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Triclosan exposure reduces thyroxine levels in pregnant and lactating rat dams and in directly exposed offspring
Food and Chemical Toxicology 59 (2013) 534–540
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Triclosan exposure reduces thyroxine levels in pregnant and lactating rat dams and in directly exposed offspring Marta Axelstad ⇑, Julie Boberg, Anne Marie Vinggaard, Sofie Christiansen, Ulla Hass National Food Institute, Technical University of Denmark, Division of Toxicology and Risk Assessment, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark
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Article history: Received 18 April 2013 Accepted 25 June 2013 Available online 4 July 2013 Keywords: Triclosan Rat Thyroxine (T4) Developmental Thyroid disrupting chemical (TDC)
a b s t r a c t Thyroid disrupting chemicals can potentially disrupt brain development. Two studies investigating the effect of the antibacterial compound triclosan on thyroxine (T4) levels in rats are reported. In the first, Wistar rat dams were gavaged with 75, 150 or 300 mg triclosan/kg bw/day throughout gestation and lactation. Total T4 serum levels were measured in dams and offspring, and all doses of triclosan significantly lowered T4 in dams, but no significant effects on T4 levels were seen in the offspring at the end of the lactation period. Since this lack of effect could be due to minimal exposure through maternal milk, a second study using direct per oral pup exposure from postnatal day 3–16 to 50 or 150 mg triclosan/kg bw/day was performed. This exposure pointed to significant T4 reductions in 16 day old offspring in both dose groups. These results corroborate previous studies showing that in rats lactational transfer of triclosan seems limited. Since an optimal study design for testing potential developmental neurotoxicants in rats, should include exposure during both the pre- and postnatal periods of brain development, we suggest that in the case of triclosan, direct dosing of pups may be the best way to obtain that goal. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Triclosan is a compound that has been used in consumer products for many years due to its high efficiency as an antibacterial agent and its low acute toxicity (SCCS, 2009). In the EU, about 85% of the total volume of triclosan is used in personal care products (toothpaste, soap, shampoo and cosmetics), 5% is used for textiles (antibacterial clothing) and 10% for plastics and food contact materials (SCCS, 2009). In recent years it has become evident that triclosan acts as a thyroid hormone disrupting chemical. This effect has been seen in metamorphosis studies in bullfrogs and Xenopus laevis (Helbing et al., 2011; Veldhoen et al., 2006), and the compound has also been shown to reduce thyroxine (T4) levels in a series of rat studies. In these studies, young adult rats (22–60 days of age) have been exposed to triclosan for varying periods of time (Crofton et al., 2007; Paul et al., 2010a; Stoker et al., 2010; Zorrilla et al., 2009), or rat dams have been exposed during gestation and lactation (Paul et al., 2012, 2010b; Rodriguez and Sanchez, 2010). The doses needed to observe T4 reductions in these studies have been varying between 10 and 300 mg/kg bw/day, probably reflecting variations in rat strain and study design. In humans, even mild reductions in T4 levels in pregnant women can have severe consequences for the neurological development of children, as associations with delayed or impaired ⇑ Corresponding author. Tel.: +45 35 88 75 41. E-mail address: [email protected] (M. Axelstad). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.06.050
cognitive and motor function have been reported in numerous epidemiological studies (Ghassabian et al., 2011; Haddow et al., 1999; Henrichs et al., 2010; Kooistra et al., 2006; Li et al., 2010; Pop et al., 1999, 2003). In addition, an ample number of animal studies have shown that prenatal hypothyroxinemia may significantly affect nerve cell migration and other molecular aspects of brain development in rats (Auso et al., 2004; Berbel et al., 2010; Cuevas et al., 2005; Gilbert and Sui, 2008, 2006; Lavado-Autric et al., 2003; Opazo et al., 2008; Sharlin et al., 2008). Our previous research has indicated that in rats, maternal hypothyroxinemia is only significantly correlated to altered behaviour and hearing in the offspring, if marked postnatal T4 decreases in the offspring are also present. The fact that postnatal thyroid hormone insufficiency has been present in almost all studies showing altered behaviour after developmental hypothyroidism in rats (Akaike et al., 1991; Brosvic et al., 2002; Noda et al., 2005; Provost et al., 1999) further corroborate the hypothesis that in rat dams, T4 reductions during gestation alone are not enough to induce adverse behavioural effects in the offspring (Axelstad et al., 2011a,b). Such an important species difference between humans and rats may beexplained by the fact that while both rodents and humans need thyroid hormones for differentiation and maturation of the central nervous system, important differences in timing of these events exit. Since a larger part of the brain maturation takes place prenatally in humans compared to rats (Howdeshell, 2002), prenatal T4 deficits might have more severe consequences for foetal brain development in humans than in rats.
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The aim of the present work was to develop a study design in which postnatal T4 reductions would be induced in rat offspring. For this purpose two studies were performed. In the first, pregnant rat dams were exposed to triclosan during gestation and lactation, and T4 levels in dams and offspring were examined. Due to a suspected influence of triclosan on the sex hormone balance (Kumar et al., 2009; Stoker et al., 2010), also prostate glands were examined in the offspring. In spite of marked reductions in maternal T4 levels during gestation and lactation, offspring T4 levels were not significantly reduced when measured at the end of the lactation period. As this result indicated limited triclosan excretion to the maternal milk, and consequently limited exposure in the neonatal period, a second study was performed. Here, neonatal rat offspring were exposed to triclosan through direct peroral dosing during lactation, with subsequent measurements of serum T4 levels.
2. Materials and methods 2.1. Chemicals The vehicle used was corn oil (Sigma–Aldrich, Denmark). Triclosan (purity >99.0%, CAS No. 3380-34-5, Alfa Aesar No. L18655) was from VWR-Bie & Berntsen, Herlev, Denmark. The triclosan solutions were kept dark, at room temperature, and continuously stirred during the dosing period. New solutions were prepared for each of the two studies, but no verification of dose concentrations was performed.
2.2. Animals and treatment Both studies were performed under conditions approved by the Danish Animal Experiments Inspectorate and by the in-house Animal Welfare Committee. The animals received a complete rodent diet (Altromin Standard Diet 1314) and acidified tap water ad libitum, and were housed under standard conditions as described in Christiansen et al. (2012). In the first study (study 1), 40 time-mated Wistar rats (HanTac:WH, Taconic Europe, Ejby, Denmark) were supplied at gestation day (GD) 3 of pregnancy. The study was performed using 2 blocks with one week in between, and all dose groups were equally represented in the blocks. The dams were distributed into four dose groups (0; 75; 150 or 300 mg/kg/day; n = 10 per group), and gavaged once daily from gestation day (GD) 7 to postnatal day (PND) 16 (day of delivery excluded), at a constant volume of 2 ml/kg bw/day. The individual doses were based on the body weight of the animal on the day of dosing. The dams were inspected twice a day for general toxicity including changes in clinical appearance. Body weights were recorded on GD 4 and daily during the dosing period. On GD 15 dams were anesthetized with HypnormÒ (fentanyl citrate/flunisone)/DormicumÒ (midazolam), and blood was drawn from the tail vein. The day after delivery, the pups were counted, sexed, weighed, checked for anomalies and anogenital distance (AGD). Of the 10 time-mated dams in each group, 7–9 were pregnant and gave birth to viable litters. Body weights were measured on PND 6 and 13, and offspring were examined for the presence of nipples/areolas on PND 13. On PND 16 all dams and pups were sacrificed. Dams were weighed, anaesthetized in CO2/O2, decapitated, and trunk blood was collected. The number of implantation scars in the uterus was registered and thyroid glands were excised and weighed. All offspring were weighed, decapitated and trunk blood collected. Blood samples from the offspring were pooled within litter in a male and a female sample. Prostates from 1 male per litter were excised, weighed and prepared for histopathological examinations. Thyroids from 1 to 2 males per litter were excised and weighed. Thyroids from 1 male per litter were excised together with the thyroid cartilage and prepared for histological examination. Thyroids and prostates were fixed in formalin, embedded in paraffin, and stained with haematoxylin and eosin, and histological evaluations of one section per organ from animals from the control group and the highest dose group were performed by a pathologist blinded to treatment groups. In the direct postnatal exposure study (study 2), 6 time-mated pregnant Wistar rats were supplied at GD 16, one week before expected delivery. Each dam was individually housed, and each litter remained housed with the respective dam until sacrifice. Two days after delivery (PND 3) the litters were culled to 8 offspring, with an equal representation of males and females when possible, and litters were assigned into one of three dosage groups (0, 50 or 150 mg/kg/day). The maximum dose was set at 150 mg/kg/day to avoid general toxic effects in the young pups. All offspring were dosed orally from PND 3 to PND 16 using a micropipette. The dams were not dosed, but remained in the cage to allow the pups to feed normally. Each pup was held by one hand while the other slowly allowed test solution to drip directly into the mouth, but only as fast as the pup would allow, ensuring precise and reliable dosing.
