Advancing human health risk assessment: integrating recent advisory committee recommendations.

Over the last dozen years, many national and international expert groups have considered specific improvements to risk assessment. Many of their stated recommendations are mutually supportive, but others appear conflicting, at least in an initial assessment. This review identifies areas of consensus and difference and recommends a practical, biology-centric course forward, which includes: (1) incorporating a clear problem formulation at the outset of the assessment with a level of complexity that is appropriate for informing the relevant risk management decision; (2) using toxicokinetics and toxicodynamic information to develop Chemical Specific Adjustment Factors (CSAF); (3) using mode of action (MOA) information and an understanding of the relevant biology as the key, central organizing principle for the risk assessment; (4) integrating MOA information into dose-response assessments using existing guidelines for non-cancer and cancer assessments; (5) using a tiered, iterative approach developed by the World Health Organization/International Programme on Chemical Safety (WHO/IPCS) as a scientifically robust, fit-for-purpose approach for risk assessment of combined exposures (chemical mixtures); and (6) applying all of this knowledge to enable interpretation of human biomonitoring data in a risk context. While scientifically based defaults will remain important and useful when data on CSAF or MOA to refine an assessment are absent or insufficient, assessments should always strive to use these data. The use of available 21st century knowledge of biological processes, clinical findings, chemical interactions, and dose-response at the molecular, cellular, organ and organism levels will minimize the need for extrapolation and reliance on default approaches.

Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis.

Environmental exposures during sensitive windows of development can reprogram normal physiologic responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyperresponsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein, a soy phytoestrogen, and the plasticizer bisphenol A, differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. Whereas both genistein and bisphenol A induce genomic estrogen receptor (ER) signaling in the developing uterus, only genistein induced phosphoinositide 3-kinase (PI3K)/AKT nongenomic ER signaling to the histone methyltransferase enhancer of zeste homolog 2 (EZH2). As a result, this pregenomic signaling phosphorylates and represses EZH2 and reduces levels of H3K27me3 repressive mark in chromatin. Furthermore, only genistein caused estrogen-responsive genes in the adult myometrium to become hyperresponsive to hormone; estrogen-responsive genes were repressed in bisphenol A-exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by genistein promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, whereas bisphenol A did not. These data show that environmental estrogens have distinct nongenomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis.

Xenoestrogen-induced regulation of EZH2 and histone methylation via estrogen receptor signaling to PI3K/AKT.

Although rapid, membrane-activated estrogen receptor (ER) signaling is no longer controversial, the biological function of this nongenomic signaling is not fully characterized. We found that rapid signaling from membrane-associated ER regulates the histone methyltransferase enhancer of Zeste homolog 2 (EZH2). In response to both 17beta-estradiol (E2) and the xenoestrogen diethylstilbestrol, ER signaling via phosphatidylinositol 3-kinase/protein kinase B phosphorylates EZH2 at S21, reducing levels of trimethylation of lysine 27 on histone H3 in hormone-responsive cells. During windows of uterine development that are susceptible to developmental reprogramming, activation of this ER signaling pathway by diethylstilbestrol resulted in phosphorylation of EZH2 and reduced levels of trimethylation of lysine 27 on histone H3 in chromatin of the developing uterus. Furthermore, activation of nongenomic signaling reprogrammed the expression profile of estrogen-responsive genes in uterine myometrial cells, suggesting this as a potential mechanism for developmental reprogramming caused by early-life exposure to xenoestrogens. These data demonstrate that rapid ER signaling provides a direct linkage between xenoestrogen-induced nuclear hormone receptor signaling and modulation of the epigenetic machinery during tissue development.

Mitogenic signal transduction caused by monomethylarsonous acid in human bladder cells: role in arsenic-induced carcinogenesis.

