[Neuroscience] potential for HEPES phototoxicity

Gary Zenitsky via neur-sci%40net.bio.net (by zenitsky from iastate.edu)
Thu Jul 5 11:57:15 EST 2007

Quick Questions:

Does a HEPES-only solution or a HEPES-containing solution, such as aCSF,
that's been variously exposed to light pose a risk to cells in vivo (i.e.,
no exposure to light once the aCSF has been introduced to the extracellular
environment)?  Would it be considered important to prepare, store, and
administer these solutions with minimal light exposure if the target cells
are never exposed to light?




More details related to questions:

I was hoping someone could "shed some light" on the photo-/cyto-toxic
activity of HEPES.  We've been using it for years but only recently came
across references citing its potentially toxic effects.  In our behavioral
neuroscience lab, HEPES is a component (10 mM) in artificial cerebral spinal
fluid (aCSF; recipe appended below), which we use as a vehicle for drug
delivery via intra-cranial micro-injections.  One or more of the
neuro-active agents that we dissolve in aCSF are light sensitive and we
prepare these drugs in amber vials under low light conditions.  When not
being used, we do store our aCSF in a (dark) refrigerator, though any given
batch might get drawn from on a daily basis (i.e., light exposed 15-30 min
per day) for several months before a new batch is made.


I did a quick read of a 1985 Zigler et al. paper, "ANALYSIS OF THE CYTOTOXIC
that 1) exogenous H202 added to non-irradiated medium is less damaging than
is irradiated medium with the

same H202 concentrations, and 2) irradiated HEPES-containing media had a
stronger inhibitory effect than did cultures in

which only HEPES (10X) and riboflavin (l0X) were irradiated.  We don't work
with cell/tissue cultures, but we do inject variously irradiated
(light-exposed) HEPES-containing solutions in vivo, that is, into an
extracellular environment (a non-irradiated medium) bathed in many of the
micronutrients found in benchtop culture media.


Some noteworthy differences between our work & cell cultures include: 1) in
vivo cells are never irradiated though our HEPES-containing aCSF is
routinely so; 2) we work with lower HEPES concentrations (only 10 mM vs. 25
mM); and 3) we inject very low volumes of a HEPES-containing solution (0.25
- 2+ microliters) in vivo wherein the neuro-active molecules bind to target
receptors or ion channels while the vehicle becomes diluted (or washed away)
across the extracellullar milieu.  Our animal subjects could conceivably
receive one such injection up to 5 times per week for a month or longer.
Please note that it's not uncommon for the "quality" of the injection site
to deteriorate (gradually or quickly) over time until drug effects are lost.
Conventionally, this "effect" is attributed to gliotic scarring of the
injection site caused by the repeated insertion of the needle into live
tissue.  But now I'm a bit curious if some of this local tissue damage could
result from low-level toxicity introduced in our aCSF.  I should note here
too that treatment & control injections follow identical protocols,
differing only in presence of drug; that is, we also run control experiments
(aCSF-only) that routinely show that our results can be attributed to the
drug's effect, not its vehicle.


If anyone knows of a more appropriate venue (listserv) to post this question
please let me know.

Thanks for any helpful comments,


Gary Zenitsky


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