PFCs contribute to PP by affecting endocrine disorders in girls
In this study, the levels of basal LH and FSH, estradiol, prolactin, testosterone, and DHEAS in PT and CPP indicated that the HPG axis is not activated in the PT girls but activated in the CPP girls, which was also confirmed by the GnRH stimulation test [
30]. With activation of HPG axis, hypothalamus increasingly secretes gonadotropin GnRH, anterior pituitary gland increasingly secretes FSH and LH, and gonad increasingly secretes estradiol and testosterone. The higher level of estradiol (aromatized from testosterone) in girls before puberty is associated with an earlier thelarche, and it can affect and maintain cognitive function, regulate the sexual behavior and ovulation in the brain, and regulate higher order neural function [
31,
32]. Behr et al. found that PFOA and PFOS can enhance estradiol-stimulated estrogen receptor β activity, and PFOS and PFBA can enhance dihydrotestosterone-stimulated androgen receptor activity [
33]. In this study, PFBA was positively correlated with estradiol, PFBS was negatively correlated with prolactin in the CPP girls, and PFUnDA, PFDA, and PFNA were positively correlated with prolactin in the PT girls, which indicated that PFCs mainly caused developmental and reproductive toxicity by disrupting the body’s endocrine homeostasis, thus leading to an imbalance in the body’s steroid hormone secretion.
DHEAS is a stable marker for adrenal androgenic activity. At the biological level, DHEAS has effects on brain development, sexuality, mood and cognition, cardiovascular disease, stroke, and mortality [
34]. Relevant longitudinal studies show that higher DHEAS level at the age of 8 predicts early menarche and doubles the risk of pubic hair development in girls [
35]. In this study, the higher level of DHEAS in the CPP girls may be the result of a combination of adrenaline secretion and gonadal secretion, and it further leads to significant secondary sexual characteristics of the CPP girls. On the other hand, the increased level of DHEAS may potentially provide additional energy for the metabolic costs of early brain development, and also acts as a co-factor in promoting cortical maturation, thus leading to increased capacity for mentalizing and perspective-taking before the onset of reproductive maturation. This conclusion could be supported by the WGCNA analysis, where MEyellow in CPP and MEbrown in PT driven by DHEAS showed glucose aggregation. In addition, the significantly higher level of glucose in the CPP girls than in the PT girls also indicated a more active energy metabolism in CPP.
Interestingly, in this study, the BMI of CPP was higher than that of other groups. Studies have shown that the HPG axis initiates when girls’ body reaches a certain fat and/or protein mass [
36]; therefore, the occurrence of CPP may be closely related to high BMI of girls. It is possible that obesity or overweight can promote the occurrence and development of CPP, which could be confirmed by the findings of many other scholars [
37,
38]. In addition, the relationship between PFCs and VD in CPP and PT indicated PFCs partly disturbed osteogenesis [
39], and also revealed new potential long-term PFC impacts on children.
CPP is primarily an early initiation of the HPGA axis, and hypothalamus generates a GnRH pulse that stimulates the pituitary gonadotropin secretion. Therefore, the corresponding core metabolic network mainly reflects the cause of activation of the HPGA axis and the metabolic disorder in the CPP girls. The link of clinical phenotype-PFCs-metabolic characteristics in the CPP girls indicated that PFCs may cause disturbance of metabolic network of CPP girls by disturbing endocrine homeostasis and/or directly affecting metabolite characteristics.
Tyrosine and phenylalanine are the precursor for catecholamines including tyramine, dopamine, epinephrine, and norepinephrine. The sympathetic nervous system neurotransmitters and catecholamines, especially norepinephrine, play an important role in the regulation of GnRH neurons [
40]. The downregulated levels of phenylalanine and tyrosine in serum of the CPP girls indicated that the biosynthesis of phenylalanine, tyrosine, and tryptophan was disturbed, which keeps consistent with the previous metabolomics results [
5]. The downregulation of phenylalanine and tyrosine levels may be due to the more consumption of norepinephrine for the activation of HPG axis, resulting in lower levels of its precursor substances. Such conclusion could be confirmed by the WGCNA analysis, where the modules MEblue and MEturquoise were primarily driven by prolactin and DHEAS (Fig.
