Chemico-Biological Interactions 183 (2010) 40–48 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint The food contaminant semicarbazide acts as an endocrine disrupter: Evidence from an integrated in vivo/in vitro approach Francesca Maranghi a,∗, Roberta Tassinari a, Daniele Marcoccia a, Ilaria Altieri a, Tiziana Catone b, Giovanna De Angelis b, Emanuela Testai b, Sabina Mastrangelo c, Maria Grazia Evandri c, Paola Bolle c, Stefano Lorenzetti a a Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy b Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy c Department of Physiology and Pharmacology “Vittorio Erspamer” Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy article info abstract Article history: Semicarbazide (SEM) is a by-product of the blowing agent azodicarbonamide, present in glass jar-sealed Received 3 August 2009 foodstuffs mainly baby foods. The pleiotropic in vivo SEM toxicological effects suggested to explore its Received in revised form possible role as endocrine modulator. Endocrine effects of SEM were assessed in vivo in male and female 18 September 2009 rats after oral administration for 28 days at 0, 40, 75, 140 mg/kg bw pro die during the juvenile period. Accepted 21 September 2009 Vaginal opening and preputial separation were recorded. Concentration of sex steroid in blood, the ex Available online 27 September 2009 vivo hepatic aromatase activity and testosterone catabolism were detected. The in vitro approach to test SEM role as (anti)estrogen or N-methyl-d-aspartate receptors (NMDARs)-(anti)agonist included dif- Keywords: Semicarbazide ferent assays: yeast estrogenicity, MCF-7 proliferation, stimulation of the alkaline phosphatase activity Food contaminant in Ishikawa cells and LNCaP-based NMDAR interference assay. In vivo SEM-treated female rats showed Endocrine disrupters delayed vaginal opening at all tested doses, whereas in males preputial separation was anticipated at SEM Sex steroids 40 and 75 mg/kg and delayed at 140 mg/kg, the latter effect probably due to the significantly decreased Estrogen receptor alpha body weight gain seen at the higher dose in both sexes. Serum estrogen levels were dose-dependently N-methyl-D-aspartate receptor reduced in treated females, whereas dehydrotestosterone serum levels were also decreased but a clear dose–response was not evidenced. Testosterone catabolism was altered in a gender-related way, aro- matase activity was increased in treated males at 75 and 140 mg/kg and in females in all dose groups. In the three estradiol-competitive assays, SEM showed a weak anti-estrogenic activity, whereas in the LNCaP-based NMDAR interference assay SEM activity resembled MK-801 antagonist effect. SEM appeared to act as an endocrine disrupter showing multiple and gender specific mechanisms of action(s). A possible cascade-mechanism of SEM on reproductive signalling pathways may be hypothesized. Such in vivo–in vitro approach appeared to be an useful tool to highlight SEM activity on endocrine homeostasis. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Endocrine disrupters (EDs) are a broad group of natural or Abbreviations: DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene; E2, 17␤- man-made chemicals which – together with their by-products – estradiol; ALP, alkaline phosphatase; ADC, azodicarbonamide; BW, body weight; can cause adverse health effects in adult organisms, in the off- CNS, central nervous system; DHT, dihydrotestosterone; DMEM, Dulbecco’s Min- imal Essential Medium; EDs, endocrine disrupters; ERs, estrogen receptors; ERE, spring or in susceptible (sub)populations altering the homeostasis estrogen-responsive sequences; EFSA, European Food Safety Authority; FBS, foetal of the endocrine system, mainly involving sex steroid and thy- bovine serum; G6PDH, G6P-dehydrogenase; GABa, gamma-aminobutiric acid; G6P, roid hormones [1]. Experimental studies put into evidence that EDs glucose-6-phosphate; GnRH, gonadotropin-releasing hormone; LH, luteinizing hor- act through different mechanisms and have different targets: the mone; NMDARs, N-methyl-d-aspartate receptors; PND, postnatal day; RLM, rat liver microsomes; SEM, semicarbazide; SSAOs, semicarbazide-sensitive amine oxidases; SD, standard deviation; TST, testosterone; YES, yeast estrogenicity screen; ␤-gal, ␤-galactosidase. ∗ Corresponding author. Tel.: +39 06 49902527; fax: +39 06 49902658. (T. Catone), [email protected] (G. De Angelis), E-mail addresses: [email protected] (F. Maranghi), [email protected] (E. Testai), [email protected] [email protected] (R. Tassinari), [email protected] (S. Mastrangelo), [email protected] (M.G. Evandri), (D. Marcoccia), [email protected] (I. Altieri), [email protected] [email protected] (P. Bolle), [email protected] (S. Lorenzetti). 