Serum T4 and T3 Levels
A significant interaction between age and dose (F6,48 = 21.56; p < 0.001) was observed for total serum T4 (). A post hoc test indicated no significant effects at the low dose for any time points tested. However, at PND 4, exposure to DE-71 decreased total serum T4 to 57 and 51% of control levels with exposure to 10.2 and 30.6 mg/kg/day, respectively. There was also a decrease of total serum T4 at PND 21 with levels 46 and 25% of controls with perinatal exposure to 10.2 and 30.6 mg/kg/day, respectively. By PND 60, no differences between control and dose groups were seen. Interestingly, there was no significant effect of dose on serum total T3 levels at any age group. In this study, decreases in total T4 levels were similar in both males and females. Therefore, we selected males only for understanding the mechanism as female offspring from this cohort were used for reproductive and neurotoxicological endpoints (Kodavanti et al., unpublished data).
Circulating Total T4 and T3 Levels (ng/ml) in Male Pups during Perinatal Exposure to a Penta PBDE Mixture, DE-71
Cytochrome P450s: Enzyme Activity and Gene Expression
Cyp1a1 mRNA expression and EROD activity are used as markers for Ah receptor activation (). There was a significant interaction of age and dose (F6,48 = 37.16; p < 0.0001) for Cyp1a1. A post hoc test indicates an effect at all doses tested at PND 4 and PND 21, and only for 30.6 mg/kg/day at PND 60. Cyp1a1 mRNA expression was induced at the 1.7, 10.2, and 30.6 mg/kg/day treatment doses by 16.1-, 44.3-, 154.2-fold at PND 4, and 4.6-, 10.2-, and 20.1-fold at PND 21, respectively. There remained a residual 5.6-fold increase in Cyp1a1 mRNA expression with the 30.6 mg/kg/day treatment group at PND 60. In agreement with this, EROD indicated a significant interaction of age and dose (F6,48 = 152.3; p < 0.0001) at all doses tested for PND 4 and PND 21, but not at PND 60. Hepatic EROD increased at the 1.7, 10.2, and 30.6 mg/kg/day doses by 23.3-, 49.5-, and 215.3-fold at PND 4, and 4.1-, 17.1-, and 31.9-fold at PND 21, respectively.
Effect of DE-71 on Hepatic Cytochrome P450 Gene Expression and Protein Activity
Cyp2b1 and Cyp2b2 mRNA expression and PROD activity are used as markers for CAR receptor activation (Maglich et al., 2002
; Waxman, 1999
; Yamada et al., 2006
). There was a significant interaction of age and dose for Cyp2b1 (F6,48
= 33.23; p
< 0.0001) and for Cyp2b2 (F6,48
= 41.28; p
< 0.0001). A post hoc
test indicated an effect at all doses tested at PND 4 and 21 but not at PND 60. Hepatic Cyp2b1 mRNA expression increased at the 1.7, 10.2, and 30.6 mg/kg/day doses by 4-, 13-, and 20.2-fold at PND 4, and 6.8-, 16.3-, and 26.8-fold at PND 21, respectively. Hepatic Cyp2b2 mRNA expression also increased significantly at the 1.7, 10.2, and 30.6 mg/kg/day doses by 1.9-, 5.6-, and 7-fold at PND 4, and 2.7-, 6.6-, and 10.6-fold at PND 21, respectively. In agreement, PROD showed a significant interaction of age and dose (F6,48
= 33.53; p
< 0.0001) with at all doses tested at PND 4 and PND 21, but not PND 60. Hepatic PROD, a marker for overall CYP2B activity, increased at the 1.7, 10.2, and 30.6 mg/kg/day doses by 8.8-, 47.2-, and 55.5-fold at PND 4, and 5.1-, 10.4-, and 9.6-fold at PND 21, respectively.
