Publications by authors named "David C Spink"

25 Publications

  • Page 1 of 1

Legacy and Emerging Per- and Polyfluoroalkyl Substances: Analytical Techniques, Environmental Fate, and Health Effects.

Int J Mol Sci 2021 Jan 20;22(3). Epub 2021 Jan 20.

Laboratory of Organic Analytical Chemistry, Wadsworth Center, New York State Department of Health, Albany, NY 12237, USA.

Due to their unique chemical properties, per- and polyfluoroalkyl substances (PFAS) have been used extensively as industrial surfactants and processing aids. While several types of PFAS have been voluntarily phased out by their manufacturers, these chemicals continue to be of ecological and public health concern due to their persistence in the environment and their presence in living organisms. Moreover, while the compounds referred to as "legacy" PFAS remain in the environment, alternative compounds have emerged as replacements for their legacy predecessors and are now detected in numerous matrices. In this review, we discuss the historical uses of PFAS, recent advances in analytical techniques for analysis of these compounds, and the fate of PFAS in the environment. In addition, we evaluate current biomonitoring studies of human exposure to legacy and emerging PFAS and examine the associations of PFAS exposure with human health impacts, including cancer- and non-cancer-related outcomes. Special focus is given to short-chain perfluoroalkyl acids (PFAAs) and ether-substituted, polyfluoroalkyl alternatives including hexafluoropropylene oxide dimer acid (HFPO-DA; tradename GenX), 4,8-dioxa-3H-perfluorononanoic acid (DONA), and 6:2 chlorinated polyfluoroethersulfonic acid (6:2 Cl-PFESA; tradename F-53B).
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http://dx.doi.org/10.3390/ijms22030995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863963PMC
January 2021

Enhanced Sensitivity for the Analysis of Perfluoroethercarboxylic Acids Using LC-ESI-MS/MS: Effects of Probe Position, Mobile Phase Additive, and Capillary Voltage.

J Am Soc Mass Spectrom 2020 Oct 31;31(10):2124-2132. Epub 2020 Aug 31.

Laboratory of Organic Analytical Chemistry, Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, University at Albany, State University of New York, Albany, New York 12237, United States.

Perfluoroethercarboxylic acids (PFECAs) have recently emerged as replacements for toxic per- and polyfluorinated alkyl substances (PFAS) including perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS). Compared with other PFAS, many PFECAs including hexafluoropropylene oxide dimer acid (HFPO-DA, trade name GenX) exhibit poor sensitivity during analysis using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and are therefore often difficult to quantify. This study examined changes in ESI probe position, mobile phase additive, and capillary voltage with the goal of enhancing PFECA sensitivity. In addition, the relative contributions of existing mechanistic theories for PFAS ionization during ESI are discussed. Results indicated that the LC-ESI-MS/MS sensitivity for 9 PFECAs can be improved significantly by altering the ESI probe position. At the optimal probe position, lowering the capillary voltage from 2.0 to 0.5 kV universally enhanced the LC-ESI-MS/MS sensitivity for PFAS analysis. For most analytes, the use of ammonium bicarbonate rather than ammonium acetate as a mobile phase additive also enhanced the analytical response. These effects have not been previously reported and suggest that many laboratories may be conducting analyses of PFECAs under suboptimal conditions. Using the strategies outlined in this study, PFECAs can be more easily incorporated into comprehensive methods for PFAS analysis. Here, we describe analytical parameters that enhance the sensitivity for some PFECAs by up to 36-fold while maintaining high sensitivity for legacy PFAS. This work not only highlights solutions to mitigate inadequate PFECA sensitivity but also provides insight into the mechanisms underlying PFAS ionization efficiency during LC-ESI-MS/MS.
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http://dx.doi.org/10.1021/jasms.0c00244DOI Listing
October 2020

Kratom Adulterated with Phenylethylamine and Associated Intracerebral Hemorrhage: Linking Toxicologists and Public Health Officials to Identify Dangerous Adulterants.

J Med Toxicol 2020 01 11;16(1):71-74. Epub 2019 Nov 11.

Upstate New York Poison Center, Syracuse, NY, USA.

Introduction: Kratom is derived from the plant Mitragyna speciosa which is indigenous to Southeast Asia. Active compounds, mitragynine and 7-hydroxymitragynine, cause mild stimulant and opioid agonist effects. Although reported to have potential benefits in the treatment of opioid use disorder, efficacy remains uncertain while adverse health effects have been reported. A compounding concern is the presence of adulterants given that this is an unregulated product.