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On each day of dosing, the entire litter (8 pups) was weighed, and the average pup weight was calculated. All pups in the litter were dosed according to the average weight receiving 2 ll of test solution per gram body weight. Thus they received from approximately 12 ll to 90 ll test solution per day in the dosing period. On PND 16 the offspring were sacrificed by decapitation and trunk blood was collected for total T4 analyses. The trunk blood was collected and analyzed individually for each pup. 2.3. Thyroxine immunoassay Blood samples from dams (GD 15 and PND 16) and offspring (PND 16) in the first study, and from offspring (PND 16) in the second study, were analyzed for total thyroxine (T4) concentration in plasma. Trunk blood was used for the analysis. A Delfia time-resolved fluoroimmunoassay kit from Perkin Elmer (cat. No. 1244030, Wallac Oy, Turku, Finland) was modified and developed specifically for analysis of T4 in rat samples. Instead of human T4 standards and T4 antibody supplied in the Delfia kits, T4 standards in T4-free rat serum (cat. Nos. 30042 and 30041, respectively) as well as rat specific biotinylated T4 (30,039) antibody from Biovian Ltd. (Finland) were used. Otherwise the assay was run as outlined in the manufacturer’s protocol and described in Axelstad et al. (2008). The analytical sensitivity is around 10 nM. Historic control values for T4 levels in PND 16 males and females were recorded being 40 ± 13 nM and 38 ± 8 nM (n = 5), respectively. Thus, the CV% between in vivo studies (n = 5) were 32% and 22% for males and females, respectively. 2.4. Statistical analysis Statistical analysis of data with normal distribution and homogeneity of variance were analysed using analysis of variance (ANOVA), followed by Dunnett’s post hoc test. In study 1, when more than one pup from each litter was examined, statistical analyses were adjusted using litter as an independent, random and nested factor in ANOVA, or analysis were done using litter means. Furthermore, since study 1 was performed in two blocks, block was included as an independent random and nested factor in the analysis however no significant effects of block were seen. In cases where normal distribution and homogeneity of variance could not be obtained by data transformation, a non-parametric Kruskal–Wallis test was used. Trend analysis on dose–response relations for hormone levels, body- and organ weights were performed using Spearman’s test. In study 2, litter was not used as the statistical unit, as triclosan exposure was direct, and not through the dam.
3. Results In study 1, maternal body weight gain was not significantly affected by triclosan exposure when weight gain was calculated from the beginning of the dosing period to the day before birth (GD7 to 21). However, when maternal body weight gain was measured from GD7 to the day after birth (PND 1), a significant dosedependent downward trend was seen (p = 0.001) and a statistically significant decrease was seen in the highest dose group compared to control (p = 0.007), indicating that 300 mg triclosan/kg/day caused a moderate degree of maternal toxicity during gestation (Table 1). Gestation length, gender distribution, postimplantation loss and litter size were unaffected by the exposure and so were maternal body weight gains, neonatal deaths and offspring body weights in the postnatal period. Furthermore, no effect was seen on male or female anogenital distance or on nipple retention (Table 1). Triclosan exposure had a marked effect on total T4 levels in dam serum. On GD 15, a significant dose-dependent downwards trend was seen (p < 0.001) and the T4 levels were decreased by 59%, 72%, and 72% in the three triclosan groups respectively (p = 0.028, p = 0.0001, p = 0.0005) (Fig. 1A and Table 2). The effect of triclosan on maternal T4 levels was also present on PND 16, where the dose-dependent downwards trend was also significant (p < 0.001) and the T4 levels were decreased by 38%, 55% and 58% in the three triclosan groups respectively (p = 0.032, p = 0.001, p = 0.0006) (Fig. 1B and Table 2). Offspring total T4 levels on PND 16 (study 1) showed no statistically significant dose-dependent trends and group means did not differ significantly from controls in any dose group (Table 2). Both absolute and relative thyroid gland weights were unaffected by triclosan exposure in dams and offspring, as no dosedependent trends and no significant differences between groups
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Table 1 Reproductive and developmental data from study 1 and 2. The table shows pregnancy and litter data, including body weights (bw) from dams and offspring exposed indirectly to 0, 75, 150 or 300 mg triclosan/kg bw/day from GD 7 to PND 16 (study 1), and offspring body weights in pups exposed directly to triclosan at doses of 0, 50 or 150 mg/kg bw/day from PND 3–16 (study 2). Data represent group means based on litter means ± SD.
**
Study 1 (indirect dosing of pups)
Control
75 mg/kg/day
150 mg/kg/day
300 mg/kg/day
Dams and litters Dam bw gain, GD 7-GD 21 Dam bw gain, GD 7-PND 1 Dam bw gain, PND 1–16 Gestation length (days) % Postimplantation loss % Perinatal loss Litter size % Perinatal deaths % Males Offspring Mean birth weight AGD males (mm) AGD females (mm) Nipples males Nipples females Mean pup bw PND 6 Mean pup bw PND13 Mean pup bw PND 16
n=9 80.8 ± 8.3 12.9 ± 5.5 39.3 ± 10.0 22.89 ± 0.33 8.19 ± 6.7 12.5 ± 7.2 10.67 ± 2.7 4.67 ± 4.5 52.6 ± 21.2
n=7 78.6 ± 14 15.1 ± 7.0 36.4 ± 5.2 23.14 ± 0.38 28.4 ± 41.7 35.5 ± 40.6 9.00 ± 3.8 6.59 ± 17.4 45.6 ± 8.9
n=8 77.6 ± 16 8.7 ± 7.1 33.8 ± 10.8 23.00 ± 0.0 19.5 ± 32.2 19.5 ± 32.2 11.00 ± 3.9 0.00 ± 0.0 48.8 ± 12.3
n=8 72 .8 ± 16 2.9 ± 5.2** 38.3 ± 12.8 23.00 ± 0.0 22.4 ± 41.2 22.4 ± 41.2 9.67 ± 4.8 0.00 ± 0.0 59.5 ± 22.7
6.23 ± 0.5 4.03 ± 0.1 2.21 ± 0.2 0.00 ± 0.0 12.4 ± 0.3 13.11 ± 1.5 26.31 ± 4.0 31.72 ± 5.3
6.12 ± 0.2 3.98 ± 0.1 2.14 ± 0.1 0.00 ± 0.0 12.3 ± 0.2 12.62 ± 0.6 26.06 ± 1.5 31.84 ± 2.4
6.08 ± 0.4 4.00 ± 0.1 2.19 ± 0.1 0.12 ± 0.2 12.2 ± 0.2 11.83 ± 1.5 23.93 ± 3.9 28.15 ± 4.8
6.16 ± 0.4 3.97 ± 0.1 2.19 ± 0.1 0.06 ± 0.1 12.4 ± 0.2 12.19 ± 1.6 24.42 ± 3.7 29.65 ± 4.9
Study 2 (direct dosing of pups)
Control
50 mg/kg/day
150 mg/kg/day
No. of litters Litter size (culled to 8 at PND 3) Mean pup bw PND 6 Mean pup bw PND13 Mean pup bw PND 16
n = 2 (1 after day 7) 8 12.1 ± 0.5 26.29 ± 0.0 32.58 ± 0.0
n=2 8 13.3 ± 0.8 29.95 ± 2.4 37.16 ± 2.9
n=2 8 and 6 13.8 ± 0.5 32.44 ± 2.2 39.91 ± 3.4
p < 0.01.
