Genetic Differences in Lethality of Newborn Mice Treated In Utero with
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《毒物学科学杂志》
Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati OH 45267–0056
Division of Neurology, Children's Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio
2 To whom correspondence should be addressed at Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati OH 45267–0056. Fax: 513–558–0925. E-mail: dan.nebert@uc.edu
ABSTRACT
Polybrominated biphenyl (PBB) exposure in humans is known to cause immunotoxicity and disorders related to the central nervous system. Coplanar PBBs bind to the aryl hydrocarbon receptor (AHR) in vertebrates. We compared the coplanar PBB, 3,3',4,4',5,5'-hexabromobiphenyl (cHBB), with its stereoisomer, the non-coplanar PBB, 2,2',4,4'6,6'-hexabromobiphenyl (ncHBB), using C57BL/6J (B6) inbred mice (having the high-affinity AHR) and congenic B6.D2-Ahrd mice (having the low-affinity AHR in a >99.8% C57BL/6J genetic background). Pregnant dams were treated i.p. with vehicle alone, cHBB, or ncHBB on gestational day 5 (GD 5). Unexpectedly, neonatal lethality within the first 72 h postpartum was significant in cHBB-treated B6 mice at doses as low as 2.5 mg/kg, whereas no deaths were seen in B6 pups whose mother had received ncHBB 100 mg/kg or in either B6.D2-Ahrd or Ahr(–/–) knockout mice whose mother had received cHBB 100 mg/kg. Histological and gross anatomical analyses of a battery of tissues in the mother or fetus at GD 18, as well as 24 h postpartum, revealed no significant differences, except for decreased thymus and spleen weights in cHBB-treated B6 GD 18 fetuses. Cross-fostering and genetics experiments confirmed the association of neonatal deaths principally with in utero (rather than lactational) exposure to cHBB, and also no paternal effect. For the end points of mouse neonatal lethality and immunotoxicity, cHBB appears to act through the high-affinity AHR receptor. Although dioxin in utero is well known to cause AHR-dependent cleft palate and hydronephrosis, cHBB did not; thus, chronic activation of the AHR appears to be necessary but not sufficient for AHR-mediated teratogenicity.
Key Words: 3,3'4,4',5,5'-(coplanar)-hexabromobiphenyl; 2,2',4,4',6,6'-(non-coplanar)-hexabromobiphenyl; newborn lethality; in utero toxicity; immunotoxicity; aryl hydrocarbon receptor (AHR).
INTRODUCTION
Polyhalogenated biphenyls represent a class of ubiquitous environmental pollutants (Headrick et al., 1999; Zabik and Zabik, 1999) and include polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). These chemicals are similar in their structure–activity relationships and share similar toxicity profiles with regard to their carcinogenicity, teratogenicity, immunotoxicity, and general health effects (Safe, 1984; Silberhorn et al., 1990; Hakk and Letcher, 2003; Darnerud, 2003; Birnbaum and Staskal, 2004). Although PCBs have been studied more than PBBs, PBBs are more toxic. The global market demand and production for brominated flame retardants (BFRs) has increased dramatically during the past 20 years, especially in Asia; more than 200,000 metric tons of BFRs are produced each year (Birnbaum and Staskal, 2004). There is increasing concern about the contamination of brominated flame retardants in our environment, and that these represent developmental neurotoxicants (Eriksson et al., 2001).
Polybrominated biphenyls and PCBs are known to elicit a potent biochemical response, principally as a function of their affinity for several types of intracellular receptors (Safe, 1984; Silberhorn et al., 1990). The number of halogens, and their positioning around the biphenyl structure, is critical in determining the activity of these halogenated hydrocarbons. For example, on the one hand, halogens in the meta- and/or para-positions (Fig. 1, top) allow the molecule to remain coplanar, show very high affinity in binding to the AHR, and elicit effects similar to those seen for AHR ligands such as polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). On the other hand, halogens in the ortho-positions (Fig. 1, bottom) cause intramolecular torsion that makes the molecule non-coplanar and limits binding to the AHR; the induction response to non-coplanar halogenated biphenyls is similar to that seen for phenobarbital (Safe, 1984).
In the 1973 environmental disaster in Southern Michigan, hundreds of individuals (on 308 farms) were accidentally exposed to about 1000 pounds of PBBs (Dunckel, 1975; Carter, 1976; Kay, 1977; Anderson et al., 1978). On a shipping dock, the fire retardant FireMaster was mistaken for the agricultural food supplement NutriMaster. Thousands of cows and pigs, hundreds of sheep, and millions of chickens were contaminated and had to be destroyed. Humans consumed the meat, milk, and eggs before the magnitude of the danger had become clear (Carter, 1976). Today, PBB levels (370 times higher in adipose tissue than in blood) continue to decrease slowly in this exposed population. Follow-up studies have estimated the PBB half-life at 10.8 years and predicted it will take more than 60 years for serum levels to fall below the limits of detection in the most highly exposed group (Rosen et al., 1995). Intriguingly, the earliest clinical signs that a disaster had occurred included central nervous system (CNS) symptoms (Valciukas et al., 1978) of amnesia, confusion, and somnolence (farmers lost their tractors, were unable to find their way home at the end of the day, and fell asleep in the fields) and leukocytopenia (i.e., immunosuppression). Also reported was chloracne (Aust et al., 1987)—which is the sentinel sign of dioxin (AHR-mediated) toxicity in exposed workers (Birnbaum, 1994). Lowered birth weights (Sunahara et al., 1987), increased respiratory illnesses (Weil et al., 1981), and the possible lowering of I.Q. among some children born to Michigan mothers exposed in 1973–1975 (Seagull, 1983; Nebert et al., 1983) have been reported, and the mental development of these children continues to be followed into adulthood. Hyperactivity and alterations in behavior have been reported with massive PCB exposures: Yusho children whose Japanese mothers were exposed in 1968 and Yu-cheng children whose Taiwanese mothers were exposed in 1979 to contaminated rice bran oil (Rogan et al., 1988; Jacobson et al., 1990; Chen et al., 1994), as well as monkeys whose mothers were treated with PCBs (Schantz et al., 1989) or dioxin (Schantz and Bowman, 1989).
Polybrominated biphenyl and PCB levels in any particular subject are not always correlated with the degree of CNS impairment—suggesting a genetic component. Mice are known to exhibit 15- to more than 20-fold differences between inbred strains in AHR ligand affinity; humans also show more than 12-fold differences in AHR ligand affinity (Nebert et al., 2004). Our hypothesis, therefore, was that an underlying genetic variability in the AHR phenotype might have accounted for interindividual differences in response to the PBB-caused CNS toxicity in Michigan. Because such a study in humans is not ethically possible, we have used a mouse model. We chose to compare the effects of the coplanar and non-coplanar HBB stereoisomers (Fig. 1) in C57BL/6J inbred mice (B6, having the high-affinity AHR) and in congenic B6.D2-Ahrd mice [having the DBA/2J (D2) poor-affinity receptor in a genetic background of >99.8% B6]. Our original experimental protocol was to administer cHBB or ncHBB to the pregnant mouse on gestational day 5 (GD 5) and then perform behavioral phenotyping tests on the offspring at 60 days postpartum. Unexpectedly, we found a striking difference in neonatal lethality—depending on both the Ahr genotype and the HBB stereoisomer administered.
MATERIALS AND METHODS
Chemicals.
Both 3,3',4,4',5,5'-hexabromobiphenyl (cHBB) and 2,2',4,4',6,6'-hexabromobiphenyl (ncHBB) were purchased from Ultra Scientific (North Kingstown, RI). Unfortunately, to our knowledge, these chemicals are no longer commercially available.
Animals and treatment.
All experiments with mice were conducted in accordance with the National Institutes of Health standards for care and use of experimental animals, the University of Cincinnati Institutional Animal Care and Use Committee (IACUC), and the Cincinnati Children's Research Foundation IACUC. Male and female mice of the inbred strain C57BL/6J were purchased from The Jackson Laboratory (Bar Harbor, ME). Male and female mice of the congenic B6.D2-Ahrd line were obtained from the Nebert mouse colony at the University Cincinnati Medical Center, and some were purchased from The Jackson Laboratory. The mice were given access to standard rodent chow and water ad libitum; the colony was maintained with a 12-h light/dark cycle. Mice were bred on a 4-day cycle, and a finding of a vaginal plug in the morning was evidence of copulation; this was labeled as gestational day 0.5 (GD 0.5).
