Genetic Differences Affecting the Potency of Stereoisomers of Isoflurane

The methods for growing nematodes and performing genetic crosses was first described by Brenner [15]and expanded by Wood. [16]Certain strains were obtained from the Caenorhabditis Genetics Center (Minneapolis, MN), including N2, unc-9 (e101), and unc-49 (e362). The double mutant unc-79 *symbol* c-9 was constructed by standard techniques. [15,16].

Ninety-nine percent pure stereoisomers of isoflurane were donated by Rod Eckenhoff (Philadelphia, PA) and Anaquest (Madison, WI).

Nematodes were exposed to the volatile anesthetics in closed glass chambers. Anesthetic was injected in liquid form and allowed to vaporize. Initial injections for N2, unc-9, unc-49, unc-79, and unc-79; unc-9 were estimated to be 20–30% below the EC⁵⁰(the effective concentration of anesthetic at which 50% of the animals are immobilized for 10 s) of racemic isoflurane for N2, the nonmutated worm. The initial concentrations of anesthetic for fc20, fc21, and fc34 were established at about 50% of the EC⁵⁰of racemic isoflurane for fc21. The animals were exposed to one concentration for 2 h with agitation, during which time a steady-state response was reached. Experiments were carried out at room temperature, which was maintained at 22–3 degrees C and checked before and during each experiment. Nematodes were then scored as immobile if they did not move for 10 s when observed through a dissecting microscope. After scoring the cultures for immobility, a sample of gas was taken from the chamber for measurement of the anesthetic concentration by gas chromatography. Either a second injection of anesthetic was added to the chamber to increase the anesthetic concentration, or some gas was withdrawn from the chamber and replaced with air to decrease the anesthetic concentration. The chambers were then allowed to equilibrate again for 2 h. After scoring the culture for immobility and obtaining a second gas sample, the procedure was repeated a third time. After the third concentration point the chambers were opened and the nematodes were allowed to recover. In all cases, the animals fully recovered after returning to room air.

The technique of adding anesthetic to chambers already containing anesthetic differs from that used in most of our previous studies, but was described in the studies with isomers of halothane. [2]Previously, each concentration was obtained by a single injection of anesthetic into a chamber. [9,10]Control experiments were done using this procedure with racemic isoflurane and the strains of C. elegans used in this study. No difference in the EC⁵⁰s or dose-response curves for these cultures in racemic isoflurane was noted between the two techniques.

EC⁵⁰s and slope constants were determined using the technique of Waud. [17]Waud compares values from different curves using a normal distribution; thus, comparisons between different EC⁵⁰s were done using analysis of variance. [17,18]Statistical methods are described in detail elsewhere. [18,19]Significance was defined as a P < 0.01 to avoid type 1 errors. Dose-response curves were constructed using at least 30 concentration points, scoring at least 50 animals at each point. Each EC⁵⁰s then represents scoring at least 1,500 animals. Because the standard errors are dependent on the number of animals scored, they became quite small. The statistical approach of Waud assumes that each measurement is independent. In this study, we used the same animals for two to three measurements (as the doses of anesthetic were changed). We have previously constructed dose-response curves using a few (5–10) animals measured multiple times and found no difference in the EC⁵⁰s from studies in which each animal is only studied once. Because these animals are isogenic, age synchronized and grown under identical conditions, they are all equally likely to move in the 10-s period used to determine immobility. Thus, repeated measurements are independent and this approach remains valid.

The (+) and (-) forms of isoflurane differed in their potencies for the wild type C. elegans, N2. The (+) form showed a 23% increase in potency compared with the (-) form. Interestingly, the racemate had an EC (⁵⁰) similar to that of the less potent isomer, the (-) form (Figure 2and Table 1).

Both unc-79 and the suppressor strain, unc-9, possess EC⁵⁰s in racemic isoflurane identical to that of N2. The N2 pattern for enantiomers also was repeated in both unc-9 (e101) and unc-79 (ec1). In each case the (+) isomer of isoflurane was more potent than the (-) isomer or the racemic mixture (Table 1).

We also studied the effects of the isomers on double mutant, unc-79 *symbol* c-9. The (+) form of isoflurane showed a 41% increase in potency compared with the (-) form and the racemate. Thus, the double mutant showed a larger difference between isomers than seen in N2, unc-79, or unc-9 (Table 1).

