(Received for publication, June 5, 1995; and in revised form, July 17, 1995)
From the
Adrenocortical mitochondrial cytochrome P450 isozymes of the
Cyp11 family normally synthesize steroids with a very strict substrate
specificity. However, for the first time, P450c11 was additionally
shown to metabolize and bioactivate the adrenotoxic environmental
pollutant 3-methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene
(MeSO-DDE). This conclusion is based on a striking
correlation between inductions of MeSO
-DDE and
deoxycorticosterone metabolism by forskolin in the adrenocortical cell
lines Y1 and Kin-8, inhibition of P450c11-dependent activities in Y1
cells by MeSO
-DDE, and metabolism of MeSO
-DDE
by non-steroidogenic COS cells after transfection with a cDNA encoding
P450c11. The interaction between xenobiotics and glucocorticoid
synthesis should focus more attention to xenobiotic-induced hormonal
disturbances.
MeSO-DDE
(3-methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene) (
)is a metabolite of the still extensively used insecticide
DDT and is therefore a global environmental pollutant. Because of its
lipophilicity and persistence, MeSO
-DDE is bioaccumulated
in the food chain, even leading to pollution of human breast
milk(1, 2) . Animal experiments have shown
MeSO
-DDE to be adrenotoxic to several species after
bioactivation in adrenal cortex
mitochondria(3, 4, 5, 6, 7) .
Furthermore, human adrenal mitochondria have also been demonstrated to
bioactivate MeSO
-DDE in vitro(8) . It has
been suggested, but never demonstrated, that the bioactivation is
mediated by mitochondrial cytochrome P450(7) . The adrenal
gland contains several P450 isozymes, but in contrast to hepatic P450s,
adrenal P450s are generally not active in xenobiotic metabolism. The
genes encoding the mitochondrial P450 11
-hydroxylase (P450c11),
cholesterol side-chain cleavage (P450scc), and aldosterone synthase
(P450aldo) belong to the Cyp11 subfamily of the P450 superfamily of
genes(9) . These enzymes show a strict substrate specificity
and are therefore not believed to metabolize xenobiotics. P450scc is
the first and rate-limiting enzyme in steroid hormone synthesis,
localized in all steroidogenic tissues, whereas P450c11 and P450aldo
are specifically localized in the adrenal cortex. The latter two
participate as the last enzymes in the synthesis of glucocorticoids and
mineralocorticoids, respectively. Given the specific mitochondrial
damage in the adrenal cortex after MeSO
-DDE exposure, we
decided to investigate the possible involvement of steroidogenic
mitochondrial P450s in the metabolism of MeSO
-DDE. For this
purpose we studied the bioactivation of MeSO
-DDE, i.e. the metabolic formation of reactive MeSO
-DDE
intermediates capable of binding irreversibly to macromolecules or
cellular antioxidants, in the mouse adrenocortical tumor cell line Y1.
We also studied the bioactivation of MeSO
-DDE in a mutant
of the Y1 cell line, Kin-8, that harbors a defect in the cAMP-dependent
protein kinase(10) , which normally participates in the
hormonal regulation of the steroid-metabolizing enzymes. In Kin-8
cells, as opposed to Y1 cells, ACTH, forskolin, or cAMP are inefficient
inducers of the P450 isozymes expressed in these cells, i.e. mitochondrial P450scc, P450c11, and P450aldo. To unambiguously
demonstrate the relative importance of the adrenal mitochondrial P450s
in the bioactivation of MeSO
-DDE, experiments were finally
conducted in monkey kidney COS cells. These cells do not normally
metabolize steroids or MeSO
-DDE, so by investigating the
MeSO
-DDE-metabolizing capacity in COS cells transfected
with expression plasmids encoding the three mitochondrial P450s we
tried to identify the enzyme responsible for bioactivation, and
presumably toxicity, of MeSO
-DDE.
