of Targretin (15 mg/kg diet, which still prevented 30% of cancers) had no effect on apoptosis but did decrease cell proliferation. Determination of serum IGF1 levels showed that treatment of rats with highly effective doses of Targretin at 272 mg/kg diet or at 60 or 20 mg/kg body wt/day by gavage caused significantly decreased serum IGF1 levels.     

The effects of ellagic acid and 13-cis-retinoic acid on N-nitrosobenzylmethylamine-induced esophageal tumorigenesis in rats

Ellagic acid (EA) and 13-cis-retinoic acid (CRA), both alone and in combination, were tested for their ability to inhibit N-nitrosobenzylmethylamine-induced tumors in the rat esophagus. Groups of male rats were fed AIN-76A diet containing EA (4 g/kg), CRA (240 mg/kg), or a combination of EA and CRA (4 g/kg and 240 mg/kg), respectively, for 25 weeks. Two weeks after initiation of the diets, NBMA (0.5 mg/kg per injection) was administered s.c. once weekly for 15 weeks. After 25 weeks on the diets, the animals were necropsied. The incidence of esophageal tumors was 97-100% in all NBMA-treated groups. The multiplicity of tumors in NBMA-treated groups was reduced significantly by EA (60%), but not by CRA, or by EA + CRA. These results demonstrate that EA and CRA do not act synergistically to inhibit NBMA-induced esophageal tumorigenesis.     

Effects of butylated hydroxyanisole pretreatment on low dose N-methyl-N'-nitro-N-nitrosoguanidine- or N,N-dibutylnitrosamine-induced rat forestomach or esophageal carcinogenesis

The effects of butylated hydroxyanisole (BHA) pretreatment on subsequent low dose N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N,N-dibutylnitrosamine (DBN) treatment on forestomach or esophageal carcinogenesis were investigated in male F344 rats. Groups of animals were pretreated with 2% BHA or basal diet alone for 24 weeks and then were given 20 mg/kg body wt MNNG once every 2 weeks, 0.025% DBN in drinking water continuously or basal diet alone for the subsequent 24 weeks. Further groups of rats were similarly treated with BHA or basal diet alone for 24 weeks, placed on basal diet for the next 24 weeks and then treated with MNNG, DBN or basal diet alone for the subsequent 24 weeks. Animals were killed 48 or 72 weeks after the beginning of the experiment. Histopathological examination showed that the incidence of forestomach tumors was not significantly affected by the BHA pretreatment in the MNNG-treated groups. On the other hand, the incidence of esophageal squamous cell carcinomas was lower in the group pretreated with BHA followed by DBN than in that treated with basal diet followed by DBN (48 week experiment). There was no significant difference in esophageal tumor incidence in the 72 week experiment. The results thus indicate that continuous treatment with 2% BHA for 24 weeks does not exert initiating activity on forestomach and esophageal epithelia.     

Effect of vitamin A deficiency on rat colon carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine.

The effect of vitamin A deficiency on the sensitivity of the colon to the carcinogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a direct-acting carcinogen, given intrarectally was studied in female Fischer rats. Animals maintained on Purina laboratory chow, semipurified vitamin A-free diet, or semipurified vitamine A-supplemented diet were given intrarectally 1.25, 0.63, or 0.31 mg MNNG 3 times weekly for 30 weeks and autopsied at the 45th week. The number of large bowel tumors per tumor-bearing rat was higher in animals receiving 1.25 mg MNNG compared to those given 0.63 or 0.31 mg. Vitamin A deficiency in rats given 1.25 mg MNNG significantly suppressed the large bowel tumor induction compared to rats fed adequate vitamin A. A high incidence of squamous cell papillomatosis of the urinary bladder was observed in rats fed vitamin A-free diet and given 1.25 mg MNNG. The present experiment suggests that the large intestine has a susceptibility that is different from that of the respiratory and urinary tracts to tumorigenic stimulation in vitamin A-deficient status.     

