Publications by authors named "Jay P Tiesman"

26 Publications

  • Page 1 of 1

Histological and Gene Expression Analysis of the Effects of Menopause Status and Hormone Therapy on the Vaginal Introitus and Labia Majora.

J Clin Med Res 2019 Nov 29;11(11):745-759. Epub 2019 Oct 29.

The Procter & Gamble Company, Cincinnati, OH, USA.

Background: The study aimed to determine the effect of menopausal status and hormone therapy on the introitus and labia majora at the levels of histology and gene expression.

Methods: Three cohorts of 10 women each (pre-menopause, post-menopause and post-menopause + hormone therapy) were selected based on the presentation of clinical atrophy and vaginal pH. Biopsies were obtained from the introitus (fourchette) and labia majora and processed for histology and gene expression analyses with microarrays. Other data collected included self-assessed symptoms, serum estradiol, testosterone, serum hormone binding globulin and the pH of the vagina and labia majora.

Results: The introitus appears exquisitely sensitive to hormone status. Dramatic changes were observed in histology including a thinning of the epithelium in post-menopausal subjects with vaginal atrophy. Furthermore, there was differential expression of many genes that may contribute to tissue remodeling in the atrophic introitus. Levels of expression of genes associated with wound healing, angiogenesis, cell migration/locomotion, dermal structure, apoptosis, inflammation, epithelial cell differentiation, fatty acid, carbohydrate and steroid metabolism were significantly different in the cohort exhibiting atrophy of the introitus. While changes were also observed at the labia, that site was considerably less sensitive to hormone status. The gene expression changes observed at the introitus in this study were very similar to those reported previously in the atrophic vagina providing further evidence that these changes are associated with atrophy.

Conclusions: The histological and gene expression changes occurring within the introitus after menopause may contribute to the constellation of symptoms that constitute the genitourinary syndrome of menopause.
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http://dx.doi.org/10.14740/jocmr4006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879024PMC
November 2019

Spike-in genomic DNA for validating performance of metagenomics workflows.

Biotechniques 2018 12 17;65(6):315-321. Epub 2018 Sep 17.

The Procter & Gamble Company, 8700 Mason Montgomery Road, Mason, OH 45040, USA.

Shotgun metagenomics is a powerful platform to characterize human microbiomes. However, to translate such survey data into consumer-relevant products or services, it is critical to have a robust metagenomics workflow. We present a tool - spike-in DNA - to assess performance of metagenomics workflows. The spike-in is DNA from two organisms - Alivibrio fischeri and Rhodopseudomonas palustris, in a ratio of 4:1 added to samples before DNA extraction. With a valid workflow, the output ratio of relative abundances of these organisms should be close to 4. This expectation was tested in samples of varying diversities (n = 110), and the mean ratio was 4.73 (99% CI [4.0, 5.24]). We anticipate this tool to be a relevant community resource for assessing the quality of shotgun metagenomics workflows and thereby enable robust characterization of microbiomes.
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http://dx.doi.org/10.2144/btn-2018-0089DOI Listing
December 2018

Age-induced and photoinduced changes in gene expression profiles in facial skin of Caucasian females across 6 decades of age.

J Am Acad Dermatol 2018 Jan 14;78(1):29-39.e7. Epub 2017 Nov 14.

The Procter & Gamble Company, Cincinnati, Ohio.

Background: Intrinsic and extrinsic factors, including ultraviolet irradiation, lead to visible signs of skin aging.

Objective: We evaluated molecular changes occurring in photoexposed and photoprotected skin of white women 20 to 74 years of age, some of whom appeared substantially younger than their chronologic age.

Methods: Histologic and transcriptomics profiling were conducted on skin biopsy samples of photoexposed (face and dorsal forearm) or photoprotected (buttocks) body sites from 158 women. 23andMe genotyping determined genetic ancestry.

Results: Gene expression and ontologic analysis revealed progressive changes from the 20s to the 70s in pathways related to oxidative stress, energy metabolism, senescence, and epidermal barrier; these changes were accelerated in the 60s and 70s. The gene expression patterns from the subset of women who were younger-appearing were similar to those in women who were actually younger.

