* Neurotoxicology Division, MD-74B, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and
The University of Albany, School of Public Health, Albany, New York
Received March 6, 2000; accepted May 24, 2000
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ABSTRACT |
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Key Words: polychlorinated aromatic hydrocarbons; polychlorinated biphenyls; thyroid-axis development; circulating T4 concentrations; lactation exposure; maternal exposure.
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INTRODUCTION |
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We reported previously that perinatal exposure of pregnant rats to a commercial PCB mixture, Aroclor 1254 (A1254) resulted in low-frequency hearing loss in adult offspring (Crofton et al., 2000; Goldey and Crofton, 1998
; Goldey et al., 1995b
; Herr et al., 1996
). We hypothesized that this ototoxicity resulted from A1254-induced hypothyroxinemia during development (Goldey and Crofton, 1998
). Evidence for this hypothesis included a correlation between the severity of functional auditory damage and the degree of postnatal thyroid hormone depletion (Goldey et al., 1995a
, b
) and amelioration of the hearing loss following postnatal thyroxine replacement (Goldey and Crofton, 1998
). Furthermore, this hearing deficit was shown to be caused by loss of hair cells in the area of the cochlea, which develops postnatally and transponds low-frequency sounds (Crofton et al., 2000
). To date, auditory function in children of exposed mothers has not been tested.
The auditory system of the rat is vulnerable to disruption of thyroid hormones during the late prenatal and early postnatal periods (Puel and Uziel, 1987; Uziel, 1986
), a time that corresponds to ongoing cochlear development (Rubel, 1978
, 1984
). The early postnatal period is also a time of peak effects of PCBs on thyroid hormones (Morse et al., 1993
, 1996
; Goldey et al., 1995b
, 1998
). We hypothesized that postnatal exposure to PCBs via lactation is the major route of exposure responsible for the decreases in circulating thyroid hormone concentrations during the early postnatal period and the corollary outcome of ototoxicity. We tested this hypothesis using a cross-fostering design that assessed a number of endpoints previously shown to be sensitive to developmental A1254 exposure (e.g., total serum T4 and T3 concentrations, eye opening, body weights, startle amplitudes, and auditory thresholds). In addition, PCB concentrations in fetal and postnatal liver tissue were assessed at multiple ages as a measure of delivered dose.
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MATERIALS AND METHODS |
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The dams were administered either 0 or 6 mg/kg Aroclor 1254 (AccuStandard, Inc., Lot# 124191) in corn oil via gavage (1.0 ml/kg) from GD 6 to postnatal day (PND) 21. Dams were assigned to treatment groups by balancing body weight gain from GD 2 to GD 6. A total of 71 dams were assigned as follows: 11 litters were used for the fetal time point on GD 21 (n = 5 controls and n = 6 A1254), and the remaining litters were used for postnatal time points and behavioral testing (n = 29 for control and n = 31 for A1254). On the day of birth, approximately half of the treated litters and half of the control litters were cross-fostered, resulting in the groups outlined in Table 1. The remaining litters were left with their original dams.
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Thyroid hormones.
Serum concentrations of total thyroxine (T4) and triiodothyronine (T3) were measured as previously described (Goldey and Crofton, 1998; Goldey et al., 1995b
). TSH was not measured because previous reports failed to find any effect of developmental A1254 exposure on this hormone (Goldey et al., 1995b
; Morse et al., 1996
). Trunk blood (from fetus on GD 20, pups on PND 3, 7, 14, and 21, and dams on GD 20 and PND 21) was allowed to clot on ice for a minimum of 30 min. Serum was collected via centrifugation of clotted samples and stored at 80°C for later analyses using radioimmunoassay kits (Diagnostic Products Corp., Los Angeles). To increase the sensitivity of the assays for the GD 20 fetal time point, an additional 25 µl of fetal serum was added to assay tubes and 25 µl of T4 free serum was added to the calibration curve tubes to correct for total volume. All samples were run in duplicate. The intra- and inter-assay variations were below 10% for both hormones.
PCB congener specific analysis.
