Toxicology and Applied Pharmacology168,253–267 (2000)doi:10.1006/taap.2000.9020, available online at http://www.idealibrary.com on
Development of Phase II Xenobiotic Metabolizing Enzymesin Differentiating Murine Clara Cells
Michelle V. Fanucchi,*,1 Alan R. Buckpitt,† Mary E. Murphy,* David H. Storms,‡Bruce D. Hammock,‡ and Charles G. Plopper*
*Department of Veterinary Anatomy, Physiology, and Cell Biology, and†Department of Molecular Bioscience, School of Veterinary Medicine;and ‡Department of Entomology, University of California, Davis, California 95616
Received June 13, 2000; accepted July 14, 2000
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Development of Phase II Xenobiotic Metabolizing Enzymes inDifferentiating Murine Clara Cells. Fanucchi, M. V., Buckpitt,A. R., Murphy, M. E., Storms, D. H., Hammock, B. D., andPlopper, C. G. (2000). Toxicol. Appl. Pharmacol. 168, 253–267.
Glutathione S-transferases (GSTs) and epoxide hydrolasesEHs) protect cells from exogenous insult by detoxifying electro-hilic compounds. Little is known about these enzyme systemsuring postnatal lung development. This study was designed toelp establish whether the heightened neonatal susceptibility ofhe lung to bioactivated cytotoxicants is the result of inadequatebility to detoxify reactive intermediates. We compared the dis-ribution of immunoreactive protein and enzymatic activity ofSTs and EHs in isolated distal airways during pre- and postnatalevelopment in lungs of mice from 16 days gestation to 9 weeksostnatal age (adult). GST alpha, mu, and pi class protein expres-ion in fetal and postnatal lung varied by isozyme and age.sozymes alpha and mu are expressed at low levels before birth,igh levels on postnatal day 7, low levels between postnatal days4 and 21, high levels at postnatal day 28, and slightly lower levelsn adults. Immunoreactive protein of isozyme pi has a peak ex-ression on gestational day 18 and again on postnatal day 4, isndetectable at postnatal day 21, and is at peak levels in the adultouse lung. GST activity in distal airways increased with age.icrosomal EH protein expression increased in intensity with age,hile activity was similar in airways from all ages. We conclude
hat in the mouse lung (1) cellular expression of glutathione-transferase varies by age and isozyme and does not increase with
1 To whom correspondence should be addressed at University of Califchool of Veterinary Medicine, Department of Anatomy, Physiology, andiology, One Shields Avenue, Davis, CA 95616. Fax: (530) 752-7690. [email protected].
253
Lung disease isthe leading cause of death in infants undeyear of age (American Lung Association, 1999). Epidemioical studies implicate exposure of infants to cigarette smoa cause in the increase of childhood respiratory diseaseset al., 1984); in the decrease in pulmonary function, whpersists into adulthood (Berkeyet al.,1986; Cunninghamet al.,1994, 1995, 1996; Hanrahan and Halonen, 1998; Wanget al.,1994); and in an increased risk for sudden infant deathdrome (Elliotet al., 1998; Klonoff-Cohenet al., 1995). Cigaette smoke contains many compounds, many of whichioactivated toxicants (Witschiet al., 1997). However, therave been no definitive studies as yet that identify wompounds or what mechanism may be the cause ofhildhood pulmonary problems.Recent studies with laboratory animals have establishe
eonates are much more susceptible to pulmonary injuryioactivated environmental toxicants than are adults. Neoabbits are much more vulnerable to pulmonary injury by450-activated furan 4-ipomeanol than are adult rabbitsose- and age-dependent manner (Plopperet al.,1994; Smiley
Jewell et al., 2000). This single acute injury to bronchioepithelial cells early during neonatal differentiation resultabnormal small airway structure that is still evident in arabbits (Smiley-Jewellet al., 1998). There is also an agelated increase in susceptibility to pulmonary injury by450-activated polyaromatic hydrocarbon naphthalene in
Fanucchiet al.,1997a). Seven-day-old mice are more susible to pulmonary injury than are 14-day-old mice, whichurn are more susceptible than are adult mice. Heighteonatal pulmonary susceptibility occurs, even though lef P450 activity are low in the developing neonatal lung wompared to the high P450 activity of the mature adultFanucchiet al., 1997b; Jiet al., 1995; Plopperet al., 1993).his suggests that the increased susceptibility of neonat
mals could be the result of an imbalance of activatingetoxifying enzymes, resulting in a decreased ability to de
fy even small amounts of electrophilic intermediates.One cellular mechanism to detoxify electrophilic interm
tes is conjugation to glutathione. There are many insta
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254 FANUCCHI ET AL.
