Post-carcinogen Treatment Research Articles

Chemoprevention of Lung Cancer with a Combination of Mitochondria-Targeted Compounds.

Simple SummaryPrevious reports showed that mitochondria-targeted honokiol and mitochondria-targeted lonidamine potently inhibit complex-I- and complexes-I/II-induced respiration and cancer cell proliferation. In this study, we investigated the efficacy of combining mitochondria-targeted honokiol and mitochondria-targeted lonidamine treatments for lung cancer prevention. We found that their combination exhibited striking tumor inhibition in the benzo[a]pyrene-induced murine lung tumor model without causing detectable side effects. Using single-cell RNA sequencing, we found combined treatment has a clear advantage in that it can significantly inhibit two oncogenic pathways—STAT3 signaling and AKT/mTOR/p70S6K signaling. Such dual inhibition may contribute to the greater efficacy of the combined drug treatment. Therefore, the combination provides a novel option for lung cancer chemoprevention.Combined treatment targeting mitochondria may improve the efficacy of lung cancer chemoprevention. Here, mitochondria-targeted honokiol (Mito-HNK), an inhibitor of mitochondrial complex I and STAT3 phosphorylation, and mitochondria-targeted lonidamine (Mito-LND), an inhibitor of mitochondrial complexes I/II and AKT/mTOR/p70S6K signaling, were evaluated for their combinational chemopreventive efficacy on mouse lung carcinogenesis. All chemopreventive treatments began one-week post-carcinogen treatment and continued daily for 24 weeks. No evidence of toxicity (including liver toxicity) was detected by monitoring serum levels of alanine aminotransferase and aspartate aminotransferase enzymes. Mito-HNK or Mito-LND treatment alone reduced tumor load by 56% and 48%, respectively, whereas the combination of Mito-HNK and Mito-LND reduced tumor load by 83%. To understand the potential mechanism(s) of action for the observed combinatorial effects, single-cell RNA sequencing was performed using mouse tumors treated with Mito-HNK, Mito-LND, and their combination. In lung tumor cells, Mito-HNK treatment blocked the expression of genes involved in mitochondrial complex ǀ, oxidative phosphorylation, glycolysis, and STAT3 signaling. Mito-LND inhibited the expression of genes for mitochondrial complexes I/II, oxidative phosphorylation, and AKT/mTOR/p70S6K signaling in lung tumor cells. In addition to these changes, a combination of Mito-HNK with Mito-LND decreased arginine and proline metabolism, N-glycan biosynthesis, and tryptophan metabolism in lung tumor cells. Our results demonstrate that Mito-LND enhanced the antitumor efficacy of Mito-HNK, where both compounds inhibited common targets (oxidative phosphorylation) as well as unique targets for each agent (STAT3 and mTOR signaling). Therefore, the combination of Mito-HNK with Mito-LND may present an effective strategy for lung cancer chemoprevention.

Abstract P5-05-15: Pregnancy inhibits mammary carcinogenesis by persistently altering the hypothalamic-pituitary axis

