Rheumatoid arthritis molecular targets and their importance to flavonoid-based therapy.

Rheumatoid arthritis (RA) is a progressive, chronic, autoimmune, inflammatory, and systemic condition that primarily affects the synovial joints and adjacent tissues, including bone, muscle, and tendons. The World Health Organization recognizes RA as one of the most prevalent chronic inflammatory diseases. In the last decade, there was an expansion on the available RA therapeutic options which aimed to improve patient's quality of life. Despite the extensive research and the emergence of new therapeutic approaches and drugs, there are still significant unwanted side effects associated to these drugs and still a vast number of patients that do not respond positively to the existing therapeutic strategies. Over the years, several references to the use of flavonoids in the quest for new treatments for RA have emerged. This review aimed to summarize the existing literature about the flavonoids' effects on the major pathogenic/molecular targets of RA and their potential use as lead compounds for the development of new effective molecules for RA treatment. It is demonstrated that flavonoids can modulate various players in synovial inflammation, regulate immune cell function, decrease synoviocytes proliferation and balance the apoptotic process, decrease angiogenesis, and stop/prevent bone and cartilage degradation, which are all dominant features of RA. Although further investigation is necessary to determine the effectiveness of flavonoids in humans, the available data from in vitro and in vivo models suggest their potential as new disease-modifying anti-rheumatic drugs. This review highlights the use of flavonoids as a promising avenue for future research in the treatment of RA.

The role of flavonoids in the regulation of epithelial-mesenchymal transition in cancer: A review on targeting signaling pathways and metastasis.

One of the hallmarks of cancer is metastasis, a process that entails the spread of cancer cells to distant regions in the body, culminating in tumor formation in secondary organs. Importantly, the proinflammatory environment surrounding cancer cells further contributes to cancer cell transformation and extracellular matrix destruction. During metastasis, front-rear polarity and emergence of migratory and invasive features are manifestations of epithelial-mesenchymal transition (EMT). A variety of transcription factors (TFs) are implicated in the execution of EMT, the most prominent belonging to the Snail Family Transcriptional Repressor (SNAI) and Zinc Finger E-Box Binding Homeobox (ZEB) families of TFs. These TFs are regulated by interaction with specific microRNAs (miRNAs), as miR34 and miR200. Among the several secondary metabolites produced in plants, flavonoids constitute a major group of bioactive molecules, with several described effects including antioxidant, antiinflammatory, antidiabetic, antiobesogenic, and anticancer effects. This review scrutinizes the modulatory role of flavonoids on the activity of SNAI/ZEB TFs and on their regulatory miRNAs, miR-34, and miR-200. The modulatory role of flavonoids can attenuate mesenchymal features and stimulate epithelial features, thereby inhibiting and reversing EMT. Moreover, this modulation is concomitant with the attenuation of signaling pathways involved in diverse processes as cell proliferation, cell growth, cell cycle progression, apoptosis inhibition, morphogenesis, cell fate, cell migration, cell polarity, and wound healing. The antimetastatic potential of these versatile compounds is emerging and represents an opportunity for the synthesis of more specific and potent agents.

Silver nanoparticles exert toxic effects in human monocytes and macrophages associated with the disruption of Δψm and release of pro-inflammatory cytokines.

Silver nanoparticles (AgNP) are the most widely produced type of nanoparticles due to their antimicrobial and preservative properties. However, their systemic bioavailability may be considered a potential hazard. When AgNP reach the bloodstream, they interact with the immune cells, contributing to the onset and development of an inflammatory response. Monocytes and macrophages play a pivotal role in our defense system, but the interaction of AgNP with these cells is still not clear. Therefore, the main objective of this work was to assess the cytotoxic and pro-inflammatory effects induced by 5, 10, and 50 nm AgNP coated with polyvinylpyrrolidone (PVP) and citrate, in concentrations that could be attained in vivo (0-25 µg/mL), in human monocytes isolated from human blood and human macrophages derived from a monocytic cell line (THP-1). The effects of PVP and citrate-coated AgNP on cell viability, mitochondrial membrane potential, and cytokines release were evaluated. The results evidenced that AgNP exert strong harmful effects in both monocytes and macrophages, through the establishment of a strong pro-inflammatory response that culminates in cell death. The observed effects were dependent on the AgNP concentration, size and coating, being observed more pronounced cytotoxic effects with smaller PVP coated AgNP. The results showed that human monocytes seem to be more sensitive to AgNP exposure than human macrophages. Considering the increased daily use of AgNP, it is imperative to further explore the adverse outcomes and mechanistic pathways leading to AgNP-induced pro-inflammatory effects to deep insight into the molecular mechanism involved in this effect.

