Sex- and age-dependent effects of maternal organophosphate flame-retardant exposure on neonatal hypothalamic and hepatic gene expression

Abstract

After the phase-out of polybrominated diphenyl ethers, their replacement compounds, organophosphate flame retardants (OPFRs) became ubiquitous in home and work environments. OPFRs, which may act as endocrine disruptors, are detectable in human urine, breast milk, and blood samples collected from pregnant women. However, the effects of perinatal OPFR exposure on offspring homeostasis and gene expression remain largely underexplored. To address this knowledge gap, virgin female mice were mated and dosed with either a sesame oil vehicle or an OPFR mixture (tris(1,3-dichloro-2-propyl)phosphate, tricresyl phosphate, and triphenyl phosphate, 1 mg/kg each) from gestational day (GD) 7 to postnatal day (PND) 14. Hypothalamic and hepatic tissues were collected from one female and one male pup per litter on PND 0 and PND 14. Expression of genes involved in energy homeostasis, reproduction, glucose metabolism, and xenobiotic metabolism were analyzed using quantitative real-time PCR. In the mediobasal hypothalamus, OPFR increased Pdyn, Tac2, Esr1, and Pparg in PND 14 females. In the liver, OPFR increased Pparg and suppressed Insr, G6pc, and Fasn in PND 14 males and increased Esr1, Foxo1, Dgat2, Fasn, and Cyb2b10 in PND 14 females. We also observed striking sex differences in gene expression that were dependent on the age of the pup. Collectively, these data suggest that maternal OPFR exposure alters hypothalamic and hepatic development by influencing neonatal gene expression in a sex-dependent manner. The long-lasting consequences of these changes in expression may disrupt puberty, hormone sensitivity, and metabolism of glucose, fatty acids, and triglycerides in the maturing juvenile.

Keywords: Endocrine disruptors; Gene expression; Hypothalamus; Liver; Maternal exposure; Organophosphate flame retardants; Sex differences.

Figure 1.. Body weight and anogenital distance.

A) Body weights (BW) in PND 0 and PND 14 female and male pups from dams orally dosed with vehicle or OPFR mixture starting on GD7. Sample size of 6 pups per group, one from each litter at each age. B) Anogenital distance (AGD) normalized to cubed root of body weight at PND 7 in female and male pups. Lowercase letters denote age differences, lowercase letters with capped bar underneath denote effects of OPFR, and uppercase letters denote sex differences (a = p < 0.05; b = p < 0.01; D/d = p < 0.0001). Data are represented as mean ± SEM, A: n = 6; B: n = 7–9 litters.

Figure 2.. Hypothalamic gene expression in PND 0 and PND 14 neonates - Neuropeptides.

Relative gene expression in PND 0 and PND 14 female and male pups from dams orally dosed with vehicle or OPFR mixture. A) Pomc; B) Cart; C) Npy; and D) Agrp. Lowercase letters denote sex differences and lowercase letters with capped bar underneath denote OPFR effects (a = p < 0.05). Data are represented as mean ± SEM, n = 5–6.

Figure 3.. Hypothalamic gene expression in PND 0 and PND 14 neonates - KNDy neurons.

Relative gene expression in PND 0 and PND 14 female and male pups from dams orally dosed with vehicle or OPFR mixture. A) Kiss1; B) Pdyn; C) Tac2; D) Bdnf; and E) Foxo1. Lowercase letters denote sex differences and lowercase letters with capped bar underneath denote OPFR effects (a = p < 0.05; b = p < 0.01). Data are represented as mean ± SEM, n = 5–6.

Figure 4.. Hypothalamic gene expression in PND 0 and PND 14 neonates - Receptors.

Relative gene expression in PND 0 and PND 14 female and male pups from dams orally dosed with vehicle or OPFR mixture. A) Esr1; B) Esr2; C) Pparg; D) Lepr; E) Insr; and F) Ghsr. Lowercase letters with capped bar underneath denote OPFR effects (a = p < 0.05; b = p < 0.01). Data are represented as mean ± SEM, n = 5–6.

Figure 5.. Hepatic gene expression in PND 0 and PND 14 neonates - Receptors.

Relative gene expression in PND 0 and PND 14 female and male pups from dams orally dosed with vehicle or OPFR mixture. A) Ppara; B) Pparg; C) Esr1; D) Insr; E) Lepr; and F) Kiss1. Lowercase letters denote sex differences and lowercase letters with capped bar underneath denote OPFR effects (a = p < 0.05; b = p < 0.01; d = p <. 0001). Data are represented as mean ± SEM, n = 5–6.

Figure 6.. Hepatic gene expression in PND 0 and PND 14 neonates - Enzymes.

Relative gene expression in PND 0 and PND 14 female and male pups from dams orally dosed with vehicle or OPFR mixture. A) G6pc; B) Pepck; C) Foxo1; D) Dgat2; and E) Fasn. Lowercase letters denote sex differences and lowercase letters with capped bar underneath denote OPFR effects (a = p < 0.05; b = p < 0.01; c = p < 0.001; d = p <. 0001). Data are represented as mean ± SEM, n = 5–6.

Figure 7.. Hepatic gene expression in PND 0 and PND 14 neonates - Xenobiotic Targets.

Relative gene expression in PND 0 and PND 14 female and male pups from dams orally dosed with vehicle or OPFR mixture. A) Bsep; B) Cd36; C) Cyp2b10; D) Cyp3a11; E) Cyp4a10; and F) Cyp7a1. Lowercase letters denote sex differences and lowercase letters with capped bar underneath denote OPFR effects (a = p < 0.05; b = p < 0.01).Data are represented as mean ± SEM, n = 5–6.