krkrkr13
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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.
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bnull
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Mood: Taking a few days off. Damned AI leeches.
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Welcome to the forum.
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.
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krkrkr13
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No, no experience with either. I'll admit I'm pretty new to actual performative chemistry. I did really well in high school and tested out of all my
chemistry requirements at uni, and at least the theoretical knowledge has come back quickly, but procedure is a big weak spot of mine.
So far I've made some chloroform and ethoxide (with CuSO4 as my dehydrating agent), along with a few more generic distillations. I've learned a lot
from both syntheses.. moving straight up to alkylating agents seems like quite an abrupt step.
Emergency protocol for something like that... all I can think of would be immediately sticking my finger in quenching solution to minimize systemic
absorption/detoxify the site.
I like the thought of the hypospray! I still need to run a couple scenarios for how much I'll need... mutating a single batch of cells takes about
0.16 mL, so we're talking about a very very tiny amount. Ideally, it would be nice to dilute the solution down to minimize small measuring errors.
It's not miscible with water so I need at least some DMSO to help with penetration, and that would help minimize evaporative loss in storage as well.
Is there any chance the DMSO would start to attack and break down the molecule?
And I'll definitely post pictures if I can get past this hump :slightly_smiling_face: I've got some eggplants on callus induction media and a few
other plants lined up for the same, so hopefully I can get a stable cell line over the next few months.
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pesco
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How about giving up on alkylating agents which can be difficult to control and use radiation instead?
Chemicals, especially volatile pose more risk than radioactive solid material.
Thoriated tungsten welding rods is one of cheap radiation sources. Thorium is weakly radioactive and most of the radiation is alpha, so the safest
form. Still mutagenic. Tiny bit of gamma can be contained with a bit of lead sheeting.
Weak, but still mutagenic. Because its weak, there us less chance to cause DNA damage in multiple loci and kill the cell.
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krkrkr13
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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
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pesco
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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
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