Fig. 1. Total thyroxine (T4) levels (nM) in dams on gestation day (GD) 15 (A) and postnatal day (PND) 16 (B), after exposure to 0, 75, 150 or 300 mg triclosan/kg bw/ day from GD 7 – PND 16 (study 1). Data represent group means based on litter means + SEM, n = 7–9. *p < 0.05; ***p < 0.001.
were seen (Table 3). Also no histopathological effects on the offspring thyroids on PND 16 were seen in the high dose group (data not shown). Statistical analysis of the both relative and absolute prostate weights (analysed with body weight as covariate) indicated no differences between treatment groups, and no dose-dependent trends were seen (Table 3). Histopathology of the prostates showed no differences between controls and the high dose group at PND 16 (data not shown). In the direct exposure study (study 2) one of the two litters in the control group had to be sacrificed on PND 7, because the dam did not take care of her offspring. Furthermore, two pups from one of the litters in the high dose group died on day PND 6. This is sometimes seen in rat litters, and was not attributed to triclosan exposure. The triclosan dosing did not cause any general toxicity effects in the exposed offspring, and no significant effects on pup body weights or body weight gains were seen during the exposure period (PND 3–16), compared to controls (Table 1). On days 13 and 16, the pups in the direct exposure study (study 2) weighed more than offspring of the same age in the indirect exposure study, and the differences were statistically significant (p = 0.0051 and p = 0.0037 on PND 13 and 16 respectively). This difference was probably due to a combination of the pups receiving oil gavage and the fact that there were fewer offspring in this study in each litter, because of the culling performed on day 3. T4 levels in the pups that had been directly exposed to triclosan at doses of 50 and 150 mg/kg/day were significantly decreased by 16% (p = 0.029) and 39% (p < 0.001) in the two dose groups respectively, when measured on PND 16 (Table 2), and a significant dose dependent trend was also seen (p < 0.001). However, an important limitation in study 2 was that all the control pups were from the same litter. Therefore there was a possible risk that due to genetic similarities, the offspring may have had high T4 levels, which could have caused the significant T4 reductions in both dose groups. To further complicate interpretation of the data, the T4 values from the pups in this litter were in the high end of our previous control values (Axelstad et al., 2008, 2011a,b).
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Table 2 T4 levels in dams and offspring from study 1 and 2. The table shows total T4 levels (nM) in dams and offspring, after exposure to 0, 75, 150 or 300 mg triclosan/kg bw/day from GD7 to PND 16 in study 1, and in pups exposed directly to triclosan at doses of 0, 50 or 150 mg/kg/day from PND 3 to 16 in study 2. Data represent group means ± SD.
*
Study 1 (indirect dosing of pups)
Control
75 mg/kg/day
150 mg/kg/day
300 mg/kg/day
No. of litters T4 in dams GD 15 T4 in dams PND 16 T4 in offs. PND 16 (males) T4 in offs. PND 16 (females)
9 38.7 ± 20.4 17.5 ± 6.9 34.9 ± 9.2 40.9 ± 6.3
7 15.8 ± 5.4* 10.9 ± 4.7* 26.0 ± 4.0 35.4 ± 5.2
8 10.7 ± 7.7*** 7.9 ± 3.7*** 34.9 ± 9.9 38.5 ± 13.8
8 10.8 ± 4.5*** 7.4 ± 2.7*** 27.4 ± 8.1 33.6 ± 5.2
Study 2 (direct dosing of pups)
Control
50 mg/kg/day
150 mg/kg/day
No. of samples T4 in offs. PND 16 (males) T4 in offs. PND 16 (females)
8 (1 l) 45.5 ± 6.8 46.6 ± 5.4
16 (2 l) 38.0 ± 5.0* 39.2 ± 4.9*
14 (2 l) 27.7 ± 2.7*** 28.3 ± 3.7***
p < 0.05. p < 0.001.
***
Table 3 Dam and offspring organ weights from study 1. The table shows absolute and relative thyroid and prostate weights in dams and male pups exposed indirectly to 0, 75, 150 or 300 mg triclosan/kg bw/day from GD7 to PND 16 (study 1). Data represent group means ± SD. No significant differences between dose groups were observed. Study 1
Control
75 mg/kg/day
150 mg/kg/day
300 mg/kg/day
Dams Dam BW, PND 16 Thyroid weight dams PND 16 Rel. thyroid weight/100 g bw
282 ± 25 0.017 ± 0.003 0.591 ± 0.09
282 ± 11 0.021 ± 0.011 0.746 ± 0.37
266 ± 13 0.020 ± 0.007 0.741 ± 0.29
272 ± 12 0.016 ± 0.003 0.576 ± 0.12
Offspring Mean male pup bw PND 16 Thyroid weight PND 16 Rel. thyroid weight/100 g bw Prostate weight PND 16 Rel. prostate weight/100 g bw
31.92 ± 5.1 0.47 ± 0.05 1.50 ± 0.16 1.19 ± 0.2 0.038 ± 0.005
31.32 ± 3.3 0.48 ± 0.06 1.52 ± 0.11 1.31 ± 0.4 0.041 ± 0.01
30.70 ± 5.0 0.40 ± 0.06 1.30 ± 0.15 1.19 ± 0.3 0.039 ± 0.007
30.03 ± 4.7 0.45 ± 0.06 1.54 ± 0.26 1.01 ± 0.3 0.034 ± 0.009
4. Discussion The present studies confirm that triclosan can disrupt thyroid hormone levels, as total T4 levels were markedly decreased in rat dams on both GD15 and PND 16 after exposure to 75; 150 and 300 mg triclosan/kg bw/day during gestation and lactation. After indirect exposure through placenta and maternal milk (study 1), the offspring T4 levels on PND 16 were not significantly decreased, whereas direct pup exposure to 50 and 150 mg/kg bw/day from PND 3–16 (study 2) appeared to decrease offspring T4 levels. Since previous studies have indicated that triclosan does not pass to the milk in sufficient amounts to significantly reduce T4 levels during the late lactation period (Paul et al., 2012), and the results from study 1 corroborate this finding, the present results taken together indicate that triclosan can probably only significantly reduce T4 levels in the offspring during the entire lactation period, if the chemical is directly dosed. T4 levels have previously been measured in a number of triclosan studies in rats, and the present results corroborate and add to these findings. In female Long-Evans (LE) rats, dosed daily with triclosan either from PND 28–31 or during gestation and lactation, doses of 100 mg/kg bw/day and above significantly lowered serum levels of T4 in the dams (Crofton et al., 2007; Paul et al., 2010a, 2012). A number of studies performed in Wistar rats have furthermore shown that even lower doses of triclosan can induce T4 reductions in this strain. Here doses of 30 mg/kg bw/day and above caused T4 levels to decrease markedly in young Wistar males dosed daily from PND 23–58 (Zorrilla et al., 2009), whereas doses of 37.5 mg/kg bw/day and above resulted in decreased total serum T4 levels in young Wistar females dosed from PND 22–43 (Stoker et al., 2010). In developmental studies performed by Paul et al. (2010b, 2012), 300 mg/kg caused serum T4 levels in offspring to decrease
on GD 20 and PND 4, while no significant reductions were seen on PND 14 or PND 21. The authors suggested that the T4 reductions observed on PND 4 pups could have resulted from transplacental exposure to triclosan and that toxicokinetic factors probably affected maternal transfer of triclosan into milk and thereby limited lactation exposure to the pups. The present T4 results (study 1) fit well with results from Paul et al. (2010b, 2012), showing no significant effects on offspring T4 levels after approximately two weeks of lactational exposure. To further test whether the lack of T4 reduction in the 16-day old offspring was due to limited triclosan excretion in the milk, the direct exposure study (study 2) was performed. Due to the quite time consuming dosing method, this study was only performed on a limited number of litters. However, the blood samples from each of the eight dosed pups in each litter were not pooled, yielding between 8 and 16 samples in each dose group. The conclusion in study 2 was that since T4 levels in the directly exposed pups seemed decreased compared to controls the lack of significant effect seen in any dose group in the offspring from study 1, was probably due to limited exposure through maternal milk. In future developmental studies of triclosan, including direct measurements of triclosan levels in the dam’s milk at different post-natal times, as well as performing a larger study with direct postnatal dosing of the offspring, would more definitively determine if the different sensitivities observed between neonatal and 14–16 day old pups, are caused by changes in exposure to triclosan or by changes in triclosan catabolism. It seemed quite clear that the T4 reductions seen in dams dosed with the 75 and 150 mg/kg from GD7-15 (59 and 72% reduction in T4, respectively) were more marked than seen in pups dosed directly from PND 3–16 with 50 and 150 mg/kg (16% and 39% reduction in T4 respectively), indicating that triclosan also may not have triggered the same degree of toxicodynamic effects in the offspring as seen in more mature animals. This could be explained by the
