In the present study, we have shown that genetic differences in AHR affinity, as well as the presence or absence of the CYP1A2 enzyme, influence the maternal-fetal unit—with regard to PCB congener pharmacokinetics and degrees of toxic response to the developing neonate and weanling mouse. Our initial goal was to decide upon a PCB mixture that not only reflected that found in human breast milk and other tissues and a dose that would activate AHR but also a PCB dosage exposure that was neither substantially lethal nor causing overt birth defects such as cleft palate and hydronephrosis. Our long-range goal was to study behavioral phenotypes in these offspring when they reached adulthood at PND60, and this is the subject of the next paper (Curran, Genter, Patel, Vorhees, Williams, and Nebert, in preparation).
We were aware of studies using Aroclor mixtures that have repeatedly described neurological deficits across rodent species (Chishti et al., 1996
; Kang et al., 2002
). However, when attempts were made to identify individual congeners responsible for these effects, it became clear that single congeners did not produce the wide-ranging effects reported when studying Aroclor mixtures in laboratory animal studies, as well as effects observed in human cohorts (Ulbrich and Stahlmann, 2004
); this suggests that a mixture of congeners, rather than individual congeners, might be responsible for PCB-induced developmental neurotoxicity. In fact, other researchers reported that a mixture of coplanar plus noncoplanar PCB congeners was required to elicit changes in thyroid hormone signaling in the developing brain (Gauger et al., 2004
). For all these reasons, we decided to use the eight PCB complex mixture listed in , with chemical structures depicted in .
Preliminary studies also confirmed that we needed to provide a booster dose of the PCB mixture—in order to maintain high CYP1A1 mRNA inducibility (as a measure of chronic AHR activation) and sufficient PCB congener concentrations in the various tissues of the pup. Hence, we chose the regimen of one dose of PCBs given on GD10.5 and a second dose on PND5; collection time points for analysis included GD11.5, GD18.5, PND6, PND13, and PND28. The time line for our entire experimental paradigm is summarized in .
We then wished to test differences in response to the PCB mixture in mice having the high-affinity versus poor-affinity AHR combined with the presence versus absence of CYP1A2. Previous work from this laboratory had convincingly shown that presence of maternal hepatic CYP1A2 was able to sequester TCDD such that the fetus was protected approximately sixfold more than fetuses from mothers lacking hepatic CYP1A2; this led, in Cyp1a2(−/−)
mothers, to an approximately six times larger dose of TCDD required to cause cleft palate and hydronephrosis (Dragin et al., 2006
). It is also well known that Ahrd
-containing mice require a 15- to 20-fold higher dose of TCDD in order to achieve the same level of CYP1 inducibility as that seen in Ahrb1
-containing mice (Poland et al., 1974
). Combining these facts, we hypothesized that the Ahrb1_Cyp1a2(−/−)
mother-fetal unit would be most vulnerable, the Ahrd_Cyp1a2(−/−)
unit would be almost as vulnerable, the Ahrb1_Cyp1a2(+/+)
unit would be most resistant, and the Ahrd_Cyp1a2(+/+)
would be almost as resistant—with regard to toxicity induced by the PCB mixture ().
True to our predictions, small but statistically significant PCB-induced decreases in newborn birth weights were found in both Cyp1a2(−/−)-containing lines () and a small but statistically significant PCB-induced decreased growth rate was seen in Ahrb1_Cyp1a2(−/−) pups between PND1 and PND28 (). In general, noncoplanar congener concentrations in mother and offspring tissues between GD11.5 and PND28 were not associated with the Ahr or Cyp1a2 genotype (–)—except for Ahrb1_Cyp1a2(−/−) PND13 pups exhibiting higher levels in all tissues (), all five noncoplanar congeners highest in Ahrb1_Cyp1a2(−/−) PND28 pup inguinal fat pad (), and PCB 105 and PCB 118 highest in Ahrd_Cyp1a2(−/−) PND28 pup subcutaneous fat ().
However, the coplanar PCB congeners did show an intriguing pattern: at various collection time points, the Ahrd_Cyp1a2(−/−)
maternal brain, liver, adipose, and mammary tissues (as well as placenta, fetal, and neonatal tissues) retained the most coplanar PCB 77—until PND28 when most coplanar congeners had been metabolically cleared from the tissues (). Thus, protection of the fetus by maternal hepatic CYP1A2 in Ahrb1_Cyp1a2(+/+)
mice, after the PND5 dose given to the mother, continues through lactation and is associated with the high-affinity AHR. Our data are consistent with previous findings that the greatest exposure to pups is during lactation—not during gestation (Seegal et al., 1997
depicts CYP1A1 and CYP1A2 mRNA levels—at the same five collection time points as carried out for congener analysis in –—as quantified by qRT-PCR. CYP1A1 mRNA was highly induced in Ahrb1
maternal liver on GD11.5, GD18.5, PND6, and PND13 but not PND28; disappearance on PND28 is consistent with the coplanar PCBs being mostly sequestered by maternal liver CYP1A2. In support of this hypothesis, CYP1A1 mRNA was highly induced in Ahrb1
maternal liver at all five time points, including PND28, because of no maternal liver CYP1A2 to tie up the coplanar PCB inducers. CYP1A1 mRNA was not significantly induced in Ahrd
maternal liver at any of the five time points; these observations are consistent with what is generally seen in Ahrd
-containing mouse lines (Nebert et al., 1972
Western blots of CYP1A1 and CYP1A2 protein could have been carried out in all the tissues in which qRT-PCR measurements of CYP1A1 and CYP1A2 mRNA levels were obtained because it is always possible that protein levels might not reflect mRNA levels. However, in several dozen studies from this laboratory (Uno et al., 2008
and references therein), there has never been an instance in which CYP1 protein levels did not accurately reflect CYP1 mRNA levels.
