Enantiomeric enrichment of 2,2′,3,3′,6,6′- hexachlorobiphenyl (PCB 136) in mice after induction of CYP enzymes

Article abstract of DOI:10.1007/s00244-007-9111-4

Several PCB congeners, present in commercial PCB formulations, are chiral. These PCBs can undergo enantiomeric enrichment in many animal species and in humans due to currently uncharacterized enantioselective biotransformation processes. To investigate if certain cytochrome P-450 enzymes (CYPs), such as CYP2B's, are responsible for this enantiomeric enrichment, we investigated the enantioselective disposition of (±)-PCB 136 in female mice after induction of different CYP enzymes by pretreatment with corn oil alone, β-naphthoflavone (CYP1A's), phenobarbital (CYP2B's), or dexamethasone (2B's and 3A's), followed by oral PCB administration. PCB 136 levels were significantly lower in phenobarbital- and, to a lesser extent, in dexamethasone-pretreated animals, presumably due to the induction of PCB 136 metabolizing enzymes. Although (+)-PCB 136 was enriched in all tissues, none of the pretreatments altered the enantioselective disposition of PCB 136 in a manner that suggests a particular CYP subfamily as the cause of the enrichment of (+)-PCB 136. Fecal PCB levels and enantiomeric fraction values changed over time in a manner consistent with slower digestive motility in the mice pretreated with phenobarbital and dexamethasone. Overall, this study does not support the hypothesis that metabolism by CYP2B enzymes is responsible for the enrichment of (+)-PCB 136 in mice.

Full text of DOI:10.1007/s00244-007-9111-4

Arch Environ Contam Toxicol (2008) 55:510–517
DOI 10.1007/s00244-007-9111-4
0
0
0
Enantiomeric Enrichment of 2,2 ,3,3 ,6,6 -Hexachlorobiphenyl
PCB 136) in Mice After Induction of CYP Enzymes
(
Izabela Kania-Korwel Æ Wei Xie Æ Keri C. Hornbuckle Æ
Larry W. Robertson Æ Hans-Joachim Lehmler
Received: 31 August 2007 / Accepted: 3 December 2007 / Published online: 24 April 2008
Ó Springer Science+Business Media, LLC 2008
Abstract Several PCB congeners, present in commercial
PCB formulations, are chiral. These PCBs can undergo
enantiomeric enrichment in many animal species and in
humans due to currently uncharacterized enantioselective
biotransformation processes. To investigate if certain
cytochrome P-450 enzymes (CYPs), such as CYP2B’s, are
responsible for this enantiomeric enrichment, we investi-
gated the enantioselective disposition of (± )-PCB 136 in
female mice after induction of different CYP enzymes by
pretreatment with corn oil alone, b-naphthoflavone
enrichment of (+)-PCB 136. Fecal PCB levels and enan-
tiomeric fraction values changed over time in a manner
consistent with slower digestive motility in the mice pre-
treated with phenobarbital and dexamethasone. Overall,
this study does not support the hypothesis that metabolism
by CYP2B enzymes is responsible for the enrichment of
(+)-PCB 136 in mice.
(
CYP1A’s), phenobarbital (CYP2B’s), or dexamethasone
(
2B’s and 3A’s), followed by oral PCB administration.
Polychlorinated biphenyls (PCBs) are an important class of
manmade, persistent organic pollutants with the general
PCB 136 levels were significantly lower in phenobarbital-
and, to a lesser extent, in dexamethasone-pretreated ani-
mals, presumably due to the induction of PCB 136
metabolizing enzymes. Although (+)-PCB 136 was enri-
ched in all tissues, none of the pretreatments altered the
enantioselective disposition of PCB 136 in a manner that
suggests a particular CYP subfamily as the cause of the
structure C12H Cl10-n (n = 0–9). Both technical and envi-
n
ronmental PCB mixtures contain more than 100 compounds
(or congeners) with different chemical structures. Depend-
ing on their degree of chlorination and their substitution
pattern, PCB congeners have physicochemical properties
that can differ by several orders of magnitude (Hornbuckle
et al. 2006). They are semivolatile and are transported over
long distances in the environment. All PCB congeners are
lipophilic and many PCB congeners are resistant to chem-
ical and biological degradation in the environment. As a
result, they tend to bioaccumulate in organisms and to
biomagnify in the food chain, thus resulting in high and
potentially toxic PCB levels in humans. Animal and epi-
demiologic studies have implicated PCBs in adverse health
effects such as carcinogenesis, heart disease, and immu-
nological dysfunctions (Robertson and Hansen 2001).