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mode of action for triclosan, as the compound is suspected of affecting the thyroid hormone system by causing induction of phase II liver enzymes (sulfonation or glucuronidation), and thereby upregulating thyroid hormone catabolism (Crofton et al., 2007; Paul et al., 2010a, 2012). This mode of action is indicated by the observation of increased liver weights (Crofton et al., 2007; Paul et al., 2010a; Zorrilla et al., 2009), increased PROD activity in the liver (a marker of Cyp2b activity) (Paul et al., 2010b, 2012; Zorrilla et al., 2009) and upregulated mRNA expression and activity of some phase I and phase II hepatic enzymes (Paul et al., 2010a, 2012). Since hepatic excretory function in rats develops postnatally to reach maximum capacity at an age of approximately 30 days (Klinger, 2005), and due to this reduced activity of many phase II enzymes, neonatal rats have a decreased capacity to metabolize and excrete (Suchy et al., 2007). A possible explanation for the smaller T4 reductions seen in the pups compared to adults might be that triclosan exposure did not lead to the same degree of induction of liver enzymes as seen in adult animals, and consequently only induced smaller T4 reductions. However, this is only one explanation for the observed results and more studies are needed to confirm the hypothesis that thyroid disrupting compounds acting by increasing liver metabolism may not affect thyroid hormone levels in neonatal rats as much as seen in older animals. In the present study no thyroid weight increases were observed in dams, despite a considerable drop in T4 levels. Normally thyroid weight increases are considered mediated through an increase of TSH, in response to decreased thyroid hormone levels. TSH levels were however not measured, because previous triclosan studies have shown that TSH levels were not affected at dose levels where significant T4 decreases were seen (Zorrilla et al., 2009; Paul et al., 2010a). Histological examination of thyroids was only performed in pre- and postnatally exposed male offspring showing no changes, whereas no examination was performed in dams or in pups exposed directly to triclosan. In the present developmental toxicity study (study 1) neither anogenital distance, nipple retention, prostate weight nor prostate histology were affected by exposure to triclosan. These endpoints are typically affected by perinatal exposure to anti-androgenic chemicals (Christiansen et al., 2010; McIntyre et al., 2001), indicating that triclosan exposure at the tested dose levels did not affect male reproductive development in an anti-androgenic manner. Adverse reproductive effects have previously been reported in males rats exposed during adulthood (Kumar et al., 2009). These included decreased weights of several reproductive organs, histopathological changes in these, and decreased levels of FSH, LH and testosterone level after two months of exposure to 20 mg/kg bw/day. It is however possible that the effects seen by Kumar et al., could be due to impurities in the triclosan used for the studies, as dioxin and furan contamination has been seen in several different triclosan samples produced in India and China (Menoutis and Parisi, 2002). In a study using post-weaning male rats exposed to up to 300 mg/kg/day from PND 23–58, no significant effects on timing of sexual maturation or reproductive organ weight were seen (Zorrilla et al., 2009). In studies focusing on reproductive effects in female offspring, a triclosan doses of 150 mg/kg/day lowered the age of vaginal opening and caused increased uterine weights (Stoker et al., 2010), while much lower doses of triclosan potentiated the effects of estradiol treatment on uterine weight (Louis et al., 2013; Stoker et al., 2010). These effects were compatible with an estrogenic mode of action, which was not tested in the present study. A number of in vitro studies examining the endocrine disrupting modes of action of triclosan have also been performed during recent years. Antagonistic activity in both ER- and AR-responsive bioassays have been shown in several studies (Ahn et al., 2008; Chen et al., 2007; Gee et al., 2008; Christen et al., 2010; Vinggaard
et al., 2008), and taken together the in vivo and in vitro data indicate that triclosan could affect the reproductive hormone axis. The lack of adverse effects in the few reproductive endpoints tested in the present study (AGD, nipple retention, prostate weight and histology) could reflect that these endpoints are more sensitive to anti-androgenic chemicals, and it is possible that triclosan is too weak an androgen receptor antagonist to cause anyantiandrogenic effects in vivo. Based on what is presently known on the effects of triclosan, an adverse outcome pathway for the effects of triclosan on the thyroid hormone system has been proposed (US EPA, 2011). Here activation of the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) in rat liver by triclosan is an initiating event, leading to up regulation of hepatic phase I and phase II enzymes, which could increase catabolism of thyroid hormones and consequently lower serum levels of these – effects that could potentially lead to altered neurodevelopment. Furthermore, triclosan has recently been shown also to affect the thyroid system by inhibiting deoidinase activity (Butt et al., 2011). Uncertainty exists, as to whether this adverse outcome pathway is also relevant for humans, because species differences with regard to activation and amino acid sequence of the PXR exist (Jones et al., 2000), which make it difficult to extrapolate results obtained with rodent PXR directly to humans (Dybdahl et al., 2012). However in vitro data show that triclosan is a moderate inducer of human PXR activity in human hepatoma cells (Jacobs et al., 2005), indicating that this could also be an initiating event in an adverse outcome pathway in humans.
5. Conclusions In conclusion, triclosan markedly lowered maternal T4 levels in rat dams during gestation and lactation, and nine days of exposure resulted in a LOAEL of 75 mg/kg bw/day, corroborating effects seen in previous rat studies. In humans, correct maternal T4 levels during pregnancy are crucial for fetal brain development, and even slight maternal hypothyroxinemia can result in adverse effects on the cognitive and motor function of children (Ghassabian et al., 2011; Henrichs et al., 2010; Kooistra et al., 2006; Li et al., 2010; Pop et al., 1999, 2003). Based on its thyroid disrupting properties there might be a need for further assessment of triclosan as a potential developmental neurotoxicant. Since the first ten postnatal days in the rat approximate the last trimester of human pregnancy with regard to the development of the central nervous system (Howdeshell, 2002), the results presented here imply that in a developmental toxicity study of triclosan, direct postnatal exposure would be the optimal study design to use, for successfully covering the entire period of human brain development during pregnancy. It is furthermore important to bear in mind that humans are exposed to a variety of thyroid disrupters, all probably acting in a dose-additive manner (Flippin et al., 2009). Since triclosan may be a potential contributor to thyroid disruption in humans, exposure should be carefully regulated in order to protect pregnant women and their children from excessive exposure to thyroid disrupting chemicals.
Funding This work was supported financially by the Danish Environmental Protection Agency.
Conflict of Interest The authors declare that there are no conflicts of interest.
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Acknowledgements Dorte Hansen, Lillian Sztuk, Bo Herbst, Sarah Simonsen, Kenneth Worm, Heidi Letting, Birgitte Møller Plesning, Vibeke Kjær and Ulla El-Baroudy are thanked for their excellent technical assistance.
