Predictive Environmental Risk Assessment of Chemical Mixtures: A Conceptual Framework

Environmental risks of chemicals are still often assessed substance-by-substance, neglecting mixture effects. This may result in risk underestimations, as the typical exposure is toward multicomponent chemical “cocktails”. We use the two well established mixture toxicity concepts (Concentration Addition (CA) and Independent Action (IA)) for providing a tiered outline for environmental hazard and risk assessments of mixtures, focusing on general industrial chemicals and assuming that the “base set” of data (EC50s for algae, crustaceans, fish) is available. As mixture toxicities higher than predicted by CA are rare findings, we suggest applying CA as a precautious first tier, irrespective of the modes/mechanisms of action of the mixture components. In particular, we prove that summing up PEC/PNEC ratios might serve as a justifiable CA-approximation, in order to estimate in a first tier assessment whether there is a potential risk for an exposed ecosystem if only base-set data are available. This makes optimum use of existing single substance assessments as more demanding mixture investigations are requested only if there are first indications of an environmental risk. Finally we suggest to call for mode-of-action driven analyses only if error estimations indicate the possibility for substantial differences between CA- and IA-based assessments

wnt5a and sFRP5 serum concentrations in lean control subjects and patients with obesity.

<p>Serum samples were taken after an overnight fast and wnt5a and sFRP5 serum concentrations were measured by ELISA. Shown are means±SEM for n = 12 lean control subjects and n = 23 patients with obesity. In order to test for statistical significance, student's t-test was used, ns = not significant.</p

Adipokine, wnt5a and sFRP5 serum concentrations before, during (1 month) and after 3 months of caloric restriction.

<p>Adipokine, wnt5a and sFRP5 serum concentrations before, during (1 month) and after 3 months of caloric restriction.</p

Anthropometric measurements before, during (1 month) and after 3 months of caloric restriction: Body composition was estimated by impedance analysis.

<p>Shown are means±SEM of n = 23 obese human subjects during a VLCD. Statistical significance was tested by student's t-test, ns = not significant.</p

Is a specific mixture assessment factor (MAF) the right way forward for regulatory risk assessment?

<p>Copy of a presentation from the SETAC annual meeting in Barcelona in 2015. A detailed report on the issue will be published by the Swedish Chemicals Agency in the near future, check for download link at kemi.se or thomasbackhaus.eu from roughly June 2015 onward.</p> <p>Comments and questions are welcome!</p

(A+B) Insulin resistance before, during (1 month) and after 3 month of caloric restriction: In the present study insulin resistance was measured by the HOMA-IR index (A) and the leptin-to-adiponectin ration (LAR) (B) since the later parameter has been shown to be closely correlated to measures of insulin resistance by hyperinsulinemic euglycemic clamp experiments [<b>16</b>].

<p>Data are given as means±SEM of n = 23 obese human subjects during a VLCD. Significance was tested by student's t-test. (<b>C</b>) <i>sFRP serum concentrations before, during (1 month) and after 3 months of caloric restriction</i>: sFRP5 serum concentrations were determined before (0 month), during (1 month) and after (3 months) of a VLCD. In this figure the levels are shown as fold increase compared to 0 month. The raw data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032437#pone-0032437-t002" target="_blank">table 2</a>. Shown are means±SEM of n = 23 obese human subjects during a VLCD. t-test was used to test for statistical significance.</p

G- and F-Actin in RP1-expressing cells.

<p><b>3A</b> G- and F-actin content of 10×10⁵ constitutively RP1 expressing cells were measured by FACS analysis. G-Actin (green) was measured in the FL-1 channel (Fluor 488, green) and F-Actin in the FL-3 channel (phalloidin rodamine staining, red). The upper left quadrant of each panel represents the F-actin pool, the upper right quadrant the G-Actin pool. The top three panels are the controls: Upper left panel, unstained control cells. Upper middle panel: Boiled fluoresceine conjugated DNAse I unable to bind G-Actin serving as a negative control. Upper right panel: Double staining of HEK293 cells with Fluor 488 conjugated DNAse I and Phalloidin-Rhodamine carrying the empty pEAK8 vector to determine the general content of G-actin and F-actin pools as reference. The bottom panels show the respective degree of G-actin decrease seen in the RP1-wt, ALA, ASP containing cell lines. <b>3B</b> Quantification of G-Actin content in RP1 expressing HEK293 cells compared to mock transfected cells from <b>3A</b>. Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p = 0.0001;*p = 0.0003).</p

Shear Stress experiments.

