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Annual report : National Institute of Environmental Health Sciences (Volume 1987) online

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first be unraveled before we will be in a position to define how signal
transduction can be affected by xenobiotics.



121



PROJECT NOMBES
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE

NOTICE OF INTRAMURAL RESEARCH PROJECT I ZQl ES 80001-15 LP



PERIOD COVERED

October 1, 1986 to September 30, 1987



Tr"L£ OF 'ROvECT <30 cmracTers or ess. 770e must fit on ore ima oet^aen »e DorOersj

Microsomal Mixed-Function Oxidase System: Specificity and Function



PRINCIPAL INVESTIGATOR (Ust omer professional personnel oeiow me Pnnapai Investigator.) (Hame, title. laDorator/. ana institute atfiliaoan!

PI: f^.M. Philpot Research Chemist LP NIEHS

Others: R. Gasser Visiting Fellow LP NIEHS

i^- Tynes Guest Worker LP NIEHS



COOPERATING UNITS rt a/iy;

University of California, Davis, CA; Scripps Institute and Research Foundation



maBRA.\CH

Laboratory of Pharmacology



SECTION

Molecular and Comparative Pharmacology



INSTTTUTE AND LOCATION

NIEHS, NIH, Research Triangle Park. North Carolina 77709



TOTAL S^AN-YEARS: PROFESSIONAL: , OTHER;



CHECK APPROPRIATE BOXt^S)

n (a) Human subjects ZI! (b) Human tissues O (c) Neither

1_ (al) Minors
C (a2) Inten^iews

SUMMARY OF WORK Use standard unrecuced Type. Do net exceed the space provided.)

Rabbit cytochrome P-450 Isozymes 2 and 5 are present in lung and liver. In the
liver, but not the lung, the concentrations of these isozymes are increased by
treatment of rabbits with phenobarbital . Homologues of isozymes 2 and 5 have been
detected in lungs of mice, rats, hamsters, guinea pigs and monkeys. Although
homologues of isozyme 2 are also present in livers of these species, hepatic homo-
logues of isozyme 5 are not detected in any species except the hamster. However,
treatment of hamsters with phenobarbital does not increase the concentration of
this isozyme in liver. Rabbit liver expresses three forms of Isozyme 2, two of
which are also present in rabbit lung. These forms have been defined on the basis
of restriction mapping and sequencing of cDNAs derived from mRNA isolated from
niver and lung. A second form present in both tissues differs by 5 out of 491
ammo acid residues. A third varient, which is present only in liver, differs at
11 and 15 positions from the other two. The expression of the three forms of iso-
zyme 2 in liver appears to be under independent control with respect to induction
by phenobarbital. Results with oligo-specific probes indicate that one form is
not induced, one is induced initially and is then repressed, and the third under-
goes induction only following prolonged treatment. Although the total mRNA for
isozyme 2 in lung is not affected by phenobarbital, it is not clear whether or not
the relative proportions of the two forms present are altered. Partial sequence
analysis of cDNAs for isozyme 5 indicate that the pulmonary and hepatic forms are
the same. Contrary to reports in the literature, we find that pulmonary mRNAs for
isozymes 2 and 5 are confined entirely to the fraction that binds to oligo-dT.
The reason for this difference appears to reside in the methods used for RNA iso-
lation. We have found that great care must be exercised in order to Isolate
intact, poly-A mRNA from lung. A reproducible method, which involves centrifuga-
tion in cesium chloride followed by precipitation, has been developed for the
purification of pulmonary mRNA in high yield.

P-^S 6&10 Rev 1/84) 1 on aP0 9i4-ei»



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



PERIOD COVERED



PROJECT NUMBER



ZOl ES 80007-16 LP



October 1, 1986 to September 30. 1987



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

Coniuqatinn and Oxidation Pathways for X enobiotic Metabolism —

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

PI: Cosette J. Serabjit-Singh Research Chemist LP NIEHS
Others:



COOPERATING UNITS (if any)

J.R. Bend, University of Western Ontario, London, Ontario, Canada



LAB/BRANCH

Laboratory of Pharmacology -

SECTION

Mnlpcular and Compa rative Pharmacology

INSTITUTE AND LOCATION

NIEHS. NTH. Research Triangle Park. North Carolina 27 709

TOTAL MAN-YEARS: PROFESSIONAL: | OTHER:



J^fl_



JUL



CHECK APPROPRIATE BOX(ES)

