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Annual report : National Institute of Environmental Health Sciences (Volume 1985) online

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present in BBM. Thus, the secretory component of the mammalian kidney is
mechanistically analogous to that of the marine teleost. Future work must



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address the control of the relative magnitude of secretory and reabsorption
fluxes and the influence of the renal handling of sulfate on the size of
the sulfate pool in control and xenobiotic stressed animals.

The extensive renal portal system of the marine teleost provides an experi-
mental system in which the role of tubular secretion on the handling of
foreign compounds may be readily studied in vivo . Results obtained for
benzo(a)pyrene (BP) and several of its phase I metabolites demonstrated the
impact of both organic anion transport and sulfate conjugation. The effi-
cacy of excretion was shown to depend upon their conversion to anions which
were substrates for the organic anion system, primarily sulfate and glu-
curonide conjugates. Although both conjugates were transported and elimi-
nated much more rapidly than the parent molecules, the sulfate conjugates
were clearly better substrates for organic anion transport and excretion
than the corresponding glucuronides. Furthermore, isolated renal tubules
were capable of effective sulfate conjugation. Thus, both regulation of
sulfate availability and sulfate conjugation occur at the same site, the
kidney.

Our final project dealt with epithelial transport of another anion,
chloride. Chloride transport by the mammalian kidney and the gill of
aquatic organisms plays a primary role in osmotic and ionic regulation.
Thus, its disruption by disease or xenobiotics has far reaching consequen-
ces. Unfortunately, the mechanism(s) of chloride transport are only par-
tially understood. Mammalian kidney has a furosemide sensitive, electro-
neutral Na/K/2C1 cotransport system. Teleost and crustacean gills have a
second system which is not directly related to sodium. We have prepared
isolated membrane vesicles from blue crab gills. These membranes transport
chloride by an anion exchange mechanism which is not influenced by sodium
or furosemide. Is is inhibited by SITS and phloretin - inhibitors of
anion exchange in many tissues - and is capable of moving chloride in
response to bicarbonate or hydroxy! gradients. The means by which
exchange is coupled to metabolic energy to produce accumulation of Cl~
against its concentration gradient has not yet been established. However,
in view of the sensitivity of both anion exchange and anion-stimulated
ATPase (An"-ATPase) to thiocyanate, it is possible that An~-ATPase might
couple CI" flux to ATP hydrolysis in analogy to the Na,K-ATPase which
energizes Na + transport. We have begun to approach this latter possibility
from two directions. First, we have shown that ATP, but not its nonhydro-
lysable analogs, will accellerate CI" transport by isolated vesicles.
Second, we have partially purified the An~-ATPase and prepared monoclonal
antibodies against it. The antibodies will be used to study its localiza-
tion within the cell (a matter of significant controversey at present), its
induction in response to increased osmoregulatory stress, and its involve-
ment in chloride transport.



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2. Plans for Future

Development of isolated membrane vesicle techniques has facilitated charac-
terization of the individual transport events and disclosed the complexity
of the events which couple transport of a given solute to the metabolic
energy and lead to net solute movement across the epithelium. We propose
to use a combination of vesicle and intact tubule techniques to address the
renal handling of both organic and inorganic solutes. Our focus will be
twofold: a) to characterize the membrane basis for the handling of anions
which play a role in xenobiotic toxicity (sulfate, organic anions), and b)
to determine the extent to which toxicity may be related to alteration in
membrane function in epithelial systems.

Many of the characteristics of the individual carriers responsible for
anion transport (organic anions, sulfate, and chloride) have been largely
determined. However, other important features including the means of
coupling to metabolic energy, the coordination of events at opposite cellu-
lar poles, and the modulation of transport in response to physiological
state or xenobiotic stress remain to be clarified. Initially, membrane
vesicle and intact cell preparations will be used to evaluate possible
means of energy coupling, including direct coupling to ATP hydrolysis or
indirect coupling via ion (particularly Na + ) or potential gradients. In
addition, purification and immunochemistry will be utilized to evaluate the
possible role of An"-ATPase in transport. Finally, we will attempt to
modify techniques developed to measure intracellular solute activities in
individual oocytes for application to the smaller cells of epithelia. If
successful, these techniques will allow us to examine directly the interre-
lationships between membrane events and cellular ultrastructure and com-
partmental ization.

Current emphasis will remain on sulfate transport in teleost and mammal
since we have the most complete picture of its handling in both species.
However, since many of its features are shared by the organic anion system,
we will attempt to extend our findings to this system as well. Here our
aim will be not only to understand the mechanism of organic anion transport
and its interactions with sulfate, but also to understand those features of
the organic anion system which are critical in determining its efficacy in
the elimination of foreign compounds.

