Fatrick Nance
Inhibition of Carbonic Anhydrase
by DDT and DDE
Herbert Keller (1) found that carbonic anhydrase was inhibited
by very small amounts of DDT (1,1,1-trichloro-2,2-bis (p-chlorophenyl)
ethane). Keller's method has been used in measuring small amounts
of DDT by a comparative assay to known amounts of DDT.
Now DDT and its derivatives are being detected throughout the
ecosystem (2,3,4), often at concentrations high enough to produce
inhibition of carbonic anhydrase according to the findings of this
report.
Carbonic anhydrase is found in all vertebrates and many invert¬
ebrates (5). It maintains acid base equilibria (5), promotes C0
transmission in respiration (5), and possibly is essential to some
calcification processes in birds (5,6,7,8,9,10), in mollusks (11,12),
arthropods (13) and coelenterata (corals) (14,15). A suggested mechansim
(14,15) is the following: Ca* + 2H00, Cac+ 0 0
The role of carbonic anhydrase in the calcification of mammalian bone
has been reviewed by Ellison (16) with the conclusion that the enzyme
is not important intthis process.
DDT could cause reproductive failure in birds due to thin egg
shells (3,17). No correlation has been demonstrated between DDT
inhibition of carbonic anhydrase and thin egg shells. DDE (1,1-dichloro¬
2,2-bis (p-chlorophenyl) ethylene) is the commonest insecticide analog
now being found in avian tissue (2,3,17) and is common in other species (2).
It has been suggested that DDE and DDT induce hepatic enzyme which
degrade steroid sex hormones in birds causing thin egg shells (3,17).
I hoped to demonstrate that DDE is an inhibitor of carbonic
anhydrase and that dorbonic anhydrase of species other tharthe bovine
préeparation used by Keller is inhibited by DDT and DDE. I thought
variations of the inhibition of carbonic anhydrase between different
species might correlate with their sensitivity to DDT and DDE.
Procedure
A modification of the colormetric carbonic anhydrase assay of
Roughton and Booth (18) was used. Among various buffers, tris (hydrox¬
ymethyl) aminomethane (tris) appears the least inhibitory (19).
A .02M solution adjusted to pH 8.20 with HCl was used. All purified
enzymes and homogenates were in this buffer solution. DDT and DDE
were dissolved in dimethylformamide. Sulfanilamide was dissolved in
tris buffer. The indicator used was .003% bromothymol blue added to
CO saturated distilled water prepared with a dry ice generator. All
reagents were maintained in an ice bath. All assays were similarly
carried out at 0'c.
The following ritual was used for all assays. 9.5ml of buffer
or enzyme-buffer solution was placed in a test tube. ml of
dimethylformamide solution of DDT or DDE was added; the tube was stop-
pered, and mixed with a vortex mixer (Vortex-Genie) for 5 seconds.
The tube was incubated for 15 minutes in an ice bath to allow for rea¬
tion between enzyme and inhibitor. 5ml of C0,-indicator mixture was
quickly added using a 10 ml pipette, the tube restopped, mixed, and
returned to the ice bath. Mixing was by inverting the stoppered tube
quickly three times. The tube was mixed at the beginning of each
minute until the end point. The pipette was precooled by filling to
lOml, allowing it to drain, refilling and allowing all but 5 ml to drain
into the assay tube. The end point was equivalent to a .001% bromo¬
thymol blue solution at ph 6.4. The activity unit chosen for conven-
ience is
Activity Units (AU) s Lo-t x 100
time from addition of C mixture to end point of
uncatalysed reaction
time from addition of CO, mixture to end point of cata-
lysed reaction.
It was very important that reagents and glassware be fully chilled
and that all manipulations be consis tent. For example, the assay was
sensitive to the way in which the tube was held during mixing,
probably due to heat transferred from the hands.
Because of inconsistencies, the assay was run in sets of 5 tubes:
4 identical catalysed tubes and a control. Control time varied from
180 to 225 seconds depending on how the apparatus was handled, but
with consistent handling, consistencies within 5 seconds were obtainable.
The me an tine of the 4 catalysed tubes was taken as thet for a
pa rticular concentration of enzyme and inhibitor. te was determined
in a similar way.
Attempts at enzyme purification using treatment with ethanol and
chloroform (Roughton and Booth, 18) yielded inactive preparations
from homogenates of the mantle. and gonad tissue of Mytilus californianus
and whole Tegula funebralis. Trace amounts of activity, i.e. 160 AU/ml,
were found in the non aqueous phase. Masurements reported here used
a crude 25% homogenate prepared by grinding the tissue in a mortar
and pestle or in a Waring blender. Waring blender prepared homogenates
were generally 3 times more active.
Mytilus used in these experiments were collected at Hopkins
Marine Station, Pacific Grove, California. Homogenates were immediately
prepared and stored at 0C. Approximately 50% activity was lost in
twenty days. Results are presented in fig. 1,2, and 3.
Discussion
Both DDT and DDE inhibit bovine red cell carbonic anhydrase.
Between 3x10 and 15x10° moles in the assay tube produced 50% inhib¬
ition. Homogenated Mytilus mantle appeared to be unixhibited by
as high as 3x10-5 moles of DDT. Mytilus californianus taken from
Monterey Bay, California were found to contain 84 ppb of DDT and
residues (2). Sulfanilamide, an additional inhibitor of carbonic
anhydrase (5,20,21), was capable of inhibiting the mussel enzyme.
Only crude homogenates were used in the study. Inhibition of mussel
carbonic anhydrase should be studied using purified enzyme preparations.
DDT (at3x10-3 M) did not inhibit carbonic anhydrase activity
in crude homogenates of the mantle tissue of Mytilus californianus.
Sulfanilamide at 3x10-2M inhibited the Mytilus homogenates. DDT and
DDE at 10 M produced 50% inhibition of bovine red cell carbonic an¬
hydrase.
This work has been supported by the U.S. National Science
Foundation. I want to thank the faculty and staff at Hopkins Marine
Station where this work was done.
Patrick Nance
Hopkins Marine Station
Pacific Grove, California
fig. 1
fig. 2
fig. 3
Legend
Activity (AU) vs. ml. of enzyme solution.
O—0 ml. of 25% homogenate of Mytilus mantle.
ml. of .O2mg/ml bovine red cell carbonic
anhydrase (Cal Biochem).
Activity (AU) vs. moles of inhibitor.
OObovine red cell carbonic anhydrase (9800 AU/mg).
Inhibitor: DDT
2Abovine red cell carbonic anhydrase (9800 AU/mg).
Inhibitor: DDE
Activity (AU) vs. moles of inhibitor.
0O homogenate of Mytilus mantle (1060 AU/ml tissue).
Inhibitor: DDT
homogenate of Mytilus mantle (785 AU/ml tissue).
Inhibitor: DDT
O—homogenate of Mytilus mantle (465 AU/ml tissue).
Inhibitor: Sulfanilamide
L 8
de
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2

8
9
88
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e
3
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