Longitudinal follow-up of health effects among workers handling engineered nanomaterials: a panel study.

BACKGROUND: Although no human illness to date is confirmed to be attributed to engineered nanoparticles, occupational epidemiological studies are needed to verify the health effects of nanoparticles. This study used a repeated measures design to explore the potential adverse health effects of workers handling nanomaterials. METHODS: Study population was 206 nanomaterial-handling workers and 108 unexposed controls, who were recruited from 14 nanotechnology plants. They were followed up no less than two times in four years. A questionnaire was used to collect potential confounders and detailed work conditions. Control banding was adopted to categorize risk level for each participant as a surrogate marker of exposure. Health hazard markers include cardiopulmonary dysfunction markers, inflammation and oxidative damage markers, antioxidant enzymes activity, and genotoxicity markers. The Generalized Estimating Equation model was applied to analyze repeated measurements. RESULTS: In comparison to the controls, a significant dose-dependent increase on risk levels for the change of superoxide dismutase (p<0.01) and a significant increase of glutathione peroxidase change in risk level 1 was found for nanomaterial-handling workers. However, the change of cardiovascular dysfunction, lung damages, inflammation, oxidative damages, neurobehavioral and genotoxic markers were not found to be significantly associated with nanomaterials handling in this panel study. CONCLUSIONS: This repeated measurement study suggests that there was no evidence of potential adverse health effects under the existing workplace exposure levels among nanomaterials handling workers, except for the increase of antioxidant enzymes.

Estrogen and cigarette sidestream smoke particulate matter exhibit ERα-dependent tumor-promoting effects in lung adenocarcinoma cells.

Estrogen and secondhand smoke are key risk factors for nonsmoking female lung cancer patients who frequently have lung adenocarcinoma and show tumor estrogen receptor α (ERα) expression. We speculated that estrogen and secondhand smoke might cause harmful effects via ERα signaling. Our results showed that 17ß-estradiol (E2), the primary form of endogenous estrogen, exacerbated proliferation, migration, and granzyme B resistance of lung adenocarcinoma cells in an ERα-dependent manner. Cigarette sidestream smoke particulate matter (CSSP), the major component of secondhand smoke, could activate ERα activity dose dependently in human lung adenocarcinoma cells. The estrogenic activity of CSSP was abolished by an ERα-selective antagonist. CSSP regulated the nuclear entry, phosphorylation, and turnover of ERα similarly to E2. Furthermore, CSSP enhanced E2-stimulated ERα activity and Ser118 phosphorylation even when ERα became saturated with E2. Activation of ERα by CSSP required GSK3ß activity, but not involving polycyclic aromatic hydrocarbons, reactive oxygen species, calcium, epidermal growth factor receptor, and PI3K/Akt. Although CSSP possessed cytotoxicity, ERα-expressing cells grew and migrated faster than nonexpressing cells on recovery from CSSP exposure as observed in E2-pretreated cells. Knockdown of ERα by siRNA diminished E2- and CSSP-stimulated cell migration. Twenty-one genes, including SERPINB9, were identified to be upregulated by both E2 and CSSP via ERα. Increased SERPINB9 expression was accompanied with increased resistance to granzyme B-mediated apoptosis. This study demonstrates that estrogen has ERα-dependent tumor-promoting activity. CSSP acts like estrogen and shows a potential to enhance estrogen-induced ERα action.

Berberine Activates Aryl Hydrocarbon Receptor but Suppresses CYP1A1 Induction through miR-21-3p Stimulation in MCF-7 Breast Cancer Cells.

Berberine and the methylenedioxy ring-opening derivatives palmatine and jatrorrhizine are active ingredients in immunomodulatory plants, such as goldenseal. This study aimed to illustrate the effects of protoberberines on aryl hydrocarbon receptor (AhR) activation and cytochrome P450 (CYP) 1 in the estrogen receptor (ER)α(+) MCF-7 breast cancer cells. Among protoberberines at non-cytotoxic concentrations (≤10 µM), berberine had the most potent and statistically significant effects on AhR activation and CYP1A1/1A2/1B1 mRNA induction. The 24-h exposure to 10 µM berberine did not change CYP1A1 mRNA stability, protein level and function. Berberine significantly increased micro RNA (miR)-21-3p by 36% and the transfection of an inhibitor of miR-21-3p restored the induction of CYP1A1 protein with a 50% increase. These findings demonstrate that the ring opening of the methylenedioxyl moiety in berberine decreased AhR activation in MCF-7 cells. While CYP1A1 mRNA was elevated, berberine-induced miR-21-3p suppressed the increase of functional CYP1A1 protein expression.