Case Details: A 54-year-old fitness instructor who used an online purchased kratom product regularly for one year developed stimulatory effects and suffered a large hemorrhagic stroke with a close temporal relationship to ingestion of a different kratom product from the one he regularly used. A collaborative investigation by medical toxicologists, a regional poison center, the state public health laboratory, and public health officials determined that his new kratom product was adulterated with phenylethylamine (PEA).

Discussion: We report a case of PEA adulterated kratom purchased and used with resultant adverse effects. PEA is structurally similar to amphetamine and is known to produce sympathomimetic effects. It is possible the stimulatory effect of PEA resulted in a marked and transient increase in blood pressure resulting in hemorrhagic stroke.

Conclusion: Medical toxicologists should form working relationships with laboratories and public health officials to aid in early identification of adulterated products that carry risk to the general population.
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http://dx.doi.org/10.1007/s13181-019-00741-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942072PMC
January 2020

Potency Analysis of Medical Marijuana Products from New York State.

Cannabis Cannabinoid Res 2019 23;4(3):195-203. Epub 2019 Sep 23.

New York State Department of Health, Wadsworth Center, Albany, New York.

In the United States, medical marijuana programs have been established in 29 states and the District of Columbia. In 2014, New York State (NYS) approved medical marijuana legislation, and its program became fully operational in January of 2016. Products manufactured under the auspices of the program may be used by certified patients in NYS for the treatment of 1 of 12 qualifying medical conditions. The NYS statute requires rigorous testing of each product lot manufactured in the state for its cannabinoid profile, bacterial and fungal contamination, mycotoxins, heavy metals, plant-growth regulators, and pesticides. Here, we report on the analysis of product cannabinoid profiles. A method employing a simple extraction/dilution technique and reversed-phase high-performance liquid chromatography with photodiode array detection (HPLC-PDA) was developed for the analysis of 10 cannabinoids: cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol (CBD), tetrahydrocannabivarin, cannabinol, Δ-tetrahydrocannabinol (Δ-THC), cannabichromene, cannabidivarin, and Δ-tetrahydrocannabinolic acid-A. The method employed internal standard quantitation and incorporated a surrogate to monitor extraction efficiency and analytical recovery. The HPLC-PDA method was validated using sample matrices composed of medium-chain triglycerides, hemp oil, sesame oil, and an ethanol-propylene glycol tincture. Limits of detection, limits of quantitation, accuracy, precision, and inter- and intraday reproducibility were found to be highly satisfactory. The validated method has been used to analyze over 3500 samples from over 700 lots of medical marijuana products manufactured in NYS from January 2016 through April 2018. Quality-control data showed quantitative spike recoveries and, for the analysis of samples from the same lot, the coefficients of variation for the principal analytes, Δ-THC and CBD, averaged <3%. Using the HPLC-PDA method, the NYS medical marijuana products were analyzed to verify the potencies on the product labels and to determine the stability of the products. An HPLC-PDA-based method was developed, validated, and employed to analyze 10 cannabinoids in a variety of medical marijuana products. The method has proven to be accurate, precise, stable, and very robust. Its use is an integral part of the NYS Medical Marijuana program for validation of the content and consistency of medical marijuana products.
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http://dx.doi.org/10.1089/can.2018.0037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757241PMC
September 2019

Noninvasive determination of human cortisol and dehydroepiandrosterone sulfate using liquid chromatography-tandem mass spectrometry.

Anal Bioanal Chem 2019 Feb 5;411(6):1203-1210. Epub 2019 Jan 5.

