Effects of dietary supplementation of N-acetylcysteine on cigarette smoke-related DNA adducts in rat tissues

Cigarette smoking plays a major role in the etiology of several human cancers. It is believed that formation of DNA adducts is an initial step in the carcinogenic process. In this study, we have examined the ability of dietary N-acetylcysteine (NAG) to inhibit the formation of cigarette smoke-related DNA adducts in various tissues of rats. Female Sprague-Dawley rats were exposed to cigarette smoke (10 mg TPM/m(3)) in a whole-body exposure chamber for 6 h per day, seven days a week for four weeks. The smoke-exposed groups were provided either an unrefined diet or diets supplemented with low (5,000 ppm) or high (20,000 ppm) dose of NAG. A sham group was given control diet and maintained on filtered ambient air. Tissue DNA analysis of smoke-exposed rats by nuclease P1-version of the P-32-postlabeling assay showed up to 6 adducts in the following descending order expressed as total adducts/10(10) nucleotides: 1 predominant (no. 5) and 4 (no. 1-no. 4) minor adducts in the (219 +/- 36), 6 minor adducts in the heart (93 +/- 11), 5 adducts in the trachea (50 +/- 16), and 4 adducts in the bladder (50 +/- 3.5); sham-treated animals showed 2 or 3 adducts in each tissue but at 4-20-fold lower levels. Dietary intervention with either high or low dose of NAC did not affect the levels of most adducts, except for the following: a 30-40% increase (P<0.05) for adducts 3 and 4 in the lung; a 40-50% decrease (P<0.05) for adduct 2 in the trachea; and a 30% increase (P<0.05) for adduct 2 in the bladder. In a second experiment conducted under identical conditions, most major and minor adducts remained unaffected with NAC intervention, except for adduct 2 in the trachea which was somewhat diminished. These results suggest that dietary NAC intervention does not significantly influence the levels of most major and minor adducts. However, some minor adducts in the lung, trachea and bladder were modulated differentially.     

Formation of cigarette smoke-induced DNA adducts in the rat lung and nasal mucosa

The formation of DNA adducts in the nasal, lung, and liver tissues of rats exposed daily to fresh smoke from a University of Kentucky reference cigarette (2R1) for up to 40 weeks was examined. The amount of smoke total particulate matter (TPM) inhaled and the blood carboxyhemoglobin (COHb) values averaged 5-5.5 mg smoke TPM/day/rat and 5.5%, respectively. The pulmonary AHH activity measured at the termination of each experiment showed an average increase of about two- to threefold in the smoke-exposed groups. These observations suggested that animals effectively inhaled both gaseous and particulate phase constituents of cigarette smoke. DNAs from nasal, lung, and liver tissue were extracted and analyzed by an improved 32P-postlabeling procedure. The results showed that the mainstream cigarette smoke induced a spectrum of at least four new DNA adducts in the nasal mucosa of the exposed rats and the magnitude of these adducts increased with the duration of exposure. In the lung tissue, the smoke exposure induced an accumulation of one DNA adduct, which upon cessation of exposure for 19 weeks was reduced by about 75%. Smoke-related adducts were not detected in the liver, a nontarget tissue. Selective chromatography and butanol extractability suggested that the nasal and lung DNA adducts are aromatic and/or hydrophobic in nature and that the smoke-related lung DNA-adduct may contain polar group(s). These data demonstrate the DNA-damaging potential of long term fresh cigarette smoke exposure and suggest the ability of the tissue to partially recover from such damage following cessation of the exposure.     