where conjugation is nonenzymatic (e.g., 4-ipomeanol), bsome cases, such as for naphthalene, this conjugation ismatically mediated by glutathioneS-transferases, which arefamily of dimeric cytosolic enzymes grouped into three clabased on isoelectric points: alpha (basic), mu (near-neuand pi (acidic) (for reviews see Mannervik, 1985; Manneand Danielson, 1988). A small number of studies desincreases in the catalytic activity of glutathioneS-transferasein whole lung perinatally in both humans and mice (Fryeet
l., 1986; Rouetet al.,1984), but whether these increases on sites where reactive intermediates are likely to be formeot been addressed.Another critical enzyme involved in the detoxification
lectrophilic metabolites is epoxide hydrolase. There areeneral forms of epoxide hydrolase, each with a diffeubstrate specificity and tissue distribution (Wixtrom and Hock, 1985), but all produce 1,2-dihydrodiols from epoxihe microsomal form is involved in the conversion of cypoxides and is found in high levels in the smooth endoplaeticulum and in lower levels in other membranous organeytosolic epoxide hydrolase hydrates aliphatic epoxides a
ound in the cytosol and peroxisomes. Cholesterol epoydrolase is in the microsomal fraction and specifically c
yzes the hydration of cholesterol epoxides. As with glutane S-transferase, data from humans and mice sugges
fetal lung contains much less epoxide hydrolase potentialadult lungs (Kaplowitzet al., 1985), although where this epression occurs has not been evaluated.
This study was designed to help establish whetherheightened neonatal susceptibility to bioactivated cytocants in the lung may be the result of an inadequate abildetoxify reactive intermediates. To determine the patterphase II xenobiotic metabolizing enzyme expression, weamined the following during pre- and postnatal developm(1) the intracellular expression of pulmonary glutathioneS-transferase isozymes alpha, mu, and pi; (2) the intraceexpression of microsomal and cytosolic epoxide hydrolaand (3) the activities of these enzymes in isolated airexplants.
MATERIALS AND METHODS
Animals and lung preparation. Female timed-pregnant and male SwWebster mice were obtained from Charles River Breeding Laboratoriesmington, MA). All animals were housed at least 7 days in laminar flow hin AAALAC-approved animal facilities at the University of California afreceipt from the suppliers before being used in experiments. Free accfood and water was provided. Animals were anesthetized with pentobasodium (60 mg/kg) and killed by exsanguination. Gender was determingrossly examining gonads from all animals younger than 1 month. For imnohistochemical studies, a cannula was inserted in the trachea and thwere inflation-fixed for 1 h with 1% paraformaldehyde (Electron MicroscoSciences, Fort Washington, PA) in 0.1 M phosphate buffer at 30 cm preThe lungs were removed, sliced, and embedded in paraffin within 24harvesting the tissue. For enzymatic assays, the lungs were removed frchest cavity, inflated with 1% Compatigel agarose (FMC BioProducts, R
inzy-
sl),
ke
ras
eet-.
ics.ise-
i-at
an
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totalbyu-ngs
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land, ME) in Waymouth’s MB/752/1 medium (Life Technologies, GrIsland, NY) at 37°C, and plunged into ice-cold Ham’s F12 nutrient mix(Life Technologies, Grand Island, NY) for 30 min. The terminal bronchiwere isolated by blunt dissection under a Wild M-8 stereomicroscopeplaced in fresh F12.
Immunocytochemistry. Paraffin sections (5–6 microns thick) from thmice per age group were labeled for immunoreactive proteins of glutatS-transferase using antibodies produced in rabbits against purified halpha, mu, and pi class isozymes (Novocastra Laboratories, UK). Antibodrecombinant rat microsomal epoxide hydrolase (a gift from Franz Oeschrecombinant mouse cytosolic epoxide hydrolase were produced in rSections from each age group were run together to eliminate variaHydrated sections were treated with 3% H2O2 to block endogenous peroxidaand were then incubated for 24 h at 4°C with the above-mentioned antibDilutions of these antibodies ranged from 1:500 to 1:1250. Immunoreaprotein was visualized with the Vectastain ABC kit (Vector LaboratoBurlingame, CA) using nickel-enhanced 39,39-diaminobenzidine tetrahydrchloride (Sigma Chemical, St. Louis, MO) as a chromagen. Controls incthe substitution of primary antibody with sera from nonimmunized rabbiwith phosphate-buffered saline. Fields were recorded on an OlympusA052 microscope with a Sony digital photo camera attached to a PMacintosh. Images were composed in Adobe Photoshop and printedCodonics NP-1600 printer.
