United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park, NC 27711
Research and Development
EPA/600/S1-85/013 July 1986
Project Summary
An Isotopic Study of the
Inhalation Toxicology of
Oxidants
John M. Hayes, and Jeffrey Santrock
The purpose of these studies was to
develop novel methods to investigate
the biological fate of inhaled ozone and
other oxygen-containing pollutants in
animal and human tissues using the
heavy isotope of oxygen, oxygen-18
(18O). Methods were developed that
facilitated the conversion of tissue
oxygen to CO2 and the subsequent
trapping of the CO2 so that it could be
subjected to isotope-ratio mass spec-
trometry. The ratios of the various
masses of evolved CO2 were used to
calculate the 18O content of the original
tissues, thus enabling the detection of
isotopic enrichments as small as 0.4%.
The above procedures were performed
by modification of a commercially avail-
able elemental analyzer to include ef-
fluent columns and trapping devices,
development of oxygen isotopic stand-
ards, and by derivation of mathematical
models for correction of blank and
memory effects originating during sam-
ple pyrolysis.
These procedures were applied to
detecting reaction products of inhaled
ozone r'Oa), and in measuring tissue
oxidation which occurs during exposure
to the hepatotoxin, carbon tetrachlo-
ride. Mice which were exposed to 1
ppm 18O3 showed measurable levels of
18O in their lungs by 20 minutes of
exposure, then increasing to higher
levels as exposure was continued. The
label was found in all biochemical
fractions of the tissue: lipid, solute, and
macromolecule. It was detectable in
trachea! and nasopharyngeal tissue of
mice but not in blood. Rats and rabbits
also showed excess 18O in their lungs
following exposure to 1803, however, it
was only possible to detect it in other
tissues when excised epithelium of the
nasal and tracheal airways was sampled.
The levels of 18O in the epithelium
appeared to far exceed those seen in
homogenates of the whole lung (on a
per gram basis).
The experiments in which carbon
tetrachloride was used demonstrated
for the first time that lipid peroxidation
is detectable in vivo as incorporation of
oxygen-18. Rats exposed to an artificial
air mixture made from 18O2 showed
substantial amounts of 18O in the lipids
as well as in the solute and macro-
molecular fractions of the whole tissue.
The amount of 18O incorporation ap-
peared to be proportional to the activity
of the cytochrome-P450 monooxygen-
ase system which metabolizes the car-
bon tetrachloride.
These results confirm that 18O tracing
studies can be applied to at least two
important problems in inhalation toxi-
cology, and suggest the need for further
studies in this area.
This Project Summary was developed
by EPA's Health effects Research Lab-
oratory, Research Triangle Park, NC, to
announce key findings of the research
projects that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Inroduction
Physiological tracing studies using
oxygen have been severely limited by the
short half lives of all radioactive isotopes
of this atom. Since oxygen-17 and oxygen-
18 are already present in all normal bio-
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logical material, tracing studies must
focus on detecting the excess over the
natural abundance of these isotopes,
which at lower levels of detectability
requires the technique of isotope ratio
mass-spectrometry. Oxygen-18 is the
isotope which is most readily available
commercially, therefore, it is most com-
monly used for tracing studies.
All techniques fortracing 18O in biolog-
ical material require purification of a
molecule of known mass which contains
oxygen that can be subjected to mass
spectrometry. Earlier techniques which
have been described for preparation of
carbon dioxide from oxygen in organic
material suffered from two problems.
First, inaccuracies were encountered due
to fractionation of oxygen isotopes be-
tween two or more intermediates in the
conversion pathway. Second, interfer-
ences arose due to oxygen contamina-
tion. Techniques which have successfully
overcome these problems have required
a great deal of skilled manipulation and
have been useful only with samples con-
taining little or no contaminating ele-
ments besides carbon and hydrogen.
The present study developed an im-
proved procedure for oxygen isotopic
analysis of physiological samples which
involves (1) pyrolysis of the dried tissue
sample, (2) conversion of the oxygen-
containing pyrolysis products to carbon
monoxide, (3) oxidation of the carbon
monoxide to carbon dioxide by iodine pent-
oxide, and (4) analysis of the carbon diox-
ide to determine 18O content in the
samples.
Specifically, the methods developed
involved determining total oxygen and
fractional abundance of 18O in 1-2 mg
samples of dried biological tissues. Sam-
ples were weighed into silver cups and
placed in the sample head of an elemen-
tal analyzer (Carlo Erba Instruments).*
They were pyrolyzed in a stream of helium
where the oxygen was quantitatively
converted to CO by passing through a
column of Ni-coated carbon. The CO was
separated from other gases chromato-
graphically and quantified by the analyzer
to determine the wt % of oxygen in the
sample. The CO effluent from the ele-
mental analyzer was captured and oxid-
ized to C02 by a l2O5-containing column
added to the instrument. The C02 was
collected and purified cryogenically and
the fractional abundance of 180 was
determined using an isotope-ratio mass
•Mention of trademarks or commercial products
does not constitute endorsement or recommenda-
tion for use.
spectrometer (Finnigan, Inc.). Although
the possible masses of the C02 ranged
from 44 to 49 atomic mass units, ion cur-
rents large enough to allow rapid mea-
surement of oxygen species from tissue
samples usually occurred only at masses
44, 45, and 46. Mathematical and theo-
retical models were employed to convert
the isotope ratios to oxygen-18 fractional
abundances, and corrections were made
in the data to account for blank and
memory effects arising during the anal-
ysis. Such corrections were made possi-
ble using oxygen isotopic standards which
were also developed and improved as part
of this project. These standards were pre-
pared by exchange reactions of benzoic
acid with water samples that differed in
their isotopic composition. The benzoic
acid could be either directly decarbox-
ylated to CO2 or passed through the ele-
mental analyzer the same as a tissue
sample. The use of benzoic acid stand-
ards made possible the determination of
the correction parameters to be used for
whole tissues.