Initial enzyme induction and mRNA induction experiments were performed on cHBB- and ncHBB-treated nonpregnant 6-week-old female B6 mice. Subsequently, pregnant dams were administered i.p. various doses (ranging from 2.5 to 100 mg/kg body weight) of either cHBB or ncHBB, or an equivalent volume of the vehicle (corn oil; 25 ml/kg) on GD 5. This regimen was chosen because treatment on GD 5 is beyond the implantation period and, therefore, the treatment time would avoid toxicity associated with the implantation process. In addition, GD 5 treatment ensures that the chemical is present during the earliest days of ectoderm and neural crest development. Intraperitoneal treatment of the mother means the drug enters the splanchnic venous system directly and then travels to the liver, lungs, and arterial circulation including to the uterus. This is more direct than oral treatment, which might involve maternal "first-pass elimination kinetics" (Lukas et al., 1971), i.e. rate of absorption from the gut, combined with HBB-mediated induction of enzymes in the GI tract and liver, all of which might affect distribution of the chemicals to other tissues including the uterine contents.
Adult mice were sacrificed by carbon dioxide asphyxiation, followed by cervical dislocation. Newborn mice were killed by decapitation. Fetuses were also taken at GD 18, and whole liver and brain were examined from the mother and fetus; newborn tissues were also examined, including spleen and thymus wet weights.
AHRE activation.
AHRD-tk-LUC cells are mouse hepatoma Hepa-1c1c7 cells that have been stably transformed with the reporter-gene construct containing the luciferase reporter gene (LUC) driven by the thymidine kinase promoter and the mouse Cyp1a1 5'-flanking region as an enhancer (Maier et al., 2000). Cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum (FBS; Life Technologies, Rockville, MD). Cells during their logarithmic growth phase were treated with 1000x concentrated stocks of either cHBB or ncHBB dissolved in dimethylsulfoxide, and the compounds were added directly to medium. Cells were treated for 12 h, after which they were harvested. Luminescence was determined at 495 nm (Maier et al., 2000).
CYP1A1 enzyme induction and mRNA accumulation.
CYP1A1 [aryl hydrocarbon hydroxylase, benzo[a]pyrene (BaP) hydroxylase] enzyme activity, using BaP as substrate, was determined as described elsewhere (Nebert and Gelboin, 1968). Total RNA from liver and brain was extracted, size-separated, blotted on nylon membranes, and probed with CYP1A1-specific cDNA for Northern blot analysis by standard methods (Chomczynski and Sacchi, 1987). CYP1A1 mRNA was also measured, using the reverse transcriptase polymerase chain reaction (RT-PCR) technique previously described (Jiang et al., 2005).
Neonatal toxicity.
Newborn deaths were recorded for the first 72 h following birth. In cross-fostering experiments, newborns from cHBB-treated mothers were nursed by vehicle-treated mothers, and vehicle-treated newborns were nursed by cHBB-treated mothers.
Histology and anatomy.
Liver and brain from GD 18 fetuses, newborn mice, and mothers were prepared for routine light- and electron-microscopic histology and morphometry (Dalton et al., 2000). In liver, phase-contrast microscopy of toluidine blue-stained 1.5-micron-thick plastic sections was used to quantify cytoplasmic, nuclear and nucleolar areas. At 40x, the areas of hematopoietic cells and of liver were determined, and the percent hematopoiesis was calculated by division of the former value by the total area times 100. A grid of 75 intersections was visualized over the light-microscopic image, using a Zeiss Photomik light-microscope and camera lucida, and the number of positive intersections lying over hematopoietic cells, and over hepatocytes, was used as a second method for quantifying the volume density of hematopoiesis in the liver. Birth defects (especially cleft palate and hydronephrosis) were looked for; gross examination of all internal organs and histology of the kidney were also carried out.
Oxidative stress parameters.
Liver and brain from GD 18 fetuses, newborn mice, and mothers were harvested and frozen on dry ice; tissues were then stored at –70°C. Reduced (GSH) and oxidized (GSSG) glutathione levels were measured spectrophotofluorometrically using the o-phthalaldehyde method, as previously described (Senft et al., 2000). Ascorbate levels were measured by the procedure described by Zannoni et al. (1974).
Immunotoxicity.
Newborn spleen and thymus were isolated, and organ wet weights were determined.
cHBB treatment of Ahr(–/–) mice.
Generation of the Ahr(–/–) knockout mouse line has been described (Fernandez-Salguero et al., 1995), and these mice are maintained on the C57BL/6J background in the Nebert mouse colony. Untreated Ahr(–/–) females and males were bred, and viability versus deaths of the first litter were recorded. For the second pregnancies, cHBB 100 mg/kg body weight was given to pregnant mothers at GD 5. More than 2 weeks later, neonatal survival during the next 72 h was recorded and compared with what had been observed for that mother in its first pregnancy. This method was used to control for the well-known fertility problems associated with Ahr(–/–) mice (Abbott et al., 1999; Benedict et al., 2000, 2003).
Distribution of radiolabeled polyhalogenated biphenyl in mother and fetus.
14C-PCB-169 (3,3',4,4',5,5'-hexachlorobiphenyl; 100 μCi/1.3 mg of chemical in toluene) was purchased from Larry Hansen (College of Veterinary Medicine, University of Illinois, Urbana). This radiolabel was evaporated with a gentle stream of nitrogen, taken up in corn oil, and used to spike the cHBB in corn oil given to the pregnant mother at GD 5 at a dose of 100 mg/kg. At GD 18, radioactivity in the liver and brain of the fetus and mother was determined.
Statistical analyses.
Means and standard errors of the means (S.E.M.) were obtained from the morphometric data, using the General Linear Model of SAS 9.1. Data were analyzed by analysis of variance (ANOVA), followed by group comparisons by Student's t-test. A value of p < 0.05 was regarded as statistically significant.
RESULTS
Dose-Dependent Activation of the AHR
A reporter cell line (AHRD-tk-LUC) for activation of the high-affinity mouse AHR has previously been reported (Maier et al., 2000). Luciferase activity in AHRD-tk-LUC cells reflects AHR activation, because these cells are stably transfected with a construct containing four aryl hydrocarbon response elements (AHREs) linked to the thymidine kinase promoter and driving the luciferase reporter gene (Maier et al., 2000). Figure 2 shows a dose-dependent increase in luminescence in cHBB-treated but not in ncHBB-treated cell cultures. These data confirm that the structural characteristics of coplanar hexabromobiphenyl render it capable of activating the AHR, whereas the non-coplanar stereoisomer cannot. This experiment also assures the purity of these HBB congeners.
CYP1A1 Enzyme Activity and mRNA Induction by cHBB
Cyp1a1 has been demonstrated in many studies to provide an accurate surrogate for AHR activation. Figure 3 shows that cHBB caused the parallel induction of hepatic CYP1A1 enzyme activity and mRNA levels in B6 mice. Accumulation of both was maximal at 3 mg/kg. In contrast, CYP1A1 was not induced in liver from B6.D2-Ahrd mice, even at 100 mg/kg (Fig. 3, middle panel). This experiment justifies use of the B6 inbred strain—having the high-affinity AHR—in comparison with the congenic B6.D2-Ahrd line—having the poor-affinity D2 receptor in a >99.8% B6 genetic background—so that we might compare the toxicity of cHBB versus ncHBB in newborn mice. Moreover, cHBB, when administered to dams at GD 5 of pregnancy, was capable of persistent (measured at GD 18) Cyp1a1 induction in the fetal liver and brain (Fig. 3, lower panel); these data confirm that cHBB crosses the placenta and may persist in fetal tissues. Finally, ncHBB at 100 mg/kg did not induce liver CYP1A1 enzyme activity or mRNA levels in B6 or B6.D2-Ahrd mice (data not shown).
Neonatal Lethality
When B6 and B6.D2-Ahrd mice were treated on GD 5 with cHBB or ncHBB (100 mg/kg), the number of live healthy-appearing pups per litter from both strains did not differ statistically (data not shown). Unexpectedly, however, we found that this dose of cHBB but not ncHBB, caused 100% deaths in all B6 pups within 72 h postpartum (Table 1). To examine further this toxicity to B6 pups, we continued to lower the prescribed amount of cHBB and found that—even at doses of 2.5 mg/kg and 5 mg/kg—there was significant lethality of B6 pups. These data show that the LD50 value, for B6 neonates whose mother had received cHBB on GD 5, is between 5 mg/kg and 10 mg/kg and that lethality appears to depend on the high-affinity AHR.
Backcross Experiments
To determine if lethality occurs as a consequence of the fetal or maternal AHR, we conducted backcross experiments. We crossed the B6.D2-Ahrd female with the B6 male, and the B6 female with the B6.D2-Ahrd male, and assessed for cHBB-induced lethality (Table 2). The pups in either cross have identical genetic makeup, i.e., Ahrb1/d heterozygotes for the high- versus poor-affinity receptor. Complete lethality occurred in the neonates whose B6 mother had received the cHBB 100 mg/kg at GD 5; in contrast, the pups whose B6.D2-Ahrd mother had received the cHBB 100 mg/kg at GD 5 exhibited 100% survival. We conclude that only GD 5 cHBB treatment in utero, in the high-affinity-AHR mother, leads to neonatal death within the first 72 h postpartum, and there is no paternal contribution. For the remainder of this study, we sought to determine the cause of this neonatal death.