All the tissue samples were frozen on dry ice, and stored at 80°C until analysis of PCBs, using high resolution gas chromatography with electron capture detection (Bush et al., 1985, 1989
). Briefly, PCB congeners were extracted by grinding the tissue in a mixture of hexane:acetone (1:1). The extract was cleaned up by passing through a chromatography column containing calibrated 4% deactivated FlorisilR with a layer of sodium sulfate on top. The column was eluted with 50 ml of hexane and the eluate containing PCBs was concentrated to 1 ml. The concentrated samples were analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a 63Ni electron capture detector with a 30-m fused silica Ultra II (DB-5) capillary column (5% phenylmethylsilicone coating, 0.33 mm film thickness, 0.25 mm internal diameter). Approximately 113 PCB congeners were identified and verified using a separate Apiezon L capillary column (Bush et al., 1985
). The microprocessor of the gas chromatograph was calibrated with a previously characterized solution of Aroclors 1221, 1016, 1254, and 1260 (200 ng/ml of each) fortified with HCB, DDE, and mirex at 5, 10, and 10 ng/ml, respectively, and 3,3',4,4'-tetrachlorobiphenyl at 20 ng/ml (Bush et al., 1985
; Schulz et al., 1989
).
Quality control for the analysis was ensured by running replicates of a sample in every batch of 10 samples to ensure the consistency of sample extraction. The samples were spiked with DDE to standardize retention times and check percent recovery of the extraction process. The average minimum detectable level (MDL) (p < 0.05 that the reported level is zero) (Type 1 error) for a sample weighing, 1 g was 0.02 ng/g. The results of PCB analysis were expressed as ppb PCB, based on tissue wet-weight.
Behavioral testing.
One male and one female per litter were repeatedly tested as previously described (Goldey et al., 1995b) for startle habituation on PND 28 and 65 and auditory thresholds for 1 and 40 kHz tones on PND 8595.
Acoustic startle responses were measured using the method of Ruppert et al. (1984). Testing was conducted in sound-attenuated chambers, each containing a wire mesh plastic-framed test cage, mounted on a load-cell/force-transducer assembly. After 5 min of acclimation, each animal received 50 trials (eliciting stimulus = 120-dB, 40-ms burst of white noise). Peak response amplitude on each trial was recorded, from which an average startle response was calculated for each animal.
Auditory thresholds were tested using reflex modification audiometry (RMA). Testing was conducted in the apparatus described above for the acoustic startle response. Auditory thresholds for 5- and 40-kHz tones were determined using the reflex modification audiometry procedure of Young and Fechter (1983) as modified by Crofton et al. (1990). After a 10-min acclimation period each rat was tested for 240 trials per frequency (eliciting stimulus = 120-dB, 40-msec burst of white noise; prepulse stimulus = 40-ms tone of variable intensity; interstimulus interval = 90 ms). RMA thresholds (auditory thresholds) were defined as the prepulse intensity above which the response was inhibited (Crofton, 1992 and Crofton et al., 1990 for details).
Data analyses.
ANOVAs and repeated measures ANOVAs were used where appropriate (SAS, 1989). Gender was nested under litter (to control for possible "litter effects") where a male and female from the same litter were tested (behavioral tests). Treatment and age were between subject factors for the hormone analyses. Age (for body weights) and startle habituation block were within subject-repeated variables. Body weights for the dams used for the GD 21 time points were analyzed separately. Auditory thresholds were estimated using a nonlinear regression procedure (see Crofton, 1992; Crofton et al., 1990 for details). Significant interactions were followed by step-down ANOVAs, as appropriate. Mean contrast comparisons between each dosage group and the vehicle-control group were made using Tukey's studentized range test (SAS, 1989). An alpha level of 0.05 was used for all tests. For the nonlinear analyses, failure to achieve a significant fit led to retesting of that animal at that frequency. Data from animals that failed to fit after 2 tests were not used. The total number of animals that did not fit for each treatment and frequency were: Ctrl/Ctrl = 3 for both frequencies; A1254/A1254 = 2 for both frequencies; A1254/Ctrl = 1 animal at 1 kHz and 2 animal for both frequencies; and Ctrl/A1254 = none.
