In Vitro Human Dermal Absorption Studies on Pesticides in Complex Mixtures: Investigation of Guidance Criteria and Possible Impact Parameters.

Pesticides must not pose unacceptable risks to human health, so risk assessments are conducted before products are authorised. Dermal exposure is often the main route of intake, so estimating realistic and trustworthy dermal absorption values is crucial for risk assessment. Although there are agreed test guidelines for in vitro dermal absorption studies, not every product is tested due to cost reasons. The present dataset consists of 945 individual in vitro experiments on the dermal absorption of human skin with 179 active substances of pesticides in 353 different mixtures, including concentrates and dilutions. The dataset was evaluated to identify the possible impacts of experimental conditions and physico-chemical properties on dermal absorption. The dataset was also analysed to assess the appropriateness of the pro rata correction for untested dilutions, and the set concentration cut-off to decide on the dilution status for choosing a default value on dermal absorption. The study found that the implementation of specific guidelines improved the harmonisation of study conduct, with support for approaches such as pro rata correction and default values. Further analysis of the specific co-formulants may identify influencing factors that may be more important than the experimental variables.

A comparative assessment of the CLP calculation method and in vivo testing for the classification of plant protection products.

In Europe, animal testing for the purpose of regulatory plant protection product (PPP) assessment should be undertaken only as a last resort. Nevertheless, there is a need to improve the acceptance of alternative methods, which has been slow due to a lack of data regarding the predictivity of in vivo effects. The CLP calculation method is an alternative method based on the concentration addition of all adverse substances in a mixture. It is often applied as a conservative approach for the estimation of toxicodynamic interactions. However, PPPs consist of pesticides and co-formulants, which in combination can also exhibit altered toxicokinetic properties. Our analysis revealed that oral and inhalation toxicity was underestimated for approximately 45% of the in vivo classified products by the CLP calculation method as compared to in vivo testing. With regard to skin and eye irritation, the CLP calculation method underestimated the irritating potential in 22% and 6% of PPPs, respectively. Based on specific concentration limits, skin sensitisation was underestimated in 34% of PPPs. Similar false negative rates have been reported for PPP in vitro testing. Hence, we suggest the development of an integrated assessment strategy, weighing all available information and considering relevant parameters influencing predictivity and uncertainty.

Illustrative case using the RISK21 roadmap and matrix: prioritization for evaluation of chemicals found in drinking water.

The HESI-led RISK21 effort has developed a framework supporting the use of twenty-first century technology in obtaining and using information for chemical risk assessment. This framework represents a problem formulation-based, exposure-driven, tiered data acquisition approach that leads to an informed decision on human health safety to be made when sufficient evidence is available. It provides a transparent and consistent approach to evaluate information in order to maximize the ability of assessments to inform decisions and to optimize the use of resources. To demonstrate the application of the framework's roadmap and matrix, this case study evaluates a large number of chemicals that could be present in drinking water. The focus is to prioritize which of these should be considered for human health risk as individual contaminants. The example evaluates 20 potential drinking water contaminants, using the tiered RISK21 approach in combination with graphical representation of information at each step, using the RISK21 matrix. Utilizing the framework, 11 of the 20 chemicals were assigned low priority based on available exposure data alone, which demonstrated that exposure was extremely low. The remaining nine chemicals were further evaluated, using refined estimates of toxicity based on readily available data, with three deemed high priority for further evaluation. In the present case study, it was determined that the greatest value of additional information would be from improved exposure models and not from additional hazard characterization.

WNT10B/ß-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer.

Wnt/ß-catenin signalling has been suggested to be active in basal-like breast cancer. However, in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of ß-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active ß-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical ß-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/ß-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.

ERα regulates lipid metabolism in bone through ATGL and perilipin.

A decrease in bone mineral density during menopause is accompanied by an increase in adipocytes in the bone marrow space. Ovariectomy also leads to accumulation of fat in the bone marrow. Herein we show increased lipid accumulation in bone marrow from estrogen receptor alpha (ERα) knockout (ERαKO) mice compared to wild-type (WT) mice or estrogen receptor beta (ERß) knockout (ERßKO) mice. Similarly, bone marrow cells from ERαKO mice differentiated to adipocytes in culture also have increased lipid accumulation compared to cells from WT mice or ERßKO mice. Analysis of individual adipocytes shows that WT mice have fewer, but larger, lipid droplets per cell than adipocytes from ERαKO or ERßKO animals. Furthermore, higher levels of adipose triglyceride lipase (ATGL) protein in WT adipocytes correlate with increased lipolysis and fewer lipid droplets per cell and treatment with 17ß-estradiol (E2) potentiates this response. In contrast, cells from ERαKO mice display higher perilipin protein levels, promoting lipogenesis. Together these results demonstrate that E2 signals via ERα to regulate lipid droplet size and total lipid accumulation in the bone marrow space in vivo.

ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis.

The benefits of estrogens on bone health are well established; how estrogens signal to regulate bone formation and resorption is less well understood. We show here that 17ß-estradiol (E2)-induced apoptosis of bone-resorbing osteoclasts is mediated by cleavage and solubilization of osteoblast-expressed Fas ligand (FasL). U2OS-ERα osteoblast-like cells expressing an EGFP-tagged FasL at the C-terminus showed decreased fluorescence after E2 treatment, indicative of a cleavage event. Treatment of U2OS-ERα cultures with a specific MMP3 inhibitor in the presence of E2 blocked FasL cleavage and showed an increase in the number of EGFP-FasL+ cells. siRNA experiments successfully knocked down MMP3 expression and restored full-length FasL to basal levels. E2 treatment of both human and murine primary osteoblasts showed upregulation of MMP3 mRNA expression, and calvarial organ cultures showed increased expression of MMP3 protein and colocalization with the osteoblast-specific RUNX2 after E2 treatment. In addition, osteoblast cell cultures derived from ERαKO mice showed decreased expression of MMP3 but not MMP7 and ADAM10, two known FasL proteases, demonstrating that ERα signaling regulates MMP3. Also, conditioned media of E2-treated calvarial osteoblasts showed an approximate sixfold increase in the concentration of soluble FasL, indicating extensive cleavage, and soluble FasL concentrations were reduced in the presence of a specific MMP3 inhibitor. Finally, to show the role of soluble FasL in osteoclast apoptosis, human osteoclasts were cocultured with MC3T3 osteoblasts. Both a specific MMP3 inhibitor and an MMP inhibitor cocktail preserved osteoclast differentiation and survival in the presence of E2 and demonstrate the necessity of MMP3 for E2-induced osteoclast apoptosis. These experiments further define the molecular mechanism of estrogen's bone-protective effects by inducing osteoclast apoptosis through upregulation of MMP3 and FasL cleavage.

Tissue-Specific Effects of Loss of Estrogen during Menopause and Aging.

The roles of estrogens have been best studied in the breast, breast cancers, and in the female reproductive tract. However, estrogens have important functions in almost every tissue in the body. Recent clinical trials such as the Women's Health Initiative have highlighted both the importance of estrogens and how little we know about the molecular mechanism of estrogens in these other tissues. In this review, we illustrate the diverse functions of estrogens in the bone, adipose tissue, skin, hair, brain, skeletal muscle and cardiovascular system, and how the loss of estrogens during aging affects these tissues. Early transcriptional targets of estrogen are reviewed in each tissue. We also describe the tissue-specific effects of selective estrogen receptor modulators (SERMs) used for the treatment of breast cancers and postmenopausal symptoms.

Altered tissue distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts in mice transgenic for human sulfotransferases 1A1 and 1A2.

Soluble sulfotransferases (SULTs) generate electrophilically reactive metabolites from numerous food-borne compounds, environmental contaminants and drugs, often resulting in mutagenicity and carcinogenicity. Substrate specificity, regulation and tissue distribution of SULTs show large interspecies differences. In humans, therefore, SULTs may be involved in the induction of cancer in different tissues than in standard animal models. To construct a rodent model taking some species differences into account, we transferred a 68.5 kb human (h) genomic sequence that comprised the transcribed and long flanking regions of SULT1A1 and 1A2 into murine oocytes. This approach resulted in several mouse lines expressing these human genes in a copy number-dependent manner with a tissue distribution similar to that in humans. In previous in vitro studies, we had demonstrated that human SULT1A1 and 1A2 efficiently catalyze the terminal activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a mutagen. The transgenic mice were used to study the hSULT1A1/1A2-mediated activation. Tissue distribution and levels of DNA adducts were determined in hSULT1A1/1A2 transgenic and wild-type mice after an oral dosage of PhIP. Transgenic mice exhibited significantly elevated PhIP-DNA adduct levels compared with the wild-type in liver (13-fold), lung (3.8-fold), colon (2-fold), kidney (1.6-fold) and cecum (1.5-fold). Moreover, among the eight tissues examined, liver was the one with the lowest and highest adduct levels in wild-type and transgenic mice, respectively. Hence, expression of hSULT1A1/1A2 not only enhanced the genotoxicity but also substantially changed the organotropism of PhIP.