* Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire, SK10 4TJ, United Kingdom;
General Electric Company, Pittsfield, Massachusetts 01201; and
National Institute of Environmental Health Sciences, Alexander Drive, Research Triangle Park, North Carolina 27709
Received October 30, 2002; accepted March 21, 2003
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ABSTRACT |
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Key Words: bisphenol A; sperm production; testis.
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INTRODUCTION |
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The fact that Sakaue et al. had observed ED effects for BPA at such a low dose, and in sexually mature animals, led us to mount an independent repeat of their study. As in all previous cases of us repeating reports of low dose effects, the objective was to establish a model of such effects in order to study their mechanism at the molecular level. Pursuant to the negative outcome of our first experiment we discussed with Sakaue et al. the precise details of their test protocol, and our subsequent experiments were designed to simulate more closely the experimental conditions that they had employed.
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MATERIALS AND METHODS |
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Analysis of dosing solutions and stability measurements.
Each dosing solution was analyzed using reverse-phase high-pressure liquid chromatography (HPLC). Stability measurements were performed by analyzing samples of the dosing solutions on the first day of dosing, storing these samples at room temperature, and then reanalyzing them 24 h after the end of dosing. Samples were diluted with solvent [methanol/tetrahydrofuran (1:2; v/v)] to give concentrations within the range of the calibration standards selected. Samples were analyzed using a 2487 Series UV detector (Waters) and a flow rate of 1 ml/min. The limit of detection was determined to be 0.2 µg/ml BPA (equivalent to a 1 µg/kg dosing solution).
Dietary analysis.
The phytoestrogen aglycone content of the diets was determined as described in detail by Wiseman et al.(2002). Portions of the diets (200 mg) were extracted by shaking with aqueous methanol at 60°C for 1 h. The extracts were defatted with hexane and hydrolyzed to the aglycones with dilute HCl. The aglycones were then extracted with ether. Daidzein, genistein, and coumestrol were detected and quantified against reference samples by liquid chromatographymass spectometry (LC-MS). Data were corrected for extraction efficiency. Quality control was determined by the concurrent analysis of a soya flour of known daidzein and genistein content, results were <9% different from those expected. The limits of detection for daidzein and genistein were 0.05 µg/g diet and for coumestrol were 0.1 µg/g diet.
Animals and housing.
Young adult (12.5 weeks old) SD (International Genetic Standard) male rats were obtained from Charles River U.K. (350450 g) and allowed 4 or 5 days for acclimatization prior to dosing. All animals were housed in stainless steel cages and were subjected to a 12-h light/dark schedule. Five independent experiments were performed, with Experiments 14 involving the exposure of animals to either vehicle or BPA (20 µg/kg; 2 mg/kg; 200 mg/kg). In the final study animals were exposed to vehicle only. Slight modifications to animal husbandry and/or diet occurred with each subsequent study. Thus, in the first experiment, animals were housed three to a cage, which was reduced to two per cage as the experiment progressed. The animals were given bedding and received Rat and Mouse No. 3 (RM3) breeding diet (pelleted; Special Diet Services Ltd., Witham, Essex, U.K.) and water (via an automatic feeding system) ad libitum. In the other four experiments, the animals were housed two/cage in stainless steel cages without any bedding and received water via glass water bottles. These experimental conditions were based on those used by Sakaue et al. (2001 and personal communication). In Experiments 2 and 3 the animals received 5002 diet (powdered; Purina Mills, Inc., Nottingham, U.K.); in Experiment 4 they were given the same diet as was used in the original Japanese study reported by Sakaue et al., 2001
(CE2 diet, pelleted; CLEA, Tokyo, Japan). In the final experiment, the animals received RM3, 5002, or CE2 diet. Group sizes of 10 were used in all experiments with the exception of the control groups in Experiments 3 and 4. In these two experiments the control group size was increased to n = 20. These larger control groups were randomly split into two identical subgroups of n = 10 prior to the start of dosing to determine any interanimal variability. The data generated from each subgroup were pooled prior to the analysis of the test data.
Dosing.
Animals (age ~13 weeks) were exposed to the appropriate compound by oral gavage using a dose volume of 5 ml/kg body weight. In Experiments 14 each animal received a single dose of either CO/EtOH or BPA at the appropriate concentration in CO/EtOH (200 mg/kg, 2 mg/kg, or 20 µg/kg) on each of 6 days. In the final experiment, animals were exposed to CO/EtOH only for 6 days.
Body weight measurements.
All animals were weighed on a daily basis during dosing and then weighed on a weekly basis until termination.
Termination.
