pharmaceuticals Article Influence of Estrogenic Metabolic Pathway Genes Polymorphisms on Postmenopausal Breast Cancer Risk Micaela Almeida 1 , Mafalda Soares 1 , José Fonseca-Moutinho 1,2, Ana Cristina Ramalhinho 1,2 and Luiza Breitenfeld 1,* 1 Health Sciences Research Centre (CICS), Faculty of Health Sciences, University of Beira Interior (UBI), Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal; [email protected] (M.A.); [email protected] (M.S.); [email protected] (J.F.-M.); [email protected] (A.C.R.) 2 Academic Hospital of Cova da Beira (CHUCB), Quinta do Alvito, 6200-251 Covilhã, Portugal * Correspondence: [email protected]; Tel.: +351-2753-290-51 Abstract: Estrogen metabolism plays an important role in tumor initiation and development. Lifetime exposure to high estrogens levels and deregulation of enzymes involved in estrogen biosynthetic and metabolic pathway are considered risk factors for breast cancer. The present study aimed to evaluate the impact of mutations acquisition during the lifetime in low penetrance genes that codify enzymes responsible for estrogen detoxification. Genotype analysis of GSTM1 and GSTT1 null polymorphisms, CYP1B1 Val432Leu and MTHFR C677T polymorphisms was performed in 157 samples of women with hormone-dependent breast cancer and correlated with the age at diagnosis. The majority of patients with GSTT1 null genotype and with both GSTM1 and GSTT1 null genotypes were 50 years old or more at the diagnosis (p-value = 0.021 and 0.018, respectively). Older women with GSTM1 CYP1B1 p GSTT1 Citation: Almeida, M.; Soares, M.; null genotype were also carriers of the Val allele ( -value = 0.012). As well, null Fonseca-Moutinho, J.; Ramalhinho, and CYP1B1Val genotypes were correlated with diagnosis at later ages (p-value = 0.022). Similar A.C.; Breitenfeld, L. Influence of results were found associating MTHFR C677T and GSTT1 null polymorphism (p-value = 0.034). Our Estrogenic Metabolic Pathway Genes results suggest that estrogen metabolic pathway polymorphisms constitute a factor to be considered Polymorphisms on Postmenopausal simultaneously with models for breast cancer risk assessment. Breast Cancer Risk. Pharmaceuticals 2021, 14, 94. https://doi.org/ Keywords: breast cancer; GSTM1; GSTT1; CYP1B1; MTHFR 10.3390/ph14020094 Academic Editors: Raquel Lima and Marcelo Correia 1. Introduction Received: 21 January 2021 Breast cancer is the most common cancer in women, counting 2.1 million cases di- Accepted: 23 January 2021 Published: 27 January 2021 agnosed in 2018 [1]. Breast cancer risk has long been associated with reproductive and hormonal history, lifestyle and hereditary [2]. Mainly, these risk factors are related to exposure to high levels of endogenous or exogenous estrogens and to mutations inherited Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in or acquired during lifetime [3]. published maps and institutional affil- Estrogens metabolism plays an important role in tumor initiation and development, iations. estrogen being considered a carcinogen [4]. This fact is not only due to the exposure to high levels of estrogen and to estrogen receptor (ER) status, to which estrogen binds to exerts its actions, but also to the possible deregulation of the enzymes involved in the estrogen biosyn- thetic and metabolic pathway, such as Cytochrome P450, family 1, subfamily B, polypep- tide 1 (CYP1B1), glutathione S-transferases (GSTs) and 5,10-methylenetetrahydrofolate Copyright: © 2021 by the authors. reductase (MTHFR) [5–8]. Licensee MDPI, Basel, Switzerland. This article is an open access article CYP1B1, codified by the CYP1B1 gene, located on chromosome 2p21–p22, is a Phase I distributed under the terms and enzyme, responsible for generating 4-hydroxyestradiol (4-OH-E2), a catechol estrogen conditions of the Creative Commons metabolite (Figure1)[9]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Pharmaceuticals 2021, 14, 94. https://doi.org/10.3390/ph14020094 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 9 Pharmaceuticals 2021, 14, 94 2 of 9 Figure 1. Schematic representation of the metabolic pathway of Oestradiol. CYP1B1 CYP1B1,, a Phase I enzyme, codified by the CYP1B1 gene,gene, leads leads to 4-OH-E2to 4-OH production.