Abstract Pregnancy, carried to term at an early age, is probably the best natural protection against breast cancer development. The relative life-long breast cancer risk for women that give birth to their first child before the age of 20 years is approximately half that of nulliparous women. In contrast, if a woman undergoes her first full-term pregnancy after the age of 35, her risk for breast cancer is increased even more than nulliparous women. In the current generation many women are career oriented and have children later in life. Universally the average age at first birth is on the rise. It is critical to understand the underlying mechanism of this protective effect of pregnancy against breast cancer to develop novel prevention strategies to reduce the risk of breast cancer without women having to undergo pregnancy early in life. Earlier, we and others have demonstrated that post-pregnancy there were persistent changes in circulating levels of hormones. In order to understand the significance of these systemic changes we determined alterations in the hypothalamic-pituitary axis in parous rats. In particular, we examined the static and dynamic alterations in the hypothalamic-pituitary axis in response to pregnancy. Seven weeks old female Lewis rats were injected with the chemical carcinogen N-methyl-N-nitrosourea (MNU) at a dose of 50mg/kg body weight intraperitoneally. Two weeks post-carcinogen treatment these rats were housed with a male rat. On the observation of the vaginal plug the male was removed from the cage. Once the rats gave birth they nursed the pups for three weeks and were weaned after that period. Mammary tumorigenesis was monitored through weekly palpation for a period of nine months. A subset of rats at 6, 12 and 24 weeks post-weaning were used to study static and dynamic changes in the level of hormones. We investigated the static alterations in the hypothalamic-pituitary axis in response to pregnancy by measuring the levels of thyrotropin releasing hormone (TRH), growth hormone releasing hormone (GHRH), somatostatin (SS), dopamine (DA), growth hormone (GH) and prolactin (PRL). Next we investigated if the dynamic alterations in the hypothalamic-pituitary axis in response to pregnancy. Control and parous animals were subjected to secretogogue treatments (Growth Hormone Related Peptide 6 for GH and Perphenazine for PRL) and the levels of GH and PRL were measured. We also isolated the pituitary and treated them with the secretogogues and measured the levels of GH and PRL. Our data demonstrated that pregnancy resulted in persistent static and dynamic alterations in circulating levels of hormones. Parous rats response to the secretogogues was severely blunted compared to the control nulliparous rats. The levels of TRH, GHRH, GH and PRL were significantly lowered in parous rats while DA and SS levels were higher in nulliparous rats. As expected mammary carcinogenesis was significantly inhibited in parous rats. Overall, these preliminary results suggest that pregnancy induces persistent changes in hypothalamic-pituitary axis, which results in a lowered hormonal promotion environment resulting in inhibition of mammary carcinogenesis. Citation Format: Subramani R, Galvez A, Pedroza D, Lakshmanaswamy R. Pregnancy inhibits mammary carcinogenesis by persistently altering the hypothalamic-pituitary axis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-15.

Roflumilast treatment inhibits lung carcinogenesis in benzo(a)pyrene-induced murine lung cancer model

Roflumilast, a potent and selective inhibitor of phosphodiesterase-4 (PDE4), has been used in treatment of COPD. PDE4 inhibitor is associated with inhibition of chronic airway inflammation, oxidative stress, and mesenchymal markers in B(a)P-induced lung tumors. The aim of this study was to assess whether roflumilast alone or added to inhaled budesonide might have dose-dependent inhibition on lung carcinogenesis induced by carcinogen B(a)P in mice. Female A/J mice were given a single dose of benzo(a)pyrene. Administration of roflumilast (1mg/kg or 5mg/kg) via oral gavage and aerosolized budesonide (2.25mg/ml) began 2 weeks post-carcinogen treatment and continued for 26 weeks. Tumor load was determined by averaging the total tumor volume in each group. Benzo(a)pyrene induced an average tumor size of 9.38 ± 1.75 tumors per mouse, with an average tumor load of 19.53 ± 3.81mm3. Roflumilast 5mg/kg treatment decreased (P < 0.05) tumor load per mouse compared to the B(a)P group. Roflumilast 5mg/kg treatment significantly increased the levels of cAMP in tumors with adjacent lung tissues (P < 0.05). The expression level of PDE4D gene was decreased by roflumilast 5mg/kg treatment, significantly (P < 0.05). Compared to the B(a)P exposure group, expression levels of HIF-1α and VEGFA were attenuated by roflumilast 5mg/kg treatment (P < 0.05). High-dose roflumilast can attenuate lung carcinogenesis in B(a)P-induced murine lung cancer model. The chemopreventive effect of roflumilast might be associated with inhibition of increased cAMP-mediated inflammatory process and markers of angiogenesis in tumor tissues.

