Gravidity influences distinct transcriptional profiles of maternal and fetal placental macrophages at term.

Introduction: Maternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Maternal reproductive history is associated with differential risk of pregnancy complications. The molecular phenotypes and roles of these distinct monocyte/macrophage populations and the influence of gravidity on these phenotypes has not been systematically investigated. Methods: Here, we used RNA sequencing to study the transcriptional profiles of MIMs and HBCs in normal term pregnancies. Results: Our analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidae compared to primigravidae. In HBCs, multigravidae displayed enrichment of gene pathways involved in cell-cell signaling and differentiation. Discussion: Our results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Furthermore, maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidae to pregnancy complications.

Rapid identification of reproductive toxicants among environmental chemicals using an in vivo evaluation of gametogenesis in budding yeast Saccharomyces cerevisiae.

Infertility affects ∼12 % of couples, with environmental chemical exposure as a potential contributor. Of the chemicals that are actively manufactured, very few are assessed for reproductive health effects. Rodents are commonly used to evaluate reproductive effects, which is both costly and time consuming. Thus, there is a pressing need for rapid methods to test a broader range of chemicals. Here, we developed a strategy to evaluate large numbers of chemicals for reproductive toxicity via a yeast, S. cerevisiae high-throughput assay to assess gametogenesis as a potential new approach method (NAM). By simultaneously assessing chemicals for growth effects, we can distinguish if a chemical affects gametogenesis only, proliferative growth only or both. We identified a well-known mammalian reproductive toxicant, bisphenol A (BPA) and ranked 19 BPA analogs for reproductive harm. By testing mixtures of BPA and its analogs, we found that BPE and 17 ß-estradiol each together with BPA showed synergistic effects that worsened reproductive outcome. We examined an additional 179 environmental chemicals including phthalates, pesticides, quaternary ammonium compounds and per- and polyfluoroalkyl substances and found 57 with reproductive effects. Many of the chemicals were found to be strong reproductive toxicants that have yet to be tested in mammals. Chemicals having affect before meiosis I division vs. meiosis II division were identified for 16 gametogenesis-specific chemicals. Finally, we demonstrate that in general yeast reproductive toxicity correlates well with published reproductive toxicity in mammals illustrating the promise of this NAM to quickly assess chemicals to prioritize the evaluation for human reproductive harm.

A science-based agenda for health-protective chemical assessments and decisions: overview and consensus statement.

The manufacture and production of industrial chemicals continues to increase, with hundreds of thousands of chemicals and chemical mixtures used worldwide, leading to widespread population exposures and resultant health impacts. Low-wealth communities and communities of color often bear disproportionate burdens of exposure and impact; all compounded by regulatory delays to the detriment of public health. Multiple authoritative bodies and scientific consensus groups have called for actions to prevent harmful exposures via improved policy approaches. We worked across multiple disciplines to develop consensus recommendations for health-protective, scientific approaches to reduce harmful chemical exposures, which can be applied to current US policies governing industrial chemicals and environmental pollutants. This consensus identifies five principles and scientific recommendations for improving how agencies like the US Environmental Protection Agency (EPA) approach and conduct hazard and risk assessment and risk management analyses: (1) the financial burden of data generation for any given chemical on (or to be introduced to) the market should be on the chemical producers that benefit from their production and use; (2) lack of data does not equate to lack of hazard, exposure, or risk; (3) populations at greater risk, including those that are more susceptible or more highly exposed, must be better identified and protected to account for their real-world risks; (4) hazard and risk assessments should not assume existence of a "safe" or "no-risk" level of chemical exposure in the diverse general population; and (5) hazard and risk assessments must evaluate and account for financial conflicts of interest in the body of evidence. While many of these recommendations focus specifically on the EPA, they are general principles for environmental health that could be adopted by any agency or entity engaged in exposure, hazard, and risk assessment. We also detail recommendations for four priority areas in companion papers (exposure assessment methods, human variability assessment, methods for quantifying non-cancer health outcomes, and a framework for defining chemical classes). These recommendations constitute key steps for improved evidence-based environmental health decision-making and public health protection.

Wildfire Smoke Exposure during Pregnancy: A Review of Potential Mechanisms of Placental Toxicity, Impact on Obstetric Outcomes, and Strategies to Reduce Exposure.

