Development and qualification of a machine learning algorithm for automated hair counting.

OBJECTIVE: Determining the amount of hair on the scalp has always been an important metric of patient satisfaction for hair growth and hair retention technologies. While simple in concept, this measurement is a difficult, resource intensive task for the dermatologist and the research scientist. Specifically, counting and measuring hair in phototrichogram images is very time consuming and labour intensive. Due to cost, often only a fraction of available images is manually analysed. There is a need for an automated method that can significantly increase speed and throughput while reducing the cost of counting and measuring hair in phototrichogram images. METHODS: Recent advances in machine learning and deep convolutional neural networks (deep learning) have led to a revolution in the analysis of image, video, speech, text and other sensor data. Image diagnostics have seen remarkable improvements with completely automated methods outperforming both human experts and human-engineered analysis methods. Deep learning methods can also provide speed and cost benefits. To enable use of a deep learning, we created a data set of 288 manually annotated phototrichogram images with marked location and length of each hair (the training dataset). We designed a custom neural network architecture and custom image processing algorithms to best utilize the available training data and to maximize performance for hair counting and length measurement. The performance of the algorithm was qualified by comparing hair count and length measurements to an independent ground truth method, the semi-manual Canfield's Hair Metrix method. RESULTS: Leveraging deep neural networks, we have developed capability to apply machine learning to reduce the time needed to acquire data from phototrichograms of patients' scalp from months to seconds. Our algorithm enables fast and fully automated hair counting and length measurement. The algorithm shows high agreement with human manually assisted analysis (ground truth). CONCLUSIONS: We have trained and deployed an algorithm utilizing this technology and have demonstrated the reproducibility, accuracy and speed of this algorithm that, once deployed, requires little to no recurring cost or manual intervention for its operation. The method allows fast analysis of large number of images, reducing study cost and significantly reducing study analysis time.

Scalp application of antioxidants improves scalp condition and reduces hair shedding in a 24-week randomized, double-blind, placebo-controlled clinical trial.

OBJECTIVE: Increasing hair fullness is a global unmet need for many men and women. An approach to the problem is to decrease hair fall or shedding by reducing scalp stratum corneum oxidation and barrier damage to increase hair retention. This study evaluated a combination of functional antioxidants and barrier-enhancing cosmetic ingredients to improve scalp condition thereby enabling stronger hair anchorage and longer retention. METHODS: Male and female subjects with normal scalp condition and self-perceived hair thinning participated in a 24-week, double-blind, placebo-controlled, randomized clinical study assessing either a regimen of treatment shampoo and leave-on treatment containing functional antioxidant and barrier-enhancing agents or an identical placebo chassis shampoo control. The functional ingredients were piroctone olamine, zinc pyrithione, zinc carbonate, niacinamide, panthenol and caffeine. At baseline and after 8, 16 and 24 weeks of product use, several measurements were taken: hair shedding, total hair count (by phototrichogram), hair samples, TEWL and evaluation of biomarkers of scalp and hair conditions. Subjects also completed self-assessment questionnaires. RESULTS: Statistically significant effects for functional ingredient-containing treatment regimen versus a placebo control shampoo formulation were observed for reduced hair shedding, increased total hair count, reduced TEWL and improvement in scalp biomarker values. Subjects also noticed these improvements assessed via self-assessment questionnaires. CONCLUSIONS: These results establish that the use of functional antioxidant and barrier-enhancing agents to further improve scalp condition can enable a reduction in hair shedding and thus an increase in perceived hair fullness. The underlying improvements in scalp condition suggest the hair benefits were achieved as a result of improved scalp skin barrier and scalp condition leading to a viable preventative approach for hair thinning.

"Quantifying the precision of ecological risk: Misunderstandings and errors in the methods for assessment factors versus species sensitivity distributions".

The science of species sensitivity distributions (SSDs) is a blend of statistical theory, ecotoxicological testing, study reliability, and biodiversity. The utility of SSDs has been well reviewed and they are viewed as a high tier assessment tool in environmental risk assessment and other disciplines. SSDs seek to improve upon probabilistic extrapolation of laboratory (and sometimes field) collected ecotoxicity data for environmental protection by modeling the diversity of multiple experimental results in the form of a single statistical distribution which reduces or eliminates the need for extrapolation with deterministic assessment factors. SSDs thus depend heavily on both statistical and biological knowledge. In this commentary we review recently published literature identifying areas of improvement based on fundamental statistical theory or application in environmental assessment contexts. We reveal that sound application of SSDs relies heavily upon a grasp of probability distributions, how asymmetric confidence intervals are derived for distributions common to SSDs, the influence of sample size on parameter estimation, and how these are collectively applied across the myriad of regulatory systems globally. Statisticians and ecotoxicologists are inextricably bound together in the goal of actually improving hazard assessment using both probabilistic and deterministic methodologies.