3. Publ ications (past 18 months)

Little, P.J., James, M.O., Pritchard, J.B., and Bend, J.R.: Benzo(a)pyrene
metabolism in hepatic microsomes from untreated and 3-methylcholanthrene-
treated southern flounder, Paralichthys lethostigma . J. Environ. Pathol . ,
Toxicol., and Oncol. 5: 309-320, 1984.



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Pritchard, J.B., and Renfro, J.L.: Interactions of xenobiotics with renal
function. In Weber, L.J. (Ed.): Aquatic Toxicology , New York, Raven
Press, 1984, Vol. 2, pp. 51-106.

Squibb, K.S., Pritchard, J.B., and Fowler, B.A.: Cadmium-metallothionein
nephrotoxicity I. Correlative, morphological and functional studies. J.
Pharmacol. Exptl . Ther. 299: 311-321, 1984.

Pritchard, J.B., and Bend, J.R.: Mechanisms controlling the renal excre-
tion of xenobiotics in fish: Effects of chemical structure. Drug Metab.
Rev. 15: 655-671, 1984.

Little, P.J., James, M.O., Pritchard, J.B., and Bend, J.R.: Temperature-
dependent disposition of 14 C-benzo(a)pyrene in the spiny lobster, Panul irus
argus . Toxicol. Appl . Pharmacol. 77: 325-333, 1985.

Lee, S.-H., and Pritchard, J.B.: Bicarbonate/chloride exchange in gill
plasma membranes of the blue crab. Am. J. Physiol . , in press.

Pritchard, J.B., and Bend, J.R.: Role of secretory transport in the renal
excretion of benzo(a)pyrene and its metabolites. Proc. Third International
Symposium on "Responses of Marine Organisms to Pollution," in press.



367



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



Z01 ES 80038-02 LP



PERIOD COVERED

October 1, 1984 to September 30, 1985



TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders )

Suicide Inhibitors of Cytochrome P-450: Isozyme and Tissue/Cell Selectivity



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)

PI: John R. Bend Chief LP NIEHS

Others: J. Mathews Staff Fellow LP NIEHS

G. Parker Chemist LP NIEHS



COOPERATING UNITS (if any)



LAB/BRANCH

Laboratory of Pharmacology



SECTION

Molecular and Comparative Pharmacology



INSTITUTE AND LOCATION

NIEHS/NIH, Research Triangle Park, North Carolina 27709



TOTAL MAN-YEARS

2.2



PROFESSIONAL:

1.2



OTHER



1.0



CHECK APPROPRIATE BOX(ES)

□ (a) Human subjects

□ (a1) Minors

□ (a2) Interviews



□ (b) Human tissues (c) Neither



SUMMARY OF WORK (Use standard unreduced type Do not exceed the space orovided )

Microsomal monooxygenase systems contain multiple isozymes of cytochrome P-450
which contribute differentially to the oxidative metabolism of endogenous and ex-
ogenous substrates; isozyme differences in K , V , regioselectivity and stereo-
selectivity are common. Hence, modulation of the Relative amounts of various P-450
isozymes can have pronounced effects on chemical metabolism and toxicity. For this
reason we are studying isozyme selectivity and tissue/cell selectivity of suicide
inhibitors of cytochrome P-450. The suicide inhibition by 1-aminobenzotriazole



(ABT) and some of its novel ^-alkylated derivatives, which we synthesized and
characterized, is being studied in rabbit lung and liver. Although ABT is a potent
suicide inhibitor, it shows little P-450 isozyme selectivity. N-benzyl-ABT, on the
other hand, is both potent and highly selective (but still not specific; it destroys
isozymes 2 and 6 associated enzyme activity but does not significantly affect iso-
zyme 5 catalyzed activity). In a related project, the chemical nature of the alkyl -
benzene metabolites, which selectively destroy pulmonary (versus hepatic) cytochrome
P-450, and the biochemical nature (e.g., isozyme specificity, involvement of other
enzymes and cofactors) of the pathways involved are being elucidated. Results to
date demonstrate at least two distinct pathways for metabolic activation, one rely-
ing only on the presence of NADPH for suicide destruction, the second relying both
on the presence of NADPH and alcohol dehydrogenase.



