Paula M. Kramer

Chemoprevention by Lipoxygenase and Leukotriene Pathway Inhibitors of Vinyl Carbamate-induced Lung Tumors in Mice

5-Leukotriene pathway inhibitors, Accolate, MK-886, and Zileuton, were evaluated as chemopreventive agents in female strain A mice. The mice were administered by injection vinyl carbamate (2 x 16 mg/kg) to induce lung tumors. Two weeks later, they received in their diet Accolate (270 and 540 mg/kg), MK-886 (30 mg/kg), Zileuton (600 and 1200 mg/kg), or combinations containing the lower concentration of two agents. Thirteen weeks later, Accolate, Zileuton (only the high concentration), and combinations of Zileuton with either Accolate or MK-886 reduced lung tumor multiplicity. At week 43, MK-886, Accolate, and Zileuton reduced lung tumor multiplicity by 37.8, 29.5, and 28.1%, respectively. They also decreased the size of the tumors and the yield of carcinomas. These results demonstrate that leukotriene inhibitors prevent lung tumors and slow the growth and progression of adenomas to carcinoma; leukotriene inhibitors warrant further consideration for potential use in humans.

Reversed-phase high-performance liquid chromatography procedure for the simultaneous determination of S-adenosyl-L-methionine and S-adenosyl-L-homocysteine in mouse liver and the effect of methionine on their concentrations.

An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-microl perchloric acid extract of the tissue. The limits of detection in 25-microl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-microl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55-11000 pmol for SAM and 50-10000 pmol for SAH. The intra-day precision and accuracy were < or =6.4% relative standard deviation (RSD) and 99.9-100.0% for SAH and < or =6.7% RSD and 100.0-100.1% for SAM. The inter-day precision and accuracy were < or =5.9% RSD and 99.9-100.6% for SAH and < or =7.0% RSD and 99.5-100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body.

Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are mouse liver carcinogens. Methylation of the c-jun and c-myc genes, expression of both genes and DNA methyltransferase (DNA MTase) activity were determined in liver tumors initiated by N-methyl-N-nitrosourea and promoted by DCA and TCA in female B6C3F1 mice. Hypomethylated and over-expression of c-jun and c-myc genes were found in DCA- and TCA-promoted liver tumors. DNA MTase activity was increased in tumors while decreased in non-involved liver. Thus, DCA- and TCA-promoted carcinogenesis appears to include decreased methylation and increased expression of c-jun and c-myc genes in the presence of increased DNA MTase activity.

Effect of trichloroethylene on DNA methylation and expression of early‐intermediate protooncogenes in the liver of B6C3F1 mice

Trichloroethylene (TCE) is a multimedia environmental pollution that is carcinogenic in mouse liver. The ability of TCE to modulate DNA methylation and the expression of immediate‐early protooncogenes was evaluated. Female B6C3F1 mice were administered 1000 mg/kg TCE by gavage 5 days/week and killed after 5, 12, or 33 days of exposure. Methylation of DNA as 5‐methylcytosine was decreased by 5 days of treatment with TCE and remained reduced for 33 days. TCE also decreased the methylation of the promoter regions for the protooncogenes, c‐jun and c‐myc. The expression of the mRNA for the two protooncogenes was increased between 60 and 120 minutes after administering the last dose of TCE and returned to control level by 24 hours. The expression of the mRNA for c‐fos remained undetectable after administering TCE. Hence, TCE decreased the methylation both of total DNA and the promoters for the c‐jun and c‐myc genes and increased the expression of their mRNA. The decreased methylation and increased expression of the two immediate‐early protooncogenes might be associated with TCE‐induced increase in cell proliferation and promotion of tumors.

Promotion by mixtures of dichloroacetic acid and trichloroacetic acid of N-methyl-N-nitrosourea-initiated cancer in the liver of female B6C3F1 mice

Hepatic tumor promoting activity was determined for mixtures of dichloroacetic acid (DCA) and trichloroacetic acid (TCA) in female B6C3F1 mice initiated on day 15 of age with 25 mg/kg N-methyl-N-nitrosourea. The mice received in their drinking water from 6 to 50 weeks of age either DCA (7.8, 15.6, or 25 mmol/l) with/without 6.0 mmol/l TCA or TCA (6.0 or 25 mmol/l) with/without 15.6 mmol/l DCA. Proliferative lesions (foci of altered hepatocytes and hepatocellular adenomas) promoted by TCA increased linearly with its concentration and were predominantly basophilic and negative for glutathione S-transferase-pi (GST-pi), while those promoted by DCA increased exponentially with its concentration and were eosinophilic and positive for GST-pi. The promoting activity of DCA and TCA in mixtures was at least additive. The proliferative lesions resulting from exposure to the mixtures were predominately similar to those promoted by DCA, i.e. contained eosinophilic and GST-pi-positive hepatocytes.

