ToxSci Advance Access originally published online on June 1, 2007
Toxicological Sciences 2007 99(1):224-233; doi:10.1093/toxsci/kfm141
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Published by Oxford University Press 2007.
Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Developing Male Wistar(Han) Rat. II: Chronic Dosing Causes Developmental Delay
* School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD, UK
Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire, HG3 1PY UK
National Institute of Environmental Health Sciences, PO Box 12233 (MD E1-06), 111 TW Alexander Drive, Research Triangle Park, North Carolina 27709
Health and Safety Laboratory, Harpur Hill, Buxton, Derbyshire, SK17 9JN, UK
¶ Central Science Laboratory, Environment, Food and Health, Sand Hutton, York, YO41 1LZ, UK
|| Institute of Occupational Medicine, Research Park North, Riccarton, Edinburgh, EH14 4AP, UK
1 To whom correspondence should be addressed. Fax: +44-115-9513251. E-mail: david.bell{at}nottingham.ac.uk.
Received March 16, 2007; accepted May 25, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
We have investigated whether fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat using chronically exposed rats to ensure continuous exposure of the fetus. Five- to six-week-old rats were exposed to control diet, or diet containing TCDD, to attain an average dose of 2.4, 8, and 46 ng TCDD/kg/day for 12 weeks, whereupon the rats were mated and allowed to litter; rats were switched to control diet after parturition. Male offsprings were allowed to develop until kills on PND70 (25 per group) or PND120 (all remaining animals). Offspring from the high-dose group showed an increase in total litter loss, and the number of animals alive on postnatal day (PND)4 in the high-dose group was
26% less than control. The high and medium dose offsprings showed decreased weights at various ages. Balano-preputial separation (BPS) was significantly delayed in all three dose groups compared to control. There were no significant effects of maternal treatment when the offsprings were subjected to a functional observational battery or learning tests, with the exception that the high-dose group showed a deficit in motor activity. Twenty rats per group were mated to females, and there were no significant effects of maternal treatment on the fertility of these rats or on the F1 or F2 sex ratio. Sperm parameters at PND70 and 120 showed no significant effect of maternal treatment, with the exception that there was an increase in the proportion of abnormal sperm in the high-dose group at PND70; this is associated with the developmental delay in puberty in this dose group. There were no remarkable findings of maternal treatment on organ weights, with the exception that testis weights were reduced by 10% at PND70 (but not PND120), and although the experiment was sufficiently powered to detect small changes, ventral prostate weight was not reduced. There were no significant effects of maternal treatment upon histopathological comparison of high-dose and control group organs. These data confirm that developmental exposure to TCDD shows no potent effect on adult sperm parameters or accessory sexual organs, but show that delay in BPS occurs after exposure to low doses of TCDD, and this is dependent upon whether TCDD is administered acutely or chronically. Key Words: dioxin; sperm; developmental toxicity.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous toxin and prototypical representative of a series of chemicals which effect toxicity through a common mechanism, binding to the AhR Aryl Hydrocarbon Receptor (AHR) (Poland and Knutson, 1982
). Much investigation has focused on the toxicity of TCDD, on the basis that other chemical congeners will show the same toxicity as TCDD but with altered potency determined by their relative agonism of the AhR and pharmacokinetics (Haws et al., 2006; Van den Berg et al., 2006). There have been reports that one of the most potent toxic effects of TCDD occur after exposure of the developing rat fetus by dosing of the pregnant dam on GD15, leading to a spectrum of effects in the reproductive system of the male offspring, principally decreased sperm count in the cauda epididymis, but including decreased weight of the seminal vesicles, prostate, and epididymis (Faqi et al., 1998; Gray et al., 1995, 1997a; Mably et al., 1992a,b,c). These effects are remarkably potent with statistically significant effects after a single maternal dose of 64 ng TCDD/kg bodyweight. In view of the consistent reports of developmental effects of TCDD on male epididymal sperm counts from three laboratories, these data have been used to set a tolerable daily intake for TCDD and related compounds by the U.K. Committee on Toxicity (COT, 2001), the WHO (JECFA, 2001), and the European Union Scientific Committee on Food (SCF, 2001).
However, the inability of low doses of developmental TCDD exposure to cause a decrease in offspring epididymal sperm counts is a finding common to later studies (Ikeda et al., 2005; Ohsako et al., 2001, 2002; Simanainen et al., 2004; Wilker et al., 1996; Yonemoto et al., 2005). Sperm counts are a highly variable end point (e.g., Ashby et al., 2003), yet several studies use small group size and manual sperm counting in concert with a nonblinded analysis. In order to resolve the discordant results in the literature, studies should be implemented using Good Laboratory Practice-compliant (GLP) methodology and large group sizes to increase the statistical power and reliability of the analysis, with the explicit prior aim of measuring epididymal sperm levels.
