The still-face effect in Chinese and Canadian 3- to 6-month-old infants

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.     

(-)-Epigallocatechin-3-gallate inhibition of ultraviolet B-induced AP-1 activity

Green tea polyphenols have been shown to inhibit cancer in a variety of tumor models, including ultraviolet B (UVB)-induced non-melanoma skin cancer. In green tea extracts, the major dry mass constituent is the family of catechins, of which (-)-epigallocatechin-(3)-gallate (EGCG) is considered to be important for the chemopreventive activity. EGCG has been shown to have antioxidant properties, but there has been little progress toward identifying the specific targets and mechanisms of its action. Using cultured human keratinocytes, we show that UVB-induced AP-1 activity is inhibited by EGCG in a dose range of 5.45 nM to 54.5 microM. EGCG is effective at inhibiting AP-1 activity when applied before, after or both before and after UVB irradiation. EGCG also inhibits AP-1 activity in the epidermis of a transgenic mouse model. This work begins to define a mechanism by which EGCG could be acting to inhibit UVB-induced tumor formation.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone mouse lung tumorigenesis by arylalkynes, mechanism-based inactivators of cytochrome P450

Arylalkynes such as 4-phenyl-1-butyne (PBY), 5-phenyl-1-pentyne (PPY) and 2-ethynylnaphthalene (2-EN) are suicide inhibitors of cytochrome P450 enzymes. Arylalkyl isothiocyanates such as 6-phenylhexyl isothiocyanate (PHITC) are structurally related to arylalkynes and are known to inhibit the cytochrome P450 mediated metabolic activation and tumorigenicity of a tobacco-specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we compared the ability of PBY, PPY, 2-EN and PHITC to inhibit A/J mouse lung tumorigenesis by NNK. Groups of 20 female mice were gavaged with 5 mumol of arylalkyne or PHITC in corn oil. Two hours later they were given a single i.p. injection of 10 mumol NNK. The mice were killed 16 weeks later. PPY and PHITC were both potent inhibitors of tumorigenesis by NNK, reducing lung tumor multiplicity from 8.35 tumors per mouse to 0.40 and 0.35 respectively. PBY and 2-EN also significantly inhibited tumor multiplicity. The results of this study demonstrate that arylalkynes and PHITC are potent inhibitors of NNK induced lung tumorigenesis in A/J mice, consistent with the hypothesis that inhibition of specific cytochrome P450 enzymes is involved in inhibition of tumorigenesis.     

Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions

We investigated clinical efficacy of green tea extracts (polyphenon E; poly E and (-)-epigallocatechin-3-gallate [EGCG]) delivered in a form of ointment or capsule in patients with human papilloma virus (HPV) infected cervical lesions. Fifty-one patients with cervical lesions (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) were divided into four groups, as compared with 39 untreated patients as a control. Poly E ointment was applied locally to 27 patients twice a week. For oral delivery, a 200 mg of poly E or EGCG capsule was taken orally every day for eight to 12 weeks. In the study, 20 out of 27 patients (74%) under poly E ointment therapy showed a response. Six out of eight patients under poly E ointment plus poly E capsule therapy (75%) showed a response, and three out of six patients (50%) under poly E capsule therapy showed a response. Six out of 10 patients (60%) under EGCG capsule therapy showed a response. Overall, a 69% response rate (35/51) was noted for treatment with green tea extracts, as compared with a 10% response rate (4/39) in untreated controls (P<0.05). Thus, the data collected here demonstrated that green tea extracts in a form of ointment and capsule are effective for treating cervical lesions, suggesting that green tea extracts can be a potential therapy regimen for patients with HPV infected cervical lesions.     

Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in patas monkey lung and liver microsomes

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.     