Several epidemiological studies have shown that PCB
exposure in utero results in adverse neurodevelopmental
effects (Schantz et al. 2003). Both in vitro and in vivo
studies have linked PCB congeners with multiple ortho
substituents to these adverse neurodevelopmental outcomes
in humans (Kodavanti 2004; Lehmler et al. 2005).
I. Kania-Korwel ꢀ W. Xie ꢀ L. W. Robertson ꢀ
H.-J. Lehmler (&)
Department of Occupational and Environmental Health,
University of Iowa, College of Public Health, 100 Oakdale
Campus, 114 IREH, Iowa City, IA 52242, USA
e-mail: hans-joachim-lehmler@uiowa.edu
I. Kania-Korwel
Department of Environmental Chemistry and Technology,
University of Silesia, Szkolna 9, 40-006 Katowice, Poland
K. C. Hornbuckle
Department of Civil and Environmental Engineering, University
of Iowa, Iowa City, IA 52242, USA
L. W. Robertson ꢀ H.-J. Lehmler
The Interdisciplinary Graduate Program in Human Toxicology,
University of Iowa, Iowa City, IA 52242, USA
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Arch Environ Contam Toxicol (2008) 55:510–517
511
Many congeners with three or more ortho chlorines not
only have been shown to be neurotoxic, but —depending
on their substitution pattern—can form stable rotational
isomers that display axial chirality (Lehmler and Robertson
Materials and Methods
Chemicals
2
001). This raises the question of enantiospecific differ-
A detailed description of the PCB 136 sample used in this
study has been provided elsewhere (Shaikh et al. 2006). In
short, PCB 136 was synthesized by the Ullmann coupling
of 2,3,6-trichloro-1-iodobenzene and purified using char-
coal to remove trace amounts of halogenated dibenzofurans
([99% purity).
ences in the disposition and, ultimately, the toxicity of
these chiral PCB congeners. Indeed, a small number of
studies have shown that pure PCB atropisomers have dif-
ferent biological effects (P u¨ ttmann et al. 1989; Lehmler
et al. 2005). Furthermore, biotransformation processes can
result in the enantiomeric enrichment of chiral PCBs in a
wide range of environmental matrices (Muller and Kohler
Animal Treatment
2
004) and in humans (Chu et al. 2003; Harrad et al. 2006).
For example, the enantioselective biotransformation of
several chiral PCB congeners by PCB-degrading bacteria
has been documented (Singer et al. 2002).
The animal experiments and all animal procedures were
approved by the Institutional Animal Care and Use Com-
mittee at the University of Iowa. Twenty-six female C57BL/
6 mice were obtained from Harlan (Indianapolis, IN). The
mice were 7 weeks old and had an average weight of
19.3 ± 1.2 g. The animals were allowed to acclimatize for
1 week before they were randomly assigned to five groups
(Fig. 1). The treatment groups received intraperitoneal
injections of corn oil alone (CO; n = 4 animals; 10 ml/kg
body weight [bw]), NF (n = 6 animals; 2.73 mg/ml CO,
100 lmol/kg bw), PB (n = 6 animals; 10.2 mg/ml saline,
400 lmol/kg bw), or DEX (n = 6 animals; 5 mg/ml CO,
123 lmol/kg bw) for 3 consecutive days. Blank samples
were obtained from a separate group of four animals that
received intraperitoneal (ip) injections of CO alone (10 ml/
kg bw) (Robertson et al. 1984; Schramm et al. 1985). The
three inducers are well investigated and have been used in
our laboratory to study the effects of CYP enzymes on the
biotransformation and disposition of individual PCB cong-
eners in vivo (Pereg et al. 2001; Tampal et al. 2002).