Previous studies have shown that human bladder cells (UROtsa), a target of arsenic-induced cancer, can biotransform arsenite to monomethylarsonous acid (MMA(III)), which is more cytotoxic and capable of transforming the UROtsa cells following long-term, low-level exposure. Cyclooxygenase-2 (COX-2) causes hyperplasia in bladder cells and is considered a key biomarker in bladder cancer. To investigate the role of mitogenic pathway stimulation in MMA(III)-induced transformation, UROtsa cells were treated with 50nM MMA(III) for 12, 24, or 52 weeks and analyzed by Western blot for COX-2 expression. Elevations in COX-2 expression were noted following chronic MMA(III) exposure, and this induction increased with duration of exposure, suggesting that COX-2 or the signal transduction pathways responsible for COX-2 protein expression may play a role in MMA(III)-induced transformation. Acute exposure studies found MMA(III) treatment (10, 50, and 100nM, 4 h) induced COX-2 in UROtsa cells with the lowest doses (10 and 50nM) causing the strongest induction. Using pharmacological inhibitors of various pathways, it was shown that epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK-1/-2), phosphoinositide 3-kinase (PI3K), and src were important in the induction of COX-2 by MMA(III). ERK-2 phosphorylation was verified by Western blot analysis with a peak at 15 min, and c-jun was translocated to the nucleus following 50nM MMA(III) treatment. To determine MMA(III) targets, receptors of the erythroblastosis oncogene family (ErbB) family were further investigated. Chronic MMA(III) exposure led to upregulation of the EGFR or ErbB1. Short-term MMA(III) treatment caused the phosphorylation of ErbB2 in its autophosphorylation site. To verify the importance of these signaling pathways to the growth of the MMA(III)-transformed UROtsa cells in soft agar, various inhibitors were used to block pathways and monitor cells growth. Pathways of importance in anchorage-independent growth of UROtsa cells chronically exposed to MMA(III) are src, PI3K, and COX-1 and -2. As COX-2 is an important mediator that contributes to carcinogenesis via promotion of cell proliferation, inhibition of cell death, induction of angiogenesis, and facilitation of invasion, and it is highly upregulated both acutely and chronically in the MMA(III)-transformed cells, it is likely that activation of the mitogen-activated protein kinase pathway and increased COX-2 expression is a plausible mechanism for MMA(III) bladder carcinogenesis.

Monomethylarsonous acid induces transformation of human bladder cells.

Arsenic is a human bladder carcinogen. Arsenic is methylated to both monomethyl and dimethyl metabolites which have been detected in human urine. The trivalent methylated arsenicals are more toxic than inorganic arsenic. It is unknown if these trivalent methylated metabolites can directly cause malignant transformation in human cells. The goal of this study is determine if monomethylarsonous acid (MMA(III)) can induce malignant transformation in a human bladder urothelial cell line. To address this goal, a non-tumorigenic human urothelial cell line (UROtsa) was continuously exposed to 0.05 muM MMA(III) for 52 weeks. Hyperproliferation was the first phenotypic change observed in exposed UROtsa (URO-MSC). After 12 weeks of exposure, doubling time had decreased from 42 h in unexposed control cells to 27 h in URO-MSC. Hyperproliferation continued to be a quality possessed by the URO-MSC cells after both 24 and 52 weeks of exposure to MMA(III), which had a 40-50% reduction in doubling time. Throughout the 52-week exposure, URO-MSC cells retained an epithelial morphology with subtle morphological differences from control cells. 24 weeks of MMA(III) exposure was required to induce anchorage-independent growth as detected by colony formation in soft agar, a characteristic not found in UROtsa cells. To further substantiate that malignant transformation had occurred, URO-MSC cells were tested after 24 and 52 weeks of exposure to MMA(III) for the ability to form tumors in SCID mice. Enhanced tumorigenicity in SCID mouse xenografts was observed after 52 weeks of treatment with MMA(III). These observations are the first demonstration of MMA(III)-induced malignant transformation in a human bladder urothelial cell line and provide important evidence that MMA(III) may be carcinogenic in human tissues.

Effects of arsenite on UROtsa cells: low-level arsenite causes accumulation of ubiquitinated proteins that is enhanced by reduction in cellular glutathione levels.

Chronic arsenic exposure increases risk for the development of diabetes, vascular disease, and cancers of the skin, lung, kidney, and bladder. This study investigates the effects of arsenite [As(III)] on human urothelial cells (UROtsa). As(III) toxicity was determined by exposing confluent UROtsa cells to As(III) (0.5-200 microM). Depleting cellular glutathione levels with buthionine sulfoximine (BSO) potentiated the toxicity of As(III). Cell viability was assessed with the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. UROtsa cell ability to biotransform As(III) was determined by dosing cells with environmentally relevant concentrations of As(III) followed by HPLC/ICP-MS analysis of cell media and lysate. Both pentavalent and trivalent monomethylated products were detected. Although cytotoxicity was observed at high doses of As(III) (approximately 100 microM) in UROtsa cells, perturbations of a variety of molecular processes occurred at much lower doses. Exposure to low-level As(III) (0.5-25 microM) causes an accumulation of ubiquitin (Ub)-conjugated proteins. This effect is enhanced when cellular glutathione levels have been reduced with BSO treatment. Because As(III) has many effects on UROtsa cells, a greater understanding of how As(III) is affecting cellular proteins in a target tissue will lead to a better understanding of the mechanism of toxicity and pathogenesis for low-level As(III).