Limitations: Broader application of these findings (eg, across races and Fitzpatrick skin types) will require further studies.

Conclusions: This study demonstrates a wide range of molecular processes in skin affected by aging, providing relevant targets for improving the condition of aging skin at different life stages and defining a molecular pattern of epidermal gene expression in women who appear younger than their chronologic age.
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http://dx.doi.org/10.1016/j.jaad.2017.09.012DOI Listing
January 2018

Grouping 34 Chemicals Based on Mode of Action Using Connectivity Mapping.

Toxicol Sci 2016 06 29;151(2):447-61. Epub 2016 Mar 29.

*Mason Business Center, The Procter & Gamble Company, Cincinnati, Ohio 45040 and.

Connectivity mapping is a method used in the pharmaceutical industry to find connections between small molecules, disease states, and genes. The concept can be applied to a predictive toxicology paradigm to find connections between chemicals, adverse events, and genes. In order to assess the applicability of the technique for predictive toxicology purposes, we performed gene array experiments on 34 different chemicals: bisphenol A, genistein, ethinyl-estradiol, tamoxifen, clofibrate, dehydorepiandrosterone, troglitazone, diethylhexyl phthalate, flutamide, trenbolone, phenobarbital, retinoic acid, thyroxine, 1α,25-dihydroxyvitamin D3, clobetasol, farnesol, chenodeoxycholic acid, progesterone, RU486, ketoconazole, valproic acid, desferrioxamine, amoxicillin, 6-aminonicotinamide, metformin, phenformin, methotrexate, vinblastine, ANIT (1-naphthyl isothiocyanate), griseofulvin, nicotine, imidacloprid, vorinostat, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) at the 6-, 24-, and 48-hour time points for 3 different concentrations in the 4 cell lines: MCF7, Ishikawa, HepaRG, and HepG2 GEO (super series accession no.: GSE69851). The 34 chemicals were grouped in to predefined mode of action (MOA)-based chemical classes based on current literature. Connectivity mapping was used to find linkages between each chemical and between chemical classes. Cell line-specific linkages were compared with each other and to test whether the method was platform and user independent, a similar analysis was performed against publicly available data. The study showed that the method can group chemicals based on MOAs and the inter-chemical class comparison alluded to connections between MOAs that were not predefined. Comparison to the publicly available data showed that the method is user and platform independent. The results provide an example of an alternate data analysis process for high-content data, beneficial for predictive toxicology, especially when grouping chemicals for read across purposes.
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http://dx.doi.org/10.1093/toxsci/kfw058DOI Listing
June 2016

Dose- and Time-Dependent Transcriptional Response of Ishikawa Cells Exposed to Genistein.

Toxicol Sci 2016 05 10;151(1):71-87. Epub 2016 Feb 10.

*Mason Business Center, The Procter and Gamble Company, Mason, Ohio 45040.

To further define the utility of the Ishikawa cells as a reliable in vitro model to determine the potential estrogenic activity of chemicals of interest, transcriptional changes induced by genistein (GES) in Ishikawa cells at various doses (10 pM, 1 nM, 100 nM, and 10 μM) and time points (8, 24, and 48 h) were identified using a comprehensive microarray approach. Trend analysis indicated that the expression of 5342 unique genes was modified by GES in a dose- and time-dependent manner (P ≤ 0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest dose of GES evaluated (10 μM). The GES' estrogenic activity was identified by comparing the Ishikawa cells' response to GES versus 17 α-ethynyl estradiol (EE, at equipotent doses, ie, 10 μM vs 1 μM, respectively) and was defined by changes in the expression of 284 unique genes elicited by GES and EE in the same direction, although the magnitude of the change for some genes was different. Further, comparing the response of the Ishikawa cells exposed to high doses of GES and EE versus the response of the juvenile rat uterus exposed to EE, we identified 66 unique genes which were up- or down regulated in a similar manner in vivo as well as in vitro Genistein elicits changes in multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response and offer an in vitro model to assess this mode of action.
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http://dx.doi.org/10.1093/toxsci/kfw024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914796PMC
May 2016

A systems approach to understanding human rhinovirus and influenza virus infection.