Laboratory studies using individual PCB congeners in
rainbow trout (Wong et al. 2002; Buckman et al. 2006),
rats (P u¨ ttmann et al. 1989; Norstr o¨ m et al. 2006), and mice
(
Lehmler et al. 2003; Kania-Korwel et al. 2007) have
shown a significant enantiomeric enrichment. Although
enantioselective metabolism is thought to be responsible
for the enantiomeric enrichment in these animal species
and in humans (Harrad et al. 2006; Norstr o¨ m et al. 2006;
Kania-Korwel et al. 2007), the specific biotransformation
processes responsible for the enantiomeric enrichment of
PCBs have not been identified. Cytochrome P-450 (CYP)
2
B enzymes are likely candidates for the enantioselective
transformation of PCBs, because PCBs with multiple ortho
substituents and vicinal hydrogen atoms are more readily
metabolized by these enzymes in many different species
(
Kaminski et al. 1981; Brown 1994; Buckman et al. 2006).
In the present study we investigated whether the
induction of CYP enzymes alters the enantioselective dis-
position of an individual PCB congener in female mice in a
similar manner. PCB 136 was selected as a model com-
pound because its disposition in several species has been
well investigated (Mizutani et al. 1980; Birnbaum 1983;
Buckman et al. 2006; Kania-Korwel et al. 2007). Further-
more, we used b-naphthoflavone (NF), phenobarbital (PB),
and dexamethasone (DEX) instead of complex PCB mix-
tures to induce CYP1A, CYP2B, and CYP2B/3A enzymes,
respectively (Robertson et al. 1984; Schramm et al. 1985).
All four treatment groups received a single oral dose of
PCB 136 (50 mg/kg bw) on a Keebler Vanilla Wafer
cookie on day 4. A detailed description of vehicle prepa-
ration is provided elsewhere (Kania-Korwel et al. 2006).
After PCB treatment, animals were housed in metabolic
cages with three (NF, DEX, or PB treatment groups) or
four (CO treatment group and animals used to obtain
sample blanks) animals per cage. Feces and urine samples
for each cage were collected daily and stored at -20°C in
glass vials prior to PCB analysis. Animals were euthanized
Fig. 1 Experimental design to
study whether the induction of
different CYP enzymes alters
the enantiomeric enrichment of
PCB 136 in female mice.
bw = body weight
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Arch Environ Contam Toxicol (2008) 55:510–517
3
days after the PCB administration by asphyxiation with
used as a recovery standard and added to each sample
before extraction. The recovery rate was 95 ± 15%
(n = 381). Corrections were made for recoveries \100%.
The limit of detection of the method was calculated from
blank samples according to IUPAC as MDL ¼ xb þ k ꢀ sb
carbon dioxide followed by cervical dislocation. Blood was
collected by cardiac puncture and stored at -20°C in glass
vials. Tissues (abdominal adipose, brain, heart, intestines,
kidney, liver, lungs, right thigh muscles, skin, spleen,
thymus, uterus) were excised en bloc and their wet weight
was determined. All tissues were stored in aluminum foil at
(x is the mean of the 30 blank measures, k is Student’s
b
t-value for n–1 degrees of freedom at 99% confidence level,
and s is the standard deviation of the blank measures) and
is equal to 4.6 ng of PCB 136. The limit of quantification
-
20°C.
b
(
LOQ) was conservatively calculated as LOQ = 10ꢀLOD
Tissue Extraction Procedure
and is equal to 46 ng of PCB 136. No detectable levels
of PCB 136 were found in blank tissue samples.
As described previously, PCB 136 was extracted from
tissue samples and precleaned on Florisil in a single step
with hexane-acetone (1:1, v/v) using a Dionex ASE 200
system (Dionex, Sunnyvale, CA) and subsequently under-
went sulfur cleanup (Kania-Korwel et al. 2006).
Statistical Analysis
The statistical analyses in this study were performed using
SAS version 9.1 (SAS Institute, Cary, NC) (Kania-Korwel
et al. 2007). The data are presented as mean ± standard
deviation. Data points recognized by SAS as outliers were
removed before the statistical analysis. Differences in PCB
136 levels and EF values between treatment groups were
analyzed by ANOVA with Bonferroni multiple-compari-
son test at a = 0.05. PCB 136 level and EF differences
within treatment groups were analyzed for significant dif-
ferences by ANOVA with Tukey multiple-comparison test
at a = 0.05. Differences between EF values compared to
the racemic mixture (0.50 ± 0.01; see above) were ana-
lyzed by one-sample t-test with a = 0.05. The overall
significance level for all statistical analyses was set at 0.05.
Since blood concentrations were below the detection limit
in PB-pretreated animals, the MDL was used for this group
to analyze the %TD data.