In the accompanying paper, we have used a robust design to show that a single acute dose of TCDD to the pregnant CRL:WI(Han) rat on GD15 fails to decrease epididymal sperm levels in male offspring but that TCDD is a potent toxin that induces lethality and a delay in balano-preputial separation (BPS) in offspring. A limitation of this study is that the TCDD was given as a single acute dose; however, it is possible that there could be a narrow temporal window of susceptibility to these effects and that no effect was seen since the dose missed the window of susceptibility. Therefore, a study was undertaken with chronic maternal dosing of TCDD to ensure continuous exposure of the fetus to TCDD; this chronic exposure to TCDD is also more representative of human patterns of exposure to TCDD through the diet and from lactational transfer (COT, 2001; Fries, 1995). The pharmacokinetics of TCDD is complex with multiple uptake and elimination phases (Weber et al., 1993), and thus the disposition of TCDD at 24 h after a single dose on GD15 is likely to vary from steady state. Indeed, it has been reported that there is induction of metabolism of TCDD during chronic exposure (Fries and Marrow, 1975), and it takes 13 weeks to attain steady-state levels of tissue TCDD in the rat (Rose et al., 1976). This is important to understand reported effects of TCDD that have a narrow temporal window of susceptibility (Ohsako et al., 2002) between GD15 and GD18. Indeed, detailed pharmacokinetic studies show that there are clear differences in disposition at GD16 between acute and chronic doses that give similar TCDD concentration in liver (Hurst et al., 2000a, 2000b). Thus, it is possible that the mode of administration (i.e., acute vs. chronic dosing protocol) may be a determinant of sensitivity of the developing fetus to TCDD.
We have therefore undertaken to repeat our previous study but using a chronic dosing protocol instead of a single acute dose of TCDD. This experiment serves both to compare against our previous work using an acute dose of TCDD and additionally to compare the effect of acute versus chronic administration on toxicological end points. Delay in puberty is an adverse effect and occurs after very low doses of TCDD during development in rats.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
Materials.
TCDD was obtained from Cambridge Isotope laboratories, MA, and purity (99% vol/vol) was verified by High Resolution Mass-Spectrometry. All other chemicals were of the highest quality available.
Animal study.
The animal studies were performed at Covance (Harrogate, UK) and were GLP compliant; the full report on this study is published as supplementary material. CRL:WI(Han) rats were housed at a temperature of 19–25°C, with two brief excursions with the lowest to 16.4°C. Animals were provided food (SQC rat and mouse breeder diet No. 3, expanded; Special Diets Services Ltd, Witham) and water ad libitum and were housed singly (the parental generation postpairing) or in groups of five for the parental generation prepairing and the F1 generation, with a 12-h light/darkness cycle. Animals of 5–6 weeks of age (100–146 g) were assigned to treatment groups using a randomization procedure based on body weight. Animals (75, 65, 65, and 65 rats, respectively) were provided with diet containing 0 (acetone alone), 28, 93, and 530 ng TCDD/kg diet (the TCDD was dissolved in acetone) ad libitum; food intake per cage was measured weekly. Exposure to TCDD in the diet was maintained for a 12-week acclimatization period and during mating and pregnancy, and dams were switched to control diet after parturition. After 12 weeks of treatment of the Parental generation females, one female was housed with one untreated male for up to 15 days and mating confirmed by a vaginal plug. The concentration of TCDD in the diet was verified by gas chromatography-mass spectrometry. Five and ten animals per group were killed in weeks 10 and 12 after starting on the diet, and on gestation day (GD)16 and 21, 15 animals from the control group and 10 animals from the treated group were killed; tissue samples from these culls were used for TCDD and RNA analyses (David R. Bell et al., in preparation). The remaining females were allowed to litter and rear their offsprings until weaning (PND21) and killed on PND21. Litters were reduced to a maximum size of eight on PND4 and to five males (where possible) on PND21. Males were then maintained untreated, until killed (25 per group, one per litter) at PND70, and all remaining animals at PND120. Although kill days are referred to as PND70 and 120, the number of animals involved required that the kills were conducted during postnatal weeks 10 and 17. During postnatal weeks 12 and 13, 20 animals from each group were tested for learning ability (swimming maze), motor activity (every 2 min for 30 min), and in week 13, a functional observation battery. During postnatal week 16, 20 males per group were paired with untreated virgin females for up to 7 days; mated females were killed on GD16 and examined for terminal body weight, pregnancy status, number of corpora lutea, number and intrauterine position of implantations, which were subdivided into live embryos, and early and late intrauterine deaths, and sex of embryos.
Necropsy and seminology.
At necropsy, animals were weighed and weights of seminal vesicles (with coagulating glands), brain, epididymes (total), liver, ventral prostate, thymus, spleen, kidneys, and testes recorded. Testis, epididymis, liver, thymus, and prostate from control and high-dose groups were fixed, embedded, sectioned at 5 µm, stained with hemotoxylin and eosin, and examined by a pathologist. Sperm counts and viability were assessed from one epididymis from each male killed in postnatal weeks 10 and 17 and samples examined microscopically for morphology. Briefly, the cauda epididymis is dissected free and the mid-distal cauda pierced two/three times with a scalpel blade. The cauda is placed into 5 ml of phosphate-buffered saline containing 0.57% (wt/vol) bovine serum albumin, preheated to 37°C. The left testis of males was frozen, pending enumeration of homogenization-resistant spermatids. Sperm number, motility, and velocity were recorded by computer-assisted sperm analysis with a Hamilton-Thorne TOX-IVOS instrument examining n = 10 fields per sample. Five hundred sperm per animal were examined microscopically, and the number of morphologically abnormal sperm was recorded to give the percent abnormal sperm.
TCDD analysis.
Samples were stored frozen until analyzed. Adipose tissue and liver samples were analyzed individually, and fetus samples from individual females were combined, but the volumes of blood samples were too low for individual analysis and were pooled. The tissue samples were homogenized and an aliquot taken for analysis. Sample aliquots were fortified with 13carbon-labeled dioxins and exhaustively extracted using mixed solvents. The extracts were initially purified by acid hydrolysis, fractionated on activated carbon, and further purified using adsorption chromatography on alumina. The eluent was concentrated under nitrogen and sensitivity standardized for measurement using additional 13carbon-labeled dioxins. TCDD was measured using high-resolution gas chromatography with high-resolution mass spectrometric detection at a resolution of 10,000 (defined at 10% of peak height). Instrument performance was monitored during the measurement interval by the use of a calibrant (perfluorokerosene) lock mass, and ions corresponding to native and [13C]-labeled dioxins were recorded. Data were processed using Masslynx and Microsoft Excel software to provide tissue concentration data. The analytical data met published acceptance criteria (Ambidge et al., 1990) for dioxins. The method used is accredited to the ISO17025 standard and has been validated and published after peer review (Fernandes et al., 2004). Each batch of samples analyzed incorporated a full reagent blank, and analytical results were validated by the analysis of an in-batch reference material (Maier et al., 1995), for which results were compared with certified or assigned data. The contribution from the batch blanks was found to be negligible.