Virology 2015 Dec 6;486:146-57. Epub 2015 Oct 6.

The Institute for Systems Biology, Seattle, WA 98109, USA. Electronic address:

Human rhinovirus and influenza virus infections of the upper airway lead to colds and the flu and can trigger exacerbations of lower airway diseases including asthma and chronic obstructive pulmonary disease. Novel diagnostic and therapeutic targets are still needed to differentiate between the cold and the flu, since the clinical course of influenza can be severe while that of rhinovirus is usually more mild. In our investigation of influenza and rhinovirus infection of human respiratory epithelial cells, we used a systems approach to identify the temporally changing patterns of host gene expression from these viruses. After infection of human bronchial epithelial cells (BEAS-2B) with rhinovirus, influenza virus or co-infection with both viruses, we studied the time-course of host gene expression changes over three days. We modeled host responses to these viral infections with time and documented the qualitative and quantitative differences in innate immune activation and regulation.
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http://dx.doi.org/10.1016/j.virol.2015.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111289PMC
December 2015

A novel transcriptomics based in vitro method to compare and predict hepatotoxicity based on mode of action.

Toxicology 2015 Feb 2;328:29-39. Epub 2014 Dec 2.

Mason Business Center, The Procter & Gamble Company, Cincinnati, OH 45040, USA.

High-content data have the potential to inform mechanism of action for toxicants. However, most data to support this notion have been generated in vivo. Because many cell lines and primary cells maintain a differentiated cell phenotype, it is possible that cells grown in culture may also be useful in predictive toxicology via high-content approaches such as whole-genome microarray. We evaluated global changes in gene expression in primary rat hepatocytes exposed to two concentrations of ten hepatotoxicants: acetaminophen (APAP), β-naphthoflavone (BNF), chlorpromazine (CPZ), clofibrate (CLO), bis(2-ethylhexyl)phthalate (DEHP), diisononyl phthalate (DINP), methapyrilene (MP), valproic acid (VPA), phenobarbital (PB) and WY14643 at two separate time points. These compounds were selected to cover a range of mechanisms of toxicity, with some overlap in expected mechanism to address the question of how predictive gene expression analysis is, for a given mode of action. Gene expression microarray analysis was performed on cells after 24h and 48h of exposure to each chemical using Affymetrix microarrays. Cluster analysis suggests that the primary hepatocyte model was capable of responding to these hepatotoxicants, with changes in gene expression that appear to be mode of action-specific. Among the different methods used for analysis of the data, a combination method that used pathways (MOAs) to filter total probesets provided the most robust analysis. The analysis resulted in the phthalates clustering closely together, with the two other peroxisome proliferators, CLO and WY14643, eliciting similar responses at the whole-genome and pathway levels. The Cyp inducers PB, MP, CPZ and BNF also clustered together. VPA and APAP had profiles that were unique. A similar analysis was performed on externally available (TG-GATES) in vivo data for 6 of the chemicals (APAP, CLO, CPZ, MP, MP and WY14643) and compared to the in vitro result. These results indicate that transcription profiling using an in vitro assay may offer pertinent biological data to support predictions of in vivo hepatotoxicity potential.
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http://dx.doi.org/10.1016/j.tox.2014.11.008DOI Listing
February 2015

Transcriptional profiling of epidermal barrier formation in vitro.

J Dermatol Sci 2014 Mar 11;73(3):187-97. Epub 2013 Nov 11.

The Procter & Gamble Company, Mason Business Center, Cincinnati, OH 45040, USA. Electronic address:

Background: Barrier function is integral to the health of epithelial tissues. Currently, there is a broad need to develop and improve our knowledge with regard to barrier function for reversal of mild skin irritation and dryness. However, there are few in vitro models that incorporate modulations of both lipids and epidermal differentiation programs for pre-clinical testing to aid in the understanding of barrier health.

Objective: We have generated a reconstituted epidermis on a decellularized dermis (DED) and characterized its barrier properties relative to human epidermis in order to determine its utility for modeling barrier formation and repair.