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B. PROJECT DESCRIPTION

Suicide Inhibitors of Cytochrome P-450: Isozyme and Tissue/Cell

Selectivity

1. Research Project

Nature of problem : Microsomal monooxygenase systems generally contain
multiple forms of cytochrome P-450, each of which may contribute to the
overall oxidative metabolism of a substrate in a different manner; isozyme
differences in regioselectivity, stereoselectivity, Km and V max are common.
Consequently, modulation of the relative amounts of P-450 isozymes can have
pronounced effects on chemical metabolism, and on toxicity. The admini-
stration of different enzyme inducers to animals results in the selective
induction of certain P-450 isozymes and the repression of others. Suicide
inhibition of cytochrome P-450 offers another potential method for affect-
ing this monooxygenase system in an isozyme and tissue selective manner.
Such inhibition, if isozyme specific, will allow the assessment of the
catalytic functions of the remaining P-450 isozymes for both endogenous and
exogenous substrates. This experimental approach should be equally valid
in systems with intact cellular structure and in microsomal preparations.

Objectives in near term (hypothesis tested) :

1. To determine the isozyme and tissue/cell selectivity of the suicide
inhibition of cytochrome P-450 mediated by the metabolism of 1-aminobenzo-
triazole and some of its N-substituted derivatives, and to utilize such
isozyme selective inhibitTon to study xenobiotic metabolism in systems with
intact cellular structure. (Specific, non-toxic suicide inhibitors offer
the potential for protection of humans against specific chemicals to which
they are exposed, or by co-administration, for enhanced toxicity of pesti-
cides. In any event this experimental approach will be useful for deli-
neating physiological functions of cytochrome P-450 at the isozyme level,
our primary interest).

2. To delineate the chemical nature of the reactive metabol ite(s) of
alkyl benzenes, such as p-xylene, which selectively destroy pulmonary ver-
sus hepatic cytochrome "P~-450, and to determine which P-450 isozymes are
responsible for this suicide catalysis (alkylated benzenes are common chem-
icals of commerce and chemical /biochemical aspects of their metabolic
activation/detoxication pathways require thorough investigation).

Experimental approach and scientific justification :

An integrated experimental approach is used and studies are conducted with
purified P-450 isozymes, subcellular fractions of various tissues, freshly
isolated or cultured cells, isolated perfused organs and with intact ani-
mals. Required novel inhibitors and metabolites are synthesized in the



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laboratory, and they are characterized by NMR and mass spectroscopy.
Metabolite isolation, identification and quantitation are most frequently
accomplished by high performance liquid chromatography and scintillation
counting or by gas chromatography. Cytochrome P-450 loss is determined by
spectral measurement and inhibition of catalytic activity is determined by
the use of isozyme selective or specific substrates (e.g., benzphetamine N_-
demethylation for isozyme 2, N-hydroxyl ation of 2-aminofluorene for isozyme
5 and 7-ethoxyresorufin deethylation for isozyme 6 in rabbit lung). This
experimental approach allows us to investigate the chemical and biochemical
basis of P-450 isozyme selective suicide inhibition.

Recent accompl ishments/significance :

1. ABT and its analogues. To better understand the mechanism of destruc-
tion of cytochrome P-450 by 1-aminobenzotriazole (ABT) in the perfused rab-
bit lung, several experiments were performed with 14 C-ABT. We demonstrated
that the pulmonary uptake of ABT from perfusion medium is low as is its
metabolism in the isolated organ (more than 95% of the parent compound was
recovered after a 60 min perfusion). After perfusion, approximately 0.3
nmol ABT equivalent/mg microsomal protein of covalently bound radioactivity
was found which approximates the loss of cytochrome P-450 (0.3 nmol/mg
microsomal protein). No ABT-mediated effect was noted on the flavin-
containing monooxygenase system (FCM) even after perfusion with 1 mM ABT
for 60 min, conditions which destroy up to 90% of the total P-450 present.
Collectively, results of our experiments demonstrate that even in the
absence of facilitative transport into the lung, ABT is an effective
suicide inhibitior of P-450, that ABT can effectively inhibit the
p_450-dependent monooxygenase system without affecting the FCM, and that
ABT metabolism by non-P-450 pathways in perfused lung is minimal.