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Lavado-Autric, R., Ausó, E., García-Velasco, J.V., Arufe Mdel, C., Escobar del Rey, F., Berbel, P., Morreale de Escobar, G., 2003. Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny. J. Clin. Invest. 111, 1073–1082. Li, Y., Shan, Z., Teng, W., Yu, X., Li, Y., Fan, C., Teng, X., Guo, R., Wang, H., Li, J., Chen, Y., Wang, W., Chawinga, M., Zhang, L., Yang, L., Zhao, Y., Hua, T., 2010. Abnormalities of maternal thyroid function during pregnancy affect neuropsychological development of their children at 25–30 months. Clin. Endocrinol. (Oxf.) 72, 825–829. Louis, G.W., Hallinger, D.R., Stoker, T.E., 2013. The effect of triclosan on the uterotrophic response to extended doses of ethinyl estradiol in the weanling rat. Reprod. Toxicol. 36, 71–77. McIntyre, B.S., Barlow, N.J., Foster, P.M., 2001. 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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Triclosan exposure reduces thyroxine levels in pregnant and lactating rat dams and in directly exposed offspring Marta Axelstad ⇑, Julie Boberg, Anne Marie Vinggaard, Sofie Christiansen, Ulla Hass National Food Institute, Technical University of Denmark, Division of Toxicology and Risk Assessment, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark
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Article history: Received 18 April 2013 Accepted 25 June 2013 Available online 4 July 2013 Keywords: Triclosan Rat Thyroxine (T4) Developmental Thyroid disrupting chemical (TDC)
a b s t r a c t Thyroid disrupting chemicals can potentially disrupt brain development. Two studies investigating the effect of the antibacterial compound triclosan on thyroxine (T4) levels in rats are reported. In the first, Wistar rat dams were gavaged with 75, 150 or 300 mg triclosan/kg bw/day throughout gestation and lactation. Total T4 serum levels were measured in dams and offspring, and all doses of triclosan significantly lowered T4 in dams, but no significant effects on T4 levels were seen in the offspring at the end of the lactation period. Since this lack of effect could be due to minimal exposure through maternal milk, a second study using direct per oral pup exposure from postnatal day 3–16 to 50 or 150 mg triclosan/kg bw/day was performed. This exposure pointed to significant T4 reductions in 16 day old offspring in both dose groups. These results corroborate previous studies showing that in rats lactational transfer of triclosan seems limited. Since an optimal study design for testing potential developmental neurotoxicants in rats, should include exposure during both the pre- and postnatal periods of brain development, we suggest that in the case of triclosan, direct dosing of pups may be the best way to obtain that goal. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Triclosan is a compound that has been used in consumer products for many years due to its high efficiency as an antibacterial agent and its low acute toxicity (SCCS, 2009). In the EU, about 85% of the total volume of triclosan is used in personal care products (toothpaste, soap, shampoo and cosmetics), 5% is used for textiles (antibacterial clothing) and 10% for plastics and food contact materials (SCCS, 2009). In recent years it has become evident that triclosan acts as a thyroid hormone disrupting chemical. This effect has been seen in metamorphosis studies in bullfrogs and Xenopus laevis (Helbing et al., 2011; Veldhoen et al., 2006), and the compound has also been shown to reduce thyroxine (T4) levels in a series of rat studies. In these studies, young adult rats (22–60 days of age) have been exposed to triclosan for varying periods of time (Crofton et al., 2007; Paul et al., 2010a; Stoker et al., 2010; Zorrilla et al., 2009), or rat dams have been exposed during gestation and lactation (Paul et al., 2012, 2010b; Rodriguez and Sanchez, 2010). The doses needed to observe T4 reductions in these studies have been varying between 10 and 300 mg/kg bw/day, probably reflecting variations in rat strain and study design. In humans, even mild reductions in T4 levels in pregnant women can have severe consequences for the neurological development of children, as associations with delayed or impaired ⇑ Corresponding author. Tel.: +45 35 88 75 41. E-mail address: [email protected] (M. Axelstad). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.06.050
cognitive and motor function have been reported in numerous epidemiological studies (Ghassabian et al., 2011; Haddow et al., 1999; Henrichs et al., 2010; Kooistra et al., 2006; Li et al., 2010; Pop et al., 1999, 2003). In addition, an ample number of animal studies have shown that prenatal hypothyroxinemia may significantly affect nerve cell migration and other molecular aspects of brain development in rats (Auso et al., 2004; Berbel et al., 2010; Cuevas et al., 2005; Gilbert and Sui, 2008, 2006; Lavado-Autric et al., 2003; Opazo et al., 2008; Sharlin et al., 2008). Our previous research has indicated that in rats, maternal hypothyroxinemia is only significantly correlated to altered behaviour and hearing in the offspring, if marked postnatal T4 decreases in the offspring are also present. The fact that postnatal thyroid hormone insufficiency has been present in almost all studies showing altered behaviour after developmental hypothyroidism in rats (Akaike et al., 1991; Brosvic et al., 2002; Noda et al., 2005; Provost et al., 1999) further corroborate the hypothesis that in rat dams, T4 reductions during gestation alone are not enough to induce adverse behavioural effects in the offspring (Axelstad et al., 2011a,b). Such an important species difference between humans and rats may beexplained by the fact that while both rodents and humans need thyroid hormones for differentiation and maturation of the central nervous system, important differences in timing of these events exit. Since a larger part of the brain maturation takes place prenatally in humans compared to rats (Howdeshell, 2002), prenatal T4 deficits might have more severe consequences for foetal brain development in humans than in rats.
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The aim of the present work was to develop a study design in which postnatal T4 reductions would be induced in rat offspring. For this purpose two studies were performed. In the first, pregnant rat dams were exposed to triclosan during gestation and lactation, and T4 levels in dams and offspring were examined. Due to a suspected influence of triclosan on the sex hormone balance (Kumar et al., 2009; Stoker et al., 2010), also prostate glands were examined in the offspring. In spite of marked reductions in maternal T4 levels during gestation and lactation, offspring T4 levels were not significantly reduced when measured at the end of the lactation period. As this result indicated limited triclosan excretion to the maternal milk, and consequently limited exposure in the neonatal period, a second study was performed. Here, neonatal rat offspring were exposed to triclosan through direct peroral dosing during lactation, with subsequent measurements of serum T4 levels.
2. Materials and methods 2.1. Chemicals The vehicle used was corn oil (Sigma–Aldrich, Denmark). Triclosan (purity >99.0%, CAS No. 3380-34-5, Alfa Aesar No. L18655) was from VWR-Bie & Berntsen, Herlev, Denmark. The triclosan solutions were kept dark, at room temperature, and continuously stirred during the dosing period. New solutions were prepared for each of the two studies, but no verification of dose concentrations was performed.
2.2. Animals and treatment Both studies were performed under conditions approved by the Danish Animal Experiments Inspectorate and by the in-house Animal Welfare Committee. The animals received a complete rodent diet (Altromin Standard Diet 1314) and acidified tap water ad libitum, and were housed under standard conditions as described in Christiansen et al. (2012). In the first study (study 1), 40 time-mated Wistar rats (HanTac:WH, Taconic Europe, Ejby, Denmark) were supplied at gestation day (GD) 3 of pregnancy. The study was performed using 2 blocks with one week in between, and all dose groups were equally represented in the blocks. The dams were distributed into four dose groups (0; 75; 150 or 300 mg/kg/day; n = 10 per group), and gavaged once daily from gestation day (GD) 7 to postnatal day (PND) 16 (day of delivery excluded), at a constant volume of 2 ml/kg bw/day. The individual doses were based on the body weight of the animal on the day of dosing. The dams were inspected twice a day for general toxicity including changes in clinical appearance. Body weights were recorded on GD 4 and daily during the dosing period. On GD 15 dams were anesthetized with HypnormÒ (fentanyl citrate/flunisone)/DormicumÒ (midazolam), and blood was drawn from the tail vein. The day after delivery, the pups were counted, sexed, weighed, checked for anomalies and anogenital distance (AGD). Of the 10 time-mated dams in each group, 7–9 were pregnant and gave birth to viable litters. Body weights were measured on PND 6 and 13, and offspring were examined for the presence of nipples/areolas on PND 13. On PND 16 all dams and pups were sacrificed. Dams were weighed, anaesthetized in CO2/O2, decapitated, and trunk blood was collected. The number of implantation scars in the uterus was registered and thyroid glands were excised and weighed. All offspring were weighed, decapitated and trunk blood collected. Blood samples from the offspring were pooled within litter in a male and a female sample. Prostates from 1 male per litter were excised, weighed and prepared for histopathological examinations. Thyroids from 1 to 2 males per litter were excised and weighed. Thyroids from 1 male per litter were excised together with the thyroid cartilage and prepared for histological examination. Thyroids and prostates were fixed in formalin, embedded in paraffin, and stained with haematoxylin and eosin, and histological evaluations of one section per organ from animals from the control group and the highest dose group were performed by a pathologist blinded to treatment groups. In the direct postnatal exposure study (study 2), 6 time-mated pregnant Wistar rats were supplied at GD 16, one week before expected delivery. Each dam was individually housed, and each litter remained housed with the respective dam until sacrifice. Two days after delivery (PND 3) the litters were culled to 8 offspring, with an equal representation of males and females when possible, and litters were assigned into one of three dosage groups (0, 50 or 150 mg/kg/day). The maximum dose was set at 150 mg/kg/day to avoid general toxic effects in the young pups. All offspring were dosed orally from PND 3 to PND 16 using a micropipette. The dams were not dosed, but remained in the cage to allow the pups to feed normally. Each pup was held by one hand while the other slowly allowed test solution to drip directly into the mouth, but only as fast as the pup would allow, ensuring precise and reliable dosing.