Methods: We followed the process of epidermal differentiation and barrier formation through immunocytochemistry and transcriptional profiling. We examined barrier functionality through measurements of surface pH, lipid composition, stratum corneum water content, and the ability to demonstrate topical dose-dependent exclusion of surfactant.

Results: Transcriptional profiling of the epidermal model during its formation reveals temporal patterns of gene expression associated with processes regulating barrier function. The profiling is supported by gradual formation and maturation of a stratum corneum and expression of appropriate markers of epidermis development. The model displays a functional barrier and a water gradient between the stratum corneum and viable layers, as determined by confocal Raman spectroscopy. The stratum corneum layer displays a normal acidic pH and an appropriate composition of barrier lipids.

Conclusion: The epidermal model demonstrates its utility as an investigative tool for barrier health and provides a window into the transcriptional regulation of multiple aspects of barrier formation.
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http://dx.doi.org/10.1016/j.jdermsci.2013.11.004DOI Listing
March 2014

Zinc pyrithione inhibits yeast growth through copper influx and inactivation of iron-sulfur proteins.

Antimicrob Agents Chemother 2011 Dec 26;55(12):5753-60. Epub 2011 Sep 26.

Procter & Gamble Co, Miami Valley Innovation Center, Cincinnati, Ohio 45253-8707, USA.

Zinc pyrithione (ZPT) is an antimicrobial material with widespread use in antidandruff shampoos and antifouling paints. Despite decades of commercial use, there is little understanding of its antimicrobial mechanism of action. We used a combination of genome-wide approaches (yeast deletion mutants and microarrays) and traditional methods (gene constructs and atomic emission) to characterize the activity of ZPT against a model yeast, Saccharomyces cerevisiae. ZPT acts through an increase in cellular copper levels that leads to loss of activity of iron-sulfur cluster-containing proteins. ZPT was also found to mediate growth inhibition through an increase in copper in the scalp fungus Malassezia globosa. A model is presented in which pyrithione acts as a copper ionophore, enabling copper to enter cells and distribute across intracellular membranes. This is the first report of a metal-ligand complex that inhibits fungal growth by increasing the cellular level of a different metal.
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http://dx.doi.org/10.1128/AAC.00724-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3232817PMC
December 2011

The genomic response of Ishikawa cells to bisphenol A exposure is dose- and time-dependent.

Toxicology 2010 Apr 17;270(2-3):137-49. Epub 2010 Feb 17.

Miami Valley Innovation Center, The Procter and Gamble Company, PO Box 538707 #805, Cincinnati, OH 45253-8707, United States.

A reliable in vitro model to determine the potential estrogenic activity of chemicals of interest is still unavailable. To further investigate the usefulness of a human-derived cell line, we determined the transcriptional changes induced by bisphenol A (BPA) in Ishikawa cells at various doses (1 nM, 100 nM, 10 microM, and 100 microM) and time points (8, 24 and 48 h) by comparing the response of approximately 38,500 human genes and ESTs between treatment groups and controls (vehicle-treated). By trend analysis, we determined that the expression of 2794 genes was modified by BPA in a dose- and time-dependent manner (p< or =0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest doses of BPA evaluated (10-100 microM), while the genomic response of the cells exposed to low doses of BPA was essentially negligible. By comparing the Ishikawa cells' response to BPA vs.17 alpha-ethynyl estradiol we determined that the change in the expression of 307 genes was identical in the direction of the change, although the magnitude of the change for some genes was different. Further, the response of Ishikawa cells to high doses of BPA shared similarities to the estrogenic response of the rat uterus, specifically, 362 genes were regulated in a similar manner in vivo as well as in vitro. Gene ontology analysis indicated that BPA results in changes to multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response after exposure to chemicals with varied estrogenic activity, and offer an in vitro model to assess this mode of action.
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http://dx.doi.org/10.1016/j.tox.2010.02.008DOI Listing
April 2010

Gingival transcriptome patterns during induction and resolution of experimental gingivitis in humans.

J Periodontol 2009 Dec;80(12):1963-82

Department of Periodontology, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27709, USA.