Gas Chromatography Analysis
PCB 136 was determined on an HP 6890 gas chromato-
6
3
graph with a Ni l-ECD as described previously (Kania-
Korwel et al. 2006). A CP Chirasil-Dex CB (b-cyclodex-
trin chemically bonded to dimethylpolysiloxane;
2
5 m 9 0.25-mm i.d 9 0.25-lm film thickness) chiral
column was used for the chiral analysis. The resolution of
PCB 136 atropisomers on the columns was 0.64 (average
of three columns). The enantiomeric fraction (EF) was
defined as
Area
/Area
ðþÞꢁPCB136
þ AreaðꢁÞꢁPCB136
ðþÞꢁPCB136
(
Harner et al. 2000; Kania-Korwel et al. 2006). PCB 136
atropisomers were identified based on their published elu-
tion order on a CP Chirasil-Dex CB column (Haglund and
Wiberg 1996). The EF of the racemic standard mixture was
0
.50 ± 0.01 (average of 58 measurements; 95% confidence
Results and Discussion
interval, 0.498 to 0.504).
PCB 136 Levels in Tissues
Lipid Analysis
The dose of PCB 136 chosen in this study resembled
typical environmental exposures (Kania-Korwel et al.
Lipid contents of tissue, feces, kidney, and liver were
determined gravimetrically as described elsewhere (Kania-
Korwel et al. 2006). The nonvolatile residue present in
solvent blanks was on average 0.28 mg (n = 7). Lipid
weights in adipose tissue, feces, kidney, and liver were
2005) and had no adverse effect on body or organ weights
in PCB 136-treated animals. Comparison of PCB 136 tis-
sue levels within each group revealed general differences
that are in agreement with previous studies from our lab-
oratory (Kania-Korwel et al. 2007). Based on the
percentage of the total dose (Table 1) and the lipid or tissue
wet weight-adjusted PCB levels (Table 2 and Fig. 2,
respectively), the highest levels of PCB 136 in all treatment
groups were found in the adipose tissue. The muscle and
skin also accumulated a large fraction of the dosed PCB
relative to other, less lipid-rich tissues. The lowest PCB
levels were found in the brain and the heart.
7
5 ± 33 mg (n = 24), 38 ± 11 mg (n = 24), 11 ± 2 mg
n = 20), and 34 ± 14 mg (n = 22), respectively.
(
Quality Assurance
PCB 30 (2,4,6-trichlorobiphenyl) and PCB 204 (2,2 ,
0
0
0
3
,4,4 ,5,6,6 -octachlorobiphenyl) were used as internal
0
standards. PCB 166 (2,3,4,4 ,5,6-hexachlorobiphenyl) was
1
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Arch Environ Contam Toxicol (2008) 55:510–517
513
a
Table 1 Tissue concentrations of PCB 136 expressed as a percentage of the total dose
Tissue
Corn oil
b-Naphthoflavone
Dexamethasone
Phenobarbital
b,c
b,c
Adipose
Blood
Brain
7.2 ± 0.4
7.1 ± 0.9
2.9 ± 0.5
2.4 ± 0.5
b
b
0.32 ± 0.32
0.007 ± 0.001
0.004 ± 0.001
0.20 ± 0.06
0.04 ± 0.01
0.06 ± 0.01
0.04 ± 0.03
3.2 ± 2.6
0.06 ± 0.05
0.02 ± 0.01
0.003 ± 0.001
0.002 ± 0.000
0.001 ± 0.000
b
b,c
b,c
0.004 ± 0.000
0.003 ± 0.000
Heart
0.005 ± 0.004
0.17 ± 0.10
0.06 ± 0.03
0.05 ± 0.02
0.02 ± 0.01
6.9 ± 3.8
0.002 ± 0.002
b
0.06 ± 0.04
b
Intestines
Kidney
Liver
0.04 ± 0.01
c
b,c
b,c
b
0.02 ± 0.01
0.004 ± 0.002
0.009 ± 0.003
0.005 ± 0.004
0.04 ± 0.02
0.02 ± 0.01
2.7 ± 1.5
Lung
c
Muscles
Spleen
Skin
1.3 ± 0.9
b
b
0.006 ± 0.002
5.1 ± 1.9
0.003 ± 0.001
6.8 ± 2.7
0.002 ± 0.001
c
2.7 ± 0.4
0.002 ± 0.001
c
2.2 ± 0.8
Uterus
0.006 ± 0.007
16 ± 4
0.006 ± 0.007
21 ± 4
0.003 ± 0.002
b,c
8.5 ± 2.1
0.001 ± 0.001
b,c
5.9 ± 1.7
Total
d
Feces
2.3
0.90
1.6
1.7
Note. Values are mean ± standard deviation. The percentage of body weight was assumed to be 5.85% for blood (Dr. Larry Hansen, personal
communication), 5.9% for adipose tissue, 38.4% for muscles, and 16.5% for skin (Brown et al. 1997). The weights of all other organs were the
a
b
c
d