Biodistribution and intestinal inflammatory response following voluntary oral intake of silver nanoparticles by C57BL/6J mice.

Silver nanoparticles (AgNP) are among the most widely commercialized nanomaterials globally, with applications in medicine and the food industry. Consequently, the increased use of AgNP in the food industry has led to an unavoidable rise  in human exposure to these nanoparticles. Their widespread use raises concerns about potential hazards to human health, specifically their intestinal pro-inflammatory effects. Thus, the main objective of this study was to evaluate the biological effects of two subacute doses of 5 nm polyvinylpyrrolidone (PVP)-AgNP in C57BL/6J mice. One mg/kg body weight or 10 mg/kg bw was provided once a day for 14 days, using a new technology (HaPILLness) that allows voluntary, stress-free, and accurate oral dosing. It was observed that after oral ingestion, while AgNP is biodistributed throughout the entire organism, most of the ingested dose is excreted in the feces. The passage and accumulation of AgNP throughout the intestine instigated a prominent inflammatory response, marked by significant histological, vascular, and cellular transformations. This response was driven by the activation of the nuclear factor-кB (NF-кB) inflammatory pathway, ultimately leading to the generation of multiple cytokines and chemokines.

Flavonoids as potential agents in the management of type 2 diabetes through the modulation of α-amylase and α-glucosidase activity: a review.

Type 2 diabetes (T2D) is one of the most prevalent metabolic diseases worldwide and is characterized by increased postprandial hyperglycemia (PPHG). α-Amylase and α-glucosidase inhibitors have been shown to slow the release of glucose from starch and oligosaccharides, resulting in a delay of glucose absorption and a reduction in postprandial blood glucose levels. Since current α-glucosidase inhibitors used in the management of T2D, such as acarbose, have been associated to strong gastrointestinal side effects, the search for novel and safer drugs is considered a hot topic of research. Flavonoids are phenolic compounds widely distributed in the Plant Kingdom and important components of the human diet. These compounds have shown promising antidiabetic activities, including the inhibition of α-amylase and α-glucosidase. The aim of this review is to provide an overview on the scientific literature concerning the structure-activity relationship of flavonoids in inhibiting α-amylase and α-glucosidase, including their type of inhibition and experimental procedures applied. For this purpose, a total of 500 compounds is covered in this review. Available data may be considered of high value for the design and development of novel flavonoid derivatives with effective and potent inhibitory activity against those carbohydrate-hydrolyzing enzymes, to be possibly used as safer alternatives for the regulation of PPHG in T2D.

A comprehensive review on the antidiabetic activity of flavonoids targeting PTP1B and DPP-4: a structure-activity relationship analysis.

Type 2 diabetes (T2D) is an expanding global health problem, resulting from defects in insulin secretion and/or insulin resistance. In the past few years, both protein tyrosine phosphatase 1B (PTP1B) and dipeptidyl peptidase-4 (DPP-4), as well as their role in T2D, have attracted the attention of the scientific community. PTP1B plays an important role in insulin resistance and is currently one of the most promising targets for the treatment of T2D, since no available PTP1B inhibitors were still approved. DPP-4 inhibitors are among the most recent agents used in the treatment of T2D (although its use has been associated with possible cardiovascular adverse events). The antidiabetic properties of flavonoids are well-recognized, and include inhibitory effects on the above enzymes, although hitherto not therapeutically explored. In the present study, a comprehensive review of the literature of both synthetic and natural isolated flavonoids as inhibitors of PTP1B and DPP-4 activities is made, including their type of inhibition and experimental conditions, and structure-activity relationship, covering a total of 351 compounds. We intend to provide the most favorable chemical features of flavonoids for the inhibition of PTP1B and DPP-4, gathering information for the future development of compounds with improved potential as T2D therapeutic agents.