Inhibition by dietary oltipraz of experimental intestinal carcinogenesis induced by azoxymethane in male F344 rats

Epidemiological studies suggest that consumption of cruciferous vegetables rich in dithiolethiones is associated with a reduction in the incidence of cancer in man. The effect of two dose levels of dietary oltipraz [5-(2-pyrazinyl)-4-methyl-1, 2- dithiole-3-thione], a substituted dithiolethione, on azoxymethane (AOM)-induced intestinal carcinogenesis and on serum levels was studied in male F344 rats. The maximum tolerated dose (MTD) of oltipraz was determined in male F344 rats and found to be 500 p.p.m. Oltipraz at levels of 200 p.p.m. (40% MTD) and 400 p.p.m. (80% MTD) diet was tested as inhibitor of intestinal carcinogenesis. At 5 weeks of age, animals were fed the modified AIN-76A (control) diet and experimental diets containing oltipraz. At 7 weeks of age, all animals except the vehicle-treated animals were administered s.c. injection of AOM (15 mg/kg body wt/week for 2 weeks). Animals intended for vehicle treatment were administered s.c. with an equal volume of normal saline. Fifty-two weeks later, all animals were killed and colon and small intestinal tumor incidences and multiplicity were compared among the dietary groups. The results indicate that feeding of 200 and 400 p.p.m. of oltipraz significantly inhibited the incidence of adenocarcinomas in colon and small intestine and multiplicity of colon adenomas and small intestinal adenocarcinomas. Animals fed 400 p.p.m. oltipraz showed increased levels of oltipraz in the serum as compared to those fed 200 p.p.m. oltipraz. The results of this study indicate that dietary oltipraz inhibits intestinal carcinogenesis.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary tumors and DNA adducts by dietary selenite

The present studies were designed to examine the influence of dietary selenite supplementation on the initiation phase of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis and to correlate selenite-induced changes in the binding of DMBA metabolites to rat mammary cell DNA with the ultimate tumor incidence. Diets formulated to contain selenium, as sodium selenite at 0.1, 0.5, 1, 2, or 4 micrograms/g were fed for 2 weeks prior to and 2 weeks following treatment with DMBA (5 mg/kg body weight). Food intake and weight gain did not differ among treatments. Tumor incidence correlated inversely to the quantity of selenium consumed (r = -0.99). Final tumor incidences were 52, 32, 24, 14, and 10% for rats fed 0.1, 0.5, 1, and 4 micrograms selenium/g, respectively. In a separate group of rats fed a diet containing 4 micrograms selenium/g during both the initiation and promotion stages the final tumor incidence was 4.8%. Selenite supplementation for 2 weeks markedly depressed the occurrence of individual and total DMBA-DNA adducts. The final mammary tumor incidence correlated positively with total DMBA-DNA adducts (r = 0.99). These studies clearly demonstrate that selenite can inhibit the initiation stage of mammary carcinogenesis. This reduction in tumor incidence is likely due to a reduction in carcinogen metabolism and ultimately adduct formation.     

Immunocytochemical localization of DNA adducts in rat tissues following treatment with N-nitrosomethylbenzylamine

Immunocytochemical visualization of O6-methylguanosine (meGua) and 7-meGua shows that DNA methylation by N-nitrosomethylbenzylamine(NMBzA) occurs not only in the target organs for tumour induction by this nitrosamine, the oesophagus and (occasionally) the tongue, but also in other tissues (liver, lung, trachea, tracheal glands and nasal cavity) for which no tumour induction by NMBzA has been reported. Thus, the organotropic carcinogenic action of NMBzA cannot be exclusively ascribed to differences in levels of DNA methylation. Additional determinants of the cancer risk in extra-oesophageal tissues could be the small size of the NMBzA-activating target cell population and a low proliferative activity.     

Chemoprevention of smoke-related DNA adduct formation in rat lung and heart.

