Profiling of Protein Adducts of Estrogen Quinones in 5-Year Survivors of Breast Cancer Without Recurrence.

AIMS: The aim of this study was to simultaneously analyze estrogen quinone-derived adducts, including 17ß-estradiol-2,3-quinone (E2-2,3-Q) and 17ß-estradiol-3,4-quinone (E2-3,4-Q), in human albumin (Alb) and hemoglobin (Hb) derived from breast cancer patients with five-year postoperative treatment without recurrence in Taiwan and to evaluate the treatment-related effects on the production of these adducts. SETTINGS AND DESIGN: CohortMethods and Material: Blood samples derived from breast cancer 5-year survivors without recurrence were collected. Albumin and hemoglobin adducts of E2-3,4-Q and E2-2,3-Q were analyzed to evaluate the degree of disposition of estrogen to quinones and to compare these adduct levels with those in patients before treatment. STATISTICAL ANALYSIS: All data are expressed as mean ± standard deviation of three determinations. We used Student's t-test to examine subgroups. Data were transformed to the natural logarithm and tested for normal distribution for parametric analyses. Linear correlations were investigated between individual adduct levels by simple regression. Statistical analysis was performed using the SPSS Statistics 20.0. RESULTS: Result confirmed that logged levels of E2-2,3-Q-derived adducts correlated significantly with those of E2-3,4-Q-derived adducts (correlation coefficient r=.336-.624). Mean levels of E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb in 5-year survivors were reduced by 60-70% when compared to those in the breast cancer patients with less than one year of diagnosis/preoperative treatment (P<.001). CONCLUSIONS: Our findings add support to the theme that hormonal therapy including aromatase inhibitors and Tamoxifen may dramatically reduce burden of estrogen quinones. We hypothesize that combination of treatment-related effects and environmental factors may modulate estrogen homeostasis and diminish the production of estrogen quinones in breast cancer patients.

Cumulative body burdens of polycyclic aromatic hydrocarbons associated with estrogen bioactivation in pregnant women: protein adducts as biomarkers of exposure.

The objective of this research was to simultaneously analyze protein adducts of quinonoid metabolites of naphthalene and endogenous estrogen in serum albumin (Alb) derived from healthy pregnant women in Taiwan and to explore the correlations among them. The isomeric forms of cysteinyl adducts of naphthoquinones, including 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) as well as estrogen quinones, including estrogen-2,3-quinones (E2-2,3-Q) and estrogen-3,4-quinones (E2-3,4-Q), are characterized after adduct cleavage. Results showed that the median levels of cysteinyl adducts of 1,2-NPQ and 1,4-NPQ on serum albumin were 249-390 and 16.0-24.8 pmol g(-1), respectively. Logged levels of 1,2-NPQ-Alb were correlated with logged levels of 1,4-NPQ-Alb (correlation coefficient r = 0.551, P < 0.001). Cysteinyl adducts of E2-2,3-Q-1-S-Alb, E2-2,3-Q-4-S-Alb, and E2-3,4-Q-2-S-Alb were detected in all subjects with median levels at 275-435, 162-288, and 197-254 pmol g(-1), respectively. We also found a positive relationship between logged levels of E2-2,3-Q-4-S-Alb and those of E2-3,4-Q-2-S-Alb (r = 0.770, P < 0.001).We noticed that median levels of E2-2,3-Q-derived adducts (E2-2,3-Q-1-S-Alb plus E2-2,3-Q-4-S-Alb) in pregnant women were greater than those of E2-3,4-Q-2-S-Alb (∼2-3-fold). Taken together, this evidence lends further support to the theme that cumulative concentration of E2-3,4-Q is a significant predictor of the risk of breast cancer. Furthermore, we noticed that levels of 1,2-NPQ-Alb are positively associated with levels of E2-3,4-Q-2-S-Alb (r = 0.522, P < 0.001) and those of E2-2,3-Q-4-S-Alb (r = 0.484, P < 0.001). Overall, this evidence suggests that environmental exposure to polycyclic aromatic hydrocarbons may modulate estrogen homeostasis and enhance the production of reactive quinone species of endogenous estrogen in humans.

Investigation of the abasic sites induced by hydrogen peroxide and methyl methanesulfonate in calf thymus DNA and BEAS-2B cells.