-E2 production. The Phase The IIPhase enzymes, II enzymes, COMT, codified COMT, by codified the COMT bygene, the andCOMT GSTM1/GSTT1, gene, and GSTM1/GSTT1,codified by GSTM1 codifiedand GSTT1 by GSTM1genes, and respectively, GSTT1 genes,inactivate respectively, the estrogen inactivate catechol, the semiquinoneestrogen cate andchol, quinone, semiquinone diminish- and quinone,ing DNA diminishing adducts formation. DNA adducts MTHFR, formation. an enzyme MTHFR, of the an folate enzyme metabolism, of the folate catalyzes metabolism,5,10-methylenetetrahydrofolate catalyzes 5,10-methylene- tetrahydrofolateto 5-methyltetrahydrofolate, to 5-methyltetrahydrofolate, which allows which the remethylation allows the remethylation of homocysteine of homocysteine to methionine, to methionine, a precursor a precursor of S- of S-adenosylmethionine (SAM). SAM is the methyl donor for COMT catalyzed reactions, allowing the inactivation of adenosylmethionine (SAM). SAM is the methyl donor for COMT catalyzed reactions, allowing the inactivation of cate- catechol estrogens. chol estrogens. AnAn important polymorphism in in CYP1B1 isis Val432Leu polymorphism, in in exon 3 (rs1056836;(rs1056836; location chromosome 2:38071060) [10]. [10]. Th Thisis polymorphism leads to an amino acidacid substitution ofof LeucineLeucine toto Valine,Valine, Val432 Val432 allele allele was was found found to to increase increase the the 4-hydroxilase 4-hydroxi- laseactivity activity ofCYP1B1 of CYP1B1 [11 [11].]. A A lack lack or or lowlow level of of Phase Phase II II detoxifying detoxifying enzymes, enzymes, such such as catecholas catechol-O-methyltransferase-O-methyltransferase (COMT) (COMT) and andGSTs GSTs,, might might lead leadto quinones to quinones accumulation, accumu- resultinglation, resulting in DNA inadducts DNA adductsformation formation and tumor and initiation tumor [3]. initiation Also, MTHFR [3]. Also, is involved MTHFR in is theinvolved conjugation in the and conjugation inactivation and of inactivation catechol estrogens of catechol by COMT. estrogens MTHFR by COMT. is the MTHFRkey en- zymeis the of key folate enzyme metabolism, of folate catalyzing metabolism, 5,10 catalyzing-methylenetetrahydrofolate 5,10-methylenetetrahydrofolate to 5-methyltetra- to hydrofolate,5-methyltetrahydrofolate, which allows the which remethylation allows the remethylationof homocysteine of to homocysteine methionine, toa precursor methion- ofine, S- aadenosylmethionine precursor of S-adenosylmethionine (SAM) [12]. In turn, (SAM) SAM [12 is]. the In turn,methyl SAM donor is the for methyl COMT donor cata- lyzedfor COMT reactions, catalyzed which reactions, allow inactivation which allow of inactivation catechol estrogens of catechol [12]. estrogens In this [regard,12]. In MTHFRthis regard, activity MTHFR alterations activity will alterations affect indirectly will affect the indirectly inactivation the of inactivation catechol estrogens of catechol by COMT.estrogens MTHFR by COMT. is codified MTHFR by the is codified MTHFR bygene, the locatedMTHFR ongene, chromosome located on1p36.3 chromosome [13]. One of1p36.3 the well [13].-studied One of the polymorphisms well-studied polymorphisms of MTHFR is the of MTHFR polymorphismis the polymorphism C677T (rs1801133, C677T location(rs1801133, chromosome location chromosome 1:11796321), 1:11796321), which leads which to a leads substitution to a substitution of alanine of alaninewith valine, with beingvaline, correlated being correlated with lower with activity lower activityof MTHFR of MTHFR[13,14]. Thus, [13,14 this]. Thus, polymorphism this polymorphism possibly affectspossibly COMT affects catalyzed COMT catalyzed reactions, reactions, compromi compromisingsing the catalyzation the catalyzation of 4-OH- ofE2 4-OH-E2 to 4-meth- to oxyestrogens4-methoxyestrogens (4-MeOE2) (4-MeOE2) [12]. [12]. TheThe GSTs, GSTs, a a superfamily superfamily of of Phase Phase II II enzymes, enzymes, are are responsible responsible for for metabolic metabolic detoxifi- detoxi- cationfication of of estrogen, estrogen, playing playing a akey key role role in in the the catalysis catalysis of glutathione (GSH) conjugation with catechol estrogen quinones, which are rapidly excreted by the cell [[3].3]. There are sevenseven classes classes of of cytosolic cytosolic GSTs: GSTs: alpha alpha (α), (α), mu mu (μ), (µ), kappa kappa (κ), (κ), pi pi (π), (π), theta theta (θ), (θ), omega (Ω) (W) andand zeta (ζ) (ζ)[ [15].15]. Among the polymorphisms studied in GSTs, Glutathione S-transferaseS-transferase ThetTheta1a1 ( (GSTT1GSTT1)) and and Glutathione Glutathione S- S-transferasetransferase Mu1 Mu1 (GSTM1 (GSTM1) null) null polymorphisms, polymorphisms, the ho- the mozygoushomozygous genotype genotype of which of which implies implies the total the total absence absence of the of enzyme, the enzyme, has previously has previously been relatedbeen related to breast to breast cancer cancer risk by risk our by research our research team team [16,17 [16]., GSTM117]. GSTM1 and andGSTT1 GSTT1 enzymes enzymes are are codified by the GSTM1 gene located on chromosome 1p13.3 and by the GSTT1 gene Pharmaceuticals 2021, 14, 94 3 of 9 located on chromosome 22q11.2, respectively [18–20].