The formation of smoke-related DNA adducts and their chemoprevention were investigated in tissues of male Sprague-Dawley rats, by testing a total of 132 DNA samples by synchronous fluorescence spectrophotometry (SFS), which mainly detects benzo[a]pyrene diolepoxide (BPDE)-DNA adducts. Groups of six animals each were exposed whole-body to mainstream cigarette smoke, once daily, for up to 40 consecutive days. No adduct was revealed in liver DNA, whereas smoke-related DNA adducts were detectable in the lung from the 8th day of exposure and continued to increase until the 40th day. Adducts to heart DNA, which were monitored after 28 and 40 days of exposure, attained even higher levels than those detected in the lungs of the same animals. A high correlation existed between the amounts of smoke-related DNA adducts measured in these two organs. The daily administration by gavage of the thiol N-acetyl-L-cysteine (NAC), an effective mutation and cancer chemopreventive agent, which had been previously shown to inhibit the formation of SFS-positive DNA adducts in rats receiving intratracheal instillations of benzo[a]pyrene, significantly prevented occurrence of the same adducts in both heart and lungs of smoke-exposed rats. No fluorescence signal was observed in liver, lung, or heart DNA of sham-exposed animals. The findings of this molecular dosimetry study complement the results of parallel histopathologic, cytogenetic, biochemical and metabolic analyses of tissues and cells from the same rats, providing evidence for a variety of significant alterations produced by exposure to cigarette smoke and for the specific protective role of NAC.     

Smoking-related DNA and protein adducts in human tissues

Tobacco smoking causes not only lung cancer but also cancer of the oral and nasal cavities, oesophagus, larynx, pharynx, pancreas, liver, kidney, stomach, urinary tract and cervix. Tobacco smoke contains many carcinogens that exert their biological effects through interaction of reactive intermediates with DNA to form DNA adducts. The same electrophilic species also react with cellular proteins. The effects of smoking are evident by the detection of elevated levels of carcinogen-DNA adducts in many human tissues and of carcinogen-protein adducts in blood. Components of tobacco smoke also induce oxidative DNA damage. Systemic exposure to tobacco-derived carcinogens is demonstrated by the observation of elevated levels of DNA adducts in tissues not directly exposed to tobacco smoke. For many of these tissues there is epidemiological evidence, varying from comprehensive to preliminary, that smoking is a causative factor in cancer of that site. The effects of passive smoking, which also causes lung cancer in non-smokers, is also evident in elevated levels of protein adducts in exposed non-smokers so exposed, relative to non-exposed non-smokers. This paper reviews the literature on smoking-related DNA and protein adducts in human tissues and shows how such studies have provided mechanistic insight into the epidemiological associations between smoking and cancer.     

Inhibition by N-acetylcysteine of carcinogen-DNA adducts in the tracheal epithelium of rats exposed to cigarette smoke

The ability of the aminothiol N-acetylcysteine (NAC) to preventthe formation of carcinogen-DNA adducts in tracheal epithelialcells was investigated in Sprague-Dawley rats exposed whole-bodyto mainstream cigarette smoke for either 40 or 100 consecutivedays. ³²P-Postlabelling analyses showed the occurrence of DNAadducts (12.49 adducts/10⁸ nucleotides) after 40 days of exposure,with a trend to formation of characteristic diagonal radioactivezones. Total adduct levels were not further enhanced after 100days of exposure to smoke, although significant changes occurredin the amounts of individual adducts. NAC, given by gavage inthe 40 day study and in drinking water in the 100 day study,significantly inhibited the formation of smoke-related carcinogen-DNAadducts in the tracheal epithelium, to such an extent that adductlevels were not significantly higher than those detected insham-exposed control rats. Together with a variety of othermolecular, clastogenicity, metabolic, cytological and histopathologicalend-points investigated in rodents and with the preliminaryevidence arising from a study in humans, these results documentthe considerable efficacy of oral NAC in inhibiting smoke-relatedcarcinogen-DNA adducts.     

DNA adducts in different tissues of smokers and non-smokers

Purified DNA from human lung, liver, bladder, pancreas, breast and cervix has been analysed for DNA adducts using the nuclease P1 modification of the 32P post-labelling technique. Tissues were obtained at autopsy from 13 men and 6 women. Relatives were asked to provide information on smoking history for deceased subjects. All tissues examined except the breast had detectable adducts. In lung, bladder and pancreatic tissue a characteristic pattern of adducts was seen which has previously been reported as typical of cigarette-smoke-induced damage. Smokers and former smokers tended to have higher adduct levels than non-smokers in the tissues examined but this was only significant for the lung. There appeared to be considerable variation in adduct levels among smokers which could not be accounted for by duration or daily consumption level. Certain smokers had high adduct levels in all tissues examined, whilst in others high levels were only seen in some tissues. All cervical samples examined had detectable adducts. These results confirm the finding that cigarette smoking is associated with DNA damage in the lung and suggest that similar damage may be related to tobacco-induced neoplasms of other tissues.     

