Dose-response study of myo-inositol as an inhibitor of lung tumorigenesis induced in A/J mice by benzo.

Dietary myo-inositol is an effective inhibitor of lung tumor induction in mice, but no dose-response studies have been reported. We assessed the ability of various doses of dietary myo-inositol to inhibit lung tumor induction in female A/J mice treated with eight weekly doses of benzo[a]pyrene (BaP) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (3 micromol of each by gavage), then killed 18 weeks later. In Expt. 1, groups of 20 mice each were treated with myo-inositol at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0% in AIN-93 diet for 1 week prior to, during, and for 1 week after the carcinogen administration period. In Expt. 2, groups of 20 mice each were treated with the same concentrations of myo-inositol in the diet as in Expt. 1, except this diet was administered from 1 week after carcinogen administration until termination. There were no effects of myo-inositol on lung tumor incidence, which was 100% in all groups treated with BaP plus NNK. However, myo-inositol significantly decreased lung tumor multiplicity in both experiments. In Expt. 1, significant reductions of 28.9 and 33.0% were observed at the 1 and 0.5% doses of myo-inositol, but not at the lower doses. In Expt. 2, a significant reduction of 48.4% was observed at the 1% dose. In both Expts. 1 and 2, there was a significant dose trend for inhibition (P<0.0001). No toxicity was observed at any dose. These results firmly establish myo-inositol as a chemopreventive agent against lung tumor induction in A/J mice, at doses that can be envisioned for human use.

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on benzo[a]pyrene metabolism and DNA adduct formation in the A/J mouse.

Benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in A/J mice by benzo[a]pyrene (B[a]P). In contrast, phenethyl isothiocyanate (PEITC) does not. We tested the hypothesis that BITC inhibits B[a]P tumorigenicity in mouse lung by inhibiting DNA adduct formation, and compared the effects of BITC and PEITC. In mouse liver or lung microsomal incubations, BITC and PEITC inhibited formation of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (B[a]P-7, 8-diol) and some other B[a]P metabolites. The metabolism of B[a]P was compared in mouse lung and liver microsomes, 6 or 24h after treatment with BITC or PEITC. In lung, 6 h after treatment, B[a]P-7, 8-diol and some other metabolites were inhibited by BITC and PEITC. However, 24 h after treatment, no inhibition of B[a]P-7,8-diol was observed in microsomes from BITC-treated mice, whereas it was substantially increased in mice treated with PEITC. Effects on B[a]P metabolism in liver microsomes were generally modest. Conversion of B[a]P-7,8-diol to mutagens by mouse liver microsomes was more strongly inhibited by BITC than PEITC. Effects on 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA adduct formation were evaluated in DNA from mice treated with isothiocyanates and B[a]P, and killed 2-120h later. The area under the curve (AUC) for BPDE-DNA adducts in lung was 29.5% less (P = 0. 001) in the BITC-B[a]P treated mice and 19.0% less (P = 0.02) in the PEITC-B[a]P mice than in the mice treated with B[a]P alone. Similar results were obtained in liver DNA. There were no significant differences between the reduction of BPDE-DNA AUC values by BITC versus PEITC. The results of this study support the hypothesis that BITC inhibits B[a]P-induced lung tumorigenesis in A/J mice by inhibiting the metabolic activation of B[a]P to BPDE-DNA adducts. However, differences in BPDE-DNA adduct formation do not appear to explain fully the contrasting effects of BITC and PEITC on B[a]P-induced lung tumorigenesis.

