thanks for your time
for correspondence:
Email: info@felixmay.com

Why the gap between in vitro and in vivo observations?

Interpreting scientific data from biological systems demands a degree of head room as, unlike classical physical systems, the intricacy of biological machinery is usually well beyond even the most observant scientist. We are inevitably left to piece together the clues we are given, first using those most reliable parts, and ending with the more tenuous ones. This experiment represents just such a puzzle, where it would seem many possible arrangements of pieces are possible, budding several explanations.

Diverse dosing

When competitive effects were observed in vitro high doses of pyrethroid were used, whereas comparatively small ones were used in this study. It could be that the observed effect on sodium channels becomes more pronounced at greater doses. Thus the competitive actions could be dose responsive, simply not appearing at the range of 1.5x the LD50.

Although it is possible that an additive effect was masked by a ceiling effect on the IC50 measurement, this is unlikely, as deltamethrin alone was able to produce some extremely high results (+350ms). A dose response curve for cismethrin, a Type I pyrethroid, reveals that there is a lot of scope for change at doses around 1.5 of the LD50.

Alternatively it could be that competition was occurring, but that other effects of the pyrethroids were confounding this. For instance actions on other channels, and on intracellular targets, could be masking or moderating the effects on sodium channels.

Experimental caveats

While this in vivo model is significantly closer to a real life poisoning situation than any cellular model, there are some points which should be kept in mind when trying to extrapolate the experimental findings to real life situations.

Firstly the rats whom were being dosed with pyrethroid were under deep anaesthesia with urethane. The effect of anaesthesia is to increase the dose of pyrethroid needed to evoke most effects, including overt and electrophysiological measures. It is clear that anesthesia affects neuronal signaling, though not how to interpret it, except as a general restraint on firing activity.

Intravenous injections of pyrethroid were used in this study, as was necessary to achieve intoxication rapidly. Occupational pyrethroid exposure occurs primarily as transcutaneous absorption, and to a lesser extent as ingestion, as discussed in toxic effects .

Relating results to humans

Studies observing humans are the ultimate test for this kind of research; there are too many variables to include in a model, giving real life exposure an additional level of significance. Any findings from rat based models, including those described here, should be integrated and compared with clinical data at the first opportunity.

Subject numbers

In the experiment there were only 8 subjects in the esbiolethrin group, and of these only 6 provided full results; while in the other two groups were 12 subjects, reducing the reliability of the data.
More onerous was the large deviations in the deltamethrin group. As the deltamethrin results were crucial for to make comparisons and to reveal competitive or additive effects, this fragile data may have undermined the quality of the experiment.

Compensating for duration of action

Esbiolethrin's effects were very short lived compared with deltamethrin, meaning that it had to be administered late, so as to coincide with he peak effect of deltamethrin. So as to fully probe the system it would have been interesting to have used an intravenous infusion of esbiolethrin to achieve a prolonged exposure before deltamethrin was added into the system. Thus as well as looking at esbiolethrin 'on top' of deltamethrin, we could see deltamethrin 'on top' of esbiolethrin.

 

Intracellular signaling

Pyrethroids directly stimulate protein kinase C-dependent protein phosphorylation as one of the first effects on cells [Enan & Matsumura, 1993]. This effect isn't necessary to produce some effects, as pyrethroids can affect isolated channels, however higher doses are required to see similar effects. BOth chloride and sodium channels can be modulated by phosphorylation state, suggesting it is an important mechanism for the hyperexcitablility symptoms.

In addition affecting the phosphorylation activity inside cells may confer other affects, some of which could be masked or dormant, only precipitating when intracellular signals are ideal. Protein kinase C is involved in gene transcription, and in modulating traffic of proteins in cells, in turn affecting calcium signaling and a host of other factors. It is possible that these kind of intracellular interactions cause effects beyond simple modulation of channel kinetics. If this is the case, interactions between pyrethroids are not limited to binding sites on sodium channels.

 

Delayed effects - artifact or key information?

It would be interesting to determine why deltamethrin takes so long to exert its maximal effect. By the time esbiolethrin had reached maximum and almost returned to baseline again, deltamethrin was only beginning to come into effect. This could be for pharmacokinetic reasons, perhaps due to a slow perfusion into the necessary areas; however the massive lipophilicity of these compounds would grant them rapid access to most organs.

Instead it could be that deltamethrin exerts its effects in part through modulation of intracellular signaling through more than simple ligand-protein association. Indeed deltamethrin might enhance excitability by facilitating existing cellular mechanisms, which take time to mobilise fully, but are also slower to depreciate.

Perhaps then in vivo deltamethrin has, by the time esbiolethrin is administered, exerted a number of actions upon cells both immediate (ion channel binding) and chronic (protein trafficking and signaling). Esbiolethrin is less effective at stimulating these processes, perhaps due to ligand affinity, and perhaps due to its shorter pharmacokinetics. When esbiolethrin is introduced it will bind to ion channels, producing it's characteristic effects there; but it will not dramatically affect the induction of intracellular mechanisms.

Intracellular signaling mechanisms might be crucial to exerting some effects of pyrethroids, and possibly also a domain for interactions between them. Even though there is no clear ceiling for the IC50 from administration of one pyrethroid (see above diagram) it is possible that administering one pyrethroid altered the system so as to create a kind of ceiling for the other pyrethroid. If such interactions were occurring, this might explain why competition can be seen in single channels and small preparations, but isn't so apparent in vivo.

Traditional binding to sodium channels, or novel lipid interactions

Another idea is that pyrethroids exert some of their effects from within the membrane lipid layer, interacting with the channel protein; a more thermodynamically stable point of access that attaching to aqueous portion of the channel. The role membrane lipids in protein function is crucial and determines many of their operating characteristics, presumably including channel kinetics [Engelman, 2005]. It is feasible that a highly lipophillic molecule like a pyrethroid might integrate into the lipid-protein phase and affect the dynamics of the protein’s movements.

If pyrethroids do interact with channel proteins from the lipid membrane, this would be an almost unexplored avenue for pharmacology. The tight restrictions in form and composition of the plasmalemma lipid bilayer are a totally different arena to the aqueous matrix in and around cells. This field is young and promises to be a fruitful area of research in the future.