Culture of the adrenocortical cell lines Y1 and Kin-8, as
well as of COS cells, were performed in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 5%
fetal calf serum, 50 units/ml penicillin, and 50 µg/ml
streptomycin. Experiments were conducted using subconfluent cultures
containing approximately 2 10
cells/30-mm plate. In
some experiments Y1 and Kin-8 cells were treated with forskolin (10
µM, in Me
SO, Sigma) for 18 h before other
additions. Steroid substrates (20 µM, Sigma) and
MeSO
-DDE (synthesized according to (4) ) were added
from 1000-fold stocksolutions in Me
SO.
Enzyme assays
were performed after growing cells in the presence of steroid substrate
(deoxycorticosterone, corticosterone, or 22-hydroxycholesterol). A
500-µl aliquot of the medium was removed and extracted with
chloroform:methanol (2:1). After one additional extraction of the
medium with chloroform, the chloroform phases were pooled, evaporated,
and dissolved in 50% acetonitrile for HPLC analyses of corticosterone
and aldosterone as described previously (11) . Pregnenolone in
the medium was quantitatively converted to progesterone with
3-hydroxysteroid dehydrogenase (Sigma) according to Talalay and
Dobson (12) before extraction of the medium. Formed
progesterone was then quantified by HPLC as above.
Metabolism of
MeSO-DDE was examined after growing cells in the presence
of [
C]MeSO
-DDE (13.4 mCi/mmol). The
medium was removed, and the cells washed with phosphate-buffered saline
and scraped into 500 µl of cold phosphate-buffered saline. The
culture medium and the cell suspensions were immediately extracted with
three volumes of chloroform:methanol (2:1), followed by centrifugation
(300
g, 15 min). The medium was extracted once more
with chloroform, and the concentration of hydrophilic
[
C]MeSO
-DDE metabolites in the
hydrophilic phase was measured by liquid scintillation. The protein
pellet was exhaustively extracted by repeatedly solubilizing the
protein in 1% sodium dodecyl sulfate and precipitating with acetone as
described by Baker and Van Dyke(13) . The protein was finally
dissolved in NaOH, and aliquots were assayed for protein (14) and irreversibly bound radioactivity.
Transfection
studies were performed on COS cells grown on 30-mm plates until almost
confluent, when they received new medium containing 3 µg of DNA (2
µg of P450 +1 µg of adrenodoxin (ADX)) in the transfection
reagent DOTAP (Boehringer Mannheim). After a 20-h transfection period,
the cells were grown for 24 h in fresh medium and then new medium
containing 20 µM steroid substrate or 4 µM
[C]MeSO
-DDE was added. Medium and
cells were harvested after 24 h of incubation, and enzyme activities as
well as [
C]MeSO
-DDE metabolism
assayed as described above.
Upon treatment of adrenocortical Y1 cells with forskolin,
which stimulates adenylate cyclase, these cells metabolized
MeSO-DDE to intermediates that reacted with cellular
protein (Fig. 1a) and gave rise to hydrophilic
MeSO
-DDE metabolites (Fig. 1b). By thin
layer chromatography analyses of the hydrophilic MeSO
-DDE
metabolites, the presence of ninhydrin-positive MeSO
-DDE
metabolites, presumably glutathione conjugates, was indicated (data not
shown). The lack of MeSO
-DDE metabolism in unstimulated
cells and the minimal MeSO
-DDE metabolism in
forskolin-stimulated Kin-8 cells (Fig. 1) suggested the
involvement of cAMP-inducible steroidogenic enzymes in the metabolism
of MeSO
-DDE. A similar inducibility of MeSO
-DDE
metabolism and one steroid hydroxylase, P450c11, by forskolin in Y1 and
Kin-8 cells (Fig. 2) further supported the involvement of
steroidogenic P450 in MeSO
-DDE metabolism.
Figure 1:
Irreversible binding of
[C]MeSO
-DDE to protein (a)
and formation of hydrophilic
[
C]MeSO
-DDE metabolites in the
culture medium (b) of normal and forskolin-induced Y1 and
Kin-8 cells. Cells were grown in the presence of 3 µM [
C]MeSO
-DDE for 6-24 h,
whereafter medium and cells were separated. After extraction with
organic solvents,
C-labeled metabolites irreversibly bound
to protein and hydrophilic
C-metabolites remaining in the
medium were measured. Each point represents mean of duplicate samples.
Mean variation between duplicates was 11%.