P1.01-011 Roflumilast Attenuates Benzo(a)Pyrene-Induced Lung Cancer via Suppression of Airway Inflammation in Murine Model

Chronic airway inflammation has been emerging targets for lung cancer chemoprevention as well as treatment of COPD. The aim of the present study was to determine the role of roflumilast and aerosolized budesonide in benzo(a)pyrene-induced lung cancer in mice and to elucidate the possible their mechanisms. Female A/J mice were given a single dose of benzo(a)pyrene. Intraperitoneal administration of roflumilast (1mg/kg, 5mg/kg) began 2 weeks post-carcinogen treatment and continued tri-weekly for 28 weeks. Aerosolized budesonide was administered by aerosol delivery for 2 min/day and 5 days/week. Tumor load was determined by averaging the total tumor volume in each group. Benzo(a)pyrene induced an average tumor size of 9.4 ± 1.8 tumors per mouse, with an average tumor load of 19.5 ± 3.8mm3. Roflumilast treatment at 1 and 5 mg/kg did not inhibit tumor number, however, reduced tumor load, an average of 8.8 ± 2.0 mm3 at 5mg/kg treatment, significantly. Aerosolized budesonide administration did not show reductions of tumor number or load. The decreased expressions of cyclic AMP and protein kinase A caused by benzo(a)pyrene were increased by roflumilast treatment. NF-κB expression in tumor tissues was lower in the roflumilast group than the place group. In vivo experiments in the benzo(a)pyrene-induced model of lung cancer show that roflumilast significant inhibits tumorigenicity via suppression of inflammation. Possible mechanisms between cAMP pathway and lung cancer development will needed to be determined.

Polymeric black tea polyphenols (PBPs) inhibit benzo(a)pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone-induced lung carcinogenesis potentially through down-regulation of p38 and Akt phosphorylation in A/J mice.

The aim of our study was to evaluate chemopreventive efficacy and possible mechanism of most abundant polyphenolic fraction in black tea, polymeric black tea polyphenols (PBPs), in experimental lung carcinogenesis model. Effect of 1.5% black tea derived PBPs on benzo(a)pyrene [B(a)P] and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced lung lesions were studied over 28 wks. Chemopreventive efficacy was studied using decrease in tumor incidence and/or multiplicity and/or delay in the latency period in A/J mice. Histopathological analysis of lung was carried out post-carcinogen treatment weeks to analyze the microscopic lung lesions. Inflammation, cell proliferation, and apoptosis markers along with signaling kinases like p38, Akt, and their phosphorylated forms were studied using immunoblotting and immunohistochemistry at 4th, 10th, and 18th wk post-carcinogen treatment. Administration of PBPs throughout the treatment period significantly decreased the multiplicity of surface tumors as well as microscopic lung lesions, including adenomas. Although tumor incidence and latency period remains unaffected, histopathological evaluation of lung at 6, 10, and 18 wks post- carcinogen treatment period showed decrease in tumor multiplicity which was also correlated with different molecular markers. Anti- inflammatory action of PBPs was demonstrated by reduced Cox-2 expression. PBPs down-regulated the B(a)P and NNK-induced cell proliferation (diminished PCNA expression, proliferation index, and Bcl-2 expression) and enhanced apoptosis (increased Bax expression and apoptotic index) potentially through phosphorylation of p38 and Akt. PBPs, most abundant polyphenolic component in the black tea, have chemopreventive effect through inhibition of inflammation, cellular proliferation, and induction of apoptosis possibly via modulation of signaling kinases. © 2016 Wiley Periodicals, Inc.

Chemopreventive effect of phosphodieasterase-4 inhibition in benzo(a)pyrene-induced murine lung cancer model