Gel electrophoresis and immunoblotting. Fresh lungs and livers wehomogenized in 4 volumes of Tris-buffered saline suspension buffer coing 1 mg/ml aprotinin and centrifuged at 9000g. The protein concentration fthe liver supernatant was 34mg per lane, and lung supernatant was run in tlanes: 17, 28, and 57mg per lane. Supernatant proteins were electropho
n 15% Instacryl gels (Eastman Kodak, Rochester, NY) in the presenodium dodecyl sulfate, transferred to Immobilon (BioRad, Hercules, CAmmunoblotted with anti-GST and anti-EH antibodies described earlier.ng of the primary antibody was revealed using rabbit anti-goat peroxintiperoxidase (Cappel, Durham, NC), as described previously (Dominet al.,984).
Glutathione S-transferase assay. GlutathioneS-transferase activity waeasured in 9000g supernatant fractions from microdissected distal airw
n 5 5 per age group). Conjugation of glutathione to 2,4-chlorodinitrobenCDNB) was measured by the method of Habiget al. (1974) and normalizeo total protein content using bovine serum albumin as a standard (Lowryet al.,951). One unit of CDNB activity equals 1mmol of glutathione adduct formeer minute.
Epoxide hydrolase assay. Microsomal epoxide hydrolase was measun 9000g supernatant fractions from microdissected distal airways (n 5 3 perge group). Hydrolysis ofcis-stilbene oxide (CSO) was determined by raetric partition assays (Gillet al., 1983) with an incubation time of 60 miata were normalized to total protein content measured with NanoO
Molecular Probes, Eugene, OR) using bovine serum albumin as a sta
Statistics. Quantitative metabolism data were evaluated for potentiaffects using a one-way analysis of variance (ANOVA). Significant differeere determined by using a post hoc multiple comparison test (Bonnfeunn) to identify the source of variance. Statistical analyses were perfosing the SigmaStat software program (Jandel Scientific, San Rafael, CAata are expressed as the group means6 SD.
RESULTS
Glutathione S-Transferase Isozyme mu
In the pseudoglandular stage of lung development (16estational age [DGA]), there is light labeling over every
n the lung, with a few airway cells more heavily labeled (FA). In the canalicular (18 DGA) and saccular (19 DG
sabeatanavatth
mim
lingnatal2E)t 3the
Fig.tedvilythan
gs ofll ages
i DGA l( rr
255POSTNATAL MOUSE LUNG PHASE II ENZYME DEVELOPMENT
stages, the mesenchyme is also lightly labeled, whereanuclei and apical cytoplasm of the airway epithelial cells lslightly more intensely (Figs. 1B and 1C). In the early postnperiod (1–7 days), there is positive labeling in both ciliatednonciliated cells (Figs. 2A–2C, 3A–3C). Most of the cells hheavily labeled nuclei and lighter labeled cytoplasm, butdays some of the cells have cytoplasm that is darker thanin the nuclei. At 10 days postnatal the labeling is predonantly on the basal side of the cells and the nuclei in prox
FIG. 1. Immunocytochemical localization of glutathioneS-transferase (fetal mice at three stages of lung development: pseudoglandular (A, Dncubated at the same time with GST antibodies (1:500). Immunoreactarrowheads). Fetal cells labeled positively for all GST isozymes. No labepresents 35mm.
thel
alde7at
i-al
airways (Fig. 4D). In distal airways, cells have less labethan the early postnatal lungs (Fig. 2D). At 2 weeks postthere is very little positive staining in the distal cells (Fig.and very light labeling in the proximal airways (Fig. 4E). Aweeks the distal airways are lightly positive (Fig. 2F) andproximal cells have more labeling on the luminal sides (4F). Nonciliated cells are more heavily labeled than ciliacells. At 4 weeks the proximal airways cells are very healabeled (Fig. 4G). The distal nonciliated cells are darker
T) isozyme proteins mu (A, B, C), alpha (D, E, F), and pi (G, H, I) in lun, canalicular (B, E, H), and saccular (C, F, I). Paraffin sections from awereprotein was detected earliest in nonciliated cuboidal airway cells in 16ungsd cells were detected in the phosphate-buffered saline control (J). Magnification ba
GS, G)iveele
tedFig.