The primary goals of this study were to investigate the formation of abasic sites (AP sites) induced by methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2), and to characterize specific types of these pro-mutagenic DNA lesions in calf thymus DNA (CT-DNA), and BEAS-2B human lung normal cell line. Furthermore, these profiles were compared with those observed in leukocytes derived from healthy controls (HC), breast cancer patients (BCP) before treatment, and 5-year survivors. Results indicated that both H2O2 and MMS induced the concentration- and time-dependent formation of AP sites in CT-DNA. To characterize the specific types of AP sites induced by H2O2 or MMS, we performed AP site cleavage assay using putrescine, T7 exonuclease (T7 Exo), and exonuclease III (Exo III). Results showed that the AP sites induced by H2O2 in CT-DNA were predominantly 5'-and 3'-nicked AP sites and no intact AP sites were detected. By contrast, the majority of AP sites generated by MMS in CT-DNA are not excisable and are classified as residual and intact AP sites. Similar approaches were performed in human BEAS-2B cells and comparable observations were confirmed in the cell-based model. Further investigation indicated that the profile of the AP sites observed in Taiwanese HC is identical to that of BEAS-2B cells treated with H2O2 whereas the pattern of AP sites detected in BCP is similar to that of CT-DNA exposed to H2O2, suggesting that these AP sites were produced primarily through reactive oxygen species (ROS) generation. More than 70 % of the AP sites in leukocytes derived from BCP were 5'-nicked and residual AP sites. Furthermore, the characteristics of the AP sites detected in 5-year survivors are comparable with the ones in HC by using putrescine cleavage assay. Overall, we speculate that deficiency in the DNA repair cascade may play a role in mediating the formation of specific types of AP sites detected in BCP.

Airborne particulate collected from central Taiwan induces DNA strand breaks, Poly(ADP-ribose) polymerase-1 activation, and estrogen-disrupting activity in human breast carcinoma cell lines.

The objectives of this investigation were to examine whether airborne particles induce DNA damaging and estrogen-disrupting effects and to explore the relationships between them. In this study, airborne particulate was collected at an urban, a suburban, and a rural site in central Taiwan. The organic solvent extracts of airborne particulate were examined in human MCF-7 and T47D-KBluc breast cancer cells. We observed significant increases in reactive oxygen species (ROS) generation in MCF-7 cells after treatment with the particulate extracts whereas aryl hydrocarbon receptor (AhR) antagonist blocked the particulate-induced ROS generation in cells. Further, induction of CYP1A1 protein expression was confirmed by immunoblots in cells treated with airborne particles, suggesting the roles of AhR status in mediating the particulate-induced toxicity. In addition, we observed that at non-cytotoxic concentration (∼0.01 m(3) air equivalent), airborne particles induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 cells. These decreases were completely blocked by three types of poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors. Moreover, we demonstrated increases in the number of DNA strand breaks in MCF-7 cells exposed to airborne particles as measured by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that airborne particles induce decreases in intracellular NAD(P)H and NAD(+) through PARP-1 activation mediated by formation of DNA strand breaks. Furthermore, we confirmed that with series dilution airborne particles (∼10(-7)-10(-2) m(3) air equivalent) possess both estrogenic and anti-estrogenic activities as determined by the ERα-mediated reporter gene assay in human T47D-KBluc breast cancer cells. In conclusions, we confirmed that the DNA-damaging activity and estrogenicity of airborne particles varied considerably with concentration (air equivalent). Our findings add further support to the theme that ROS formation is a significant determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to airborne particles and that oxidative stress and the subsequent induction of DNA damage may, in part, contribute to airborne particle-induced carcinogenesis.

Imbalances in the disposition of estrogen and naphthalene in breast cancer patients: a potential biomarker of breast cancer risk.

Elevation of naphthoquinones and estrogen quinones, which are reactive metabolites of naphthalene and estrogen, is thought to be an important indicator of naphthalene- and estrogen-induced carcinogenesis. We compared background levels of naphthalene and estrogen quinone-derived adducts in serum albumin (Alb) from 143 women with breast cancer and 119 healthy controls. Cysteinyl adducts of naphthoquinones, including 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ), and estrogen quinones, including estrogen-2,3-quinones (E2-2,3-Q) and estrogen-3,4-quinones (E2-3,4-Q), were characterized after adduct cleavage. Levels of estrogen quinones and naphthoquinones were positively correlated in healthy controls, but not in breast cancer patients (p < 0.05). Compared with controls, levels of 1,2-NPQ and E2-3,4-Q were elevated by two- to ten-fold in cancer patients (p < 0.001). To explore the correlation between estrogen- and naphthalene-derived quinone adducts and disease status, we performed linear discriminant analysis of the ratio of 1,2-NPQ-Alb to (1,2-NPQ-Alb plus 1,4-NPQ-Alb) versus the ratio of E2-3,4-Q-2-S-Alb to (E2-2,3-Q-4-S-Alb plus E2-3,4-Q-2-S-Alb) in patients and controls. These two groups were separable using albumin adducts of estrogen quinones and naphthoquinones, with 99.6% overall correct classification rate (overall accuracy). The findings of this study suggest that differences in the disposition of estrogen and naphthalene, and the subsequent elevation of cumulative E2-3,4-Q and 1,2-NPQ may serve as biomarkers of breast cancer risk.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates the induction of DNA strand breaks and poly(ADP-ribose) polymerase-1 activation by 17beta-estradiol in human breast carcinoma cells through alteration of CYP1A1 and CYP1B1 expression.