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On each day of dosing, the entire litter (8 pups) was weighed, and the average pup weight was calculated. All pups in the litter were dosed according to the average weight receiving 2 ll of test solution per gram body weight. Thus they received from approximately 12 ll to 90 ll test solution per day in the dosing period. On PND 16 the offspring were sacrificed by decapitation and trunk blood was collected for total T4 analyses. The trunk blood was collected and analyzed individually for each pup. 2.3. Thyroxine immunoassay Blood samples from dams (GD 15 and PND 16) and offspring (PND 16) in the first study, and from offspring (PND 16) in the second study, were analyzed for total thyroxine (T4) concentration in plasma. Trunk blood was used for the analysis. A Delfia time-resolved fluoroimmunoassay kit from Perkin Elmer (cat. No. 1244030, Wallac Oy, Turku, Finland) was modified and developed specifically for analysis of T4 in rat samples. Instead of human T4 standards and T4 antibody supplied in the Delfia kits, T4 standards in T4-free rat serum (cat. Nos. 30042 and 30041, respectively) as well as rat specific biotinylated T4 (30,039) antibody from Biovian Ltd. (Finland) were used. Otherwise the assay was run as outlined in the manufacturer’s protocol and described in Axelstad et al. (2008). The analytical sensitivity is around 10 nM. Historic control values for T4 levels in PND 16 males and females were recorded being 40 ± 13 nM and 38 ± 8 nM (n = 5), respectively. Thus, the CV% between in vivo studies (n = 5) were 32% and 22% for males and females, respectively. 2.4. Statistical analysis Statistical analysis of data with normal distribution and homogeneity of variance were analysed using analysis of variance (ANOVA), followed by Dunnett’s post hoc test. In study 1, when more than one pup from each litter was examined, statistical analyses were adjusted using litter as an independent, random and nested factor in ANOVA, or analysis were done using litter means. Furthermore, since study 1 was performed in two blocks, block was included as an independent random and nested factor in the analysis however no significant effects of block were seen. In cases where normal distribution and homogeneity of variance could not be obtained by data transformation, a non-parametric Kruskal–Wallis test was used. Trend analysis on dose–response relations for hormone levels, body- and organ weights were performed using Spearman’s test. In study 2, litter was not used as the statistical unit, as triclosan exposure was direct, and not through the dam.
3. Results In study 1, maternal body weight gain was not significantly affected by triclosan exposure when weight gain was calculated from the beginning of the dosing period to the day before birth (GD7 to 21). However, when maternal body weight gain was measured from GD7 to the day after birth (PND 1), a significant dosedependent downward trend was seen (p = 0.001) and a statistically significant decrease was seen in the highest dose group compared to control (p = 0.007), indicating that 300 mg triclosan/kg/day caused a moderate degree of maternal toxicity during gestation (Table 1). Gestation length, gender distribution, postimplantation loss and litter size were unaffected by the exposure and so were maternal body weight gains, neonatal deaths and offspring body weights in the postnatal period. Furthermore, no effect was seen on male or female anogenital distance or on nipple retention (Table 1). Triclosan exposure had a marked effect on total T4 levels in dam serum. On GD 15, a significant dose-dependent downwards trend was seen (p < 0.001) and the T4 levels were decreased by 59%, 72%, and 72% in the three triclosan groups respectively (p = 0.028, p = 0.0001, p = 0.0005) (Fig. 1A and Table 2). The effect of triclosan on maternal T4 levels was also present on PND 16, where the dose-dependent downwards trend was also significant (p < 0.001) and the T4 levels were decreased by 38%, 55% and 58% in the three triclosan groups respectively (p = 0.032, p = 0.001, p = 0.0006) (Fig. 1B and Table 2). Offspring total T4 levels on PND 16 (study 1) showed no statistically significant dose-dependent trends and group means did not differ significantly from controls in any dose group (Table 2). Both absolute and relative thyroid gland weights were unaffected by triclosan exposure in dams and offspring, as no dosedependent trends and no significant differences between groups
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Table 1 Reproductive and developmental data from study 1 and 2. The table shows pregnancy and litter data, including body weights (bw) from dams and offspring exposed indirectly to 0, 75, 150 or 300 mg triclosan/kg bw/day from GD 7 to PND 16 (study 1), and offspring body weights in pups exposed directly to triclosan at doses of 0, 50 or 150 mg/kg bw/day from PND 3–16 (study 2). Data represent group means based on litter means ± SD.
**
Study 1 (indirect dosing of pups)
Control
75 mg/kg/day
150 mg/kg/day
300 mg/kg/day
Dams and litters Dam bw gain, GD 7-GD 21 Dam bw gain, GD 7-PND 1 Dam bw gain, PND 1–16 Gestation length (days) % Postimplantation loss % Perinatal loss Litter size % Perinatal deaths % Males Offspring Mean birth weight AGD males (mm) AGD females (mm) Nipples males Nipples females Mean pup bw PND 6 Mean pup bw PND13 Mean pup bw PND 16
n=9 80.8 ± 8.3 12.9 ± 5.5 39.3 ± 10.0 22.89 ± 0.33 8.19 ± 6.7 12.5 ± 7.2 10.67 ± 2.7 4.67 ± 4.5 52.6 ± 21.2
n=7 78.6 ± 14 15.1 ± 7.0 36.4 ± 5.2 23.14 ± 0.38 28.4 ± 41.7 35.5 ± 40.6 9.00 ± 3.8 6.59 ± 17.4 45.6 ± 8.9
n=8 77.6 ± 16 8.7 ± 7.1 33.8 ± 10.8 23.00 ± 0.0 19.5 ± 32.2 19.5 ± 32.2 11.00 ± 3.9 0.00 ± 0.0 48.8 ± 12.3
n=8 72 .8 ± 16 2.9 ± 5.2** 38.3 ± 12.8 23.00 ± 0.0 22.4 ± 41.2 22.4 ± 41.2 9.67 ± 4.8 0.00 ± 0.0 59.5 ± 22.7
6.23 ± 0.5 4.03 ± 0.1 2.21 ± 0.2 0.00 ± 0.0 12.4 ± 0.3 13.11 ± 1.5 26.31 ± 4.0 31.72 ± 5.3
6.12 ± 0.2 3.98 ± 0.1 2.14 ± 0.1 0.00 ± 0.0 12.3 ± 0.2 12.62 ± 0.6 26.06 ± 1.5 31.84 ± 2.4
6.08 ± 0.4 4.00 ± 0.1 2.19 ± 0.1 0.12 ± 0.2 12.2 ± 0.2 11.83 ± 1.5 23.93 ± 3.9 28.15 ± 4.8
6.16 ± 0.4 3.97 ± 0.1 2.19 ± 0.1 0.06 ± 0.1 12.4 ± 0.2 12.19 ± 1.6 24.42 ± 3.7 29.65 ± 4.9
Study 2 (direct dosing of pups)
Control
50 mg/kg/day
150 mg/kg/day
No. of litters Litter size (culled to 8 at PND 3) Mean pup bw PND 6 Mean pup bw PND13 Mean pup bw PND 16
n = 2 (1 after day 7) 8 12.1 ± 0.5 26.29 ± 0.0 32.58 ± 0.0
n=2 8 13.3 ± 0.8 29.95 ± 2.4 37.16 ± 2.9
n=2 8 and 6 13.8 ± 0.5 32.44 ± 2.2 39.91 ± 3.4
p < 0.01.
