Contribution of ATM and ATR kinase pathways to p53-mediated response in etoposide and methyl methanesulfonate induced DNA damage.

p53 is a key integrator of cellular response to DNA damage, supporting post-translational repair and driving transcription-mediated responses including cell cycle arrest, apoptosis, and repair. DNA damage sensing kinases recognize different types of DNA damage and initiate specific responses through various post-translational modifications of p53. This study evaluated chemical specificity of the p53 pathway response by manipulating p53 or its upstream kinases and assessing the effect on DNA damage and cellular responses to prototype chemicals: etoposide (ETP, topoisomerase II inhibitor) and methyl methane sulfonate (MMS, alkylating agent). p53-deficient cells demonstrated reduced accumulation of the p53 target proteins MDM2, p21, and Wip1; reduced apoptotic response; and increased DNA damage (p-H2AX and micronuclei) with both chemicals. However, p53 was not essential for cell cycle arrest in HT1080 or HCT116 cells. The two chemicals induced different patterns of kinase activation, particularly in terms of Chk 1, Chk 2, p38, and ERK 1/2. However, inhibition of the ATM pathway showed a greater effect on p53 activtation, apoptosis, and accumulation of DNA damage than ATR-Chk 1 or the MAP kinases regardless of the chemical used. These results indicate that ATM is the predominant upstream kinase responsible for activation of the p53-mediated DNA damage response for both MMS and ETP, though the downstream kinase response is markedly different. Environ. Mol. Mutagen. 58:72-83, 2017. © 2017 Wiley Periodicals, Inc.

Editor's Highlight: Development of an In vitro Assay Measuring Uterine-Specific Estrogenic Responses for Use in Chemical Safety Assessment.

A toxicity pathway approach was taken to develop an in vitro assay using human uterine epithelial adenocarcinoma (Ishikawa) cells as a replacement for measuring an in vivo uterotrophic response to estrogens. The Ishikawa cell was determined to be fit for the purpose of recapitulating in vivo uterine response by verifying fidelity of the biological pathway components and the dose-response predictions to women of child-bearing age. Expression of the suite of estrogen receptors that control uterine proliferation (ERα66, ERα46, ERα36, ERß, G-protein coupled estrogen receptor (GPER)) were confirmed across passages and treatment conditions. Phenotypic responses to ethinyl estradiol (EE) from transcriptional activation of ER-mediated genes, to ALP enzyme induction and cellular proliferation occurred at concentrations consistent with estrogenic activity in adult women (low picomolar). To confirm utility of this model to predict concentration-response for uterine proliferation with xenobiotics, we tested the concentration-response for compounds with known uterine estrogenic activity in humans and compared the results to assays from the ToxCast and Tox21 suite of estrogen assays. The Ishikawa proliferation assay was consistent with in vivo responses and was a more sensitive measure of uterine response. Because this assay was constructed by first mapping the key molecular events for cellular response, and then ensuring that the assay incorporated these events, the resulting cellular assay should be a reliable tool for identifying estrogenic compounds and may provide improved quantitation of chemical concentration response for in vitro-based safety assessments.

A map of the PPARα transcription regulatory network for primary human hepatocytes.

Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARα) in primary human hepatocytes using the selective PPARα ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIP-seq studies at 2 and 24h to assess genomic binding of PPARα. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARα binding-either directly at PPARα response elements or via alternative mechanisms. Almost half of regulated genes had no PPARα binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARα and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERα, and HNF4α. The linkage from PPARα binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10 µM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways.

Assessing dose-dependent differences in DNA-damage, p53 response and genotoxicity for quercetin and curcumin.

As part of a longer-term goal to create a quantitative mechanistic model of the p53-Mdm2 DNA-damage pathway, we are studying cellular responses to compounds causing DNA-damage by various modes-of action, including two natural polyphenols: quercetin (QUE) and curcumin (CUR). QUE and CUR are weak mutagens in some in vitro assays and possess both anti- or pro-oxidant effects depending on dose. This study examines the dose-response of DNA-damage pathway to these compounds in HT1080 cells (a human cell line with wild-type p53) at doses relevant to human exposure. CUR was more potent in causing reactive oxygen species, DNA damage (measured as phospho-H2AX) and p53 induction, with lowest observed effect levels (LOELs; 3-8 µM) approximately three-fold lower than QUE (20-30 µM). CUR showed a strong G2/M arrest and apoptosis at ≈ 10 µM. QUE caused S phase arrest at low doses (8 µM) and apoptosis was only induced at much higher doses (60 µM). At concentrations with similar levels of p-H2AX and p53 biomarkers, CUR caused greater micronuclei frequency. CUR induced clear increases micronuclei at 3-6 µM, while QUE had a weaker micronuclei response even at the highest doses. Thus, even with two compounds sharing common chemistries, DNA-damage response patterns differed significantly in terms of dose and cell fate.