The primary purpose of this research is to investigate the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) pretreatment and estrogen receptors-alpha (ER alpha) status on the induction of DNA damage by 17beta-estradiol (E 2) in human ER alpha(+)/MCF-7 and ER alpha(-)/MDA-MB-231 breast cancer cells. Results indicated that E 2 (0.1-100 nM) alone induced significant increases in cytotoxic response, reactive oxygen species (ROS) generation, and glutathione depletion in MDA-MB-231 cells but not in MCF-7 cells. At noncytotoxic concentrations, E 2 induced dose-related reduction in intracellular NAD(P)H in MDA-MB-231 cells through decreases in intracellular NAD (+) mediated by poly(ADP-ribose) polymerase-1 (PARP-1) activation as determined by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Further investigation using the single-cell gel electrophoresis (Comet) assay confirmed that the PARP-1 activation induced by estrogen in MDA-MB-231 was due to increases in the number of DNA strand breaks. This evidence indicates that E 2 induces decreases in intracellular NAD(P)H and NAD (+) in MDA-MB-231 cells through PARP-1 activation mediated by the formation of DNA strand breaks. Further investigation indicated that the cytotoxic and DNA-damaging effects induced by E 2 in MDA-MB-231 cells were completely blocked by pretreatment of TCDD (10 nM for 72 h). In contrast, with TCDD pretreatment, significant increases in cytotoxic response, ROS generation, glutathione (GSH) depletion, DNA strand breaks, and PARP-1 activation were detected in MCF-7 cells exposed to E 2. We demonstrated that TCDD modulated the differential induction of DNA damage by estrogen in MDA-MB-231 and MCF-7 cells primarily through the inducibility of cytochrome P450 1A1 and 1B1 expression. Overall, this evidence suggests that TCDD is capable of inducing imbalances in the expression of enzymes responsible for the bioactivation of estrogen leading to the subsequent accumulation of DNA damage and initiation of DNA repair in MDA-MB-231 and MCF-7 cells. Furthermore, we confirmed that ER alpha plays a protective role in modulating the induction of DNA damage by E 2 in human breast cancer cells.

Albumin and hemoglobin adducts of estrogen quinone as biomarkers for early detection of breast cancer.

Cumulative estrogen concentration is an important determinant of the risk of developing breast cancer. Estrogen carcinogenesis is attributed to the combination of receptor-driven mitogenesis and DNA damage induced by quinonoid metabolites of estrogen. The present study was focused on developing an improved breast cancer prediction model using estrogen quinone-protein adduct concentrations. Blood samples from 152 breast cancer patients and 71 healthy women were collected, and albumin (Alb) and hemoglobin (Hb) adducts of estrogen-3,4-quinone and estrogen-2,3-quinone were extracted and evaluated as potential biomarkers of breast cancer. A multilayer perceptron (MLP) was used as the predictor model and the resultant prediction of breast cancer was more accurate than other existing detection methods. A MLP using the logarithm of the concentrations of the estrogen quinone-derived adducts (four input nodes, 10 hidden nodes, and one output node) was used to predict breast cancer risk with accuracy close to 100% and area under curve (AUC) close to one. The AUC value of one showed that both data sets were separable. We conclude that Alb and Hb adducts of estrogen quinones are promising biomarkers for the early detection of breast cancer.

Differentiating between local and remote pollution over Taiwan.