Climate change is accelerating the intensity and frequency of wildfires globally. Understanding how wildfire smoke (WS) may lead to adverse pregnancy outcomes and alterations in placental function via biological mechanisms is critical to mitigate the harms of exposure. We aim to review the literature surrounding WS, placental biology, biological mechanisms underlying adverse pregnancy outcomes as well as interventions and strategies to avoid WS exposure in pregnancy. This review includes epidemiologic and experimental laboratory-based studies of WS, air pollution, particulate matter (PM), and other chemicals related to combustion in relation to obstetric outcomes and placental biology. We summarized the available clinical, animal, and placental studies with WS and other combustion products such as tobacco, diesel, and wood smoke. Additionally, we reviewed current recommendations for prevention of WS exposure. We found that there is limited data specific to WS; however, studies on air pollution and other combustion sources suggest a link to inflammation, oxidative stress, endocrine disruption, DNA damage, telomere shortening, epigenetic changes, as well as metabolic, vascular, and endothelial dysregulation in the maternal-fetal unit. These alterations in placental biology contribute to adverse obstetric outcomes that disproportionally affect the most vulnerable. Limiting time outdoors, wearing N95 respirator face masks and using high quality indoor air filters during wildfire events reduces exposure to related environmental exposures and may mitigate morbidities attributable to WS.

Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα.

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000  g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000  g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000  g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.

Racial/ethnic and geographic differences in polybrominated diphenyl ether (PBDE) levels across maternal, placental, and fetal tissues during mid-gestation.

Prenatal polybrominated diphenyl ether (PBDE) exposures are a public health concern due to their persistence and potential for reproductive and developmental harm. However, we have little information about the extent of fetal exposures during critical developmental periods and the variation in exposures for groups that may be more highly exposed, such as communities of color and lower socioeconomic status (SES). To characterize maternal-fetal PBDE exposures among potentially vulnerable groups, PBDE levels were examined in the largest sample of matched maternal serum, placenta, and fetal liver tissues during mid-gestation among a geographically, racially/ethnically, and socially diverse population of pregnant women from Northern California and the Central Valley (n = 180; 2014-16). Maternal-fetal PBDE levels were compared to population characteristics using censored Kendall's tau correlation and linear regression. PBDEs were commonly detected in all biomatrices. Before lipid adjustment, wet-weight levels of all four PBDE congeners were highest in the fetal liver (p < 0.001), whereas median PBDE levels were significantly higher in maternal serum than in the fetal liver or placenta after lipid-adjustment (p < 0.001). We also found evidence of racial/ethnic disparities in PBDE exposures (Non-Hispanic Black > Latina/Hispanic > Non-Hispanic White > Asian/Pacific Islander/Other; p < 0.01), with higher levels of BDE-100 and BDE-153 among non-Hispanic Black women compared to the referent group (Latina/Hispanic women). In addition, participants living in Fresno/South Central Valley had 34% (95% CI: - 2.4 to 84%, p = 0.07) higher wet-weight levels of BDE-47 than residents living in the San Francisco Bay Area. PBDEs are widely detected and differentially distributed in maternal-fetal compartments. Non-Hispanic Black pregnant women and women from Southern Central Valley geographical populations may be more highly exposed to PBDEs. Further research is needed to identify sources that may be contributing to differential exposures and associated health risks among these vulnerable populations.

Up-regulated cytotrophoblast DOCK4 contributes to over-invasion in placenta accreta spectrum.

In humans, a subset of placental cytotrophoblasts (CTBs) invades the uterus and its vasculature, anchoring the pregnancy and ensuring adequate blood flow to the fetus. Appropriate depth is critical. Shallow invasion increases the risk of pregnancy complications, e.g., severe preeclampsia. Overly deep invasion, the hallmark of placenta accreta spectrum (PAS), increases the risk of preterm delivery, hemorrhage, and death. Previously a rare condition, the incidence of PAS has increased to 1:731 pregnancies, likely due to the rise in uterine surgeries (e.g., Cesarean sections). CTBs track along scars deep into the myometrium and beyond. Here we compared the global gene expression patterns of CTBs from PAS cases to gestational age-matched control cells that invaded to the normal depth from preterm birth (PTB) deliveries. The messenger RNA (mRNA) encoding the guanine nucleotide exchange factor, DOCK4, mutations of which promote cancer cell invasion and angiogenesis, was the most highly up-regulated molecule in PAS samples. Overexpression of DOCK4 increased CTB invasiveness, consistent with the PAS phenotype. Also, this analysis identified other genes with significantly altered expression in this disorder, potential biomarkers. These data suggest that CTBs from PAS cases up-regulate a cancer-like proinvasion mechanism, suggesting molecular as well as phenotypic similarities in the two pathologies.

Association of polybrominated diphenyl ether (PBDE) levels with biomarkers of placental development and disease during mid-gestation.