Determination of narcotic potency using a neurobehavioral assay with larval zebrafish.

BACKGROUND: Identifying chemicals with narcotic potency is an important aspect of assessing the safety of consumer products that may be accidentally ingested. A rapid and efficient assay of narcotic potency is desired for assessing chemicals with such suspected activity. OBJECTIVES: This purpose of this research was to develop a non-mammalian vertebrate, high throughput, neurobehavioral method to assess the narcotic potency of chemicals using larval zebrafish. METHODS: Larval zebrafish were acutely exposed to chemicals beginning at 5 days post fertilization (5 dpf). Locomotor activity, elicited by regular, periodic photostimulation, was quantified using a video tracking apparatus. Narcotic potency was determined as the molar concentration at which photostimulated locomotor activity was reduced by 50% (IC50). Toxicity was assessed based on observations of morbidity or mortality. Recovery was assessed following removal of test material by serial dilution and reassessment of photostimulated behavior 24 hr later (6 dpf). RESULTS: A total of 21 chemicals were assessed. Etomidate, a human narcotic analgesic agent, was used as a reference material. Investigating a series of eleven linear, primary alcohols (C6 to C16), a relationship between narcotic potency and carbon number was observed; narcotic potency increased with carbon number up to C12, consistent with historical studies. For a set of technical grade surfactants, nonionic surfactants (i.e., alcohol ethoxylates) were observed to be narcotic agents while anionic surfactants produced evidence of reduced locomotor activity only in combination with toxicity. Of the solvents evaluated, only ethanol exhibited narcotic activity with an IC50 of 261 mM and was the least potent of the chemicals investigated. Etomidate was the most potent material evaluated with an IC50 of 0.39 µM. CONCLUSIONS: The larval zebrafish neurobehavioral assay provides a method capable of estimating the narcotic potency of chemicals and can identify if toxicity contributes to observed neurobehavioral effects in the test organism.

Fish embryo tests and acute fish toxicity tests are interchangeable in the application of the threshold approach.

A database was compiled for algal Organisation for Economic Co-operation and Development (OECD) test guideline 201, for Daphnia magna OECD test guideline 202, for the acute fish toxicity (AFT) OECD test guideline 203, and for the fish embryo toxicity (FET) OECD test guideline 236 to assess the suitability and applicability of the FET test in a threshold approach context. In the threshold approach, algal and Daphnia toxicity are assessed first, after which a limit test is conducted at the lower of the 2 toxicity values using fish. If potential fish toxicity is indicated, a full median lethal concentration assay is performed. This tiered testing strategy can significantly reduce the number of fish used in toxicity testing because algae or Daphnia are typically more sensitive than fish. A total of 165 compounds had AFT and FET data available, and of these, 82 had algal and Daphnia acute toxicity data available. Algae and Daphnia were more sensitive 75 to 80% of the time. Fish or FET tests were most sensitive 20 and 16% of the time, respectively, when considered as the sole fish toxicity indicator and 27% of the time when both were considered simultaneously. When fish were the most sensitive trophic level, different compounds were identified as the most toxic in FET and to AFT tests; however, the differences were not so large that they resulted in substantially different outcomes when potencies were binned using the United Nations categories of aquatic toxicity under the Globally Harmonized System for classification and labeling. It is recommended that the FET test could be used to directly replace the AFT test in the threshold approach or could be used as the definitive test if an AFT limit test indicated toxicity potential for a chemical. Environ Toxicol Chem 2019;38:671-681. © 2019 SETAC.

Development of growth equations from longitudinal studies of body weight and height in the full term and preterm neonate: From birth to four years postnatal age.