ABSTRACTPurpose: Cigarette smoking increases chronic airway inflammation, oxidative stress, and epithelial mesenchymal transition (EMT), which may result in chronic obstructive pulmonary disease (COPD) and tumor growth in the lung. Phosphodiesterase 4 (PDE4) inhibitors are known to reduce inflammation, and they have recently been introduced for the treatment of COPD. We assessed the impact of rolipram, a selective PDE4 inhibitor, on chemoprevention in benzo(a)pyrene-induced lung cancer in mice. Materials and Methods: Female A/J mice were given a single dose of benzo(a)pyrene. Intraperitoneal administration of rolipram began 2 weeks post-carcinogen treatment and continued tri-weekly for 28 weeks. Tumor load was determined by averaging the total tumor volume in each group. Results: Benzo(a)pyrene induced an average tumor size of 10.4 ± 1.7 tumors per mouse, with an average tumor load of 25.9 ± 3.8 mm3. Rolipram significantly decreased tumor number, by 45.1%, and tumor load, by 52.9%, compared with the benzo(a)pyrene group. Ki67 staining was reduced in rolipram-treated mice compared with benzo(a)pyrene-treated mice. The increased expression of EMT markers caused by benzo(a)pyrene was inhibited by rolipram. Rolipram significantly attenuated NF-κB and Nrf2 expression in benzo(a)pyrene-induced lung cancer tissues. Conclusions: In vivo experiments in the benzo(a)pyrene-induced model of lung cancer show that PDE4 inhibition significant inhibits lung carcinogenesis. Our results provide evidence that PDE4 inhibitors may be suitable for the prevention of the lung cancer in high-risk groups, for example, heavy smokers and patients with COPD.

Chemoprevention of lung carcinogenesis by the combination of aerosolized budesonide and oral pioglitazone in A/J mice

Budesonide, a synthetic glucocorticoid used for treating asthma, and pioglitazone, a synthetic peroxisome proliferator-activated receptors γ ligand used for the treatment of diabetes, were evaluated for their combinational chemopreventive efficacy on mouse lung cancer using female A/J mice with benzo(a)pyrene used as the carcinogen. All chemopreventive treatments began 2-wk post-carcinogen treatment and continued daily for 20 wk. Budesonide was administered by the aerosol route using an improved aerosol delivery system. Pioglitazone was introduced by oral gavage. The characterization of drug distribution showed that budesonide introduced by aerosol delivery accumulated only in the lung. Budesonide alone reduced tumor load by 78% and pioglitazone alone reduced tumor load by 63%. By combining aerosolized budesonide with pioglitazone, the inhibition on tumor load was 90%. In vitro experiments using human cancer cells showed that budesonide and pioglitazone exhibited independent, additive inhibitory effects on cell growth. Our results provide evidence that aerosolized budesonide and oral pioglitazone could be a promising drug combination for lung cancer chemoprevention.

Long-term hormonal promotion overcomes genetic resistance to mammary cancer

It is well known that ovarian steroids estradiol and progesterone play a vital role in the development of mammary cancer. Here, using the genetically highly resistant Copenhagen rats we demonstrate that sustained exogenous treatment with estradiol and progesterone overcomes genetic resistance to mammary cancer. It has been demonstrated that Copenhagen rats develop preneoplastic lesions upon exposure to carcinogens. However, these preneoplastic lesions fail to progress to ductal carcinomas in situ or overt mammary carcinomas. The preneoplastic lesions eventually decrease in number and are absent by 60 days post-carcinogen treatment. In the present study, we exposed 7-week-old female Copenhagen rats to N-methyl-N-nitrosourea (MNU; 50 mg/kg BW). Immediately after MNU treatment the rats were divided into the following groups: (1) control; (2) 30 mg estradiol 17β; (3) 30 mg progesterone; and (4) 30 mg estradiol 17β plus 30 mg progesterone. All hormone treatments were administered via individual silastic pellets for a period of 9 months post-carcinogen treatment. The control animals displayed a low incidence of mammary cancer (10%). Hormone treatments produced significantly higher incidences of mammary cancer, with estradiol at 50%, progesterone at 65% and estradiol plus progesterone at 90%. Hormone treatment sustained the growth of the lesions induced by MNU by increasing expression of Areg, Bcl-2, Ccnd-1 and Vegf genes, while decreasing expression of Bad, Bax, Casp 3, 8, 9 and p53 genes. Furthermore, hormone treatment increased CCND-1 and PARP proteins levels. The data clearly demonstrates that hormonal environment supports mammary cancer progression by increasing cell proliferation, and angiogenesis while inhibiting apoptosis.