Effect of nanoparticles exposure on fractional exhaled nitric oxide (FENO) in workers exposed to nanomaterials.

Fractional exhaled nitric oxide (FENO) measurement is a useful diagnostic test of airway inflammation. However, there have been few studies of FENO in workers exposed to nanomaterials. The purpose of this study was to examine the effect of nanoparticle (NP) exposure on FENO and to assess whether the FENO is increased in workers exposed to nanomaterials (NM). In this study, both exposed workers and non-exposed controls were recruited from NM handling plants in Taiwan. A total of 437 subjects (exposed group = 241, non-exposed group = 196) completed the FENO and spirometric measurements from 2009-2011. The authors used a control-banding (CB) matrix to categorize the risk level of each participant. In a multivariate linear regression analysis, this study found a significant association between risk level 2 of NP exposure and FENO. Furthermore, asthma, allergic rhinitis, peak expiratory flow rate (PEFR), and NF-κB were also significantly associated with FENO. When the multivariate logistic regression model was adjusted for confounders, nano-TiO2 in all of the NM exposed categories had a significantly increased risk in FENO > 35 ppb. This study found associations between the risk level of NP exposure and FENO (particularly noteworthy for Nano-TiO2). Monitoring FENO in the lung could open up a window into the role nitric oxide (NO) may play in pathogenesis.

Dioxin and estrogen signaling in lung adenocarcinoma cells with different aryl hydrocarbon receptor/estrogen receptor α phenotypes.

Evidence suggests that estrogen affects the pulmonary response to carcinogenic pollutants, such as dioxins. In this study, we examined dioxin and estrogen signaling cross-talk in lung adenocarcinoma cell lines that were engineered to exhibit different aryl hydrocarbon receptor (AhR)/estrogen receptor (ER) α phenotypes. Data showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) weakly antagonized estrogen-activated ERα activity in cells expressing abundant ERα, but little AhR. Increase of AhR expression or presence of a dioxin-responsive element in proximity silenced the antiestrogenic effect of TCDD. AhR was bound to dioxin-responsive element and transcriptionally active in both TCDD-untreated and -treated lung adenocarcinoma cells. 17ß-estradiol (E2) reduced basal and TCDD-induced AhR activity only in ERα-positive cells. AhR and ERα exhibited a protein-protein interaction in the presence of E2. Cotreatment with TCDD moderated this protein interaction. Colocalization of ERα and AhR at the estrogen-responsive site under E2 and TCDD/E2 treatments implied that E2 ∣ ERα might hijack AhR away from the dioxin-responsive site. Increasing the relative expression of AhR to ERα counteracted inhibition of AhR activity by E2 ∣ ERα. When AhR and ERα were both highly expressed, TCDD and E2 up-regulated expression of dual-responsive genes cytochrome P450 (CYP) 1A1 and CYP1B1 in a cumulative manner, increasing the danger of metabolic activation of carcinogens. Whereas TCDD ∣ AhR and E2 ∣ ERα appeared to regulate CYP1B1 separately through their binding sites, E2 ∣ ERα increased the TCDD responsiveness and mRNA expression of CYP1A1 in a noncanonical way. In conclusion, AhR/ERα expression pattern, estrogen level, and promoter context determine the genomic action of dioxin in lung adenocarcinoma cells.

Aryl hydrocarbon receptor is a target of 17-Allylamino-17-demethoxygeldanamycin and enhances its anticancer activity in lung adenocarcinoma cells.