Fig. 1. Total thyroxine (T4) levels (nM) in dams on gestation day (GD) 15 (A) and postnatal day (PND) 16 (B), after exposure to 0, 75, 150 or 300 mg triclosan/kg bw/ day from GD 7 – PND 16 (study 1). Data represent group means based on litter means + SEM, n = 7–9. *p < 0.05; ***p < 0.001.
were seen (Table 3). Also no histopathological effects on the offspring thyroids on PND 16 were seen in the high dose group (data not shown). Statistical analysis of the both relative and absolute prostate weights (analysed with body weight as covariate) indicated no differences between treatment groups, and no dose-dependent trends were seen (Table 3). Histopathology of the prostates showed no differences between controls and the high dose group at PND 16 (data not shown). In the direct exposure study (study 2) one of the two litters in the control group had to be sacrificed on PND 7, because the dam did not take care of her offspring. Furthermore, two pups from one of the litters in the high dose group died on day PND 6. This is sometimes seen in rat litters, and was not attributed to triclosan exposure. The triclosan dosing did not cause any general toxicity effects in the exposed offspring, and no significant effects on pup body weights or body weight gains were seen during the exposure period (PND 3–16), compared to controls (Table 1). On days 13 and 16, the pups in the direct exposure study (study 2) weighed more than offspring of the same age in the indirect exposure study, and the differences were statistically significant (p = 0.0051 and p = 0.0037 on PND 13 and 16 respectively). This difference was probably due to a combination of the pups receiving oil gavage and the fact that there were fewer offspring in this study in each litter, because of the culling performed on day 3. T4 levels in the pups that had been directly exposed to triclosan at doses of 50 and 150 mg/kg/day were significantly decreased by 16% (p = 0.029) and 39% (p < 0.001) in the two dose groups respectively, when measured on PND 16 (Table 2), and a significant dose dependent trend was also seen (p < 0.001). However, an important limitation in study 2 was that all the control pups were from the same litter. Therefore there was a possible risk that due to genetic similarities, the offspring may have had high T4 levels, which could have caused the significant T4 reductions in both dose groups. To further complicate interpretation of the data, the T4 values from the pups in this litter were in the high end of our previous control values (Axelstad et al., 2008, 2011a,b).
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Table 2 T4 levels in dams and offspring from study 1 and 2. The table shows total T4 levels (nM) in dams and offspring, after exposure to 0, 75, 150 or 300 mg triclosan/kg bw/day from GD7 to PND 16 in study 1, and in pups exposed directly to triclosan at doses of 0, 50 or 150 mg/kg/day from PND 3 to 16 in study 2. Data represent group means ± SD.
*
Study 1 (indirect dosing of pups)
Control
75 mg/kg/day
150 mg/kg/day
300 mg/kg/day
No. of litters T4 in dams GD 15 T4 in dams PND 16 T4 in offs. PND 16 (males) T4 in offs. PND 16 (females)
9 38.7 ± 20.4 17.5 ± 6.9 34.9 ± 9.2 40.9 ± 6.3
7 15.8 ± 5.4* 10.9 ± 4.7* 26.0 ± 4.0 35.4 ± 5.2
8 10.7 ± 7.7*** 7.9 ± 3.7*** 34.9 ± 9.9 38.5 ± 13.8
8 10.8 ± 4.5*** 7.4 ± 2.7*** 27.4 ± 8.1 33.6 ± 5.2
Study 2 (direct dosing of pups)
Control
50 mg/kg/day
150 mg/kg/day
No. of samples T4 in offs. PND 16 (males) T4 in offs. PND 16 (females)
8 (1 l) 45.5 ± 6.8 46.6 ± 5.4
16 (2 l) 38.0 ± 5.0* 39.2 ± 4.9*
14 (2 l) 27.7 ± 2.7*** 28.3 ± 3.7***
p < 0.05. p < 0.001.
***
Table 3 Dam and offspring organ weights from study 1. The table shows absolute and relative thyroid and prostate weights in dams and male pups exposed indirectly to 0, 75, 150 or 300 mg triclosan/kg bw/day from GD7 to PND 16 (study 1). Data represent group means ± SD. No significant differences between dose groups were observed. Study 1
Control
75 mg/kg/day
150 mg/kg/day
300 mg/kg/day
Dams Dam BW, PND 16 Thyroid weight dams PND 16 Rel. thyroid weight/100 g bw
282 ± 25 0.017 ± 0.003 0.591 ± 0.09
282 ± 11 0.021 ± 0.011 0.746 ± 0.37
266 ± 13 0.020 ± 0.007 0.741 ± 0.29
272 ± 12 0.016 ± 0.003 0.576 ± 0.12
Offspring Mean male pup bw PND 16 Thyroid weight PND 16 Rel. thyroid weight/100 g bw Prostate weight PND 16 Rel. prostate weight/100 g bw
31.92 ± 5.1 0.47 ± 0.05 1.50 ± 0.16 1.19 ± 0.2 0.038 ± 0.005
31.32 ± 3.3 0.48 ± 0.06 1.52 ± 0.11 1.31 ± 0.4 0.041 ± 0.01
30.70 ± 5.0 0.40 ± 0.06 1.30 ± 0.15 1.19 ± 0.3 0.039 ± 0.007
30.03 ± 4.7 0.45 ± 0.06 1.54 ± 0.26 1.01 ± 0.3 0.034 ± 0.009
4. Discussion The present studies confirm that triclosan can disrupt thyroid hormone levels, as total T4 levels were markedly decreased in rat dams on both GD15 and PND 16 after exposure to 75; 150 and 300 mg triclosan/kg bw/day during gestation and lactation. After indirect exposure through placenta and maternal milk (study 1), the offspring T4 levels on PND 16 were not significantly decreased, whereas direct pup exposure to 50 and 150 mg/kg bw/day from PND 3–16 (study 2) appeared to decrease offspring T4 levels. Since previous studies have indicated that triclosan does not pass to the milk in sufficient amounts to significantly reduce T4 levels during the late lactation period (Paul et al., 2012), and the results from study 1 corroborate this finding, the present results taken together indicate that triclosan can probably only significantly reduce T4 levels in the offspring during the entire lactation period, if the chemical is directly dosed. T4 levels have previously been measured in a number of triclosan studies in rats, and the present results corroborate and add to these findings. In female Long-Evans (LE) rats, dosed daily with triclosan either from PND 28–31 or during gestation and lactation, doses of 100 mg/kg bw/day and above significantly lowered serum levels of T4 in the dams (Crofton et al., 2007; Paul et al., 2010a, 2012). A number of studies performed in Wistar rats have furthermore shown that even lower doses of triclosan can induce T4 reductions in this strain. Here doses of 30 mg/kg bw/day and above caused T4 levels to decrease markedly in young Wistar males dosed daily from PND 23–58 (Zorrilla et al., 2009), whereas doses of 37.5 mg/kg bw/day and above resulted in decreased total serum T4 levels in young Wistar females dosed from PND 22–43 (Stoker et al., 2010). In developmental studies performed by Paul et al. (2010b, 2012), 300 mg/kg caused serum T4 levels in offspring to decrease
on GD 20 and PND 4, while no significant reductions were seen on PND 14 or PND 21. The authors suggested that the T4 reductions observed on PND 4 pups could have resulted from transplacental exposure to triclosan and that toxicokinetic factors probably affected maternal transfer of triclosan into milk and thereby limited lactation exposure to the pups. The present T4 results (study 1) fit well with results from Paul et al. (2010b, 2012), showing no significant effects on offspring T4 levels after approximately two weeks of lactational exposure. To further test whether the lack of T4 reduction in the 16-day old offspring was due to limited triclosan excretion in the milk, the direct exposure study (study 2) was performed. Due to the quite time consuming dosing method, this study was only performed on a limited number of litters. However, the blood samples from each of the eight dosed pups in each litter were not pooled, yielding between 8 and 16 samples in each dose group. The conclusion in study 2 was that since T4 levels in the directly exposed pups seemed decreased compared to controls the lack of significant effect seen in any dose group in the offspring from study 1, was probably due to limited exposure through maternal milk. In future developmental studies of triclosan, including direct measurements of triclosan levels in the dam’s milk at different post-natal times, as well as performing a larger study with direct postnatal dosing of the offspring, would more definitively determine if the different sensitivities observed between neonatal and 14–16 day old pups, are caused by changes in exposure to triclosan or by changes in triclosan catabolism. It seemed quite clear that the T4 reductions seen in dams dosed with the 75 and 150 mg/kg from GD7-15 (59 and 72% reduction in T4, respectively) were more marked than seen in pups dosed directly from PND 3–16 with 50 and 150 mg/kg (16% and 39% reduction in T4 respectively), indicating that triclosan also may not have triggered the same degree of toxicodynamic effects in the offspring as seen in more mature animals. This could be explained by the