University at Albany, State University of New York, 1400 Washington Avenue, Albany, NY, 12222, USA.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurements of steroids in human saliva has garnered increased interest in the area of clinical psychoneuroendocrinological research. However, performance characteristics of LC-MS/MS methods for the analysis of steroids in saliva are limited. Human saliva samples were collected via passive drool. Cortisol and dehydroepiandrosterone sulfate (DHEA-S) in the samples were extracted together, resolved on a C18-A column, and analyzed using tandem mass spectrometry. The LC-MS/MS method had limits of quantitation of 0.03 and 0.06 ng/mL for DHEA-S and cortisol, respectively. Method evaluations showed coefficient variation (%CV) of inter-assay ranging 4.6-17.9% for DHEA-S and cortisol, recoveries of 102.4-109.5% for DHEA-S and 94.6-98.3% for cortisol, and assay linearity with R = 0.9964 for DHEA-S (1.0-25.0 ng/mL) and R = 0.997 (1.0-25.0 ng/mL) for cortisol. No cross contamination among samples was observed. Human saliva showed 20% and 18% ion enhancement effect for DHEA-S and cortisol assay, respectively. No interference by ten common steroids was detected. Regression analysis of method comparisons with laboratory-developed test (LDT) method revealed R = 0.9688 (LC-MS/MS = 0.9665 LDT-LC-MS/MS - 0.7355) for cortisol, and R = 0.9039 (LC-MS/MS = 1.0173 LDT-LC-MS/MS + 3.6797) for DHEA-S. Reference ranges for young adults were determined to be 0.3-5.9 ng/mL for females and 0.1-5.6 ng/mL for males for salivary cortisol, and 0.6-7.4 ng/mL for females and 0.6-10.1 ng/mL for males for salivary DHEA-S. An LC-MS/MS method for quantifying cortisol and DHEA-S in human saliva was developed and validated for clinical and psychoneuroendocrinological research that require noninvasive means of measuring these hormones.
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http://dx.doi.org/10.1007/s00216-018-1549-xDOI Listing
February 2019

Liquid chromatography-tandem mass spectrometry analysis of 17-hydroxyprogesterone in dried blood spots revealed matrix effect on immunoassay.

Anal Bioanal Chem 2019 Jan 20;411(2):395-402. Epub 2018 Nov 20.

New York State Department of Health, Wadsworth Center, Empire State Plaza, Albany, NY, USA.

Immunoassays for measuring 17-hydroxyprogesterone (17-OHP) produce high rates of false positives that impact the identification of congenital adrenal hyperplasia (CAH) in neonates. A confirmatory test with high analytical specificity employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology is needed in newborn screening for CAH. 17-OHP and cortisol were extracted from dried blood spot (DBS) samples, resolved on a C18 column, and measured using tandem mass spectrometry. The results were compared with those determined using the AutoDELFIA immunoassay. The LC-MS/MS method had a limit of quantitation of 10.0 and 5.0 ng/mL for 17-OHP and cortisol, respectively. The method characteristics showed coefficient variation (%CV) ≤ 11.9% for both 17-OHP and cortisol, recoveries ranging from 83.1 to 101.5% for 17-OHP and from 95.1 to 102.8% for cortisol, and linearity with R = 0.9994 for 17-OHP and R = 0.9996 for cortisol, clinical sensitivity of 100.0% and a specificity of 96.4% as obtained by receiver operating characteristic analysis on 45 patient samples when 17-OHP > 39.1 ng/mL was selected as the cutoff value. Comparison between the LC-MS/MS and the AutoDELFIA immunoassay methods revealed a poor correlation for patient DBS samples (R = 0.6784); however, an excellent correlation was obtained for QC and proficiency test (PT) DBS samples (R = 0.9797). The LC-MS/MS method produced reliable results for 17-OHP and cortisol for the diagnosis of CAH. The AutoDELFIA immunoassay appears to be subject to matrix effects in the analysis for 17-OHP in DBS patient samples. The DBS samples of non-patient origin may not be suitable for assessing analytical accuracy of immunoassays.
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http://dx.doi.org/10.1007/s00216-018-1449-0DOI Listing
January 2019

Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer.

Toxicol Appl Pharmacol 2015 Jan 4;282(1):30-41. Epub 2014 Nov 4.

Wadsworth Center, New York State Department of Health, Albany, NY 12201, United States; Department of Environmental Health Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY 12201, United States. Electronic address:

The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.
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http://dx.doi.org/10.1016/j.taap.2014.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404625PMC
January 2015

Measurement of unconjugated estriol in serum by liquid chromatography-tandem mass spectrometry and assessment of the accuracy of chemiluminescent immunoassays.

Clin Chem 2014 Jan 19;60(1):260-8. Epub 2013 Nov 19.

Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China;

Background: Unconjugated estriol (uE3) is routinely analyzed in clinical laboratories as risk assessment for Down syndrome. Immunoassays of various types are the most commonly used methods. The accuracies of RIAs and ELISAs for uE3 have been questioned, and to date there have been no independent studies investigating the accuracy of the relatively new chemiluminescent immunoassays. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for uE3 measurements in serum.

Methods: Serum samples from patients in the second trimester of pregnancy were used, and uE3 concentrations were measured by LC-MS/MS and the Beckman Coulter Access® 2 and Siemens IMMULITE 2000 automatic chemiluminescent immunoassay analyzers.