BACKGROUND: Polybrominated diphenyl ether (PBDE) exposures have been associated with adverse pregnancy outcomes. A hypothesized mechanism is via alterations in placental development and function. However, we lack biomarkers that can be used as early indicators of maternal/fetal response to PBDE exposures and/or perturbations in placental development or function. METHODS: To evaluate the relationship between PBDE levels and placental biomarkers during mid-gestation of human pregnancy (n = 62), we immunolocalized three molecules that play key roles in cytotrophoblast (CTB) differentiation and interstitial/endovascular uterine invasion-integrin alpha-1 (ITGA1), vascular endothelial-cadherin (CDH5), and metalloproteinase-1 (MMP1)-and assessed three morphological parameters as potential indicators of pathological alterations using H&E-stained tissues-leukocyte infiltration, fibrinoid deposition, and CTB endovascular invasion. We evaluated associations between placental PBDE levels and of biomarkers of placental development and disease using censored Kendall's tau correlation and linear regression methods. RESULTS: PBDEs were detected in all placental samples. We observed substantial variation in antigen expression and morphological endpoints across placental regions. We observed an association between PBDE concentrations and immunoreactivity of endovascular CTB staining with anti-ITGA1 (inverse) or interstitial CTBs staining with anti-CDH5 (positive). CONCLUSIONS: We found several molecular markers that may be sensitive placental indicators of PBDE exposure. Further, this indicates that placental biomarkers of development and disease could be useful barometers of exposure to PBDEs, a paradigm that could be extended to other environmental chemicals and placental stage-specific antigens.

Trisomy 21 is Associated with Caspase-2 Upregulation in Cytotrophoblasts at the Maternal-Fetal Interface.

Impaired placentation is implicated in poor perinatal outcomes associated with Trisomy 21. Earlier studies revealed abnormal cytotrophoblast differentiation along the invasive pathway as a contributing mechanism. To further elucidate the causes, we evaluated Caspase-2 expression at the protein level (immunolocalization and immunoblot) in samples from Trisomy 21 (n = 9) and euploid (n = 4) age-matched placentas. Apoptosis was investigated via the TUNEL assay. An immunolocalization approach was used to characterize Caspase-3, Fas (CD95), and Fas ligand in the same samples. Caspase-2 was significantly overexpressed in Trisomy 21 placentas, with the highest expression in villous cores and invasive cytotrophoblasts. Immunolocalization showed that Caspase-3 had a similar expression pattern as Caspase-2. Using the TUNEL approach, we observed high variability in the number of apoptotic cells in biopsies from different regions of the same placenta and among different placentas. However, Trisomy 21 placentas had more apoptotic cells, specifically in cell columns and basal plates. Furthermore, Caspase-2 co-immunolocalized with Fas (CD95) and FasL in TUNEL-positive extravillous cytotrophoblasts, but not in villous cores. These results help explain the higher levels of apoptosis among placental cells of Trisomy 21 pregnancies in molecular terms. Specifically, the co-expression of Caspase-2 and Caspase-3 with other regulators of the apoptotic process in TUNEL-positive cells suggests these molecules may cooperate in launching the observed apoptosis. Among trophoblasts, only the invasive subpopulation showed this pattern, which could help explain the higher rates of adverse outcomes in these pregnancies. In future experiments, this relationship will be further examined at a functional level in cultured human trophoblasts.

Cadmium induced p53-dependent activation of stress signaling, accumulation of ubiquitinated proteins, and apoptosis in mouse embryonic fibroblast cells.

The tumor suppressor oncoprotein, p53, is a critical regulator of stress-induced growth arrest and apoptosis. p53 activity is regulated through the ubiquitin proteasome system (UPS) with stress-induced disruption leading to increased accumulation of p53, resulting in growth arrest. In the present study, we investigate the role of p53 to determine sensitivity to cadmium (Cd) and whether induction of stress signaling responses and perturbation of the UPS are involved in Cd-induced cytotoxicity and apoptosis. We treated synchronously cultured p53 transgenic mouse embryonic fibroblasts, both wild-type p53+/+ and knockout p53-/- cells, with cadmium chloride (Cd, 0.5-20µM) for 24 h. Cd-induced cytotoxicity was assessed by cellular morphology disruption and neutral red dye uptake assay. Proteins in the stress signaling pathway, including p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK); ubiquitination, such as high-molecular weight of polyubiquitinated proteins (HMW-polyUb); and apoptotic pathways, were all measured. We found that Cd induced p53-dependent cytotoxicity in the p53+/+ cells, which exhibited a twofold greater sensitivity. We observed a dose-dependent stimulation of p38 MAPK and SAPK/JNK phosphorylation that corresponded to accumulation of HMW-polyUb conjugates and lead to the induction of apoptosis, as evidenced by the elevation of cleaved caspase-3. Our study suggests that Cd-mediated cytotoxicity and induction of stress signaling responses, elevated accumulation of HMW-polyUb conjugates, and resulting apoptosis are all dependent on p53 status.