Chemopreventive Effect of Kava on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone plus Benzo[a]pyrene–Induced Lung Tumorigenesis in A/J Mice

Lung cancer is the leading cause of cancer death, and chemoprevention is a potential strategy to help control this disease. Epidemiologic survey indicates that kava may be chemopreventive for lung cancer, but there is a concern about its potential hepatotoxicity. In this study, we evaluated whether oral kava could prevent 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo[a]pyrene (B[a]P)-induced lung tumorigenesis in A/J mice. We also studied the effect of kava to liver. At a dose of 10 mg/g diet, 30-week kava treatment (8 weeks concurrent with NNK and B[a]P treatment followed by 22 weeks post-carcinogen treatment) effectively reduced lung tumor multiplicity by 56%. Kava also reduced lung tumor multiplicity by 47% when administered concurrently with NNK and B[a]P for 8 weeks. Perhaps most importantly, kava reduced lung tumor multiplicity by 49% when administered after the final NNK and B[a]P treatment. These results show for the first time the chemopreventive potential of kava against lung tumorigenesis. Mechanistically, kava inhibited proliferation and enhanced apoptosis in lung tumors, as shown by a reduction in proliferating cell nuclear antigen (PCNA), an increase in caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP). Kava treatment also inhibited the activation of nuclear factor kappaBNF-kappaB, a potential upstream mechanism of kava chemoprevention. Although not rigorously evaluated in this study, our preliminary data were not suggestive of hepatotoxicity. Based on these results, further studies are warranted to explore the chemopreventive potential and safety of kava.

Dietary effects of soy isoflavones daidzein and genistein on 7,12-dimethylbenz[a]anthracene-induced mammary mutagenesis and carcinogenesis in ovariectomized Big Blue(R) transgenic rats

The major constituents of isoflavones, daidzein (DZ) and genistein (GE) are known to interact with the alpha and beta estrogen receptors (ERalpha/beta) in several tissues including mammary. In this study, we used ovariectomy (OVX) to model menopause and determined the effects of DZ, GE or 17beta-estradiol (E2) exposures on chemically induced mutagenesis and carcinogenesis in the mammary glands of female Big Blue (BB) transgenic rats. The rats were fed control diet containing the isoflavones and E2 and treated with a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at PND 50. Animals were sacrificed at 16 or 20 weeks post-carcinogen treatment to assess mutant frequencies (MFs) and histopathological parameters, respectively. The isoflavones or E2 supplementation alone resulted in modest increases in the lacI MF that were not significantly different from the MFs measured in rats fed the control diet alone. DMBA exposure, however, induced significant increases in the lacI MFs in the mammary of both OVX and ovary intact (INT) rats and Hprt MFs in spleen lymphocytes (P<or=0.01). In general, feeding the isoflavones or E2 separately did not cause any significant changes in DMBA-induced mutagenicity in the mammary. However, feeding the isoflavone mixture (DZG) resulted in a significant reduction in the DMBA-induced lacI MFs (P<or=0.05). Cell proliferation as measured by PCNA immunohistochemistry was increased in both OVX and INT rats exposed to DMBA as compared with rats fed control diet (P<or=0.05). Mammary histology indicated that hyperplasia was induced in most of the treatment groups including control. Although DMBA treatment did not induce mammary tumors in the OVX rats, adenoma and adenocarcinoma were detected in the mammary glands of INT rats.