Animals were terminated 5 weeks after the start of dosing (i.e., 18 weeks of age) by an overdose of Halothane followed by cervical dislocation. Each rat was weighed just prior to termination. Liver, kidney, testes, epididymides, seminal vesicles, and ventral prostate were removed and weighed. Both testes as well as the prostate were flash frozen in liquid nitrogen and stored at -80°C for further investigation.
Estimation of daily sperm production.
Homogenization resistant sperm counts were determined as described previously (Ashby et al., 1997) using the method of Blazak et al.(1993)
. Each frozen right testis (decapsulated) was homogenized in 50 ml 0.9% (w/v) NaCl containing merthiolate (0.01% w/v) and Triton X-100 (0.05%, v/v) using a Waring blender. The number of released sperm heads was determined in duplicate using an improved Neubauer haemocytometer. The number of sperm present in the area of the counting chamber used was equivalent, when multiplied by 104, to the number of sperm/ml homogenate. The variation between duplicate readings was less than 10%. Daily sperm production (DSP) values were obtained using the transit time factor of 6.1 (Blazak et al., 1993
).
Statistical analyses.
For each of the four experiments in which animals were exposed to BPA, organ weights and sperm data were evaluated by ANOVA procedures to determine the significance of differences among the four treatment (three BPA and one control) groups. For organ weights, body weight was also included as a covariable (ANCOVA). Pairwise comparisons of BPA groups vs. controls were made by Dunnetts test. In addition, in order to increase the power of the study for detecting any subtle BPA effects, an ANOVA was carried out using the data from all four experiments together in a single analysis to determine the significance of any BPA effects, experiment-to-experiment variability, and the interaction between these two factors. In the final study, (different diets) Fishers least significant difference test (LSD test) was used to make all possible pairwise comparisons among the different diet groups.
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RESULTS |
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Dietary Analysis
The phytoestrogen content of the batches of diet used in the present studies is shown in Table 1. CE2 diet had the highest phytoestrogen content when the daidzein, genistein, and coumestrol contents were combined and expressed as total genistein equivalents. The results of these analyses were compared to those obtained for diets in the OECD program to validate the rat uterotrophic bioassay (Phase Two; Owens et al., 2003
) where RM3, 5002, and CE2 were also used (Table 2
). The phytoestrogen contents of RM3 and CE2 were similar between the two studies whereas 5002 had a much lower phytoestrogen content in the present study. The diet analysis for the two studies was carried out by different laboratories but a previous comparison of the analysis methods using a standard soya flour gave comparable results (Dr. D. Clarke, personal communication). We conclude therefore that the difference for 5002 is due to batch variability in manufacture.
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Exposure to BPA
The group mean (± SD) data for the terminal body and organ weights generated in Experiments 14 are presented in Table 3. These data indicated that liver and kidney weights were significantly correlated (p < 0.01) with body weight, but that other organ weights were not. More importantly, there were no significant effects on either body or organ weights induced by BPA at any of the dose levels tested in any of the four studies. Analysis of the sperm data (Table 4
) gave a similar picture in that BPA did not significantly affect sperm counts or DSP at any of the doses tested in any of the studies conducted. Experiments 3 and 4 had 20 control animals divided at random into two groups of 10. These groups were pooled after preliminary analyses revealed no biologically important differences between them.
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Interexperiment Variability
Although it is clear that BPA did not affect any of the parameters measured in any of the four studies (Tables 3 and 4
), it was apparent that there was significant experiment-to-experiment variability as described above. For example, consideration of the control data highlights significant variation for particular parameters. Specifically, a comparison of the control sperm count and DSP data from Experiments 1 and 2 (carried out before completion of Experiments 3 and 4) showed that the two experiments were significantly (p < 0.01) different. As it was thought that such differences were unlikely to be due to random variability, attempts to address these differences were made in subsequent studies.
One obvious variable between the first two studies was diet (RM3 was used in Experiment 1 and 5002 was used in Experiment 2). Thus, in the third experiment animals were again fed 5002 diet and the control group was increased to n = 20, which was randomly split (prior to dosing) into two subgroups of n = 10. All other experimental conditions were identical to those of the second study. Thus, if diet was the causal agent for the significant variation, then it was expected that the control response in this third study would be similar to that observed in Experiment 2 and would be significantly different from that of Experiment 1. Comparison of the two control subgroups of 10 animals showed that they were not significantly different, so they were pooled prior to evaluating the data from all three studies. Comparison of the sperm count and DSP data measured for the three control groups showed that the data from Experiment 3 were, in fact, intermediate between the controls of Experiments 1 and 2 (Table 4), a response that, despite increased sample size, did not differ significantly from either of the other control groups.