Pro-inflammatory effects of silver nanoparticles in the intestine.

Nanotechnology is a promising technology of the twenty-first century, being a rapidly evolving field of research and industrial innovation widely applied in our everyday life. Silver nanoparticles (AgNP) are considered the most commercialized nanosystems worldwide, being applied in diverse sectors, from medicine to the food industry. Considering their unique physical, chemical and biological properties, AgNP have gained access into our daily life, with an exponential use in food industry, leading to an increased inevitable human oral exposure. With the growing use of AgNP, several concerns have been raised, in recent years, about their potential hazards to human health, more precisely their pro-inflammatory effects within the gastrointestinal system. Therefore a review of the literature has been undertaken to understand the pro-inflammatory potential of AgNP, after human oral exposure, in the intestine. Despite the paucity of information reported in the literature about this issue, existing studies indicate that AgNP exert a pro-inflammatory action, through generation of oxidative stress, accompanied by mitochondrial dysfunction, interference with transcription factors and production of cytokines. However, further studies are needed to elucidate the mechanistic pathways and molecular targets involved in the intestinal pro-inflammatory effects of AgNP.

Nano-based drug delivery systems used as vehicles to enhance polyphenols therapeutic effect for diabetes mellitus treatment.

Diabetes mellitus is one of the biggest health emergencies of the 21st century worldwide, characterized by deficiency in insulin secretion and/or action, leading to hyperglycemia. Despite the currently available antidiabetic therapeutic options, 4.2 million people died in 2019 due to diabetes. Thus, new effective interventions are required. Polyphenols are plant secondary metabolites and have been recognized for their vast number of biological activities, including potential antidiabetic effects. However, the poor bioavailability and high metabolization of polyphenols restrict their biological effects in vivo. Nanotechnology is a promising area of research to improve the therapeutic effect of several compounds. Therefore, this review provides an overview of the literature about the utility of nano-based drug delivery systems as vehicles of polyphenols in diabetes treatment. It was possible to conclude that, in general, nano-based drug delivery systems can potentiate the beneficial antidiabetic properties of polyphenols, when compared with the free compounds, opening a new field of research in diabetology.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Protective Role of Flavonoids against Intestinal Pro-Inflammatory Effects of Silver Nanoparticles.

Silver nanoparticles (AgNP) have been increasingly incorporated into food-related and hygiene products for their unique antimicrobial and preservative properties. The consequent oral exposure may then result in unpredicted harmful effects in the gastrointestinal tract (GIT), which should be considered in the risk assessment and risk management of these materials. In the present study, the toxic effects of polyethyleneimine (PEI)-coated AgNP (4 and 19 nm) were evaluated in GIT-relevant cells (Caco-2 cell line as a model of human intestinal cells, and neutrophils as a model of the intestinal inflammatory response). This study also evaluated the putative protective action of dietary flavonoids against such harmful effects. The obtained results showed that AgNP of 4 and 19 nm effectively induced Caco-2 cell death by apoptosis with concomitant production of nitric oxide, irrespective of the size. It was also observed that AgNP induced human neutrophil oxidative burst. Interestingly, some flavonoids, namely quercetin and quercetagetin, prevented the deleterious effects of AgNP in both cell types. Overall, the data of the present study provide a first insight into the promising protective role of flavonoids against the potentially toxic effects of AgNP at the intestinal level.

Chalcones as Modulators of Neutrophil Oxidative Burst under Physiological and High Glucose Conditions.