Cross-Fostering Experiments
In order to distinguish between in utero effects of cHBB versus maternal fostering behavior or cHBB in milk, we switched litters at birth between cHBB-treated and control mothers (Table 3). B6 pups, whose mothers had received cHBB 100 mg/kg at GD 5, did not survive the first 72 h postpartum; if cross-fostered to corn oil-treated dams within 6 h of birth, 21% of the cHBB-treated B6 pups survived. Control B6 pups—whose mothers received corn oil injections on GD 5—all survived the first 72 h postpartum when cross-fostered to cHBB-treated dams (100 mg/kg at GD 5). The dams' behavior was monitored three times a day during the 72-h postpartum window to determine if pups were being nursed and groomed normally. We found no significant differences in the behavior of treated or control dams of either line (Table 3). We therefore conclude that it is, in large part, the in utero cHBB treatment of the high-affinity AHR mouse that leads to death within the first 72 h postpartum, but that cHBB received via the milk might be a contributory factor.
In control B6 pups nursing on cHBB dams, weight loss and lethality were noted beginning at 9 days postpartum. This finding supports our conclusion (above) that persistent (defined here as greater than 1 week) activation of the AHR in the pup by cHBB, distributed to the pup in the mother's milk, appears to be a contributory factor to neonatal lethality.
Histology
Light- and electron-microscopy of the liver and brain and morphometrics of the liver in the GD 18 fetus, its mother, and newborns of all groups were carried out in the six (3 x 2) possible groups: cHBB 100 mg/kg, ncHBB 100 mg/kg, and vehicle-treated controls in B6 mice versus and B6.D2-Ahrd mice. Despite the fact that cHBB administered on GD 5 produced residual effects at GD 18 that could be seen in the fetal liver and brain in the form of increased CYP1A1 mRNA levels (Fig. 2), no statistically significant morphological differences in the livers or brains could be detected among any of the groups. Morphometry on GD 18 fetal livers suggested a slight increase in the volume density (Vd) of hemopoietic islands was higher in both cHBB- and ncHBB-treated groups (100 mg/kg on GD 5) and in both B6 and B6.D2-Ahrd mice, compared with that in control groups; in contrast, no difference between cHBB and ncHBB was seen in either mouse line. The response (i.e., elevation in hemopoietic Vd) was greater in cHBB-treated B6 than in cHBB-treated B6.D2-Ahrd fetal livers (p = 0.08). The same trend appeared when the sizes of hemopoietic sites were quantified: cHBB-treated B6 showed greater sizes than cHBB-treated B6.D2-Ahrd fetal livers (p = 0.04).
Because of questionable teratogenesis of PCBs in fish (Meyer and Di Giulio, 2002), we examined the pups specifically for signs of birth defects—including especially cleft palate and hydronephrosis, two hallmarks of dioxin-induced AHR-mediated teratogenesis in mice—and found none in these newborns. We did, however, note cHBB-induced spleen and thymus atrophy (another hallmark of dioxin-induced AHR-mediated toxicity in laboratory animals), which was found in the B6 neonates but not the B6.D2-Ahrd newborns; this finding is discussed below. No gross abnormalities of any other internal organs were seen.
AHR Dependence of cHBB-Induced Neonatal Lethality
If cHBB-induced neonatal lethality is indeed mediated by the AHR, would Ahr(–/–) homozygous knockout pups be protected in utero We first allowed four Ahr(–/–) nulliparous females to mate with Ahr(–/–) males and deliver one litter without treatment (Table 4), because of the decreased fecundity of this knockout line (Abbot et al., 1999); three of four dams produced viable pups. In the second round of breeding, the mothers were treated with cHBB (100 mg/kg) at GD 5. Of 17 newborns from two mothers, 16 survived the newborn period. Dissection of the uteri showed no implantations in the two mothers not having any pups in the second round; because cHBB was given at GD 5, which is after implantation, we conclude that the AHR-null property and not the cHBB was the cause of no pregnancies in these two animals. The findings in Table 4 therefore provide support for dependence of the AHR in cHBB-caused neonatal lethality.
Pharmacokinetics of 14C-PCB 169 in Mother and Fetus
It was not possible to obtain radiolabeled PBBs, but we were able to buy 14C-PCB-169 to carry out pharmacokinetics with a radiolabeled similar polyhalogenated biphenyl. The similarity in physicochemical properties of cHCB and cHBB (Table 5) justifies the use of 14C-HCB as a surrogate for 14C-HBB in the pharmacokinetic studies; pharmacokinetics for halogenated biphenyls depends primarily upon size, charge, and the rate of metabolism. Table 5 shows that the two chemicals have nearly identical molecular dimensions, hydrophobicity properties, and dipole moments. The extreme hydrophobicities for both molecules render each almost completely water-insoluble; the differences in the log p values of 6.84 versus 8.48 are trivial, when molecules are so extremely hydrophobic. The lack of any adjacent carbons having hydrogen atoms renders either chemical highly resistant to biotransformation.
We therefore studied the uptake and distribution of this label, when it accompanied the usual high cHBB dose at GD 5 in B6 and B6.D2-Ahrd mice (Table 6). The maternal and GD 18 fetal livers of B6 exhibited >2-fold and >4-fold more polyhalogenated biphenyl, respectively, than that of B6.D2-Ahrd. The maternal and GD 18 fetal brains of B6 showed 75% less and no significant difference in polyhalogenated biphenyl, respectively, than that of B6.D2-Ahrd. These data are consistent with the likelihood that either PCBs or PBBs, given on GD 5, remain at detectable levels in maternal and fetal liver and brain 13 days later.
Oxidative Stress Parameters
Glutathione (GSH) is one of the major antioxidant molecules of the cell and most sensitive to change—if intracellular oxidative stress occurs in response to any exogenous or endogenous stimulus (Dalton et al., 2002). If cHBB causes oxidative stress in B6 but not B6.D2-Ahrd and ncHBB causes no oxidative stress in either B6 but not B6.D2-Ahrd, then we would expect to see lowered GSH levels and an elevated GSSG/(GSSG + GSH) ratio in cHBB-treated B6 but not in any of the other groups. This trend was not observed in maternal (not shown) or fetal liver or brain at GD 18 (Fig. 4) after HBB treatment at GD 5. Ascorbate levels were also determined, and no significant lowering of hepatic ascorbate levels was seen in cHBB-treated B6 mice, as compared with cHBB-treated B6.D2-Ahrd, ncHBB-treated B6 or B6.D2-Ahrd, or controls (data not shown).
Immunotoxicity
Polybrominated biphenyls are known to be toxic to the immune system. One of the most obvious signs of immunotoxicity is atrophy of the thymus and spleen, which had been noted during the anatomical and histological examinations of newborns. Figure 5 shows that the thymus and spleen weights were significantly decreased in cHBB-treated B6 newborns as compared with cHBB-treated B6.D2-Ahrd newborns, ncHBB-treated newborns of either mouse line, and controls of either mouse line. Although we may conclude that AHR-mediated immunotoxicity occurs in newborn mice whose mother received cHBB 100 mg/kg at GD 5, the relationship of this to the specific cause of death during the first 72 h postpartum remains unclear.
DISCUSSION
Differences in AHR-binding affinity of polyhalogenated biphenyls have previously been associated with varying degrees of toxicity and carcinogenicity (Safe, 1984; Silberhorn et al., 1990). We have shown in this study that cHBB but not ncHBB, given to the pregnant mother on GD 5, is responsible for a number of neonatal effects in B6 mice having the high-affinity AHR—but not the B6.D2-Ahrd congenic mice having the poor-affinity AHR. Numerous lines of evidence support this conclusion. (1) The cHBB treatment of AHRD-tk-LUC cells in culture (Fig. 2) confirms that the AHRE is activated in a dose-dependent fashion, whereas ncHBB treatment does not, thereby implicating cHBB in activating the AHR. (2) cHBB treatment of B6 mice, but not the B6.D2-Ahrd congenic mice, leads to the upregulation of hepatic CYP1A1 enzyme activity and mRNA levels. In fact, persistence of CYP1A1 mRNA induction was detectable in both maternal and fetal liver and brain on GD 18, 13 days after the single dose of cHBB had been given (Fig. 3). (3) Neonatal lethality within 72 h postpartum occurred in B6 but not B6.D2-Ahrd pups whose mothers had received cHBB (Table 1). In fact, cHBB is lethal at doses more than 20 times lower than ncHBB doses. No lethality was observed in any animal treated with ncHBB (100 mg/kg). (4) The cHBB-induced lethality in B6 is maternal in origin (Table 2) and is primarily the effect of in utero GD 5 exposure rather than via mother's milk (Table 3). (5) Protection against neonatal lethality is statistically significantly associated with the lack of the AHR in cHBB-treated Ahr(–/–) knockout mice (Table 4), providing further support for involvement of the AHR in these neonatal deaths. (6) Decreased thymus and spleen weights in cHBB-treated B6 mice—as compared with cHBB-treated B6.D2-Ahrd, ncHBB-treated B6 or B6.D2-Ahrd, or controls (Fig. 5)—confirms the presence of cHBB-induced AHR-mediated immunotoxicity.