Absence of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by flavin-containing monooxygenase (FMO)

The N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco and tobacco smoke. Carbonyl reduction, alpha-carbon hydroxylation (activation) and N-oxidation of the pyridyl ring (detoxification) are the three main pathways of metabolism of NNK. In this study, metabolism of NNK was studied with lung and liver microsomes from F344 rats, Syrian golden hamsters and pigs and cloned flavin-containing monooxygenases (FMOs) from human and rabbit liver. Thermal inactivation at 45 degrees C for 2 min reduced FMO S-oxygenating activity but did not affect N-oxidation of NNK, leading to the conclusion that FMOs are not implicated in the detoxification of NNK. Detoxification of NNK was not increased by n-octylamine or by incubation at pH 8.4, supporting the conclusion that FMOs are not involved in the metabolism of NNK. SKF-525A (1 mM) significantly reduced N-oxidation and alpha-carbon hydroxylation, suggesting that these two pathways were catalyzed by cytochromes P450. Metabolism of NNK was lower with lung microsomes than with liver microsomes. Inhibition of metabolism of NNK by SKF-525A was also observed with rat lung microsomes, leading to the conclusion that cytochromes P450 are involved in pulmonary metabolism of NNK. Cloned FMOs did not metabolize NNK. In conclusion, cytochromes P450 rather than FMOs are involved in N-oxidation of NNK. The high capacity of hamster liver microsomes to activate NNK does not correlate with the resistance of this tissue to NNK-induced hepatocarcinogenesis.     

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p-XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.     

Microbiological activity of whole and fractionated crude extracts of tea (Camellia sinensis), and of tea components

Aqueous extracts of teas (Camellia sinensis) of different types and from various sources inhibited a wide range of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus. Tea extracts were bactericidal to staphylococci and Yersinia enterocolitica at well below 'cup of tea' concentrations. Activity was confined to one of four fractions obtained from a green tea extract by partition chromatography. Testing of pure tea compounds and closely related chemicals suggested that the antibacterial activity of extracts of green tea can be explained by its content of epigallocatechin, epigallocatechin gallate and epicatechin gallate. In black tea extracts, theaflavin and its gallates are additional antibacterially active components.     

Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung tumor formation by FGN-1 (sulindac sulfone)

The sulfone derivative of the non-steroidal anti-inflammatory drug (NSAID), sulindac, has been reported to inhibit mammary and colon tumor formation in rodent models of chemically-induced carcinogenesis. Unlike its parent compound, this metabolite lacks cyclo-oxygenase inhibitory activity. A tumor induction protocol, consisting of NNK administration in the drinking water over several weeks to model chronic human exposure, was used to test whether the sulfone (called FGN-1) could inhibit the formation of primary lung tumors in mice. A total of 150 female, AIN76A-fed, A/J mice received 9 mg of NNK each. Concentrations of FGN-1 that had been previously determined not to affect body weight gain were added to the food at levels of 0, 250, 500 and 750 mg/kg of diet (30 mice/group) starting 2 weeks before NNK administration and continuing for 22 weeks. At that time pleural surface tumors were counted. Tumor incidence decreased significantly from 96 % in the control diet and 93% in the 250 FGN-1 mg/kg diet to 63 and 67% in the 500 and 750 mg FGN-1/kg diet groups, respectively (P < 0.001 by chi-square analysis). Lung tumor multiplicity decreased from 18.1+/-3 tumors/ mouse (mean+/-SEM, control diet) to 12.3+/-3 (250), 5.3+/-1 (500) and 2.1+/-1 (750) (P < 0.0005 by post hoc ANOVA). In previous studies using this carcinogenesis protocol, the maximum tolerated dose of sulindac inhibited lung tumor multiplicity by no more than 50% with no effect on incidence. This dose-dependent reduction in tumorigenesis by a non-toxic dose of FGN-1 indicates a strong chemopreventive activity against experimental induction of lung carcinogenesis. The greater potency of the sulfone over sulindac and its lack of toxic side effects because of its inability to affect cyclo-oxygenase activity suggests that clinical testing in individuals at high risk for lung cancer should be considered.     