Results: The LC-MS/MS method was validated and showed limit of detection 0.05 ng/mL; limit of quantification 0.2 ng/mL; linearity of response to 32 ng/mL; total imprecision of 16.2%, 10.4%, and 8.2% for uE3 at 1.10, 4.18, and 8.32 ng/mL, respectively; and analytical recoveries of 95.9%-104.2%. ANOVA of the correlation for LC-MS/MS results vs chemiluminescent immunoassays results showed R(2) = 0.9678 (Access 2 = 0.9305 LC-MS/MS + 0.2177, Sy|x = 0.1786, P < 0.0001), and R(2) = 0.9663 (IMMULITE 2000 = 0.8849 LC-MS/MS - 0.0403, Sy|x = 0.1738, P < 0.0001). Bland-Altman plots of uE3 results revealed concentration-dependent immunoassay biases. Mock risk analysis for Down syndrome showed no apparent difference in the risk assessment outcomes if the adjusted method-specific multiples of the median were used, and the assay imprecision was <10% CV.

Conclusions: Standardization of immunoassay methods for uE3 analysis is needed to improve the accuracy of the measurements.
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http://dx.doi.org/10.1373/clinchem.2013.212126DOI Listing
January 2014

Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

Biochem Pharmacol 2012 Sep 21;84(5):722-35. Epub 2012 Jun 21.

Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.
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http://dx.doi.org/10.1016/j.bcp.2012.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965201PMC
September 2012

Potential biological functions of cytochrome P450 reductase-dependent enzymes in small intestine: novel link to expression of major histocompatibility complex class II genes.

J Biol Chem 2012 May 27;287(21):17777-17788. Epub 2012 Mar 27.

Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York, Albany, New York 12201-0509. Electronic address:

NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine.
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http://dx.doi.org/10.1074/jbc.M112.354274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366852PMC
May 2012

Expression of the aryl hydrocarbon receptor is not required for the proliferation, migration, invasion, or estrogen-dependent tumorigenesis of MCF-7 breast cancer cells.

Mol Carcinog 2013 Jul 2;52(7):544-54. Epub 2012 Mar 2.

Laboratory of Molecular Toxicology, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.

The AhR was initially identified as a ligand-activated transcription factor mediating effects of chlorinated dioxins and polycyclic aromatic hydrocarbons on cytochrome P450 1 (CYP1) expression. Recently, evidence supporting involvement of the AhR in cell-cycle regulation and tumorigenesis has been presented. To further define the roles of the AhR in cancer, we investigated the effects of AhR expression on cell proliferation, migration, invasion, and tumorigenesis of MCF-7 human breast cancer cells. In these studies, the properties of MCF-7 cells were compared with those of two MCF-7-derived sublines: AH(R100) , which express minimal AhR, and AhR(exp) , which overexpress AhR. Quantitative PCR, Western immunoblots, 17β-estradiol (E2 ) metabolism assays, and ethoxyresorufin O-deethylase assays showed the lack of AhR expression and AhR-regulated CYP1 expression in AH(R100) cells, and enhanced AhR and CYP1 expression in AhR(exp) cells. In the presence of 1 nM E2 , rates of cell proliferation of the three cell lines showed an inverse correlation with the levels of AhR mRNA. In comparison with MCF-7 and AhR(exp) cells, AH(R100) cells produced more colonies in soft agar and showed enhanced migration and invasion in chamber assays with E2 as the chemoattractant. Despite the lack of significant AhR expression, AH(R100) cells retained the ability to form tumors in severe combined immunodeficient mice when supplemented with E2 , producing mean tumor volumes comparable to those observed with MCF-7 cells. These studies indicate that, while CYP1 expression and inducibility are highly dependent on AhR expression, the proliferation, invasion, migration, anchorage-independent growth, and estrogen-stimulated tumor formation of MCF-7 cells do not require the AhR.
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http://dx.doi.org/10.1002/mc.21889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433635PMC
July 2013

Persistent and non-persistent changes in gene expression result from long-term estrogen exposure of MCF-7 breast cancer cells.

J Steroid Biochem Mol Biol 2011 Feb 23;123(3-5):140-50. Epub 2010 Dec 23.