Effects of phenethyl isothiocyanate and benzyl isothiocyanate, individually and in combination, on lung tumorigenesis induced in A/J mice by benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Phenethyl isothiocyanate (PEITC) is an effective inhibitor of lung tumorigenesis induced in rats and mice by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) while benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in mice by another tobacco smoke carcinogen, benzo[a]pyrene (BaP). However, little is known about the inhibitory effects of PEITC and BITC in combination, or about the effects of PEITC or BITC on tumorigenesis by a mixture of NNK and BaP. In this study, we carried out a series of experiments pertinent to these questions. In Experiment 1, treatment of A/J mice with PEITC (6 micromol), BITC (6 micromol), or a combination of the two (6 micromol each) by gavage, 2 h prior to each of eight weekly gavage treatments with a mixture of BaP and NNK (3 micromol of each), had no effect on lung tumor multiplicity. In Experiment 2, we evaluated the inhibitory potential of four different mixtures of PEITC and BITC, administered by gavage 2 h prior to each of eight weekly doses of BaP and NNK, as given in Experiment 1. Mixtures of PEITC and BITC (12 micromol of each, or 12 micromol PEITC and 9 micromol BITC) significantly reduced lung tumorigenesis induced by a mixture of BaP and NNK. In Experiment 3, we investigated the effects of dietary PEITC (3 micromol/g diet), BITC (1 micromol/g diet), or a mixture of PEITC (3 micromol/g diet) and BITC (1 micromol/g diet). These compounds were started 1 week before, and continued through to 1 week after the eight weekly treatments with BaP and NNK. PEITC, and PEITC plus BITC, both significantly inhibited lung tumor multiplicity; inhibition was due mainly to PEITC. In Experiment 4, we tested dietary PEITC (3, 1, or 0.3 micromol/g diet) as an inhibitor of lung tumorigenesis induced by BaP, NNK, or BaP plus NNK using a protocol identical to that in Experiment 3. PEITC was an effective inhibitor of lung tumor multiplicity induced by NNK and a mixture of BaP plus NNK, but not by BaP. Dietary PEITC, or PEITC plus BITC, was more effective in these experiments than the compounds given by gavage. The results of this study demonstrate that proper doses of dietary PEITC and dietary as well as gavaged PEITC plus BITC are effective inhibitors of lung tumorigenesis induced in A/J mice by a mixture of BaP and NNK.

Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has a chiral center but the tumorigenicity of the NNAL enantiomers has not been previously examined. In this study, we assessed the relative tumorigenic activities in the A/J mouse of NNK, racemic NNAL, (R)-NNAL, (S)-NNAL and several NNAL metabolites, including [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] beta-O-D-gluco-siduronic acid [(S)-NNAL-Gluc], 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran, 4-(3-pyridyl)butane-1,4-diol and 2-(3-pyridyl) tetrahydrofuran. We also quantified urinary metabolites of racemic NNAL and its enantiomers and investigated their metabolism with A/J mouse liver and lung microsomes. Groups of female A/J mice were given a single i.p. injection of 20 micromol of each compound and killed 16 weeks later. Based on lung tumor multiplicity, (R)-NNAL (25.6 +/- 7.5 lung tumors/mouse) was as tumorigenic as NNK (25.3 +/- 9.8) and significantly more tumorigenic than racemic NNAL (12.1 +/- 5.6) or (S)-NNAL (8.2 +/- 3.3) (P < 0. 0001). None of the NNAL metabolites was tumorigenic. The major urinary metabolites of racemic NNAL and the NNAL enantiomers were 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), NNAL-N-oxide and NNAL-Gluc, in addition to unchanged NNAL. Treatment with (R)-NNAL or (S)-NNAL gave predominantly (R)-hydroxy acid or (S)-hydroxy acid, respectively, as urinary metabolites. While treatment of mice with racemic or (S)-NNAL resulted in urinary excretion of (S)-NNAL-Gluc, treatment with (R)-NNAL gave both (R)-NNAL-Gluc and (S)-NNAL-Gluc in urine, apparently through the metabolic intermediacy of NNK. (S)-NNAL appeared to be a better substrate for glucuronidation than (R)-NNAL in the A/J mouse. Mouse liver and lung microsomes converted NNAL to products of alpha-hydroxylation, to NNAL-N-oxide, to adenosine dinucleotide phosphate adducts and to NNK. In lung microsomes, metabolic activation by alpha-hydroxylation of (R)-NNAL was significantly greater than that of (S)-NNAL. The results of this study provide a metabolic basis for the higher tumorigenicity of (R)-NNAL than (S)-NNAL in A/J mouse lung, namely preferential metabolic activation of (R)-NNAL in lung and preferential glucuronidation of (S)-NNAL.

Evaluation of butylated hydroxyanisole, myo-inositol, curcumin, esculetin, resveratrol and lycopene as inhibitors of benzo[a]pyrene plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice.