Abnormal appearance of lymphocytes in the Michigan dairy farmers exposed to PBBs in FireMaster has been reported (Bekesi et al., 1978), and PBB-induced transplacental immunotoxicity has been noted in swine (Howard et al., 1980). Further, PBBs in mice cause lowered serum immunoglobulin-M levels and a compromised immune response (Loose et al., 1981). Despite these signs and symptoms of immunotoxicity, no clearly established anatomical birth defects were ever described in the PBB-exposed Southern Michigan cohort (Nebert et al., 1983); in contrast, cognitive effects have been observed (vide infra).
TCDD (dioxin) mediates teratogenic effects via the AHR. TCDD is well known to cause AHR-mediated cleft palate and hydronephrosis in the rodent (Poland and Knutson, 1982), but these appear to be species-specific effects—because no signs of these abnormalities have been seen in humans heavily exposed to dioxin, PCBs, or PBBs (Couture et al., 1990). Curiously, our laboratory has found cleft palate and hydronephrosis in B6 newborns whose mothers were treated at GD 10 with TCDD at a dose of 25 μg/kg (data not shown), whereas these abnormalities were not seen in pups of cHBB-treated mothers in the present study. Therefore, although both TCDD and cHBB mediate immunotoxicity (and other toxicity including neonatal lethality) via the AHR, TCDD but not cHBB appears to cause these birth defects specific to the rodent. Why does this discrepancy exist One can only conclude that, during teratogenesis caused by TCDD, the high-affinity AHR appears to be necessary but not sufficient for inducing cleft palate and hydronephrosis in the mouse. An additional factor(s), or pathway, is used by TCDD (but not by cHBB) to cause this teratogenic outcome; it is likely that not only cHBB, but other PCB and PBB congeners, would behave similarly.
Whether damage to the newborn's immune system is responsible for lethality during the first 72 h postpartum is open to question. It has been previously reported that the cHBB causes a significant decrease in thymus and spleen weights in adult Wistar rats (Robertson et al., 1982). Gross autopsies—as well as histology of GD 18 fetuses, maternal and newborn liver and brain—showed no morphological differences. Cross-fostering experiments confirmed that lethality within 72 h postpartum was primarily associated with the cHBB exposure in utero, because most pups from a cHBB-treated B6 dam cannot be rescued by cross-fostering to an untreated dam; furthermore, control B6 pups transferred to cHBB-treated lactating dams displayed 100% survival (Table 3). However, cHBB-treated pups that showed no survival when taking milk from their cHBB-treated mother, exhibited 21% survival when taking milk from an untreated mother; this finding suggests that there is a toxic component in cHBB-treated mother's milk that contributes to neonatal lethality.
The effect is clearly maternal, as shown in the backcross experiments (Table 2): lethality of Ahrb1/d heterozygotes during 72 h postpartum occurs if the mother receiving the cHBB at GD 5 is B6, but lethality of Ahrb1/d heterozygotes during 72 h postpartum does not occur if the mother receiving the cHBB is B6.D2-Ahrd. TCDD-induced AHR activation during pregnancy has been shown to disrupt mammary gland differentiation (Vorderstrasse et al., 2004); might cHBB in B6 mothers therefore adversely affect breast tissue development and lactation Although Vorderstrasse et al. (2004) have demonstrated that neonatal lethality occurs in a similar time frame in TCDD-exposed mouse litters, their proposed mechanism of lactational failure cannot explain our results in the present study: control B6 pups, cross-fostered to cHBB-treated dams, all grew at a normal rate for the first week postpartum. This is strong evidence that the cHBB-treated dams were able to produce adequate amounts of milk.
That the polyhalogenated biphenyl can cross the placenta and persist in fetal liver and brain (Table 6) supports our findings that the cHBB-induced AHR-mediated effects of CYP1A1 mRNA upregulation in fetal liver and brain (Fig. 3) are caused by the persistence of this chemical in those tissues 13 days after cHBB treatment of the mother. Previous studies in rats, as well as with mice in the present study (Table 5), have shown that in utero exposure to PBBs leads to the accumulation of PBBs in the brains of offspring postpartum (Rickert, 1978); approximately 5% of the intraperitoneal dose administered is stored in the maternal liver. In fact, whereas the maternal polyhalogenated biphenyl accumulates to greater amounts in B6 liver than B6.D2-Ahrd liver (Table 6), the polyhalogenated biphenyl accumulates to significantly lesser amounts in B6 brain than B6.D2-Ahrd brain. This effect is most likely related to inducible CYP1A2 in liver as the "sink" for dioxin (Kuroki et al., 1986; Voorman and Aust, 1987; Poland et al., 1989a, 1989b; Kedderis et al., 1991, 1993; Chen et al., 2003; Shertzer et al., 2004). In other words, cHBB treatment of the B6 mother results in highly induced levels of hepatic CYP1A2 (as well as liver CYP1A1 and extrahepatic CYP1A1, CYP1A2, and CYP1B1 in many tissues; reviewed in Nebert et al., 2004); liver CYP1A2 has the unusual property of sequestering polyhalogenated biphenyls and dioxins—thereby preventing them from distribution to extrahepatic tissues (Shertzer et al., 2004) such as maternal brain.
It should be noted that the cHBB and ncHBB used in the present study have no adjacent unsubstituted carbon atoms and therefore are very poorly metabolized or excreted (Kohli et al., 1978). This property may very well help account for our findings of the persistent CYP1A1 induction in fetal and maternal liver and brain at GD 18, (Fig. 3), and persistent radiolabeled polyhalogenated biphenyl in these tissues (Table 6), 13 days after the single dose that had been given at GD 5.
Many PBB and PCB congeners are noteworthy environmental toxicants because they persist for years in animals and in the soil (Hakk and Letcher, 2003). These chemicals are also genotoxic (Kohli et al., 1978). The toxicity and carcinogenicity of specific isomers are inversely proportional to their capacity to form DNA adducts, suggesting the possible importance of a nongenotoxic (perhaps receptor-mediated) rather than a genotoxic mechanism of action (Goldstein et al., 1979; Kimbrough et al., 1981; Gupta et al., 1983). Halogenated biphenyls have also been shown to be potent tumor promoters (Preston et al., 1981; Jensen et al., 1982) when, for example, diethylnitrosamine is given as the tumor initiator. PBBs and PCBs are highly immunotoxic, and they disrupt sex hormone and cholesterol pathways and thyroid functions (Bahn et al., 1980; Darnerud, 2003). Although these environmental chemicals cause toxicity to, and subtle effects on, the CNS of laboratory animals and humans (Brown et al., 1981; Pantaleoni et al., 1988; Kodavanti et al., 1995; Dellovade et al., 1996; Jacobson and Jacobson, 1996), the mechanisms of this neurodevelopmental toxicity remain completely unknown. Whether the >20-fold difference in cHBB-induced neonatal lethality in high-affinity-AHR mice, compared with that in poor-affinity-AHR mice, that we have found herein, can be explained by toxicities noted in any of these above-mentioned studies remains to be experimentally demonstrated.
NOTES
1 These two coauthors contributed equally to this work.
ACKNOWLEDGMENTS
We thank our colleagues for valuable suggestions and critical discussions of this project. We appreciate the work of Kathy LaDow for animal husbandry, and Stacey Andringa for assistance with microscopy. This work was supported in part by National Institutes of Health (NIH) grants P30 ES06096 and R01 ES08147.
We just became aware of a previous publication (Aulerich et al., 1985) in which adult female mink were fed diets containing various concentrations of PCBs. All mink fed the coplanar 3,3',4,4',5,5'-hexachlorobiphenyl (0.5 ppm) died within 60 days, whereas no deaths or reproductive defects were seen in mink fed the non-coplanar 2,2',4,4',5,5'-hexachlorobiphenyl or the non-coplanar 2,2',3,3',6,6'-hexachlorobiphenyl. These data–in a completely randombred animal--are not only consistent with the AHR-mediated effects seen with genetically different mouse lines reported herein, but also underscore the relevance of these data to randombred human populations. This publication also confirms that these coplanar versus non-coplanar effects with PCBs are similar to that reported herein with PBBs.