We have demonstrated that aryl hydrocarbon receptor (AhR) is overexpressed in lung adenocarcinoma (AD). AhR is usually associated with heat shock protein 90 (Hsp90) in the cytoplasm. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, is currently under evaluation for its anticancer activity in clinical trials. Here we investigated whether AhR plays a role in 17-AAG-mediated anticancer activity by functioning as a downstream target or by modulating its anticancer efficacy. AhR expression in lung AD cells was modulated by siRNA interference or overexpression. Tumor growth was determined with colony formation in vitro or in vivo. Anticancer activity of 17-AAG was determined by measuring cell viability, cell cycle distribution, and expression of cell cycle regulators. Proteins and mRNA levels were examined by immunoblotting and the real-time reverse transcription-polymerase chain reaction, respectively. In this study, AhR overexpression positively modulated growth of lung AD cells, at least partially, via RelA-dependent mechanisms. Although treatment with 17-AAG reduced AhR levels and AhR-regulated gene expression in lung AD cells, AhR expression increased anticancer activity of 17-AAG. In addition, 17-AAG treatment reduced cell viability, CDK2, CDK4, cyclin E, cyclin D1, and phosphorylated Rb levels in AhR-expressing lung AD cells. NAD(P)H:quinone oxidoreductase (NQO1), which is regulated by AhR, was shown to increase anticancer activity of 17-AAG in cells. Knockdown of NQO1 expression attenuated the reduction of cell cycle regulators by 17-AAG treatment in AhR overexpressed cells. We demonstrated that AhR protein not only functions as a downstream target of 17-AAG, but also enhances anticancer activity of 17-AAG in lung AD cells.

Flavonoids exhibit diverse effects on CYP11B1 expression and cortisol synthesis.

CYP11B1 catalyzes the final step of cortisol biosynthesis. The effects of flavonoids on transcriptional expression and enzyme activity of CYP11B1 were investigated using the human adrenocortical H295R cell model. All tested nonhydroxylated flavones including 3',4'-dimethoxyflavone, α-naphthoflavone, and ß-naphthoflavone upregulated CYP11B1 expression and cortisol production, whereas apigenin and quercetin exhibited potent cytotoxicity and CYP11B1 repression at high concentrations. Nonhydroxylated flavones stimulated CYP11B1-catalyzed cortisol formation at transcriptional level. Resveratrol increased endogenous and substrate-supported cortisol production like nonhydroxylated flavones tested, but it had no effect on CYP11B1 gene expression and enzyme activity. Resveratrol appeared to alter cortisol biosynthesis at an earlier step. The Ad5 element situated in the -121/-106 region was required for basal and flavone-induced CYP11B1 expression. Overexpression of COUP-TFI did not improve the responsiveness of Ad5 to nonhydroxylated flavones. Although COUP-TFI overexpression increased CYP11B1 and CYP11B2 promoter activation, its effect was not mediated through the common Ad5 element. Treating cells with PD98059 (a flavone-type MEK1 inhibitor) increased CYP11B1 promoter activity, but not involving ERK signaling because phosphorylation of ERK1/2 remained unvarying throughout the course of treatment. Likewise, AhR was not responsible for the CYP11B1-modulating effects of flavonoids because inconsistency with their effects on AhR activation. 3',4'-dimethoxyflavone and 8-Br-cAMP additively activated CYP11B1 promoter activity. H-89 reduced 3',4'-dimethoxyflavone-induced CYP11B1 promoter activation but to a lesser extent as compared to its inhibition on cAMP-induced transactivation. Our data suggest that constant exposure to nonhydroxylated flavones raises a potential risk of high basal and cAMP-induced cortisol synthesis in consequence of increased CYP11B1 expression.

Aryl hydrocarbon receptor protects lung adenocarcinoma cells against cigarette sidestream smoke particulates-induced oxidative stress.

Environmental cigarette smoke has been suggested to promote lung adenocarcinoma progression through aryl hydrocarbon receptor (AhR)-signaled metabolism. However, whether AhR facilitates metabolic activation or detoxification in exposed adenocarcinoma cells remains ambiguous. To address this question, we have modified the expression level of AhR in two human lung adenocarcinoma cell lines and examined their response to an extract of cigarette sidestream smoke particulates (CSSP). We found that overexpression of AhR in the CL1-5 cell line reduced CSSP-induced ROS production and oxidative DNA damage, whereas knockdown of AhR expression increased ROS level in CSSP-exposed H1355 cells. Oxidative stress sensor Nrf2 and its target gene NQO1 were insensitive to AhR expression level and CSSP treatment in human lung adenocarcinoma cells. In contrast, induction of AhR expression concurrently increased mRNA expression of xenobiotic-metabolizing genes CYP1B1, UGT1A8, and UGT1A10 in a ligand-independent manner. It appeared that AhR accelerated xenobiotic clearing and diminished associated oxidative stress by coordinate regulation of a set of phase I and II metabolizing genes. However, the AhR-signaled protection could not shield cells from constant oxidative stress. Prolonged exposure to high concentrations of CSSP induced G0/G1 cell cycle arrest via the p53-p21-Rb1 signaling pathway. Despite no effect on DNA repair rate, AhR facilitated the recovery of cells from growth arrest when CSSP exposure ended. AhR-overexpressing lung adenocarcinoma cells exhibited an increased anchorage-dependent and independent proliferation when recovery from exposure. In summary, our data demonstrated that AhR protected lung adenocarcinoma cells against CSSP-induced oxidative stress and promoted post-exposure clonogenicity.

Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation.

Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3ß, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation.

ERα-dependent estrogen-TNFα signaling crosstalk increases cisplatin tolerance and migration of lung adenocarcinoma cells.

Lung adenocarcinoma is the most common type of lung cancer in women. Our previous studies demonstrated that 17ß-estradiol (E2) promoted lung adenocarcinoma cell proliferation and tumor growth through estrogen receptor ERα. Transcriptomic analysis suggested that E2 potentiated TNFα-NFκB signaling in ERα-expressing lung adenocarcinoma cells. This study further demonstrated that E2 increased TNFα receptor expression and TNFα-triggered NFκB activity in ERα-expressing cells. E2-activated ERα had no physical association with NFκB p65/p50 heterodimer but facilitated TNFα-initiated IκBα degradation, NFκB nuclear translocation, and S468/S536 phosphorylation of p65 essential for NFκB activity. While knockdown of ERα prevented E2 from boosting NFκB activity, antiestrogen ICI 182,780 stimulated NFκB activity like E2. Inhibition of GSK3ß hampered E2:ERα-promoted NFκB activity and abolished S468 phosphorylation of p65, suggesting that GSK3ß played a role in the E2-TNFα signaling crosstalk. In ERα-expressing cells, E2 and TNFα synergistically regulated many genes that were not typically responsive to either E2 or TNFα. Functional analysis of microarray data inferred that E2/TNFα-induced transcriptomic changes improved cell survival and movement. Viability and colony formation assays validated that E2 and TNFα together increased cisplatin tolerance of ERα-expressing cells. Wound healing assays also confirmed that E2/TNFα cotreatment increased cell migration in an ERα-dependent manner. E2/TNFα-induced dysregulation of genes such as cell survival and movement-associated genes, proto-oncogenes, metallothioneins and histone core genes was correlated with poor overall survival in patients. In summary, E2 and TNFα engaged in an ERα-dependent positive crosstalk in lung adenocarcinoma cells, consequently increasing NFκB activation, cisplatin tolerance and cell migration and worsening prognosis.

Exposure profiles of workers from indium tin oxide target manufacturing and recycling factories in Taiwan.

Indium tin oxide exposure poses a potential health risk, but the exposure assessment in occupational setting remains incomplete and continues to be a significant challenge. To this end, we investigated the association of work type, airborne indium concentration, respirable fraction of total indium, and cumulative indium exposure index (CEI) with the levels of plasma indium (P-In) and urinary indium (U-In) among 302 indium tin oxide target manufacturing and recycling workers in Taiwan. We observed that recycling-crushing produced the highest concentrations of total indium (area: 2084.8 µg/m3; personal: 3494.5 µg/m3) and respirable indium (area: 533.4 µg/m3; personal: 742.0 µg/m3). Powdering produced the highest respirable fraction of total indium (area: 58.6%; personal: 81.5%), where the workers had the highest levels of P-In (geometric mean: 2.0 µg/L) and U-In (1.0 µg/g creatinine). After adjusting for the confounder, the CEIs of powdering (ßPR = 0.78; ßPR = 0.44), bonding (ßPT = 0.61; ßPT = 0.37), and processing workers (ßPT = 0.43; ßPT = 0.28) showed significant associations with P-In and U-In, validating its utility in monitoring the exposure. Also, the respirable fraction of total indium significantly contributed to the increased levels of P-In and U-In among workers. The varying levels of relationship noted between indium exposure and the levels of P-In and U-In among workers with different work types suggested that setting the exposure limits among different work types is warranted.

Health risk of metal exposure via inhalation of cigarette sidestream smoke particulate matter.