Both fc20 and fc21 represent loss-of-function mutations, which unmask sites of anesthetic action more sensitive to all volatile anesthetics than seen in N2. As candidates for final common sites of action for these anesthetics, they also may present different relationships between the enantiomers of isoflurane.

The mutant fc20 showed an increase in sensitivity to the (+) isomer compared with the racemate of isoflurane (Table 2). This pattern is similar to that seen in N2 and the other high-dose requiring strains. fc21, conversely, demonstrated a 38% increase in sensitivity to the (+) isomer compared with the (-) isomer (Figure 3and Table 2). In addition, the pattern of the differences in sensitivities for fc21 was not like that seen in N2, unc-9, unc-79, and unc-79 *symbol* c-9. In fc21, the potency of the racemate mimics that of the more potent isomer, the (+) form.

fc34 and unc-49 represent unique opportunities to compare the enantiomers. The mutation fc34 gives rise to a contractile response to volatile anesthetics. These animals shrink when exposed to any volatile anesthetic and have an EC⁵⁰in racemic isoflurane about 30% of that of N2. fc34 did not show different sensitivities to the enantiomers of isoflurane (Table 2).

unc-49 will isolate any response remaining after the gamma aminobutyric acid (type A) channel is removed, and has been noted to be resistant to racemic halothane compared with N2. [2]Also a shrinker, unc-49 was not resistant, compared with N2, to the racemate form of isoflurane (Table 1). unc-49 showed increased potency of the (+) form of isoflurane compared to the (-) form and the racemate, a pattern similar to that seen with N2 (Table 1).

No significant differences were found in the slope constants between the enantiomers or the racemate in any strain and were similar to those reported earlier for the racemate. [9,10]In addition, dose-response curves repeated 2 or 3 times in successive weeks and by 2 or 3 independent observers did not differ (data not shown).

N2, the wild type C. elegans, contains all the possible sites of volatile anesthetic action in C. elegans and serves as a control for animals in which potential sites of action have been removed or altered. The increase in sensitivity of N2 to the (+) isomer of isoflurane compared with the (-) isomer and racemate was similar to the pattern of sensitivities for N2 to isomers of halothane. Two possibilities exist for the difference in potencies between the (+) and (-) forms of isoflurane in N2. An inhibitory site of action may be more sensitive to the (+) form; this would result in a greater inhibitory effect by the (+) isomer than by the (-) isomer and cause a leftward shift of the dose-response curve of the (+) isomer relative to the (-) isomer. The other possibility is that an excitatory site may be more sensitive to the (-) form; this would result in a greater excitatory effect by the (-) isomer and a net rightward shift of the dose-response curve of the (-) isomer relative to the (+) isomer. Of course, combinations of these two effects are possible.

Loss-of-function mutations may eliminate or unmask gene products that confer stereospecific differences in potency, resulting in specific changes in potency of stereoisomers of volatile anesthetics. Therefore, we tested such mutations from each step in the pathway from Figure 1, along with unc-49, for sensitivities to stereoisomers of isoflurane.

As described in a previous article, the genes listed in Figure 1were ordered by their interactions with each other. [9]If two mutations work along separate pathways then an animal carrying both mutations will show characteristics of both mutations. If the mutations represent early and late steps in a common pathway, the doubly mutant animal will exhibit the behavior of the mutation that represents the later step. The earlier acting mutation requires the activity of the second step to exert its effect.

By the above criterion, mutations in the gene unc-79 occupy the most upstream position in the pathway in Figure 1. However, they do not affect sensitivity to isoflurane. We expected, therefore, that the pattern of differences seen in N2 with respect to enantiomers of isoflurane would be similar to that of unc-79. A similar argument can be made for the gene unc-9. The N2 pattern of sensitivity to the stereoisomers of isoflurane was repeated in unc-79 and unc-9 as well as the double mutant unc-79; unc-9. The pattern of sensitivities of a unc-79 to isomers of isoflurane was different than that seen to isomers of halothane (Figure 4). We interpret these data to indicate that a gene product other than those eliminated in unc-9 or unc-79 is responsible for the difference in potencies of the (+) and (-) forms of isoflurane in N2. It is noteworthy that the difference between the enantiomers was increased in the double mutant. Such a result indicates that these two genes in their normal (i.e., nonmutated) form actually mask part of the ability to distinguish between the stereoisomers. We see a similar, although less dramatic difference between stereoisomers in the mutant unc-49. Taken together, these results indicate the presence of at least one site of action for anesthetics not dependent on the genes unc-79, unc-9, or unc-49. This site confers the stereospecific differences still present in these mutant strains.