Background: To our knowledge, changes in the patterns of whole-transcriptome gene expression that occur during the induction and resolution of experimental gingivitis in humans were not previously explored using bioinformatic tools.

Methods: Gingival biopsy samples collected from 14 subjects during a 28-day stent-induced experimental gingivitis model, followed by treatment, and resolution at days 28 through 35 were analyzed using gene-expression arrays. Biopsy samples were collected at different sites within each subject at baseline (day 0), at the peak of gingivitis (day 28), and at resolution (day 35) and processed using whole-transcriptome gene-expression arrays. Gene-expression data were analyzed to identify biologic themes and pathways associated with changes in gene-expression profiles that occur during the induction and resolution of experimental gingivitis using bioinformatic tools.

Results: During disease induction and resolution, the dominant expression pathway was the immune response, with 131 immune response genes significantly up- or downregulated during induction, during resolution, or during both at P <0.05. During induction, there was significant transient increase in the expression of inflammatory and oxidative stress mediators, including interleukin (IL)-1 alpha (IL1A), IL-1 beta (IL1B), IL8, RANTES, colony stimulating factor 3 (CSF3), and superoxide dismutase 2 (SOD2), and a decreased expression of IP10, interferon inducible T-cell alpha chemoattractant (ITAC), matrix metalloproteinase 10 (MMP10), and beta 4 defensin (DEFB4). These genes reversed expression patterns upon resolution in parallel with the reversal of gingival inflammation.

Conclusions: A relatively small subset (11.9%) of the immune response genes analyzed by array was transiently activated in response to biofilm overgrowth, suggesting a degree of specificity in the transcriptome-expression response. The fact that this same subset demonstrates a reversal in expression patterns during clinical resolution implicates these genes as being critical for maintaining tissue homeostasis at the biofilm-gingival interface. In addition to the immune response pathway as the dominant response theme, new candidate genes and pathways were identified as being selectively modulated in experimental gingivitis, including neural processes, epithelial defenses, angiogenesis, and wound healing.
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http://dx.doi.org/10.1902/jop.2009.080645DOI Listing
December 2009

From bench to beauty counter: using genomics to drive technology development for skin care.

Authors:
Jay P Tiesman

J Drugs Dermatol 2009 Jul;8(7 Suppl):s12-4

The Procter & Gamble Company, Miami Valley Innovation Center, Cincinnati, OH 45253-8707, USA.

The skin is not only the body's largest organ but is widely considered to be the sentinel organ of the human body as it is often the first line of defense from environmental insults. Acting as both an environmental sensor and guardian, the skin responds to external cues and helps us adapt to our surroundings in many ways, such as tanning to adapt to repeated ultraviolet (UV) exposure. In order to better understand the molecular events that occur as the skin adapts to its environment, we need a deeper understanding of how the genes in the cells comprising the skin are regulated and how this regulation leads to changes in biological response. Regulation of messenger RNA (mRNA) ultimately results in changes in protein production, which is responsible for carrying out the physical and chemical reactions responsible for the skin's adaptation response. Each step in this cellular response is the subject of intense research. In fact, scientific sub-disciplines have developed around understanding each of these steps: genomics, proteomics and metabonomics. There are a myriad of applications for genomics in skin. Since capabilities, such as gene chips, provide fundamental molecular insights into skin cell biology that can be exploited for the development of new products, it is anticipated that genomics will continue to play an ever-increasing role in skin care.
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July 2009

Genomics of skin aging: practical applications.

J Drugs Dermatol 2009 Jul;8(7 Suppl):s3

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July 2009

The genomic response of a human uterine endometrial adenocarcinoma cell line to 17alpha-ethynyl estradiol.

Toxicol Sci 2009 Jan 20;107(1):40-55. Epub 2008 Oct 20.

Miami Valley Innovation Center, The Procter and Gamble Company, Cincinnati, Ohio 45253, USA.

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p
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http://dx.doi.org/10.1093/toxsci/kfn219DOI Listing
January 2009

Gene expression profiles during in vivo human rhinovirus infection: insights into the host response.

Am J Respir Crit Care Med 2008 Nov 24;178(9):962-8. Epub 2008 Jul 24.