In this study, an approach has been developed for differentiating between local and remote pollution over Taiwan, based on homogeneity perspective (variations of the standard deviation) of both AERONET measurements and NASA MERRA aerosol reanalysis (version 2, MERRA-2) over a 15-year period (2002 - 2017). The analysis of seasonal variations of the standard deviation of aerosol optical depth (AOD) measurements at six AERONET sites and MERRA AOD data in Taiwan showed that, in spring when remote aerosols dominate, the standard deviation is almost three times lower than that in autumn, when aerosols from local sources dominate. This finding was supported by MERRA AOD over the open ocean area: total AOD data were used to differentiate between local and remote pollution over both Taiwan and the open ocean area in the vicinity of Taiwan. Over Taiwan, MERRA total AOD showed a primary maximum in spring and a secondary one in autumn. Over the open ocean area, where there are no local sources of anthropogenic aerosols, MERRA total AOD showed only one maximum in spring and no maximum in autumn. This suggests that, in Taiwan, the maximum in autumn is attributed to local air pollution, while the pronounced maximum in spring is mainly caused by air pollution from continental Asia. The analyses of spatial distribution of 15-year monthly mean MERRA winds confirmed the above-mentioned results. Furthermore, similar to total AOD, MERRA sulfate AOD peaked in autumn over Taiwan, but not over the oceanic area: this indicates the contribution of local emissions of anthropogenic aerosols from the industrial sector. The standard deviation of MERRA sulfate AOD in spring is two-three times lower than the standard deviation in autumn: this is additional evidence that, in spring, sulfate aerosols from remote sources are predominant; while in autumn sulfate aerosols from local sources dominate.

Disparity in the induction of glutathione depletion, ROS formation, poly(ADP-ribose) polymerase-1 activation, and apoptosis by quinonoid derivatives of naphthalene in human cultured cells.

The purpose of this study is to examine the differences in the induction of cytotoxic effects and poly(ADP-ribose) polymerase-1 activation in human MCF-7 breast cancer cells by quinonoid derivatives of naphthalene, including 1,2-naphthalenediol (NCAT), 1,4-naphthalenediol (NHQ), 1,2-naphthoquinone (1,2-NQ), and 1,4-naphthoquinone (1,4-NQ). Results from the cytotoxic response analyses in cells indicated that all naphthalene quinonoids induced cell death in MCF-7 cells at concentrations ranging from 0.1 to 100microM where NHQ and 1,4-NQ were more efficient than NCAT and 1,2-NQ in the induction of cell death. Results from Western blot analyses confirmed that treatment of cells with NCAT and NHQ resulted in up-regulation of p53 protein expression and a significant shift in bax/bcl2 ratio, suggesting the induction of p53-dependent apoptosis in MCF-7 cells. Additionally, we observed that all naphthalene quinonoids induced increases in reactive oxygen species (ROS) formation and glutathione (GSH) depletion in MCF-7 cells. The induction of ROS formation and GSH depletion in cells by naphthalene quinonoids decreases in the rank order 1,4-NQ>NHQ>1,2-NQ approximately equal to NCAT. Further investigation indicated that least-squares estimates of the overall rates of elimination (k(e)) of naphthalene quinonoids in MCF-7 cells decreased in the rank order 1,4-NQ>1,2-NQ>NHQ>NCAT. Values of k(e) were estimated to be between 0.280h(-1)(T(1/2)=151min) and 13.8h(-1)(T(1/2)=3.05min). These results provide evidence that the para-isomeric form of naphthalene quinonoids tend to induce acute production of ROS and alterations in intracellular redox status in cells, leading to the subsequent cell death. Further, all naphthalene quinonoids induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 cells at non-cytotoxic concentrations. The reduction of intracellular NAD(P)H in cells exposed to NCAT and 1,2-NQ was blocked by two types of poly(ADP-ribose) polymerase (PARP) inhibitors whereas PARP inhibitors did not prevent the reduction of NAD(P)H in cells exposed to NHQ and 1,4-NQ. Further investigation confirmed that increases in the number of DNA single-strand breaks were detected in MCF-7 cells exposed to NCAT and 1,2-NQ as measured by the single-cell gel electrophoresis (Comet) assay whereas NHQ and 1,4-NQ did not induce increases in the number of single-strand breaks in MCF-7 cells. Overall, results from our investigation suggest that while NHQ and 1,4-NQ are more efficient in the induction of cell death, NCAT and 1,2-NQ are prone to induce depletion of NAD(P)H and NAD(+) mediated by PARP-1 activation through formation of DNA single-strand breaks in human cultured cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces oxidative stress, DNA strand breaks, and poly(ADP-ribose) polymerase-1 activation in human breast carcinoma cell lines.

