The toxicity of triclosan, bisphenol A, bisphenol A ... - Springer Link

Mol Cell Toxicol (2012) 8:209-216 DOI 10.1007/s13273-012-0026-4

ORIGINAL PAPER

The toxicity of triclosan, bisphenol A, bisphenol A diglycidyl ether to the regeneration of cnidarian, Hydra magnipapillata Hyung-Geun Park1 & Min-Kyeong Yeo1 Received: 3 August 2011 / Accepted: 16 September 2011 �The Korean Society of Toxicogenomics and Toxicoproteomics and Springer 2012

Abstract Triclosan, bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) are widely used in cosmetics, cleaners and plastic bowls. However, triclosan has been detected in sewage samples after treatment. Moreover, BPA and BADGE are thought to be endocrine disruptors, and these chemicals bio-accumulate in aquatic living organisms. Human skin and oral tissues are commonly exposed to phenolic chemicals such as triclosan, BPA and BADGE. These types of cells have an excellent regeneration capacity. Hydra magnipapillata inhabits rivers and ponds, and is widely used as a test species because of its ability to rapidly regenerate. Therefore, we investigated the biological toxicity of phenolic chemicals (triclosan, BPA and BADGE) using the freshwater cnidarian, Hydra magnipapillata. We observed severe biological damage after exposure to 1-5 ppm of each of the phenolic chemicals tested. In the Hydra magnipapillata exposed to triclosan (1 ppm, 4 h), there was epidermal tissue and nematocyst damage. Although we did not observe significant biological toxicity in regenerated tissue from Hydra magnipapillata treated with BPA and BADGE, the regeneration capacity was inhibited in the group exposed to triclosan. Hydra tentacles that were treated with phenolic chemicals (1 ppm, 4 h) were moved to a control medium in order to assess recovery after exposure to triclosan, BPA and BADGE. There was no significant difference between the treated and control groups. Moreover, there was no difference in apoptosis, as viewed with a confocal laser microscope, between the endocrine-disrupting phenolic chemical1

Department of Environmental Science and Environmental Research Center, College of Engineering, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446701, Korea Correspondence and requests for materials should be addressed to M.-K. Yeo ( [email protected])

exposed groups and regeneration groups. The results of this study suggest that BPA and BADGE do not exhibit biological toxicity, but triclosan has toxic effects on the cellular reproduction process that is part of regeneration in Hydra magnipapillata. Keywords Hydra magnipapillata, Triclosan, Bisphenol A, Bisphenol A diglycidyl ether, Regeneration There are a number of chemicals in products that are used by consumers every day. For example, triclosan is widely used in products such as toothpaste, cosmetics, detergents, textile fabrics and plastics1. However, triclosan has been detected in samples collected from wastewater treatment plants and is a suspected endocrine disrupter2,3. Since treated wastewater is discharged into rivers, chemicals such as triclosan can have a significant influence on aquatic organisms in the ecosystem. Bisphenol A (BPA) is consumed in large amounts in consumer products. It is used in polycarbonate plastic products, and in the epoxy resin of metallic pipe borders, toys, water pipes, drink containers and baby bottles4. BPA adversely affects the reproductive tract, mammary gland development and sex-specific functions, and has been shown to reduce fertility5. Bisphenol A diglycidyl ether (BADGE) is a substance that is used as a precursor to the epoxy used to coat food cans, storage containers and acrylic/epoxy glue. BADGE is used in the manufacture of epoxy resin, and is synthesized by reacting epichlorohydrin with BPA6. The estrogenic activity of BADGE is 1/100 times that of BPA, but BADGE is a component of BPA that can disrupt the endocrine system. Recently, BPA was shown to be an endocrine disruptor, so stronger regulations have been introduced to promote the restriction and prohibition of BPA use.

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Mol Cell Toxicol (2012) 8:209-216

Gastric region

Regeneration

Section

Figure 1. Diagram of the cutting location for the regeneration experiment in Hydra magnipapillata.