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RESULTS |
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Thyroid Hormones
Maternal exposure to A1254 caused hypothyroxinemia in the dams, GD 20 fetuses, and early postnatal offspring (Fig. 5). Maternal T4 was depressed by 32% relative to controls on GD 20 (Fig. 5
, right panel). On PND 22, maternal T4 was decreased 40% in the A1254/A1254 group and 39% in the Ctrl/A1254 group. There was no significant effect of A1254 on maternal T3 concentrations in any of the treated groups (p > 0.05, data not shown). For the GD 20 data, these results were supported by a significant hormone by treatment interaction on GD 20 [F(1, 18) = 9.27, p < 0.0070], a main effect of treatment on maternal T4 [F(1, 9) = 9.39, p < 0.0135], and no main effect of treatment on maternal T3 [F(1, 9) = 0.21, p < 0.6569]. For PND 22, there was a significant hormone by treatment interaction [F(3, 92) = 14.75, p < 0.0001], a main effect of treatment on maternal T4 [F(3, 46) = 14.72, p < 0.0001], and no main effect of treatment on maternal T3 [F(3, 46) = 0.63, p < 0.5966]. Mean contrast comparisons revealed the treatment-dependent differences discussed above and illustrated in Figure 5
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PCB Tissue Concentrations
Concentrations of total PCBs for liver and frontal cortex are illustrated in Figure 6. In the perinatal exposed group there was very sharp increase in liver concentration of PCBs following birth which peaked at around 70 ppm on PND 7 and declined to 30 ppm by PND 21 (Fig. 6
, left panel). The prenatal-only group, A1254/Ctrl, had much lower concentrations on PND 3, approximately 4 ppm, and declined steadily to 99 ppb by PND 21. The postnatal-only exposure, Ctrl/A1254 group, had PCB concentrations that were only slightly less than those seen in the A1254/A1254 group. Interestingly, PCB concentrations in the unexposed group, Ctrl/Ctrl, rose sharply after birth and then steadily decreased through PND 21. PCB concentrations in the frontal cortex on PND 21 (Fig. 6
, right panel) reflected the same pattern seen in the liver, i.e., postnatal exposure accounted for the bulk of the PCBs found in postnatal tissue on PND 21, and tissue concentrations in the prenatal-only group had declined to control levels. Due to limited sample sizes (n = 3 per treatment group per time point) no statistical analyses were conducted on these data.
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Auditory Thresholds
Results of cross fostering clearly demonstrate that the low-frequency hearing deficit seen after perinatal exposure to A1254 is the result of postnatal exposure (Fig. 7). Perinatal A1254 exposure caused a low-frequency specific hearing deficit, with a 19 dB increase in threshold for the 1-kHz tone. There was no effect of prenatal-only exposure on either the high- or low-frequency tones. The postnatal-only exposure demonstrated the same magnitude (21 dB) and frequency specificity of effect seen in the perinatal exposed group. These results are supported by a significant frequency by treatment interaction [F(3, 38) = 6.89, p < 0.0008], a significant main effect of treatment for 1 kHz [F(3, 42) = 13.72, p < 0.0001], and a nonsignificant main effect of treatment for 40 kHz [F(3, 42) = 0.53, p < 0.6636]. There was no main effect of gender, nor any interaction of gender with any other variables (all p > 0.1).
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DISCUSSION |
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The data presented herein clearly demonstrate that the critical period for the ototoxicity resulting from developmental A1254 exposure is postnatal (Fig. 7). The degree of hearing deficit seen in the cross-fostered postnatal group was similar to the animals that were not cross-fostered and received perinatal exposure. These results also demonstrate that the fetal period is not a sensitive period for A1254-induced ototoxicity in the rat, since there was no detectable effect of A1254 on auditory function in the cross-fostered prenatal group compared to controls. The postnatal window of vulnerability to the ototoxic effects of A1254 is consistent with the ontogeny of the cochlea in the rat, the known periods of sensitivity to hypothyroidism, and the effects of A1254 on thyroid hormones. Cochlear development is tonotopic in that basal regions (which develop early) are sensitive to higher frequencies (e.g., 40 kHz) and apical regions (which mature later) are sensitive to lower frequencies (e.g., 1 kHz) (Müller, 1991a
,b
; Rubel, 1978
, 1984
). The cochlear sensitive period for hypothyroidism in rats varies from about GD 19 to PND 15 (Deol, 1973
; Hebert et al., 1985
; Uziel et al., 1985a
,b
, 1986
). Hypothyroidism during this period results in permanent malformation of cochlear structures and functional hearing loss, and T4 replacement therapy during the postnatal period allows for more normal functional and anatomical development (Deol, 1973
, 1976
; Meza et al., 1996
; Uziel et al., 1980
, 1981
, 1985a
, 1985b
). The fact that low-frequency hearing and apical hair cells are affected (Crofton et al., 2000
; Goldey and Crofton, 1998
; Goldey et al., 1995b
; Herr et al., 1996
) following the hypothyroxinemia induced by A1254, which is maximal during the second and third postnatal week (Goldey and Crofton, 1998
; Goldey et al., 1995b
; Morse et al., 1993
), is reasonably consistent with the known basal-to-apical direction of postnatal cochlear ontogeny (Müller, 1991a
,b
; Rubel, 1978
, 1984
). Furthermore, recent work has demonstrated that postnatal thyroxine administration will partially alleviate the low-frequency loss caused by A1254 exposure (Goldey et al., 1998).