Inhibition of initiator-promoter-induced skin tumorigenesis in female SENCAR mice fed a vitamin A-deficient diet and reappearance of tumors in mice fed a diet adequate in retinoid or beta-carotene

Retinoids have chemopreventive activity for epithelial tumors in a variety of systems, including the two-stage tumorigenesis system of mouse skin in which only the promotion stage is inhibited. We asked whether dietary vitamin A deficiency could affect the skin tumorigenic response, prior to major changes in body weight or general health of the animals. Two regimens were tested to induce vitamin A deficiency. SENCAR mice were either (a) fed a vitamin A-deficient diet from 4 or 9 weeks of age or (b) their mothers were fed the diet from the time of birth of the experimental animals which were then weaned on the same diet. The latter regimen produced typical symptoms of vitamin A deficiency in the offspring by Weeks 12-14 and all the mice died by Week 19; the former regimen permitted sufficient accumulation of retinol and its esters to sustain life for up to 45 and 75 weeks, respectively, in the majority of mice. For our experiments, vitamin A depletion was produced by placing the mothers on the deficient diet at birth of the experimental animals. A single topical dose of 20 micrograms of 7,12-dimethylbenz(a)anthracene (DMBA) was used as the initiator at 3 weeks of age and 1 to 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) once weekly as the tumor promoter for 10 weeks (from Week 4 through 13 of the experiment). Fifty-five % of mice (n = 40) on Purina laboratory chow (mean body weight, 31.4 g) developed skin tumors (2.58 per mouse) at 12 weeks, versus 2.5% (0.05 papillomas per mouse) of mice (n = 40) kept on the purified vitamin A-deficient diet (mean body weight, 30.3 g), a 98% decrease in tumor/mouse. Retinoic acid (RA) (1-3 micrograms/g diet) supplementation after Week 12 caused a rapid tumorigenic response in 95% of the mice by week 22. This tumor response occurred to a reduced extent in the absence of continued TPA treatment up to Week 13. Even though tumor incidence increased within 1 week of RA and 95% of the mice showed the tumorigenic response, the number of tumors per mouse was about 50% of that observed in mice maintained on standard Purina diet. This was confirmed in an experiment in which the mice were maintained for life either on Purina or on the RA (3 micrograms/g) containing purified diet, the latter being the control group for the effect of vitamin A deficiency on skin tumorigenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Procarbazine carcinogenicity in methotrexate-treated or lipotrope-deficient male rats

Procarbazine hydrochloride (PCZ), a chemotherapeutic agent used extensively to treat Hodgkins disease and other tumors, induces leukemia, lymphoma, mammary gland and other solid tumors in rodents and non-human primates and is strongly implicated as a leukemogen in humans. Lipotrope (choline and methionine) deficiency is a powerful potentiator of chemical carcinogenesis in liver and, under some conditions, in other tissues in rodents. Methotrexate (MTX), another commonly used chemotherapeutic agent, interferes with one-carbon metabolism and limits availability of lipotropes. Studies of PCZ carcinogenesis in lipotrope-deficient or MTX-treated male rats are reported, showing that both deficiency and MTX increased PCZ carcinogenicity in the mammary gland. In addition, PCZ was found to induce abnormalities of hepatic choline metabolism. Weanling male Sprague-Dawley rats were fed control (C) or lipotrope-deficient (D) diet. After 3 weeks, C and D rats were given PCZ, MTX, the two drugs together or 0.9% saline by i.p. injection. Doses were 0.2 or 0.5 mg MTX/kg or 25 mg PCZ/kg, given 2 or 3 days per week for 5 or 14 weeks. After 5 weeks of drug treatment livers were assayed for choline, phosphatidylcholine, phosphocholine (PCho), glycerophosphocholine and betaine. PCZ perturbed choline metabolism, increasing hepatic choline and PCho in deficient or MTX-treated rats and, to a smaller extent, in rats fed control diet. MTX markedly enhanced the effect of PCZ on choline metabolism. PCZ-induced mammary tumor incidence was increased 50-70% by lipotrope deficiency or by MTX. In PCZ-treated rats, cumulative probability of bearing a mammary tumor was significantly increased by lipotrope deficiency (P = 0.05), and was increased similarly but not significantly by MTX (P = 0.1). Cumulative tumor numbers per group in PCZ-treated rats were significantly greater in both deficient and MTX-treated rats compared to rats fed control diet (P less than 0.005). Incidences of leukemia, lymphoma and Zymbal's gland tumors induced by PCZ were not significantly altered by diet or MTX.     

Vitamin E protection against nitrosamine-induced esophageal tumor incidence in mice immunocompromised by retroviral infection.

