Effects of Epigallocatechin Gallate,L-Ascorbic Acid, α-Tocopherol,and Dihydrolipoic Acid on the Formation of Deoxyguanosine Adducts Derived From Lipid Peroxidation

Oxidation of polyunsaturated fatty acids (PUFAs) releases α,β-unsaturated aldehydes that modify deoxyguanosine (dG) to form cyclic 1,N ²-propanodeoxyguanosine adducts. One of the major adducts detected in vivo is acrolein (Acr)-derived 1,N ²-propanodeoxyguanosine (Acr-dG). We used a chemical model system to examine the effects of 4 antioxidants known to inhibit fatty acid oxidation on the formation of Acr-dG and 8-oxodeoxyguaonsine (8-oxodG) from the PUFA docosahexaenoic acid (DHA) under oxidative conditions. We found that epigallocatechin gallate (EGCG) and dihydrolipoic acid (DHLA) inhibit both Acr-dG and 8-oxodG formation. In contrast, ascorbic acid and α-tocopherol actually increase Acr-dG at high concentrations and do not show a concentration-dependant inhibition of 8-oxodG. We also studied their effects on blocking Acr-dG formation directly from Acr. EGCG and DHLA can both effectively block Acr-dG formation, but ascorbic acid and α-tocopherol show weak or little effect. These results highlight the complexity of antioxidant mechanisms and also reveal that EGCG and DHLA are effective at suppressing lipid peroxidation-induced Acr-dG and 8-oxodG formation as well as blocking the reaction of dG with Acr.     

Chemopreventive Effects of 3-Phenylpropyl Isothiocyanate on Hamster Lung Tumorigenesis Initiated with N-Nitrosobis(2-oxopropyl)amine

The chemopreventive effects of 3-phenylpropyl isothiocyanate (PPITC) were investigated in N -nitrosobis(2-oxopropyl)amine (BOP)-initiated hamsters. A total of 120 female 5-week-old hamsters were divided into 6 groups. Animals in groups 1–3, each consisting of 30 hamsters, were twice sc injected 7 days apart as an initiation treatment. Hamsters in groups 1 and 2 were respectively given 100 μmol and 10 μmol of PPITC by gavage 2 h prior to each BOP treatment. Animals in group 3 were treated with BOP alone, serving as an initiation-positive control. Animals in groups 4–6, each consisting of 10 hamsters, were given 100 μmol or 10 μmol of PPITC alone, or non-treated, thus being available as matched negative controls to groups 1–3. At termination (experimental week 51 after the first BOP injection), the incidences of lung adenomas and/or adenocarcinomas were significantly decreased in groups 1 and 2 as compared to the group 3 value (P<0.01). The combined lung tumor incidences were inhibited by 94% and 59% at 100 and 10 μmol doses, respectively. The inhibitory effects of PPITC were thus dose-dependent. The data for multiplicity of lung tumors dramatically illustrated the inhibitory effects of PPITC, and there were also statistically significant differences in the chemopreventive effect between 100 μmol and 10 μmol PPITC treatments. On the other hand, the PPITC treatments did not significantly modulate the development of neoplastic lesions in the pancreas, liver and kidney, although the treatments did show inhibitory tendencies, except on the liver lesions. Under the present experimental conditions, PPITC itself did not exhibit tumorigenicity or apparent toxicity. The results in the present study thus clearly indicate that PPITC has an effective chemopreventive action on BOP-induced lung tumorigenesis in hamsters.     

Targeting cell cycle machinery as a molecular mechanism of sulforaphane in prostate cancer prevention

Epidemiological studies recently concluded that consumption of cruciferous vegetables such as broccoli, cabbage, and cauliflower, etc. is inversely related to prostate cancer risk, although the mechanism of prevention and the responsible phytochemicals are unknown. Since clinically significant prostate cancer eventually can grow independent of androgen, the association of the growth and tumorigenesis of such prostate cancer cells with sulforaphane (SFN) which is a predominant isothiocyanate in cruciferous vegetables, investigated. These vegetables contain high concentrations of glucosinolate glucoraphanin, which yield sulforaphane when hydrolyzed by the plant enzyme myrosinase. This study showed that exposure of human androgen-independent DU-145 prostate cancer cells to SFN resulted in the inhibition of growth and tumorigenesis, as revealed by a reduction in cell density, DNA synthesis, and clonogenesis. Analyses of the mechanism revealed that SFN mediated cell cycle arrest by modulating the expression and functions of cell cycle regulators. SFN induced signals that inhibited the activity of cyclin-dependent kinase cdk4 with an up-stream induction of cdk inhibitor p21WAF-1/Cip-1, and reduced cyclin D1. The inhibition of cdk kinase activity could be affected with <1 micro M SFN within 24 h. As a result, phosphorylation of Rb proteins, which activates the transition from G1- to S-phase, was significantly decreased and the cell cycle progression retarded. SFN also down-regulated the expression of bcl-2, a suppressor of apoptosis, and activated caspases to execute apoptosis in the prostate cancer cells. The regulators of cell cycle have thus been revealed as targets of sulforaphane for growth arrest and apoptosis induction. The potential of SFN, as an active dietary factor to inhibit initiation and post-initiation of prostate cancer carcinogenesis is discussed.     