The effects of phenethyl isothiocyanate, N-acetylcysteine and green tea on tobacco smoke-induced lung tumors in strain A/J mice

Male and female strain A/J mice were exposed to a mixture of cigarette sidestream and mainstream smoke at a chamber concentration of total suspended particulates of 82.5 mg/m3. Exposure time was 6 h/day, 5 days/week for 5 months. The animals were allowed to recover for another 4 months in filtered air before sacrifice and lung tumor count. Male animals were fed either 0.2% N-acetylcysteine (NAC) or 0.05% phenethyl isothiocyanate (PEITC) in diet AIN-76A with 5% corn oil added. Female animals received normal laboratory chow and were given a 1.25% extract of green tea in the drinking water. Corresponding control groups were fed diets without NAC or PEITC or given plain tap water. Exposure to tobacco smoke increased lung tumor multiplicity to 1.1-1.6 tumors/lung, significantly higher than control values (0.5-1.0 tumors/lung). None of the putative chemopreventive agents (NAC, PEITC or green tea extract) had a protective effect. In positive control experiments, PEITC significantly reduced both lung tumor multiplicity and incidence in mice treated with the tobacco smoke-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In mice treated with three different doses of urethan and fed NAC in the diet, a significant reduction in lung tumor multiplicity was found only at one dose level. Green tea extract did not reduce lung tumor multiplicity in animals treated with a single dose of NNK. It was concluded that successful chemoprevention of tobacco smoke-induced lung tumorigenesis might require administration of several chemopreventive agents rather than just a single one.     

Effects of 1,4-phenylenebis(methylene)selenocyanate and selenomethionine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorigenesis in A/J mouse lung

We reported earlier that continuous feeding of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibited lung tumor induction by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the A/J mouse (El-Bayoumy et al., Carcinogenesis, 14, 1111-1113, 1993). The present investigation was designed to determine whether p-XSC inhibits pulmonary neoplasia induced by NNK in female A/J mice during the initiation phase of carcinogenesis or during the post-initiation phase. The naturally occurring selenomethionine was also included in this study. Doses higher than 4 p.p.m. of selenomethionine can induce toxic effects, therefore, dietary supplementation of this compound was selected at a dose level of 3.75 p.p.m. However, we were able to give p-XSC at selenium levels of 7.5 and 15 p.p.m., as mice can tolerate such doses in this form without any adverse effects. NNK was given by a single i.p. injection at dose of 10 micromol in 0.1 ml of saline. Selenomethionine did not show chemopreventive activity when administered in either phase of tumorigenesis. In contrast, p-XSC significantly reduced lung tumor multiplicity regardless of whether it was given during the initiation phase of tumorigenesis (P = 0.0009 at both levels of selenium) or post-initiation (P = 0.0009 at 15 p.p.m. and P = 0.036 for 7.5 p.p.m.). This is the first report describing that the synthetic organoselenium compound, p-XSC, can effectively block and suppress chemically (NNK)-induced lung tumor development in mice.     

Biodistribution of, antimutagenic efficacies in Salmonella typhimurium of, and inhibition of P450 activities by ellagic acid and one analogue

