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Reprod Toxicol
2024 Jan 01;123:108496. doi: 10.1016/j.reprotox.2023.108496.
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Teratogenic and neuro-behavioural toxic effects of bisphenol A (BPA) and B (BPB) on Xenopus laevis development.
Metruccio F
,
Battistoni M
,
Di Renzo F
,
Bacchetta R
,
Santo N
,
Menegola E
.
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Bisphenol A (BPA) is a plastic additive with endocrine disruptive activity, classified in 2017 by EU ECHA as substance of very high concern. A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species, including the amphibian Xenopus laevis. Among BPA analogues, bisphenol B (BPB) is used as alternative in different not-EU countries, including US, but seems to share with BPA its endocrine disruptor properties. Aim of the present work is the evaluation of the effects of BPB versus BPA exposure in a X. laevis developmental model. A windowed exposure (R-FETAX method) was applied covering the developmental phylotypic period (teratogenicity window), or the late tailbud stages (neuro-behavioural toxicity window, corresponding to the spontaneous swimming acquisition period). Samples were monitored for lethal effects during the full test period. External morphology evaluation and deglutition functional test were applied in any group. Abnormal tadpoles were also processed for cartilage staining. In groups exposed during neuro-behavioural toxicity window the swimming test was also applied. Lethality and malformations were obtained only in samples exposed during the teratogenicity window; these data were modelled using PROAST software and BPB relative potency resulted about 3 times higher than BPA. The day-by-day evaluation revealed that lethality was correlated to embryonic abnormal development of gills and apoptosis in gillprimordia. Teratogenicity was never detected in groups exposed during the neuro-behavioural toxicity window, where some significant neuro-behavioural deficits were detected in tadpoles exposed to the highest tested concentrations of BPA and BPB.
Fig. 1. BPA and BPB chemical structures. Note the extreme similarity between the two molecules.
Fig. 2. R-FETAX protocol: grey boxes represent the exposure windows, white boxes the maintenance in FETAX solution, the red cross the timing of observed BP-related lethality (approximately at NF 42/44 stages) after exposure during the phylotypic period (NF 10–26). PH= phylotypic period; LTB= late tailbud stages; TP= tadpole.
Fig. 3. Main experiment: phenotypes observed at the end of R-FETAX in groups exposed at phylotypic stages (NF 10–26, teratogenicity window).
External morphology: a-a’) normal phenotype of NF 46 tadpole. Note the linear encephalon (dotted line), the eye (*) representing the limit border between the anterior craniofacial region (#) and the branchial basket (+), the coiled intestine (>, index of NF 46 developmental stage reached) and the tail (ç). b) abnormal phenotype showing round head (dotted line) and ventral oedema (E). Normal coiling intestine (>). c) severe abnormal phenotypes with multiple defects, including round head, oedema and bent (B) or wavy (w) tail. Normal intestine coiling (>) confirms NF 46 stage reached. a-a’) DMSO-exposed tadpole; b) BPB 7.5 μM-exposed tadpole; c) BPB 7.5 μM-exposed tadpole. Red stain in the intestine= deglutition test positive. a-c) Magnification 8x; a’-b) Magnification 20x.
Cartilage evaluation d-e (flat-mount technique, magnification 40x): structure of facial and branchial skeletal elements in a DMSO-exposed tadpole (d) and in a BPB 7.5 μM-exposed tadpole classified as abnormal at the external evaluation (e). In e facial elements are reduced but not fused (= maxilla, ° mandible, ce ceratohyal cartilage); gill basket (gb) is shorter and smaller than normal.
Fig. 4. Superfast- R-FETAX. Morphology of tailbud exposed during the teratogenicity window (NF 10–26) and evaluated 1.5 days later (at NF 40) using a cold-light stereomicroscope (a, b). By definition, NF 40 stage is characterised by “mouth broken through, length of gills about twice their breadth, the anterior branched and posterior one sometimes also showing a branch, blood circulation in gills beginning, outlines of proctodeum and tailmyotomes forming angles of 90 degrees (>)”. A strict correlation between abnormal gillprimordia and apoptosis was evident: acridine orange staining shows fluorescent bright green apoptotic areas in atypical gillprimordia (b’). Unspecific yolk auto-fluorescence was visible at the ventral region (*, where the yolk mass (a-b, #) is abundant in intestines) in all samples. a-a’, DMSO normal sample, b-b’ BPA 25 μM sample with reduced gillprimordia, characterised by marked fluorescence. Dotted boxes indicate the branchial region. Magnification: a-b 20x; a’-b’ 80x.
Fig. 5. Superfast- R-FETAX. SEM fine morphology of tailbud exposed during the teratogenicity window (NF 10–26) and evaluated 1.5 days later (at NF 40). a-a’) normal gillprimordia: both gill rudiments (A, B) are typically branched and nipple-shaped, the branches of the anterior one well elongated. Extremely shortened and unbranched (b) or absent (c-c’) gillprimordia were observed in samples exposed at NF 10–26 to BPA 25 μM or BPB 7.5–10 μM. High magnification SEM images show the normal skin at NF 40 stage with three cell lines visible: keratinocytes (*), mucosal cells (#) and ciliated cells (@) both in DMSO-exposed normal samples (A’’) and in BPs-exposed samples (c’’). In c’’ (sample with gillprimordium agenesis) note an apoptotic cellular debris (+) about to be extruded from the surface. Dotted boxes indicate the gillprimordium regions and the area of subsequent magnification. a-a’-a’’), DMSO normal sample; b) BPB 7.5 μM; c-c’-c’’) BPB 10 μM. Magnification: a-b-c) 250x; a’-c’) 1k x; a’’-c’’) 10k x.
Fig. 6. Exponential models showing teratogenicity dose-response curves of BPA (a) and BPB (b) and curves modelled fixing c= 0 to derive the relative potency factor (RPF, c), indicating BPB (red line, cross) nearly three times more potent than BPA (black line, triangles). X axis= log10 dose; Y axis= % total effect (dead + malformed).
Fig. 7. Exemplificative swimming profiles of tadpoles exposed during the neuro-behavioural toxicity window (tailbud stages, NF 38–46) to DMSO alone (left) or to BPA 10 μM (right). Green circle represents the arena, light blue dotted circle represents the 0.75 inner circle area, set for data analysis. Yellow lines: swimming tracking elaboration. Note, in the left image, the normal behaviour with the tracking profile mostly limited to the outer ring and, in the right image, the disorganised swimming profile typical of BPA 10 μM tadpoles.