wet weight determined in this study. Statistically different from CO group, from NF group, and from DEX group. Excreted over a 3-day
period
Table 2 Comparison of lipid-adjusted PCB 136 levels in adipose
tissue, kidney, and liver of mice from different treatment groups: lg/g
lipid, mean ± standard deviation
The mice pretreated with DEX or PB had lower body
burdens of PCB 136 than the mice pretreated with CO
alone or NF, relative to the total dose. This was also
reflected in significantly lower percentages of the total dose
and/or PCB levels in many tissues (e.g., adipose tissue,
blood, intestine, kidney, liver, lung, muscle, skin, and
spleen) in the DEX and PB groups compared with the CO
and/or NF animals. The liver-to-adipose tissue ratio in the
PB group (0.005 ± 0.001) was significantly lower than that
in the CO (0.013 ± 0.001) and DEX (0.012 ± 0.001)
groups and usually lower than that in the NF group
Treatment group
Adipose tissue
Kidney
Liver
CO
NF
99 ± 21
77 ± 7
17 ± 6
9.4 ± 1.1
7.2 ± 0.8
2.8 ± 2.2
1.6 ± 0.9
23 ± 10
5.2 ± 2.4
2.1 ± 0.8
a,b
b
a,b
a,b
DEX
PB
40 ± 9
a,b
30 ± 11
a,b
CO, corn oil; NF, b-naphtoflavone; DEX, dexamethasone; PB, phe-
a
b
nobarbital. Statistically different from CO group and from NF
group
Fig. 2 PCB 136 levels in tissues
of mice from CO (control), NF
(b-naphthoflavone), DEX
(dexamethasone), and PB
(phenobarbital) treatment
groups. Bar represents mean;
error represents standard
#
deviation. Different from CO
group; * different from NF group
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(
0.012 ± 0.001). This low ratio suggests relatively low
liver. This interpretation is supported by the low levels of
PCB 136 (i.e., below the MDL) in blood from PB-pre-
treated animals. NF, on the other hand, is known to induce
primarily CYP1A activity in mice. Since CYP1A’s are not
able to bind or metabolize PCB 136, pretreatment with NF
did not alter the disposition of PCB 136 in a significant
manner, and PCB 136 tissue levels and the remaining
percentage of the total dose were typically comparable to
those in the CO group.
levels of PCB 136 in livers from PB animals. Although the
total dose of PCB 136 was usually higher in the NF group
than in CO animals, the levels in individual tissues in both
groups were comparable. The brain was the only notable
exception and had significantly lower PCB levels in the NF
group. No statistically significant differences in PCB levels
were observed between the DEX and the PB groups.
We hypothesized that the induction of enzymes involved
in the metabolism of PCB 136 would increase the total
metabolism of PCB 136. PCB 136 has four ortho chlorine
substituents and free vicinal positions in each phenyl ring.
Because of this structure, PCB 136 is readily metabolized
by CYP2B (Schnellmann et al. 1983; Waller et al. 1999)
and, possibly, CYP3A enzymes (Hrycay and Bandiera
Enantiomeric Fraction of PCB 136 in Tissues
In agreement with an earlier study from our laboratory
(Kania-Korwel et al. 2007), an enrichment of (+)-PCB 136
was observed in all tissues in all four treatment groups
(Fig. 3). The EFs (defined as EF = Area(+)-PCB136/Area(+)-
2
003), which explains the short half-life of PCB 136 in
mice (Mizutani et al. 1980). In contrast, PCB 136, like
other multiple ortho substituted PCB congeners, most
likely does not have a high affinity and is not a substrate for
CYP1A enzymes (Hrycay and Bandiera 2003). Based on
these structure-activity relationships, induction of enzymes
of the CYP2B and 3A subfamilies would, therefore, be
expected to increase the biotransformation of PCB 136 and
to reduce its half-life in mice.