Cyp3a1 mRNA expression and BROD activity were used as markers for PXR receptor activation (Xie et al., 2000
). There was a significant interaction of age and dose for Cyp3a1 (F6,48
= 17.66, p
< 0.0001). A post hoc
test indicated a significant effect at all doses tested at PND 4 and PND 21, but not at PND 60. Hepatic Cyp3a1 mRNA expression increased with 30.6 mg/kg/day treatment by 2.6-fold at PND 4 and 6.9 and 14.7-fold at PND 21 with 10.2 and 30.6 mg/kg/day treatment, respectively. In agreement with this, a significant interaction of age and dose (F6,48
= 64.90; p
< 0.0001) was observed for BROD at all doses tested at PND 4 and 21. BROD increased 9.8- and 13.3-fold at PND 4 with treatments of 10.2 and 30.6 mg/kg/day, and 5.8-, 34.9-, and 40.7-fold at PND 21 with treatments of 1.7, 10.2, and 30.6 mg/kg/day, respectively.
UGTs: Enzyme Activity and Gene Expression
A significant interaction of age and dose (F6,48 = 12.81; p < 0.0001) was detected for UGT-T4 enzyme activity (). A post hoc test indicated a significant effect of DE-71 at the highest dose with a 2.5- and 2.8-fold increase at PND 4 and 21, respectively. No induction at PND 60 was observed. To determine which UGT isoform(s) may contribute to the overall enzyme activity, hepatic UGT mRNA expressions were further examined.
Effect of DE-71 on Hepatic UGT-T4 Protein Activity and Gene Expression
For Ugt1a1 mRNA expression, a significant interaction of age (F6,48 = 4.755; p < 0.0001) but not dose was observed at every dose group. However, a significant interaction of age and dose for Ugt1a6 mRNA expression (F6,48 = 4.755; p < 0.0001) was identified at 10.6 and 30.6 mg/kg/day, with an increase of 7.2- and 19.6-fold at PND 4, respectively. Additionally, there was a significant interaction of age and dose for Ugt1a7 mRNA expression (F6,48 = 3.479; p < 0.0062) with an increase of 1.6- and 2.1-fold at 10.6 and 30 mg/kg/day, respectively, on PND 4 and 2.6-fold at the high dose on PND 21. Lastly, a significant effect of age for Ugt2b mRNA expression (F6,48 = 10.34; p < 0.0001) was identified. The large increase in developmental expression observed for Ugt2b may possibly mask a dose effect; therefore a one-way ANOVA was performed which identified a significant increase with 30.6 mg/kg/day treatment of 5.5- and 67.6-fold at PND 4 and 21, respectively.
Thus, although a significant induction of gene expression was observed at the middle dose, there is no concomitant increase in UGT-T4 activity suggesting the enzyme assay used may not be sensitive enough to reflect the observed changes in gene expression.
SULTs: Enzyme Activity and Gene Expression
Exposure to DE-71 led to a significant effect on age for SULT-T4 (F6,48 = 11.25; p < 0.0001), Sult1a1 (F6,48 = 154.4; p < 0.0001) and Sult1c1 (F6,48 = 31.39; p < 0.0001) with no dose related changes (). However, a significant interaction of age and dose was identified for Sult1b1 mRNA expression (F6,48 = 7.540; p < 0.0001). A post hoc test identified a 2.8-fold increase on PND 4 with exposure to the high dose, and 2.1- and 4.1-fold increase at 10.2 and 30.6 mg/kg/day on PND 21, respectively.
Effect of DE-71 on Hepatic SULT-T4 Protein Activity and Gene Expression
Efflux Transporters: Gene Expression
A significant interaction between age and dose for the efflux transporter Mdr1 (F6,48 = 10.34; p < 0.0001) was observed (). A post hoc test indicated an effect of DE-71 at all doses for PND 4 and 21, but not at PND 60. There was a dose-dependent increase in hepatic Mdr1 mRNA expression of 1.5-, 1.8-, and 2.4-fold at PND 4 and 1.5-, 1.8-, and 2.2-fold at PND 21. Similarly, a significant interaction of age and dose for Mrp2 mRNA expression (F6,48 = 27.65; p < 0.0001) was also observed. Mrp2 mRNA gene expression levels increased 1.6-fold at the 30.6 mg/kg/day treatment group for PND 4. The expression of Mrp2 at PND 21 increased 1.5-, 1.9-, and 3.4-fold, respectively. At PND 60, a significant 30 and 40% reduction at 10.2 and 30.6 mg/kg/day was also found. Lastly, a significant interaction of age and dose was observed for Mrp3 mRNA expression (F6,48 = 17.62; p < 0.0001). A post hoc test indicated an effect for all doses at PND 4 and 21, but not at PND 60. The expression of hepatic Mrp3 mRNA, a major sinusoidal efflux transporter of glucuronides, indicated a 1.6-, 2.5-, and 4.1-fold increase at PND 4 and a 2.2-, 2.8-, and 5.1-fold increase at PND 21.