Laboratory of Molecular Toxicology, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

Life-long estrogen exposure is recognized as a major risk factor for the development of breast cancer. While the initial events in the regulation of gene expression by estrogen have been described in detail, far less is known of the role of estrogen in the long-term regulation of gene expression. In this study, we investigated the effects of long-term exposure of MCF-7 breast cancer cells to 1nM 17β-estradiol on gene expression with the goal of distinguishing between gene expression that is continually reliant on estrogen receptor (ER) function as opposed to secondary and persistent effects that are downstream of ER. To assess the direct involvement of ER in the differential gene expression of long-term estrogen exposed (LTEE) cells in comparison with that of control cells, we exposed cultures to the selective estrogen receptor modulator raloxifene (RAL). cDNA microarray analysis showed that exposure to RAL inhibited expression of numerous characterized estrogen-regulated genes, including PGR, GREB1, and PDZK1. Genes that were increased in expression in LTEE cells yet were unaffected by RAL exposure included the aryl hydrocarbon receptor (AHR) and numerous other genes that were not previously reported to be regulated by estrogen. Epigenetic regulation was evident for the AHR gene; AhR transcript levels remained elevated for several cell passages after the removal of estrogen. Signal transducer and activator of transcription 1 (STAT1); STAT1-regulated genes including ISG15, IFI27, and IFIT1; and MHC class I genes were also up-regulated in LTEE cells and were unaffected by RAL exposure. STAT1 is commonly overexpressed in breast and other cancers, and is associated with increased resistance to radiation and chemotherapy. This is the first study to relate estrogen exposure to increased STAT1 expression in breast cancer cells, an effect that may represent an additional role of estrogen in the pathogenesis of breast cancer.
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http://dx.doi.org/10.1016/j.jsbmb.2010.12.010DOI Listing
February 2011

Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Anal Biochem 2010 Jul 31;402(2):121-8. Epub 2010 Mar 31.

Wadsworth Center, New York State Department of Health, State University of New York at Albany, 12201, USA.

A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards 17alpha-methyl-T and 17alpha-methyl-DHT, were extracted from tissues and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in product ion spectra of HTP derivatives of both T and DHT that showed analyte-specific fragmentations; the latter fragmentations were characterized by the use of high-resolution Orbitrap MS/MS. These specific fragmentations enabled quantitation of T and DHT in the multiple-reaction monitoring (MRM) mode. The method was validated with charcoal-stripped serum as the matrix. The lower limit of quantitation (LLOQ) was 0.10ng/ml for T and 0.50ng/ml for DHT. The method was then used for determination of serum and tissue levels of T and DHT in transgenic mice carrying a hypomorphic NADPH-cytochrome P450 reductase gene (Cpr-low mice). Remarkably, ovarian T levels in Cpr-low mice were found to be 25-fold higher than those in wild-type mice, a finding that at least partly explains the female infertility seen in the Cpr-low mice. In conclusion, our method provides excellent sensitivity and selectivity for determination of endogenous levels of T and DHT in mouse tissues.
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http://dx.doi.org/10.1016/j.ab.2010.03.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876209PMC
July 2010

Long-term estrogen exposure promotes carcinogen bioactivation, induces persistent changes in gene expression, and enhances the tumorigenicity of MCF-7 human breast cancer cells.

Toxicol Appl Pharmacol 2009 Nov 18;240(3):355-66. Epub 2009 Jul 18.

Laboratory of Molecular Toxicology, Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.

The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor alpha (ERalpha) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERalpha and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERalpha- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17beta-estradiol (E(2)). With these LTEE cells and with parallel control cells cultured without E(2) supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E(2)-dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E(2).
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http://dx.doi.org/10.1016/j.taap.2009.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180932PMC
November 2009

Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry.

Authors:
Li Xu David C Spink

Anal Biochem 2008 Apr 28;375(1):105-14. Epub 2007 Nov 28.

Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.

