Professor Pang Guangchang, Tianjin University of Commerce et al.: Application potential of G protein-coupled estrogen receptor in dairy estrogen detection

Milk and dairy products contain a variety of sources of estrogen, such as estrogen secreted by cows during milk production, and hormones and drugs artificially added during cow feeding
.
When estrogen in the environment, water and food exceeds a certain limit, estrogen may be enriched in the human body through the food chain, affecting human health
.
Estrogen is lipophilic and more susceptible to residue in foods of animal origin, which may lead to excessive estrogen levels in foods such as dairy products
.
Long-term intake of such dairy products or foods will cause a large amount of exogenous estrogen to enter the human body and accumulate in the body, which may endanger human health, so the analysis and detection of estrogen in dairy products is very important
.

A large number of experiments have found that estrogen interacts with G protein-coupled estrogen receptors (GPERs) to transmit rapid non-genomic signals, thereby exerting multiple types of biological effects, but the estrogen interference effects mediated by membrane receptor GPER have rarely been reported
.
Most of the research on GPER focuses on the biological and pharmaceutical fields, and there are few
reports in the field of food safety testing.
GPER research in the field of food safety testing involves the application of
estrogen receptor (ER) and aromatic hydrocarbon receptor (AHR).
Feng Chunlei, Lu Dingqiang* and Pang Guangchang* from the School of Biotechnology and Food Science, Tianjin University of Commerce, focused on the application potential of GPER in dairy estrogen detection, elaborated on the relationship between GPER and estrogen and estrogen detection in dairy products, and focused on the research progress
of biological detection methods based on estrogen signaling pathway.

1.
Estrogen in dairy products

Detection of estrogen in dairy products

The significance of estrogen detection in dairy products: all stages of dairy processing technology may be contaminated by chemical pollutants, especially the contamination of steroid hormones, which will affect human health after being ingested by the human body through the food chain and continuously accumulated, such as residual estrogen will have a certain damage to the human reproductive organs and liver, and may also induce cancer
.
Studies have shown that milk contains relatively high levels of estrogen and progesterone metabolites, especially from pregnant cows, which may increase the risk of
breast cancer.
In addition, diethylstilbestrol (DES) present in milk and dairy products may induce precocious puberty in children, uterine cancer and male feminization, and estrogen bisphenol A (BPA) present in milk and dairy product packaging can damage human fertility, which may cause recurrent miscarriage or premature birth in women and decreased semen quality and sperm DNA damage
in men.
Therefore, the detection of estrogen in dairy products is of great significance
.

Source and type of estrogen in dairy products: There are roughly two types of dairy estrogen: one is the natural estrogen secreted by dairy cows in the process of milk production, natural estrogen is naturally occurring estrogen in animals and humans, generally refers to E1, E2 and E3, of which E2 has the strongest effect, the chemical structure of E2 makes it biodegradable and fat-soluble, easy to enrich in the organism, the harm to the individual level mainly includes inducing hypertension, coronary heart disease and carcinogenic, Clinical basic studies have shown that E2 can greatly increase the incidence of gynecological tumors, and can also have a congenital carcinogenic effect on infants through the placenta, and also have toxic effects on the reproductive system and immune system; The second is exogenous addition, mainly including artificially added hormones, such as injecting hormone-containing milk-stimulating liquid into infertile cows, or using exogenous estrogen as an animal feed additive, or using feed containing fungal estrogen to feed cows
.
Exogenous addition of estrogen will interfere with the synthesis, secretion, binding, metabolism, digestion or production of similar to the organism's own estrogen, which will adversely affect
the normal estrogen dynamic balance, reproduction and growth of the organism.
The sources and types of different estrogens in dairy products are shown
in Table 1.

Problems with hormone testing in dairy products: dairy products are an important food
widely consumed by people.
Due to the estrogenic effects of estrogen-like compounds in milk and dairy products, there is increasing attention to the detection of estrogens in dairy products, but due to the complexity of dairy products, the analysis of estrogen-like compounds in dairy products has not been widely used compared to simple sample matrices (e.
g.
, water samples), although a few studies have analyzed natural and synthetic estrogens
in cheese samples.

