Alkyl Sulfonates (Am I Crazy?)

Long story short, I'm running an amateur tissue culture lab, and one of the long term experiments I want to run involves mutagenic culturing of various plant cells.

An important part of this process is being able to, well, mutate the cell DNA... which means alkylating agents. The standard one used (at least for plants) is EMS, ethyl methanesulfonate.

This has given me quite a bit of pause. I have decent equipment, including a class II laminar flow (not hooked up to vent externally, unfortunately), and I'm willing to acquire more. I fully agree with the maxim that 'if you can't afford to do it safely, you can't afford to do it,' and I want to make sure that I have all necessary precautions in place to do this well before doing it all. EMS is not as dangerous as initial impulse might suggest, but it's still quite nasty... a NOAEL (no observed adverse effect level) of 25 mg/kg/day would give a standard TDI (tolerable daily intake) of about 17.5 mg <=> 1.5 mL a day for a typical size person.

To pre-empt the coming information dump, I want a sanity check to see if what I'm doing is even sane/reasonable, and if so, whether my protocols make sense and would keep me safe under a worst-case scenario

There are two discrete problems I need to solve here. The first is synthesis and storage, the second is active use.

Problem 1: Synthesis and storage.
The reaction itself is pretty bland. Chloroform + ethoxide -> Triethyl orthoformate (TEOF), and then TEOF + methanesulfonic acid to yield EMS. The reaction is water sensitive, so I'm using CuSO4 to remove water from the system. At least from the procedure on Erowid and a similar patent that I read, the most dangerous step I'm foreseeing is glassware shattering under vacuum distillation... which a few dry-run distillations should help give me some confidence won't happen. My fume hood is just a homemade box coated in butyl epoxy (yikes), but I at least have a moderately sensitive anemometer to check my face velocity and some sotolon to do a qualitative sanity check. I have a PAPR rated for volatiles as a backup that I'm planning on using too, just to make sure I'm covered, so I don't foresee fumes being a problem.

Penetration time is about 20 min with nitrile gloves, so splash contact is covered. I have butyl gloves I could use as well.

EMS hydrolyzes readily within minutes to hours in a mix of NaOH and thiosulfate, so once I'm done everything would get a nice long overnight soak . I have a vented storage cabinet (with fans hooked up), so I'm okay with storing it in a standard borosilicate reagent bottle, with secondary containment... presumably I shouldn't have any issues when the storage cabinet freezes over the winter?

I do also plan on creating little pharmaceutical vials (10 ml) and crimping them, so that I can handle smaller volumes when it comes time to using them. Other than a simple visual leak-test, I don't have the equipment right now to actually validate the seal (realistically, that would require a vacuum-decay chamber), but even if there were micro-leaks I imagine the exposure level would be so diluted/small over a short handling period that I'm not too worried about this right now (maybe I should be...).

Problem 2: Active Use
This is where things start to get less clear in my mind. The steps, at least at a conceptual level, are pretty simple: mix cell suspension with appropriate level of EMS (usually 30 mM), let it mutate for a certain amount of time (usually one to a few hours), start the inactivation with thiosulfate, and then pellet and wash down the cells a couple times to get rid of any traces of EMS.

The first problem is getting the EMS from the vial to the mutation chamber (probably going to be a falcon tube). There are apparently closed-transfer systems you can set up with vial adapters and cannulas.. nothing I have experience with, but maybe that's a question for a pharmacist? Either way I would prefer to avoid any open transfers (or bare needles) if possible.

Centrifugation is where I start to get more concerned. I just have a cheap centrifuge, and there's always the risk of falcon tubes cracking or leaking fluid. All I'm doing is pelleting cells, so the speed is low (about 200g range), for maybe 5-15 minutes, but splashing large amounts of EMS fluid everywhere would be nightmare material. I think my only options here would be to either totally inactivate the solution before centrifuging (a protocol/dose that could be inactivated in-situ over 24 hours would probably lower the total EMS concentrations, too, which makes handling easier), or buy a centrifuge with biocontainment lid add-ons (something like a TX-150 is pricey but not too bad used)... with either approach, once the cells were pelleted, inactivating/disposing of the solution and washing the cells doesn't seem to difficult.