The formation of reactive oxygen species (ROS) plays a critical role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicities in mammalian cells since it promotes cell proliferation, growth arrest, and apoptosis. In this study, we investigated whether TCDD induces oxidative stress and DNA damage in human ERalpha(+)/MCF-7 and ERalpha(-)/MDA-MB-231 breast cancer cells and whether this is accompanied by the initiation of DNA repair events. Results indicated that viability of MCF-7 and MDA-MB-231 cells was concentration- and time-dependently reduced by TCDD. Further, we observed significant increases in ROS formation and decreases in intracellular glutathione (GSH) in these two cell lines after TCDD treatment. Overall, the extent of cell death was greater in MCF-7 cells than in MDA-MB-231 cells whereas the magnitude of ROS formation and GSH depletion was greater in MDA-MB-231 cells than in MCF-7 cells. In addition, we observed that at non-cytotoxic concentration (1nM for 5h), TCDD induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 and MDA-MB-231 cells. These decreases were completely blocked by three types of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. The catalytic activation of PARP-1 in cells treated with TCDD was confirmed by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Moreover, we demonstrated increases in the number of DNA strand breaks in MCF-7 and MDA-MB-231 cells exposed to TCDD as measured by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that TCDD induces decreases in intracellular NAD(P)H and NAD(+) through PARP-1 activation mediated by formation of DNA strand breaks. In addition, we demonstrated that the extent of oxidative stress and DNA damage was greater in MDA-MB-231 cells than in MCF-7 cells, with a strong correlation to estrogen receptor (ER) status. In conclusions, our findings add further support to the theme that ROS formation is a significant determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to TCDD and that the TCDD-induced oxidative stress and DNA damage may, in part, contribute to TCDD-induced carcinogenesis.

Consecutive evaluation of graphene oxide and reduced graphene oxide nanoplatelets immunotoxicity on monocytes.

The biocompatibilities of graphene-family nanomaterials (GFNs) should be thoroughly evaluated before their application in drug delivery and anticancer therapy. The present study aimed to consecutively assess the immunotoxicity of graphene oxide nanoplatelets (GONPs) and reduced GONPs (rGONPs) on THP-1 cells, a human acute monocytic leukemia cell line. GONPs induced the expression of antioxidative enzymes and inflammatory factors, whereas rGONPs had substantially higher cellular uptake rate, higher levels of NF-κB expression. These distinct toxic mechanisms were observed because the two nanomaterials differ in their oxidation state, which imparts different affinities for the cell membrane. Because GONPs have a higher cell membrane affinity and higher impact on membrane proteins compared with rGONPs, macrophages (THP-1a) derived from GONPs treated THP-1cells showed a severer effect on phagocytosis. By consecutive evaluation the effects of GONPs and rGONPs on THP-1 and THP-1a, we demonstrated that their surface oxidation states may cause GFNs to behave differently and cause different immunotoxic effects.

Genetic polymorphisms in APE1 Asp148Glu(rs3136820) as a modifier of the background levels of abasic sites in human leukocytes derived from breast cancer patients and controls.

BACKGROUND: Apurinic/apyrimidinic (abasic/AP) sites are among the most common endogenous DNA lesions. AP sites, if not repaired, could result in genomic instability as well as chromosome aberrations. Information regarding the direct assay of the number of abasic sites in human leukocytes and its association with risk of breast cancer has not been reported. METHODS: In this study, we investigated the association between certain risk factors for breast cancer and the background levels of AP sites in leukocytes derived from 148 Taiwanese women with breast cancer and 140 cancer-free controls. The risk factors studied include age, body mass index (BMI), and polymorphisms of apurinic/apyrimidinic endonuclease (APE1) [APE1 Asp148Glu(rs3136820)]. RESULTS: Mean levels of AP sites were estimated to be 23.3 and 50.3 per 106 nucleotides in controls and breast cancer patients, respectively (~twofold, p < 0.001). In subjects with age <50 or BMI < 27 (kg/m2), the levels of AP sites in breast cancer patients were ~2-3-fold greater than those of controls (p < 0.05). Additionally, results from the AP site 3'-cleavage assay indicated that the AP sites detected in both controls and patients were likely to be oxidant-mediated 5'-cleaved AP sites (~61-64 %). The number of AP sites in breast cancer patients was ~twofold greater in subjects with Asp/Glu + Glu/Glugenotypes than those with Asp/Asp genotype (p < 0.001). CONCLUSIONS: We confirmed that cumulative body burden of AP sites is a significant predictor of the risk of developing breast cancer and that genetic predisposition and environment factors may modulate the induction of oxidative DNA lesions in breast cancer patients.

Induction of ROS formation, poly(ADP-ribose) polymerase-1 activation, and cell death by PCB126 and PCB153 in human T47D and MDA-MB-231 breast cancer cells.