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Division of Neurology, Children's Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio
2 To whom correspondence should be addressed at Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati OH 45267–0056. Fax: 513–558–0925. E-mail: dan.nebert@uc.edu
ABSTRACT
Polybrominated biphenyl (PBB) exposure in humans is known to cause immunotoxicity and disorders related to the central nervous system. Coplanar PBBs bind to the aryl hydrocarbon receptor (AHR) in vertebrates. We compared the coplanar PBB, 3,3',4,4',5,5'-hexabromobiphenyl (cHBB), with its stereoisomer, the non-coplanar PBB, 2,2',4,4'6,6'-hexabromobiphenyl (ncHBB), using C57BL/6J (B6) inbred mice (having the high-affinity AHR) and congenic B6.D2-Ahrd mice (having the low-affinity AHR in a >99.8% C57BL/6J genetic background). Pregnant dams were treated i.p. with vehicle alone, cHBB, or ncHBB on gestational day 5 (GD 5). Unexpectedly, neonatal lethality within the first 72 h postpartum was significant in cHBB-treated B6 mice at doses as low as 2.5 mg/kg, whereas no deaths were seen in B6 pups whose mother had received ncHBB 100 mg/kg or in either B6.D2-Ahrd or Ahr(–/–) knockout mice whose mother had received cHBB 100 mg/kg. Histological and gross anatomical analyses of a battery of tissues in the mother or fetus at GD 18, as well as 24 h postpartum, revealed no significant differences, except for decreased thymus and spleen weights in cHBB-treated B6 GD 18 fetuses. Cross-fostering and genetics experiments confirmed the association of neonatal deaths principally with in utero (rather than lactational) exposure to cHBB, and also no paternal effect. For the end points of mouse neonatal lethality and immunotoxicity, cHBB appears to act through the high-affinity AHR receptor. Although dioxin in utero is well known to cause AHR-dependent cleft palate and hydronephrosis, cHBB did not; thus, chronic activation of the AHR appears to be necessary but not sufficient for AHR-mediated teratogenicity.
Key Words: 3,3'4,4',5,5'-(coplanar)-hexabromobiphenyl; 2,2',4,4',6,6'-(non-coplanar)-hexabromobiphenyl; newborn lethality; in utero toxicity; immunotoxicity; aryl hydrocarbon receptor (AHR).
INTRODUCTION
Polyhalogenated biphenyls represent a class of ubiquitous environmental pollutants (Headrick et al., 1999; Zabik and Zabik, 1999) and include polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). These chemicals are similar in their structure–activity relationships and share similar toxicity profiles with regard to their carcinogenicity, teratogenicity, immunotoxicity, and general health effects (Safe, 1984; Silberhorn et al., 1990; Hakk and Letcher, 2003; Darnerud, 2003; Birnbaum and Staskal, 2004). Although PCBs have been studied more than PBBs, PBBs are more toxic. The global market demand and production for brominated flame retardants (BFRs) has increased dramatically during the past 20 years, especially in Asia; more than 200,000 metric tons of BFRs are produced each year (Birnbaum and Staskal, 2004). There is increasing concern about the contamination of brominated flame retardants in our environment, and that these represent developmental neurotoxicants (Eriksson et al., 2001).
Polybrominated biphenyls and PCBs are known to elicit a potent biochemical response, principally as a function of their affinity for several types of intracellular receptors (Safe, 1984; Silberhorn et al., 1990). The number of halogens, and their positioning around the biphenyl structure, is critical in determining the activity of these halogenated hydrocarbons. For example, on the one hand, halogens in the meta- and/or para-positions (Fig. 1, top) allow the molecule to remain coplanar, show very high affinity in binding to the AHR, and elicit effects similar to those seen for AHR ligands such as polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). On the other hand, halogens in the ortho-positions (Fig. 1, bottom) cause intramolecular torsion that makes the molecule non-coplanar and limits binding to the AHR; the induction response to non-coplanar halogenated biphenyls is similar to that seen for phenobarbital (Safe, 1984).
In the 1973 environmental disaster in Southern Michigan, hundreds of individuals (on 308 farms) were accidentally exposed to about 1000 pounds of PBBs (Dunckel, 1975; Carter, 1976; Kay, 1977; Anderson et al., 1978). On a shipping dock, the fire retardant FireMaster was mistaken for the agricultural food supplement NutriMaster. Thousands of cows and pigs, hundreds of sheep, and millions of chickens were contaminated and had to be destroyed. Humans consumed the meat, milk, and eggs before the magnitude of the danger had become clear (Carter, 1976). Today, PBB levels (370 times higher in adipose tissue than in blood) continue to decrease slowly in this exposed population. Follow-up studies have estimated the PBB half-life at 10.8 years and predicted it will take more than 60 years for serum levels to fall below the limits of detection in the most highly exposed group (Rosen et al., 1995). Intriguingly, the earliest clinical signs that a disaster had occurred included central nervous system (CNS) symptoms (Valciukas et al., 1978) of amnesia, confusion, and somnolence (farmers lost their tractors, were unable to find their way home at the end of the day, and fell asleep in the fields) and leukocytopenia (i.e., immunosuppression). Also reported was chloracne (Aust et al., 1987)—which is the sentinel sign of dioxin (AHR-mediated) toxicity in exposed workers (Birnbaum, 1994). Lowered birth weights (Sunahara et al., 1987), increased respiratory illnesses (Weil et al., 1981), and the possible lowering of I.Q. among some children born to Michigan mothers exposed in 1973–1975 (Seagull, 1983; Nebert et al., 1983) have been reported, and the mental development of these children continues to be followed into adulthood. Hyperactivity and alterations in behavior have been reported with massive PCB exposures: Yusho children whose Japanese mothers were exposed in 1968 and Yu-cheng children whose Taiwanese mothers were exposed in 1979 to contaminated rice bran oil (Rogan et al., 1988; Jacobson et al., 1990; Chen et al., 1994), as well as monkeys whose mothers were treated with PCBs (Schantz et al., 1989) or dioxin (Schantz and Bowman, 1989).
Polybrominated biphenyl and PCB levels in any particular subject are not always correlated with the degree of CNS impairment—suggesting a genetic component. Mice are known to exhibit 15- to more than 20-fold differences between inbred strains in AHR ligand affinity; humans also show more than 12-fold differences in AHR ligand affinity (Nebert et al., 2004). Our hypothesis, therefore, was that an underlying genetic variability in the AHR phenotype might have accounted for interindividual differences in response to the PBB-caused CNS toxicity in Michigan. Because such a study in humans is not ethically possible, we have used a mouse model. We chose to compare the effects of the coplanar and non-coplanar HBB stereoisomers (Fig. 1) in C57BL/6J inbred mice (B6, having the high-affinity AHR) and in congenic B6.D2-Ahrd mice [having the DBA/2J (D2) poor-affinity receptor in a genetic background of >99.8% B6]. Our original experimental protocol was to administer cHBB or ncHBB to the pregnant mouse on gestational day 5 (GD 5) and then perform behavioral phenotyping tests on the offspring at 60 days postpartum. Unexpectedly, we found a striking difference in neonatal lethality—depending on both the Ahr genotype and the HBB stereoisomer administered.
MATERIALS AND METHODS
Chemicals.
Both 3,3',4,4',5,5'-hexabromobiphenyl (cHBB) and 2,2',4,4',6,6'-hexabromobiphenyl (ncHBB) were purchased from Ultra Scientific (North Kingstown, RI). Unfortunately, to our knowledge, these chemicals are no longer commercially available.
Animals and treatment.
All experiments with mice were conducted in accordance with the National Institutes of Health standards for care and use of experimental animals, the University of Cincinnati Institutional Animal Care and Use Committee (IACUC), and the Cincinnati Children's Research Foundation IACUC. Male and female mice of the inbred strain C57BL/6J were purchased from The Jackson Laboratory (Bar Harbor, ME). Male and female mice of the congenic B6.D2-Ahrd line were obtained from the Nebert mouse colony at the University Cincinnati Medical Center, and some were purchased from The Jackson Laboratory. The mice were given access to standard rodent chow and water ad libitum; the colony was maintained with a 12-h light/dark cycle. Mice were bred on a 4-day cycle, and a finding of a vaginal plug in the morning was evidence of copulation; this was labeled as gestational day 0.5 (GD 0.5).
