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Berries are gaining increasing importance lately for their chemopreventive and therapeutic potential against several cancers. In earlier studies, a blueberry-supplemented diet has shown protection against 17β-estradiol (E2)-mediated mammary tumorigenesis. This study tested both preventive and therapeutic activities of diet supplemented with whole blueberry powder (50:50 blend of Tifblue and Rubel). Animals received 5% blueberry diet, either 2 weeks prior to or 12 weeks after E2 treatment in preventive and therapeutic groups, respectively. Both interventions delayed the tumor latency for palpable mammary tumors by 28 and 37 days, respectively. Tumor volume and multiplicity were also reduced significantly in both modes. The effect on mammary tumorigenesis was largely due to down-regulation of CYP 1A1 and ER-α gene expression and also favorable modulation of microRNA (miR-18a and miR-34c) levels. These data suggest that the blueberry blend tested is effective in inhibiting E2-mediated mammary tumorigenesis in both preventive and therapeutic modes.

Dietary polyphenols may contribute to the prevention of several degenerative diseases, including cancer. Anthocyanins have been shown to possess potential anticancer activity. The aim of this study was to determine anthocyanin bioavailability in lung tissue of mice fed a blueberry diet (5% w/w) for 10 days or a bolus dose (10 mg/mouse; po) of a native mixture of bilberry anthocyanidins. All five anthocyanidins present in the blueberry were detected in the lung tissue using improved methods. The effect of various solvents on the stability of anthocyanins and their recovery from the biomatrix was analyzed. Detection of anthocyanins and their metabolites was performed by UPLC and LC-MS. Although anthocyanins were not detected, cyanidin was detected by UPLC-PDA and other anthocyanidins were detected by LC-MS, following conversion to anthocyanidins and selective extraction in isoamyl alcohol. The results show that anthocyanins can be detected in lung tissue of blueberry-fed mice and thus are bioavailable beyond the gastrointestinal tract.

Cigarette smoke contains a variety of carcinogens, cocarcinogens, mutagens, and tumor promoters. In addition to polycyclic aromatic carcinogens and tobacco-specific nitrosamines, cigarette smoke also contains an abundance of catechols, aldehydes, and other constituents, which are DNA damaging directly or indirectly; therefore, they can also contribute to cigarette smoke-mediated carcinogenicity. In this study, we investigated the potential of cigarette smoke constituents to induce oxidative damage to DNA through their capacity to redox cycle. When DNA (300 μg/mL) was incubated with cigarette smoke condensate (0.2 mg of tobacco particulate matter/mL) and CuCl(2) as a catalyst (50-100 μM), a variety of oxidative DNA adducts were detected by (32)P-postlabeling/TLC. Of the total adduct burden (2114 ± 419 adducts/10(6) nucleotides), over 40% of all adducts were attributed to the benchmark oxidative DNA lesion, 8-oxodeoxyguanosine (8-oxodG). Adducts were formed dose dependently. Essentially, similar adduct profiles were obtained when cigarette smoke condensate was substituted with ortho- and para-dihydroxybenzenes. Vehicle treatment with Cu(2+) or CSC alone did not induce any significant amount of oxidative DNA damage. Furthermore, coincubation of cigarette smoke condensate and ortho-dihydroxybenzene with DNA resulted in a higher amount of oxidative DNA adducts than obtained with the individual entity, suggesting that adducts presumably originated from catechols or catechol-like compounds in cigarette smoke condensate. Adducts resulting from both cigarette smoke condensate and pure dihydroxybenzenes were chromatographically identical to adducts formed by reaction of DNA with H(2)O(2), which is known to produce 8-oxodG, and many other oxidative DNA adducts. When the cigarette smoke condensate-DNA reaction was performed in the presence of ellagic acid, a known antioxidant, the adduct formation was inhibited dose dependently, further suggesting that adducts originated from oxidative pathway. Our data thus provide evidence of the capacity of catechols or catechol-like constituents in cigarette smoke to produce oxidative DNA damage, which may contribute to the tumor-promoting activity of cigarette smoke.

We previously demonstrated the protective effects of blueberry (BB) and black raspberry (BRB) supplemented at 2.5% dose in an ACI rat mammary tumor model. Here, we assessed a dose-related alteration in tumor indices with diet supplemented with 5% BB or BRB powder. The diet was well tolerated. Tumor palpation from 12 weeks revealed first tumor appearance by 84 days in the control group, that was delayed by 24 and 39 days with the BB and BRB diets, respectively (p = 0.04). Ellagic acid detected in the plasma of rats fed the BRB diet was in the range of 96.6-294.2 ng/mL. While the BB diet showed better efficacy in reducing mammary tissue proliferation and tumor burden, tumor latency was delayed efficiently by BRB. Furthermore, BB was effective in downregulating CYP1A1 expression, while BRB downregulated ERα expression effectively. Distinct anticarcinogenic effects of the two berries correspond to their distinct phytochemical signatures.