Ellagic acid (EA) is generated by hydrolysis of ellagitannins present in fruit berries and edible nuts and grapes. Large doses of EA prevent lung tumorigenesis induced by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. In this study, we document the efficacies of the EA structural analogue (3,4,7,8-tetrahydroxy-6H-benzo[b,d]pyran-6-one) (analogue 1) to inhibit specific P450 activities, pulmonary metabolism of NNK in A/J mice, and NNK-induced mutations in Salmonella typhimurium. Mouse lung microsomes metabolized benzyloxyresorufin, a marker of cytochrome P450 2B1 activity, more extensively than methoxyresorufin or ethoxyresorufin. The EA analogue was more effective than EA in inhibiting dealkylation of the three alkoxyresorufins, suggesting that it is a nonspecific inhibitor of P450s. Mouse lung microsomes hydroxylate testosterone in the 7alpha and 6beta positions, suggesting contributions of P450 2A1 and P450 3A2 isozymes, respectively. Inhibition of both pathways was more effective with the EA analogue than with EA. Mouse lung explants metabolized NNK by alpha-carbon hydroxylation (activation) and pyridine N-oxidation (deactivation). Both pathways were inhibited when 100 microM EA was added to the culture medium. The EA analogue was a better inhibitor of the activation of NNK to electrophilic species than EA. Mouse lung microsomes activate NNK to intermediates mutagenic to S. typhimurium. Inhibition of NNK mutagenicity by EA or the EA analogue was 20 or 65%, respectively. The distribution of the EA analogue in lung and liver was determined following gavage with 1.7 mmol of the EA analogue. In the lung, a maximal level of EA analogue corresponding to 105 nmol was observed 30 min after administration of the analogue. The level in liver tissues was 4-fold lower than in the lung. Results of this study demonstrate that the EA analogue is more effective than EA in inhibiting the pulmonary activation of NNK and suggest that the EA analogue could be effective in preventing lung tumorigenesis.     

Recovery from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced immunosuppression in A/J mice by treatment with nonsteroidal anti-inflammatory drugs

BACKGROUND: We have previously reported that nonsteroidal anti-inflammatory drugs inhibit lung tumorigenesis induced by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. PURPOSE: The aims of this study were to determine if NNK suppresses humoral (i.e., antibody) and cellular immune responses in mice and if nonsteroidal anti-inflammatory drugs could attenuate these immune responses. METHODS: Female A/J mice (7-8 weeks old) were fed nonsteroidal anti-inflammatory drugs starting 2 weeks before the beginning of NNK treatment (9.1 mg per mouse in total) and continuing through the 7 weeks of NNK treatment. Eight groups (two control groups and six experimental groups) of 10 mice each were used per experiment. Animals in the two control groups received the same diet and water as animals in the six experimental groups; one control group received no nonsteroidal anti-inflammatory drugs or NNK and the other control group received only NNK. The primary humoral and cellular immune responses to the various treatments were assayed by the plaque-forming cell technique and by measurement of natural killer cell cytotoxic activity, respectively. At the end of each experiment, the animals were killed, blood was collected, plasma was prepared, and levels of the immune system modulator prostaglandin E2 were measured. RESULTS: NNK treatment inhibited the plaque-forming cell response by approximately 50%; this inhibition was attenuated by treatment with sulindac or acetylsalicylic acid (P = .0001 for both). In contrast, treatment with naproxen, which had no chemopreventive (i.e., tumor inhibitory) efficacy, further increased by 26% (P = .05) the immunosuppressive effect of NNK. The cytotoxic activity of splenic natural killer cells against YAC-1 cells was reduced by 60% (P = .002); treatment with acetylsalicylic acid (254 mg/kg of diet) reduced the NNK-induced natural killer cell cytotoxicity inhibition by 50% (P = .02), whereas the administration of the specific cyclooxygenase-2 inhibitor NS-398 (7 mg/kg of diet) resulted in an almost complete recovery (approximately 95%, P = .04) of natural killer cell activity. The prostaglandin E2 plasma concentration was approximately 100% greater in NNK-treated mice than in untreated mice. Treatment of the mice with nonsteroidal anti-inflammatory drugs attenuated this elevation (from approximately 25% to 100%), and NS-398 (7 mg/kg of diet) was the most effective (100%). CONCLUSIONS AND IMPLICATIONS: The ability of various nonsteroidal anti-inflammatory drugs to inhibit NNK-induced carcinogenesis appears to be directly related to the ability of these drugs to inhibit NNK-induced immunosuppression. Our results suggest that the chemopreventive effect of nonsteroidal anti-inflammatory drugs may be mediated through the modulation of prostaglandin E2 synthesis.     