The present data also demonstrate that the timing of the hypothyroxinemia induced by developmental exposure to A1254 is dependent on the timing of exposure. Prenatal-only exposure resulted in prenatal hypothyroxinemia, as evidenced by the 42% depression in T4 on GD 20. However, this effect rapidly diminished and recovered to control levels during the postnatal period, when exposure was limited to only that A1254 still remaining in the offspring from fetal exposure. In contrast, postnatal-only exposure resulted in the rapid development of hypothyroxinemia that was similar to that found following perinatal exposure. These effects are consistent with two important features of developmental exposure to polyhalogenated hydrocarbons. The first is the kinetics of highly lipid-soluble chemicals during development. As seen in the present study (Fig. 6.) and in a number of other reports (Masuda et al., 1978
; Takagi et al., 1986; Vodicnik and Lech, 1980
), compounds like TCDD and PCBs are transferred to the fetus in limited quantities compared to the amount delivered via lactation. Thus, the time of greatest exposure to highly lipophilic chemicals during development in mammals is postnatal. Furthermore, the rapid decline in PCB tissue concentrations following a prenatal-only exposure is also consistent with other studies showing that the rapid decline in postnatal tissue concentrations is due to declines in milk concentrations (McCormack et al., 1979
; Vodicnik and Lech, 1980
) and dilution of tissue concentrations due to the rapid body weight gain of the offspring (Young et al., 1997
). The second important feature of developmental exposure is the ontogeny and induction of hepatic uridine diphosphoglucuronosyl transferases (UDPGTs). Induction of hepatic UDPGT isoforms that glucuronidate thyroxine is known to be, at least partially, responsible for the declines in circulating T4 concentrations following exposure to PCBs (Barter and Klaassen, 1992
, 1994
; Bastomsky and Murthy, 1976
; Visser et al., 1993
; Volp, 1988
). The greater effects of A1254 on postnatal T4 concentrations is consistent with the mostly postnatal exposure and with inducibility of UDPGT isoforms during the period right after birth (Lucier et al., 1976; Morse et al., 1993
, 1996
). Alternatively, data from in vitro studies suggest that PCBs and hydroxy metabolites of PCBs may displace T4 from serum transport proteins (Cheek et al., 1999
; Chauhan et al., 2000
; Lans et al., 1993
). Whether or not the displacement of T4 from serum proteins plays an in vivo role during development remains to be determined.
The impact of the current A1254 exposure regimen to the dams appeared to be minimal. There was no apparent maternal toxicity associated with the 6 mg/kg/day dosage of A1254 as evidenced by a lack of difference in maternal body weights. There was, however, a mild hypothyroxinemia present in the exposed dams. There was a significant depression in T4 for both the fetal (32%) and weaning (39%) time points. This maternal hypothyroxinemia is consistent with previous observations (Morse et al., 1993, 1996
) and is consistent with increased glucuronidation of T4 in maternal liver following A1254 exposure (Morse et al., 1993
, 1996
). The mild hypothyroxinemia, coupled with the lack of effect on maternal weight gain and no impact on fetal or postnatal survival, suggest minimal maternal toxicity compared to effects in offspring.
