Genetic variability affects absolute and relative potencies and kinetics of the anesthetics isoflurane and sevoflurane in Drosophila melanogaster

Abstract

Genetic variability affects the response to numerous xenobiotics but its role in the clinically-observed irregular responses to general anesthetics remains uncertain. To investigate the pharmacogenetics of volatile general anesthetics (VGAs), we developed a Serial Anesthesia Array apparatus to expose multiple Drosophila melanogaster samples to VGAs and behavioral assays to determine pharmacokinetic and pharmacodynamic properties of VGAs. We studied the VGAs isoflurane and sevoflurane in four wild type strains from the Drosophila Genetic Reference Panel, two commonly used laboratory strains (Canton S and w ¹¹¹⁸ ), and a mutant in Complex I of the mitochondrial electron transport chain (ND23 ⁶⁰¹¹⁴ ). In all seven strains, isoflurane was more potent than sevoflurane, as predicted by their relative lipid solubilities, and emergence from isoflurane was slower than from sevoflurane, reproducing cardinal pharmacokinetic and pharmacodynamic properties in mammals. In addition, ND23 ⁶⁰¹¹⁴ flies were more sensitive to both agents, as observed in worms, mice, and humans carrying Complex I mutations. Moreover, we found substantial variability among the fly strains both in absolute and in relative pharmacokinetic and pharmacodynamic profiles of isoflurane and sevoflurane. These data indicate that naturally occurring genetic variations measurably influence cardinal pharmacologic properties of VGAs and that flies can be used to identify relevant genetic variations.

Figure 1

The Serial Anesthesia Array. (A) Main components of the array are an agent-specific anesthetic vaporizer, flow meters, and a serial array of anesthetizing positions. The small footprint allows housing within a conventional fume hood. A compressed air cylinder supplies air via a pressure regulator to the flow meter. The vaporizer controls the vaporization of liquid anesthetics and accurately delivers a given concentration to the air flowing from the air cylinder. Anesthetizing positions are assembled out of 50 mL conical tubes with customized caps fitted with input and output ports made of polystyrene pipettes. The tubes are interconnected via the external pipette ends with Tygon tubing. Within each tube the upstream (inflow) port opening is positioned closer to the bottom than the downstream (outflow) port to facilitate complete gas exchange (inset diagram). A fine mesh covers outflow ports to prevent flies from escaping. The number of positions in the array is flexible. The standard 8 + 1 configuration is shown. (B) The first position in the array contains water to humidify the mixture of air and VGA, and the remaining positions are available for experimental samples. A flow indicator at the downstream end of the array monitors the activity of the system. (C) Quantification of the rise in ISO concentration in the downstream tube 8 with air flowing at 1.5 L/min. Black circles indicate gas chromatograph measurements of ISO concentration in tube 8 over time (n ≥ 2, R² = 0.95). Gray triangles indicate the theoretical change in anesthetic concentration calculated using the equation C_Array = C_AF(1-e^−T/τ) (see Materials and Methods for details), assuming a single compartment of equivalent volume.

Figure 2

All positions in the Serial Anesthesia Array receive an equivalent concentration of VGA. (A) The Percent Active of male w¹¹¹⁸ flies exposed to 0.4% ISO at 1–8 days of age for 10 min in the most upstream (tube 1) and downstream (tube 8) positions (n = 9) (P = 0.77, unpaired equal variance two-tail t-test). (B) The Early Recovery Time of mixed sex w¹¹¹⁸ flies exposed to 2% ISO for 1 h at 0–7 days of age in tubes 1–8 (n = 2) (P = 0.36, one-way ANOVA).

Figure 3

Sex but not age influence the speed of recovery from anesthesia. (A) The Early Recovery Time of mixed sex w¹¹¹⁸ flies exposed to anesthesia at the indicated age ranges (n = 5) (ISO P = 0.98 and SEVO P = 0.89, one-way ANOVA). (B) The Early Recovery Time of w¹¹¹⁸ male (M) and female (F) flies exposed to anesthesia at 1–8 days old (n = 3) (**P < 0.01, ***P < 0.001, unpaired equal variance two-tail t-test).

Figure 4

Deep anesthesia does not affect lifespan. Survival curves for mixed sex w¹¹¹⁸ flies exposed to (A) 2% ISO (264–289 flies) or (B) 3.5% SEVO (264–285 flies) for 1 hr (red) or 5 hr (green) at 0–7 days old. (C) Median and (D) maximum lifespans for exposure times from 0.5 to 5.0 hr (n = 5). (E) Median and (F) maximum lifespans for 1–8 day old males and female flies exposed to 2% ISO or 3.5% SEVO for 2 hr (n = 3) (not significant (ns), unpaired equal variance two-tail t-test).

Figure 5

Genetic background modulates the PK and PD properties of VGAs. Panels A-E: Dose-response relationships for ISO and SEVO used to calculate EC₅₀s. The Percent Active was measured in 1–8 day-old male flies at different concentrations (n = 3–5 per genotype and agent). Panels F-J: Time course of recovery from anesthesia (2% ISO or 3.5% SEVO for 1 hr) for the same genotypes as in panels A-E. (n = 3–7 per genotype and agent).

Figure 6

The ND23⁶⁰¹¹⁴ mutation in Complex I of the mitochondrial electron transport chain increases the potency of ISO and SEVO. The Percent Active of 2–4 day old male Canton S and ND23⁶⁰¹¹⁴ flies exposed to different concentration of (A) ISO (n = 3) or (B) SEVO (n = 3). (C) EC₅₀s for Canton S and ND23⁶⁰¹¹⁴ flies, derived from panels A and B, respectively (**P < 0.01, equal variance unpaired two-tail test).

Figure 7

Natural Drosophila populations harbor a variety of responses to VGAs. Summary of EC₅₀s (A) and TtR₅₀s (B) from all tested fly lines (Figs 5 and 6) indicates that PD and PK vary unpredictably. (C) The EC₅₀ values of one agent (ISO or SEVO) is a poor predictor of the EC₅₀ of the other (R² = 0.34). (D) The potency of an agent (ISO or SEVO) does not correlate with its rate of emergence: R² = 0.069 and 0.001 for ISO and SEVO, respectively. Data are derived from experiments shown in Figs 5 and 6 and Supplemental Fig. 6 (see Table 1 for numerical values).