The three mutants with low dose sensitivities to both isoflurane and halothane are also interesting. fc20 was able to distinguish between stereoisomers of isoflurane in a pattern similar to the pattern seen in N2. This response was different than that of fc20 to halothane (Figure 4). fc21 and fc34 also showed strikingly different patterns of responses to isomers of isoflurane compared to their responses to isomers of halothane. fc21 shows a 60% difference in sensitivities to the (+) and (-) forms of isoflurane, whereas fc34 is unable to distinguish between the isomers. This is in contrast to the effects of these two mutations on sensitivities to enantiomers of halothane (Figure 4). fc34 showed a threefold difference in sensitivities to the stereoisomers of halothane whereas fc21 had identical sensitivities to both enantiomers and the racemate of halothane. In isoflurane fc21 also shows a pattern for the racemate that is different than the expected pattern described earlier. The dose-response curve of the racemate is close to the more potent isomer, rather than shifted toward the less potent isomer. One possible explanation for this is that the isomers slow synergy; that is, in combination they have an interactive effect that makes the mixture more potent than predicted.

The results with the three very sensitive strains are interesting for two reasons. First, they demonstrate that the ability to distinguish between stereoisomers of one volatile anesthetic does not imply the ability to distinguish between others. Second, because these three mutations lead to patterns of sensitivity different than the wild type worm, they are candidates for the normal gene product(s) that give rise to stereospecific effects of isoflurane seen in N2.

The different potencies of the enantiomers observed in this study are unlikely to be caused by differences in their metabolism, because we allow the animals to come to a steady-state response. However, the metabolism of isoflurane has not been measured in C. elegans. In studies by Eckenhoff, the amount of racemate halothane was decreased in unc-79 and unc-80 when compared to N2 at identical concentrations.** If decreased metabolism, even of only one isomer, caused the difference in sensitivities, one would expect the opposite result. It also seems unlikely that the varied responses of the mutant strains to stereoisomers of isoflurane all arise from different changes in metabolism. However, we cannot formally rule this out at this time.

As mentioned earlier, several researchers have studied the relative potencies of stereoisomers of volatile anesthetics. Franks and Lieb exposed an isolated neuron from the molluscan nervous system to stereoisomers of isoflurane. [4]They found that the (+) isomer was twice as potent as the (-) isomer at eliciting a response from a novel anesthetic activated Potassium⁺channel. Jones and Harrison also recently showed that the (+) isomer of isoflurane produced greater increases in inhibitory postsynaptic currents than the (-) isomer in rat hippocampal neurons. [6].

Harris et al. studied the effects of stereoisomers of volatile anesthetics in whole animals. [1]They found a 20–40% increase in sleep time of the (+) isomer compared with the (-) isomer. However, insufficient drug precluded the determination of the EC⁵⁰s. Lysko et al. determined minimum alveolar concentrations of isomers of isoflurane in rats. [3]They found a similar increase in the potency of the (+) isomer as did Harris et al. [1]They also noted that the EC⁵⁰of the racemate was intermediate between the two isomers, a result differing from that found in C. elegans. It is interesting that in all these studies that have found a difference in the potencies of stereoisomers of isoflurane, the (+) isomer has been more potent than the (-) isomer. In general, we also find the (+) isomer to be more potent in C. elegans. However, in tadpoles, Firestone and colleagues were unable to identify a difference between stereoisomers of isoflurane.*** Thus, while it is certainly possible to show a twofold or threefold difference in potencies between stereoisomers of volatile anesthetics, not all systems will exhibit different responses to the stereoisomers of each volatile anesthetic.

The previous studies indicate that the precise three-dimensional structure of a volatile anesthetic can change its potency, and that multiple sites of action exist. It seems reasonable to expect that each volatile anesthetic will fit in a site of action differently. Thus, steric differences in potency for one anesthetic should not necessarily imply differences for all of them. By isolating the effects of several components controlling sensitivity to volatile anesthetics, it may be possible to identify different types of interaction with stereoisomers. We chose mutations germane to each step in the genetic pathway affecting sensitivity (Figure 1) to examine differences among the stereospecific effects of different volatile anesthetics.