D (a) Human subjects K (b) Human tissues U (c) Neither

□ (a1) Minors
n (a2) Interviews



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

The cytosolic glutathione S-transferase (GST) of the rabbit lung is composed
primarily of two equally abundant isozymes. These isozymes, GST 25kd and GST
24kd. are homodimers that are distinguishable by 1) the mobilities of their sub-
units 25 and 27kdaltons, respectively, in SDS polyacryl amide electrophoretic
gels; 2) their isoelectric points, pi 7.4 and 9.1, respectively, by 2-dimensio-
nal gel electrophoresis; 3) by distinct immunoreactivity with polyclonal antisera
elicited against each isozyme and; 4) differences in the N-terminus (GST 27kd ap-
pears to have a blocked N-terminus similar to the alpha class of GST, and GST
25kd has 15 of 17 residues in common with the pi class isozymes of other spe-
cies) The specific activity of GST 27kd is half that of GST 25kd with chloro-
2,4-dinitrobenzene as substrate, but is twice that of GST 25kd with pyrene 4,5-
oxide (PO) as substrate. The activity of both isozymes toward PO is almost non-
stereoselective in contrast to the stereoselectivity of the cytosol for the S-
configured prochiral carbon. The total PO activity of the purified isozymes
represents at best 10% of the cytosolic activity. The subumts of GST 27kd and
GST 25kd are present in heptic cytosol, which is stereospecific for the S-con-
figured prochiral carbon of PO, but were not purified as homogeneous isozymes by
the methods used for the pulmonary cytosol. However, similar to the pulmonary
isozymes, the stereospeci ficity and the specific activity towards PO was not
maintained during purification of the hepatic GST. The specific activity toward
PO of the rabbit hepatic and pulmonary preparations were comparable to those of
hepatic GST preparations from other species, except for the rat and human GST ^,
which were 10 times more active. Whether there is an endogenous cytosolic fac-
tor that modulates the PO activity of some GST isozymes remains to be determined,
in addition to studying the cellular distribution and genetic regulation of GST
and the relationship, if any, to the susceptibility of the lung to toxicants/

carcinogens .

123— ^ r— —



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



PROJECT NUMBER



ZOl ES 80031-11 LP



PERIOD COVERED

October 1. 1986 to September 30. 1987



TITLE OF PROJECT (80 characters or less. Title must tit on one line fiefween the Ixrders.)

Role of Altered Membrane Function in Xenobiotic Toxicity



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

PI: J.B. Pritchard Research Physiologist LP NIEHS



Others;



D.S. Miller
P.M. Smith



Expert
Visiting Fellow



LP NIEHS
LP NIEHS



COOPERATING UNITS (if any)

University of Florida; Duke University; University of North Carolina



UVB/BRANCH

Laboratory of Pharmacology



SECTION

Molecular and Comparative Pharmacology



INSTITUTE AND LOCATION

NIEHS, NIH. Research Triangle Park. North Carolina 27709



TOTAL MAN-YEARS:
6.0



PROFESSIONAL:



3.0



OTHER:



3.0



CHECK APPROPRIATE BOX(ES)

n (a) Human subjects
n (a1) Minors
n (32) Interviews



n (b) Human tissues C3 (c) Neither



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

The ability to transport solutes across epithelial membranes is a vital function
of many organs, e.g., kidney. In turn, epithelial transport depends upon individ-
ual transport systems located in apical (BBM) and basolateral (BLM) membranes.
Because of their complex organization, functional importance, and exposed loca-
tion, epithelial membranes are particularly susceptible to toxic effects of
foreign chemicals. Recent work has focussed on the renal organic anion transport
system, since this system determines the extent of elimination of toxic xenobio-
tics. Using p-aminohippuric acid (PAH), a model substrate for this system, it
was shown that transport requires the coordinated action of two distinct carrier
proteins. One mediates exchange of external PAH for internal glutaric acid (or
d-ketoglutarate). The second taps energy stored in the Na gradient to drive glu-
tarate back into the cell, maintaining the steep (in>out) glutarate gradient
needed to drive PAH uptake. Together the two systems indirectly couple BLM PAH
transport to the sodium gradient and metabolic energy stores. BBM are unable to
couple the two processes. Thus, PAH secretion requires BLM uptake and intracel-
ular accumulation, followed by exit of PAH down its electrochemical gradient at
the BBM. Studies using electrophysiological and radiochemical techniques to ex-
amine organic cation transport (the second major xenobiotic excretory pathway)
show that the basolateral step in secretion of the model organic cation, tetra-
ethyl ammonium (TEA), is carrier-mediated and strongly potential dependent. To
focus on intracellular events, including binding, subcellular compartmental iza-
tion, and information transfer between the surface membrane and intracellular
organelles, cryomicrodissection and microinjection techniques were developed in
amphibian oocytes . Particularly striking was the observation that intracellular
insulin , at doses (0.5-5 pmoles) too low to alter surface membrane transport,
caused marked changes in both protein and RNA synthesis rates. These prelimi-
nary results argue strongly for a physiological role of intracellular insulin
receptors.