The primary purpose of this research is to investigate whether exposure to polychlorinated biphenyls (PCBs), i.e. PCB153 and PCB126, is associated with induction of reactive oxygen species (ROS), poly(ADP-ribose) polymerase-1 (PARP-1) activation, and cell death in human T47D and MDA-MB-231 breast cancer cells. Results indicated that PCB153 and PCB126 induced concentration- and time-dependent increases in cytotoxic response and ROS formation in both T47D and MDA-MB-231 cells. At non-cytotoxic concentrations both PCB153 and PCB126 induced decreases in intracellular NAD(P)H and NAD+ in T47D and MDA-MB-231 cells where T47D cells were more resistant to PCB-induced reduction in intracellular NAD(P)H than MDA-MB-231 cells. Further investigation indicated that three specific PARP inhibitors completely blocked PCB-induced decreases in intracellular NAD(P)H in both T47D and MDA-MB-231 cells. These results imply that decreases in intracellular NAD(P)H in PCB-treated cells may be, in part, due to depletion of intracellular NAD+ pool mediated by PARP-1 activation through formation of DNA strand breaks. Overall, the extent of cytotoxic response, ROS formation, and PARP-1 activation generated in T47D and MDA-MB-231 cells was greater for PCB153 than for PCB126. In addition, the cytotoxicity induced by PCB153 and PCB126 in both T47D and MDA-MB-231 cells was completely blocked by co-treatment of catalase, dimethylsulfoxide, cupper (I)-/iron (II)-specific chelators, and CYP1A/2B inhibitors. This evidence suggests the involvement of ROS, Cu(I), Fe(II), and CYP1A/2B enzymes in mediating the induction of cell death by PCB153 and PCB126. Further, antagonism was observed between PCB126 and PCB153 for effects on cytotoxic response and ROS formation in T47D and MDA-MB-231 cells. Antagonism was also observed between PCB153 and PCB126 in the induction of NAD(P)H depletion at lower concentration (<10 microM) in T47D cells, but not in MDA-MB-231 cells. In conclusions, results from our investigation suggest that ROS formation induced by PCBs is a significant determinant factor in mediating the DNA damage and cell death in human breast cancer cells. The data also suggests that the status of estrogen receptor alpha may play a role in modulating the PCB-induced oxidative DNA damage and cell death in human breast cancer cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates estradiol-induced aldehydic DNA lesions in human breast cancer cells through alteration of CYP1A1 and CYP1B1 expression.

BACKGROUND: Many genes responsible for the bioactivation of endogenous estrogen to reactive quinonoid metabolites, including cytochrome P450 (CYP) 1A1, 1A2, and 1B1, are well-known target genes of the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). METHODS: The purpose of this research was to investigate the roles of TCDD-mediated altered gene expression in the induction of aldehydic DNA lesions (ADLs) by 17ß-estradiol (E2) in human MDA-MB-231 and MCF-7 breast cancer cells. RESULTS: We demonstrated that increases in the number of oxidant-mediated ADLs, including abasic sites and aldehydic base/sugar lesions, were detected in MDA-MB-231 cells exposed to E2. The DNA-damaging effects of E2 in MDA-MB-231 cells were prevented by pretreatment of cells with TCDD. In contrast, we did not observe statistically significant increases in the number of ADLs in MCF-7 cells exposed to E2. However, with TCDD pretreatment, an approximately twofold increase in the number of ADLs was detected in MCF-7 cells exposed to E2. CONCLUSIONS: TCDD pretreatment induces disparity in the disposition of E2 to reactive quinonoid metabolites and the subsequent formation of oxidative DNA lesions through alteration of CYP1A1 and CYP1B1 expression in human breast cancer cells.

Formation and removal of pentachlorophenol-derived protein adducts in rodent liver under acute, multiple, and chronic dosing regimens.

We investigated the kinetics of production and elimination of chlorinated quinone adducts of liver cytosolic proteins derived from pentachlorophenol (PCP), following oral administration under acute dosing (0-40 mg/kg body weight [bw] in Sprague-Dawley rats, 0-120 mg/kg bw in F344 rats, and 0-60 mg/kg bw in B6C3F1 mice), multiple dosing (0-60 mg/kg bw/day for 5 days in F344 rats and B6C3F1 mice), and chronic feeding (60 mg/kg bw/day for 6 months in F344 rats). We measured adducts of both tetrachloro-1,2-benzoquinone (Cl4-1,2-BQ) and tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) following reduction of cysteinyl adducts by Raney nickel and gas chromatography-mass spectrometry. Ratios of Cl4-1,2-BQ to Cl4-1,4-BQ adducts were much greater in mice (0.8-2) than in F344 rats (0.04-0.07), indicating that Cl4-1,2-BQ is an important PCP-binding species in mice but not rats. Following acute administration of 20 mg PCP/kg bw to Sprague-Dawley rats and B6C3F1 mice, the time course of adduct elimination over 14 days followed biphasic kinetics, with a rapid phase representing at least 92% of the adduct burden. Using data from acute experiments, we predicted adduct levels in rats and mice after the multiple- and chronic-dosing regimens. The agreement between predicted and observed levels was good (intraclass correlation coefficients of predicted and observed pairs of logged adduct levels were 0.812-0.921). These results provide evidence that the kinetics of liver protein adducts were not influenced by the dosing regimen of PCP, a recognized toxicant of the liver.

