Joint Action Toxicity of Arsenic (As) and Lead (Pb) Mixtures in Developing Zebrafish

Abstract

Arsenic (As) and lead (Pb) are environmental pollutants found in common sites and linked to similar adverse health effects. Multiple studies have investigated the toxicity of each metal individually or in complex mixtures. Studies defining the joint interaction of a binary exposure to As and Pb, especially during the earliest stages of development, are limited and lack confirmation of the predicted mixture interaction. We hypothesized that a mixture of As (iAsIII) and Pb will have a concentration addition (CA) interaction informed by common pathways of toxicity of the two metals. To test this hypothesis, developing zebrafish (1-120 h post fertilization; hpf) were first exposed to a wide range of concentrations of As or Pb separately to determine 120 hpf lethal concentrations. These data were then used in the CA and independent action (IA) models to predict the type of mixture interaction from a co-exposure to As and Pb. Three titration mixture experiments were completed to test prediction of observed As and Pb mixture interaction by keeping the Pb concentration constant and varying As concentrations in each experiment. The prediction accuracy of the two models was then calculated using the prediction deviation ratio (PDR) and Chi-square test and regression modeling applied to determine type of interaction. Individual metal exposures determined As and Pb concentrations at which 25% (39.0 ppm Pb, 40.2 ppm As), 50% (73.8 ppm Pb, 55.4 ppm As), 75% (99.9 ppm Pb, 66.6 ppm As), and 100% (121.7 ppm Pb, 77.3 ppm As) lethality was observed at 120 hpf. These data were used to graph the predicted mixture interaction using the CA and IA models. The titration experiments provided experimental observational data to assess the prediction. PDR values showed the CA model approached 1, whereas all PDR values for the IA model had large deviations from predicted data. In addition, the Chi-square test showed most observed results were significantly different from the predictions, except in the first experiment (Pb LC₂₅ held constant) with the CA model. Regression modeling for the IA model showed primarily a synergistic response among all exposure scenarios, whereas the CA model indicated additive response at lower exposure concentrations and synergism at higher exposure concentrations. The CA model was a better predictor of the Pb and As binary mixture interaction compared to the IA model and was able to delineate types of mixture interactions among different binary exposure scenarios.

Keywords: arsenic; developmental toxicity; lead; metal; mixtures; zebrafish.

Figure 2

Percent mortality assessed every 24 h from 1–120 h post fertilization (hpf) for Pb and As. Single chemical exposures for Pb (A) and As (B). N = 4 biological replicates for each treatment group with 50 subsamples per each treatment group. Data is presented as mean ± standard deviation. * p < 0.05.

Figure 1

Experimental overview of study. Zebrafish embryos were dosed with a concentration range of each metal to determine lethal concentrations (LC) for benchmark treatment groups in the mixture titration experiments and for predictive mathematical modeling. The expected and observed results were compared among the mathematical models using the prediction deviation ratio and Chi-square test. Type of mixture interaction was determined using regression modeling.

Figure 3

Percent mortality for mixture experiments assessed every 24 h from 1–120 h post fertilization (hpf). Mixture experiments in which Pb was held constant at the 120 hpf-LC₂₅ (39.0 ppm) in experiment 1 (A) and in which Pb was held constant at the 120 hpf-LC₅₀ (73.76 ppm) in experiment 2 (B), while As was varied to include the 120 hpf-LC₂₅, 120 hpf-LC₅₀, and 120 hpf-LC₇₅. N = 3 biological replicates for each treatment group with 50 subsamples per each treatment group. Data is presented as mean ± standard deviation. * p < 0.05.

Figure 4

Comparison of prediction deviation ratio (PDR) of mixtures for the CA and IA models. For the CA model (A), each exposure scenario in each experiment was evaluated for PDR values that approached 1. The IA model (B) is limited to evaluating metal mixtures at the same LC percentage for approaching 1. N = 3 biological replicates for each treatment group with 50 subsamples per each treatment group.

Figure 5

Comparison of predicted lethality using the Independent Action (IA) and Concentration Addition (CA) models. The log of the prediction deviation ratios (PDR) of mixtures for the CA and IA models. N = 3 biological replicates for each treatment group with 50 subsamples per each treatment group.

Figure 6

Observed and predicted mortality using the concentration addition (CA) model. Developing zebrafish were exposed to the metal mixtures from 1 to 120 h post fertilization (hpf) and mortality recorded. Results were compared to predicted mortality based on CA models when the Pb LC₂₅ (39.0 ppm, mg/L) concentration was held constant (A) or when the Pb LC₅₀ (73.76 ppm) concentration was held constant (B). Similarity of predicted and observed curves indicates additive interaction (within in shaded region) (A), where upper deviation from predicted curve suggests synergistic response (B). N = 3 biological replicates per treatment group with 50 subsamples per biological replicate.

Figure 7

Observed and predicted mortality using the independent action (IA) model. Developing zebrafish were exposed to the metal mixtures from 1 to 120 h post fertilization (hpf) and mortality recorded. Results were compared to predicted mortality based on the IA model, where the predicted LC of each metal is equal. The upper deviation of the observed curve compared to the predicted curve suggests a synergistic response. N = 3 biological replicates per treatment group with 50 subsamples per biological replicate.