inataighma
s intheostrklyareilia
stalareeks
ges: 1e time with
256 FANUCCHI ET AL.
the ciliated cells (Fig. 2G). In the adult the proximal ciliacells are darker than the lightly labeled nonciliated cells (4H). Distal airway cells are moderately positive (Fig. 2H)
Glutathione S-Transferase Isozyme alpha
During fetal lung development, there is light labelingevery cell in the lung (Figs. 1D–1F). In the early postnperiod the airway epithelial cells changed from having lpositive labeling over the cells in the distal and proxi
FIG. 2. Immunocytochemical localization of glutathioneS-transferase (G(A), 4 (B), 7 (C), 10 (D), 14 (E), 21 (F), and 28 (G) days postnatal age,GST mu antibodies (1:500). Immunoreactive protein was detected in n
.
ltl
regions to having some dark labeling over apical regiondistal airways (Figs. 3A–3C) and all cells positive inproximal airways (Figs. 5A–5C). At 10 days postnatal mdistal and proximal airway cells are positive and have dalabeled nuclei (Figs. 3D, 5D). At 2 weeks the distal airwaysmoderately positive (Fig. 3E). In the proximal airways the care very dark (Fig. 3E). At 3 weeks the cells of the diairway are lightly positive (Fig. 3F). The proximal cellsalso lightly positive in the apical region (Fig. 5F). By 4 we
) isozyme proteins mu in terminal bronchioles of mice at the following ad adults (H). Paraffin sections from all ages were incubated at the samiliated cells (arrowheads). Magnification bar represents 35mm.
STan
onc
im
ovetly
ageibe
riodeliallialely.od-
iss inest
have
g ages:e time withag
257POSTNATAL MOUSE LUNG PHASE II ENZYME DEVELOPMENT
the labeling increased again in both the distal and proxairways (Figs. 3G, 5G).
Glutathione S-Transferase Isozyme pi
In the pseudoglandular stage there is light labelingevery cell in the lung, while the airway cells labeled slighdarker (Fig. 1G). Through the canalicular and saccular stthe mesenchymal labeling decreases, whereas the nuclapical cytoplasm of the airway epithelial cells start to la
FIG. 3. Immunocytochemical localization of glutathioneS-transferase (G1 (A), 4 (B), 7 (C), 10 (D), 14 (E), 21 (F), and 28 (G) days postnatal ageGST mu antibodies (1:500). Immunoreactive protein was detected in nonbar represents 35mm.
al
r
es,andl
more intensely (Figs. 1H and 1I). In the early postnatal pe(1–7 days) there is positive labeling in type 2 alveolar epithcells (Figs. 6A–6C, 7A–7C). Ciliated and nonciliated epithecells in both distal and proximal airways are labeled positivAt 10 days postnatal, the proximal airway epithelium is merately positive (Fig. 7D) and the distal airway epitheliumlightly positive (Fig. 6D). At 2 weeks the labeling decreaseboth distal and proximal airways (Figs. 6E, 7E). The lowamount of labeling occurs at 3 weeks. The distal airways
) isozyme proteins alpha in terminal bronchioles of mice at the followinnd adults (H). Paraffin sections from all ages were incubated at the samated cells (arrowheads) and in an occasional alveolar macrophage (*). Mnification
ST, acili
ight ond
a
n thtab
asng in
dro-ex-
table
(A), 4ith GST mu
r res
258 FANUCCHI ET AL.
no labeling (Fig. 6F) and the proximal airways have very llabeling, mostly in the apical regions (Fig. 7F). The amounimmunoreactive pi protein starts to increase at 4 weeks apresent in even greater abundance in the adult (Figs. 6G6H, 7G and 7H).
Cytosolic Epoxide Hydrolase
Cytosolic epoxide hydrolase protein was detected only ismooth muscle of pulmonary veins. There was no detec
FIG. 4. Immunocytochemical localization of glutathioneS-transferase (G(B), 7 (C), 10 (D), 14 (E), 21 (F), and 28 (G) days postnatal age, and adantibodies (1:500). Immunoreactive protein was detected in nonciliate35 mm.
tfisnd
ele
protein found in pulmonary epithelial cells. Liver tissue wused as a positive control, and there was positive stainiliver cells (data not shown).