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.09.016 F. Maranghi et al. / Chemico-Biological Interactions 183 (2010) 40–48 41 homeostasis of sex steroids, thyroid hormones and their cascade 2. Materials and methods effects on reproduction and fertility being better characterised. The most vulnerable and susceptible phases of life cycle to ED adverse 2.1. Chemicals, reagents and cell culture media effects are pregnancy, childhood and puberty when the endocrine system plays a key role in the development and differentiation of Chemicals and reagents, including SEM hydrochloride (CAS no. the whole organism. In humans, EDs have been mainly associated 563-41-7, purity ≥98–99%), ␤-galactosidase (␤-gal) used in the YES with apparent changes in both male and female reproductive health assay, NMDA (CAS no. 6384-92-5), glycine (CAS no. 54-40-6) and [2]. (+)-MK-801 hydrogen maleate (CAS no. 77086-22-7) used in the Several potential food contaminants have been shown to act NMDAR-interference assay, were purchased from Sigma–Aldrich as EDs. Semicarbazide (SEM) is a by-product of azodicarbonamide (Milan, Italy). All the ingredients for yeast media, unless otherwise (ADC), which is used to foam the plastic gaskets of metal lids. stated, were research grade biochemicals suitable for cell culture In the food-production chain, SEM is released during manufac- and purchased from Sigma–Aldrich (Milan, Italy): yeast minimal ture when packaged foods are heated to ensure a tight seal and medium and yeast growth medium preparations have been previ- it is present as food contaminant in baby foods, fruit juices, jams ously described [17]. Chlorophenol red-␤-d-galactopyranoside was and conserves. Moreover, SEM was also found in different food- obtained from Roche Diagnostics (Monza, Milan, Italy). Cell culture stuffs such as powder and liquid milk, egg and whey powder media, supplements for MCF-7 and Ishikawa cells were purchased [3,4]. from Sigma–Aldrich (Karlsruhe, Germany), whereas RPMI1640 w/o Following the European Food Safety Authority (EFSA) warning, phenol red and foetal bovine serum (FBS) to grow LNCaP cells have the European Commission banned ADC as blowing agent in plastics been provided by Invitrogen (S. Giuliano Milanese, Italy). Estrogen- since August 2005 [5] asking for further data on potential toxicolog- free FBS used in YES, ALP and E-screen assays were obtained from ical effects of SEM. However, ADC is still used in some countries and Hyclone (Logan, Utah, USA) and charcoal-stripped FBS to perform this may explain the presence of SEM in a wide range of breaded NMDAR-interference assay by Bio-Whittaker (Lonza Milano srl, food products [6]. Other sources of SEM include the carrageenan Treviglio, Italy). Cell culture lysis buffer (CyLyse®) and staining extraction process and, in some countries, hypochlorite treatments solution (containing 4,6-diamidino-2-phenylindole/DAPI) were [6–8]. EFSA indicated that risks for consumers appear to be low purchased by Partec GmbH (Münster, Germany). The cell prolif- (SEM levels measured in foods up to 25 ␮g/kg = 25 ppb): never- eration kit “Cell Titer 96 AQueous One Solution Cell Proliferation theless, infants may be a sub-population at increased risk of SEM Assay” to perform the LNCaP-based NMDAR-interference assay exposure [9]. (MTS assay) has been purchased by Promega (Promega Italia, SEM is known to inhibit the following enzymes: (i) Milano, Italy). semicarbazide-sensitive amine oxidases (SSAOs), involved in Detection kits for sex steroid hormones (E2 and dihydrotestos- the oxidative metabolism of dietary and environmental xeno- terone—DHT) have been purchased from Perkin–Elmer (DELFIA® biotics [10–12]; (ii) lysyl oxidase, a key enzyme involved in the Estradiol Kit #1244-056) and Alpha Diagnostic International USA stabilization of extracellular matrix by cross-linking of proteins (DHT ELISA Kit #1940) respectively. such as collagen and elastin [13] and (iii) glutamic acid decarboxy- Roche GmbH (Mannheim, Germany) supplied NADPH, NADP, lase, synthesizing gamma-aminobutiric acid (GABA), the principal glucose-6-phosphate (G6P), and G6P-dehydrogenase (G6PDH). non-peptidal neurotransmitter in the central nervous system Defatted bovine serum albumin was from Serva, Feinbiochemicsa (CNS) [14,15]. (Heidelberg, Germany). Testosterone (TST), corticosterone (as Targets of SEM toxicological effects include skeletal, cardio- internal standard), 4-androsten-3,17-dione