Arsenic- and cadmium-induced toxicogenomic response in mouse embryos undergoing neurulation.

Arsenic (As) and cadmium (Cd) are well-characterized teratogens in animal models inducing embryotoxicity and neural tube defects (NTDs) when exposed during neurulation. Toxicological research is needed to resolve the specific biological processes and associated molecular pathways underlying metal-induced toxicity during this timeframe in gestational development. In this study, we investigated the dose-dependent effects of As and Cd on gene expression in C57BL/6J mouse embryos exposed in utero during neurulation (GD8) to identify significantly altered genes and corresponding biological processes associated with embryotoxicity. We quantitatively examined the toxicogenomic dose-response relationship at the gene level. Our results suggest that As and Cd induce dose-dependent gene expression alterations representing shared (cell cycle, response to UV, glutathione metabolism, RNA processing) and unique (alcohol/sugar metabolism) biological processes, which serve as robust indicators of metal-induced developmental toxicity and indicate underlying embryotoxic effects. Our observations also correlate well with previously identified impacts of As and Cd on specific genes associated with metal-induced toxicity (Cdkn1a, Mt1). In summary, we have identified in a quantitative manner As and Cd induced dose-dependent effects on gene expression in mouse embryos during a peak window of sensitivity to embryotoxicity and NTDs in the sensitive C57BL/6J strain.

Integrating genetic and toxicogenomic information for determining underlying susceptibility to developmental disorders.

To understand the complex etiology of developmental disorders, an understanding of both genetic and environmental risk factors is needed. Human and rodent genetic studies have identified a multitude of gene candidates for specific developmental disorders such as neural tube defects (NTDs). With the emergence of toxicogenomic-based assessments, scientists now also have the ability to compare and understand the expression of thousands of genes simultaneously across strain, time, and exposure in developmental models. Using a systems-based approach in which we are able to evaluate information from various parts and levels of the developing organism, we propose a framework for integrating genetic information with toxicogenomic-based studies to better understand gene-environmental interactions critical for developmental disorders. This approach has allowed us to characterize candidate genes in the context of variables critical for determining susceptibility such as strain, time, and exposure. Using a combination of toxicogenomic studies and complementary bioinformatic tools, we characterize NTD candidate genes during normal development by function (gene ontology), linked phenotype (disease outcome), location, and expression (temporally and strain-dependent). In addition, we show how environmental exposures (cadmium, methylmercury) can influence expression of these genes in a strain-dependent manner. Using NTDs as an example of developmental disorder, we show how simple integration of genetic information from previous studies into the standard microarray design can enhance analysis of gene-environment interactions to better define environmental exposure-disease pathways in sensitive and resistant mouse strains.

Methylmercury induced toxicogenomic response in C57 and SWV mouse embryos undergoing neural tube closure.

Methylmercury (MeHg) is a developmental neurotoxicant and teratogen and is hypothesized to perturb a wide range of biological processes, like other metals including arsenic (As) and cadmium (Cd). Common inbred mouse strains including C57 (sensitive) and SWV (resistant) display differences in sensitivity to metals such as As and Cd when exposed during neurulation. In this study, we investigated the impact of MeHg on neurulation, assessing for potential differences in sensitivity and associated toxicogenomic response in C57 and SWV mouse embryos. Parallel with morphological assessments of neural tube closure, we evaluated quantitative differences in MeHg-induced alterations in expression between strains at the gene level and within gene-enriched biological processes. Specifically, we observed differing sensitivities to MeHg-induced impacts on neural tube closure between C57 and SWV embryos in a time-dependent manner. These observations correlated with greater impact on the expression of genes associated with development and environmental stress-related pathways in the C57 compared to the SWV. Additional developmental parameters (e.g. mortality, growth effects) evaluated showed mixed significant effects across the two strains and did not support observations of differential sensitivity to MeHg. This study provides potential insights into MeHg-induced mechanisms of developmental toxicity, alterations associated with increased MeHg sensitivity and common biological processes affected by metals in embryos undergoing neurulation.

A system-based comparison of gene expression reveals alterations in oxidative stress, disruption of ubiquitin-proteasome system and altered cell cycle regulation after exposure to cadmium and methylmercury in mouse embryonic fibroblast.