Dietary effects of soy isoflavones daidzein and genistein on 7,12-dimethylbenz[a]anthracene-induced mammary mutagenesis and carcinogenesis in ovariectomized Big Blue transgenic rats

The major constituents of isoflavones daidzein (DZ) and genistein (GE) interact with the and estrogen receptors in several tissues including mammary tissues. In this study, we used ovariectomy (OVX) to model menopause and determined the effects of DZ, GE or 17beta-estradiol (E(2)) exposures on chemically induced mutagenesis and carcinogenesis in the mammary glands of female Big Blue transgenic rats. The rats were fed control diet containing the isoflavones and E(2) and treated with a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at PND50. Animals were euthanized at 16 or 20 weeks post-carcinogen treatment to assess mutant frequencies (MFs) and histopathological parameters, respectively. The isoflavones or E(2) supplementation alone resulted in the lac I MFs that were not significantly different from the MFs measured in rats fed the control diet alone. DMBA exposure, however, induced significant increases in the lac I MFs in the mammary tissues of both OVX and INT rats and Hprt MFs in spleen lymphocytes (P < 0.01). In general, feeding the isoflavones or E(2) did not cause any significant changes in DMBA-induced mutagenicity in the mammary tissues. However, feeding the isoflavone mixture (daidzein + genistein; DZG) resulted in a significant reduction in the DMBA-induced lac I MFs (P < 0.05). Cell proliferation as measured by PCNA immunohistochemistry was increased in both OVX and INT rats exposed to DMBA as compared with rats fed control diet (P < 0.05). Mammary histology indicated that hyperplasia was induced in most of the treatment groups including control. Although DMBA did not induce mammary tumors in the OVX rats, adenoma and adenocarcinoma were detected in the mammary glands of INT rats.

Inhibition of lung tumorigenesis in A/J mice by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine and myo-inositol, individually and in combination.

Isothiocyanates, their N-acetylcysteine conjugates, and myo-inositol (MI) are inhibitors of lung tumorigenesis in A/J mice. However, chemoprevention by combinations of these compounds in different temporal sequences has not been examined. This is important for developing practical approaches to lung cancer chemoprevention in smokers and ex-smokers. We used a tumor model in which A/J mice are treated with 8 weekly doses of benzo[a]pyrene (B[a]P) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and killed 19 weeks after the final treatment. In Experiment 1, isothiocyanates or their N-acetylcysteine conjugates were added to the diet (1 or 3 micro mol/g) from 1 week before until 1 week after carcinogen treatment. The compounds were 2-phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), N-acetyl-S-(N-benzyl-thiocarbamoyl)-L-cysteine (BITC-NAC), N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC), and N-acetyl-S-(N-3-phenylpropylthiocarbamoyl)-L-cysteine (PPITC-NAC). Significant reductions in lung tumor multiplicity were observed in mice treated with PEITC, PEITC-NAC, PPITC and PPITC-NAC. PEITC-NAC was chosen for combination studies with MI (Experiment 2). Mice were treated with B[a]P plus NNK without or with PEITC-NAC (3 micro mol/g diet), MI (55.5 micro mol/g diet), or PEITC-NAC plus MI (3 micro mol plus 55.5 micro mol/g diet). Different temporal sequences of dietary additions were investigated: carcinogen treatment phase; post-carcinogen treatment phase; entire experiment; 50% of carcinogen treatment phase until termination; and 75% of carcinogen treatment phase until termination. All treatments reduced lung tumor multiplicity except PEITC-NAC post-carcinogen or from 75% of the carcinogen treatment phase. Reduction of lung tumor multiplicity by PEITC-NAC plus MI was greater than that in the mice treated with the agents alone in all temporal sequences. When all results were combined, PEITC-NAC plus MI was significantly more effective than the agents alone. There was a significant trend for reduction in lung tumor multiplicity with increased duration of treatment by the chemopreventive agents. These results provide a basis for further development of mixtures of PEITC-NAC and MI for chemoprevention of lung cancer.