Sulfonyl chlorides substituted with functional groups having high proton affinity can serve as derivatization reagents to enhance the sensitivity for steroidal estrogens in liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The most commonly used reagent for derivatization of estrogens for LC-ESI-MS/MS is dansyl chloride. In this study, we compared dansyl chloride, 1,2-dimethylimidazole-4-sulfonyl (DMIS) chloride, pyridine-3-sulfonyl (PS) chloride, and 4-(1H-pyrazol-1-yl)benzenesulfonyl (PBS) chloride for derivatization of 17beta-estradiol (E2) prior to LC-ESI-MS/MS. The product ion spectra of the dansyl and DMIS derivatives were dominated by ions representing derivatization reagent moieties. In contrast, the product ion spectrum of the PS derivative of E2 and, to a lesser extent, the PBS derivative, showed analyte-specific fragment ions. Derivatization with PS chloride was therefore chosen for further investigation. The product ion spectrum of the PS derivative of E2 showed intense ions at m/z 272, assigned to the radical E2 cation, and at m/z 350, attributed to the loss of SO(2) from the [M+H](+) ion. Third-stage mass spectrometry of the PS derivative of E2 with isolation and collisional activation of the m/z 272 ion resulted in steroid C and D ring cleavages analogous to those observed in electron ionization mass spectrometry. The product ion spectra of the PS derivatives of estrone, 17alpha-ethinylestradiol, equilin, and equilenin showed similar estrogen-specific ions. Using derivatization with PS chloride, we developed an LC-ESI-MS/MS method with multiple reaction monitoring of primary and confirmatory precursor-to-product ion transitions for the determination of E2 in serum.
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http://dx.doi.org/10.1016/j.ab.2007.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675187PMC
April 2008

Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites.

Toxicol Appl Pharmacol 2008 Feb 5;226(3):213-24. Epub 2007 Sep 5.

Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.

The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 microM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17beta-estradiol (E(2)) metabolism, whereas BKF levels greater than 1 muM inhibited E(2) metabolism. Time course studies showed that induction of CYP1-catalyzed E(2) metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.
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http://dx.doi.org/10.1016/j.taap.2007.08.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423327PMC
February 2008

1,2-Dimethylimidazole-4-sulfonyl chloride, a novel derivatization reagent for the analysis of phenolic compounds by liquid chromatography electrospray tandem mass spectrometry: application to 1-hydroxypyrene in human urine.

Authors:
Li Xu David C Spink

J Chromatogr B Analyt Technol Biomed Life Sci 2007 Aug 10;855(2):159-65. Epub 2007 May 10.

Wadsworth Center, New York State Department of Health, Albany, NY 12201-0599, USA.

A novel derivatization method employing 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) to improve the mass spectrometric response for phenolic compounds in liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and tandem mass spectrometry (LC-ESI-MS/MS) is described. Several environmentally relevant compounds, including chloro-, aryl- and alkylphenols, steroidal estrogens, and hydroxy-polycyclic aromatic hydrocarbons (OHPAHs), were selected to evaluate this technique. A facile derivatization procedure employing DMISC results in dimethylimidazolesulfonyl (DMIS) derivatives that are stable in aqueous solution. These DMIS derivatives produced intense [M+H](+) ions in positive-ion LC-ESI-MS. The product ion spectra of the [M+H](+) ions of simple phenols were dominated by ions representing the DMIS and dimethylimidazole moieties, whereas product ion spectra of the DMIS derivatives of OHPAHs with three or more fused aromatic rings showed prominent ArO(+) ions, the relative intensity of which increased with the number of rings. The DMIS derivatives of the selected phenolic compounds showed excellent chromatographic properties. To substantiate the utility of derivatization with DMISC, an analytical method employing enzyme hydrolysis, solid phase extraction, derivatization with DMISC, and analysis by LC-ESI-MS/MS with multiple reaction monitoring for determination in human urine of 1-hydroxypyrene, a widely used biomarker for the assessment of human exposure to PAHs, was developed and validated.
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http://dx.doi.org/10.1016/j.jchromb.2007.04.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2041808PMC
August 2007

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture.

Cell Biol Int 2006 Mar 19;30(3):227-38. Epub 2006 Jan 19.

Wadsworth Center, New York State Department of Health, PO Box 509, Albany, NY 12201-0509, USA.

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.
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http://dx.doi.org/10.1016/j.cellbi.2005.11.006DOI Listing
March 2006

TCDD and PCBs inhibit breast cancer cell proliferation in vitro.

Toxicol In Vitro 2004 Dec;18(6):811-9

Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.

The effects on cell proliferation of arylhydrocarbon receptor (AhR) agonists in estrogen-responsive T47D and ZR-75-1 cells were investigated. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the non-ortho-substituted polychlorinated biphenyl (PCB) congeners, PCB 77, PCB 81, PCB 126, and PCB 169 each inhibited 17beta-estradiol (E(2))-stimulated cell proliferation in a dose-responsive manner. In the absence of added E(2), TCDD, PCB 77, PCB 81, and PCB 169 had no significant effect on cell proliferation, while PCB 126 at high concentrations caused slight elevations. The order of effective inhibition of E(2)-stimulated cell proliferation by the PCB congeners was: PCB 81>PCB 126 approximately = PCB 169>PCB 77. In the comparative literature, mammalian TEFs for these congeners toxic potency are in the order: PCB 126>PCB 169>PCB 81 approximately = PCB 77 [Organohalogen Compd. 34 (1997) 237]. Our results thus show an unexpected different pattern for the inhibitory effects of PCBs congeners on E(2)-mediated cell proliferation.
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http://dx.doi.org/10.1016/j.tiv.2004.04.004DOI Listing
December 2004

Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin.