CHEMOPREVENTION OF VINYL CARBAMATE-INDUCED LUNG TUMORS IN STRAIN A MICE

The ability of potential chemopreventive agents to prevent vinyl carbamate-induced lung tumors was determined in 2 different experiments. Female strain A mice administered intraperitoneally either a single injection of 60 mg/kg vinyl carbamate that induced 24.0 +/- 1.72 tumors/mouse at 24 weeks or 2 injections of 16 mg/kg vinyl carbamate each (32 mg/kg total dose) that induced 43.2 +/- 3.2 tumors/mouse at 20 weeks. Lung carcinomas were found as early as 16 weeks. Dexamethasone and piroxicam provided in the diet were found to significantly inhibit lung tumors induced by 60 mg/kg vinyl carbamate at 24 weeks whereas myo-inositol also provided in the diet, did not significantly inhibit tumor formation. In animals given 6 16-mg/kg doses of vinyl carbamate, tumor multiplicity was reduced roughly 25% by alpha-difluoromethylornithine and green tea and reduced 50% by dexamethasone and piroxicam. Combinations of these agents were also tested using a total dose of 32 mg/kg of vinyl carbamate. Although alpha-difluoromethylornithine and green tea did not result in a significant inhibition of lung tumor formation if used alone, the combination of alpha-difluoromethylornithine and green tea resulted in a significant reduction of tumor multiplicity. The combinations of alpha-difluoromethylornithine or green tea with either dexamethasone or piroxicam or the combination of dexamethasone and piroxicam did not decrease tumor multiplicity greater than achieved by dexamethasone and piroxicam alone. In summary, selected chemopreventive agents previously shown to inhibit lung tumors by other chemical carcinogens also inhibited vinyl carbamate-induced lung tumors.

Effect of calcium on azoxymethane-induced aberrant crypt foci and cell proliferation in the colon of rats

Calcium has been proposed for prevention of colon cancer. The effects of calcium on azoxymethane (AOM)-induced aberrant crypt foci (ACP), a putative precancerous lesion, and cell proliferation were determined in rat colon. Male F344 rats were given AIN-76A diet that contained calcium at concentrations of 0.5, 1.0, 2.5, 5.0, 10.0 and 15.0 g/kg in experiment 1 and 0.2, 2.0 and 6.0 g/kg in experiment 2. One week after the rats received these diets, they were given the first of two weekly 15 mg/kg injections of AOM. The rats were killed after 35 days of exposure to the different diets. In experiment 1, exposure to either low (0.5 and 1.0 g/kg) or high (10.0 and 15.0 g/kg) concentrations of calcium reduced the yield of ACF relative to 5.0 g/kg calcium. In experiment 2, exposure to 0.2 and 2.0 g/kg calcium resulted in a lower yield of ACF than 6.0 g/kg. Cell proliferation in ACF and non-involved crypts was reduced in animals that received 0.5 or 15.0 mg/Kg relative to 5.0 mg/kg diet calcium. Our results indicate that both lower and higher concentrations of calcium relative to its standard concentration in AIN-76A diet can prevent ACF and reduce the extent of cell proliferation in the lesion which would likely lead to the prevention of colon cancer.

Prevention of mouse lung tumors by targretin

Targretin has indicated chemotherapeutic activity against nonsmall‐cell lung cancer and chemoprevention in rat mammary gland. Therefore, targretin was evaluated for the prevention of 4‐(methylnitrosoamino)‐1‐(3‐pyridyl)‐1‐butanol (NNK) and vinyl carbamate‐induced lung tumors in female strain A mice. Three experiments were performed: (i) a dose‐response study with vinyl carbamate‐induced tumors; (ii) a limited treatment study also with vinyl carbamate and (iii) prevention of NNK‐induced tumors. In the dose‐response study, 0, 10, 30, 100 and 300 mg/kg targretin were administered after vinyl carbamate. Dose levels of 30 mg/kg and greater significantly decreased tumor multiplicity by >19%. However, the efficacy of 30 and 300 mg/kg was not significantly different demonstrating a shallow dose‐response relationship. In the limited treatment study, 200 mg/kg targretin was administered to the mice from 4–13, 4–19, 4–25 and 23–25 weeks after vinyl carbamate. Administering targretin from weeks 4–19 and 4–25 decreased the multiplicity of tumors from 35.3 ± 1.43 to 29.1 ± 1.51 and 25.0 ± 0.93, respectively, and along with administering it from weeks 23–25 decreased tumor size. In the third study, when targretin (100 and 300 mg/kg) was administered for 3 weeks after NNK followed by a 20 weeks holding period, tumor multiplicity was reduced from 10.6 ± 1.13 to 6.38 ± 0.75 and 4.60 ± 0.70, respectively. Hence, targretin demonstrated both preventive and therapeutic activity with respect to mouse lung tumors supporting its further development as a preventive and therapeutic agent for lung cancer.