Statistical analysis.
Data were first analyzed at Covance, as they were collected, with their standard statistical package. Continuous outcomes were analyzed using one-way ANOVA or ANCOVA, after log transformation where necessary. Pairwise comparisons with control were made using Dunnett's test, and a linear trend test was applied. Data measured as proportions of animals were analyzed using the Cochran-Armitage test for dose response and Fisher's exact test for pairwise comparisons. These tests were interpreted with one-sided risk for increased incidence with increasing dose.
Further analyses of selected variables were carried out in the package GenStat (Payne, 2004) and included terms for random variation between litters. F1 body weights were analyzed by a mixed model ANOVA, with a one-way treatment group structure and a normally distributed random term for litters. In most analyses litter effects were significant, with the effect that estimated SEs were larger than in the simple ANOVA model. Comparisons between treated groups and control used Williams' test (Williams, 1972). Early body weights were analyzed with a two-way (dose group x day) ANOVA mixed model with random litter effects. From PND21 onward, when the pups were individually identified in the data, a repeated measures model was applied, with random terms for litter and pup differences. Time to BPS was analyzed by a proportional hazards mixed model with a random term for litter effects (Lee et al., 2006) and with body weight as a covariate.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
Dietary levels of TCDD were targeted to achieve comparable levels of TCDD in the liver to that seen in GD16 liver during the acute study (Bell et al., 2007
; Bell et al., in preparation), extrapolating from previous comparisons of TCDD tissue burden using acute and chronic dosing regimes (Hurst et al., 2000a,b); the diet was made up at 28, 93, and 530 ng TCDD/kg and assuming a food intake of 10% of body weight per day, a dose of 2.8, 9.3, and 53 ng TCDD/kg/day. The concentration of TCDD in the diet was verified by measuring TCDD in ten 25-g aliquots of low-dose diet; the determined concentration was 29 ng TCDD/kg, and the coefficient of variation was 1.5%. Feed stability was also tested; after a 2-month interval, four samples of the same diet were found to contain 30 ng TCDD/kg with a coefficient of variation of 4.2%. Duplicate feed samples (25 g) were tested on two occasions, yielding the following TCDD concentrations (all in ng TCDD/kg): control 0.6, 0.1; low dose 30, 24; medium dose 68, 102; high dose 560, 371.
Parental Health
One animal in the high-dose group died on day 2 of lactation, but all other animals survived. Females in the high-dose group gained slightly more weight in the first 5 weeks of the prepairing period than controls, but over the whole of the prepairing period (and at all other times), there was no significant difference in weight gain between groups. During the prepairing period, the high-dose group ate more food than controls, but there was no difference in food intake between groups during the lactation period (see Supplementary data). The average doses based on nominal feed concentration and average food intake were 2.4, 8, and 46 ng TCDD/kg/day.
Littering and Offspring
In all groups, the precoital time was 2–3 days, and the mating, fertility, and fecundity indices were similar in all groups. In the animals killed on GD16, there was a significant dose-response relationship to show fewer implantations and fetuses with TCDD dose, but there were no significant pairwise comparisons with control, and the pooled GD16 and 21 data showed no significant effect of TCDD dose on the uterine/implantation data (see Supplementary data). At littering, three females in the control group, four females in the low and medium dose group, and eight females in the high-dose group showed total litter loss (Table 1). The number of pups alive on day 1, expressed as a ratio to the number of pups born, was significantly decreased in the high-dose group, and the number of pups surviving between days 1 and 4 (as a ratio of number of pups alive day 1) was also statistically significantly reduced in the high-dose group.
|
F1 Body Weight Gain and BPS
There was a dose-related reduction in mean pup body weight on PND1, and this remained so throughout the lactation period (Fig. 1). The decrease in body weight, relative to control, was most marked at PND4 and was apparent in all three dose groups. Males in the low and medium dose groups had similar weight to control at PND21 but showed a dose-related trend to being lighter than the control group until PND120. The high-dose group males were lighter than controls at PND21 and gained less weight than controls over the course of the study, with a marked reduction in weight gain immediately after weaning in the high-dose group offspring. BPS was significantly delayed in all three treatment groups (by 1.8, 1.9, and 4.4 days for low, medium and high dose, respectively), and this was dose dependent (Fig. 2). The delay in BPS was analyzed by fitting a proportional hazards model to the incidence rates, and the rate ratios relative to control were 54% (95% confidence interval 38–75%), 49% (35–69%), and 26% (17–38%) for the low, medium, and high-dose groups, respectively. The day of BPS was analyzed with body weight on PND21 or 42 as covariates and with a random effect for litter differences; while the effects of body weight fell short of significance, there was highly significant between-litter variation. However, the adjustments to the mean BPS were minimal, and the treated groups remained significantly different from control; thus the body weight at PND21 or 42 does not affect delay in BPS.
|
|
Learning and Motor Activity
There were no adverse effects of maternal treatment on learning and memory in the swimming maze or in the performance in the functional observational battery. However, the offsprings of animals in the high-dose group were less active than the controls (p < 0.05) when subject to a test of motor activity over 30 min (see Supplementary data).