+ Area(-)-PCB136) in all tissues in all four treatment
PCB136
groups were statistically different from (± )-PCB 136
(EF = 0.50) and ranged from EF = 0.57 (spleen in PB
mice) to EF = 0.74 (liver in PB mice). In the NF, DEX,
and PB groups, the liver showed the highest enantiomeric
enrichment (0.71 ± 0.04, 0.71 ± 0.01, and 0.74 ± 0.04,
respectively). In the CO group the highest enrichment was
found in the brain (EF = 0.68 ± 0.03) and the liver
(EF = 0.62 ± 0.002). Overall, the EF values in tissues
from the CO group were in good agreement with the EF
values in an analogous study from our laboratory (Kania-
Korwel et al. 2007).
PB is a potent inducer of CYP2B-type enzymes,
whereas DEX is known to mostly induce both CYP2B and
CYP3A enzymes. Confirming our hypothesis, PCB 136
levels in the DEX and PB groups were usually lower
compared to those in CO animals, which received no
inducer as part of their pretreatment (i.e., CO alone). The
liver-to-adipose tissue ratio of the PB group was compar-
atively low, which suggests that biotransformation by
CYP2B enzymes resulted in low levels of PCB 136 in the
We hypothesized that the induction of enzymes involved
in the metabolism of PCB 136 would change the EFs of
PCB 136 in the tissues and in feces. Specifically, we
expected that an increase in the levels of different CYP
enzymes (and, thus, the number of active sites responsible
Fig. 3 Enantiomeric fraction
(EF) values of PCB 136 in tissues
of mice from CO (control), NF
(b-naphthoflavone), DEX
(dexamethasone), and PB
(phenobarbital) treatment
groups. Bar represents mean;
error represents standard
deviation. Two brain samples
with no error bars were analyzed
as a pooled sample for the group.
#
ND, not detected. Different
from CO group
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Arch Environ Contam Toxicol (2008) 55:510–517
515
for the enantioselective biotransformation of PCB 136)
would result in a more pronounced enantiomeric enrich-
ment of (+)-PCB 136 in all tissues and in the feces.
However, with the exception of the liver, comparison of the
EF values in tissues revealed no differences in the enan-
tioselective disposition of PCB 136 among groups.
Specifically, induction of CYP2B-type enzymes did not
result in a more pronounced enantiomeric enrichment in
the PB group. This result is in agreement with our earlier
observation that rats with increased CYP2B activity did not
display a significant enantiomeric enrichment of several
chiral PCB congeners (Kania-Korwel et al. 2006). Both
studies provide evidence that metabolism of PCB 136 by
CYP2B’s in the liver is not a likely cause of the enantio-
meric enrichment of chiral PCBs in rodents. At the same
time, the present study does not confirm a correlation
between increased levels of CYP1A-type enzymes and an
increased enantiomeric enrichment of chiral PCBs such as
PCB 136. Although we currently cannot explain this dif-
ference between these studies, it is not surprising that
induction of CYP1A does not increase the enantioselective
biotransformation of PCB 136. It is surprising, however,
that induction of CYP2B had no effect.
hepatic protein content. Studies to explore this hypothesis
are currently under way.
Fecal PCB 136 Levels and EF Values
To assess whether the induction of hepatic enzymes prior to
administration of PCB 136 influenced its (enantioselective)
excretion, urine and feces were collected each day from
animals in all treatment groups and the PCB levels and
enantiomeric fractions were determined. In agreement with
previous animal studies (Matthews and Tuey 1980;
Birnbaum 1983), the urine and the feces were minor routes of
elimination compared to the biotransformation of PCB 136
in the liver. Specifically, the urine contained no detectable
amount of PCB 136, while the feces contained 7–10 lg/g (or
1–2% of the total dose) of PCB 136 on day 1 and the level
decreased thereafter in all four groups (Fig. 4A).