Effect of budesonide on the methylation and mRNA expression of the insulin-like growth factor 2 and c-myc genes in mouse lung tumors

The use of surrogate end‐point biomarkers could help in the development of chemopreventive agents. To define potential surrogate end‐point biomarkers, the ability of budesonide to decrease mRNA expression of the insulin‐like growth factor‐2 (Igf‐II) and c‐myc genes and to cause the remethylation of the genes was investigated in lung tumors. Lung tumors were induced in female strain A mice by administering i.p. 16 mg/kg vinyl carbamate for 2 consecutive wk or by a single dose of 100 mg/kg benzo[a]pyrene (B[a]P). Thirty‐four weeks later, the mice given vinyl carbamate received budesonide (0.6 or 2.4 mg/kg diet) for 7 d and then were killed. Mice were killed 24 wk after administration of B[a]P. The mRNA expression of the Igf‐II and c‐myc genes was increased in lung tumors relative to normal lung tissue. Budesonide decreased mRNA expression of both genes in tumors. The methylation status of 27 CpG sites in the differentially methylated region 2 in the Igf‐II gene was determined with the bisulfite‐treated DNA‐sequencing procedure. The numbers of methylated CpG sites were 17–21 in normal lung (70.4 ± 2.6%); 0–2, and 1–2 in lung tumors induced by vinyl carbamate and B[a]P (4.9 ± 1.2% and 4.6 ± 1.2%, respectively); and 4–5 or 7–16 in tumors after treatment with 0.6 or 2.4 mg/kg budesonide (16.0 ± 1.2% and 46.2 ± 5.1%, respectively). Thus, lung tumors had strikingly less methylated CpG sites than normal lung tissue, while even limited treatment with budesonide resulted in remethylation of the CpG sites in tumors. With HpaII digestion followed by Southern blot analysis, the internal cytosine of CCGG sites in the c‐myc gene was found to be methylated in normal lung tissue, whereas some of the sites were unmethylated in lung tumors. Treatment for 7 d with budesonide resulted in the remethylation of these sites. In conclusion, mouse lung tumors showed decreased methylation of the Igf‐II and c‐myc genes that was associated with increased expression of these genes. Budesonide treatment caused remethylation and decreased expression of both genes. The results support the possibility of using decreased mRNA expression and remethylation of the Igf‐II and c‐myc genes as biomarkers for the efficacy of budesonide.

Termination of piroxicam treatment and the occurrence of azoxymethane-induced colon cancer in rats

Piroxicam has been shown to prevent azoxymethane (AOM)-induced colon cancer when administered during the promotion/ progression phase. The requirement for continued treatment with piroxicam in order to maintain prevention of colon cancer was investigated. Male F344 rats were administered 15 mg/kg AOM at 7 and 8 weeks of age and started to receive piroxicam (200 mg/kg) in their diet at 11 weeks after the second dose of AOM. Piroxicam was removed from the diet of some of the rats at weeks 19 and 28 and the animals were held until week 47. Other rats continued to receive piroxicam until sacrificed at week 47. Treatment with piroxicam from week 11-47 reduced the yield of colon tumors. When treatment was terminated at week 19 or 28 the yield of tumors at week 47 was not reduced. Within 1 week of the start of piroxicam treatment, the number of aberrant crypt foci (ACF)/animal was decreased. Termination of treatment resulted in the recurrence of ACF. Apoptosis in adenomas was increased when piroxicam treatment was continued to week 47 but not when treatment was terminated earlier at week 19 or 28. The proliferating cell nuclear antigen-labeling index in adenomas was not affected by piroxicam even when treatment was from week 11 to 47. In summary, termination of treatment resulted in the occurrence of ACF and colon cancer indicating that prevention by piroxicam was reversible. Furthermore, enhancement of apoptosis and not decreased cell proliferation correlated with prevention of colon cancer.