Analysis of Reproductive Capacity of F1 Males
Twenty F1 males were mated during postnatal week 16, and the median precoital time was 2–3 days in all groups. The uterine/implantation data were similar in all groups, and there was no significant difference in the proportion of male offspring between groups (Table 2).
|
In the F1 males killed in postnatal week 10, epididymal sperm counts were not significantly different between dose groups or were parameters describing sperm motility (Table 3). However, the proportion of abnormal sperm was significantly elevated in the high-dose group compared to control. The mean number of spermatids in the high-dose group was 14% lower than control, and this difference was statistically significant. However, in the males killed in postnatal week 17, mean seminology data were unaffected by maternal treatment. Neither epididymal sperm levels nor testicular spermatids were affected by treatment (Table 3). Moreover, comparison of the control epididymal sperm levels with our previous data from CRL:WI(Han) rats showed that the absolute value of sperm counts were similar between the two studies (Fig. 3), confirming the consistency of these estimations.
|
|
Body Weight and Pathology
Terminal body weights were not significantly different between the four groups at PND70 but were significantly decreased in the high-dose group (6.9%) , relative to control, at PND120; a 5.5% decrease in the medium dose group was just outside statistical significance at p < 0.05. At PND70, the high-dose group testes were lighter than control as both an absolute and ratio to body weight (
12 and 8%, respectively). Spleen weight was significantly elevated (by 8%) in the high-dose group animals at PND70 and was elevated (by 1.3–3.4%) in all three dose groups at PND120. At PND120, kidneys of the low and medium dose group were statistically significantly greater than control (2%), ventral prostate of the medium dose group was greater than control (9.4%), liver to body weight ratios from the low and high-dose group were significantly greater than control (4.5%), and absolute brain weight was less than control in the medium dose group (2.2%) and greater than control as a ratio to body weight in the medium and high-dose groups. Histological examination of organs revealed solely minor findings that were consistent with the usual pattern of findings in animals of this strain and age; there were no findings that were associated with maternal TCDD treatment. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
TCDD was administered in the diet at a constant amount per kilogram of diet; the TCDD was stable in the diet, and there was batch to batch variation of up to 30% in TCDD concentration. Dietary administration of TCDD in this manner results in variation in the administered dose on a dose per kilogram basis, as animals eat more food as a fraction of bodyweight when they are younger (see Supplementary data); thus, maternal dietary intake of TCDD was
150% of average at the start of the study and 70% of average at the end of gestation. Over the course of the study, average food intake was slightly less than the nominal 10%/kg/day, and hence the administered dose was 10% less than planned. A further source of variation arises from the fact that animals were housed in groups of five animals; it is not clear that all rats consumed equal amounts of food. Analysis of tissue TCDD concentrations and mRNA levels confirm that TCDD was adequately dosed and will be reported in detail elsewhere (Bell et al., in preparation).
There was no evidence of direct maternal toxicity of the TCDD, although the high-dose group ate more food than controls during the prepairing period. However, the high-dose group had 8% fewer pups alive on day 1 (as a ratio of pups born), and the number surviving from PND1–4 was 18% lower than control. Thus, the TCDD treatment in the high-dose group reduced pup numbers by 26% by PND4. This effect of TCDD on increased pup lethality is consistent with other work (Bjerke and Peterson, 1994; Bjerke et al., 1994; Gray et al., 1995, 1997a; Mably et al., 1992c; Roman et al., 1995, 1998; Sommer et al., 1996) and with the accompanying study in the CRL:WI(Han) rat (Bell et al., 2007). Whereas there were 15% fewer pups alive on PND4 after an acute maternal dose of 1000 ng TCDD/kg (Bell et al., 2007), there were 26% fewer pups alive on PND4 after a chronic maternal dose of 46 ng TCDD/kg/day (Table 1). Moreover, these data conceal a much stronger effect on postnatal lethality after chronic administration of TCDD, since the acute dose study lethality consisted of 12% fewer pups born and 3% of pups dying after birth and before PND4, consistent with a higher peak concentration of TCDD in utero arising from the single acute gavage dose by comparison with the chronic dosing regime. It is of interest that this postnatal lethality is coincident with a severe depression in weight in the treated groups (Fig. 1). The substantial postnatal lethality confirms the extraordinary potency of TCDD as a developmental toxicant.
In agreement with previous results in the same (Bell et al., 2007) and other rat strains (Bjerke and Peterson, 1994; Bjerke et al., 1994; Faqi et al., 1998; Gray et al., 1997a; Roman et al., 1995, 1998; Sommer et al., 1996; Yonemoto et al., 2005), maternal TCDD treatment led to a delay in BPS (Fig. 2). Whereas an acute dose of 1000 ng TCDD/kg led to a significant average delay in puberty of 2.8 days (Bell et al., 2007), chronic doses of 2.4, 8, and 46 ng TCDD/kg/day led to average delays of 1.8, 1.9, and 4.4 days, respectively (Fig. 2). The more potent effect of TCDD after chronic dosing is unlikely to be attributable to an unlikely sampling of the control group that resulted in an artefactually low control value, since our previous study found a mean day of BPS in the control group of PND45.8 (Bell et al., 2007), as against PND45.4 in this study (Supplementary data, Fig. 2), and these two values are not significantly different from each other. Thus, developmental delay in puberty is the most sensitive adverse effect of TCDD in this study.
