Anna Mattsson

The plastic component bisphenol A (BPA) impairs reproductive organ development in various experimental animal species. In birds, effects are similar to those caused by other xenoestrogens. Because of its endocrine disrupting activity, BPA is being substituted with other bisphenols in many applications. Using the chicken embryo model, we explored whether the BPA alternatives bisphenol AF (BPAF), bisphenol F (BPF), and bisphenol S (BPS) can induce effects on reproductive organ development similar to those induced by BPA. Embryos were exposed in ovo from embryonic day 4 (E4) to vehicle, BPAF at 2.1, 21, 210, and 520 nmol/g egg, or to BPA, BPF, or BPS at 210 nmol/g egg and were dissected on embryonic day 19. Similar to BPA, BPAF and BPF induced testis feminization, manifested as eg testis-size asymmetry and ovarian-like cortex in the left testis. In the BPS-group, too few males were alive on day 19 to evaluate any effects on testis development. We found no effects by any treatment on ovaries or Müllerian ducts. BPAF and BPS increased the gallbladder-somatic index and BPAF, BPF and BPS caused increased embryo mortality. The overall lowest-observed-adverse-effect level for BPAF was 210 nmol/g egg based on increased mortality, increased gallbladder-somatic index, and various signs of testis feminization. This study demonstrates that the BPA replacements BPAF, BPF, and BPS are embryotoxic and suggests that BPAF is at least as potent as BPA in inducing estrogen-like effects in chicken embryos. Our results support the notion that these bisphenols are not safe alternatives to BPA.

Bisphenol A (BPA) and phthalate diesters are ubiquitous environmental contaminants. While these compounds have been reported as reproductive toxicants, their effects may partially be attributed to metabolites. The aim of this study was to examine reproductive organ development in chicken embryos exposed to the BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP; 100 µg/g egg) or a human-relevant mixture of 4 phthalate monoesters (85 µg/g egg). The mixture was designed within the EU project EDC-MixRisk based upon a negative association with anogenital distance in boys at 21 months of age in a Swedish pregnancy cohort. Chicken embryos were exposed in ovo from an initial stage of gonad differentiation (embryonic day 4) and dissected two days prior to anticipated hatching (embryonic day 19). No discernible effects were noted on reproductive organs in embryos exposed to the mixture. MBP-treated males exhibited retention of Müllerian ducts and feminization of the left testicle, while MBP-administered females displayed a diminished the left ovary. In the left testicle of MBP-treated males, mRNA expression of female-associated genes was upregulated while the testicular marker gene SOX9 was downregulated, corroborating a feminizing effect by MBP. Our results demonstrate that MBP, but not the phthalate monoester mixture, disrupts both male and female reproductive organ development in an avian embryo model.

Perfluoroalkyl acids (PFAAs) are ubiquitous environmental contaminants and eggs and nestlings of raptors and fish-eating birds often contain high levels of PFAAs. We studied developmental effects of a mixture of ten PFAAs by exposing chicken embryos to 0.5 or 3 μg/g egg of each compound in the mixture. Histological changes of the thyroid gland were noted at both doses and increased expression of mRNA coding for type III deiodinase was found at 0.5 μg/g egg. Serum concentrations of the free fraction of thyroid hormones (T3 and T4) were reduced by the PFAA mixture at 3 µg/g egg, which is in line with a decreased synthesis and increased turnover of thyroid hormones as indicated by our histological findings and the decreased mRNA expression of type III deiodinase. The relative weight of the bursa of Fabricius increased at a dose of 3 μg/g egg in females. The bursa is the site of B-cell development in birds and is crucial for the avian adaptive immune system. Analysis of plasma and liver concentrations of the mixture components showed differences depending on chain length and functional group. Our results highlight the vulnerability of the thyroid hormone and immune systems to PFAAs.

Excess estrogen exposure of avian embryos perturbs reproductive organ development in both sexes and demasculinizes the reproductive behaviors of adult males. We have previously shown that these characteristic effects on the reproductive organs also can be induced by exposure of Japanese quail (Coturnix japonica) embryos to selective agonists of estrogen receptor alpha (ERα). In contrast, the male copulatory behavior is only weakly affected by developmental exposure to an ERα agonist. To further elucidate the respective roles of ERα and ERβ in estrogen-induced disruption of sexual differentiation, we exposed Japanese quail embryos in ovo to the selective ERα agonist 16α-lactone-estradiol (16αLE₂), the selective ERβ agonist WAY-200070, or both substances in combination. The ERα agonist feminized the testes in male embryos and reduced cloacal gland size in adult males. Furthermore, anomalous retention and malformations of the Müllerian ducts/oviducts were seen in embryos and juveniles of both sexes. The ERβ agonist did not induce any of these effects and did not influence the action of the ERα agonist. Male copulatory behavior was not affected by embryonic exposure to either the ERα- or the ERβ-selective agonist but was slightly suppressed by treatment with the two compounds combined. Our results suggest that the reproductive organs become sexually differentiated consequent to activation of ERα by endogenous estrogens; excessive activation of ERα, but not ERβ, during embryonic development may disrupt this process. Our results also suggest that the demasculinizing effect of estrogens on male copulatory behavior is only partly mediated by ERα and ERβ, and may rather involve other estrogen-responsive pathways.

Untargeted metabolic profiling of body fluids in experimental animals and humans exposed to chemicals may reveal early signs of toxicity and indicate toxicity pathways. Avian embryos develop separately from their mothers, which gives unique possibilities to study effects of chemicals during embryo development with minimal confounding factors from the mother. In this study we explored blood plasma and allantoic fluid from chicken embryos as matrices for revealing metabolic changes caused by exposure to chemicals during embryonic development. Embryos were exposed via egg injection on day 7 to the environmental pollutant perfluorooctanoic acid (PFOA), and effects on the metabolic profile on day 12 were compared with those caused by GW7647 and rosiglitazone, which are selective agonists to peroxisome-proliferator activated receptor α (PPARα) and PPARγ, respectively. Analysis of the metabolite concentrations from allantoic fluid by Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) showed clear separation between the embryos exposed to GW7647, rosiglitazone, and vehicle control, respectively. In blood plasma only GW7647 caused a significant effect on the metabolic profile. PFOA induced embryo mortality and increased relative liver weight at the highest dose. Sublethal doses of PFOA did not significantly affect the metabolic profile in either matrix, although single metabolites appeared to be altered. Neonatal mortality by PFOA in the mouse has been suggested to be mediated via activation of PPARα. However, we found no similarity in the metabolite profile of chicken embryos exposed to PFOA with those of embryos exposed to PPAR agonists. This indicates that PFOA does not activate PPAR pathways in our model at concentrations in eggs and embryos well above those found in wild birds. The present study suggests that allantoic fluid and plasma from chicken embryos are useful and complementary matrices for exploring effects on the metabolic profile resulting from chemical exposure during embryonic development.