Hemoglobin adducts as biomarkers of estrogen homeostasis: elevation of estrogenquinones as a risk factor for developing breast cancer in Taiwanese women.

The aim of this study was to establish a methodology to analyze estrogen quinone-derived adducts, including 17ß-estradiol-2,3-quinone (E2-2,3-Q) and 17ß-estradiol-3,4-quinone (E2-3,4-Q), in human hemoglobin (Hb). The methodology was then used to measure the levels of these adducts in Hb derived from female breast cancer patients (n=143) as well as controls (n=147) in Taiwan. Our result confirmed that both E2-2,3-Q- and E2-3,4-Q-derived adducts, including E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb, were detected in all breast cancer patients with median levels at 434 (215-1472) and 913 (559-2384) (pmol/g), respectively. Levels of E2-2,3-Q-4-S-Hb correlated significantly with those of E2-3,4-Q-2-S-Hb (r=0.622-0.628, p<0.001). By contrast, median levels of these same estrogen quinone-derived adducts in healthy controls were 71.8 (35.7-292) and 139 (69.1-453) (pmol/g). This translated to ~6-fold increase in mean values of E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb in breast cancer patients compared to those in the controls (p<0.001). Our findings add further support to the theme that cumulative body burden of estrogen quinones is an important indicator of breast cancer risk. We hypothesize that combination of genetic events and environmental factors may modulate estrogen homeostasis and enhance the production of estrogen quinones which lead to subsequent generation of pro-mutagenic DNA lesions in breast cancer patients.

Investigation of the cumulative body burden of estrogen-3,4-quinone in breast cancer patients and controls using albumin adducts as biomarkers.

Both 17ß-estradiol-2,3-quinone (E2-2,3-Q) and 17ß-estradiol-3,4-quinone (E2-3,4-Q) are reactive metabolites of estrogen. Elevation of E2-3,4-Q to E2-2,3-Q ratio is thought to be an important indicator of estrogen-induced carcinogenesis. Our current study compared the cumulative body burden of these estrogen quinones in serum samples taken from Taiwanese women with breast cancer (n=152) vs healthy controls (n=75) by using albumin (Alb) adducts as biomarkers. Results clearly demonstrated the presence of cysteinyl adducts of E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb in all study population at levels ranging from 61.7-1330 to 66.6-1,590 pmol/g, respectively. Correlation coefficient between E2-2,3-Q-4-S-Alb and E2-3,4-Q-2-S-Alb was 0.610 for controls and 0.767 for breast cancer patients (p<0.001). We also noticed that in premenopausal subjects with body mass index (BMI) less than 27, background levels of E2-3,4-Q-2-S-Alb was inversely proportional to BMI with about 25% increase in E2-3,4-Q-2-S-Alb per 5 kg/m(2) decrease in BMI (p<0.001). In addition, we confirmed that mean levels of E2-3,4-Q-2-S-Alb in breast cancer patients were ∼5-fold greater than in those of controls (p<0.001). Overall, this evidence suggests that disparity in estrogen disposition and the subsequent elevation of cumulative body burden of E2-3,4-Q may play a role in the development of breast cancer.

Observations of ozone and carbon monoxide at Mei-Feng mountain site (2269 m a.s.l.) in Central Taiwan: seasonal variations and influence of Asian continental outflow.

Continuous measurements of ozone (O(3)) and carbon monoxide (CO) were carried out at Mei-Feng (24.05°N, 120.10°E, 2269 m above sea level), a remote mountain site in central Taiwan, to investigate the influence of long-range transported air pollution on O(3) and CO variations in the subtropical Pacific region. Data collected from March 2009 to September 2010 revealed average mixing ratios of 37±14 ppb for O(3) and 188±82 ppb for CO at this remote site. Diurnal variations for both O(3) and CO were observed as well in all seasons. The higher levels for O(3) and CO in the afternoon were attributed to transport of boundary layer pollution to the site during daytime upslope flow. Monthly means of both O(3) and CO showed maxima in spring and in the continental air masses from Southeast Asia, coastal China, and Korea/Japan. On the contrary, the lower O(3) and CO levels found in summer were due to the marine air masses originating from the Philippine Sea and Pacific Ocean. The relationship between O(3) and CO was analyzed, using nighttime data to minimize any local influence. The results showed a fairly good correlation between O(3) and CO from March to September. The contribution of CO from the Asian outflow reached a maximum in spring (88 ppb) and had a minimum in summer (27 ppb). The photochemical buildup of O(3) resulting from anthropogenic emissions in continental Asia was estimated to be 15 ppb in spring, while its production was insignificant, with an average of 4 ppb, in summer. A positive correlation between O(3) and CO plus high ozone levels in springtime suggested that the enhancements of O(3) were likely due to O(3) which was photochemically produced over this region.