The greater sensitivity of rats to chronic, as opposed to acute, dosing of TCDD is unexpected. Our data show that acute exposure on GD15 gives a markedly less sensitive effect of TCDD on BPS compared to chronic exposure (Bell et al., 2007) (Fig. 2); thus, it is likely that GDs 15–21 are not a period when the fetus is especially sensitive to this effect of TCDD. It is not clear when chronic maternal exposure to TCDD exerts the toxic effect in offspring leading to delayed BPS; it could be during gestation or it could result from lactational transfer of TCDD to the offspring. There are differences in disposition arising from acute versus chronic exposure to TCDD, and so it would follow that a high-dose acute exposure may well be uninformative for determining whether intrauterine or lactational transfer of TCDD is important during low-dose chronic exposure. Lactational transfer of TCDD accounts for the majority (>90%) of pup TCDD after an acute dose of TCDD on GD18 (Li et al., 1995) or GD15 (Nishimura et al., 2005), and a chronic dosing regimen can achieve high concentrations of TCDD in the offspring (Hurst et al., 2000a; Korte et al., 1992). Indeed, it has been shown that high maternal doses of TCDD cause hypothyroidism and hydronephrosis in F1 rats via lactational transfer of TCDD (Nishimura et al., 2003, 2005, 2006). Further, the ability of TCDD to directly suppress testosterone levels in adult rats is well established (Kleeman et al., 1990; Mandal et al., 2001; Moore et al., 1985). We therefore propose that the effect of chronic administration of TCDD to the dam resulting in lethality and delayed BPS in the offspring is mediated via lactational transfer of TCDD.
There were no significant effects of maternal TCDD treatment on learning and memory end points, with the exception that the offsprings from high-dose group animals were less active than the controls in a test of motor activity over 30 min (Supplementary data). This is in general agreement with the lack of effect seen on these end points in the acute dose study; however, the effect at high dose is associated with a >25% incidence of pup lethality, and it is not clear whether this is a nonspecific effect associated with the high dose of TCDD.
Seminological investigations failed to reveal any remarkable findings, with the exception that the percent of abnormal sperm was elevated and testicular spermatids were decreased in the high-dose group at PND70 (Table 3). However, given the size of the delay in puberty in the high-dose group (Fig. 2), this increase in abnormal sperm can be parsimoniously explained by a delay in puberty retarding the start of spermatogenesis and the consequent delayed onset of spermatogenesis yielding high numbers of abnormal sperm (Creasy, 2003). Notably, there was no significant difference in the effects of maternal treatment on epididymal sperm number (Table 3). This data were consistent with the data after acute exposure to TCDD, and direct comparison of the absolute sperm counts in the control groups (Fig. 3) showed that the studies had good consistency. These studies were performed to determine if previous reports of gestational TCDD exposure causing decreases in epididymal sperm levels were reproducible (Faqi et al., 1998; Gray et al., 1997a; Mably et al., 1992a); although our previous single-dose study failed to show this effect (Bell et al., 2007), it remained possible that this result was due to a failure to expose the developing fetuses at the correct developmental stage. However, exposure of the fetus with a chronic dosing protocol excludes this possibility for the failure to show a decrease in epididymal sperm levels, and the experiment has a 95% power for detecting a 30% difference in control means at p < 0.05 based on the data from the control group (Table 3). In agreement with these data, a functional test of mating ability of the F1 males showed no significant difference in the reproductive function of control or treated animals (Table 2). Although there have been sporadic reports of alterations in sex ratios after parental TCDD exposure (Ikeda et al., 2005; Mocarelli et al., 2000), these reports are difficult to interpret since they are mechanistically inconsistent (maternal vs. paternal exposure), and there is no precedent for either finding. We therefore set out an explicit, prior hypothesis that TCDD would alter sex ratios in the F1 or F2 animals in a well-powered study; our data show no significant effect of TCDD treatment on sex ratio (Table 2), consistent with other animal studies (Rowlands et al., 2006). In this experiment, the weight of ventral prostate was directly measured, and this showed no significant difference from control (Table 4). Given the variability of the control animals, our experiment has a 90% power for detecting a 10% decrease from control values in ventral prostate weight, which would be sufficient to detect the 40% decrease in prostate weight described by Mably et al. (1992c) and Ohsako et al. (2001, 2002).
|
This study fails to show developmental toxicity of maternal TCDD on F1 epididymal sperm levels, and this finding is difficult to explain by appealing to strain differences, since, Holtzmann rats (Ikeda et al., 2005
; Mably et al., 1992a; Ohsako et al., 2001), Long-Evans rats (Gray et al., 1997b; Yonemoto et al., 2005), and Wistar/Wistar(Han) (this study, Bell et al., 2007; Faqi et al., 1998) have all been used, and in each case, the repeat studies showed that developmental exposure to TCDD (at less than 500 ng/kg) causes no decrease in F1 epididymal sperm levels. This study used the outbred CRL:WI(Han) rat for comparison with the Bor Wistar strain used by Faqi et al. (1998) since this laboratory has background data for this strain, and this is essential for putting the results into the context of historical control data (Ashby et al., 2003). It is of note that a Wistar(Han) line shows resistance to TCDD toxicity (Pohjanvirta et al., 1987; Tuomisto et al., 1999). However, susceptibility to acute lethality of TCDD varies between Wistar(Han) substrains (Pohjanvirta and Tuomisto, 1990), and Table 1 and Bell et al. (2007) demonstrate that the CRL:WI(Han) rat is sensitive to TCDD-induced lethality as a consequence of maternal exposure. We are currently characterizing the molecular basis and prevalence of the AhR (Tuomisto et al., 1999) in CRL:WI(Han) and in Wistar strains (David R. Bell, unpublished data). The function of the AhR in mediating TCDD toxicity is paramount, and understanding the role of this rat AhR, and indeed, the human AhR (Connor and Aylward, 2006), in mediating toxicity is a key component for risk assessment.