a-Methyl benzyl -1-aminobenzotriazole (a-MB), an N-substituted analogue of
ABT that is sterically more hindered about the nitrogen atom that is a
target for oxidation than N-benzyl-ABT (BBT), was synthesized and charac-
terized by exact mass spectrometry and NMR. Dose-response data were
obtained for the suicidal destruction of cytochrome P-450 and inactivation
of P-450 isozyme 2-dependent benzphetamine N-demethyl ation and isozyme
6-dependent 7-ethoxyresorufin 0-deethylation in pulmonary microsomes pre-
pared from 6-naphthoflavone-induced rabbits with ABT, BBT and a-MB. We
found that ABT inactivated all three major P-450 isozymes (forms 2, 5 and
6) present in these microsomes. BBT was more potent than ABT (up to
100-fold), and was completely and equally effective at destroying form 2
and form 6 catalyzed activity; BBT did not inactivate isozyme 5 even at a
concentration of 1 mM. a-MB is the most potent and selective suicide inhi-
bitor of this series of chemicals. At a concentration of 1 yM, it destroys
approximately 80% of isozyme 2, about 20% isozyme 6 and does not affect
isozyme 5. At higher concentrations (up to 1 mM), more than 90% of iso-
zymes 2 and 6 are inactivated but isozyme 5 is spared. Preliminary results



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with BBT administered via the lateral ear vein of rabbits at a dose of 10
ymol/kg demonstrated that up to 90% of P-450 form 2 was destroyed in lung.
Our results indicate that BBT and especially a-MB have potential as isozyme
and tissue-selective suicide inhibitors of cytochrome P-450.

2. Alkyl benzenes. Methylated benzenes with 1 to 6 methyl groups were
tested for their ability to destroy (spectrally) rabbit pulmonary cyto-
chrome P-450 in the presence of NADPH. Of the many congeners tested, only
1,2,3-trimethylbenzene was a potent suicide inhibitor (40-80% destruction).

2. Plans for Future

1. ABT and its analogues. The alkylated porphyrin resulting from heme
modification during destruction of cytochrome P-450 by BBT and will be
isolated and chemically characterized to delineate the mechanism of suicide
inhibition. One additional compound, the very sterically hindered N-t-
butyl ABT will be synthesized and tested for its relative ability to Inac-
tivate rabbit P-450 forms 2 and 6. From data obtained with BBT and a-MB,
we predict that the _t-butyl derivative will be more selective than BBT or
a-MB for the destruction of form 2 vs. 5. We also intend to resolve
(±)-a-MB to determine the relative activities of the two enantiomers as
suicide inhibitors of P-450 isozymes 2 and 6. ABT, BBT and a-MB will be
tested for inhibitory characteristics with FCM purified from pig liver.
Finally, the ABT analogue which shows the highest P-450 isozyme selectivity
will be tested for its ability to inhibit the functions of individual
isozymes in vivo or in perfused organ preparations.

2. Alkyl benzenes. The major 1,2,3-trimethylbenzene metabolites formed in
the pulmonary monooxygenase system will be chemically characterized to
determine the fate of the ring and methyl groups as either free metabolites
or covalent adducts to the heme prosthetic group of P-450 during the inac-
tivation of pulmonary P-450.

C. Publications (past 18 months)

Mathews, J.M., and Bend, J.R.: Analogs of 1-aminobenzotriazole (ABT) as
isozyme selective inhibitors of rabbit pulmonary cytochrome P-450 (P-450).
Fed. Proc. 44: 1466, 1985.

Mathews, J.M., Dostal , L.A., and Bend, J.R.: Inactivation of rabbit pulmo-
nary cytochrome P-450 in microsomes and isolated perfused lungs by the
suicide substrate 1-aminobenzotriazole. J. Pharmacol. Expt. Ther. , in
press.



371



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



Z01 ES 80039-02 LP



PERIOD COVERED

October 1, 1984 to September 30, 1985



TITLE OF PROJECT (80 characters or less Title must lit on one line between the borders )

Xenobiotic Transformation in Isolated Cells



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)



PI: James R. Fouts
Others: Theodora Devereux
Thomas Massey
Janet Diliberto
Blair Hoyle
Thomas El ing
Richard Phil pot



Research Pharmacologist
Research Biologist
Visiting Fellow
Biological Lab. Technician
Biological Lab. Technician
Research Chemist
Research Chemist



LP


NIEHS


LP


NIEHS


LP


NIEHS


LP


NIEHS


LP


NIEHS


LMB


NIEHS


LP


NIEHS



£iome¥ry ancf rYsk. Assessment Program (BRAP); Histology, NIEHS; Department of Pulmo-
nary Medicine, University of North Carolina School of Medicine, Chapel Hill, N.C.
(human tissues)



.LAB/BRANCH . _, n

Laboratory of Pharmacology



Cell Pharmacology



r^ffs/W^e search Triangle Park, North Carolina 27709



TOTAL MAN-YEARS



PROFESSIONAL



4.6



2.0



OTHER



2.6



CHECK APPROPRIATE BOX(ES)

□ (a) Human subjects

□ (a1) Minors

□ (a2) Interviews



Ell (b) Human tissues □ (c) Neither



SUMMARY OF WORK (Use standard unreduced type Do not exc.eed the. space provided .) .