Microsomal Epoxide Hydrolase
In general there is always more microsomal epoxide hylase protein expressed in the proximal airways than ispressed in the distal airways. Unlike the glutathioneS-trans-ferase, microsomal epoxide hydrolase protein is not detec
) isozyme proteins mu in lobar bronchi of mice at the following ages: 1(H). Paraffin sections from all ages were incubated at the same time w
ells (arrowheads) and in some ciliated cells (arrow). Magnification bapresent
STultsd c
ataedtheley limarethe9B
in
ays,s are
isimal
alFig.ainsrox-ssionex-
: 1 (A),4 e with GSTm rs3
259POSTNATAL MOUSE LUNG PHASE II ENZYME DEVELOPMENT
in fetal lung (Fig. 8A). It is detected earliest at 1 day postnIn proximal airways, airway epithelial cells are lightly labelwith the edges of the cells slightly darker (Fig. 8B). Interminal bronchioles, airway epithelial cells are lightly labe(Fig. 9A). Pulmonary alveolar macrophages are intenselbeled. At 4 days postnatal the airway cells in the proxairways are still only lightly labeled, but labeling is modiffuse than that at 1 day (Fig. 8C). Labeling in cells ofterminal bronchioles increases slightly at 4 days (Fig.Labeling for the microsomal epoxide hydrolase protein
FIG. 5. Immunocytochemical localization of glutathioneS-transferase (G(B), 7 (C), 10 (D), 14 (E), 21 (F), and 28 (G) days postnatal age, andu antibodies (1:500). Immunoreactive protein was detected in noncilia5 mm.
l.,
da-l
).-
creases dramatically at 7 days postnatal. In proximal airwthe apex of nonciliated cells labeled intensely. Ciliated celllightly labeled (Fig. 8D). In terminal bronchioles, theresimilar staining, but it is less intense than that in the proxairways (Fig. 9C). At 14 days, the labeling in the proximairways is so intense that it covers all of the airway cells (8E). Labeling in the terminal bronchioles, however, remconstant (Fig. 9D). At 21 days postnatal labeling in the pimal airways reaches adult levels and patterns of expre(Figs. 8F–8H). In terminal bronchioles adult patterns and
) isozyme proteins alpha in lobar bronchi of mice at the following agesults (H). Paraffin sections from all ages were incubated at the same timcells (arrowheads) and in some ciliated cells (arrow). Magnification barepresent
STadted
Fig
ig.ntwch
m ng
B GSTm ass-s male hichif ep-o mu-n nd at5 ctedi
ges: 1e time with
260 FANUCCHI ET AL.
pression are not reached until after 28 days postnatal (9F–9H).
Specificity of Antibodies
Immunoblots of the glutathioneS-transferase isozymes (F10) and epoxide hydrolases show only one band preseeach antibody, and the intensity of the band increasedincreasing protein. Liver 9000g supernatants contain mu
ore glutathioneS-transferase protein than does the lu
FIG. 6. Immunocytochemical localization of glutathioneS-transferase ((A), 4 (B), 7 (C), 10 (D), 14 (E), 21 (F), and 28 (G) days postnatal age,GST mu antibodies (1:500). Immunoreactive protein was detected in n
s.
forith
.
ands were observed at 25 kDa (GST alpha), 26 kDa (u), and 22.5 kDa (GST pi). The antibodies were cl
pecific as shown in Fig. 10. The immunoblot of microsopoxide hydrolase showed a single band at 49 kDa, w
ncreased with increasing protein. Unlike glutathioneS-trans-erase, the liver contained only slightly more microsomalxide hydrolase per mg protein than did the lung. The imoblot of cytosolic epoxide hydrolase showed a single ba9 kDa in the lane containing liver, but no bands were dete
n any of the lung lanes.
T) isozyme proteins pi in terminal bronchioles of mice at the following ad adults (H). Paraffin sections from all ages were incubated at the samiliated cells (arrowheads). Magnification bar represents 35mm.
GSan
onc
tioto
g a
dith
i inm tedf ice(
thetoxi-
(A), 4ith GST mu
r res3
261POSTNATAL MOUSE LUNG PHASE II ENZYME DEVELOPMENT
Glutathione S-Transferase Activity
The activity of GST as measured by the conjugaof CDNB in microdissected distal airways from 7-14-day postnatal and adult mice increased with increasin(Fig. 11A).