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mode of action for triclosan, as the compound is suspected of affecting the thyroid hormone system by causing induction of phase II liver enzymes (sulfonation or glucuronidation), and thereby upregulating thyroid hormone catabolism (Crofton et al., 2007; Paul et al., 2010a, 2012). This mode of action is indicated by the observation of increased liver weights (Crofton et al., 2007; Paul et al., 2010a; Zorrilla et al., 2009), increased PROD activity in the liver (a marker of Cyp2b activity) (Paul et al., 2010b, 2012; Zorrilla et al., 2009) and upregulated mRNA expression and activity of some phase I and phase II hepatic enzymes (Paul et al., 2010a, 2012). Since hepatic excretory function in rats develops postnatally to reach maximum capacity at an age of approximately 30 days (Klinger, 2005), and due to this reduced activity of many phase II enzymes, neonatal rats have a decreased capacity to metabolize and excrete (Suchy et al., 2007). A possible explanation for the smaller T4 reductions seen in the pups compared to adults might be that triclosan exposure did not lead to the same degree of induction of liver enzymes as seen in adult animals, and consequently only induced smaller T4 reductions. However, this is only one explanation for the observed results and more studies are needed to confirm the hypothesis that thyroid disrupting compounds acting by increasing liver metabolism may not affect thyroid hormone levels in neonatal rats as much as seen in older animals. In the present study no thyroid weight increases were observed in dams, despite a considerable drop in T4 levels. Normally thyroid weight increases are considered mediated through an increase of TSH, in response to decreased thyroid hormone levels. TSH levels were however not measured, because previous triclosan studies have shown that TSH levels were not affected at dose levels where significant T4 decreases were seen (Zorrilla et al., 2009; Paul et al., 2010a). Histological examination of thyroids was only performed in pre- and postnatally exposed male offspring showing no changes, whereas no examination was performed in dams or in pups exposed directly to triclosan. In the present developmental toxicity study (study 1) neither anogenital distance, nipple retention, prostate weight nor prostate histology were affected by exposure to triclosan. These endpoints are typically affected by perinatal exposure to anti-androgenic chemicals (Christiansen et al., 2010; McIntyre et al., 2001), indicating that triclosan exposure at the tested dose levels did not affect male reproductive development in an anti-androgenic manner. Adverse reproductive effects have previously been reported in males rats exposed during adulthood (Kumar et al., 2009). These included decreased weights of several reproductive organs, histopathological changes in these, and decreased levels of FSH, LH and testosterone level after two months of exposure to 20 mg/kg bw/day. It is however possible that the effects seen by Kumar et al., could be due to impurities in the triclosan used for the studies, as dioxin and furan contamination has been seen in several different triclosan samples produced in India and China (Menoutis and Parisi, 2002). In a study using post-weaning male rats exposed to up to 300 mg/kg/day from PND 23–58, no significant effects on timing of sexual maturation or reproductive organ weight were seen (Zorrilla et al., 2009). In studies focusing on reproductive effects in female offspring, a triclosan doses of 150 mg/kg/day lowered the age of vaginal opening and caused increased uterine weights (Stoker et al., 2010), while much lower doses of triclosan potentiated the effects of estradiol treatment on uterine weight (Louis et al., 2013; Stoker et al., 2010). These effects were compatible with an estrogenic mode of action, which was not tested in the present study. A number of in vitro studies examining the endocrine disrupting modes of action of triclosan have also been performed during recent years. Antagonistic activity in both ER- and AR-responsive bioassays have been shown in several studies (Ahn et al., 2008; Chen et al., 2007; Gee et al., 2008; Christen et al., 2010; Vinggaard
et al., 2008), and taken together the in vivo and in vitro data indicate that triclosan could affect the reproductive hormone axis. The lack of adverse effects in the few reproductive endpoints tested in the present study (AGD, nipple retention, prostate weight and histology) could reflect that these endpoints are more sensitive to anti-androgenic chemicals, and it is possible that triclosan is too weak an androgen receptor antagonist to cause anyantiandrogenic effects in vivo. Based on what is presently known on the effects of triclosan, an adverse outcome pathway for the effects of triclosan on the thyroid hormone system has been proposed (US EPA, 2011). Here activation of the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) in rat liver by triclosan is an initiating event, leading to up regulation of hepatic phase I and phase II enzymes, which could increase catabolism of thyroid hormones and consequently lower serum levels of these – effects that could potentially lead to altered neurodevelopment. Furthermore, triclosan has recently been shown also to affect the thyroid system by inhibiting deoidinase activity (Butt et al., 2011). Uncertainty exists, as to whether this adverse outcome pathway is also relevant for humans, because species differences with regard to activation and amino acid sequence of the PXR exist (Jones et al., 2000), which make it difficult to extrapolate results obtained with rodent PXR directly to humans (Dybdahl et al., 2012). However in vitro data show that triclosan is a moderate inducer of human PXR activity in human hepatoma cells (Jacobs et al., 2005), indicating that this could also be an initiating event in an adverse outcome pathway in humans.
5. Conclusions In conclusion, triclosan markedly lowered maternal T4 levels in rat dams during gestation and lactation, and nine days of exposure resulted in a LOAEL of 75 mg/kg bw/day, corroborating effects seen in previous rat studies. In humans, correct maternal T4 levels during pregnancy are crucial for fetal brain development, and even slight maternal hypothyroxinemia can result in adverse effects on the cognitive and motor function of children (Ghassabian et al., 2011; Henrichs et al., 2010; Kooistra et al., 2006; Li et al., 2010; Pop et al., 1999, 2003). Based on its thyroid disrupting properties there might be a need for further assessment of triclosan as a potential developmental neurotoxicant. Since the first ten postnatal days in the rat approximate the last trimester of human pregnancy with regard to the development of the central nervous system (Howdeshell, 2002), the results presented here imply that in a developmental toxicity study of triclosan, direct postnatal exposure would be the optimal study design to use, for successfully covering the entire period of human brain development during pregnancy. It is furthermore important to bear in mind that humans are exposed to a variety of thyroid disrupters, all probably acting in a dose-additive manner (Flippin et al., 2009). Since triclosan may be a potential contributor to thyroid disruption in humans, exposure should be carefully regulated in order to protect pregnant women and their children from excessive exposure to thyroid disrupting chemicals.
Funding This work was supported financially by the Danish Environmental Protection Agency.
Conflict of Interest The authors declare that there are no conflicts of interest.
M. Axelstad et al. / Food and Chemical Toxicology 59 (2013) 534–540
Acknowledgements Dorte Hansen, Lillian Sztuk, Bo Herbst, Sarah Simonsen, Kenneth Worm, Heidi Letting, Birgitte Møller Plesning, Vibeke Kjær and Ulla El-Baroudy are thanked for their excellent technical assistance.
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