Initial enzyme induction and mRNA induction experiments were performed on cHBB- and ncHBB-treated nonpregnant 6-week-old female B6 mice. Subsequently, pregnant dams were administered i.p. various doses (ranging from 2.5 to 100 mg/kg body weight) of either cHBB or ncHBB, or an equivalent volume of the vehicle (corn oil; 25 ml/kg) on GD 5. This regimen was chosen because treatment on GD 5 is beyond the implantation period and, therefore, the treatment time would avoid toxicity associated with the implantation process. In addition, GD 5 treatment ensures that the chemical is present during the earliest days of ectoderm and neural crest development. Intraperitoneal treatment of the mother means the drug enters the splanchnic venous system directly and then travels to the liver, lungs, and arterial circulation including to the uterus. This is more direct than oral treatment, which might involve maternal "first-pass elimination kinetics" (Lukas et al., 1971), i.e. rate of absorption from the gut, combined with HBB-mediated induction of enzymes in the GI tract and liver, all of which might affect distribution of the chemicals to other tissues including the uterine contents.
Adult mice were sacrificed by carbon dioxide asphyxiation, followed by cervical dislocation. Newborn mice were killed by decapitation. Fetuses were also taken at GD 18, and whole liver and brain were examined from the mother and fetus; newborn tissues were also examined, including spleen and thymus wet weights.
AHRE activation.
AHRD-tk-LUC cells are mouse hepatoma Hepa-1c1c7 cells that have been stably transformed with the reporter-gene construct containing the luciferase reporter gene (LUC) driven by the thymidine kinase promoter and the mouse Cyp1a1 5'-flanking region as an enhancer (Maier et al., 2000). Cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum (FBS; Life Technologies, Rockville, MD). Cells during their logarithmic growth phase were treated with 1000x concentrated stocks of either cHBB or ncHBB dissolved in dimethylsulfoxide, and the compounds were added directly to medium. Cells were treated for 12 h, after which they were harvested. Luminescence was determined at 495 nm (Maier et al., 2000).
CYP1A1 enzyme induction and mRNA accumulation.
CYP1A1 [aryl hydrocarbon hydroxylase, benzo[a]pyrene (BaP) hydroxylase] enzyme activity, using BaP as substrate, was determined as described elsewhere (Nebert and Gelboin, 1968). Total RNA from liver and brain was extracted, size-separated, blotted on nylon membranes, and probed with CYP1A1-specific cDNA for Northern blot analysis by standard methods (Chomczynski and Sacchi, 1987). CYP1A1 mRNA was also measured, using the reverse transcriptase polymerase chain reaction (RT-PCR) technique previously described (Jiang et al., 2005).
Neonatal toxicity.
Newborn deaths were recorded for the first 72 h following birth. In cross-fostering experiments, newborns from cHBB-treated mothers were nursed by vehicle-treated mothers, and vehicle-treated newborns were nursed by cHBB-treated mothers.
Histology and anatomy.
Liver and brain from GD 18 fetuses, newborn mice, and mothers were prepared for routine light- and electron-microscopic histology and morphometry (Dalton et al., 2000). In liver, phase-contrast microscopy of toluidine blue-stained 1.5-micron-thick plastic sections was used to quantify cytoplasmic, nuclear and nucleolar areas. At 40x, the areas of hematopoietic cells and of liver were determined, and the percent hematopoiesis was calculated by division of the former value by the total area times 100. A grid of 75 intersections was visualized over the light-microscopic image, using a Zeiss Photomik light-microscope and camera lucida, and the number of positive intersections lying over hematopoietic cells, and over hepatocytes, was used as a second method for quantifying the volume density of hematopoiesis in the liver. Birth defects (especially cleft palate and hydronephrosis) were looked for; gross examination of all internal organs and histology of the kidney were also carried out.
Oxidative stress parameters.
Liver and brain from GD 18 fetuses, newborn mice, and mothers were harvested and frozen on dry ice; tissues were then stored at –70°C. Reduced (GSH) and oxidized (GSSG) glutathione levels were measured spectrophotofluorometrically using the o-phthalaldehyde method, as previously described (Senft et al., 2000). Ascorbate levels were measured by the procedure described by Zannoni et al. (1974).
Immunotoxicity.
Newborn spleen and thymus were isolated, and organ wet weights were determined.
cHBB treatment of Ahr(–/–) mice.
Generation of the Ahr(–/–) knockout mouse line has been described (Fernandez-Salguero et al., 1995), and these mice are maintained on the C57BL/6J background in the Nebert mouse colony. Untreated Ahr(–/–) females and males were bred, and viability versus deaths of the first litter were recorded. For the second pregnancies, cHBB 100 mg/kg body weight was given to pregnant mothers at GD 5. More than 2 weeks later, neonatal survival during the next 72 h was recorded and compared with what had been observed for that mother in its first pregnancy. This method was used to control for the well-known fertility problems associated with Ahr(–/–) mice (Abbott et al., 1999; Benedict et al., 2000, 2003).
Distribution of radiolabeled polyhalogenated biphenyl in mother and fetus.
14C-PCB-169 (3,3',4,4',5,5'-hexachlorobiphenyl; 100 μCi/1.3 mg of chemical in toluene) was purchased from Larry Hansen (College of Veterinary Medicine, University of Illinois, Urbana). This radiolabel was evaporated with a gentle stream of nitrogen, taken up in corn oil, and used to spike the cHBB in corn oil given to the pregnant mother at GD 5 at a dose of 100 mg/kg. At GD 18, radioactivity in the liver and brain of the fetus and mother was determined.
Statistical analyses.
Means and standard errors of the means (S.E.M.) were obtained from the morphometric data, using the General Linear Model of SAS 9.1. Data were analyzed by analysis of variance (ANOVA), followed by group comparisons by Student's t-test. A value of p < 0.05 was regarded as statistically significant.
RESULTS
Dose-Dependent Activation of the AHR
A reporter cell line (AHRD-tk-LUC) for activation of the high-affinity mouse AHR has previously been reported (Maier et al., 2000). Luciferase activity in AHRD-tk-LUC cells reflects AHR activation, because these cells are stably transfected with a construct containing four aryl hydrocarbon response elements (AHREs) linked to the thymidine kinase promoter and driving the luciferase reporter gene (Maier et al., 2000). Figure 2 shows a dose-dependent increase in luminescence in cHBB-treated but not in ncHBB-treated cell cultures. These data confirm that the structural characteristics of coplanar hexabromobiphenyl render it capable of activating the AHR, whereas the non-coplanar stereoisomer cannot. This experiment also assures the purity of these HBB congeners.
CYP1A1 Enzyme Activity and mRNA Induction by cHBB
Cyp1a1 has been demonstrated in many studies to provide an accurate surrogate for AHR activation. Figure 3 shows that cHBB caused the parallel induction of hepatic CYP1A1 enzyme activity and mRNA levels in B6 mice. Accumulation of both was maximal at 3 mg/kg. In contrast, CYP1A1 was not induced in liver from B6.D2-Ahrd mice, even at 100 mg/kg (Fig. 3, middle panel). This experiment justifies use of the B6 inbred strain—having the high-affinity AHR—in comparison with the congenic B6.D2-Ahrd line—having the poor-affinity D2 receptor in a >99.8% B6 genetic background—so that we might compare the toxicity of cHBB versus ncHBB in newborn mice. Moreover, cHBB, when administered to dams at GD 5 of pregnancy, was capable of persistent (measured at GD 18) Cyp1a1 induction in the fetal liver and brain (Fig. 3, lower panel); these data confirm that cHBB crosses the placenta and may persist in fetal tissues. Finally, ncHBB at 100 mg/kg did not induce liver CYP1A1 enzyme activity or mRNA levels in B6 or B6.D2-Ahrd mice (data not shown).
Neonatal Lethality
When B6 and B6.D2-Ahrd mice were treated on GD 5 with cHBB or ncHBB (100 mg/kg), the number of live healthy-appearing pups per litter from both strains did not differ statistically (data not shown). Unexpectedly, however, we found that this dose of cHBB but not ncHBB, caused 100% deaths in all B6 pups within 72 h postpartum (Table 1). To examine further this toxicity to B6 pups, we continued to lower the prescribed amount of cHBB and found that—even at doses of 2.5 mg/kg and 5 mg/kg—there was significant lethality of B6 pups. These data show that the LD50 value, for B6 neonates whose mother had received cHBB on GD 5, is between 5 mg/kg and 10 mg/kg and that lethality appears to depend on the high-affinity AHR.
Backcross Experiments
To determine if lethality occurs as a consequence of the fetal or maternal AHR, we conducted backcross experiments. We crossed the B6.D2-Ahrd female with the B6 male, and the B6 female with the B6.D2-Ahrd male, and assessed for cHBB-induced lethality (Table 2). The pups in either cross have identical genetic makeup, i.e., Ahrb1/d heterozygotes for the high- versus poor-affinity receptor. Complete lethality occurred in the neonates whose B6 mother had received the cHBB 100 mg/kg at GD 5; in contrast, the pups whose B6.D2-Ahrd mother had received the cHBB 100 mg/kg at GD 5 exhibited 100% survival. We conclude that only GD 5 cHBB treatment in utero, in the high-affinity-AHR mother, leads to neonatal death within the first 72 h postpartum, and there is no paternal contribution. For the remainder of this study, we sought to determine the cause of this neonatal death.