Characterization of estrogen quinone-derived protein adducts and their identification in human serum albumin derived from breast cancer patients and healthy controls.

Both 17ß-estradiol-2,3-quinone (E2-2,3-Q) and 17ß-estradiol-3,4-quinone (E2-3,4-Q) are reactive metabolites of estrogen that are thought to be responsible for the estrogen-induced genotoxicity. The aim of this study was to establish a methodology to analyze estrogen quinone-derived protein adducts and to measure the background levels of these adducts in human serum albumin (Alb) derived from female blood donors in Taiwan. Results from in vitro experiments confirmed that the production of estrogen quinone-derived adducts on serum Alb increased with increased concentration of estrogen quinones. Time-course experiments suggested that both E2-2,3-Q- and E2-3,4-Q-derived adducts rapidly reached maximum values at 10 min mark and remained constant thereafter for up to 24 h. Additionally, with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) pretreatment, the production of estrogen quinone-derived protein adducts was detected in human MCF-7 breast cancer cells exposed to estrogen. Co-treatment of a catechol-O-methyl transferase inhibitor further enhanced the production of estrogen quinone-derived adducts in all cases. When we investigated the levels of estrogen quinone-derived adducts in human serum Alb, cysteinyl adducts of E2-2,3-Q-1-S-Alb, E2-2,3-Q-4-S-Alb, and E2-3,4-Q-2-S-Alb were detected in all healthy female controls (n=10) with median levels at 147 (range 14.1-533), 197 (range 30.0-777), and 65.6 (range 17.6-1360) (pmol/g), respectively. We noticed that levels of E2-2,3-Q-derived adducts were 2-fold greater than those of E2-3,4-Q-2-S-Alb in controls whereas levels of E2-3,4-Q-2-S-Alb were 2-fold higher than those of E2-2,3-Q-derived adducts in patients (n = 20). Additionally, levels of E2-2,3-Q-4-S-Alb correlated significantly with those of E2-3,4-Q-2-S-Alb (correlation coefficient r = 0.684-0.850, p < 0.05). Overall, we conclude that cumulative body burden of E2-3,4-Q is a significant predictor of breast cancer.

Investigation of the cumulative tissue doses of naphthoquinones in human serum using protein adducts as biomarker of exposure.

Both 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are reactive metabolites of naphthalene that are thought to be responsible for the naphthalene-induced cytotoxicity and genotoxicity. The aim of this study was to investigate the cumulative tissue dose of 1,2-NPQ and 1,4-NPQ in human serum derived from blood donors in Taiwan via measurements of albumin adducts by a methodology, which employs trifluoroacetic acid anhydride and methanesulfonic acid to selectively cleave cysteinyl adducts on proteins. Both 1,2-NPQ and 1,4-NPQ adducts were detected in all male and female subjects (n=22). The median levels of 1,2-NPQ adduct in human subjects were estimated to be 268 (range 139-857) and 203 (range 128-1352) (pmol/g) in male (n=11) and female (n=11) subjects, respectively. In contrast, the median levels of 1,4-NPQ adduct were estimated to be 45.0 (range 22.0-117) and 38.9 (range 21.5-172) (pmol/g) in male and female subjects, respectively. We noticed that levels of 1,2-NPQ adduct were significantly correlated with those of 1,4-NPQ adduct (correlation coefficient r=0.643, p<0.01). Results from in vitro experiments confirmed that the production of naphthoquinones-derived adducts on serum albumin increased with increased concentration of naphthoquinones (0-100 microM). Linear relationships were observed over the range of concentration. Time-course experiments suggested that both 1,2-NPQ and 1,4-NPQ-derived adducts rapidly reached maximum values at 10 min mark and remained constant thereafter. The reaction rate constant analyses indicated that the second-order rate constants, representing in vitro reactions between naphthoquinones and cysteine residues of serum albumin, were estimated to be 0.0044/0.0002L(gprotein)(-1)h(-1), respectively. Overall, the cumulative tissue doses of 1,4-NPQ (217-316 nM h) in male and female subjects were approximately 3-fold greater than those of 1,2-NPQ (76-98 nM h) in the study population. The initial concentrations of serum 1,2-NPQ and 1,4-NPQ in the study population were estimated to be between 145-188 and 807-1175 nM, respectively. We conclude that the relatively large amounts of naphthoquinones present in human serum may point to toxicological consequences.