Urinary isothiocyanate levels,brassica, and human breast cancer

Brassica vegetable consumption (e.g., Chinese cabbage) provides isothiocyanates (ITC) and other glucosinolate derivatives capable of inducing Phase II enzymes [e.g., glutathione S-transferases (GSTM1, GSTT1, and GSTP1) and NADPH quinine oxidoreductase] and apoptosis, altering steroid hormone metabolism, regulating estrogen receptor response, and stabilizing cellular proliferation. Asian populations consuming large amounts of Brassica have a lower breast cancer incidence compared with Western populations; however, the association between Brassica consumption and breast cancer risk is uncertain. It is difficult to estimate glucosinolate exposure and degradation in humans, possibly limiting epidemiological investigations of Brassica and cancer associations. We conducted a case control investigation of breast cancer in Shanghai, China, using urinary ITC levels as a biological measure of glucosinolate intake and degradation in populations with habitual Brassica intake. A representative subgroup of 337 cases providing presurgery, fasting, and first-morning urine specimens was one-to-one matched (age, menopausal status, date of urine collection, and day of laboratory assay) to population controls. Urinary ITC levels were inversely associated with breast cancer [odds ratio (OR) (Quartile 1) = 1 (ref); OR(Q2) = 0.9, 95% confidence interval (0.6, 1.4); OR(Q3) = 0.7, (0.5, 1.1); OR(Q4) = 0.5, (0.3, 0.8), adjusted for age, menopausal status, soy protein, fibroadenoma history, family breast cancer, physical activity, waist-to-hip ratio, body mass index, age at menarche, and parity in conditional logistic model]. This protective association persisted within post and premenopausal women. In contrast, total Brassica intake estimated from a food frequency questionnaire was not associated with breast cancer. Trends in the association between urinary ITC and breast cancer were more consistent with homozygous deletion of GSTM1 or GSTT1, the AAgenotype of GSTP1 (A313G), or with the C allele of NADPH quinine oxidoreductase (C609T), although interactions were not statistically significant. In conclusion, greater Brassica vegetable consumption, as measured by the urinary ITC biomarker, was associated with significantly reduced breast cancer risk among Chinese women.     

Evidence of alterations in base excision repair of oxidative DNA damage during spontaneous hepatocarcinogenesis in Long Evans Cinnamon rats

The Long-Evans Cinnamon (LEC) rat, an animal model for Wilson's disease, is an inbred mutant strain, which because of the genetic copper metabolism disorder develops hepatitis approximately 4 months after birth, followed by chronic hepatitis later in life, and eventually all of the surviving animals from liver injury and hepatitis develop spontaneous hepatocellular carcinomas. This animal model also shows that the generation of reactive oxygen species and the accumulation of oxidative damage in the liver DNA has significantly increased over the lifetime of LEC versus the wild-type Long-Evans Agouti (LEA) rats. Thus, the LEC rats having this genetically induced oxidative condition are proved to be very useful model for the study of endogenous DNA lesions and their relation to spontaneous carcinogenesis. In this study, we tested the hypothesis that differences do exist between these two rat strains in respect to their capacity to repair oxidative DNA base modification, which could explain the elevation of endogenous oxidative damage in the LEC rat liver DNA. We found that both the activity and expression at the protein and RNA levels of major DNA glycosylases, endonuclease III and 8-oxoguanine DNA-glycosylase, which initiate the excision and repair of oxidized bases, were significantly altered during the acute (16-18 weeks) and early chronic (24 weeks) phases of hepatitis. Enzyme levels were restored in the later period of chronic hepatitis (week 40) in the LEC rat liver as compared with the age-matched LEA rats. This early reduction in the capacity to repair oxidative DNA base damage could have contributed to the accumulation of mutagenic adducts in liver DNA. These findings show for the first time in an animal model that acute hepatitis impairs the repair of oxidative DNA base damage and strongly suggest that the repair of endogenous DNA adducts plays a critical role in the development of spontaneous hepatocellular carcinoma in LEC rats.     