DNA adducts, protein adducts, and sister chromatid exchange in cigarette smokers and nonsmokers

In order to validate markers of internal dose and biologically effective dose of carcinogens, a battery of measurements was made on blood samples from 22 smokers and 24 nonsmokers. The markers included immunoreactivity in an enzyme-linked immunosorbent assay (ELISA) quantified in white blood cells with the use of a polyclonal anti-benzo[a]pyrene diol epoxide-I-DNA antibody, 4-aminobiphenyl hemoglobin (4-ABP-Hb) adducts measured by negative chemical ionization mass spectrometry, sister chromatid exchange (SCE) in cultured lymphocytes, and cotinine in plasma measured by radioimmunoassay. Several blood samples were drawn from each subject. In blood samples 1 and 3 having detectable levels of DNA adducts, mean femtomole-per-microgram levels were consistently higher among smokers compared to nonsmokers. The borderline significance of this difference may be attributable to the small numbers of subjects. Consistently higher adduct levels were seen in females compared to males. In sample 3, adduct levels were significantly correlated with measurements of active smoking in smokers and with passive smoking in nonsmokers. By contrast to the ELISA data, which may reflect cumulative exposure from multiple background sources, the 4-ABP-Hb assay was able to distinguish clearly between smokers and nonsmokers. SCEs were significantly elevated in the smokers compared to nonsmokers. Also observed were significant correlations between 4-ABP-Hb and both cotinine and SCEs, as well as a positive correlation between the 4-ABP-Hb and DNA adduct levels (sample 3) that was highly significant. The correlation between DNA and 4-ABP-Hb adducts was significant in smokers but not nonsmokers (sample 3). These results support the need for batteries of markers to detect and to quantify the carcinogenic dose to humans resulting from both specific and "background" environmental exposures.     

Linear relationship between DNA adducts in human lung and cigarette smoking

Human lung and bladder DNA has been isolated and purified from either surgical or autopsy specimens. Smoking history details were obtained from patients or their close relatives. Each DNA sample was investigated using the nuclease P1 digestion modification of the 32P-postlabelling procedure. Data are presented for 48 lung and 19 bladder specimens. The samples were subdivided into three groups for data analysis, viz. smokers, former smokers and nonsmokers. The mean adduct levels (adducts per 10(8) nucleotides) in lung samples were: [see text] The chromatographic pattern of bladder DNA adducts for smokers was similar to that for smokers' lung DNA, although less intense. Adduct levels in former smokers tended to be lower than in smokers, although loss of adducts appeared to require several years after cessation of smoking. These findings support a link between DNA adduct levels and cigarette smoking, for both the lung and the bladder. For the former tissue there was a strong linear correlation between adduct levels and the number of cigarettes smoked.     

DNA adducts formed by the comutagens harman and norharman in various tissues of mice

Covalent modifications of DNA in various tissues of mice with harman or norharman were analyzed by 32P-postlabeling assay. Administration of 0.1% harman to mice in their diet for 4 weeks resulted in DNA adducts in the liver and kidney. No specific DNA adduct was detected in other tissues, such as the glandular stomach, large intestine and brain. Similar treatment of mice with norharman resulted in DNA adducts in the kidney, glandular stomach and large intestine, but not in the liver or brain. These results suggests the in vivo genotoxicities of harman and norharman.     

