Protective effects of sulforaphane against toxic substances and contaminants: a systematic review

Phytomedicine 130 (2024) 155731 Available online 19 May 2024 0944-7113/© 2024 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Review Protective effects of sulforaphane against toxic substances and contaminants: A systematic review ☆ Antonio Cascajosa-Lira, Ana I Prieto * , Silvia Pichardo , Angeles Jos , Ana M Came´ an ´ Area de Toxicología, Facultad de Farmacia, Universidad de Sevilla, Profesor García Gonz´ alez n 2, 41012 Seville, Espa˜ na ARTICLE INFO Keywords: Sulforaphane Toxicity Protective Anti-inflammatory Antioxidant ABSTRACT Background: Sulforaphane (SFN) is a dietary isothiocyanate, derived from glucoraphanin, present in cruciferous vegetables belonging to the Brassica genus. It is a biologically active phytochemical that acts as a nuclear factor erythroid 2-related factor 2 (Nrf2) inducer. Thus, it has been reported to have multiple protective functions including anticancer responses and protection against a toxic agent’s action. Purpose: The present work systematically reviewed and synthesised the protective properties of sulforaphane against a toxic agent. This review reveals the mechanism of the action of SFN in each organ or system. Methods: The PRISMA guideline was followed in this sequence: researched literature, organised retrieved documents, abstracted relevant information, assessed study quality and bias, synthesised data, and prepared a ☆ Abbreviations: 4-HNE: 4-Hydroxynonenal; 6-HITC: 6-(methylsulifnyl) hexyl isotiocyanate; ADMA: asymmetric dimethylarginine; ASA: Acetylsalicylic acid; AHR: aryl hydrocarbon receptor; Akt: serine/threonine protein kinase; ALT: alanine aminotransferase; AMPK: adenosine 5 ′ -monophosphate (AMP)-activated protein kinase; Ang II: Angiotensin II; ARE: antioxidant responsive element; ARPE-19: retinal cells; As: Arsenic; AST: aspartate aminotransferase; BaP: Benzo(a)pyren; Bcl-2: B- cell lymphoma 2 protein family; Bax: bcl-2-like protein 4; BEAS-2BR cells: human bronchial epithelial cell line; BRL-3A: fibroblast-like cell isolated from the liver of a rat; BSO: L-buthionine-sulfoximine; b.w.: body weight; C2C12: myoblast cell line; CAT: catalase; Cd: Cadmium; CPF: chlorpyrifos; CPT-1: carnitine palmitoyl transferase-1; CUS: chronic unpredictable stress; D3T: 3-H-1,2.dithiole-3-thione; DBC: Dibenzo[def,p]chrysene; DDAH: dimethylaminohydrolase; DSS: Dextran sodium sulphate; E2: 17-β-estradiol; EdU: the 5-ethynyl-2 ′ -deoxyuridine assay; EMT: epithelial-mesenchymal transition; ER: Estrogen Receptor; ERK1/2: extracellular signal-regulated kinases 1 and 2; Erod: ethoxyresorufin O-deethylase; fEPSP: total field excitatory postsynaptic potential; GalN: D-galactosamine; G6PDH: Glucose-6- phosphate de-hydrogenase; GCLM: glutamate cysteine ligase modifier subunit; G-CSF: granulocyte colony-stimulating factor; GLC cells: Granulosa-lutein cell line; gclc: gluta-mate-cysteine ligase catalytic subunit; GM-CSF: granulocyte-macrophage colony-stimulating factor; GPx: glutathione peroxidase; GR: glutathione reductase; GSK-3b: glycogen synthase kinase 3 beta; GST: glutathione-S-transferase; GSTM3: Glutathione S-transferases mu3 γ-GCS: γ-glutamylcysteine synthetase; GU: Gastric Ulcer; gst pi: glutathione S-transferase pi; HaCaT: HepaRG: human hepatoma-derived cell line; HBMEC-3: Human Brain Microvascular Endothelial Cells; HepG2: human liver cancer cell line; HMOX1: heme oxygenase 1 gene; GSH: glutathione; HO-1H3K4me3: histone H3 lysine 4; HUVEC: EndoGRO Human Umbilical Vein Endothelial Cells; I3C: Indole-3-carbinol; ICAM-1: Intercellular Adhesion Molecule 1; IL-8: Interleukin 8; IL-9: Interleukin 9; i.p.: intraperitoneal; IFN-γ: Interferon gamma; iNOS: ´ oxido nítrico sintasa indicible; IR: Irradiation; Keap1: Kelch-like ECH-associated protein 1; LDH: Lactate dehydrogenase; LLC-PK1: Lilly Laboratories Cul-ture-Porcine Kidney 1; LPS: bacterial lipopolysaccharide; LPO: lipid peroxidation; LORR: Loss of righting reflex; LX-2: Hepatic stellate cells; M1: proinflammatory phenotype of microglia; MA: Methamphetamine; miR-19A: MicroRNA 19a; miR-19b: MicroRNA 19b; MCF-7: Breast Cancer Cells; MC-LR: Microcystin- LR; MCP-1: Monocyte chemoattractant protein-1; MDA: malondialdehyde; mTOR: mammalian target of rapamycin; MIP-1β: macrophage inflammatory pro-tein-1β; MN: micronucleus; MPO: Myeloperoxidase; NAFLD: Nonalcoholic Fatty Liver Disease; NCCs: Neural Crest Cells; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; NIH 3 T3: embryonic mouse fibroblast cell line; NLRP3: NOD-, LRR- and pyrin domain-containing 3; NSAID: Nonsteroidal anti-inflammatory drugs; NEC: Necrotizing enterocolitis; NO: nitric oxide; NOX1: NADPH oxidase 1; NOX4: NADPH oxidase 4; NQO1: NAD(P)H dehydrogenase [quinone] 1; Nrf2: nuclear factor erythroid 2-related factor 2; OACs: Osteoarthritic articular chondrocytes; OPZ: Oltipraz; p21: cyclin dependent kinase inhibitor 1A; PBS: Phosphate-buffered saline; PDGF: Platelet-derived growth factor; PGC-1: peroxisome proliferator-activated receptor-gamma coactivator; PHA: Polyhydroxyalkanoates; PTEN: phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase; POR: Cytochrome P450 Oxidoreductase; PRDX1: Peroxiredoxin 1; QUIN: Quinolinic acid; ROS: reactive oxygen species; SH-SY5Y: neuroblastoma cell line; STZ: Streptozotocin; T-AOC: total antioxidant capacity; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SOD: Superoxide dismutase; TG: Triglyceride; TNF α : tumor necrosis factor alpha; TFAM: mitochondrial transcription factor A; TLR4: toll-like receptor 4; TNFRSF1A: Tumor necrosis factor receptor superfamily member 1 A; TNFSF10: Tumor necrosis factor ligand superfamily member 10; T-SOD: Total Superoxide Dismutase; TrxR-1: thioredoxin reductase-1; TXR1: human thioredoxin; VCAM-1: Vascular cell adhesion protein 1; Vero cells: monkey kidney epithelial cell line; VLDL: VLDL: very low density lipoprotein; γ-GCL: γ Glutamate–cysteine ligase. * Corresponding author at: ´ Area de Toxicología, Facultad de Farmacia. Universidad de Sevilla. C/ Profesor García Gonz´ alez, nº 2. C.P. 41012, Seville Spain. E-mail addresses: [email protected] (A. Cascajosa-Lira), [email protected] (A.I. Prieto), [email protected] (S. Pichardo), [email protected] (A. Jos), [email protected] (A.M. Came´ an). Contents lists available at ScienceDirect Phytomedicine journal homepage: www.elsevier.com/locate/phymed https://doi.org/10.1016/j.phymed.2024.155731 Received 29 February 2024; Received in revised form 3 May 2024; Accepted 10 May 2024 Phytomedicine 130 (2024) 155731 2 comprehensive report. Searches were conducted on Science Direct and PubMed using the keywords "Sulforaphane" AND (“protective effects” OR “protection against”). Results: Reports showed that liver and the nervous system are the target organs on which attention was focused, and this might be due to the key role of oxidative stress in liver and neurodegenerative diseases. However, protective activities have also been demonstrated in the lungs, heart, immune system, kidneys, and endocrine system. SFN exerts its protective effects by activating the Nrf2 pathway, which enhances antioxidant defenses and reduces oxidative stress. It also suppresses inflammation by decreasing interleukin production. Moreover, SFN inhibits apoptosis by preventing caspase 3 cleavage and increasing Bcl2 levels. Overall, SFN demonstrates multifaceted mechanisms to counteract the adverse effects of toxic agents. Conclusion: SFN has potential clinical applications as a chemoprotective agent. Nevertheless, more studies are necessary to set the safe doses of SFN in humans. Introduction Sulforaphane (SFN) is a natural compound within the isothiocyanate group of organosulfur compounds. The chemical formula correspond to 1-isothiocyanate-(4R)-(methylsulfinyl)butane (Fig. 1). SFN has undergone extensive investigation in recent years for its protective efficacy across various in vivo pathologies, alongside in vitro studies conducted on experimental models. SFN exerts influence on oxidative stress and antioxidant capacity, neuroinflammation, and numerous other biochemical irregularities (Baralic et al., 2024). SFN is a dietary isothiocyanate derived from glucosinolates which is present in several cruciferous vegetables belonging to the Brassica genus. These vegetables include cauliflower, broccoli, kale, cole crops, cabbage, collards, Brussels sprouts, as well as other genera such as radish, mustard, and cress (Fahey et al., 2001). SFN is produced by the action of the enzyme β-thioglucoside glucohydrolase or myrosinase on glucosinolates. However, this enzyme is physically separated from the substrate, so it is necessary for the plant to suffer previous aggression processes to generate the enzymatic hydrolysis and the production of SFN, which is the primary product of the reaction (Shapiro et al., 2001). Consuming a diet rich in fruits and vegetables has been associated with a reduced risk of developing metabolic diseases. However, not all fruits and vegetables exhibit uniform effectiveness in this regard (Padayachee et al., 2017). In particular, the consumption of cruciferous vegetables, such as broccoli, has shown greater potential to mitigate the risk of metabolic disorders, including cancer and diabetes, compared to other vegetables (Latt´ e et al., 2011; Marshall et al., 2023). This positive impact is attributed to the content of glucosinolates, with glucoraphanin (4-methylsulfinylbutyl glucosinolate) being a major component. Young broccoli has been found to contain significantly higher levels of glucosinolates, particularly glucoraphanin, with concentrations 20–50 times higher than those found in mature broccoli (Vanegas et al., 2022). Under specific reaction conditions, such as pH, temperature, and presence of iron, other reaction products different from glucosinolates, such as thiocyanates and nitriles, can also be generated. These nonenzymatic, intramolecular rearrangements contribute to the formation of the aforementioned additional products (Hayes et al., 2008; Guerrero-Beltr´ an et al., 2012). Usually, the precursor of SFN is found in broccoli in high concentration: 0.8–21.7 µmol/g of dry weight (Guerrero-Beltr´ an et al., 2012). The consumption of 200 mg of broccoli can result in approximately 2 µM SFN in the plasma after about 2 h (Gasper et al., 2005). Furthermore, the highest level reported in plasma was 7.3 µM after consumption of 100 g of high glucosinolate broccoli containing 345 µmol SFN and its metabolites (Ye et al., 2002). Although SFN and other isothiocyanates (ITCs) from cruciferous vegetables are recognised for their beneficial effects, it is important to note that moderate intakes, typically within the range of 100 to 200 gs of fresh cruciferous vegetables per day, are considered chemoprotective agents. However, elevated levels of ITCs can potentially induce stress-related cytotoxicity. A study proposed that SFN, when administered at concentrations ranging from 10 to 30 μ M, led to the induction of DNA single-strand breaks in cultured human HUVEC cells (Sestili et al., 2010). SFN is an inducer of nuclear factor erythroid 2-related factor 2 (Nrf2) and produce powerful cytoprotective effects (Dinkova-kostova et al., 2017; Kubo et al., 2017). Moreover, this transcription factor plays a key role in redox homeostasis (Hashimoto, 2018; Yamamoto et al., 2018, Yu and Xiao, 2021). SFN has been shown to induce many health benefits (Juge et al., 2007), including anti-inflammatory properties. It inhibits the production of pro-inflammatory cytokines, such as IL-6, in various human cell lines and in vivo models (Kawarazaki et al., 2017; Burnett et al., 2017; Folkard et al., 2014). The first known chemotherapeutic properties of SFN were antiproliferative and anticancer. However, nowadays, research on the new protective effects of SFN has been increased. Current research has also focused on the positive impact of SFN on pathologies such as brain, liver, kidney, cardiovascular system, lungs, and muscle among others (Klomparens and Ding, 2019; Aranda-Rivera et al., 2022; Chang R., 2022; Brasil et al., 2023). Due to these reported beneficial effects, SFN has also been used to protect against toxic agents (Guerrero-Beltr´ an et al., 2012). Humans are exposed to an increasing number and diversity of chemicals from the environment (Sturla and Wang, 2023); therefore, to reveal chemoprotectants such as SFN that could prevent or reverse the potential toxicity induced by toxic agents is of interest. The purpose of the present systematic review is: 1) to comprehensively and rigorously evaluate existing literature on sulforaphane’s protective effects and the mechanisms of actions in different organs following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines; 2) to analyze the risk of bias present in the selected literature; 3) to know the future perspectives and limitations of SFN with regard to its potential therapeutic applications against different toxic agents. Fig. 1. Metabolism and chemical structure of glucoraphanin and sulforaphane. Created with BioRender.com. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 3 Materials and methods The present investigation constituted a systematic review conducted in accordance with the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines, as outlined by Cascajosa-Lira et al. (2022). These guidelines serve as a structured framework for the planning and execution of systematic review studies. Our methodology started with a comprehensive literature search, proceeded with the organization of retrieved documents, abstracting pertinent information, and assessing the quality and bias of each individual study. Subsequently, data synthesis was conducted, culminating in the preparation of a comprehensive report. Information sources and search strategy Two electronic research databases, Science Direct (https://www.sci encedirect.com/) and PubMed (https://pubmed.ncbi.nlm.nih.gov/), were searched on April 1, 2023. The keywords strings chosen were: ("Sulforaphane") AND (“protective effects” OR “protection against”). The searches included works published in all languages. The Science Direct option search was ‘all fields except full text (NOFT)’ and the PubMed option search was ‘all fields’. Study selection. Eligibility and exclusion criteria Once the exploration had been performed, a three-step process was carried out to review all records according to the eligibility criteria: first, Fig. 2. PRISMA flowchart of studies selection. * These sections share one or more records. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 4 the title was read, second the abstract, and third the entire text of the publication. The works obtained by the two databases were crossed with EndNote X9 software to identify possible duplicates and to classify the works according to the exclusion and inclusion criteria. Conflicts about whether a given reference should be incorporated were determined by agreement of the authors. Also, some of the records include information from two models, so they were classified in both sections. The details of the search method and the classification of records are presented in Fig. 2. All international studies were considered. The eligibility criteria for inclusion in this work were the following: 1) articles available in English; 2) articles published prior to April 1, 2023 and after January 1, 2000; 3) studies conducted in animal or in vitro models; 4) research investigating the effects of SFN as a protective agent against toxicity induced by chemical substances, environmental pollutants, or naturally occurring toxic agents; and 5) studies providing relevant information on the mechanisms of action of SFN in protection against toxicity. The following exclusion criteria were applied: 1) non-systematic and narrative reviews; 2) articles published in languages other than English; 3) proceedings and dissertations 4) books or book chapters; 5) editorial material; 6) studies not focusing on SFN as a protective agent against toxic agents; 7) studies in which SFN is combined with other compounds without specifically evaluating its protective effect and 8) studies that do not provide relevant information on the protective activity of SFN in relation to toxicity. Criteria and exclusions are visually represented in the PRISMA diagram of the study selection process (Fig. 2). Data extraction and data items After a comprehensive reading of each of the articles selected for the review, the following items were established: Toxic substance; Sulforaphane concentration or dose; Experimental model; Exposure condition, and Main results. Risk of bias The authors meticulously assessed the risk of bias in selected studies by rigorously evaluating several key criteria. First, they scrutinized whether the studies had a clear objective, ensuring that the research goals were well-defined and articulated. Second, the authors examined whether the product under investigation was adequately characterised, ensuring clarity regarding its composition and properties. Third, they assessed the reproducibility of the assay employed in the studies, verifying whether the experimental procedures could be reliably replicated to yield consistent results. Additionally, the authors evaluated the comparability of the experimental groups, ensuring that any differences observed were attributable to the interventions being studied rather than extraneous factors. Finally, they scrutinised the statistical analyses performed in the studies, ensuring that appropriate statistical methods were employed to accurately interpret the data and draw valid conclusions. This comprehensive approach allowed the authors to thoroughly assess the risk of bias across multiple dimensions of the selected studies. The results of this preliminary assessment are represented in table S1, in which the risks of bias of the selected studies are included. Most of the studies selected presented low risk of bias. Kinetic and mechanism of action of sulforaphane Kinetic and distribution of sulforaphane Sulforaphane kinetic and distribution studies show a wide organ distribution (Veeranki et al., 2013), even crossing the blood-brain barrier (Jazwa et al., 2011). Although the cross-through of the placental barrier has not been studied as far as we know, there is a study that reported the presence of SFN metabolites in the plasma of newborns (Shorey et al., 2013). Therefore, SFN can exert its beneficial effects in multiple organs as represented in the results of the present review. Mechanism of action of sulforaphane The main mechanism of action of SFN is as an indirect antioxidant (Fig. 2), and this fact has been outlined in numerous works included in this review (Tables 1-8). SFN initiates the expression of detoxification enzymes through the Nrf2/ Keap1/ARE signaling pathway when exposed to oxidative and/or electrophilic conditions. Moreover, it indirectly influences Nrf2 by promoting its translocation and accumulation in the nucleus, potentially phosphorylating Nrf2 through activation of various kinases, including MAP (mitogen-activated protein kinase), PKB/Akt (protein kinase B) and PKC (protein kinase C). Studies have reported the activation of more than 500 genes by SFN via the Nrf2/ARE signaling pathway (Ruhee et al., 2020). ARE, which acts as a cis-acting enhancer sequence, regulates the basal expression of phase 2 detoxification and antioxidant genes. In addition to its antioxidant activity primarily through Nrf2 activation, SFN demonstrates the ability to mitigate inflammation reducing phase 1 cytochrome P450 enzymes, improves phase II enzymes, and decreases HIF alpha and COX 2, among other enzymes (Zhou et al., 2014). Furthermore, Nrf2 significantly contributes to the inhibition of the nuclear factor-kappa beta (NF-κB) signaling pathway, which is pivotal in the regulation of inflammation (Russo et al., 2018). In relation to carcinogenesis, SFN-induced activation of Nrf2 leads to the upregulation of various cytoprotective genes recognised for their anticarcinogenic effects (Russo et al., 2018). In terms of its antidiabetic properties, SFN has been observed to mitigate insulin resistance through modulation of the PI3K/Akt and JNK/IKK, AMPK/mTOR pathways. Furthermore, it enhances glucose transport via the IRS-1/Akt/GLUT4 and PPAR/GLUT4 pathways, while also improving blood glucose levels through the PPAR/GSK/GS pathway (Wang et al., 2022a). Hepatoprotective effects of sulforaphane The hepatoprotective effects of SFN reported in the selected studies are summarised in Table 1. Its efficiency has been demonstrated using both in vitro and in vivo models. The effects of SFN are mainly mediated by its antioxidant, anticarcinogenic, anti-endoplasmic reticulum (ER) stress and lipid and alcoholic metabolism-regulating activity. According to the findings obtained after the revision, the following whole mechanism of action is proposed (Fig. 3). The protective action of SFN has been demonstrated in vitro in the following cell lines: HepaRG, LX-2, LO2, HepG2, and HHL5, in addition to primary cultures. The concentrations tested ranged from 2 to 100 µM, but the concentration most commonly used to demonstrate a protective effect was 5 µM. These studies have tested SFN against the toxic mechanism of lipopolysaccharides (LPS) (Al-Bakheit et al., 2020; Ishida et al., 2021), medications such as acetaminophen (Noh et al., 2015), plasticisers such as bisphenol-A (Hong et al., 2023), metals such as cadmium (He et al., 2021) or vanadium (Visalli et al., 2017), H 2 O 2 (Li et al., 2012; Liu et al., 2019), ethanol (Zhou et al., 2014) and cyanotoxins such as MC-LR (Gan et al., 2010). The mechanism of action of these toxins is mainly to create an inflammatory reaction (LPS and Bisphenol-A) and to promote oxidative stress (H 2 O 2 , metals, Ethanol, and Acetaminophen). In addition, MC-LR is able to inhibit hepatic phosphatases (PP1 and PP2A), resulting in necrosis and apoptosis. According to the findings obtained after the revision, the following whole mechanism of action is proposed (Fig. 4). The mechanism of action of SFN is mainly due to its antioxidant activity as an inducer of the Nrf2 factor, which increases the amount of GSH available and thus reduces the oxidative stress that can be caused by toxicants (Gan et al., 2010). Furthermore, some antioxidant genes seem to be up-regulated in the presence of SFN such as HMOX1, NQO1, GSTM3, TrxR-1, and OH-1 (Li et al., 2012; Noh et al., 2015; Ishida et al., 2021). On the other hand, in vitro anti-inflammatory activity has been demonstrated by reducing A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 5 Table 1 Overview of studies reporting the protective effects of SFN in liver. Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference Acetaldehyde Lipopolysaccharide (LPS) Ethanol/ CCl 4 0–40 μ M 5 μ moL/d/b.w. Cells were treated with different concentrations of acetaldehyde (0–200 μ M) and SFN (0–40 μ M) for 12 to 48 h. LX-2 cells were exposed to 5 or 20 μ M of SFN and 100 ng / ml of LPS for 12 h. SFN was administered orally as a mixture of diet (5 μ moL/d/b.w.) once a day for 14 days. On the 13th day, mice were fed a 2.5 % liquid diet and received i.p. injections of CCl4 twice weekly (1 ml/kg b.w.) HepaRG and LX-2 cells, human hepatoma, and liver stellate cell lines. Mouse model of alcoholic liver fibrosis induced by EtOH/CCl 4 . SFN induced ADH activity of ADH in HepaRG cells and suppressed ADH- induced proliferation and profibrogenic activity in LX-2 cells with up-regulation of Nrf2-regulated antioxidant genes (HMOX1, NQO1 and GSTM3). SFN attenuated the LPS/toll-like receptor 4-mediated sensitisation to transforming growth factorβ with down-regulation of NOX1 and NOX4. In EtOH/CCl 4 treated mice, SFN significantly inhibited Kupffer cell infiltration and fibrosis, decreased fat accumulation and LPO, and induced Nrf2-regulated antioxidant response genes. Ishida et al., 2021 Acetaminophen (APAP) 10 µM for hepatocytes. 5 mg/kg pretreatmen in mice Primary hepatocytes were pretreated with SFN for 6 h, then with APAP (15 mM) and incubated for 14 h. Hepatic injury was induced in mice by injection of 300 mg / kg of APAP after SFN treatment (5 mg/kg) and sacrificed after 6 h. Primary hepatocytes and also mice In primary hepatocytes: SFN pretreatment reduced cell death and MDA production after APAP exposure; attenuated intracellular GSH and preserved genes associated with GSH synthesis; increased Nrf2 target genes expression, especially HMOX-1. In the mouse model, SFN pretreatment: lowered AST and ALT levels and inhibited APAP-induced liver histological damage induced by APAP; blocked LPO and ROS production in the liver; regulated Nrf2 target gene expression and inhibited GSH depletion in the liver. Noh et al., 2015 Arsenic (As) 20, 40, 80 mg/ kg b.w. of SFN Six groups were established: control, As (5 mg / kg b.w.), SFN (20, 40, 80 mg/ kg b.w.) +As (5 mg/kg b.w.); and Vit. C (100 mg/kg b.w.); +5 mg / kg b.w.). Male Wistar rats The As-induced oxidative damage was confirmed by a significant increase in the levels of δ-aminolevulinic acid dehydratase, and depletion in antioxidant content. As also increased the pro-apoptotic marker (Bax) and DNA damage, with decreased Nrf2 protein responsible for liver protection. SFN provided therapeutic and prophylactic efficacy against As-induced oxidative hepatic damage through its strong antioxidant property. SFN ameliorated As-induced alternations in liver through the activation of Nrf2 by the PI3K / Akt-mediated pathway. Thangapandiyan et al., 2019 Bisphenol-A (BPA) 100 µM 10 mg/kg b.w. LO2 cells were treated with 100 nM BPA and 0, 0.5, 1, 10 and 100 µM of SFN for 24 h. Mice were exposed to 10 mg / kg b. w. of SFN, along with 100 μ g/kg b. w. of BPA ip for 6 weeks. LO2 cell line (Human hepatocyte cells) C57/BL6J mice SFN improves BPA-induced lipid metabolic abnormalities (number of lipid droplets, levels of triglycerides and mRNA expression of lipogenesisrelated genes) and stress of the reticulum endoplasmic (ER) in both LO2 cells and in mice. Hong et al., 2023 Cadmium (Cd) 0–80 µM for 24 h. 0.5, 1 and 2 mg/ kg/b.w. daily for 6 weeks Mice were orally exposed to 0.5, 1 or 2 mg / kg / b.w. daily of SFN for 6 weeks +10 mg / kg / b.w. CdCl 2 during the last 4 weeks. HepG2 cells Kunming mice SFN decreased Cd cytotoxicity in HepG2 cells and induced liver damage in mice (such as haemorrhage in the liver parenchyma, cell degeneration, focal hepatocyte necrosis, hepatocyte disarrangement, and congestion of portal vein congestion) in a dosedependent manner. SFN (dose of 2 mg / kg / b.w. mainly) restores AST, ALT, TBil, SOD, CAT, GST, GSH, MDA, and T-AOC levels to control values. SFN showed antioxidant and antiinflammatory effects against Cdinduced liver damage, associated He et al., 2021 (continued on next page) A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 6 Table 1 (continued) Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference with the modulation of the intrinsic Nrf2/ARE and NF-κB pathway. Cisplatin 500 µg/kg/d for 3 days Rats were pretreated i.p. with SFN (500 µg for 3 days) and then exposed to cisplatin (single i.p. injection, 10 mg/kg) and sacrificed 3 days later. Wistar rats SFN prevented cisplatin-induced liver damage and attenuates histopathological alterations of the ALT and AST enzymes observed in exposed rats. SFN protected against cisplatininduced oxidative stress in the rat liver by reversing the increase in LPO and protein carbonyl content and the decrease in GSH and antioxidant enzymes (CAT, SOD, GPx, GR). SFN has a high scavenging capacity for free radicals such as peroxynitrite anion, superoxide anion, singlet oxygen, and peroxyl radicals, and prevented mitochondrial alteration of oxygen uptake and decreased complex I activity. Gaona-gaona et al., 2011 Cuprizone (CPZ) 2 mg/kg/day for two weeks Rats were fed a CPZ-contained diet (0.2 %) for four weeks after which they were SFN i.p. administrated (2 mg/kg/day) for two weeks. Wistar rats SFN treatment reversed all the biochemical alterations produced by CPZ such as ALT and AST. In addition, SFN produced in decrease a LPO and enhanced T-AOC levels and CAT activities in rats pretreated with CPZ, reducing CPZ-induced oxidative stress in hepatic tissue. Fouad et al., 2023 D-galactosamine (GalN) and lipopolysaccharide (LPS) 3 mg/kg The rats were divided into 4 groups: control, SFN treated and 2 groups i. p. injected with GalN (300 mg/kg) and LPS (30 μ g/kg), one of them also exposed i.p. to SFN. Male Wistar rats SFN provided protection against GalN/LPS-induced fulminant liver failure. This was demonstrated with reduced mortality rates, reduced serum AST and ALT activities, restored SOD, CAT, GPx and Oh-1 activities, and improved pathological liver changes. Sayed et al., 2014 Ethanol In vitro: 6 μ M In vivo: 0.05 g/kg In vitro: cells were treated with or without 100 mM ethanol in the absence or presence of SFN. In vivo: mice were gavaged twice a day with 30 % ethanol at a dose of 3 g/kg b.w. for 5 days. Some mice were injected i.p. with SFN, at the dose of 0.05 g/kg once a day for 5 days. In vitro: HepG2, E47 cells In vivo: SV129 humanised male CYP2E1 knockinknocking mice In vitro SFN elevated Nrf2 levels and decreased lipid accumulation in cells cultured with ethanol. In vivo, SFN activated Nrf2, increased levels of Nrf2 target heme oxygenase- 1 and decreased oxidant stress as shown by the decrease in LPO and 3- nitrotyrosine protein adducts and increased GSH levels. It decreased the levels of triglycerides and cholesterol, and Oil Red O staining. It proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice. Similarly, in vitro, SFN elevated Nrf2 levels and decreased lipid accumulation in cells cultured with ethanol. Zhou et al., 2014 Ethanol 0.1 ml/g b.w. SFN Six groups of mice were established: normal control group (A), model control (B), bifendate pill (BDP) intervention group (C,) aerobic exercise intervention group (D), SFN intervention group (E) and group treated with SFN integrated with aerobic exercise (F). Mice in groups A and B were intragastrically administrated with 50 % ethanol for 8 ml/kg b.w. for 12 weeks. From the fifth week, the intervention group C was given 0.1 ml/g b.w. of BDP before ethanol, while the same amount of SFN solution was given to intervention groups E and F; and group B received an equal amount of distilled water. The training time, in groups D and F, was 60 min a day, 5 days a week and 8 weeks in total. Mice SPF SFN administration has a stronger treatment effect than aerobic exercise alone, and the combination of SFN and aerobic exercise has the strongest protective effect on acute alcoholic hepatic injury in mice. The SOD activity in liver experienced significant increases and the MDA and triglyceride (TG) experienced significant decreases in the SFN group and the group treated with SFN administration integrated with aerobic exercise. VLDL in the serum of mice also experienced significant decreases in this latter group. Thus, both SFN and aerobic exercise can impose a certain protective effect on acute alcoholic liver injury in mice and help reducing the damage caused by alcohol in liver cells. Wang and Zhou, 2020 (continued on next page) A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 7 Table 1 (continued) Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference Hydrogen peroxide (H 2 O 2 ) 2,5–80 µM Cells were exposed to several concentrations of SFN and / or Selenium (Se) for different times. HHL-5 cells SFN and Se synergistically induced TrxR-1 expression in HHL-5 cells. SFN can protect against H 2 O 2 -induced cell death, and this protection was also synergically enhanced with Se. By using siRNA to kill TrxR-1 or Nrf2, the protection of SFN (5 µM) was reduced. SFN-induced TrxR-1expression was associated with significant levels of Nrf2 translocation into the nucleus. Li et al., 2012 Hydrogen peroxide (H 2 O 2 ) 5 µM Cells were exposed to several concentrations of SFN for 24 h and were treated with 700 µM H 2 O 2 for another 24 h. HHL5 and HepG2 cells Pretreatment with low concentration of SFN (≤5 µM) reduced the cytotoxicity induced by H 2 O 2; moreover SFN reduced DNA damage caused by H 2 O 2 in HepG2 but not in HHL5 cells. The Nrf2 / GSH signaling pathways play a crucial role in the protective effects of SFN against H 2 O 2. Liu et al., 2019 Lipopolysaccharide (LPS) 0.13- 0.39 mg/kg Mice were treated with LPS (15 mg / kg i.p.) and with SFN at different concentrations (0.13 or 0.26 or 0.39 mg / kg i.v.) 12 h after LPS injection. Male c57BL/6 mice SFN decreased the mortality and the lethal liver injury previously increased by LPS at the highest concentration tested. SFN decreased serum levels of ALT and AST. Furthermore, SFN decreased the expression of inflammatory cytokines and the expression of the toll-like receptor 4 (TLR4) protein. Lee et al., 2020 Lipopolysaccharide (LPS) 2 µM Cells were treated with SFN for 24 h and subsequently they were exposed to 1 μ g/ml LPS for 24 h. Hep G2 cells SFN suppressed hepcidin secretion LPS-induced transcription and interleukin-6 (IL-6) reducing the inflammatory responses in these cells without decreasing the cells viability. Al-Bakheit and Abu-Qatouseh, 2020 Microcystin-LR (MC-LR) 10 μ M HepG2, BRL-3A and NIH 3 T3 cells were treated with 10, 5 or 40 μ M MC-LR for 24 h with and without pretreatment with SFN (1–20 μ M) for 12 h. HepG2, BRL-3A and NIH 3 T3 cell lines SFN protects in a time- and concentrationdependent way against the cytotoxicity caused by MC-LR. SFN produced a significant increase in intracellular GSH levels in HepG2 cells, increased the number of cell colonies, and reduced cell death by apoptosis in all cell lines tested. Gan et al., 2010 Microcystin-LR (MC-LR) 5 μ mol/animal Mice were injected daily i.p. for 10 days with 40 and 50 MC-LR; with and without SFN (12 h prior to exposure to MC-LR). Male BALB/c mice SFN prevents MC-LR-induced hepatotoxicity and animal death in mice. In fact, it can block MC-LR- induced apoptosis. Furthermore, SFN stabilised Nrf2 in vivo and inhibited LPO and GSH reduction. Sun et al., 2011 Olanzapine (OLZ) 90 mg/kg/d Mice were treated with 8 mg/kg/ d of OLZ by osmotic minipump for 4 weeks and SFN (90 mg/kg/d) by gavage starting 1 week prior to administration of OLZ. C57BL/6 mice SFN can partially prevent the dysregulated glucose and lipid metabolism produced by OLZ together with a high-fat diet, representing protection against liver injury. SFN increased Nrf2 target gene (NQO1) and decreased OLZ-produced 4-HNE adducts produced by OLZ. Isaacson et al., 2020 Vanadium (VOSO4) 5 µM Cells were exposed to VOSO4, SFN, and a mixture of both. HepG2, Caco-2, and Vero cells Intracellular ROS, DNA, and lysosomal oxidative damage underlined the indirect antioxidant activity of SFN, confirmed by the increase in GSH. SFN neutralised the metal-induced imbalance of redox homoeostasis Visalli et al., 2017 4-HNE: 4-Hydroxynonenal; ADH: aldehyde dehydrogenase; Akt: serine/threonine protein kinase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; APAP: Acetaminophen; Bax: bcl-2-like protein 4; BRL-3A: fibroblast-like cell isolated from the liver of a rat; Caco-2: human colon adenocarcinoma; CAT: catalase; galN: D-galactosamine; GPx: glutathione peroxidase; GR: glutathione reductase; GSH: glutathione; GST: glutathione-S-transferase; GSTM3: Glutathione S-transferases mu3; HepaRG: human hepatoma-derived cell line; HepG2: human liver cancer cell line; HMOX1: heme oxygenase-1; IL: interleukin; LPS: bacterial lipopolysaccharide; LPO: lipid peroxidation; LO2 cells: Human hepatocyte cells; LX-2: Hepatic stellate cells; MC-LR: Microcystin-LR; MDA: malondialdehyde; mTOR: mammalian target of rapamycin; NF-κB: Nuclear factor kappa light chain enhancer of activated B cells; NIH 3 T3: embryonic mouse fibroblast cell line; Nrf2: nuclear factor erythroid 2- related factor 2; NOX1: NADPH oxidase 1; NOX4: NADPH oxidase 4; NQO1: NAD(P)H dehydrogenase [quinone] 1; OLZ: olanzapine; ROS: reactive oxygen species; SOD: superoxide dismutase; T-AOC: total antioxidant capacity; Tbil: Total bilirrubine; TLR4: toll-like receptor 4; TNF α : tumour necrosis factor alpha; Vero cells: monkey kidney epithelial cell line. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 8 hepcidin and IL-6; consequently, the inflammatory response and cancer progression could be inhibited (Al-Bakheit et al., 2020). Additionally, SFN has been able to repair DNA and lysosomal oxidative damage (Visalli et al., 2017). SFN has been shown to inhibit the catalytic activity in isolated microsomes (oxidation of para nitrophenol) and to block the genotoxicity of nitrosodimethylamine, a substrate for oxidation by CYP2E1. Therefore, Nrf2 activation coupled with possible inhibition of CYP2E1 was believed to make SFN an attractive chemical to blunt the toxic actions associated with CYP2E1 (Zhou et al., 2014) (Fig. 4). Regarding in vivo studies, SFN has been assayed in the following models: Wistar rats (mainly males), SFP mice, C57BL/6 J mice, Kunming mice, BALB/C mice. The doses tested varied widely from 500 µg/kg to 100 mg/kg. These studies have investigated the effects of SFN on countering the toxic actions of d-galactosamine and lipopolysaccharide (LPS) (Sayed et al., 2014; Lee et al., 2019), drugs such as acetaminophen (Noh et al., 2015), cuprizone (Fouad et al., 2023), olanzapine (Isaacson et al., 2020) and cisplatinum (Gaona-Gaona et al., 2011); metalloids such as arsenic (Thanga-pandiyan et al., 2019), plasticisers such as bisphenol-A (Hong et al., 2023), metals such as cadmiun (he et al., 2021), ethanol (Zhou et al., 2014; Wang and Zhou, 2020), cyanotoxins such as Microcystin-LR (Sun et al., 2011). Similar to the in vitro findings, these toxics induced an inflammatory responses accompanied by pro-oxidant conditions in vivo models. Furthermore, MC-LR primarily elicits toxic effects in the liver due to the use of specific transporters. The primary route of administration for SFN was oral, with intraperitoneal (i.p.) injection being the secondary option. In vivo research validates the mechanism of action, with its primary attributes being the antioxidant and anti-inflammatory activities, which are responsible for the protective effects. When exposed to certain toxins, cells can become more vulnerable to increased permeability, leading to elevated levels of plasma enzymes from the liver such as alanine aminotransferase (ALT), aspartate aminotransferase (ASP), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total bilirubin, and gamma-glutamyl transferase (GGT). Furthermore, the intestinal membrane becomes more susceptible Fig. 3. SFN acts as an indirect antioxidant through the Nrf2/ Keap1/ARE signaling pathway. Created with BioRender.com. CAT – Catalase; GSH – Glutathione; GPx - Glutathione Peroxidase; GR - Glutathione Reductase; GST - Glutathione S-Transferase; HMOX-1 - Heme Oxygenase-1; NQO1 - NAD(P)H Quinone Dehydrogenase 1; SOD - Superoxide Dismutase. Created with BioRender.com. Fig. 4. Mechanism of action of SFN in liver. Created with BioRender.com. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 9 to potential transfer of LPS from the intestinal microbiota to the liver, thereby triggering inflammatory processes. SFN can effectively decrease permeability, restore cell membrane integrity, and decrease the infiltration of enzymes and inflammatory substances. On the other hand, SFN could restore antioxidant enzyme levels when they are affected by toxics, primarily mediated by the increase in Nrf2 factor. Regarding histopathological studies, SFN-pretreated animals exposed to Cd show markedly attenuated abnormalities such as vacuolization, inflammatory cell infiltration, sinusoidal dilatation, and distinct damage of cytoplasmic organelles (He et al., 2021). Recently, Hong et al. (2023) reported that SFN demonstrated a mitigating effect on lipid metabolism disorder and stress of the ER induced by BPA in both in vitro and in vivo models. In vitro findings suggested that SFN reduced lipid accumulation in hepatocytes and lowered the levels of crucial lipogenic enzymes by inhibiting the ER pathways. However, in vivo, SFN did not cause a reduction in the liver/body weight ratio in animals treated with BPA. New research in order to elucidate the role of ER stress in vivo is needed. Neuroprotective effects of sulforaphane The main neuroprotective effects of SFN reported in the selected literature are reviewed in Table 2. Its efficacy has been demonstrated using both in vitro and in vivo models, in vitro models being the most common. The effects of SFN are mainly related with the antiinflammatory and antioxidant activity that prevents neuronal degeneration. Studies carried out in vitro have been performed by SFN exposure to several cell lines and primary cultures from different parts of the nervous system: SH-SY5Y (Lee et al., 2014; Zhou et al., 2016; Brasil et al., 2023), neural crest cells (Li et al., 2019), BV-2 cells (Konwinski et al., 2014; Wang et al., 2020; Yang et al., 2023), N2A cells (Kwak et al., 2007), primary microglial and cortical neurons (Yang et al., 2023), or primary astrocytes (Bergstrom et al., 2011). The in vitro protective effects of SFN are investigated against proteins such as amyloid beta (Yang et al., 2023) or prion (Lee et al., 2014), insecticides such as chlorpyrifos (Brasil et al., 2023) or rotenone (Zhou et al., 2016), herbicides such as paraquat (Mizumo et al., 2011), chemicals such as H 2 O 2 (Konwinski et al., 2004; Kwak et al., 2007; Bergmenton et al., 2011; Mizuno et al., 2011) and Lipopolysaccharide (Holloway et al., 2016; Wang et al., 2020). The majority of neurotoxic agents under investigation act as acetylcholinesterase (AchE) inhibitors, encompassing insecticides and herbicides. Additionally, research has also focused on toxics implicated in cognitive dysfunction, such as amyloid beta and prions. There is only one study using an ex vivo model, with organotypic cultured rat hippocampal tissue from rats exposed to scopolamine (SCOP) and SFN (Park et al., 2021). In this study, the authors showed that SFN exhibited a concentration-dependent increase in overall fEPSP (field excitatory postsynaptic potential) after high-frequency stimulation and mitigated the interference of SCOP-induced fEPSP in the CA1 area of the hippocampus. Furthermore, SFN prevented the long-term potentiation (LTP) and cognitive abilities induced by cholinergic and muscarinic receptor blockade. These findings indicate that SFN mitigates the decline induced by SCOP in short-term working memory, long-term