Assessing the relevance of in vitro measures of phthalate inhibition of steroidogenesis for in vivo response.

Human phthalate exposure occurs as mixtures of diesters with varying activity towards testosterone-dependent development. Dibutyl (DBP), diethylhexyl (DEHP) and butylbenzyl (BBP) phthalate disrupt sexual development in the fetal rat. Dimethyl (DMP) and diethyl (DEP) phthalate do not. These differences in potency may result from differential delivery of the monophthalates to the testes or from variation in the abilities of the compounds to alter steroidogenesis. We tested five phthalates in pregnant rats (500mg/kg-d, GD12-19) and analyzed the fetal testes for corresponding monoesters (MMP, MEP, MBP, MEHP, MBeP). Testes MMP and MEP levels were 2-40-fold higher than the active monoesters, MBP and MEHP. BBP exposure led to low concentrations of MBeP, but similar MBP levels to DBP. An in vitro MA-10 cell assay measured the direct effect of monophthalates on testosterone production. MEHP inhibited LH-stimulated testosterone production at 1microM. RT-PCR confirmed down-regulation of genes associated with cholesterol transport and steroid synthesis and metabolism by MEHP. Five additional phthalates were tested for testosterone inhibition. MBP and mono-n-octyl phthalate were similar to MEHP; MMP, MEP and MBeP were poor inhibitors of testosterone production. Based on these results, differences in the phthalates' ability to interfere with sexual development in vivo appears to be more associated with differential potency for testosterone inhibition than differences in tissue doses.

Reproductive toxicity and pharmacokinetics of di-n-butyl phthalate (DBP) following dietary exposure of pregnant rats.

Most rodent developmental toxicity studies of dibutylphthalate (DBP) have relied on bolus gavage dosing. This study characterized the developmental toxicity of dietary DBP. Pregnant CD rats were given nominal doses of 0, 100, or 500 mg DBP/kg/day in diet (actual intake 0, 112, and 582 mg/kg/day) from gestational day (GD) 12 through the morning of GD 19. Rats were killed 4 or 24 hr thereafter. DBP dietary exposure resulted in significant dose-dependent reductions in testicular mRNA concentration of scavenger receptor class B, member 1; steroidogenic acute regulatory protein; cytochrome P450, family 11, subfamily a, polypeptide 1; and cytochrome P450 family 17, subfamily a, polypeptide 1. These effects were most pronounced 4 hr after the end of exposure. Testicular testosterone was reduced 24 hr post-exposure in both DBP dose groups and 4 hr after termination of the 500-mg DBP/kg/day exposure. Maternal exposure to 500 mg DBP/kg/day induced a significant reduction in male offspring's anogenital distance indicating in utero disruption of androgen function. Leydig cell aggregates, increased cord diameters, and multinucleated gonocytes were present in DBP-treated rats. Monobutyl phthalate, the developmentally toxic metabolite of DBP, and its glucuronide conjugate were found in maternal and fetal plasma, amniotic fluid, and maternal urine. Our results, when compared to previously conducted gavage studies, indicate that approximately equal doses of oral DBP exposure of pregnant rats, from diet or gavage, result in similar responses in male offspring.

Using a comparative in vivo DNase I footprinting technique to analyze changes in protein-DNA interactions following phthalate exposure.