Epoxide Hydrolase Activity
Bronchiolar microsomal epoxide hydrolase (as measurethe hydrolysis ofcis-stilbene oxide) did not increase w
FIG. 7. Immunocytochemical localization of glutathioneS-transferase ((B), 7 (C), 10 (D), 14 (E), 21 (F), and 28 (G) days postnatal age, and adantibodies (1:500). Immunoreactive protein was detected in nonciliate
5 mm.
n
ge
by
ncreasing age. There was no significant differenceicrosomal epoxide hydrolase activity in airways isola
rom postnatal mice compared to airways from adult mFig. 11B).
DISCUSSION
The purpose of this study was to establish whetherheightened neonatal susceptibility to bioactivated cyto
T) isozyme proteins pi in lobar bronchi of mice at the following ages: 1(H). Paraffin sections from all ages were incubated at the same time w
ells (arrowheads) and in some ciliated cells (arrow). Magnification bapresent
GSultsd c
y thaan
ssithilum
g thislaseoso-m inuscle
, bio-
), 1 (B),4 EH antibody(
262 FANUCCHI ET AL.
cants in the lung is the result of an inadequate abilitdetoxify reactive intermediates by defining the pattern of pII xenobiotic metabolizing enzyme expression during pre-postnatal development. We found that the cellular expreof immunoreactive protein and enzymatic activity for glutaoneS-transferases increase during the postnatal period ofdevelopment in mice. The expression of immunoreactive
FIG. 8. Immunocytochemical localization of microsomal epoxide hyd(C), 7 (D), 14 (E), 21 (F), and 28 (G) days postnatal age, and adults (
1:1250). Immunoreactive protein was detected in nonciliated cells (arr
osedon-ngi-
crosomal epoxide hydrolase protein also increases durintime, although the level of microsomal epoxide hydroactivity does not increase. Immunoreactive protein for cytlic epoxide hydrolase was not detected in airway epitheliumice of any age studied, but was detected in the smooth mof pulmonary veins.
In the few mammalian species that have been studied
ase (mEH) protein in lobar bronchi of mice at the following ages: fetal (AParaffin sections from all ages were incubated at the same time with meads) and in ciliated cells (arrows). Magnification bar represents 20mm.
rolH).owh
ueededuow
ofto
ce ofbjit-in
: 1 (A), 4( e with mEHa ificat
263POSTNATAL MOUSE LUNG PHASE II ENZYME DEVELOPMENT
transformation activity tends to be low in embryonic tissand then increases postnatally to a level that is maintainadult life (reviewed in Fanucchi and Plopper, 1997). It is wrecognized that the Clara cell is the primary target in the alung for a large number of environmental contaminants. H
FIG. 9. Immunocytochemical localization of microsomal epoxide hydB), 7 (C), 10 (D), 14 (E), 21 (F), and 28 (G) days postnatal age, and antibody (1:1250). Immunoreactive protein was detected in nonciliated
represents 20mm.
sin
lllt-
ever, little information exists regarding the susceptibilityClara cells during differentiation. Clara cell susceptibilityCYP-activated toxicants is accounted for by the abundanimmunoreactive CYP protein found in this cell type (SeraSingh et al., 1988) and by the high level of CYP activity
ase (mEH) protein in terminal bronchioles of mice at the following ageslts (H). Paraffin sections from all ages were incubated at the same timlls (arrowheads) and in an occasional alveolar macrophage (*). Magnion bar
rolduce
-v ands e (C1 nad tivo i-
itydedt-ooxY
lts.th
ndsheencaerede
aseted
aoflan
dptisexp
r weight
1 te.