The potential activities of butylated hydroxyanisole (BHA), myo-inositol, curcumin, esculetin, resveratrol and lycopene-enriched tomato oleoresin (LTO) as chemopreventive agents against lung tumor induction in A/J mice by the tobacco smoke carcinogens benzo[a]pyrene (BaP) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were evaluated. Groups of 20 A/J mice were treated weekly by gavage with a mixture of BaP and NNK (3 micromol each) for 8 weeks, then sacrificed 26 weeks after the first carcinogen treatment. Mice treated with BHA (20 or 40 micromol) by gavage 2 h before each dose of BaP and NNK had significantly reduced lung tumor multiplicity. Treatment with BHA (20 or 40 micromol) by gavage weekly or with dietary BHA (2000 ppm), curcumin (2000 ppm) or resveratrol (500 ppm) from 1 week after carcinogen treatment until termination had no effect on lung tumor multiplicity. Treatment with dietary myo-inositol (30,000 ppm) or esculetin (2000 ppm) from 1 week after carcinogen treatment until termination significantly reduced lung tumor multiplicity, with the effect of myo-inositol being significantly greater than that of esculetin. Treatment with dietary LTO (167, 1667 or 8333 ppm) from 1 week before carcinogen treatment until termination had no effect on lung tumor multiplicity. The results of this study demonstrate that BHA is an effective inhibitor of BaP plus NNK-induced lung tumorigenesis in A/J mice when administered during the period of carcinogen treatment and that, among the compounds tested, myo-inositol is most effective after carcinogen treatment.

Human bone marrow heparan sulfate induces leukemia cell differentiation.

The proliferation and development of hematopoietic cells occurs in close association with bone marrow stroma. Heparan sulfate is a major component of the stroma. We have isolated a form of heparan sulfate proteoglycan from a human stromal cell line grown in vitro in the presence of [35S]sulfate. This proteoglycan contains a phosphatidylinositol component which likely anchors it to the stromal cell membrane. The glycosaminoglycan chains of this proteoglycan could induce maturation of the HL-60 myeloid leukemia cell line. A less hydrophobic heparan sulfate proteoglycan was also present in the stroma, but could not induce HL-60 maturation. The two heparan sulfates had glycosaminoglycan chains that were similar in size (36 Kd) and charge density. Structural studies suggested only minor but perhaps significant differences in the carbohydrate sequences of the two heparan sulfates. The relationship of these subtle structural differences to the difference observed in differentiation-inducing activity remains to be elucidated.

Chemoprevention of benzo[a]pyrene-induced forestomach cancer in mice by natural phthalides from celery seed oil.

Bioassay-directed fractionation of celery seed oil from the plant Apium graveolens (Umbelliferae) led to the isolation of five natural products, including d-limonene, p-mentha-2,8-dien-1-ol, p-mentha-8(9)-en-1,2-diol, 3-n-butyl phthalide, and sedanolide. Of these compounds p-mentha-2,8-dien-1-ol,3-n-butyl phthalide, and sedanolide exhibited high activities to induce the detoxifying enzyme glutathione S-transferase (GST) in the target tissues of female A/J mice. 3-n-Butyl phthalide and sedanolide (20 mg/dose every two days for a total of 3 doses) increased GST activity 4.5-5.9 and 3.2-5.2 times over the controls in the mouse liver and small intestinal mucosa, respectively. At the same dose, p-mentha-2,8-dien-1-ol induced GST activity about 3.7-fold above that of the controls. Thus, these compounds were further tested for their ability to inhibit benzo[a]pyrene- (BP) induced tumorigenesis in mice. After treatment with 3-n-butyl phthalide and sedanolide, the tumor incidence was reduced from 68% to 30% and 11%, respectively. About 67% and 83% reduction in tumor multiplicity was also observed with 3-n-butyl phthalide and sedanolide. p-Mentha-2,8-dien-1-ol produced only a small or no significant reduction of forestomach tumor formation. The data indicating that 3-n-butyl phthalide and sedanolide were both active in tumor inhibition and GST assays suggested a correlation between the inhibitory activity and the GST-inducing ability. The phthalides are known to determine the characteristic odor of celery. The results suggest that phthalides, as a class of bioactive natural products occurring in edible umbelliferous plants, may be effective chemopreventive agents.

Inhibition of benzo[a]pyrene-induced tumorigenesis by myristicin, a volatile aroma constituent of parsley leaf oil.

Glutathione S-transferase (GST) assay-guided fractionation of parsley leaf oil from the edible plant Petroselinum sativum Hoffm. (Umbelliferae) led to the isolation of myristicin. Myristicin showed high activity as an inducer of the detoxifying enzyme GST in the liver and small intestinal mucosa of female A/J mice. Reduction of myristicin yielded dihydromyristicin that retained the GST-inducing activity. Myristicin and dihydromyristicin were tested for their capacity to inhibit benzo[a]pyrene (B[a]P)-induced tumor formation in female A/J mice. A 65% inhibition of the tumor multiplicity in the lung was observed as the result of treatment of myristicin. Dihydromyristicin produced small or insignificant reduction of lung tumor formation. In the forestomach, myristicin showed a 31% inhibition of tumor formation; while dihydromyristicin exhibited a 27% inhibition. Comparison of the structures and activities indicated that the saturation of the isolated double bond in myristicin resulted in a significant decrease in the inhibitory activity against B[a]P-induced tumorigenesis. The present results showed that myristicin, an active inducer of GST activity, is an effective inhibitor of B[a]P-induced tumorigenesis in mice. Stimulation of GST activity by myristicin could be a major mechanism for its inhibition of B[a]P or other carcinogens that may be detoxified in the same manner. As a culinary herb parsley is regularly consumed by humans. Parsley leaf oil is also used extensively for garnishing and seasoning. The results of this study indicate that as a major volatile aroma constituent of parsley myristicin may be an effective cancer chemopreventive agent.