Effect of DE-71 on Hepatic Efflux Transporter Gene Expression
Influx Transporters: Gene Expression
A significant effect of age was observed for Oat2 (F6,48 = 270.5; p < 0.0001) and Ntcp (F6,48 = 42.79; p < 0.0001) but no dose effect was present (). However, a significant interaction of age and dose for Oatp1a4 mRNA expression exists (F6,48 = 7.540; p < 0.0001). A post hoc test identified a 1.3-fold increase for 30.6 mg/kg/day at PND 21. No effect at PND 4 and 60 were observed for Oatp1a4.
Effect of DE-71 on Hepatic Influx Transporter Gene Expression
Transthyretin: Gene Expression
There was a significant effect of age (F6,48 = 205.7; p < 0.0001) and dose (F6,48 = 3.02; p < 0.039) observed for Ttr (). A post hoc test indicated that Ttr mRNA expression was significantly decreased 20% at the highest dose tested, 30.6 mg/kg/day, on PND 21. No effects were seen at PND 4 or 60.
Effect of DE-71 on Hepatic Transthyretin Gene Expression
Deiodinase 1: Enzyme Activity and Gene Expression
D1 is a marker for acute changes in TH metabolism (Zoeller et al., 2006
) (). A significant interaction of age and dose for D1 enzyme activity (F6,48
= 15.02; p
< 0.0001) was observed for all doses tested at PND 4 and for 30.6 mg/kg/day at PND 21. At PND 4, D1 activity decreased 60% at all doses, whereas at PND 21, a significant 70% decrease in activity was observed only at the highest dose. There was also a significant interaction of age and dose for d1 mRNA expression (F6,48
= 5.978; p
< 0.0001). A post hoc
test indicated an effect at doses of 10.2 and 30.6 mg/kg/day for PND 4 and at 30.6 mg/kg/day for PND 21, but not at PND 60. At PND 4, expression levels were decreased in a dose related fashion to 40 and 50% at 10.2 and 30.6 mg/kg/day, respectively. However, on PND 21, the only decrease in d1 expression was at the high dose. No changes were observed at PND 60.
Effect of DE-71 on Hepatic Deiodinase 1 Protein Activity and Gene Expression
Previous hypotheses on perinatal TH disruption by DE-71 have focused on induction of hepatic UGT-T4–mediated TH catabolism resulting in decreased circulating T4 levels. It has also been hypothesized that competitive binding between PBDE metabolites and TH to serum transport proteins affects thyroid homeostasis. This study further investigates parameters involved in the alteration of TH levels with a focus on nuclear receptor–mediated activation during development, in the presence of a PBDE mixture. Specifically, this study aims to further identify genes activated by AhR, CAR, and PXR during perinatal exposure to DE-71. This information will further the risk assessment and provide information as to the mechanisms by which this commercial penta mixture exerts its effects. Concomitant with a decrease in T4, we observed an increase in hepatic UGT-T4 activity and UGT mRNA isozyme gene expression, a decrease in D1 enzyme activity and mRNA expression, and increases in the transcription of Sult1b1 and transporters of TH or glucuronides in neonatal and juvenile male rats following maternal PBDE treatment.