Major cell types of trie Tung are being isolated and studied for their
of xenobiotics and selected lipids (in comparison with liver cells),
ies are being made with Clara, type II and ciliated cells of the lung,
the effects of the techniques used in isolating the enriched populatio
from tissues are being made with various antibodies to selected cytoch
isozymes and Western blotting/microdots for quantifying these isozymes
peptides in cells at various stages in the isolation procedures. A mi
photofluorometer is being used to quantify xenobiotic metabolism in si
and to study variations in this metabolism among cells of an "homogeno
tion. Variations in enzyme activity in periportal and centrolobular 1
the perinatal period are compared with these activities in the adult 1
cells from dogs and humans are being prepared and analyzed for P-450 i
xenobiotic metabolism in comparison with cells from the rabbit.



metabol isms
Special stud-
Studies of
ns of cells
rome P-450

and related
crospectro-
ngle cells
us" popula-
iver cells in
iver. Lung
sozymes and



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B. PROJECT DESCRIPTION

XENOBIOTIC BIOTR ANSFORMATION IN ISOLATED CELLS

1. Research Project

Nature of problem: Damage to tissues by chemicals is often localized in
one part ofThe~Ti ssue or varies in intensity from one area of the tissue
to another. With many chemicals, cellular damage is dependent on the meta-
bolism of the chemical by enzymes in the target cells. Localized or area-
specific damage implies that different cells or cells in different parts of
the organ may have different chemical metabolizing systems. Our research
is directed to analyzing and quantifying chemical metabolisms in different
cell types and among cells of one type. We have been studying these chemi-
cal metabolizing systems in 3 organs: lung, skin, and liver, and have
shown that our basic hypothesis is true in each organ - different types of
cells do have different amounts of chemical metabolizing enzymes. The pre-
sent work will continue these studies and also correlate the chemical
metabolizing activities with specific damages to cells by selected chemi-
cals (whose effects depend on metabolism). Studies of effectors of the
type and amount of chemical metabolism in specific cell types and organs
are also being made (e.g., animal pretreatments with chemicals/drugs to
induce or inhibit metabolism; variations in metabolism in the perinatal
period). Our recent studies have mostly concerned metabolizing systems in
isolated cells of the lung. Isolated cells may be able to more exactly
answer questions about individual cell metabolic capacities and regulations
than studies on the whole tissue (as with hi stochemical methods), and cell-
cell interactions may be studied with greater control of variables. Iso-
lated cells do quickly change in enzyme composition (especially with regard
to chemical metabolizing systems of certain types), and it is necessary to
study such changes and how these may obscure the comparison of results with
the in vivo situation. Our studies therefore focus on these problems too:
1) isolating enriched populations of specific cell types and 2) studies of
changes in enzymes in isolated cells and comparison with cells in the
tissue.

There have been three major sub-projects active in the last year: 1)
characterization of chemical metabolizing systems in Clara and type II
cells of the rabbit lung; 2) development of methods for isolating ciliated
cells and Clara cells from upper airways of rabbits and a study of their
chemical metabolisms; 3) developing the microspectrophotometer for study of
metabolism of benzo(a)pyrene by single isolated liver cells. A fourth pro-
ject is just beginning - developing methods for isolating type II (and, if
possible, Clara cells) from human and dog lung - as provided by collabora-
tors in the pulmonary function division at N.C. Memorial Hospital. This
project, if successful, will then continue by studying xenobiotic metabo-
lisms and P-450 isozyme contents in the isolated cells, and comparison with
rabbit cells. With enough samples, it may be possible to correlate disease



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and/or smoking history with cell enzyme activity in the human materials. A
collaboration with Dr. Klinger from the Institute for Pharmacology and
Toxicology in Jena (East Germany) continues. This project began during Dr.
Klinger 1 s study here in 1983. The thesis is that cells of the periportal
versus centrolobul ar areas of liver will develop xenobiotic metabolizing
systems at different rates in the perinatal period. Successful development
of the microspectrophotometer as a tool to study xenobiotic metabolism by
single liver cells will help in this project.

Object ives in near term :

a ) The lung cells:

1. Characterize the xenobiotic and endogenous substrate metabolizing
enzymes of the Clara and alveolar type II cells:

aa) Determine and quantify cytochrome P-450 isozymes in these cells in
the rabbit lung.

bb) Study steroid and lipid metabolisms (progesterone, glucocorti-



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1985) → online text (page 38 of 114)