PHS 6040 (Rev 1/84)



124



SPO SI 4-9 < a



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



PROJECT NUMBER



ZOl ES 80040-04 LP



PERIOD COVERED

October 1, 1986 to September 30. 1987



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

Developmental Pharmacogenetics of Liver Microsomal Testosterone Hydroxylases

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

PI: M. Negishi Visiting Scientist LP NIEHS

Others: M. Noshiro Visiting Associate LP NIEHS

R. Masaki Visiting Associate LP NIEHS

J. Squires Visiting Fellow LP NIEHS

T. Ichikawa Visiting Fellow LP NIEHS

R. Lindber Visiting Fellow LP NIEHS

B. Burkhart Biologist 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: PROFESSIONAL;

6^ L 5.0



OTHER:



1.5



CHECK APPROPRIATE BOX(ES)

D (a) Human subjects D (b) Human tissues Kl (c) Neither

n (a1) Minors
n (a2) Interviews



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

This project is aimed at understanding the mechanisms of sex-dependent gene regu-
lation of steroid hydroxylases in liver and kidney. Full-length cDNAs encoding
for three sex-specific hepatic testosterone hydroxylases in mice were isolated
and sequenced. With the use of the cDNAs, the following have been uncovered.
I-P-450]^5 (female-specific 16a-hydroxylase) : The specific expression in females
is regulated by a single locus named Rip, and its repression in males is under
the control of the locus named Ripr. Both loci are closely localized on chromo-
some 7, along with the I-P-450i5jj structural gene which is greater than 35kb in
size and has 9 exons. It was found that growth hormone and estrogen are repres-
sors of I-P-450i5 in males. Genetic evidence suggests that estrogen acts on
Ripr locus. C-P-450i5(j (male-specific 16a-hydroxylase) : The comparisons of mRNA
and activity levels in livers from androgen-treated 129/J mice, which had been
castrated at day one or at adulthood, provided the evidence that there are at
least two differentially regulated C-P-450]^5jj's in livers from adult males; one
is neonatally imprinted and the other is reversibly regulated by androgen. The
expression of C-P-450i6(i in males is also regulated by growth hormone. It is,
therefore, interesting to see how the pituitary and sex hormones are cooperating
to regulate this hydroxylase gene. The cDNA of C-P-450]^5^ was ligated to pcD
vector and transfected in COS cells, resulting in expression of 16a-hydroxylase
activity. The gene for reversibly regulated C-P-450]^5jj was characterized to be
about 5kb with nine exons. P-^50i5^ (female-specific 15a-hydroxylase) : Two
Two highly homologous, but differentially regulated genes (Types I and II) were
identified and isolated. The type I gene is predominantly expressed in female
but not male livers, but expressed only in male but not female kidney. The re-
pression of P-450i5jj in male liver and the expression in male kidney are reci-
procally regulated by androgen. Type II genes are female-specific in both liver
and kidney, although the level of expression in kidney is minimal.

125 ~ '



PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE



NOTICE OF INTRAMURAL RESEARCH PROJECT



ZOl ES 80042-01 LP



PERIOD COVERED

October 1, 1986 to September 30. 1987



TITLE OF PROJECT (80 characters or lass. Title must lit on one line tietween the borders.)

Calcium Regulation and Signal Transduction Mechanisms



PRINCIPAL INVESTIGATOR (Ust other professional personnel Ss/ow the Principal Investigator.) (Name, title, laboratory, and institute alfillation)

PI: James W. Putney, Jr. Pharmacologist LP NIEHS

Others: Arlene Hughes Staff Fellow LP NIEHS

Hiroshi Sugiya Visiting Associate LP NIEHS



COOPERATING UNITS (if any)

None



LAB/BRANCH

Laboratory of Pharmacology



SECTION



Calcium Regulation Section



INSTITUTE AND LOCATION



NIEHS. NIH. Research Triangle Park. North Carolina 27709



TOTAL MAN-YEARS:
5.0



PROFESSIONAL:
3.0



OTHER:

2.0



CHECK APPROPRIATE BOX(ES)

D (a) Human subjects D (b) Human tissues Q (c) Neither

D (a1) Minors
n (a2) Interviews



SUMMARY OF WORK (Use standani unreduced type. Do not exceed the space provided.)