As a model, C. elegans has many outstanding features; however, it is not without its limitations. The normal animal is relatively resistant to volatile anesthetics in comparison to other species, with an EC⁵⁰5–10 times that of minimum alveolar concentrations in humans. This may represent an evolutionary adaptation of this ancient organism to living at cool temperatures and being exposed to organic compounds like alcohols and alkanes within its environment, or the choice of endpoint defining anesthesia in worms. In addition, the relative rank of potencies of isoflurane and enflurane is slightly different in worms than in humans. This difference is a result of the failure of mammals to adhere to the Meyer-Overton relationship in regard to the chemical isomers, enflurane and isoflurane. Enflurane is slightly more lipid soluble than isoflurane (oil-gas partition coefficient of 96 vs. 91 for isoflurane), yet is less potent than isoflurane in humans, dogs, and mice. [20]Unlike their more complicated animals, C. elegans generates the rank order of volatile anesthetic potencies predicted by the Meyer-Overton relationship. Several studies indicate there are multiple sites of action for volatile anesthetics. It seems likely that enflurane has an excitatory effect in mammals that does not exist in C. elegans; this in turn causes its more perfect adherence to the Meyer-Overton relationship. Obviously, simple models are simpler than complex ones. Far from eliminating C. elegans as a model, we believe this strengthens it as a manageable system, without the complicating response to enflurane. Overall, we think that the drawbacks of C. elegans are outweighed by the animal's impeccable adherence to the Meyer-Overton relationship [21]the reversibility of our anesthetic endpoint, the qualitative behavior of the animals in various anesthetic agents, and the maintenance of the cutoff effect. [22].

This study demonstrates two important points. As seen most clearly in the case of fc34 (but also noted with unc-79), the ability to distinguish between stereoisomers of halothane does not imply the ability to distinguish between stereoisomers of isoflurane. In the case of fc21, we see the reverse effect: fc21 can distinguish between the enantiomers of isoflurane but not halothane. These results strengthen the case that these volatile anesthetics do have specific steric constraints to their effects on an anesthetic site of action. The patterns of such differences also are consistent with our earlier genetic studies indicating at least two distinct sites of action for volatile anesthetics. These distinct sites, which change sensitivity to specific groups of anesthetics, also show different stereospecific responses to the different anesthetics. However, it should be remembered that it is the varied response of mutants that leads to this conclusion. The altered proteins in fc34 and fc21 lead to the difference in responses to different stereoisomers. Thus, the different altered proteins each have separate effects on these volatile anesthetics. Of course, such different sites may reside on one common protein target or even one phospholipid; however, they are separable at least by genetics. These results raise the possibility of identifying more ideal volatile anesthetics. [8].

A second important point can be found in the strains requiring high doses of anesthetics, specifically N2, unc-9, unc-79, unc-49, and unc-79; unc-9. With both halothane and isoflurane we see a similar pattern. The potencies of the racemate are always similar to the less potent stereoisomer. Standard models predict that the dose-response curves of the racemates be about halfway between those of the isomers, but shifted from the more potent isomer toward that of the less potent isomer. [23,24]Our dose-response curves lie, in general, to the right of these predictions. Such a shift would conventionally be interpreted as an antagonistic interaction between the two isomers. This antagonism could arise from direct interaction or steric interference between the isomers. However, the model assumes that these drugs are mutually at their site of action. [24]In our opinion, it also assumes similar mechanisms of action by the drugs. Because we do not know if these assumptions hold, the rightward shift of curves generated by the racemate may arise from different modes of action, and not from antagonism.

The authors thank Ms. Susan Thomas for her technical assistance in the preparation and care of the C. elegans strains, and Ms. Sharon Peskin and Ms. DaLee Lambert for their critical reading and aid in the preparation of the manuscript; Sandor Kiss who isolated fc34 and Helen Salz for her discussions concerning these mutations; Helmut F. Cascorbi for suggestions concerning the manuscript and ongoing work; and the faculty members of the Department of Anesthesiology at University Hospitals.

* Bamber B, Jorgensen E: University of Utah, Salt Lake City, UT: personal communication.

** Eckenhoff RG: Subcellular distribution of halothane in anesthetic sensitive mutants of C. elegans (abstract). FASEB J. 1991; 5: A868.

*** Firestone S, Ferguson C, Firestone L: Isoflurane's optical isomers are equipotent in Rana pipiens tadpoles (abstract). ANESTHESIOLOGY 1992; 77:A758.