For example, there is a restriction on importing BPAcontaining infant bottles into Canada, the EU and some other countries. In Korea, the packaging criteria for utensils and containers have been revised to prohibit the use of BPA (http://www.kfda.go.kr/)7. The EPA, FDA and CPSC have not determined that there is enough evidence to restrict the use of triclosan, and thus, there are no legal restrictions on the use of triclosan in the USA or Korea. All three of these phenolic chemicals (triclosan, BPA and BADGE) are thought to be endocrine disruptors, and endocrine disruptors bioaccumulate in aquatic living organisms. Hydra magnipapillata is a hydrozoan that inhabits rivers and ponds and is widely used as a test species because of its ability to rapidly regenerate; any part of the body can be regenerated in just 36 h. Human skin and oral cells also have an excellent regeneration capacity, and these types of cells are commonly exposed to phenolic chemicals such as triclosan, BPA and BADGE. Therefore, we investigated the biological toxicity of phenolic chemicals (triclosan, BPA and BADGE) using the freshwater cnidarian, Hydra magnipapillata, as our model organism. We observed morphological denaturation and the lethal dose during 96 h of exposure to 1-5 ppm of each of the phenolic chemicals. In addition, in order to assess the regeneration capacity of gastric sections under exposure to phenolic chemicals (1 ppm, 96 h), we used the scoring methods described by Wilby et al. (1988)8. We also sought to determine the residual effect of these phenolic chemicals (5 ppm, after an exposure time of 4 hours) on regeneration capacity, and we assessed population numbers in the third generation. Using the histology method, we observed nematocysts, a type of inner cell, after 4 h of exposure to the different phenolic chemicals at a concentration of 1 ppm. In addition, using confocal laser microscopy, we investigated survival toxicity, apoptosis and necrosis in Hydra magnipapil-

Table 1. a) Scoring system for damaged phenotype based on the Wilby Morphology Score (1988). b) Graded scoring of Hydra magnipapillata after triclosan, BPA and BADGE exposure. a) Score

Morphological Status of Polyp

10 9 8 7 6 5 4 3 2 1 0

Mouth, 4-6 tentacles, peduncle Mouth, 4-6 tentacles Mouth, ⁄4 tentacles, basal disk Mouth, ⁄4 tentacles Tentacle buds and basal disk Tentacle buds only Basal disk only Normal wounding healing Healed but expanded Open ends, not healed or dead Disintegrated

b) Group Control Triclosan (1 ppm) Triclosan (2.5 ppm) Triclosan (5 ppm) BPA (1 ppm) BPA (2.5 ppm) BPA (5 ppm) BADGE (1 ppm) BADGE (2.5 ppm) BADGE (5 ppm)

Hours 0

4

8

24

48

72

96

10 10 10 10 10 10 10 10 10 10

10 9.2 8.8 7.6 9.8 9.4 8.6 9.8 9.6 8.8

10 6.5 5.4 4.8 9.7 9.4 8.5 9.8 9.6 8.6

10 3.2 2.2 1.8 9.6 9.4 8.2 9.8 9.6 8.4

10 2.4 1.9 1.3 9.5 9.3 8.2 9.7 9.6 8.4

10 1 0 0 9.3 9.2 8.1 9.6 9.6 8.4

10 0 0 0 9.2 9 8 9.6 9.6 8.4

lata and found that the phenolic chemicals had a significant effect on cell death in Hydra magnipapillata. The effects of triclosan, BPA and BADGE on Hydra magnipapillata

The Hydra magnipapillata cells were exposed to triclo-


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0h

4h

24 h

48 h

72 h

96 h

A Control

B Triclosan

C BPA

D BADGE

A-1

B-1

Figure 2. Regeneration in Hydra magnipapillata exposed to 1 ppm of phenolic compounds. The groups are A: control group, B: triclosan-exposed group, C: BPA-exposed group, and D: BADGE-exposed group. A comparison of Hydra magnipapillata in the control group with that in the triclosan-exposed group (control group (A-1) and triclosan exposed group (B-1)) 24 h after initiation of treatment. Arrows indicate abnormal hydra and hydra death.