ndfromch
0
264 FANUCCHI ET AL.
isolated Clara cell populations (Chichesteret al., 1991; Deereuxet al., 1985). In addition, distinct species-specificite-specific differences in acute toxicity to naphthalenYP-activated toxicant) have been reported (Buckpittet al.,992, 1995; Plopperet al.,1992a,b). The species and regioifferences correlate closely with the rate and stereoselecf naphthalene epoxidation (Buckpittet al., 1992). The pos
tive correlation between metabolic activation and toxichowever, does not hold true in neonatal rabbits expose4-ipomeanol (Plopperet al.,1994) or in neonatal mice exposto naphthalene (Fanucchiet al.,1997a). Although differentiaing Clara cells in neonates have low levels of CYP monygenase activity, they are much more susceptible to Cactivated compounds than are mature Clara cells of adu
One explanation for this apparent mismatch betweenlevel of P450 activity and toxicity of bioactivated compouis that toxic cell injury can occur whenever there is a higlevel of activating versus detoxifying enzyme activity presOur current study suggests that this may indeed be theThe phase II metabolizing enzyme glutathioneS-transferasactivity is lower in distal airways of postnatal mice compato the activity in adult mice. This decreased potential totoxify reactive intermediates may contribute to the incresusceptibility of differentiating Clara cells to CYP-activatoxicants. In contrast, the microsomal epoxide hydrolasetivity of distal airways is similar in the distal airwayspostnatal and adult mice. This would suggest that the baof activation versus detoxification in the neonates favorstoxification and should result in neonates being less susceto bioactivated compounds. This, however, is not the cadiscrepancy which may be addressed by two possible e
FIG. 10. Western blots of glutathioneS-transferase isozymes alpha, mstandards. Lanes 2, 7, and 12 contain 34mg protein of mouse liver homoge
0, 15) protein of mouse lung homogenate. (B) Lanes 1 and 6 are molecThe remaining lanes contain 17mg (3, 8), 28mg (4, 9), or 57mg (5, 10) pro
u, pi (A) and epoxide hydrolase (B). (A) Lanes 1, 6, and 11 are moleculanate. The remaining lanes contain 17mg (3, 8, 13), 28mg (4, 9, 14), or 57mg (5,ular weight standards. Lanes 2 and 7 contain 34mg protein of mouse liver homogenatein of mouse lung homogenate.
a
lity
,to
-P-
e
rt.se.
d-d
c-
cee-ble, ala-
FIG. 11. Enzyme activity of glutathioneS-transferase isozymes (A) amicrosomal epoxide hydrolase (B) in microdissected distal airwayspostnatal and adult mice. Results are means6 SD of at least three mice at eatime point. *, indicates activity is significantly lower than that of adult (p ,
.05).
ror (orr
redb aysf inc dh oneS taa
tanecidlutanlutoun
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lua
reeloeveem
ny
n thnen
ou
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em
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luta-bothmen-rvedages,d muelingnce ofcom-
ithfromthan
ane,tains
allysper,pox-asesethanoliceins,theion of. We
nd inast 10the
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is not
265POSTNATAL MOUSE LUNG PHASE II ENZYME DEVELOPMENT
nations: (1) epoxide hydrolase may not play an importantin deactivating reactive intermediates in neonatal mice, othe areas of highest epoxide hydrolase activity may not clate with the areas of highest P450 activity.
In this study, glutathioneS-transferase activity (as measuy the conjugation of CDNB to glutathione) in distal airw
rom lungs of neonatal mice increases progressively withreasing age. This is different from what has been describeumans, in whom the total activity of pulmonary glutathi-transferase decreases fivefold between 13 weeks gesnd birth and then remains constant postnatally (Fryeret al.,
1986). This decrease is associated with a loss in detecprotein (Cossaret al., 1990). More than 90% of glutathioS-transferase activity in lungs of humans results from the a(pi) isoenzyme throughout development. The remaining gthioneS-transferase activity is made up of the basic (alpha)near-neutral (mu) isoenzymes. The decline in the total gthioneS-transferase activity results from a loss in the amof acidic isoenzyme present (Beckettet al.,1990; Fryeret al.,1986; Strangeet al., 1985).
There is a mismatch between specific activity for glutathS-transferases evaluated by conjugation to CDNB in bronoles of postnatal mice protein expression detected in this sCatalytic activity toward CDNB is only one assessment ofdetoxification potential of the glutathioneS-transferases anmay not be totally representative (CDNB is a general substhat allows evaluation of total glutathioneS-transferase activity, but may over- or underrepresent specific isozymes). Hever, because of the small sample size of distal airways thabe obtained from neonatal mice, we were able to evacatalytic activity with CDNB only.
In the present study, we found that cellular protein expsion for isozymes alpha and mu varies throughout devment. Compared to steady-state levels in adults, protein lare low before birth, high on postnatal day 7, low betwpostnatal days 14 and 21, and high at postnatal day 28. Imnoreactive protein of isozyme pi has a peak expressiogestational day 18 and declines to a low at postnatal da(little to no detectable protein) and is again at peak levels iadult mouse lung. Postnatal variation in the expressioindividual isozymes of glutathioneS-transferases has also bepreviously observed in the liver. The mRNA levels of varisubunits of glutathioneS-transferase (Yb1, Yb2, Yb3, Ya, andYp) in the liver of postnatal rats vary throughout postndevelopment (Abramovitz and Listowsky, 1988). A sudtransient decrease in glutathioneS-transferase activity durinthe fifth postnatal week of liver development in rats hasbeen observed (Baarset al., 1980).