Several epidemiological studies indicate that neutrophils, under hyperglycemic conditions, are involved in the perpetuation of the inflammatory status, a characteristic of diabetes mellitus, leading to the production of prodigious quantities of reactive species and the release of neutrophil extracellular traps (NETs). Accordingly, our aim was to study the ability of a panel of 25 structurally related chalcones to modulate human neutrophil oxidative burst and the production of NETs under physiological and high glucose conditions. In general, all chalcones presented similar effects under physiological and high glucose conditions. 2',4-Dihydroxy-3-methoxychalcone (3), here studied for the first time, was the most active (IC50 ≤ 5 µM) on the inhibition of neutrophil oxidative burst, showing the importance of the presence of hydroxy substituents at the C-2' and C-4 positions of the A and B rings, respectively, and a 3-methoxy substituent at B ring of the chalcone scaffold. In the present experimental conditions, NETs release only occurred under high glucose levels. The pentahydroxylated chalcone 1 was the only one that was able to modulate the NETs release. This study provided important considerations about the chalcones' scaffold and their modulatory effect on human neutrophil activities at physiological and high glucose conditions, evidencing their potential use as complementary antidiabetic agents.

Structural Specificity of Flavonoids in the Inhibition of Human Fructose 1,6-Bisphosphatase.

Liver fructose 1,6-bisphosphatase (FBPase) is a recognized regulatory enzyme of the gluconeogenesis pathway, which has emerged as a valid target to control gluconeogenesis-mediated overproduction of glucose. As such, the management of diabetes with FBPase inhibitors represents a potential alternative for the currently used antidiabetic agents. In this study, the FBPase inhibition of a panel of 55 structurally related flavonoids was tested, through a microanalysis screening system. Then, a subset of seven active inhibitors and their close chemical relatives were further evaluated by molecular dynamics (MD) simulations using a linear interaction energy (LIE) approach. The results obtained showed that D14 (herbacetin) was the most potent inhibitor, suggesting that the presence of -OH groups at the C-3, C-4', C-5, C-7, and C-8 positions, as well as the double bond between C-2 and C-3 and the 4-oxo function at the pyrone ring, are favorable for the intended effect. Furthermore, D14 (herbacetin) is stabilized by a strong interaction with the Glu30 side chain and the Thr24 backbone of FBPase. This is the first investigation studying the in vitro inhibitory effect of a panel of flavonoids against human liver FBPase, thus representing a potentially important step for the search and design of novel inhibitors of this enzyme.

Evaluation of a flavonoids library for inhibition of pancreatic α-amylase towards a structure-activity relationship.

α-Amylase has been considered an important therapeutic target for the management of type 2 diabetes mellitus (T2DM), decreasing postprandial hyperglycaemia (PPHG). In the present work, a panel of 40 structurally related flavonoids was tested, concerning their ability to inhibit α-amylase activity, using a microanalysis screening system, an inhibitory kinetic analysis and molecular docking calculations. From the obtained results, it was possible to observe that the flavone with a -Cl ion at 3-position of C-ring, an -OH group at 3'- and 4'- positions of B-ring and at 5- and 7- positions of A-ring and the C2 = C3 double bond, was the most active tested flavonoid, through competitive inhibition. In conclusion, some of the tested flavonoids have shown promising inhibition of α-amylase and may be considered as possible alternatives to the modulation of T2DM.

α-Glucosidase inhibition by flavonoids: an in vitro and in silico structure-activity relationship study.

α-Glucosidase inhibitors are described as the most effective in reducing post-prandial hyperglycaemia (PPHG) from all available anti-diabetic drugs used in the management of type 2 diabetes mellitus. As flavonoids are promising modulators of this enzyme's activity, a panel of 44 flavonoids, organised in five groups, was screened for their inhibitory activity of α-glucosidase, based on in vitro structure-activity relationship studies. Inhibitory kinetic analysis and molecular docking calculations were also applied for selected compounds. A flavonoid with two catechol groups in A- and B-rings, together with a 3-OH group at C-ring, was the most active, presenting an IC50 much lower than the one found for the most widely prescribed α-glucosidase inhibitor, acarbose. The present work suggests that several of the studied flavonoids have the potential to be used as alternatives for the regulation of PPHG.

Effects of Chrysosporum (Aphanizomenon) ovalisporum extracts containing cylindrospermopsin on growth, photosynthetic capacity, and mineral content of carrots (Daucus carota).