Urinary isothiocyanate levels and lung cancer risk among non-smoking women: a prospective investigation

Background

Aside from tobacco carcinogen metabolism, isothiocyanates (ITC) from cruciferous vegetables may induce apoptosis or steroid metabolism to reduce lung cancer risk. To separate the effect of these divergent mechanisms of action, we investigated the association between urinary ITC levels and lung cancer risk among non-smoking women.

Methods

We conducted a nested case-control within the Shanghai Women's Health Study. Subjects included 209 incident lung cancer cases who never used tobacco, and 787 individually matched non-smoking controls. Conditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI) summarizing the association between urinary ITC levels and lung cancer. Secondary analyses stratified the ITC-lung cancer analyses by menopausal status, exposure to environmental tobacco smoke, and GSTM1 and GSTT1 genotypes.

Results

Urinary ITC levels were not significantly associated with lower lung cancer risk among non-smoking women, regardless of exposure to environmental tobacco smoke or menopausal status. Furthermore, this association was not modified by GSTT1 genotype. However, an inverse association was suggested among women with a GSTM1-positive genotype (Q1: OR = 1.0 (reference); Q2: OR = 0.35 (0.14, 0.89); Q3: OR = 0.47 (0.20, 1.10); Q4: OR = 0.63 (0.35, 1.54), p-trend = 0.38). In contrast, lung cancer risk was positively associated with urinary ITC levels among women with the GSTM1-null genotype (Q1: OR = 1.0 (reference); Q2: OR = 1.67 (0.80, 3.50); Q3: OR = 1.54 (0.71, 3.33); Q4: OR = 2.22 (1.05, 4.67), p-trend = 0.06).

Conclusion

Urinary ITC levels were not associated overall with lower lung cancer risk among non-smoking women, but secondary analyses suggested an interaction between urinary ITC levels, GSTM1 genotype, and lung cancer risk.     

Isothiocyanates inhibit proteasome activity and proliferation of multiple myeloma cells

Isothiocyanates (ITCs), including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and sulforaphane, compounds found in cruciferous vegetable, are highly effective in inducing cell cycle arrest and apoptosis in a variety of cancer cells and animal models. Although some studies indicate that ITC-induced reactive oxygen species (ROS) generation may underlie apoptosis induction, our recent studies show that covalent binding to target proteins may be an important event triggering apoptosis. In this study, we report that BITC and PEITC significantly inhibit proteasome activity in a variety of cell types. Further studies show that ITCs inhibit both the 26S and 20S proteasomes, presumably through direct binding, and that this inhibition is unrelated to either ROS generation or ITC-induced protein aggregation. The potency of ITC-induced proteasome inhibition correlates with the rapid accumulation of p53 (tumor suppressor) and IκB nuclear factor-kappaB (nuclear factor-kappaB inhibitor). Finally, our results demonstrate that BITC and PEITC, the two strongest proteasome inhibitors, significantly suppress growth of multiple myeloma (MM) cells through induction of cell cycle arrest at G?/M phase and apoptosis. This study suggests that proteasome, like tubulin, is a potential molecular target of ITCs, thus providing a novel mechanism by which ITCs strongly inhibit growth of MM cells and new leads in identifying compounds with therapeutic and preventative efficacies for MM. It also supports the future studies of ITCs as therapeutic and preventive agents for MM.     

Rapid single-dose model for lung tumor induction in A/J mice by 4-(methylnitrosainino)-1-(3-pyridyl)-1-butanone and the effect of diet

This paper describes the development of a relatively rapid single-dosemodel for induction of lung adenomas in female A/J mice by thetobacco-specific nitrosamine 4-(methylnitros- amino)-1-(3-pyridyl)-1-butanone(NNK). Mice maintained on AIN-76A semi-synthetic diet were givena single ip. dose of 2.5, 5 or 10 µmol NNK in saline andkilled 3–7 months later. Maximum lung tumor induction,measured by lung tumors per mouse (tumor multiplicity), occurredin 3.5 months. There was no significant increase in tumor multiplicitybetween 3.5 and 7 months. Four months after treatment, numbersof lung tumors per mouse were 11.9 ? 1.0(10 µmol NNK),3.6 ? 0.4 (5 µmol), 0.9 ? 0.4 (2.5 µmol) and 0.07? 0.1 (saline). Lung tumor multiplicity in mice treated witha single dose of 10 µmol NNK and maintained on AIN-76Adiet was significantly hIgher (8.3 ? 0.5) than in mice treatedwith NNK and maintained on NIH-07 diet (2.5 ? 0.3). The resultsof this study establish a useful bioassay for identifi cationof compounds that can modify NM lung tuinorigenesis.