spatial memory, and avoidance memory in rats. These effects are correlated with the induction of brain-derived neurotrophic factor (BDNF) and cAMP response element binding protein (CREB) expression in the hippocampus, along with the enhancement of synaptic activity. Therefore, SFN merits further investigation as a potential agent for preventing and treating Alzheimer’s disease (AD) or disorders related to learning and memory deficits in individuals affected by neurodegenerative disorders (Park et al., 2021) (Fig. 5). In terms of in vivo studies, only three different models have been used: C57BL/6 mice, Wistar and Sprague-Daley rats. The in vivo protective effects of SFN were investigated against 6-hydroxydopamine (Morroni et al., 2013), cis-platinum (Fouad et al., 2022), ethanol (Li et al., 2019; Xu et al., 2020), drugs such as pilcarpine (Folbergrov´ a et al., 2023), and lipopolysaccharide (LPO) (Holloway et al., 2016; Wang et al., 2020). Similar to what has been studied in vitro, toxic agents studied in vivo also caused mainly cognitive and coordination dysfunctions. The protective effect of SFN in vivo has been shown to have 3 main pathways: 1) by antioxidant effects, 2) by anti-inflammatory effects, and 3) by improving motor coordination and reflexes. SFN exhibited a multifaceted protective role in various contexts. In the context of neurodegenerative oxidative damage to the basal ganglia, SFN restored nigral GSH levels and enhanced GST and GR, thus increasing the antioxidant potential (Morroni et al., 2023). Furthermore, SFN inhibited ROS production and MDA accumulation in mice with rotenone-induced dopaminergic neural loss (Zhou et al., 2016). Consequently, SFN could serve as a prevention of neurodegenerative processes such as Parkinson’s disease and improve locomotor activity. Furthermore, SFN exerted anti-inflammatory effects on LPS by suppressing NFκB signaling, as reported by Wang et al. (2020). Additionally, SFN demonstrated its efficacy in mitigating alcohol-induced loss of righting reflex (LORR) duration without affecting latency, as observed in the study by Xu et al. (2020). Taking into account the in vitro and in vivo information reported, the entire mechanism of action is proposed in Fig. 5. Lastly, SFN is involved in the prevention of adverse effects caused by cis-platinum. SFN increased in AchE activity and restored redox status by regulating LPO, NO, and GSH levels (Fouad et al., 2022) after exposure to pilocarpine and LiCl. The protective effects of SFN extended to the hippocampal and the dentate gyrus, where it prevented damage induced by oxidative stress (Folbergrov´ a et al., 2023). Nephroprotective effects of sulforaphane Table 3 includes the nephroprotective effects of SFN found in the scientific literature, in both in vitro and in vivo models. The antiinflammatory and antioxidant activities of SFN may be responsible of its efficient prevention of renal injury. In relation to this protective effect, more in vivo studies have been found than in vitro ones, in contrast to what has been found in the previous tissues. Only one in vitro study has been found that attributes protective effects of SFN using LLC-PK1 cells against the toxic effects of cis-platinum by restoring mitochondrial membrane potential (Guerrero-Beltr´ an et al., 2010). The rest of the studies have been carried out in Wistar rats, mainly testing protective effects against metals and their compounds: cadmium (Li et al., 2015), mercury (Guo, 2016), cis-platinum (Guerrero-Beltr´ an et al., 2010; 2012) and calcium oxalate (Liu et al., 2012). The toxic effects induced by these metals at the renal level caused mainly renal failure, glomerulopathies, and tubulopathies. All the studies highlight a common mechanism of action: SFN restores the levels of urinary blood urea nitrogen (BUN) and restores the enzymatic activities of NAG, LDH, ALP, SOD, and GSH-Px. Additionally, because of its antioxidant effects, it is able to decrease the high levels of MDA produced by toxic substances. Cardioprotective effects of sulforaphane Table 4 presents a synthesis of the cardioprotective effects of SFN, including evidence derived from both in vitro and in vivo models. Table 4 consolidates the key findings from the selected studies to provide a comprehensive overview of SFN’s impact on cardiovascular health. These effects are mainly ascribed to the antioxidant activity of SFN, demonstrating its efficacy in preventing damage to the heart and aorta. There is only one in vitro study using primary cultured cells from rat. A mechanism of action against H 2 O 2 -produced oxidative stress has been demonstrated in which the presence of antioxidant enzymes is increased when cells are treated with SFN and estradiol (E2) (Angeloni et al., 2017). All the in vivo studies have been conducted in C57BL/6 J mice. The toxic substances assayed were as follows: cuprizone (CPZ), methamphetamine (MA) and angiotensin II (Ang II) to induce hypertension. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 16 Table 6 Overview of studies reporting the protective effects of SFN on toxicants with gastrointestinal effects. Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference Acetic acid 15 mg/kg Intracolonic single injection of 2 ml of 4 % acetic acid and SFN by oral gavage daily for two weeks. Sprague Dawley rats All morphological changes in the colon were improved by treating with SFN. Significantly increased expression of PGC- 1, TFAM, Nrf2, and HO-1 associated with a reduction in the expression of mTOR, cyclin D1, and PCNA in the liver. Alattar et al., 2022 Acetylsalicylic acid (ASA) 5 mg/kg Induction of gastric ulcer (GU) was undertaken using 1-ml ASA (150 mg / kg of body weight, i.g.). SFN was administered by orogastric gavage 1 h before induction of GU. Male Wistar Albino rats SFN reduced the number of TUNEL positive cells and iNOS-positive cells. It decreased TOS and OSI, as well as increased TAS and TT levels. It increased the activities of SOD and GPX. It decreased TNF- α and ADMA levels and increased NO levels. Increased mRNA expression of DDAH-1 and DDAH-2 and reduced NF-kB mRNA in comparison with those of the ASA group. Zeren et al., 2016 Dextran sodium sulphate (DSS) 25 mg / kg b.w. SFN Mice orally received phosphate buffered saline as control or SFN for 7 days. Acute colitis was induced by ingestion of 4 % DSS through drinking water for 5 days. Subsequently, both groups of animals received PBS for a further 7 days. C57BL/6 J mice Pretreatment with SFN before inducing colitis significantly minimised both body weight loss and disease activity index when compared to control mice. Furthermore, SFN-pretreated mice exhibited significantly longer colon lengths than control mice. Both macroscopic and microscopic examinations of the colon showed reduced inflammation in SFN pretreated animals. mRNA analysis of distal colon samples confirmed decreased expression of inflammatory markers and increased expression of Nrf2-dependent genes in SFN pretreated mice. Wagner et al., 2013 Dextran sodium sulphate (DSS) Broccoli seed extract (BSE): 212.0 ±41.0 nmol/ g SFN (concentration detected in mice colonic contents) The first week: 0.2 ml of skim milk and the second week: 0.2 ml of skim milk + 2.5 % DSS or 0.2 ml of 370 mg/kg BSE- dissolved skim milk +2.5 % DSS C57BL/6 J mice The administration of broccoli seed extract demonstrated a potential to alleviate the development of colitis in mice subjected to DSS treatment. In particular, the BSE intervention led to a significant reduction in the production of inflammatory markers, including IL-6, IL- 1β, and TNF- α , within the colon. Furthermore, BSE treatment diminished MDA activity. Furthermore, the intervention with broccoli seed extract improved colon injury, as evidenced by the reduction of tissue edema and substantial inflammatory cell infiltration within the mucous and submucous layers. Furthermore, it mitigated colonic structural damage, including crypt disruption, goblet cell depletion, and intestinal epithelial cells. Interestingly, Broccoli seed extract contributed to the restoration of intestinal barrier function, as indicated by enhanced expression of ZO-1 and claudin-1 proteins. Wu et al., 2023 Dextran sodium sulfate (DSS) Broccoli extract (nonstandardised) The authors attribute the effects to the SFN Mice were fed a 10 % steamed broccoli sprout diet and a three-cycle regimen of 2.5 % DSS in drinking water over a 34- day experiment to simulate chronic relapsing ulcerative colitis. C57BL/6 J mice Recovery of intestinal microbiota levels. Holman et al., 2023 Dextran sulphate sodium (DSS) Steamed broccoli sprout diet (5 %) containing a low level of SFN (<25 µg/g diet) and a high level of glucoraphanin Prepared diet for 2 weeks and then treated with 2 % DSS C57BL/6 J female and male mice The broccoli sprouts reduced chemically induced colitis. This protective effect was dependent on the presence of an intact microbiota. Zhang et al., 2023 N- nitrosobis(2- oxopropyl) amine (BOP) 80 ppm The hamsters were s.c. injected with BOP (10 mg/kgb.w) 4 times a week, and fed a diet supplemented with 80 ppm SFN during the initiation (3 weeks including 1 week before and after the injection of BPO) or post initiation stages (basal diet after the last BOP ingestion and fed for 14 weeks with SFN) Male 6-week-old Syriam hamsters SFN blocked the initiation of BOP of hamster pancreatic carcinogens. SFN decreased atypical hyperplasias in pancreatic ducts, and multiplicity of adenocarcinomas in the initiation stage but not in the post-initiation stage. Kuroiwa et al., 2006 A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 17 Some studies also demonstrated the protective effects of SFN during pregnancy in embryos exposed to 2-amino-1-methyl-6-phenylimid-azo [4,5-b] pyrimidine (Zhang et al., 2021) and on placental cell lines against the inflammatory effect of TNF- α (Cox et al., 2019). The safety of SFN and broccoli sprout supplements during pregnancy is warranted given the commercial availability. Furthermore, no effects on the incidence of sperm abnormalities were reported in mice treated with up to 10 g/kg b.w. (Zhou et al., 2015). However, it is important to note that ADMA: asymmetric dimethylarginine; ASA: Acetylsalicylic acid; BOP: N-nitrosobis(2-oxopropyl)amine; DDAH: dime-thylaminohydrolase; DSS: Dextran sodium sulphate; GPx: glutathione peroxidase; HMOX1: heme oxygenase-1 (HO-1H3K4me3: histone H3 lysine 4; IL: interleukin; iNOS: inducible nitric oxide sinthase; MDA: malondialdehyde; mTOR: mammalian target of rapamycin; NF-kB: nuclear factor kappa light chain enhancer of activated B cells; Nrf2: nuclear factor erythroid 2- related factor 2; NO: nitric oxide; OSI: oxidative stress index; PCNA: proliferating cell nuclear antigen; PGC-1: peroxisome proliferator activated receptor-gamma coactivator; s.c.: subcutaneous; SOD: Superoxide dismutase; TAS: total antioxidant status; TFAM: mitochondrial transcription factor A; TNF α : tumour necrosis factor alpha; TOS: total oxidative stress; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labelling. Table 7 Overview of the studies reporting the protective effects of SFN against toxicants with immunitary effects. Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference Butylbenzyl Phthalate (BBP) 5 μ M 10 −5 М of BBP +SFN (the medium was replaced every two days during 4-day treatment) MCF-7 cells The growth promoting effect of BBP could be mitigated by SFN, accompanied by a reversal of altered expression of miR-19a, miR-19b, PTEN, and p21. SFN also suppressed the binding of upregulated miR-19 with PTEN induced by BBP. Cao et al., 2023 Cd 20–100 µM 24 h with IC 10 , IC 25 , IC 50 of Cd (data not shown) and SFN for 24 and 48 h. Human lymphocytes and monocytes SFN decreased Cd-induced cytotoxicity in a concentration- and time-dependent manner Alkharashi et al., 2017 Co-60 γ-radiation, doxorubicin or bleomycin 400 nM Whole blood or lymphocytes were exposed to 0–2 Gy Co-60 gamma ray radiation or to carcinogen (doxorubcin or bleomycin) for 1 h before PHA stimulation. Subsequently, 400 nM of SFN was added in phase G0 or G1 of the cell cycle (2 or 20 h after PHA stimulation, respectively). Blood from volunteers Reduction of MN% (up to 70 %) in peripheral blood lymphocytes of individuals exposed to γ- and β-radiation, doxorubicin or bleomycin. SFN reduces both acute and late effects related to radiation-induced genomic damage. Katoch et al., 2013 Hydrogen peroxide (H 2 O 2 ) 10 µM SFN for 24 h Cells were exposed for 22 h to SFN and then 2 h to 200 µM of H 2 O 2 for inducing oxidative stress. Granulosa-lutein cells (GLCs) were collected from women 20 to 38 years old with normal menstrual cycles with 25–35- day periods Intracellular ROS and apoptosis rose dramatically in GLC with enhanced oxidative stress. SFN therapy decreased ROS and apoptosis levels and increased overexpression of AMPK, AKT, and NRF2 genes and proteins. Taheri et al., 2022 Lipopolysaccharide (E. coli serotype 0111:B4) 5 and 50 mg / kg i.p. 1, 10 and 100 μ M In vivo: Mice were exposed to 5 mg/ kg and 50 mg/kg of SFN dissolved in corn oil 24 h by injection prior to exposure to 0.5 mg/kg of LPS. In vitro: Cells were treated with SFN (1, 10 and 100 μ M) 24 h before adding LPS (500 ng/ml and 10 µg/ ml) for 4 h. Wild-type (WT) C57BL/6 J mice and Nfe2l2tm1Ywk knockout (Nrf2-KO) mice. HBMEC-3 cell line In vivo, SFN reduced increased LPS in brain and serum inflammatory cytokines (IFN-γ, MCP-1 and TNF- α ) whilst also increasing IL-10 expression and reduced MPO levels. In vitro SFN reduced neutrophil recruitment through down-regulation of E-selectin and vascular cell adhesion molecule 1 (VCAM-1) and decreased ROS induced by LPS. SFN may reduce cerebral inflammation in sepsis produced by LPS by mediating its antiinflammatory and antioxidant effects through Keap1/Nrf2 transcriptional activation of the antioxidant system and via the NFκB pathway. Holloway et al., 2016 Lipopolysaccharide (LPS) 0–50 µM Macrophages were preincubated for 6 h with and without SFN and then treated with LPS for 24 h. Raw 264.7 murine macrophages LPS significantly increased the expression of inflammatory cytokines and the concentration of NO in nontreated cells. SFN prevented the expression of NO and cytokines by regulating inflammatory enzyme iNOS and activating the Nrf2 / HO-1 signal transduction pathway. Ruhee et al., 2019 Akt: serine/threonine protein kinase; AMPK: adenosine 5 ′ -monophosphate (AMP)-activated protein kinase; GLC cells: Granulosa-lutein cell line; GU: Gastric Ulcer; HBMEC-3: Human Brain Microvascular Endothelial Cells; HMOX1: heme oxygenase-1 (HO-1H3K4me3: histone H3 lysine 4; IFN-γ: Interferon gamma; iNOS: ´ oxido ntrico sintasa indicible; Keap1: Kelch-like ECH-associated protein 1; LPS: bacterial lipopolysaccharide; MCF-7: Breast Cancer Cells; MCP-1: Monocyte chemoattractant protein-1; miR-19A: MicroRNA 19a; miR-19b: MicroRNA 19b; MN: micronuclei; NSAID: Non-steroidal anti-inflammatory drugs; Nrf2: erythroid 2-related factor 2; NO: nitric oxide; p21: tumour protein; PTEN: phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase; ROS: reactive oxygen species; TNF α : tumour necrosis factor alpha; VCAM-1: Vascular cell adhesion protein 1. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 18 Table 8 Overview of the other studies reporting the protective effects of SFN against toxic substances in several models. Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference 2-Amino-1-methyl-6- phenylimidazo[4,5-b] pyrimidine (PhIP) 5 µM Nano-SFN 200 µM PhIP +5 µM Nano-SFN for 36h Early chick culture (embryos) Nano-SFN can effectively alleviate the PhIP microenvironment-induced abnormal development of the embryonic nervous system and has a protective effect on embryonic development. Thus, it reversed PhIP-inhibited neural cell differentiation in the neural tube. Improved generation and migration. Zhang et al., 2021 2-chloroethyl ethyl sulfide 5 µM 48 h with SFN and 5 min with 2 mM of 2-chloroethyl ethyl sulfide NCTC2544 human keratinocytes SFN restored toxicity by upregulating the gene expression of GCLM and Nrf2. Moreover, SFN increased the GHS content. Abel et al., 2011 4-hydroxy-2-nonenal (4- (HNE) 5.0 mg/kg Mice were administered SFN or carnosic acid (CA) (1 mg / kg) (or respective vehicles) 48 h before cortical mitochondria. The purified mitochondria were then exposed ex vivo to 4-HNE and incubated for 15 min at 37 ◦C. The samples were analyzed for mitochondrial bioenergetics. Male CF-1 mice Administration of either compound (SFN or CA) significantly increased heme oxygenase-1 mRNA in cortical tissue 48 h after administration, verifying that both were capable of inducing the Nrf2–ARE pathway. Furthermore, the prior in vivo administration of SFP and CA attenuated 4-HNE-induced inhibition of mitochondrial respiration for complex I, but only CA acted to protect complex II. SFN and CA reduced the amount of 4-HNE bound to mitochondrial proteins determined by Western blot. Miller et al., 2013 5-Fluorouracil 2 and 20 mg/kg Negative control group, solvent control, 5-Fu+SFN-l (2 mg/kg) and 5-Fu+SFN – H (20 mg/kg). Exposure by gavage for 11 days. Intestinal mucositis was induced in the 5-Fu, SFN-l and SFN – H groups by injection of 5-Fu (300 mg/kg) on sixth day. Male Balb/c mice SFN was able to attenuate 5-Fu- induced intestinal injury by restricting related clinical symptoms, regulating inflammatory homeostasis, and maintained intestinal permeability. SFN at high doses was more effective in enhancing antioxidant defence and tight junction in jejunum segment at the molecular level. Wei et al., 2020 Benzo (a) pyrene (BaP) 9 mM/mice/d Mice were treated with BaP (100 mg / kg b.w. i.p.) three times a week and kept for 20 weeks. Previously, animals were administered orally with SFN (9 mM/mice/d) on alternate days for two weeks prior to first dose of BaP and continuously treated until the 12th week and sacrificed later. Swiss Albino mice SFN had inhibitory effects on B[a]P- induced aryl hydrocarbon receptor (AHR) activation resulting in decreased Phase I enzyme activities in mice. SFN enhanced Nrf2 transcription together with phase II enzymes resulting in reduced carcinogen-induced stress. Kalpana Deepa Priya et al., 2011 Benzo(a)pyren (BaP) 0.55 mg / kg of SFN or 30 % dry broccoli (substituting alfalfa) The fish were fed over 30 days with three different diets, one containing broccoli, another containing SFN, and controls containing alfalfa instead. Subsequently, 100 µg/g b.w. BaP was injected i.p. to all fish being sacrificed at different times (0, 12, 24, 36, 48 and 72 h). Tilapia fish (Oreochromis niloticus) The presence of broccoli or SFN significantly increased the basal levels of CYP450 activity, which could improve detoxification capacity. After exposure to BaP, less aromatic metabolites were found in bile in fish fed with diets enriched with broccoli or SFN compared with control fish. Thus, these diets induce changes in the enzymatic systems involved in the detoxification metabolism of fish. Villa-Cruz et al., 2009 Blue light (400 nm) 5–25 μ M Cells were pretreated with SFN for 3 h and then exposed to blue light at intensity of 2000 ±500 lx for 24h Human RPE cell line ARPE-19 SFN protected against blue light–induced oxidative injury, inflammation, and apoptosis. Molecular mechanisms may involve increased antioxidative, autophagy, and PGC-1 α related mitoprotective properties. It maintains the Nrf2- related redox state and upregulates the expression and autophagy of SIRT1 and PGC-1 α expression and autophagy. Yang et al., 2021 (continued on next page) A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 19 Table 8 (continued) Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference Cadmium (Cd) 20–100 µM 24 h with 6, 13.65 and 39.8 μ M of Cd and SFN for 24 and 48 h Human mesenchymal stem cells (hMSCs) Reduced necrotic cells. Alleviated the acidified cytoplasm. Attenuated the decrease in MMP. The expression pattern of the POR, TNFRSF1A and TNFSF10 genes was significantly stabilized. Alkharashi et al., 2019 Cadmium (Cd) 5 µg/g b.w. Shrimps were injected with Nrf2 targeting sdRNA (4 µg/g b.w.) or SFN (5 µg/g b.w.) and then exposed to 40 mg / l of Cd Cl 2 for 48 h. Marsupenarus japonicus SFN decreased the number of apoptotic cells and alleviated the oxidative stress: increased T-AOC, SOD activity, SOD mRNA expression, and reduced MDA content. Detoxified enzymes, such as GST, Erod, and their corresponding gene expressions, were elevated in the SFN group. Ren et al., 2021 Cadmium (Cd) 50 μ M SFN Zebrafish larvae were incubated with SFN or 0.01 % DMSO control for 24 h at 28 ◦C. Subsequently, they were exposed to 5 and 25 µM Cd. Zebrafish larvae (EKW strain). Pre-incubation of zebrafish larvae with SFN increased gst pi, gclc, and prdx1 expression levels, which were associated with protection against Cd-mediated damage to the olfactory epithelium, supporting Nrf2 ′ s protective role against Cdinduced oxidative stress. Furthermore, SFN pre-incubation afforded protection against cell damage, thus supporting a protective role for an Nrf2-regulated cellular. Wang and Gallagher, 2013 Cisplatin 0.5–5 µM for 24 h 500 µg/kg Cells were treated with SFN (0–5 µM) for 24 h and then 40 µM of cisplatin was added for another 24 h. Rats were exposed to a single dose of cisplatin by injection (7.5 mg/kg). SFN (500 µg/kg i.v.) was administered twice: 24 h before exposure and also 24 h after toxicant exposure and the animals were sacrificed three days after injection of cisplatin. LLC-PK1 cells Wistar rats SFN prevented cisplatin-induced cell death. SFN improved renal dysfunction, structural damage, oxidative/ nitrosative stress, GSH depletion, increased urinary excretion of hydrogen peroxide, and decreased antioxidant enzymes (CAT, GPx, and GST) activities. Guerrero-Beltr´ an et al., 2010 Dexamethasone (DEX) 5 μ M The C2C12 myoblasts were treated with 5 μ M SFN in the presence of 5 μ M DEX for 24 h. C2C12 myoblasts SFN treatment improved protein synthesis by activating Akt and prevented DEX-mediated upregulations of the myostatin and Atrogin-1 mRNA, increasing the mRNA level of MyoD and reducing protein degradation. Son et al., 2017 Dibenzo[def,p]chrysene (DBC) 400 ppm SFN alone 600 ppm SFN +500 ppm indole-3-carbinol (I3C) Pregnant mice were exposed to different diets from day 9 of gestation until weaning (postnatal day 21). They were exposed to SFN alone or in combination with I3C. Female mice B6129SF1 Purified SFN, incorporated into the maternal diet at 400 ppm, decreased the latency of DBC-dependent morbidity. However, when I3C and SFN were administered in equimolar combination, no protective effect was observed. SFN metabolites measured in the plasma of newborns were positively correlated with exposure levels through the maternal diet but not with offspring mortality. Shorey et al., 2013 H 2 O 2 7 µM The OACs were incubated with 200 μ M H 2 O 2 for 8 h. Subsequently, the OAC and osteochondral composites were treated with SFN for 72 h. Osteoarthritic articular chondrocytes (OACs) SFN ameliorates H 2 O 2 -induced oxidative stress and cartilage matrix through suppressing inflammatory cytokines and activating the Keap1 / Nrf2 pathway. Yang et al., 2020 High-fat and highfructose (HFHFr) diet 15 and 30 mg/kg Control group, HFHFr group, HFHFr +low-dose SFN and HFHFr +high-dose SFN for 12 weeks. SFN was administered to mice every two days by gavage. Male C57BL/6 mice Long-term SFN supplementation improved the intestinal microbial composition in HFHFr diet-induced NAFLD mice. SFN improved tight junction proteins in the colon, inhibited the LPS/TLR4 signalling pathway and endoplasmic reticulum stress in the intestine, and improves intestinal inflammation. As a consequence, it maintained Xu et al., 2023 (continued on next page) A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 20 Table 8 (continued) Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference intestinal barrier integrity, reduced intestinal-derived LPS, and inhibited the liver LPS/TLR4 signalling pathway to improve liver steatosis and steatohepatitis. Ionizing radiation 0–30 µM Single treatment: cells were incubated 4 h with SFN. Repeated treatment: cells were treated for 4 h with SFN for 3 consecutive days prior to radiation exposure. Primary human skin fibroblasts Fibroblasts exposed to repeated SFN showed a more pronounced dosedependent induction of Nrf2- regulated mRNA and a reduced amount of radiation-induced free radicals compared to cells treated once with SFN. Cellular protection from ionizing radiation measured by the EdU assay was increased, specifically in cells exposed to repeated SFN treatment. SFN was unable to protect Nrf2- knockout mouse embryonic fibroblasts, indicating that radioprotection was Nrf2- dependent. Furthermore, radioprotection by repeated SFN treatment was dose dependent with an optimal effect at 10 uM, Mathew et al., 2014 Maleic acid 1 mg/kg 1 dose of 400 mg / kg maleic acid and SFN each day for four days Male Wistar rats SFN prevented proteinuria, increased urinary excretion of N- acetyl-β-D-glucosaminidase, and decreased plasma GPx activity, renal blood flow, and oxygenation and perfusion of the renal cortex. Briones-Herrera et al., 2018 Methyl methanesulfonate (MMS), urethane (URE), 4-NQO and H 2 O 2 0.14, 0.28, 0.56 mM (corresponding to 25, 50, 100 % lyophilized broccoli) in a DMSO/ Tw80–EtOH mixture (DTE) The third instar larvae were fed SFN (0.14, 0.28, 0.56 mM)/DTE plus mutagens to give a final concentration of MMS (0.5 mM), URE (20 mM), 4-NQO (2 mM) or H 2 O 2 (20 mM) in triplicate. Standard and high bioactivation (HB) crosses of Drosophila melanogaster SFN did not show clear protective effects against the genotoxicity of the compounds tested. Although SFN showed a tendency to reduce the genotoxicity produced by MMS, this effect could be due to the action of the solvent (DTE mixture). Additionally, SFN showed per se genotoxic effects and increased the standard cross of genotoxicity of H 2 O 2 in the D. melanogaster and HB cross at 0.28 mM and 0.56 mM, respectively. The results were inconclusive for the protective effects of SFN against the genotoxicity of MMS, URE, 4-NQO, and H 2 O 2 . Due˜ nas-García et al., 2012 Paraquat 1 µM 22–24 h of cotreatment with 250 μ M of paraquat and SF Bovine in vitromatured oocytes Inhibition of increased levels of ROS content. Rescue of GSH and T-SOD levels. Cotreatment with SFN elevated the low ratio of matured oocytes induced by paraquat. The application of SFN markedly facilitated the transcriptions of GCLC, GCLM, HO-1, NQO1, and TXN1 under paraquat exposure, but the expressions of the mRNA TXNRD1 and PRDX1 mRNA had no marked difference. Feng et al., 2023 Quinolinic acid (QUIN) 5 mg/kg Rats received i.p. SFN 24 h and 5 min before the intrastriatal infusion of QUIN. Male Wistar rats SFN enhanced reduced GSH levels and increased GPx and GR activities. SFN also prevented QUIN-induced oxidative stress (measured by oxidised proteins levels), histological damage, and the circling behaviour. Santana-Martínez et al., 2014 Sodium arsenite 40 μ M Pretreatment with SFN (40 mМ) for 12 h was performed in larvae for 12 h before arsenite treatment (2 mM from 3.5 to 4 days after fertilisation), and SFN was then withdrawn by exchanging E3+medium Zebrafish larvae Pretreatment with SFN improved the survival of zebrafish larvae after arsenic exposure. Fuse et al., 2016 (continued on next page) A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 21 Table 8 (continued) Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference containing arsenite. The solutions were changed every 2 days during the tests. Streptozotocin (STZ) 40 μ g/kg Mice received i.p. injections of 40 μ g/kg SFN daily for 3 days prior to the administration of 80 mg of STZ / kg b.w. (to induce diabetes). Apart from that group, there were 3 more: one nontreated group and two only receiving SFN or STZ. Five days after the first STZ injection, all animals were sacrificed. Male ICR mice Pretreatment with SFN blocked STZ- induced islet destruction and restored the number of islet cells secreting insulin to the level of the control. Song et al., 2009 Tert‑butyl hydroperoxide 0.5 µM 24 h of SFN pretreatment and 24 h of 0.1, 0.5, 1, 5, or 10 mM tert‑butyl hydroperoxide ARPE-19 cells SFN attenuated ROS production, decreased mitochondrial dysfunction, activated phase II detoxification enzymes, and suppressed pro-inflammatory mediators (cytokines ICAM-1 and MCP-1). 