Natural toxins produced by freshwater cyanobacteria, such as cylindrospermopsin, have been regarded as an emergent environmental threat. Despite the risks for food safety, the impact of these water contaminants in agriculture is not yet fully understood. Carrots (Daucus carota) are root vegetables, extensively consumed worldwide with great importance for human nourishment and economy. It is, therefore, important to evaluate the possible effects of using water contaminated with cyanotoxins on carrot cultivation. The aim of this work was to investigate cylindrospermopsin effects on D. carota grown in soil and irrigated for 30 days, with a Chrysosporum ovalisporum extract containing environmentally relevant concentrations of cylindrospermopsin (10 and 50 µg/L). The parameters evaluated were plant growth, photosynthetic capacity, and nutritional value (mineral content) in roots of carrots, as these are the edible parts of this plant crop. The results show that, exposure to cylindrospermopsin did not have a clear negative effect on growth or photosynthesis of D. carota, even leading to an increase of both parameters. However, alterations in mineral contents were detected after exposure to crude extracts of C. ovalisporum containing cylindrospermopsin. A general decline was observed for most minerals (Ca, Mg, Na, Fe, Mn, Zn, Mo, and P), although an increase was shown in the case of K and Cu, pointing to a possible interference of the cyanobacterial extract in mineral uptake. This study is the first to evaluate the effects of C. ovalisporum extracts on a root vegetable, however, more research is necessary to understand the effects of this toxin in environmentally relevant scenarios.

Combined dual effect of modulation of human neutrophils' oxidative burst and inhibition of colon cancer cells proliferation by hydroxycinnamic acid derivatives.

Colon cancer is one of the most incident cancers in the Western World. While both genetic and epigenetic factors may contribute to the development of colon cancer, it is known that chronic inflammation associated to excessive production of reactive oxygen and nitrogen species by phagocytes may ultimately initiate the multistep process of colon cancer development. Phenolic compounds, which reveal antioxidant and antiproliferative activities in colon cancer cells, can be a good approach to surpass this problem. In this work, hydroxycinnamic amides and the respective acid precursors were tested in vitro for their capacity to modulate human neutrophils' oxidative burst and simultaneously to inhibit growth of colon cancer cells. A phenolic amide derivative, caffeic acid hexylamide (CAHA) (4) was found to be the most active compound in both assays, inhibiting human neutrophils' oxidative burst, restraining the inflammatory process, inhibiting growth of colon cancer cells and triggering mitochondrial dysfunction that leads cancer cells to apoptosis. Altogether, these achievements can contribute to the understanding of the relationship between antioxidant and anticancer activities and based on the structure-activity relationships (SAR) established can be the starting point to find more effective phenolic compounds as anticancer agents.

Biodistribution of polyacrylic acid-coated iron oxide nanoparticles is associated with proinflammatory activation and liver toxicity.

Iron oxide nanoparticles (IONs) have physical and chemical properties that render them useful for several new biomedical applications. Still, so far, in vivo safety studies of IONs with coatings of biomedical interest are still scarce. The aim of this study, therefore, was to clarify the acute biological effects of polyacrylic acid (PAA)-coated IONs, by determining their biodistribution and their potential proinflammatory and toxic effects in CD-1 mice. The biodistribution of PAA-coated IONs in several organs (liver, spleen, kidneys, brain, heart, testes and lungs), the plasma cytokines, chemokine and aminotransferases levels, white blood cell count, oxidative stress parameters, adenosine triphosphate and histologic features of liver, spleen and kidneys were evaluated 24 h after a single acute (8, 20 or 50 mg kg(-1) ) intravenous administration of PAA-coated IONs in magnetite form. The obtained results showed that these IONs accumulate mainly in the liver and spleen and, to a lesser extent, in the lungs. Although our data showed that PAA-coated IONs do not cause severe organ damage, an inflammatory process was triggered in vivo, as evidenced by as evidenced by increased neutrophils and large lymphocytes in the differential blood count. Moreover, an accumulation of iron in macrophages of the liver and spleen was observed and hepatic lipid peroxidation was elicited, showing that the IONs are able to induce oxidative stress. The effects of these nanoparticles need to be further investigated regarding the mechanisms involved and the long-term consequences of intravenous administration of PAA-coated IONs. Copyright © 2016 John Wiley & Sons, Ltd.