Prevention of heterocyclic amine formation by tea and tea polyphenols

Heterocyclic amines (HCAs), formed during the cooking of meats and fish, are thought to be the genotoxic carcinogens associated with important types of human cancer in meat-eating populations, such as cancer of the breast, colon or pancreas. We studied the effect of black or green tea, and of the tea polyphenols theaflavine gallate (TFG, black tea) and epigallocatechin gallate (EGCG, green tea) on the formation of typical HCAs, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), using the model in vitro systems of J?gerstad. Green tea and black tea and solutions of TFG and EGCG lower the formation of PhIP by 62-85% during 1 h heating at 160 degrees C of 1 mmol creatinine, 1 mmol phenylalanine and 0.5 mmol glucose in 3.3 ml diethylene glycol-water (10:1), where the inhibitors replaced 0.3 ml of the water. The production of MeIQx and related HCAs, in the same system but with 1 mmol glycine instead of phenylalanine was likewise reduced, determined by amounts of mutagens formed. In the latter systems, the teas were not, or less effective, but the polyphenols were inhibitory. Thus, the tea products represent another approach to lower the formation of HCAs.     

Biliary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the rat

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pancreas carcinogen in rats. The biliary excretion of NNK was therefore studied in anesthetized female Sprague-Dawley rats following i.p. administration of 0.7 mumol/kg [carbonyl-14C]NNK. The concentration of radioactivity peaked within 30 min and decreased thereafter exponentially. Cumulative excretion of radioactivity reached a plateau at 6-9% of the total dose. HPLC analysis revealed the presence of 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl)-butyric acid (keto acid), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl beta-D-glucopyranosiduronic acid (NNAL Glu), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNK. NNAL Glu was the major metabolite contributing 34 +/- 4% of total radioactivity in bile at 30 min and 58 +/- 4% at 5 h. The percentage of acidic metabolites remained constant at approximately 20%. In contrast, the percentage of NNK and NNAL decreased within the first 2 h to < 5% and < 10% respectively. The elimination kinetics of NNK and its metabolites fitted into a one-compartment model with a half-life of 37 min for NNK, 52 min for NNAL and 110 min for NNAL Glu and acidic metabolites. In three rats dosed with 240 mumol/kg NNK i.p., the concentration of radioactivity peaked after 1-2 h and decreased very slowly thereafter. After 5-8 h a total of 12-17% of the dose has been excreted in the bile with no indication of a plateau. At all time points NNAL Glu was the major metabolite contributing up to 95% of total radioactivity in bile. The percentage of acidic metabolites was < 5% throughout the experiment. Whereas NNK contributed one-third of the radioactivity at 30 min and decreased rapidly, the percentage of NNAL in bile remained rather constant at approximately 5-10%. In conclusion, the detection of NNK, NNAL and NNAL Glu gives support to the hypothesis that tobacco-specific carcinogens could reach the pancreas retrograde from the bile, especially at high NNK concentrations.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis by dietary olive oil and squalene

Epidemiological studies have suggested that frequent olive oil consumption may be a protective factor against lung cancer formation. Squalene, a characteristic compound in olive oil, is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and has been proposed to inhibit the farnesylation of ras oncoproteins. The present study investigated the effect of dietary olive oil and squalene in a mouse lung tumorigenesis model. Female A/J mice were fed AIN-76A diets containing 5% corn oil (control), 19.6% olive oil, or 2% squalene starting at 3 weeks before a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (103 mg/kg, i.p.). Animals were maintained on their respective diets throughout the study. At 16 weeks after NNK administration, 100% of the mice in the control group had lung tumors with a tumor multiplicity of 16 tumors per mouse. The olive oil and squalene diets significantly (P < 0.05) decreased the lung tumor multiplicity by 46 and 58%, respectively. The squalene diet significantly (P < 0.05) decreased lung hyperplasia by 70%. In mice fed a diet containing 2% squalene for 3 weeks, the activation of NNK was increased by 1.4- and 2.0-fold in lung and liver microsomes, respectively, but its relationship to the inhibition of carcinogenesis is not clear. These results demonstrate that dietary olive oil and squalene can effectively inhibit NNK-induced lung tumorigenesis.     