The broad aim of this project is to understand at the cellular and molecular
level, the mechanisms by which surface membrane receptors for hormoniss, neuro-
transmitters and growth factors modify cellular responses through mobilization
of cellular calcium. Recent findings indicate that an early event in the action
of receptors of this class is the hydrolysis of a membrane phospholipid, phospha-
tidylinositol 4,5-bisphosphate, to yield two putative second messenger molecules,
diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DG is believed to
activate a specific kinase in cells (protein kinase £) and IP3 is believed to act
by releasing stored calcium. IP3 is subsequently metabolized by two distinct
pathways, one involving dephosphorylation, and the other phosphorylation and de-
phosphorylation; the result is a complex mixture of different inositol phosphates
which might have significant biological actions. The formation and metabolism
of these compounds is being investigated in intact and broken cell systems to
determine the quantities formed of each of these substances, and the kinetics of
their formation and degradation. Some of the studies involve the use of tissues
from euthanized laboratory rats, while others use immortal cell lines (rat pan-
reatoma). These techniques involve the use of tritium-labeled precursors, and
eparation of compounds by a combination of anion exchange chromatography and
PLC. Cellular calcium is being investigated in the same model cell systems
ith fluorescent indicators to determine correlations between calcium and the
arious inositol phosphates. These studies should provide insights into mecha-
hisms of action of calcium mobilizing agents, and may indicate vulnerable sites
of interaction with environmental toxins such as heavy metals.



PHS 6040 (Rev 1/84) 1 pp. GPO »14-»I»



THE LABORATORY OF PULMONARY PATHOBIOLOGY
Summary Statement

Two broad research topics are being pursued in the Laboratory of Pulmonary
Pathobiology (LPP). One is concerned with pulmonary cell biology and mechanisms
of pulmonary disease, the other with mechanisms of carcinogenesis.

I . Pulmonary Studies

A major part of LPP's research efforts is concerned with the exploration of
mechanisms of particle and fiber toxicity for the respiratory tract. Upon
deposition in the distal lung, particles such as silica and asbestos cause a
multitude of cellular and biochemical reactions which ultimately result in
pulmonary fibrosis (silicosis and asbestosis). Our studies have shown that
within hours after particle exposure, macrophages are attracted to the sites of
deposition and a wave of cell proliferation is triggered in the epithelium of
the bronchioles and the alveoli, as well as in the pulmonary interstitium. It
is not clear which of these cellular reactions are important in the pathogenesis
of pulmonary fibrosis. Since the recruitment of macrophages to the sites of
particle deposition is such an early and prominent event, the hypothesis has
been advanced, that many of the cellular changes, particularly the proliferation
of interstitial fibroblasts are mediated by factors elaborated and released from
macrophages during phagocytosis of the particles.

The studies carried out to date on the role of macrophages have produced the
following major findings. 1) Within a few hours of exposure to a variety of
inhaled particles, a complement-dependent chemotactic factor for macrophages is
activated in the alveolar lining layer. 2) Consequently, macrophages are
attracted to the sites of deposition and phagocytize the particles. 3) During
particle binding and phagocytosis, macrophages release a number of arachidonic
acid metabolites of both the cyclooxygenase and lipoxygenase pathways. Several
of these are known to be powerful mediators of inflammation. 4) In addition,
the macrophages produce growth factor{s) which stimulate proliferation of
pulmonary fibroblasts. This growth factor is similar to platelet-derived growth
factor (PDGF) which is known to be encoded by the c- cis proto-oncogene. Current
studies are aimed at quantifying this growth factor and establishing its
identity and biological activity in vivo .

Another series of studies is concerned with the response of alveolar epithelial
type II cells to toxic particles, namely silica which is known to induce
silicosis. Type II cells are the producers of pulmonary surfactant, a mixture
of lipids and proteins which prevents the collapse of distal airspaces. Silica
exposure causes a massive accumulation of surfactant in the lungs of rabbits,
producing a disease state reminiscent of alveolar proteinosis. Both the
intra-and extra-cellular surfactant pools are increased and we found that the
normal equilibrium between synthesis, secretion and clearance of surfactant from
the alveolar spaces is disrupted. Hypertrophic type II cells have been isolated
from silica exposed lungs to identify precisely which steps in the biosynthesis
and secretion of surfactant are affected. Whether the type II cell response
(hyperplasia, hypertrophy and increased surfactant production) is the result of
a direct interaction of silica particles with the type II cell membrane or
whether it is mediated by some factor released from other cells, is the subject
of future studies.