Results
The above techniques were applied to
detect 03 reaction products in laboratory
animals following exposure to 1.0 ppm
1803. Mice exposed to 18O3 showed an
increase in 1803 above the natural back-
ground level that became significant after
20 minutes of exposure. A 30-min expo-
sure resulted in about 10 nmoles of O3-
derived oxygen in the total respiratory
tract, of which 56% was present in the
nasopharynx, 5% in the trachea, and 39%
in the lungs. No increase in blood 18O was
detected. Lung tissue which was frac-
tionated into lipid, macromolecules, and
methanol-water soluble phases showed
an approximately equal uptake of 1803-
derived 18O into the different fractions
when expressed on a per dry weight
basis. Preliminary studies to determine
the persistence of the 180 label in the lung
following exposure to 18O3 showed that
18O content was diminished to near con-
trol values by 12 hr post-exposure.
Rats and rabbits exposed to 1803
showed about half the enrichment of 180
in the lungs as did the mice, while
enrichments in the head and blood were
below the level of detectability of the
assay. The low level of 18O in the head of
the larger species was believed to be the
result of the large amounts of bone in the
pulverized tissues which contained such
large amounts of natural 180 as to make
detectability of small enrichments more
difficult. In an attempt to eliminate the
isotope dilution problem, freeze-drie
epithelium from 1803-exposed rabbit
was sampled, and levels of excess 18(
compared in different regions of the res
piratory tract. Much higher enrichment!
of 18O were seen in the epithelial tissues
indicating that O3 reaction products an
mainly localized in the respiration linint
layers. 03-derived 18O was most concen
trated in the nasal passages and uppe
tracheal epithelium, becoming less con
centrated in the peripheral portions of thi
lung.
The other application that was made o
the 180 tracing techniques was in detec
tion of tissue oxidation using a mode
lipid peroxidation initiator, carbon tetra
chloride (CCU). The involvement of oxida
tion reactions in CCUtoxicity, particularly
lipid peroxidation, has been reported b]
such indirect measures as formation o
conjugated dienes, fluorescent pigments
malonaldehyde, and volatile alkanes ir
intoxicated tissues. However, direct in-
corporation of oxygen has not beer
reported previously. The cytochrome P-
450 monoxygenase system of the livei
normally oxidizes lipid-soluble xenobiot
ics to more polar compounds, thus facili-
tating their excretion. However in the
case of CCU, toxic metabolites formed b\
the cytochrome P-450 system initiate i
variety of pathological reactions includ
ing inhibition of lipoprotein secretion intc
the plasma, swelling of mitochondria
and decreased enzyme activities and pro-
tein synthesis. Induction or inhibition o
the cytochrome P-450 system by pheno
barbital or piperonyl butoxide causes ar
increase or decrease, respectively, in the
toxic effects of CCU.
In order to test the hypothesis thai
bound oxygen could be detected in liver
during in vivo CCU exposure, rats were
injected with CCU(lg/kg) and maintainec
for one hour on a closed respirator sys-
tem in which they breathed an artificia
air mixture containing 21% 1802 in N2
Rats exposed to 18O2 alone incorporatec
650 nmoles of excess 180/g liver anc
CCU-treated animals exposed to 1802 hac
an excess 180 of 1230 nmoles/g livei
which was further elevated to 198C
nmoles/g by phenobarbital pretreatment
Rats pretreated with piperonyl butoxide
showed excess 18O similar to groups noi
treated with CCU. A significant portion o
the excess 18O was found in each of the
major liver fractions examined: metha
nol-water soluble, chloroform soluble
and pellet. These results suggest tha
CCU initiates autooxidation of non-lipic
as well as lipid constituents of liver.
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Conclusions
A newtechniquefortracing oxygen-18
in whole animals and complex biological
samples has been developed. The method
involves quantitative conversion of tissue
oxygen to carbon dioxide, and evaluation
of the masses of carbon dioxide evolved
to afford a measurement of 180 content.
These techniques have been applied with
success to the determination of the bio-
logical fate of inhaled ozone, and to the
measurement of tissue oxidation induced
by a model peroxidation initiator, carbon
tetrachloride.
John M. Hayes and Jeffrey Santrock are with Indiana University, Bloomington, IN
47405.
Gary E. Hatch is the EPA Project Officer (see below).
The complete report, entitled "An Isotopic Study of the Inhalation Toxicology of
Oxidants, "(Order No. PB86-109 485/AS; Cost: $ 16.95, subject to change) will
be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA-22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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