Lung tumor induction in A/J mice by the tobacco smoke carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene: a potentially useful model for evaluation of chemopreventive agents

The purpose of this study was to establish a lung tumor model for the evaluation of chemopreventive agents against lung cancer in smokers. Lung tumor induction in A/J mice by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) was studied using protocols in which these two tobacco smoke carcinogens were given individually or in combination. Groups of female A/J mice were treated by either intragastric gavage (i.g.) or by intraperitoneal injection (i.p.) with various doses of NNK and/or BaP for 8 consecutive weeks. The mice were killed either 9 or 19 weeks later and tumors of the lung and forestomach were counted. The i.g. route of administration proved to be more satisfactory than i.p. administration, because it avoided complications due to tumor formation at the injection site and associated mortality. A dose-response relationship for lung tumor induction by i.g. administration of NNK and BaP in combination was established in the mice killed 9 or 19 weeks after completion of carcinogen treatment. The highest total doses of NNK and BaP (a total of 24 mumol of each) induced more lung tumors than would have been expected by extrapolation from the lower doses. Comparisons of NNK and BaP given individually showed that BaP was more tumorigenic to the lung than NNK when given by the i.g. route; i.p. administrations of BaP were complicated by local tumor formation and mortality. The most favorable dosing regimen of NNK and BaP for evaluation of chemopreventive agents appears to be a total dose of 24 mumol of each, administered in eight weekly subdoses i.g., with sacrifice 9 weeks after completion of dosing. This regimen induced 10.5 +/- 4.4 lung adenomas/mouse. A combination of benzyl isothiocyanate and phenethyl isothiocyanate, given 2 h prior to each gavage of NNK and BaP, was found to be an effective inhibitor of lung tumor formation, reducing the tumor multiplicity to 5.9 +/- 5.7 lung adenomas/mouse (P < 0.001) and completely inhibiting forestomach tumor development. The results of this study provide a convenient model for assessing the efficacy of chemopreventive agents against lung cancer induction by tobacco smoke carcinogens.     

Consumption of green or black tea does not increase resistance of low-density lipoprotein to oxidation in humans

Epidemiologic studies indicated that tea consumption reduces the risk of cardiovascular disease. We assessed the effect of green or black tea consumption on resistance of low-density lipoprotein (LDL) to oxidation ex vivo and on serum lipid concentrations in healthy volunteers. In a 4-wk parallel comparison trial, 45 volunteers consumed 900 mL (6 cups) mineral water, green tea, or black tea/d. Blood samples drawn while subjects were fasting were obtained before and after the study. The effect on resistance of subsequently isolated LDL to oxidation of adding green or black tea extract to plasma was investigated in an in vitro experiment. Consumption of 900 mL (6 cups) green or black tea/d did not affect serum lipid concentrations, resistance of LDL to oxidation, or markers of oxidative damage to lipids in vivo, although consumption of green tea slightly increased total antioxidant activity of plasma. The in vitro experiment showed that resistance of isolated LDL to oxidation increased only after incubation of plasma with very high amounts of green or black tea. These amounts, when converted to tea catechin concentrations, were much higher than those expected in vivo. We conclude that daily consumption of 900 mL (6 cups) green or black tea/d for 4 wk had no effect on serum lipid concentrations or resistance of LDL to oxidation ex vivo. Future research should focus on mechanisms by which tea flavonoids may reduce the risk of cardiovascular disease other than by increasing the intrinsic antioxidant status of LDL.