Inhibition of DES-induced DNA adducts by diallyl sulfide: implications in liver cancer prevention

Diethylstilbesterol (DES) is known to cause cancer in humans and animals. Diallyl sulfide (DAS), a component of garlic, has been shown to prevent various types of cancer, presumably via metabolic modulation. Previously, we have demonstrated that DAS prevents the oxidation and reduction of DES in vitro. We hypothesize that DAS will inhibit the metabolism of DES in vivo thus preventing the formation of DES-induced DNA adducts. To test this hypothesis, five groups of five male Sprague-Dawley rats were treated as follows: the control received 0.5 ml of corn oil daily for four days. The second group received 50 mg/kg DAS daily for four days. The third group received 50 mg/kg DAS daily for four days followed by 150 mg/kg DES on day five. The fourth group received 400 mg/kg DAS on day five followed by 150 mg/kg DES. The fifth group received 150 mg/kg DES on day five. All of the rats were sacrificed on day five, 4 h after DES treatment. DNA was isolated from the liver and analyzed by 32P-post-labeling for DNA adducts. The in vitro study was performed utilizing four reactions described as follows: the control reaction contained 200 microg DNA, microsomes (346 microg protein/ml), and 10 mM DES; no oxidation co-factor (cumen hydroperoxide) was added. The second reaction, a complete oxidation system, contained 200 microg DNA, microsomes (346 microg protein/ml), 30 mM cumen hydroperoxide, and 10 mM DES. The third reaction contained 200 microg DNA, microsomes (346 microg protein/ml), 30 mM cumen hydroperoxide, 50 mM DAS, and 10 mM DES. The fourth reaction contained 200 microg DNA, microsomes (346 microg protein/ml), 30 mM cumen hydroperoxide, 100 mM DAS, and 10 mM DES. All of the in vitro reactions were buffered with 100 mM KPO4 pH 7.4 and incubated for 30 min at 37 degrees C. DNA was extracted and analyzed by 32P-post-labeling. We found that DAS inhibited the formation of DES-induced DNA adducts in a dose-dependent fashion. We have shown that DES-induced DNA adducts were inhibited in rats that received DAS pre-treatment and co-treatment with DES. These results suggest that DAS directly inhibits the metabolism of DES thus preventing the formation of DNA adducts. In addition to directly inhibiting the metabolism of DES, DAS appears to alter the expression of the metabolic machinery such that DES-induced adducts are inhibited. The inhibition of DES-induced DNA adducts by DAS may prevent the initiation of estrogen-induced cancer.     

DNA adducts of cisplatin and carboplatin in tissues of cancer patients

An enzyme-linked immunosorbent assay (ELISA) has been developed with an antiserum elicited against cisplatin-modified DNA and used to quantify the intrastrand bidentate d(GpG)- and d(ApG)-diammineplatinum adducts in DNA samples prepared from nucleated blood cells and tissues of cancer patients receiving cisplatin or carboplatin chemotherapy. In nucleated blood cell DNA, adducts accumulated with increasing dose administered over a period of months, and a correlation was observed between the ability of a patient to form high levels of adduct and the frequency of tumour remission. Thus, many patients who did not form adducts also did not respond to therapy. Adduct distribution was shown to be widespread in many human tissues, and similar quantities of adducts were formed in peripheral blood cell DNA and tumour tissue. In addition, evidence suggests that residues of persistent adducts remain in many tissues weeks and even months after treatment. All of the above observations were obtained with the cisplatin-DNA ELISA; however, in comparison with other published data, the adduct levels reported are low. It now appears certain that the cisplatin-DNA ELISA results in an underestimation of adduct values in biological samples, since some human samples have been assayed by both this and two other procedures--the G-Pt-GMP ELISA and atomic absorbance spectroscopy. Values obtained with the two other procedures compare well with each other, but those obtained with the cisplatin-DNA ELISA for three human samples are 10-300-fold lower. The factors that result in this discrepancy are still under investigation.     

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.     