127



Another major research effort of the Laboratory 1s to study specific pulmonary
cell types, in order to elucidate biochemical and molecular mechanisms
regulating their differentiation as well as various other functions. Under
intensive investigation is the pulmonary Clara cell, a cell type located
preferentially (but not exclusively) in the bronchioles. Clara cells are known
to be rich in drug metabolizing enzymes and are suspected of secreting
proteinaceous products of an undefined nature. This cell also is believed to
play a major role in the maintenance and repair of the bronchiolar epithelium
and to have stem-cell functions. Using highly purified Clara cell suspensions
it was shown that their major secretory product is a 12 kOa protein which reacts
with Clara cell antisera and is localized in osmiophilic granules. The same
protein is also found in pulmonary lavage fluid supporting the notion that the
12 kOa protein is a secretory product. Messenger RNA was isolated from p-rified
Clara cells and it was demonstrated that the primary translation product encodes
a protein with a molecular weight about 1 kOa larger than the secreted 12 kOa
protein and is therefore believed to be a signal peptide. Studies are currently
underway to determine the function(s) of this low molecular weight protein.

In vivo culture studies with highly purified Clara cells have shown that these
cells have a high proliferative potential, considerable self-renewal capacity
and can give rise to terminally differentiated ciliated cells. Current studies
are designed to examine the relationship between Clara cells and other secretory
cells in the conducting airways.

To study the biochemical and molecular mechanisms of differentiation of
epithelial cells from the conducting airways, an epithelial cell culture system
has been developed over the last several years. Using this _in vitro model of
tracheo-bronchial cell differentiation, it was shown that these cells have a
dual differentiation potential. When grown on collagen gels in the presence of
retinoids they differentiate as secretory cells. Analysis of the
^H-glucosamine-labeled secretory products revealed that the cells secrete mucous
glycoproteins under these conditions. In the absence of retinoids the cells
instead undergo squamous differentiation and acquire many features typical of
epidermal keratinocytes. The pathway of keratinocyte differentiation occurs in
atleast three discrete steps: a) terminal cell division with loss of clongenic
potential which is induced by removal of E6F from the medium or by the addition
of TGFp ; b) expression of the keratinocyte phenotype (which is measured by
increase in epidermal transglutaminase, increase in cholesterol sulfate, and
increase in specific, large molecular weight keratins) and c) the formation of
cross-linked envelopes. Expression of the keratinocyte can be enhanced by
addition of Ca"*""*" to the media and inhibited by retinoids. cDNA clones from
squamous differentiated cells were obtained for the purpose of isolating the
genes controlling keratinocyte differentiation in airway epithelial cells.

II. Carcinogenesis Studies

The development of cancer is believed to occur in multiple steps. According to
the most widely accepted model of carcinogenesis, the process is initiated by
the interaction of a carcinogen with normal stem cells, converting them to
preneoplastic cells. These heritably altered cells have an Increased
probability of generating neoplastic offspring. Through a series of secondary
changes which may either be spontaneous or induced (e.g. by carcinogens or tumor
promoters), preneoplastic cells are converted to neoplastic and malignant cells.



128



Our carcinogenesis studies have focused on the following major problems: 1) the
importance of chromosome mutations in neoplastic transformation; 2) the role of
proto-oncogenes and tumor suppressor genes in different stages of neoplastic
transformation and 3) the mechanism by which the c- src gene product transforms
Syrian hamster embryo (SHE) cells.

A large number of carcinogens induce DNA damage and gene mutations at specific
loci and their carcinogenicity has often been linked to their mutagenic
activity. However, a number of carcinogens have been found, which have little
or no demonstrable mutagenic activity. One of these is the synthetic estrogen
diethyl stilbestrol (DES), which we showed is able to transform SHE cells without
causing any detectable point mutations. But DES does disrupt microtubule
organization and causes numerical chromosome changes i.e. aneuploidy. Other
agents which are inactive as gene mutagens but active as chromosomal mutagens
and induce neoplastic transformation are sodium arsenite, sodium arsenate and



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1987) → online text (page 14 of 31)