Isolation and biological evaluation of potential cancer chemopreventive agents from ambrette musk residue.

Two nitroaromatic compounds, 1-(1,1-dimethylethyl)-3,5-dimethyl-2,4-dinitrobenzene (1) and 1-[4-(1,1-dimethylethyl)-2,6-dimethyl-3,5-dinitrophenyl]ethanone or musk ketone (2), were isolated from ambrette musk residue, which is widely used in the food and cosmetics industry. The ability of 1 and 2 to induce increased activity of the detoxifying enzyme glutathione S-transferase was tested in A/J mice. Enzyme induction in the cytosols of liver, forestomach, lung, colon, and small intestinal mucosa was determined. Biological evaluation revealed that both compounds exhibit high activity as glutathione S-transferase inducers in liver and small intestinal mucosa. The effects of 1 and 2 on the levels of acid-soluble sulfhydryl in the five mouse tissues were also determined. Both compounds slightly elevated sulfhydryl levels in the small intestinal mucosa but significantly decreased the sulfhydryl levels in the other tissues. Because the ability of anticarcinogenic compounds to induce an increase in the detoxifying enzyme activity correlates with their tumor inhibitory activity, 1 and 2 may be potential cancer chemopreventive agents.

Anethofuran, carvone, and limonene: potential cancer chemopreventive agents from dill weed oil and caraway oil.

Bioassay-directed fractionation of dill weed oil and caraway oil, respectively, from the plants Anethum graveolens L. and Carum carvi L. (Umbelliferae) has led to the isolation of three monoterpenes, anethofuran (1), carvone (2), and limonene (3). Their structures were determined on the basis of spectral analysis. These compounds induced the detoxifying enzyme glutathione S-transferase in several mouse target tissues. The alpha,beta-unsaturated ketone system in carvone appeared to be critical for the high enzyme-inducing activity.

Sesquiterpenes from clove (Eugenia caryophyllata) as potential anticarcinogenic agents.

Bioassay-directed fractionation of clove terpenes from the plant Eugenia caryophyllata has led to the isolation of the following five active known compounds: beta-caryophyllene [1], beta-caryophyllene oxide [2], alpha-humulene [3], alpha-humulene epoxide I [4], and eugenol [5]. Their structures were determined on the basis of spectral analysis (hreims, 1H and 13C nmr). These compounds showed significant activity as inducers of the detoxifying enzyme glutathione S-transferase in the mouse liver and small intestine. The ability of natural anticarcinogens to induce detoxifying enzymes has been found to correlate with their activity in the inhibition of chemical carcinogenesis. Thus, these sesquiterpenes show promise as potential anticarcinogenic agents.

Phenylalkyl isothiocyanate-cysteine conjugates as glutathione S-transferase stimulating agents.

To develop analogues of phenylalkyl isothiocyanate with less toxicity and better biological activity, two water-soluble phenylalkyl isothiocyanate-cysteine conjugates, S-[N-benzyl(thiocarbamoyl)]-L-cysteine (1) and S-[N-(3-phenylpropyl)(thiocarbamoyl)]-L-cysteine (2), were synthesized. The induction of increased activity of the detoxifying enzyme glutathione S-transferase by the conjugates and their parent compounds was determined and compared in several tissues of A/J mice. The biological evaluation revealed that the conjugates as GST enzyme inducers appeared to be less toxic and even more potent than the parent compounds in the mouse bladder. Compounds 1 was much more active than 2 in all the tissues examined, while their parent compounds showed an inverse order of activity. Thus, an increase in the alkyl chain length of the parent isothiocyanates or a decrease in the alkyl length of the conjugates could result in higher enzyme-inducing activity in the same compound series. Since a number of nitrosamines have been identified as prime bladder carcinogens and phenylalkyl isothiocyanates have been reported to inhibit a wide range of carcinogenic nitrosamines, the corresponding conjugates may serve as prodrugs to protect against nitrosamine-induced urinary bladder carcinogenesis once they are delivered to the target organ.