2020 TNF α 5–20 μ M SFN and 1–100 ng/ml of TNF α for 24h Placentae, umbilical cords, and serum. HUVEC cells. Reduced TNF- α mediated HUVEC secretion of endothelin-1, VCAM-1, ICAM-1, and E-selectin, and prevented increased endothelial permeability. In placental explants, reduced the secretion of soluble Flt- 1, soluble endoglin, and activin A. Cox et al., 2019 Type 2 diabetes mellitus model induced by a high-fat diet for 3 months and streptozotocin 0.5 mg/kg 4-month SFN given s.c. daily five days a week Male C57BL/6 J mice SFN may prevent inhibition of the LKB1/AMPK pathway DM-derived oxidative stress of the LKB1 / AMPK pathway, and subsequently influence the master regulators of lipid metabolism, including the degradation of intracellular LDs to fatty acids by lipophagy, and the increase in mitochondrial fatty acid uptake and oxidation by upregulated CPT-1 and PGC-1 α . This results in the prevention of T2DM- induced lipotoxicity and, hence, cardiomyopathy. Zhang et al., 2014 UV irradiation (UVR) 5, 10, 25 µM Cells were pre-incubated with 5, 10 or 25 µM of SFN for 24 h before UV irradiation (300 mJ/ cm 2 ). HaCaT cell line (keratinocytes) SFN, mainly at 10 µM up-regulated the gene for the enzyme CAT and the Nrf2 target genes, γ-GCS, HO-1 and NQO1 in UV irradiated skin cells. SFN reduced UVR-induced structural damage in the epidermis 48 h after UV-irradiation in a concentration-dependent manner (5 and 10 µM). However, the higher concentration of SFN (25 µM) led to a significant increase in sunburn cells compared to non-SF treated skin. Kleszczy´ nski et al., 2013 Vinclozolin 45 mg/kg One dose of 125 mg/kg vinclozolin and one dose of SFN 48 h later CD-1 mice SFN produced a dose-dependent rescue (increase) in masculinisation of anogenital distance. Amato et al., 2022 X-ray Irradiation 0.5 mg/kg b.w. Control, SFN, IR, and IR+SFN groups. SFN and IR+SFN groups were injected i.v. once, 5 times a week for 4 weeks. Later, in the IR and IR-SFN groups, the left thigh of mice was treated with a single X-ray irradiation of 40 Gy at a dose rate of 200 cGy/min. The mice were sacrificed after 1 month of feeding. SPF C5BL/6 mice SFN prevented radiation-induced muscle fibrosis. It inhibited the expression of components of the IR- induced extracellular matrix and oxidative stress in the skeletal muscle. Also, it increased Nrf2 expression and function through AKT/GSK-3β/Fyn pathway, and downregulated TGF-β1/Smad pathway. Wang et al., 2022c 5-Fu: 5-Fluorouracil; Akt: serine/threonine protein kinase; AHR: aryl hydrocarbon receptor; AMPK: adenosine 5 ′ -monophosphate (AMP)-activated protein kinase; ARPE-19: spontaneously arising retinal pigment epithelia; BaP: Benzo (a) pyrene; CA: carnosic acid; DBC: Dibenzo[def,p]chrysene; DEX: Dexamethasone; EdU: 5-ethy- nyl 2´-deoxyuridine; Flt-1: vascular endothelial growth factor receptor 1; Fyn: tyrosine-specific phospho-transferase; GCLM: glutamate cysteine ligase modifier subunit; GPx: glutathione peroxidase; GR: glutathione reductase; GSH: glutathione; GSK-3b: glycogen synthase kinase 3 beta; HaCaT cell: keratinocytes; hMSCs: human mesenchymal stem cells; HNE: 4-hydroxy-2-nonenal; HMOX1: heme oxygenase-1 (HO-1H3K4me3: histone H3 lysine 4; ICAM-1: Intercellular Adhesion Molecule 1; I3C: Indole-3-Carbino; i.v.: intravenous; LLC-PK1: Lilly Laboratories Culture-Porcine Kidney 1; HUVEC: EndoGRO Hu-man Umbilical Vein Endothelial Cells; LPS: bacterial lipopolysaccharide; NAFLD: Non-alcoholic fatty liver disease; NCCs: Neural Crest Cells; NCTC2544: human keratinocytes; Nrf2: nuclear factor erythroid 2-related A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 22 the concentrations used in these studies far exceed what would be achieved with typical consumption of fresh vegetables or recommended doses of available supplements (Shorey et al., 2013). Toxicity of sulforaphane Although SFN has numerous beneficial and protective effects against toxins, no substance is exempt from having adverse or toxic effects at high doses. The toxicity of SFN has been tested both acutely and subchronically. Scola et al. (2017) observed severe toxicity, such as deep sedation, ataxia, ptosis, and tremors in mice acutely exposed to high doses of SFN intraperitoneally. In this study, doses of 300 mg SFN/kg resulted in death for all animals within 180 min. Similar symptoms were reported at doses of 250 mg/kg, with seven out of twelve mice dying within 240 min. At a dose of 200 mg/kg, sedation and ptosis were observed in all animals, with six out of twelve mice dying within the first night. At lower doses, such as 150 mg/kg, sedation persisted, but mortality was not observed. Additionally, authors determined the LD 50 in 212.67 mg/kg. On the other hand, Zhou et al. (2015) studied the effects of a glucoraphanin-rich broccoli extract in rats orally exposed for 30 days at doses of 3 g/kg/day (equivalent to 390 mg/kg/day of glucoraphanin, SFN precursor). During the 30-day feeding study, no mortality or treatment-related adverse clinical findings were observed. Animals in all groups displayed normal activities and growth. Body weights did not significantly differ between the SFN-treated and control groups, although there was a slight decrease in food consumption in high-dose males. However, this decrease was associated with an increase in food utilization rate, and body weight gain remained similar to control groups. Minor fluctuations in haematology and clinical chemistry parameters were observed, but they fell within the historical control range and showed no clear dose-response relationships. Clinical chemistry parameters showed minimal changes within the historical control range of the testing laboratory, with no significant differences in liver or kidney toxicity indicators. Organ weights increased in high-dose males for the spleen and kidneys, while the absolute weight of the testes was slightly reduced. However, all organ weight values remained within the historical control range. Macroscopic evaluations at necropsy revealed no abnormalities, and histological evaluations of various tissues showed normal structure without observable abnormalities related to SFN treatment. Additionally, mutagenicity/genotoxicity studies have been performed to evaluate the glucoraphanin-rich broccoli extract (GBE). Thus, three mutagenic/genotoxic experiments, including an Ames test, an in vivo mouse micronucleus, and an in vivo mouse sperm abnormality (Zhou et al., 015). In the Ames test, four Salmonella typhimurium histidine-deficient test strains (TA97, TA98, TA100 and TA102) were exposed up to 5000 µg SFN/plate in presence and absence of S9 metabolic activation system. In the two in vivo tests, mice were exposed up to 10 g/Kg b.w of BGE by oral gavage. The results showed no mutagenic activity in the Ames assay and no evidence of genotoxic potential in the in vivo assays at any of the doses tested. In general, the toxic effects of SFN have been shown to occur at very high doses. However, there are few studies on this subject, and more investigations are needed to establish safe therapeutic doses of SFN. Concluding remarks The chemoprotective effects of SFN have gained toxicological relevance due to the large number of xenobiotics against which it exerts protective properties, its contribution against the progression of different diseases, and its beneficial effects on health. Thus, several mechanisms of action have been reported for SFN such as antioxidant, anti-inflammatory, anticancer, immunomodulatory, metabolic regulator, or protective against endocrine disruption, as described in the present study. The concentration used in the in vitro assays ranges between 0.5–160 µM based on cytotoxicity studies performed by some authors in previous assays, although the concentration most commonly used in these experimental models with a protective effect is 5 µM. On the other hand, in vivo studies, doses from 5 to 30 mg/kg have been used, 5 mg/kg being the dose usually chosen as effective. As outlined in the previous section, doses associated with toxic effects significantly exceed those involved in protective effects by a factor of approximately 50 (250 mg/kg versus 5 mg/kg). Consequently, a safe dosage range of SFN could be established in which only beneficial and protective effects are expected to occur. In relation to kinetic studies, its distribution in most of the tissues has been demonstrated, while its potential uptake through the placental barrier remains to be elucidated. In general, the main mechanism of action of SFN against toxic agents is antioxidant, being a modulator of apoptosis and to have anticancer effects. SFN causes the activation of Nrf2 factor, which in turn increases the content of GSH and the activity of several antioxidant enzymes, CAT, SOD, GPx, and GR, which reduces biomarkers of oxidative stress (Santana-Martnez et al., 2014; Feng et al., 2023). Additionally, potentiation of the antioxidant response by SFN restores S-glutathionylation in the mitochondrial fraction (Aranda-R- ivero et al., 2023). Activation of Nrf2 also reduces inflammation by decreasing interleukin production (i.e. IL-1β) and pyrin domain 3 of the nucleotide-binding domain-like receptor family (Aranda-Rivero et al., 2023). Reduction of oxidative stress and inflammation avoid apoptosis by preventing caspase 3 cleavage and increased levels of B-cell lymphoma 2 (Bcl2) (Kalpana Deepa Priya et al., 2011). Taken together, the present review shows that SFN decreases oxidative stress, prevents inflammation and cell death by apoptosis after exposure to toxic agents. SFN has proven to be effective in different tissues and cellular types (liver, kidney, nervous system, etc.). In relation to the liver, one of the hepatoprotective effects of SFN involves its ability to shield the liver against carcinogens, thereby preventing tumour formation. An ideal chemopreventive agent is expected to exert minimal impact on normal cells while demonstrating potent inhibitory effects on cell proliferation and carcinogenic pathways in cancer cells. Despite numerous studies that investigated both the protective and cytotoxic effects of SFN, the data comparing its impact on normal cells versus cancer cells are still very scarce (Liu et al., 2019). Furthermore, ongoing research aims to explore the effects of SFN on lipogenic enzymes, transcription factors, cytokines such as TNF alpha, mitogen-activated kinases such as JNK, and mitochondrial dysfunction, representing future directions to improve our understanding of the protective role of SFN in the liver (Zhou et al., 2014). In addition, SFN has shown neuroprotector effects against substances capable of inducing neurodegenerative effects. This issue is of great interest due to the increase of this type of diseases and the great concern on the part of the population. However, additional research employing experimental animal models is necessary to assess the influence of SFN bioavailability on its potential mitochondrial protection and exert anti-inflammatory actions in brain cells. Globally, liver and nervous system are by far the most investigated target organs in which SFN protective effects have been studied (approximately 38 % of the studies). This could be due to its mechanism factor 2; MCP-1: Monocyte chemoattractant protein-1; MMP: Ma-trix metalloproteinases; MMS: Methyl methanesulfonate; MyoD: myogenic differentiation protein 1; PGC-1 α : peroxisome proliferator activated receptor-alpha coactivator; PhIP: 2-Amino-1-methyl-6-phenylimid-azo[4,5-b]pyrimidine; POR: Cytochrome P450 Oxidoreductase; s.c.: subcutaneously; Smad: mothers against decapentaplegic; T-AOC: total antioxidant capacity; TGF-β1: transforming growth factor beta 1; TLR4: toll-like receptor 4; TNF α : tumour necrosis factor alpha; TNFRSF1A: Tumour necrosis factor receptor superfamily member 1 A; TNFSF10: Tumour necrosis factor ligand superfamily member 10; TrxR-1: thioredoxin reductase-1; TXR1: human thioredoxin; PRDX1: Peroxiredoxin 1; SOD: Total Superoxide Dismutase; URE: urethane; VCAM- 1: Vascular cell adhesion protein 1; γ-GCL: γ Glutamate–cysteine ligase. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 23 of action, as it is well known that oxidative stress is a crucial factor in liver diseases (Cicho˙ z-Lach and Michalak, 2014) and it is also a key modulator in many neurodegenerative diseases (Singh et al., 2019). Numerous potential clinical applications in humans have been attributed to SFN. In this respect, it has demonstrated efficacy as a chemoprophylactic agent against various cancer types including stomach, breast, colon, and prostate cancers. Furthermore, SFN has shown to hold promise in mitigating hepatic insufficiency, as well as improving cognitive and locomotor functions. Moreover, SFN has been implicated in ameliorating complications associated with type II diabetes by regulating signalling pathways in organs such as the pancreas, kidney, heart, skeletal muscle, brain, and others. Additionally, there is an emerging interest in the chronic use of SFN as a novel therapeutic approach for preventing muscle damage in athletes undergoing daily high-intensity exercise (Sato et al., 2021). Despite its potential clinical applications, there are not many studies that demonstrate the protective effect of SFN on the kidney, considering that it is a target organ of multiple toxic substances (Guerrero-Beltr´ an et al., 2012). Similarly, studies dealing with lungs, heart, or immune system are also limited. Moreover, although it has recently been demonstrated that SFN has beneficial effects on the intestinal microbiota (Marshall et al., 2023), the role of the microbiota in the mechanism of action of SFN against toxic gastrointestinal agents has not been extensively addressed, taking into account that the microbiota performs various crucial functions for the well-being of the organism (Marshall et al., 2023). It is worth noting the current interest to explore the beneficial effects of SFN at the endocrine level, particularly in the context of therapeutic, improving diabetes mellitus and metabolism complications induced by xenobiotics (Mthembu et al., 2023). These complications include diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, as well as other metabolic problems such as non-alcoholic fatty liver disease and skeletal muscle. However, similarly to previous instances, the precise mechanism of action is not yet understood. Some researchers propose that SFN may play an active role in activating the nuclear factor erythroid 2-related factor 2 or effectively modulating AMP-activated protein kinase to offer protection against diabetic complications (Mthembu et al., 2023). A potential limitation of a study investigating the protective effects of SFN against toxic substances is the extrapolation of findings from experimental models to clinical settings. While the present in vitro and in vivo reported studies provide valuable insights into the mechanisms and efficacy of SFN, translating these results to human populations may pose challenges due to differences in metabolism, dosage requirements, and individual variability. Regarding the evaluation of risk of bias, the present study has considered several variables, including the clarity of the objectives, the characterization of the product under investigation, the reproducibility of the assay, comparability, and the adequacy of statistical analysis. Out of the 87 studies included, only two exhibited a medium risk of bias across these parameters, while the remaining studies demonstrated a low risk. This indicates a generally robust methodological quality across the majority of the selected studies, enhancing the reliability of the findings. Consequently, the potential SFN protection mechanism holds promise in countering the impact of new toxic agents and presents a great potential for therapeutic applications as an antioxidant, antiinflammatory, antidiabetic, and anticancer substance. However, although there is a large body of research on liver and cerebral protective effects, there are still studies that do not clarify the mechanisms of action of SFN against metabolic disorders or endocrine disruption. Therefore, further research is needed to exploit the mechanisms of action of SFN and thus to broaden the potential safe therapeutic applications of this substance. Funding This research was funded by the Consejería de Economía y Conocimiento of Junta de Andalucía (P1 8 -TP -2147) and Ministeri o de Universidades (grant FPU number 2019 /01247). CRediT authorship contribution statement Antonio Cascajosa-Lira: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Ana I Prieto: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Silvia Pichardo: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Angeles Jos: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Ana M Came´ an: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors wish to thank the Consejería de Economía y Conocimiento of Junta de Andalucía (P18-TP-2147) for the financial support. Antonio Cascajosa Lira thanks the Spanish Ministerio de Universidades for the funding FPU grant (FPU2019/01247). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2024.155731. References Abel, E.L., Bubel, J.D., Simper, M.S., Powell, L., McClellan, S.A., Andreeff, M., MacLeod, M.C., DiGiovanni, J., 2011. Protection against 2-chloroethyl ethyl sulfide (CEES) — Induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway. Toxicol. Appl. Pharmacol. 255 (2), 176–183. Al-Bakheit, A., Abu-Qatouseh, L., 2020. Sulforaphane from broccoli attenuates inflammatory hepcidin by reducing IL-6 secretion in human HepG2 cells. J. Funct. Foods 75, 104210. Alkharashi, N.A.O., Periasamy, V.S., Athinarayanan, J., Alshatwi, A.A., 2017. Sulforaphane mitigates cadmium-induced toxicity pattern in human peripheral blood lymphocytes and monocytes. Environ. Toxicol. Pharmacol. 55, 223–239. Alkharashi, N.A.O., Periasamy, V.S., Athinarayanan, J., Alshatwi, A., 2019. Sulforaphane alleviates cadmium-induced toxicity in human mesenchymal stem cells through POR and TNFSF10 genes expression. Biomed. pharmacother. 115, 108896. Amato, C.M., Fricke, A., Marella, S., Magus, J.P., Bereman, M., McCoy, K.A., 2022. An experimental evaluation of the efficacy of perinatal sulforaphane supplementation to decrease the incidence and severity of vinclozolin-induced hypospadias in the mouse model. Toxicol. Appl. Pharmacol. 451, 116177. Angeloni, C., Teti, G., Barbalace, M.C., Malaguti, M., Falconi, M., Hrelia, S., 2017. 17β- Estradiol enhances sulforaphane cardioprotection against oxidative stress. J. Nutr. Biochem. 42, 26–36. Aranda-Rivera, A.K., Cruz-Gregorio, A., Aparicio-Trejo, O.E., Tapia, E., S´ anchez- Lozada, L.G., García-Arroyo, F.E., Amador-Martínez, I., Orozco-Ibarra, M., Fern´ andez-Valverde, F., Pedraza-Chaverri, J., 2022. Sulforaphane Protects against A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 24 Unilateral Ureteral Obstruction-Induced Renal Damage in Rats by Alleviating Mitochondrial and Lipid Metabolism Impairment. Antioxidants 11, 1854. Baralic, K., ˇ Zivanovic, J., Maríc, Ð., Bozic, D., Grahovac, L., Antonijevic Miljakovic, E., Curcic, M., Buha Djordjevic, A., Bulat, Z., Antonijevic, B., Ðuki Cosic, D., 2024. Sulforaphane—A Compound with Potential Health Benefits for Disease Prevention and Treatment: Insights from Pharmacological and Toxicological Experimental Studies. Antioxidants 13, 147. Bergstr¨ om, P., Andersson, H.C., Gao, Y., Karlsson, J.O., Nodin, C., Anderson, M., Nilsson, M., Hammarsten, O., 2011. Repeated transient sulforaphane stimulation in astrocytes leads to prolonged Nrf2-mediated gene expression and protection from superoxide-induced damage. Neuropharmacol 60, 343–353. Brasil, F.B., Almelda, F.J.S., Luckachaki, M.D., Dall’Oglio, E.L., Oliveira, M.R., 2023. The isothiocyanate sulforaphane prevents mitochondrial impairment and neuroinflammation in the human dopaminergic SHSY5Y and in the mouse microglial BV2 cells: role for heme oxygenase1. Metab. Brain Dis. 38, 419–435. Briones-Herrera, A., Avila-Rojas, S.H., Aparicio-Trejo, O.E., Cristobal, M., Le´ on- Contreras, J.C., Hern´ andez-Pando, R., Pinz´ on, E., Pedraza-Chaverri, J., S´ anchez- Lozada, L.G., Tapia, E., 2018. Sulforaphane prevents maleic acid-induced nephropathy by modulating renal hemodynamics, mitochondrial bioenergetics and oxidative stress. Food Chem. Toxicol. 115, 185–197. Burnett, J.P., Lim, G., Li, Y., Shah, R.B., Lim, R., Paholak, H.J., McDermott, S.P., Sun, L., Tsume, Y., Bai, S., Wicha, M.S., Sun, D., Zhang, T., 2017. Sulforaphane enhances the anticancer activity of taxanes against triple negative breast cancer by killing cancer stem cells. Cancer Lett. 394, 52–64. Cao, W., Lu, X., Zhong, C., Wu, J., 2023. Sulforaphane Suppresses MCF-7 Breast Cancer Cells Growth via miR-19/PTEN Axis to Antagonize the Effect of Butyl Benzyl Phthalate. Nutri. Cancer 75 (3), 980–991. Cascajosa-Lira, A., Andreo-Martínez, P., Prieto, A.I., Ba˜ nos, A., Guillam´ on, E., Jos, A., Came´ an, A.M., 2022. In Vitro Toxicity Studies of Bioactive Organosulfur Compounds from Allium spp. with Potential Application in the Agri-Food Industry: A Review. Foods. 11 (17), 2620. Chang, H.Y., Lin, C.W., Yang, C.M., Yang, C.H., 2020. Nrf-2 activator sulforaphane protects retinal cells from oxidative stress-induced retinal injury. J. Funct. Foods 71, 104023. Chang, R., 2022. Research advances in the protective effect of sulforaphane against kidney injury and related mechanisms. BIO Web of Conferences 55, 01006. Cicho˙ z-Lach, H., Michalak, A., 2014. Oxidative stress as a crucial factor in liver diseases. World J. Gastroenterol. 20 (25), 8082–8091. Cox, A.G., Gurusinghe, S., Abd Rahman, R., Leaw, B., Chan, S.T., Mockler, J.C., Murthi, P., Marshall, S.A., Lim, R., Wallace, E.M., 2019. Sulforaphane improves endothelial function and reduces placental oxidative stress in vitro. Pregnancy Hypertens. 16, 1–10. Dinkova-Kostova, A.T., Fahey, J.W., Kostov, R.V., Kensler, T.W., 2017. KEAP1 and done? Targeting the NRF2 pathway with sulforaphane. Food Sci. Technol. 69, 257–269. Due˜ nas-García, I.E., Santos-Cruz, L.F., Casta˜ neda-Partida, L., Casta˜ neda-Sortibr´ an, A.N., Ordaz-T´ ellez, M.G., S´ anchez-Santos, A., Dur´ an-Díaz, A., Rodríguez-Arnaiz, R., Heres- Pulido, M.E., 2012. Interactions of sulforaphane and dimethyl sulfoxide with methyl methanesulfonate, urethane, 4-nitroquinoline-1-oxide and hydrogen peroxide in the Drosophila melanogaster wing spot test. Food Chem. Toxicol. 50 (12), 4479–4486. Fahey, J.W., Zalcmann, A.T., Talalay, P., 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochem 56, 5e51. Feng, Z., Wang, T., Sun, Y., Chen, S., Hao, H., Du, W., Zou, H., Yu, D., Zhu, H., Pang, Y., 2023. Sulforaphane suppresses paraquat-induced oxidative damage in bovine in vitro-matured oocytes through Nrf2 transduction pathway. Ecotoxicol. Environ Saf. 254, 114747. Folkard, D.L., Melchini, A., Traka, M.H., Al-Bakheit, A.A., Saha, S., Mulholland, F., Watson, A., Mithen, R.F., 2014. Suppression of LPS-induced transcription and cytokine secretion by the dietary isothiocyanate sulforaphane. Mol. Nutr. Food Res. 58, 2286–2296. Fouad, I.G., El-Sayed, S.A.M., Mabrouk, M., Ahmed, K.A., Beherei, H.H., 2022. Neuroprotective Potential of Intranasally Delivered Sulforaphane-Loaded Iron Oxide Nanoparticles Against Cisplatin-Induced Neurotoxicity. Neurotox. Res. 40 (5), 1479–1498. Fouad, I.G., 2023. Sulforaphane, an Nrf-2 Agonist, Modulates Oxidative Stress and Inflammation in a Rat Model of Cuprizone-Induced Cardiotoxicity and Hepatotoxicity. Cardiovasc. Toxicol. 23 (1), 46–60. Fuse, Y., Nguyen, V.T., Kobayashi, M., 2016. Nrf2-dependent protection against acute sodium arsenite toxicity in zebrafish. Toxicol. Appl. Pharmacol. 305, 136–142. Gan, N., Mi, L., Sun, X., Dai, G., Chung, F.L., Song, L., 2010. Sulforaphane protects Microcystin-LR-induced toxicity through activation of the Nrf2-mediated defensive response. Toxicol. Appl. Pharmacol. 247 (2), 129–137. Gasparello, J., D’Aversa, E., Papi, C., Gambari, L., Grigolo, B., Borgatti, M., Finotti, A., Gambari, R., 2021. Sulforaphane inhibits the expression of interleukin-6 and interleukin-8 induced in bronchial epithelial IB3-1 cells by exposure to the SARS- CoV-2 Spike protein. Phytomedicine 87, 153583. Gasper, A.V., Al-Janobi, A., Smith, J.A., Bacon, J.R., Fortun, P., Atherton, C., Taylor, M. A., Hawkey, C.J., Barrett, D.A., Mithen, R.F., 2005. Glutathione S-transferase M1 polymorphism and metabolism of sulforaphane from standard and highglucosinolate broccoli. Am. J. Clin. Nutr. 82 (6), 1283–1291. Gaona-Gaona, L., Molina-Jij´ on, E., Tapia, E., Zazueta, C., Hern´ andez-Pando, R., Calder´ on-Oliver, M., Zarco-M´ arquez, G., Pinz´ on, E., Pedraza-Chaverri, J., 2011. Protective effect of sulforaphane pretreatment against cisplatin-induced liver and mitochondrial oxidant damage in rats. Toxicol 286 (1-3), 20–27. Guerrero-Beltr´ an, C.E., Calder´ on-Oliver, M., Tapia, E., Medina-Campos, O.N., S´ anchez- Gonz´ alez, D.J., Martínez-Martínez, C.M., Ortiz-Vega, K.M., Franco, M., Pedraza- Chaverri, J., 2010. Sulforaphane protects against cisplatin-induced nephrotoxicity. Toxicol. Lett. 192 (3), 278–285. Guerrero-Beltr´ an, C.E., Calder´ on-Oliver, M., Pedraza-Chaverri, J., Chirino, Y.I., 2012. Protective effect of sulforaphane against oxidative stress: recent advances. Exp. Toxicol. Pathol. 64 (5), 503–508. Guo, M., 2016. Protective effect of sulforaphane on mercuryinduced acute renal injury in rats. Prev. Med. 23 (12), 1461–1464. Hashimoto, K., 2018. Essential role of Keap1-Nrf2 signaling in mood disorders: overview and future perspective. Front. Pharmacol. 9, 1182. Hayes, J.D., Kelleher, M.O., Eggleston, I.M., 2008. The cancer chemopreventive actions of phytochemicals derived from glucosinolates. Eur. J. Nutr. 47, 73–88. He, Q., Luo, Y., Xie, Z., 2021. Sulforaphane ameliorates cadmium induced hepatotoxicity through the up-regulation of /Nrf2/ARE pathway and the inactivation of NF-κB. J. Func. Food 77, 104297. Holloway, P.M., Gillespie, S., Becker, F., Vital, S.A., Nguyen, V., Alexander, J.S., Evans, P.C., Gavins, F.N.E., 2016. Sulforaphane induces neurovascular protection against a systemic inflammatory challenge via both Nrf2-dependent and independent pathways. Vascul. Pharmacol. 85, 29–38. Holman, J.M., Colucci, L., Baudewyns, D., Balkan, J., Hunt, T., Hunt, B., Kinney, M., Holcomb, L., Stratigakis, A., Chen, G., Moses, P.L., Mawe, G.M., Zhang, T., Li, Y., Ishaq, S.L., 2023. Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice. mSystems. 8 (5), e0053223. Hong, L., Xu, Y., Wang, D., Zhang, Q., Li, X., Xie, C., Wu, J., Zhong, C., Fu, J., Geng, S., 2023. Sulforaphane ameliorates bisphenol A-induced hepatic lipid accumulation by inhibiting endoplasmic reticulum stress. Sci. Rep. 13 (1), 1147. Isaacson, R.H., Beier, J.I., Khoo, N.K., Freeman, B.A., Freyberg, Z., Arteel, G.E., 2020. Olanzapine-induced liver injury in mice: aggravation by high-fat diet and protection with sulforaphane. J. Nutr. Biochem. 81, 108399. Ishida, K., Kaji, K., Sato, S., Ogawa, H., Takagi, H., Takaya, H., Kawaratani, H., Moriya, K., Namisaki, T., Akahane, T., Yoshiji, H., 2021. Sulforaphane ameliorates ethanol plus carbon tetrachloride-induced liver fibrosis in mice through the Nrf2- mediated antioxidant response and acetaldehyde metabolization with inhibition of the LPS/TLR4 signaling pathway. J. Nutr. Biochem. 89, 108573. Jazwa, A., Rojo, A.I., Innamorato, N.G., Hesse, M., Fernandez-Ruiz, J., Cuadrado, A., 2011. Pharmacological targeting of the transcription factor Nrf2 at the basal ganglia provides disease modifying therapy for experimental parkinsonism. Antioxid. Redox Signal. 14, 2347–2360. Juge, N., Mithen, R.F., Traka, M., 2007. Molecular basis for chemoprevention by sulforaphane: a comprehensive review. Cell. Mol. Life Sci. 64, 1105–1127. Kalpana Deepa Priya, D., Gayathri, R., Sakthisekaran, D., 2011. Role of sulforaphane in the anti-initiating mechanism of lung carcinogenesis in vivo by modulating the metabolic activation and detoxification of benzo(a)pyrene. Biomed. Pharmacother. 65 (1), 9–16. Katoch, O., Kumar, A., Adhikari, J.S., Dwarakanath, B.S., Agrawala, P.K., 2013. Sulforaphane mitigates genotoxicity induced by radiation and anticancer drugs in human lymphocytes. Mutat. Res. - Genet. Toxicol. Environ. 