Covalent DNA damage in tissues of cigarette smokers as determined by 32P-postlabeling assay

Covalent DNA addition products (adducts) formed by the reaction of chemical carcinogens or their metabolites with DNA are critically involved in the initiation of chemical carcinogenesis and may serve as molecular markers and dosimeters for environmental carcinogen exposures. Using a highly sensitive 32P-postlabeling assay for DNA adduct analysis, we studied DNA damage elicited by cigarette smoke in tissues of smokers. A multitude of characteristic smoking-induced, presumably aromatic DNA adducts were found to occur in a dose- and time-dependent manner in the lung, bronchus, and larynx of smokers with cancer of these organs and to decline only slowly after cessation of smoking. Low levels of adducts appeared to persist for up to 14 years in the lungs of exsmokers with high previous exposures. These results corroborate data of epidemiological studies showing that the lung cancer risk and mortality of smokers increase with the intensity and duration of smoking and decline only slowly after cessation of smoking. Tissue distribution studies in autopsy samples revealed the presence of smoking-associated DNA lesions also in the kidney, bladder, esophagus, heart, ascending aorta, and liver. The most extensive DNA damage was found in lung and heart, i.e., 1 aromatic adduct in about 10(7) DNA nucleotides. Our results suggest that cigarette smoking-induced DNA adduct formation is causally related to cancer in the target organs.     

Immunocytochemical localization of DNA adducts induced by a single dose of N-nitroso-N-methylbenzylamine in target and non-target tissues of tumor formation in the rat

The formation and short-term persistence of O6-methylguanine (O6-meGua) and 7-methylguanine (7-meGua) in individual cells of various target and non-target tissues for tumor induction in rats were examined after a single dose of N-nitroso-N-methylbenzylamine (NMBzA). In the principal target organ, the esophagus, both adducts were observed at 6 h after 0.5, 1.0 and 2.5 mg NMBzA/kg in a dose-dependent manner in nuclei of epithelial cells only. Nuclear staining in this organ had apparently declined by 72 h and modified nuclei were found in the more differentiated cells located closer to the lumen. In epithelial cells of the tongue, another target organ of NMBzA, methylation at 6 h was also dose dependent. At 72 h nuclear staining was lower and again largely located in differentiated cells. In the liver, a non-target organ, O6-meGua was not detectable and 7-meGua-specific staining was weak, being only observed at 6 h after the highest dose. Dose-dependent DNA methylation was seen, both at 6 and 72 h, in other non-target organs such as lung (bronchiolar epithelial cells), trachea (epithelial and glandular cells) and nasal cavity (respiratory epithelial cells, ductal cells of the respiratory lamina propria and cells of Bowman glands of the olfactory lamina propria); the nuclei of the glandular cells were highly methylated. Visual inspection of lung, trachea and nasal cavity indicated no or only minor losses of O6-meGua and 7-meGua between 6 and 72 h. Microdensitometric determination of the nuclear staining at 6 and 72 h indicated that the promutagenic O6-meGua was partially lost from cells of the tongue epithelium but did persist in esophageal epithelial cells; 7-meGua was lost to a substantial extent from both tongue and esophagus. The present results imply that the organotropism of NMBzA is not uniquely determined either by the initial level or the short-term persistence of DNA methylation.     

Inhibition of N-nitrosomethylbenzylamine-induced tumorigenesis in the rat esophagus by dietary freeze-dried strawberries

In the present study, we examined the ability of dietary freeze-dried strawberries to inhibit N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. Initially, we conducted a bioassay to determine the effects of dietary freeze-dried strawberries on esophageal tumor development. Two weeks prior to NMBA treatment, animals were placed on a control diet or diets containing 5 and 10% freeze-dried strawberries. NMBA treatment was once per week for 15 weeks. At 30 weeks, 5 and 10% freeze-dried strawberries in the diet caused significant reductions in esophageal tumor multiplicity of 24 and 56%, respectively. Based on these results, we conducted studies to determine potential mechanisms by which freeze-dried strawberries inhibit tumorigenesis. In a short-term bioassay, we evaluated the effects of dietary freeze-dried strawberries on the formation of O6-methylguanine in the rat esophagus. Animals were placed on control diet or diets containing 5 and 10% freeze-dried strawberries for two weeks. At the end of this period, animals received a single subcutaneous dose of NMBA and were killed 24 h later. A significant decrease in O6-methylguanine levels was observed in the esophageal DNA of animals fed strawberries, suggesting that one or more components in strawberries influence the metabolism of NMBA to DNA-damaging species. Finally, in order to evaluate post-initiation effects, we conducted a study where freeze-dried strawberries were administered in the diet only following NMBA treatment. Animals were placed on control diet and dosed with NMBA three times per week for 5 weeks. Immediately following NMBA treatment, animals were placed on control diet or diets containing 5 and 10% freeze-dried strawberries. At 25 weeks, 5 and 10% freeze-dried strawberries in the diet significantly reduced tumor multiplicity by 38 and 31%, respectively. Our data suggest that dietary freeze-dried strawberries effectively inhibit NMBA-induced tumorigenesis in the rat esophagus.     