In summary, this study and the accompanying paper (Bell et al., 2007) demonstrate a consistent pattern of TCDD toxicity in the CRL:WI(Han) rat, including perinatal lethality, weight loss, and delay in BPS, but our data show no potent effect of TCDD on F1 epididymal sperm levels or accessory sexual organ weight, despite having adequate power to detect these effect. Our data identify delay in puberty as the most sensitive adverse effect of maternal TCDD toxicity and show that chronic dosing dramatically alters the incidence of this effect. It will be relevant to determine if this effect is mediated via lactational transfer of TCDD to the pups.
|
SUPPLEMENTARY DATA |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
The full study report with individual animal data is provided as an appendix. Supplementary data are available online at http://toxsci.oxfordjournals.org/.
|
FUNDING |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
UK Food Standards Agency (T01034).
|
ACKNOWLEDGMENTS |
|---|
The authors wish to thank the Food Standards Agency and expert reviewers (Professors G. Gibson, A.G. Renwick, and Dr A.G. Smith) for helpful comments and guidance.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
|---|
Ambidge PF, Cox EA, Creaser CS, Greenberg M, Gem MGD, Gilbert J, Jones PW, Kibblewhite MG, Levey J, Lisseter SG, et al. Acceptance criteria for analytical data on polychlorinated dibenzo-para-dioxins and polychlorinated dibenzofurans. Chemosphere (1990) 21:999–1006.
Ashby J, Tinwell H, Lefevre PA, Joiner R, Haseman J. The effect on sperm production in adult Sprague-Dawley rats exposed by gavage to bisphenol a between postnatal days 91–97. Toxicol. Sci. (2003) 74:129–138.
Bell DR, Clode S, Fan MQ, Fernandes A, Foster PMD, Jiang T, Loizou G, MacNicoll A, Miller B, Rose M, et al. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the developing male Wistar(Han) rat I: no decrease in epididymal sperm count after a single acute dose. Toxicol. Sci. (2007) 99(1):224–233.
Bjerke DL, Peterson RE. Reproductive toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male-rats—different effects of in-utero versus lactational exposure. Toxicol. Appl. Pharmacol. (1994) 127:241–249.[CrossRef][Web of Science][Medline]
Bjerke DL, Sommer RJ, Moore RW, Peterson RE. Effects of in-utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on responsiveness of the male-rat reproductive-system to testosterone stimulation in adulthood. Toxicol. Appl. Pharmacol. (1994) 127:250–257.[CrossRef][Web of Science][Medline]
Connor KT, Aylward LL. Human response to dioxin: aryl hydrocarbon receptor (AhR) molecular structure, function, and dose-response data for enzyme induction indicate an impaired human AhR. J. Toxicol. Environ. Health B Crit. Rev. (2006) 9:147–171.[CrossRef][Web of Science][Medline]
COT. Statement on the Tolerable Daily Intake for Dioxins and Dioxinlike Polychlorinated Biphenyls (2001) Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment, Food Standards Agency, Available at: http://www.food.gov.uk/science/ouradvisors/toxicity/statements/cotstatements2001/dioxinsstate.
Creasy DM. Evaluation of testicular toxicology: a synopsis and discussion of the recommendations proposed by the society of toxicologic pathology. Birth Defects Res. B Dev. Reprod. Toxicol. (2003) 68:408–415.[CrossRef][Web of Science][Medline]
Faqi AS, Dalsenter PR, Merker HJ, Chahoud I. Reproductive toxicity and tissue concentrations of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring rats exposed throughout pregnancy and lactation. Toxicol. Appl. Pharmacol. (1998) 150:383–392.[CrossRef][Web of Science][Medline]
Fernandes A, DSilva K, Rose M. Simultaneous determination of PCDDs, PCDFs, PCBs and PBDEs in food. Talanta (2004) 63:1147–1155.
Fries GF. A review of the significance of animal food-products as potential pathways of human exposures to dioxins. J. Anim. Sci. (1995) 73:1639–1650.[Abstract]
Fries GF, Marrow GS. Retention and excretion of 2,3,7,8-tetrachlorodibenzo-para-dioxin by rats. J. Agric. Food Chem. (1975) 23:265–269.[CrossRef][Web of Science][Medline]
Gray LE, Kelce WR, Monosson E, Ostby JS, Birnbaum LS. Exposure to TCDD during development permanently alters reproductive function in male Long-Evans rats and hamsters—reduced ejaculated and epididymal sperm numbers and sex accessory-gland weights in offspring with normal androgenic status. Toxicol. Appl. Pharmacol. (1995) 131:108–118.[CrossRef][Web of Science][Medline]
Gray LE, Ostby JS, Kelce WR. A dose-response analysis of the reproductive effects of a single gestational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male Long Evans hooded rat offspring. Toxicol. Appl. Pharmacol. (1997a) 146:11–20.[CrossRef][Web of Science][Medline]
Gray LE, Wolf C, Mann P, Ostby JS. In utero exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters reproductive development of female long evans hooded rat offspring. Toxicol. Appl. Pharmacol. (1997b) 146:237–244.[CrossRef][Web of Science][Medline]
Haws LC, Su SH, Harris M, DeVito MJ, Walker NJ, Farland WH, Finley B, Birnbaum LS. Development of a refined database of mammalian relative potency estimates for dioxin-like compounds. Toxicol. Sci. (2006) 89:4–30.
