Effects of carbamazepine on the central nervous system of zebrafish at human therapeutic plasma levels

Abstract

The fish plasma model (FPM) facilitated the environmental risk assessment of human drugs by using existing data on human therapeutic plasma concentrations (H_TPCs) and predicted fish plasma concentrations (FPCs). However, studies on carbamazepine (CMZ) with both the mode of action (MOA) based biological effects at molecular level (such as neurotransmitter and gene level) and measured FPCs are lacking. Bioconcentration of CMZ in adult zebrafish demonstrated that the FPM underestimated the bioconcentration factors (BCFs) in plasma at environmental CMZ exposure concentrations (1-100 μg/L). CMZ significantly increased Glu and GABA, decreased ACh and AChE as well as inhibited the transcription levels of gabra1, grin1b, grin2b, gad1b, and abat when the actual FPCs were in the ranges of 1/1000 H_TPC to H_TPC. It is the first read-across study of CMZ integrating MOA-based biological effects at molecular level and FPCs. This study facilitates model performance against a range of different drug classes.

Keywords: Animal physiology; Biochemistry; Biological sciences; Physiology.

Graphical abstract

Figure 1

Fish-muscle uptake of CMZ in 7-day exposure at various concentrations of CMZ All the CMZ concentrations were detected by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After reaching the first peak at 6–12 h, the concentration of CMZ decreased at 12–24 h. After that, the concentration rised again, probably to a second peak. Error bars represent as mean ± SD (n = 3).

Figure 2

Bioconcentration of CMZ in fish plasma (μg/L) and muscle (μg/kg) after 7-day exposure with various external concentrations (10, 100, and 1000 μg/L) All the CMZ concentrations were detected by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The CMZ concentrations in the muscle increased with the corresponding measured plasma concentrations, but were significantly lower than those in plasma. Error bars represent as mean ± SD (n = 3).

Figure 3

Relationship between predicted and measured fish plasma concentrations (FPCs) of CMZ in zebrafish following a 7-day study The predicted plasma concentration is based on Equations 1 and 2. All the plasma concentrations were detected by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). (A) Relationship between predicted and measured FPC. The predicted FPCs at 1, 10, and 100 μg/L were lower than the actual measured FPCs. The measured FPCs were less than the maximum human therapeutic concentration (H_TPC, 12000 μg/L). The gray area in figure (A) indicates the H_TPC range: 4000–12000 μg/L. (B) Relationship between log predicted FPC and log measured FPC. The measured FPCs were in the range of 10 times the predicted FPCs. The red dashed line denotes log unit deviations. The black dashed line is 1:1 line. External exposure concentration for each plasma concentration: □: 1 μg/L; : 10 μg/L; : 100 μg/L; : 1000 μg/L. Error bars represent as mean ± SD (n = 3).

Figure 4

Neurotransmitters and AChE activity in zebrafish were affected after 7-day exposure in four concentration levels of CMZ Neurotransmitters and AChE activity in zebrafish were affected after 7-day exposure in four concentration levels of CMZ. Neurotransmitters (A–C) and AChE activity (D) were evaluated via enzyme-linked immunosorbent assay (ELISA). CMZ significantly decreased ACh (A) and AChE activity (D), increased Glu (B) and GABA(C). External concentration: EC; the corresponding fish plasma concentration: FPC. Error bars represent as mean ± SD. (n = 3; Duncan test: ∗: p < 0.05, ∗∗: p < 0.01).

Figure 5

CMZ inhibited the expression of neurotransmitter-related genes when the actual FPCs were in the ranges of 1/1000 H_TPC to H_TPC CMZ inhibited the expression of neurotransmitter-related genes when the actual FPCs were in the ranges of 1/1000 H_TPC to H_TPC. The expression of abat (A), gabra1 (B), gad1b (C), grin1b (D), and grin2b (E) genes in zebrafish after 7-day exposure at four concentration levels of CMZ were determined by real-time PCR. External concentrations: ECs; the corresponding fish plasma concentrations: FPCs. Error bars represent as mean ± SD. (n = 3; Duncan test ∗: p < 0.05, ∗∗: p < 0.01).