Table 2. Scoring key for assessing toxic effects on the regeneration of gastric sections over time (based on Wilby et al. (1988)). Scoring of regeneration capacity in Hydra magnipapillata after exposure to triclosan (1 ppm), BPA (1 ppm) and BADGE (1 ppm). Group

0h

4h

Control Triclosan BPA BADGE

3 (3) 3 (3) 3 (3) 3 (3)

3 (3) 3 (3) 3 (3) 3 (3)

8h 4.5 (2-6) 0.2 (0-1.3) 3.1 (3-4) 3.2 (3-4)

san, BPA and BADGE, and then scored based on the methods reported by Wilby et al. (1988) (see Table 1-b for scores). The toxicity in the triclosan-exposed group

24 h

48 h

72 h

96 h

4.6 (2-6) 0.1 (0-1) 3.4 (3-4) 3.5 (3-4)

8.2 (6-8) 0 5.7 (4-7) 5.9 (4-7)

8.9 (7-10) 0 7.1 (6-9) 7.6 (6-9)

9.5 (9-10) 0 8.1 (7-9) 8.7 (8-9)

was greater than that in both the BPA- and BADGEexposed groups. Specifically, the scores were ⁄3.2 24 h after exposure to 1, 2.5 and 5 ppm of triclosan. How-

212


(I) A

45 Control Triclosan BPA BADGE

40 35 Population number

ever, the scores were ¤8.2 in the BPA- and BADGEexposed groups. The effects of the phenolic chemicals (triclosan, BPA and BADGE) on the regeneration capacity of Hydra magnipapillata over a 96 h period are shown in Figure 2 and Table 2. We observed rapid regeneration over time in the control group. In contrast, the triclosan-exposed group had some alterations in the bumpy parts of the body 4 h after exposure. Compared with the control group, the triclosan-exposed group displayed significantly different regeneration capabilities. Entire hydras were degraded in the triclosan-exposed group, while some individuals were regenerated in the BPA-exposed group. However, tentacle regeneration proceeded more slowly in the BPA-exposed group compared to the control group. Most of the hydras exposed to triclosan were dead 24 h after the initiation of exposure, and most of the experimental BPAand BADGE-treated groups exhibited regeneration progression, even though regeneration in both the BPA and BADGE groups was slower than that in the control group (Table 2). These results indicate that triclosan is more toxic than the other phenolic chemicals that were tested. Hydra tentacles treated with phenolic chemicals

30 25 20 15 10 5 0 0

2

4

6

8

12

16

18

20

Days

Group

Generation

Control Triclosan

Parent regeneration (~4 day) 1st generation (~11 day) 2nd generation (~18 day) 3rd generation (~20 day) Total numbers

BPA BADGE

1 13 35 39

1 11 32 36

1 12 32 35

1 9 27 31

39

36

35

31

Figure 3. The population number of Hydra magnipapillata after recovery from exposure to phenolic chemicals (5 ppm, 4 h).

(II) thread spines

B

lid

stiletto shaft capsule body

1. desmoneme 2. isorhiza

C

D

3. anisorhiza 4. mastigophore 5. microbasic eurytele 6. macrobasic eurytele

7. stenotele (Citation in Peter Schuchert, 2005, Hydrozoa Directory)

Figure 4. Nematocysts in Hydra magnipapillata. (I) Our observations: A) control hydra nematocysts, 1000×, B) triclosan-exposed hydra nematocysts (1 ppm, 4 hours), 1000×, C) BPA-exposed hydra nematocysts (1 ppm, 4 h), 1000×, D) BADGE exposed hydra nematocysts (1 ppm, 4 h), 1000×; and (II) Reference figures of the types of hydra nematocysts (Schuchert, 2005)9. Abbreviations: D, desmoneme; S, stenotele.