Proinflammatory Pathways: The Modulation by Flavonoids.

Inflammation is a natural, carefully orchestrated response of the organism to tissue damage, involving various signaling systems and the recruitment of inflammatory cells. These cells are stimulated to release a myriad of mediators that amplify the inflammatory response and recruit additional cells. These mediators present numerous redundancies of functions, allowing a broad and effective inflammatory response, but simultaneously make the understanding of inflammation pathways much difficult. The extent of the inflammatory response is usually self-limited, although it depends on the balance between the pro- and anti-inflammatory signals. When that equilibrium is dislocated, a more widespread inflammatory response may take place. Flavonoids have been shown to be possible alternatives to the traditionally molecules used as anti-inflammatory agents. In fact, the biological activities of flavonoids include the modulation of the diverse phases of inflammatory processes, from the gene transcription and expression to the inhibition of the enzymatic activities and the scavenging of the reactive species. In the present review, the inflammatory network is widely revised and the flavonoids' broad spectrum of action in many of the analyzed inflammatory pathways is revised. This kind of integrated revision is original in the field, providing the reader the simultaneous comprehension of the inflammatory process and the potential beneficial activities of flavonoids.

Polyacrylic acid-coated and non-coated iron oxide nanoparticles induce cytokine activation in human blood cells through TAK1, p38 MAPK and JNK pro-inflammatory pathways.

Iron oxide nanoparticles (ION) can have a wide scope of applications in biomedicine, namely in magnetic resonance imaging, tissue repair, drug delivery, hyperthermia, transfection, tissue soldering, and as antimicrobial agents. The safety of these nanoparticles, however, is not completely established, namely concerning their effect on immune system and inflammatory pathways. The aim of this study was to evaluate the in vitro effect of polyacrylic acid (PAA)-coated ION and non-coated ION on the production of six cytokines [interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), interferon gamma (IFN-γ) and interleukin 10 (IL-10)] by human peripheral blood cells, and to determine the inflammatory pathways involved in this production. The obtained results showed that PAA-coated and non-coated ION were able to induce all the tested cytokines and that activation of transforming growth factor beta (TGF-ß)-activated kinase (TAK1), p38 mitogen-activated protein kinases (p38 MAPK) and c-Jun N-terminal kinases (JNK) were involved in this effect.

Effects of microcystin-LR, cylindrospermopsin and a microcystin-LR/cylindrospermopsin mixture on growth, oxidative stress and mineral content in lettuce plants (Lactuca sativa L.).

Toxic cyanobacterial blooms are documented worldwide as an emerging environmental concern. Recent studies support the hypothesis that microcystin-LR (MC-LR) and cylindrospermopsin (CYN) produce toxic effects in crop plants. Lettuce (Lactuca sativa L.) is an important commercial leafy vegetable that supplies essential elements for human nutrition; thus, the study of its sensitivity to MC-LR, CYN and a MC-LR/CYN mixture is of major relevance. This study aimed to assess the effects of environmentally relevant concentrations (1, 10 and 100 µg/L) of MC-LR, CYN and a MC-LR/CYN mixture on growth, antioxidant defense system and mineral content in lettuce plants. In almost all treatments, an increase in root fresh weight was obtained; however, the fresh weight of leaves was significantly decreased in plants exposed to 100 µg/L concentrations of each toxin and the toxin mixture. Overall, GST activity was significantly increased in roots, contrary to GPx activity, which decreased in roots and leaves. The mineral content in lettuce leaves changed due to its exposure to cyanotoxins; in general, the mineral content decreased with MC-LR and increased with CYN, and apparently these effects are time and concentration-dependent. The effects of the MC-LR/CYN mixture were almost always similar to the single cyanotoxins, although MC-LR seems to be more toxic than CYN. Our results suggest that lettuce plants in non-early stages of development are able to cope with lower concentrations of MC-LR, CYN and the MC-LR/CYN mixture; however, higher concentrations (100 µg/L) can affect both lettuce yield and nutritional quality.