Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents.

The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 µg/L or 59 µM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (-4.10 kcal mol-1) compared to estradiol (-25.79 kcal mol-1), which suggests that activation of this receptor by glyphosate is via a ligand-independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERα in vitro, albeit at relatively high concentrations.

Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells.

Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in "BPA-free" products. We have compared estrogenic activity of BPA with 6 bisphenol analogues [bisphenol S (BPS); bisphenol F (BPF); bisphenol AP (BPAP); bisphenol AF (BPAF); bisphenol Z (BPZ); bisphenol B (BPB)] in 3 human breast cancer cell lines. Estrogenicity was assessed (10-11-10-4 M) by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element-mediated transcription in a luciferase assay. BPAF was the most potent bisphenol, followed by BPB > BPZ ∼ BPA > BPF ∼ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs. Data mining of ToxCast high-throughput screening assays confirm our results but also show divergence in the sensitivities of the assays. Gene expression profiles were determined in MCF-7 cells by microarray analysis. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα gene expression biomarker further indicates that all BPA alternatives act as ERα agonists in MCF-7 cells. These results were confirmed by Illumina-based RNA sequencing. In conclusion, BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. These findings point to the importance of better understanding the risk of adverse effects from exposure to BPA alternatives, including hormone-dependent breast cancer.

Variations in the 3'UTR of the CYP21A2 Gene in Heterozygous Females with Hyperandrogenaemia.

Heterozygosity for CYP21A2 mutations in females is possibly related to increased risk of developing clinical hyperandrogenism. The present study was designed to seek evidence on the phenotype-genotype correlation in female children, adolescents, and women with CYP21A2 mutations and variants in the 3'UTR region of the gene. Sixty-six patients out of the 169 were identified as carriers of CYP21A2 mutations. Higher values of stimulated 17 hydroxyprogesterone (17-OHP) levels were found in the carriers of the p.Val281Leu mutation compared to the carriers of other mutations (mean: 24.7 nmol/l versus 15.6 nmol/l). The haplotype of the ∗52C>T, ∗440C>T, and ∗443T>C in the 3'UTR was identical in all heterozygous patients with p.Val281Leu and the haplotype of the ∗12C>T and ∗52C>T was identical in all heterozygous patients with the p.Gln318∗. In conclusion, hyperandrogenaemic females are likely to bear heterozygous CYP21A2 mutations. Carriers of the mild p.Val281Leu mutation are at higher risk of developing hyperandrogenism than the carriers of more severe mutations. The identification of variants in the 3'UTR of CYP21A2 in combination with the heterozygous mutation may be associated with the mild form of nonclassic congenital adrenal hyperplasia and reveal the importance of analyzing the CYP21A2 untranslated regions for the appropriate management of this category of patients.

Phenotypic variability of hyperandrogenemia in females heterozygous for CYP21A2 mutations.

OBJECTIVES: The objective was to seek evidence on the prevalence and consequences of heterozygous CYP21A2 mutations in girls, adolescent, and adult females with clinical manifestation of androgen excess. PATIENTS AND METHODS: The study included 64 girls diagnosed with premature adrenarche (PA) in childhood and 141 females with clinical hyperandrogenemia manifested in adolescence or adulthood. Direct DNA sequencing and multiplex ligation-dependent probe amplification analysis were used to identify mutations in the CYP21A2 gene. RESULTS: (1) Thirty-four patients were diagnosed with nonclassical-congenital adrenal hyperplasia (NC-CAH) based on the 17-hydroxyprogesterone (17-OHP) levels and the presence of two mutations in CYP21A2 and therefore were excluded from the study, 66 were found to be heterozygotes and finally 105 had no identifiable mutations. The most frequent mutations among the carriers were the mild p.Val281 Leu and p.Qln318stop. Higher levels of mean stimulated 17-OHP were found in the carriers of the p.Val281 Leu. (2) A notable increased allelic frequency for the known p.Asn493 Ser polymorphism was observed in the pool of females with hyperandrogenemia in whom no mutation was identified. (3) In girls, who presented early with PA, 26.6% were diagnosed with NC-CAH and carried two mutations, 28.7% were identified as heterozygotes 43.7% had no identifiable genetic defect in the translated region of the CYP21A2 gene. On the contrary, in the group of 141 females with late onset hyperandrogenemia, the presence of 2 mutations was detected in 12%, 1 mutation in 33.4% and no mutation in 54.6%. CONCLUSIONS: The carrier status for 21-OHD, may be an important factor in the variable phenotype of hyperandrogenism and may be a contributing factor for the early manifestation of the disease.