Biochem Pharmacol 2003 Dec;66(12):2313-21

New York State Department of Health, Wadsworth Center, Albany, NY 12201-0509, USA.

The aryl hydrocarbon receptor (AhR), when activated by exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulates expression of several phase I and phase II enzymes and is also involved in the regulation of cell proliferation. Several studies suggest that endogenous AhR ligand(s) may exist. One putative endogenous ligand is indirubin, which was recently identified in human urine and bovine serum. We determined the effect of indirubin in MCF-7 breast cancer cells on induction of the activities of cytochromes P450 (CYP) 1A1 and 1B1, as measured by estradiol and ethoxyresorufin metabolism, and on induction of the CYP1A1 and CYP1B1 mRNAs. With 4-hr exposure, the effects of indirubin and TCDD at 10nM on CYP activity were comparable, but the effects of indirubin, unlike those of TCDD, were transitory. Indirubin-induced ethoxyresorufin-O-deethylase activity was maximal by 6-9 hr post-exposure and had disappeared by 24 hr, whereas TCDD-induced activities remained elevated for at least 72 hr. The effects of indirubin on CYP mRNA induction were maximal at 3 hr. Indirubin was metabolized by microsomes containing cDNA-expressed human CYP1A1 or CYP1B1. The potency of indirubin was comparable to that of TCDD in a CYP1B1-promoter-driven luciferase assay, when MCF-7 cells were co-exposed to the AhR ligands together with the CYP inhibitor, ellipticine. Thus, if indirubin is an endogenous AhR ligand, then AhR-mediated signaling by indirubin is likely to be transient and tightly controlled by the ability of indirubin to induce CYP1A1 and CYP1B1, and hence its own metabolism.
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http://dx.doi.org/10.1016/j.bcp.2003.08.019DOI Listing
December 2003

Estrogen regulates Ah responsiveness in MCF-7 breast cancer cells.

Carcinogenesis 2003 Dec 11;24(12):1941-50. Epub 2003 Sep 11.

Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.

Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor alpha (ERalpha)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E2 exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E2 elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced E2 metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9-12 months) in low-E2 medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E2 metabolism and ethoxyresorufin O-deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E2-deprived cells showed elevated levels of ERalpha mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1-promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E2-deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E2-deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E2; ERalpha and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E2 metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.
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http://dx.doi.org/10.1093/carcin/bgg162DOI Listing
December 2003

Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection.

Anal Biochem 2003 Jan;312(2):162-6

New York State Department of Health, Wadsworth Center, PO Box 509, Albany 12201-0509, USA.

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.
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http://dx.doi.org/10.1016/s0003-2697(02)00444-xDOI Listing
January 2003

Stimulatory effect of cigarette smoking on the 15 alpha-hydroxylation of estradiol by human term placenta.

Clin Pharmacol Ther 2002 May;71(5):311-24

Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC, USA.

Objective: Our objective was to characterize the oxidative metabolism of estradiol by human term placenta and its modulation by cigarette smoking.

Methods: Placental microsomes were prepared from term placentas obtained from 13 cigarette smokers (20 to 30 cigarettes per day until the time of delivery) and 13 control subjects who were nonsmokers. Estrogen metabolism was studied by incubation of 250 nmol/L [(3)H]estradiol with placental microsomes and NADPH, and the estrogen metabolites were determined by HPLC and gas chromatography-mass spectrometry.