<p>2A Analysis of shear stress-dependent adhesion of RP1 mutants on endothelial cells under flow 1×10⁵ HEK293 cells stably transfected with different RP1 mutants were allowed to settle for 3 min on parallel plate flow chambers with pre grown confluent HUVECs. Subsequently, preheated HBSS/0.1% BSA was flushed through the chambers at the indicated calculated shear stress, and shear stress levels were increased every 30 s. Photographs were taken and adherent cells were counted in four fields for every condition. Cell line with empty vector (black squares), RP1 wild type (wt) (black circles), RP1-ALA²³⁶ (ALA) mutant (white circles), RP1-ASP²³⁶ (ASP) mutant (white squares). Values are means of n = 5–6+/− SEM. Asterisks denote statistically significant differences *p<0.05 or **p<0.01 between parental cell line and ASP mutant as determined by a two-tailed t-test. <b>2B Analysis of RP1 expression under fluid shear stress</b> Native HEK293 cells were exposed to fluid shear stress or simply cultured (control). Thereafter, cells were lysed and total protein from the lysates was employed in immunoprecipitation of RP1. Endogenous RP1 was detected by an RP1 specific antibody. α-tubulin served as a loading control. RP-1protein detected by Western blot was quantified using the ImageJ software. Asterisks mark statistically significant differences **p<0.01 between sheared and non-sheared cells as determined by a two-tailed t-test. <b>2C </b><b>Analysis of RP1 phosphorylation under fluid shear stress</b> HEK293 cells overexpressing RP1-wt were exposed to 1dynes/cm² shear stress. From cell lysate RP1 was immunoprecipitated and subjected to Western blotting. In parallel the phosphorylation status of RP1 was detected with an anti phospho-serine antibody (anti PS). As control HEK293 cells overexpressing RP1-wt were cultured without shear stress and otherwise processed alike. Total RP1 (RP1) expression served as loading control. The difference between phosphorylation intensity in sheared versus control cells was statistically significant (**, p<0.05). <b>2D Analysis of RP1 shRNA regulated cells and RP1 phosphor-mutants under fluid shear stress</b> HEK293 containing empty vector or various mutants were exposed to increasing shear stress. The curves show the percentage of adhering cells under different shear stress intensity (0, 1.5, 4.5 and 8.5 dynes/cm2) on control cells transfected with irrelevant shRNA served as a reference (black boxes) and were compared to RP1 specific shRNA bearing cells and the mutant cell lines RP1-ALA236, RP1-ASP236. A significant gain of cell adhesion is seen for RP1 specific shRNAs (white boxes). The statistical differences of adherent cells between the depicted cell lines and the control cells are marked with Asterisks *p<0.05 or **p<0.01.</p

Cadherin expression in RP1-mutants.

<p><b>4A</b> N-cadherin levels of HEK293 cells containing empty vector (c), wt, ALA and ASP were determined by immunoblotting with an N-terminal N-cadherin antibody. The middle panel shows a processed N-cadherin fragment (named CTF1) detected by a fragment specific antibody in respective lysates. The lower panel shows the β-tubulin loading control. <b>4B</b> The Western blot signals of <b>4A</b> were quantified using the Image-J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997–2008). Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001) comparing control versus wt and mutants regarding complete N-cadherin and comparing wt versus mutants regarding N-cadherin cleavage fragment (CTF1). In the right panel, the empty c lane indicates no detectable CTF in control cells. <b>4C</b> N-cadherin levels were measured by incubation with a monoclonal antibody directed against the cytoplasmic tail and subsequent FACS analysis. The negative control (yellow line) was incubated with secondary antibody only. The positive control (red) was empty vector containing HEK293 cells. The results for HEK293 expressing RP1-wt are depicted in black, RP1-ALA²³⁶ in green and RP1-ASP²³⁶ in blue. <b>4D</b> Quantification of N-cadherin levels from FACS analysis<b>.</b> Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001).</p

Binding and Phosphorylation of RP1 by CK2.

<p><b>1A</b> Identification of CK2 phosphorylation site - RP1-sequence (amino acid), three potential CK2 kinase sites S59, S72, S236 (underlined) were identified by prosite scan (<a href="http://www.expasy.ch" target="_blank">www.expasy.ch</a>). The peptides used for in vitro experiments (1C) are marked in bold. S236 the actual CK2 phosphorylation site is shown in red. <b>1B</b> Interaction–assay RP1/CK2 - Endogenous RP1 (first panel) was co-precipitated with its potential binding partners. RP1/CK2 kinase interaction could be detected by specific α/ß CK2 subunit antibodies. The black wedges in this panel indicate increasing stringency of washing procedure (% Tween20/PBS). In a reverse experiment (right side panel), endogenous RP1 was verified as genuine CK2 binding substrate. By using CK2 subunits as baits, RP1 could be detected in the pulldowns by its specific RP1 antibody (right panel). No signal was seen when an insignificant IgG antibody was used. On the far right 1/10 of cell lysate of the foregoing experiments is depicted as an input control. The black wedges in this panel indicate stringency of the washing procedure (0.01% and 0.3% Tween/PBS). <b>1C</b> Biotinylated peptides (A: aa54–65, B: aa70–80, C: aa229–240) containing the potential CK2 phosphorylation sites S<i>⁵⁹</i>, S<i>⁷²</i>, S<i>²³⁶ were</i> synthesized and tested as CK2 phosphorylation substrates (A, B, C, 3 µg each) in an <i>in vitro</i> phosphorylation assay. A known positive CK2 kinase site peptide (DDDDSDDDDD, 3 µg) served as a control. The black wedge indicates incubation times (minutes). <b>1D</b> CK2 kinase assay - Recombinant CK2 and ³³P-gamma-ATP were incubated in vitro with different amounts of RP1-wt protein (first panel shows a coomassie stain of his-tagged purified RP1 protein used for the assay) and phosphorylation was measured by autoradiography (middle panel). The amounts of RP1 protein used are indicated above the middle panel. Autophosphorylation of CK2 at its subunit ß served as positive control RP1-ALA236 mutated protein (ALA) was almost non-phosphorylated in comparison to the wild type protein (right side upper panel). The lower panel on the right side shows a coomassie stain representing the amount of RP1 used for this experiment.</p