6b1, c1).
Serine and glycine are connected through biosynthesis to provide necessary synthetic precursors of proteins, nucleic acids, and lipids. At the same time, serine homeostasis plays a vital role in maintaining brain energy metabolism [
41]. The homeostasis of serine/glycine is essential for the proliferation of human primary muscle progenitor cells and efficient skeletal muscle regeneration [
42]. Therefore, the decreased serine level in the CPP girls may be one of the reasons for the lifelong high short stature in adults, but further research is needed to confirm it. Creatine is essential in maintaining human growth, development, and health and can improve skin and bone health [
43]. Creatinine was selected as a biomarker for predict CPP in Qi’s study [
5], and it was regarded as one of the specific biomarkers for CPP in this study. Creatinine is linked to muscle mass, and the lower level of serum creatinine observed in the CPP girls could be a result of the excessive weight and relative low muscle/fat ratio caused by low physical activity. In the MEyellow and MEblue, creatine and creatinine are mainly related to amino acids and driven by multiple PFCs (PFOA, PFNA, PFDA), DHEAS, and prolactin (Fig.
6b1, c1), indicated that exposure to PFCs and/or endocrine disturbance will cause amino acid metabolism disturbance, thus affecting the bone growth of girls.
The increased levels of α-&β-glucose and lactose and decreased level of hypoxanthine in the CPP girls suggested the disturbed glycolysis/gluconeogenesis (energy metabolism). PFOA, PFDA, and PFNA were positively correlated with PFOS and mainly drove MEyellow containing α-&β-glucose. Studies have shown that the accumulation of PFCs, especially PFOS, contributes to the disorder of lipid and glucose metabolisms in children, but the mechanism is still in the initial stage of exploration [
12,
44]. Compared with adolescent girls, the CPP girls had lower insulin sensitivity, glucose and lipid metabolism profile, and body composition, and the metabolic disturbance remained unchanged even after 1 year of GnRH treatment [
45,
46]. The accumulation of PFCs may be a crucial reason for the decreased insulin sensitivity and high level of serum glucose in the CPP girls [
47].
Glutamate and GABA are the principal excitatory and inhibitory neurotransmitters. Their interactions with GnRH neurons, including the regulation of GnRH gene and protein expression, hormone release, and modulation by estrogen, are critical to age-appropriate changes in reproductive function [
48]. Glutamate is also crucial for bone growth and reconstruction [
49]. In our study, the significantly decreased glutamate level in the CPP girls implies its dynamic change in the development of CPP. We speculated that the increased glutamate level stimulated the HPG axis and induced CPP in the early stage. However, the occurrence of CPP is often accompanied with rapid height growth, and subsequently the glutamate in bone and blood is excessively consumed at a certain stage of CPP.
In addition, the downregulated levels of isoleucine, alanine, valine, methionine, histidine, and α-ketoisovalerate in the CPP girls indicated the disturbed aminoacyl-tRNA biosynthesis. The disruption of aminoacyl-tRNA biosynthesis further supports our inference that body needs to consume large amounts of amino acids during the initial growth spurt and rapid bone maturation stage of the CPP girls. The significantly reduced level of VD in the CPP girls further indicated the affected bone growth of the CPP girls. It is possible that the lack of VD affected the absorption and deposition of calcium in the bone, thus affecting the health and growth of bones. Balance of aminoacyl-tRNA biosynthesis is closely related to bone health, and its disruption can lead to osteocyte protein synthesis dysfunction, marrow hypoplasia, and osteoporosis [
50]. This may also be the reason for the short height of CPP girls at adult. However, whether appropriate supplementation of amino acids in diet can improve the adult height, or whether aminoacyl-tRNA biosynthesis can become a targeted therapeutic metabolic pathway for CPP girls is worthy of further research and discussion.