The subcellular distribution of glutathioneS-transferasisozymes has been described in the lungs of adult rats and(Coursinet al., 1992; Forkertet al., 1999; Lee and Dinsdal1994). Labeling of immunoreactive protein was documenteboth cytoplasmic and nuclear compartments of Clara aniated cells. A similar labeling pattern was reported in deve
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ing as well as adult kidneys in hamsters (Oberleyet al.,1991).In the present study, we also observed labeling of the gthioneS-transferase isozymes’ immunoreactive protein inthe nuclear and cytoplasmic compartments. The comparttalization did vary with postnatal age, however. We obsenuclear labeling of all three isozymes at most postnatalbut at 7 days postnatal the labeling for isozymes alpha anwas shifted to the cytoplasmic compartment. Nuclear labwas again present at 10 days postnatal. The consequeprotein expression in the cytoplasmic versus the nuclearpartment is not clear.
Although glutathioneS-transferase activity increases wincreasing age, epoxide hydrolase activity does not. Datahumans suggest that fetal lung contains more cytosolicmicrosomal epoxide hydrolase activity (Pacifici and R1982) and that, compared with adult lungs, fetal lung conmuch less epoxide hydrolase potential (Kaplowitzet al.,1985).In the lung, epoxide hydrolase mRNA expression graduincreases to adult levels by 65 days (Simmons and Ka1989). Increases in fetal human pulmonary microsomal eide hydrolase activity, however, do not correlate with increin gestational age (Omiecinskiet al., 1994). In this study, wfound that mice have greater expression of microsomalcytosolic epoxide hydrolase in airway epithelium. Cytosepoxide hydrolase protein is detected only in pulmonary vnot in airway epithelium. This may not be in contrast tohuman data, though, because the site-specific expressepoxide hydrolases has not been evaluated in humansmeasured epoxide hydrolase activity in isolated airways aisolated blood vessels. The blood vessels contained at letimes more cytosolic epoxide hydrolase activity than didairways (data not shown). This may indicate that, by evaluwhole lung, the contribution of epoxide hydrolase activity frnonairway tissue can profoundly influence the assay.
In addition to the balance between activation and detocation enzymes such as glutathioneS-transferases and epoxhydrolases, inadequate or reduced glutathione levels oinability to generate additional glutathione may be importaClara cell toxicity, particularly in differentiating cells. Strikispecies and airway-level differences in the steady-stateand rates of resynthesis of glutathione have been rep(Duan et al., 1996). Without an adequate amount of reduglutathione available, no amount of glutathione transfeactivity will protect cells from injury. Steady-state levels arates of glutathione synthesis have not been examineddifferentiating Clara cells of neonates.
Although steady-state levels of the activating and detocation enzymes may appear to be in balance in airway eplium of distal lung in neonatal mice, the detoxification enzymay be depleted before all of the toxicant is removed.longed exposure to low concentrations of toxicant may meffectively deplete glutathione levels as well as phase Izymes in target cell populations. The putative reactive inmediate of naphthalene metabolism, naphthalene oxide,
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cytotoxic when added as a bolus to isolated Clara cells,is cytotoxic when infused slowly to the cells (Chichesteret al.,1994). The same may be true for differentiating Clara cellungs of neonatal mice. There may be sufficient activatioCYP monooxygenases to sustain a low concentration ofintermediate within the target cell that may eventually owhelm the phase II detoxification system.
In summary, the present study demonstrates age-depepatterns in microsomal epoxide hydrolase and glutathionS-transferase expression and activity in mouse lung. This pais different from what has been described for humans, alththese differences may exist because very little human tissavailable to study in the postnatal period of lung developmThe discordance between cellular expression of proteindetectable glutathioneS-transferase activity, as opposed toclose correlation observed for microsomal epoxide hydroin the mouse, emphasizes the need to carefully characteriactivity of an enzyme within the subcompartment in whichcellular expression is being defined.
ACKNOWLEDGMENTS
The University of California at Davis is an NIEHS Center for EnvironmeHealth Sciences (05707) and support for core facilities used in this wgratefully acknowledged. These studies were supported in part by NIH GES06700, ES04311, ES04699, and ES02710 and by EPA Grant R827442
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