Cross-Fostering Experiments
In order to distinguish between in utero effects of cHBB versus maternal fostering behavior or cHBB in milk, we switched litters at birth between cHBB-treated and control mothers (Table 3). B6 pups, whose mothers had received cHBB 100 mg/kg at GD 5, did not survive the first 72 h postpartum; if cross-fostered to corn oil-treated dams within 6 h of birth, 21% of the cHBB-treated B6 pups survived. Control B6 pups—whose mothers received corn oil injections on GD 5—all survived the first 72 h postpartum when cross-fostered to cHBB-treated dams (100 mg/kg at GD 5). The dams' behavior was monitored three times a day during the 72-h postpartum window to determine if pups were being nursed and groomed normally. We found no significant differences in the behavior of treated or control dams of either line (Table 3). We therefore conclude that it is, in large part, the in utero cHBB treatment of the high-affinity AHR mouse that leads to death within the first 72 h postpartum, but that cHBB received via the milk might be a contributory factor.
In control B6 pups nursing on cHBB dams, weight loss and lethality were noted beginning at 9 days postpartum. This finding supports our conclusion (above) that persistent (defined here as greater than 1 week) activation of the AHR in the pup by cHBB, distributed to the pup in the mother's milk, appears to be a contributory factor to neonatal lethality.
Histology
Light- and electron-microscopy of the liver and brain and morphometrics of the liver in the GD 18 fetus, its mother, and newborns of all groups were carried out in the six (3 x 2) possible groups: cHBB 100 mg/kg, ncHBB 100 mg/kg, and vehicle-treated controls in B6 mice versus and B6.D2-Ahrd mice. Despite the fact that cHBB administered on GD 5 produced residual effects at GD 18 that could be seen in the fetal liver and brain in the form of increased CYP1A1 mRNA levels (Fig. 2), no statistically significant morphological differences in the livers or brains could be detected among any of the groups. Morphometry on GD 18 fetal livers suggested a slight increase in the volume density (Vd) of hemopoietic islands was higher in both cHBB- and ncHBB-treated groups (100 mg/kg on GD 5) and in both B6 and B6.D2-Ahrd mice, compared with that in control groups; in contrast, no difference between cHBB and ncHBB was seen in either mouse line. The response (i.e., elevation in hemopoietic Vd) was greater in cHBB-treated B6 than in cHBB-treated B6.D2-Ahrd fetal livers (p = 0.08). The same trend appeared when the sizes of hemopoietic sites were quantified: cHBB-treated B6 showed greater sizes than cHBB-treated B6.D2-Ahrd fetal livers (p = 0.04).
Because of questionable teratogenesis of PCBs in fish (Meyer and Di Giulio, 2002), we examined the pups specifically for signs of birth defects—including especially cleft palate and hydronephrosis, two hallmarks of dioxin-induced AHR-mediated teratogenesis in mice—and found none in these newborns. We did, however, note cHBB-induced spleen and thymus atrophy (another hallmark of dioxin-induced AHR-mediated toxicity in laboratory animals), which was found in the B6 neonates but not the B6.D2-Ahrd newborns; this finding is discussed below. No gross abnormalities of any other internal organs were seen.
AHR Dependence of cHBB-Induced Neonatal Lethality
If cHBB-induced neonatal lethality is indeed mediated by the AHR, would Ahr(–/–) homozygous knockout pups be protected in utero We first allowed four Ahr(–/–) nulliparous females to mate with Ahr(–/–) males and deliver one litter without treatment (Table 4), because of the decreased fecundity of this knockout line (Abbot et al., 1999); three of four dams produced viable pups. In the second round of breeding, the mothers were treated with cHBB (100 mg/kg) at GD 5. Of 17 newborns from two mothers, 16 survived the newborn period. Dissection of the uteri showed no implantations in the two mothers not having any pups in the second round; because cHBB was given at GD 5, which is after implantation, we conclude that the AHR-null property and not the cHBB was the cause of no pregnancies in these two animals. The findings in Table 4 therefore provide support for dependence of the AHR in cHBB-caused neonatal lethality.
Pharmacokinetics of 14C-PCB 169 in Mother and Fetus
It was not possible to obtain radiolabeled PBBs, but we were able to buy 14C-PCB-169 to carry out pharmacokinetics with a radiolabeled similar polyhalogenated biphenyl. The similarity in physicochemical properties of cHCB and cHBB (Table 5) justifies the use of 14C-HCB as a surrogate for 14C-HBB in the pharmacokinetic studies; pharmacokinetics for halogenated biphenyls depends primarily upon size, charge, and the rate of metabolism. Table 5 shows that the two chemicals have nearly identical molecular dimensions, hydrophobicity properties, and dipole moments. The extreme hydrophobicities for both molecules render each almost completely water-insoluble; the differences in the log p values of 6.84 versus 8.48 are trivial, when molecules are so extremely hydrophobic. The lack of any adjacent carbons having hydrogen atoms renders either chemical highly resistant to biotransformation.
We therefore studied the uptake and distribution of this label, when it accompanied the usual high cHBB dose at GD 5 in B6 and B6.D2-Ahrd mice (Table 6). The maternal and GD 18 fetal livers of B6 exhibited >2-fold and >4-fold more polyhalogenated biphenyl, respectively, than that of B6.D2-Ahrd. The maternal and GD 18 fetal brains of B6 showed 75% less and no significant difference in polyhalogenated biphenyl, respectively, than that of B6.D2-Ahrd. These data are consistent with the likelihood that either PCBs or PBBs, given on GD 5, remain at detectable levels in maternal and fetal liver and brain 13 days later.
Oxidative Stress Parameters
Glutathione (GSH) is one of the major antioxidant molecules of the cell and most sensitive to change—if intracellular oxidative stress occurs in response to any exogenous or endogenous stimulus (Dalton et al., 2002). If cHBB causes oxidative stress in B6 but not B6.D2-Ahrd and ncHBB causes no oxidative stress in either B6 but not B6.D2-Ahrd, then we would expect to see lowered GSH levels and an elevated GSSG/(GSSG + GSH) ratio in cHBB-treated B6 but not in any of the other groups. This trend was not observed in maternal (not shown) or fetal liver or brain at GD 18 (Fig. 4) after HBB treatment at GD 5. Ascorbate levels were also determined, and no significant lowering of hepatic ascorbate levels was seen in cHBB-treated B6 mice, as compared with cHBB-treated B6.D2-Ahrd, ncHBB-treated B6 or B6.D2-Ahrd, or controls (data not shown).
Immunotoxicity
Polybrominated biphenyls are known to be toxic to the immune system. One of the most obvious signs of immunotoxicity is atrophy of the thymus and spleen, which had been noted during the anatomical and histological examinations of newborns. Figure 5 shows that the thymus and spleen weights were significantly decreased in cHBB-treated B6 newborns as compared with cHBB-treated B6.D2-Ahrd newborns, ncHBB-treated newborns of either mouse line, and controls of either mouse line. Although we may conclude that AHR-mediated immunotoxicity occurs in newborn mice whose mother received cHBB 100 mg/kg at GD 5, the relationship of this to the specific cause of death during the first 72 h postpartum remains unclear.
DISCUSSION
Differences in AHR-binding affinity of polyhalogenated biphenyls have previously been associated with varying degrees of toxicity and carcinogenicity (Safe, 1984; Silberhorn et al., 1990). We have shown in this study that cHBB but not ncHBB, given to the pregnant mother on GD 5, is responsible for a number of neonatal effects in B6 mice having the high-affinity AHR—but not the B6.D2-Ahrd congenic mice having the poor-affinity AHR. Numerous lines of evidence support this conclusion. (1) The cHBB treatment of AHRD-tk-LUC cells in culture (Fig. 2) confirms that the AHRE is activated in a dose-dependent fashion, whereas ncHBB treatment does not, thereby implicating cHBB in activating the AHR. (2) cHBB treatment of B6 mice, but not the B6.D2-Ahrd congenic mice, leads to the upregulation of hepatic CYP1A1 enzyme activity and mRNA levels. In fact, persistence of CYP1A1 mRNA induction was detectable in both maternal and fetal liver and brain on GD 18, 13 days after the single dose of cHBB had been given (Fig. 3). (3) Neonatal lethality within 72 h postpartum occurred in B6 but not B6.D2-Ahrd pups whose mothers had received cHBB (Table 1). In fact, cHBB is lethal at doses more than 20 times lower than ncHBB doses. No lethality was observed in any animal treated with ncHBB (100 mg/kg). (4) The cHBB-induced lethality in B6 is maternal in origin (Table 2) and is primarily the effect of in utero GD 5 exposure rather than via mother's milk (Table 3). (5) Protection against neonatal lethality is statistically significantly associated with the lack of the AHR in cHBB-treated Ahr(–/–) knockout mice (Table 4), providing further support for involvement of the AHR in these neonatal deaths. (6) Decreased thymus and spleen weights in cHBB-treated B6 mice—as compared with cHBB-treated B6.D2-Ahrd, ncHBB-treated B6 or B6.D2-Ahrd, or controls (Fig. 5)—confirms the presence of cHBB-induced AHR-mediated immunotoxicity.