After perinatal exposure to DE-71, a dose-dependent induction of CYP1A1 and CYP2B demonstrates a similar induction pattern as previously reported (Zhou et al., 2002
), whereas we observe for the first time a dose-dependent induction of CYP3A. This is in agreement with a 28-day oral exposure study using a purified DE-71 mixture in adult Wistar rats (van der Ven et al., 2008
). This is also the first report measuring the induction of CYP450 mRNA expression after perinatal exposure to DE-71. The induction of CYP1A1, CYP2B, and CYP3A along with their respective mRNA transcripts signifies DE-71 is an agonist for AhR, CAR, and PXR, respectively. The increased induction seen in CYP2B as compared with CYP3A supports the suggestion that PBDE congeners have a preference for CAR over PXR (Pacyniak et al., 2007
; Sanders et al., 2005
; Richardson et al., 2008
). Although individual PBDE congeners have been shown not be AhR agonists (Peters et al., 2006
), induction of CYP1A1 gene expression and protein further supports the presence of dioxin-like contaminants in the DE-71 mixture (Hanari et al., 2006
) as being responsible for the Ah receptor-based responses (Sanders et al., 2005
Our data correlate with previous UGT developmental enzyme activity studies in Long-Evans male rats which also demonstrated decreases in TH concentrations along with increases in hepatic UGT-T4
activity with exposure to DE-71 (Zhou et al., 2002
). Different UGT isoforms have unique developmental patterns and their regulation is thought to be nuclear receptor specific. In this study, perinatal DE-71 exposure increased AhR mediated Ugt1a6 (Auyeung et al., 2003
; Nishimura et al., 2005
) and Ugt1a7 (Metz and Ritter, 1998
) along with CAR mediated Ugt2b (Zhou et al. 2005
) mRNA expression. Members of the UGT1A family specifically glucuronidate T4
, whereas members of the UGT2b family glucuronidate T3
(Vansell and Klaassen, 2002). The significant increase in hepatic Ugt1a6 and Ugt1a7 mRNA expression further supports contaminants in DE-71 as agonists for the AhR. In addition, PBDE induction of Ugt2b at the high dose parallels the UGT induction pattern and is likely CAR mediated.
In this study, there is a lack of DE-71 effect on SULT-T4
, Sult1a1, and Sult1c1, whereas an increase in Sult1b1 mRNA expression was identified. SULT1A1, 1B1, and 1C1 have enzymatic activity towards T3
, whereas SULT1C1 exhibits higher expression in males. The lack of a sex difference in Sult1b1 expression suggests it has a similar role in both sexes. For this reason it has been hypothesized that SULT1B1 may be more important for TH homeostasis than other isoforms (Dunn et al., 1999
; Fujita et al., 1997
). Considering sulfation is a reversible pathway of TH metabolism which depends on the free hormone recovery by sulfatases (Darras et al., 1999
), specific induction of Sult1b1 may be important in TH homeostasis during exposure to endocrine disrupting compounds such as PBDEs. In vitro
rodent experiments have demonstrated hepatic SULTs having different conjugation affinities for iodothyronines with T4
(Kaptein et al., 1997
). Although the Km of rodent SULT-T4
is high in relation to other iodothyronines, T4
comprises the fractional majority of TH at any given time in circulation and can possibly contribute to a large fraction of T4
for sulfation. The lack of effect observed on hepatic SULT-T4
activity after exposure to DE-71 contrasts with the observed increases in Sult1b1 mRNA expression. This could be due to SULT-T4
enzyme activity assay not evaluating the activity of specific SULT isoforms. Alternatively, increases in Sult mRNA expression may not result in marked increases of the respective enzymes.
The efflux transporters Mdr1, Mrp2, and Mrp3 are members of the ABC binding cassette superfamily (Borst and Elferink, 2002
; Dean and Allikmets, 2001
) and are regulated by AhR, CAR and PXR (Cherrington et al., 2002
; Geick et al., 2001
; Johnson et al., 2002
; Kast et al., 2002
; Maglich et al., 2002
; Teng et al., 2003
; Xiong et al., 2002
). Mrp2 resides at the canalicular membrane and secretes its substrates into bile (Müller et al., 1996
). Mrp3 is present at low levels at the basolateral membrane for export of substrates into sinusoidal blood. The expression of Mdr1 and Mrp2 are inducible by hormones and steroids and their activity may change during development (Courtois et al., 1999
; Demeule et al., 1999
). The induction of Mdr1 and Mrp2 both occurred at PND 4 and 21 with Mdr1 having a greater sensitivity at PND 4. Although Mrp2 and Mdr1 both have a high sensitivity to PBDEs later in development, Mdr1 efflux mechanisms appear to be involved in elimination and detoxification during early postnatal development to a greater extent than Mrp2. The developmental expression of the basolateral efflux transporter Mrp3 has been previously documented in mice (Maher et al., 2005
) and correlates with our findings in rats. Its age and dose-dependent sensitivity to PBDEs appear to be similar to the canalicular efflux transporter Mdr1; however Mrp3 is induced to a greater degree.