Purines and pyrimidines give prominent contributions in the development of the central nervous system, but the exact molecular mechanisms remain unclear [
51]. PP girls have an increased risk of precocious sexual behavior and an increased prevalence of mental disorders such as depression and anxiety in adulthood [
52,
53]. In this study, the downregulated serum levels of hypoxanthine, dihydrothymine, and uridine were observed in the CPP girls. Dihydrothymine belongs to MEblack jointly driven by PFOA, PFNA, DHEAS, FT4, and VD. This suggested that the disturbed pyrimidine metabolic pathway may be caused by the joint action of external environment and internal factors. CPP girls need to advance adaption to the cognitive, emotional, and changes of puberty prematurely, therefore, the sense of anxiety and depression increases significantly, resulting in a certain disorder of pyrimidine metabolism.
PT does not involve the early initiation of HPGA axis and keep normal growth of height and maturation of bone age. Its occurrence is mainly due to the breast enlargement caused by exogenous hormone intake. In Qi et al. study [
5], upregulated levels of succinate and 1-methylhistidine in urine can effectively predict PT. In this study, it was found that most amino acids showed upregulated in PT girls, including the specific biomarker 1-methylhistidine and the potential biomarker succinate. Amino acids are not only the building blocks of proteins and an indispensable component of cells, but also play versatile roles in regulating cell metabolism, proliferation, differentiation, and growth by themselves or their derivatives. Their requirements vary at the various stages of children’s growth and development [
54,
55]. Studies have shown that arginine and ornithine supplementation can promote the secretion of growth hormone and insulin growth factor-1 [
56]. We speculated that the nutritional diets promote the high level of amino acid in the PT girls for providing energy for the growth and development. But the more relevant reason may be that exposure to PFCs disturbs the body’s amino acid metabolism, endocrine homeostasis, and vitamin level, which will affect the girl’s bone health and growth.
In this study, the metabolites in glycerolipid metabolic pathway and galactose metabolic pathway including
N,N-dimethylglycine, ethanolamine, glycerol, dihydrothymine, and
myo-inositol were upregulated in the PT girls. Studies have shown that thyroid dysfunction will affect the body’s glycerol metabolism and gluconeogenesis pathway [
57,
58]. In this study, FT4 and TFHSA mainly drive MEpink containing glycerol, and PFHpA mainly drives MEred containing low-density lipoprotein,
myo-inositol, phosphocholine, and glycerophosphorycholine, suggesting that the PFCs exposure and high level FT4 may be the main reason contributed to the disorder of serum glycerolipid metabolic and galactose metabolism in PT. However, this study cannot directly prove the causation between the elevated FT4 level and PFCs exposure in PT.
Interestingly, the disordered trend of pyruvate metabolism and butyrate metabolism kept almost consistent between CPP and PT (Fig.
5), indicating that pyruvate metabolism and butyrate metabolism disorder are possibly a common phenomenon of PP. In other words, simultaneously elevated levels in serum formate, ethanol, and 3-hydroxybutyrate may serve as the early diagnostic indicators for CPP and PT, i.e., precocious puberty in girls, but the stratification of PP still needs to be further determined based on the serum-specific biomarkers. Ethanol may act on bone remodeling including osteocyte apoptosis [
59,
60]. Elevated level of 3-hydroxybutyrate will inhibit the differentiation and growth of (pre)-chondrocytes [
61] and participate in the process of osteoporosis [
62]. There is a strong correlation between PFOA and PFNA in CPP and PT. Ethanol and 3-hydroxybutyrate belong to MEred which were jointly driven by PFNA and FT4 in CPP. In PT, ethanol and 3-hydroxybutyrate belong to the MEblack which were jointly driven by PFOA and FT4. It is suggested that PFC exposure and/or fluctuations in FT4 level will lead to elevated ethanol and 3-hydroxybutyrate in PP and thus affects bone growth and development, but more evidence is needed to prove this point.
It should be pointed out that this study had several limitations. Firstly, the results were obtained from a smaller-sample-size cohort, and measurements in an expanded cohort are necessary to validate the disease-specific biomarkers and determine their universality. Secondly, clinical reports have revealed the gender-related differences of PP not only in symptoms but also in pathophysiology. Therefore, the present findings could not naturally extend to boys, and further study on the sex-specific metabolic characteristics of PP is needed to comprehensively understand the underlying pathogenic mechanisms of PP. Finally, the detailed mechanism of how PFCs lead to endocrine homeostasis imbalance still keeps unclear and needs further study.