The lack of effect of any of the exposures to A1254 on the age of eye opening was unexpected, based on our previous work. Goldey et al. (1995b) and Goldey and Crofton (1998) demonstrated an accelerated age of eye opening following perinatal exposure to A1254. There may be a number of reasons for this discrepancy. First and foremost is dose, the previous work used a dose of 8 mg/kg/day, whereas the present work used only 6 mg/kg/day. This dose may be below that needed to induce an effect. An alternative explanation may involve a combination of the dose and the manufacturer's batch number for the A1254. Recent work by Frame and colleagues (Frame et al., 1996) has demonstrated considerable differences in congener composition for some commercial batches of Aroclor mixtures. The previous work used a batch of A1254 (Accustandard Lot #6024) that has been shown to contain higher concentrations of dibenzofurans and coplanar PCB congeners compared to the batch (Accustandard Lot#124191) used in the present work (Burgin et al., 1999
). The total aryl hydrocarbon toxic-equivalent (AH-TEQ) for batch #6024 is 10-fold higher compared to batch #124191 (Burgin et al., 1999
). As postulated previously (Goldey and Crofton, 1998
), an Ah receptor-based mechanism may explain the early eye opening seen with developmental A1254 exposure. Early eye opening in TCDD-exposed rats has been postulated to result from Ah receptor activation and subsequent alterations in epidermal growth factor (EGF) signaling pathways (Madhukar et al., 1988
). Activation of EGF receptors during development is well known to induce early eye opening (Aulerich et al., 1988
; Cohen, 1962
; Lakshmanan et al., 1985
; Schlessinger et al., 1983
). Thus, the batch with the higher AH-TEQ would be a more potent mixture for induction of early eye opening compared to the lot used in the present work.
Maternal A1254 exposure at 6 mg/kg/day did affect offspring growth. This effect was less pronounced and more transient compared to that previously reported from this lab (Goldey and Crofton, 1998; Goldey et al., 1995b
). In addition, we found no increase in postnatal mortality, as was previously reported (Goldey and Crofton, 1998
; Goldey et al., 1995b
). This difference is most likely to be due to the lower dosage used (i.e., 6 mg/kg/day), as well as to the difference in lot number as discussed above.
The results of this cross-fostering experiment support some important principles for developmental studies. First, it underscores that the timing of exposure and the duration of effect are integrally linked. In the present study, measurement of T4 during the late postnatal period would underestimate or, even miss, the hypothyroxinemic effect of prenatal-only exposure to A1254. This is due to the transient nature of the effect. If the effect of exposure is permanent, then measurement can be taken at any time post-exposure. Admittedly, maternal exposure during only the prenatal period is not the same as a prenatal-only cohort in a cross-fostering study. In the former, the pups are weaned on the exposed dams, so there will be a postnatal exposure component for lipophilic xenobiotics. However, this exposure will not be of the same magnitude as pups reared with a dam that had been exposed perinatally. Second, caution is urged in interpreting results from studies where exposure was only prenatal. The kinetics of the compound(s) and the windows of vulnerability of the endpoint of concern must be taken into account prior to concluding negative findings.
The present findings may have relevance for predicting the effects of PCB exposure on human health, clearly indicating that the postnatal period is the vulnerable window of exposure for the ototoxicity of developmental exposure to A1254 in rats. This is consistent with the degree of exposure, the adverse impact on thyroxine, and the postnatal ontogeny of the cochlear structures that are damaged (Crofton et al., 2000). However, in comparing cochlear ontogeny between rats and humans, one major difference is readily apparent: cochlear tissue in humans develops almost entirely prenatally (cf., Sulik, 1995). This does not rule out effects of PCBs on human cochlear structure and/or function. However, dose-response data for hypothyroxinemia in the rat, gathered from previous work in this laboratory (Goldey et al., 1995a
, 1995b
; Goldey and Crofton, 1998
), suggest that in the rat hypothyroxinemia must exceed approximately 60% before detectable auditory dysfunction occurs. A considerable degree of fetal hypothyroxinemia may need to occur before human cochlear development is adversely affected. Clearly, human epidemiological studies that track fetal thyroid hormone concentrations as well as auditory function are needed to definitively address this issue.
In summary, the present findings demonstrated that the critical period for the ototoxicity of developmental A1254 exposure is within the first few postnatal weeks in the rat. This effect is consistent with the greater magnitude of postnatal exposure via lactation, as well as the greater degree of postnatal hypothyroxinemia that occurs during a critical window of vulnerability for the developing rat cochlea.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Phone: (919) 541-2672. Fax: (919) 541-4849. E-mail: crofton.kevin{at}epa.gov.
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