In rat liver, basolateral uptake systems include the sodium taurocholate cotransport protein (NTCP) and the OATPs. The basolateral Na+
-dependent bile salt transporter, NTCP, is specific to hepatocytes and is distributed homogeneously throughout the liver (Stieger et al., 1994
). Bile salts are the major substrate for NTCP, however, other compounds such as estrogen conjugates, TH, and xenobiotics that are covalently bound to taurocholate can also be transported (Kouzuki et al., 2000
). DE-71 did not alter mRNA expression levels of Ntcp. The lack of effect seen with exposure to this commercial mixture implies Ntcp is not activated by AhR/CAR/PXR. In addition, full maturation of NTCP transport activity is delayed until 4 weeks of age due to incomplete glycosylation (Kühlkamp et al., 2005
). This delay may contribute to the lack of Ntcp mRNA expression alterations during perinatal exposure seen here.
Although NTCP represents the major hepatocellular uptake system for conjugated bile salts, OATPs mediate sodium-independent uptake of a large variety of substrates. Specifically, OATP1a4 actively transports bile acids, xenobiotics, and TH. OATP1a4 belongs to the ABC cassette superfamily and is regulated by CAR and PXR (Wagner et al., 2005
). Because OATP1a4 is a basolateral hepatic influx transporter, the increased expression levels seen here may indicate a demand for sequestration of PBDEs and T4
into the liver for biotransformation and elimination.
Oat2 is found in higher concentrations in adult male rat liver than in kidney, however, the opposite is true for females (Buist et al., 2002
; Pavlova et al., 2000
). In this study, the expression levels of Oat2 in male Long-Evans rats were low at PND 4 and increased at PND 21, and then remained high through PND 60. This developmental pattern is consistent with previous findings for rat Oat2 in the liver (Simonson et al., 1994
). The level of transcript increases dramatically within two days post-partum and continues until the level stabilizes within the second week of postnatal development.
D1 is mainly expressed in the liver, thyroid, and kidney. Evidence suggests D1 is regulated directly or indirectly by CAR (Tien et al., 2007
) however time and dose response relationship observed here suggests D1 is regulated by CAR and/or AhR but not PXR. Raasmaja et al. (1996)
suggest a possible mechanism for the reduction in T4
to be due to increased tissue-specific deiodinase activity converting T4
. In this study, hepatic D1 activity decreased 60% at all doses on PND 4, and 70% with the highest dose on PND 21. Similar decreased serum T4
and hepatic D1 enzyme activity were seen with the PCB mixture, Aroclor 1254 (Hood and Klaassen, 2000
). Considering the similarities in PCB and PBDE structures and effects, common mechanisms are likely involved in D1 reduction. Interestingly, decreases in rat hepatic D1 have also been observed following exposure to 2,3,7,8-tetrachlorodibenzo-p
-dioxin (TCDD) and suggests D1 effects seen here may be attributed, at least in part, to the dioxin-like contaminants (Viluksela et al., 2004
). However, decreases in D1 were a secondary effect to circulating T4 reduction after TCDD exposure. Future studies are needed to determine whether the sensitive decrease in D1 activity is also observed with exposure to purified PBDE congeners.