213

Total

Regeneration

A-1

A-2

B-1

B-2

C-1

C-2

D-1

D-2

A Control

B Triclosan

C BPA

D BADGE

Figure 5. Confocal microscopy data. 1) Total (whole body) after 4 h: A-1, B-1, C-1, D-1; and 2) regeneration (gastric section region) after 4 h: A-2, B-2, C-2, D-2. *Markers: (1) YO-PRO-1 (green) and (2) PI (red); merged images.

were moved to a control medium in order to assess their recovery after exposure to triclosan, BPA and BADGE. After 8 h in the control medium, the hydra’s tentacles slowly recovered. After four days of exposure, we observed hydra that had recovered and formed complete adults. While the phenolic chemicals were toxic, the exposed hydras were still able to recover their regenerative capabilities. We also investigated the reproductive capacity of the hydras by assessing population numbers in the third generation (Figure 3). Although there were differences according to the phenolic chemical used, all experimental groups had a similar reproductive capacity. Microscopic observation of cellular effects and cell death in Hydra magnipapillata

In this study, we was microscopically investigated cel-

lular differences in the treated groups compared to the control group. We observed nematocysts in the tissue after 4 h of exposure to the different phenolic chemicals at a concentration of 1 ppm (Figure 4). There are several types of nematocysts in Hydra magnipapillata9, and the stenotele type of nematocyst appeared in the control and BADGE-exposed groups. The desmoneme type of nematocyst appeared in the BPAexposed group. There were no significantly damaged nematocysts in the BPA- and BADGE-exposed groups, and the injured nematocysts in the triclosan-exposed group were broader and had unclear capsular bodies. We also performed confocal microscopy to assess the extent of apoptosis and necrosis after exposure to the phenolic chemicals (Figure 5). Our results showed that apoptosis occurred in the groups treated with BPA or BADGE, but there was less apoptosis in the group treated with triclosan. The triclosan-treated group had

214

decreased sample amounts because degradation started 4 h after the initiation of treatment. Apoptosis was observed in hydra exposed to BPA, both after whole body exposure and after exposure of the gastric section. Hydra exposed to BADGE did not show any morphological damage, but exhibited apoptosis and cellular necrosis.

Discussion In this study, we assessed the toxicities of several phenolic chemicals on Hydra magnipapillata regeneration. Hydra exposed to a high concentration (5 ppm) of BPA and BADGE, appeared to maintain a very strong regeneration capacity. Additionally, the lethal dose of triclosan was lower than that of the other chemicals (BPA or BADGE) (Table 1). There are several types of nematocysts in Hydra magnipapillata, two of which are the stenotele and desmoneme10,11. The number of stenoteles increases in the S/G2 phase of the cell cycle12,13. In this study, the shapes of the stenoteles were altered in the triclosan-treated group compared to the control group (Figure 4-B). Moreover, the regeneration capacity in Hydra magnipapillata after exposure to triclosan (1 ppm) was significantly reduced (Table 2 and Figure 2). This finding is consistent with those of previous reports indicating that the number of stenoteles depends on the regeneration capacity14,15. The desmoneme type of nematocyst appeared, but we were unable to observe stenoteles in the BPA-exposed group. No seriously damaged hydra were seen in the BPA-exposed group, but hydra exposed to BPA constricted more slowly than hydra in the control group. Therefore, we suspect that BPA exposure affects the hydra cell cycle or injured nerve cells related to constriction. We confirmed that triclosan impaired the regeneration capabilities of Hydra magnipapillata. However, we did not observe any morphological problems in the hydra in the BADGEtreated group. This is in contrast to previous work in which BPA and BADGE seriously damaged the reproductive systems and endocrine systems in humans, human cells, mice and zebrafish16-19. Hydra has been shown to generate very specific epidermal layers and have reportedly lost genes related to membrane attack complex/perforin domain (MAC/PF) proteins20. Most living organisms have the MAC/PF protein, the function of which is to attach a signal included in a non-self protein to the receptor in a cell membrane. We hypothesize that hydra have a specific membrane structure that protects against estrogenic chemicals. The specific functions of hydra proteins deserve future study.