Exposure to environmental chemicals often induces changes in gene expression leading to a variety of developmental and physiological problems. Understanding the underlying mechanism of these changes will aid in assessing human risk to these chemicals. Traditional methods for analyzing protein-DNA interactions include in vivo footprinting and chromatin immunoprecipitation (ChIP). However, ChIP does not provide binding location, and conventional footprinting is too subjective and time consuming for comparing protein binding in toxicological studies. Here, in vivo DNase I footprinting is adapted for use with the automated DNA sequencer to provide a semiquantitative map of changes in DNA-protein interactions in the promoter of steroidogenic acute regulatory (StAR) protein. StAR is the rate-limiting step in testosterone biosynthesis and is downregulated following in utero di-butyl phthalate (DBP) treatment in rats through an unknown mechanism. In vivo footprinting identified three regions of altered DNase digestibility following DBP treatment, and EMSA identified the corresponding transcription factors as SF-1, c/ebp beta, and GATA4. ChIP assays confirmed changes in protein-binding activity of SF-1 and c/ebp beta, but only c/ebp beta gesponds to only DBP. This suggests that c/ebp beta ginding is involved in DBP-induced transcriptional changes. By tailoring in vivo footprinting for toxicological studies, it can provide a detailed and accurate map of protein-DNA interactions and is an excellent first step in determining the changes in the structure of transcriptional machinery following an exogenous chemical treatment.

CCAAT/enhancer binding protein beta, but not steroidogenic factor-1, modulates the phthalate-induced dysregulation of rat fetal testicular steroidogenesis.

Prolonged in utero exposure of fetal male rats to dibutyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples, among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes and, therefore, reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semiquantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including steroidogenic acute regulatory, scavenger receptor B-1, cytochrome P450 side chain cleavage, and cytochrome P450 17A1, that are down-regulated after an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by EMSA, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein beta (c/ebp beta) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp beta in DBP treated animals, whereas SF-1 was reduced in both diethyl phthalate (nontoxic) and DBP (toxic) treatments. These results suggest that inhibition of c/ebp beta, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. In addition, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.

Differential steroidogenic gene expression in the fetal adrenal gland versus the testis and rapid and dynamic response of the fetal testis to di(n-butyl) phthalate.

The phthalate ester di(n-butyl) phthalate (DBP) causes feminization of male rats upon in utero exposure by repressing expression of genes required for testicular steroidogenesis. Previous work in our laboratory has shown that repression of gene expression and steroidogenesis in the fetal testis is apparent within a few hours of DBP exposure. The purpose of this study was to determine the precise timing of DBP-associated gene expression changes in the fetal testis using transcriptional profiling and to determine whether DBP exerts similar effects on steroidogenesis in the fetal adrenal. A DBP time-course experiment showed that testicular steroidogenesis was decreased within 1 h of DBP exposure and that this decrease preceded the repressed transcription of Star (steroidogenic acute regulatory protein); Scarb1 (scavenger receptor class B, member 1; also know as Sr-b1); Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1; also known as P450SCC); and Cyp17a1 (cytochrome P450 family 17, subfamily a, polypeptide 1; also known as Cyp17). Gene expression profiling demonstrated rapid (within 1 to 3 h) and transient induction of immediate early genes in the fetal testis after administration of DBP to the pregnant dam. There was a statistically insignificant decrease in corticosterone production by the fetal adrenal after in utero exposure to DBP from Gestation Day 12 to Gestation Day 19. The extent of steroidogenesis diminution was much less in the adrenal than in the testis (approximately 45% decrease in the adrenal versus 87% decrease in the testis) and expression of genes required for steroidogenesis in the adrenal was unaffected by DBP. Together, these studies demonstrate that DBP initiates a rapid and dynamic change in gene expression in the fetal testis that likely plays a role in the reduction in steroidogenesis that is unique to the fetal testis relative to the steroidogenically active fetal adrenal.

Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanism.

In utero exposure to di(n-butyl) phthalate (DBP) leads to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists. DBP demonstrates no affinity for the androgen receptor, but rather leads to diminished testosterone production by the fetal testis. The purpose of this study was to determine the onset and reversibility of DBP effects on the fetal testis and to determine at a functional level the points in the cholesterol transport and steroidogenesis pathways affected by DBP. Starting at gestational day (gd) 12, pregnant rats were gavaged daily with 500 mg/kg DBP or corn oil control. Significant decreases in testosterone production and mRNA expression of scavenger receptor B1, P450(SCC), steroidogenic acute regulatory protein, and cytochrome p450c17 were observed as early as gd 17. Testosterone, mRNA, and protein levels remained low 24 h after withdrawal of DBP treatment but increased 48 h after cessation of DBP exposure. In another experiment, pregnant dams were treated with DBP until gd 19, with the start of DBP treatment moved 1 d later into gestation for each treatment group, with the final group dosed only on gd 19. Significant decreases in testosterone, mRNA expression, and protein expression were evident as early as 3 h after treatment with DBP, with full repression apparent 24 h after treatment. Using a testis explant system, we determined that DBP treatment led to diminished transport of cholesterol across the mitochondrial membrane as well as diminished function at each point in the testosterone biosynthesis pathway except 17 beta-hydroxysteroid dehydrogenase. The transcriptional repression caused by DBP does not appear to be mediated via interference with steroidogenic factor-1 as determined by reporter assays. We conclude that high-dose DBP exposure leads to rapid and reversible diminution of the expression of several proteins required for cholesterol transport and steroidogenesis in the fetal testis, resulting in decreased testosterone synthesis and consequent male reproductive maldevelopment.