The reduction in D1 activity and mRNA message levels after perinatal exposure to the penta PBDE commercial mixture has proven challenging to explain. D1 is thought to be responsible for the major portion of T3
production peripherally. However, during hypothyroidism, plasma T4
is reduced and peripheral T3
conversion from T4
is believed to be sustained by upregulation of extrahepatic D2 and downregulation of D1 (Zavacki et al., 2005
). D2 is a more catalytically efficient enzyme, maintaining T3
levels by increasing the fractional conversion of T4
to active T3
as compared with the equal conversion of active T3
and inactive rT3
In addition to D2, SULTs have been reported to work with D1 in the regulation of TH homeostasis. Sulfation in the human fetus has been proposed to be a protective mechanism regulating T3
levels (Santini et al., 1993
). This regulation is mediated by deiodinases (T4
S to T3
S), and furthermore by sulfatases (T3
S to T3
), during periods of high T3
demands (Chopra, 1994
; Santini et al., 1992
). In this study, rat hepatic Sult1b1 was increased. It has been shown that deiodination rates by D1 of T4
to inactive rT3
are increased nearly 200 times by sulfation (T4
S to rT3
S), whereas deiodination of T4
to active T3
is completely lost after sulfation (T4
S to T3
S) (Visser et al.
, 1993). Therefore, the decreases in rat hepatic D1 activity observed after exposure to xenobiotics may act as a protective mechanism to reduce the conversion of T4
S to inactive rT3
S. Favorably, this reduction of D1 serves to maintain T3
Risk assessment of the PBDE commercial mixtures is ongoing and this in vivo
study aids in a better evaluation of the possible risks for human beings. Although the administered doses used here may seem high, this study as well as others, have shown that effects of PBDEs are seen in animal models at concentrations within 10-fold of the high end of the human population in North America (McDonald, 2005
). This study demonstrates perinatal exposure as low as 1.7 mg DE-71/kg/day is sufficient to alter hepatic enzyme activity measured as early as PND 4 (). The most sensitive endpoints in terms of both mRNA and enzyme activity were a reduction of D1 and induction of CYP1A1, 2B1/2, and 3A. These endpoints were all more sensitive at PND 4 than PND 21 with the exception of Cyp3a1/CYP3A. In contrast, Ugt2b, Sult1b1, and Mrp2 expression are all higher on PND 21 than PND 4, but these endpoints were less sensitive than CYP3A. Expression of Mdr1 and Mrp3 are also extremely sensitive at PND 4. In contrast, UGT mRNA effects were only seen at 10.2 mg/kg/day or greater and enzyme activity at 30.6 mg/kg/day, indicating UGT-T4
is not the most sensitive marker for this PBDE mixture. All effects were largely reversible by PND 60. The induction of hepatic Sult1b1 mRNA expression seen here along with decreases of D1 may work together to maintain serum T3
and reduce T4
. In addition, the hepatic efflux transporters Mdr1, Mrp2, and Mrp3 may be involved. However, studies to identify sulfated TH specific transporters and whether the alterations in mRNA levels seen in this study are reflected in protein levels and enzyme activity are clearly needed.
Summary on the Effect of DE-71 on THs, Hepatic Protein Activity, and Gene Expressiona
The T4 depleting effects of DE-71 are likely to involve multiple mechanisms of action including Phase II glucoronidation and sulfation, transthyretin displacement, decreased hepatic deiodinase 1 activity, and increases in hepatic transporter phase III elimination (). This study has demonstrated coordinate modification in the expression of transport proteins and detoxification enzymes in the postnatal period of development after low perinatal exposure to DE-71, a commercial PBDE mixture. These alterations could be responsible for the important and rapid changes in TH observed during this period of life. Because DE-71 contains both PBDEs and PBDDs/PBDFs, it is difficult to attribute every effect measured in this study to the sole activation of CAR/PXR or AhR, respectively. Considering household dust contains both PBDEs and dioxin-like compounds and is suspected to be a major source of exposure for humans and other animals, this commercial mixture study is important to the understanding of its real world toxic effects. In furthering the risk assessment of this commercial PBDE mixture, this data demonstrates that DE-71 disrupts TH homeostasis in rats during development via perinatal exposure; however, the mechanism(s) of action appear complex.
FIG. 1. Possible mechanisms of TH disruption after perinatal exposure to the commercial “penta” PBDE flame-retardant mixture, DE-71. (1) PBDE congeners and dioxin-like (DX) contaminants enter the circulation. (2) PBDE (parent or hydroxylated metabolite) (more ...)