Hurst CH, DeVito MJ, Birnbaum LS. Tissue disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in maternal and developing Long-Evans rats following subchronic exposure. Toxicol. Sci. (2000a) 57:275–283.
Hurst CH, DeVito MJ, Setzer RW, Birnbaum LS. Acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in pregnant Long Evans rats: association of measured tissue concentrations with developmental effects. Toxicol. Sci. (2000b) 53:411–420.
Ikeda M, Tamura M, Yamashita J, Suzuki C, Tomita T. Repeated in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure affects male gonads in offspring, leading to sex ratio changes in F-2 progeny. Toxicol. Appl. Pharmacol. (2005) 206:351–355.[CrossRef][Web of Science][Medline]
JECFA. Joint FAO/WHO Expert Committee on Food Additives, Fifty-seventh meeting, Rome, 5–14 June 2001. Summary and Conclusions (2001) Vol. 909. Rome: Joint FAO/WHO Expert Committee on Food Additives.
Kleeman JM, Moore RW, Peterson RE. Inhibition of testicular steroidogenesis in 2,3,7,8-tetrachlorodibenzo-para-dioxin-treated rats—evidence that the key lesion occurs prior to or during pregnenolone formation. Toxicol. Appl. Pharmacol. (1990) 106:112–125.[CrossRef][Web of Science][Medline]
Korte M, Thiel R, Koch E, Stahlmann R, Neubert D. Tissue concentrations of 2,3,7,8-TCDD in rats and offspring after continuous exposure during the late fetal and postnatal-period. Chemosphere (1992) 25:1183–1188.
Lee Y, Nelder JA, Pawitan Y. Generalized Linear Models with Random Effects: Unified Analysis via H-likelihood (2006) Boca Raton, FL: Chapman & Hall/CRC.
Li XL, Weber LWD, Rozman KK. Toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats including placental and lactational transfer to fetuses and neonates. Fundam. Appl. Toxicol. (1995) 27:70–76.[CrossRef][Web of Science][Medline]
Mably TA, Bjerke DL, Moore RW, Gendronfitzpatrick A, Peterson RE. Inutero and lactational exposure of male-rats to 2,3,7,8-tetrachlorodibenzo-para-dioxin.3. Effects on spermatogenesis and reproductive capability. Toxicol. Appl. Pharmacol. (1992a) 114:118–126.[CrossRef][Web of Science][Medline]
Mably TA, Moore RW, Goy RW, Peterson RE. Inutero and lactational exposure of male-rats to 2,3,7,8-tetrachlorodibenzo-para-dioxin.2. effects on sexual-behavior and the regulation of luteinizing-hormone secretion in adulthood. Toxicol. Appl. Pharmacol. (1992b) 114:108–117.[CrossRef][Web of Science][Medline]
Mably TA, Moore RW, Peterson RE. Inutero and lactational exposure of male-rats to 2,3,7,8-tetrachlorodibenzo-para-dioxin.1. effects on androgenic status. Toxicol. Appl. Pharmacol. (1992c) 114:97–107.[CrossRef][Web of Science][Medline]
Maier EA, Vancleuvenbergen R, Kramer GN, Tuinstra L, Pauwels J. Bcr (non-certified) reference materials for dioxins and furans in milk powder. Fresenius J. Anal. Chem. (1995) 352:179–183.[CrossRef]
Mandal PK, McDaniel LR, Prough RA, Clark BJ. 7,12-Dimethylbenz[a]anthracene inhibition of steroid production in MA-10 mouse leydig tumor cells is not directly linked to induction of CYP1B1. Toxicol. Appl. Pharmacol. (2001) 175:200–208.[CrossRef][Web of Science][Medline]
Mocarelli P, Gerthoux PM, Ferrari E, Patterson DG, Kieszak SM, Brambilla P, Vincoli N, Signorini S, Tramacere P, Carreri V, et al. Paternal concentrations of dioxin and sex ratio of offspring. Lancet (2000) 355:1858–1863.[CrossRef][Web of Science][Medline]
Moore RW, Potter CL, Theobald HM, Robinson JA, Peterson RE. Androgenic deficiency in male-rats treated with 2,3,7,8-tetrachlorodibenzo-para-dioxin. Toxicol. Appl. Pharmacol. (1985) 79:99–111.[CrossRef][Web of Science][Medline]
Nishimura N, Yonemoto J, Miyabara Y, Sato M, Tohyama C. Rat thyroid hyperplasia induced by gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Endocrinology (2003) 144:2075–2083.
Nishimura N, Yonemoto J, Nishimura H, Ikushiro S, Tohyama C. Disruption of thyroid hormone homeostasis at weaning of holtzman rats by lactational but not in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Sci. (2005) 85:607–614.
Nishimura N, Yonemoto J, Nishimura H, Tohyama C. Localization of cytochrome P450 1A1 in a specific region of hydronephrotic kidney of. rat neonates lactationally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology (2006) 227:117–126.[CrossRef][Web of Science][Medline]
Ohsako S, Miyabara Y, Nishimura N, Kurosawa S, Sakaue M, Ishimura R, Sato M, Takeda K, Aoki Y, Sone H, et al. Maternal exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the development of reproductive organs of male rats: dose-dependent increase of mRNA levels of 5 alpha-reductase type 2 in contrast to decrease of androgen receptor in the pubertal ventral prostate. Toxicol. Sci. (2001) 60:132–143.
Ohsako S, Miyabara Y, Sakaue M, Ishimura R, Kakeyama M, Izumi H, Yonemoto J, Tohyama C. Developmental stage-specific effects of perinatal 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on reproductive organs of male rat offspring. Toxicol. Sci. (2002) 66:283–292.