Tea and Tea Polyphenols Inhibit Cell Hyperproliferation, Lung Tumorigenesis, and Tumor Progression

Both green and black tea have been shown to inhibit lung tumorigenesis in laboratory animal experiments. Green tea inhibited N-nitrosodiethylamine-induced lung tumor incidence and multiplicity in female A/J mice when tea was given either during the carcinogen treatment period or during the post-carcinogen treatment period. In a separate tumorigenesis model, both decaffeinated black tea and decaffeinated green tea inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor formation. Studies in which tea was administered during different time periods in relation to the NNK suggest that tea can inhibit lung tumorigenesis at both the initiation and promotion stages. The antiproliferative effects of tea may be responsible for these anti-carcinogenic actions. Black tea polyphenol preparations decreased NNK-induced hyperproliferation. Black tea also inhibited the progression of pulmonary adenomas to adenocarcinomas and the formation of spontaneous lung tumors in A/J mice. Growth inhibition by various tea polyphenols has been demonstrated in human lung H661 and H1299 cells. Although inhibition of cell growth and signal transduction pathways by tea components have been demonstrated, the concentrations required to produce the effect are higher than achievable in tissues in vivo. More research is necessary to translate these laboratory results to applications in human chemoprevention.

Effect of tamoxifen and D,L-2-difluoromethylornithine on the growth, ornithine decarboxylase activity and polyamine content of mammary carcinomas induced by 1-methyl-1-nitrosourea.

The effect of combined treatment with D,L-2-difluoromethylornithine (DFMO) and tamoxifen on the growth status, ornithine decarboxylase (ODC) activity and polyamine content of established 1-methyl-1-nitrosourea (MNU)-induced mammary tumors was investigated. DFMO treatment, a 0.125% solution provided as drinking water, inhibited the rate of tumor occurrence and reduced the number of mammary tumors induced by a high dose of MNU (50 mg/kg body weight) during the first 120 days post-carcinogen treatment. Tamoxifen was administered daily via s.c. injection (25 micrograms/100 g body weight) to tumor-bearing rats in both treatment groups, i.e. control and DFMO-treated, for a 30-day period beginning 120 days after carcinogen. Tamoxifen treatment induced tumor regression but the percentage of regressing, static or growing tumors was no different in the presence or absence of DFMO. Whereas the mammary tumors of DFMO-treated rats had reduced ODC activity and lower polyamine concentrations in comparison to the tumors of untreated animals, tamoxifen had no effect on these parameters independent of its effect on tumor growth status. DFMO did not increase the efficacy of tamoxifen in inducing tumor regression.

Hepatocarcinogenicity of Diethylnitrosamine to the Self-Fertilizing Hermaphroditic Fish &lt;italic&gt;Rivulus marmoratus&lt;/italic&gt; (Teleostomi: Cyprinodontidae)&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

The larvae, 3 days post hatching, of self-fertilizing hermaphroditic fish Rivulus marmoratus were exposed to diethylnitrosamine (DENA) (CAS: 55-18-5; N-nitrosodiethylamine) at levels of 50-400 ppm for 2 hours. At 3-month intervals, the exposed fish were sampled until 9 months postcarcinogen treatment. Hepatomas were induced in 6- and 9- month groups of the 100-ppm-treated and 200-ppm-treated fish. An exposure of fish larvae to 400 ppm DENA also produced similar tumors on 3-, 6-, and 9-month groups. The tumor incidences were clearly dose dependent. These results offered the possibility of the use of R. marmoratus as a suitable model for experimental carcinogenesis.

Null effects of vitamin A analogs on the dimethylnitrosamine kidney tumor model.

The effect of retinoids on the induction of both epithelial and mesenchymal tumors of the rat kidney by a single dose of 40 mg/kg dimethylnitrosamine was tested using 13-cis-retinoic acid and the trimethylmethoxy phenyl analog of retinoic acid ethylamide. 13-cis-retinoic acid was administered for 3 weeks, 10 weeks, or 26 weeks, post-carcinogen treatment, in order to coincide with known morphological phases occurring within the kidney which culminated in the development of macroscopic tumors. The ethylamide analog and placebo beadlets were administered for the 26 week period only. None of the retinoid schedules significantly influenced the survival of the animals, nor the incidences of renal mesenchymal tumors or renal cell adenomas/adenocarcinomas. Tumor latency, histological grade or frequency of metastatic invasion were also unaltered by the treatments. Possible reasons for the observed lack of effect are discussed, including speculation regarding the potency of this single-dose model of chemical carcinogenesis.