An important next step in future studies would be to determine the concentration, identity, and transfer of PCB metabolites produced during gestation and lactation. Does the reduction in lower molecular weight congeners represent true clearance or simply the biotransformation into potentially toxic metabolites (Kimura-Kuroda et al., 2005
)? This is an important question because hydroxylated and methylsulfonated metabolites can cross the placenta (Park et al., 2009
; Soechitram et al., 2004
), and some have reported half-lives nearly as long as the parent congeners (Hovander et al., 2006
; Linderholm et al., 2010
). The small amounts of tissue available in a rodent study precluded the analysis at this time, but the use of radiolabeled congeners offers an option for tracking such metabolites more effectively.
CYP1A1 mRNA showed an increased trend that was not statistically significant (p
> 0.05), in Ahrb1
pup brain at PND28, whereas CYP1A1 mRNA was still statistically significantly induced in Ahrb1
pup brain at PND28 (). This observation is consistent with hepatic CYP1A2 acting as a “sink” (Dragin et al., 2006
)—when maternal CYP1A2 is absent, more PCBs reach the offspring.
The data confirm that the effect of CYP1A induction by coplanar PCBs can be seen not only in maternal liver but also in embryonic, fetal, and neonatal liver, as well as in PND28 brain. In other words, we achieved our goal of the PCB mixture regimen having a significant effect on the maternal-fetal unit—including AHR activation in postnatal brain. These data give us encouragement to proceed with the behavioral studies (to be described elsewhere).
Interestingly, decreases in PND13 and PND28 spleen weight ()—as well as PND6, PND13, and PND28 thymus weight ()—are more closely related to the Ahr
genotype than the presence or absence of CYP1A2. This finding is consistent with recent studies (Shi et al., 2010
) showing that intestinal inducible CYP1A1 is by far the most critical in detoxifying oral BaP—thereby preventing this PAH from distributing itself to distal tissues. Distal tissues, in this case, would include the intrauterine contents as well as pups receiving PCB-laced milk via lactation when the mother receives oral PAHs; distal tissues thus would also include pup spleen. Our present study indicates that this phenomenon extends from BaP to PCBs that are able to be metabolized by CYP1 enzymes; this finding further indicates that intestinal AHR affinity (and therefore inducible gut CYP1A1) is more crucial than the effect of maternal liver CYP1A2 acting as a sink to sequester AHR ligands such as coplanar PCBs. Unlike BaP, however, some more highly chlorinated PCBs (e.g., PCB 126) are not good CYP1 substrates, although they are excellent inducers of the CYP1 enzymes. Thus, an alternative explanation includes the possibility that persistent induction of CYP1 enzymes could lead to oxidative stress, thereby imposing more toxicity to the mice.
That pup splenic atrophy is more closely related to the Ahr
genotype—than the presence or absence of CYP1A2—would further suggest that both Cyp1a2(+/+)
mothers received sufficient amounts of coplanar PCBs to elicit immunosuppression and activate the AHR receptor. Presumably, consistent with the (Dragin et al., 2006
) study, at some lower levels of coplanar PCBs being administered, we would be able to see an effect in pups of Cyp1a2(−/−)
mothers but no effect in pups of Cyp1a2(+/+)
mothers. But, perhaps not; a dose-response study would clarify this question.
The liver weight to total body weight ratio was dramatically increased—a sign of chronic AHR activation—in all four PCB-treated genotypes at PND6 compared with controls receiving no PCBs (). The liver weight to total body weight ratio was significantly increased in the two Ahrb1-containing mouse lines at PND13 but only in Ahrb1_Cyp1a2(−/−) weanlings at PND28. Intriguingly, this is the mouse line that we had predicted would be most vulnerable to the PCBs' regimen (), and this is the same mouse line that had the highest amounts of coplanar PCB 126 and PCB 169 in PND28 pup inguinal fat ( and ).
Finally, can we extrapolate our mouse data to human populations? One might query whether the combination—of amount of exposure to coplanar PCBs, > 60-fold variation in basal and inducible hepatic CYP1A2 levels, and > 12-fold differences in AHR affinity—might be relevant to human risk assessment of PCB-induced birth defects. The answer to this question is not yet proven, but the present study, especially combined with the previous study (Dragin et al., 2006
), provides the basis for speculation about an “at-risk” subset in human populations. For example, the genotype of the affected fetus need not necessarily carry a teratogenic risk; rather, instead, a susceptible maternal genotype might be more crucial to risk of birth defects.
Hence, the highest risk for PCB-induced birth defects is likely to be in one whose maternal liver has genetically very low CYP1A2 activity, combined with a fetus who expresses the highest AHR inducibility. This notion follows from the observation that, although fetal CYP1A2 does not contribute to PCB-induced teratogenesis because it is not expressed in the embryo or fetus (Nebert, 1989
), fetal high-affinity AHR is probably essential for coplanar PCB-induced toxicity and teratogenesis, just as it is for TCDD-mediated teratogenesis (Peters et al., 1999
; Thomae et al., 2004
It should be noted that, for human cohort studies, determination of the precise level of environmental PCB exposure would be expected to be a confounding factor in such genotype-phenotype association studies. In addition, to date, no DNA variant sites in or near either the human CYP1A2
or the AHR
gene have been shown unequivocally to reflect variations in the CYP1A2 or AHR phenotype (Jiang et al., 2006
; Nebert et al., 2004