Hydras in the triclosan-treated group exhibited degraded epidermal cells. Perhaps the cause of the abnormal phenotype in the triclosan-treated group was the result of damage to the protective system in the gastric region of the hydra. Our findings suggest that the transport systems of hydra membranes are affected differently by triclosan and BPA or BADGE. In conclusion, our results demonstrate that triclosan, among the many phenolic chemicals, has a toxic effect on regeneration in Hydra magnipapillata. However, BPA and BADGE do not appear to cause serious morphological damage. These results suggest that more research should be conducted to clearly determine the toxic effects of phenolic chemicals in living organisms.

Materials & Methods Test organism

Hydra magnipapillata were kindly donated by Dr. Seungshic Yum of the Korea Ocean Research and Development Institute, Korea. Hydra magnipapillata cultures were maintained and grown in glass bowls containing 0.5 L of hydra medium (110 mg/L TES [NTris(hydroxymethyl) methyl L-2-aminoethanesulfonic acid], 147 mg/L CaCl2∙2H2O, pH 7) at 20±1� C, with 16 h light and 8 h dark photoperiods, following a procedure adapted from Trottier et al. (1997)21. Test chemicals and treatment conditions

Triclosan (Sigma, USA, Lot # 0001412854), Bisphenol A (BPA) 99%+(Aldrich, USA, Lot # MKAA2480) and Bisphenol A diglycidyl ether (BADGE) (Sigma, USA, Batch # MKBB6775) were prepared from 100 mg/L stock solutions using the hydra medium as a diluent. The final triclosan, BPA and BADGE exposure concentrations were 1 ppm, 2.5 ppm and 5 ppm. Ten hydras were tested at each concentration and for the control (hydra medium-only), and the tests were carried out at 20±1� C with a 16 : 8 h light : dark regime. The test continued for 96 h and was repeated three times. The experimental groups were as follows: Group 1 was the general control group, Group 2 was exposed to triclosan, Group 3 was exposed to BPA and Group 4 was exposed to BADGE. Hydras were observed at 0, 4, 8, 24, 48, 72 and 96 h after exposure (Table 2). Biological effects of triclosan, BPA and BADGE on regeneration

We cut the body of the hydras in order to determine how exposure to 1 ppm of each of the three endocrinedisrupting phenolic chemicals affected the regenera-


tion capacity. Our experimental approach was based on modifications of the methods of Pachura-Bouchet et al. (2000)22, and the location of the cut is shown in Figure 1. Samples were taken from each group and treated as described below. We assessed toxicity in individual animals by microscopic observation, and recorded the effects on (i) survival toxicity and (ii) the numerical score devised by Wilby et al. (1988)8, which was assigned according to the morphological and physiological condition of the animal from 4 to 96 h post-exposure. A score of 10 was assigned if normal and 0 if the polyp had disintegrated (Table 1-a). The median scores for each of the morphological conditions were compared using non-parametric Friedman and KruskalWallis tests23. In addition, we investigated the ability of Hydra magnipapillata to recover after exposure to triclosan, BPA and BADGE by analyzing population numbers in the third generation. To determine the regeneration numbers, we counted the number of completely separate hydra bodies after bud formation over 20 days, and repeated this test three times. We also investigated the effects of the chemicals on inner tissues, including the nematocysts, by cutting and staining the hydra with 0.1% Safranin O solution and comparing the internal cellular forms. We observed the internal cells of Hydra magnipapillata using a phase-contrast microscope (Leitz Biomed, Leica, Germany). Cell death in Hydra magnipapillata observed via confocal laser microscopy

Apoptotic and necrotic cell death in Hydra magnipapillata were investigated using confocal laser scanning microscopy (LSM 510 META, Zeiss Co., Germany). We prepared cells for using a modified method. The sample volume in the final step was standing with 20 μL, and the marker for apoptosis was 20 μM YOPRO-1 (YO-PRO-1 iodide, Molecular Probes Co., USA). The necrosis marker was propidium iodide (PI) stain. Hydra magnipapillata cells were treated with 1 μL of 2 μM PI and 1 μL of 1 μM YO-PRO-1. The reaction was incubated on ice for five minutes, and then the fluorescent cells were observed under a 488nm Ar laser, a 543-nm HeNe laser and a 633-nm HeNe laser24.

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