Cigarette sidestream smoke particulate matter (CSSP) is a major source of airborne metals in the indoor environment. However, the health impacts of inhalation of CSSP-bound metals are rarely studied. In this study, we quantify the amount of 37 metals discharged through CSSP from a leading Taiwan brand of cigarette, Long Life. We also estimate cancer and non-cancer risks due to inhalation of these metals and investigate possible modes of toxic action. Long Life CSSP exhibits a distinctive carcinogenic metal profile compared with Western brands. When released to a 60-m3 poorly ventilated room, Long Life CSSP metals increase the risk for cancer by a 9.26 or 20.90 in a million chance and the hazard quotient for non-cancer toxicity by 0.496 or 0.286 per cigarette depending on risk estimation system. Cd accounts for more than 90% and 80% of cancer and non-cancer risk, respectively. Long Life CSSP also contains considerable amounts of Al, Ba, and Fe. Metals are not responsible for CSSP-induced cytotoxicity, oxidative stress, and transactivation activity of AhR, Nrf2, and ERα. However, they diminish resveratrol-activated Nrf2 activity and downstream antioxidant gene expression in low-AhR-expressing lung cells. Our results suggest that chronic exposure to Long Life CSSP elevates Cd-associated cancer and non-cancer risks. Furthermore, exposure to Long Life CSSP metals may impair Nrf2-mediated antioxidant protection.

Polychlorinated biphenyl exposure and CYP19 gene regulation in testicular and adrenocortical cell lines.

Exposure to polychlorinated biphenyls (PCBs) disturbs many estrogen-mediated biochemical processes. PCBs may cause these abnormalities by altering expression of the aromatase gene CYP19. This study demonstrated that high concentrations of PCB126 increased basal CYP19 mRNA abundance in mouse testicular Leydig I-10 cells and human adrenocortical H295R cells. Stimulating the cells with chorionic gonadotropin or 8-Br-cAMP concealed the estrogenic effect of PCB126. PCB126 is a powerful ligand for nuclear receptor AhR. Antagonizing the AhR activity of H295R by an inhibitor abolished PCB126-elicited CYP19 induction. However, PCB126 elevated basal CYP19 expression and aromatase activity in a slow progressive manner contrary to the sharp induction of the classic AhR target gene CYP1A1. Exposure of H295R to PCBs with different AhR activation abilities also varied CYP19 and CYP1A1 expression in dissimilar patterns, although the CYP19 mRNA levels were in line with the AhR activation abilities of the congeners. In contrast to PCB126, PCB39, which could not activate AhR and lacked effect on CYP1A1, significantly reduced CYP19 mRNA expression. AhR apparently played an important role in CYP19 gene regulation, but it might regulate CYP19 differently from CYP1A1 in the adrenocortical cells. Regardless of the action mechanism, PCB exposure increases risk for CYP19 dysregulation.

Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells.

In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10nM TCDD in the presence of different concentrations of arecoline (50-300 microM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver.

Mechanistic study of polychlorinated biphenyl 126-induced CYP11B1 and CYP11B2 up-regulation.

Although polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal, little is known about the effects of these endocrine disruptors on adrenal steroidogenesis. Our previous studies showed that high concentrations of PCB126 stimulated CYP11B1 and CYP11B2 mRNA expression and consequently raised cortisol and aldosterone synthesis in the human adrenocortical H295R cells, respectively. In this study, we further investigated the mechanism underlying the PCB126-induced steroidogenic alterations. We first examined the role of the PCB126 nuclear receptor aryl hydrocarbon receptor (AhR) using a potent antagonist 3',4'-dimethoxyflavone (3',4'-DMF). Although 3',4'-DMF abolished AhR-dependent transcriptional activity, it could not block PCB126-stimulated CYP11B1 and CYP11B2 induction. Conversely, 3',4'-DMF synergistically increased the stimulatory effects of PCB126. Furthermore, PCB39, -77, -132, -156, and -169, whether AhR ligands or not, all could increase CYP11B1 and CYP11B2 mRNA accumulation. Promoter analyses demonstrated that PCB126 had little effects on the transcription rate of both genes, whereas RNA degradation assays showed that PCB126 protected both transcripts from degradation. In contrast, 3',4'-DMF exhibited positive effects on transcription but no influence on transcript stability. The synergistic induction of CYP11B1 and CYP11B2 mRNA levels by the PCB126/3',4'-DMF cotreatment might result from the combination of transcriptional regulation by 3',4'-DMF and posttranscriptional regulation by PCB126. This study also demonstrated that an internal region of CYP11B1 mRNA (nucleotides 881-1285) was important for PCB126-mediated transcript stabilization. From these findings, we concluded that PCB126 up-regulated steroidogenic CYP11B1 and CYP11B2 mRNA expression not via AhR-mediated transcriptional activation but by increasing posttranscriptional mRNA stability.