Abnormal appearance of lymphocytes in the Michigan dairy farmers exposed to PBBs in FireMaster has been reported (Bekesi et al., 1978), and PBB-induced transplacental immunotoxicity has been noted in swine (Howard et al., 1980). Further, PBBs in mice cause lowered serum immunoglobulin-M levels and a compromised immune response (Loose et al., 1981). Despite these signs and symptoms of immunotoxicity, no clearly established anatomical birth defects were ever described in the PBB-exposed Southern Michigan cohort (Nebert et al., 1983); in contrast, cognitive effects have been observed (vide infra).
TCDD (dioxin) mediates teratogenic effects via the AHR. TCDD is well known to cause AHR-mediated cleft palate and hydronephrosis in the rodent (Poland and Knutson, 1982), but these appear to be species-specific effects—because no signs of these abnormalities have been seen in humans heavily exposed to dioxin, PCBs, or PBBs (Couture et al., 1990). Curiously, our laboratory has found cleft palate and hydronephrosis in B6 newborns whose mothers were treated at GD 10 with TCDD at a dose of 25 μg/kg (data not shown), whereas these abnormalities were not seen in pups of cHBB-treated mothers in the present study. Therefore, although both TCDD and cHBB mediate immunotoxicity (and other toxicity including neonatal lethality) via the AHR, TCDD but not cHBB appears to cause these birth defects specific to the rodent. Why does this discrepancy exist One can only conclude that, during teratogenesis caused by TCDD, the high-affinity AHR appears to be necessary but not sufficient for inducing cleft palate and hydronephrosis in the mouse. An additional factor(s), or pathway, is used by TCDD (but not by cHBB) to cause this teratogenic outcome; it is likely that not only cHBB, but other PCB and PBB congeners, would behave similarly.
Whether damage to the newborn's immune system is responsible for lethality during the first 72 h postpartum is open to question. It has been previously reported that the cHBB causes a significant decrease in thymus and spleen weights in adult Wistar rats (Robertson et al., 1982). Gross autopsies—as well as histology of GD 18 fetuses, maternal and newborn liver and brain—showed no morphological differences. Cross-fostering experiments confirmed that lethality within 72 h postpartum was primarily associated with the cHBB exposure in utero, because most pups from a cHBB-treated B6 dam cannot be rescued by cross-fostering to an untreated dam; furthermore, control B6 pups transferred to cHBB-treated lactating dams displayed 100% survival (Table 3). However, cHBB-treated pups that showed no survival when taking milk from their cHBB-treated mother, exhibited 21% survival when taking milk from an untreated mother; this finding suggests that there is a toxic component in cHBB-treated mother's milk that contributes to neonatal lethality.
The effect is clearly maternal, as shown in the backcross experiments (Table 2): lethality of Ahrb1/d heterozygotes during 72 h postpartum occurs if the mother receiving the cHBB at GD 5 is B6, but lethality of Ahrb1/d heterozygotes during 72 h postpartum does not occur if the mother receiving the cHBB is B6.D2-Ahrd. TCDD-induced AHR activation during pregnancy has been shown to disrupt mammary gland differentiation (Vorderstrasse et al., 2004); might cHBB in B6 mothers therefore adversely affect breast tissue development and lactation Although Vorderstrasse et al. (2004) have demonstrated that neonatal lethality occurs in a similar time frame in TCDD-exposed mouse litters, their proposed mechanism of lactational failure cannot explain our results in the present study: control B6 pups, cross-fostered to cHBB-treated dams, all grew at a normal rate for the first week postpartum. This is strong evidence that the cHBB-treated dams were able to produce adequate amounts of milk.
That the polyhalogenated biphenyl can cross the placenta and persist in fetal liver and brain (Table 6) supports our findings that the cHBB-induced AHR-mediated effects of CYP1A1 mRNA upregulation in fetal liver and brain (Fig. 3) are caused by the persistence of this chemical in those tissues 13 days after cHBB treatment of the mother. Previous studies in rats, as well as with mice in the present study (Table 5), have shown that in utero exposure to PBBs leads to the accumulation of PBBs in the brains of offspring postpartum (Rickert, 1978); approximately 5% of the intraperitoneal dose administered is stored in the maternal liver. In fact, whereas the maternal polyhalogenated biphenyl accumulates to greater amounts in B6 liver than B6.D2-Ahrd liver (Table 6), the polyhalogenated biphenyl accumulates to significantly lesser amounts in B6 brain than B6.D2-Ahrd brain. This effect is most likely related to inducible CYP1A2 in liver as the "sink" for dioxin (Kuroki et al., 1986; Voorman and Aust, 1987; Poland et al., 1989a, 1989b; Kedderis et al., 1991, 1993; Chen et al., 2003; Shertzer et al., 2004). In other words, cHBB treatment of the B6 mother results in highly induced levels of hepatic CYP1A2 (as well as liver CYP1A1 and extrahepatic CYP1A1, CYP1A2, and CYP1B1 in many tissues; reviewed in Nebert et al., 2004); liver CYP1A2 has the unusual property of sequestering polyhalogenated biphenyls and dioxins—thereby preventing them from distribution to extrahepatic tissues (Shertzer et al., 2004) such as maternal brain.
It should be noted that the cHBB and ncHBB used in the present study have no adjacent unsubstituted carbon atoms and therefore are very poorly metabolized or excreted (Kohli et al., 1978). This property may very well help account for our findings of the persistent CYP1A1 induction in fetal and maternal liver and brain at GD 18, (Fig. 3), and persistent radiolabeled polyhalogenated biphenyl in these tissues (Table 6), 13 days after the single dose that had been given at GD 5.
Many PBB and PCB congeners are noteworthy environmental toxicants because they persist for years in animals and in the soil (Hakk and Letcher, 2003). These chemicals are also genotoxic (Kohli et al., 1978). The toxicity and carcinogenicity of specific isomers are inversely proportional to their capacity to form DNA adducts, suggesting the possible importance of a nongenotoxic (perhaps receptor-mediated) rather than a genotoxic mechanism of action (Goldstein et al., 1979; Kimbrough et al., 1981; Gupta et al., 1983). Halogenated biphenyls have also been shown to be potent tumor promoters (Preston et al., 1981; Jensen et al., 1982) when, for example, diethylnitrosamine is given as the tumor initiator. PBBs and PCBs are highly immunotoxic, and they disrupt sex hormone and cholesterol pathways and thyroid functions (Bahn et al., 1980; Darnerud, 2003). Although these environmental chemicals cause toxicity to, and subtle effects on, the CNS of laboratory animals and humans (Brown et al., 1981; Pantaleoni et al., 1988; Kodavanti et al., 1995; Dellovade et al., 1996; Jacobson and Jacobson, 1996), the mechanisms of this neurodevelopmental toxicity remain completely unknown. Whether the >20-fold difference in cHBB-induced neonatal lethality in high-affinity-AHR mice, compared with that in poor-affinity-AHR mice, that we have found herein, can be explained by toxicities noted in any of these above-mentioned studies remains to be experimentally demonstrated.
NOTES
1 These two coauthors contributed equally to this work.
ACKNOWLEDGMENTS
We thank our colleagues for valuable suggestions and critical discussions of this project. We appreciate the work of Kathy LaDow for animal husbandry, and Stacey Andringa for assistance with microscopy. This work was supported in part by National Institutes of Health (NIH) grants P30 ES06096 and R01 ES08147.
We just became aware of a previous publication (Aulerich et al., 1985) in which adult female mink were fed diets containing various concentrations of PCBs. All mink fed the coplanar 3,3',4,4',5,5'-hexachlorobiphenyl (0.5 ppm) died within 60 days, whereas no deaths or reproductive defects were seen in mink fed the non-coplanar 2,2',4,4',5,5'-hexachlorobiphenyl or the non-coplanar 2,2',3,3',6,6'-hexachlorobiphenyl. These data–in a completely randombred animal--are not only consistent with the AHR-mediated effects seen with genetically different mouse lines reported herein, but also underscore the relevance of these data to randombred human populations. This publication also confirms that these coplanar versus non-coplanar effects with PCBs are similar to that reported herein with PBBs.
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