A fourth experiment was conducted, which was identical to the third experiment with the exception that the animals received CE2 diet (as used by Sakaue et al., 2001) rather than 5002 or RM3 diet. In this study the two control subgroups did differ significantly (p < 0.05) with regard to sperm/testis and DSP/testis (Table 4
). However, this "stray" significance is consistent with chance expectations given the number of variables assessed and could, therefore, be attributed to no more than interanimal variation and/or random variability, as there were no differences between the two subgroups in terms of animal husbandry and compound exposure. Thus, for the purpose of comparing the control and test data, the two subsets were pooled (as was done in Experiment 3).
When all four control groups were considered, the experiment-to-experiment variability for body weight, all sperm and DSP parameters, and epididymis weight were significant (p < 0.05). However, since Experiments 14 were carried out over a nine month period, this interexperiment variation may have been due to some other confounding factors (e.g., different animal shipments, subtle differences in animal husbandry) of which diet could have been one. Thus, one final experiment was conducted to evaluate any possible diet effect (Table 6), in which the only difference between the groups was the diet (RM3, 5002, CE2) the animals received. Comparison of these data showed that there were no significant differences in all parameters measured, with the exception of the right epididymis weight in the CE2 diet group, which was significantly (p < 0.05) reduced (by ~9%) relative to the other two diet groups. However, such an observation is more than likely to be spurious for three reasons. First, given the number of variables evaluated, one spurious significant finding is consistent with chance expectation. Second neither the left epididymis nor total epididymides weight was affected. Finally, this finding is not supported by the results in Experiments 14 in which (for example) the RM3 diet was associated with right epididymis weights that were lower, not higher, than that seen for the CE2 diet (see Table 3
).
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DISCUSSION |
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The results of the present studies, together with the original data reported by Sakaue et al.(2001) are shown in Figure 1
. Three points of interest emerge. First, the DSP values for all of our test and control groups were in the same range as the significantly reduced DSP values reported by Sakaue et al.(2001)
. Second, although the decline in DSP induced by BPA (Sakaue et al., 2001
, Fig. 1
) was dose-related over the range 20 ng/kg20 µg/kg, there was a plateau for the depressed DSP values over the dose range 20 µg/kg200 mg/kg BPA (five orders of magnitude). Third, there were higher SDs for the DSP values in the control and the low (unaffected) BPA dose groups in the studies reported by Sakaue et al. than for those groups where DSP was reduced by BPA. The lower SD values in the last groups were also similar in magnitude to all of the DSP SD values seen in the present study (Fig. 1
). These observations led us to compare our DSP values, and those of Sakaue et al.(2001)
, with rat DSP values reported by our two laboratories, and by other investigators, over the past seven years (Fig. 2
and Table 7
). Included in Table 7
are the results from a preliminary evaluation of BPA using Holtzman rats (Sakaue et al., 1999
). The report of that study was only available as an abstract in Japanese; however, the data were made available to us for use in Table 7
and Figure 2
by the authors.
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Other instances of unexplained variability in control animal ED parameters possibly affecting the outcome of ED studies have been described (e.g., Ashby, 2001; Ashby et al., 1999
; Odum and Ashby, 2000
; Sharpe et al., 1998
; Tinwell et al., 1999, 2002
; Welsch et al., 2001
). Probably the most relevant of these citations concerns the reported trophic effect of BPA on the mouse prostate gland. In that case the weight of the control prostate glands in the original study (Nagel et al., 1997
) were lower than in our repeat study that failed to confirm this property of BPA (Ashby et al., 1999
). However, in an earlier study from the laboratory of Nagel et al., control prostate weights higher than those reported by Ashby et al. had been described (Ashby, 2001
; Nonneman et al., 1992
,). Resolution of such inconsistencies among control groups should perhaps act as a prelude to resolution of different test outcomes from studies of low-dose chemical-induced ED effects.
We currently have no explanation for the different test outcomes between the present studies and those of Sakaue et al. (2001). The only possibility that we have not explored (for practical reasons) is that the SD rats used in our two laboratories have subtly different genetic make-ups leading to different sensitivities to BPA. Charles River IGS SD rats were used in the present study. These IGS SD rats are available in Japan, but the studies by Sakaue et al. employed Jcl:SD rats obtained from Japan CLEA. These latter were derived over 30 years ago by Japan CLEA from the original Charles River Cr:CD(SD) colony. If such subtle intrastrain differences were shown to account for the different assay outcomes on BPA it would solve one problem, but create a more serious one for the conduct and international comparability of chemical toxicity evaluations.
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ACKNOWLEDGMENTS |
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NOTES |
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The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.
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