Body burdens of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls and their relations to estrogen metabolism in pregnant women.

Polychlorinated dibenzo-p-dioxins (PCDDs, dioxins), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) are environmental endocrine disruptors that have half-lives of 7-10 years in the human body and have toxicities that probably include carcinogenesis. A high ratio of 4-hydroxyl estradiol (4-OH-E2) to 2-hydroxyl estradiol (2-OH-E2) has been suggested as a potential biomarker for estrogen-dependent neoplasms. In this cohort study of maternal-fetal pairs, we examined the relationship of PCDD/PCDF and PCB exposure to levels of estrogen metabolites in the sera of 50 pregnant women 25-34 years of age from central Taiwan. Maternal blood was collected during the third trimester, and the placenta was collected at delivery. We measured 17 dioxin congeners, 12 dioxin-like PCBs, and 6 indicator PCBs in placenta using gas chromatography coupled with high-resolution mass spectrometry. Estrogen metabolites in maternal serum were analyzed by liquid chromatography tandem mass spectrometry. The ratio of 4-OH-E2:2-OH-E2 decreased with increasing exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (beta=-0.124, p=0.004 by the general linear regression model, R=0.4). Meanwhile, serum levels of 4-OH-E2 increased with increasing concentrations of high-chlorinated PCDFs (i.e., 1,2,3,4,6,7,8-hepta-CDF: beta=0.454, p=0.03, R=0.30). Altered estrogen catabolism might be associated with body burdens of PCDDs/PCDFs. Our study suggests that exposure to PCDDs/PCDFs significantly affects estrogen metabolism. Therefore, PCDD/PCDF exposure must be considered when using the OH-E2 ratio as a breast cancer marker.

Arsenic inhibits induction of cytochrome P450 1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human hepatoma cells.

The aim of this study was to examine the arsenic effect on activation of aryl hydrocarbon receptor (AhR)-mediated gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in human hepatoma cells. The human hepatoma Huh7 cells were treated with sodium arsenite (NaAsO2) from 0.5 to 20 microM for 24 h. Our data revealed that NaAsO2 < or = 10 microM caused no significant cytotoxic effect on Huh7 cells (p>0.05). We also established a dioxin-responsive element (DRE)-mediated Chemical Activated LUciferase eXpression (CALUX) cell line, Huh7-DRE-Luc, by stable transfection of Huh7 with a DRE-driven firefly luciferase reporter plasmid (4xDRE-TATA-Luc). Treatments of Huh7-DRE-Luc and Huh7 with NaAsO2 attenuated the 2,3,7,8-TCDD-induced DRE-CALUX and cytochrome P450 1A1 (CYP1A1) activations, respectively, in a dose-dependent manner. We found that the calculated CALUX-toxic equivalent (TEQ) levels induced by cotreatment of NaAsO2 > or = 3.0 microM and 10 nM 2,3,7,8-TCDD were significantly lower than that induced by 2,3,7,8-TCDD alone (p<0.05). In the present study, we demonstrated that arsenic not only inhibited the TCDD-induced CYP1A1 activation but also interfered with DRE-CALUX bioassay in human hepatoma cells. Our finding also suggests that extensive cleanup of sample for removal of any possible interfering factor is critical to guarantee the accuracy of dioxin-TEQ levels using DRE-CALUX bioassay.

PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.