Department of Physiology and Biophysics, Health Sciences 1626, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1 Canada.

Rationale: Human rhinovirus infections cause colds and trigger exacerbations of lower airway diseases.

Objectives: To define changes in gene expression profiles during in vivo rhinovirus infections.

Methods: Nasal epithelial scrapings were obtained before and during experimental rhinovirus infection, and gene expression was evaluated by microarray. Naturally acquired rhinovirus infections, cultured human epithelial cells, and short interfering RNA knockdown were used to further evaluate the role of viperin in rhinovirus infections.

Measurements And Main Results: Symptom scores and viral titers were measured in subjects inoculated with rhinovirus or sham control, and changes in gene expression were assessed 8 and 48 hours after inoculation. Real-time reverse transcription-polymerase chain reaction for viperin and rhinoviruses was used in naturally acquired infections, and viperin mRNA levels and viral titers were measured in cultured cells. Rhinovirus-induced changes in gene expression were not observed 8 hours after viral infection, but 11,887 gene transcripts were significantly altered in scrapings obtained 2 days postinoculation. Major groups of up-regulated genes included chemokines, signaling molecules, interferon-responsive genes, and antivirals. Viperin expression was further examined and also was increased in naturally acquired rhinovirus infections, as well as in cultured human epithelial cells infected with intact, but not replication-deficient, rhinovirus. Knockdown of viperin with short interfering RNA increased rhinovirus replication in infected epithelial cells.

Conclusions: Rhinovirus infection significantly alters the expression of many genes associated with the immune response, including chemokines and antivirals. The data obtained provide insights into the host response to rhinovirus infection and identify potential novel targets for further evaluation.
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http://dx.doi.org/10.1164/rccm.200805-670OCDOI Listing
November 2008

Genomic expression changes induced by topical N-acetyl glucosamine in skin equivalent cultures in vitro.

J Cosmet Dermatol 2007 Dec;6(4):232-8

Procter & Gamble Company, Cincinnati, OH, USA.

N-acetyl glucosamine (NAG) has been shown to be effective in reducing the appearance of hyperpigmented spots. From published in vitro mechanistic testing, glucosamine inhibits enzymatic glycosylation, a required processing step in converting inactive human pro-tyrosinase to the active tyrosinase, a key enzyme in the production of melanin. There is also published literature discussing the anti-inflammatory and antioxidant properties of glucosamine compounds. To identify additional mechanisms by which NAG might affect melanin production, an in vitro genomics experiment was conducted in SkinEthic skin equivalent cultures, which were topically dosed with NAG vs. a vehicle control. Relative to vehicle, NAG reduced melanin production, and the expression of several pigmentation-relevant genes were affected (down-regulated or up-regulated) by NAG treatment. Thus, there are several mechanisms that may be operative in the observed pigmentation effects.
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http://dx.doi.org/10.1111/j.1473-2165.2007.00339.xDOI Listing
December 2007

Uterine temporal response to acute exposure to 17alpha-ethinyl estradiol in the immature rat.

Toxicol Sci 2007 Jun 9;97(2):467-90. Epub 2007 Mar 9.

The Procter and Gamble Company, Miami Valley Innovation Center, Cincinnati, OH 45253, USA.

The rat uterus responds to acute estrogen treatment with a series of well-characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized. In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 alpha-ethinyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes. Immature 20-day-old rats were exposed to a single dose of EE (10 microg/kg), and the effects on uterine histology, weight, and gene expression were determined after 1, 2, 8, 24, 48, 72, and 96 h. EE induced changes in the expression of 3867 genes, at least at one time point (p < or = 0.0001), and at least 1.5-fold (up- or downregulated). Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72, or 96 h after exposure to EE, respectively (p < or = 0.0001, t-test). At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways. Using the Gene Ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.
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http://dx.doi.org/10.1093/toxsci/kfm046DOI Listing
June 2007

The ABRF MARG microarray survey 2005: taking the pulse of the microarray field.

J Biomol Tech 2006 Apr;17(2):176-86

University of Iowa, DNA Facility, 323 EMRB, Iowa City, IA 52242, USA.

Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291780PMC
April 2006

Analysis of Affymetrix GeneChip data using amplified RNA.

Biotechniques 2006 Feb;40(2):165-6, 168, 170

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD 21205, USA.

When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5' end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic. Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting.
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http://dx.doi.org/10.2144/000112057DOI Listing
February 2006

Design of a microsphere-based high-throughput gene expression assay to determine estrogenic potential.

Environ Health Perspect 2005 Sep;113(9):1164-71

Miami Valley Innovation Center, Procter & Gamble Company, Cincinnati, OH 45253, USA.

Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 microg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1280396PMC
http://dx.doi.org/10.1289/ehp.7843DOI Listing
September 2005

Gene expression changes induced in the testis by transplacental exposure to high and low doses of 17{alpha}-ethynyl estradiol, genistein, or bisphenol A.

Toxicol Sci 2005 Aug 18;86(2):396-416. Epub 2005 May 18.

Miami Valley Innovation Center, The Procter and Gamble Company, Cincinnati, Ohio 45253, USA.

The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 alpha-ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001-10 microg EE/kg/day, 0.001-100 mg Ges/kg/day, or 0.002-400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.
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http://dx.doi.org/10.1093/toxsci/kfi198DOI Listing
August 2005

Evaluation of the gene expression changes induced by 17-alpha-ethynyl estradiol in the immature uterus/ovaries of the rat using high density oligonucleotide arrays.

Birth Defects Res B Dev Reprod Toxicol 2005 Apr;74(2):164-84

Miami Valley Innovation Center, The Procter and Gamble Company, Cincinnati, Ohio 45253-8707, USA.

Background: In a previous study, we determined the effects of 17-alpha-ethynyl estradiol (EE) on gene expression using microarrays that represented approximately 9,000 genes, which was the state of-the-art. Higher content arrays with almost double the number of genes have since become available. In order to better determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have replicated the previous experiment using the more comprehensive microarray.

Methods: Immature 20-day-old Sprague-Dawley rats were exposed to 0.1, 1, and 10 microg EE/kg/day ( subcutaneously [s.c.]), for four days, dosing from postnatal day (PND) 20-23). Changes in a more comprehensive expression level of 15,923 rat annotated genes and expressed sequence tags were evaluated on PND 24.

Results: By comparing the response of the treatment groups versus controls using various statistical parameters, we determined that the expression of 1,394 genes showed a significant change with respect to control (p< or =0.0001), to at least one of the EE dosages. The tissues from animals exposed to 0.1 microg EE/kg/day showed changes in the expression of only 33 genes, whereas when they were exposed to 1 or 10 microg EE/kg/day, the expression of 409 and 548 genes was modified, respectively. A dose-dependent analysis indicated that 592 genes showed a robust and significant response to EE exposure (increased or decreased). Our analysis confirmed the regulation of previously identified estrogen-sensitive genes, and clearly identified novel mediators of estrogen actions, both in the uterus as well as in the ovary.

Conclusions: This compendium of genes represents the largest compilation of estrogen-responsive genes that has ever been identified for the immature uterus and ovary of any species, and can be used to generate testable hypothesis to improve the understanding of the molecular pathways associated with physiological and pathophysiological responses to exposure to chemicals with estrogenic properties.
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http://dx.doi.org/10.1002/bdrb.20032DOI Listing
April 2005

Impact of the phytoestrogen content of laboratory animal feed on the gene expression profile of the reproductive system in the immature female rat.

Environ Health Perspect 2004 Nov;112(15):1519-26

Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, Ohio 45253-8707, USA.

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 microg/kg/day 17alpha-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase-polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1247616PMC
http://dx.doi.org/10.1289/ehp.6848DOI Listing
November 2004

Identification of a gene expression profile that discriminates indirect-acting genotoxins from direct-acting genotoxins.

Mutat Res 2004 May;549(1-2):5-27

The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 538707 09, Cincinnati, OH 45253-8707, USA.

During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK(+/-) mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals when their mechanism of action is unknown.
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http://dx.doi.org/10.1016/j.mrfmmm.2003.11.012DOI Listing
May 2004

Gene expression profile induced by 17 alpha-ethynyl estradiol in the prepubertal female reproductive system of the rat.