Overview of genetic defects in endocrinopathies in the island of Cyprus; evidence of a founder effect.

AIM: Hereditary endocrinopathies in Cyprus exhibit evidence of a founder effect and display the influence of past migration patterns. The genetic frequency and mutation pattern of a specific disorder of sex development (DSD), which is classified as 46,XX DSD or 46,XY DSD, and the non-classic form of congenital adrenal hyperplasia (NC-CAH) outline a type of genetic drift. RESULTS: Not only the high prevalence of the NC-CAH p.V281L mutation but also the rarity of CAH large lesions present a genetic diversity similar to that observed in the Middle Eastern countries. In addition, both the high frequency of the 5-alpha steroid reductase deficiency (5αSRD) IVS1-2A>G mutation and the carrier frequency of the 17-beta hydroxysteroid dehydrogenase 3 (17ß-HSD-3) p.R80Q mutation are good examples of a founder effect. p.R80Q can be considered a founder mutation, even though it has been identified in patients of Dutch, Brazilian, and Portuguese origin. This has led to the speculation that it has a Phoenician origin. Phoenicians as ancient traders migrated around 750 BC from present day Syria, Lebanon, and Israel toward Portugal, Spain, and also to nearby Cyprus. While the 5αSRD IVS1-2A>G mutation has already been extensively reported in Turkish patients, it is very common in the Eastern Mediterranean region. CONCLUSION: This short article portrays clearly, through specific endocrine genetic disorders, the past migration trends in Cyprus that shaped the present-day gene pool of the Greek-Cypriot population.

Characterization of antioxidant/anti-inflammatory properties and apoA-I-containing subpopulations of HDL from family subjects with monogenic low HDL disorders.

BACKGROUND: Genetic factors regulate both high-density lipoprotein (HDL) levels and functionality, thus affecting HDL antiatherogenic properties. We characterized the HDL antioxidant/anti-inflammatory properties and apoA-I-containing subpopulations in families with monogenic low HDL disorders. METHODS: Subjects with mutations in apolipoprotein A-I (apoA-I), ATP-binding cassette transporter A1 (ABCA1) or lecithin:cholesterol acyltransferase (LCAT) and family controls were studied. HDL antioxidant/anti-inflammatory properties were assayed by an in vitro fluorometric method and HDL-associated paraoxonase-1 (PON1), platelet activating factor-acetylhydrolase (PAF-AH), LCAT, malondialdehyde (MDA), PAF and serum amyloid A (SAA) were measured. ApoA-I-containing HDL subpopulations were analyzed by two-dimensional non-denaturing gel electrophoresis. RESULTS: ApoA-I heterozygotes and subjects with partial or complete ABCA1 or LCAT deficiency had HDL with reduced antioxidant/anti-inflammatory properties and increased MDA levels. HDL-PON1 activity was reduced in apoA-I heterozygotes and in subjects with complete ABCA1 deficiency. HDL-PAF-AH activity was reduced in subjects with partial or complete ABCA1 deficiency or complete LCAT deficiency. HDL-LCAT activity was reduced in all LCAT mutation carriers. HDL-PAF levels were increased in apoA-I heterozygotes. HDL-SAA levels were increased in subjects with complete ABCA1 deficiency. ApoA-I, ABCA1 and LCAT heterozygotes were depleted of the large α1 HDL subpopulation. Subjects with complete LCAT deficiency showed mostly the small α4 HDL subpopulation and subjects with complete ABCA1 deficiency the α4 and preß HDL subpopulations. CONCLUSIONS: This study shows that mutations in apoA-I, ABCA1 and LCAT have direct effect on the antioxidant/anti-inflammatory properties of HDL. Furthermore, our study shows the effect of specific mutations on the apoA-I-containing HDL subpopulation profiles.