Dioxins and polychlorinated biphenyls (PCBs) have been shown to accumulate in the adrenal glands when incorporated into the body. However, the impacts of exposure on adrenal steroidogenesis have not been thoroughly investigated. In this study, we demonstrated that dioxin-like PCB126 altered androgen, cortisol, and aldosterone biosynthesis differentially in human adrenocortical H295R cells. PCB126 diminished basal and cAMP-induced androstenedione production as well as CYP17 mRNA expression in a dose-dependent and time-dependent manner. The CYP17 repression was accompanied with decreases in the encoded 17 alpha-hydroxylase and 17,20-lyase activities, particularly the latter. In contrast, high concentrations of PCB126 stimulated basal cortisol and aldosterone biosynthesis, including induction of CYP21B, CYP11B1, and CYP11B2 mRNA expression and elevation of the conversion of cortisol from 17-OH-progesterone and aldosterone from progesterone. cAMP abolished the positive effect of PCB126 on cortisol synthesis, while it synergistically enhanced PCB126 stimulation on CYP11B2 expression and aldosterone production. It seemed likely that the downregulation of CYP21B caused by the combination of PCB126 and cAMP counteracted the CYP11B1 induction stimulated by the co-treatment. In addition, high concentrations of PCB126 might sensitize the regulation of adrenocorticotropin (ACTH) on the adrenocortical cells by increasing ACTH receptor levels. Because adrenal steroids have profound influences on glucose tolerance, insulin sensitivity, lipid metabolism, obesity, vascular function, and cardiac remodeling, this article also discusses the potential association of the detected adrenocortical alterations with increased diabetic and cardiovascular risk found among highly exposed people.

Steroidogenic factor 1 differentially regulates basal and inducible steroidogenic gene expression and steroid synthesis in human adrenocortical H295R cells.

The significance of steroidogenic factor 1 (SF-1) in adrenal steroidogenesis was studied using adrenocortical cell lines transformed with a dominant negative mutant of SF-1. Constitutive expression of the mutant did not only impair the activity of endogenous SF-1 but also diminish its own expression, suggesting that SF-1 was under autoregulation. Inhibition of the endogenous SF-1 activity significantly reduced basal and inducible transcription of CYP17, CYP21B and CYP11B1, but exhibited little effects on StAR and CYP11A1 expression. Stimulating the transformed cells with potassium and cAMP freed CYP11B2 from the mutant-caused transcriptional inhibition, whereas the transformation abolished induction of CYP17 by both stimulants. Consistent with the transcriptional changes of steroidogenic genes, basal and inducible synthesis of cortisol and androgens drastically declined in the transformed cell lines. The relief of CYP11B2 repression following the potassium and cAMP stimulation removed the restraint the mutant exerted on aldosterone synthesis, and resulted in aldosterone overproduction in the stimulated transformed cells. SF-1 also plays a role in regulating the adrenocorticotrophic hormone (ACTH) responsiveness of the adrenocortical cells. Inhibition of SF-1 activity significantly decreased basal expression of ACTH receptor and its induction by potassium and cAMP.

Assessment of the potential of polyphenols as a CYP17 inhibitor free of adverse corticosteroid elevation.

Inhibition of 17α-hydroxylase/17,20-lyase (CYP17), which dictates the proceeding of androgen biosynthesis, is recommended as an effective treatment for androgen-dependent diseases. However, androgen depletion by selective CYP17 inhibition is accompanied with corticosteroid elevation, which increases risk of cardiovascular diseases. In this study, we evaluated the likelihood of polyphenols as a CYP17 inhibitor without cardiovascular complications. All examined polyphenols significantly inhibited CYP17 in human adrenocortical H295R cells, but their effects on androgen and cortisol biosynthesis were diverse. Resveratrol was the most potent CYP17 inhibitor with an approximate IC50 of 4 µM, and the inhibition might weigh on the 17α-hydroxylase activity more than the 17,20-lyase activity. Resveratrol also inhibited 21α-hydroxylase (CYP21) essential for corticosteroid biosynthesis but to a lesser extent, thus preventing the occurrence of cortisol elevation following CYP17 blockade. Although transcriptional down-regulation was important for α-naphthoflavone-mediated CYP17 inhibition, resveratrol inhibited CYP17 and CYP21 mainly at the level of enzyme activity rather than enzyme abundance and cytochrome P450 electron transfer. Daidzein also inhibited CYP17 and CYP21 although less potent than resveratrol. Daidzein was the only polyphenol showing inhibition of 3ß-hydroxysteroid dehydrogenase type II (3ßHSD2). The exceptional 3ßHSD2 inhibition led to dehydroepiandrosterone accumulation alongside daidzein-caused androgen biosynthetic impairment. In contrast, androgen and cortisol secretion was increased or remained normal under α-naphthoflavone and ß-naphthoflavone treatments, suggesting that CYP17 inhibition was counteracted by increased substrate generation. α-naphthoflavone and ß-naphthoflavone also enhanced the formation of cortisol from 17-hydroxyprogesterone and testosterone from androstenedione. Our findings suggest a potential application of resveratrol in androgen deprivation therapy.