758 (1-2), 29–34. Kawarazaki, A., Horinaka, M., Yasuda, S., Numajiri, T., Nishino, K., Sakai, T., 2017. Sulforaphane suppresses cell growth and collagen expression of keloid fibroblasts. Wound Repair. Regen. 5 (2), 224–233. Kleszczy´ nski, K., Ernst, I.M., Wagner, A.E., Kruse, N., Zillikens, D., Rimbach, G., Fischer, T.W., 2013. Sulforaphane and phenylethyl isothiocyanate protect human skin against UVR-induced oxidative stress and apoptosis: role of Nrf2-dependent gene expression and antioxidant enzymes. Pharmacol. Res. 78, 28–40. Klomparens, E., Ding, Y., 2019. The neuroprotective mechanisms and effects of sulforaphane. Brain Circ. 5 (2), 74–83. Konwinski, R.R., Haddad, R., Chun, J.A., Klenow, S., Larson, S.C., Haab, B.B., Furge, L.L., 2004. Oltipraz, 3H-1,2-dithiole-3-thione, and sulforaphane induce overlapping and protective antioxidant responses in murine microglial cells. Toxicol. Lett. 153 (3), 343–355. Kubo, E., Chhunchha, B., Singh, P., Sasaki, H., Singh, D.P., 2017. Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress. Sci. Report 7, 14130. Kuroiwa, Y., Nishikawa, A., Kitamura, Y., Kanki, K., Ishii, Y., Umemura, T., Hirose, M., 2006. Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters. Cancer Lett. 241 (2), 275–280. Kwak, M.K., Cho, J.M., Huang, B., Shin, S., Kensler, T.W., 2007. Role of increased expression of the proteasome in the protective effects of sulforaphane against hydrogen peroxide-mediated cytotoxicity in murine neuroblastoma cells. Free Radic. Biol. Med. 43 (5), 809–817. Latt´ e, K.P., Appel, K.E., Lampen, A., 2011. Health benefits and possible risks of broccolian overview. Food Chem. Toxicol. 49 (12), 3287–3309. Lee, J.H., Jeong, J.K., Park, S.Y., 2014. Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway. NeuroSci. 278, 31–39. Lee, C., Yang, S., Lee, B.S., Jeong, S.Y., Kim, K.M., Ku, S.K., Bae, J.S., 2020. Hepatic protective effects of sulforaphane through the modulation of inflammatory pathways. J. Asian Nat. Prod. Res. 22 (4), 386–396. Li, D., Wang, W., Shan, Y., Barrera, L.N., Howie, A.F., Beckett, G.J., Wu, K., Bao, Y., 2012. Synergy between sulforaphane and selenium in the up-regulation of thioredoxin reductase and protection against hydrogen peroxide-induced cell death in human hepatocytes. Food Chem. 133 (2), 300–307. Li, J., 2015. Protective effect of sulforaphane on cadmium induced renal damage in rats. Chin. Med. J. 28 (1), 11–13. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 25 Liu, P., Wang, W., Tang, J., Bowater, R.P., Bao, Y., 2019. Antioxidant effects of sulforaphane in human HepG2 cells and immortalised hepatocytes. Food Chem. Toxicol. 128, 129–136. Liu, H., Yang, X., Tang, K., Ye, T., Duan, C., Lv, P., Yan, L., Wu, X., Chen, Z., Liu, J., Deng, Y., Zeng, G., Xing, J., Ye, Z., Xu, H., 2020. Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis. Theranostics. 10 (16), 7319–7334. Lv, X., Meng, G., Li, W., Fan, D., Wang, X., Espinoza-Pinochet, C.A., Cespedes-Acu˜ na, C. L., 2020. Sulforaphane and its antioxidative effects in broccoli seeds and sprouts of different cultivars. Food Chem. 316, 126216. Marshall, S.A., Young, R.B., Lewis, J.M., Rutten, E.L., Gould, J., Barlow, C.K., Giogha, C., Marcelino, V.R., Fields, N., Schittenhelm, R.B., Hartland, E.L., Scott, N.E., Forster, S. C., Gulliver, E.L., 2023. The broccoli-derived antioxidant sulforaphane changes the growth of gastrointestinal microbiota, allowing for the production of antiinflammatory metabolites. J. Funct. Foods 107, 105645. Mathew, S.T., Bergstr¨ om, P., Hammarsten, O., 2014. Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation. Toxicol. Appl. Pharmacol. 276 (3), 188–194. Mthembu, S.X.H., Mazibuko-Mbeje, S.E., Moetlediwa, M.T., Muvhulawa, N., Silvestri, S., Orlando, P., Nkambule, B.B., Muller, C.J.F., Ndwandwe, D., Basson, A.K., Tiano, L., Dludla, P.V., 2023. Sulforaphane: A nutraceutical against diabetes-related complications. Pharmacol. Res. 196, 106918. Miller, D.M., Singh, I.N., Wang, J.A., Hall, E.D., 2013. Administration of the Nrf2-ARE activators sulforaphane and carnosic acid attenuates 4-hydroxy-2-nonenal-induced mitochondrial dysfunction ex vivo. Free Radic. Biol. Med. 57, 1–9. Morroni, F., Tarozzi, A., Sita, G., Bolondi, C., Zolezzi, Moraga, J.M., Cantelli-Forti, G., Hrelia, P., 2013. Neuroprotective effect of sulforaphane in 6-hydroxydopamine- lesioned mouse model of Parkinson’s disease. Neurotoxicol 36, 63–71. Noh, J.R., Kim, Y.H., Hwang, J.H., Choi, D.H., Kim, K.S., Oh, W.K., Lee, C.H., 2015. Sulforaphane protects against acetaminophen-induced hepatotoxicity. Food Chem. Toxicol. 80, 193–200. Padayachee, A., Day, L., Howell, K., Gidley, M.J., 2017. Complexity and health functionality of plant cell wall fibers from fruits and vegetables. Crit. Rev. Food Sci. Nutr. 57 (1), 59–81. Park, H.S., Hwang, E.S., Choi, G.Y., Kim, H.B., Park, K.S., Sul, J.Y., Hwang, Y., Choi, G. W., Kim, B.I., Park, H., Maeng, S., Park, J.H., 2021. Sulforaphane enhances longterm potentiation and ameliorate scopolamine-induced memory impairment. Physiol. Behav. 238, 113467. Qin, Y., Zhang, H., Liu, Q., Jiang, B., Chen, J., Zhang, T., 2021. Sulforaphane attenuates oxidative stress and inflammation induced by fine particulate matter in human bronchial epithelial cells. J. Funct. Foods 81, 104460. Ruhee, R.T., Ma, S., Suzuki, K., 2020. Protective Effects of Sulforaphane on Exercise- Induced Organ Damage via Inducing Antioxidant Defense Responses. Antioxidants 9 (2), 136. Russo, M., Spagnuolo, C., Russo, G.L., Skalicka-Wo´zniak, K., Daglia, M., Sobarzo- S´ anchez, E., Nabavi, S.F., Nabavi, S.M., 2018. Nrf2 Targeting by Sulforaphane: A Potential Therapy for Cancer Treatment. Crit. Rev. Food Sci. Nutr. 58, 1391–1405. Sayed, R.H., Khalil, W.K., Salem, H.A., Kenawy, S.A., El-Sayeh, B.M., 2014. Sulforaphane increases the survival rate in rats with fulminant hepatic failure induced by D- galactosamine and lipopolysaccharide. Nutr. Res. 34 (11), 982–989. Sato, K., Kihara, H., Kumazawa, Y., Tatara, K., 2021. Oral chronic sulforaphane effects on heavy resistance exercise: Implications for inflammatory and muscle damage parameters in young practitioners. Nutrition 90, 111266. Sestili, P., Paolillo, M., Lenzi, M., Colombo, E., Vallorani, L., Casadei, L., Martinelli, C., Fimognari, C., 2010. Sulforaphane induces DNA single strand breaks in cultured human cells. Mut. Res. 689 (1-2), 65–73. Singh, A., Kukreti, R., Saso, L., Kukreti, S., 2019. Oxidative Stress: A Key Modulator in Neurodegenerative Diseases. Molecules 22 24 (8), 1583. Shapiro, T.A., Fahey, J.W., Wade, K.L., Stephenson, K.K., Talalay, P., 2001. Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans. Cancer Epidemiol. Biomarkers Prev. 10, 501–508. Shorey, L.E., Madeen, E.P., Atwell, L.L., Ho, E., L¨ ohr, C.V., Pereira, C.B., Dashwood, R.H., Williams, D.E., 2013. Differential modulation of dibenzo[def,p]chrysene transplacental carcinogenesis: maternal diets rich in indole-3-carbinol versus sulforaphane. Toxicol. Appl. Pharmacol. 270 (1), 60–69. Son, Y.H., Jang, E.J., Kim, Y.W., Lee, J.H., 2017. Sulforaphane prevents dexamethasoneinduced muscle atrophy via regulation of the Akt/Foxo1 axis in C2C12 myotubes. Biomed. Pharmacother. 95, 1486–1492. Song, M.Y., Kim, E.K., Moon, W.S., Park, J.W., Kim, H.J., So, H.S., Park, R., Kwon, K.B., Park, B.H., 2009. Sulforaphane protects against cytokine- and streptozotocininduced beta-cell damage by suppressing the NF-kappaB pathway. Toxicol. Appl. Pharmacol. 235 (1), 57–67. Sun, X., Mi, L., Liu, J., Song, L., Chung, F.L., Gan, N., 2011. Sulforaphane prevents microcystin-LR-induced oxidative damage and apoptosis in BALB/c mice. Toxicol. Appl. Pharmacol. 255 (1), 9–17. Taheri, M., Roudbari, N.H., Amidi, F., Parivar, K., 2022. Investigating the effect of Sulforaphane on AMPK/AKT/NRF2 pathway in human granulosa-lutein cells under H2O2-induced oxidative stress. Eur. J. Obstet. Gynecol. Reprod. Biol. 276, 125–133. Thangapandiyan, S., Ramesh, M., Miltonprabu, S., Hema, T., Jothi, G.B., Nandhini, V., 2019. Sulforaphane potentially attenuates arsenic-induced nephrotoxicity via the PI3K/Akt/Nrf2 pathway in albino Wistar rats. Environ. Sci. Pollut. Res. Int. 26 (12), 12247–12263. Vanegas, T.A., Tish, N., Rodov, V., 2022. Enhancement of glucosinolate formation in broccoli sprouts by hydrogen peroxide treatment. Foods. 11 (5), 655–670. Veeranki, O.L., Bhattacharya, A., Marshall, J.R., Hang, Y., 2013. Organ-specific exposure and response to sulforaphane, a key chemopreventive ingredient in broccoli: implications for cancer prevention. Br. J. Nutr. 109 (1), 25–32. Villa-Cruz, V., Davila, J., Viana, M.T., Vazquez-Duhalt, R., 2009. Effect of broccoli (Brassica oleracea) and its phytochemical sulforaphane in balanced diets on the detoxification enzymes levels of tilapia (Oreochromis niloticus) exposed to a carcinogenic and mutagenic pollutant. Chemosphere 74 (9), 1145–1151. Visalli, G., Facciol` a, A., Bertuccio, M.P., Picerno, I., Di Pietro, A., 2017. In vitro assessment of the indirect antioxidant activity of Sulforaphane in redox imbalance vanadium-induced. Nat. Prod. Res. 31 (22), 2612–2620. Wang, L., Gallagher, E.P., 2013. Role of Nrf2 antioxidant defense in mitigating cadmiuminduced oxidative stress in the olfactory system of zebrafish. Toxicol. Appl. Pharmacol. 266 (2), 177–186. Wang, Y., Mandal, A.K., Son, Y.O., Pratheeshkumar, P., Wise, J.T.F., Wang, L., Zhang, Z., Shi, X., Chen, Z., 2018. Roles of ROS, Nrf2, and autophagy in cadmiumcarcinogenesis and its prevention by sulforaphane. Toxicol. Appl. Pharmacol. 353, 23–30. Wang, J., Zhou, H., 2020. Protective effects of sulforaphane and aerobic exercise on acute alcoholic hepatic injury in mice. Saudi J. Biol. Sci. 27 (11), 3145–3149. Wang, Z.C., Chen, Q., Wang, J., Yu, L.S., Chen, L.W., 2020. Sulforaphane mitigates LPS- induced neuroinflammation through modulation of Cezanne/NF-κB signalling. Life Sci. 262, 118519. Wang, X., Chen, X., Zhou, W., Men, H., Bao, T., Sun, Y., Wang, Q., Tan, Y., Keller, B.B., Tong, Q., Zheng, Y., Cai, L., 2022a. Ferroptosis Is Essential for Diabetic Cardiomyopathy and Is Prevented by Sulforaphane via AMPK/NRF2 Pathways. Acta Pharm. Sin. B 12, 708–722. Wang, H., Tian, Y., Zhang, Q., Liu, W., Meng, L., Jiang, X., Xin, Y., 2022b. Essential role of Nrf2 in sulforaphane-induced protection against angiotensin II-induced aortic injury. Life Sci. 306, 120780. Wang, H., Wang, B., Wei, J., Zheng, Z., Su, J., Bian, C., Xin, Y., Jiang, X., 2022c. Sulforaphane regulates Nrf2-mediated antioxidant activity and downregulates TGF- β1/Smad pathways to prevent radiation-induced muscle fibrosis. Life Sci. 311 (Pt B), 121197. Wagner, A.E., Will, O., Sturm, C., Lipinski, S., Rosenstiel, P., Rimbach, G., 2013. DSS- induced acute colitis in C57BL/6 mice is mitigated by sulforaphane pre-treatment. J. Nutr. Biochem. 224 (12), 2085–2091. Wei, L., Wang, J., Yan, L., Shui, S., Wang, L., Zheng, W., Liu, S., Liu, C., Zheng, L., 2020. Sulforaphane attenuates 5-fluorouracil induced intestinal injury in mice. J. Funct. Foods 69, 103965. Wu, J., Guo, W., Cui, S., Tang, X., Zhang, Q., Lu, W., Jin, Y., Zhao, J., Mao, B., Chen, W., 2023. Broccoli seed extract rich in polysaccharides and glucoraphanin ameliorates DSS-induced colitis via intestinal barrier protection and gut microbiota modulation in mice. J. Sci. Food Agric. 103 (4), 1749–1760. Xin, Y., Bai, Y., Jiang, X., Zhou, S., Wang, Y., Wintergerst, K.A., Cui, T., Ji, H., Tan, Y., Cai, L., 2018. Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway. Redox. Biol. 15, 405–417. Xu, J.F., Lu, J.J., Cao, Y., Wang, W., Li, H.H., Chen, J.G., Wang, F., Wu, P.F., 2020. Sulforaphane alleviates ethanol-mediated central inhibition and reverses chronic stress-induced aggravation of acute alcoholism via targeting Nrf2-regulated catalase expression. Neuropharmacol 176, 108235. Xu, Y., Huang, X., Huangfu, B., Hu, Y., Xu, J., Gao, R., Huang, K., He, X., 2023. Sulforaphane Ameliorates Nonalcoholic Fatty Liver Disease Induced by High-Fat and High-Fructose Diet via LPS/TLR4 in the Gut-Liver Axis. Nutrients. 15 (3), 743. Xu, R., Wu, Y., Xiang, X., Lv, X., He, M., Xu, C., Lai, G., Xiang, T., 2024. Sulforaphane effectively inhibits HBV by altering Treg/Th17 immune balance and the MIF- macrophages polarizing axis in vitro and in vivo. Virus. Res. 341, 199316. Yamamoto, M., Kensler, T.W., Motohashi, H., 2018. The KEAP1-NRF2 system: a thiolbased sensor-effector apparatus for maintaining redox homeostasis. Physiol. Rev. 98, 1169–1203. Yang, J., Song, X., Feng, Y., Liu, N., Fu, Z., Wu, J., Li, T., Chen, H., Chen, J., Chen, C., Yang, L., 2020. Natural ingredients-derived antioxidants attenuate H2O2-induced oxidative stress and have chondroprotective effects on human osteoarthritic chondrocytes via Keap1/Nrf2 pathway. Free Radic. Biol. Med. 152, 854–864. Yang, P.M., Cheng, K.C., Huang, J.Y., Wang, S.Y., Lin, Y.N., Tseng, Y.T., Hsieh, C.W., Wung, B.S., 2021. Sulforaphane inhibits blue light-induced inflammation and apoptosis by upregulating the SIRT1/PGC-1 α /Nrf2 pathway and autophagy in retinal pigment epithelial cells. Toxicol. Appl. Pharmacol. 421, 115545. Yang, Y., Zhang, J., Yang, C., Dong, B., Fu, Y., Wang, Y., Gong, M., Qiu, P., Xie, W., Lu, T., 2023. Sulforaphane attenuates microglia-mediated neuronal damage by downregulating the ROS/autophagy/NLRP3 signal axis in fibrillar Aβ-activated microglia. Brain Res. 1801, 148206. Ye, L., Dinkova-Kostova, A.T., Wade, K.L., Zhang, Y., Shapiro, T.A., Talalay, P., 2002. Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans. Clin. Chim. Acta 316 (1–2), 43–53. Yu, C., Xiao, J.H., 2021. The Keap1-Nrf2 system: a mediator between oxidative stress and aging. Oxid. Med. Cell. Longev. 19, 6635460. Yu, H., Peng, Y., Dong, W., Shen, B., Yang, G., Nie, Q., Tian, Y., Qin, L., Song, C., Chen, B., Zhao, Y., Li, L., Hong, S., 2023. Nrf2 attenuates methamphetamine-induced myocardial injury by regulating oxidative stress and apoptosis in mice. Hum. Exp. Toxicol. 42, 9603271231219488. Zeren, S., Bayhan, Z., Kocak, F.E., Kocak, C., Akcılar, R., Bayat, Z., Simsek, H., Duzgun, S. A., 2016. Gastroprotective effects of sulforaphane and thymoquinone against acetylsalicylic acid-induced gastric ulcer in rats. J. Surg. Res. 203 (2), 348–359. A. Cascajosa-Lira et al.