Figure 2: P450c11 activity in normal and forskolin-induced Y1 and Kin-8 cells. The mutant Kin-8 has a defect cAMP-dependent protein kinase, strongly decreasing the expression and inducibility of P450scc and P450c11(8) . Cells were grown in the presence of deoxycorticosterone for 6 or 12 h, whereafter an aliquot of the medium was extracted and analyzed for corticosterone by HPLC. Each point represents mean of duplicate samples. Mean variation between duplicates was 11%.
We next
tested the ability of the three mitochondrial P450s present in
adrenocortical Y1 cells to metabolize MeSO-DDE. This
metabolism was assessed in COS cells transfected with expression
plasmids encoding mouse P450c11, P450scc, and P450aldo, respectively,
either alone or in combination with a plasmid encoding bovine
adrenocortical ADX(15, 16) . Adrenodoxin is an
iron-sulfur protein that participates in the transfer of electrons from
NADPH to the mitochondrial P450s, a process previously shown to be
rate-limiting for steroid hydroxylations in transfected COS
cells(16) . As can be seen in Table 1, the transfections
were successful, although cotransfection with the ADX plasmid was
necessary to obtain high P450 activities with the respective
substrates. Only cells transfected with P450c11 metabolized
MeSO
-DDE to any significant extent, as determined by the
formation of hydrophilic MeSO
-DDE metabolites in the medium (Table 1), and irreversible binding of MeSO
-DDE to
protein (Fig. 3). Thus, both formation of hydrophilic
metabolites and irreversible protein binding of MeSO
-DDE is
mediated by the same isozyme, i.e. P450c11.
Figure 3:
Irreversible binding of
[C]MeSO
-DDE to protein in COS cells
transfected with plasmids encoding P450aldo, P450c11, and P450scc,
respectively, either alone or in combination with a plasmid encoding
ADX. Transfected COS cells were grown in the presence of
[
C]MeSO
-DDE for 24 h, whereafter
[
C]MeSO
-DDE irreversibly bound to
protein was measured. Figure shows mean ± S.D. (n = 3).
In vivo exposure of mice to MeSO-DDE and in vitro experiments with subcellular adrenal homogenates have shown a
selective irreversible binding of MeSO
-DDE in the adrenal
cortex(4, 5, 7) . The present studies provide
direct evidence for an involvement of adrenocortical mitochondrial
P450s in the bioactivation of MeSO
-DDE, by showing that
minimal MeSO
-DDE or deoxycorticosterone metabolism occurred
in uninduced adrenocortical cells and that forskolin induction
increased the metabolism of both substrates to the same extent.
Furthermore, we unambiguously ascribe the metabolism of
MeSO
-DDE to P450c11 through transfection studies in COS
cells. To our knowledge, this is the first example of
xenobiotic-induced toxicity mediated by mitochondrial
steroid-metabolizing P450s.
In addition, at concentrations only
slightly affecting the cell number (assessed as amount of protein per
plate), MeSO-DDE was a potent inhibitor of the
P450c11-activity in the Y1 cells (Fig. 4). In contrast, equal
concentrations of the non-adrenotoxic DDE did not affect P450c11 or
cell number (data not shown). This finding may provide an explanation
to the previously observed reduced capacity of mice exposed to
MeSO
-DDE to synthesize glucocorticoids(6) .
Figure 4:
Effect of MeSO-DDE on
P450c11-activity in Y1 cells and amount of protein per plate remaining
after culture with MeSO
-DDE. Y1 cells were grown in the
presence of forskolin and MeSO
-DDE for 20 h, whereafter
deoxycorticosterone was added. Three hours later culture medium was
assayed for corticosterone and cells for protein content. Figure shows
mean ± S.D. (n = 3-5).
This study thus illustrates two mechanisms, bioactivation of xenobiotics and inhibition of steroid hydroxylases, whereby xenobiotics may interfere with glucocorticoid synthesis. Because the adrenal gland generally accumulates high levels of lipophilic xenobiotics(17, 18) , and since a normal capacity to produce glucocorticoids is vital for organisms during stressful periods, the ability of certain xenobiotics to interfere with steroid hormone synthesis may have great significance and deserves further attention.