Payne R. The Guide to GenStat Release 8. Part 2: Statistics (2004) Rothamsted, UK: Lawes Agricultural Trust.
Pohjanvirta R, Tuomisto J. Letter to the editor. Toxicol. Appl. Pharmacol. (1990) 105:508–509.[CrossRef][Medline]
Pohjanvirta R, Tuomisto J, Vartiainen T, Rozman K. Han/Wistar rats are exceptionally resistant to TCDD-I. Pharmacol. Toxicol. (1987) 60:145–150.[Web of Science][Medline]
Poland A, Knutson JC. 2,3,7,8-Tetrachlorodibenzo-Para-dioxin and related halogenated aromatic-hydrocarbons—examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. (1982) 22:517–554.[CrossRef][Web of Science][Medline]
Roman BL, Sommer RJ, Shinomiya K, Peterson RE. In-utero and lactational exposure of the male-rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin—impaired prostate growth and development without inhibited androgen production. Toxicol. Appl. Pharmacol. (1995) 134:241–250.[CrossRef][Web of Science][Medline]
Roman BL, Timms BG, Prins GS, Peterson RE. In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development—2. Effects on growth and cytodifferentiation. Toxicol. Appl. Pharmacol. (1998) 150:254–270.[CrossRef][Web of Science][Medline]
Rose JQ, Ramsey JC, Wentzler TH, Hummel RA, Gehring PJ. Fate of 2,3,7,8-tetrachlorodibenzo-para-dioxin following single and repeated oral doses to Rat. Toxicol. Appl. Pharmacol. (1976) 36:209–226.[CrossRef][Web of Science][Medline]
Rowlands JC, Budinsky RA, Aylward LL, Faqi AS, Carney EW. Sex ratio of the offspring of Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study. Toxicol Appl Pharmacol (2006) 216:29–33.[CrossRef][Web of Science][Medline]
SCF. Opinion of the Scientific Committee on Food on the Risk Assessment of Dioxins and Dioxin-like PCBs in Food (2001) Available at: http://europa.eu.int/comm/food/fs/sc/scf/outcome_en.html. Accessed May 30, 2001.
Simanainen U, Haavisto T, Tuomisto JT, Paranko J, Toppari J, Tuomisto J, Peterson RE, Viluksela M. Pattern of male reproductive system effects after in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in three differentially TCDD-sensitive rat lines. Toxicol. Sci. (2004) 80:101–108.
Sommer RJ, Ippolito DL, Peterson RE. In utero and lactational exposure of the male Holtzman rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: decreased epididymal and ejaculated sperm numbers without alterations in sperm transit rate. Toxicol. Appl. Pharmacol. (1996) 140:146–153.[CrossRef][Web of Science][Medline]
Tuomisto JT, Viluksela M, Pohjanvirta R, Tuomisto J. The AH receptor and a novel gene determine acute toxic responses to TCDD: segregation of the resistant alleles to different rat lines. Toxicol. Appl. Pharmacol. (1999) 155:71–81.[CrossRef][Web of Science][Medline]
Van den Berg M, Birnbaum LS, Denison M, De Vito M, Farland W, Feeley M, Fiedler H, Hakansson H, Hanberg A, Haws L, et al. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. (2006) 93:223–241.
Weber LWD, Ernst SW, Stahl BU, Rozman K. Tissue distribution and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats after intravenous-injection. Fundam. Appl. Toxicol. (1993) 21:523–534.[CrossRef][Web of Science][Medline]
Wilker C, Johnson L, Safe S. Effects of developmental exposure to indole-3-carbinol or 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring. Toxicol. Appl. Pharmacol. (1996) 141:68–75.[Web of Science][Medline]
Williams DA. Comparison of several dose levels with a zero dose control. Biometrics (1972) 28:519–531.[CrossRef][Web of Science][Medline]
Yonemoto J, Ichiki T, Takei T, Tohyama C. Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and the body burden in offspring of Long-Evans rats. Environ. Health Prev. Med. (2005) 10:21–32.[CrossRef]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Simon, L. L. Aylward, C. R. Kirman, J. C. Rowlands, and R. A. Budinsky Estimates of Cancer Potency of 2,3,7,8-Tetrachlorodibenzo(p)dioxin Using Linear and Nonlinear Dose-Response Modeling and Toxicokinetics Toxicol. Sci., December 1, 2009; 112(2): 490 - 506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Jiang, D. R. Bell, S. Clode, M. Q. Fan, A. Fernandes, P. M. D. Foster, G. Loizou, A. MacNicoll, B. G. Miller, M. Rose, et al. A Truncation in the Aryl Hydrocarbon Receptor of the CRL:WI(Han) Rat Does Not Affect the Developmental Toxicity of TCDD Toxicol. Sci., February 1, 2009; 107(2): 512 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Hotchkiss, C. V. Rider, C. R. Blystone, V. S. Wilson, P. C. Hartig, G. T. Ankley, P. M. Foster, C. L. Gray, and L. E. Gray Fifteen Years after "Wingspread"--Environmental Endocrine Disrupters and Human and Wildlife Health: Where We are Today and Where We Need to Go Toxicol. Sci., October 1, 2008; 105(2): 235 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Bell, S. Clode, M. Q. Fan, A. Fernandes, P. M. D. Foster, T. Jiang, G. Loizou, A. MacNicoll, B. G. Miller, M. Rose, et al. Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Developing Male Wistar(Han) Rat. II: Chronic Dosing Causes Developmental Delay Toxicol. Sci., September 1, 2007; 99(1): 224 - 233. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||