Obviously I still have a few details to work out, but are there any glaring flaws in my protocols? Am I even able to safely do this? Or do I accept that this is just beyond the scope of what I can reasonably do as an amateur, and look for alternative less effective but safer ways to try accomplishing the same thing.

Probably, yes. Who isn't these days?

Do you have any previous experience with mutagenics or at least with alkylating agents?

What is your (detailed) emergency protocol? What would you do if, say, one of the vials breaks and a glass shard slices open one of the fingers of the glove and your skin, dousing your fresh wound with EMS, thereby exposing your cells to the mutagenic? I know it is a bizarre, very unlikely scenario but you must be ready for that kind of accident. I think that some trials using dyed water should help you to adjust your protocol.

For the transfer, why don't you build something similar to a hypospray? Find a tube (PVC should do) slightly wider than the vial but about 90% its length. There must be a cap for the tube, either threaded or to be glue onto it. Attach two needles to the cap so their sharp ends are inside the tube, one needle for a measured quantity of air and another for EMS extraction. Use different types of needle or paint them different colors. Assuming you do not put your finger inside the tube, you simply take the vial, push it inside the tube, then squeeze the hypospray trigger to dispense a predetermined quantity of mutagenic. I suppose you can adapt an automatic pipette to provide the measured quantity of air.

Don't forget that gloves sometimes have tiny holes.

If you manage to conduct these experiments exclusively in vitro, not in vivo with your body as substrate, please post a report and some pictures.

I'd thought about that. Different agents have different types of damage that they induce, and that leads to different structural changes as well.

Alpha and gamma radiation tend to induce double-stranded breaks, which when they're repaired often lead to insertions, deletions, translocations, and other large-scale shifts in the genome. They can have dramatic effects, but also tend to be very lethal in the short-term and decrease genomic stability in the long-term. With alpha, the very thing that makes it safer (low penetration) also makes it less effective, because it will only mutate surface cells. and cause chimeric effects. Granted, I would have to deal with those anyways. But even, it might be tricky to be able to expose the tissue to the radiation without losing my sterile environment, depending on how exactly I would approach dosing.

I have access now to a 254nm UV lamp, which in my reading tends to induce T->C mutations. It suffers from the same problem as alpha radiation, mere microns worth of penetration, although it does tend to lead to higher genomic stability because the changes are less structural.

Point mutators, especially liquid ones, have great penetration, and usually induce C->T mutations. In practice they also have very high genomic stability in the resulting plants, and I belive that's why they're used. Perhaps,

I'm open to try radiation, especially if I can run some experiemnts with my UV light. But, if it is going to decrease long-term evolvability, I'm only getting superficial changes that look pretty but won't go anywhere, is my thinking. Although, recently, I also learned that primarily stress responses are regulated epigenetically, and so perhaps I could use methylation inhibitors to try altering those instead, which would be a degree of safety easier to work with. OR, perhaps, using a pro-mutagen (a furanocoumarin, say) along with targeted UV light, to add an extra layer of protection.


It's actually really encouraging to me to see someone respond after so many months! I'm having some troubles with getting my plants to grow, but I've been honing in my protocols, and I'm hoping to have some results to share soon. Still no EMS, but I've been building a few potential systems for closed-transfers (auto-sampler first try, although I do like the simplicity of the hypospray still). Once the weather warms up and I get my new fume hood installed, I'm hoping to go ahead with at least the first few steps of the synthesis, and try to get a few mL of TEOF

Can't say much about specific modes of mutation by radiation. Long out of uni and that topic wasn't my main interest anyway

In regards to sterile environment radiation should not pose more contamination risk than alkyls. You have to introduce alkyls to the culture, just as you would do with radioactive material if you want to play with alpha. In case of beta and gamma risk of contamination is by far lower, as sources of this radiation can be placed outside of petri dish/test tube.

Also, there's a host of other chemical mutagenic agents easier to get/synthesize and far safer than alkyls. Proflavine for example. Added bonus - it is bactetiostatic.

Anyway. I would explore all the options ang go for alkyls only if nothing else would work. They are very nasty