Interaction of organophosphate pesticides and related compounds with the androgen receptor.

Identification of several environmental chemicals capable of binding to the androgen receptor (AR) and interfering with its normal function has heightened concern about adverse effects across a broad spectrum of environmental chemicals. We previously demonstrated AR antagonist activity of the organophosphate (OP) pesticide fenitrothion. In this study, we characterized AR activity of analogues of fenitrothion to probe the structural requirements for AR activity among related chemicals. AR activity was measured using HepG2 human hepatoma cells transfected with human AR plus an androgen-responsive luciferase reporter gene, MMTV-luc. AR antagonist activity decreased as alkyl chain length of the phosphoester increased, whereas electron-donating properties of phenyl substituents of the tested compounds did not influence AR activity. Oxon derivatives of fenitrothion, which are more likely to undergo hydrolytic degradation, had no detectable AR antagonist activity. Molecular modeling results suggest that hydrogen-bond energies and the maximum achievable interatomic distance between two terminal H-bond capable sites may influence both the potential to interact with the AR and the nature of the interaction (agonist vs. antagonist) within this series of chemicals. This hypothesis is supported by the results of recent AR homology modeling and crystallographic studies relative to agonist- and antagonist-bound AR complexes. The present results are placed in the context of structure-activity knowledge derived from previous modeling studies as well as studies aimed toward designing nonsteroidal antiandrogen pharmaceuticals. Present results extend understanding of the structural requirements for AR activity to a new class of nonsteroidal, environmental, OP-related chemicals.

Combined effects of dietary phytoestrogen and synthetic endocrine-active compound on reproductive development in Sprague-Dawley rats: genistein and methoxychlor.

Humans and wildlife are frequently exposed to mixtures of endocrine active-compounds (EAC). The objective of the present study was to investigate the potential of the phytoestrogen genistein to influence the reproductive developmental toxicity of the endocrine-active pesticide methoxychlor. Three levels of genistein (0, 300, or 800 ppm) and two levels of methoxychlor (0 or 800 ppm) were used in this study. Sprague-Dawley rats were exposed to the two compounds, either alone or in combinations, through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning. Both compounds, methoxychlor in particular, were associated with reduced body growth at 800 ppm, but pregnancy outcome was not affected by either treatment. An acceleration of vaginal opening (VO) in the exposed female offspring was the only observed effect of genistein at 300 ppm. Exposure to 800 ppm genistein or 800 ppm methoxychlor caused accelerated VO and also altered estrous cyclicity toward persistent estrus in the female offspring. The estrogenic responses to genistein and methoxychlor administered together were apparently accumulative of the effects associated with each compound alone. Methoxychlor, but not genistein, delayed preputial separation (PPS) in the male rats. When administered with methoxychlor, genistein at 800 ppm enhanced the effect of methoxychlor on delaying PPS. Genistein and methoxychlor treatment did not change gender-specific motor activity patterns in either sex. To explore possible mechanisms for interaction between the two compounds on development, we performed estrogen receptor (ER)- and androgen receptor (AR)-based in vitro transcriptional activation assays using genistein and the primary methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). While the in vitro assays supported the estrogenic effects of genistein and methoxychlor and the antiandrogenic effects of methoxychlor, the reactivity of these compounds with ERs alpha and beta could not predict the greater in vivo estrogenic potency of methoxychlor over genistein; nor could the potentiation of the methoxychlor effect on PPS by genistein be predicted based on in vitro HPTE and genistein reactions with the AR. Data from this study indicate that phytoestrogens are capable of altering the toxicological behaviors of other EACs, and the interactions of these compounds may involve complexities that are difficult to predict based on their in vitro steroid receptor reactivities.