Physiologically based pharmacokinetic (PBPK) models are developed from compound-independent information to describe important anatomical and physiological characteristics of an individual or population of interest. Modeling pediatric populations is challenging because of the rapid changes that occur during growth, particularly in the first few weeks and months after birth. Neonates who are born premature pose several unique challenges in PBPK model development. To provide appropriate descriptions for body weight (BW) and height (Ht) for age and appropriate incremental gains in PBPK models of the developing preterm and full term neonate, anthropometric measurements collected longitudinally from 1,063 preterm and 158 full term neonates were combined with 2,872 cross-sectional measurements obtained from the NHANES 2007-2010 survey. Age-specific polynomial growth equations for BW and Ht were created for male and female neonates with corresponding gestational birth ages of 25, 28, 31, 34, and 40 weeks. Model-predicted weights at birth were within 20% of published fetal/neonatal reference standards. In comparison to full term neonates, postnatal gains in BW and Ht were slower in preterm subgroups, particularly in those born at earlier gestational ages. Catch up growth for BW in neonates born at 25, 28, 31, and 34 weeks gestational age was complete by 13, 8, 6, and 2 months of life (males) and by 10, 6, 5, and 2 months of life (females), respectively. The polynomial growth equations reported in this paper represent extrauterine growth in full term and preterm neonates and differ from the intrauterine growth standards that were developed for the healthy unborn fetus.

On the impact of sample size on median lethal concentration estimation in acute fish toxicity testing: Is n = 7/group enough?

The fish acute toxicity test method is foundational to aquatic toxicity testing strategies, yet the literature lacks a concise sample size assessment. Although various sources address sample size, historical precedent seems to play a larger role than objective measures. We present a novel and comprehensive quantification of the effect of sample size on estimation of the median lethal concentration (LC50), covering a wide range of scenarios. The results put into perspective the practical differences across a range of sample sizes, from n = 5/concentration up to n = 23/concentration. We also provide a framework for setting sample size guidance illustrating ways to quantify the performance of LC50 estimation, which can be used to set sample size guidance given reasonably difficult (or worst-case) scenarios. There is a clear benefit to larger sample size studies: they reduce error in the determination of LC50s, and lead to more robust safe environmental concentration determinations, particularly in cases likely to be called worst-case (shallow slope and true LC50 near the edges of the concentration range). Given that the use of well-justified sample sizes is crucial to reducing uncertainty in toxicity estimates, these results lead us to recommend a reconsideration of the current de minimis 7/concentration sample size for critical studies (e.g., studies needed for a chemical registration, which are being tested for the first time, or involving difficult test substances). Environ Toxicol Chem 2018;37:1565-1578. © 2018 SETAC.

Probabilistic Monte Carlo estimation for quantitative exposure assessment of lotion transfer via baby wipes usage.

Unique aspects of childhood exposure to products need childs specific exposure data. This study developed a probabilistic exposure model for lotion transfer to diapered skin through normal use of baby wipes in children up to 48 months of age. Monte Carlo simulations used baby wipe diary data from the US, Germany and the UK, body weight data from the US, and lotion transfer data from single and multiple wipes adjusting for separate diaper changes. The models predicted a declining number of wipes used/day with a reduction in lotion transfer as age and body weight increased. Experimental testing on multiple sequential wipes used on an overlapping area showed a reduction in lotion deposition by 23.9% after the first wipe. Overall, the weighted population average over the approximate diapering period of 0-36 months across the three geographies at 50th, 90th, & 95th percentiles, were between 130, 230, 260 mg/kg/day, respectively, and 150, 270, 310 mg/kg/day depending on whether a reduction due to overlap is implemented. The statistical model represents an effective strategy to determine exposure to baby wipes lotion for exposure based risk assessment.

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

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.

Quantitation of baby wipes lotion transfer to premature and neonatal skin.

Exposure to topically applied substances occurs routinely in premature and hospitalized infant care. Safety determinations are most accurate when exposures are based on appropriately designed studies to capture variations in practice patterns and population heterogeneity. Current safety assessments may not reflect actual practice resulting in overly conservative or understated default assumptions for toxicological determinations. We quantified the amount of baby wipes lotion transferred to premature and term neonatal skin as grams/kg body weight/day. We observed the soil type and number of wipes used for skin cleansing and measured lotion transfer from one wipe applied to freshly clean, dry skin. A Bayesian imputation approach was applied to compute lotion exposure and produce summary statistics. Model covariates were age and weight at evaluation, gender, soil type, soil amount, and number of diaper changes per day. Lotion transfer was measured for 66 premature and 55 term neonates with 449 and 254 evaluations, respectively. The wipes per day was 12.52 overall (all infants and soils), 12.78 for premature and 12.21 for term neonates. Lotion transfer was 0.20 g/kg/day (95th percentile) overall, 0.21 for premature and 0.19 for term neonates. The statistical and experimental methodology represents an effective strategy for determining exposure and assessing risk.

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

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), ß-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.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals.

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals.