In addition, in the European regulation on the use of hormones as animal growth promoters, the amendment directive states that the use of hormone-active substances in livestock fattening is prohibited to ensure the health of
residents within the EU.
Despite this legislation, there is no specific MRP for estrogenic compounds in dairy products, and hormones can still be abused
.
Studies have shown that exogenous estrogens such as BPA migrate from packaging materials to milk or dairy products, and although many studies report BPA in milk consumed by humans, there is little
research data on BPA content and contamination pathways in dairy products.
In addition, there are many problems in the current detection standards of estrogen in dairy products, such as the narrow coverage of estrogen types, the detection method is not novel, the sample processing method is complex, and the new estrogen structural analogues or their metabolites cannot be detected
.

Estrogen transmits signals through GPER receptors

Estrogen receptors: Estrogen performs physiological functions through genomic and non-genomic pathways mediated by ER and GPER, respectively (Figure 1).

The classical genomic way in which estrogen performs its physiological function is by binding to ER, which then forms ER dimers, which then bind to estrogen response elements (ERE) in the promoter sequence of the target gene to induce target gene expression
.
The non-genomic pathway is a rapid signaling pathway mediated by GPER, including cyclic AMP (cAMP) pathway, mitogen-activated protein kinase (MAPK) pathway, calcium ion (Ca2+) pathway, etc.
, the main pathway is to open or close^Ca2+ ion channels to rapidly control nerve and endocrine responses
.
With the discovery of ligands that selectively regulate GPER function in vitro and clinical studies, as well as the application of GPER knockout mice, more potential roles of GPER have attracted extensive attention
from researchers.

Estrogen classification of the action with GPER: The ligand species of GPER are widely distributed and are still in the research stage
.
Many ligands acting on ERα or ERβ can also act on GPER
.
The most widely used GPER-specific agonist today is G-1
.
Common GPER non-specific agonists contain natural ligand E2, it is reported that E2 has binding affinity for GPER in the range of 3~6 nmol/L, in addition, a series of exogenous estrogens synthesized (industrial, agricultural and pharmacological) and natural (plant and fungal estrogens) can also play a role as GPER agonists, such as phytoestrogens daidzein, resveratrol, genistein and environmental estrogens such as BPA and chemical raw materials nonylphenol (NP) commonly found in plastic products, etc.
Notably, tamoxifen and raloxifene are also GPER agonists
.
Specific antagonists of GPER include G-15 and G-36, and nonspecific antagonists include the intermediate metabolite of estrogen in vivo 2-hydroxy-17β-E2
.

2.
Detection method of estrogen in dairy products

In vivo experiments and cell proliferation experiments

The earliest estrogen biological detection method is in vivo experiment, among which uterine nutrition experiment and hepatocyte yolk protogen (VTG) production experiment is a conventional method for screening estrogen and estrogen analogues in the environment in China, and later in order to improve the detection efficiency of estrogen and estrogen analogues, a cell proliferation assay (E-Screen method) based on the effect of estrogen and estrogen analogues on target cell proliferation was established
。

In vivo experiments and E-Screen methods for estrogen detection have their own advantages and disadvantages, and although uterine nutrition experiments have the advantages of high reliability and simple operation, they take a long time, are not suitable for large-scale screening, and have poor
reproducibility.
In addition, hepatocyte VTG generation experiment has the advantages of high sensitivity and convenient operation, but its application will be limited
by the presence of natural yolk in female animals.
Compared with the above methods, the E-Screen method is more sensitive, usually combined with in vivo experiments to ensure the reliability of the results, the detection results obtained by cell proliferation experiments with MCF7 cells are highly reliable, because MCF7 cells are derived from humans, which excludes the uncertainty of animal experiments, but the experimental conditions, medium and serum required by this method have special requirements, so the experimental conditions and costs are higher
.

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