Environmental and occupational exposures to heavy metals such as methylmercury (MeHg) and cadmium (Cd) pose significant health risks to humans, including neurotoxicity. The underlying mechanisms of their toxicity, however, remain to be fully characterized. Our previous studies with Cd and MeHg have demonstrated that the perturbation of the ubiquitin-proteasome system (UPS) was associated with metal-induced cytotoxicity and apoptosis. We conducted a microarray-based gene expression analysis to compare metal-altered gene expression patterns with a classical proteasome inhibitor, MG132 (0.5 microM), to determine whether the disruption of the UPS is a critical mechanism of metal-induced toxicity. We treated mouse embryonic fibroblast cells at doses of MeHg (2.5 microM) and Cd (5.0 microM) for 24 h. The doses selected were based on the neutral red-based cell viability assay where initial statistically significant decreases in variability were detected. Following normalization of the array data, we employed multilevel analysis tools to explore the data, including group comparisons, cluster analysis, gene annotations analysis (gene ontology analysis), and pathway analysis using GenMAPP and Ingenuity Pathway Analysis (IPA). Using these integrated approaches, we identified significant gene expression changes across treatments within the UPS (Uchl1 and Ube2c), antioxidant and phase II enzymes (Gsta2, Gsta4, and Noq1), and genes involved in cell cycle regulation pathways (ccnb1, cdc2a, and cdc25c). Furthermore, pathway analysis revealed significant alterations in genes implicated in Parkinson's disease pathogenesis following metal exposure. This study suggests that these pathways play a critical role in the development of adverse effects associated with metal exposures.

Embryonic toxicokinetic and dynamic differences underlying strain sensitivity to cadmium during neurulation.

Differences in sensitivity are observed between mouse strains, C57 (sensitive) and SWV (resistant) when exposed to cadmium (Cd) during the neurulation period. In this study, we investigated the toxicokinetics of Cd in relation with toxicodynamic responses to identify factors affecting differential Cd-sensitivity in C57 and SWV. Using a level of exposure which induced developmental toxicity and differential effects between strains, we assessed maternal and embryonic Cd uptake and evaluated biomarkers of response previously linked with Cd exposure, specifically metal ion regulators (Mt1, Mt2, DMT1) and markers of cell cycle arrest/apoptosis induction (p53, Cdkn1a, c-Casp3). Greater Cd uptake was observed in C57 embryos compared to SWV and these observations of differential uptake were associated with increased alterations in expression of biomarkers of metal response (e.g. c-Casp3) and strain sensitivity. Using sensitive and resistant mouse strains, we have identified toxicokinetic and dynamic differences which underlie observed differences in Cd embryonic sensitivity and response.

Cadmium-induced differential toxicogenomic response in resistant and sensitive mouse strains undergoing neurulation.

Common inbred mouse strains, such as the C57BL/6 (C57) and the SWV, display differences in sensitivity to environmental teratogens during gestation. For example, the C57 is more sensitive than the SWV to cadmium (Cd) exposure during neurulation, inducing a higher incidence of neural tube defects (NTDs). Here, we report, using Cd as a model teratogen, the first large scale toxicogenomic study to compare teratogen-induced gene expression alterations in C57 and SWV embryos undergoing neurulation, identifying toxicogenomic responses that associate with developmental toxicity and differential sensitivity. Using a systems-based toxicogenomic approach, comparing Cd-exposed and control C57 and SWV embryos (12- and 24-h postinjection [p.i.] [gestational day 8.0, ip]), we examined differentially expressed genes at multiple levels (biological process, pathway, gene) using Gene Ontology (GO) analysis, pathway mapping and cross-scatter plots. In both C57 and SWV embryos, we observed several gene expression alterations linked with cell cycle-related classifications, however, only in the C57 we observed upregulation of p53-dependent mediators Ccng1 and Pmaip1, previously associated with cell cycle arrest, apoptosis and NTD formation. In addition, we also identified a greater reduction in expression of nervous system development-related genes (e.g., Zic1, En2, Neurog1, Elavl4, Metrn, Nr2f1, Nr2f2) in the C57 compared to the SWV (12-h p.i.). In summary, our results indicate that differences in Cd-induced gene expression profiles between NTD resistant and sensitive strains within enriched biological processes (including developmental and cell cycle-related categories) associate with increased sensitivity to developmental toxicity as determined by observations of increased NTD formation, mortality (resorptions) and reduced fetal growth. Such observations may provide more detailed and useful mechanistic clues for identification of differences in life-stage specific teratogenic response.

Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways.

Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As(3+)) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53(+/+) and p53(-/-) mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53(-/-) cells than in the p53(+/+) cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As(3+). A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53(+/+) MEFs, As(3+) induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53(-/-) MEFs, As(3+) induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.