Spin Traps: In Vitro Toxicity and Stability of Radical Adducts*
Nadeem Khan1, Carmen M. Wilmot1,
Gerald M. Rosen2,3, Eugene Demidenko
4, Jie Sun5, Joy Joseph6, Julia O
Hara1, B Kalyanaraman6 and Harold M. Swartz1
1Department of Diagnostic
Radiology, EPR Center, Dartmouth Medical School, Hanover, NH 03755 USA;
2Department
of Pharmaceutical Sciences, University of Maryland School of Pharmacy, and
Medical Biotechnology Center,
University of Maryland Biotechnology Institute,
Baltimore, MD 21201 USA;
3Center for Low Frequency EPR for In Vivo
Physiology, University of Maryland School of Pharmacy,
Baltimore, MD 21201 USA;
4Section of Biostatistics and Epidemiology, Dartmouth Medical
School, Hanover, NH 03756 USA;
5Jie Sun, Oxis International
Incorporated, 6040 N. Cutter Circle, Suite 317, Portland, OR 97217 USA;
6
Biophysics Research Institute and Free Radical Research Center, The Medical
College of Wisconsin, Milwaukee, WI USA
Introduction
The technique of spin
trapping of short-lived free radical intermediates has become a valuable tool
in the study
of free radical processes occurring in chemical and biological
environments. Recently several new spin traps have been
developed that provide
enhanced stability of spin adducts and therefore potentially greater
applicability for biomedical studies,
especially studies in intact animals and
eventually, in human subjects. However, in order to use spin traps effectively
in functioning biological systems, it is essential to understand their
biological interactions. Such data are needed to predict
potential
physiological or pathological effects associated with the use of spin traps and
to devise experiments that overcome
these unwanted side effects. We therefore
have carried out a study on the effect of some recently developed nitrone
spin
traps (DMPO, CMPO, EMPO, BMPO and DEPMPO) on Chinese Hamster Ovary (CHO) and 9L
tumor cells,
and also measured the effects of the CHO cells on the stability of
the various spin trapped adducts.
METHODS
Trypan Blue Exclusion Test
This test is a measure of the integrity
of the mechanisms for maintaining the membrane and provides a convenient
method
to measure a parameter that often is related to cell viability.
Clonogenicity Assay
The ability to form a clone
from a single cell over a period of seven days after treated with spin traps
for 24 hours,
is a rigorous test of the ultimate viability of a cell.
Measurement of Oxygen Consumption Rates
The rate of oxygen consumption is a
convenient and valid method to evaluate acute effects on normal biochemical
and
physiological functions.
Production and Measurement of Half-life of the Spin Trapped Adducts
The sulfite, hydroxyl, and
methyl spin adducts were produced by well-established methods. The decay
kinetics
of the spin trapped adducts were measured by recording the EPR
spectrum every 60 seconds, and plotting the signal intensity
of the low-field
peak of the EPR spectrum against time. The decay of the spin trapped adducts
followed first order kinetics.
The decay constant (Kd) was determined
by fitting the data to the function: SI = SI0 x exp(-Kdt),
where SI0 is the signal
intensity at time zero, Kd is the
rate constant for the decay, and t is the time in seconds.
Results
Toxicity varied with the type
of cell line and the parameter that was measured. In aqueous solutions the
order
of stability for all spin adducts was SO3> OH>
CH3, while in cell suspensions it was SO3>
OH ≈ CH3. The radical
adducts of the
new spin traps had significantly increased stability as compared to DMPO. These results indicate
that the new spin traps
potentially offer increased stability of spin adducts in functioning cells.
Discussion
The new spin traps appear to
be potentially useful for studies in living cells. There are some effects on
cells
from these spin traps. Whether the observed effects would be
experimentally significant would depend on the particular
experiment (e.g. if
oxygen consumption was an important parameter in the experiment, then the data
on these effects might
be useful in choosing the spin trap to be used). There
was little toxicity at 2.5 mM of most spin traps. At 25 mM and 50 mM,
the
extent of the effects varied among spin traps and the assay that was used.
These effects are unlikely to compromise
many studies, but this will depend on
the goals and nature of the experiment. Results obtained from CHO and 9L cells
indicate that the structure of the spin trap that is used with a particular
cell line might be critical for the experiments.
The overall message seems
clear: each type of application is likely to need preliminary studies of
stability and toxicity to
determine which spin trap is the most appropriate for
the experimental goal.
CONCLUSIONS
These results indicate that
with appropriate controls and selections of spin traps, reactive free radicals
in functioning
biological systems can be followed by EPR. This should lead to
very effective and unique studies of free radicals in vivo,
making it
feasible to test directly hypotheses about the pathophysiological roles of free
radicals in specific disease states.
* This work is accepted for publication in Free Radical Biology and Medicine.