Experimental and epidemiological data associate the exposure of estrogens to cancer development in several tissues, particularly, the breast, endometrium, liver, and kidney. One plausible mechanism of estrogen-mediated carcinogenicity is DNA damage by redox cycling of estrogen catechols. Reports have shown that metabolism of estrogens results in 2- and 4-hydroxylation to catechol metabolites which can then redox cycle. We examined the capacity of the endogenous estrogen, 17β-estradiol, and two equine estrogens which formulate a significant proportion of hormone replacement drugs, equilenin and equilin, to induce oxidatively generated DNA damage. Microsome/Cu(II)-mediated activation of all three estrogens resulted in numerous oxidation DNA adducts, as detected by (32)P-postlabeling/TLC. Essentially the same DNA oxidation pattern was also found when catechol estrogens were incubated with DNA in the presence of Cu(II) suggesting that redox cycling of catechol estrogens mediates the formation of these DNA adducts. Since the oxidation patterns induced by estrogen catechols and other chemically diverse catechols were chromatographically identical to those generated by Fenton-type chemistry and these adducts were inhibited by known ROS modifiers (up to 96%), this oxidatively generated DNA damage is believed to be the product of the attack of free radicals on DNA, rather than direct addition of the estrogen quinones. These data support a mechanistic role by endogenous and synthetic estrogens to induce oxidative DNA damage in addition to specific DNA adducts.

Many carcinogenesis and tumorigenesis studies reported in the past several decades have relied upon bolus dose(s) of test compounds to determine their DNA damage and carcinogenic potential. The high doses are far from the human scenario where exposure is almost always to low doses and for long duration. In this study, we report a novel polymeric implant system that provides continuous ("24/7") exposure to low doses using benzo[a]pyrene (BP) as a model carcinogen. Cylindrical implants (1 cm length, 3.2 mm diameter; 10 mg BP/100 mg implant) prepared from polycaprolactone:F68 (9:1) showed controlled release in vitro for long duration. To determine the rate of release and biochemical effects in vivo, groups of female Sprague-Dawley rats received either no treatment or subcutaneous sham or BP implants (1 cm, 10% load) and were euthanized after 6, 15, 30, and 180 days; the average dose of BP by the implant route was 16.7 ± 3 μg/rat. For comparison, rats were also treated with a single bolus dose of BP intraperitoneally (10 mg/rat) and euthanized at 6, 15, and 30 days. DNA adducts analyzed by (32)P-postlabeling in the lung and liver increased steadily with time with levels reaching 31 ± 3 and 17 ± 6 adducts/10(9) nucleotides, respectively, after 25 weeks; the adduct burden in the mammary tissue initially increased but then declined with time presumably due to high cell turn over. In contrast, the bolus dose treatment showed the highest DNA adduct levels after 6 days, followed by a steady decline. The steady accumulation of tissue DNA adducts in the implant groups corroborates the sustained overexpression of CYP1A1 and 1B1, the cytochrome P450s involved in the conversion of BP to its electrophilic metabolites. In contrast, the overexpression of CYP1A1 and 1B1 resulting from the bolus dose of BP lasted only for a few days. This is the first demonstration revealing that low-dose, continuous exposure to environmental polycyclic aromatic hydrocarbons such as BP can render sustained expression of CYPs and steady accumulation of tissue DNA adducts. On the basis of our recent study in which we showed the presence of 17β-estradiol in the lung, the sustained overexpression of CYP1A1 and 1B1 due to continuous exposure to BP may increase the susceptibility to estrogen-mediated carcinogenicity.

The polyphenolics in green tea are believed to be the bioactive components. However, poor bioavailability following ingestion limits their efficacy in vivo. In this study, polyphenon E (poly E), a standardized green tea extract, was administered by sustained-release polycaprolactone implants (two, 2-cm implants; 20% drug load) grafted subcutaneously or via drinking water (0.8% w/v) to female S/D rats. Animals were treated with continuous low dose of benzo[a]pyrene (BP) via subcutaneous polymeric implants (2 cm; 10% load) and euthanized after 1 and 4 weeks. Analysis of lung DNA by (32)P-postlabeling resulted in a statistically significant reduction (50%; p = 0.023) of BP-induced DNA adducts in the implant group; however, only a modest (34%) but statistically insignificant reduction occurred in the drinking water group at 1 week. The implant delivery system also showed significant reduction (35%; p = 0.044) of the known BP diolepoxide-derived DNA adduct after 4 weeks. Notably, the total dose of poly E administered was >100-fold lower in the implant group than the drinking water group (15.7 versus 1,632 mg, respectively). Analysis of selected phase I, phase II, and nucleotide excision repair enzymes at both mRNA and protein levels showed no significant modulation by poly E, suggesting that the reduction in the BP-induced DNA adducts occurred presumably due to known scavenging of the antidiolepoxide of BP by the poly E catechins. In conclusion, our study demonstrated that sustained systemic delivery of poly E significantly reduced BP-induced DNA adducts in spite of its poor bioavailability following oral administration.