Retrovirally induced immunosuppression may elevate the incidence of chemically induced cancers. A proposed hypothesis to explain this relationship is the increased free radical activity observed during retroviral infection and carcinogen activation. We previously found that vitamin E retarded growth of esophageal tumors accompanied by reductions of free radical products. This study investigated the contribution that retroviral immunosuppression has on esophageal cancer induced by the carcinogen N-nitrosomethylbenzylamine (NMBzA), and the response that increased levels of dietary vitamin E has on this induced carcinogenesis. Female C57BL/6 mice received NMBzA or vehicle (corn oil) i.p. weekly for 3 weeks. Then some of the mice were infected with LP-BM5 murine retrovirus and fed diets containing 30 IU vitamin E or 172 IU vitamin E/kg of diet. As an assessment of free radical activity, exhaled ethane was measured prior to killing the animals at 26 weeks. Esophagi from the various mice groups were assessed for size and frequency of tumors. Livers homogenates were analyzed for vitamins A and E, lipid fluorescence, conjugated dienes and malondialdehyde. Hepatic levels of vitamin A and E were decreased (P < 0.05) and indices of lipid peroxidation were greater (P < 0.05) in NMBzA-treated mice relative to controls. Lipid peroxidation and serum transaminases (ALT and AST) were greatest in mice given NMBzA and infected with the retroviruses. Incidence of esophageal tumors were also greatest in the NMBzA-treated, immunocompromised animals. Mice fed vitamin E-supplemented diets showed increased (P < 0.05) hepatic concentrations of vitamin E and vitamin A, decreased activities of serum transaminases, decreased indices of lipid peroxidation, and decreased size and frequency of esophageal tumors in both the immunocompromised and non-immunocompromised mice. These results suggest that vitamin E plays an antioxidant function that retards the incidence of esophageal cancers in immunocompromised and non-immunocompromised animals.     

Dietary folate deficiency suppresses N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats

Epidemiologic studies have suggested that dietary folate intake is inversely related to breast cancer risk. However, epidemiologic evidence has not been consistent nor has it provided unequivocal support for this purported inverse relationship. This study investigated the effect of dietary folate on N-methyl-N-nitrosourea (MNU)-induced mammary tumorigenesis in rats. Weanling, female Sprague-Dawley rats were fed diets containing either 0 (deficient; n = 22), 2 (basal dietary requirement, control; n = 20) or 8 mg (supplemented; n = 20) folate/kg diet for 30 weeks. At 50 days of age, rats received an i.p. injection of MNU (50 mg/kg body wt). At necropsy, all macroscopic mammary tumors were identified and examined microscopically. The effect of dietary folate on genomic DNA methylation in mammary tumorigenesis was determined by the in vitro methyl acceptance assay. The incidence of mammary adenoma and adenocarcinoma in the folate-deficient group was lower than that of the control and folate-supplemented groups (55 versus 90 and 75%, respectively, P = 0.043). Kaplan-Meier analyses also demonstrated a similar trend in the rates of appearance of either adenoma or adenocarcinoma (P = 0.06). In contrast, folate supplementation did not significantly modulate mammary tumorigenesis compared with the control group. Although mammary tumors were significantly hypomethylated compared with non-neoplastic mammary tissues in each dietary group (P < 0.03), folate status did not significantly affect the extent of DNA methylation. The data suggest that dietary folate deficiency of a moderate degree suppresses, whereas folate supplementation at four times the basal dietary requirement does not significantly modulate, mammary tumorigenesis in this model. The role of folate in mammary tumorigenesis needs to be clarified for safe and effective prevention of breast cancer.     

Dietary zinc deficiency enhances esophageal cell proliferation and N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor incidence in C57BL/6 mouse.

The effect of zinc deficiency on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor formation in rats has been well documented. Our previous work showed that zinc deficiency and its associated increased esophageal cell proliferation were of paramount importance in esophageal tumor development in the NMBA-rat model. However, there has been no report concerning zinc deficiency and NMBA-induced esophageal tumor formation in mice. In this study, weanling C57BL/6 mice were fed ad libitum with either a zinc-sufficient or a zinc-deficient diet containing 3-4 ppm of zinc, and received six intragastric doses of NMBA (2 mg/kg; twice weekly for 3 weeks). The animals were sacrificed 46 weeks later after in vivo bromodeoxyuridine (BrDU) labeling followed by immunohistochemical detection of cells in S-phase. At 46 weeks, the tumor incidences in zinc-deficient mice were 57, 100, and 100% respectively, in the esophagus, forestomach and squamocolumnar junction with the glandular stomach (SCJ), as compared to 17, 39, and 67% in the corresponding tissue of zinc-sufficient mice. The difference between the two dietary groups was significant at P < 0.02 for the esophagus, and P < 0.001 for the forestomach and the SCJ. BrDU labeling revealed that the esophageal labeling index and the number of labeled cells were increased by zinc deficiency. These results support a role of increased cell proliferation in esophageal carcinogenesis in the mouse.