SHORT COMMUNICATION: Inhibition of rat mammary tumorigenesis by dietary cholesterol

The effects of dietary cholesterol and oxidized cholesterolon mammary tumor development were examined in female Sprague-Dawleyrats exposed to the carcinogen N-methyl-N-nitrosourea (MNU).Animals were administered 50 mg/kg MNU at 50 days of age andfed either a control (AIN-76) diet or the control diet supplementedwith 0.3% cholesterol or 0.3% oxidized cholesterol for up to26 weeks. The oxidized cholesterol was prepared by heating cholesterolat 110°C for 48 h. Gas chromatographic analysis of the oxidizedcholesterol revealed a 2% yield of oxidation products in additionto a large amount of unchanged cholesterol (>96%). Tumorincidence in the cholesterol group (67%) was significantly lowerthan in the control group (96%, P <0.05), but the oxidizedcholesterol group (79%) was not significantly different fromthe control or cholesterol groups. Average number of tumorsper animal was lower in the cholesterol group (1.5) than inthe control (2.8) or oxidized cholesterol groups (2.3, P <0.005).Serum low density lipoprotein (LDL) cholesterol was greaterin the cholesterol (185± 38 mg/dl) and the oxidized cholesterolgroups (160± 34 mg/dl) than in the control (55±4 mg/ dl, P <0.05), although there was no difference betweenthe cholesterol and the oxidized cholesterol groups. These resultsshow that dietary cholesterol inhibits mammary tumor developmentin this model. Elevated serum LDL cholesterol may inhibit denovo cholesterol synthesis in preneoplastic and/or tumor cells,thereby inhibiting their proliferation.     

Formation of DNA adducts in mouse tissues after intratracheal instillation of 1-nitropyrene

1-Nitropyrene (1-NP), a ubiquitous environmental pollutant,is a mammalian mutagen and causes cancer in animals. The abilityof the lung, liver and kidney to form 1-NP-DNA adducts was determinedin adult male B6C3F₁ mice following a single intratracheal instillationof 1-NP. 1-NP-DNA adducts were isolated and characterized inmouse lung, liver and kidney by HPLC analysis of the enzymaticallydigested DNA. Multiple DNA adducts were present in lung, liverand kidney at 1 day after administration. One of the major adductsin lung (20% of the total eluted radioactivity) coeluted withthe synthetic marker, N-(deoxyguanosin-8-yl)-1-amino-pyrene(C8-dG-AP). This adduct (10% of total eluted radio-activity)and others were still present in the lung at 28 days after administrationof 1-NP. One of the adducts in liver and kidney DNA digestsalso coeluted with C8-dG-AP. Treatment of the adducts with 0.3M NaOH resulting in earlier eluting peaks containing radioactivity,indicative of an imidazole ring-opening adduct. A portion ofthe original peak of radioactivity that coeluted with C8-dG-APand other adducts, however, was not affected by 0.3 M NaOH.Thus, the chromatographic properties and chemical behavior ofthe adducts formed in vivo suggest that one of the adducts inthe lung is C8-dG-AP which is formed by nitroreduction of 1-NP.Other adducts may be formed via ring-oxidation followed in someinstances by nitroreduction. These data indicate that DNA adductsof 1-NP metabolites may be formed in the lung (a primary sitefor inhaled particles), liver and kidney following inhalationof airborne particles containing 1-NP.