Enantiomeric enrichment of (+)-PCB 136 was observed
in feces collected during the entire study (Fig. 4B). On Day
1, the EF was only slightly higher (0.52 to 0.56, depending
on the group) than the racemic mixture (EF = 0.50) in all
groups. The EF increased over time and was approximately
0.64 to 0.71 on day 3 in all treatment groups. These EF
values are comparable to the EF values observed in the
liver. The rates at which the EF values changed were dif-
ferent depending on the pretreatment. The EF value in
feces from CO and NF mice increased from day 1 to day 2
and almost plateaued on days 2 and 3. In contrast, the EF
value of PCB 136 in feces from PB mice showed a slight
increase, from 0.52 to 0.58, from day 1 to day 2, followed
by a more pronounced increase on day 3. In contrast, the
fecal EF value from DEX mice hardly increased from day 1
to day 2 (0.54 and 0.55, respectively) but markedly
increased from day 2 to day 3, to reach an enrichment
The only difference in EF values between groups was
the significantly lower EF value of PCB 136 in liver from
the CO group compared to the NF, DEX, and PB groups
(
Fig. 3). We currently cannot explain this difference in
hepatic EF values. A different rate of enantioselective
biotransformation due to the induction of different CYPs
(
e.g., CYP1A’s versus CYP2B’s) is not a likely explanation
for this difference because the hepatic EF values in the NF,
DEX, and PB groups are comparable. It is more likely that
the more pronounced enrichment of (+)-PCB 136 in the
NF, DEX, and PB livers is related to a transient increase in
Fig. 4 PCB 136 level (A)
and enantiomeric fraction
(
EF; B) in feces collected on
each day of the study from
mice in the CO (control), NF
(b-naphthoflavone), DEX
(dexamethasone), and PB
(phenobarbital) treatment
groups. Data represent the
average PCB levels and EF
values from two cages (NF, PB,
and DEX treatment groups)
with three animals or one cage
(CO treatment group) with four
animals
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comparable to that in the other three groups. This change in
EF values over time mirrors the decrease in PCB levels
observed for the DEX group.
not result in the expected increase in EF values. Instead, the
extent of the enantiomeric enrichment of (+)-PCB 136 was
similar among the four treatment groups. These findings do
not suggest enantioselective biotransformation (e.g., by
CYP2B enzymes) as the cause of the enrichment of
(+)-PCB 136 in mice and, most likely, of other chiral PCB
congeners in other animal species and in humans. Instead,
other enantioselective processes, for example, enantiose-
lective binding to (hepatic) enzymes, should be
investigated as the potential cause of the enantiomeric
enrichment of PCB 136 and other PCB congeners in vivo.
The high amount of nearly racemic PCB 136 excreted
within the first 24 h after administration in the CO, NF, and
PB groups is mostly due to nonabsorbed, racemic PCB 136
(
Norstr o¨ m et al. 2006). The slight enantiomeric enrichment
at this time point is due to a small amount of previously
absorbed PCB 136 (i.e., racemic PCB 136 that is absorbed
in the gastrointestinal tract, undergoes enantiomeric
enrichment, and, subsequently, is excreted back into the
gastrointestinal tract). The comparatively small quantities
of PCB 136 excreted 2 and 3 days after PCB administration
were due to previously absorbed PCB 136. This previously
absorbed PCB 136 underwent enantiomeric enrichment
and, thus, has an EF value that represents the enantiomeric
enrichment observed in the tissues at day 3 after PCB
administration. The different excretion profile of PCB 136
in the DEX group, with regard to both PCB level and EF
values, is due to decreased intestinal motility resulting from
the dexamethasone pretreatment (Persico et al. 1991) and,
thus, a longer retention of racemic, nonabsorbed PCB 136 in
the gastrointestinal tract. Similarly, the time-dependent
changes in fecal EF values in the PB group, which are
almost identical to the changes observed in the DEX group,
are probably also due to decreased intestinal motility caused
by a direct effect of phenobarbital on the gastrointestinal
tract (Holzer et al. 1987). Overall, the altered fecal excre-
tion of PCB 136 suggests that the pharmacological effect of
both DEX and PB may play a minor role in the overall
disposition of PCB 136 in DEX and PB animals compared
to the other two groups.
Acknowledgments We thank Dr. Regine Garcia Boy for assistance
with the animal procedures, Collin Just for help with the GC analysis,
Holly Moriarty and Allison Smith for help with the analytical work,
and Dr. Botond Banfi for use of his metabolism cages. This research
was supported by Grants ES05605, ES013661, and ES012475 from
the National Institute of Environmental Health Sciences, NIH, and
Major Research Instrumentation Grant BES-0420378 form the
National Science Foundation.
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