Active and passive smoking are major risk factors for lung cancer. Pro-oxidants in tobacco smoke have been implicated in smoking-associated disease development due to their potential role in inducing oxidative stress. Previous studies have failed to associate increased levels of oxidative damage to DNA with the formation of the potentially mutagenic lesion, 8-oxo-2-deoxyguanosine (8-oxodG), probably due to repair of this lesion. However, no systematic studies have been performed to assess the dose- and time-dependent formation and removal of this lesion by cigarette smoke exposure. In the present study, female A/J mice were exposed to side-stream cigarette smoke in a whole body exposure chamber for 6 h a day, 5 days a week for up to 6 weeks. Age-matched controls were maintained in filtered ambient air. Lung tissues were harvested from 2, 4, and 6 weeks smoke-exposed mice after 1, 3, 6, and 20 h, following the cessation of smoking. A significant increase in the levels of 8-oxodG in lung DNA was observed in 10 day smoke-exposed mice at 1 (11.5+/-1.1/10(6) nucleotides), 3 (20.2+/-2.7/10(6) nucleotides; p=0.0008), and 6 h (17.2+/-1.0/10(6) nucleotides; p<0.005) postcessation, as compared with age-matched sham treatment (8.8+/-2.3/10(6) nucleotides) (mean+/-SD). The levels significantly declined 20 h after the cessation of smoke exposure (14.0+/-1.6/10(6) nucleotides), although they were still higher than the control. Our results strongly suggest that there is a significant increase in the 8-oxodG levels immediately after the cessation of smoking, which is repaired over time. This initial increase in 8-oxodG levels may lead to gene mutations, and accumulation of such mutations over time can eventually lead to malignant transformation of the cells.

Studies were undertaken to determine the formation of benzylic-DNA adducts in rats administered 7,12-dimethylbenz[a]anthracene (DMBA) and its meso-region metabolites by subcutaneous injection. Here, we show that 7-hydroxymethyl-12-methylbenz[a]anthracene (7-HMBA) and 7-sulfoxymethyl-12-methylbenz[a]anthracene (7-SMBA) gave rise to some benzylic-DNA adducts indistinguishable from adducts formed from DMBA. Adducts were analyzed by butanol enrichment-mediated 32P-postlabeling assay. Female Sprague-Dawley rats given a combined dose of 420 micromol DMBA/kg b. wt resulted in two major and up to nine minor adducts in the subcutaneous tissue, with chromatographic resemblance to benzylic-DNA adducts prepared in vitro. Subcutaneous administration of 7-HMBA, 7-SMBA, and 7-methyl-12-hydroxymethylbenz[a]anthracene (12-HMBA) (210, 42, and 210 micromol/kg b. wt, respectively) each resulted in one major and several minor benzylic-DNA adducts. From cochromatography with reference adducts, it was concluded that the benzylic DNA adduct 4, derived from the parent compound, comigrates with the major adduct from 7-HMBA and 7-SMBA, whereas adducts 2 and 3 comigrate with adducts resulting from 12-HMBA and 7-methyl-12-sulfooxymethylbenz[a]anthracene, respectively. These data suggest that 7-sulfooxymethyl- and 12-sulfooxymethy derivatives produce distinct adducts. Several major and minor diol epoxide-related DNA adducts were also detected. The diol epoxide- and benzylic-DNA adducts were found in a 2:1 ratio. The oral, intraperitoneal, and intramammiliary treatments with DMBA showed no detectable benzylic adducts in the liver and mammary glands 24 h after the last treatment, although the adduct formation was clearly evident with SMBA and/or HMBA treatments, suggesting that hydroxylation of DMBA to form HMBA may be the rate-limiting step for the meso-methyl substitution pathway. The present study clearly demonstrates the in vivo formation of benzylic-DNA adducts from DMBA. The data also reveal the involvement of the 12-methyl group of DMBA in adduct formation.