Toxicol Sci 2003 Apr 7;72(2):314-30. Epub 2003 Mar 7.

Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, Ohio 45253, USA.

The profound effects of 17beta-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems, are mediated mostly by regulation of temporal and cell type-specific expression of different genes. In order to understand better the molecular events associated with the activation of the estrogen receptor (ER), we have used microarray technology to determine the transcriptional program and dose-response characteristics of exposure to a potent synthetic estrogen, 17 alpha-ethynyl estradiol (EE), during prepubertal development. Changes in patterns of gene expression were determined in the immature uterus and ovaries of Sprague-Dawley rats on postnatal day (PND) 24, 24 h after exposure to EE, at 0.001, 0.01, 0.1, 1 and 10 micro g EE/kg/day (sc), for four days (dosing from PND 20 to 23). The transcript profiles were compared between treatment groups and controls using oligonucleotide arrays to determine the expression level of approximately 7000 annotated rat genes and over 1740 expressed sequence tags (ESTs). Quantification of the number of genes whose expression was modified by the treatment, for each of the various doses of EE tested, showed clear evidence of a dose-dependent treatment effect that follows a monotonic response, concordant with the dose-response pattern of uterine wet-weight gain and luminal epithelial cell height. The number of genes whose expression is affected by EE exposure increases according to dose. At the highest dose tested of EE, we determined that the expression level of over 300 genes was modified significantly (p < or = 0.0001). A dose-dependent analysis of the transcript profile revealed a set of 88 genes whose expression is significantly and reproducibly modified (increased or decreased) by EE exposure (p < or = 0.0001). The results of this study demonstrate that, exposure to a potent estrogenic chemical during prepubertal maturation changes the gene expression profile of estrogen-sensitive tissues. Furthermore, the products of the EE-regulated genes identified in these tissues have a physiological role in different intracellular pathways, information that will be valuable to determine the mechanism of action of estrogens. Moreover, those genes could be used as biomarkers to identify chemicals with estrogenic activity.
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http://dx.doi.org/10.1093/toxsci/kfg037DOI Listing
April 2003

Gene expression profile induced by 17alpha-ethynyl estradiol, bisphenol A, and genistein in the developing female reproductive system of the rat.

Toxicol Sci 2002 Jul;68(1):184-99

The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 538707, No. 805, Cincinnati, OH 45253-8707, USA.

Exposure to some compounds with estrogenic activity, during fetal development, has been shown to alter development of reproductive organs, leading to abnormal function and disease either after birth or during adulthood. In order to understand the molecular events associated with the estrogenicity of different chemicals and to determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have used microarray technology to determine the transcriptional program influenced by exposure to this class of compounds during organogenesis and development. Changes in patterns of gene expression were determined in the developing uterus and ovaries of Sprague-Dawley rats on GD 20, exposed to graded dosages (sc) of 17alpha-ethynyl estradiol (EE), genistein, or bisphenol A (BPA) from GD 11 to GD 20. Dose levels were roughly equipotent in estrogenic activity. We compared the transcript profiles between treatment groups and controls, using oligonucleotide arrays to determine the expression level of approximately 7000 rat genes and over 1000 expressed squence tags (ESTs). At the highest tested doses of EE, BPA, or genistein, we determined that less than 2% of the mRNA detected by the array showed a 2-fold or greater change in their expression level (increase or decrease). A dose-dependent analysis of the transcript profile revealed a common set of genes whose expression is significantly and reproducibly modified in the same way by each of the 3 chemicals tested. Additionally, each compound induces changes in the expression of other transcripts that are not in common with the others, which indicated not all compounds with estrogenic activity act alike. The results of this study demonstrate that transplacental exposure to chemicals with estrogenic activity changes the gene expression profile of estrogen-sensitive tissues, and that the analysis of the transcript profile of these tissues could be a valuable approach to determining the estrogenicity of different compounds.
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http://dx.doi.org/10.1093/toxsci/68.1.184DOI Listing
July 2002