The fish embryo test (FET) is a potential animal alternative for the acute fish toxicity (AFT) test. A comprehensive validation program assessed 20 different chemicals to understand intra- and interlaboratory variability for the FET. The FET had sufficient reproducibility across a range of potencies and modes of action. In the present study, the suitability of the FET as an alternative model is reviewed by relating FET and AFT. In total, 985 FET studies and 1531 AFT studies were summarized. The authors performed FET-AFT regressions to understand potential relationships based on physical-chemical properties, species choices, duration of exposure, chemical classes, chemical functional uses, and modes of action. The FET-AFT relationships are very robust (slopes near 1.0, intercepts near 0) across 9 orders of magnitude in potency. A recommendation for the predictive regression relationship is based on 96-h FET and AFT data: log FET median lethal concentration (LC50) = (0.989 × log fish LC50) - 0.195; n = 72 chemicals, r = 0.95, p < 0.001, LC50 in mg/L. A similar, not statistically different regression was developed for the entire data set (n = 144 chemicals, unreliable studies deleted). The FET-AFT regressions were robust for major chemical classes with suitably large data sets. Furthermore, regressions were similar to those for large groups of functional chemical categories such as pesticides, surfactants, and industrial organics. Pharmaceutical regressions (n = 8 studies only) were directionally correct. The FET-AFT relationships were not quantitatively different from acute fish-acute fish toxicity relationships with the following species: fathead minnow, rainbow trout, bluegill sunfish, Japanese medaka, and zebrafish. The FET is scientifically supportable as a rational animal alternative model for ecotoxicological testing of acute toxicity of chemicals to fish.

Correlation of chemical structure with reproductive and developmental toxicity as it relates to the use of the threshold of toxicological concern.

In the absence of toxicological data on a chemical, the threshold of toxicological concern (TTC) approach provides a system to estimate a conservative exposure below which there is a low probability of risk for adverse health effects. The original toxicology dataset underlying the TTC was based on NOELs from repeat dose studies. Subsequently there have been several efforts to assess whether or not these limits are also protective for reproductive/developmental effects. This work expands the database of chemicals with reproductive and developmental data, presents these data in a comprehensive and transparent format and groups the chemicals according to the TTC "Cramer Class" rules. Distributions of NOAELs from each of these classes were used to assess whether the previously proposed TTC values based on repeat dose data are protective for reproductive/developmental toxicity endpoints as well. The present analysis indicates that, for each Cramer Class, the reproductive and developmental endpoints would be protected at the corresponding general TTC tiers derived by Munro et al. (1996).

Assessing the risk of type 1 allergy to enzymes present in laundry and cleaning products: evidence from the clinical data.

Microbial enzymes have been used in laundry detergent products for several decades. These enzymes have also long been known to have the potential to give rise to occupational type 1 allergic responses. A few cases of allergy among consumers using dusty enzyme detergents were reported in the early 1970s. Encapsulation of the enzymes along with other formula changes were made to ensure that consumer exposure levels were sufficiently low that the likelihood of either the induction of IgE antibody (sensitization) or the elicitation of clinical symptoms be highly improbable. Understanding the consumer exposure to enzymes which are used in laundry and cleaning products is a key step to the risk management process. Validation of the risk assessment conclusions and the risk management process only comes with practical experience and evidence from the marketplace. In the present work, clinical data from a range of sources collected over the past 40 years have been analysed. These include data from peer reviewed literature and enzyme specific IgE antibody test results in detergent manufacturers' employees and from clinical study subjects. In total, enzyme specific IgE antibody data were available on 15,765 individuals. There were 37 individuals with IgE antibody. The majority of these cases were from the 1970s where 23 of 4687 subjects (0.49%) were IgE positive and 15 of the 23 were reported to have symptoms of allergy. The remaining 14 cases were identified post-1977 for a prevalence of 0.126% (14/11,078). No symptoms were reported and no relationship to exposure to laundry and cleaning products was found. There was a significant difference between the pre- and post-1977 cohorts in that the higher rates of sensitization with symptoms were associated with higher exposure to enzyme. The clinical testing revealed that the prevalence of enzyme specific IgE in the population is very rare (0.126% since 1977). This demonstrates that exposure to these strong respiratory allergens via use of laundry and cleaning products does not lead to the development of sensitization and disease. These data confirm that the risk to consumers has been properly assessed and managed and support the concept that thresholds of exposure exist for respiratory allergy. Expansion of enzyme use into new consumer product categories should follow completion of robust risk assessments in order to continue ensuring the safe use of enzymes among consumers.

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

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.

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

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

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

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.