Results: 2-Hydroxyestradiol was the major hydroxyestrogen detected, followed by 6alpha-hydroxyestradiol. Small amounts of several other hydroxyestrogen metabolites (4-hydroxyestradiol, 6beta-hydroxyestradiol, 7alpha-hydroxyestradiol, and 16alpha-hydroxyestradiol) were also detected. Large amounts of estrone plus small amounts of 2-hydroxyestrone and unidentified nonpolar metabolites were formed. Cigarette smoking stimulated the placental hydroxylation of benzo[a ]pyrene by about 16-fold. Cigarette smoking had little or no effect on the overall rate of placental estradiol metabolism or on the formation of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, or 16alpha-hydroxyestradiol. However, placental formation of 4-hydroxyestradiol and 7alpha-hydroxyestradiol was increased 38% (P =.08) and 150% (P =.05), respectively, in cigarette smokers. The formation of 6alpha-hydroxyestradiol was decreased 33% (P =.04). Metabolic formation of 15alpha-hydroxyestradiol was observed during incubations of estradiol with placental microsomes from 11 of the 13 cigarette smokers, but this metabolite was not detected during incubations with placental microsomes from any of the 13 nonsmokers. Analysis of data from all 26 placentas showed that the 15alpha-hydroxylation of estradiol was highly correlated with benzo[a ]pyrene hydroxylation (r = 0.93; P <.001).

Conclusions: Many hydroxylated estradiol metabolites were formed by placental microsomes from cigarette smokers and nonsmokers. 15alpha-Hydroxylation of estradiol was markedly stimulated in the placentas of cigarette smokers.
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http://dx.doi.org/10.1067/mcp.2002.122500DOI Listing
May 2002

Induction of CYP1A1 and CYP1B1 in T-47D human breast cancer cells by benzo[a]pyrene is diminished by arsenite.

Drug Metab Dispos 2002 Mar;30(3):262-9

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.

Polycyclic aromatic hydrocarbons (PAHs) and metals are often environmental cocontaminants, yet there have been relatively few studies of combined effects of PAHs and metals on cytochrome P450 (P450)-catalyzed metabolism. We examined the effects of NaAsO(2) in combination with benzo[a]pyrene (BAP) on CYP1A1 and CYP1B1 in T-47D human breast cancer cells by using estrogen metabolism as a probe of their activities. Exposure to BAP caused elevated rates of the 2- and 4-hydroxylation pathways of estrogen metabolism, indicating induction of both CYP1A1, an estradiol 2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-induced metabolism peaked 9 to 16 h after exposure and returned to near-basal levels by 48 h. Concentration-response studies showed maximal induction of the 2- and 4-hydroxylation pathways at 3 microM BAP; higher levels caused reduced rates of metabolism due to inhibition of CYP1A1 and CYP1B1. NaAsO(2) caused pronounced decreases in the induction of CYP1A1 and CYP1B1 by 3 microM BAP because cotreatment with 10 microM NaAsO(2) inhibited the rates of the 2- and 4-hydroxylation pathways by 86 and 92%, respectively. Western immunoblots showed diminished levels of BAP-induced CYP1A1 by coexposure to NaAsO(2). The levels of the CYP1A1 and CYP1B1 mRNAs induced by BAP were not significantly affected by coexposure to NaAsO(2); however, heme oxygenase 1 mRNA levels were markedly induced by coexposure to BAP and NaAsO(2). These results indicate a post-transcriptional inhibitory effect of arsenite on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly resulting from reduced heme availability.
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http://dx.doi.org/10.1124/dmd.30.3.262DOI Listing
March 2002

Understanding the human health effects of chemical mixtures.

Environ Health Perspect 2002 Feb;110 Suppl 1:25-42

School of Public Health, Department of Environmental Health and Toxiciology, University at Albany, State University of New York, Rensselaer 12144, USA.

Most research on the effects of chemicals on biologic systems is conducted on one chemical at a time. However, in the real world people are exposed to mixtures, not single chemicals. Although various substances may have totally independent actions, in many cases two substances may act at the same site in ways that can be either additive or nonadditive. Many even more complex interactions may occur if two chemicals act at different but related targets. In the extreme case there may be synergistic effects, in which case the effects of two substances together are greater than the sum of either effect alone. In reality, most persons are exposed to many chemicals, not just one or two, and therefore the effects of a chemical mixture are extremely complex and may differ for each mixture depending on the chemical composition. This complexity is a major reason why mixtures have not been well studied. In this review we attempt to illustrate some of the principles and approaches that can be used to study effects of mixtures. By the nature of the state of the science, this discussion is more a presentation of what we do not know than of what we do know about mixtures. We approach the study of mixtures at three levels, using specific examples. First, we discuss several human diseases in relation to a variety of environmental agents believed to influence the development and progression of the disease. We present results of selected cellular and animal studies in which simple mixtures have been investigated. Finally, we discuss some of the effects of mixtures at a molecular level.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1241145PMC
http://dx.doi.org/10.1289/ehp.02110s125DOI Listing
February 2002